Blige

oh rs ret red

Sree hie

 

cll ee ete

 

 

a
Sc
Bee ten te ee
y teeta

 

 

 

 

 

 

Ceres ass

et Seca
eee its

fob

 

 

   

 

sil cas

toe ee oT
R. B. HINMAN
COLLECTION

 

PROFESSOR OF ANIMAL HUSBANDRY
1921-1943

New York
State College of Agriculture
At Cornell University
. Ithaca, N. Y.
Cornell University Library

nA
PHYSIOLOGICAL
CHEMISTRY

A TEXT-BOOK AND MANUAL FOR
STUDENTS

BY
ALBERT P. MATHEWS, Ph.D.

PROFESSOR OF BIOCHEMISTRY, THE UNIVERSITY OF CINCINNATI

THIRD EDITION

ILLUSTRATED to

NEW YORK
WILLIAM WOOD AND COMPANY
CopyrricxT, 1920
By WILLIAM WOOD & COMPANY

 

First Edition, September, 1915
Reprinted, February, 1916
Reprinted, March, 1916

 

Second Edition, September, 1916
Reprinted, August, 1917
Reprinted, -sptember, 1918
Reprinted, Augnet. 1919

 

Third Edition, September, 1020
Reprinted, July, 1921
Renrinted. October. ro21
Reprinted, August, 1923
Reprinted, October, 1923
Reprinted, July, 1924

Printed in the United States of America
TO
MY WIFE
PREFACE

I hope that this book may raise in the minds of those who read it
more questions than it answers. Enormous as the science of physio-
logical chemistry, or bio-chemistry, has grown to be, covering: as it
does the whole of the chemical and physico-chemical phenomena of
living nature, only a beginning has as yet been made in it. To few
of its fundamental questions can we now give an answer. The great
discoveries remain for the future. To arouse interest in the subject,
to stimulate curiosity and inquiry, are the main objects of every teacher.
I hope that in the pages which follow I have not hit too wide of this
mark,

-Of so large a subject one can be personally familiar with but a small
part. It is difficult to estimate the value of work done in fields other
than those in which one has worked. It may be that the emphasis has
‘not always been put in the right place. Some parts of the subject have
been treated far more fully than others, and, possibly, more fully than
their importance deserves. The chapters on the chemistry of the carbo-
hydrates, fats and proteins and the physical chemistry of the cell are
longer than is usual. But a thorough knowledge of this part of the
subject is essential to a comprehension of physiology and pathology. On
the other hand, this has necessitated a briefer treatment than they de-
served of some other matters. I have not been able to consult the whole
of the vast literature of biochemistry and I know that many valuable
and suggestive papers have probably escaped my attention. At the end
of each chapter there will be found a short list of papers bearing on
the subject dealt with in that chapter. Many of these should be read
by students. and material may be taken from them for conferences. Most
of these papers are recent. They have been chosen not because they are
necessarily better than older papers, for the reverse may be the case,
but because in them the older literature is cited and they reflect the
more modern point of view. While I have expressed opinions here and
there, I have, as far as space permits, given definite experiments rather
vi PREFACE

than conclusions only, so that the reader may judge the evidence for
himself.

In the preparation of the practical work I have been assisted by my
colleague, Professor F. C. Koch, whose aid is gratefully acknowledged.
For the derivation of the scientific words and their meanings I have
relied on the excellent Medical Dictionary of Stedman. I have drawn
freely for tables and cuts on other works. .

UNIVERSITY oF CHICAGO,
May, 1915.

PREFACE TO THE THIRD EDITION

In the third edition the practical part has been rearranged, largely
rewritten, many: new and important methods, such as those of blood
analysis, have been added, and many revisions made in the text ‘required
by the development of knowledge since the second edition was published.
This revision has been most extensive in the chapter on vitamines.

Cincinnati. May, 1920.
CONTENTS.
PART 1. é

THE CHEMISTRY OF PROTOPLASM AND THE CELL

CHAPTER

I.

THE GENERAL PROPERTIES OF LIVING MATTER

Definition of living. Difference between living and lifeless.
Lavoisier. Protoplasm. Physical appearance. Origin of living
things. Energy changes. Psychic phenomena. Origin of living
energy. Combustive processes. Hydrations. Dehydrations. Con-
densations. Speed of living reactions. Catalysis. Enzymes.
Orderliness of reactions. Organization of cell. Colloids. General
chemical composition. Water. Inorganic salts. Organic sub-
stances.

ff. THE CARBOHYDRATES

{II.

Occurrence. Definition. Classification Monosaccharides;
isomerism Optical properties. Molecular form. Specific rotatory
power. Polariscope. Structural formulas of hexoses and pen-
toses. Dissociation of monosaccharides. Reactions of biological
interest. Action of alkali on monosaccharides; on di- and
polysaccharides. Action of acids on monosaccharides. On poly-
saccharides. Oxidation. Fehling’s solution. By copper acetate.
Reduction of carbohydrates. Reaction with hydrocyanic acid.
Oximes. Osazones. With ammonia. Synthesis in plants Special
properties of various carbohydrates. Levulose. Mutarotation.
Glucose or Dextrose. Galactose. Glucosides. Disaccharides.
Cane sugar, or saccharose. Lactose. Maltose. Colloidal poly-
saccharides. Starch. Cellulose. References.

THE LIPINS. FATS. OILS. WAXES. PHOSPHATIDES.

STEROLS

Properties. Classification. Historical. Amount. Fats and
fatty oils. Composition. Physical properties. Glycerol. Fatty
oils. Resemblance of chemistry of painting to biological processes.
Methods of identification of fats and oils. Melting points Iodine
number. Iodine value of various fats, oils, and waxes. Hydrogen
number. Ozonides. Saponification. Saponification number.
Reichert-Meissl number. Acetyl number. Separation of fatty
acids. Physiological value of fats. Oxidation. Origin of fats.
Essential oils. Waxes. Composition of waxes. Sterols. Choles-
terol. Reactions: Salkowski, Liebermann-Burchard; Schifi’s, oxy-
cholesterol; methyl furfural reaction. Quantitative determination.
Amount in different tissues. Chemistry of cholesterol. Physio-
logical importance. Other sterols. Phospholipins or phosphatides.
Definition. Classification. Method of separation. Lecithin.
Choline Quantitative determination of choline. Amount of
choline in various tissues. Cuorin. Physical properties of phos-
pholipins. Auto-oxidation. Other functions. Hydrolysis of phos-
pholipins. Other phospholipins. Glycolipins. Localization of
phospholipins in cells. References,

IV. THE PROTEINS ; :

Occurrence. Methods of extraction. Composition. Properties.
Definition. Classification. Decomposition products. Important

1X

PAGE

16

61

104
x

OHAPTER

CONTENTS

properties of amino-acids. Union with salts, acids, alkalies, alde-
hydes. Carbamino reaction. Deamidization. Lactams and
piperazine nuclei. Taste. Uptical properties. Amounts of amino-
acids in different proteins. Structure of protein molecule Syn-
thesis. Number of free amino and carboxyl groups. Molecular
weight. Distribution of nitrogen. Color reactions. Precipitation
reactions. Chromoproteins. Distribution of protein substances
between nucleus and cytoplasm. Chemistry of cell nucleus.

‘Methods of obtaining nuclei. Composition of chromatin. Nucleic

acid. Basic constituents of nucleus. Protamines. Enzymes in
nucleus. Formation and destruction of nuclear material. Refer-
ences.

V. THE PHYSICAL CHEMISTRY OF PROTOPLASM .

Water. Salts. Ionic theory. Osmotic pressure. Surface
tension. Surface films. Colloids. Gels. Water absorption. Os-
motic pressure of colloidal solutions. Electrical phenomena. Re-
action. Catalysis. Oxidation. Summary. References.

PART IL

THE MAMMALIAN BODY CONSIDERED AS A MACHINE.
ITS GROWTH, MAINTENANCE, ENERGY TRANSFOR-
MATIONS AND WASTE SUBSTANCES

VI.

VII.

VIII.

The body resembles a magnet.

ANIMAL HEAT .

History. Lavoisier. Origin of heat. Conservation of energy.
Dulong and Depretz. Respiratory quotient. Regnault and
Reiset. Rubner. Atwater-Rosa-Benedict calorimeter. Income
and outgo of energy. Growth. Summary References.

THE RAW MATERIALS OR FOODS

Definition. Water. Importance of diversity. Amount re-
quired. Composition of some foodstuffs. Milk. Proteins in milk.
Lipins. Lactose. Other organic constituents. Inorganic constit-
uents. Milk glands. Enzymes. Souring. Foreign substances
in milk. Various kinds of milk. Eggs. Egg white, composition.
Yolk, composition. References.

SALIVARY DIGESTION

Digestion in general. Saliva, origin. Nervous control of
secretion. Composition. Amount secreted. Functions. Chem-
istry of mucin. Chondroitie acid. Preparation of mucin. Diges-
tive action of saliva> Starch digestion. Ptyalin. Determination
of activity of amylase. Conditions of activity. Inhibition by
products of digestion. Law of action of ptyalin. Time of appear-
ance in development. Variation in different animals. Other
enzymes of saliva. Importance of salivary digestion. Composi-
tion of salivary glands. References.

IX. DIGESTION IN THE STOMACH .

Morphology. General physiology of human stomach. Manner
of obtaining gastric juice. Pure gastric juice. composition.
Amount. Variation of character with diet. Gastric hormones.
Gastrin. Digestive actions of juice. Pepsinogen. Pepsin. Con-
ditions of activity. Law of action. Products of peptic digestion.
Character of linkings attacked by pepsin. Energy changes in

PAGE

190

266

269

300

319
CHAPTER

CONTENTS

digestion. Fate of pepsin. Hydrochloric acid. Methods of quan-
titative determination. Variation of secretion in disease. Origin
of acid. Rennin and its action. Salivary and intestinal digestion
in the stomach. Summary of gastric digestion. References.

X. DIGESTION IN THE INTESTINE

XI.

XII.

XIII.

Duodenal secretion. Enterokinase. Enzymes in duodenal
juice. Other functions of the duodenum. Pancreatic juice. Com-
position. Control of secretion. Secretin. Digestive action on
fats. Steapsin. Conditions of action. On carbohydrates. Amyl-
opsin, properties. Lactase and maltase. On proteins. Trypsin.
Law of action. Nuclease. Summary of pancreatic juice. The
bile. Coimposition. Secretion. Amount. Functions. Circulation.
Influence on putrefaction. Chemistry of bile pigments. Bilirubin,
ete. Where are pigments made? Transformation of hemoglobin
to pigments. Bile salts. Preparation and properties. Glycocholie
acid. Taurocholic acid. Sulphur in bile. Cholie acid. Glyco-
choleic, taurocholeic and choleic acids. Soaps. Cholesterol in bile.
Stercorin. Phospholipins in bile. Mucin. Bacterial decomposi-
tion of food in intestine. Feces. Decomposition of proteins,
amines and aporrhegmas Methods of reducing putrefaction.
Summary of digestion. References.

ABSORPTION

Fats. Carbohydrates. Proteins. In different parts of tract.
Conclusion. References.

THE CIRCULATING TISSUE. THE BLOOD

Lymph. Functions Composition in general. Blood a living
tissue. White corpuscles. Red corpuscles, or erythrocytes.
Platelets. Plasma. Vivi-diffusion. Non-protein nitrogen, amount.
Respiratory function. Amount of gases in blood. Oxygen, how
carried. Carbon dioxide. Mechanism of exchange in lungs. Nature
of union of hemoglobin with oxygen. Dissociation, conditions of.
Temperature, acids and alkalies, salts and other factors. Bio-
logical significance. Exchange of oxygen in tissues. Respiration
of blood itself. Evolution of hemoglobin. Compounds of hemo-
globin with other gases. Summary of oxygen. carrying power.
Laking. Composition of red corpuscles. Chemistry of hemo-
globin. Occurrence. Crystalline forms. Oxyhemoglobin, prop-
erties. Hemoglobin. Globin. Hematin and hemin. Hematopor-
phyrin. Hemochromogen. Blood as carrier of waste products. As
distributor of internal secretions. Viscosity. Coagulation of
blood. Alkalinity of blood. Hydrogen ion content and method of
determination. Gas chain. Indicator method. Osmotic pressure
of blood. Conductivity. Enzymes in blood. Proteins of plasma,
amount. Fibrinogen. Serum globulin. Serum albumin. Origin
of proteins. Functions. Function of endothelium. References,

THE MASTER TISSUE OF THE BODY. THE BRAIN

Chemistry and metabolism. Structure. Chemistry. Thu-
dichum. Separation of phospholipins. Lecithin Kephalin.
Paramyelin. Myelin. Diamino-mono-phosphatides. Amidomyelin.
Sphingomyelin Diamino-diphosphatides. Assurin. Cerebrosides
or galactosides. Phrenosin and kerasin. Protagon. Cerebro-
sulphatides. Sulpholipins. Amido-lipins. Krinosin. Bregenin.
Sterols. Extractives. Caprine. Inosite. Distribution of sub-
stance between gray and white matter. Cerebro spinal fluid.
Physiological interpretation of chemical composition. Medullary
sheaths, function. Memory, physical basis of. Oxygen exchange
of brain. Summary. References.

PAGE

384

451

458

563
xii CONTENTS

CHAPTER PAGE
XIV. THE CONTRACTILE TISSUES. MUSCLE bp siaten 18 ; 597

Amount. General composition. Proteins of plasma, iaealli,
myogen, etc. Stroma proteins. Proteins of smooth muscle. Ex-
tractives. Creatine. Carnosine. Ignotine. Carnitine. Novain.
Taurine. Purines. Glycocoll. Inosine. Inosinic acid. Inosite.
Mytilite. Xylose. Carnie and phosphocarnic acid. Succinie acid.
Lipins. Inorganic. Internal secretion. Formative metabolism.
Energy metabolism. Glycogen content. Glycolytic power. Lactic
acid. Rdle of nucleus. Mechanism of contraction. References.

XV. THE CONNECTIVE, OR SUPPORTING TISSUES. THE BONES.
CARTILAGE. TEETH. CONNECTIVE TISSUE 63)

White tissue. Tendo Achillis. Composition. Yellow connec-
tive tissue. Ligamentum nuchae. Composition. Cartilage.
Chondroitice acid. Bones, organic; inorganic. Teeth.

XVI. THE CRYPTORRHETIC TISSUES. THE THYROID. PARA-
vw  'THYROID. HYPOPHYSIS. SUPRA-RENAL. REPRODUC-
a TIVE GLANDS. PINEAL GLAND. THYMUS : 640

Hypophysis. Structure. Acromegaly. Extirpation. Pitui-
trin. References. Parathyroids, or epithelial bodies. Thyroids.
Histology, function. Myxedema. Cretinism. Basedow’s disease.
Extirpation effects. Nitrile reaction. Active principle. Iodo-
thyrin. Thyreoglobulin. Supra-renal capsules. Anatomy and
histology. Embryology. Functions. Effects of extirpation.
Adrenaline, or adrenine. Amount in glands. Secretion. Composi-
tion. Sexual glands. Thymus. Pineal gland. References.

XVII. THE EXCRETIONS OF THE BODY. URINE. ... 681

Amount of various excretions. Secretion of urine’ Amount,
specific gravity. Acidity. Osmotic pressure. General composi-
tion. Nitrogenous constituents. Urea, composition, chemistry,
origin, and variation. Creatinine and creatine, Chemistry.
Amount secreted. Origin. Purine bodies and allantoine. Uric
acid, chemistry, amount secreted, origin. Allantoine. Nucleic
acid metabolism. Hippuric acid. Ammonia. Amino acids and
peptides. Urocanic acid. Various bases. Aromatic oxy acids,
phenol, indoxyl, scatoxyl, phenyl acetic, etc. Ethereal sulphates.
Homogentisie acid. Sulphur of urine. Chlorides. Calcium and
magnesium. Pathological constituents. Protein. Carbohydrates.
Acetone and diacetic acid. Hydroxybutyric acid. Metabolism of
various substances not foods. Pairing with glycuronic acid; with
glycine; with sulphuric acid; with ornithine; uramido acids;
nitriles and cyanides; methylation and demethylation. Conelu-
sion. Pigments. References.

XVIII. THE METABOLISM OF THE BODY CONSIDERED AS A
WHOLE. CARBOHYDRATE METABOLISM 787

Claude Bernard. Diabetes. Glycogen function of liver.
Sugar puncture. Origin of glycogen in carbohydrates and pro-
teins. Glycogen to glucose. Influence of pancreas. Internal se-
cretion of pancreas. Glyoxalase. Cause of diabetes. Summary
of réle of liver. Conditions of sugar burning in muscle. D:N
ratio. Phlorhizin diabetes. References.

XIX. PROTEIN METABOLISM OF BODY . ; ise 795

Amount of protein needed. Cornaro and Fletcher. “Is mini-
mum protein desirable? Protein storage. Catabolism of proteins.
Course of oxidation of various amino acids. Origin of aceto-
acetic acid. Sulphur metabolism. Synthesis of amino acids in
body. References.
CHAPTER
XX.

CONTENTS

“METABOLISM UNDER VARIOUS CONDITIONS. -VITAMINES.
RESPIRATION AND CONCLUSION ._.

Metabolism in starvation. Lack of water nid, inineral abs
stances. Vitamines. Beri-beri. Pellagra. ‘Scurvy. Vitamines
in growth. Tissue respiration.’ References. ;

PART III.

PRACTICAL WORK AND METHODS. . . . . .

_ XXL

XXII.

XXITI.

XXIV.

XXV.

XXVI.

XXVII.
XXVIII.

XXIX.

XXX.

EQUIPMENT OF THE, LABORATORY

Desk reagents. A word to the student. Side shelf reagents,
Special apparatus. Desk outfits and blanks. General directions
for work. Filtering. . Pipettes.

QUANTITATIVE ANALYSIS .

Equivalent solutions. Normal solutions. Acid. Normal solu-

tions. Alkalies. Indicators. Quantitative determination of nitrogen.

THE CARBOHYDRATES BR RS OE NB SG

Tests for detection. Reducing powers of carbohydrates.
Methods for the identification of particular carbohydrates De-
composition of carbohydrates by enzymes; by acids. Methods for
quantitative estimation.

-THE FATS .

Physical properties of oils sin fats, Saponification. Methods
of identification. Preparation and properties of phospholipins,
sterols, etc.

THE PROTEINS

Elementary composition anil teas for detection, " Methods
of precipitating. Methods of isolating and preparing different
kinds. Quantitative determination of nitrogen and phosphorus
in. Van Slyke amino nitrogen method Preparation of trypto-
phane. Isolation of amino acids by Dakin method. Prepara-
tion of tyrosine and cystine. Preparation of proline and glutamic
acid.

COMPOSITION OF THE FOODS ee Ue GR a, eg
Milk. Meat. Bone. Potato. Bread.
SALIVARY DIGESTION Pe ee ee ee

GASTRIC DIGESTION... es
Pepsin, pepsinogen, rennin and hydrochloric mre Quantite-
tive methods for determining pepsin. Examination of gastric
contents.
INTESTINAL INDIGESTION
Pancreas and doudenal secretion. Pancreatic amylase, lipase
and protease. Erepsin. The bile. The feces.

THE BLOOD

Determination of corpuscles. Determination ae hemoglobin.

Laking. Hemoglobin, decomposition products, spectra, etc. Co-
agulation. Quantitative chemical analysis Non-protein nitrogen
by Folin and Wu method. Urea nitrogen by Folin and Wu method.
Creatinine by Folin and Wu method. Creatine by Folin and Wu
method. Uric acid by Folin and Wu method. Sugar by Folin and
Wu method. Non protein nitrogen by Greenwald method. Creati-

xiii
PAGH

829

858

858

864

870

904

915

949

962
965

977

990
xiv

CHAPTER

XXXI.

CONTENTS

nine and sugar by Lewis-Benedict method. Uric acid by Benedict’s
modification of Folin’s. Urea by Van Slyke modification of Mar-
shall. Clorides in blood. . Sodium and potassium in blood.
Calcium and magnesium in blood. Alkali reserve by Van Slyke and
Cullen, Hydrogen ion concentration. :

THE URINE a ee ec a

Preparation and properties of urea and uric acid. Creatinine,
hippuric acid and indican. Detection of pathological constituents.
Albumin. Dextrose. Lactose. Acetone, acetoacetic acid and
hydroxybutyric. Glycuronic acid and pentoses. Bile. Blood.
Quantitative determination of urinary constituents. Total nitro-
gen. Urea. Ammonia. Creatinine. Uric acid. Hippuric acid.
Allantoine. Total purines. Total acidity. Amino acids. Total
sulphuric acid. Conjugated sulphates. Total sulphur. Chlorides.
Calcium. Phosphates Glucose by various methods. Acetone. Di-
acetic acid. Organic acids by titration (diacetic, ete.). Saccharose.
Hydrogen ion concentration. Adrenaline.

1060
PART I

THE CHEMISTRY OF PROTOPLASM
AND THE CELL
CHAPTER I.
THE GENERAL PROPERTIES OF LIVING MATTER.

The various objects on the surface of the earth may be divided into
two great classes, the living and the lifeless: the former being char-
acterized by the possession of certain properties which the latter lack.
The first of the distinctive properties of living matter is the power of
movement; and of movement having an internal rather than an external
origin. These movements are either from place to place as in animals;
or movements of growth and foliage as in plants. It is by the property .
of movement that we instinctively distinguish living and lifeless. A
second property is that of growth; growth not by the apposition of
particles to the outside of the living thing, but growth from within, by
the intercalation of substances within the organism. Another, the most
characteristic, and the only property it is certainly known that some
of the simpler organisms possess, organisms too small to be seen, is that
of reproduction. Such organisms are called living because they are
capable of indefinite multiplication. Finally we have two properties
which often require special apparatus for their detection, but which are,
none the less, fully as fundamental as the others, the properties, namely, :
of respiration and irritability. All living things respire, that is they
consume oxygen, liberate energy by combustion or oxidative changes,
and they give off a gas, carbon dioxide; and they are irritable; that is
they respond in some way, either by a change in the rate of reproduction,
in movement, in growth, or in some other of their functions when their
surroundings change. We cannot directly observe that many of the
smaller forms of life are irritable, but we believe from analogy that they.
must be so.

These five properties, movement, growth, reproduction, respiration
and irritability, are, hence, those properties possessed by living things,
and not possessed, or at least not all of them, by any non-living thing.
Their possession defines a living thing. When we speak of life we
mean this peculiar group of phenomena; and when we speak of explain.
ing life, we mean the explanation of these phenomena in the terms of
better known processes in the non-living.

How it happens that living things have these properties which are
lacking in the non-living has only within comparatively recent times be-
come a subject of scientific investigation. For many centuries the,
4 PHYSIOLOGICAL CHEMISTRY

problem was regarded as solved. Since living things are apparently
lifeless things plus something else, it was assumed that there was in
living things a spirit, an energy, an entelechy, or a demon, which did
not exist in lifeless matter, and to this spirit, or entelechy, all of these
peculiar vital properties were ascribed. It was not until the end of the
eighteenth and the beginning of the nineteenth century that this explana-
tion was doubted, and only since then has the attempt been made to
discover the origin of the vital properties.

To the solution of this problem many men have contributed and it is
perhaps invidious to pick out anyone for special mention, but physio-
logical chemistry certainly took a long stride forward, if indeed it may
not be said to have originated, about 1775-1793 in the work of that
great man of science, Lavoisier. In that beautiful series of papers
published in the Memoirs of the French Academy, papers which should
be read by every student of the science as true examples of real scientific
work, embodying the happiest combination of imagination and experi-
mental verification, Lavoisier showed that the heat of the body, that
peculiar property of the living body, was due to the burning, or com-
bustion, of its substances,—a burning analogous in all respects to the
combustion of a candle, or of a piece of coal. Animal heat and animal
respiration were thus correlated, and the living energy was seen to have
its origin in the combustion of hydrogen and carbon.

It remained, however, for the histologists to show what was the real
physical substratum of the living phenomena, and this grew immediately
out of the discovery of the compound microscope. Living things, in their
outward form, are extremely diverse, but when they are examined
microscopically it is found that all are composed of microscopic units
called cells. Within these cells there is a substance of a peculiar and
unique nature found nowhere else; a substance called by Dujardin, who
first described it in animals, sarcode; and by von Mohl, who saw it in
plants, protoplasm * (protos, first; plasma, form). This sarcode, or proto-
plasm, Dujardin described as a sticky, viscid, clear, or slightly granular,
substance, which would adhere to a glass rod and could be pulled out
in long thin strands, much as candy can be pulled out. In it was a more
refractive, spherical body called the nucleus, discovered by Robert Brown
in 1831. It was not, however, until about 1861 that sarcode and proto-
plasm were recognized as essentially identical in all plants and all ani-
mals, and the conclusion drawn that it was the real living basis, the
physical basis of life. Max Schulze especially contributed to the estab-
lishment of this conception.

The recognition of the fact that all living things had in them a sub-
stance essentially identical in its main features in all cells provided at
once a basis for those peculiar and common properties of living things.

*The name was given by Purkinje in 1839.
THE GENERAL PROPERT1&S OF LIVING MATTER 5

Irritability, respiration, growth, metabolism, movement are the properties
of living matter, or protoplasm. It is the chemistry of this substance and
its products with which the science of physiological chemistry, or bio-
chemistry, has to deal.

The physical appearance and consistence of this living matter varies
in different cells, sometimes being jelly-like in its rigidity; at other
times, or in other locations, decidedly fluid. It may be seen in many
vegetable cells, such for example as the fine stamen hairs of the spider-
lily, Tradescantia, or in Nitella, to be in active movement, the proto-
plasm keeping up a circulation within the cells; its flowings may carry
unicellular organisms from place to place; even in the cells of higher
animals, as in the eggs of one of the tunicates, the external layer of the
protoplasm appears fluid and may flow about the egg; and in the nerve
cell of the vertebrate brain its movements are supposed to make and
break those fine, inter-cellular connections at the basis of memory, asso-
ciation and thought. On the other hand, protoplasm may be quite jelly-
like and semi-rigid and highly elastic, as in the epithelial and muscle
cells of vertebrates; and it may be now rigid and now fluid as its state
changes with its condition of activity. These facts have been established,
in part, by Kite’s and Chambers’ microscopic dissection of cells by very
fine glass needles.

The optical appearance of living matter is that of a clear, trans-
parent ground substance in which are imbedded a great number of
granules of different sizes and often of different densities and: different
tints. It is generally believed, because of its uniformity and universality,
that the clear ground substance with the nucleus is the living substance
itself, and that the granules represent raw materials, or secretory, or
waste substances. The granules are generally colorless, but they may be
colored as in pigment cells, or in the blood cells of the sea-urchin,
Arbacia, where they are a beautiful deep red. They may be either
spherical, or rod shaped, ellipsoidal, or crystalline. When stains enter
living matter they may combine with and color the granules, but the
ground substance does not appear ever to color while it is living. Finally
living matter is always probably very slightly alkaline in reaction, but
it becomes acid on dying.

Living matter, therefore, is a substance found in all living things,
essentially the same in all, but differing somewhat in its physical appear-
ance and chemical’ composition in each particular kind of cell.
The physical and psychological complex of phenomena to which is
given the collective name of ‘‘ life ’’ is associated always, so far as we
know, with this substance, although each individual property may
be independent of it; and it is the problem of the science of physi-
ology to discover, to analyze these phenomena and, if possible, to find
6 PHYSIOLOGICAL CHEMISTRY

how they arise from the physical-chemical-psychic constitution of
protoplasm.

How the differentiation into living and lifeless arose on the earth
is still unknown, but most physiological chemists are of the opinion that
since living matter is to-day being constantly made out of lifeless, and
we have no reason to believe that the course of events was different in
this respect in the past, that living originated from lifeless; and, prob-
ably, not at one step, but as the result of a series of transformations
taking in the first instance a very long time. It must be remembered,
too, in considering the gap between living and lifeless, that while this
appears to be wide and profound, if we consider the higher organisms
such as man himself, it is not so profound if we consider the very sim-
plest forms of life. Living forms exist so minute as to be almost, or quite,
beyond the realm of microscopic vision; such forms can have only the
simplest structure, since their volume is so small that they can contain
only a small number of molecules of the size of those in living matter.
The difference between these forms and lifeless matter would seem to be
reduced almost to a simple chemical difference. In fact, the differences
between living and lifeless appear on closer examination to be quantita-
tive rather than qualitative.

Living matter is nearly always in movement, movements of growth,
of active streaming or of changes of shape; and since to move objects,
such as nuclei, requires that work be done, and since energy is that which
does work, living matter must be the seat of energy transformations.
It might be supposed that this energy, or capacity for work, was due
to some peculiar, non-physical, vital force or spirit, but experiment has
now clearly demonstrated that this is not the case, but that this energy
comes ultimately from light and immediately from the union of the living
matter, or its constituents, with oxygen. The law of conservation of
energy in living things is the most fundamental law of biology. Living
matter is, indeed, a machine for the transformation of chemical and other
forms of potential energy into various forms of kinetic energy, or into
the chemical energy of new compounds.

The kinetic energy of living things may appear as heat, as mass
movements, as light or as electrical energy. Thus all forms of living
matter are exothermic; they constantly produce heat, so that their
temperature is more or less above that of their environment. The
chemical transformations of living things are necessarily, for the most
part, exothermic. In some cases, however,.the energy appears as light
rather than heat. This is the case, for example, in the luminous organs
of the fire-fly; and probably in the phosphorescent organs of the
Ctenophores and in Noctiluca; in these forms combustion produces light,
and the liberation of heat is reduced to a minimum, so that the light
 

THE GENERAL PROPERTIES OF LIVING MATTER : 7

of the fire-fly may be said to be the most efficient lamp in existence, in
the sense of there being least waste of energy as heat.

Another form of energy set free by living things is electrical. Elec-
trical disturbances occur in all cells when combustion takes place in
them, but in some instances nearly the whole of the energy appears to
take this form instead of heat. This is well illustrated in the electrical
organ of the Torpedo, in which stimulation causes a strong electrical
current, so that this organ, made of modified muscle, is a very efficient ©
battery and a study of its physiology may ultimately show how fats,
sugars or other carbon compounds, or carbon itself, may be burnt with
the liberation of electrical energy in place of heat. But the most striking
example of this kind is found, probably, in the nerve impulse, which
though it is accompanied by, or is due to, the production of a large
amount of carbon dioxide, and is hence a direct or indirect oxidation,
nevertheless appears to generate no heat, but only a well-marked elec-
trical current of momentary duration. On the other hand, the muscle
cell has developed a mechanism by which much of the energy appears
to be used in producing molar movements; although here the larger
proportion still appears as heat.

Finally in all these cases some of the energy is re-transformed, with
some consumption of heat, into the potential energy of new chemical
compounds, forming thus new combustible substances.

Thus far a very important manifestation of living things has been
omitted, namely, the psychical phenomena which accompany the energy
transformations in our brains, and which we must believe arise in some
way from simple phenomena of the same kind perhaps occurring in every
chemical transformation. These psychical phenomena are omitted be-
cause it has not yet been possible to show that consciousness, or intel-
lectual activities, represent any portion of the transformed energy; and
they are, at present, not supposed to be in the chain of physical cause
and effect. They are generally regarded, in other words, as outside, or
concomitant, or epiphenomena, which occur parallel with the physical
changes, and which appear to be dependent upon them, but which do not
themselves produce or influence such changes. It cannot be denied, how-
ever, that this is a most unsatisfactory solution of the most interesting
of all problems, since if consciousness has this position it becomes difficult
to attack the problem as all other physical problems have been attacked.
It is perhaps wiser to wait until more light has been thrown upon this
subject. Negative evidence, the failure to detect loss of energy accom-
panying consciousness changes, is not a satisfactory basis for any firm
conclusion. It:may prove to be. the case, although the evidence is cer-
tainly not favorable at present, that consciousness, or rather the psychical
basis of it, should be put together with heat, light and electricity as one
8 . PHYSIOLOGICAL CHEMISTRY

of the accompanying manifestations of energy transformations in living
and, presumably, in lifeless things also.

‘It is very important to remember in the course of the transformation
of potential into kinetic energy in living matter that the kinetic energy
may appear in various forms, and that if it appears in some other form
than heat, the heat which one might expect to appear does not do so,
but it is replaced by light, electrical currents, movements, possibly
psychic energy, if there is such a thing, or some other form of energy
of movement.

Since living matter is constantly giving off energy in these different
forms, it must be receiving it from some source, or creating it. Careful
experiments, which will be cited later in the book, prove that living
matter does not create energy, but that in it energy is simply trans-
formed from one kind to another, as it is elsewhere in the universe.
Living matter must then get its energy from some source. This source
is the food and the oxygen of the air. The chemical system consisting
of oxygen and foods contains potential energy. This system is formed,
with its potential energy, by the action of chlorophyll, the green coloring
matter of plants and the protoplasm of plants. Sunlight acting on
these green plant parts in the presence of carbon dioxide and water
brings about a separation of the carbon and oxygen of the carbon
dioxide. The energy of the sunlight is transformed in this process. The
carbon, with a small] part of the oxygen, becomes converted into various
food substances (carbohydrates, etc.) and the oxygen accumulates in
the air. This separation of carbon and oxygen requires that work
should be done and consequently the expenditure of energy, and this
energy is obtained from the light absorbed by the green leaves. All the
energy of living things comes, therefore, in the long run from the sun.
The food and oxygen thus separated contain between them potential
energy, since, under favorable conditions, not well understood but such
as exist in living matter, they will combine again to form carbon dioxide
and water and set free, in so doing, the energy required for their
previous separation. The energy of living things, whether it appears
as heat, light, electrical disturbances or movements of masses, is due then
directly, or indirectly, to the combustion of the carbon and hydrogen
of the body by the oxygen of the air. Living matter is a combustion
engine, with cylinders and connecting rods of molecular dimensions
and provided, possibly, with an electrical sparking device not so dis-
similar in principle from that of an internal combustion or explosion
engine. The discovery of the origin of the energy of living protoplasm
in the combustion of carbon and hydrogen was one of the greatest, if
not the greatest and most fundamental, discovery in chemical biology;
and‘it is considered more at length in Chapter VI.
THE GENERAL PROPERTIES OF LIVING MATTER ‘9

But it must not be thought from what has preceded that combustive
changes are the only kinds of chemical changes occurring in living
matter. The fact is quite otherwise. There are also, in the first place,
reducing reactions. In order that any substance may oxidize it must
also be reducing. A reducing substance is one which has the power
of combining with oxygen. Now all the food and organic substances
of protoplasm have the power of combining with oxygen under appro-
priate conditions, hence living matter is seen to be made of reducing
substances. If it happens that there is not sufficient free oxygen for
these reducing substances to unite with when they enter into an actively
reducing condition, and how they come to enter such a condition will be
considered presently, those which are the stronger reducing steal the
oxygen away from other weaker reducing bodies which have got a little;
or the reducing particles, finding no oxygen to unite with and being in
a condition to unite with something, join, or condense, together, two
or more parts of molecules uniting to form new substances; and in this
way, probably, the fats are formed from the sugars. Since no cell ever
has a sufficient supply of oxygen to oxidize all the reducing substances
set free or active, and since, indeed, it cannot continue to exist if ever
the oxygen becomes thus plentiful, all living matter has a steady reduc-
ing action and there are a great many reducing reactions, as well as
oxidations, going on in cells. ‘It is, indeed, as we shall see, this play
of oxidation and reduction which accounts for many of the synthetic
transformations in protoplasm. Furthermore, since the absorption of
oxygen must be proportional to the surface of the cell, whereas the
requirement goes proportional to the mass, the size of cells must be
regulated or fixed in some way to secure the proper balance between
oxidation and reduction.

A very large class of chemical transformations in protoplasm con-
sists of hydrations, as would be anticipated in a medium containing,
as protoplasm does, 80 per cent. of water. By a hydration is meant
the union of water with a substance. When this union takes place
some substances become unstable, for some reason not understood by
the writer, and fall into fragments. This process of decomposition with
the taking on of water is called hydrolytic decomposition, or cleavage
(Gr. hydor, water; lysis, separation). And among the disintegrative,
or catabolic (Kata, down) chemical changes, this is one of the most
important. All digestive changes are of this kind.

Besides oxidations and reductions, condensations and hydrolyses,
‘there is finally another great class of chemical reactions known as
dehydration syntheses. It is a singular fact that protoplasm. although
it is four-fifths water, nevertheless synthesizes complex substances such
as proteins, carbohydrates and fats by a process which involves the
ic PHYSIOLOGICAL CHEMISTRY

liberation of water and which is ordinarily duplicated outside the cell
by means of high temperature, or by strong water-attracting substances,
such as phosphorus pentoxide or sulphuric acid. These dehydration syn-
theses taking place in such a wonderfully aqueous medium have been a
great puzzle. It has been suggested by Drechsel that many of them are
dehydrations produced not by a simple taking out of water, but by a
reduction followed by an oxidation. There is reason to believe this
explanation in some instances to be well founded, although syntheses
of the more complex of these bodies by this method have not yet been
produced outside the cell. The subject requires further investigation.
One fact strongly in its favor is that such syntheses are retarded if the
respiration of the cell is reduced by deprivation of oxygen, by anesthetics
or in other ways; or if-the reducing power of the cell is destroyed by the
supply of too much oxygen.

There is still another feature of cell chemistry which must strike
even the most superficial observer, and that is the speed with which
growth and the chemical reactions occur in it. Everyone knows that
sugar dissolved in water does not rapidly oxidize to carbon dioxide,
but remains intact for a long period; but in the cell it oxidizes with
surprising speed, liberating heat, light, or doing work by the energy
set free. It has been found that if glucose is dissolved in water and
exposed to air, particularly in the light, it undergoes a very slow oxida-
tion and decomposition. The difference between its behavior in and
out of the cell is a difference of speed of decomposition, rather than
a difference in kind. A similar fact is seen in the behavior of starch.
Starch boiled with water does not easily take on water and split into
sweet glucose, but in the plant cell it changes into sugar under appro-
priate conditions very rapidly. How does it happen then that the
chemical changes of the foods go on so rapidly in living matter and
so slowly outside? This is owing to the fact, as we now know, that
living matter always contains a large number of substances, or com-
pounds, called enzymes (Gr. en, in; zymé, yeast; in yeast) because they
occur in a striking way in yeast. These enzymes, which are probably
organic bodies, but of which the exact composition is as yet unknown,
have the property of greatly hastening, or as is generally said, catalyz-
ing, various chemical reactions. The word catalytic (Kata, down; lysis,
‘separation) means literally a down separation or decomposition, but it
is used to designate any reaction which is hastened by a third substance,
this third substance not appearing much, if at all, changed in amount
at the end of the reaction. Living matter is hence peculiar in the speed
with which these hydrolytic, oxidative, reduction or condensation reac-
tions occur in it; and it owes this property to various substances, cata-
ivtie agents, or enzymes, found in it everywhere. Were it not for these
THE GENERAL PROPERTIES OF LIVING MATTER ll

substances reactions would go on so slowly that the phenomena of life
would be quite different from what they are. Since these catalytic sub-
stances are themselves produced by a chemical change preceding that
which they catalyze, we might, perhaps, call them the memories of those
former chemical reactions, and it is by means of these memories, or
enzymes, that cells become teachable in a chemical sense and capable of
transacting their chemical affairs with greater efficiency. Whether all

Attraction-sphere enclosing two centrosomes.

Plastids lying in the
Plasmosome or cytoplasm
true

nucleolus

Chromatin-
network
Nucleus / _..
Linin-network

  
 

Karyosome,
net-knot, or 4
chromatin-
nucleolus

 

Vacuole

Passive bodies (meta-
plasm or paraplasm)
suspended in the cy-
toplasmic meshwork

Fic. 1.—Diagram of a cell according to Wilson, illustrating the organization and
specialization of the cell.

our memories have some such basis as this we cannot at present say,
since we do not yet know anything of the physical basis of memory.
Living reactions have one other important peculiarity besides speed,
and that is their ‘‘ orderliness.’’ The cell is not a homogeneous mixture
in which reactions take place haphazard, but it is a well-ordered chemical
factory with specialized reactions occurring in various parts. If proto-
plasm be ground up, thus causing a thorough intermixing of its parts,
it can no longer live, but there results a mutual destruction of its
various structures and substances. The orderliness of the chemical
reactions is. due to the cell structure; and for the phenomena of life
to persist in their entirety that structure must be preserved. It is true
that in such a ground-up mass many of the chemical reactions are
presumably the same as those which went on while structure persisted,
but they no longer occur in a well-regulated manner; some have been
12 PHYSIOLOGICAL CHEMISTRY

checked, others greatly increased by the intermixing. This orderliness
of reactions in living protoplasm is produced by the specialization of
the cell in different parts shown in Figures 1 and 2. Thus the nuclear
wall, or membrane, marks off one very important cell region and keeps the
nuclear sap from interacting with the protoplasm. Profound, and often
fatal, changes sometimes occur in cells when an admixture of nuclear
and cytoplasmic elements is artificially produced by rupture of this
membrane. Other localizations and organizations are due to the colloidal

 

 

 

Fic. 2.—Section of a dividing egg cell (Lillie) showing alveolar or granular structure
of protoplasm at 3, the spindle with chromatin at 1, and finely granular protoplasm at 2.
The peripheral layer at 5 is different from the parts lying inward.

nature of the cell protoplasm and possibly to its lipoid character. By a
colloid is meant, literally, a giue-like body; a substance which will not
diffuse through membranes and which forms with water a kind of tissue,
or gel. “It is by means of the colloids of a protein, lipoid or carbohydrate
nature which make up the substratum of the cell that this localization
of chemical reactions is produced; the colloids furnish the basis for the
organization or machinery of the cell; and in their absence there could
be nothing more than a homogeneons conglomeration of reactions. The
properties of colloids become, therefore, of the greatest importance in
interpreting cell life, and it is for this reason that they have been
studied so keenly in the past ten years. The colloids localize the cell
reactions and furnish the physical basis of its physiology ; they form the
cell machinery.

The general chemical composition of living matter. Water.—It is
little short of astounding that living matter with all its wonderful
THE GENERAL PROPERTIES OF LIVING MATTER 13

properties of growth, movement, memory, intelligence, devotion, suffer-
ing and happiness should be composed to the extent of from 70 to 90
per cent. of nothing more complex or mysterious than water. Such a
fact as this is most perplexing, especially when all experiment shows
that this water is playing a profoundly important part in the generation
of the vital phenomena. Any interference with the amount normally
present makes a change at once in the activities of the cell. In fact,
we might say that all living matter lives in water, as Claude Bernard
put it. For not only is this obviously true in the lower and simpler
forms of animals and plants, which are little more than naked masses
of protoplasm living in water, but it is no less true of the higher forms,
since in all of them an internal medium, or environment, of a liquid
nature, the lymph, the blood, or sap, is found which is the immediate’
environment of the cells. Water is the largest and one. of the most
important constituents of living matter; and if organisms are carefully
examined the most various devices are found to assure the regulation
of the water content of the cells of the body. The younger, the more
vigorous, the more alive, the more actively growing, the more impres-
sionable cells are, the more watery are they. Perhaps more than anyone
else the French physiologist, Dubois, has emphasized the important réle
of water in life. Table I gives the proportion of water found in various
kinds of tissues.

TaBLe I. AMOUNT OF WATER IN VARIOUS TISSUES.

orzen oe Orgen ee
Brain. White matter ........... 68 Liver (human) .............0006 76
Brain. Gray matter ............ 84 Cartilage (hyaline) ............. 67
Brain Embryonic .............. 91 Thymus - (calf) ...........--005. 77
Musele (mammalian) ........... 73 Kidney (child) .................. 78
Muscle (fish) ...........ceeeeee 80 Suprarenal gland ............... 80
Electrical organ ..........0..006 92 Dentine: ears ct tirana heen: shaseeend eves 10

Salts, and inorganic elements.—One would very naturally expect
that living matter might contain some very rare, peculiar and costly
metal, or substance, like radium, to which its properties might be
attributed. But quite the contrary seems to be the case. Besides water,
the inorganic constituents of protoplasm are salts, and they are among
the commonest salts on the surface of the earth. Sodium, potassium,
magnesium, calcium, iron, sulphates, chlorides, phosphates and ear-
bonates are essential to life and are found in practically all living matter.
The amount of these various inorganic elements differs somewhat in
different cells and tissues, but they occur in all. Other common elements
are sometimes present, such as iodine, manganese, copper, zinc, barium
14 PHYSIOLOGICAL CHEMISTRY

or silicon, but these are generally confined to special plants and animals.
About 1 per cent. of the weight of the protoplasm is composed of the
salts or inorganic metals and acids mentioned (Figure 3). Furthermore,
these salts are not mere inert substances, they are not simply absorbed
with the water and tolerated, but they are in combination, in part at
least, with the organic matter of the protoplasm. They are not simply
clinkers clogging the grates of the protoplasmic fires, but they are active
in the production of the vital phenomena. Indeed, some have gone so

    

a

Fic. 8.—The distribution of potassium in cells after Macallum. (qa) striated muscle;
(b) nucleated blood corpuscles; (c) nerve fiber. ‘The black precipitate represents the
potassium.

far as to believe, as we shall see, that by means of the electrical charges :

they bear when in solution they vitalize the colloidal, organic substratum
of the cell and make it alive. Any change in their relative proportions
at once affects the activity of the cell; thus by increasing or diminishing
the proportion of sodium, calcium or potassium skeletal muscle may be
made to twitch rhythmically or to remain at rest; nerve impulses may be
set up in motor nerves, or the irritability of the nerve raised or lowered ;
chromophores of fish scales may be contracted or expanded; and the
activities of all cells increased or diminished. Magnesium sulphate acts
much as an anesthetic on mammals, but paralyzes, also, the endings of
the motor nerves in the muscles. Furthermore, by increasing the total
amount of salt in protoplasm many cells may be stimulated and egg
cells of some animals caused to develop parthenogenetically without the
aid of sperm.
THE GENERAL PROPERTIES OF LIVING MATTER 16

Thus in some instances 94 per cent. of living matter consists of noth-
ing more unusual or remarkable than water and the commonest salts.

It is certainly not without significance that living matter is so watery
and contains the salts of the sea. It would appear probable from this
that living matter originated either in the sea itself or, perhaps, in some
pool of water which contained, possibly in dilute form, the common salts.
It has been suggested that it was in some slowly-drying volcanic pool
where concentration could take place, and where cyanides and other
similar reactive organic compounds might have been formed by the
vigorous electrical discharges accompanying the eruptions, that living
matter first appeared. We would thus have sprung from the thunder-
bolts of Jove, if this theory is true; but we are, at any rate, the children
ef the sun and the sea, of Apollo and Aphrodite.

The organic matter.—The remainder of living matter, 10 to 25 per
cent. by weight, is organic. This organic matter is found to consist
of, or may be divided for purposes of convenience into, four great groups
of substances: 1, substances of the fat group soluble in alcohol and ether,
called lipins; 2, substances of the sugar group, carbohydrates; 3, sub-
stances containing nitrogen, carbon, hydrogen and oxygen, called pro.
teins; 4, various simple substances such as urea, creatinine, inosite,
phenols, etc., called extractives, because they are soluble in water by
which they may be extracted from the cell when the latter is first
coagulated. In muscle the relative proportion of these substances is as
follows: protein, 19 per cent.; carbohydrate, 0.3 per cent.; lipin, 3
per cent.; salts, 3 per cent.; water, 75 per cent. In the following chap-
ters the chemistry of each of these great groups of organic substances.
beginning with the carbohydrates, will be discussed.
CHAPTER II.
THE CARBOHYDRATES.

Occurrence.—All living organisms, except the most simple, which
are nothing more than naked masses of protoplasm, consist of both
living and lifeless matter, the lifeless having been formed or secreted
by the living. This lifeless matter forms the greater part of the sup-
porting framework, or serves as reserve food. In plants these support-
ing tissues, or reserve foods, consisting of the cellulose or woody parts,
the starches, mucilages or gums, such as that which exudes from the
bark of the cherry-tree, are composed of the elements carbon, hydro-
gen and oxygen and belong to a great group of substances known as
sugars or carbohydrates. The supporting tissues of animals, unlike
those of plants, contain a large proportion of nitrogen and belong gen-
erally to the group of proteins, although chitin, which forms the hard
shell of crabs and other invertebrates, contains a large amount of carbo-
hydrate (glucosamine). But it is not only as the supporting tissues
of plants and animals that carbohydrates occur. They are found, also,
in the living matter itself, making part of the chromatin of the nucleus,
or distributed as glycogen or sugar, free or combined, through the
cytoplasm ; and it is, indeed, largely by the combustion of carbohydrate
that we derive our energy. Since substances of this class are the simplest
of the colloidal materials of cells, and are among the most abundant
organic constituents of living things; since they are formed from the
inorganic compounds of carbon dioxide and water, and in the long run ~
all the energy of living matter comes from them, and since both the
fats and proteins originate from them, a study of the organic constitu-
ents of protoplasm may best begin by a study of their composition and
chemical nature.

Definition.—The carbohydrates are compounds of carbon, hydrogen
and oxygen occurring in animals and plants. They get their name from
the fact that in the majority, though not in all, the hydrogen and oxygen
are in the proportion of two atoms to oue, that is, they are in the same
proportion as in water; and, indeed, by the action of heat, or of strong
dehydrating agents, they are split into carbon and water, as in the
process of making charcoal or in the charring of sugar. The formula
of glucose, a typical carbohydrate, is C,H,,0O,. But while in the ma-
jority of the naturally occurring members of this group the hydrogen

16
| THE CARBOHYDRATES 17

and oxygen are in this proportion, in some cases, as in rhamnose,
C,H,,0,, a methyl pentose, they have not this proportion. Many sub-
stances, also, have hydrogen and oxygen in this proportion which are
not carbohydrates, such as lactic acid, C,H,O,, or acetic acid, C,H,0,,
which differ from the carbohydrates in their chemical properties. Many
of the carbohydrates have a sweet taste, although some substituted mem-
bers of the group among the glucosides are intensely bitter, and polysac-
charides may be tasteless. When pure they are white; some, like cane
sugar, crystallize; others, like starch, are colloidal and do not
crystallize.

The chemical properties of carbohydrates characterize them as well
as, or better than, their composition. All of the simpler ones readily
oxidize. They are, hence, reducing substances and a large part of the
reducing powers of protoplasm are due, in the long run, to these sub-
stances. They reduce ammoniacal silver nitrate, or alkaline solutions
of mercury, copper, gold or bismuth salts. On the other hand, they have
oxidizing properties too. They will absorb nascent hydrogen, uniting
with it and oxidizing the substance from which the hydrogen is taken.
The simultaneous possession of these and other properties shows that

H

|
they contain aldehyde, or ketone, groups, —C=O or =C=O, in the
molecule. Hither of these groups can take up hydrogen yielding an

alcohol; or by oxidation go over inte a carboxyl group, HG 08
S
O

The simplest carbohydrates, therefore, are aldehydes or ketones, and
they form accordingly two groups: aldoses and ketoses. Their reaction
in aqueous solution is neutral to the usual indicators, but they possess,
nevertheless, very weak acid and basic characters, being very weak
amphoteric compounds. Thus they contain some hydrogen which may
be replaced by a metal, such as lead, or sodium, and they are thus able
to neutralize, to a slight extent, the causticity of sodium hydrate. They
are to this extent acids, though they lack the acid taste. This acid
property is due to the fact that they contain alcohol groups, all alcohols
behaving like very weak acids, since the alcohol hydrogen may be, in
part, replaced by a metal. They are, however, very weak acids. The
number of hydrogen ions in their solutions is very small, smaller than
in solutions of carbon dioxide of equal concentration. The dissociation
constant of every sugar is very small. By the dissociation constant is

< :
meant the value K, where ka C, is the concentration of hydro-
3

gen ions, C, the concentration of the sugar anion and C, the concentra-
tion of the undissociated molecule. The dissociation constant of glucose
18 PHYSIOLOGICAL CHEMISTRY

at 18° is 5.9X10—3 (Osaka) or 3.6X10— (Madsden) ; that of sae-
charose is 1.1410—"8 (Madsden), or 2.410— (Michaelis and Rona) ;
maltose is 1810-18 (Michaelis and Rona) ; and levulose is 8.8x10—*
(Michaelis and Rona). With bases such a sugar as glucose will react
according to the following equation:

C,H,,0, + NaOH = C,H, 0.Na + H,0

The sugars are, then, alcohols as well as aldehydes or ketones. They are
polyhydric alcohols having one alcohol group attached to each carbon
atom, but that of the aldehyde or ketone group.

Their basic properties are due to the oxygen of the aldehyde. By
the aldehyde oxygen they have the property of uniting with acids to form
so-called oxonium salts, but this union is easily dissociated, the basicity
being very weak.

C,H,,0, + HCl =.C,H,,0,.HCI

6 12 6 12 6
The carbohydrates may, then, be defined thus: They are compounds
of carbon, hydrogen and oxygen, the oxygen and hydrogen being often
but not always in the proportion to form water; and, further, they are
aldehyde or ketone derivatives of polyhydrie alcohols. Their properties
are probably due to the juxtaposition of an alcohol and an aldehyde or
ketone group.
The aldehyde structural formula for dextrose is
OH OH OH OH OH H

a a ee
uu uu

and the formula for the ketose, levulose, is
OH . OH OH O FH

| | ft
— Ze c—c —b—b—on.

=

Classification.—It is convenient to divide the carbohydrates into
three great classes according as their molecules contain one, two or
several saccharide (simple carbohydrate) groups. ‘These classes are
the monosaccharides, the disaccharides and the polysaccharides. The
members of the first two groups are generally crystalline bodies; but
many, though not all, of the last group are colloidal in aqueous solu-
tion. The more important monosaccharides found in nature are
d-glucose, or grape sugar, or dextrose as it is also called; d-levulose, or
fruit sugar; galactose; xylose; arabinose; mannose; and d-ribose. The
disaccharides are saccharose, or sucrose, as cane sugar is also called;
lactose, or milk sugar; and maltose, or malt sugar. The common polysac-
charides are cellulose, gums, dextrins, starches and glycogen.

The monosaccharides are in their turn classified by the number of
carbon atoms, or more properly by the aldehyde, ketone and alcohol
THE CARBOHYDRATES 19

groups they contain into bioses, trioses, tetroses, pentoses, hexoses,
heptoses, octoses, nonoses, etc. Of these the first six are found in
nature, but the hexoses are the more abundant. Each of these groups
from the trioses on is subdivided into two groups, the aldoses and ketoses,
according as they are aldehydes or ketones. Thus mannose, dextrose
and galactose are hexose aldoses having the general formula, C,H,,0,;
levulose is a ketose hexose; ribose and xylose are pentose aldoses,
C,H,,O,; of the trioses, glycerose, C,H,O;, is an aldose, while dioxyace-
tone is a ketose.

f 1. Bioses. Aldose. Glycolaldehyde.
: Aldoses, Glycerose.
2. Trioses. tease: Dinmyacetone.
Aldoses. Hrythrose.
8. Tetroses, — { Rutones: d-Erythrulose.
I, MONOSACCHARIDES { Aldoses, Arabinose, xylose,
4. Pentoses. ribose.
Ketoses. t-Arabinulose.
Aldoses. Dextrose, galactose.
5. Hexoses. mannose.
Ketoses. Levulose, sorbose.
L 6. Heptoses. Aldoses. d-Mannoheptose.
f 1. Lactose. (Glucose + galactose.)
2. Maltose. (Glacoge -}+ glucose.)
CARBOHYDRATES II. DISACCHARIDES 4 8. Saccharose. (Glucose + levulose.)
4. Trehalose. (Glucose + glucose.)

\ 5. Melibiose. (Galactose + glucose.)

( { Melitose (Raffinoge) in mo-
lasses.
. Trisaccharides. { Melizitose. (Pinus larix.)
1 (Levulose + glucose -}-
: galactose.)
. Tetrasaccharides. Lupeose in peas; stachyose,
(Lupeose consists of two
molecules of galactose,
Ill. POLYSACCHARIDES one of glucose, and one
of levulose.)
Dextrine,
Glycogen.
. Celtulose.
. 38. Colloidal polysaccharides. 4 Starch.
| Mucilages.
Gums

=

n

\

 

\ Inulin.

 

L L
Monosaccharides. Structural formulas. Isomerism. Optical
properties.

a. Hezoses.
Analysis of glucose, galactose and mannose shows that they all con-

tain the same proportion of carbon, hydrogen and oxygen; a propor-
tion corresponding to the formula: C,H,,0,. They have also the same
chemical properties showing that all of them are aldehydes and poly-
hydric alcohols. When chemical compounds have the same chemical
atoms in their molecules in the same proportions they are called isomers;
or are said to be isomeric with each other. Thus lactic acid, C,H,0,,
and dioxyacetone, C,H,O,, are isomers, When, in addition to having
20 PHYSIOLOGICAL CHEMISTRY

the same number of atoms of the same kind in the molecule, these atoms
are arranged in the same general way so that the chemical nature of
the substances is the same, then those substances are said to be stereo-
isomeric, a word which means ‘“‘ having a like form ’’ (Greek, stereos,
solid). Since mannose, galactose and dextrose are all of them aldoses and
polyhydric alcohols.their molecules must be, on the whole, very similar ; they
are, therefore, stereo-isomers. Their molecules differ only in their forms
and we may now examine how these molecules may differ in their shape.
This brings us to one of the most important subjects in the whole of
physiological chemistry, namely, the subject of the shapes of molecules;
in the pages which follow we shall find many examples illustrating the
importance of molecular form in vital processes of all kinds.

The proof that the atoms in a molecule occupy definite positions,
so that the molecule has a definite shape, was one of the most beau-
tiful and fundamental discoveries of Pasteur, made while he was still
a very young man, in 1848; and since this discovery is at the bottom
of all the beautiful science of molecular form which has been built
upon it, and as the importance of this molecular property is showing
itself in every field of biological work, it is fitting that we consider
Pasteur’s work at some length. Pasteur had been greatly interested
in crystalline form. Why do substances crystallize in definite shapes?
Among the substances of an organic nature which gave very fine, large
crystals, tartaric acid and its salts were noteworthy. Now there were
two kinds of tartaric acid known to Pasteur, the ordinary tartaric
acid, the acid of wine, which Biot had shown to be dextro-rotatory,
ie., its solutions had the property of rotating the plane of polarization
of polarized light to the right; and another kind of tartaric acid found
by Kastner and called racemic acid (ZL. racemus, a bunch of grapes)
of the same composition as the other but which had no action at all
on polarized light. It and its salts were inactive. Pasteur undertook
to study the crystalline forms of these two acids. He expected to find
that racemic acid would have a different erystalline form from the
ordinary dextro-rotatory tartaric acid. He found, however, that when
the sodium-ammonium salt of the inactive (racemic) acid was crystal-
lized below 28° crystals of the same shape as those of the correspond-
ing salt of the dextro acid appeared. On looking at the crystals
more closely, however, he found that there were in reality among the
crystals of sodium-ammonium racemate crystals of two different kinds
which are illustrated in Figure 4. These crystals were exactly alike
with the exception of a small facet, 0’, and the corresponding facet
diagonally opposite to it. These two facets were so placed in these two
kinds of crystals that the crystals would not correspond if superimposed
one on the other. In the one kind of crystal the facet was on the right
THE CARBOHYDRATES 21

side as it was in the dextro-tartaric acid; while in the other form of
erystal it was on the left side. The crystals were not symmetric, they
were asymmetric and, as it were, mirror images of each other.

He separated these two forms of crystals and thinking that they
might show different optical properties he dissolved them and exam-
ined the solutions in the polariscope. To his great joy, he found that
the solution of the one form now rotated the plane of polarization to
the right ; while the solution of the other form rotated it to the left. This
great discovery showed at once that crystalline form must depend on
molecular form, because in the solution the molecules were separated and
the crystalline form had disappeared, but the asymmetrical action on
light persisted. The action of the solution on light showed that the indi-

 

 

 

 

 

 

 

 

c e
6 ;
q gy
dv p| pia b p | pa
rT L
Fic. 4.—Two forms of crystals of levo and dextro tartaric acid (Landolt).

vidual molecules must be of two different forms, a dextro-rotatory and a
levo-rotatory form. The molecules of tartaric acid must be asymmetrical,
just as the crystals were asymmetrical. The discovery, of course, cleared
up at once the difference between the two kinds of tartaric acid. It
showed that there were at least three different forms of tartaric acid, the
dextro-rotatory, the levo-rotatory and the third, or racemic, form which
was composed of equal amounts of tne other two kinds and which was
inactive on light. Pasteur afterwards discovered a fourth, the meso-
tartaric acid. By this discovery of Pasteur we know that the shapes of
molecules may be asymmetric, and that the atoms of these molecules do
not easily rearrange themselves, for if they did the molecule would
readily pass from the one form to the other. It is one of the most funda-
mental discoveries in physics or chemistry.

The difference in shape of the molecules of the two forms of
tartaric acid was made more precise many years later, practically
coincidently, in 1874 by LeBel and van ’t Hoff. They actually pictured
the possible arrangement of the atoms in the molecule by which the
asymmetry was produced. If the carbon atom is represented as lying
at the center of a tetrahedron, of which the apices represent the position
22 PHYSIOLOGICAL CHEMISTRY

of the four atoms attached to the carbon atom, it becomes possible to
picture the different arrangements of the atoms causing the asymmetry.
This is illustrated in Figure 5. If the four atoms or atomic groups
attached to the carbon atom are all different, as they are in the case of
iodo, chlor, brom, methane, CHICIBr, then it is possible to arrange these
atoms in two different ways, as is shown in the figure, the two tetrahe-
drons not being superimposable, but being mirror images of each other.
If, however, two of the atom groups attached to the carbon are the same,

H

cu I I cu

Br Br
Fie. 5.

then it is impossible so to arrange them that the tetrahedrons will not
be superimposable. Methane, chlor- or dichlorbrom-methane can have
but one form, a symmetrical one. A carbon atom, then, with four
different atoms or atomic groups attached to it is said to be asymmetrical,
since it produces an asymmetrical crystalline and molecular form, and
an asymmetrical action on polarized light. The atomic groups about
such a carbon atom may have two different arrangements. Asymmetric
carbon atoms in the sugar molecules illustrated on page 28 are printed
in black-face type. Not all compounds with asymmetric carbon atoms
rotate the plane of polarized light, since in some, of which mesotartaric
acid is an example, compensation may occur, some atoms rotating the
plane of polarized light in one direction; while others rotate it in an
opposite direction: the total effect of the molecule on light being nil.
Most compounds with asymmetric carbon atoms, however, exist in two
forms, one dextro- the other levo-rotatory.

The various forms of tartaric acid (stereo-isomers) may be repre-
sented as follows, the asymmetric carbon atoms being printed in black-
face type:

CcooH COOH COOH
u—¢_—on HO—¢—H u—¢—on
|
HO—C—H u—b_on u—¢—on
boon boon boon
d—Tartaric acid 1—Tartaric acid Meso-tartaric acid (Inactive).

 

Racemic acid (Inactive).

All compounds having an asymmetric carbon atom in them may
exist, therefore. in two different forms, these forms being stereo-isomeric
THE CARBOHYDRATES 23

forms and also optical antipodes. One of these optical isomers rotates
the plane of polarized light in the one direction, just as much as its
antipode rotates it in the other direction. The physical and chemical
properties, such as the melting points and solubility in symmetrical
solvents, of these two antipodes are almost or quite the same. Stereo-
isomers which are not optical antipodes generally have different melting
and boiling points and solubilities. The separation of the optical
antipodes can be accomplished by picking out the crystals in the way
Pasteur did in a few instances; or by the different solubilities of their
compounds with other optically active substances; or by the action of
moulds, yeasts or other living organisms which often destroy one, but
not the other antipode. The mould, penicillium glaucum, destroys the
dextro- but not the levo-tartaric acid.

In the figure which has been given of the possible shape of the
molecule (Figure 5), one might suppose that the atoms in the molecule
were far apart, in which case it would be difficult to see why the
molecule should keep its form. The figure is, however, probably incor-
rect in this particular. The attraction between the atoms of a molecule
is so great that they probably lie closely packed together and with
very little freedom of movement beyond that of minute vibration about
a center. The amount of this vibration and the space at the disposal
of the atoms becomes somewhat greater as the temperature rises, since
there is good reason for believing that molecules expand with a rise in
temperature, although the expansion is not very great. The pressures
due to molecular and atomic attractions on the surfaces of molecules
are enormous. Thus the pressure called the internal pressure of a liquid
or a gas, which is due to molecular cohesion, or the attraction between
the molecules, is, at zero centigrade in ether, about 2,000 kilograms
per square centimeter, and it increases considerably at temperatures
below this. Now the attraction between the atoms within the molecule
is certainly many times greater than the attraction between the molecules,
although it is not yet known just how great it is. By this attraction,
therefore, the atoms within the molecules will be under a compression
certainly of many thousands of kilograms per square centimeter in
addition to the cohesive pressure. It is not impossible that the pressure
driving together the atoms of a molecule may be more than a hundred
thousand kilograms per square centimeter. It is not probable that this
pressure is distributed evenly over the molecule, since some atoms are
held more firmly than others. So great a pressure as this must cer-
tainly drive the atoms of the molecule very close together so that, at
relatively low temperatures at least, the molecules must have the prop-
erties of rigid solids with the atoms having very little power of move-
ment. Theoretically, however, they will always have some movement
24 PHYSIOLOGICAL CHEMISTRY

at temperatures above absolute zero, and so the molecules above this
temperature are not absolutely incompressible. Of course at higher
temperatures as the molecules separate this pressure is reduced and
in some cases the attraction between particular atoms of a compound
is less than that stated. Greater mobility of the atoms exists in such
molecules so that the atoms may shift their positions, undergoing what
is known as a tautomeric change. It is not surprising, however, that
subjected to such high pressures the atoms of a molecule generally
arrange themselves in the position of greatest stability and if, tempo-
rarily, they take unstable positions, they may undergo rearrangement.
Such molecular rearrangements are by no means uncommon. The
racemization of optically active compounds is such a process of atomic
rearrangement.

Molecular form is of fundamental importance throughout living na-
ture. Most naturally occurring organic compounds are asymmetric and
usually only one of two possible isomers occurs in any organism. Of
the optical isomers of any amino acid or carbohydrate only one gen-
erally will serve to nourish an organism, or, if both are foods, one is
usually better used than the other. The enzymes, or catalytic agents,
will only act on compounds of a very particular molecular form. Yeast
will ferment d-glucose, d-mannose or d-fructose, all of which have the
same configuration of the last three carbon atoms, but it will not ferment
1-fructose, or l-glucose, or ]-mannose, or l-galactose. The phenomena of
immunity, such as specific antitoxins, precipitins and anaphylaxis, also
involve molecular form. Protein which has been racemized by the
action of sodium hydrate will no longer cause anaphylaxis. In the
very accurate and specific adjustment of the spermatozoon to the ovum,
an adjustment so accurate that a spermatozoon will usually only fer-
tilize the eggs of its own species, it is probable that the form of the
molecules of sperm and eggs are in some manner related or adjusted
to each other. In fact, the whole living world is an asymmetric world;
the development of different species and varieties probably depends
on asymmetric molecules, since animal forms, like the forms of erystals,
must, in the last analysis, be but the expression of the forms of the
molecules of which the protoplasm is composed.

Molecular asymmetry may be most easily detected by means of
the action of the molecules on polarized light. When polarized light,
that is light which has passed through a Nicol’s prism, passes through
a solution of a substance of which the molecules are asymmetrical it
is acted upon, so that the plane of polarization of the light on emer-
gence from the solution does not coincide with the plane of polarization
of the entering light. The plane of polarization has been rotated to
one side or the other, the degree to which it is rotated depending on
THE CARBOHYDRATES 25

the kind of molecules and the number of molecules the light has passed.
It is dependent, in other words, upon the concentration and the length
of the tube. It is also dependent upon the wave length of the light. The
plane of polarization of blue light is rotated, for some substances, about
twice as much as that of yellow light by the same molecules. Hence one
uses always monochromatic light and the degree of rotation is gener-
ally expressed for sodium light for a concentration of one gram of
substance in a cubic centimeter of solution and for a tube one decimeter
in length. This angle is called the specific rotatory power of the sub-
stance. Temperature also affects the degree of rotation. In general the
higher the temperature, the lower the rotation. It is usual to give the
specific rotation at or near 20° C. The specific rotatory power as just
described (@) is written as follows:

@)p

Since for many substances the specific rotatory power varies, also,
with the concentration of the solute and the character of the solvent,
it is desirable to give these data also. (@) in the above formula is
the angle of rotation which the plane of polarization of the D line of
the spectrum (sodium) would undergo in passing through 1 dm. of a
solution containing one gram of substance to one eubic centimeter at
20° C.

The specific rotatory power is calculated from the angle of rotation
produced by a solution of known strength in a tube of known length.
The formula is as follows:

20°__@.100 i (a)? __2,100
Le.’ D = Lp.d.
a being the observed angle of rotation at 20° C.; 1, the length of the
tube in decimeters; c, the number of grams of active substance in 100
c.c. of solution ; p, the number of grams of active substance in 100 grams
of solution; and d, the density. pd=c. (qa) is the specific rotatory
power.

Just how molecules with asymmetric carbon atoms rotate the plane
of polarization of light is not yet understood. It would seem necessary
for the light to pass through all the molecules in one direction, in order
that the actions of the different molecules should coincide and not neu-
tralize each other. If this is so, polarized light must orient the molecules
and perhaps the molecular asymmetry enables the light waves to do this.
If the molecules of an asymmetric substance in solution, or in a liquid,
are thus oriented by light so that all the molecular axes coincide, then
the conditions in such a solution might approximate to those in a erystal ;
the magnetic properties of the molecules, if they have any, should coin-
cide and might be detectable. This very interesting and fundamental

 

    
26 PHYSIOLOGICAL CHEMISTRY

problem remains for future investigation. The reason why a rise in
temperature diminishes the rotatory power would also be clear; since
by heat the molecular vibration increases and presumably it would be
more difficult to hold the molecular axes in line. Thus increasing the
temperature should diminish the rotatory power for the same reason
that increasing the temperature of iron diminishes its magnetism, i.e.,
by destroying molecular orientation.

The rotation of the plane of polarization may be due to the fact
that the vibrations of some of the valence electrons occur more easily
in some planes than in others.

The Polariscope.—The Polariseope is used to measure the rotatory power.
Figure 6. In this instrument the light of a sodium flame, produced by heating
sodium chloride or bromide, is first passed through a light filter of potassium

0S
F -E

 

Fic. 6.—Polariscope (Landolt). A, lens; B, polarizing Nicol prism; 0, arm to rotate
the polarizer; D, quartz plate; #, analyzing prism mounted so that it rotates with the
circle G which is marked in degrees; F, the observing telescope; J, the vernier for reading
the rotation, and K, telescopes for increasing the accuracy of reading. The tube con-
taining the solution goes between the analyzer and polarizer, the cover of this space being
shown open. The quartz plate, D, is replaced in the Lippich type polarimeters by the
small Nicol’s, B and O, shown in Fi@. 6a.
bichromate to remove extraneous rays, and then is plane polarized by passing
through a Nicol prism or a Glan-Thompson prism of Iceland spar, called the
polarizer (B, Figure 6). The light then passes through the solution and then through
another Nicol prism, E, called the analyzer, which is so mounted that it can be
rotated about an axis. The light on emerging. from the polarizer is plane polarized
in a plane at right angles to the optical section of the Nicol prism. When this light
passes through a solution of an active substance such as glucose, the plane of polar-
ization is rotated, or bent, at an angle to the right or to the left. If the analyzing
prism is so placed that its optical section corresponds to that of the polarizer, the
light passes through it to the eve without change; if, however, its optical section is
THE CARBOHYDRATES 27

ut an angle with that of the polarizer the light from the latter is split into two
rays, one of which is reflected, so that only part of the light passes to the eve and
the field is less light than when the optical sections coincided; and when the angle
of the optical section of the analyzer is at right angles to that of the polarizer, no
light at all comes through it, all being reflected, at the plane of section of the prism,
to the side where it is absorbed by black surfaces. The field is then dark. If the

 

 

 

 

OA

 

Fic. 6a.—Arrangement of the polarizer with two accessory Nicols to give a three
divided ficld (Landolt). (Lippich type polarimeter).

analyzer is placed at the point of total absorption of the light this may Be taken
ag the zero point. Jf now an active solution is placed between the analyzer and the
polarizer the plane of polarization of the light ernerging from the polarizer is twisted
to one side, hence the vibrations of the light entering the analyzer are no longer in
the plane of the optical axis, in which case they would be totally reflected, but they
are at an angle with that so that more or less of the light comes through. It is neces-
sary to rotate the analyzer to one side or the other to again produce the complete
absorption of the light. If it is necessary to rotate to the right, the substance is said
to be dextro-rotatory. In order to make the polariscope more sensitive it is common,
in the better instruments, to introduce close to the polarizer and between it and the
solution two small prisms, Nicols, so placed that they project with a sharp edge
partly across the circular field. These prisms are fixed, and their edges are focussed
by the observing telescope. This has the effect of dividing the field of view into
three parts as shown in Figure 6a. At the zero point these three fields should have
the same illumination. The advantage of this is that the zero end point is more
sharply determined, since the shade of the three fields may be matched very exactly.
Some instruments have three prisms in addition to the Nicol polarizer, giving a
four divided field. These instruments are called two, three or four shadow instru-
ments respectively. In using the polariscope it is essential that the light should be
uniform in the field, of « maximum brightness, it should be carefully centered
through the apparatus and into the eye, and the polarizer, C, should be turned to a
28 PHYSIOLOGICAL CHEMISTRY

ininimum angle which it is possible to read clearly. If colored solutions are to be
examined, it is necessary to select a colored light which is not absorbed by the solu-
tion. A mercury lamp is useful as a source of light when combined with the proper
light filters.

We may now return to the problem of the way in which we shall
represent on a plane surface the fact that several aldose sugars of the
general formula C,H,,0, are known. How shall the different structures
of these molecules be pictured? A careful study of the possible arrange-
ments of the atoms in the molecule shows that there are eight different
aldose, hexose, stereo-isomeric carbohydrates possible, depending on the
arrangement of the hydrogens and hydroxyls in the chain, and that
there are two optical antipodes of each of these stereo-isomers, making
sixteen possible aldose hexoses in all. Not all of these have been found
in nature. It will be seen that there are four asymmetric carbon atoms
in each hexose molecule. The number of possible stereo-isomers of any
substance may be found from the formula: Number=2°, where n is the
number of asymmetric carbon atoms in the molecule. Some of the
structural formulas of the sixteen aldose hexoses and ketose hexoses are
given below. Their different structures are represented on a plane
surface by writing the formulas with the aldose group at the top and
the alcohol and hydrogen atoms variously placed at the sides of the
zarbon atoms.

COH ° CoH COH CoH
HOH HOoH GOH HOoH
HOCH HGOH HOoH HOOH
HOOH HOCH HOUH HOOH
HOH HOoH HOH HOGH
buon bu ,0H CH,OH du,on
d-glucose. l-glucose. d-galactose. galactose.
COH CoH CoH CoH
HOOR HOH node HCOH
HOOH HOOH HOCH HGOH
HGOH HOOH HGOH HOOH
OCH HOH HOH HOCH
éu,on bar on bir. on du,on
1-talose. d-talose. d-mannose. 1-mannose,
CoH

HOH

HOOH
HOoH
HOOH

bu 20H

l-gulose.

CHO
H oda
ndoH
HOdH
don
cu,on

lL-idose.

CH,OH
bo
HOUH
HOH

|
HOCH

da_on

1-sorbose.

b. Pentoses.

THE CARBOHYDRATES

cOH
HOdH
HOdH
nGOH
HOdH 2
du,oH
d-gulose.
CHO
HGOH
HOUH
H¢oH
HOCH
da,ox

d-idose.

CH,OH
bo
HOOH

HOCH
HOOH
don

d-sorbose.

Isomerism.

CH,OH
bo
HoOH
HOOH

HOOH
bu on

l-tagatose.

CH,OH
bo
HObH
HOH
HoH
don

d-levulose.

29

CH,OH
co
HOoH
HOoH
GOH
da 20H

d-tagatose.

CH,OH
bo
HOOH

HOCH
HOCH
by OH

1-levulose.

There are three asymmetric carbon atoms in each pentose, so that
there are possible 2%, or eight possible isomers of the aldoses. The

structural formulas are as follows:

COH
HO—t—H
n—d_on
H—G—OH

|
CH,OH
d-arabinose.

COH
n_d_on

no—¢_H

nod

|
CH,OH

{-arabjnose,

COH
HO-6_H
u_b_on
Hoban
CH,OH
d-xylose.

re

H—C—OH
]
HO—C—H

|

H—C—OH

|
CH,OH

I-xylose,
30 PHYSIOLOGICAL CHEMISTRY

coH cOH coH e
H—¢_OH HO—¢—H HOCH H—C—OH
u-d-on Hod HO-O-H H—C—OH
n—t_on Ho—¢_H u—d_on HO—¢_—H

ba,on da OH CH,O# (H,0H
d-ribose. L-ribose. d-lyxose. 1-lyxose.

Of these pentoses, d-ribose, xylose and arabinose are of most interest
to biologists, d-ribose being found in some nucleic acids (guanylic and
yeast) ; and arabinose occurring in the gum associated with, or making
part of, the enzyme, amylase. Xylose (Gr. Xylon, wood) is a pentose
obtained by the hydrolysis of straw or wood. The pentoses generally
occur in nature in gums and tetra, or polysaccharides. Xylose has been
found as a constituent of the cephalopod muscle and other tissues
(Henze). There are also ketose pentoses.

ce. Heptoses.

The alcohol of a heptose, d-mannoheptose, called volemite, was found
by Bourquelot in Persea gratissima and the fungus, Lactarius volemus,
and by Bougault and Allard in the dry residue of Primulacee. An
unknown heptose, osazone melting at 195° and formed only on long
heating, was isolated from human urine (Rosenbreger).

Dissociation of the monosaccharides. Reactions of the monosac-
charides of biological interest——The monosaccharides, while compara-
tively stable in the test tube, are very unstable in living matter. They
break up there and are converted into fats, proteins and other sub-
stances. How they are rendered so unstable by the protoplasm is
unknown and is a very interesting problem on which many men are at
present working. It will help us to understand the possible causes of
instability in living matter, if we study how this instability or decom-
position may be produced outside the body; and into what kinds of
substances the carbohydrates break up when they are thus decomposed.
Among the agents which we may use to produce decomposition of the
mono- or disaccharides, alkalies and acids are the simplest.

Action of alkalies on monosaccharides.—All the monosaccharides
and some of the disaccharides are unstable in alkaline solution and
decompose into a variety of substances. Ifa solution of glucose, levulose,
galactose, maltose or lactose is made alkaline, it turns a yellowish-brown
color and acquires a smell of caramel. If heated, this change goes on
more, rapidly and the solution quickly turns brown in some cases, or
yellow in others. This behavior is the basis of Moore’s test for sugars.
The stronger the alkali the more rapid is the change. If, however, air
has free access to the alkaline solution being shaken with it or drawn
THE CARBOHYDRATES 31

rapidly through it, and if the alkali is not too strong, the brown color
does not develop, but a rapid oxidation occurs, causing at times a faint
phosphorescence and always liberating heat.

Chemical examination of the brown liquid shows that the monosac-
charides and many of the disaccharides have undergone profound decom-
position even if the amount of alkali is small. In strong alkali a great
number of acids are produced having six, five, four, three, two or one
carbon atoms in them. Moreover, volatile substances appear in the
absence of oxygen, which give the iodoform test like ethyl alcohol but
which are more probably glycolaldehyde, or oxyacetone, or glyoxal.
Condensation products are also formed, in the absence of oxygen, lead-
ing to the development of the brown color due to humus and caramel
substances.

H HH’ 1
| |
H—C—C—0O; Bo ta age
| |
bu H du Oo i | \
Glycolaldehyde. Oxyacetone. Glyoxal.

If the alkali is very weak a molecular (tautomeric) rearrangement
of the sugar molecule occurs, accompanied by very little or no decom-
position of the carbon chains (Nef). Thus d-glucose, d-mannose or
d-levulose have the same configuration of the molecule except in the
first two carbon atoms of the chain, as may be seen in the structural
formule on page 28. If any one of these sugars is dissolved in weak
alkali and allowed to stand, all the other sugars of this group appear
in time in the solution. Thus there is the formation of a ketose,
d-levulose, from an aldose, d-glucose, sugar. On the other hand, the
sugars of the galactose series, such as tagatose, sorbose or talose, do
not appear. Only those sugars appear which involve a change in
structure of the first two or three carbon atoms of the chain, thus show-
ing that the molecule is most unstable and reactive at this end. There
is, as it were, a gradient of reactivity in the molecule from the aldehyde
end extending downward, resembling, superficially at any rate, the
gradient in reactivity in an earthworm, which is most.reactive at the
head end. The transformation of an aldehyde to a ketose sugar, and
from the one isomer to the other, probably takes place with the inter-
mediate formation of an enol modification as follows:

H # H H H H

I 4
r—t—¢ — Oo +H,0 —— R—C—C—OH + NaOH ——— R—C—C—O0H +H, 0

bx bu On bu bna
Aldose.
32 PHYSIOLOGICAL CHEMISTRY

OH H ae

eta — ie = Rb — bon =< — R—C—C_OH
OH ONa '¢ ik

Their great instability in alkaline solutions makes it necessary in evapo-
rating sugar solutions to be sure that the solution is exactly neutral.

The explanation of this behavior of the sugars is very interesting.
The decomposition of glucose may be taken as a type of all. The first
thing which happens when mixed with an alkali like sodium hydrate is
that a union occurs and a salt is formed. One of the hydrogens of the
glucose behaves as an acid hydrogen and is, hence, believed to be slightly
ionized. It is this hydrogen which is replaced by sodium. The hydrogen
thus replaced may be the hydroxyl. hydrogen just behind the aldol
group, the @ hydroxyl, or else one of the hydrogens of the aldol group.
An aldehyde easily opens up its double bonds between carbon and
oxygen and adds water to form a polyhydric alcohol, as follows:

R-C=0 —- R-0- —~ R- 0-08

i a“ i

With sodium hydrate there is formed the salt either:

H H H H
rbd =0; or, n—¢_d_o—na.

ba bu bx
This salt is unstable and the molecule now first forms enols and then
breaks apart into a number of pieces, double bonds appearing first
between the carbons in the manner described on page 86 and then
disruption occurring at the double bonds, thus:
H H OH OH

not_$—bo= d-c_o_na;

hk ton |

and this is perhaps followed by subsequent decomposition into such
pieces as:

Enol. Ketose.

OH H OH H
ais , at-d=; =b- Lom,
k ou
1. 2. 3.
H H OH H OH OH

HOtbo¢ Y= ilies b_d_o—ne.
x hu

4, 5.
By this dissociation pieces of varying numbers of carbon atoms are
THE CARBOHYDRATES 33

probably formed. These dissociated pieces are very reactive in their
nascent state when the free bonds on the carbon are open. They undergo
intermolecular changes into acids, aldehydes or alcohols; they have
strong reducing properties and if oxygen is present they unite with it
to form aldehydes and acids; but if sufficient oxygen is not present to
oxidize each piece as rapidly as it is set free, the particles interact, con-
densation occurs, caramel and resinous substances are produced which
cause the brown color.

The part which is hypothetical in the foregoing explanation is the
composition of the fragments which are first formed under the action of
the alkali. There is no doubt that a salt is first formed and that this salt
is unstable and decomposes with an unsaturated enol state intervening.
We infer the nature of the fragments from the composition of the final
products.

This decomposition, or fragmentation, is probably closely similar to
the decomposition of the sugars in living matter; and there, as here, if
sufficient oxygen is present, as it probably is on the periphery of cells,
sugar will be burned or oxidized to lactic, carbonic, formic, glyceric,
tartaric or tartronic acids; while if oxygen is not present in sufficient
amounts to burn these reactive pieces as rapidly as they are set free,
and this will probably be the case in the interior of the cells, the pieces
will reduce substances near them or each other, or they will condense
with ammonia or with each other transforming into amino acids, aro-
matie substances, fatty acids and other products of the metabolism of
the sugars. The important thing, however, to note in this and to
remember, for we shall return to it in discussing the metabolism of the
sugars and indeed of other substances in the body, is that the decom-
position or rearrangement of the molecule into reactive pieces is a pre-
liminary to metabolic transformations.

SoME ACIDS FORMED FROM THE CARBOHYDRATES BY OXIDATION.

HCOOH Formic
HOCOOH Carbonic
COOH—COOH Oxalic
CH,—CHOH—COOH Laetie
CH,—CO—COOH Pyruvic
COOH—CHOH—COOH Tartronic
COOH—CH,—CH,— COOH Succinie
COOH—CH,—CHOH—COOH Malie
COOH—CHOH—CHOH—COOH Tartarie
COOH—CHOH—-CHOH—CHOH—CH ,0H Ribonie

COOH—-CHOH—CHOH—CHOH—CHOH—COOH Saccharic
COOH—CHOH—CHOH—CHOH-—-CHOH—CH, 0H Gluconiec

The ionic theory explains the reason why the molecule is so unstable
in the salt form, whereas it is so stable in the form of the free monosac-
34 PHYSIOLOGICAL CHEMISTRY

charide. This explanation is as follows: The sugar molecule itself ionizes
in aqueous solution very little. This is shown by the fact that solutions
of the sugars in water are non-conductors; the avidity of the sugar as
an acid is very low. There are, hence, at any instant of time in the
solution of a monosaccharide very few C,H,,O; ions. The sugar is a
weaker acid than carbonic; it is about as weak as boric, or hydrocyanic
acid. The salt formed by the addition of sodium hydrate, however,
ionizes easily, hence the salt is widely dissociated, just as sodium acetate
is much more widely dissociated than acetic acid. Just why the sodium
salt ionizes more than the hydrogen salt is not yet known, but it may
be that it is connected with the power of the sodium to unite with water
molecules through its reserve or extra valences, this power being absent
in the hydrogen which apparently has very few such reserve valences.
At any rate, whatever the reason may be, sodium does ionize more. As
a result the oxygen atom of the carbohydrate from which the sodium
is separated is left with a free negative charge and this may be supposed
to exert an influence over the whole molecule, since the bonds between
the atoms are electrical in nature, but the effect is strongest in the
two or three terminal carbons. As a result the molecule loses water
and double bonds appear at several places in the molecule. The double
bond between carbon atoms is not stronger than a single bond, but it
is weaker. Why it is weaker is not certainly known, but it may be
because when two bonds are present the atoms can separate without
electro-static stresses being set up between them, since each atom takes
a positive and negative charge with it thus: C7 +C—- C+, and Cz. We
shall find the same facts of the instability of unsaturated carbon com-
pounds illustrated in the fats and indeed in other cases, double bonded
compounds being generally more reactive than single bonded. <A very
striking example of a molecular rearrangement due to ionization is
shown by the indicator phenol-phthalein in which the negative ion under-
goes rearrangement to a red quinonoid substance.

If the alkali is very weak so that only the terminal and @ carbon
atoms are involved (Nef), condensation of the monosaccharides to form
di- and polysaccharides may occur. Thus cane sugar may be synthesized
from d-glucose. There is at first under the influence of the alkali a
transformation of some of the glucose to d-fructose and then the con-
densation of some of these molecules with glucose to make cane sugar.

Action of alkalies on di- and polysaccharides.—The action of alka-
lies on disaccharides varies with the nature of the sugar. Cane sugar,
for example, is a very weak acid, it has no free aldehyde group and
it is stable in an alkaline solution. Consequently it does not break into
fragments and accordingly cane sugar reduces an alkaline copper salt
solution only at a very slow rate. The case is, however, quite different
THE CARBOHYDRATES 35

ter those disaccharides like lactose and maltose which contain free
aldehyde groups. They are almost as unstable as glucose in alkaline
solution; their solutions turn brown or yellow very quickly. This is
. probably the reason, for example, that soda biscuits become yellow if too
much soda is used in their preparation. The decomposition of the lactose
of the milk by the alkali causes a yellow color. Solutions of lactose and
glucose and maltose of the same molecular concentration and the same
alkalinity absorb oxygen at almost the same rate. The character of the
fragments into which the disaccharides decompose is still unknown.
Many of the polysaccharides are quite stable in alkaline solution. This
is the case, for example, with glycogen or animal starch. It is pre-
pared by destroying all the protein matter'of the cells. by cooking the
tissue with 30 to 40 per cent. potassium hydrate, a procedure which
leaves the glycogen quite unaffected. Starch also is quite resistant to
alkalies. :

Action of acids on monosaccharides.—Not only do the carbohydrates
decompose spontaneously in alkaline solution, but they do so in acid
as well. The molecule is, indeed, most stable in the neutral form and
least stable as a salt. The decomposition of the monosaccharides in acid
solution is slower than in alkali and the decomposition is not so complete,
so that the various steps can be followed and the intermediate products,
or some of them, can be isolated. But it is as yet uncertain whether
the decomposition in the acid is in all particulars identical with the early
stages of the alkaline decomposition or not. Advantage is taken of the
difference in the decomposition of hexose and pentose sugars in acid
solution tc distinguish between them.

If acid is added to the solution of a hexose and if the reaction is
quickened hy boiling, it will be found that the hexose decomposes. If
the acid is strong, brown or black humus substances are produced ; if the
distillate is collected it is found to contain formic acid, carbon monoxide,
a little hydroxymethylfurfural, and in the solution remaining in the flask

O

considerable quantities of levulinic acid, CH,—C_—CH,—CH,—COOH,
are to be found. If, however, a pentose is subjected to the same treat-
ment, it distills over almost quantitatively as furfuraldehyde or furfural,
and this we detect by the colored condensation products it yields with ani-
line acetate, orcine, phloroglucine, resorcine and other substances. Pen-
toses may, therefore, be readily distinguished from hexoses by the large
quantity of furfural yielded on distilling the former with acid, and by the
levulinie acid formed under the same circumstances from a hexose.
Berthelot gives the following figures illustrating the decomposition of
hexoses on heating with phosphoric acid:
36 PHYSIOLOGICAL CHEMISTRY

Glucose heated with phosphoric acid for several hours:

co, a names eee 2.07%
COO saerncineteaes 1.19
Formie acid ...... 11.90
Levulinie acid .... 39.88
Humic acid ...... 23.60

 

78.64
Loss (in part H,0) 21.36

This different behavior of hexoses and pentoses is the basis of the
orcine, aniline acetate and most tests for pentoses. Bran and straw
contain large amounts of polysaccharides containing a pentose (xylose),
which are hydrolyzed by the acid. The free pentose is then converted
into furfural. Bran heated with acid yields accordingly a great deal of
furfural.

As has already been stated the decomposition of the monosaccharides
by acids is closely analogous to that by alkalies, but is not so rapid or
profound, and some of the intermediate products may be isolated. The
acid first unites with the carbohydrate molecule, forming a salt. This
is shown by the fact that the addition of a carbohydrate solution to a
weak acid solution diminishes its acidity. The acids probably unite with
the double bonded oxygen of the aldehyde, if this is present, or with
the oxygen of the alcohol groups. It is well known that the aldehydes,
ketones and other organic compounds containing oxygen have very weak
basic properties, due to the oxygen they contain. They form with acids
what are known as ozonium salts, analogous to the ammonium salts of
nitrogen. Oxygen is at times tetravalent; it opens up two residual
valences and is able thereby to unite with acids just as ammonia does.
Thus we probably have in the sugars when treated with hydrochloric
acid solution the compounds:

H
R-C=0; R-0= 0-01; R= +l
H i i 8
The chloride thus formed ionizes, the chlorine being negative, the rest
of the molecule positive and, possibly as a result of this free positive
charge, molecular rearrangement takes place, water is eliminated and,

in the case of pentose, furfural is formed thus:
OHH OH OH H H

ptt ¢4oW 6 te ee wb do c—C = 0+ 8H,0 + HCl

bh bak 4d L—o—! 4

In alkaline solution furfural is unstable and rapidly decomposes as
already described, but in acid solution it does not readily decompose,
1

THE CARBOHYDRATES 37

, but may be distilled. In thé case of a hexose the reaction may go in
various ways, methylhydroxyfurfural being formed in small quantities,

. perhaps as an unstable intermediate product and many decomposition
and condensation products, i.e., levulinic acid, humic acid, ete. Methyl-
hydroxyfurfural has the following formula:

H H OH H
ec = bd = ed =O ~
| os

Mcthylhydroxyfurfural.
It is probably the substance which is responsible for the color in the
Molisch test and in the Seliwanoff reaction.

The behavior of the carbohydrates in acids and alkalies shows that
there are two points of attack in the molecule, two points at which
the molecule may unite with protoplasm or enzymes, namely, the residual
or extra valences on the oxygen, where acids unite; or the double bonds
in the aldehyde, where the alkali unites. It is possible, perhaps probable,
that the decomposition of a monosaccharide by an enzyme is analogous
to that produced by acids and alkalies, the enzyme upsetting the equi-
librium of the molecule in the same manner as it is upset by acids or
alkalies, the easiest change produced being in the aldehyde or @ carbon
groups. .

It must not be concluded from this discussion that substances are
always more unstable in the ionic or salt form than they are in the
undissociated form. In some cases, at any rate, the reverse is the case.
Thus cysteine, one of the amino acids, unites with oxygen with great
speed in the neutral or undissociated form, whereas it is very stable in
the acid solution where it exists as a salt, and oxidizes at a slow rate
in an alkaline solution. And many other examples of this sort might be
given. The free organic acids, undissociated, are indeed often less stable
than their salts.

Action of acids on polysaccharides.—The di- and polysaccharides
decompose readily into monosaccharides by hydrolysis when treated with
acids, and in this respect they are less stable in acid than in alkaline
solution. The various disaccharides break up with varying speed in acid
solution and those which are the most resistant to the action of alkali
are the most sensitive to the action of acid. Thus saccharose, which
decomposes very slowly in the alkali, is the easiest of the disaccharides
to invert with acid; and lactose and maltose, which are so unstable in
alkali,-hydrolyze much more slowly than cane sugar in acids. Starch and
glycogen are quickly hydrolyzed by acid, but are very resistant to
alkalies. It is by the action of acids on starch that commercial glucose
is prepared. The probable reason why the polysaccharides are so sensi-
tive to acids and so inert to alkalies:is that, as their acidity is reduced
38 PHYSIOLOGICAL CHEMISTRY

by polymerization, they react very little with alkalies to form salts;
on the other hand, their basicity is increased, causing them to unite more
readily with acids. The reactions are written as follows:

C,,H,,0,, +H,0 + HCl—- 2C,H, 0, + HCl.

12 #22 11 6 12

(C,H 0.), + 2H,0 + HCl 20H Oo, + HCl.

6 10 12 6

Oxidation.—The monosaccharides are readily oxidized; and in acid,
but particularly in alkaline, solution they are fairly strong reducing
agents, oxidizing themselves to acids. Their reducing powers furnish
one means of detecting them and measuring the quantity present in a
solution. An alkaline solution of any monosaccharide will reduce
methylene blue, cupric, silver, ferric, mercuric, gold or bismuth salts;
and either in acid or alkaline solution they will reduce bromine, chlorine,
permanganates, peroxides or atmospheric oxygen. They burn sponta-
neously with the liberation of heat, or even some light, in alkaline solu-
tions, and with the liberation of electrical energy, heat and possibly
light in protoplasm. By their combustion various acids are formed,
carbonic, lactic, tartaric, malic, malonic, tartronic, oxalic, gluconic and
saccharic. By very mild oxidation the osone is first formed, p. 43.

Fehling’s Solution. They are quantitatively estimated, or qualita-
tively detected, by their reducing action on alkaline cupric tartrate
solutions: Such a solution is that of Fehling. This consists of two
solutions, A and B, which are kept separate until they are used. A
consists of an aqueous solution of cupric sulphate containing 34.64 grams,
CuS0O,.5H,0, in 500 ¢.c.; B is made by dissolving 125 grams of potas-
sium hydrate and 173 grams of sodium potassium tartrate (Rochelle
Salts) in water and making up to 500 ec.c. Just before using, equal
quantities of the two solutions are mixed and heated to boiling and an
equal, or smaller, quantity of the solution supposed to contain the sugar
is added and the solution boiled in a qualitative test for two or three
minutes. The solutions are kept separate until the time of using because
they interact slowly, the tartrate slowly reducing the copper. This fact
suffices to show the incorrectness of the statement sometimes made that
the reducing action of the monosaccharides is due entirely to the aldehyde
groups they contain. There are no such groups in the tartrates.

The various constituents of Fehling’s solution act as follows: The
sodium hydrate unites with the carbohydrate, decomposing it, in the
manner already discussed, into a number (8—4) of reactive fragments,
these fragments being the actual reducing substances. These fragments
and the remaining original molecules are then oxidized to various acids,
such as oxalic, malonic, tartronic, carbonic, gluconic and so on. The
reaction by which gluconic acid is formed may be written as follows:

C,H,,0, + 2Cu(OH), = C,H,,0,+ Cu,O + 2H,0

Gluconic Cuprous
acid. oxide.
THE CARBOHYDRATES 39

The tartrate is added to unite with the cupric hydrate formed by the
interaction of sodium hydrate and cupric sulphate, which in the absence
of the tartrate would be precipitated as a blue, gelatinous precipitate,
turning into the black oxide on heating. This precipitate would obscure
any cuprous oxide which might be formed by reduction. The tartrate
by uniting with the cupric hydrate holds it in solution; the following
reaction taking place:
Na—O—C—O Na0—C=—O

|
HC—OH t_o—cuoH
-HC—OH + 2Cu(OH) —nt_o_cuor + 2H,0

Kobo Kobo
Rochelle salt.

The cupric hydrate-tartrate compound is a deep-blue color and is
soluble. It is in equilibrium with a very small quantity of Cu(OH),
which remains in solution ; and this is in equilibrium with a very minute
amount of free Cu ions and OH ions. The cupric ion having two
free positive charges, which it gives up very readily, is the oxidizing
agent and it is reduced to the cuprous state by the active reducing frag-
ments formed from the sugar molecule by the alkali. Cuprous hydrate,
which is thus formed, is a yellow substance which is very insoluble. It
either precipitates as such or it loses water and forms the bright red,
insoluble cuprous oxide, Cu,0. This cuprous oxide may be filtered from
the solution by an asbestos filter, washed and weighed direct, or it may
be oxidized to the black cupric oxide and weighed as such; or dissolved
in nitric acid and the copper determined by electrolytic deposition ; or
dissolved in ferric alum [NH,), SO,, Fe,(SO,) 3-24H,0] and the fer-
rous salt formed titrated by permanganate. In this way the amount: of
reduction occurring in the solution may be determined. . The best method
is the last. (often called Bertrand’s). It will be seen that the tartrate-
copper-hydrate acts as a reservoir of cuprie hydrate, which is dis-
tributed all through the solution, and as soon as the cupric ions are
reduced and the equilibrium thus upset, new cupric hydrate is at. once
dissociated from the tartrate compound. It thus happens that although
at any instant of time there is only a minute amount of the intermediate
substance, the cupric ion, present, yet since the ion. is formed. instan-
taneously the reaction can proceed at. a rapid rate. If it. took, an _appre-
ciable time for the. dissociation. of. “the copper tartrate compound this
solution. could not be. used in the way. it. is. This i is a. very good. example,
in. all likelihood, of. the manner in which. a large transformation. can
take place through an intermediate. stage, ‘yet the intermediate stage
itself be present at.any instant of time only i in the most: minute amounts.
One molecule of .glucose will under certain . conditions reduce four or
five molecules of cupric hydrate.
40 PHYSIOLOGICAL CHEMISTRY

Inasmuch as the reaction is not complete, but the oxidation con-
tinues at a slow rate for a long time, the malonic, tartaric and gluconie
acids being slowly oxidized, the quantitative determinations have to be
made under strictly comparable conditions of concentration, time of
heating, etc., and the method is at best rather unsatisfactory. For the
quantitative determination more in detail, see Part III, experiment 241.

Glucose, levulose or galactose will reduce copper sulphate without
being made alkaline, but the time required is much longer, the reason
being that the number of active sugar particles in such solutions and
the number of hydroxyl ions is enormously less than in the alkaline solu-
tions. On the other hand, the number of cupric ions is larger in the acid
solution, but this is not sufficient to counterbalance the diminution in
the active reducing particles formed from the sugar. If cupric sulphate
is heated with levulose for some time the cupric oxide is reduced in
large part to metallic copper, beautiful reddish crystals of copper being
formed. The reaction in this case goes so slowly that the cuprous oxide
is not formed rapidly enough to precipitate, but remains in solution
and is further reduced to the metallic form. Copper acetate solutions
with more or less acetic acid added to them may also be used in place
of Fehling’s solution. One such mixture is that of Barfoed, which is
incorrectly used sometimes to distinguish between monosaccharide and
disaccharide sugars. All sugars, however, reduce copper acetate with
or without the addition of acetic acid if given time enough, and the
separation of the different sugars by this test is purely quantitative,
depending on the velocity with which various sugars oxidize. It is not
qualitative. Of the various sugars levulose reduces in acid solution the
most rapidly, since it is the strongest acid among the sugars; then
galactose, glucose, maltose, lactose and cane sugar follow in the order
named. The initial rate of oxidation of the various sugars in mixtures
of cupric acetate and acetic acid is as follows: lactose, 1; maltose, 1.15;
glucose, 5.71; galactose, 8.72; mannose, 8.72; levulose, 55.13. Since
there is quite a decrease in acidity or stability between glucose and
maltose a glucose solution reduces Barfoed’s quite a good deal faster
than maltose under similar conditions of concentration and heating, so
that with a strict control of these factors, Barfoed’s solution may be used
to distinguish qualitatively between these two groups of sugars, the
monosaccharides on the one hand and the di- and polysaccharide sugars
on the other; but a strong solution of maltose will reduce more rapidly
than a weak solution of dextrose, so that as the test is ordinarily per-
formed without control of the concentration of the sugar used, and time
of heating, it is indecisive. It is sometimes stated that the disaccharides
must be inverted first before reducing, but the reducing action -of the
disaccharides in alkaline solution, in which they are not inverted, shows
that inversion is not a necessary prerequisite for reduction. The weak
THE CARBOHYDRATES 41

reducing powers of saccharose are due to the stability of the sugar in
alkaline solution, but it will reduce Fehling’s solution at a very slow
rate. Glycerine also will reduce Fehling’s solution, but the rate is so
slow as not to introduce an appreciable error as the estimation is ordi-
narily carried out.

By oxidation of glucose by bromine in acid solution the sugar is
converted quantitatively into gluconic acid, the monocarboxylic hexonic
acid, and by further oxidation into saccharic acid. By nitric acid both
of these acids are obtained from glucose, and galactonic and mucic acids
from galactose.

Other substances reduced by the carbohydrates with a color change
are described in the practical part. Among these may be mentioned
white bismuth subnitrate which is reduced to black bismuth; picrie acid
which is reduced to red picramic acid:

C,H,.0H.(NO,), C,H,.0H.NH,. (NO,),
Picric acid. Picramic acid.

 

silver nitrate or mercury salts to black metals or mirrors. All of these
changes are produced by reduction and may be caused by other sub-
stances than sugars.

The reduction of carbohydrates.—Carbohydrates not only have the
power of oxidizing themselves at the expense of other bodies, thus acting
as reducing substances, but they also may oxidize other substances, being
themselves reduced thereby. When oxidized the reactions liberate heat :
they are exothermic and it is by means of such reactions that the body
gets its energy. But if the carbohydrates are reduced, the reactions are
endothermic; heat is absorbed to be given out again when the substance
is finally burned. Both kinds of reactions go on in living matter and
in our own bodies in the decomposition of the carbohydrates of the
food. The greater part of the carbohydrate is oxidized and the heat
and energy are set free by which we live, but some of the fragments of
the carbohydrate molecule do not oxidize at once; they are reduced,
not oxidized; and by this means various important substances, pre-
eminently the fats, are formed in the body. The carbohydrates have
the power of absorbing nascent hydrogen and they are changed thereby
in part to alcohols. Such alcohols are quite widespread in nature. Thus
levulose and mannose are transformed into d-mannitol, a hexatomic
alcohol; d-glucose into d-sorbite. Similarly the fragments into which
the glucose molecule falls may take up hydrogen and be converted to
the long carbon chains of fatty acids such as palmitic, C,,H,,0,, and
stearic, C,,H,,0,, acids. But the exact steps of this process are still
unknown. By means of this oxidizing property the carbohydrates play
a great part in what is known as anerobic respiration. Many bacteria
and animal tissues have the property of continuing to live and give off
42 PHYSIOLOGICAL CHEMISTRY

carbon dioxide in the absence of air. They can carry out a great number
of reactions which are in part oxidative without atmospheric oxygen.
It has been found that the injection of glucose enables fish and other
animals to get on for a much longer time without atmospheric oxygen
than when they have no glucose; and glucose is particularly favorable
for the anerobic life of many bacteria. Glucose thus plays, in virtue of
its oxidizing properties, a very important part in cell life. Tissues
which contain glycogen or glucose will generally live longer in the
absence of air than tissues which do not contain these substances. It has
been suggested that in these cases the tissue oxidizes itself from the
oxygen of the water, the nascent hydrogen set free being taken up by
the glucose. Whether this explanation of the favorable action of glucose
on angrobic respiration is correct or not is doubtful. It may be that
one molecule is reduced to an alcohol, while the other sugar molecule is
oxidized to an acid. This would be possible if the amount of heat set
free by the oxidation of aldehyde to acid was greater than the amount
of heat rendered latent by the reduction of aldehyde to alcohol.

Relation to hydrocyanic acid.—An interesting property of the car-
bohydrates is their power of uniting with hydrocyanic acid. If glucose
be dissolved in water and potassium cyanide added to it, not all the
_bydrocyaniec acid can be distilled off from the glucose on acidification.
‘Some has combined with the glucose. Glucose exists, for example, in the
white of hen’s eggs to about 0.5 per cent. It was found that the alco-
holie extract of egg-white had the power of binding hydrocyanic acid,
due to the presence of this glucose. The importance of this power of
binding hydrocyanic acid from a chemical point of view is that it en-
ables one to build up the sugars carbon atom by carbon atom. A
heptose may in this way be formed from glucose as follows:

CH,OH—CHOH—CHOH—CHOH—CHOH—CHO + HCN——
CH ,OH -CHOH—CHOH—CHOH—CHOH—CHOH—CN

CH,OH.(CHOH) (CN 42H, O —+ CH,OH. (CHOH) ,.COOH + NH,
CH 0H. (CHOH) *COOH 43H —_ CH: 20H (CHOH ) COH +H, 0
Heptose.

By the’ action of acids the nitrile is saponified to the acid which by
‘reduction yields the aldehyde, glucoheptose. Physiologically the reac-
tion is of interest because hydrocyanie acid unites with some substance
in the cell which is of great importance in respiration; and it indicates
that glucose should be a good antagonist to the cyanides. Whether this
reaction plays any part in building up sugar in plant cells is very
doubtful.

Oximes.—Just as by the action of hydrocyanic acid the sugars may
be built up carbon atom by carbon atom, so by the action of hydrox-
ylamine the sugar of the next smaller number of carbon atoms may
THE CARBOHYDRATES 43

be obtained. Hydroxylamine, HNHOH, which corresponds to hydrogen
peroxide, HOOH, and like it has reducing and oxidizing properties, acts
on glucose to form the glucose-oxime thus:
CH,OH (CHOH) ,.COH + HNHO!]—~ « MOH (CHOH), CH:NOH -+- H,0
Glucose oxime.

The oxime when heated with acetic anhydride loses water and is con.
verted into the acetylated glucosenitrile; which when warmed with am-
moniacal silver nitrate loses hydrocyanic acid and is converted into an
acetylated pentose from which the pentose may be set free.

Osazones.—A reaction very important for distinguishing between
different mono- and disaccharides is the formation of osazones. If
phenyl hydrazine and glucose solution be warmed the following reaction
takes place:

1. 0,H, 0 is +C et, NH.NH, _—_ CH,OH. (CHOH) .CH = N.NHC,H, +H,0
Phenyl hydrazine. Phenyl-gluco-hydrazone.
2. CH,OH. (CHOH) cH =N.NHC,H, + 2C,H NH.NH, =
Cc et NH, + CH,OH. (CHOH) gC ( = N.NHC et, ) CH = N.NHC.H, +NH,
Aniline. + Phenyl-glucosazone.
The phenyl osazones are generally needle-shaped small crystals, yellow
in color. The melting points of the different osazones differ somewhat
as follows: glucosazone, 205° C.; galactosazone, 192-195°; maltosazone,
206° ; lactosazone, 200°.

The hydrazones which are formed in the reaction are generally not
crystalline, but in the case of mannose the hydrazone is insoluble and
forms colorless, plate-like crystals (m. p. 195°) which make the identifi-
cation of this sugar very easy. If heated long enough, however, mannose
will form the yellow osazone also. The crystals of the various osazones
cannot be distinguished easily by their shapes. Some of the substi-
tuted phenyl hydrazines give more insoluble osazones, the methyl or
ethyl-phenyl hydrazine being often employed. Since the sugars may
be regenerated from their osazones, this is one method which may be
used for their separation from a solution. It will be found that the
speed with which the various sugars react with phenyl hydrazine differs,
being in the order of their ease of oxidation, levulose reacting with the
greatest ease, and lactose most slowly. Levulose, mannose and glucose
give identical osazones, showing that the last four carbon groups in
each chain are the same in configuration. (See page 28.) Glucosamine
readily yields glucosazone. In reaction 2 above the sugar is oxidized to
an osone which condenses with the third molecule of phenyl hydrazine.

If glucosazone is treated with hydrochloric acid and water, glucosone
ig first formed, which on reduction yields d-fructose. Glucosone is an
aldehyde ketone of the formula: CH,OH.(CHOH),.CO.CHO. By this
means one can convert an aldose into a ketose. Glucosone is one of the
first products of oxidation of glucose by mild oxidizing agents.
44 PHYSIOLOGICAL CHEMISTRY

The formation of the osazones is made in practice by using pheny] hydrazine
hydrochloride in place of the free phenyl hydrazine. The hydrochloride is used for
two reasons: both because the salt is more stable than the free base and because
it is more soluble. Owing to hydrolytic dissociation of the hydrochloride, free
hydrochloric acid is produced. This checks the speed of the reaction by reducing
the dissociation of the sugar and, moreover, the reaction of the phenyl hydrazine
with the sugar involves the free base of the hydrazine and not the ion of the salt.
Accordingly to reduce the acidity of the hydrochloride of phenyl hydrazine and to
inerease the amount of free base it is customary to add sodium acetate to the phenyl
hydrazine hydrochloride. This makes sodium chloride and phenyl hydrazine acetate,
and since acetic acid is a very weak acid the phenyl hydrazine acetate undergoes
hydrolytic dissociation into the acid and free base and the reaction goes much better
than if the sodium acetate is not used.

By the use of methyl-phenyl hydrazine (Neuberg) the ketoses can be
distinguished from the aldoses. The ketoses form osazones with this
reagent, while the aldoses form only hydrazones. According to Betti
the aldoses may also be separated from the ketoses by the use of
#-naphthol-benzylamine,

C,H,
. es,
10 6
This combines easily with the aldoses to form a crystalline product, but
not with ketoses. Glucose can thus be readily separated from levulose.

Reactions of carbohydrates with ammonia to form nitrogen con-
taining compounds.—Glucose and other carbohydrates react readily with
ammonia in weakly alkaline solutions to form nitrogen-containing sub-
stances such as the amino-acids. See page 186. By this reaction the
proteins probably originate from the carbohydrates.

Synthesis in Plants—All the energy of the animal body has come
in the long run from the sun in the form of light energy. We are the
children of the sun. The great synthetic agency on the earth’s surface
has not been heat, but light. Living matter could not have come into
existence when the temperature of the earth was very different from
what it is at present, for the activities of all living things are limited
practically to the very narrow temperature range from about zero
centigrade to about 70°. Light is to-day actively bringing about the
syntheses underlying all vital processes and, in the absence of evidence
to the contrary, we may believe that this has been the case since before,
the origin of living matter. Heat produces its syntheses in a very rough
manner by the mechanical shocks of the rapidly vibrating molecules,
or by the vibrations of the atoms in the molecules; but light, by a finer
mechanism, its shorter waves, picks out the very bonds of the atoms
themselves, the valence electrons, and tears them away from or in-
jects them into atoms. Light is thus an enormously more powerful
chemical agent than heat. It seems from the work of Drnde and his
THE CARBOHYDRATES 46

successors that it is the valence electrons which have the property of
vibrating with light, and these are responsible for the refractive and
color properties of substances. Of the light waves it is particularly the
short ultra-violet rays which are most powerful.

The synthesis of the monosaccharides from carbon dioxide and
water is at present carried on only in the chlorophyll or other pigment-
hearing plants such as:the red or blue-green alge; but, as we shall see,
chlorophyll is not necessary for this synthesis; it only makes possible
the utilization of parts of the spectrum which otherwise cannot be used.
The synthesis occurs in the chlorophyll bodies composed of chlorophyll
and protoplasm ; in these bodies carbon dioxide and water unite in the
sunlight to form.carbohydrates and liberate at the same time oxygen.
If green leaves are exposed to carbon dioxide and sunlight starch appears
in them. The same leaves in the dark make no starch and that which
existed already disappears. Chlorophyll is not necessary for the trans-
formation of a monosaccharide into a polysaccharide since glucose turns
into starch in the roots, or tubers.

The method of the synthesis of the monosaccharides while in some
particulars still obscure has been greatly illuminated by the discovery
that light itself, and particularly ultra-violet light, even in the absence
of all chlorophyll or protoplasm, will make formaldehyde and oxygen
from a mixture of carbon dioxide and water. This has been the work of
Berthelot and Gaudichon. For this synthesis ultra-violet light, that is
light of very short wave length, beyond the spectrum visible to our
eyes, is most effective. If a mixture of carbon dioxide and water is
illuminated by the light of a mercury arc in a tube of pure silica, this
light being particularly rich in ultra-violet rays and silica not absorbing
them as does glass, it has been found that the mixture very quickly
contains hydrogen, oxygen, carbon monoxide, carbon dioxide, formalde-
hyde and hydrogen peroxide. The reaction goes to the point of equi-
librium ; it is never complete. If one start with a carbohydrate solution,
or with formaldehyde and oxygen, the reaction is reversed to the same
mixture, as if one started with carbon dioxide and water. The reaction
which ensues may be written thus:

Light + 2CO, + 2H,0 —~ 0, + CO +CH,04H,0

The energy of the ultra-violet light has been absorbed and appears as
the potential energy of the system ‘‘ oxygen-formaldehyde.’’ It is gen-
erally stated that the energy goes into the formaldehyde as potential
chemical energy, but this is not strictly true: the energy is really
represented by the system, ‘‘ O,—H,,’’ and by the system, ‘‘ Oxygen-
formaldehyde,’’ for by the reunion of these substances the energy is set
free. In a still narrower sense the energy may be said to be locked up
in the oxygen molecule; the oxygen atom in an elemental condition being
astaally less stablo (in the presence of water) owing to its having one
46 PHYSIOLOGICAL CHEMISTRY

negative electron less than in the ionic oe or when it is united with
hydrogen or carbon.

Oxygen seems particularly sensitive to the action of these light waves,
a great many organic compounds being easily oxidized in light and in
the presence of oxygen, but being quite stable in the dark in the presence
of oxygen; or in light, in the absence of oxygen. Cholesterol, for ex-
ample, behaves in this way. The light syntheses are also independent,
or largely independent, of temperature, so that light syntheses and de-
compositions will occur at low as well as at high temperatures. In short,
the chemist has in light an enormously more efficient and finer agent for
chemical syntheses than he has in heat.

How very powerful these ultra-violet radiations are’is made apparent
by their action on the skin. An exposure of the skin to the light of a
quartz mercury tube for thirty seconds is often sufficient to cause as
severe a sunburn as one will ordinarily have after a day’s exposure to
the sun; and the destructive action of the rays on the retina or cornea
is said £6 be so great that glass spectacles, preferably darkened, must
always be worn in conducting these experiments. Blindness is: easily
produced by the rays. Their powerful irritant action on the skin is
made use of therapeutically in treating skin diseases by sunlight or by
the Finsen method. The irritant action of these rays greatly aggravates
the skin eruption in smallpox. It is an interesting fact that although
these rays irritate the cornea in so destructive a manner they do not
give rise to a sensation of light. We cannot see by them. There are,
however, creatures which can. Ants perceive the longer of these ultra-
violet radiations; but on the other hand they cannot perceive light at
the other end of the spectrum which is readily perceived by us, conse-
quently ants under a brownish-red glass which absorbs the violet end
of the spectrum are in darkness to them, whereas we can watch them
without difficulty. The ultra-violet rays from the sun are for the most
part absorbed in passing through the atmosphere, being absorbed by the
oxygen and moisture of the air. But since carbon dioxide is always
present in the air it is interesting to reflect that the fundamental syn-
thesis of formaldehyde is probably going on all the time in the air at
the expense of these rays; and H,O, and hydrogen are constantly form-
ing. Before the carboniferous period, when the coal now locked in the
earth was partly in the air as carbon dioxide, this synthesis may have
been far more extensive than it is to-day. Ultra-violet light is then
absorbed by carbon dioxide and water and the energy thus absorbed
becomes potential energy represented chiefly by the system oxygen-
formaldehyde.

The amount of ultra-violet light reaching the earth’s surface after
passing the atmosphere is small, and the advantage of chlorophyll

“a
THE CARBOHYDRATES 47

appears to be that it makes possible the utilization of the longer light
vibrations, which reach the earth in greater abundance, or with more
energy in them. Chlorophyll absorbs in the red or orange end of the
spectrum and has hence a green color. The energy of the absorbed red
light is then used in the synthesis of formaldehyde, just as the ultra-
violet light is used in the absence of chlorophyll. It has been suggested
that cholorophyll acts the part of a transformer, absorbing the long waves
and either giving off shorter ultra-violet vibrations or in an indirect
way bringing the same synthesis to pass. Chlorophyll seems to act much
the part of the light sensitizers added to photographic plates to make
them sensitive to the red end of the spectrum. The exact manner of
action of the chlorophyll is, however, not yet clear. It has been shown
by Priestly and Usher that chlorophyll extracted from leaves has the
property of forming formaldehyde from sunlight, carbon dioxide and
water, so that living matter is not necessary for this fundamental: syn-
thesis. It is, no doubt, not without significance that chlorophyll solutions
are fluorescent and that uranium salts seem to have a similar Biol:
dynamic action on carbon dioxide.

Formaldehyde, CH,O, thus formed is itself the simplest of the carbo-
‘hydrates. If made very faintly alkaline it has been found that it trans:
forms itself spontaneously by condensation into a mixture of sugars
called formose or acrose. This condensation goes probably as follows :

2H,C = O —~ CHO.

2 oF
Further condensation leads to the hexoses. This transformation is all
‘down hill as far as the energy goes. That is, the reaction is exothermic,
‘one molecule of a hexose liberates less energy on oxidation than do six
molecules of formaldehyde.

The transformation of formaldehyde into a true monosaccharide is
not, however, produced in plants by an alkaline reaction for plant
protoplasm is almost neutral in reaction. It has a hydrogen ion con-
centration of about 210-8 normal. The condensation of formal would
dé ‘slow under these conditions. Moreover plants make different sugars
‘so that the transformation must be directed or regulated in some way.
It'is necessary, in order that the light synthesis should go steadily for-
ward, that the oxygen and the formaldehyde should be removed from
the reaction as quickly as possible so that equilibrium cannot be estab-
lished. An additional reason for the removal of the formal is that the
latter is véry toxic and it must be changed to some non-toxic substance.
Plants possess, to accomplish these ends, a catalytic agerit of some kind
as yet unknown which converts the formaldehyde almost as quickly-as
it is formed into hexose or pentose, so that at any instant of time there
‘is not more than a trace of the aldehyde-in the leaves, As has been said,
48 PHYSIOLOGICAL CHEMISTRY

hydrogen peroxide is formed in the light reaction, but as all cells possess
a catalase to convert this into oxygen and water, oxygen is at once freed
and escapes. Hydrogen peroxide, if it accumulates, would destroy the
chlorophyll. It is probable that some animal cells, as well as plants,
are able to convert formaldehyde into a sugar, since some animals have
chlorophyll and can form starch. Volvox and Euglena viridis have this
power. It has recently been stated that turtle’s liver can convert for-
maldehyde into glycogen, but the proof is not yet convincing.

It is probable that not all the formal produced by light synthesis,
or which may be produced in the reverse process of the decomposition
of the carbohydrates, is used in making carbohydrate. Formal is ex-
tremely reactive, combining with amino-groups (NH,) wherever they
are unsubstituted to make methylene derivatives, R—N=CH,, which,
by reduction, become methyl derivatives. Many methylated bases are
found in plants and some also in animals. Such bases are choline,
stachydrine, betaine, creatine. It is very interesting, also, that similar
methylations happen in the animal body when pyridine and some other
compounds are ingested, a fact which would indicate that formaldehyde
might be an intermediate product of carbohydrate metabolism in ani-
mals, as well as in plants. So far as the author knows it has not yet
been isolated from animal tissues as it has from plants, but formic acid
is found in the brain and elsewhere.

The chemical composition of chlorophyll, which has this wonderful
function in the world, is not yet known. It is easily extracted from
leaves by ether, but is then mixed with various lipin impurities. When
decomposed it yields pyrrol derivatives like hemoglobin, the coloring
matter of the blood. It is evidently related more or less closely to the
hematin of hemoglobin, hemopyrrol being identical with phytopyrrol.
Unlike hemoglobin it contains no iron, but the plant must have iron
for its synthesis. Its close relationship to hemoglobin is further estab-
lished by the discovery of the plant chromoproteins, phycoerythrin
and phycocyan, which are crystalline conjugated proteins like hemo-
globin, but they are found in plants and are closely related to their
chlorophyll. Chlorophyll contains magnesium in its molecule, and it
has been suggested that its powers depend on the presence of this ele-
ment. But there is no good reason for this supposition except that
magnesium is used by chemists for certain condensations, although
under widely different conditions from those prevailing in plant tissues.
Hemoglobin also absorbs light, but it is the light chiefly at the violet end
of the spectrum, although there are some bands in the green. By this
absorption the blood pigment is supposed to protect the delicate tissues
from the irritant action of the more refractive rays. It has recently
been suggested that the iron which is always present in the chloroplasts
THE CARBOHYDRATES 49

of plant cells plays a very important part in the synthesis of for-
maldehyde as well as of the chlorophyll. There can be no doubt that
many colorless plant tissues, if exposed to light and if they contain iron,
are able to synthesize chlorophyll.

Special properties of various carbohydrates. A. monosaccharides.
—d-Levulose or d-Fructose. This is one of the most unstable of the
monosaccharides. It is a levo-rotatory, ketose hexose. It shows the prop-
erty of mutarotation. The d- does not mean that it is dextro-rotatory,
but that it is related to d-glucose, which is dextro-rotatory. "When first
dissolved the solution exhibits a much greater rotatory power than after
standing. The specific rotation of the fresh solution is [a] —=—133.5°
but it falls gradually to [a]% =—92.0°. The final rotation is obtained
quickly by the addition of a small amount of sodium carbonate, or other
weak alkali. It is one of the sweetest of the sugars, being sweeter than
glucose. It is widespread in nature, being a constituent of many di- and
polysaccharides. It is one of the constituents of cane sugar, melitose
and lupeose, and inulin (C,H,,0,;) ,, a polysaccharide of the dahlia bulb,
dandelion root, burdock, chicory, etc., is composed of levulose. It re-
duces alkaline, or neutral, solutions of copper salts at a rate faster than
does glucose, but the total amount of cuprous oxide formed per molecule
of sugar is less than in glucose. It is prepared readily by the hydration
of inulin by water at 100°, or by the hydrolysis of cane sugar by sul-
phurie acid. To prepare it from cane sugar the mixture of glucose and
levulose known as invert sugar is freed from acid, concentrated, cooled
and calcium hydrate added. Levulose is precipitated as the calcium
salt, which is filtered, suspended in water and decomposed by carbonic
acid. Pure levulose is white and crystallizes in small needles (m. p. 95°),
or in a dense mass, which, on standing, slowly decomposes, particularly
in the light, and becomes a faint yellow color. Levulose is easily fer-
mentable by yeast, since the last four carbon atoms have the same con-
figuration as d-glucose, and for this reason it was called by Fischer
d-levulose, although levo-rotatory. d- and l-levulose have been syn-
thesized by the action of weak alkali upon formaldehyde. The mixture
was called w-acrose. When heated with hydrochloric acid, solutions of
levulose turn a deep orange-red and by this reaction they may be easily
distinguished from glucose solutions. Levulose may also be distin-
guished by the reaction of Seliwanoff described on page 865. It forms
glucosazone, m. p. 205° C. Levulose together with other sugars is formed
spontaneously from glucose by the action of very weak alkali.

The mutarotation of levulose is probably due, like that of glucose,
to the fact that it exists in solution in two forms, an @ and # lactone
form, the beta form being very much stronger rotatory than the other.
The ketone and lactone forms suggested are the following:
50 PHYSIOLOGICAL CHEMISTRY

 

CH 20H eo
b =O HO—C
Ho—¢_H HO—¢_H
H—b—on H—G_OH
H—b_oH H—¢—0—
bx 20H bao
Ketone form. Lactone form.

d-Glucose or Dextrose. Also called grape sugar. C©,H,,0,. This
is the chief constituent of commercial glucose and is formed by
the action of dilute acids on starch; it is one of the constituents
of cane sugar and is found free in the sap of most plants, in the
juice of many fruits (cherry 11%) and in the blood of many ani-
mals. Together with levulose it occurs in fruits and it may undoubtedly
he called the most important monosaccharide. The glucose found in
nature is d-glucose; it is dextro-rotatory, the specific rotatory power being
either +113.4°, or +19°, as it exists in two varieties. Ordinary glucose
in water solution has the specific rotatory power of +52.2°, being a
mixture of the other two varieties, a glucose and & e@lucose. This mix-
ture is sometimes called y -glucose. In a fresh solution only the first,
or «, variety exists, but it slowly transforms itself into the & variety
until a point of equilibrium is reached. This happens when there is
present .368 parts of the first and .632 parts of the second. This is the
explanation of the mutarotation which such solutions show. The point
of equilibrium is reached only after a day at ordinary temperature and
in neutral reaction; but the addition of even a small amount of alkali
brings it to pass in a few minutes. The two forms of the glucose are
supposed to have the structural formulas shown as follows:

H—C—0H
u—¢—on}

HO—¢—H
ud
u_¢_on

bir,on

 

8-d-glucose. a-d-glucose.

The osazone melts at 205°C. Dextrose crystallizes with one molecule
of water at ordinary temperatures having a melting point of 86°; from
concentrated solutions at higher temperatures it crystallizes anhydrous,
m. p. 146°.

Commercial glucose, or corn syrup, as it is often called in America,

is made by the action of dilute acid, generally hydrochloric, on potato
THE CARBOHYDRATES 61

starch abroad, but out of corn starch in America. Sulphuric acid was
at one time used in the hydrolysis and still is in some localities. The
syrup known as commercial glucose contains often a large proportion
of dextrins as well as glucose. There have been in the past cases
of arsenical poisoning from the use of glucose prepared from sulphuric
acid which contained arsenic. One such case occurred in England and
resulted in the death of several persons. If commercial glucose con-
tained only dextrose and dextrins, no objection could be taken to its use
as a food, except on the ground that it is less sweet than cane sugar,
and it may be used as an adulterant of cane syrup. Unfortunately,
however, the purity of the commercial article, as is shown by the acci-
dent just referred to where it was used in brewing, is not always above
suspicion. A further difficulty arises from the fact that purified starch
may not be used as the raw product, and the possibility exists that other
substances of a non-carbohydrate nature may find entrance to the final
product.

The fermentation of glucose by yeast, with the production of alcohol,
carbon dioxide and some other substances in small amounts, is one of
the most important reactions with which the physiological chemist has
to deal and it has of recent years been the subject of many investigations,
but its mechanism is still obscure. The discovery of the mechanism
of this process would possibly reveal the manner in which the dextrose
molecule is broken down in the course of metabolism and the deter-
mination of this fact is one of the most fundamental problems of
metabolism and nutriticn. Many practical advantages would probably
come from its discovery. The fermentation is brought about by an
‘ enzyme, zymase, which does not dissolve out of the yeast cell as long
as it is alive and is accordingly called an endoenzyme. It was obtained
by Buchner by grinding the yeast with sand, mixing it with diatomaceous
earth and pressing the liquid out with an hydraulic press. It may also
be obtained by treating yeast with acetone, or the vapors of methyl
aleohol, and in other ways which cause the discharge of the fluid con-
tents of the yeast cell. The nature of this fermentation change in glucose
will be referred to again in the chapter on the metabolism of the carbo-
hydrates.

d-Galactose. This monosaccharide, a hexose aldose, is found both in
animals and plants. In the animal body it is made in the mammary
glands and forms one of the constituents of lactose, or milk sugar, the
other constituent being glucose; it possibly occurs also in the glyco-
protein, mucin, of the saliva; and it is an important constituent of
phrenosin and kerasin, substances making part of the myelin sheaths
of nerves. It appears in the nervous system at the time myelination
begins. In plants it occurs in the hemi-cellulose of the endosperm of
62 PHYSIOLOGICAL CHEMISTRY

Molinia cerulea which yields on hydrolysis galactose, mannose and
arabinose ; and also in the seed coats of peas and garden beans (Phaseolus
vulgaris) which yield fructose, galactose and arabinose. Schulze states
that carbohydrates yielding mucic acid when oxidized, and, hence, con-
taining galactose, are almost as widespread in plant seeds as cane sugar.
Lupeose, which is probably a tetrasaccharide from the seeds of Lupinus
luteus and angustifolius, by hydrolysis yields one-half of the sugar as
galactose. Lupeose is closely similar to stachyose and to a carbohydrate
from the seeds of Cicer aretrium. Galactose (m. p. 162-164") is a weaker
acid than levulose, but a little stronger than glucose. In a copper acetate
solution the oxidation goes at a slightly greater speed than that of glu-
cose, but far slower than levulose. (See page 40.) On oxidation in an
acid medium by bromine, or chlorine, or nitrie acid, it yields galactonic
acid, C,H,,0,, and mucie acid, C,H,,0,. It is by the latter reaction
that it can be distinguished. It is fermented very slowly by ordinary
yeast. It is interesting that mucie acid corrodes teeth in a manner
closely similar to the corrosion occurring in the mouths of some people.
It may be the active principle of this corrosion. Galactosazone melts
at 192-195°. The specific rotation of galactose is

(a)? — + 80.5 (10% solution) ; when first dissolved (a)9= + 144°.

~ Glucosides. These are bodies widespread in nature, found both in
animals and plants, which are characterized by the fact that on hydroly-
sis by acids they yield glucose, or some other monosaccharide. They
are in reality ethers, that is compounds of some alcohol with the aldehyde
group of the glucose or carbohydrate. Since they contain no free alde-
hyde they yield no osazones, nor do they reduce Fehling’s solution with-
out previous inversion. The simplest glucosides are the methyl and ethyl
glucosides produced by the union of methyl or ethyl alcohol and glucose,
in the presence of hydrochloric acid. Other sugars as well as glucose
yield such unions. Thus there are pentosides in some of the simpler
nucleic acids and glucosides in the more complex. The pentose in the
former case is d-ribose and the other component guanine or adenine.
The formula of methyl glucoside is the following (Fischer) :

eee CH yO:CH
HCO Hon]
HO—C—H Ho—b_H
i ‘
H— HC———O
; 4. |
H—C—OH H—C—OH
by on én,on
2
B-Methyl-glucoside. a-Methyl-glucoside.
THE CARBOHYDRATES 53

There are, as will be seen from the formula, two classes of glucosides:
the a and the & The former are split by maltase; the latter by the
enzyme, emulsin. Lactose is a f-glucoside. The following table? will
give some idea of the variety and importance of this group of substances.
Many of them form the active principles of some of the best-known
drugs. The formation of these glucosides in plants is probably in the
nature of a detoxication of the poisonous alcohols, such as salicylic or
hydroquinone, by their union with glucose. The glucosides are inert in

the plants (Ciamician and Ravenna).
1. Ethylene derivatives.

Sinigrin. The postassium salt of myronic acid, C 10H 16 NS 2kO, H,0. Found in
black mustard and horse radish. Has a burning taste. Becomposes on
hydrolysis into glucose, allyl mustard oil, and potassium bisulphate. Fer-
mented by the enzyme myrosin, found in mustard. The formula is as
follows:

C,H, ,9, S.CN—C.H, +H,0 —+ 0,H,,0, + C,H.NCS + KHSO,
O—SO,OK Dextrose. Allyl mustard oil
(Allyl-iso-thiocyanate )

Sinalbin, C,H, N,8,0, . Found in white mustard (Sinapis alba). It hy-
drolyses into mustard oil, HO.C gt,CH,-NCS, glucose and sinapin sulphate,
a compound of choline and sinapinic acid and sulphuric acid. Its formula
is as follows:

0,H,,0, 8..NCH, C,H,OH
“0.80, 00, a, On

Sinapin is (CH. 39) 05H, CH:CH: c0.0.6, Hi, N(GH, ) OH

a nas
0 ees, Choline.

Sinapinic acid.

2. Benzene derivatives. Arbutin, C_,H,.O,, composed of glucose and hydroquinone,
_ is found in the bearberry. It has a diuretic action, and some antiseptic
"powers.

Salicin, also called saligenin, is found in the willow. C,,H,,0O,. Ptyalin and
emulsin hydrolyse it to glucose and saligenin, ortho-oxybenzylalcohol,
C,H, (OH).CH,OH. Populin, C,,H,,0,, a benzoyl salicin, is found in the
bark of the poplar, Populus tremula.

3. Styrolene derivatives. Derivatives of styrolene, C sH,-CH:CH,.

Coniferin, C,,H,,0O,. Cambium of conifers. By emulsin hydrolyzed to, glucose
and coniferyl alcohol.

Phlorhizin, C,H, 42 . Root bark of cherry and other fruit trees. Yields
glucose and phloretin, C,H ,0,, the phloroglucin ester of paraoxyhy-
dratropic acid. Produces glucosuria in mammals. C,H, 0, +H,0=
OH.C,H,.CH (COOH) .CH, + C,H,0..

p-oxyhydratropic acid. Phloroglucin.

4. Anthracene derivatives.

In this group occur many of the purgatives. The sugar may be rhamnose
in place of glucose. Chrysophanic acid and emoidine of rhubarb occur as
glucosides, or rhamnosides. Similar substances are found in Frangula and
Jalap. Digitoxin, saponin and strophanthin are also glucosides.

1 Abbreviated from a similar table in the Encyclopedia Britannica, article

“ Glucosides.”

 

coat oes

Le

 

 
64 PHYSIOLOGICAL CHEMISTRY

Amygdalin, C,,H,,NO,,, a glucoside in the bitter almond, is decom-
posed by maltase into glucose and mandelic nitrile glucose, and the latter
substance by emulsin, a ferment found with the amygdalin, into glucose,
benzaldehyde and hydrocyanic acid.

Saponin, a glucoside from Sapindus utilis, yields on hydrolysis
d-fructose, arabinose, rhamnose and sapogenin, C,,H,,0,. The saponin
of horse-chestnuts yields sapogenin and arabinose, d-glucose and
d-fructose (Winterstein and Blau).

We may mention also the fact that glucose enters in similar glucoside
union to make many important animal substances, as, for example, some
of the phosphatides which we shall consider more at length.

TaBte I]. InLusrratine THE HyproLyzine Acrion or VaRious ENZYMES oN Dir-
FERENT GLUCOSIDES.
Glucosides acted upon and hydrolyzed by

I 0g Tir
Invertin Maltase Emulsin
Saccharose Maltose Amygdaline
Raffinose Methyl-d-glucoside-a Coniferine
Gentianose Ethy]-d-glucoside-a Piceine
Benzyl glucoside Salicine
Glycerine glucoside-a Helicine
Amygdaline Esculine
Trehalose Arbutine
Methy]-d-fructoside Lactose
Methyl-d-galactoside-8

Benzyl-glucoside (one isomer)
Glyceryl-glucoside “ “
Methyl-d-glucoside_s
B. Disaccharides.—Hexose disaccharides have the formula C,.H,.0,,
and they are characterized by yielding two molecules of monosaccharides
when hydrolyzed. The hydrolysis may be produced either by proper
ferments or by the action of acids. The following are some of the hexosc
disaccharides which have been obtained, together with their place of
occurrence and the monosaccharides which they yield:

Disaccharide Occurrence fcuk _

Cane sugar. (Sucrose) Sugar cane; beets; maple tree sap Levulose
(Saccharose) (Saccharum officinarum ) Dextrose

Maltose Germinating barley Dextrose
Digestion of starch Dextrose

Lactose Milk Dextrose
Galactose

Trehalose Various fungi. Boletus Dextrose
edulis. Ergot. Trehala Dextrose

Melibiose From melitose in molasses Galactose

Australian manzs Dextrose
THE CARBOHYDRATES 55

Disaccharides have been artificially synthesized. Thus two molecules
of glucose unite in the presence of acids to form a small amount of
isomaltose, a disaccharide which is like maltose but, differs from it in
being amorphous and not fermenting with yeast. Cane sugar has been
synthesized by Marchlewski from potassium fructosate and aceto-chloro-
glucose, and also by Nef from feebly alkaline glucose solutions. In
fact, it is probable that in every solution of glucose a small amount of
maltose or isomaltose is formed spontaneously, but in the absence of.
acid, or the ferment maltase, the reaction goes very slowly. ;

Cane sugar. Beet sugar. Sucrose or Saccharose. C,,H,.0,,. This
is commercially the most important of the disaccharides. It is a erystal-
line sugar, very sweet to taste, which reduces Fehling’s solution. so
slowly that it is generally said not to reduce it at all. It occurs in
sugar cane, beets, sap of maple trees, etc. It is readily oxidized in acid
solution. It is dextro-rotatory, the specific rotatory power being
[a]? =+66.67° (varies with concentration). It melts at 160°, forming
barley sugar, and at 200° is changed to a brown mass of caramel by
the loss of water. It: yields saccharates with lime, strontia or barium
hydrate. Cane sugar is inverted, that is changed into a mixture of
glucose and levulose, by the action of acids, or the enzyme invertin,
which is found in beets, in the intestinal secretions of mammals, in
many plants and in yeast, Saccharomyces cerevisiae. The fact that it
does not reduce Fehling’s solution and forms neither a hydrazone nor
an osazone, leads to the conclusion that the aldehyde and ketone groups
cannot be free but must be substituted, and the following structural
formula has been proposed for it:

 

 

 

 

CH. O CH,OH
| a¢-on '——=6
; | H¢—oH | eon
C—H HCOH
HOH bo dn
(u,oF éH,0H

Fischer formula for cane sugar.

When cane sugar is hydrolyzed the superior rotating power of the levu-
lose, as compared with the glucose, causes the total rotation of the mix-
ture, which is called. invert sugar, to be levo-rotatory, in place of dextro-
rotatory. For this reason the sugar is said to be inverted. In studying
the rate of rotation in the polariscope it is important to remember
that dextrose has a very great mutarotation. The glucose first
set free from the levulose, «-glucose, has a very high dextro-rotation,
so that the inversion progresses faster than one would suppose from the
56 PHYSIOLOGICAL CHEMISTRY

polarimetrie determination. This error can be avoided by adding to
the invert mixture a small amount of sodium carbonate, which causes
the glucose to take its stable rotatory power almost at once, and the
reading will, therefore, enable one to determine the amount of inversion
which has taken place before the alkali is added. The specific rotatory
power of invert sugar is [a] =—19.84° (e=5).

Lactose. C,,H,,0,,+H,O. This is the sugar of milk, present to the
extent of about 5% in cow’s milk, but it is also found at times in the
urine of pregnant animals and in the amniotic liquid of the cow. It is
formed in the mammary glands. It is not nearly so sweet as cane sugar
and unlike it, it reduces Fehling’s solution as readily as glucose. It
forms, also, an osazone melting at 200° C. When hydrolyzed by acids it
breaks into galactose and glucose, and it hydrolyzes at a much slower
rate than does cane sugar. It is also hydrolyzed by an enzyme, lactase,
found in the intestinal mucosa. It shows mutarotation. When first
dissolved [@]%° —+87° (82.97). Its final specific rotatory power is
[a]}%° —+52.53°. Lactose does not ferment with yeast nor can it be
used as a food by animal tissues. It must be hydrolyzed first. It is
hydrolyzed by lactase in the alimentary canal, and fermented by several
of the bacteria, for example by the bacillus coli communis, but not by
the typhoid bacillus, and this constitutes, therefore, one way of distin-
guishing these two species. It is an interesting question whether lactose
is a glucose-galactoside or a galactose-glucoside. In other words, is the
aldehyde group of the galactose or of the glucose concerned in the union
of the two molecules. This point can be determined in two ways. We
may oxidize the lactose gently. By this the free aldehyde group will be
oxidized, but not the substituted one. Then by hydrolysis there will be
obtained either galactonic acid, if the galactose aldehyde was free; or
gluconie acid, if the glucose aldehyde was free. The other component
will be present as a hexose and it can be separated as the osazone.
Another method is to form the osazone of the sugar and then thé osone
and to hydrolyze. The osone obtained indicates in which part of the
molecule the free aldose group was (Fischer and Armstrong). By these
methods it has: been found that lactose is a glucose-galactoside. That is,
the aldehyde of the galactose is substituted by the glucose molecule, the
free aldose being the glucose. On the other hand, melibiose is a galactose-
glucoside. The structural formula of lactose is hence probably that
shown on the next page.

Lactose is easily distinguished from glucose and maltose by its non-
fermentability by yeast and by its yielding mucic acid when heated with
nitric acid. It is split by emulsin, but not by ptyalin. Since the free
aldehyde group of lactose is in the glucose molecule, bromine water
THE CARBOHYDRATES 57

oxidizes this group, forming lactobionic acid, C,,H,,0,., which on hy-
drolysis is converted into d-gluconic acid and galactose.

H H

|
O———_—-C—H
H—C—OH Ho—d_H
| HO—C—H Ho—d_H

ee |
0—C—H H—C—OH
H—d_on HOCH
don CHO
d-galactose. d-glucose.

Formula of lactose.

Maltose. C,,H,,0,,+H,0. This is one of the commonest and most
important of the disaccharide sugars. It is formed by the action of
amylase on starch, and since both the enzyme and starch are almost
universal in the plant world, and the enzyme and animal starch, or
glycogen, widespread in the animal world, maltose is very common in
animal and plant tissues. It crystallizes readily in white needles which
contain one molecule of water. It reduces Fehling’s solution, but a given
weight of glucose reduces more copper hydrate than the same weight of
maltose. It forms an osazone, more soluble than glucosazone, which melts
close to that of glucose at 206° C. Maltose is dextro-rotatory, its specific
rotation being [a]*°”—+ 136, computed for C,,H,,0,, and after stand-
ing. The beginning rotation is +.118. On hydrolysis by acids, or by the
enzyme maltase, it splits into two molecules of dextrose. It, like cane
sugar, is an a-glucoside and is, accordingly, not hydrolyzed by emulsin.
(Contrary to lactose.) Maltose may be distinguished from glucose by
the fact that when heated with dilute acids the reducing power of the
maltose solution increases, whereas that of glucose undergoes no change.
Maltose is not so sweet as cane sugar and it readily ferments with yeast,
showing that yeast must have a maltase in it. It is possible that maltose '
may be directly utilizable by animal tissues, but this is still in doubt.
The formula of maltose is

H
yd» oo
| Ss
HO—C—H a
HOt HO—C—H
non 0
no—b_a Pace
Tae CH,OH

d-glucose. d-glucose.
58 PHYSIOLOGICAL CHEMISTRY

C. Polysaccharides which are colloidal in aqueous solution, or
which are insoluble.—The more important of the polysaccharides are
starch, or amylum, cellulose, glycogen, dextrins, various gums, mucilages
and inulin. They are all soluble in water with the exception of
cellulose, and they form emulsoid colloids, which may be precipitated by
salts. Only a very brief account of some of their properties need be
given here.

The formation of polysaccharides from monosaccharides is probably
an attribute of all living matter, just as the formation of proteins from
amino acids is an attribute of all living matter without exception. This
transformation apparently takes place very easily. Thus a small amount
of alkali will cause a condensation of some molecules of monosaccharides,
and small amounts of acid will make some iso-maltose from glucose. We
do not know, however, how this transformation is produced in living
matter. Whether the condensation is due to a simple dehydration, or
whether the phenomena of oxidation and reduction play a part in it. It
seems in animal tissue, at any rate, that the synthesis of glycogen from
glucose cannot take place if the cell is anesthetized, a fact which is taken
‘by some to indicate that the respiration of ‘the cell is in some way
involved in ‘this condensation. The whole subject is a fertile’ field for
aves tiEg tit: e Fe

-- Starch. Starch, or amylum, is a polysaccharide of dextrose which it
Heide cake drolpea with acids. It may be composed of maltose groups,
since it yields maltose on hydrolysis by ptyalin, an enzyme. The for-
mula is generally given as (C,H,,0,),, but it is very hard to prepare
starch entirely free from phosphoric acid. It is possible that phosphoric
acid: is ‘in union with the starch molecule. The size and composition of
the’ ‘starch molecule is still very uncertain. A recent determination by
3 -colérimetric method of the number of hexose molecules in ‘the molecule

of-s some of ‘the polysaccharides gave the following results:
wee Hye «

      

 

Starch ccscaceaeu: hexose groups.
“Glycogen “8-9 ie
2 2. 2.0. Brythtodextrin 2-4 #8. .o i a
Achroodextrin .... 4 7) 0" .2°. SO. wut
Raffinose ........ 3) ne
Maltose ......... 2h SY ay. oe
‘Dacfose- + errr 2 a sa 88 °

Soluble starch, which: “is formed from cee ies the action of hot water,
dilute acids or amylase, i ig dextro-rotatory, . {ols . += +190.24 (e=3.995).
Brown, Morris and Millar g give [alo =+202% —

Wheat flour contaifs-a small amount “Of saccharose and raffinose and
also some amylase. .The-amylase attacks:the-_starch in dough, setting
free maltose which is then fermented by the yeast with the liberation
THE CARBOHYDRATES 50

of carbon dioxide. Yeast alone is unable to ferment starch since it
_ contains no amylase.

Gums. These are white, gummy substances soluble in water but gen-
erally precipitated by Fehling’s solution and very widespread in nature.
‘They nearly always contain phosphoric acid, which it is impossible to get
entirely separated from the organic matter without hydrolysis, and also
some organic acid in addition to the polysaccharide. The phosphoric
acid appears to be in union with the gum molecule and it is not impos-
sible that it plays a very important part in the synthesis of the gum.
These may be complex phosphatides. The gums, like the starches, form
electro-negative colloidal solutions. Gums are found chiefly in the plant
world, although there is some evidence that they occur also in the ani-
mal world. There is one always associated with the enzyme, invertin.
It is a white gum, easily obtained from brewer’s yeast. It yields on
hydrolysis both mannose and glucose. Many of the gums contain either -
rhamnose, or arabinose, or other pentoses. Thus gum arabic and the
gum associated with the enzyme amylase yield arabinose on hydroly-
sis. The mucilages resemble the gums except that they are more
hygroscopic and their solutions will not filter. Gum arabic is one of
the best-known gums. Many of the gums contain galactose.

Cellulose. This is the main constituent of wood. This polysaccharide
forms the main part of the wall of plant cells. It is also found in some
animals such as the tunicates. There are probably many different cellu-
loses, but the composition of the more complex members of the group
is still unknown. Cellulose is insoluble in all ordinary solvents, but dis-
solves in ammoniacal copper sulphate solution and in zine chloride solu-
tion in hydrochloric acid. Cellulose does not reduce Fehling’s solution,
but on hydrolysis with acid it yields glucose and some other sugars
which do reduce. Nitric acid converts cellulose into the nitro-derivative
known as guncotton, a very explosive substance. Hemi-celluloses are
found in many seeds and young plant tissues and they serve either as
reserve foods or as supporting tissues. They yield on hydrolysis
galactose, arabinose or rhamnose, mannose and, some of them, fructose.
They are probably simpler in composition than the celluloses. Con-
centrated sulphuric acid dissolves cellulose, which may be precipitated
from it by the addition of water. The cellulose is changed by this
process into a compound which gives a blue color with iodine (amyloid).

REFERENCES.

Photosynthesis. B. J. Moore: The presence of inorganic iron compounds in the
chloroplasts of the green cells of plants considered in relation to natural
photosynthesis and the origin of life. Proceedings of the Royal Society,
London, Series B, 87, p. 557, 1914. Ibid., 87, p. 163, 1913. References will
60 PHYSIOLOGICAL CHEMISTRY

be found to the work of Berthelot and Gaudichon, Priestly and Usher and
others.

Products of decomposition of various sugars. J. U. Nef: Dissoziationsvorgiinge
in der Zuckergruppe Annalen der Chemie, 403, p. 204, 1913. References
to Nef’s earlier work will be found here, and also to that of Lobry de
Bruyn and Alberda van Ekenstein.

Rate of oxidation of various sugars in alkalies and acids. Mathews: The spon-
taneous oxidation of sugars. Jour. Biol. Chem., Vol. VI, p. 3, 109.

Mathews and Bunzel: The mechanism of the oxidation of glucose by bromine
in neutral and acid solutions. Journal of the American Chemical Society,
Vol. XXXI, p. 464, 1909.

Articles in the Encyclopzdia Britannica on Sugar and Glucosides.

Molecular form. Life of Louis Pasteur by Vallery-Radot. Translation. Pasteur:
Recherches sur la dissymétrie moléculaire des produits organiques naturels
Lecons de chimie professée en 1860. Paris, 1861. (Translated into German.
Ostwald’s Classiker der exakten Wissenschaften. No. 28, Leipzig, 1891.)

Le Bel: Sur les relations qui existent entre les formules atomiques des corps
organiques et le pouvoir rotatoire de leur dissolutions. Bull. Soc Chem.
(2), 22, p. 337, 1874. Various other papers later in the same publication.
Also in the Comptes rendus, pp. 114, 304 and 417, 1892.

Simple sugars and glucosides. EH. Frankland Armstrong. In the Series of Bio-
chemical monographs. Longmans, Green & Co., London.

Betti: Sulla distinzioni degli aldosi dei chetosi. Gazz. chim. Ital., 1912, p.
288. Abstract. J. de Phar. et de Chim. (7), 6, p. 222, 1912.

Neuberg: Berichte, 34, p. 2626. Hill: A method of isolating maltose when
mixed with glucose. Pro. Chem. Soc., 17, 1901, p. 45.

Hudson and Yanovsky: Mutarotation. J. Amer. Chem. Soc., 39, 1917, p. 1013.
CHAPTER III.

THE LIPINS. FATS, OILS, WAXES, PHOSPHATIDES,
STEROLS.

It will be recalled that all living matter contains a larger or smaller
amount of organic substances which are soluble in alcohol, ether and
other fat solvents. These substances help to give to protoplasm its prop-
erties of containing large amounts of water but not dissolving; and also
the power of taking up readily and in large amounts chloroform, ether
and other substances soluble in fats but not readily soluble in water.
They are among the fundamental and ever-present constituents of living
matter. They may be given the group name of lipins (Greek, lipos, fat).
In this chapter the amount, chemical nature, origin and some of the
general properties of the more important of these substances will be
considered, while certain of them found chiefly in some special tissue
like the brain will be dealt with more completely in the chapters treating
of the chemistry of the organs.

Properties.—While the group of lipins contains such widely dif-
ferent chemical substances as the aromatic essential oils, like clove oil;
the true neutral fats, like mutton tallow; the sterols, which are aromatic
aleohols, and the phosphatides, or phospholipins, which contain large
amounts of phosphoric acid, the members of the group all possess two
or three properties in virtue of which they are called lipins. These
properties are their greasy, or fat-like feel, their solubility in chloroform
and fat solvents, and their insolubility in water. They constitute, then,
a very heterogeneous group, chemically and physiologically. The fol-
lowing classification based on that proposed by Gies will give a general
view of these bodies: :

LIPINS. CLASSIFICATION.

LIPINS. Constituents of protoplasm having a greasy feel; soluble in alcohol-ether
1. (a) Fats. Neutral esters of glycerol and fatty acids which are solid at 20° C.
(b) Fatty acids.

2. Fatty oils. Neutral esters of glycerol.and fatty acids liquid at 20° C

A. Drying oils. Harden on exposure to light and air. Linseed oil.
B. Semi-drying oils. Thicken slowly on exposure to light and air.

Cottonseed oil.

C. Non-drying oils. Remain liquid on exposure to light and air. Olive cil.

61
62 PHYSIOLOGICAL CHEMISTRY

3. Essential oils. Volatile, generally odoriferous substances of oily and of
varied chemical nature, being aldehydes, acids, terpenes, alcohols, etc.
Oil of cloves, turpentine, wintergreen, etc.

4. Waxes. Esters of sterols and fatty acids. Beeswax, carnauba wax. Sperm
oil. Spermaceti.

5. Sterols. Alcohols, generally of terpene group, solid at ordinary tempera-
tures. Cholesterol, phytosterol, cetyl aleohol, myricy] alcohol, ete.
(Oxidation products terpenie acids.)

6. Phospho-lipins. Phosphatides. Fatty substances yielding on hydrolysis
phosphoric acid and fatty acids. :

A. Mono-amino-monophospholipins. Lecithin, cephalin.
B. Di-amino-monophospholipins,
C. Mono-amino-diphospholipins.

7. Glyco-lipins. Fatty substances yielding on hydrolysis fatty acids and a
carbohydrate, generally glucose or galactose. Contain no phosphorus.
Cerebron. Kerasin. - Phrenosin.

8. Sulpho-lipins. Fatty substances yielding on hydrolysis fatty acids and
sulphuric acid. Sulphatide of brain. Protagon. Nature still undetermined.

9. Amino-lipins. Fatty substances containing amino nitrogen and fatty acids.
Contain no phosphorus. Not well characterized. Bregenin.

Historical_—The fats were the first of the three great classes of food.
stuffs to have their composition determined. This was the work of the
French chemist, Chevreul, in 1814. He found that fats were saponifiable
by alkalies into glycerol and soap. He identified and named cholesterin,
or solid bile, which was a non-saponifiable, fat-like substance. Chevreul
is notable, also, for living longer than any scientist since Aristotle, dying
at the great age of 102, in the year 1889.

Amount.—The amount of lipins, that is of ether-aleohol soluble, fat-
like substances, found in different cells and tissues is widely variable,
and the character of the lipin is peculiar to each tissue, but in general
a tissue composed chiefly of living matter, but not serving as a depot
of fats, contains from one to ten per cent. by weight of lipins. Of
the total organic matter of a rapidly-growing tissue, such for example
as an embryo pig, about 1.6 per cent. is lipin. In some tissues, how-
ever, the amount is much greater. In the sperm, eggs, brain and supra-
renal capsules of mammals, for example, the lipin makes about 7.58 to
19.51 per cent. of the fresh tissue; and in those tissues. which are
the storehouse of fat, such as the subcutaneous fatty tissue, the
mesenteric fat, or bone marrow, even as much as 90 per cent. by weight
may be lipin.

The fats and fatty oils——Composition. The fats and fatty oils are
the neutral esters of the tri-hydric alcohol, glycerol, C,H,O,, and certain
higher fatty acids such as palmitic, C,,H,,0,, stearic, C,,H,,0., oleie,
C,,H,,0., ete. They are, therefore, compounds of carbon, hydrogen and
oxygen, but they contain far less oxygen in proportion to the carbon
than do the carbohydrates. The fats differ from the oils physically in
THE LIPINS. FATS, OILS, WAXES, PHOSPHATIDES, STEROLS 63

that they are solid at ordinary temperatures, whereas the oils are liquid
at ordinary (18-25° C.*‘ temperatures; and chemically they differ in that
the fatty acids of the cats are, for the most part or wholly, saturated ;
while in the oils some of them are unsaturated. A typical fat is tri-
palmitin, or tri-stearin. These two constitute the chief parts of the fats
of mammals. These fats have the following composition:

7 oO H 0

ll | I
H—C—O—C— (CH 2) ; 4, CH, H—C—O0—C—-(CH,) “ «CH,

| |

ea ee ( CH, ) a CH Z i a ( CH, ) , « CH,

oO 0
H—C—0—C— (CH,) 4 a CH, ‘3 H—C—0—C—(CH,), «CH x

| Il | I}

H O H oO

Tri-palmitin, or palmitin. Tri-stearin, or stearin.

A typical oil is tri-olein, which is the chief constituent of olive oil and
which is composed of glycerol and three molecules of the unsaturated
acid, oleic acid.

 

H HHHHHH4H HHHHHHH4HE
a Pde de ie, eae Petre foe
H—C—0—C—C—C—C—-C_—-C—00-6 = C-C—C—C_-C— C000

ll | | | | | | |
SHAHHHHHH HHHUHHHEH
HHHHHHHH HHHHHHHHSE
fas wes ne a teed oe on eee eal.
H—(—0—C—C—-C—-G—G_G 006 = 0-006 CCC 00
a eine fa | |
(HHHHUUA HHHHHHUH
HHAH HAHA SA HHHAHHHH HH
itp) cal iid aero BN Che hs al
Boo 0=7 00205006 0 6-6 0

h OHHHHH HEH HHHHHHHE

It is obvious that by substituting other fatty acids many different
fats can be made. A great number of these exist in nature; and in fact
the different tissues of the same animal, or the corresponding fats of
different animals, differ either in the nature or proportion of the fatty
acids in the fats. Thus cold-blooded animals, such as fishes and
amphibia, have fats which are fluid at ordinary temperatures. They
are, in reality, oils and contain much unsaturated fatty acid, either oleic
or an analogous acid. The fat of the earthworm contains, for example,
butyrin, 4.47 per cent.: olein, 87.42 per cent.: stearin and palmitin, 8.11
per cent. In naturally occurring fats. such as lard, lard oil, tallow or
milk fat, or vegetable oils such as those of the peanut, olive, etc., there
are always present the glycerides of various fatty acids. No natural fat
as it oceurs in the tissue contains only a single glyceride, and the
glycerides may contain more than one kind of fatty acid. In lard and
tallow, the glycerides are chiefly those of stearic and palmitic acid
but there is also present some olein. Lard oil, which is obtained
_ 64 PHYSIOLOGICAL CHEMISTRY

from lard by cooling until the tristearin and palmitin have partially

crystallized out and then pressing the warm mass, thus expressing the

oil, consists largely of triolein. From beef fat, or tallow, a similar oil ’
is obtained in the same way and this is called oleo oil, or oleomargarine,

and is used in the manufacture of artificial butter.

It is important to remember that most naturally occurring oils, or
oils pressed from fats, contain some cholesterol, or phytosterol, and often
phospho-lipins, They are not pure oils.

Butter yields about 7 per cent. of volatile fatty acids which are
mainly butyric, C,H,O,, and caproic acid, C,H,,0,; but there are also
small amounts of caprylic, C,H,,0,, and capric, C,,H,.0,, acids. Of the
non-volatile fat of butter about 30 to 40 per cent. is olein and 60 to 70 per
cent. palmitin, with a little stearin. Butter also contains a small amount
of a phospho-lipin and some cholesterol. Small amounts of other fatty
acids, possibly derived from the phosphatide, such as arachidic and
laurie, C,,H,,O, and C,,H,,0,, have also been found in butter. The
natural yellow coloring matter of butter is mainly carotin, with some
xanthophyll, and is derived from the green fodder.

Oleomargarine, or butterine, is a buttery substance made mainly from:
oleomargarine oil, which is generally churned with milk. When made
from clean materials it is a wholesome food, but the experiments of
Mendel and Osborne suggest that its nutritive value is not equal to that
of butter, since it did not promote growth of young rats as did butter.

Table IV contains the formulas, boiling and melting points of various
fatty acids occurring in the natural fats. There are many others which
are not included in this table.’

Taste IV.
1. Acids of acetic series, C,H,,0, ‘
Pressure _—Boiliug oe Found in
Acetic C,H,0, 760 119° 17 Spindle tree oil.
Butyrie C,H,0, 760 162.3° -6.5 Butter fat.
Isovalerice C,H,,O, 760 173.79 -51 Porpoise; dolphin.
Caproic C,H,,0 760 202° 8
Caprylic CH’o? 761 236° 16.5 Butter fat. Cocoanut oil
816 " i
Caprie C.H.,0, 760  268°-270° 31.3 ) Palm nut oil.
Laurie C,,H,,0, 100 225° 43.6 Laurel oil. Cocoanut.
Myristic C,,H,,0, 100 250.5° 53.8 Mace butter. Nutmeg.
Palmitie C,,H,,0, 100 271.5° 62.62 Palm oil. Lard.
Stearic C,,H,,0, 100 291° 69.32 Tallow.
Arachidic C,H, O 17 Arachis oil
2 20, 40 2
Behenie C,H, A 35-84 Ben oil.
Lignoceric Cc, sH,,0, 80.5 Arachis oil.

1 For other data see table pp. 10-11. Leathes, The Fats. Longmans, Green, 1910,
THE LIPINS. FATS, OILS, WAXES, PHOSPHATIDES, STEROLS 65

2. Aeryllie or olete acid group. CH 0

nD gn— 9 2

Tiglic C,H,0, 760 =—-1998.5° 64.5 Croton oil.
Physetoleic C,.H,.°, 30 Caspian seal oil.
, 100 %85.5° 14 Most oils.
Oleie 0,,H1,.0, | ae
Rapie C,H, 0°, Rape.
Erucic CoH. 30 281° 35-34 Rape; fish oils.
3. Linolic series. C,H, ais
Linolie C, H,,0, 50.5 Maize; cottonseed.
Tariric sega Oil of Pic.
Eleomargaric ee 48 Tung oil.
4. Linolenio acid series. C,H, 0,
Linolenic C, ,H,,0, Linseed oil.
Iso “ “ “ oc ce
5. Series C,H, 0,
Clupanodonic C, ,H,,°, Fish liver; biubber.
6. Hydroaxylated acids.
Ricinoleic C_ HO 15 250° 4-5 Castor oil.
; ‘I acid ae
Quince oil aci 19! 3498
7. Dihydroaxylated acids.
Dihydroxy stearic .
acid C, ,H,,0, 141-143 Castor oil.

Phy: ical Properties. While the neutral fats and oils are not crystal-
line in the cells, the temperature being high enough to keep the fat liquid,
they may often be crystallized on cooling their. hot benzene solutions.
The crystals are long needles. They are quite insoluble in water for
some reason as yet unknown. The affinity of fat and water is small. The
molecular weight is large, for tristearin being 896. Fats and the fatty
acids and soaps reduce the surface tension of water. The lipin collects
-in the surface film so that the concentration in the film is greater than
in the water as a whole. For this reason an old surface of water con-
taminated by oil or soap contains more soap or oil than a new surface
and has a lower surface tension. The oils have the property of spreading
over water in an extremely thin layer of molecular dimensions. This
layer, according to Lord Rayleigh, has a thickness of only 2.7107 ems.,
a diameter but little greater. than that of a fat molecule. It is possible
that this property of collecting in surface films and of lowering the sur-
face tension may be of value in some vital mechanics of protoplasm; but
the whole matter of surface tension and, above all, the surface tension
of the surface of contact of water and such a mixture as that of proto-
plasm is still too obscure to permit of any definite statement concerning
this possible function of the fats.

The specific gravities of all oils and fats are lower than that of water.
06 PHYSIOLOGICAL CHEMISTRY

Rape oil having a specific gravity of 0.915 is one of the lightest. The non-
drying oils, like olive or sweet oil, range from 0.916-0.920; the drying oils
are about 0.930; the ordinary fats 0.930; castor oil and cocoa butter have
the highest specific gravity, i.e., 0.970. The lower the cohesion the more
liquid will be the fat, the larger will be the space at the disposal of its
molecules, the greater their freedom of movement and hence the lower
the specific gravity.

Fats have no sharp melting points, since none of them are pure
glycerides, all being mixtures. But even the pure glyceride tri-stearin
shows a curious behavior. It melts at 55°, solidifies and then again melts
at 71.5-75°. This is due possibly to a tautomeric rearrangement of the
molecule. The drying oil, linseed oil, remains fluid to the lowest tem-
. perature —28° C. On heating, oils slowly decompose and at about
300° C. acrolein is given off. It is by the appearance of acrolein on heat-
ing that neutral fats may be distinguished from fatty acids.

Glycerol. There are only two or three reactions of glycerol which
need mention at this time. One of the most important of its reactions
is the formation of acrolein from it by heating. If glycerol is heated
to 300°, and particularly if it is heated with some substance such as acid
potassium sulphate, or P.O;, which has an affinity for water, it loses two
molecules of water and is converted into the unsaturated aldehyde,
acrolein. Acrolein has a very irritating action on the mucous membrane
and may be detected by its sharp odor. The reaction of its formation may
be written as follows:

eae 1

CHOH — —+ CH + 2H,0
|

da,on CHO

Glycerol. Acrolein,

By oxidation glycerol is converted into glycerose, a mixture of dioxyace-
tone and glycerine aldehyde, and finally into glyceric acid, CH;OH-
CHOH-COOH. Glycerol has a sweet taste. It reduces Fehling’s solu-
tion only at a very slow rate. It readily esterifies with phosphoric and
other acids.

cH,OH CH,0H CH,OH CH,OH COOH COOH
|
dnox _ buon ‘ co ~~ bnon —_— éHOH _—_ b(0H) ‘
| |
ba, OH CHO buon COOH boo doou
Glycerol. Glycerylalde- Dioxyace- Glyceric Tartronic Mesoxalic
hyde. tone. acid. acid. acid.

Fatty oils. The true fatty oils are divided into three general classes:
into those which harden on exposure to: air, light and moisture; those
which dry slowly, and those which do not dry. The first are known as
THE LIPINS. FATS, OILS, WAXES, PHOSPHATIDES, STEROLS 67

drying oils; the second as semi-drying; the last as permanent or non
drying oils. The first are used in painting, and linseed oil is a typical,
and probably the most valuable, drying oil; while cottonseed oil dries
very much more slowly, and olive oil is a non-drying oil. The chemical
difference between these kinds of oils lies in the different stabilities of
the fatty acids they contain. The drying oils have very unstable fatty
acids; while the non-drying have relatively stable acids. The very
unstable fatty acids of the drying oils are very reactive and as might
be expected they are found, among other places, in the lipins of the brain,
heart, liver (cod-liver oil) ; and in other organs of which the metabolism
is very high ; whereas the stable acids of the fais and non-drying oils are
found in locations of lowered metabolism and in the depots of fats in
fat tissues. Owing to their reactivity, the drying oils absorb oxygen
and become rancid much more rapidly than the non-drying. The dif-
ference in stability of the fatty acids in the drying oils, such as linseed
oil, and the non-drying, such as oleic, is due to the presenée of more
double-bonded carbon atoms, called unsaturated carbon atoms, in the
linseed-oil group. Oleic acid contains only a single double-bonded
couple of carbon atoms, whereas the drying oils contain acids having
two or three such couples. The formula of oleic acid is C,,H,,0., or
CH,.CH,.CH,.CH, .CH,.CH,.CH,.CH,.CH=CH.CH,.CH.,.CH,.CH,.CH,
.CH,CH,.COOH, the double bond being in the middle of the chain. This
is shown by the fact that on oxidation it yields n-nonoie acid, C,H,,0.,
and azelaie acid, C,H,,0,. In linseed oil there occur such unsaturated
acids as linolenic, C,,H,,0,, with three pairs of double-bonded carbons,
and linolic, C,,H,,0,, with two pairs of double-bonded carbons.
Resemblance of the chemistry of painting to some biological processes.
It is interesting to consider the many curious resemblances of the
chemical processes involved in painting with protoplasmic respiration,
memory and growth. The use of linseed oil in painting depends on the
fact that it spontaneously oxidizes in the air, especially in the light, and
decomposes, the decomposition products forming a resinous hard mass
of a composition still largely undetermined. Linseed oil has the power,
then, of spontaneous oxidation; it respires. It takes up oxygen and it
gives off carbon dioxide and other volatile substances. Light, and par-
ticularly ultra-violet light, accelerates this respiration just as it does that
of protoplasm. Moreover growth, or rather synthesis, occurs, for in the
condensation following the decomposition substances are formed more
complex than the linolenic acid. Heat moreover is set free. It is, in
other words, a veritable metabolism which the linseed oil undergoes. But
the relationship to protoplasmic metabolism does not end here. In living
matter there are substances which hasten the oxidation, catalytic sub-
stances. or oxidases as they are called. In the presence of these sub-
68 PHYSIOLOGICAL CHEMISTRY

stances oxidation goes on much more rapidly than in their absence. Cells
use various substances as oxidative accelerators. Manganese salts are
used by some; copper or iron salts by others; or organic oxides and
peroxides, like the quinones, by others. Thé painter uses similar sub-
stances to accelerate the decomposition and drying of the oil. The oil is
sometimes boiled in iron or copper vessels and dryers of various kinds
are added to help the oxidation, such as manganese dioxide, litharge,
manganous borate or iron salts; or he uses an organic oxidizer, turpen-
tine. which in the light picks up oxygen with great ease and carries it

 

 

= T T
Q AssonrTtion

 

 

 

— Fine
1 1 1

ie. i = 1 i

 

Fia. 7.—Curve showing the rate of absorption of oxygen by linseed oil. Ordinate:
mms. of negative pressure due to absorption of oxygen; abscissa: time in days.

over to the oil. In this respect, then, the metabolism of paint resembles
that of protoplasm.

But most remarkable of all is the fact that the oil may be taught
to oxidize itself and it remembers its lesson for some time. If linseed oil
is exposed to light, or ultra-violet rays, in the presence of air in a closed
flask provided with a mercury manometer, for the first 24 to 36 hours
nothing seems to happen; but then slowly the oil begins to oxidize and it
oxidizes at a constantly accelerating pace so that the oxygen is used up
in the flask and the negative pressure may be measured by the mercury
manometer. The curve of the rate of absorption of oxygen is at first
convex downward, not, like most chemical reactions, concave. Figure 7.
It is the curve of an autocatalysis. It is as if the oil had to be taught
by the light to oxidize itself and learned to oxidize better and better.
THE LIPINS. FATS, OILS, WAXES, PHOSPHATIDES, STEROLS 69

Now it may be shown that the oil remembers. If after 60 hours’ illu-
mination when the oil is oxidizing let us say at a fairly rapid rate,
the illumination is discontinued and the oil put in the dark, the oxidation
goes on at a slower pace. If after a period of a few hours in the dark
the oil is again illuminated by the light, it will be found that the oxida
tion no longer waits 24 hours before beginning, but now the stimulation
by the lamp is effective within an hour or more; the oil acts as if it
remembered the teaching by a previous illumination and now oxidizes
at a more rapid rate. However, oil can also forget. If left in the dark
24 hours or more after being taught to oxidize, it has forgotten and
now. the teaching must be done all over again, a long illumination being
necessary before the oxidation begins. We do not usually speak: of the
long latent period of the oxidation as a period of teaching, but it is called
in chémistry a period of ‘‘ inductance ’’; and we do not say that the
oil is learning to oxidize itself, and doing it better and better, but we
say that it shows phenomena of autocatalysis; nor do we say that it
forgets again in the dark, but that the intermediary, autocatalytic agent
has disappeared ; but when organisms show the same kind of phenomena
we speak of teaching, of latent periods, of stupidity, of good or bad
memories. And it is not impossible by any means that the phenomena of
memory, shown in greatest perfection by the mammalian cerebrum, may
have at the bottom some such basis as this, and the persistence within
certain cells of substances of an autocatalytic nature which have re-
mained from a previous stimulation. Perhaps the brain cells remember
longest because they most carefully maintain intact, or preserve,’ these
labile autocatalytic substances. It may be mentioned that the whole of
growth is an autocatalytic process. There are always left over in the
cell, at the close of a period of feeding, substances, enzymes, derived from
the metabolism of the foods, which hasten the metabolism of the next
succeeding feeding and hasten growth. It is because of the presence of
these autocatalytic substances that foods change into protoplasm so much
more rapidly in cells than outside of them.

Methods of identification of oils and fats—The identification of
the oils and fats is a matter of commercial as well as of scientific in-
terest. A partial identification can be made by means of the melting
point and by the determination of the iodine, hydrogen, oxygen, saponi-
fication, Reichert-Meiss] and acetyl numbers.

The melting points, as already stated, are not sharp and definite,
as fats are not pure substances but always mixtures, but nevertheless
something may be learned from the melting points. A low melting point
means either that the fats contain saturated acids of short carbon chains,
or that there are long but unsaturated chains, as in oils. The melting
point of a fat as it occurs in the tissue is generally, or always, lower than
70 PHYSIOLOGICAL CHEMISTRY

the usual temperature of the tissue in which it is found. Thus in cold-
hlooded animals, such as fishes, the fats are found to be really oils, liquid
at 20°. In mammals the fat of the hoofs which are exposed to cold is
generally oil (Neat’s foot oil), while that about the kidneys melts at
a high temperature and contains more tri-stearin.
The melting point of tri-stearin is 55°, permanent 71.5°
tri-palmitin 50.5°, « 66.5°
tri-olein -6°
Lodine number. The unsaturated fatty acids have the property of
adding atoms at the double bond. Thus iodine, bromine, oxygen and
hydrogen may be added here. Hence these fats have reducing powers,
in that they readily oxidize themselves at the double bond going
over into the hydroxy-acid, or even into a ketone acid; they also have
oxidizing powers, in that they will take up hydrogen and be converted
into the more stable saturated fats. By reduction or by oxidation with
bromine or iodine, the number of unsaturated bonds may be discovered,
since each such bond adds two atoms of iodine or bromine, two of hydro-
gen and presumably two oxygen atoms.

ri
C,H,,—C =C—C,H,,0,+1, = o,,,t-d-o H_.O..

8 15 8 15 2

HoH aH

Thus if it is desired to know whether linseed oil has been adulterated
with cottonseed oil, it is only necessary to determine how much
iodine in centigrams a gram of the oil will take up from a solution
of iodine; or how much hydrogen or oxygen it will unite with.
The first figure is especially important and is easily determined (see
page 908) ; it is called the iodine number; but recently the two last, the
hydrogen and oxygen numbers, have also become important aids in the
identification of the fats.

TABLE V. IoprnE VaLuEs or Various O1Ls, Fats aND WAXES.

I. Vegetable. Rape 94-102
1. Drying oils. Brazil nut 90-106
Linseed 175-205 .
3. Non-drying.
Hempseed = Apricot kerne 96-108
Weta Ago Peach “ 93-109
Sunflower 119-135 Almond 93-100
: x Olive 79-88
2. 9 ei ioe Grape seed 96
Casto -86
Maize, corn 113-125 ea as
Beech nut 111-120 II. Animal oils.
Cottonseed 108-110 Fish 140-175

Sesame 103-108 Menhaden 161
THE LIPINS. FATS, OILS, WAXES, PHOSPHATIDES, STEROLS 71

Sardine 161-193 2. Semi-drying.
Herring 124-142 Horse 75-85
Cod liver 167 :
Shark “ 115 3. Non-drying.
Whale oil 121-136 Goose fat 70
Dolphin 99-126 Lard 50-70
Horses’ foot 74-90 Bone 45-56
Neat’s foot ‘ 67-73 Beef tallow 38-46
Egg oil 68-82 Mutton tallow 25-46
Butter 26-38
III. Vegetable fats.
Laurel oil 68-80 V. Waxes.
Palm oil 53 Sperm oil 81-80
Mace butter 40-52 Carnauba wax 13
Cacao butter 32-41 Wool 102
Borneo tallow 15-31 Bees * 8-11
Palm kernel oil 13-14 Spermaceti (Cetin) 0-4
Cocoanut oil 8-9 Insect wax (Coccus ceriferus) 0-1.4
Japan wax 4-10
Iodine number of various fatty acids.
IV. Animal fats. Oleic acid 90.07
1. Drying. Erucic 75.15
Ice bear 147 Linolic 181.42
Rattlesnake 106 Linolenie 274.1
Clupanodonic 367.7

The hydrogen number. The discovery of a practicable method of
adding hydrogen to unsaturated oils is a matter of great commercial
importance, since by this means ill-smelling, or ill-tasting, or cheap
vegetable oils, such as cottonseed or even linseed oils, may be converted
into substances’ closely resembling true stable animal fats, which at
present are far more expensive. The ill-taste and smell of the oil is due
‘to the decomposition products which arise from the fatty acids in con-
sequence of their unstable nature. By hydrogenation the true fats are
formed. A method recently employed on a commercial scale for this
hydrogenation has been by the use of hydrogen and finely divided nickel
oxide which has the important advantage over finely divided nickel, in
that it is said not to be so easily poisoned by the impurities in the oils.
The only disadvantage in the use as foods of these cheap vegetable lards
is that they usually contain small amounts of nickel which it has been
impossible thus far to eliminate. Since nickel, when absorbed, is a toxic
substance, the presence of even very small amounts of nickel in any food
must be regarded as undesirable. -

Ozonides. The unsaturated fats absorb oxygen. The heat generated
by this process may cause the spontaneous inflammation of cotton. In
an acetic acid solution oleic acid absorbs an equimolecular quantity of
ozone. An ozonide is thereby formed, C,,H,,0,, a viscid, almost color-
12 PHYSIOLOGICAL CHEMISTRY

less, transparent liquid, heavier than water, which does not combine with
‘iodine and which is stable at 80-90°. At 120° this decomposes; 5 per cent.
being converted into a gaseous mixture of the following composition:
CO,, 2.7-7 per cent.; CO, 71-88 per cent.; CH,, 16.5 per cent.; H,, 5.4
per cent. There is also formed a mobile oil (Molinari and Soncini).
Triolein forms the ozonide, C,,H,,,0,.30, ; each molecule of the fatty acid
adding a molecule of ozone at the double bond. This is a viscid, colorless
oil decomposing at 136°, and when saponified by potassium alcoholate it
yields glycerol, azelaic, C,H,,O0,, and nonylic, C,H,,0,, acids, and also
oxystearic acids, C,,H,,0, and C,,H,,0,.

‘Fatty acids are also rendered unstable by partial oxidation, and when
m this condition they have a more intense physiological action. Thus
castoy oil may owe its cathartic action to the ricinoleic acid, C,,H,,03,
an oxidized oleic acid which is especially active when in a free state,
as it is in the intestine. An oxidized stearic acid, ketostearic acid, is
found in mushrooms of the genus Lactarius, CH,(CH,),,—CO—(CH,),
COOH (Bonzant and Charaux), and dihydroxy stearic acid, possibly
farmed by oxidation of oleic acid, in castor oil.

Saponification. The property of fats of being hydrolyzed by water
and alkalies is of practical importance. That fats when treated with
alkalies make soaps is generally known, but the exact mechanism of the
process is not yet clear. If a neutral fat is heated with acid, preferably
in an alcoholic solution, or with alkali, or even with superheated steam,
it is split into the fatty acids of which it was composed and glycerol.
This is the process of saponification.

H Oo H
I get |
a—¢_o¢_(cn,),—on H—C—OH
8
a
i i
HH 00 (CH) ,¢—CH, +3KOH —~ H—C—OH + 3KOC—(CH,),,—CH,
! |
| \
H—C—O0—C— ( CH, ) 1s OH, EO Oe

tr tk
If alkalies are used, the fatty acids set free at once unite with the alkali
to form alkali salts of the fatty acids which are called soaps. The
characteristic of a soap is its slippery feel and its property of making
a foam when dissolved in water and shaken. Not all substances with
these physical properties are soaps, however. Many plants, such as the
soap-bark tree, contain saponaceous or soap-like substances, as far as
‘these physical properties are concerned, but which are not soaps in a
chemical sense. A soap, therefore, is a salt of a higher fatty acid which,
when dissolved in water, foams on shaking and the solution has a low
THE LIPINS. FATS, OILS, WAXES, PHOSPHATIDES, STEROLS 73

surface tension. Sodium acetate is the salt of a fatty acid, but it is not
a soap because it lacks the physical properties of soap.

The speed of saponification of a fat has been found to be propor-
tional to the number of hydroxyl, or hydrogen ions in the solution.
For this reason saponification by ammonium hydrate is much slower
than by sodium hydrate; and by water, C2. —.8X10~, is still slower.
The process of saponification forms then one method of measuring the
concentration of such ions.

But while the fact of hydrolysis or saponification is quite evident,
when we ask the further question of the mechanism of the process we get
at. once into very badly-known territory. What is the point of attack
of the enzyme, or the alkali, or the acid on the fat molecule? The most
probable answer is that it is either the doubly bonded oxygen atom, or
that linking glycerol and fatty acid together, which is the point at which
union with the saponifying agent takes place. It seems that one molecule
‘can influence another only when it is united with it. If this is true, the
saponifying agent must unite with the fat molecule. Since one of the
oxygen atoms of esters is almost certainly quadrivalent, there being two
extra valences in such molecules, union probably occurs here, at least
in the case of lipase. Union with acids probably occurs as follows:

R—O—C —R’+ HCl =" R—O—C—R’
II

II
O= HOCI

Probably the last compound is unstable and breaks into RC] and
R’-COOH. RCI reacts with the water to form ROH and HCl.

The hydrogen or hydroxyl ions appear to act catalytically because
they are left at the end of the reaction with their concentration un-
changed, but in reality they have united with the ester, decomposed it
and are again set free.

Saponification numbers of various fats. The saponification number
is the number of milligrams of KOH required to neutralize the fatty
acids contained in one gram of oil or fat. It serves to tell whether the
fatty acids in the fat have a high or low molecular weight, for it is clear
that the smaller the molecular weight the more molecules of the acids
there will be in the gram of fat. Most fats and oils having chiefly
palmitic, oleic or stearic acid in them have saponification numbers of
about 195, but some which have more complex acids are lower than this.
Thus for the rape-oil group, rape oil containing erucic acid, C,.FI,,0,,
the saponification number is about 175. Similarly, lipins which contain
earnaubie acid. C,,H,,0,, will have still lower saponification numbers.
On the other hand, butter which contains caproic, caprylic and butyric
acids, all of these of low molecular weight, has a high saponification
74 PHYSIOLOGICAL CHEMISTRY

number, the saponification number of butter fat being 227. The saponi-
fication number of oleomargarine is about 195, so that it constitutes an
easy method of distinguishing between butter and oleomargarine. Cocoa-
nut and palm oil and some of the blubber oils, such as porpoise-jaw oils,
have also a considerable quantity of volatile acid of low molecular weight
and the saponification number is between 240 and 260.

Reichert-Meissl number. Another useful method of discovering the
true nature of a fat or oil is to determine the amount of volatile fatty
acid in it. For this determination a weighed amount of the fat is saponi-
fied with alcoholic potash or by potassium hydrate, the mixture is acidi-
fied to set free the fatty acids, phosphoric or sulphurie acid being used,
as they are non-volatile, and then the mixture after adding pumice stone
is distilled. The amount of fatty acid distilling over is titrated with
N/10 KOH. The number of c.c. of N/10 KOH required to neutralize the
volatile fatty acids from five grams of oil or fat is called the Reichert-
Meissel number. Ordinary fats have a Reichert number of 0 when the
saponification number is 195. For butter fat the number is 25-30; cocoa-
nut oil 6-7; and for palm kernel oil 5-6. If the saponification number
is high, the Reichert number is, also, generally high.

Acetyl number. Some oils, such as castor oil, contain oxidized fatty
acids and, since the number of these hydroxy! groups is known if the oil
is pure, the determination of this number is of use in detecting adultera-
tion. These hydroxyl groups are detected by acetylation by means of
acetic anhydride, and hence we have the acetyl number, which is the
number of milligrams of KOH necessary to neutralize the acetic acid
saponified from 1 gram of acetylated fat.

Separation of the fatty acids. Another not difficult method for learn-
ing something of the chemical nature of a fat consists in saponifying the
fat and then determining the proportion of oleic acid present. After
saponification the fatty acids are dissolved in alcohol and lead acetate in
alcoholic solution is added. The lead salts of the fatty acids are pre-
cipitated, filtered, dried and extracted with ether. Lead oleate is soluble
in ether, while the palmitate and the stearate are insoluble. The deter-
mination of the relative amount of oleic acid present is thus simple. By
treating the ether solution of the lead oleate with hydrochloric acid, lead
chloride is precipitated. On evaporating the ether oleic acid remains
behind.

Physiological value of fats. Fats are of use to the body in several
ways. Being poor heat conductors, a layer of fat beneath the skin helps
to conserve bodily heat; by their physical properties they contribute to
the physical constitution of protoplasm; and finally they are the heat-
producing foods par excellence. They are burned or oxidized in the
cells, setting free much heat, nine large calories per gram of fat. An
understanding of their oxidation is, therefore, very important in under-
standing fat metabolism.
THE LIPINS. FATS, OILS, WAXES, PHOSPHATIDES, STEROLS 75

Oxidation. The fats burn in the body with ease, but outside the body
the saturated fats are decidedly inactive at body temperature. Oils,
however, when air is blown through them, ‘‘ blown oils,’’ become ulti-
mately thick, gelatinous and dark colored. They oxidize even at room
temperature. It is not yet certainly known whether the fatty acids are
burned in metabolism only after they are set free from the glycerol, or
whether they can be burned best when in ester union. The general pres-
ence of lipases, enzymes for hydrolyzing fats, in all cells and the fact that
the form of any substance which accumulates in cells is generally the
stable form, and it is neutral fat which is stored, leads to the inference
that they are hydrolized before oxidizing. The fatty acids are, then,
probably united to some substance. which hastens their oxidation.

It is not certainly known how the higher fatty acids are oxidized, but
it is believed that the oxidation occurs first in the & carbon atom, i.e,
the second from the carboxyl, where it has been shown to occur in the
simpler acids, such as butyric. It has been found by Dakin that hydro-
gen peroxide produces much the same kind of oxidations as the body.
Both hydrogen peroxide and living matter oxidize the fatty acids by
stages. In butyric acid the beta carbon atom is first oxidized, giving
rise to a ketone acid. This is then hydrolysed to acetic acid which in its
turn is oxidized to.carbon dioxide and water. The reactions may be
represented as follows:

rs a , ?

CH, co COH _7 COOH

[. a Oy | + 4,6

= a —_— e NCH,

COOH COOH COOH COOH
Butyric acid. Acetoacetic. Enol form. Acetic acid.

8-Ketobutyric acid. 8-Oxycrotonic acid.
: B-Oxybutyric acid.
2-Butanon acid.

Fatty acids oxidized in the # carbon are very unstable and in the
free state readily decompose; they are far less stable than those oxidized
either in the v or y carbons. By reduction the enol form will go over
into the 6-hydroxybutyric acid as follows:

le i
COH CHOH
| +3,-—|

CH CH,

| |
COOH cooH

Enolform. $-hydroxybutyric
acid.
76 : PHYSIOLOGICAL CHEMISTRY

The oxidation of the long carbon-chain saturated acids is believed to
follow this same process, the # carbon being oxidized each time to the
ketone and then this splitting-off acetic acid leaving the fatty acid with
two less carbons. The long chain is thus split up, two carbons going
each time. Every fatty acid with an even number of carbons in the
chain will thus ultimately produce the #-ketone butyric acid. It is not
certain, however, that oxidations may not occur, also, in other carbons of
the chain. The process may be pictured as follows:

CH CH CH
| 8
tH,),, (Gi) (CH,) 10
| |
das, +90, —* co +H,0 —~ CH, + 7
bs 4 da, bu COOH
|
boon boon COOH
Palmitie acid. B -Keto-palmitic. Myristic acid. Acetic acid.

It is not impossible that the synthesis of the fatty acids may be brought
about by a reverse process of reduction, two carbon atoms being added
each time. This would explain why it is that all the naturally occurring
fatty acids have an even number of carbon atoms. The oxidation of the
unsaturated fatty acids follows a more complicated course and a variety
of splitting products are formed, some of them volatile.

The course of the oxidation of the fatty acids will well repay
further study. The aceto acetic ester, the ester of diacetic acid, is a
very reactive substance widely used by chemists in synthesizing a great
variety of substances. It is by no means impossible that some ester of
diacetic acid is also widely used by nature in the synthesis of the sub-
stances in the cell.

Origin of fats. There appear to be two distinct problems in the
formation of the fats: the first is the origin of the long carbon chains
making the fatty acids; the second, the nature of the synthesis of these
chains with glycerol. While neither of these problems has is yet been
solved, we may consider what is known about them.

From the fact that the long carbon chains in the natural fats always
contain an even number of carbon atoms, there can hardly be any other
conclusion than that the synthesis is not made carbon atom by carbon
atom, but by the addition of two, four or six carbon atoms at a time.
From the decomposition by oxidation where two carbons are split off
at a time the inference may be drawn that the synthesis also involves
pieces of two carbons. Since the fats are formed by reduction from the
sugars, the synthesis obviously involves reductions. What the pieces are
which are thus condensed it is impossible to say positively at the present
THE LIPINS. FATS, OILS, WAXES, PHOSPHATIDES, STEROLS 77

time, but they may possibly be either ethylidene or glyoxal, glycolic
aldehyde or acet aldehyde. But this is not the only possibility.

Whatever may be the nature of the carbon fragments giving rise to
the fatty acids, there is no doubt of two facts: that they arise by the
process of the fermentative decomposition of the sugars common to all
tissues ; and, in the second place, that these pieces act as oxidizing agents,
being themselves reduced. The fats accordingly represent, in essentials,
reduced, condensed carbohydrates. That processes of reduction play a
part in fat formation is shown by the chemical composition of the fats.
The fatty acids contain little oxygen; they are carbon-hydrogen com-
pounds except for the carboxyl group. The system oxygen-fatty acid
contains, therefore, far more potential energy than the system oxygen-
carbohydrate. One gram of fat burned produces nine calories of heat as
compared with four for a gram of carbohydrate. That the fats origi-
nate from the carbohydrates is shown not only by practical human
experience that a carbohydrate diet is fattening, but by direct experi-.
ment. The transformation of starch into oil takes place commonly in
plants and many bacteria form acetic, propionic, lactic or butyric acid
by carbohydrate fermentation.

Since the fats when oxidized yield more energy than an equal weight
of the sugars from which they are derived, it is probable, since most
reactions are in their entirety exothermic, that the reduction of some
of the sugar to fat coincides with the oxidation of some of the sugar to
carbon dioxide. In this way the gain in energy in the system fat-oxygen
represents some of the energy set free by the total combustion of some
of the sugar. Thus it may be inferred that to make one molecule of
palmitic acid, C,,H,,0,, perhaps four, or more, molecules of C,H,,0,
are destroyed, some being oxidized as shown by the schematic reaction:

6 C,H,,0, + 130, —_- 2000, + C, ,H,,0, + 20H,O +n Cals.

6 12 16 32

The condensation of the fatty acid thus formed with glycerol to make
neutral fat is one of the condensations so common in protoplasm and of
which the synthesis of the polysaccharides from the monosaccharides is
a type, but the nature of which is still so obscure. If glycerol and fatty
acid are mixed, some spontaneous condensation occurs with the elimi-
nation of water. The rate at which this spontaneous condensation occurs
is hastened by lipase, the fat-splitting ferment. It has accordingly been
suggested that the synthesis of the fats from glycerol and fatty acids
in the cells may be brought to pass by the action of the lipase. However
reasonable this explanation appears at first glance, there are certain grave
objections to it. The point of equilibrium of fatty acid, glycerol and
neutral fat is one in which very little neutral fat coexists with a great
deal of glycerol and fatty acids. Now in protoplasm much neutral fat
78 . PHYSIOLOGICAL CHEMISTRY

is found and only traces of the glycerol and fatty acid, unless these are
formed by autolysis. This fact alone makes it impossible to ascribe the
synthesis to lipase unless some other condition be also assumed by which
the neutral fat is removed as quickly as it is formed from the influence
of the lipase and away from the system in which it has been part. A
second reason is the fact that the synthesis goes on most rapidly in those
cells which contain little or no lipase. There seems to be no lipase, for
example, in the mammary glands in which fats are rapidly formed.
Practically conclusive evidence against the lipase theory is the fact that
the synthesis of the fats, like other syntheses, is dependent upon oxygen,
and the lipase action is independent of oxygen. The syntheses depend
on the vitality of the cell. They do not take place in an etherized cell.
It is clear that this synthesis must be left for future work to decipher.
The origin of the glycerol is not difficult. It is closely related to the
carbohydrates, and glycerose is one of the products of their decomposi-
tion. By reduction glycerose yields glycerol. Glycerose is a mixture of
dioxyacetone and glycerine aldehyde, CH,OH-CHOH-COH.

The question may finally be asked of the location in the cell of the
neutral fat or oil. In many cells the fat or oil may be seen in the form
of fine round droplets distributed through the cytoplasm: This is the case
in egg cells and in some plant cells. In fat tissue the cell is almost com-
posed of one large droplet of fat, inclosed in the cell membrane. But
in living protoplasm itself fat also occurs, but in such a fine state of
subdivision, or union with the other constituents, presumably phospho-
lipins, that it cannot be detected microscopically. That the fat is there
is shown by chemical examination and by its appearance in cells, par-
ticularly after they have been exposed to chloroform vapors. After
anesthesia by chloroform of only one hour’s duration, the liver, kidney
and heart cells may show by staining with Sudan 3 an abundant pres-
ence of fat in small droplets, whereas the total fat is found by analysis
to be no greater than before. The anesthetic evidently causes a change
in its distribution and perhaps in its saturation, so that it aggregates
into visible droplets. Fat may be stained in cells by Sudan 3, or by Nile
blue. Only lipins with unsaturated acids stain with osmic acid.

Essential oils—These oils are a heterogeneous chemical group with
widely different. compositions but having three properties in common:
they are volatilu, they make a temporary grease spot on paper, and they
are combustible. Most of them also have an odor. Among these oils
are clove oil, cedar oil, oil of cardamom, oil of peppermint, oil of winter-
green, to mention only a few. They are widely distributed among plants.
Some of them, perhaps the majority, are aromatic aldehydes or ketones
belonging to the terpenes of which the oil of turpentine is an example;
others are aliphatic compounds such as the essential oil of Rita graveolus,
THE LIPINS. FATS, OILS, WAXES, PHOSPHATIDES, STEROLS 79

which is methyl-n-nonyl ketone, CH,.CO.(CH,),.CH,; others are acids.
Those of the aromatic group which are terpenes are related to the sterols
and the resins. The essential oils, while of considerable importance in
pharmacy and therapeutics and in commerce as flavoring extracts, are
of less importance in animal physiology, none having been isolated from
the human body.

The terpenes constitute a group of very great commercial and prac-
tical importance, camphor and menthol, C,,H,,0, belonging in this
group. The true terpenes have one or several hexa-carbon rings with

two side chains:
é 2 3 rt VA
— 1
7 a NXC16

Oil of turpentine is chiefly pinene, C,,H,,. The terpenes are generally
benzene derivatives, but the benzene nucleus is usually only partially
unsaturated and contains only one or two double bonds. Some of them
have two or more rings, some only one. India rubber is a complex
terpene. This can be formed by the condensation of isoprene, a diolefine:

CH, = C—CH = CH, :

 

 

 

!
CH,
Isoprene.
CH, CH CH CH CH CH CH, CH
3 2 3 3 8 8 8 8
pe AZ
y Nou Cc CH
| |
ber bis CH CH
LN
u¢ ‘UH, HC ‘oz, Bo CH, H.C 0H.OH CH,—-CH——OH,
hee oll} |
ud da nd dar, HC Ch! H,C CH, CH,C.CH,
, : \ G 5 |
YW on Yy PA CH, Cc co
| 1
biz, CH, da, CH, bs,
Limonene, Terpin, Pinene, Menthol, Camphor, C, oH ‘ o-
CAs C,H 5% C,H, C,H9-

Terpenes and some oxygen derivatives.

The aliphatic terpenes are of great theoretical importance because
they stand intermediate between the fatty acids on the one hand and the
resins and aromatic terpenes and cholesterol on the other. Such an ole-
fine terpene is myrcene, C,,H,., found in the oil of bay. It is probably
CH,—CH—CH=CH.CH,.CH,.CH:C(CH,),. While these substances
occur in plants. it is not impossible that similar substances may appear
80 PHYSIOLOGICAL CHEMISTRY

as intermediate products of metabolism in animals. Thus d-citronellol,
C,,H,,OH, is CH,.C(:CH,).CH,.(CH,),.CH(CH,).CH,OH. On oxida-
tion it yields acetone and 4-methyl adipic acid. Geraniol, C,,H,,0,
yields on oxidation such products as acetone and levulinic acids. More-
over these citrals, such as geraniol, readily condense to six carbon rings
on heating with acids, thus showing one possible origin of the aromatic
rings. Santalol is a sesquiterpene alcohol of unknown composition,
C,,H,,0. Cholesterol, other sterols and bile acids are probably terpene
derivatives, but.they are solids at ordinary temperatures and differ
widely in chemical and physical appearance from the essential oils.

The waxes.—Waxes are of both animal and vegetable origin. They
are the esters of fatty acids with a mono-hydric solid alcohol, or sterol.
They contain no glycerol. Most waxes are solids at ordinary tempera-
tures, but at least one, sperm oil, is liquid. Most naturally occurring
waxes are mixtures of the esters of various fatty acids with various
sterols. Lanolin, or wool fat, is such a wax; another is bees-wax. Bees-
wax is principally myricyl palmitate. The waxes generally have a dif-
ferent texture from the fats, but their solubilities are similar. They are
saponifiable like the fats, but often with more difficulty. The saponifica-
tion is easily carried out in petroleum ether containing a little absolute
alcohol by the addition of metallic sodium. Sodium alcoholate is formed,
which saponifies the wax. Among the waxes the cholesterol esters of the
blood deserve special mention.

Composition of the waxes. Table VI contains the formulas of various
fatty acids and alcohols which have been isolated from different waxes.

TABLE VI.
Composition of the Wazes.

Formulas of various fatty acids and alcohols which have been isolated from
different waxes.

I. Acids. Saturated. Formula Melting point Wax
Ficocerylic C,H... 57° C. Gundang.
Myristie C,H,,0, 53.8° Wool.

Palmitic C, #,,9, 62.62° Bees. Spermaceti.
Carnaubie .C, 4H,.0, 72.5° Carnauba. Wool.
Cerotic C,H, 77.8° Bees. Wool. Insect
Melissic C, Heo 91° Bees,
Psyllostearylie Cc, oft e00e 94-95° Psylla.

II. Acryllic series.

Physetoleic C, H,,9, 30° Sperm oil,
Doeglie (7) C,H, ,0, s ee
Lanopalmie C, ,H,,0, 87-88° Wool.
Coccerie C,,H,.°, 92-93°

Lanoceric C,H, Oo 104-105° Wool.
THE LIPINS, FATS, OILS, WAXES, PHOSPHATIDES, STEROLS 81

{II. Alcohols. Sterols.

Pisang ceryl C, H,,0 78° Pisang.

Cetyl (Ethal) C,H, 0 50° Spermaceti.
Octadecyl C,H, 30 59° “
Carnaubyl C,,H 0 68-69° Wool.

Ceryl C,H, ,0 79° “Chinese.
Myricyl (Melissyl) C,H 620 85-88° Bees. Carnauba.
Psyllosteary] C,H .0 68-70° Psylla.
Lanolin alcohol C,,H,,0 102-104° Wool.
Ficocery]l Cc we 3 198° Gundang.
Coceeryl C,H 529. 101-104° Cochineal wax.
Cholesterol C,,H,,0 148.4-150.8° Wool.
Tso-cholesterol C,H,,0 137-138° Wool.

The sterols.—The sterols (stereos, solid; ol, the ending signifying an
alcohol), literally solid alcohols, are a very important group of the lipins.
4s their name signifies, they are alcohols solid at ordinary temperatures.
They are readily soluble in ether and CHCI,, easily crystallized, the erys-
tals having a mother-of-pearl glance and a greasy feel. Cholesterin, or
cholesterol, as it is now more generally called, was the first member of
the group to be discovered and is the most important member. Phy-
tosterol, stercorin or coprosterin, cetyl alcohol are others.

Cholesterol.—This, the most important substance in the group, was
isolated from gall stones in 1785 by Fourcroy, when it was confounded
with adipocere. Its true nature as a non-saponifiable, fat-like body was
discovered by Chevreul in 1814, who named it cholesterin from the
Greek chole, bile; and stereos, solid. It is most readily obtained from
gall stones by pulverizing them, and extracting with boiling aleohol con-
taining a small amount of potassium alcoholate. On cooling, or if neces-
sary concentrating first by evaporation of the alcohol, cholesterol erys-
tallizes out as white, shining, plate-like rhombic crystals generally hav-
ing one corner broken. Figure 8. The angles of the sides are generally
76° 30’ or 87° 31’. The form varies with the solvent. The pure crystals
melt at 148.5° C. A'solution of the crystals in CHC1,, or ether, is levo-
rotatory | 7 |2¢°——31.12° for a 2 per cent. ether solution. Cholesterol is
volatile at 300° in vacuo, subliming unchanged.

Cholesterol is insoluble in water, acids or alkalies; slightly soluble
in soap solutions and much more soluble in solutions of bile salts. It
-dissolves readily in hot or cold ether, carbon bisulphide, chloroform, ben-
zene, various hydrocarbons, acetone and hot alcohol. It is slightly solu-
ble in cold alcohol. It is readily soluble in oleic acid and fluid fats. Its
solutions react neutral and have neither taste, color, nor smell. It cannot
be saponified, but is very slowly decomposed by concentrated alkalies.

If gall stones are not available, cholesterol can be most easily pre-
pared by extracting mammalian brains, such as sheep or hog brains, with
82 3 PHYSIOLOGICAL CHEMISTRY

cold acetone. The brains, freed as far as possible from adherent blood
vessels and membranes, are ground in a meat chopper and three times
their weight of acetone is added. After six to twelve hours of extrac-
tion the acetone, which is diluted with the water of the tissue, is filtered
off and a new portion of acetone added and extracted for twenty-four
hours or more while heating on the water bath. On filtering off the
acetone and concentrating by distillation on the steam bath impure

 

iG. x.—Crystals of cholesterol. Fic. 9.—Crystals of phytosterol from soil
(Schreiner and Shorey).
cholesterol separates out. It may be purified »y recrystallizing from
hot alcohol, or other solvents. The cholesterol may be obtained, also,
by making one extraction with cold acetone to remove most of the water
and then extracting the brain mass in a large Soxhlet with acetone.

Color reactions. Cholesterol is remarkable for its power of forming
pigments. Advantage is taken of this property for the purpose of its
qualitative detection by the Salkowski, Liebermann. Lifschiitz and Neu-
berg methods.

Salkowski reaction. Cholesterol, or the substance to be examined,
is dissolved in 5 cc. of chloroform; an equal volume of concentrated
sulphuric acid is poured underneath the chloroform. In the presence of
cholesterol the chloroform solution very soon becomes colored a cherry
red, while the sulphuric acid is dark red with a brilliant green fluorescence.
Poured into a porcelain evaporating dish, the chloroform becomes violet,
green and finally yellow. Cholesterol esters give this reaction, but only
after a delay, probably consumed in setting free the cholesterol. This
reaction is not so delicate or specific as the acetic anhydride reaction.

Acetic anhydride reaction. (Liebermann-Burchard.) A little choles-
THE LIPINS. FATS, OILS, WAXES, PHOSPHATIDES, STEROLS 83

terol is dissolved in 2 ¢.c. of chloroform, then ten drops of acetic anhy-
dride and one or two drops of concentrated sulphuric acid are added.
In the presence of cholesterol a violet, changing quickly to a blue-green,
is obtained. If more chloroform is added in proportion to the acetic
anhydride, the development of the green is delayed. This reaction is
the best and most delicate of the qualitative reactions.

Dry cholesterol reaction. (Schiff’s.) Ifa few crystals of cholesterol
in a porcelain dish are moistened with concentrated HCl, 2-3 volumes,
containing one volume of dilute FeCl,; or with a drop of concentrated
sulphuric acid containing a trace of FeCl, ; and then evaporated carefully
to dryness over a flame, a reddish violet residue changing to a bluish
violet is obtained. With concentrated nitric acid alone cholesterol crys-
tals give, on evaporation of the acid, a yellow spot changing to orange
when moistened with an alkali (xantho reaction). This is not peculiar
to cholesterol, but is given by many aromatic substances.

Oxycholesterol reaction. (Lifschitz reaction.) A few mgs. of choles-
terol are dissolved in 2-3 c¢.c. of glacial acetic acid and oxidized by the
addition of a few particles of benzoyl peroxide. The solution is brought
once or twice to the boiling point. To the cooled solution of oxycholes-
terol four drops of concentrated sulphuric acid are added and shaken.
The color becomes a bright red, then blue and finally green. Spectro-
scopically it shows the typical absorption band in the red characteristic
of oxycholesterol. One part of cholesterol in 10,000 may be detected by
this method. Oxycholesterol gives this reaction without the addition
of the benzoyl peroxide.

Methyl furfural reaction. (Neuberg and Rauschwerger’s reaction.)
—A trace of rhamnose or methyl furfural is added to the alcoholic solu-
tion of cholesterol and then concentrated sulphuric acid is cautiously
added. At the contact surface a red ring forms at once and by shaking,
while cooling the solution, the whole fluid colors itself a beautiful red
and shows, after dilution with alcohol, an absorption band which begins
before E and ends at the B line. The color is permanent for several
days. Phytosterol also gives this reaction and so do the bile acids.
(Pettenkofer’s reaction. Camphor derivatives, etc.)

Quantitative determination. For the quantitative determination of
the amount of cholesterol in a tissue, the latter is repeatedly extracted
with boiling alcohol and then by ether ; the extracts are united and evapo-
rated to dryness on the steam bath; the residue is then repeatedly
exiiacted with dry ether and the ether portions united. The ether is dis-
tilled and the residue is dissolved in benzene. To the benzene is now
added an amount of metallic sodium equal in weight to the residue and
absolute alcohol is added cautiously in small amounts until the sodium
just dissolves. When the sodium is dissolved saponification of the fats
84 PHYSIOLOGICAL CHEMISTRY

is continued on the water bath with a reflux condenser for two hours.
The solution is then cooled, and filtered from the soaps, and the soaps
carefully extracted with water-free benzene, the extracts being added
to the filtrate. The benzene is now washed repeatedly with water to
remove the sodium alcoholate until the wash water reacts neutral. The
benzene is now distilled and the residue is dissolved in hot 94 per cent.
alcohol and made up to known volume. Ten c.c. of the alcoholic solu-
tion of cholesterol are now taken and mixed hot with a 1 per cent. solution
in 90 per cent. alcohol of crystallized digitonin as long as a precipitate
forms. The precipitate of digitonin-cholesterol, after standing a few
hours, is filtered through a weighed asbestos Gooch crucible, washed
with cold aleohol and ether, dried at 100-110° and weighed. To the
weight is added .0016 gm. for each 10 c.c. of solution as a correction for
the amount of the digitonin cholesterol left in solution, the compound
not being absolutely insoluble. The united weights multiplied by the
factor 0.2431 gives the weight of cholesterol in the amount of solution
taken.

Quantitative determination. Colorimetric. (Grigant’s.)—The gravi-
metric method described is probably superior to the colorimetric,
but digitonin cannot always be easily obtained. For the colorimetric
determination the tissue is heated in 1 per cent. sodium carbonate in
50 per cent. alcohol for twenty-five minutes on the steam bath. After
cooling the dried residue is extracted with ether to remove the cholesterol,
the ether is evaporated and the cholesterol is determined colorimetrically
by the acetic anhydride method by comparing the color with standard
tubes made with known amounts of pure cholesterol at the same time.

Amount of cHolesterol in different tissues.—Cholesterol is relatively
more abundant in the brain than in any other organ of the body. It
forms an important constituent of the medullary sheaths of nerve
fibers, but it probably also occurs in the axis cylinder, since it is found
in practically all cells. More of it is found in the white matter of the
pons or corpus callosum than in the gray matter. In the human brain
Kirschbaum and Linnert found by the digitonin method in the cortex
1.15 per cent. of cholesterol; in the white matter, 2.47 per cent.; in the
cerebellum, 1.31 per cent.; in the pons and medulla oblongata, 4.03 per
cent., and in the whole brain, 2.69 per cent. These figures are computed
on the weight of the fresh undried tissue. Dimitz found 3.35-4.26
per cent. in the spinal cord. Thudichum, by measurement of the non-
saponifiable residue of the brain, found in the gray matter 1.95 per cent.;
in the white substance, 3.26 per cent. In the dry ox brain Lifschiitz got
11-12 per cent. as nearly pure cholesterol. Ninety-eight per cent. of this
was free cholesterol, the other 2 per cent. existed in the ester form. Egg-
oil, prepared from the yolks of eggs, contains about 5.5 per cent. of raw
cholesterol, one-fifth being oxycholesterol ester. Trade lecithin (Merck)
THE LIPINS. FATS, OILS, WAXES, PHOSPHATIDES, STEROLS 85

contains esters of cholesterol and oxycholesterol about 2-2.5 per cent.
Cholesterol esters are also present in the kidneys of the ox, and other
glandular organs; there is no oxycholesterol in the liver. Cholesterol is
present in the vernix caseosa, 16.8 per cent. of the total cholesterol being
oxycholesterol. In human blood serum the total cholesterol content.
varied from 1.17-2.95 mgs. per c.c. (Weston and Kent); or 1.97 gram
in the liter (Tscovesco). Esters of cholesterol form doubly refracting
masses in atheromatous arteries. Increase in the cholesterol of the food
increases that of the blood (Kundson).

Chemistry of cholesterol.—The empirical formula for cholesterol is
C.,H,,OH or C.,H,,0H (Mauthner and Suida). When dissolved in
glacial acetic or propionic acid it easily esterifies. It is, therefore, an
aleohol and there is but one alcohol group. It adds bromine, two atoms
to the molecule, and also two atoms of iodine, or hydrogen. It has,
therefore, at least one double bond. According to Mauthner, however, it
adds two molecules of ozone, or six atoms of oxygen, which would indi-
cate two double bonds. When oxidized it yields, first, a ketone, choles-
teron, a neutral body also called oxycholesterol, C,,H,,0; and on further
oxidation a series of cholesterinic acids, dicarboxylic and tricarboxylic.
It is only very slowly acted upon by potassium hydrate in solution, but
when fused with KOH it is attacked below the point of fusion. It is
readily oxidized by benzoyl peroxide, by potassium permanganate, or
bichromate in glacial acetic solution. By reduction, it absorbs two
atoms of hydrogen and goes into hydrocholesterin. It is not hydrogerated
by the ordinary process of hydrogerating oils. It is unstable in
light in the presence of oxygen and changes to a darker color, a lower
melting point, and to oxycholesterol, giving then Lifschiitz’ reaction.
(Schulze; Winterstein.) In lanolin, the fat of sheep’s wool, various
decomposition products of cholesterol as well as oxy- and unchanged
and iso-cholesterol are found. Esters are readily formed, most of them
being crystalline. The palmitic, stearic, oleic, benzoic and salicylic
esters, among others, have been prepared. By fusing cholesterol with
propionic acid, the propionic acid ester is formed. Many of these esters
when cooling after melting show a beautiful play of colors from violet,
blue-green, orange and carmine red. : This may be used for detecting
cholesterol. If gently fused with propionic anhydride, two to three drops
in a dry test tube, and a little of the fused mixture taken out on a glass
rod, the play of colors may be seen.

From a study of the decomposition products and the properties of
the substance, the conclusion has been tentatively reached that cholesterol
is a mono-hydroxy-secondary alcohol, with a terminal vinyl group; with
a double bond; and probably containing four saturated hexa-carbon
rings. The structure of the nucleus has not yet been made out. The
formula as far as known is, then, according to Mauthner:;
86 PHYSIOLOGICAL CHEMISTRY

CH.

3
aH BO, Hag A :CB,
.
HC ba.
CH (0H)

It belongs in the general group of the terpenes and it is probably closely
related to the bile acids.

Physiological importance.—Cholesterol is one of the most important
physiological substances. In pathology, also, it plays a considerable rdle.
Ii forms one of the chief constituents of gall stones, and deposits in the
walls of arteries. It may have some value as a remedy. As we have
seen, it forms a weak molecular union with saponaceous substances like
digitonin. It acts in a similar way with other hemolytic substances, such
as saponin, and other glucosides. It neutralizes their toxic action and
thus protects the blood corpuscles of the body. The red cells are con-
stantly being attacked by hemolyzing substances and dissolved. If the
rate of destruction surpasses the rate of new formation anemia is pro-
duced. Cholesterol has the property of protecting the corpuscles from
many of these dissolving substances, such as the bile salts among others.
It is, hence, being tried as a remedy in hemoglobinuria and anemia.
It probably plays a very important réle in the blood in this way.
Another property of cholesterol, possibly of no less importance, is that
it neutralizes, or checks, the action of lipolytic enzymes. It may in this
way, perhaps, protect the lipins of the cell from digestion, or help regu-
late the rate of their digestion. It has recently been found by Faust
and Abel that derivatives of cholesterol are among the most powerful of
heart poisons. These investigators have isolated from the skins of toads
crystalline substances, evidently oxidation products of cholesterol, which
have most powerful digitalis-like actions on the heart. Their artificial
formation from cholesterol may put in the hands of the physician a valu-
able remedy of the digitalis class. Cholesterol, or its degradation prod-
ucts, aids the other lipins in giving to cells their power of holding large
quantities of water without losing their peculiar semifiuid characters,
and without dissolving. As a constituent of wool fat, or lanolin, it helps
form a valuable salve menstruum for the physician, lanolin taking up
80 per cent. of water in which water soluble drugs may be dissolved
and applied as a salve. As a constituent of waxes and the sebum of the
skin it protects the epidermal structures; it forms one of the most
abundant lipins in the brain and occurs in nearly all living tissues; it is
believed to be the mother substance from which the bile acids are derived
and so plays indirectly an important part in the absorption of fats from
the intestine; it probably gives rise, also, to some of the lipochrome pig-
THE LIPINS. FATS, OILS, WAXES, PHOSPHATIDES, STEROLS 87

ments and possibly to some of the odoriferous substances of plants and
animals. :

The emulsions formed from water and cholesterol esters, or oxy-
cholesterol in union with, or mixed with, other lipins, particularly the
amino-lipins, are extremely interesting and should be carefully studied.
Some of the acids formed by the oxidation of cholesterol are poisons
having a toxic and hemolytic action comparable with some of the snake
venoms. By repeated oxidation of cholesterol Windaus obtained an acid,
C,H 0s,

(CH,), = CH.CH,.CH,.C_H,, —CO—COOH

Bene

CooH C=O

COOH
more hemolytic than the bile acids. The acid C,,H,,O, was as powerful
as many saponins in dissolving red blood cells. It caused local necrosis
like that caused by many snake poisons. Cholesterol probably plays an
important part in the Wasserman reaction. Cerebro-spinal liquid of
syphilitics generally contains more cholesterol than normal (Pighini).
Other sterols.—Other sterols isolated from animal and plant tissues

are the following:
Rotation

Substance Formula M.P. (a) 29 Where found
Phytosterolg C,H, 0 135°-144° Various plants
Sitosterol C,,H,,0 or 136.5° —26.4 (Ether) Wheat embryos

C,,H,,0OH H,0 (Ritter ) —33.9 (CHC],) Calabar bean.
137.5° (Burian)
Psylla alcohol C, H,,OH 69°-70° Psylla wax.
Bumble-bee wax
Isocholestefol C,H oo 140.1° (CHCI,)*7° -+ 59.1 Wool fat
(137°-138°) “+. 60.0
(Dried at 80°)
Taraxasterol C,H, OH Taraxicum officinale
(Power and
Browning)
Homo “ “ C osttggOH
Clutyianol CAH 2@0 (OH), Clutyia similis
(Tutin and Clewer)
Spongosterol OC,,H,,0 124° [a] 4$—19.59° Sponge
Bombicesterol 148° —34° (Ether) Silkworm chrysalis
Stigmasterol C,H 432 170° —45.0° (CHC1,) Calabar bean
Rape oil; cacao
butter
Ergostero] 160° Boletus edulis
Cueurbitol C,H 40% 260° Watermelon seed
Stercorol C,H, 30 95-96° + 24° Human feces
(Koprosterol )
Hippocopro-
sterol CHO /9.5-79.5° Horse feces
88 PHYSIOLOGICAL CHEMISTRY

Phospholipins, or phosphatides.—Although the fats have been dis-
cussed before the phosphatides because of the simple structure of the
former, were we to place them in the order of their importance in the
production of vital phenomena, there can be little doubt that the phos-
phatides should have been considered first, as among the most important
substances in living matter. For they are found in all cells, and it is
undoubtedly their function to produce, with cholesterol and proteins, the
peculiar semifluid, emulsion state of protoplasm. The latter holds mucli
water in it, but does not dissolve. Indeed it might be said that the
phosphatides with cholesterol make the essential physical substratum of
living matter.

Definition. This physical substratum of phospholipin differs in dif-
ferent cells and probably in the same type of cells in different animals,
but everywhere, from the lowest plants to the highly differentiated brain
cells of mammals and of man himself, it possesses certain fundamental
chemicai and physical properties. In all cases the phospholipin sub-
stratum is soluble in alcohol containing some water. The phospholipins
may be separated from the protein, salt and other insoluble substances
by extraction with 85 per cent. ethyl alcohol. Many. but not all, are
soluble when pure in absolute ether, or in absolute alcohol, but all, or
nearly all, may be extracted with impurities by 85 per cent. alcohol. It
is extremely difficult to remove the phospholipins completely from the
protein, with which a part appears to be in combination, but by- repeated.
fifteen to twenty, extractions with boiling 85 per cent. or 90 per cemi
alcohol and ether they may be practically completely removed.

Most of the so-called fat of tissues is in reality phospholipin and
cholesterol. It is usually obtained by ether or alcohol extraction of the
dried tissue. The total alcohol-ether extract (fat) of the pig's liver
was found by MacLean and Williams to contain 84 per cent. of phospho-
lipin and 16 per cent. only was neutral fat and cholesterci, or substances
soluble in acetone.

There is probably always more than one phospholipin in a cell and
when they are separated from each other and from fat and sterol some
of them, such as cephalin, are almost insoluble in absolute alcohol even
when boiling; and others are insoluble in ether; but in a mixture, those
which are the more soluble hold those which are iess soluble in- solution,
so that their separation by differential solubility is a matter of difficulty.

The phospholipins are so called because they always contain phos-
phoric acid; in addition they always contain some higher fatty acids;
and the typical ones an organic base, which is sometimes choline, but
in many its nature is unknown. Most, but apparently not all, rontain
also glycerol. They resemble the fats and oils, then, in containing fatty
acids and glycerol, but they differ from them in having, in addition,

 
THE LIPINS. FATS, OILS, WAXES, PHOSPHATIDES, STEROLS 89

phosphoric acid and some nitrogen base. It is just this composition which
explains their peculiar relation to water, for by their fatty parts they
are prevented from dissolving in the water, while in virtue of the choline
or other base and phosphoric acid in their molecules they have a great
affinity for it.

The exact chemical composition of none of the phospholipins has
been definitely established, but the constitution of at least two of them
is probably approximately known. These two are lecithin and cuorin.
The reason why more of them are not known is owing to the lack of accu-
rate chemical quantitative methods for their preparation and analysis,
and the great difficulty of securing stable, pure substances for analysis.
But lecithin and cuorin, may be taken as types of phospholipins which
are widespread.

Classification of the phospholipins (phosphatides).—The following
classification was suggested by Thudichum for the phosphatides and is
coming more and more into use. The phospholipins are divided into the
following groups:

1. Mono-amino-monophospholipins. Lecithin, cephalin.

2. Mono-amino-diphospholipins. Cuorin.

8. Di-amino-monophospholipins. Sphingomyelin.

4. Tri-amino-monophospholipins. Carnaubon.

This classification is of course of a temporary nature until the real
composition of the members of the group is known, when a more definite
classification can be made.

General method for the separation of phospholipins from cells.

Dry the tissue at low temperature. If possible freeze it immediately after
removal from the animal to stop at once the action of possible phosphatideases.
Shave the frozen tissue in a Kossel knife machine and allow it to dry in a frozen
state in a vacuum desiccator over Al,O, or CaCl,. Extract the dry tissue twice with
cold acetone at room temperature. This removes most of the fat and sterol. Suck
off the acetone; free the powder from acetone by evaporation at room temperature;
and extract with absolute ether at room temperature. The ether will remove any
cephalin, or lecithin. Some cerebrin and other similar cerebrosides or glycolipins,
which when pure are insoluble in ether, may also go into solution in the ether when
the latter has lecithin in it. Most of the glycolipins, however, will remain behind.
The ether extraction may be followed by a boiling alcohol extraction, which will take
out most of the glycolipins, if any are present, and the remnant of lecithin and other
phospholipins. The ether extract had best be treated separately from the alcoholic.
Evaporate the ether under diminished pressure at low temperature; take up the
extract in cold absolute ether and allow to stand twenty-four hours or longer, in a
tall cylinder until a white precipitate (glycolipin, sulphatide, etc.) has settled out.
The clear solution is then sucked off from the precipitate and to it two volumes of
good acetone are added. This precipitates the phospholipins and leaves most of the
fat and cholesterol in solution. Redissolve in ether and reprecipitate, after settling
out the white substance; and repeat this until the ether solution is entirely clear.
By the addition now of three volumes of absolute alcohol to each volume of ether the
90 PHYSIOLOGICAL CHEMISTRY

lecithin substances may be separated from the cephalin, or cuorins which are pre-
cipitated.

The substances thus separated as lecithin and cephalin are still impure mix-
tures of several phospholipins. The method for the preparation of pure lecithin is
given in the chapter on the brain. It consists in the precipitation of the lecithin
as the cadmium salt by alcoholic cadmium chloride, the fractioning of the precipitate
by means of benzene and the final separation of the lecithin as the chloride by HS
and of the free lecithin by the use of Millon’s base.

If it is not possible to freeze the tissue it should be placed at once in a large
amount of 95% alcohol, cut into small pieces and extracted twice with cold alcohol,
to remove the water, and then boiled out repeatedly with 85% alcohol. The phos-
pholipins are recovered by distilling off the alcohol and allowing to cool. They may
be separated in the method described under the brain.

Lecithin.—This is one of the phospholipins of the yolk of the hen’s
egg. The name is from the Greek, lekythos, meaning yolk of egg. It
was first isolated and the name given by Gobley' in 1846. Diakonow,
in Hoppe-Seyler’s laboratory, worked out what is believed to be its com-
position in 1867, although it is very improbable that he ever had pure
lecithin. On hydrolyzing it with barium hydrate, he obtained glycerol,
phosphoric acid, stearic acid, oleic acid and the base choline (neurine,
according to Thudichum). He wrote its formula C,,H,.NPO,+H,O
and regarded it as distearyl lecithin. Its structural formula was be-

lieved to be as follows:
H oO

nb of (CH,),,—CH,
0
|
sels (CH,) .—
Oo

|
ealueelllg: seep (CH,) OH

bh de

Hypothetical formula of lecithin.

Its molecular weight has not been directly determined. The molecular
weight with the above formula would be 807. It was very quickly found
by Hoppe-Seyler and his pupils that lecithin-like bodies were present
in all cells and that they might contain palmitic, oleic and other acids;
but until recently it was believed that the base was always choline and
the alcohol glycerol. It is now clear that there are a group of substances
differing widely in chemical composition, of which lecithin is but one.
This group we have called the phospholipins. Lecithin in ether solution
will not diffuse through a rubber membrane. Its a weight may
possibly be double that cited.

' Gobley: Journal de pharmacie et de chimie, Vol. XI, p. 409; XII, p. 5; XVII, p.
401; XVIII, p. 107.

pens
THE LIPINS. FATS, OILS, WAXES, PHOSPHATIDES, STEROLS 91

The hydrolytic decomposition of lecithin is supposed to follow the
reaction :

C,H,,NPO, +4H,0 ___ 0, ,H, 0, + C, H,,0, + O,H,0, + H,PO, + 0,1, NO,
Lecithin. Water. Oleicacid. Stearic Glycerol. Phosphoric Choline.
acid. acid.

There is one atom of nitrogen to one of phosphorus, so that lecithin
is a mono-amino-monophospholipin. N:P::1:1.

The actual analyses of egg lecithin have given figures for the fatty
acid content which correspond closely to the theoretical; and approxi-
mately the theoretical amount of phosphorus and nitrogen has been
found, but the quantitative determination of the choline is still impos-
sible and only some 80 per cent. of the theoretical amount has actually
been recovered. The chief difficulties in the way of a quantitative deter-
mination of choline have been two: the first is that choline is not.entirely
stable in barium hydrate which is generally used for the hydrolysis, and
some nitrogen of an unknown nature always remains in the fatty acid
residue when this method of hydrolysis is used; the second is that the
precipitation of choline by platinum chloride is not quantitative, par-
ticularly in the presence of other substances such as glyceryl-phosphoric
acid. Moreover, unless special methods are used, the platinum compound
nearly always contains potassium. The first difficulty can be avoided by
the use of the method of alcoholysis, that is hydrolysis by hydrochloric
acid in absolute alcohol. But the second is still unsurmounted.

Pure lecithin is a white substance, crystallizing in very thin plates
which mass together to form a waxy mass. It is readily soluble in alcohol
and ether and can be separated from other phospholipins which may
accompany it only by precipitating it with cadmium chloride and the use
of a complicated method of purification. See page 571.. Few people have
prepared pure lecithin. According to Thudichum, lecithin always con-
tains oleic acid. The point of union of the choline is still disputed and
the exact structure of the molecule is uncertain. The formula given is,
perhaps, the most probable. Thudichum believed that the fatty acids
were united with the phosphoric acid, but this does not seem very
probable.

On hydrolysis lecithin yields oleic, palmitic or stearic acids, choline
or neurine, glyceryl-phosphoric acid, and free glycerol and phosphoric
acid. Many substances have in the past been called lecithins which were
either very impure lecithin or phospholipins of a similar character.
Lecithin is fairly stable and may be kept as the cadmium salt or even
“as free lecithin for a long period unchanged. It is much more stable
than the cephalins (kephalins).

It is probable that there are a number of different ‘eatin: since
92 PHYSIOLOGICAL CHEMISTRY

the character of the fatty acid may vary in different members of the
group. A phospholipin yielding choline, fatty acids, glycerol and phos.
phoric acid may be called lecithin. Lt is doubtful, however, whether the
phospholipiu of the egg, which is properly called lecithin, is found in any
other tissue. We might call those phospholipins which yield choline the
choleo-phospholipins.

Choline. This is the base found in egg lecithin. It is trimethyl,
oxyethyl ammonium soar and has the following formula:

“ae

Choline, C,H, ,NO,.

js -CH,OH

It is related to neurine and muscarine, the latter being an oxidized
choline found in poisonous mushrooms such as Agaricus muscarius.

CH cH,
\/ CH,.CHO \/: il =CH,
CH, _¥ CH,—N
"is ff
GH.” “OH cH, Nou
Muscarine, C,H, ,NO,. Neurine, C,H, ,NO.

Choline was discovered by Strecker in ox-bile, hence the name choline,
from the Greek chole, bile. It is found in many different cells, as phas-
pholipins which are said to yield choline are widespread in nature. In
some instances, however, the substances identified micro-chemically as
choline may not have been choline, but trimethyl amine (Dorée and
Golla). Neurine is found in the nervous system of the ox, but not free.
It is probably derived from choline in the process of preparation. It is
difficult to see how neurine could form a part of a phospholipin except
as a salt through the hydroxyl. Choline is also related to the betaines.
Betaine is the anhydride of trimethyl amino-acetic acid. The formula is
as follows:

CH,

CH Xk os =0

ch 0

Betaine, O,H, ,NO,.
Betaine gets its name from the fact that it occurs free in the sap of the
sugar beet, Beta vulgaris. It is also found in many other plants and the
homologues of betaine are very common in plants, and one, the @ -Oxy-y
trimethy! butyro betaine, is found in muscle (carnutine). Large
THE LIPINS. FATS, OILS, WAXES, PHOSPHATIDES, STEROLS 93

amounts of betaine occur in the fresh muscles and salivary glands of
cephalopods.

Choline is a fairly strong base, alkaline in reaction and absorbing CO,
from the air. It crystallizes readily as the double salt of platinum
chloride (C,;H,,NOC1), PtCl, in reddish yellow plates. Thudichum
states that the corresponding neurine salt crystallizes much more beau-
tifully if there is some potassium chloride present as an impurity.
Choline is precipitated by phospho-tungstic acid. It is unstable as the
free base, but stable as the salt. It will be noticed that in lecithin, if
the ordinary formula is correct, the hydroxyl of the choline is free.
This may be substituted by other anions such as chlorine, making lecithin
chloride. This is very important from the point of view of the action
of inorganic salts on protoplasm, for at this point the anions of the salts
may act on the living matter by changing the state of aggregation of
the lecithin by substituting this hydroxyl of the lecithin. Moreover, by
the interaction of the hydrogen of the phosphoric acid with the hydroxyl
of the choline anhydrides may be formed.

Choline has a pronounced physiological action and its esters are even
more powerful. It is said by most observers to cause a lowering of the
blood pressure. Modrakowski and Popielski, however, maintain that pure
choline raises the blood pressure in non-anesthetized animals. The lat-
ter recrystallized the platinum double salt and picked out the larger,
better-formed crystals in order, as they thought, to get a pure product.
The reason for the discrepancy between these different observers in the
action of choline is not clear. Choline decomposes readily. They ascribe
the lowering of the blood pressure obtained by others to impurities
produced by decomposition. On the other hand, carefully prepared,
synthetic choline still lowers the blood pressure. Most investigators
tested the action on anesthetized animals. Perhaps the difference lies
here; possibly the larger crystals picked out by Popielski may have been
impurities or choline esters, since Thudichum has shown for neurine
that a slight impurity of potassium greatly improves the power of crys-
tallization of the platinum salt. Choline, as the free base, is said to
decompose readily, yielding trimethy] amine; and trimethyl amine has
been found to be a normal constituent of human blood, urine and other
fluids and tissues. If a lecithin, or other phospholipin, is hydrolyzed
with barium hydrate some nitrogen evaporates and part of it goes as
trimethyl amine. The curious fact is that the amount of trimethyl amine
obtained does not seem to depend on the time of heating, which would
indicate the possibility either that trimethyl amine is not derived from
the choline but from some other base, or else that it is preformed in
small amounts in the lipin. The subject needs further investigation.
As an example of the contradictory evidence on some of these points by
94 PHYSIOLOGICAL CHEMISTRY

the best investigators, the observation of Thudichum on cephalin may be
cited. According to Thudichum cephalin, which he purified with the
greatest care, more carefully than most other observers, contains quanti-
ties of neurine which was identified as the platinum double salt. He
found also amino-ethyl alcohol and another base. Now according to
Koch there is probably no neurine or choline in cephalin and this has
been confirmed by Foster. What, then, was the base which was so
much like neurine as to mislead so sharp-sighted an investigator as
Thudichum? What weight shall be attributed to the reports of others
as to the presence or absence of choline or neurine in animal tissues
or phospholipins?

The decomposition of choline by heating into trimethyl amine and
glycol is represented by the following equation:

nae 7 CH,.CH,OH ae
CH,—N —- CH,—N + CH,OH.CH,OH
ae’ oe cH.

Choline. Trimethyl “oittinis Glycol.

It is the opinion of several observers that choline and similar sub-
stances of the class of betaines, which are quite common in the plant
and probably in the animal world, originate by the methylation of some
o! the amino acids.

Quantitative determination of choline.—The following method was used by
Kinoshita for the quantitative determination of the amount of choline in tissues.
Cne-half to one kilo of the organ freed from connective tissue and fat is hashed and
mixed with a double weight of water acidified with acetic or hydrochloric acid and
boiled. The extract is filtered through a cloth and the extraction three times re-
peated. The fluid of the extract is allowed to cool, the fatty layer removed and after
acidification with hydrochloric acid to prevent the decomposition of the choline it
is concentrated first over the free flame and then on the water bath; the concentrate
is poured into a 3-liter, round-bottom, Jena glass flask and by vacuum distillation
on the water bath brought to dryness. The residue is then extracted three times in
the same flask with boiling ether and the ether poured off. The choline is then re-
moved by three extractions with boiling 95% alcohol and the united alcohol extracts
brought by distillation in vacuo on the water bath to dryness. The residue is ex-
tracted with absolute alcohol, the alcohol evaporated and again extracted with abso-
lute alcohol and the last absolute alcohol extract is filtered. The choline in the
absolute alcohol solution is now precipitated by the addition of an absolute aleohol
solution of PtCl, or HgCl,. The precipitate after twenty-four hours’ standing in the
cold is suspended in water and decomposed with H,S and then without filtering it is
evaporated to dryness in a porcelain dish on the water bath. The residue is taken up
in a little water, filtered and the residue carefully washed. The solution is evapo-
rated to a small bulk and the gold double salt formed by the addition of AuCl.. The

A A . 8
gold chloride-choline crystals are weighed.
Choline may be precipitated from its solution by KI a3 by PtCl,; or by an alcoholic
THE LIPINS. FATS, OILS, WAXES, PHOSPHATIDES, STEROLS 95

HgCl, solution. KT, will precipitate when added to an aqueous choline chloride solution.
A 10% HgCl, solution in alcohol, or a 10% solution of PtCl, in alcohol when added
to an alcoholic solution of choline chloride, are about equally efficacious as precipi-
taunt. A .002-.001% solution of choline chloride will give with either reagent a distinct
precipitate in 10 to 15 minutes; and a .0006-.0003% solution will give a noticeable
precipitate after 30 minutes. Even a .00005% solution will give a slight precipitate
after 2 hours. Sublimate solution in absolute alcohol added to the alcoholic solution

 

oi 7 B 3
Fig. 10.—A. Crystals of choline periodide. B. Crystals undergoing disintegration and
formation of oily drops (Rosenheim).

of choline chloride precipitates far more completely than the aqueous solution
(Kinoshita). :

None of the micro-chemical tests which have been proposed for the detection of
choline are reliable. Most of these reactions, such as the Florence reaction, are given
by neurine, trimethyl amine and other bodies (Kinoshita).

Amount of choline in various tissues.—The quantitative determina-
tion of the choline in various tissues has yielded the following results.
This choline is not free but is split off by heating the tissue with hydro-
chlorie acid.

Amount of choline in per cent. of the wet weight of the tissue. Direct
determination by the gold salt. (Kinoshita).

Tissurs oF Ox

Per cent. ‘Per cent.

choline. choline.
Pancreas I 015 Spleen .012-.030
“Tia .031 Muscle .016-.032
. IIb -022 Liver -010-.017
3 IIe .021 Kidney -020-.029
Small intestine .022-.030 Lung .016-.011

Cuorin.—C,,H,,;NP,0,,. This as the name implies is a phospholipin
isolated from the beef heart. In its solubilities and other properties it
resembles cephalin.

Method of preparation. The method used by Erlandsen for its prepa-
ration is as follows: The’ ox heart freed from fat, blood vessels, etc., is
96 PHYSIOLOGICAL CHEMISTRY

ground in a meat grinder and spread on glass plates before a fan to dry.
It loses about 70 per cent. of its weight as water evaporates. It is then
ground in a mill and dried in vacuo over sulphuric acid at 40°. (The
author’s experience is that it is better not to use sulphurit acid in vacuo
as a drying agent because of the vapor pressure of the acid. CaCl, or
Al,O, are not open to this objection.) The powdered mass is then ex-
tracted with ether, 1 liter to each 500 grams of powder, at room tempera-
ture, changing daily until all ether-soluble material is extracted. The
extracts are evaporated in vacuo to a syrup. The ether extract is about
7 per cent. of the dry powder. This extract is re-extracted with absolute
ether, and allowed to stand. A white substance (probably glycolipin,
A.P.M.) separates out. The ether is decanted from the white residue;
precipitated with water-free acetone. The precipitate is redissolved in
ether and reprecipitated several times. This frees the cuorin from fat
and cholesterol and some other substances such as the white substance.
It is now redissolved in ether and reprecipitated by four volumes of cold
absolute aleohol at 0°-2° C. The precipitate, which is crude cuorin, is
extracted with warm absolute alcohol; the cuorin remains insoluble.
Thus prepared it resembles cephalin, a phospholipin of the brain. It is
a yellowish brown, transparent, almost odorless, glassy substance; it is
very hygroscopic and becomes sticky and finally fluid on standing in the
air. It melts, undergoing decomposition. It contains no sulphur. It
is easily soluble in ether, chloroform, carbon bisulphide and petroleum
ether. At high temperatures it is soluble in glacial acetic acid, acetic
ester and amyl alcohol, and precipitates out on cooling. It is insoluble
in hot and cold methyl and ethyl alcohol and acetone. In water it sinks
at once to the bottom, but swells and finally dissolves to a turbid emul-
sion, which reacts neutral. The emulsion becomes quite clear on adding
alkali. Analysis of cuorin gave C, 61.63 per cent.; H, 9.03 per cent.;
N, 1.015 per cent.; P, 4.46 per cent.; O, 23.86 per cent. The ratio of
P:N::2:1. It is hence a mono-amino-diphosphatide. The iodine number
is 101. It contains three fatty acid groups per molecule of the average
composition of C,,H,,0,. Melting point 47-48°. Iodine number of the
acids, 130.1. The fatty acids contain acids of the linolic and linolinic
groups, and are, therefore, doubly or trebly unsaturated. This is a point
of much interest in view of the keen metabolism of the heart tissue.
Glyceryl-phosphorie acid was isolated. The base was not choline, but
an unknown base, the nature of which has not been determined. Cuorin
resembles cephalin in many ways. It is very improbable that the cuorin
thus prepared is a single substance and further work, using the meth-
ods of Thudichum for the separation of the cuorin into its constitu-
ents, must be done. There are other phospholipins in the heart than
cuorin. :
THE LIPINS. FATS, OILS, WAXES, PHOSPHATIDES, STEROLS 97

We shall have occasion to examine the composition of other members
_of the phospholipins in the chapter on the brain.

Physical properties of phospholipins—The physical characters of
the phospholipins, by whatever method they are prepared, are extremely
interesting. They are generally light yellow to brown, waxy or salve-
like substances, not crystalline, although some have been crystallized.
They dry in vacuo over a drying agent to a hard mass, which may easily
be pulverized. The powder so obtained is generally very hygroscopic
and when exposed to the air it takes up quantities of water and turns
to a dark-brown, thick, semifluid mass. It is probably partially oxidized
at the same time, and it is chemically changed, so that after thus becom-
ing partially fluid and standing some time, there is a change in solubility ;
generally the solubility in ether is diminished and that in water increased.
The phospholipins, unlike the fats, form permanent emulsions, or colloidal
solutions, if shaken with water and many of these emulsions are unstable,
being easily precipitated by various common salts. One of their most
striking peculiarities is that if rubbed with water or shaken with water,
but not too long, and then examined under the microscope, they will’
be seen in isolated shreds or strings. Water enters the masses of phos-
pholipin, which do not usually take a spherical shape but often the
most regular and striking forms, reminding one irresistibly of the struc-
ture of cells. Often they take the form of long fibers with a hollow core
and highly refractive walls, looking much like a medullated nerve fiber
with the axis cylinder in the center ; or they may take forms of flasks with
long perfectly regular, whip-like processes looking like enlarged, ex-
ploded nettle cells. These forms are called myelin forms because of the
resemblance they bear to the myelin sheaths of nerves. They form liquid
crystals, the molecules having a definite orientation. In this condition
they may be doubly refracting. Liquid crystals are easily deformed by
pressure and fuse when brought into contact. A mixture of cephalin,
amidolipin and cholesterol is particularly apt to assume these. forms.
Phrenosin and kerasin, two glycolipins of the brain, behave similarly.
The power of making these myelin forms differs in different phospho-
lipins, and it is possible that the presence of certain impurities, such as
cholesterol, or its esters, may influence the process. It is possible that
cells owe their hygroscopic character, their clear refractive appearance,
their organization and power of taking definite shapes in part to this
phospholipin groundwork. Indeed, if one rubs together in a mortar
some phospholipin containing cholesterol, some white of egg and some
olive oil, there is obtained a mixture of substances which mimics exactly
the physical appearance and many of the vital staining reactions of
protoplasm.

Auto-oxidation——Another very important chemical property of the
98 PHYSIOLOGICAL CHEMISTRY

-phospholipins is that they undergo auto-oxidation, that is they oxidize
spontaneously when exposed to the air. The fact that this fundamental
substratum of living matter has this power of taking up oxygen, of burn-
ing itself and, possibly, inducing oxidation in substances dissolved in
it, although this possibility has not yet been investigated, is of the
greatest importance for the theory of the mechanism of respiration, since
all protoplasm has also this power of reduction, or auto-oxidation. This
property of the phospholipins can be very easily observed, as it is accom-
panied by a darkening of the phosphatide preparations on exposure to
air, and a change in their solubilities. The phospholipin from the heart,
cuorin, and the brain cephalin are particularly active in this way, but
nearly all phospholipins show something of this same property of taking
up oxygen. Erlandsen observed the following increase in weight in some
cuorin left in a desiccator over sulphuric acid.

Weight of cuorin

December 29. 0.3705 gram.
February 6. 0.4039
November 20. 0.3949

The iodine number of the fresh cuorin was about 101, but after standing
in the desiccator it fell to 22.33. The original preparation showed, on
analysis, a composition of C,,H,,,NP,O,,; but after standing these num-
bers changed to C,,H,,,NP,0,;.. At the same time the substance became
insoluble in ether. It is this ease of oxidation which is one of the com-
plicating circumstances in the isolation of unchanged cuorin and
cephalin.

This power of auto-oxidation is due, in large measure at least, to the
unsaturated fatty acids in these phospholipins. Thus cuorin and
cephalin have acids of the linolenic acid series which contain three pairs
of unsaturated carbon atoms, C,,H,,O,; and nearly all the phospholipins
contain some oleic or other unsaturated acids. They may possible con-
tain also unsaturated groups in the basic constituents, but this, while
in some instances probable, is not yet certain.

It is very suggestive that the oxidized phospholipin has a much
greater affinity for water than the unoxidized. Possibly this process
may play a part in the cell mechanics, since most movements in cells are
apparently due to this changing affinity for water. By oxidation the
phospholipin might be made to take up water, by reduction to lose it.

Other functions of phospholipins.—Besides their fundamental fune-
tion of making, with the sterols, a water-containing, semifluid, highly-
reducing, auto-oxidizable, semilipoid crystalline substratum, which con-
‘tributes to or makes: possible the vital phenomena, the phospholipins are
also concerned in some of the phenomena of immunity. Flexner and
Noguchi observed that the hemolytic action of cobra venom toward cer-
THE LIPINS. FATS, OILS, WAXES, PHOSPHATIDES, STEROLS 39

tain red blood corpuscles was dependent upon the presence of some sub-
stance in the serum. Kyes found that this serum substance was extract-
able with cold alcohol and it proved to be a phospholipin. He then showed
that cobra venom united with lecithin (?) to form a substance which he
called a Iecithide, and these lecithides were extremely hemolytic,—they
dissolved blood corpuscles with great power. Lecithin emulsions have
also some hemolytic power themselves. The phosphatides may, therefore,
by uniting with toxins, or possibly with the foods, influence the power
of the latter to unite with and affect the activity of protoplasm. Hither
the phospholipins or glycolipins have the power of binding, or uniting
with tetanus toxin. This function of the phospholipins is being inves-
tigated at the present time. The phospholipins appear to play an impor-
tant part in the Wasserman reaction for syphilis.

Method of hydrolysis of the phospholipins——The phospholipins (phosphatides)
are generally decomposed by heating an emulsion with barium hydrate under a
condenser for several hours. By this method, however, the fatty acids or soaps are
generally « deep brown and contain some nitrogen, a partial decomposition of the
base having occurred. Other methods suggested and tried have been by using a methy]
alcohol solution of barium hydrate; or by suspending the phospholipin in water and
boiling with 5% HCl. The best and cleanest method, however, for all alcohol
soluble lipins, is to dissolve or suspend them in absolute alcohol, to saturate the
solution with dry hydrochloric acid gas, stopper the flask and allow it to stand
twenty-four hours at room temperature. Then boil on the water bath for
3-5 hours, using a reflux condenser. By this method the fatty acids are
esterified with alcohol as rapidly as they are set free, and they remain in solu-
tion. The mixture is then evaporated with the addition of water on the water
bath; the esters are insoluble in water and may be easily separated by filtration.
In the acid filtrate the base, often choline, which is more stable as the salt than the
free base, may be separated by precipitation with platinum chloride leaving the
glycerol and phosphoric acid in solution.

A useful method of determining the character of the phospholipin is that of
Herzig and Meyer as developed by Foster in the writer’s laboratory. It con-
sista in heating the phospholipin with hydriodic :cid and ammonium iodide and col-
lecting the isopropyl and methyl iodides in silver nitrate and weighing the silver
iodide. The isopropyl iodide comes over at 112-120° and represents the amount of
glycerol present. Two methyl groups come from the choline. and possibly three, at
240-310°, as methy! iodide. From the silver iodide collected at these two tempera-
tures an idea may be had of the amount of glycerol and choline present, and the
phospholipin partially identified. Cephalin contains glycerol about 10%, but no base
yielding methyl iodide, ie., neither neurine nor choline.

Other phospholipins—Among the other phospholipins a few may
be mentioned at this place, namely, heart lecithin, carnaubon, jecorin,
glucose lecithin, cephalin and sphingomyelin. The last two are found
in brain, the first in the ox heart, carnaubon in the kidney and the other
two in the liver. They will be taken up more in detail] under the chemical
composition of the organs in which they occur, and at this place we
100 PHYSIOLOGICAL CHEMISTRY

will give only a summary of their composition to give some idea of the
composition of the group. Especial attention is called to the fact that
many of the members of the group, like jecorin, contain a carbohydrate
radicle in the molecule. It is found that those which contain such a
group are more soluble in water than the rest, and they are less soluble
in ether. Some of them are quite insoluble in absolute ether, but will
dissolve in ether containing water. They are also more hygroscopic than
egg lecithin. Their chemical nature is still quite obscure. Such carbo-
hydrate-containing phospholipins have been found not only in the livers
of many animals, but also in the egg cells of Echinoderms, and they are
probably widespread in the animal and plant kingdom. Several have
been isolated from plants. Their group name is glyco-phospholipins.

The character of the base in the phospholipins is probably very vari-
able. Amino-ethyl alcohol, or hydroxyethyl amine was isolated by Thudi-
chum from cephalin, and it has since been found in many phospholipins.
Serine and oxyamino-butyric acid were isolated by MacArthur from
cephalin. Vidin, C,H,,N,O,, was obtained from a phospholipin in
Lupinus albus. Many other nitrogenous bases probably occur in the
different phospholipins and the investigation of these bodies is one of the
most promising fields of physiological chemistry.

Glycolipins.—The glycolipins, or cerebrosides, are phosphorus-free
lipins which contain a carbohydrate and fatty acids. Phrenosin, kerasin
and cerebron are typical members of the group. Phrenosin is a white,
crystalline lipin found in the medulla of the fibers of the vertebrate
nervous system. It occurs in considerable quantities in the brain after
medullation has begun. The properties of this substance or group of
substances are described in the chapter on the brain. The glycolipins on
hydrolysis yield a sugar, which is often galactose, various fatty acids
and a nitrogen-containing alcohol. The molecular weight is undeter-
mined. While these bodies have been found in the brain, it is prob-
able that they are not confined to that tissue, since phospholipins
containing a sugar are widespread. It is not impossible that these may
be compounds or mixtures of a glycolipin with a phospholipin.

Localization of the phospholipins in the cell.—The proportion of
phospholipin in the sperm is high and it is found chiefly, or altogether,
in the tails. The nuclear heads appear to be almost, or quite, free of
alcohol-ether soluble materials. The heads and tails of the herring sperm
were examined, the heads being separated by suspension of the sperm
in water and centrifugalization. The heads being heavier are thrown
off from the tails. This observation would indicate that the nucleus of
the ripe sperm had little or no lipin material in it. The conclusion that
the lipins are, for the most part, in the cytoplasm is confirmed, also,
by examination of the red blood corpuscles. Mammalian erythrocytes
THE LIPINS. FATS, OILS, WAXES, PHOSPHATIDES, STEROLS 101

have no nuclei, and yet they contain phospholipins and sterols. The
localization of the phospholipin in the cell may also be studied by the
use of staining reagents. The phospholipins are acids or salts of acids.
There is a free acid hydroxyl in the phosphoric acid, if the ordinary
formula is correct. The phospholipins are generally present in cells as
salts, a hydrogen being replaced by sodium, potassium, calcium, am-
monium or organic bases. Being salts they have the power of forming
salts with the basic dyes. They do this and they stain intravitam by
means of the basic dyes. If an emulsion is made of lecithin, or other
phosphatide, egg albumin and olive ‘oil, it will be found that the granules
of lecithin or phospholipin take up neutral red, or other basic dye,
most easily and the granules stain a deep red. It does not do to conclude
that all the granules of cells which stain red in neutral red are phos-
pholipin, because other colloids of an electro-negative, or anionic, char-
acter will stain in the same way, but certainly some of the fine granules
in cells look very much like lecithin; they stain in the same way aad
they are soluble in ether. Furthermore, the density of lecithin is greater
that that of water, so that if cells are centrifugalized we should expect
these granules to go to the base of the cell and the oil to come to the part
of the cell nearest the axis of the centrifuge where the centrifugal force
is least. It is found that these small granules which have the staining
reaction and appearance and solubility of lecithin also behave the same
way in the centrifuge. It is probable, therefore, that the phospholipin
is distributed all through the cytoplasm; and that in egg cells, in which
there is a storage of lecithin or other phospholipin as reserve food, the
lipin occurs also as granules in the cytoplasm. Many believe that the
surface of the cell has a character more particularly lipoid or lipin in
character than the interior. They consider the blood corpuscles to be
essentially but bags of lipin and sterol filled with hemoglobin, and they
attribute the ease of penetration of ether, alcohol: and other substances
into cells to their solubility in this membrane. In the author’s opinion
there is no satisfactory evidence as yet of the existence of any such
particularly lipin-like outer membrane and experiments indicate that
the lipin character of the cytoplasm extends throughout it. The
entrance of basic dyes can as easily be explained by their power of union
with protein as by their power of union with phospholipin. Kite’s work
shows that the permeability of the cell is the same all the way through.
From this it follows that if the entrance of substances into cells
depends upon the presence of lipoids in the membrane, these
same lipoids must occur all through the cell. There are, also, far too
large quantities of lipins in cells to be confined to a surface layer of
molecular thickness. Phospholipins are found, also, in many secre-
sions. such as that of the stomach glands, milk, pancreatic secretion,
102 PHYSIOLOGICAL CHEMISTRY

etc. It is not impossible that many of the granules called zymogen
granules in cells which stain readily in neutral red may owe this prop-
erty to the fact that they contain phospholipins. The mito-chondria are
probably in part phospholipin.

REFERENCES. Lyris.

Classification. Gies and Rosenbloom: Biochemical Bulletin, 1, p. 51, 1912.

Oils and fats. Encyclopedia Britannica, XI edition.

Waxes. “ ee “ “

The fats. Leathes: Longmans, Green & Co. London, 1910.

Cuorin. riandsen: Untersuchungen iiber die lecithinartigen Substanzen.

Zeits. f. physiol. Chem., 51, p. 71, 1907.

6. Egg yolk. Phospholipins in egg yolk. Stern and Thierfelder: Zeits. f.
physiol. Chem., 53, 1907, p. 371.

7. Nature of base in lecithin. MacLean: Biochemical Journal, iv, p. 455, 1900.

8. Monamino-diphosphatide in egg yolk. MacLean: Biochemical Journal, iv, p.
168, 1909.

9. Glyco-phospholipins, Winterstein: Zeitschrift f. physiol. Chem., lviii, p. 500,
1909.

10. Acid hydrolysis of phospholipins. Moruzzi: Zeit. f. physiol. Chem., lv, p.
352, 1908.

11. Choline in animal tissues and fluids. Webster: Biochem. Journal, iv, p. 331,
1909.

12. Trimethyl amine in blood, urine and cerebrospinal fluid. Dorée and
Golla: Biochem. Journal, v, p. 306, 1911.

13. Quantitative determination of choline. MacLean: Zeits. f. physiol. Chem.,
lv, p. 360, 1908.

14. Quantitative determination of choline and betaine. Stanek: Zeits. f. physiol.
Chem., xlviii, p. 334, 1906. /

15. Nature of union of choline in lecithin. Malengreau and Prigent: Zeits. f.
physiol. Chem., 77, p. 107, 1912.

16. Amino-ethyl alcohol in phospholipins. Trier: Zeits. f. physiol. Chem., 73, p.

See gs eae

383, 191).

17. Oxyethyl amine in egg lecithin. Eppler: Zeits. f. physiol. Chem., 87, p. 233,
1913.

18. Origin of betaines, Schulze and Trier: Zeits. f. physiol. Chem., 81, p. 53,
1912.

19. Vidine. Njegorau: Zeits. f. physiol. Chem., 76, p. 1, 1911.

20. Alcoholic saponification of lecithin. Rollet: Zeits. f. physiol. Chem., 61, p.
210, 1910.

21. Purification of phosphatides. MacLean: Biochem. Jour., vi, p. 355, 1912.

22. Carnaubon. MacLean: Biochem. Jour., vi, p. 354, 1912.

23. Acetyl choline in ergot. Hwins: Biochem. Jour., viii, p. 44, 1914.

24. Staining reactions of lipins. Weigert reaction. Lorrain Smith: Journal of
Pathology and Bacteriology, XI, p. 415; 12, p. 1, 1907. Biochem. Jour., 8,
1914.

25. Role of phospholipins and cholesterol in immunity and hemolysis. Brown-
ing: Jour. of Pathology and Bacteriology, xiv, p. 484, 1909; xvi, p. 225.

26. Cobra poison and lecithin. Toxolecithides. Morgenroth and Koya: Biochem.
Zeits., 25, p. 88, 1910.
28.
29.
30.
31.
32.
» 33.
34.
35.
36.
37.
38.
39.
40.

41.

42,

THE LIPINS. FATS, OILS, WAXES, PHOSPHATIDES, STEROLS 103

Wasserman’s reaction and lipins. Browning, Cruickshank and McKenzie:
Biochem. Zeitschrift, 25, 1910, p. 76.

Relation to oxidases. Vernon: Biochem. Zeits., 47, p. 374, 1912.

Physiology, etc. Wilson: Biochemical Journal, vi, p. 100, 1911.

Cholesterol. Quantitative determination. Windaus: Zeits. f. physiol. Chem.,
65, p. 110, 1910. :

Cholesterol. Quantitative determination. Ritter: Zeits. f. physiol. Chem.,
34, p. 426 1901.

Cholesterol. Quantitative determination.  Lifschiitz: Biochem. Zeits., 25
p- 426, 1910.

Cholesterol in disease. Cerebral spinal fluid. Pighini: Zeits. f. physiol.
Chem., 61, p. 508, 1909.

Cholesterol in tissues. Lapworth: Journal Pathology and Bacteriology, 15,
p. 254, 1911. :

Cholesterol in attacks of hemoglobinuria. Pringsheim: Miinchener med.
Wochenschrift, 59. p. 1757, 1912.

Chemistry. of wool fat. Darmstaedter and Lifschiitz: Berichte d. d. Chem.
Gesell., 31, p. 97, 1898.

Absorption of cholesterol and physiology. Various articles by Dorée and
Gardner in the Proceedings of the Royal Society, London, ser. B, 1913-1914.

Cholesterol in cells. Policard: Jour. de Physiol. et Pathol. gen. 16, p. 624,
1914.

Cholesterol content of cells under various conditions of diet. Terroine:
Jour. de Physio. et. Path gen. 16, pp. 583, 607, 1914.

Content of lipoids and activity. Mayer and Schaeffer: Jour. de Physiol. et
Pathol. gen., 16, p. 344, 1914. See also p. 23.

Mitochondria consist of lecitho-proteins. Mayer, Rathery and Schaeffer:
Jour. de Physiol. et Pathol. gen., 16, pp. 583, 607, 1914.

Cholesterol metabolism in hen’s egg. Mueller: Jour. Biol. Chem., 21, p. 24,
1915.
CHAPTER IV.

THE PROTEINS. .

- Occurrence.—If living matter of any kind, whether plant or animal, |

be extracted with alcohol and ether to remove the fats and lipins, the
greater part of the organic matter remains behind as a white amorphous
mass. If this mass be rapidly extracted with water, or dilute alcohol,
various salts (chlorides, phosphates, carbonates) and a small amount of
organic matter, called extractives, are removed, together with a little
of an acid, nucleic acid. There remains the greater part of the organic
matter insoluble. If this organic matter be analyzed it is found to
contain nitrogen. It consists in most instances of about 52 per cent.
of carbon; 7 per cent. hydrogen; 15 per cent. nitrogen; 1-2 per cent.
of sulphur; 0.2-3 per cent. of phosphorus; and 23 per cent. of oxygen.
This organic, nitrogenous, amorphous matter since it makes by far the
greater portion of the organic substance of living matter proper,
exclusive of cell walls, is called protein,’ a word meaning ‘‘ of the first
importance.’’ Protein substances are found, like the fats and carbohy-
drates, nowhere else in nature than in living matter, or as the products
of the action of living matter. In plants more or less carbohydrate is
associated with the protein; but in animals by far the greater part of
the tissue solids are generally protein.

The proteins, therefore, very early attracted the attention of investi-
gators, whether biologists or chemists, and the opinion has from the
uirst been held that they must play a predominant réle in the production
of the vital phenomena. Probably the fact that the composition of the
fats and carbohydrates was relatively simple, was easily understood and
early made out, in its general features at any rate, whereas the proteins
were apparently more complex and have until recently kept the mys-
tery of their composition inviolate, strengthened this view. Some have
even gone so far as to suppose that the protein in living matter is alive
and endowed with properties other than ordinary chemical and physical
properties. Whether the protein, which we are able to separate from
cells for analysis, has the same composition it had while in living matter
is a question which is unanswerable, as long as we know nothing of its

+The name protein, from the Greek -pwreio¢, pre-eminence, was given by

Mulder, Jour. f. prak. Chem., 16, p. 129, 1839. The word proteid has now been
dropped and protei™ anbatituted for it.

104
THE PROTEINS 105

composition while in the living mixture. But while the statement is,
often categorically made that the materials which we examine chemically
are dead materials, and therefore incapable of throwing light on the
composition of living matter, it must be remembered that it is the part
of those who assert that they differ to show that this is the case. And
this at present cannot be done. While, therefore, it is conceivable, and
in itself not unlikely, that the protein may. contain, while it is in the
living mixture, unstable groups, i.e., aldehyde or nitrile groups, which are
made stable in the process of extraction, no one has as yet proved this
to be the case; nor, indeed, has any strong evidence been produced show-
ing it to be the case. While some speak of living, as distinct from
lifeless protein, no one can as yet point to any single chemical property
found in living and not found in dead. protein; although the differences
between living matter and dead protein are profound. Whatever the
truth may prove to be we must, at any rate, attack the dead protein,
that is protein separated from non-protein substances, for following this
path has already led to many brilliant.and fortunate discoveries. These
discoveries have revolutionized the science of nutrition and have reacted
on all branches of biological science, from botany and anthropology to
the diagnosis and treatment of disease. Let us see, therefore, what sub-
stances have been isolated from this amorphous, colorless mixture of
protein bodies constituting so essential.a part of the framework of living
matter; and what properties the substances so isolated possess.

Methods of extraction.—The separation and exact analysis of these
protein bodies is not yet complete. It was necessary to devise methods
for the separation .of the various proteins, for in any one cell there is a
mixture of such bodies; and the elucidation of the structure of the mole-
cules was most difficult, owing to their complexity and instability. It
was found that the solubilities and some other properties of the proteins
were easily modified by acids and alkalies, by heat, by alcohol, by some
metal salts and by many enzymes and oxidizing agents. Subjected to
the action of any of these agencies many proteins change their nature,
as is evident from their change in solubility; they are said to be dena-
turized. Proteins so modified are called derived proteins. For the sepa-
ration from the cell of natural, or unmodified, protein it was found nec-
essary, in most instances, to dissolve them out with water,’ with neutral
solutions of sodium chloride, or the sulphates of the alkali metals, and
to avoid alkaline or acid reactions, high temperatures or alcohols.

It is very difficult indeed to get from living matter itself individual,
pure proteins. The living matter is such a mixture of different kinds
of proteins that it is only occasionally that a form of protoplasm is
obtained which consists largely, or exclusively, of a single protein. Oné
of the very few cases in which probably pure individual proteins have

®
106 PHYSIOLOGICAL CHEMISTRY

been separated from a living animal cell is the protamine which may be
separated from the head of the fish sperm. The spermatozoon is a very
highly differentiated cell, the head being composed of pure nuclear mat-
ter of the simplest kind known. Moreover the protein in the head is of
a very peculiar kind, being very strongly basic and soluble in strong or
dilute acids, so that its separation from other proteins is not difficult,
and there is apparently in some sperm but one protein present. Else-
where in the animal kingdom one can at times, owing to the peculiar
nature of the protein, secure proteins which are probably pure substances.
Hemoglobin, the red protein of the blood, crystallizes very easily in
characteristic crystals, so that this protein is probably pure. In the
egg cells of selachians crystalline substances exist which are probably
proteins. In animals it is only in special cells such as the cells of the thy-
roid gland (colloid matter) or the egg cells, and in very few of these, that
a storage of a particular kind of reserve protein exists, so that this one
kind of protein is in such excess that it may be separated from the
others. In general, animals do not store proteins, as they do carbohy-
drates and fats, except in the form of living matter. The condition is
different in plants and it is from plants that crystalline proteins which
are probably pure individuals may be most easily had for analysis. In
seeds and nuts there is a large amount of reserve or stored protein. Many
of these proteins are of a crystalline form in the seeds or nuts. Moreover
the living protoplasm in these structures is reduced to a minimum. It is
found chiefly in the embryo, which in some cases, as in wheat, may be
separated by milling processes from the rest of the grain; and in any
case, it is generally present in very small amount compared to the large
amount of reserve protein present. There is another feature of these
plant reserve proteins, besides their presence in large amounts and their
ease of crystallization, which makes them of particular value for the
study of the nature of the proteins. They are very stable proteins and
do not easily change their condition; or they do not change their condi-
tion and solubility so easily as the protein in living matter, in the
protoplast proper. In order that substances may accumulate in cells
they must be rather resistant; the reactive substances are short-lived.
This is a general rule for all the substances of the cells. Thus the fat
which is laid down as reserve fat is generally stable saturated fat; in
the carbohydrates it is the stable polysaccharides or the most stable
disaccharide, cane sugar, which accumulate as a reserve. The same thing
is true for the phospholipins. The phospholipin stored as a reserve
in the egg cell is the most stable form, lecithin. The phospholipins
isolated from active living matter are very unstable with highly unsatu-
rated acids in them. This same rule is true for the proteins and in seeds
and nuts the large amount of reserve protein present is unusually stable
¢
THE PROTEINS 107

and so best adapted to withstand the crude manipulation of the chemist.
The stable proteins used by animals for supporting tissues, such as
horn or elastin, are extremely insoluble. A very easily ecrystallizable
substance obtained from nuts is the protein, excelsin, of the brazil nut.
This occurs crystalline in the nut itself. Another crystalline reserve
protein is edestin, which occurs in hemp seed. And there are many
others. These proteins may be obtained crystalline by the simple expedi-
ent of dissolving them out of the seeds or nuts with 10 per cent. sodium
chloride and dialyzing the salt out of the solution. The protein will
crystallize in the dialyzing tube. The investigation of these plant pro-
teins is largelv the work of the American chemist, Osborne, who has thus
carried forward the work well begun many years ago by the German
investigator, Ritthausen.

The separation of proteins from plant seeds and nuts is generally
best made by extracting the ground nuts, or seeds, with distilled water,
or by 10 per cent. sodium chloride; or 75 per cent. alcohol. A few
proteins exist in some plant seeds which are insoluble either in water
or absolute alcohol, but which are soluble in dilute alcohol. Gliadin of
wheat and zein of corn are examples of such proteins. The separation
of the mixture of the proteins obtained by extraction with salt solution
is best made either by dialysis, which will precipitate some of them and
dialyze away impurities; or by precipitating them by saturating their
solutions with ammonium sulphate which precipitates all the proteins.
The redissolved precipitate may be separated by fractional precipitation
with ammonium sulphate, the globulins, being less soluble than the albu-
mins in ammonium sulphate, are precipitated by half saturation. Since
acids change the plant proteins very easily, both by their own action
and by their power of setting free digestive enzymes which are present
in all forms of living matter, it is often wise to add just enough dilute
barium hydrate to keep the reaction neutral to phenolphthalein during
the extraction.

While many of the proteins are thus extracted with neutral salt solu-
tions or water, there generally remains a residue which can only be dis-
solved by the use of alkali, which may and probably does alter the com-
position of the protein. In the cereals, for example, water or neutral salt
solution dissolves very little of the protein. :

The extraction of the proteins from animal tissues is a very much
more difficult matter than from vegetable. In the first place the animal
cells have more reactive protein in them and generally there are present
powerful digestive enzymes, the so-called autolytic enzymes, which are
apt in a prolonged extraction to change, more or less, the protein of
the cell. These enzymes are easily checked by the maintenance of a
neutral or faintly alkaline reaction. Extraction always removes, also,
108 PHYSIOLOGICAL CHEMISTRY

some of the phospholipins with the proteins and the separation of these
from the proteins without. coagulation of the latter is very difficult, and
it is doubtful if it is often possible. The methods of extraction employed
differ so widely in different tissues that it is hard to make any general
statements. It is sometimes advisable to extract the ground or finely-cut
tissue directly with 10 per cent. sodium chloride solution so as to bring
everything possible into solution; and sometimes it is better to dry the
tissue and after removing the lipin by some method which does not
coagulate the protein, to extract then with salt solution and fraction
the extract. In a few cases: the proteins can only be separated by the
use of alkali, as in the nucled-proteins; and in other cases, when strongly
basic proteins are present, acids must be used (protamines and
histones).

Probably the best general method for obtaining proteins in a least
modified form from animal tissues is the following: The organ or tissue,
of which it is desired to examine the proteins, is frozen while still living.
It can be frozen either by liquid carbon dioxide or liquid air or by any
other method which chills it at once to a very low temperature so that
all chemical change is at once inhibited and the development of an acid
reaction prevented. It is then quickly shaved into a snow in a Kossel
knife machine, which is a large microtome, the snow being kept below
the freezing point. The cooled snowy mass of the organ is placed in
vacuum desiccators over some good drying agent. Aluminum oxide is
one of the best, but any other non-volatile, water-absorbing medium may
be used ; and the desiccator is evacuated to a very high vacuum, .01-.001
mm. of Hg., by a good air pump. In the vacuum all conduction of heat
is prevented and the organ dries while still frozen. When thoroughly
dry the ground mass is fairly stable. It may now be extracted by a good,
dry petroleum ether. It is better to use petroleum ether rather than
ether for this purpose, since this consists chiefly of the light, saturated
hydrocarbons like pentane and these are less reactive than ether and the
chlorine substitution products of the hydrocarbons such as chloroform,
which are apt to contain impurities and reactive decomposition products.
By this extraction nearly all the phospholipins, cholesterol and fat are
taken out. The last portions of phosphatides can be removed only by
repeated boilings with absolute alcohol, a process which coagulates the
protein and hence cannot be used if it is desired to keep the proteins
soluble. After the extraction with petroleum ether the protein residue
is dried at room temperature and then finally ground in a good mill.
It is best now to sieve it through a fine wire sieve to remove fibers
of connective tissue, blood vessels, etc., if an animal organ has been
used. By this sifting process the sample is made as homogeneous as
possible. ae
THE PROTEINS 109

The further treatment of the sample will depend on what substances
are desired. One method, however, is to extract those proteins which
are soluble in dilute, 10 per cent. sodium chloride. A considerable part
of the proteins of many organs goes into solution by this process, but gen-
erally much remains undissolved. This mixture may be separated into
several fractions, for example, by half saturating it with ammonium
sulphate, or by saturating it with magnesium sulphate, a portion of the
proteins known as globulins are thrown out and may be removed by fil-
tration, redissolved in weak salt solution and reprecipitated. In some
such way as this, the exact method which it is best to follow varying
with the different organs, the various proteins making up the protein
residue of cells may be examined. It may be said that in all cells thus
far examined, a number of different bodies may thus be isolated from
each kind of living matter and the character of the proteins thus iso-
lated is peculiar to the kind of cells from which they are obtained.
Thus one finds different proteins in the liver and spleen, or in the
_ livers of different species of animals. The protein mass of any one
cell is, therefore, made up of a group of bodies, which may be sepa-
rated one from another by their solubilities in water, salt or dilute
alcohol.

Composition.—If a little of this protein mass after extraction with
alcohol and ether be mixed with soda lime in a dry test-tube and heated,
it will be found to yield ammonia, which we may detect by its odor. By
this reaction it is found that the proteins contain nitrogen. If some be
heated by itself it chars to a black mass, showing that it contains carbon
and is organic. If'the combustion is carried out in a dry tube, moisture
will condense on the walls of the test-tube, showing that the protein
contains hydrogen; if some of the mass is fused with sodium carbonate
or hydrate, or even suspended in dilute sodium hydrate and thoroughly
boiled, and to the solution lead acetate is added, it will be found that a
black precipitate of lead sulphide will be formed, proving that the pro-
teins contain unoxidized sulphur; and, finally, if another portion be
fused with sodium carbonate and potassium nitrate, the fusion mass
dissolved in nitric acid, and ammonium molybdate added and warmed,
a yellow precipitate of phosphomolybdic acid proves the presence of
phosphorus.

A quantitative estimation of the amounts of the elements in different
proteins has shown some variation, but the typical protein substances
contain, of carbon about 50 per cent.; nitrogen, 16 per cent.; hydrogen,
7 per cent.; oxygen, 22 per cent.; sulphur, 0-3 per cent.; phosphorus,
0-4 per cent.
110 PHYSIOLOGICAL CHEMISTRY

CoMPosITION ox VARIOUS PLANT AND ANIMAL PROTEINS.
c H N Ss oO

,

Amandin, almond .......... 51.30 6.90 18.90 0.48 22.47

Corylin, hazel-nut .......... 50.72 6.86 19.03 0.83 22.56

Excelsin, Brazil-nut ........ 52.23 6.95 18.26 1.09 21.47

Edestin, hemp-seed .......... 51.36 7.01 18.65 0.88 22.10

Globulin, cotton-seed ....... 51.71 6.86 18.30 0.62 22.51

Vignin, cow-pea ............ 52.64 6.95 17.25 0.42 22.74

Glycinin, soy-bean .......... 52.01 6.89 17.47 0.71 22.92

‘Legumin, lentil, vetch ....... 51.72 6.95 18.04 0.39 22.90

Phaseolin, kidney-bean ..... 52.57 6.97 15.84 0.383 24.29

(Conglutin, blue lupine ...... 51.13 6.86 18.11 0.32 23.10

Vicilin, TORCH) coaciem-dvadiguianenter 52.29 7.03 17.11 0.17 23.40

‘Legumelin, lentil ........... 53.31 6.71 16.08 0.97 22.93

Gliadin, wheat, rye ........ 52.72 6.86 17.66 1.03 21.73

Glutenin; wheat ............ 52.34 6.83 17.49 1.08 22.26

Globulin, wheat ............ 51.03 6.85 18.30 0.69 23.13

Hordein, barley ............ 54.29 6.80 17.20 0.85 20.86

Animal proteins:

COMA BEN aisles auied teat are 50.75 6.47 17.86 Hoffmeister
Gelatin (commercial) ...... 49.388 6.80 17.97 0.7 25.13 Chittenden
‘Gelatin, ligaments .......... 50.49 6.71 17.90 0.57 24.33 Richards and Gies
Elastin, yellow elastic ....... 53.95 7.03 16.67 0.38 21.97 Schwarz

-Mucin, submaxillary ........ 48.84 6.80 12.32 0.84 31.20 Hammarsten
Mucoid, tendon ............. 48.76 6.53 11.75 2.33 30.63 Hammarsten
Serum globulin ............. 52.71 7.01 15.85 1.11 23.32 Chittenden andGies
Serum albumin ............. 53.08 7.10 15.93 1.90 21.96 Michel

Fibrin: sopessonceereen areas 52.68 6.83 16.91 1.10 22.48 Hammarsten
Hemoglobin, ox ............. 54.66 7.25 17.70 0.45 19.54 Fe—0.4 Hufner
Casein, cow ............000- 53.00 7.00 15.70 0.8 2265 P—0.85
Nucleo-histone, thymus ...... 43.69 5.60 16.87 0.47 P= 5.23 i
Nucleo-protamine, herring ....41.20 5.75 21.06 0.00 25.99 P=—6.07
‘Clupein, herring ............ 47.93 7.59 31.68 0. 00 12.78

‘The composition of the plant proteins of this table have been taken from
Osborne’s work on the plant proteins.

a ‘Properties.—A further study of these substances shows that they
form colloidal solutions. If they are dissolved in water or dilute salt
‘solution and the solution is placed in a bag of parchment paper and the
bag, | is placed. in the solvent, the proteins do not pass outside the bag.
‘They cannot pass through parchment, or collodion, or animal membranes
by diffusion. Dissolved substances which do not pass through mem-
_branes of this kind were called by the English physicist, Graham, col-
‘loids,, or glue-like bodies, because glue, which is also a protein, cannot
‘diffuse. Most proteins, therefore, are colloids and we may infer from
‘the fact that they are colloids and so non-diffusible that their molecules
in aqueous solvents are large. Another property they possess, also
indicating a large molecular size when in solution at any rate, is the

opalescence of many of their solutions. Moreover they are generally
THE PROTEINS 111

amorphous, although a few, like hemoglobin, crystallize easily and others
can be made to crystallize with some difficulty.

Another property of nearly all proteins is that when they are treated
with a mixture of the digestive juices of the intestine and pancreas of
mammals, or when they are heated for some time with acids, they are
decomposed into a mixture of substances called amino-acids which. erys-
tallize. In other words proteins are digestible by certain enzymes and on
digestion yield amino-acids.

They have besides certain powers of developing colored products
when treated with various reagents, which are discussed farther on;
and they may be precipitated by the salts of heavy metals and they
form insoluble compounds with many acids such as tannic or picric or
phosphotungstic. These reactions are used for their easy detection, or
even their quantitative determination. And, finally, they will neutralize
in a marked degree the causticity of acids, and to some extent that of
alkalies, showing, in this way, that they are amphoteric bodies, being
both bases and acids. These various properties. served to define the
proteins long before we had any definite information of their chemical
composition, and we may recapitulate them here as a definition of the
proteins :

Definition.—The proteins are organic substances found in nature
in living matter, or associated with it, and always produced by it. They
consist of carbon, hydrogen, nitrogen, oxygen, generally, but not always,
some sulphur and sometimes they contain phosphorus. The proportions
of these constituents are approximately: C, 50 per cent.; H, 7 per cent.;
N, 16 per cent.; O, 25 per cent.; P, 0-3 per cent.; S, 0-3 per cent. Their
molecules are large, at least in aqueous solution, so that they are colloidal
and do not pass through parchment paper; they give certain color and
precipitation reactions, being precipitated by alkaloidal precipitating
agents; chemically they are both bases and acids uniting with acids
te diminish their acidity, and with bases to diminish their causticity;
they are digestible by various enzymes and are broken up by acids when
in solution and yield, when thus digested or. decomposed, a mixture of
crystalline, simple substances known as amino-acids and most, if not all,
of these are a@-amino acids.

All of these properties of the proteins are explained, or accounted
for, by the hypothesis that the protein molecule is composed of a series
of amino-acids united through their carboxyl and amino groups. This
hypothesis was first well grounded by Kossel by his analytical studies
‘of the simplest proteins, the protamines; and confirmed by Curtius and
Emil Fischer, who synthesized bodies having these same. properties by
the condensation of amino-acids in the manner. pupposed by the hypothe-
sis of Kossel.
112 PHYSIOLOGICAL CHEMISTRY

Classification.—By the means which have just been discussed a great
variety of protein substances, all having certain properties in common
in virtue of which they have been called proteins, have been isolated
from living cells, tissues and secretions, and separated one from another
by their solubilities and chemical properties. While the chemical com-
position of none of these substances is-as yet accurately known, it has
been found necessary and convenient to divide them into classes, the basis
of classification being chemical so far as possible, and where a chemical
classification is not possible, solubility has been made the basis. Two
such classifications have been proposed, one by the English Society of
Physiologists, the other by the American Society of Biochemists. The
two classifications are similar in their principal features.

American classification.—Proteins are defined as nitrogenous, or-
ganic substances consisting wholly, or in part, of amino-acids united by
their carboxyl and amino groups. They are divided into three main
classes :

1. Simple proteins.

2. Compound or conjugate proteins.

8. Derived proteins.
The first two classes are natural proteins; the last includes the artificial
and proteins modified by reagents.

I. THE SIMPLE PROTEINS.—These are naturally occurring
proteins which on being treated with enzymes or acids break up only
into a-amino acids or their derivatives. They differ from the conjugate
proteins in that the latter break up not only into amino-acids, but also
into other non-protein substances. The simple proteis are separated
into the following groups by their solubilities and other properties:

x <A. Albumins. Simple proteins, coagulable by heat, soluble in
water and dilute salt solutions. Ovalbumin, serum albumin.

x 8B. Globulins. Simple proteins, heat coagulable, insoluble in water,
but soluble in dilute solution of salts of strong bases and acids. Serum
globulin, edestin.

C. Glutelins. Simple proteins, heat coagulable, insoluble in water
or dilute salt, but soluble in very dilute acids or alkalis. Glutenin.

D. Prolamines. Simple proteins, insoluble in water, soluble in 80
per cent. alcohol. Gliadin, hordein, zein. Found in grains.

E. Albuminoids. Simple proteins, insoluble in dilute acid, alkali,
water or salt solutions. Elastin, keratin, collagen.

F. Histones. Simple proteins, not coagulable by heat, soluble in
water and in dilute acid; strongly basic, and insoluble in ammonia.
Histone from birds’ corpuscles and thymus.

G. Protamines. Simple proteins, strongly basic, non-coagulable by
heat, soluble in ammonia, and yielding large amounts of diamino-acids
THE PROTEINS 113

on decomposition. Sturin, salmin, clupein, ete. Found in ripe sperm
of fishes.

Il. CONJUGATED PROTEINS.—These are compounds of sim-
ple proteins with some other non-protein group. The other group is
generally acid in nature. They are subdivided into the following classes:

A. Chromoproteins." The prosthetic group (Gr. prosthesos, addi-
tional) is colored. It may be hematin as in hemoglobin, or the colored
radicles of phycoerythrin or phyecocyan. Hemoglobin, hemocyanin,
phycoerythrin, phycocyan.

B. Glyco- or glucoproteins. The prosthetic group contains a car-
bohydrate radicle. In mucin and cartilage it may be chondroitie acid.
Mucin, ichthulin, mucoids.

C. Phosphoproteins. Proteins of the cytoplasm. The prosthetic
group is not known, but it contains phosphoric acid, but not nucleic acid
or a phospholipin. Casein, vitellin.

D. Nucleoproteins. Proteins of the nucleus. The chromatin. The
prosthetic group is nucleic acid. Nuclein, nucleohistone.

E. Lecithoproteins. Found in the cytoplasm and limiting mem-
brane. Prosthetic group is lecithin or a phospholipin. No lecithoprotein
has as yet been isolated. They probably exist.

F. Lipoproteins.? Existence is still doubtful. The prosthetic
group is a higher fatty acid.

III. DERIVED PROTEINS.—This group is an artificial one, but
it includes all the various decomposition products of the naturally occur-
ring proteins which are produced by the action of reagents, or enzymes,
or physical agents such as heat; and also artificially synthesized pro-
teins. It is divided into various groups by ela biey. and also somewhat
according to the degree of decomposition.

A. PRIMARY PROTEIN DERIVATIVES.

‘~ a. Proteans. Derived proteins. The first products of the action of
acids, enzymes or water. Insoluble in water. Hdestan, myosan.

b. Metaproteins. The further action of acids and alkalies produces
metaproteins. These are soluble in weak acids or alkalies but insoluble
in neutral solutions. Acid albumin (acid metaprotein) ; alkali albumin.

e. Coagulated proteins. Insoluble protein products produced by
the action of heat or alcohol. _ .

B. SECONDARY PROTEIN DERIVATIVES.

Xa. Proteoses. Hydrolytic decomposition products of proteins. Sol-
1The subdivision here called chromoproteins is called “ Hemoglobins” in the
usual American classification. The word Chromoprotein has so many points of
superiority over that recommended by the Society that it has been adopted here. It
was adopted by the English classification.

2 Artificial lipoproteins are easily made. This subdivision has been added by the

author to the classification adopted by the Society.
114 PHYSIOLOGICAL CHEMISTRY

‘uble:in water, not coagulable by heat, precipitated by saturating their

solutions with ammonium sulphate.

‘Yb, Peptones. Hydrolytic decomposition products of proteins; solu-
‘ble ‘in water, not coagulable by heat, not precipitated by saturation with
ammonium sulphate; generally diffusible and giving the biurét reaction.
x ¢ Peptides. These are polymers of amino-acids of which the com-
position is known. Many are synthetic. The amino-acids are united
through the amino and carboxyl groups. They may or may not give
the biuret reaction. They are not heat coagulable. They are called
di-, tri-, tetra-, penta-peptides, etc., according as they contain two or sev-
eral amino-acids in the molecule.

ENGLISH CLASSIFICATION.
I. SIMPLE PROTEINS.

Protamines.

Histones.

Globulins.

Albumins.

Glutelins.

Gliadins. (Prolamins.) (Soluble in 80 per cent. alcohol;
insoluble in water.)

7. Sclero-proteins. (Forming the skeletal structure of
animals. )
8. Phosphoproteins. Caseinogen.
II. CONJUGATED PROTEINS,
‘1. Chromoproteins.
2. Nucleoproteins.
3. Glucoproteins.
III HYDROLYZED PROTEINS.
1. Metaproteins.
2. Albumoses or Proteoses.
3. Peptones.
4. Polypeptides.

The definitions adopted of these groups are essentially those already
given in the American classification. It will be noticed that the English
‘classification places the phosphoproteins among the simple proteins.

Decomposition products of the proteins.—Before we take up more in
detail the peculiar characters of the different proteins included in the
protein mass from cells, we may consider, first, those properties which
all such masses show, whatever their origin, since these are the funda-
mental or peculiar properties common to the proteins as a class, and they
were observed before the composition of the proteins was known, or
the reactions explainable. The first of these properties, namely the
chemical composition, has already been referred to. The elements com-

S2.Sh Ie eto

J
THE PROTEINS 115

posing them are among the most abundant on the earth. Besides their
elementary composition, all such protein masses show certain color reac-.
tions which are convenient for their identification; and they all yield
certain crystalline amino-acids when cooked with acid. We may begin
with a study of these decomposition products, since the way followed to
get an insight into the composition of such a large molecule as that of
protein is to break it into smaller pieces in various ways and from
the character of these pieces, which are sufficiently small to permit the
determination of their composition, to imagine how they are united in
the larger molecule; we then put this imagination to the test, as to its
validity, by artificially uniting the pieces in the way they are supposed
to occur and see if the artificial product thus obtained corresponds, in
its properties, with the natural product whose composition was sought.
If it does correspond the structure of the molecule is considered solved.
The main difficulty, or one difficulty, in such studies on chemical
structure, comes from the possibility that the substances isolated in the
decomposition may not have been preformed in the molecule, but may
have arisen secondarily during the reaction; but this difficulty can be
avoided, in part at least, by breaking up the molecule in a variety .of
ways; if the products are the same whatever method is used, it may be
confidently believed the fragments actually pre-existed in the mole-
cule. In the case of the proteins the problem was in this particular
unusually simple. The proteins may be broken up in a variety of ways,
but they always yield the same end products, from which it is certain
that these fragments pre-exist, or at least the greater part of them, in the
protein molecule. The proteins may be broken up by heating them for
several hours at a boiling temperature with 10 per cent. sulphuric acid,
or 30 per cent. hydrochloric acid; or by the action of superheated steam
acting for many days; or by the action of barium hydrate either cold
or heated ; or they may be decomposed at ordinary temperatures in a solu-
tion of nearly neutral reaction by digestive enzymes isolated from animal
or plant tissues. The result is always, in its main features, the same.
If treated in any of these ways the colloidal character of the protein
disappears and it is converted into a mass of simple substances, which
are crystallizable and which can be separated from each other. These
substances are amino-acids. From these experiments the conclusion was
drawn that the amino-acids must pre-exist in the molecule, since it is
very unlikely that in these various conditions of acidity and temperature
always the same products would be formed did they not pre-exist.
Furthermore, by oxidation of the protein by permanganate, or other
oxidizing agents, oxidized products of the amino-acids may be had. . The
conclusion that the amino-acids are preformed in the moiecule is also
strengthened by the character of the decomposition which has taken
116 PHYSIOLOGICAL CHEMISTRY

place. It is found that the total weight of the amino-acids from a given
weight of protein is greater than that of the protein from which they
are derived; the proportion of carbon and nitrogen in the decomposed
mass is less; and that of hydrogen and oxygen is more. This shows
that in the process of decomposition the elements of oxygen and hydrogen
have been added; and further study shows that water has been taken
up and combined in the process of decomposition, and that water alone
will bring the decomposition to pass. From this it is clear that all these
decompositions produced by acids, alkalies or enzymes are in reality
produced by water, they are hence called hydrolyses (Gr. Hyddr, water;
lysis, to loosen) or hydrolytic decompositions. Since this is a mild pro-
cess, no great energy transformations accompanying the decomposition,
it is an additional evidence that the amino-acids pre-exist in the protein
molecule and that they are separated from each other by the entrance of
water.

The amino-acids which have been identified are nearly all a-amino
acids, but it is not impossible that others than @ acids may still be found.
An a@-amino acid is an organic acid in which one hydrogen of the a
carbon atom, that is the carbon atom just behind the carboxyl, is substi-
tuted by an amino group. An amino group is NH,.

The following amino-acids have been isolated from the cleavage of
simple proteins: glycocoll, alanine, valine, caprine, or glycoleucine, leu-
cine, iso-leucine, aspartic acid, glutamic acid, tyrosine, pheny! alanine,
tryptophane, histidine, arginine, lysine, proline, cystine, oxyproline,
diamino-acetic acid, dioxy-diaminosuberic acid, C,H,,N,O,, diamino-
trioxydodecanoie acid, C,,H,,N,0,, and a@-amino-&-hydroxy glutaric
acid, C,H,NO,. The last three were found in caseine. The composition
of these acids is shown in the following structural formulas. A
f-alanine, that is &-amino-propionic acid has been isolated from
carnosine, a di-peptide found in muscle.

Structure of amino-acids isolated from simple proteins.

I. Aliphatic, mono-amino, mono-carboxylic acids.

H H CH; CH

H H
nna, ntn new uta aan
o= b_ox n—O_NE, ut HCH u—¢_NE,
Oo= bon n—¢_NH, n—¢-H o=¢-on
o=b_on H—G_NH,

|
O = C—OH

4. Glycocoll = ¥ Alanine —_———- ———— Valine
‘ a-aminoacetic (a-amino (a-amino (a-amino (Iso-propy!
acid) propionic butyric valerianic a@-amino acetic
acid) acid) acid) * acid)

C,H.NO,. C,H_NO.. C HNO. OH, ,NO,. C,H, ,NO,. #
THE PROTEINS Lil
H CH, CH CH H H
| a e
H—C—H ¢--H Hei cH, H—C—OH H—C—sn
| i bees
n—d_H H--C—H C—H H—C—NH, u-4 x H,
| | |
n—b_H u—d_nu, H—C—NH, O = C—0OH 0 =C—OH
| |
nbn 0 = C—OH 0 = ©—OH
|
H—C—NH,
|
0 = C—OH . ,
Glyeolencine *Kyeucirie Tso-leucine Serine Y Cysteine
Caprine (a-iso butyl (ethyl, methyl (B-livdroxy (B'thio,
(a-amino normal -amino acetic a-amino propionic a-amino a-amino
caproic acid) acid) acid) propionic propionic
: acid) acid)
0,4, ,NO,,. C,H, ,NO,. O,H, ,NO,. C,H_NO,. C,H, NSO,.
H za» aE
| of. |
H—C—S—S—C—H
t |
NH,—C—H H—C—NH,
| Ie a
0--C—OH 0 =C—OH
Y Cystine.
C,H, N 25,0:
II. Mono-amino, di-carbozxylic acids. _ III. 1so-cyclic amino-acids.
O = C—OH O=C+OH i C—UH a jn
ete | ON o a:
H—C—H H—C—H HG - Cc iC Nou, othe
{ \| ff
H—C--NH H—C—H HC CH HC CH ,
ae 5 7 W nwa
O—C—OH H—C—NH, C ‘
{ | |
O—C—OH H—C—H H—C—H
po —
H—C—NH, Ht NE,
ie
/ * Q—C—OH o= bon
Aspartic acid X Glutamic acid 'yrosine Phenyl alanine-

(a-amino succinic

acid)

C,H,NO,.

oO a

(a-amino glitaric (8-para-hydroxyphenyl. (8- phenyl]. a-amino

acid ) a-amino Hae propionic acid)
aci
CH »NO ° C,H, ,NO., C,H 4 1NG,.

9

  

Fig. 11.—Crystals of cystine.

 

Fie. 12, EGoveai of iwtoema
118 PHYSIOLOGICAL

IV. Hetero-cyclic amino-acids.
Cal H NH,O
Y
|
uc” ‘co — cdg on
|

ae ¢ cH
YOO
X Tryptophane

(a-amino, f-indole propionic acid)
C,H NO...

a a eS

H—C—NH,

Oss bon
Histidine
(a-amino. B-imidazole
prepionie acid)
¢ SH,

CHEMISTRY

Proline
(a-pyrrolidine carboxylic acid)
C,H,NO..

H, 1 — CHOH
I

a, Vo

Oxy-proline

(The position of the hydroxyl is
uncertain )
C,H,NO..

V. Diamino-mono carboxylic acids.

NH
| 2
C=NH

ta
n—é_H

nH

|
H—C—H

peat

0 = (on
X% Arginine
(e-amino, é-guanidine
valerianic acid)
C.H_N,O,.

6 14 4-2"

giles
rt
aie
n_}_wH,
c= bon

_ Lysine
a,e di-amino,
eaproic acid)
C.H NO.

a 22

Important properties of the amino-acids.—l. Solubility. Com-
pounds with acids and bases. The amino-acids are nearly all readily
soluble in water, but tyrosine is sparingly soluble in cold, but more
soluble in hot water, while cystine is soluble with difficulty in both hot
and cold water. Cysteine, on the other hand, is very soluble. They are
all readily soluble in dilute acids and alkalies except cystine, which does
THE PROTEINS 119

not dissolve easily in dilute ammonia. Leucine, while it is quite soluble
in cold water, redissolves very slowly. The amino-acids are insoluble in
ether. The reaction of all the mono-carboxylic-mono-amino acids is
amphoteric to litmus; the diamino acids and histidine and arginine react

 

Fig. 13.—Crystals of histidine dichloride (Schreiner and Shorey),

alkaline in solution and absorb CO, from the air; whereas the mono-
amino, dicarboxylic acids (aspartic, glutamic) are acid in reaction. Pos-
sessing both carboxyl and amino groups, they unite both with acids and
bases and behave thus both as bases and acids. They form two series
of salts. As bases they react like substituted ammonias to form hydro-
chlorides with hydrochloric acid. The salts thus formed ionize into the
amino-acid as the cation and chlorine as the anion. As acids they
unite with bases, such as sodium hydrate, to form the sodium salt of the
amino-acid, which on ionizing yields sodium as the cation and the
amino-acid as the anion. These reactions may be written as follows:

H : H : a
| eee .
H_¢_H H—C—H /H : H—C—H /H
| | <2 | | fae
H—C—NH, + Hcl ~*~ H—C—N—H aa H—C—N—H + Cl
: } ol —e
O—C—OH ; O—C—OH . 0O=—C—OH
Alanine: Alanine hydrochloride. Alanine Chlorine
: cation. anion.

The positive charge, it will be noticed, is on the nitrogen atom of the
amino group.
120 PHYSIOLOGICAL CHEMISTRY

With bases the reaction is as follows:

H H H
én # nd ud

| | \ +
H—O—NH, + NaOH—~ ae Si ot H,0 = H—C—NH, + Na
O—H 0—C—O—Na, oto
Alanine. Sodium alanate. Alanine Sodium
anion. cation.

The negative charge is on the oxygen atom when the amino-acid is present
as an anion.

As they are both weak acids and weak bases, both of their salts
undergo hydrolytic dissociation, so that the sodium salts are alkaline
in reaction, due to sodium hydrate; and the hydrochlorides are acid,
there being always present some free hydrochloric acid.

H oO HO
Li LI
CH 3 C—C—OH + H,0 = CH 3s -C—C—0H + HCl
a |
NH,HCl abe, H,0
Alanine hydrochloride. Alvnine: fies base.
H O H O

cu —dtl_o wa + H,0 2 CH —¢ loa + NaOH

tn An

Sodium alanate. Alanine.

This property of the amino-acids of uniting both with bases and acids
is of very great importance in determining the behavior of the proteins,
which act similarly.

2. Union with salts of metals.

By their amino groups the acids will unite, also, with such salts as
mercuric chloride or nitrate, silver nitrate, platinum chloride and cupric
chloride to form double salts, and some of these compounds are crystal-
line. All of these metals come below hydrogen in the order of their
solution tensions, that is in the electromotive series of the metals. The
reaction with cupric chloride may be written as follows:

CH,—CH,—CH.NH » COOH + CuCl .— CH,—CH,—CH.NH,CuCl » COOH.

Advantage is taken of this power of the amino-acids to combine with
copper and mercuric salts to precipitate them from their solutions. Mer-
curic acetate in the presence of carbonates is one of the best reagents to
use for this purpose, although the precipitation is probably not quanti-
tative and many other compounds than the amino-acids will precipitate
THE PROTEINS 121

with this reagent. One makes the solution weakly alkaline by the addi-
tion of sodium carbonate and then adds alternately a little of a 25 per
cent. solution of mercuric acetate, and a few drops of sd@im carbonate
to keep the reaction alkaline, as long as a white precipitate is obtained
and then sufficient of the reagents so that on stirring the precipitate has
a faint yellowish-red color. Then 5-8 volumes of 98 per cent. alcohol are
added.

The amino-acids also have the property of forming compounds with
ordinary salts such as sodium chloride.

 
   
  
 

3. Condensation of aldehydes with the amino group.

The amino groups will also condense with aldehydes, particularly
with formaldehyde, with the elimination of water according .to the
reaction :

HN 0 H CH,=N oO

ba | Lt
CH,—C-C_OH + H-C=0 —- CH,—C—C—0H+H,0

|
H rt
Alanine. Formaldehyde. Methylene alanine.
CH,=N 0 H—N—CH,

| II |
CH,—C—C—OH + 2H—~- CH,—C—C—OH
| |
H tO
‘Methylene alanine. Methyl] alanine.

By this reaction the methylene substitution products are formed and
these by reduction go over readily into the methyl-amino derivatives as
shown above. This substitution of methylene for the positive element
hydrogen in the amino group reduces the basicity of the amino group
so that the acid character of the carboxyl comes clearly into evidence,
and these substituted acids may now be titrated with sodium hydrate,
using phenol-phthalein as indicator. This reaction is extremely impor-
tant, partly because such methyl substitution products are quite com-
mon in animals and plants and they are probably produced in this way,
and partly because this reaction is the basis of the Sorensen method,
which is one of the best methods for determining in a simple way the
amount of amino-acids in a mixture. See p. 982. Other aldehydes do
not react so readily as formaldehyde with the amino group.

4. The carbamino reaction of amino-acids.

Another very interesting and important reaction of the amino-acids
is their union with calcium and carbonic acid to form carbamino com-
pounds, a reaction discovered and studied by Siegfried and a reaction
of considerable biological importance, since it occurs in the living organ-
ism. When ammonia and carbonic acid’ unite, two compounds may be
122 PHYSIOLOGICAL CHEMISTRY

formed, namely, ammonium carbonate, (NH,),CO,, and ammonium car-
bamate, the ammonium salt of carbamic acid, NH,OCO.NH,.
‘O 0
lI II
H—O—C—O—H + 2NH, —— NH,—C—O—NH aot H,0
Carbonic acid. Ammonia. Ammonium carbamate.
The esters of carbamic acid are quite important. They are called ure-
thanes. The ethyl ester, ordinary urethane, is a useful hypnotic.
1
NH to -cH—cx i
Urethane.
The carbamino reaction of the amino-acids is similar to this union of
ammonia and CO, to make carbamic acid.
Alanine, for example, unites with carbon dioxide in the presence of a
lime salt to form the compound
; CH,—CH—NH—C =0
| |
0 = C—O0—Ca—O

This reaction is of considerable importance in getting an idea of the
composition of mixtures of amino-acids and in studying the course of
the hydrolytic decomposition of the proteins. One determines the nitro-
gen and, by boiling the filtered solution containing the carbamino com-
pound, the amount of calcium carbonate precipitated. There is thus
obtained a relation between the number of nitrogen atoms and the mole-
cules of carbon dioxide which have been in union. The result is expressed
as the quotient CO,/N. In the case of monoamino acids this quotient will
be one. ‘Such a quotient will mean that all the amino-acids in the mixture
are monoamino acids and all.the amino groups are free, since the reaction:

only occurs between free.amino groups and free carboxyl groups. If
_diamino acids are present, or if the-amino-acids are in part combined
in peptide unions, the quotient will be less than one, since the amount of
nitrogen is greater than the number of free amino groups. It is possible
by studying this quotient to follow the course of the digestion of the
-proteins and determine when the reaction is complete. There are, how-
-ever, easier methods which will be shown later.

5. Deaminization by oxidation.

The amino group is detached only with. great difficulty in acid hy-
-drolysis, or by the action of alkalies, One would think it might easily be
replaced by hydroxyl to form the hydroxy acid, but this is not the case.
If it were, it would be impossible, to, isolate. the amino- -acids, from the
proteins by acid hydrolysis. The amino-acids are most stable in the form
of their acid salts, Most of them.are. quite stable, too, in alkaline solv-
THE PROTEINS 123

tion, but cystine and cysteine will lose their sulphur to a large extent
in alkaline solution and arginine decomposes into ornititiime and urea.
The fact that the amino nitrogen is so firmly attached| jhe amino-
acids so stable in acid solution enables the conclusion tome@mdrawn that
the ammonia, which always appears in small amounts when a protein is
hydrolyzed in acid solution, cannot have come from the amino acids, but
must have had some other linking in the protein molecule. As a matter
of fact it represents acid amide nitrogen and not amino-acid nitrogen.
It was in a free carboxyl group. It is called amide nitrogen. Its amount
is variable, as we shall see. But while the amino group is not readily
detached by a simple hydrolysis, it can be readily removed by an oxida-
tion. By various oxidizing agents such as hydrogen peroxide, or perman-
ganate, the amino group is displaced and the corresponding ketonic acid
is formed. This reaction is reversible. The reaction may be written as
follows:

  
  
 
 

CH, CH,

| ]
H—C—NH,+0 == C=O + NH,

o—¢_ox o = dou

Alanine. Pyruvic acid.

The reaction in the left direction goes on only in the presence of reducing

agents. Both these reactions are of very great importance, since they

occur in the metabolism of amino-acids in living matter and show the

way in which amino-acids may form from the decomposition products

of the sugars and ammonia; and how the proteins may be converted,

with the loss of ammonia, into carbohydrates. The ammonia thus formed

in the body by this oxidation of the amino-acids has a great many func-
- tions which are discussed on page 248. By further oxidation the ammonia

thus set free may be oxidized to nitrogen gas. This may be done by

bromine, or chlorine, or nitrous acid in the following reaction:

NH, +HO.NO —~ 2H,0 +N,
Nitrous acid.

This last reaction is used in determining the quantity of amino-acids or
the amino nitrogen of a protein in the van Slyke method. The nitrogen
gas evolved by treatment of the protein or its hydrolyzed products by
nitrous acid is collected and measured and the number of amino groups in
a solution of the acids is thus determined with very great ease. The same
method, using bromine in place of nitrous acid, is employed clinically
for the determination of urea and some other forms of nitrogen in the
urine. Although the amino group is replaced by oxygen with ease, it
is not replaceable with hydrogen. That is, if a protein is hydrolyzed with
hydrochloric acid and tin, so that nascent hydrogen is set free, the hydro-
124 PHYSIOLOGICAL CHEMISTRY

gen does not displace the amino group to form the fatty acid. In fact,
tin is not i uently added to reduce the decomposition caused by oxi-
dation d hydrolysis.

6. F nu of lactams and piperazine nuclei.

Another4mportant peculiarity of these a acids is the fact that they
form anhydrides, two molecules combining, with the greatest readiness.
Thus it is only necessary to evaporate solutions of leucine or other
amino-acids to produce some condensation to imides, such as leucinimide;
diketopiperazine nuclei are thus formed:

  
  
   
  
 

O
I

(CH ao= CH—CH,—CH.NH—C CH,—CH,

| |
0 = C—HN—CH—CH,—CH =(CH,), ; a Na
Z
‘ou,
Leucinimide (Diisobutyl-diketopiperazine) . Piperazine.

It is considered probable that heterocyclic rings of this kind occur in
the protein molecule, but their presence has not yet been proved. Such
a condensation may, however. give rise to some of the cyclic amino-acids
from straight chain amino-acids.

When the amino group is in the y or 6 position as compared with the

a B
carboxyl, as it is in glutamic acid, for example, COOH—CH,—-CH,—

CHNH,—COOH, an internal condensation within the molecule occurs
with the greatest ease. These anhydride substances correspond to the
lactones and are called lactams. It is an interesting fact that although
the amino-acids themselves are without special physiological or toxic
action, these lactams are powerful poisons, producing strychnine-like
convulsions. By this lactam formation some of the cyclic amino-acids
may be formed from the straight chain amino-acids.

This may, for example, be the origin of proline. Glutamic acid under-
goes a condensation of this kind to form the lactam, pyrrolidone car-
boxylic acid, which by reduction will yield pyrrolidine carboxylic acid,
or proline.

Oo= C—CH,—CH,—CH.NH,—COOH =e
ou

CH,—CH, CH a

i L +2H, —- +4H,0
o=C H—COOH du, ¢H—cOoR
Yi \x4
Pyrrolidone carboxylic acid. Proline.

7. The taste of amino-acids.
‘The amino-acids being very weak acids and having amino groups
THE PROTEINS 125

which resemble, in their properties, the hydroxyl group the carbo-
hydrates, have also, in many cases, a sweet taste. ll, as its
name implies (Gr. glykys, sweet; kolla, glue; the swe ce from
glue), is very sweet. Alanine is also sweet and so is ¢
leucine. Leucine, however, is tasteless and iso-leucine is

It is not yet known why some substances are sweet and others bitter.
The problem is identical with the general problem of the nature of stimu-
lation and the character of the irritable response of living matter. In
this case it is essentially the question of how the taste buds of the sweet-
perceiving nerves are stimulated.

8. Optical properties of amino-acids.

The amino-acids obtained from proteins by hydrolysis by acids or
enzymes are all except glycine optically active. Most are levo-rotatory.
The @ carbon is always asymmetric, except in glycine. Since the total
rotatory action of any collection of amino-acids obtained by acid
hydrolysis or by digestive enzymes is different from, and generally
greater than, the rotation of the protein from which they come, the
change in the rotatory power of a digesting protein enables us to follow
the rate of digestion.

The amino-acids obtained from the proteins by hydrolysis by alka-
lies, or which have been obtained by acid hydrolysis from protein which
has been treated for a time with dilute alkali, are for the most part
‘inactive. The amino-acids obtained from such alkali-treated proteins:
are as a rule, but not exclusively, composed of equal amounts of the
dextro- and levo-rotatory forms of the acids. Since in the proteins prob-
ably only one of the optically active forms of an amino-acid occurs, as
is shown by the activity of those acids when freed by enzymes or acid
hydrolysis, it follows that by the action of alkali the other optically active
‘form has been produced. This process by which from one optically active
isomer the opposite optically active isomer is produced is called ‘‘ racemi-
zation.’? The name is derived from racemic acid (page 22) which is
composed of equal quantities of the two optical antipodes of tartaric
acid. Now it is found that the amino-acids when free do not easily or
rapidly racemize when treated by dilute alkali. The racemization pro-
duced by the action of dilute alkali on protein must, therefore, ocqur
while the amino-acids are combined in the molecule of the protein.
The way this is produced is very interesting and very valuable infor-
mation about which amino-acids have free carboxyl groups on them
in the protein molecule can be obtained by taking advantage of this
ease of racemization by alkali of the amino-acids which have the carboxyl
combined. The explanation as given by Dakin is as follows:

The amino-acids of the proteins are linked through their amino @pd
earboxyl groups in the following way as illustrated by alanyl-alanine.

   
    
 
126 PHYSIOLOGICAL CHEMISTRY

 
  

Dipeptides like this do not easily undergo racemization; but more com-
plex pol s do.
0H HOH

|
see Ow
‘ |
bu * CH,

Alanyl-alanine. Ketone form.
This is the ketone form. Now by intramolecular rearrangement the
hydrogen of the ~ varbon atom passes over to the oxygen of the ketone
group to make the enol form, thus:

O H H OF

pe es:
|
CH, CH,
Enol peptide form.

The ketone and enol forms are probably in equilibrium with each
other, some enol going back to ketone and some ketone to enol.

By this enol formation the carbon atom of the second alanyl group
which had been asymmetrical now becomes symmetrical. When, now,
the ketone form with its asymmetric carbon is regenerated, the second
acid with the double bond will form equal amounts of the two optical
isomers, since there will be nothing to determine that one rather than
the other isomer shall be formed. It is clear that the alkali must assist in
producing the enol form in the peptide groups and in this way it produces
its racemization. Furthermore, since only those amino-acids which have
their carboxyl groups combined can undergo the enol formation, we may
find out, by studying the optical activity of various amino-acids pro-
duced from protein by acid hydrolysis after the protein had been treated
with dilute alkali, which of the acids have been racemized and which not.
Those which were racemized must have had their carboxyl groups bound ;
those which were not probably had their carboxyls free.

Amounts of various amino-acids in different proteins.—The vari-
ous proteins differ in the amounts of the different amino-acids which
they, yield on hydrolysis. While it is not yet possible to determine many
df the amino-acids quantitatively, yet this is possible for some of them
and approximations may be made for the others. The quantitative deter-
mination of arginine, lysine and histidine in a mixture of amino-acids
can be made. The protein is hydrolyzed by prolonged cooking with 10
per cent. sulphuric acid or concentrated HCl and the bases, after sepa-
ration of the humus materials by filtration and decolorization with char-
cog], are precipitated by phosphotungsfic acid. The further separation
of the three acids is accomplished by the different solubilities of the
silver salts on adding barium hydrate to neutral and alkaline reaction.
The detailed method is given elsewhere, The other amino-acids are
THE PROTEINS 127

separated by Fischer’s method of hydrolyzing with concentrated hydro-

  

chloric acid. On saturating the filtered, concentrated m mixture
with hydrochloric acid gas and allowing to stand in thé h of the
glutamic hydrochloride will usually crystallize out at be sepa-
rated. The filtrate from this acid is mixed with absolute“al@ono!l, repeat-

edly evaporated, dissolved in absolute alcohol and the ethyl Eafe form
by passing in dry hydrochloric acid gas. On standing the hydrochloride
of the ester of glycocoll may crystallize out. The reaction involved is
the formation of the ethyl esters of the amino-acids and is as @pllows:

oe ane H 7
H—C—C—OH + HCl + C,H,OH —~ n—d-c_o-4-d_u +4H,0
| I
H O i U i
Glycoeoll. Alcohol. Glycocoll-ethyl-ester hydrochloride.

The other esters are freed from the hydrochloric acid by careful neu-
tralization; the esters are removed by shaking out in ether and they
are separated into several fractions by fractional distillation at differ-
ent temperatures in a good vacuum. From these fractions the various
amino-acids may be separated more or less completely.. Although the
method of determining the amounts of the various amino-acids are thus
not quantitative,’ the results obtained from different proteins by skilled
hands are comparable and the composition of the amino-acid mixture
obtained by hydrolysis of different proteins may be compared. The ester
method is accompanied by many losses, so that the figures which follow!
representing the amount of amino-acids obtained from different proteins
by this method are, for all except histidine, arginine, lysine, tyrosine and
cystine, to be regarded as approximate and minimum values only. But
as the losses in experienced hands are probably about the same for each
protein, the figures give at least a general idea of the amount of amino-
acids thus obtained and indicate clear-cut and important differences in
the composition of the different proteins.

A new method recently introduced by Dakin which consists in con-
tinuous extraction of the monoamino acids from the hydrolysate by
butyl aleohol leaving quantitatively behind the dicarboxylic and basic
acids, promises to be much superior to any other method. By means of

1 The chief sources of loss in the method are: 1. Incomplete hydrolysis. Man

the peptide linkings are very resistant to acid hydrolysis and many hours (24 or
mort) heating with 25% hydrochloric acid are required before hydrolysis is com-
plete. 2. The formation of humus substances. These are chiefly due to decom-
positions of the carbohydrate group, when such 4 sent, and of tryptophane and
histidine. Proteins which, like zein, contain lite NN e radicals do not form black
humus substances. 3. A loss in estenification. @& =¥ in distillation of the esters.
In distilling off the ether, leucine, wee and glyceesM esters go over in part “ee
generally lost. There is also some decomposition during distillation. 5. Losség#in
separating the amino-acids in a crystalline form. From a known mixture of amino-
acids Osborne and Jones could recover by this method only 66.17%.

 
    
  
 
128 PHYSIOLOGICAL CHEMISTRY
it he was to show the presence of considerable quantities of an
@ hydroxy acid in casein and other proteins.

Whi eins yield amino-acids the relative amounts of the
differen y in each protein. Thus salmin, a protamine found
in the he he salmon sperm, yields arginine to over 85 per cent.
@ its weight; others yield large amounts of leucine, or glycocoll or
glutamic acid; some yield no tyrosine, or cystine, or tryptophane. The
proteins differ from each other chemically, therefore, in the amount of
the diffef@ht amino-acid nuclei they contain.

  
    
    
 

PROTAMINES COMPOSITION.
Per Cent. oF ToTaL NITROGEN PRESENT AS NITHOGEN OF DIFFERENT AMINO ACID8.

 

 

 

  

 

F
pt le 1 |e} 8g {e a !
3s. |A le a 5 2 le
a lesltelBslgees| Eaele (fe Ge] 22 [el be Ee
S\SHSbleSlBS/45]) S/FS,5 | ne lob) BR le) Sa [BB
BSE wos Os| o/SelasiPsl wh size fa a am
S&lso|HMalsolzalEnlas(ss| 8 | wo [se ef |82) 9a 7)
@ mslselaeSsiess clsale |S fee] #8 se] J Je
ts [alae | lS S|& |e |x w a Bo 1é we 22 Es |a3
Bale al SoS alt e531 18] se|ic| 12 |¢8| 28 £3
aslSSis flees |SITE 2it8| 8 /SS [Sel 2 (Ss zh (248
Balgses|5 o|- 5|g5\4 2 |e& Si/Jeles| £3 ($3) Be ies
& |Pos</sals" sam Sien) | aa G0] ge (Sl So [Eo
oye |8 |s |B | ae |F las jk | sa oS
ae le ‘ae |O 2
no
a B a B
Histidine............. 0 | 0 /11.8) 0 | 56) 0 | 67 0 0 0 o|;0 ;0 0:
Arginine... 88 0 | 63.5! 88.8] 78.1) 79 5/ 76.3/81.5 |86 3} 86 2% |87.3/88.9 885/67.7| 8.7 28.0/42.3
Lysine. ... 0 84/0 )/0/0;0/0)0 0 0 |;0 30.38 66/11.0
Monoamino 5 9 8) 11.01/10 7] 14.0]11.3] 9.4 4] 7.1 6.7 25.1
Tyrosine ........... 0 | 0 | 0 | 00) 06) o |+ Jo 0 0 0 2.2) 1.5/+
Alanine.... os + it it
Valine... + +)4-
Soa versa ate wera ea t sal ly
roline..... i
Ammonia.........005 0 |0 0 | 0 0;0;}0)0)]0 0 0 |0 0
Leucine ...... J+ .
Tryptophane, 0 ;0 {0 |+ 0/0 0 010 +

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1 Kossel ; Zeit. physiol. Chem., 88, 163 (1913) ; Kossel and Edlbacher : ibid. 88, 186 (1913).
2Kossel and Dakin: Zeit. physiol. Chem., 40, p. 811, 565 (1904), 41, 407 (1904),
‘Compare with A. E Taylor : Jour biol Chem., 3, 389 (1908).
4Kossel: Zeit. physiol. Chem., 44, 347 (1905),
° Kossel: Zeit. physiol. Chem., 69, 188 (1910).
In the accompanying tables it will be noticed that the different pro-
teins have a widely different composition. Thus the proteins of silk have
If their molecule composed of glycocoll and alanine; whereas in salmin
per cent. of the molecule is arginine. The alcohol soluble proteins
like zein contain very large amounts of proline and glutamic acid. Tyro-
sine, tryptophane and other amino-acids are present in some and absent
in others. The amount of ammonia which is obtained by distilling the

weakly alkaline solution; hydrolytic products is seen to be roughly
parallel to the amount mic acid in the molecule, those proteins
lid gliadin having a la ount o@™lutamic acid, have also a large

nt of ammonia, whereas the protamines which lack glutamic acid

in their molecules have no ammonia. This fact means that glutamic and

possibly aspartic acids have an amide group in the free carboxyl. Atten-
&
129

THE PROTEINS

“N SUIPISIY S19 paw : N aust] ZOOL : N GUrOIsIv 8B NT 18107 39°,
Peureyqo “(eter) reg “OT “wey ‘org mop ‘oxdIg UBA pograUT ,,dnoad,, 943 AE oc
“GO6T) TH “wayD “jorskyd -y197 : suoy pus uap/eysepgy er
“G6161) ‘968 'd ‘ET “mop ‘woyoorg *ueUte10.g (6161)
‘gee “d ‘ET “mor ‘waqo01g —oSed 908 UIABd JO HIOA Juana B10 JO “(LI61) eee ‘6
“aaqo “lOlg “dno .4sany puy ui0gs(> “BOK PE “WMaYyD ‘jorshyd “y197 : aUIOW '(ZO6T)
SS ‘wWanamolesyd 4197 +418 .:(S061) &3 ‘PH * WayD ‘[orskyd “jag -uepleysapqy er

“(906T) 898 ‘6B “prar ‘Anvog

    

puujouRags 61) OL “GG “MaTD ‘JoIsdyd eg tsiapy puY suede] ‘AOYOSIT 11
“C6D sig ‘Th yoy  WONBING or
‘(Ol6T) G22 ‘89, yoy BANG gt

(OGL) B22 ‘89 “MAYO ‘Jolsfyd “9107 : a800g +r
“GO6T) Tot ‘HB ‘toIsdqq ‘nop ‘soury souOL pus aUI0qQ30 gr

é ‘ : ; (6067) est ‘BB ‘“prqr ‘souor pus
auzogsO ‘ (6061) $8 “To1sANA nop seUrY ~ yJIOMUOALaT pus sauor ‘aus0gsO er

a. %
ed
e

we

“(bo6T) Lob ‘TH + (FOSD sag ‘tre OF" weYD ‘ToIsdud “Hog + UAE PUL [ex80y 12
(6061) #9 ‘9 “pow ‘dxq ‘lolg “90g ‘a01g seHAIS ULA pat oueae7 ot
(9061) e8@ ‘GT “Tors4yg ‘nop -roMy “WOQNY pus sus0gsO * (GO6T) E2E
9g “o0g ‘woyo ‘omy “NOL ‘s]1IBH PUB UOC : (G06) 6E ‘ge “unoyo ‘jorssyd “3197
Duane F (assoy | (Fost) ‘OF AG06D LB ane ” ‘
“(ost) 268 ‘TG | (G06T) FEF ‘LE “MeTD ‘Joreéud “107 ,, ” a
“CIGD ‘eh ‘TA as ” ” “iuepueyl ” ‘
(2061) Se ‘BG “uIEYD “foredqd “{19Z + YIAOUINOA PUB DOpjByIEpgy »

*(L061) oF ‘OB ” ” » tddep » 9
“(B06T) 298 ‘BS i ” » «$40 Oe
‘Qos 8 ‘6T oy ” » ~ 86

(061) 946 ‘6T oy 7 3 as fai
“QOGT) Ldb + (2061) 86h ‘OG “Lolsdyg “Inof ‘soMyY : dep pus eur0980 1

 

 

 

      

  

 

 

 

 

TS°@ | O'S | 80°0S | 00'6G | LZ'S9 1@'09 88°28} GOTT] e'%9 g9°@9 | 2°98 91°82 | OP'TL| 18°89 |" 1830L
GT 00 ‘sald | ‘s21q | ‘821g "so1g | “so1g 00 801d | “881d | 000 | ‘BeId | ‘BeIg | “891g |" ‘auvydo}dsay,
I9E | ~ Sot | pT | Ove | SGT |] 4 08°T 86°3 GOS ors O'S | Lh | IG | IG °° RuOWMYy
6'9 BOL | SYS | SP 18% | 948 | O40 | GOT 30 | P91 g9o°T 00 86°F 66° 000 | 00°0 | 00°0 | 00°0 *auisd'T
gT Gs vO | OIL O6T | IAT | 8ST | 9% é A ms 61'S 00 69 |. bs &h'0 | 83 | 1970 | 680 ‘ourpijslH
ast) €0 |I8s | 292 | Fg OL | 16 | GSTL) PRHL] 28 | PLT] LV HL| P'S] TAIL SOIT | Sot | 91'S | OIE | 88s soulUlaly
as gp | 00°70 | ST 26 06 | OT 286 | ZL | art | 208 9€ | 80'S sls 00 aot ors Go's. | 29T | OST | GIT *aulsol fy,
é 1200 00 | ¢0 6 é é $60 gL é oor 00 |} JPUn | YepaUn | ¢ é gr'0 é aunedyy
eee tL ao FO | 90 OL | FS é é é é TL 00 $8°0 82 eg°0 é COT é sto | 90°0 veers euldas
898 | 940 | 221s] 8st | 4t | 200 | seo | 81 S6SE] OL 6 | PRES! GBSL| SAL] PECL) FEEL) OO | 2GE9T O8'8T | AI°9¢ dad 86°GP| S28 "plow srw
; pu
“at foco | eh | OT | OL [ee 81'S | 08s | SS | OFS | GB) ase | OFF | OO} OFS | IB | TAT | ION | BGO | seO |"‘pIOR ONaVdsy
OG's | 68'S |88'E | FO | oF eT eT |&0 FG | LOG | S98 | eee 61 | so’s 60°E 00 SL's 13°3 gc'9 | 80'S | Ges | 04% | ouLURE|AUZNg
OPT | PLT 1892 | BS | Fs 40 80 |08 SL | 99'S | FHS | B'S 28 | 99's Oly OIL) soe ¥0'P #06 | ELST) 90° | BB'S [*"°*  OUlOIg
OS'TE | 881%) 16 13 | 06 40 1420 |8P 48°6 | TLOL) Sh | eB L Sat] O28 0S'FT | 00°0 00°8 08'8 ac'6t| 49& | 19'S | 089 aulonay
“""") PTL |S6L Ere ee PN aes = ra 28'l | OS'S | 9L0 | 930 GP | IGT | 61089 ep é 9e'T 88'T | STO | 180 é “* OULIBA
OPE | 899 | SBI 80 Bb | 98s | 00°06 | 86 G20 | 23 | OFT | @6oT OT | 88s 09° | 00°0 80's SUT 64'°6 | SFO | 00% | GET “aulUBTy
GO |G29¢/¢b°0 | Q9T} 000 | 0098 | 6g ee} ST 00°0 | 000 | TsO | 440 | SFO | 09°0 os'€ | 00°0 | 8&0 68°0 | 00°0 | 00°0 | z0°0 | gro *too004 1)
Q |, ° n N °
[es] Bo | 2a] @ <4 < > 2 Dou re} & wm ial re g ie} =
zs] 9 ef) re | ro lse/s08) ,8| Ze] = & | be | Bx = | me] as e | fF Q| 8
Bele [o8| 2 | BE | 2 | TE |gelee:| 25 /S2|oe|/F2/ 22/28) 28| wt | 22) $8 | a | 2 les) eB | E
Be} 5/25) & Sf) 8s] se |SsB28| 25 | os: | 98) 82/2) es) fe) ge | 25) o8 | sh | | lee] ge]
Be) | PF] = [Shi st] scizserjee/ee/ 2) Be) ee) eel ee] er | Fe) BE | PR | B [Seley fF
= ™ ~ = [es el ~ = a
ee th ee eeee ean ha a Pea | oy By ON hy

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

|

“SNIZLOUG SQOIUVA WONd GALVIOS] SCIOY-ONINY JO “LNEO aad

‘NOILISOdHOO, ‘SNITLOUd
130 PHYSIOLOGICAL CHEMISTRY

tion may be directed also to the fact that proteins corresponding in kind
have very similar but not identical compositions. The
tch and the pea illustrate this fact. The protamines are
the prote! ich have the fewest different amino-acids in their mole-
cules; and those acids which are present are chiefly basic acids.

The total amount of the amino-acids found rarely equals: 100 per
cent. of the protein molecule. It is in fact seldom more than twos
thirds of the protein. Salmin alone yields 110 per cent. of amino-acids.
The weight of the amino-acids recovered is greater than the weight of the
salmin hydrolyzed, for the reason that a molecule of water has been
added between each two amino-acids in the process of hydrolysis. Were
the methods of determination accurate all the proteins should show more
‘than a hundred per cent. of the weight of the protein as amino-acids.
The 30-40 per cent. of the protein molecule not accounted for in most
proteins might be due to the losses in an@lysis, or to the presence of other
unknown amino-acids in the decomposition products. It is the opinion
of Osborne, who has particularly studied this question, that the defi-
ciency is chiefly due to the losses in analysis, since from a known amount
of amino-acids he could recover only about 60 per cent. It is probable
also that there are some unknown amino or other acids in the residue.
There are reasons for thinking that some of the sulphur may be in another
form than cystine and probably various hydroxy amino-acids remain
undetected.

The structure of the protein molecule—Since all methods of:
hydrolysis, whether by water, by the mild action of enzymes at body or
room temperature, by acids and alkalies, yield amino-acids, it is safe
to conclude that these nuclei are not secondary products of decomposi-
tion, but that they pre-exist in the protein molecule. That the proteins
are indeed made up of amino-acids linked through the carboxyl group of
one acid and the q-amino group of another is now certain. This result
is largely due to the work of A. Kossel on the composition of the basic
proteins found in the cell nuclei of the sperm of the salmon and sturgeon.
Kossel discovered that the protamin, salmin, a strongly basic protein
which can be separated from the head of the salmon sperm, yielded on
hydrolysis nearly 90 per cent. of its weight as the single amino-acid
arginine; in the case of the sturgeon protamine, sturin, two other amino-
acids were present, namely lysine and histidine. From this and other
considerations he drew the conclusion that the proteins were made up of
amino-acids linked through their amino and carboxyl groups, many of
them at any rate having a protamine-like nucleus to which the different
amino-acids were attached, the number and kind of these amino-acids be-
igg variable in different proteins. This conception allied the proteins to
the scheme of the carbohydrates. In this view proteins corresponded to
the polysaccharides ; the amino-acids to the various monosaccharides ; and

 
  
  
THE PROTEINS 131

Kossel named those amino-acids with six carbon atoms, namely histidine,
lysine and arginine, ‘‘ hexone bases ’’ to bring out this similarity. By
the work of Emil Fischer and Curtius this conception of fovsiason
of proteins was proved to be correct by the synthesis on®the basis of
Kossel’s theory of various bodies of a protein nature.

The amino-acids are linked together in the protein molecule in the
following way through their amino and carboxyl groups. The union of
a molecule of alanine with one of leucine may be pictured as follows:

 

ae =< ye) a 7 ak
H-C_N—H H 6-H H_O_NCH H ‘6-H
| + Ea ho | | I
O = C—OH H—N—C—H 0 = C——_N. C—H
|
O = C—OH 7 o= b_on
Alanine. Iso-Leucine. Alanyl-leucine.

This leaves a free amino group at one end of the chain and a free car-
boxyl at the other, at which other amino groups can be attached. A
series of amino-acids put together in this way to form a polypeptide as
it is called, in this case a decapeptide, is shown on page 132.

The resemblance of this union to that of the polysaccharides is very
close. By looking at the formula of the disaccharide, maltose, on page 57,
it will be seen that the two monosaccharide molecules are attached to
each other through an oxygen atom. The carbons of the different mono-
saccharide groups do not unite directly with each other. In the formula
of a polypeptide just given, the different amino-acids are united through
a nitrogen atom. The carbons of the monopeptides do not unite directly
to make a polypeptide, any more than do the carbons of the monosac-
charides to make a polysaccharide. A further resemblance lies in the
fact that in each case the synthesis involves the loss of a molecule of water
between each two monopeptide groups, or monosaccharide groups. The
main difference apparently lies in the fact that there are a far larger
number of amino-acids used in the synthesis of the proteins, or polypep-
tides, than of monosaccharides to make polysaccharides. No protein has
as yet been discovered which yields only a single amino-acid, although
salmin yielding 88 per cent. of arginine does not come far from it. Inulin,
however, is supposed to yield only levulose when it is hydrolyzed; and
glycogen is supposed to yield only glucose. Many of the other carbo-
hydrates, however, are composed of several different monosaccharides.
No doubt as means of separation of the monosaccharides improve, it will
be found that the polysaccharides contain more kinds of monosaccharides
than is at present believed.

The evidence that the amino-acids in the proteins are linked through
CHEMISTRY

PHYSIOLOGICAL

132

or 6 8 4 9 g , 2 é g
uypos[s—{A[8a —[Sats4[—[ Lutes o— Aurs.1e—| Lurso143—]441edse— | A1os—ujone,—[Auery

‘HN HN HO
H e 4
| = | ON

"HNOH HN n10 On

HOH HOH HO OH
| | WV

°HO HO HOH u6 HOH 0 HO090 HO "HR "HD
H ou HOW HOW HOW HOH HOH HOH on "HD
{
THE PROTEINS 133

the amino and carboxyl groups is the fact that they have been syn-
thesized into protein-like bodies by such union, and the further fact that
the number of the free amino and carboxy! groups in a protein molecule
is very small, showing that both amino and carboxyl groups are
combined.

Synthesis of the proteins.—The synthesis of protein-like substances
from the amino-acids has been accomplished in several ways. 1. By
dehydration. By heating leucine and glycocoll in the presence of phos-
phorus pentoxide Grimaux and later Pickering obtained colloidal bodies
with many of the properties of the proteins. 2. By the condensation of
glycocoll Curtius obtained a base, the biuret base, which is now known
to be triglycyl-glycine ethyl ester. 38. The first systematic attempts at
synthesis which were successful were those of Emil Fischer on the basis
of Kossel’s theory of the nature of the protein molecule, and these
attempts have led to the successful synthesis of a great number of
artificial polypeptides, some having the general nature of the albumoses,
being digestible by trypsin and erepsin and giving the color reactions
of the proteins. The methods used in the synthesis are as follows:

The carboxyl and amino groups are not of themselves sufficiently
reactive to combine rapidly, more rapidly than they dissociate. It is
necessary to make one of them at least more reactive, so that the velocity
of the reaction which is leading to their synthesis is greater than the
velocity of their decomposition by hydrolysis. This greater reactivity
is secured for the carboxyl group by substituting the hydroxyl with
chlorine, to make the acid chloride. This can be done by treatment of
the amino-acid by phosphorus pentachloride. There is thus formed from
alanine, or glycine, the hydrochloride of the acid chloride:

H

|
CH,—C—NH, HCI

|
0=c—Cl
This will now unite with a molecule of an amino-acid, or a polypeptide,
liberating hydrochloric acid thus:

H H
bs |
cH,— NH,HCl CH ‘ CH 3 CNH, HCl CH,
Ta | | |
0=C—Cl + H—N—C—H _— Oo= Poe a ye + HCl
H |
Oo = C—OH é eee
Alanyl chloride Alanine. =

hydrochloride. Alanyl-alanine hydrochloride.

By treating the alanyl-alanine with phosphorus pentachloride it may be
converted into the acid chloride in its turn and it will then unite with
the amino group of some other amino-acid, for example, leucine:
134 PHYSIOLOGICAL CHEMISTRY

 

H H
| |
CH,—C—NH,HC] CH, CH,C,H, CH 3 CNH HCl CH, CH,C,H,
| Ze | |
Oo=C n—4 —H + CH 0 =C——_N——_—C—-H CH
i | H | | | H |
| i J H |
o=C—Cl HN—C—H —~ O= C—N—C—H
| |
OC—OH O = C—OH
Alanyl-alanine chloride. Iso-leucine. Alanyl-alanyl-leucine hydrochloride.

‘Two tri-peptides or even more complex peptides may in this way be
condensed into a hexa- or other poly-peptide.

Another method used by Fischer consisted in adding an amino-acid
to the amino group of the terminal acid of a peptide, using a bromine
substitution product of a fatty acid chloride; and then after union with
the amino group replacing the bromine by an amino group by treating
with ammonia. The process is then repeated. Suppose it is desired to
make an alanyl-leucine. The leucine is treated with brompropionyl
chloride and then the reaction product with ammonia as follows:

H CH, C,H, H CH, C,H,
| SZ | SZ
CH,—C—Br + H 1 CH,—C—Br H CH + HCl
nae | a a
o=C—Cl HN Se 0 = C——_N——C—-H
= ellen 0 = C—OH
Brompropionyl Iso-leucine. Brompropiony]-leucine.
chloride.

2. CH yCHBr—CO—NH—CH (C, H, )—COOH + NH, ——
CH _—CHNH —CO—NH—CH ( C,H,)—COOH + HBr
Brompropiony]-leucine. Alanyl- insti

The process may now be repeated with the alanyl-leucine and either
brompropionylchloride or some other similar compound may be united
to the di-peptide and converted into the amino compound by the action
of ammonia. So a tri-peptide may be made. By the use of these methods
a great number of artificial polypeptides have been made by Fischer,
Abderhalden, Curtius and their co-workers. One of the most complex
of these polypeptides contained 18 amino-acid groups, namely three leu-
cine and 15 glycocoll groups. It was l-leucyl-triglycyl-l-leucyl-triglycyl-
l-leucyloctoglycylglycine. NH,CH(C,H,)CO.(NHCH.CO),.NHCH(C,
H,) CO.(NHCH,CO) ,.NHCH(C,H,)CO.(NHCH,CO) ,NHCH,COOH.

These complex artificial polypeptides have the properties of the de-
rived proteins. They are like aloumoses. They give the biuret and other
reactions of the proteins, which are given by the various amino-acids of
THE PROTEINS 135

which they may have been composed, such as the tyrosine or tryptophane
reactions. They are precipitated by mercuric chloride and phospho-
tungstic acid. And some of them are digestible by trypsin and erepsin.
They are optically active, also, like the natural bodies. One of them
produced an anaphylaxis reaction. It has not yet been possible to
form a protein which is coagulated by heat; nor has any artificial
protein been made which is identical with the naturally occurring pro-
teins. On the other hand, many of the di- and tri-peptides which appear
in the artificial hydrolysis of the naturally occurring proteins have been
synthesized artificially. The final synthesis of the natural proteins is
probably only a question of industry and time.

Other linkings in the molecule.—It must not be supposed that the
NH—CO— grouping is the only method of linking amino-acids in the
protein molecule, although it is undoubtedly the principal one. Another
is certainly by means of the cysteine sulphur. This union is brought
about by oxidation and released again by reduction. This linking may
be of great importance in determining the reactivity of living proto-
plasm, since oxidations and reductions are constantly taking place in it.
Thus if two molecules of cysteine are oxidized, and in neutral or in the
faintest alkaline reaction the oxidation goes spontaneously very rapidly
in the air, they are converted into one molecule of cystine.

The reaction is as follows:

H H H H i
| |

| |
HC—SH HS—C—H HC—S—S—C—H
| |
HtNH, + 0 + HCNH, —~ HONH, HtNH, + H,0

| | |
boon COOH cooH cooH

Cysteine. Cysteine. Cystine.

It is possible, although it has not yet been shown to be the case, that
if two proteins each containing cysteine are oxidized, a more complex
cystine protein would be the result. By reduction this could be broken
up again. There seems to be evidence from certain color reactions with
sodium nitro-prusside, with which cysteine gives a beautiful red color,
that some natural proteins contain cysteine, while others contain cys-
tine. It would seem not impossible that this union might join mole-
cules of protein into more complex groups; and possibly the fibers of
the aster in cell division might be formed in this way. The author
found that these fibers would only form in the sea-urchin egg in the
presence of oxygen and they at once broke up and disappeared when
oxygen was withdrawn from the egg. At any rate we have in the
systeine sulphur one of the most reactive points of the protein mole-
136 PHYSIOLOGICAL CHEMISTRY

cule. Heffter and the writer have particularly tried to bring it into
rclationship with cell processes. The union is as follows:

R—CH,—S—S—CH,—R’

It would seem that the protein with cysteine in the molecule might
change its state of solution when it became cystine, and this might
alter the state of viscidity of the protoplasm, cr possibly even its
affinity for water.

Another linking which is possible is an ester union through thre:
hydroxyl of the serine, or oxyproline, or tyrosine with carboxyl.
Whether such unions exist is stiii unknown. Another linking is that
typefied by guanidine and ornithine in forming arginine. The linking
is of the following kind: NH=C(NH,)—NH—CH,—R. So far as is
known this union occurs only in arginine.

This part of the subject should not be left without reference to
another very suggestive fact. None of the artificial polypeptides are
digestible by pepsin, though many of them digest with trypsin or
erepsin. This matter is discussed on page 404. This fact may mean that
there are other kinds of unions between the polypeptide groups which
gc to make up the protein molecule than unions between the amino and
carboxyl groups as just stated. Pepsin might act on these unions. On
the other hand, it might be that the failure of pepsin to digest the
protamines or the artificial polypeptides was owing to the fact that the
pepsin acts only on certain specific amino-acid junctions and that we
have not yet happened to test these particular junctions with the
enzyme. The fact that during peptic digestion the free amino groups
increase in numbers (page 362) bears out the latter supposition.

A very curious relationship has recently been found by Kossel in
the protamines of the fish sperm and may be mentioned in this connec.
tion. He finds that in these proteins there are always approximately,
or exactly, two molecules of a basic amino-acid like arginine, histidine
or lysine, to each molecule of a mono-amino acid. This fact suggests
that possibly the protamine may be made of a series of tri-peptides
Similar tri-peptides have been isolated by Siegfried in the course of the
slow hydrolysis of various proteins and called by him, kyrins. It has
been suggested by Taylor that the protamine, salmin, may be made up
of these tri-peptides, or protones, united as follows:
| Arginine } Arginine } Arginine Arginine] (Arginine ) f{ Arginine

Serine }—j Serine {_/ Proline | Proline }—4 Proline }_j Valine
Lae, | argihine Arginine | | arginine | | depiatne | | arginine
The first cleavage of the molecule by hydrolysis would consist in the
setting free of the tri-peptides which would then be separately broken
THE PROTEINS ; 137

up. This view, while it is in consonance with many facts, cannot yet be
said to be well grounded.

Number of free amino and carboxyl groups in proteins.—That there
are only a few free amino groups in the protein molecule is shown by a
variety of reactions. Acids, for example, combine with the amino, NH,,
groups, but not with the imino, NH—, groups; or if they unite with
the latter the union is a very weak one and dissociation occurs. The
basicity of the group, NH—, is no doubt reduced by the neighboring
C=O group. At any rate, the acid-combining power of the protein
molecule is generally only two to four molecules of hydrochloric acid
to what we believe to be a single molecule of protein. Thus edestin, a
crystalline protein from hemp seed, forms two series of salts, a mono-
and a di-chloride (Osborne). As digestion takes place and the amino
groups become free, the power of taking up acid greatly increases. Kossel
has shown that the amount of acid taken up by protamine is in direct
relation to the amount of the free amino groups it has. In general, pro-
teins with more lysine and arginine combine with more acid. This indi-
cates that one of the amino groups in each of these acids is uncombined
in the molecule; in other words, that only the a-amino group is bound
in both arginyl and lysyl.

Another proof that there are few free amino groups is the power of
union with formalin. Formaldehyde unites with the free amino groups to
form water and methylene addition products (see page 121). It does not
react with the imino, NH, groups. Now it is found that the amount of
formalin bound or taken up is small in the intact proteins, but undergoes
a steady increase as hydrolysis progresses. Indeed by means of formol
titration the progress of a hydrolysis can be most easily followed. It is
found that the rate of increase of the acid-combining power and of
formalin binding in such a hydrolysis go parallel. Still another method
for the detection of the amount of free amino groups, and from a quan-
titative standpoint perhaps the best, is the method of Van Slyke, which
depends on the fact that nitrous acid reacts with free amino groups
liberating nitrogen gas which can be collected and measured (for reac-
tion see page 123). It is found that the amount of nitrogen deplaceable
from a protein by nitrous acid is a very small fraction (5 to 84) of the
total nitrogen, but that as hydrolysis proceeds and the amino groups be-
come free, the amount steadily increases. All of these methods, then,
prove beyond question that the amino-acids have most of their amino
groups combined and that they are, therefore, probably linked through
the amino groups.

That the carboxyl groups also are in combination in the protein and
few of them free is shown, in the first instance, by the fact that the
power of the protein to combine with alkali increases as the hydrolysis
138 PHYSIOLOGICAL CHEMISTRY

proceeds. It may be shown also by the method of Dakin. By the action
of dilute alkali on protein a decrease in the rotatory power results and
the subsequent acid hydrolysis of the protein thus acted upon yields the
racemic form of nearly all the amino-acids (page 126). There are only
a few amino-acids in such hydrolyses which have not been racemized by
the alkali. Now since the acids having free carboxyl groups do not
racemize, the fact that most of them are racemized by alkali treatment
of the proteins shows that the great majority of the carboxyls must have
been united with something in the molecule.

Since the great majority of both the carboxyl and amino groups of
the protein molecule are combined, it is probable that they have com-
bined with each other.

Molecular weight of the proteins —We may now ask the question
how large is the molecule of protein? How many of these amino-acids
does a molecule have in it? This is a very difficult question toe answer
for the majority of the proteins, but for a few of them it may be
answered with a considerable degree of probability. There is no doubt
that the molecular size of the great majority of the proteins, of all the
natural proteins, is very large. This is shown by the fact that they will
not diffuse through parchment paper. They are colloidal in aqueous
solution. This means that the diameter of their molecules is certainly
more than lu. Even protamine, which is in many ways the simplest of
the proteins, is colloidal. It might be, however, that the proteins were
colloidal in water but not in other solvents. Soap is colloidal in water,
but not in alcohol. The molecular size of the proteins in other solvents
than. water has hardly been investigated. It is possible that in water
several simple protein molecules might aggregate by processes known
as association to form large complexes, just as many simple substances,
such as alcohol or acetic acid, associate to form double or triple mole-
cules. The molecular size of the proteins dissolved, for example, in
formamide, if they will dissolve in it, should be investigated. While
it is possible for the reason just stated that the large molecular size of
the proteins when dissolved in water does not necessarily mean that the
individual molecules of the protein are large, there are other reasons
which make such a conclusion practically inevitable. There are several
different ways in which the molecular weight may be determined both
ky indirect and direct methods. The results obtained by these two
methods are in very good agreement. We will consider the indirect
methods first.

Calculation from the sulphur content. The crystalline form of sev-
eral of the proteins is so distinct and constant that we may assume that
these represent chemical individuals. On repeated precipitations they
do not change their form or composition. Many of these proteins have
THE PROTEINS 139

the sulphur largely in the form of cysteine. Perhaps it is altogether in
that form in some. It is probably present in other forms than cysteine,
perhaps as cystine in others. If there is one molecule of cystine in a
molecule of protein there must be two atoms of sulphur to each protein
molecule. Two atoms of sulphur have a molecular weight of 64. If
there is 1 per cent. of sulphur in the molecule, the molecular weight of
such a protein would be at least 6,400. If there was 0.5 per cent. 8, the
molecular weight would be 12,800. The following computations of the
molecular weights and formulas of various plant and animal proteins
were made by Osborne from the sulphur on the basis that there were two
or more atoms of S to the molecule.

MoLecuLarR WeiGHt, CoMPOSITION AND PossIBLE EMPIRICAL FoRMULAS OF PROTEINS.

 

 

 

 

   

 
  

 

 

Composition Formula a
Ba
5 8 'b0
c|HiN Ss | Fe| P| Oo C|H| N|s|Fe/P] 0 Se
51 30 | 6.90 | 18.90 | 0.429 |.....-]...-5- 22.471 |Amandin.............. 638 |1030) 202 | 2) 209 | 14922
51.72 | 6.95 | 18.04 | 0885 ]......]...06 22.905 | Legumin.. seoeees | 718 [1158] 214 | 2 238 | 16642
55.23 | 7.26 | 16.13 | 0.600 |......|-.---- 20.78 Zein..... 736 |1161| 184] 3) . 208 | 15983
54 29 | 6 20 ze ot Hordein. 675 [1014] 181 | 4 194 | 14880
51.36 | 7 01 Edestin. 4 201 | 14530
52.72 | 6.86 3|Gliadin . 5).. 211 | 15568
5218 | 6.92 Excelsin 5]. 198 | 14738

Animal proteins ;
34.98 | 7.20 GloDin. ....:eseeeeeeee 700 |1098] 184] 2).. 196 | 15274
52.68 | 6.83 BAD TiN ssc diene saaiaioace 645 |1004) 178 | 5 207 | 14708
52.71 | 7.01 .82 |Serglobulin, horse... 628 |1002] 160) 5j.. 209 | 14310
52.93 | 6 90 § |Wibrinogen,............ 679 | 1062] 183) 6].. 207 | 15276
52.75 | 710 Ovalbumin. . ++ | 696 |1125] 175 | 8]. 220 | 15703
52.19 | 7.18 Lactalbumin..... 644 |1064] 166 | 8/.. 214 | 14792
52.99 | 7 01 Seralbumin, horse.. 662 }1051| 171] 9].. 207 | 14989
52 25.) 6.65 Seralbumin, hnman....| 684 |1045] 178 }11) . 225 | 15697:
54.64 | 7.09 5 |Oxyhemoglobin, horse. | 758 |1181} 207] 2) 1 210 | 16655
54.57 | 7.11 Oxyhemoglobin, dog ..| 758 |1185) 195] 3) 1 219 | 16667
53.13 | 7 06 Casein... ...scceessueees 708 |1180| 180| 4]....] 4| 224 | 15982
51.56 | 7.12 Ovovitellin............ 671 |1112| 182] 5)....] 4) 227 | 15628

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

From the foregoing figures it is clear that if the proteins in question
are individuals their molecular weight is certainly high. In the case of
hemoglobin it will be seen that the computation of the molecular weight
on the assumption that there is one molecule of cystine gives the same
result for horse hemoglobin as the assumption of one atom of iron in
the molecule; for dog hemoglobin, however, it is necessary to assume
that there are three sulphur atoms in the molecule, which would mean
one molecule of cystine and one molecule of some other sulphur com-
pound, possibly cysteine. The molecular weight might, of course, be
some multiple of these figures.

Computation of the molecular weight of hemoglobin from the oxygen
compound. That the molecular weight of oxyhemoglobin is approxi-
mately that indicated in the foregoing table is shown, also, by a caleu-
lation of the molecular weight from the number of grams of oxygen or
carbon monoxide taken up by a gram of hemoglobin, assuming that each
140 PHYSIOLOGICAL CHEMISTRY

molecule of hemoglobin combines with one molecule of the gas. The
molecular weight of the carbon-monoxide hemoglobin is given by the
ratio 28:x::a:1, where a is the weight of carbon monoxide combined
in one gram of carbon-monoxide hemoglobin, and x the molecular
weight of the hemoglobin. Hiifner found that one gram of the carbon-
wonoxide hemoglobin contains 1.338 c.c. of CO computed at 0° and 760
mm. pressure, or .0016745 gram. From this the molecular weight of
the carbon-monoxide hemoglobin is computed as 16,669 (.0016745:
1::28:M). This figure agrees almost exactly with that computed from
the sulphur and iron. It is also in agreement with the direct deter-
mination of the molecular weight made from the osmotic pressure. Com-
puting from the heat of formation of one gram of oxyhemoglobin from
hemoglobin and oxygen, Barcroft and Hill found the molecular weight
tu be 15,200.

Direct determination of molecular weight by the osmotic pressure
method. The determination of the molecular weight of proteins cannot
be made by the boiling-point method because most of the proteins coagu-
late or change on boiling. The freezing-point method also is not suffi-
ciently accurate for such large molecules. There are two methods which
may be used: the osmotic-pressure method, and the measurement of the
vapor pressure at lower temperatures than boiling by the method re-
cently introduced by Menzies. The determination of the molecular
weight of hemoglobin has been made by measuring the osmotic pressure
of solutions of known strength of hemoglobin. The only real difficulty
in this method consists in getting perfectly tight membranes which are
truly semipermeable, that is membranes which readily pass the solvent
but not the solute through them. Hiifner and Gansser used the
apparatus in Figure 14. The solution of hemoglobin is brought into
the diffusion shell of Schleicher and Schull which is closed and con-
nected with a mercury manometer. The diffusion shell is then placed
in water and by osmosis the water enters the solution, forcing the
mercury up until the pressure becomes so high that it presses just as
much water out as that which enters. The principle of the method is
that a solution which contains in a liter an amount of the substance equal
in grams to the molecular weight will have a pressure at 0° of 22.41
atmospheres. A half-molecular solution which has only an amount of
substance equal to half a molecular weight has half this pressure, and
so on. It is only necessary then to measure the osmotic pressure of the
hemoglobin at 0° or some other temperature to find what fraction this is
of 22.41 atmospheres or the corresponding osmotic pressure at the tem-
perature employed for the hemoglobin, and divide the weight of hemo-
globin dissolved in one liter of solution by this fraction to get the
molecular weight. The formula is as follows;
THE PROTEINS 141

22.41 (1 4- 0.00366t) 760.¢
Sa ee

{n this formula (1-++0.00366t) is the temperature correction, since the
osmotic pressure increases with the temperature. t is the temperature
at which the determination is made; ¢ is the concentration of the solute
in grams in one liter of solution; and p’ is the osmotic pressure of the

 

 

 

 

 

Fig. 14.—Osmometer for determining the osmotic pressure of oxyhemoglobin solutions
(Hiifner and Gansser). a, diffusion cell containing oxyhemoglobin solution run in through
t, h, and r; b, manometer for measuring osmotic pressure; w, beaker containing water.
Fig. 11. Detail of cock o..

solution if it had not been diluted from the volume v to v’ by the
entrance of water. The correction is of course a small one. It took
several hours for the pressure to reach its maximum and it remained
at this maximum for several hours. Some of the results obtained are
given in the following table:
142 PHYSIOLOGICAL CHEMISTRY

p’=pv’/v M __ Kind of hemoglobin.

e t Pp v v’
mm. Hg. mm. Hg.
62.72 10° 62.7 23.5 23.6 62.97 14,780
10 58.5 68.75 15,840 Horse Hb.
108.0 1 109.0 23.7 109.9 16,790
109.2 1 114.9 115.9 16,110 Ox Hb
216.0 1 198.0 23.9 201.0 18,370 .
216.0 1 224.0 227.8 16,210

The mean value of all the determinations gave for horse hemoglobin
the molecular weight of 15,115 and for ox Hb 16,321.

This direct determination confirms fully the determinations by the
other method and leaves very little doubt that the molecular weight of
hemoglobin is really about 16,693. It should be mentioned, however,
that Weymouth Reid by the osmotic-pressure method got 48,000 and
Roaf 32,000 as the molecular weight of oxyhemoglobin. It is probable
from these numbers that in both these cases some association of the
hemoglobin had occurred giving Roaf double molecules and Reid triple
molecules. 16,693 is the minimum molecular weight if there is one atom
of iron to each molecule.

But while these results are so concordant and striking there is one
fact which is not apparently in harmony with this determination; or
at any rate it is as yet unexplained. The molecular weight of casein
when dissolved in formamide was found to be only about 400. In the
same solvent starch had a weight of 645, corresponding to a tetra-
saccharide. A molecular weight of 400 would be a tri-peptide. Further
investigation of the molecular weight of casein and other proteins in
this solvent should be made. In water there is no doubt but that the
molecular weight is far higher than this.

How many amino-acids would there be in a molecule of protein? Tf
the molecular weight of casein is 16,000 it must have at least 120 amino-
acids in it since the average weight of a molecule of amino-acid is about
130. Some 15 different acids have been separated from casein, so that
on the average there would be about seven molecules of each kind.’
If the molecule has this size and so many acids, it will be seen that
there may be an astonishing number of caseins possible. They might
differ from each other in the order or the amount in which the amino-
acids occur in the molecule; or the acids might be isomers. One might
have leucine and another iso-leucine. In fact, the number of amino-
acids is so great that by modifying the proportion of those present in
different proteins, or by modifying the arrangement of them in the mole-
cule, or by the introduction of optical isomers practically an infinite

‘Recent indirect determinations of the molecular weight of casein by Van

Slyke indicate that the molecular weight of casein is about half this amount, or
about 8,000.
THE PROTEINS 143

number of combinations is possible It is this great diversity, combines!
of course with the diversity in the lipins and carbohydrates, which has
made possible the very large number of different kinds of organisms
on the earth.

Crystallized proteins.—A very interesting crystallized protein has been ob-
tained (Katake and Knoop) from the milk of Antiaris toxicaria, the poisonous
Upas tree of Java, by extraction with 85 per cent. alcohol, drying the extract and
then cooking out the extract with 0.8 per cent. acetic acid. On evaporating the
extract the protein crystallizes out in needles and prisms. Recrystallized from hot
water, the crystals are eventually obtained of uniform appearance containing 15.73
per cent. of water. The ash-free crystals are small, solid polyhedra. They react
acid in solution. They give all protein reactions including sulphur, except Molisch.
The solution is not precipitated by picric or nitric acids, nor by ferrocyanide and
acetic acid, but is precipitated by phosphotungstie acid. DissoJved in glacial acetic
acid, the substance shows the Tyndall phenomenon of scattering a beam of light.
It is, therefore, colloidal in this solution. The rotation is (@)p) =—19.25°. The
composition was C, 48.02; H, 5.71; N, 15.65; S, 7.20; O, 23.47. It contains more
sulphur than any other protein. If there is only one molecule of cystine present in
the molecule, the minimum molecular weight would be 900. It certainly yields on
hydrolysis cystine, lysine, glycocoll, alanine, proline and valine.

Cc H N 8 0

Computed for (C, gH oN 8,0,4) n 48.27; 5.63; 15.69; 7.16; 23.25

Found 48.02; 5.71; 15.65; 7.20; 23.42

Water of crystallization found 15.73 per cent. Computed for the above formula
with 9H,O, 15.35 per cent. ,

Distribution of nitrogen in the protein molecule—The analysis of
the proteins by hydrolysis and the quantitative isolation of the various
amino-acids is exceedingly laborious and requires a very large amount
of material. Shorter methods have been devised to give a general idea of
the nitrogen distribution between various amino-acids and which are ap-
plicable to as little material as 2 grams. The best of these methods is the
group method perfected by Van Slyke. The total nitrogen of the protein
molecule may be divided into four main groups, namely, ammonia nitro-
gen, amino N, imino N and basic N. These groups are determined in ‘the
following way: During the acid hydrolysis of the proteins the acid amide
nitrogen is split off as ammonia. It is determined by getting rid of the
acid of the hydrolysate, making the solution faintly alkaline with lime,
and distilling off the NH, under diminished pressure. The material freed
from ammonia and filtered to remove excess lime and some melanineé +
is precipitated, after acidification, with phosphotungstic acid. This
precipitates the basic amino-acids, arginine, lysine and histidine, and
cystine. The nitrogen determined in this precipitate is called the basic

+The melanine is formed chiefly from tryptophane reacting with some aldehyde.
Tyrosine may contribute a little also. (Holm and Gortner.) a¢
144 PHYSIOLOGICAL CHEMISTRY

nitrogen. A portion of the filtrate from this precipitate, after removal
of excess phosphotungstic acid and neutralization, is treated with nitrous
acid by the Van Slyke method. This liberates the nitrogen present as
free amino groups. The nitrogen is collected and measured. This is the
amino N. It comes from the monoamino acids. Another portion of the
filtrate has the total nitrogen determined by Kjeldahl! and the difference
between this and the amino nitrogen gives the imino nitrogen, namely
that in proline, oxyproline and one-half of tryptophane nitrogen. The
method has been still further refined by Van Slyke to permit a determi-
nation of the different basic amino-acids and some of the others. Some of
the results he has obtained in the examination of different proteins are
embodied in the accompanying table. The nitrogen which is evolved
when non-hydrolyzed proteins are treated by nitrous acid comes from
the ¢-amino group of lysine, which is thus shown to be free in the mole-
cule. The a-amino group of lysine is combined.

PERCENTAGE OF THE TOTAL NITROGEN OF VARIOUS PROTEINS PRESENT IN VARIOUS
Amino-acips (Van Slyke).
Gliadin  Edestin Hair Gelatin Fibrin) Hemo- Ox hem-

(Dog) cyanin oglobin
Ammonia N ..........05- 25.52 9.99 10.05 2.25 832 5.95 5.24
Melanin N ........-0.0005 0.86 198 7.42 007 43.17 £2165 3.6
Cystine N 1.25 149 660 0.07 0.99 O80 0.2
Arginine N : 5.71 27.05 15.33 14.70 13.86 15.73 7.7
Histidine N f Basic N..... 5.20 5.75 348 448 483 13.23 127
Lysine N 0.75 3.86 5.37 632 1151 849 109
Amino N of the filtrate ..... 51.98 47.55 47.5 563 543 513 57.0

Non-amino N of the filtrate
(proline, oxyproline, 14
tryptophane) .......... 8.50 L7 3.1 14.9 2.7 3.8 2.9

 

SUM sacaeciwsseasences 99.77 99.37 98.85 99.u2 99.58 100.95 100.0

Color reactions of the proteins.—The proteins yield colored products
when acted upon by various reagents, and these colors are utilized in
detecting the presence of protein matter in solutions and body fluids,
and in determining easily the presence or absence of some amino-acids
from the molecule. The most characteristic of these reactions, that is
the reaction given by all native proteins and by the larger number of
the derived products, is the biuret reaction. It is not, however, so
delicate as some of the others.

The biuret reaction. If a solution of a protein is made alkaline,
preferably by sodium or potassium hydrate, and a drop or two of dilute
cupric sulphate solution is added, well mixed and allowed to stand at
room temperature, or if it is gently heated, the clear fluid above any
precipitate which may be formed has, if a protein is present, a violet
tinge. The reaction is most delicate when made at room temperature,
but it may be hastened by heating, only in some cases the color
is destroyed by heat. The shade of the color varies from a reddish violet
THE PROTEINS 145

in the case of some peptones, or simple peptides, to a blue violet in many
other proteins. Sometimes in the presence of certain gums which are
precipitated by the copper, the color may be on the precipitate, but this
is the exception.

The reaction is called the biuret reaction for the reason that it is
given, also, by biuret, a substance NH,.-CO—NH—CO—NH, formed
by the condensation of two molecules of urea (hence biurea, or biuret)
with the elimination of ammonia.

NH,— a NH, + NH,
o=0(NH, +020 /5H, e ; at
H. \nq, Al oh

Biuret is easily made by heating a few crystals of urea in a dry test-
tube to a little above their melting point and cooling when the odor of
ammonia is perceived. The biuret may be detected by the biuret test.
The fact that biuret gives this reaction shows that the reaction is not
peculiar to the proteins. Many other substances give this reaction.
Schiff has shown that any diacid amide in which the two amide groups
are not attached to the same carbon will give the reaction. Thus oxa-
mide, NH,—CO—CO—NH,, or malonamide react. One of the amide
groups must be unsubstituted, but the other may be substituted as it
always is in the protein molecule. Thus NH,—CO—CO—NHR will give
the reaction. Asparagine, the amide of aspartic acid, gives a blue-violet
biuret reaction. In this case we have COOH—CH,—CHNH,—CONH,,
which is not a diacid amide. The reaction may, however, be due to the
formation of an amino compound by a kind of lactone (lactam) forma-
tion thus
NH

|
o= box H—CONH, 2

We would thus have two acid amide groups, one of them free. Similarly
leucine amide gives with sodium hydrate and cupric sulphate a red
salt-like compound in red crystals (Bergell and Busch). Succinimide
also forms, in similar circumstances in the presence of potassium hydrate,
reddish needles fairly stable in the solid form and of the composition

co—CH,
K(x “DoH, O;
co—tn,
but which are readily decomposed in aqueous solution by acids. The

rubidium and cesium salts are red violet; the sodium salt pale blue; the
lithium salt ultramarine. All of these are supposed to be derived from

the hypothetical acid
CO—CH,
ox | *) a,
co—CH,
146 PHYSIOLOGICAL CHEMISTRY

Schiff isolated the biuret potassium compound in red needles to which
he ascribed the formula:
OH OH

se os *Sc=0
NH NH
o= a HO ee

K

 

K

Among other substances giving the reaction are urobilin, a coloring
matter derived from the bile and found in the urine (Stokvis, Salkow-
ski). It may be mentioned that strongly basic proteins which are already
alkaline in their aqueous solution, such as the protamines and pro-
tones (Goto), will give the biuret reaction without any addition of
alkali.

From the foregoing we may conclude that the proteins give this reac-
tion because they contain at least one acid amide group and other substi-
tuted amide groups attached to neighboring carbon atoms. If the pro-
teins are deamidized, that is if the free amide groups are split off by
the action of strong acid, the product which remains does not give the
biuret reaction, although it is still a protein, digestible by trypsin and
other enzymes and giving other protein reactions. All native proteins,
therefore, since they give the reaction, contain some acid amide nitrogen.
The biuret reaction, unlike all the other color reactions, is not a reaction
for any specific amino-acid, but rather is dependent on the constitution
of the proteins.

The color of the biuret test is due probably to the copper atom. Many
copper compounds are blue and others, like the metal itself or cuprous
oxide, are red. It is probable that in the blue compounds the copper
atom is in a different state from what it is in the red form, possibly
being partially reduced, consequently the valence electrons have a dif-
ferent period of vibration so that the light absorption is changed. As
this state of the atom may be induced by a great number of compounds,
it is clear that the biuret test cannot be a specific test for proteins, or
for any particular class of bodies.

Since the color change depends on an alteration of the state of the
copper atom, it may be anticipated that other metals having several
stages of oxidation and different colors and which combine with amino
groups may also give a similar reaction. This is the case. Pickering |
found that cobalt salts also might be used for the biuret test, and the
reaction is even more delicate than with copper. Zinc, iron and man-
ganese gave no color change.

Millon’s reaction. This reaction. consists in the development of a
THE PROTEINS 147

red color, when a protein is heated or allowed to stand some time in
contact with a mixture of mercuric nitrite and nitrate. If a few drops
of Millon’s reagent is added to a solution, or suspension, of many pro-
teins and this is heated, the protein is precipitated and the precipitate
after a time turns red. To make Millon’s reagent dissolve 1 part by
weight of mercury in 2 parts concentrated nitric acid and dilute with
twice its bulk of water, allow the precipitate to settle and use the super-
natant liquid. The protein does not need to be in solution for this
reaction and it may hence be used for the detection of proteins in sec-
tions of tissues. The color is not deep enough for a good microscopic
stain. In place of Millon’s solution, which contains a good deal of free
acid, Nasse recommends that an aqueous solution of mercuric acetate be
used, to which at the time of using there is added a few drops of a
1 per cent. solution of sodium or potassium nitrite. It is usually not
necessary to acidify, but the addition of a little acetic acid to the above
solution is sometimes advantageous.

The Millon reaction is given by all organic compounds containing
a monohydroxy benzene nucleus. It is hence given by phenol, salicylic
acid and many other substances. It is not given by a di- or tri-hydroxy
phenol unless one of the hydroxyls is substituted, as in esters or ethers.
Since the only group thus far recognized of the protein molecule which
contains a- monohydroxy benzene ring is the tyrosine group, this reac-
tion when applied to proteins detects the presence of this group. As not
all proteins contain tyrosine, for example pure gelatin and various pro-
tamines, not all proteins give the reaction. It is a good deal more
delicate than the biuret reaction and the presence of proteins when the
dilution is great may be detected by this and the xantho-proteic reac-
tion, when the biuret test quite fails to show their presence.

The character of the colored compound formed has been studied by
Vaubel. The color probably involves the state of oxidation of the mer-
cury atom, since many mercury compounds are red (cinnabar).

Millon’s reaction is interfered with by hydrogen peroxide, chlorides
and by alcohol. If these are present it is necessary to use an excess of
reagent.

Xantho-proteic reaction. This, as the name says, is the yellow reac-
tion of proteins (Greek, zanthos, yellow). In contact with nitric acid
most proteins develop a lemon-yellow color which changes to an orange
when the solution is-made alkaline. The protein either in solution or
suspension is heated with a few drops of concentrated nitric acid to
6 c.c. of water in the test-tube for from one to three minutes, cooled and
ammonia or sodium hydrate added to an alkaline reaction.

This reaction is due to the benzene nuclei in the molecule. The reac-
tion is given by tyrosine, phenyl alanine and by tryptophane, the three
148 PHYSIOLOGICAL CHEMISTRY

amino-acids contained in proteins having benzene nuclei. Trypto-
phane gives the reaction most intensely and easily ; then tyrosine ; whereas
phenyl] alanine requires a longer heating, or more nitric acid. Proteins
which lack these three groups, for example salmin, sturin and clupein
among the protamines, do not give the xantho-proteic reaction.

The mechanism of the reaction consists in the formation of a mono-
nitro benzene, or possible a dinitrobenzene. The nitrated benzenes such
as picric acid, C,H,(NO,),OH, are light yellow in acid solution, but a
deep orange in the salt form. Such nitro derivatives are formed in the
course of the reaction. These nitrobenzenes are all toxic and are some-
times used as dyes (Martius Yellow) for coloring macaroni and other
foodstuffs, although their use is forbidden in most countries. The yellow
color is probably due to the NO, groups (vibration periods of the elec-
trons of the valences of the nitrogen or oxygen), since some of the nitro-
gen oxides are brown or reddish yellow.

Tryptophane reactions. Tryptophane, containing as it does the
indole group, is the chromogenic radicle of the protein molecule par
excellence. Tryptophane and tyrosine are the protein nuclei which give
rise in their metabolism to most of the body pigments, such as the blood
pigment (pyrrol nucleus), bile pigments (pyrrol from tryptophane),
melanins and reds from tyrosine, etc. Tryptophane, as its name implies,
ie., the bright (Gr. phanos, bright) substance formed in the course of
tryptic digestion, readily yields, like indole, a series of bright colors,
reds, violets, blues, when oxidized. There are a number of color reactions
which depend on the presence of tryptophane and among these is the
Adamkiewicz reaction.

Adamkiewicz reaction. If to a few c.c. (2-3) of a pretein solution
one adds an equal quantity of glacial acetic acid and then 4-5 c.c. of
concentrated sulphuric acid, at the zone of contact a violet ring forms
in the presence of a protein containing tryptophane: If the tube is
shaken, the violet color generally develops all through the solution if
not too much sulphuric acid has been used. This reaction depends on
the presence of aldehydes in the glacial acetic acid. It has been found
(Hopkins and Cole) that most samples of glacial acetic acid which have
stood some time contain some glyoxylic acid, HCO.COOH. It is said
that some samples of glacial acetic acid will not give Adamkiewicz reac-
tion, although the writer has never seen any such. The test may be
performed, therefore, by using glyoxylic acid in place of glacial acetic.
The glyoxylic acid is easily made by reducing oxalic acid with powdered
magnesium. An equal volume of this acid (Hopkins-Cole reagent) is
added to the solution in the place of the glacial acetic and the test per-
formed otherwise in the same manner. The réle of the glyoxylic acid is
not explained, but it possibly consists in hastening the oxidation of the
THE PROTEINS 149

tryptophane or condensing with it in the presence of acid to give the
color. 3

Other aldehydes may be used in this test besides glyoxylic acid.
Formaldehyde has been suggested by Rosenheim and Acree. In fact,
this reaction is used for the detection of formaldehyde in milk and is
ot very great delicacy. Casein, the protein in milk, contains relatively
a large amount of tryptophane in its molecule. If a little formaldehyde
is added to milk and the milk does not stand long enough for the for-
maldehyde to have united with the free amino groups of the milk pro-
teins, the addition of strong hydrochloric acid containing a trace of
iron, or of sulphuric acid with iron, leads to the development of a violet
color. It has been recently suggested by Cole that perhaps the Adamkie-
wicz reaction is due to the presence of formaldehyde in the glacial acetic
acid rather than to the glyoxylic acid. Perhaps other aldehydes will
act similarly.

Liebermann’s reaction. Another color reaction involving trypto-
phane is that of Liebermann when carried out in the manner originally
prescribed by him. Liebermann found that protein treated first with
alcohol and ether and then with hydrochloric acid developed often ‘a
violet or bright blue color. This reaction is probably due to the pres-
ence of aldeiiydes in the alcohol and ether (Cole) which combine with
the protein and on subsequent heating with strong hydrochloric acid
develop the tryptophane reaction. If the protein contains both trypto-
phane and sugar, it is not necessary to treat it with alcohol or ether
first, since by the action of the strong acid on the carbohydrate
aldehydes are formed which give a colored reaction product with
some of the protein groups and presumably with the tryptophane. See
page 36.

Other tryptophane reactions. Bromine. Tryptophane when free,
but not when united in the protein molecule, gives in a faintly alkaline
solution with bromine or chlorine water a beautiful violet color. This
reaction was discovered by Claude Bernard as distinguishing tryptic
from peptic digestion. Adamkiewicz’ reaction is given both by the free
and linked tryptophane. The color in the bromine test is possibly due
to the formation of indigo, since indole gives a similar reaction. By
this bromine reaction one can follow the course of the splitting off of
tryptophane from the protein molecule during the process of digestion.

Tryptophane will also give colored products in the presence of aro-
matic aldehydes (Rohde). If a little p-dimethyl-amino-benzaldehyde is
dissolved in concentrated sulphuric acid and run beneath a solution of
protein in a test-tube, a red-violet ring at the zone of junction develops.
A similar reaction occurs with vanillin, or benzaldehyde sulphuric acid
and protein. These reactions are given also by free indole groups as
150 PHYSIOLOGICAL CHEMISTRY

well as by tryptophane; p-nitro- ae gives an intense, stable,

green color; vanillin,
COH

YN
OCH,
VA 3

a beautiful red, becoming violet by dilution; para-dimethyl-amino ben-
zaldehyde,
CHO

QO

N(CH,),

a red becoming violet. In the spectrum a wide absorption band in the
orange between A 615-570 and a second in the green between A 555-540
are to be seen. The method of making this test is as follows: To 6 c.c.
of the protein solution or suspension in a test-tube add 5-10 drops of
a 5 per cent. solution of p-dimethyl-amino-benzaldehyde in 10 per cent.
sulphuric acid and then add concentrated sulphuric acid drop by drop,
with frequent shaking until color appears. If. the albumin is very dilute
this method is not sensitive enough. In that case put concentrated sul-
phuric acid containing 1 per cent. dissolved aldehyde beneath the solu-
tion and see if a colored ring of contact develops. In the ring method
it is possible to detect tryptophane in 0.003 per cent. concentration.
Casein reacts in about 0.15 per cent. contentration, so that tryptophane
must make about 2 per cent. of the casein molecule. This reaction is used
in the urine, feces and bile to detect urobilinogen. | That substance proba-
bly contains scatole.

Triketo-hydrindene hydrate reaction. Ninhydrin reaction. A very
sensitive reagent for most amino-acids, proteins, peptones and some other
substances is triketo-hydrindene hydrate. A blue color develops on
boiling. The test is given by amino-acids which have at least one free
carboxyl and a free amino group. Ninhydrin is

co
H, <0 (OH),.
CO

A description of the test is given on page 919, The reaction is posi-
tive with proteins, proteoses and with all the amino-acids with the excep-
tion of proline, oxyproline, pyrrolidon carbonic acid. It is positive
also with asparagine and glutamine, amino-oxy-valerianic, diamino pro-
pionic, sarkosine and alanyl alanine. It is negative with proline, oxy-
proline, glucosamine, guanine, allantoine, leucinimide, urea. The aibu-
mins give a very blue color, as do also all polypeptides, all a-amino acids
and #-alanine. Ammonium carbonate gives a red coloration, and histi-
dine after a while hecomes a Burgundy red. Gly-occ7l will give the
THE PROTEINS: 151

reaction in 1:10,000 solution. By means of this valuable. reagent it is
possible to show the presence of amino- acids in fresh urine ee ‘in the
protein-free blood serum.
Carbohydrate reaction. Many proteins contain a. exrhonvdnats
_ nucleus in their molecule. This may be detected by Molisch’s reaction.
The principle of the reaction consists in converting the carbohydrate into
aldehyde decomposition products (furfural, formol, pyruvic aldehyde,
ete.) by the action of strong acid and then the detection of these by
some aromatic substance. The method usually employed is that of
Molisch. To the solution (5-6 ¢.c.) to be examined 1-2 drops of a 10
OH

per cent. alcohol solution of a-naphthol, CO ,are added and then a

few c.c. of concentrated sulphuric acid is be ents down the side
of the tube. A violet ring develops at the zone of contact in the pres-
ence of carbohydrates. The a-naphthol in the presence of sulphuric
acid condenses with the aldehydes formed from the carbohydrate by the
action of the acid to form colored compounds. If the protein contains
a good deal of carbohydrate and also tryptophane, it may not be neces-
sary to add the a-naphthol, the tryptophane taking its place. Thus egg-
white contains a good deal (0.5 per cent.) of glucose. If a little egg
white is boiled in water with strong hydrochloric acid a violet color
develops without any addition of a-naphthol. In this case the aldehyde
is generated from the glucose by the acid, and the proteins furnish the
tryptophane. Liebermann’s reaction is sometimes tried in this form.
Molisch’s reaction for carbohydrates appears later as Pettenkofer’s test
for bile acids. In this case the carbohydrate is added and the chromogen
is supplied by the bile acids.

Sulphur reaction. Reference may also be made here to two or three
sulphur reactions. Sulphur occurs in the protein molecule in the re-
duced form either as cysteine or cystine. If a protein containing either
cystine or cysteine is boiled with sodium hydrate, the sulphur is in part
split off as the sulphide. If a little lead acetate is added either before
or after heating, a brown or black color develops and ultimately a black
precipitate of lead sulphide settles out.

Some proteins, and particularly those from actively metabolic cells,
probably contain cysteine in place of cystine in the molecule and, as we
have already noticed elsewhere, this difference may be of great
importance in cell life (see Heffter and Arnold). If a protein which
contains cysteine is dissolved in water and 2-4 drops of a fresh 4-5
per cent. solution of sodium nitroprusside and then a few drops of
ammonia are added, an intense purple-red color appears at once. The
color disappears on the addition of acetic acid. This reaction, however,
is not specific or characteristic. The color is given by other substances
152 PHYSIOLOGICAL CHEMISTRY

than cysteine, for example by other sulphides, by acetone, creatinine,
etc., but cysteine is the only substance likely to be present in protein
which will give the reaction. Proteins of the supporting tissues of the
body generally contain cystine; those of active organs cysteine.

Precipitation reactions of the proteins——Both for the purpose of
detecting the presence of proteins in solution and of removing them
from solution their precipitation reactions are impovtant. Probably all
natural proteins contain a small number of free amino groups and free
carboxyl groups. They are hence both basic and acid. By means of
these groups they can unite and form salts, many of which are insoluble,
with both acids and bases. Among the acids giving more or less insolu-
ble compounds with proteins are tannic, metaphosphoric, picric, picro-
lonic, phosphomolybdic, phosphotungstic, tri-iodo-hydriodic, chromic and
bichromic acids, and many acid dyes; and among the bases are the metals
copper, iron, manganese, aluminum, lead, mercury, nickel, platinum,
gold; organic bases such as quinine, strychnine and many other alka-
leids, some basic proteins, such as protamines and histones; and basic
dyes such as thionin, fuchsin and methylene blue or neutral red. The
acids which precipitate are generally those which precipitate alkaloids
also. A great deal of confusion exists in the literature on this subject
of precipitation of proteins because of a failure to realize that these
precipitates are true chemical compounds. They are sometimes called
without any good reason ‘‘ adsorption ’’ compounds, indicating that they
belong to that hypothetical class-of physical unions of which so little
of a definite nature is known, but which is supposed to depend on surface
tension. The whole behavior of the proteins shows these precipitates to
be true compounds.

The reactions are as a matter of fact almost certainly simple salt
formations. Whenever the precipitation is to be made by a reagent of
which the precipitating part is in the anion or negative group of the
molecule, the solution must, for all except the basic proteins such as
histone and protamine, be acid in reaction. The basic proteins may be
precipitated either in neutral or even slightly alkaline reactions for
the reason given below. If, however, the precipitating substance is a
metal, or base, the precipitation either does not take place at all or not
so completely unless the solution be slightly alkaline. The reason for
this is as follows: The precipitating agents of the first class mentioned
are the free acids, or the salts of acids, and the part uf their molecule
which precipitates is the negative part, or the anion. In this group are
all the acids mentioned above and many others not there included, such
as bichromic, chromic, ferrocyanic, etc. The precipitates which are
formed have been found always to be the protein salts of the precipi-
tating acids. They are protein bichromate, tannate, picrate, picrolonate,
THE PROTEINS 153

ferrocyanide, etc.; and in the case where basic precipitating substances
are used the precipitates always carry down the base and they are gen-
erally the salts of the protein, such as quinine or lead proteinate. Ifa
colored base is used to precipitate, the fact that. the precipitate is col-
ored shows that the base has gone down with the protein. In a few
cases, such as precipitation with mercury, platinum or copper salts,
the union of the salt is with the amino group, as will presently be
shown.

The reason why the protein must be in an acid solution to precipitate
with the alkali salts of the acids mentioned is that the protein must be
electro-positive to unite with the electro-negative radicle of the salt;
and it must be in an alkaline medium to precipitate with the bases, be-
cause the protein must be electro-negative to unite with the electro-
positive bases.

In acid solutions proteins become electro-positive; and in alkaline
solution they become electro-negative. This was shown by Hardy. If
egg white be dialyzed against distilled water until free from salts and
then boiled, it becomes opalescent, but the protein is not precipitated ;
it remains in colloidal solution. If, now, to this solution a little acid
is added and an electric current is sent through the solution, the protein
collects in a- tough, white mass at the cathode; while, if the solution is
made very faintly alkaline, the protein collects at the anode. The fact
that the protein moves in the electric stream proves that it carries an
electric charge; that it moves to the negative electrode, or cathode, in
an acid solution shows it to be electro-positive; and to the anode in an
alkaline solution proves it is there electro-negative. The electric sign
of the protein molecule is different in an acid from what it is in an
alkaline solution.

Some rather extraordinary explanations have been given of this
change of sign, which is a matter of fundamental importance in under-
standing cell metabolism, vital and ordinary staining, ete. Thus it
was suggested that as the hydroxyl ion moves faster than the sodium
or potassium ion it hurries on ahead of the sodium and hitting the
protein molecule first buries itself in that molecule, thus making the
molecule electro-negative; and in acids, the hydrogen ion goes first,
is entombed in its turn and makes the molecule of protein electro-
positive. There is no need, however, for this fanciful explanation which
has nothing to recommend it exeept its picturesque nature. The real
explanation is probably quite different. By means of the free amino
groups the proteins are basic and they combine with the acid by these
groups, forming thereby salts like substituted ammonias thus:

R—CHNH, + HCI -—- R—CHNH,.HCI.
B is the rest of the protein molecule. The salt R—CHNH,Cl now ionizes
154 PHYSIOLOGICAL CHEMISTRY

into RCHNH,* and Cl. The chlorine is electro-negative and the rest of:
the molecule is:electro-positive. Hence in acid solutions the proteins,
with the exception of some very acid ones like casein, are always electro-
positive.
‘In alkaline solution the free carboxyls unite with the alkali to form
salts: :

RCOOH + NaOH —+ RCOONa + H,0.
RCOONa now ionizes into RCOO” and Na”. Thus the protein becomes
electro- negative.

2 The reactions with the precipitating reagents now become clear. They
are as follows: _

_ 1. Protein + CH, .COOH ——~ Protein acetate.

2. Protein acetate + Na bichromate——~ Protein bichromate -+- NaOCOcH,.
Precipitate.

yee

‘3. Protein + NaOH —— Na proteinate.
4, Na ee ++ Pb acetate —-> Lead proteinate + NaOCO. CH,.
Png e§ Precipitate.

: But, while this is the rule for most of the proteins, there are certain
ones which appear at first glance to be exceptions. For example, the
protamines and histones may be precipitated by colored acid dyes, or
by’ sddium picrate, or bichromate in neutral, or even faintly alkaline
solution: The reason for this is that these proteins are so strongly basic,
having so many basic amino-acids in their molecules, that they are electro-
positive even in a neutral solution in which they exist as the free bases.
They’ may even be positive in faintly alkaline media. They do not change
to: the electro-negative state until some excess of alkali has been added.
Similarly some of the acid proteins, such for example as some of the
vegetable proteins which contain a large amount of glutamic acid in the
molecule ‘and are hence fairly strong acids, may be precipitated in neu-
tral or even faintly acid solution by the basic precipitating reagents. For
these proteins do not at once become electro-positive as soon as the reac-
tion: becomes. ‘faintly acid. Casein is a protein of this kind. Another
complication is introduced by the affinity of all metals below hydrogen in
the scale of solution tension, such as mercury, gold, copper and platinum,
for amino groups. These metals will not only form simple salts with
the proteins by displacing the hydrogen from the carboxyl group, but
they will also form addition compounds or double salts by union with the
amino groups. It will be found, therefore, that mercuric chloride will
precipitate even in 4 faintly acid medium, and so will the others of this
group. - ‘This, however, is not an exception to the rule stated, but an
additional’ kind of chemical union between the precipitating agent and
the protein. _ In most of these cases, also, the precipitation is found to be
thore “complete-i in a faintly alkaline than in a faintly acid medium.
THE PROTEINS 155

One of the best ways of completely separating the proteins from a
sclution is by using basic lead acetate. Mercuric chloride in a faintly
alkaline solution may, however, also be used.

With this brief account of the properties of the proteins we may pass
to the consideration of some few which are of particular interest in the
cell. We shall not now consider all the different kinds of proteins, leav-
ing the individual sa ea of the group to be treated more at length
in connection with the organs or fluids of the body in which they occur.
There is one group, however, which is colored and of very general inter-
est, as members of this group are found both in plants and animals.
These are the chromoproteins. They occur in the cytoplasm of cells.

Chromoproteins.—There are two groups of chromo, or colored, pro-
teins which may occur in the cytoplasm: the hemo-chromoproteins ob-
tained from blood, of which the hemoglobins are the best examples; and,
second, the phyco-chromoproteins which are obtained from seaweed.
These latter are very interesting proteins because in a way they are
intermediate between hemoglobin and chlorophyll. The chromatic group
of hemoglobin is an iron containing pyrrol complex called hematin; and
the iron free part of hematin resembles chlorophyll, which also yields
pyrrols on decomposition. It is very interesting, therefore, as showing
the close relation between hemoglobin and chlorophyll that a chromo-
protein closely resembling hemoglobin in several ways and particularly
. in its ease of crystallization has been isolated from the red and blue-
green alge. The red coloring matter of the Floridie, phykoerythrin,
and the blue coloring matter of the blue-green alge, phycocyan (phykos,
seaweed; cyan, blue; erythros, red), crystallize most readily. The sub-
stances are obtained from seaweed just as hemoglobin is obtained from
the corpuscles of the blood by laking in distilled water. Ammonium
sulphate (30 grams to 100 c.c. solution) is then added and the phyko-
erythrin and the phykocyan precipitate. They are globulins. If the
precipitate is redissolved by the addition of water and the salt dialyzed
out, the protein crystallizes out in the dialyzing tube in microscopic
crystals. Phykoerythrin is coagulated by boiling; it is soluble in weak
alkalies and neutral salts, but insoluble in distilled water. It is pre-
cipitated by acetic acid, but redissolves in an excess. It is precipitated
by (NH,).SO,, MgSO, and alcohol. It quickly loses its color in the light,
particularly in an alkaline solution. The analyses gave C, 50.82; H, 7.01;
N, 15.87; S, 1.60; O, 25.20. It is free from ash and resembles chlorophyll
in containing no iron.

Distribution of protein substances between the cytoplasm and the
nucleus.—The proteins of the cell nucleus are sharply. differentiated from
those of the cell cytoplasm. In the nucleus many of the proteins, in some
eases all of them, are nucleoproteins, characterized by the presence in
156 PHYSIOLOGICAL CHEMISTRY

the molecule of nucleic acid. The simple proteins in the nucleus are
often more basic than the general run of proteins and sometimes they
are very basic proteins, such as the protamines and histones. The occur-
rence of these proteins is, however, the exception rather than the rule.
The composition of the nuclear proteins will be considered presently.
The proteins of the cytoplasm are less well characterized and of very
diverse character. They include both the proteins of the living proto-

 

 

 

Fig. 15.—Phycoerythrin crystals (Kylin).

plast and lifeless secretory or reserve proteins of a varied nature. They
are often globulins, that is simple proteins insoluble in water, but soluble
in dilute salt solution. Thus in the cytoplasm of muscle there are the
simple proteins, myosin and myogen and myosin fibrin; in the thyroid
gland, the thyreoglobulin of the colloid material which is found in the
cytoplasm is a globulin. On the other hand, albumins are found there
also. In the white blood corpuscles a simple protein corresponding to
serum albumin has been found. In many cells of the body there occurs
in the cytoplasm, also, a globulin coagulating at the low temperature of
56°, which is the temperature of coagulation of fibrinogen. It is gen-
erally believed, too, that phosphoproteins are found in the cytoplasm,
and this is certainly the case in some cells. Thus casein is found in the
cytoplasm of the milk glands and vitellin in the cytoplasm of the hen’s
THE PROTEINS 157

egg and some other eggs. Both of these bodies are phosphoproteins.
There is some reason for believing that in the living protoplasm the
protein may’ be in union with phospholipins, carbohydrate and possibly
fats. It is not possible, however, to make a definitc statement on this
point. The decomposition products of protein metabolism probably
also occur there.

We may then say that in the nucleus are found the nucleoproteins;
whereas in the cytoplasm of the cell these are probably lacking (see
page 173). The protoplast of the cytoplasm consists in all likelihood of a
mixture of simple albumins and globulins, coagulable by heat, and phos-
pholipins, and some of these simple proteins may be and probably are
in loose physical or chemical union with phospholipin, fat and carbo-
hydrate. In other cells one finds mucin, which is a glycoprotein. These
proteins do not occur free for the most part, but in union with inorganic
salts, salts of sodium, potassium, calcium and magnesium preponderating.
These cytoplasmic proteins in the living cell are predominantly electro-
negative, but occasionally electro-positive protein may be present, as in
the red blood corpuscles in which the hemoglobin is electro-positive.
Since the whole of the protein world is at some time in the cytoplasm of
cells, it will be seen that this part of the cel] is wonderfully diverse in
its chemical nature. The general features of the living protoplast, as
distinct from secretory granules, reserve proteins or structural elements,
are, however, so similar in all cells that it is probable that in its funda-
mental chemical constitution it is everywhere closely alike, although dif-
fering in some rarticulars. What this constitution is, is the great
unsolved problem of physiological chemistry.

CHEMISTRY OF THE CELL NUCLEUS.

Morphology. If living cells are examined under the microscope, all
except the simplest animal cells (Monera) and the bacteria may be seen
to contain within the granular protoplasm a clear, almost or quite
homogeneous, more refractive area. This area, called the nucleus and
first described by Robert Brown in 1831, is generally spherical or ellip-
soidal in shape, though at times it is quite irregular in outline. Figure
1, p. 11. Sometimes it is separated from the surrounding protoplasm
by a distinct visible membrane; at other times no membrane may be
seen in the living cell, though it is probably always present. In size, the
nucleus may fill almost the entire cell, as in cells of the thymus gland
or the sperm head, or it may be a very small part of the total bulk of
the cell, as in many eggs and muscle cells.

Generally no structure can be seen within the living nucleus, but in
some cases, as in the germinal vesicle of many eggs, there may be seen,
158 PHYSIOLOGICAL CHEMISTRY

in addition to a distinct membrane, spherical or irregularly shaped
more dense portions which are known morphologically as nucleoli. When
the cell divides by caryokinesis there may also be seen, in the most favor-
able cases, as in the testes of grasshoppers, and in some transparent eggs,
the spindle fibers and the chromatic masses called by morphologists
chromosomes. In general, however, as long as the cell is alive no other
structure may be seen within the nucleus than the nucleolus.

The physical structure. The physical consistence of the nucleus has
been found by Kite to vary greatly in different cells. By his very in-
genious method of microscopic cell dissection by means of extremely fine
glass needles (diameter 1 yw or less), Kite has found that most nuclei
are separated from the protoplasm by a very tough distinct nuclear mem-
brane. Within this membrane one generally finds either a liquid (sol)
or a fairly viscid gel in which no structure, except sometimes the nucle-
olus, is to be discovered by his methods. The nucleus of an ameba, for
example, or the nucleus of an immature starfish egg, contains a liquid,
and when the nuclear wall is ruptured the contents escape into the sur-
rounding cytoplasm, mixing with the latter and setting up most inter-
esting chemical changes within it, discussed further on page 180. But
the nuclei of most differentiated cells which he examined, such as
epithelial, liver or pancreas cells of the amphibian, Necturus, or the frog,
or rabbit, are quite jelly-like. They may be cut into several pieces, each
piece retaining its form and in this case not mixing with the cytoplasm.
It is indeed altogether probable that the physical state of the nuclear
contents is not constant in any cell, but varies from fluid to gel under
various conditions. This is indicated, for example, by the experiments
of Calkins and Miss Peebles in their cutting to pieces of infusoria. At
times the cutting could be made as if through a jelly, the pieces not losing
their contents when cut; and at other times the protoplasm was so liquid
that it readily escaped through the cut. Kite has made similar observa-
tions on Amceba proteus and they have been made also by Gruber. One
of the constituents of the nucleus is nucleic acid and this has quite
remarkable powers of forming gels; and it may be that this jelly-like
consistence of many nuclei is due to the presence of this substance.

One of the most important observations of Kite is that it is impos-
sible by his method of dissection to find in living nuclei any more dense
masses, or networks, which might correspond with the chromatin net-
work, or chromosomes to be seen in fixed and stained nuclei. Whether
these pre-exist in the cell nucleus when it is alive, or whether they first
appear as the result of the action of fixing agents, may seem doubtful
from this observation; but the extreme and detailed regularity of these
morphological pictures in fixed cells (Figure 2, p. 12), and their steady
development during karyokinesis, make it unlikely that they are pro-
THE PROTEINS 159

duced by the fixing agent. It seems more probable that they exist in the
living nucleus, though perhaps not quite in the form revealed in the
nuclear corpse, even though they can neither be seen nor found by dis-
section. It may be remarked, indeed, that the dissection method, by the
enormous stimulation of the cell which it entails and the mechanical
mixing of the parts of the cell, must render an interpretation of the
results obtained by it somewhat uncertain.?

Function. There is no question but that the nucleus, forming as it
does so universal a constituent of cells, is of fundamental importance
to cell life. The sperm head, which alone enters many eggs, the tail
being left outside, and which is able to produce the development of an
organism resembling in many most minute particulars the parent organ-
ism from which it came, is often composed exclusively of a nucleus. So
the nucleus must play a great part in inheritance. Inheritance is equally
from father and mother, and it can hardly be a coincidence that the
embryo contains an equal share of nuclear material from father and
mother, whereas the cytoplasmic material is obtained almost exclusively
from the mother.

The importance of the nucleus is shown very clearly in many experi-
ments which have been performed on unicellular organisms. If an
ameceba, or other protozoon organism, be cut into two parts, one of which
contains the nucleus, while the other lacks it, it is found that while both
pieces may continue in motion and may capture food, it is only the part
with the nucleus which is able to grow and. reconstitute the cell; the
protoplasm without the nucleus cannot regenerate the nucleus and in
a short time it dies and disintegrates. This experiment shows that both
nucleus and cytoplasm are necessary for growth and development.

Similar facts showing the great importance of the nucleus in the
growth and synthesis of new protoplasm are beautifully illustrated in
gland and vegetable cells. If vegetable cells are plasmolyzed, that is
shrunk from the cell wall by the action of hypertonic salt solutions, it
sometinies happens that the protoplasm becomes divided within the cell
into a nuclear containing and a nuclear free portion; it is only the former
which makes a new cell wall and grows to a new cell. In many gland
cells the protoplasm during glandular rest in whole, or in large part,
becomes differentiated into secretory material, generally taking the form
of granules. The nucleus remains with only a very small quantity of
cytoplasm around it. Now, when the cell secretes, these granules are dis-
charged or dissolved, and the new undifferentiated protoplasm which
takes their place appears always first close to the nucleus, as if it were
being formed here.

‘Chambers has recently found that the chromosomes may appear quite sud-
denly in nuclei.
160 PHYSIOLOGICAL CHEMISTRY

There can be no doubt from all these facts that the nucleus plays a
very important part in the synthesis of the cell protoplasm. It appears
as if under favorable conditions the nucleus might be able to make the
cytoplasm about it, but no one has as yet succeeded in proving this. It
might be tested by growing the spermatozoa in such conditions that
they would make themselves into cells provided with cytoplasm. Per-
haps it might be proved, also, by isolating nuclei by means of Kite’s
method. At present all that can be said: definitely is that both protoplasm
and nucleus appear to be necessary for growth and development. Many
chemical transformations, probably most of them, occur in the extra
nuclear part of the protoplasm. But the nucleus is nevertheless of
fundamental importance.

Chemical composition. Method of obtaining nuclei for chemical
analysis.—The chemical composition of an organ of such vital impor-
tance in inheritance and cell life is a matter of very great interest. What
knowledge we have of it is owing more particularly to Miescher and
above all to Kossel. There are several ways in which the chemical nature
of the nucleus may be studied. We may study cells consisting chiefly
of nuclei, such as leucocytes, and contrast their composition with that
of cells consisting chiefly of cytoplasm, such as muscle, or egg cells,
or red blood corpuscles of mammals. Substances which are found in
predominating amounts in the first group of cells we would be justified
in inferring came from the nuclei. Another method, although one to
be used with great caution in interpreting observations, is the use of
microchemieal stains. The best method is to separate the nucleus
from the cytoplasm and to study the chemical composition of each
separately.

The first method was that used by Miescher, with whom our knowl-
edge of the composition of the nucleus begins in 1876. It had been known
that living tissues all contained large amounts of phosphoric acid in
different combinations. This acid early attracted the attention of chem-
ists, some of whom even went so far as to say ‘‘ ohne Phosphor keine
Gedanke ’’ (‘‘ without phosphorus no ideas’’). And we are coming to
realize more and more clearly the fundamental réle phosphoric acid plays
in all vital phenomena. It was soon found that the phosphoric acid was
present in at least two forms. One part could be extracted by alcohol
and was in organic union. It was present in the lecithin discovered by
Gobley. Another part could be extracted by cold water from the tissues
already extracted with alcohol. This part consisted of inorganic
phosphates. After removing these two forms of phosphoric acid there
remained a considerable proportion of the phosphoric acid in the protein
residue of the cell. Hoppe-Seyler put his pupil Miescher at the task
of finding out what compound of phosphoric acid remained in this
THE PROTEINS 161

residue. Miescher worked chiefly with pus, which in those days of septic
surgery could be readily obtained. He found that most of this remnant
of phosphoric acid-containing material could be extracted with dilute
alkalies and reprecipitated by acetic acid. It was in organic union with
proteins. Since pus cells consisted chiefly of nuclei with very small
amounts of cytoplasm and this material constituted the greater part of
the residue, there could be little doubt that it came from the nucleus,
and for this reason it was called by Miescher ‘‘ nuclein.’’ It was quickly
found that nuclein was a constituent of all cells examined. Thus Hoppe-
Seyler found it in yeast; it was isolated from sperm, spleen and a great
variety of tissues. This nuclein contained varying amounts of phos-
phorus varying from 0.9-4 per cent. One of the easiest ways of prepar-
ing such a nucleoprotein is to extract a tissue with dilute alkali; or even
to boil it with water, some of the nuclein goes into solution in the boiling
water. Shortly after this Kossel found that if this nuclein was boiled
with acids, it yielded a number of xanthine bases, of which the formulas
will be given presently, such as xanthine, hypoxanthine, guanine and
adenine, a new base which he discovered and named adenine (Gr. adén,
gland) because he isolated it first from the pancreatic gland. The dis-
covery that the xanthine bases could be obtained from nuclein was a
discovery of fundamental importance, for it indicated that these bases,
which are found in human urine, and urie acid, which belongs in the
same group of substances, must come from the nuclein of the body and
not from the ordinary albumin, as had been supposed up to that time.
In 1887 Altmann, an histologist, took a long step forward when he suc-
ceeded in isolating from Miescher’s nuclein by digesting it with pepsin-
hydrochlorie acid an organic acid, containing 8-9 per cent. of phos-
phorus, which was free from albumin, all the albumin tests being nega-
tive. He called this acid nucleic acid.

Before examining the constitution of this important acid discovered
by Altmann, a word nay be said about another method of determining
the constitution of the nucleus. The best method is to examine the heads
of spermatozoa. These, in the fishes and most animals, consist wholly,
or almost wholly, of nuclear material; and while they undoubtedly
represent very highly specialized nuclei, nevertheless they are still
nuclei. This method of studying nuclear composition was found by
Miescher.

If the ripe testes of a fish such as the salmon, which Miescher studied,
or the herring, are taken and ground to a pulp and then strained through
cheesecloth the sperm go through; the connective tissue remains behind.
It is an additional advantage that in fishes the sperm all ripen at the
same time so that a homogeneous product is had. The unripe sperm have
a different, more complex, composition from the ripe. The sperm mass
162 PHYSIOLOGICAL CHEMISTRY

is then suspended in normal salt solution, or in a dilute magnesium sul-
phate solution, and centrifugalized. By this means they are freed from
the liquid in which they are suspended in the testes. After one or two
washings of this kind, the sperm are suspended in distilled water and
centrifugalized very rapidly. In the distilled water the tails swell, and
the heads are so much heavier and denser that they are separated from
the tails by the centrifugal force and accumulate at the bottom of the
“ tube as a pure white mass. Above this mass of heads, there may be
seen in the centrifugal tube a slimy tenacious layer of swollen tails more
gray in color than the heads. This layer of tails is coherent and may be
easily lifted out. Above this again is a layer of water, opalescent, and
containing the greater part of the lecithin, cholesterol and much protein
in solution. After several washings and centrifugalizing in distilled water
the heads are clean from tails. Under the microscope they look perfectly
normal. They are not changed in shape nor apparently in size. They
appear to have lost none of their constituents. They constitute pure
nuclear matter. It is of course possible that they have lost some material
in the washing in spite of the fact that they do not appear to have done
so. Thus far only two kinds of. sperm have been examined in this way,
the salmon by Miescher, and the herring by the author and Steudel. If
these pure white sperm heads are now extracted by alcohol and ether
only traces (.1-.01 per cent.) of alcohol-ether soluble substances are found
in them. From this it appears either that the lecithin and lipoids have
been extracted by the distilled water, or else that they are confined chiefly
to the middle pieces and tails, and that they are not found in the nucleus.
The small amount found was so variable as to suggest that it may have
come from remnants of tails, which had not been completely separated
from the heads.

The other kind of nucleus which has been obtained free and pure for
analysis is that of the red blood corpuscles of hens. The corpuscles,
treated in the same manner as the sperm, swell, they are laked, and the
nuclei become free and may be accumulated by centrifugal action. These
nuclei have been recently examined by Ackermann.

THE COMPOSITION OF CHROMATIN.—The sperm head con-
sists wholly, or almost entirely, of chromatin. This chromatin consists
of a nuclein. In the heads of salmon sperm the chromatin is salmin
nucleate ; in the herring it is clupein nucleate. See page 178. In all cells
it has been found that the chromatin consists of two parts: an acid part,
nucleic acid, discovered by Altmann, and a basic part which is always
some member of the simple proteins, but a different protein in every
kind of cell which has been examined thus far. We will consider first
the composition of the acid part of the nucleus, or nucleic acid, and then
the basic or protein part of the molecule.
THE PROTEINS 163

Nucleic acid.—Method of isolation. Nucleic acid may be obtained
from tissues without necessarily isolating the nuclei first. It is most
easily obtained by the Kossel-Neumann method. Perfectly fresh tissue
must be taken and as quickly as possible after its removal from the body
it is ground in a meat chopper and thrown into boiling water slightly
acidified with acetic acid to destroy the enzymes. The reason for the
necessity of haste is that there are present in most cells enzymes, called
nucleases, which very rapidly attack and partially decompose the nucleic
acid. The residue is ground as fine as possible and then brought into
twice its weight of a boiling solution of sodium hydrate and sodium
acetate (1.6 per cent. NaOH and 10 per cent. Na acetate) and extracted
for from %-2 hours at boiling temperature. By this treatment the
nucleic acid is dissolved and extracted from the cells. The mass is then
neutralized with acetic acid, centrifugalized and, if necessary, filtered
hot. The filtrate is now concentrated and the filtered solution is poured
into alcohol, about three volumes of 95 per cent. to one of the solution.
The nucleic acid is precipitated as the sodium salt. It may be purified
by resolution and reprecipitation. By this method (Neumann’s) from
1 kg. of dry thymus gland 180-200 grams of nucleic acid are obtained.

Nucleic acid —Physical and chemical properties. The sodium salt
of nucleic acid thus prepared is soluble in water. If dissolved in hot
water to a concentration of 5 per cent. it gelatinizes firmly, on cooling,
to a clear, slightly opalescent gel. This property has already been men-
tioned in connection with the jelly-like consistence of some nuclei, and
the solidity of the chromosomes. When dry the. salt is pure white,
amorphous, having neither taste nor smell. It gives no protein tests;
the biuret, Millon, xanthoproteic and tryptophane reactions are negative.
It added to a solution of protein containing a little free acetie acid, it
precipitates the protein, forming thereby an artificial nuclein. It does
not reduce Fehling’s solution ; it is not crystalline in any of its salts. It
is optically active, dextro-rotatory, the rotatory power being (@)p =
+154.2. The substance is fairly stable with alkalies, but on long boiling
(2 hours) in alkaline solution it goes over into a #-nucleic acid, which
no longer gelatinizes, and which has a different per cent. of composition
from the first. It is very unstable in the form of the free acid and is
readily hydrolyzed into its constituents. The free acid is white like the
salt, unstable in the light, turning a reddish or brownish red color when
exposed in the powder form. It is fairly soluble in hot water, but
much less soluble in cold. It is insoluble in alcohol, ether and similar
solvents.

Per cent. of composition of nucleic acid. The very great ease with
which the purines are split off from the molecule and the necessity of
using acid at some stage of the separation makes it very difficult to
164 PHYSIOLOGICAL CHEMISTRY

obtain nucleic acids which are entirely normal. All of the older analyses
in which the nucleic acid was precipitated by free acid are almost cer-
tainly incorrect. The following analyses are some which have been
obtained :

Origin Cc H N P Observer
Sperm of Alosa ........... 36.27 5.00 15.96 8.11 Levene and Mandel.
Human placenta .,......... 37.44 4.32 15.32 9.67 Kikkoje.

Spermatozoa (Muraenoesox). 37.50 4.36 16.04 9.73 Inouye.

Various acids have given percentages of composition which differ some-
what among themselves. The relation of P:N is as 4 atoms to 14 or 15.

The most probable formula according to Steudel is C,,H,,N,s,P,Oz0,
which requires that the molecule should be composed of four hexose
molecules, two purines, two pyrimidines and four molecules of phosphoric
acid. By the action of endocellular enzymes nucleic acid is very quickly
partially digested, which accounts for many of the discordant results of
analyses.

Decomposition. We will first consider the composition of the true
nucleic acids, or polynucleotides, as they are ealled, such as are found
in the nuclei of all cells thus far examined, leaving the simpler, or mono-
nucleotides, such as guanylic, or inosinic acid, for later consideration.

The true nucleic acids thus prepared by Neumann’s method are
extremely unstable if heated in the presence of acids; or even if left
in an acid solution for a short time at room temperature. They decom-
pose on prolonged heating with 3 per cent. sulphuric acid, or by heating
under pressure with acetic or other acids, into orthophosphorie acid,
various basic substances, i.e., guanine, adenine, cytosine, thymine, uracil
and either into a pentose or levulinic and formic acids. A method which
gives the guanine and adenine in almost quantitative amounts and which
is very simple is that of Steudel, who treats the copper salt with half-
concentrated nitric acid.

It was Kossel who showed that the nucleic acids split under acid
hydrolysis into the purine bases, orthophosphoric acid, levulinie acid,
or a pentose, and the pyrimidine bases which he discovered and named.
He found that the purine bases, some phosphoric acid and levulinic acid
appeared very easily; the remnant of the molecule consisting of phos-
phoric acid, carbohydrate and pyrimidine bases was isolated by Kossel
and Neumann and called thymic acid. The pyrimidine bases are far
more difficult to detach from the molecule than the purines.

The work of Steudel and Levene has shown that in the nucleic acid
itself there are two purine bases, adenine and guanine. These bases are
heterocyclic compounds, and may be regarded as derivatives of the sub-
stance, purine. Caffeine, the active principle of coffee and tea, is a
purine.
THE PROTEINS 165

(6)

@® N=CH
lL | w
@) HC 6)C—NII
i i @gCcHe)
8) N-(aCc — Ne
Purine.

Products of hydrolysis.—Chemistry of the purines. Guanine. This
purine base, C,H,N,O, or 2-imino-6-oxypurine, or 2-amino-6-oxypurine,
owes its name to the fact that it was first isolated from guano. Its ,
graphic formula is either

HN—C = 0 HN—C=—0
| | |
HN — d C—NH. ;0r H.N—C C—NH
ty OF “Wy yoB
HN—C—N 7 Kl G

As may be seen in the graphic formula it contains the radicles urea,
guanidine and tartronic acid. It is a fairly strong base, precipitated by
ammonia from its aqueous solutions, a peculiarity which makes it easy
to separate it from adenine. It is soluble in acids and in strong alkalies.
It is precipitated by silver nitrate either in neutral or an ammoniacal
‘solution, and forms double salts with the nitrate. Its nitrate crystallizes
readily. The nitrate is insoluble in strong (half-concentrated) nitric
acid. Guanine crystallizes readily from a dilute solution as the picrate.
It. forms a crystalline compound with bichromates.

Free guanine is found in various deposits in tissues. Thus it is found
in the free state in the concretions about the joints of hogs suffering from
so-called guanine gout. It occurs free in the scales and skins of the bony
fishes; and in the swim bladder, to which it gives the peculiar pearly-
white appearance. It is easily isolated from these sources by extracting
with dilute acid and precipitating with ammonia. On oxidation it yields
xanthine, uric acid, allantoine, urea, oxalic acid and other substances.

Guanase is an enzyme found in various organs of the body, in the
liver, spleen, lungs, ete., which hydrolyzes guanine with the formation
of ammonia and xanthine:

HN—C —O HN—C=0
fejspe as er
HN=—C C—NH. +H,O —- O=C C—NH +
Li pce i. oe 8
HN—C—N 7 HN—C—N
Guanine. Xanthine.

Adenine. This base, C,H,N,, or 6-amino purine, was discovered by
Kossel in the cleavage products of the nuclein of the ox pancreas and
called adenine (Gr. aden, gland) because of its origin from a gland. Its
empirical formula is that of a polymer of hydrocyanic acid, and indeed
hydrocyanie acid and cyanogen spontaneously change into substances
366 PHYSIOLOGICAL CHEMISTRY

which are allied to the purines. Adenine has been found to be pre-
formed in the nucleic acid molecule and it occurs in all true nucleic
acids, polynucleotides, where it has been looked for. It is not precipi-
tated by ammonia, hence its separation from guanine. Its structural
formula. is:

N=C—NH,
ud bon
t I pee
—C—n F
Adenine.

It is precipitated like guanine by picric or metaphosphoric acid and forms

‘erystalline picrates. It is usually separated in this form. The melting
point of the anhydrous base is 360-365°. It is a stronger base than
guanine. It is quite stable in the presence of mild oxidizing agents, but
is easily decomposed by acids in the presence of a reducing agent. A
far better yield is obtained by Steudel’s method of hydrolysis of the
nucleic acid by nitric acid, than by hydrolysis with hydriodic, or other
non-oxidizing or reducing acid reagents. The fact that the base is so
unstable in the presence of reducing agents may have some bearing in
cell physiology, since the nucleus is probably always situated at a point
in the cell where reductions are strongest. Hydrochloric acid at 180-200°
C. decomposes it into carbon dioxide, glycocoll, ammonia and formic
acid. Many cells, perhaps all, contain a ferment known as ‘‘ adenase,”’
discovered by Jones, which by hydrolysis converts adenine into hypoxan-
thine as follows:

N= C—NH, HN—C=0
|
ud bom \ +4H,0—- AC a +NH,
i i es:
— C—N —C-N 7
Adenine. Hypoxanthine.

Xanthine and Hypoxanthine. Besides these purines, which pre-exist
in the nucleic acid molecule, there are often found among the products
of hydrolysis of nucleic acids by acids xanthine and hypoxanthine.
These bases, however, are produced either by the action of the acid on
the guanine and adenine, or more often by the action of enzymes,
such as adenase and guanase of the tissues, which have converted the
adenine and guanine into xanthine and hypoxanthine before the nucleic
acid was prepared. Xanthine is 2,6-dioxy purine; hypoxanthine is 6-oxy-
purine.

a is Oo =e.
HC NH o= —NH
(i, 2oH al oe

Peoe iii CH aN ,03 Xanthine. O,BN,0,.
THE PROTEINS 167

Xanthine owes its name (Gr. zanthos, yellow) to the yellow reaction
its gives when gently heated to dryness in a porcelain dish with nitric
acid. The yellow spot moistened with sodium hydrate turns first red and
then purple red on heating, in distinction from uric acid. It was dis-
covered in urinary calculi in 1817 by Marcet. On dry heating it de-
composes into hydrocyanie acid, carbon dioxide and ammonia. It is both
an acid and a base. It owes its acid properties to the fact that by a
tautomeric rearrangement of the molecule the enol form appears:

HN—C=O
{| |
HO—C C—NH a
CH
{Uy 7
Enol form of xanthine.

The hydrogen of the hydroxyl is replaceable by metals.
Hypoxanthine, literally little, or less, xanthine, is a reduced xanthine.
Tt was formerly called sarkine. It is 6-oxypurine, having the following
formula:
HN—C —0

bod
HC C—NH
Il | >CH
no—n F
Hypoxanthine.

Hypoxanthine forms small colorless needles. It does not give the
xanthine reaction with nitric acid, nor does it give the Weidel reaction.
With hydrochloric acid and zinc a solution of hypoxanthine becomes first
a ruby red and then turns brownish red on addition of alkali. Hypo-
xanthine is soluble in dilute alkalies and is not precipitated by ammonia.
When treated with ammonia and an excess of silver nitrate, a crystalline
compound having, when dried at 120°, a constant composition of
2(C;H,Ag,N,O)H,O separates out. Use is made of this in the quanti-
tative separation. Hypoxanthine picrate is little soluble. Hypoxanthine
as well as other purines which have the nitrogen at number 7 or the
carbon in 8 unsubstituted give red azo compounds with diazo-benzolsul-
phonie acid in alkaline or neutral solution. The compound is probably
of the following nature: =

HN—C = 0

0¢ Cy NC_H,S0_.H

nydin Ae
Some pyrimidines give this reaction also. All purines are precipitated by
cupric: sulphate and a reducing substance such as sodium bisulphite.
They form-insoluble cuprous compounds. This is the basis.of their quan-
168 PHYSIOLOGICAL CHEMISTRY

titative determination by the Kriiger-Schmidt method. Hypoxanthine is
present in nearly all cells. It is a constituent of inosinic acid of muscle.

Pyrimidine bases.—Nucleic acid yields two or three pyrimidine bases
when it is hydrolyzed long enough, but probably only two of them are
preformed in the molecule, uracil being formed from the cytosine during
the hydrolysis. These bases were discovered by Kossel. They are thy-
mine, cystosine and uracil.

Thymine. This is 2,6-dioxy, 5-methyl pyrimidine. The structural
formula is as follows:

HN—C=—0

0 = bon, The empirical formula is C.H.N_O,.
wh—bn

It was first isolated from the hydrolytic products of thymic acid obtained
from the thymus gland, hence its name. The pyrimidines are found very
generally in cells not only in nucleic acid but as glucosides. Vicin
and convicin discovered by Ritthausen and Preuss are hexose glucosides
of pyrimidines. Thymine crystallizes from cold water, in which it is
little soluble, in the form of clusters of small leaves or needles. (m.p.
about 321°.) Thymine sublimes undecomposed. It is not readily pre-
cipitated by ammonia and silver nitrate. It is precipitated by phospho-
tungstic acid.
Cytosine. ‘This is 2-oxy, 6-amino pyrimidine or
N=C—NH,
0 =6 on or C,H,N,0
HN — C—H
The free base is little soluble in water and crystallizes in thin plates with
a mother-of-pearl glance. It is precipitated by silver nitrate in the
presence of an excess of barium hydroxide, and by phosphotungstic acid.
It gives the murexide reaction with chlorine water and ammonia. Like
uracil it also gives a violet color (dialuric acid?) when treated with bro-
mine until cloudy and then baryta water added (Wheeler and Johnson).
Uracil is 2,6-dioxy pyrimidine.
HN—C=0

ov OH
wt

The reactions of this base are much like those of cytosine, but it is not
precipitated by phosphotungstic acid. It is only imperfectly precipi-
tated by silver nitrate and baryta water. It crystallizes from water in
clusters of needles. It is nearly insoluble in alcohol and ether. Unlike
THE PROTEINS 169

thymine it does not sublime undecomposed, except on very careful heat-
ing. Generally decomposition takes place with the formation of red
vapors.

Carbohydrate group.—aAll true nucleic acids, or polynucleotides, of
animal origin thus far examined have been found to contain a hexose
group, or several of them; whereas the nucleic acid from yeast and that
from wheat, called tritico-nucleic acid, contain a pentose. Kossel dis-
covered that on hydrolysis the thymus nucleic acid yielded levulinic acid
and formic acid. It does not yield a reducing sugar. The production
of levulinic and formic acid indicated clearly the presence of a hexose,
since, as we have seen in the chapter on carbohydrates, the hexoses yield
these bodies when heated with acid. On the other hand, he found in
yeast nucleic acid on hydrolysis no levulinic acid, but a reducing sugar
which gave large quantities of furfural when distilled. This showed
the carbohydrate in this nucleic acid to be a pentose. Nucleic acids from
fish sperm, thymus, spleen, liver, testes, pancreas, supra-renals, brain,
lining of the alimentary canal and kidneys have all been found to yield
levulinic acid and hence contain hexoses in the molecule. The nature
of this hexose is still uncertain. It gives a saccharic acid (episaccharic
acid) of as yet undetermined nature when the nucleic acid is hydrolyzed
with nitric acid (Steudel). It has recently been suggested (Feulgen)
that it is of the nature of glucal, an aldehyde derivative of glucose,
C,H,,0,. Glueal is an unstable non-toxic substance. When a pentose is
present it is d-ribose.

That the substances thus obtained constitute all that there are in
the nucleic acid molecule is made probable by the recent work of Steudel
and Levene. Steudel by means of his nitric acid method of hydrolysis
obtains nearly a quantitative yield of the purine bases. The phosphoric
acid is casy to determine, but the determinations of the carbohydrate
and the pyrimidines are still far from being quantitative. Steudel gives
the following result of an attempt at a ‘quantitative analysis. It is
assumed that the molecule contains four phosphoric-acid groups; two
purines; two pyrimidines; and four carbohydrate nuclei. He found
28.95 per cent. of the total nitrogen as guanine nitrogen ; 38.42 per cent.
as adenine nitrogen; 11.47 per cent. as cytosine; and 13.11 per cent. as
thymine nitrogen, making a total of 92 per cent. of the whole nitrogen.
As the methods are not exactly quantitative, it is clear that these four
bases are probably the only ones present. The amounts of the bases
isolated and computed were as follows:

Computed s#ouna
Guanine .............. 10.72 9.01
Adenine .............. 9.58 10.68
Cystosine ............. 7.86 4.26

Thymine ...........4. 8.93 8.33
170 PHYSIOLOGICAL CHEMISTRY

As some of the cytosine is unavoidably converted into uracil by the
hydrolysis, the agreement must be considered as very satisfactory.

To determine the carbohydrate he weighed the levulinic acid formed
and computed from the figures of Conrad and Gultzeit how much carbo-
hydrate this amount of levulinic acid represented. His complete analyses
of thymus and sperm nucleic acids were as follows:

Computed for O43Hs7Nis030P,4 Found
88

 

 

GUATINE a seviiel sis oceceeck 3-04 MS FS ERO EG 10.8 8.7
PG ETING, ons sel iniav eee g inwnnan gob: tontec taco it duae trace talon Se 9.73 10.5
PDH TING:. -. ccaissvestndsdosaleanel ae o Reuade-o ote caw leds: alas 9.08 8.2
Cy GOSIMe: aig apes yeas geen eS Sa edaaestetnennmm yen RUG 9.15 4.2
Phosphoric acid .......... cece eee eect eee 20.46 20.31
Hex0se ssa nga ess ete oe eee es Ca as 51. 57.
111.20 108.9

The decomposition may be represented as follows:
Cy gP sng + 8H,0 = C,H,N,O+ CHIN, + C.H,N,O, + C,H.N.O+

42° 5715 4 30 575 6b 56 2 475 8
Nucleic acid. Guanine. Adenine. Thymine. Cytosine.
4C,H, 0 , + 4HPO,

Hexose. Metaphosphoric
acid.

The agreement is as good as could be expected. Nucleic acid consists,
then, of these few building stones and 50 per cent. of the molecule is
carbohydrate. The nature of this carbohydrate of the animal nucleic
acids has not yet been determined beyond the fact that it is a hexose.
It is possibly not always the same hexose.

Structure of the molecule-——We may now take up the problem of
the way in which these smaller molecules are united to build up the big.
Kossel very early suggested that nucleic acid was composed of a polymer-
ized metaphosphoric acid to which the bases and carbohydrates were
attached and structural formulas based on his findings were proposed by
Bang and Osborne and Harris. In these suggestions the backbone of
the molecule consisted of four molecules of phosphoric acid to which the
bases and carbohydrates were attached. The real structure of the mole-
cule has been elucidated largely by the work of Neuberg on the simple
nucleic acid, inosinie acid, and of Bang and Levene and Jacobs on
guanylic acid, and Jones and Levene on yeast nucleic acid. Since the
first two acids have contributed to our understanding of the structure of
the nucleic acid molecule, we may stop and consider them here, although
they are possibly not constituents of the nuclei.

Guanylic acid.—This is an acid belonging to the general group of
nucleic acids, but being less complex than those found in the cell nuclei.
It is a mononucleotide, and may be called guanosine phosphoric acid..
It was isolated by Bang from the ox pancreas and was found by him to
contain no other base than guanine, whence its name, phosporic acid
and a pentose. Bang thought it contained glycerol, but this was incor-
rect. This acid is found in the ox pancreas in addition to the real nucleic
THE PROTEINS 171

acid which we have been considering. It has been obtained also from
the liver and spleen and from yeast. It is best obtained from Ham-
marsten’s nucleoproteid in the following way:

If the fresh pancreas of the ox is hashed and boiled with water,
Hammarsten found that a nucleoproteid went into solution in the
water, from which it could be obtained by slightly acidifying with acetic
acid, the nucleoproteid being precipitated. The gland residue from
which this nucleoproteid has been extracted will yield the true nucleic
acid of the type of those already considered, if treated by Neumann’s
method. The guanylic acid is separated from the nucleoproteid precipi-
tate by redissolving in sodium hydrate, reacidifying, precipitating and
filtering. The filtrate is poured into alcohol. The guanylic acid precipi-
tates as a powder. This guanylic acid Steudel showed contained no
glycerol, no levulinic acid, but only guanine, phosphoric acid and a pen-
tose. Its constitution was worked out by Levene, who succeeded in
isolating from it both a compound of guanine and pentose, a pentoside,
or a nucleoside as he called it, guanosine; and on the other hand a phos-
phoric acid pentose compound. These facts showed that the pentose
was united both to the guanine and to the phosphoric acid and that its
composition was as follows:

0O= C—NH
0 H H H HH |
I elle eal tite 1 pos C—NH
HO—P—0—C—C—C—C—C—c¢ |
: | | | fort \wN  —C-—NH
| | | OH OH
OH H oO

 

Guanylic acid.

While its molecular weight has not been directly determined, the com-
pounds it forms leave little doubt that it is but a single molecule, a mono-
nucleotide as Levene and Jacobs call it. The character of the pentose was
long in doubt, but the authors just mentioned have shown that it is
d-ribose, a levo-rotatory, aldose pentose of the arabinose type not pre-
viously known to occur in animals. The point of union of the sugar
with the guanine is not yet certain, but it is either in purine 7 or 8 as
is figured, and probably the latter, although Burian thought the union
was in number 7. Gwuanosine is, therefore, a pentoside. It may be men-
tioned that the position of the attachment of the phosphoric acid in the
sugar is also uncertain. Guanosine, C,,H,,N;0,, does not reduce
Fehling’s solution until it is decomposed. [a ]*)——60.52.

Guanylic acid has also been separated from ox liver and Jones suc-
* ceeded in getting it from yeast nucleic acid by a quick digestion by an
enzyme, tetra-nucleotidase, found in the pig pancreas. Guanylic acid
is dextro-rotatory.

Inosinic acid.—This is an acid similar to guanylic acid, but it is
172 PHYSIOLOGICAL CHEMISTRY

composed of a molecule of hypoxanthine, a pentose and phosphoric acid.
It was isolated from Liebig’s beef extract and is supposed to occur in
muscle. Whether it does pre-exist in the muscle is probable, but not
certain. It was the study of this acid by Neuberg which really gave the
key to the structure of the nucleic acids. Neuberg thought it had the
formula 0

ae ee
OH ;
But Levene and Jacobs isolated from it a compound called inosine, a
union of pentose and hypoxanthine, showing that inosinic acid must have
a formula similar to guanylic acid. It is not, however, identical in its
structure. From yeast another pentoside was isolated, an adenine pen-
toside called adenosine. Guanosine had already been isolated by Schulze
from plants and called by him vernin. Uridine, C,H,O,.C,H,N,O(OH),
is the ribose uracil nucleoside.

Nucleic acid.—Levene and Jacobs have also isolated other fragments
of the molecule of yeast and thymo-nucleic acid. They conclude from
their work and that of Steudel that the structure of thymus nucleic acid
is probably

OC—NH

|
H H OH 4H _ A ae
oe a oe ean Kt
| duu du da t a
Ho—b—0
mae
O O—P OH
pelle
—G— C—C—C—C—C — Thymine
hound dad
H HH on | H
g—6—4—4_§_o_h
—C —C—C—C—C—C Cytosine.
ot Le ted
0 O—POH
au oo :
1 H.N—C=N
O OH H OH Oh H J
H— es b= bo bo La a —

bi bk fe

vie nucleic acid (Levene and Jacobs).

 
THI PROTEINS 173

This would correspond with Steudel’s formula, C,,H,,N,,O,.P,. Such
a nucleic acid would be a tetra-nucleotide.

While the facts seem to bear out this formula, in its main features
at any rate, it cannot be said that it is as yet conclusively established.
The exact point of attachment of the phosphoric acid to the sugar is still
obscure. The great difficulty of hydrolyzing the di-nucleotide, thymic
acid, seemed to indicate that the union between the pyrimidine nucleo-
tides was not through phosphoric acid, but was an ether-like union. It
will be noticed that the molecule as written in the Levene-Jacob’s formula
is hexabasic. All of the four nucleotides of yeast nucleic acid, i.e.,
adenosine phosphoric acid, cytidine phosphoric acid, guanosine phos-
phoric acid and uridine phosphoric acid, have now been obtained crys-
talline. (Jones and Kennedy; Levene.)

Another possible formula would be the following:

 

 

 

ae ges NH,C=N
H H | H H | 1 |
Pk. tor, a /NH-C CH
C= PO e006 — ee St I
| a ee ee are
OH H OH OH
O :
| H H Wi oH
fe rg A
Os P—O— C—C—C—C—C—C— Thymine
lee et lash
| k OH H OH OH H
0 |
| H H O H H
fo ba RL [lcs
0 = P—O— (0 —C—C—C— C—C— Cytosine
1 | {
| i ber hk OH OH H
0 O = C—NH
H | |
| HH O H NH—C CNH
[i ge) Ei bi es Lae a
0=P—0—Uu—c—c—c—c—c—-vq _ i tt
H OH OH OH H
OH

Nucleic acid. A possible formula.

Does nucleic acid exist outside the nucleus?—There are several very
interesting questions as yet unsolved concerning the location in the cell
of the nucleic acid. It seems probable, though there is nothing really
known about it, that guanylic and inosinie acid may be in the cytoplasm
of the cells in which they occur, though they may be in the nucleus. It
is possible that they do not exist free in the cell, but are united with the
true nucleic acid and are set free by endocellular enzymes. Nothing is
really known about their function or location. Their staining reaction
will probably resemble that of the real nucleic acids. Guanylic acid
gelatinizes much as the nucleic acids, and it was this property that caused
174 PHYSIOLOGICAL CHEMISTRY

Bang to maintain that it must be more complex than a single nucleotide.
Inosinie acid is probably the source of the hypoxanthine of muscle and
it is very interesting that this substance is increased during muscular
contraction.

There can be little doubt that the true nucleic acids, that is the poly-
nucleotides, like thymus nucleic acid, are found only in the nucleus. This
was first indicated by the work of Kossel, who determined the amount
of purine bases obtainable from different tissues. The amount ran pro-
portional to the amount of nuclear material present; it was high in
embryonic tissue; in the thymus; and low in muscle. It is shown also
by the fact that no nucleic acid is found in some unfertilized eggs where
the nuclei are very small'in proportion to the cytoplasm, and none in the
mammalian red blood cells which lack nuclei. On the other hand, nucleic
acid is found wherever nuclei occur, as in the red corpuscles of bird’s
blood which are nucleated. It has never been shown positively to be
a constituent of the cytoplasm, but it is certain that it is found in the
nucleus. It is probable, therefore, that it is confined to the nucleus, but
there are some facts which may be urged against this conclusion. For
example, some believe that nucleic acid is found in the cytoplasm, because
not all the cytoplasmic phosphoric acid in organic union is split off from
its union by sodium hydrate. If the substance in the cytoplasm was a
vitellin, or casein-like compound, it would presumably have been split
off. Nucleic acid, unlike the phosphoproteins, does not split off its phos-
phorie acid when treated by alkali hydrates. And recently nucleic
acid has been found in the sea-urchin’s egg, where the nuclei are very
small. The author got a substance with some of the properties of nucleic
acid in some quantity from unfertilized eggs of the sea-urchin. It could
not be positively identified, however, as the quantity was too small. In
all these cases, then, it is still uncertain whether the substances described
were really nucleins, and the probability is that they did not contain true
nucleic acid. Further work, however, is necessary on this subject before
a definite statement can be made that nucleic acid is never found in the
cytoplasm. It is certain, however, that most of the phosphoric acid
compounds in the cytoplasm are not nucleic acids.

Are all nucleic acids the same?—The question whether all animal
nuclei contain the same, or different, nucleic acids cannot be answered
definitely, since only two of the animal nucleic acids have been accu-
rately examined, namely that of the sperm of herring and from the
thymus gland of calves. These two acids appear to be identical. They
contain the same bases in the same proportions and they have the same
physical properties. Until the nature of the carbohydrate is discov-
ered it is impossible to say whether they contain the same carbohydrate,
but all indications are that these two nucleic acids are identical. Since
they come from such widely different sources, it would indicate that
THE PROTEINS 175

probably the same nucleic acid is found in totally different kinds of cells,
a conclusion of the utmost importance in interpreting the probable. réle
of nucleic acid in the cell. All other nucleic acids of animal origin,
except guanylic and inosinic acids, have been found to yield the same
splitting products when hydrolyzed, so that they must be closely similar
to thymus nucleic acid, if they are not identical with it.

On the other hand, only two plant nucleic acids have been carefully
examined. These are triticonucleic acid from wheat, and yeast nucleic
acid. These are apparently identical, and they differ from the animal
nucleic acids in having d-ribose, a pentose sugar, in the place of a hexose.
They may also differ in other particulars. The composition of neither
of these acids is exactly known, and particularly the molecular weight
has not been determined. Steudel’s analyses indicate that yeast nucleic
acid may be a tri-nucleotide and not a tetra-nucleotide, as Levene thinks.
No one has as yet isolated yeast nucleic acid which on analysis would
yield figures for carbon, phosphorus and nitrogen comparable with a
tetra-nucleotide. But this may be due to the fact that yeast contains a
nucleotidase, and possibly if some of the yeast cells are dead when ana-
lyzed a partial digestion of the nucleic acid may have taken place. Only
fresh, living, active yeast should be used for the preparation of this acid.

Another possibility which complicates the question of the. identity
of the nucleic acids is that in the nucleus we may have a polymer of a
tetranucleotide, as Steudel has suggested for the sperm head. He found,
namely, that the viscosity of the solution of the herring sperm heads in
alkali was greater than an equivalent solution of protamine nucleate ; and
he inferred from this a different state of aggregation of the nucleic acid
outside and inside the cell. It is of course possible that some other
factor than that suggested was responsible for the observed result.

The tentative conclusion may with all reserve be drawn from the fore-
going facts, that the nucleic acids of different nuclei of animal tissues
are certainly closely similar if they are not identical; but that they differ
in their carbohydrate radicles from such plant nucleic acids as have
been examined. It is possible that the hexose component will not be
found to be the same everywhere. Their similarity would clearly indi-
cate that nucleic acids have the same function in all cells. If they inter-
vene actively in cell metabolism, it must be in connection with some
fundamental cell property such as growth, irritability. or respiration
which is common to all cells. It may be, however, that. it has only the
function of a supporting structure, or aids in keeping the physical
viscosity of the nucleus what it has to be. In favor of this view it. may
be mentioned that it is a fairly stable substance, otherwise it could not
accumulate,. and its most probable function would appear to the writer
to be that it serves as a colloidal, gelatinous substratum in. the nature
of an organic skeleton to which the specifically active, more labile,
176 PHYSIOLOGICAL CHEMIEFhyY

albuminous constituents, possibly of a catalytic nature, may be attached.
Forming a firm union with the acid, these active substances may be thus
confined to, or located in, the nucleus from which they may at times get
free. But nothing positive as to its function can be stated without further
investigation.

It is of interest to recall, in view of the foregoing statement, that all
so-called nuclear stains of a basic nature, with the exception of the mor-
danted stains such as iron hematoxylin, combine with the nucleic acid.
In thus following the chromatin and chromosomes by means of these
stains, cytologists, if the view stated above of the significance of nucleic
acid is correct, may be following the inert skeletal material of the nucleus,
while the active albuminous material is entirely neglected for the reason
that it does not gel and does not stain with basic dyes. All theories of
inheritance based on the behavior of the nucleic acid of the nucleus, that
is the behavior and number of the chromosomes, must be accepted only
with the greatest reserve, until the function of this substance may be
shown to be something more than a skeletal substance. We have as yet
no chemical evidence that the different chromosomes have different
nucleic acids in them, but such evidence as we have is contrary to this
view. If the chromosomes do differ chemically, as perhaps their indi-
vidual and peculiar shapes and sizes may indicate, it is more probable,
as we shall shortly see, that they differ in their protein or basic rather
than in their acid moieties.

THE BASIC CONSTITUENTS OF THE NUCLEUS.—Nucleie
acid is either a hexa- or tetra-basic acid, probably the former; and it
forms a series of salts. We have now to ask the question with what basic
substances is nucleic acid united in the chromatin? Are the bases organic
or inorganic?

It is probable that some inorganic bases, i.e., calcium, are present;
it is certain that organic bases of a protein nature are always present.
The only nuclei carefully examined in a clean form, free from cytoplasm,
are the sperm heads, and possibly the nuclei of birds’ corpuscles. These
always yield some calcium phosphate when dissolved or ashed. It seems
certain that calcium is generally present. MacCallum, from cytological,
microchemical studies, has concluded that nuclei contain no potassium,
since around the outside of the nucleus he generally obtains a deposit of
potassium-cobalto nitrite by his method, but none in the nucleus. But to
his conclusion it may be objected that the place where the precipitate
forms is not necessarily indicative of the location of the soluble salt.
Tnere is, indeed, very little evidence of what inorganic salts or bases we
have in the nucleus itself. This question must be left for further work.
It appears, from some recent work, that iron, contrary to an earlier view,
is not present in all nuclei.
THE PROTEINS 177

The organic bases which occur in some chromatins are among the
most interesting substances in the cell, whether considered from the
physiological or chemical point of view. Our knowledge of these
bases, the study of which gave Kossel the clew to the constitution
of the proteins, we owe chiefly to Kossel and Miescher and pre-eminently
to the former. These bases are protein in nature and consist either
of basic proteins called protamines or histones, or of other more com-
plex proteins.

The protamines.—If the sperm heads of the salmon, sturgeon, her-
ring and other fishes are extracted with 10 per cent. sulphuric acid, or
hydrochloric acid, there goes into solution about 19 per cent. of the dry,
alcohol- and ether-extracted heads. The nucleic acid remains behind
more or less altered and insoluble. Three extractions of the heads with
10 per cent. sulphuric acid for about half an hour at a time will take out
practically all of the removable base. The substance which goes into
solution as a sulphate is of a protein nature; when precipitated by alcohol
as the sulphate it is a white, somewhat hygroscopic, amorphous powder,
giving, in the case of the herring, salmon and sturgeon sperm, no Millon,
or xanthoproteic, or tryptophane reaction, but a good biuret reaction.
This substance was named protamine by its discoverer, Miescher, who
obtained it from salmon sperm (Gr. protos, first, amine). The protamine
from salmon is called salmin.

General properties. The protamines, although individually different,
have the following properties in common: In the free state all are strong
bases, alkaline to litmus, and not precipitated by ammonia. They give
a splendid biuret test, but Millon, xanthoproteic or Adamkiewicz reac-
tions are in many cases negative, but in some protamines positive. They
are digestible by trypsin, but not by pepsin-hydrochloric acid; they are
readily soluble in water, but not in alcohol, and their sulphates separate
as an oil when the saturated aqueous solution is shaken with ether. They
are not coagulated or changed by heating. They precipitate proteins by
uniting with them in ammoniacal solution, and this is a very delicate
test for them. In this respect they act like metallic bases. Unlike most
proteins, they are precipitated from a neutral solution by neutral solu-
tions of sodium picrate, ferrocyanide or tungstate, and they may even
be precipitated in faintly alkaline solutions. The reason for this pecu-
liarity has already been explained. They are such strong bases that their
molecules are electro-positive even in faintly alkaline solutions. On
analysis they consist of carbon, hydrogen, oxygen and nitrogen, but they
contain no sulphur. The elementary analyses of some are as follows:

Cc H N oO Ft cl
Clupein ......... te. 47.93 7.59 31.68 12.78 — — Free base.
Salmin ............ 22.96 4.32 14.83 6.7 24.73 26.56 Plat. chloride salt.
Sturin .........---. 24.32 4.49 14.20 847 23.10 25.42
178 PHYSIOLOGICAL CHEMISTRY

The formula for salmin is probably C,,H,,,N,,0..; that for sturin,
CygHggN,,0,. The molecular weight is not yet determined.

The protamines differ from all other proteins in the small number of
different amino-acids they yield on hydrolysis and in the character of
these acids. Kossel found that salmin, one of the simplest, yielded 87
per cent. of its molecule as arginine, and it was this discovery which sug-
gested to him the constitution of the proteins. The composition of the
hydrolytic, cleavage products of numerous protamines is given on
page 128.

Does the sperm chromatin consist exclusively of protamine
nucleinate?—The chromatin of the sperm head is supposed to be the
bearer of the hereditary qualities and zodlogists have pictured it as com-
posed of individual units, biophores or determinants, each of which rep-
resents some specific unit-character of the adult. If this hypothesis
were true, we should expect the sperm chroriatin to be extremely com-
plex; more complex indeed than any chromatin in the body, since it is
supposed to represent them all. As a matter of fact, chemical examina-
tion shows this chromatin in the fish sperm to be the simplest found any-
where. The heads of the herring sperm do not contain any tyrosine;
they give no Millon, xanthoproteic or tryptophane test. They contain no
coagulable protein. They have the following composition after extrac-
tion with alcohol and ether:

Average
C 40.99—41.48 41.20
H 5.62— 5.83 5.75
N 20.78—21.44 21.06
P  5.87— 6.33 6.07

Steudel has recently confirmed these figures. Accepting his formula for
‘the composition of nucleic acid, C,,H,,N,,P,0,,, and Kossel’s formula for
clupein, or salmin, C,,H,,N,,0,, there would be required for protamine
nucleate:

Computed for
CysHiraNs20s5P4 Homa
C 40.97 41.24
H 5.33 5.27
N_ 20.95 21.09
P_ 5.80 6.02
O 26.95 26.37

This formula requires 64.8 per cent. nucleic acid and 35.2 per cent,
protamine. He actually isolated 93 per cent. of the calculated amounts
of each of these substances and the deficit was undoubtedly due to the
fact that the methods are not entirely exact. There can be no doubt,
therefore, that the chromatin of herring sperm when fully ripe consists
of a neutral salt of protamine nucleate. Miescher found very similar
relationships in the salmon sperm, the head consisting largely or wholly
of salmin nucleate. The white fish sperm head has the composition:
Covi ssN5s1Ov2 (CygHs2N1sP,On0) «—24H,0.
THE PROTEINS 179

Nature of the union of protamine and nucleic acid. The ease with
which the protamine may be separated from the nucleic acid by acids
or alkalies indicates clearly that the two are in a salt-like union. Prob-
ably the union is between the free amino groups at the end of the chain
of the arginine and the acid radicle of the nucleic acid (Steudel). By
extracting first with alkali these free amino groups of the arginine of
the salmin are decomposed, ammonia being set free and ornithine re-
maining. If, now, the compound is acidified a reunion of the nucleic acid
and protamin does not take place. This is the probable basis of the
Neumann method of preparing nucleic acid. But there can be equally
little doubt that we often have other than salt unions between the pro-
tein and nucleic acid. It is impossible to extract all the protein from
the nuclei of all cells by acid. The union is too firm.

Other basic constituents. Histone. In the sperm of the sea urchin,
Arbacia, the author isolated by acid extraction a basic protein resem-
bling histone in some particulars and protamine in others. About 11 per
cent. by weight of the alcohol and ether extracted, dried whole sperm
was extracted by acid. The arbacin sulphate contained 15.91 per cent.
of nitrogen, whereas protamine sulphate contains about 25.13 per cent.
In this experiment the sperm heads were not separated from the tails.
The substance was not a true histone, for it did not precipitate with
ammonia, except very incompletely. Nucleic acid was also isolated.
Arbacin was strongly basic and gave the Millon test. Only a small pro-
portion of the protein could be extracted by acid from the sperm, indi-
cating that not all of it was in a salt union, or else that the tails made
a very considerable proportion of the whole.

The chromatin of both thymus gland and bird’s blood corpuscles con-
tains a basic, simple protein, histone, in a salt union with nucleic acid.
This fact was also discovered by Kossel. These nuclei have been recently
obtained and studied by Ackermann.

The method of isolating the nuclei has already been given (page 162).
The dried nuclei after alcohol and ether extraction contained 3.93 per
cent. P; 17.20 per cent. N. If Steudel’s formula for nucleic acid is used
in place of the formula employed by Ackermann, it is computed from the
phosphorus that the nuclei contain 43.96 per cent. nucleic acid and
56.04 per cent. of histone, if they contain only histone nucleate. From
Steudel’s formula nucleic acid contains 15.18 per cent. of N. Hence in
100 grams of the nuclei containing 17.2 grams of nitrogen, 6.67 grams
are in the nucleic acid and 10.53 grams in the histone. Since histone
contains 18.3 per cent. N, the nuclei must contain 57.5 per cent. of histone.
Both nitrogen and phosphorus indicate, therefore, that the nuclei con-
tain 43-44 per cent. of nucleic acid and 56-57 per cent. of histone. Acker-
mann actually extracted by hydrochloric acid (1 per cent.) 63.9 per cent.
180 PHYSIOLOGICAL CHEMISTRY

(53.9?) of histone, leaving 46.1 per cent. insoluble nucleic acid instead
of about 44 per cent. Some purine bases undoubtedly went into solution
and the residue contained only 7.79-7.99 per cent. of P and 15 per cent.
of N, so that some histone may have been left unextracted. Although
these figures do not check exactly, the method not being quantitative, it
is clear, nevertheless, that these nuclei consist chiefly, if not entirely,
of histone nucleate, and contain no other protein substance in any quan-
tity. If the molecular weight of nucleic acid is 1,387 and that of histone
about -1,600, which is the simplest formula which can be ascribed to it,
a molecule of chromatin might be simply histone nucleate containing one
molecule of each substance.

It is greatly to be desired that studies similar to these should be made
on other tissues so that we may have a more accurate knowledge of the
composition of the chromatin of as many cells as possible. Only when
this is done will physiological chemistry be able to contribute to the vexed
and vexing question of chromosomal! inheritance.

Concerning the nature of the simple protein united with nucleic acid
in other nuclei than these few kinds, nothing is known. Basic proteins
corresponding to histone and protamine have not been isolated from other
cells than those mentioned.

Enzymes in the nucleus.—Many nuclei, and particularly the large
germinal vesicles of starfish eggs when unripe (Asterias vulgaris, etc.)
contain very little of the morphological substance called chromatin. The
greater part of these nuclei consists of a liquid sap which contains protein
matter, if we may conclude from the fine precipitate produced in it by
fixing agents such as mercuric chloride. No one has yet obtained this
nuclear sap for chemical analysis, but there is no question that its admix-
ture with the extra-nuclear cytoplasm produces marked chemical changes
in the latter and greatly stimulates cell respiration. Delage, Loeb and
the author have particularly studied the changes so produced. If unripe
or immature eggs in which the germinal vesicle is intact are placed in
sea-water, some of the eggs rupture the nuclear membrane and the
nuclear sap mixes with the cytoplasm. Some eggs do not rupture the
nucleus spontaneously, but they may be made to do so artificially by
shaking. Eggs in which the nuclear sap has penetrated the cell cyto-
plasm behave very differently from eggs in which the nuclear sap remains
in the nucleus. If rupture of the membrane takes place, the eggs become
very sensitive to oxygen and they will only live about 10-18 hours in
oxygenated sea-water. At the end of that time the protoplasm becomes
opaque and seems filled with a multitude of spherules, the protoplasm
being disintegrated into these spherules. If, however, the nuclear sap
does not penetrate the cell cytoplasm and the nuclear membrane remains
intact, or if the eggs after the nucleus and cytoplasm are mixed are
THE PROTEINS 181

placed in an atmosphere of hydrogen, or if they are slightly poisoned by
potassium cyanide which prevents oxidation, the eggs remain alive for
several days. It is very clear from this experiment that when the nuclear
wall is ruptured either naturally or by mechanical means, the eggs
become very sensitive to oxygen and, if not protected by fertilization,
they will rapidly die in the presence of oxygen. The most probable
explanation of these facts is that substances are present in the nuclear
sap which when mixed with the protoplasm cause the mixture to undergo
auto-oxidation leading, if not checked, to death. A simple, though per-
haps not a correct, way of stating these facts is that the nuclear sap
contains oxidases, or substanves which stimulate respiration.

The change in the cytoplasm produced by this admixture of nuclear
sap is also made visible in other ways than by oxidative changes. Some-
times spermatozoa penetrate eggs which do not maturate and in which
the nuclear wall remains intact. In that case no typical aster develops
about the advancing sperm. but only the faintest radiations about the
sperm nucleus. This may be the case even though the sperm is lying
close to the germinal vesicle. If, however, it enters an egg which has
lost the nuclear wall so that the nuclear sap can escape, the typical
big asters develop at once about the sperm, provided the eggs have
oxygen.

Similar facts have been recorded by Delage. If a piece of proto-
plasm cut from an egg in which the nucleus is intact be entered by a
spermatozoon, no division figure is developed. If, however, a sperm
enters a piece cut from an egg in which the nuclear membrane has been
ruptured, then the large normal sperm aster develops. It is clear that
the change in the cytoplasm produced by the nuclear admixture enables
the sperm to produce its typical effects. Inasmuch as these astral figures
are dependent for their existence upon a supply of oxygen and disappear
if the eggs are placed in hydrogen. reappearing again when they are
returned to oxygen, their behavior again indicates the important part
the nuclear sap plays in. respiration. Yatsu found that nucleus-free
pieces of Cerebratulus eggs, if cut off from the eggs before maturation
occurred, would not develop asters when treated by strong magnesium
chloride solutions, whereas similar pieces cut after maturation would
develop them. .

A very similar phenomenon illustrating the importance of the nuclear
sap is shown in the first segmentation of the egg of the sea urchins,
Arbacia and Toxopneustes. Wilson and the author observed that a
marked pause in the segmentation process occurs just before the segmen-
tation. The nuclear wall of the big segmentation nucleus is at that time
intact. The large segmentation asters fade out, except near the nucleus.
Suddenly the nuclear wall breaks at the two poles close to the asters. It
182 PHYSIOLOGICAL CHEMISTRY

uppears to be dissolved or digested away. By this means the nuclear sap
and the asters may come into contact; and coincident with this, the great
radiations of the asters burst forth in full magnificence, their streamers,
like a. miniature aurora borealis, flung wide throughout the cell, and cell
division is rapidly consummated. Just at this time, too, there is a sud-
den outburst of carbon dioxide and the cell becomes extremely sensitive
to ether, cyanides, acids and other poisons, a fact clearly indicative that
the protoplasm is in a very reactive and unstable condition.

All these facts indicate in no uncertain manner that substances are
present in the nuclear sap which on entering the cytoplasm produce chem-
ical changes there. Not only are respiratory changes stimulated many
fold, but also digestion seems to be inaugurated. Autolytic enzymes
also evidently become active, either because they are set free from the
nucleus, or because the nuclear materials activate, directly or indirectly,
the inactive enzymes of the cytoplasm. Many yolk granules are dissolved
and the nucleolus also dissolves and disappears; the nuclear membrane
suffers a like fate and the chromatin itself, which has been more
voluminous and less avid for basic dyes, diminishes in bulk and increases
its staining power as if a considerable amount of protein had been
digested or separated from it. It is also well known that the unfertilized
eggs of hens keep much better and do not undergo autolytie digestion
as do the fertilized eggs. These phenomena speak for the presence in the
nucleus of oxidases and digestive enzymes. Since during cell division
these enzymes are set free and at the same time the chromatic elements
are in many cases plainly losing substance, it is possible that these two
facts should be correlated and the conclusion drawn that in the resting
condition of the nucleus enzymes of various kinds stick to, or combine
with, the nucleic acid and are thus accumulated, made resistant, more
stable and rendered inert, and that during caryokinesis, and possibly
at other times also, they are split off from the acid, become free in the
sap, enter the cytoplasm and rejuvenate the cell by digesting its aceumu-
lated colloidal material. Possibly the guanylic acid may, as an extra
nuclear material, combine with the trypsin of the pancreas to make the
inactive trypsinogen. Possibly there are also within the nucleus some
of the nucleases which digest nucleic acid itself.

These few remarks will serve to illustrate the attractiveness, the
importance and the obscurity of the field of the composition and func-
tion of the cell nucleus. Possibly they may stimulate some to the inves-
tigation of a subject of which the importance is only commensurate with
our ignorance. We may in this connection recall the fact that it has
been suggested (Gautier) that immediately about the nucleus there takes
place something of the nature of an anerobic fermentation of the food
materials, by which CO, is produced and many active fragments are
THE PROTEINS 183

formed which later in the periphery of the cell are oxidized by the enter-
ing oxygen, or condense to other compounds.

The formation and destruction of nuclear material.—We may close
this chapter on the composition of the nucleus with a brief review of
what is known concerning the formation and destruction of nuclear
material. From what substances does a cell make nucleic acid, or prota-
mine or histone? And what are the substances formed from its
disintegration ?

Origin of the proteins and nucleic acid. We will consider first the
origin of nucleic acid, since this is the simpler problem. The question
is then this: From what substances and in what manner is nucleic acid
formed in cells? There are certain aspects of this question which can be
definitely answered. There is evidence that the source of the phosphoric
acid is inorganic phosphates. It is known that phosphates are necessary
ingredients of the foods of all animals and all plants. Indeed this acid
has quite a peculiar position in the cell. It not only enters into the com-
position of many of the proteins and of all nucleic acids of which it
appears to form the backbone as it were, but in the phospholipins it no
doubt contributes powerfully to the production of vital phenomena. It
plays an important part in the maintenance of the neutrality of the
protoplasm and in the activity of many enzymes. The acid owes its
fundamental importance in metabolism probably to its power of polymer-
izing in the form of metaphosphoric acid, HPO,, and, in the second place,
to its power of forming ester unions with carbohydrate and other sub-
stances. It has in this regard a power only second to that of boric acid.
By this power it forms the basis of nucleic acid, for at the bottom of this
acid is the ester of phosphoric acid with either a pentose or an unknown
hexose. This same property of forming esters with carbohydrates is
shown at its best in the case of inosite, which is found probably in all
cells combined with several molecules of phosphoric acid as in phytin,
which is the hexa-phosphoric acid ester. (See page 613.) This part of
the molecule of nucleic acid offers no difficulty for an understanding of
the method of its formation, although we are not yet certain of the exact
steps in the process. The formation of the purine and pyrimidine bases,
however, is a somewhat more difficult problem. It has recently been dis-
eussed by Johnson. Since the pyrimidines are the simpler bases, we will
assume that the purines are formed from them.

There is no doubt that all cells, animal as well as plant, can make
their purines without being fed purines. Whether they can all make
pyrimidine also is not entirely certain, but there is no doubt that plants
have this power and it is probable that animals have it also. In milk
or in the yolk and white of egg there are neither purines nor pyrimidines
in more than extremely small amounts, and yet the developing organism
184 ; PHYSIOLOGICaL CHEMISTRY

nourished by these foods makes both of these substances at a very rapid
rate. Birds and reptiles, too, can certainly form purine, uric acid, from
amino-acids of various kinds, so that there is no question but that they
have the power of synthesizing these bases from the simplest compounds,
and probably from carbohydrates and ammonia.

Hydrocyanic acid, HCN, is one of the most reactive of substances. It
is found combined in a great many plants. Its great importance in the
synthesis of living matter was clearly recognized by Gautier. Hydro-
cyanic acid dissolved in water and allowed to stand gives rise to many
substances found in living matter. Urea, alanine, carbamic acid, cya-
nates and, according to Gautier, substances related to xanthines or really
xanthines, although this is denied by Fischer, appear in it. It has been
repeatedly suggested that this substance may have been a very important
contributor to the formation of living matter in the first instance. Three
molecules of hydrocyanic acid will condense to form the amino-malonic
nitrile,

CN
3HCN —~+ H x tn

by

Amino-malonic nitrile.
This nitrile might condense directly with urea to form a pyrimidine; or
it might be hydrolyzed first to form the amino-malonic acid,
H,N.CH (CN), +4H,0 _— NH,.CH(COOH), + 2NH,
Amino-malonic acid.

The acid inight then condense with urea or guanidine to give a pyrimi-
dine. If the condensation is with the nitrile, a diamino pyrimidine is
the result; if with the acid, uramil, an amino-oxy-pyrimidine results:

NH, NC NH—CO
| | fee
CO +HCNH, —~* CO CNH, + NH,
| * tl
tn, NC hove,
Dioxy-diamino pyrimidine.
NH HOOC NH—CO
| Ls
bo + vse — bo oe + 2H,0
NH, HOOC Nu—do
Uramil.

The condensation of either of these bodies with another molecule of urea
to form a purine is analogous to numerous syntheses in living matter,
although we do not know just how they are produced. The reaction may
be represented as follows:
THE PROTEINS 185

NH—CO NH—CO
\ | | |
co rie +NH, —- oe Looe NH,OH
ba bo nH, >00 NH—O—N
Uric acid.

The synthesis could as readily go through alloxan which might be formed
from glyoxalcarbonic acid formed from the carbohydrate decomposi-
tion. It has been shown that glucose when it decomposes in weakly
alkaline solution forms some glyoxalcarbonic acid. With ammonia this
will condense with formic aldehyde to make an imidazole as follows:

COH HC—NH

| NH, Il cH

co + +H,co —~ C—N

| NH |

COOH 5 COOH
Glyoxalearbonic acid. Imidazolylearbonic acid.

A similar condensation might occur with urea:

COH NH, N—CH

| | od

co +co —-0CG CO+ H,0
| | | |

COOH NH, HN — CO

Trioxypyrimidine.

The trioxypyrimidine by oxidation could give alloxan which by con-
densation with urea might yield a purine.

Another possible source of pyrimidine would be by oxidation of
arginine to guanidine propionic acid and the condensation of this body
to an amino pyrimidine:

NH, COOH NH—CO
| | | |

a cH, — a ae
NH—— CH, NH—CH,

Guanidine propionic acid.

This formation would be analogous to the formation of creatinine from
creatine, page 706.

While the exact course of the formation in the cell is thus obscure,
there are no great difficulties in imagining how the condensation might
occur in the presence of ammonia, or urea or hydrocyanie acid or for-
mamide and the reactive decomposition products of the carbohydrates.
Whatever may be the exact steps in the process, it may be regarded as
probable that they, like the amino-acids, are formed by the condensation
of ammonia with the reactive decomposition products of the carbohy-
drates. Essentially, therefore, speaking broadly, the proteins and the
186 PHYSIOLOGICAL CHEMISTRY

nucleins arise by the condensation of the decomposition products of the
carbohydrates with ammonia. It may be added further that, in order
that the proper decomposition products shall be formed from the carbo-
hydrates, it is necessary that the reaction shall be guided or directed, and
that this is probably accomplished by the presence in cells of accelerating
agents, or enzymes, which hasten one reaction or another, the particular
reaction differing in different cells, so that the proper decomposition
products shall occur in the proper amounts.

Origin of the amino-acids. The amino-acids of the animal body are
obtained chiefly as the products of the digestion of plant proteins,
but the animal organism has certainly the power of making some of
them from ketonic acids, like pyruvic acid and ammonia, a subject dis-
cussed on p. 818. To what extent animals have this power of making
amino-acids from ketonie acids and ammonia, or in any other way, is
still being investigated and no certain answer to the problem can be
given at the present time. While it appears that animal protoplasm has
in general the same chemical properties as plant, there is no doubt that
this power of manufacture of amino-acids which is so noteworthy a
property of plant life is reduced certainly to a very subordinate power
in the animal, for it appears necessary to supply most animals
with ready-made amino-acids. The plant amino-acids are almost cer-
tainly derived in the long run and in large measure from ammonia and
carbohydrates. By the fermentation of glucose, or when glucose is
decomposed by alkalies and presumably by the processes of plant
metabolism, various ketonic aldehydes, such as pyruvic aldehyde, are
produced. Pyruvic acid, CH,—CO-—-COOH, is thus formed or glyoxylic
acid, HCO—COOH. Ammonia, derived from the nitrates which are
reduced in the plant protoplasm, condenses with these compounds to form
imino compounds which by reduction yield amino-acids, thus

CH,—CO—COOH + NH, ——- CH,—CNH—COOH +H 20
CH 3s -CNH—COOH + H, —~> CH 3 ~CHNH coor
Alanine.
HCO—COOH + NH, —~ HCNH—COOH + H,0
CHNH—COOH + H, — CH,NH, —COOH
Glycocoll.
By a similar reaction guanidine, one of the constituents of arginine and
guanine, may arise from urea and ammonia:
0 =C(NH,), + NH, —-HNC(NH,), + H,0
Urea. Guanidine.
The origin of proline from glutamic acid has already been indicated
(p. 124). The exact method in which the other amino-acids arise in the
plant is still uncertain, but it is probable that they are formed for the
most part from the degradation products of the sugars uniting with
THE PROTEINS 187

amnionia. Light, or at least chlorophyll, is nol necessary for this syn-
thesis, since many of the bacteria and moulds which are free from chloro-
phyll can make many different amino-acids from a single source of
ammonia such as asparagine and some carbohydrate. Imidazole groups
may be formed by long contact of ammonia, glucose and oxygen, or an
oxidizing agent, from glyoxal carbonic acid:

COH NH, HC-—NH

| + + H,CO—-~ || g CH+3H,0
c=0 NH, c—N

has l

CcooH COOH

REFERENCES. Prorerins AND CHEMISTRY OF NUCLEUS.

{. General Works: The Vegetable Proteins. Osborne: Monographs on Biochem-

istry. Longmans, Green and Co. 1909. London.

Chemical Constitution of Proteins. Plimmer: Part 1, 1912;
Part 2, 1913. Monographs on Biochemistry. Edited by
Plimmer and Hopkins. London.

General Characters of Proteins. Schryver: Monographs on
Biochemistry, 1910.

Protamines and Histones. A. Kossel: Monographs on Bio-
chemistry. Longmans, Green and Co. 1914.

Chemistry of Proteins. Mann.

2. Nucleic acid. Composition. General. Nucleic Acids. Their Chemical Prop-
erties and Physiological Behavior. W. Jones: Monographs on
Biochemistry. Longmans, Green and Co. 1914.

3. nt ae Composition. First.work, Altmann: Arch. f. Anat. u. Physiol.
p. 526, 1889. Kossel: ibid., 1893, p. 157.

4, ff “Composition. Levene: Journal of the Amer. Chem. Soc., 32, p.
231, 1910.

5. ss “ Decomposition by nitric acid. Steudel: Zeit. f. physiol. Chem.,
49, p. 406, 1906; 48, p. 425, 1906; 53, p. 14, 1907.

6. ee “Formula. Thymus nucleic acid. Levene and Jacobs: Jour. Biol.
Chem., 12, p. 411, 1912.

7. s§ s Yeast nucleic acid. Levene and Jacobs: Ber d. d. chem. Gestll.
42, p. 2474, 1909; 48, p. 3150, 1910; 44, p. 1027, 1911.
Kowalewsky: Zeit. f. physiol. Chem., 69, p. 240.

8. & “ Formation of guanylic acid from yeast nucleic acid. Jones:
Jour. Biol. Chem. 12, p. 31, 1912.

9. < “  Inosinie acid. Levene and Jacobs: Ber. d. d. chem. Gesell. 44,
p. 746, 1911.

10. $ “ — @uanylic acid. Steudel: Zeit. f. physiol. Chem. 80, p. 40, 1910.
Levene and Jacobs: Jour. Biol. Chem. 12, 1912.

11. . “« Pyrimidine nucleosides. Levene and LaFarge: Ber. d. d. chem.
Gesell. 45, p. 608. Johnson and Chernoff: Jour. Biol. Chem. 14,
p. 307, 1913.

12. « “ Purine hexose compound. Mandel and Dunham: Jour. Biol.

Chem. 11, p. 85, 1912.
“ “ Yeast nucleotides. Levene: J. Biol. Chem. 41, p. 483, 1920; Jones
and Kennedy: J. Pharm. and Expl. Therap., xiii, p. 45, 1919.

.
188

13.

14.

15.

16.

17.

18.
19.

20.

21.

22.

23.
24,

26.

27.

28.

29,

30.

31,

32,

33.

Nucleic

PHYSLOLOGICAL CHEMISTRY

acid. Guanosine hewoside from thymus nucleic acid. Levene ‘and
Jacobs: Jour. Biol. Chem. 12, p. 377, 1912.
= Carbohydrate group in molecule.. Steudel- Zeit. f. physiol. Chem.
55, p. 407, 1908; 56, p. 212, 1908. Feulgen: Zeit. f. physiol.
Chem. 92, p. 154, 1914.
Pentose group. Levene and Jacobs: Ber. d. d. chem. Gesell.
43, p. 3147, 1910.
= Wheat nucleic acid. Levene and Jacobs: Ber. d. d. chem. Gesell.
43, p. 3164, 1910.
Relation to glucosidal enzymes. Tschermorutzky: Zeit. f. phys.
Chem. 80, p. 298, 1912.
Decomposition by enzymes. See chapter on uric acid.

Nucleoproteid, of pig’s liver. Scaffadi: Zeit. f. physiol. Chem., 58, 1908-9, p.

272.

Nucleus.

“

Function. Gruber: Biologische u. expt. Untersuchungen an Amoeba
proeteus. Archiv. f. Protistenkunde 25, pp. 316-374, 1912.

Chemistry. Kossel: Ueber die chemische Beschaffenheit des Zell-
kerns. Miinchener med. Wochenschrift, 2, 1911. Nobel prize ad-
dress, 1910.

Chemistry. Mathews: Sperm nucleus. Zeits. f. physiol. Chem. 23,
1897. Birds’ blood corpuscles. Ackermann: Zeits. f. physiol. Chem.
43, 1904-5, p.299. Nuclei of thymus. Abderhalden and Kashiwado:
Ibid., 81, 1912, p. 285. Sperm. Steudel: Zeits. f. physiol. Chem.
83, 1913, p. 72.

Calcium content. Horkammer: Biochem. Zeitschrift, 39, 1912, p. 271.

Iron content. Masing: Zeits. f. physiol. Chem. 66, 1910, p. 262.

Nucleic acid in eggs. Tscherroutzky: Zeits. f. physiol. Chem., 80,
1912, p. 194. Plimmer and Scott: Journal of Physiology, 38, p. 247.
Levene and Mandel: Zeits. f. physiol. Chem. 49, p. 262. Masing:
Ibid., 75, 1911, p. 135; 67, 1910, p. 161.

Nucleic acid. Purines. Origin of purines in plants. Johnson: Jour. Amer.
Chem Soc., 36, 1914, p. 337.

Pyrimidine compounds. Physiological action. Kleiner: Jour. Biol. Chem.,
11, 1912, p. 443.

Relation of nucleic acid to stains, Feulgen: Zeits. f.. physiol. Chem., 80, 1912,
p. 73..

Nature of free amino groups in proteins. Van Slyke and Birchard: Jour.

Biol.

Chem., 16, p. 539, 1913. Kossel and Cameron: Zeits. f. physiol. Chem.,

76, 1912, p. 457. Kossel and Gawrilow: Ibid., 81, 6. 274, 1912. Skraup:
Annalen der Chemie, cccli, p. 379, 1906. Abderhalden and Van Slyke: Zeits.
f. physiol. Chem., 74, p. 505, 1911.

Racemization of proteins, Dakin and Dudley: Casein. Jour. Biol. Chem., 15,
p- 263, 1913; 13, p. 357, 1912. Kossel: Zeits. f. physiol. Chem., 72, p. 486,
1911; 78, p. 402, 1912; 84, p. 1, 1913.

Racemized protein not digestible by enzymes. Dakin and Dudley: Jour.

Biol.

Chem., 15, p. 271, 1913.

Racemized protein does not cause anaphylaxis. Ten Broeck: Jour. Biol.
Chem., 17, p. 369, 1914.

Anaphylaxis by synthetic polypeptide. Abderhalden: Zeits. f. physiol. Chem.,
81, 1912.
34,

36.

37.

38.
39.

40.
41.

42.

43.

44.
45,

46.

47.

48.

52.

54.

55.

THE PROTEINS 189

Conversion of amino-acids into ketonic aldehydes. Dakin: Jour. Chem. Soc.,
1913.

Protamin. Kossel: Zeits. f. physiol. Chem., 69, 1910, p. 138.

Amines from amino-acids. Bickel and Pawlow: p-oxypheny] ethyl amine.
Biochem. Zeitschrift 47, 1912.

Aporrhegmas. Ackermann and Kutscher: Zeits. f. physiol. Chem., 69, p. 265,
1910.

Agmatine. Kossel: Zeits. f. physiol. Chem., 68, p. 170, 1910.

p-Oxyphenyl amino butyric acid. Goldschmidt: Monatshefte f. Chemie., 38, p.
1379, 1912.

Dioxytyrosin. Guggenheim: Zeits. f. physiol. Chem., 88, 1913, p. 276.

Quantitative determination of hexone bases. Weiss: Zeits. f. physiol. Chem.,
52, p. 109, 1908. Van Slyke: Jour. Biol. Chem., 10, 1911, p. 15.

Molecular weight of hemoglobin. Hiifner and Gansser: Archiv. f. Physiol.,
1907, p. 209.

Heat coagulation of oxyhemoglobin. Chick and Martin: Journal of Physi-
ology, 40, p. 404, 1910.

Casein. Isoelectric point. Michaelis: Biochem. Zeitschrift 47, p. 260, 1912.

Gelatin. Isoelectric point. Michaelis and Grineff: Biochem. Zeits. 41, 1912,
p. 373.

Separation of gelatin from other substances. Berrar: Biochem. Zeits. 47,
1912, p. 189.

Precipitation by half saturation with (NH,),SO,. Wiener: Zeits. f. physiol.
Chem., 74, 1911, p. 29

Carbamino reaction. Siegfried: Zeit. f. physiol. Chem., 44, p. 85, 1905; 46, pp.
401-414, 1905; 54, pp. 423, 437, 1908; 58, p. 84, 1908. Ergebnisse der
Physiologie, 9, pp. 334-350, 1910.

Amides and imides of amino-acids. Bergel and Boll: Zeit. f. physiol. Chem.,
76, p. 464, 1912. (Earlier papers cited here.)

Biuret reaction. Schiff: Berichte d. d. chem. Gesell., 29, p. 298, 1896.

Anhydrides of amino-acids. Grimaux: Sur des colloides azotés. Bull. soc.
chim. 1882, (2), 38, p. 64. Schiff: Ueber Polyaspartsauren. Annalen der
Chem., 310, p. 301, 1899. Schiitzenberger: Essai sur la synthése des
materiés proteiques. C. Rend. 112, p 198, 1891.

Polypeptides by partial hydrolysis of proteins. Abderhalden: Zeits. physiol.
Chem., 65, p. 417, 1910. See also various articles by Siegfried on “ Kyrins ”
in the Zeits. f. physiol. Chem.

Physical Chemistry of Proteins. Ueber die Verbindungen der Proteine mit
anorganischen Substanzen und ihre Bedeutung fiir die Lebensvorgiinge.
Robertson: Ergebnisse der Physiol., Asher and Spiro, 10, 1910, pp. 216-361;
Physical Chemistry of the Proteins.

Diazo Reaction. Pauly: Zur Kenntnis der Diazoreaktion des Eiweisses.
Zeit. f. physiol. Chem., 94, p. 284, 1915.

New method of separation of amino-acids from hydrolysed proteins.
Dakin: Biochem. Jour., xii, 1918.
CHAPTER V.
THE PHYSICAL CHEMISTRY OF PROTOPLASM.

Thus far we have considered the general composition of living matter
and the chemical nature and origin of the carbohydrates, fats and pro-
teins which make up the larger part of the organic basis of the cell,
furnish energy for its vital activities and form its machinery. Knowl-
edge of the chemical composition of these bodies does not enable us to
understand how they can produce vital phenomena. For this it is neces-
sary to understand not only their chemical composition, but also their
physics or dynamics. In this chapter the physical chemistry of the cell
will be considered, since physical chemistry is the science which deals
with the explanations of chemical reactions. 5

Water.—The most abundant element of the cell is water. 70-93 per
cent. of the protoplasm is water. To understand vital mechanics, knowl-
edge must be had of the properties and possibilities of water. What is
it doing in the cell? What does water contribute to the complex of life?
What is water? It is a singular fact that the exact composition of this
abundant substance, a sine qua non of life, is not yet known. That water
decomposes into hydrogen and oxygen and that there are very nearly,
if not exactly, two volumes of hydrogen liberated to one of oxygen is
common knowledge. Also, it is certain that water is formed by the union
of hydrogen and oxygen. The simplest formula which can be written for
water is H,O, H—O—H, and this is generally given as its formula, but
there are many facts which show that water as it exists in the liquid
and solid form and probably in the form of its vapor even at 365°, which
is its critical temperature, has a more complex formula. Its high critical
temperature, cohesion, refractive index and boiling point all show that
the formula is more complex than H,O. The molecule of water would
be very light were the above formula true; it should boil at a low tem-
perature, and have a low surface tension. Instead it has a very high
surface tension, much higher than any of the hydrocarbons. Hence
it is certain that the formula is more complex, at least at temperatures
lower than 400° C. That the formula is some multiple of H.O is shown
also by the following circumstance: Eétvés found that if the surface ten-
sion is multiplied by the */, power of the volume of a gram mol. of a liquid
the result, which is the surface energy of a gram mol., was equal. for all
normal non-associating substances, to 2.27 (T,-T) ergs, T, being the

190
THE PHYSICAL CHEMISTRY OF PROTOPLASM 191

critical temperature and T the absolute temperature at which the sur-
face tension was measured. For all substances which associated, that is
substances in which polymerization occurred, the product was less than
2.27 (T.-T). Now water was found to have a surface tension energy
which was less than half 2.27 (T,-T) and the coefficient instead of being
2.27 fell lower and lower as the temperature was lower. Since all liquids
in which the molecules do not remain the same but coalesce to form larger
molecules as the temperature falls behave in this way, it is clear that
water is also more complex than H,O at temperatures below the critical
and that the degree of complexity increases as the temperature falls.
Ramsay and Shields computed from the surface tension that the formula
at the boiling point must be about (H,O),, and in ice about (H,O),.
Eotvos had also come to this result earlier. Determination of the freezing
point of solutions of water in other solvents leads to the formula (H,0),.
Water is indeed one of the most associated liquids known. The molecular
weight and the valence of the molecule at the critical temperature can
also be determined from the cohesion, and this determination shows that
the molecule at the critical temperature is at least (H,O),. From some
of these and other facts, Armstrong has concluded that the molecule of
water in the liquid form is probably (H,O),; and that by the condensa-
tion of the simple molecule H,O, which he has named hydrol, in.o
a ring or chain compound like the polymethylenes water is formed. It
is probable that not all the molecules are thus associated, but that some
dissociation takes place so that some free hydrol probably exists even
in liquid water. The following kinds of molecules, then, probably exist
in liquid water at 20-40° C.:

{ |
H—O—H; Bees H—O —0—H; H—O—O—H;

hh okt

H H H
H—0€ ae nH D_CH
07? io dda

\
HW “a H a SG

The cause of this great association of water is probably the extra
valences of the oxygen. Oxygen may be tetravalent here. Now, hydro-
gen differs from all other elements thus far studied in the fact that its
valence is almost ‘or entirely fixed and unchangeable; it has in it almost
none of those reserve, or extra, valences, which appear in all the other
elements. Chlorine, for example, may be univalent, trivalent, pentavalent
or heptavalent. The result is that when hydrogen is united with a single
-other atom the extra valences which may occur on the other atom cannot
be satisfied by union with those of the hydrogen; there is, hence, nothing
192 PHYSIOLOGICAL CHEMISTRY

else for them to unite with than the other similar valences on another
molecule, thus producing molecular unions and association. Oxygen is
certainly at times quadrivalent and hence the oxygen atom of hydrone
may have, in addition to the valences uniting it with the hydrogen, two
extra valences.

A physical property of water of very great biological importance,
which is probably correlated with this association, is the high specific
heat of water. It takes more heat to raise the temperature of a gram
of water one degree than is required to raise the temperature of a gram
of any other substance, either solid or liquid, one degree. This high
specific heat of water is due in part to the fact that there are in a gram
of water a large number of molecules, but chiefly to the fact that the
dissociation of the water into hydrone consumes heat and the association
accordingly liberates heat. At any rate, whatever may be its cause, this
high specific heat is of value to the cell, since when heat is liberated in
the course of the vital reactions the temperature of the cell does not
rise very greatly ; the water acts as if it were a buffer, taking up the heat
liberated and giving it off gradually. Thus this property of water is of
importance in preventing violent temperature changes which might lead
to uncontrollable decompositions in the cell.

Another very remarkable property of water is its power of solution.
No other solvent surpasses water. All kinds of substances dissolve in it:
salts, carbohydrates, proteins and even fat solvents to some extent. Its
power of solution, also, contributes much to the possibilities of life. This
power of solution has not yet been explained, but it is probable that it,
also, is correlated with, or due to, the extra valences on the oxygen atoms,
which are perhaps able to unite with the extra valences on the dissolving
molecules, and thus to produce solution.

Water has also a higher specific inductive capacity, or dielectric con-
stant, than any other liquid, except possibly hydrogen dioxide. It isa
good insulator. It does not in itself, at ordinary temperatures, conduct
the current readily. In virtue of its high specific inductive capacity it
happens that when electrical disturbances occur in a cell they are

DreLEctTRic Constants oF SoME Liqurps *

Dielectric constant or specific
inductive capacity

Wate? vicinus mearinietee er dagameeta aed 77.0
Formic acid ccsscicmse ver eaawes ners eenaaed 63.0
Methyl! alcohol 6 scccscaaiee atid ven wesw ann 33.7
tH YL, BCORON: vise: c iain ieosiel menace etiecwadena kere 25.9
BPrOpyl BICOHO! ood. cccatiannniecgenr parece annie auesiayes 22.0
Ammonia, liquid ............ 00. cece eee 22.0
Amy] alcohol 2. cotesssasaven cer saiaen wun. 16.0
ChIOrOf ORM «ii scutes Noeace Bete ea desed ctenesaaesnet 5.0
BOG pois, a thica acts ahve ducioracends auc neebshdcone oapelsoods 4.4
Carbon bisulphide ........... cece eee eens 2.6
Benzene! ocoasswi. casas met mmeioamnng ween 2.3

* Jones: Hlements of Physical Chemistry. Macmillan. 1902, p. 146.
THE PHYSICAL CHEMISTRY OF PROTOPLASM 193

not instantly compensated, so that oppositely charged particles
may coexist in water, the attraction between two oppositely charged
particles in water being only 1-77th that in air. It is probably
because of this property that water forms such a good ionizing
medium. At any rate, this property may account for the undoubted
fact, whatever explanation we may choose to give of that fact,
that substances dissolved in water interact with greater ease and speed
than when dissolved in any other medium. It has the property then, so
important for the cell, of accelerating all kinds of chemical reactions.
Thus hydrogen and oxygen will not unite, except at very high tempera-
tures, unless some water is present: hydrochloric acid and sodium
hydrate react vigorously in the presence of water, but not when they
are quite dry; chlorine and hydrogen do not form hydrochloric acid,
except at very high temperatures, unless water be present; and everyone
knows that the rusting of iron does not occur unless water is there too.
Water has, then, this fundamental property of making reactions go on
between bodies dissolved in it or wet by it. This property is believed
by many to be correlated with its ionizing powers, and with the fact
that its solutions conduct electrical current more than those of any
other solvents. And this property brings us to the consideration of the
salt solution in protoplasm.

Salts.—All protoplasm contains a solution of salts and these salts
are of the nature of those of the sea. What, then, is a salt solution?
How can that in protoplasm be assisting in the production of vital phe-
nomena? Just as it is not yet known with certainty what the composi-
tion of liquid water is, so it is not known what is the exact state of
affairs in a salt solution. No fact shows more clearly the limitations of
chemical and physical knowledge at the present time than that one can-
not say positively just wuat is a solution of common table salt in water.
It is known, howeve.., that salt solutions have certain properties and
these may be dealt with even in the absence of their explanation. One
of these properties of most fundamental importance is that aqueous
solutions of the common salts conduct the electrical current. This fact
was studied by that inspiring British physicist, Michael Faraday. He
found that if a current flows through a solution of sodium chloride, for
example, the sodium moved down with the current to the cathode, or
negative electrode, and the chlorine moved up against the current to the
positive electrode, the anode. Since the metal part of the salt moved
down with the current, he called such wandering metals cations, from
the Greek kata, down, and ion, going; and the negative, or metalloid part
of the molecule, was called an anion (Gr. ana, up). Now it is clear that
if the sodium moves down with the current it must be positively charged,
and the chlorine moving up must be negatively charged, since only par-*
ticles with charges on them move in an electrical field. Faraday did not
194 PHYSIOLOGICAL CHEMISTRY

know where the sodium got its charge. He thought that these ions did
not pre-exist in the solution, but that the action of the current separated
the neutral sodium-chloride molecule into a positive and negative par-
ticle. On the other hand, it was later suggested by Clausius that the
ions did pre-exist, since no energy seemed to be consumed in the separa-
tion. This view of Clausius was put on a much firmer foundation and
introduced as a powerful and fruitful theory into chemistry by the
Swedish physicist, Arrhenius, in the year 1881. The basis of this theory
of Arrhenius of the pre-existence of the ions, the so-called ionic theory,
was that the osmotic pressures of solutions of electrolytes was higher than
the osmotic pressure of equally concentrated solutions of non-electrolytes.
The osmotic pressure and the vapor pressure are functions of the number
of dissolved molecules in a given volume. It was found that a molar
solution of sodium chloride depressed the freezing point, or raised the
boiling point, of water more than a molar solution of sugar. Arrhenius
brought this fact into relation with the anomalous pressures of some
gases. It is found, for example, in heating nitrogen tetroxide, N,O,,
that the product of the pressure by the volume increases more than it
should, according to the gas law, PV—RT, and the explanation of this
is that some of the N,O, dissociates into two molecules of NO,.
Arrhenius suggested that the greater osmotic pressure and lower vapor
pressure of electrolyte solutions, as compared with equally concentrated
solutions of non-electrolytes, was due to the fact that the salt dissociated,
also, like vapors of chlorine, bromine or iodine, and that the pieces into
which it dissociated were the electrically charged ions of Faraday and
Clausius. This theory, it will be noticed, explained at once the anomalous
conductivity, the low freezing and high boiling points and the higher
osmotic pressure of salt solutions. The ionic theory thus introduced has
proved to be one of the most fruitful theories of chemistry. It has
explained more facts, which without it were quite unexplainable, than
probably any other chemical hypothesis except the atomic theory; and
while some are disposed to criticise it and there are some facts which are,
at first glance, difficult to explain by the theory, there can be no question
of the enormous usefulness of the theory whether in its present form it is
exactly true or not.

We may perhaps pause for a moment to consider a few of the more
important evidences of the truth of this fundamental theory so illu-
minating for physiology. It enables us to understand the avidity
of acids and bases. There was no explanation of the variation
in the strength of acids and bases before this theory.. It was known
that hydrochloric acid was much more powerful and active than
acetic or lactic acid. The ionic theory explained this at once. Acids,

"on the ionic theory, are bodies which dissociate in solution so as to
THE PHYSICAL CHEMISTRY OF PROTOPLASM 195

form hydrogen ions. This dissociation may be represented as
follows :

aS ee eS
Hol = H+0l CH,.COOH = H + 0.c0.cH,
ee CHOH =" H106c
HNO, = H+NO, HOH —— H+ 00,5,
~+- a
80, =— H+ Hso, HCN = H+ON

All acids, then, have hydrogen ions in their solutions; their acidity is due
to this; and their activity is proportional to the number of such ions
there are in unit volume. This conclusion may be tested by comparing
the conductivities of acids with their chemical or physiological activity.
The amount of current which can be ferried by the ions between the
electrodes in a solution in unit time will evidently be a function of the
number of ions and their speed. It is found that a solution of hydro-
chloric acid will carry in a given time much more electricity across than
a solution of acetic acid of the same concentration. There is no reason
to believe that the speed of the hydrogen ion differs in the two cases; and
while the acetic ion moves at a slower pace than the chlorine ion, its
velocity has been determined and it is found that the difference is not
sufficient to account for the difference in conductivity. There seems to
be but the single possibility that the number of hydrogen ions is greater
in the solution of hydrochloric acid than in that of acetic; hence, if the
strength of the acid is proportional to the number of hydrogen ions,
hydrochloric acid should be much stronger than acetic and in the same
proportion as is determined by their conductivities. This was found to
be the case. All acids split cane sugar into glucose and levulose; invert
it, in other words. The speed with which they do this is different in
different acids. It is a function of the number of hydrogen ions which
are in the solution, so that if the speed of hydrolysis is measured the
relative number of hydrogen ions in different acids of the same concen-
tration can be determined and they should be approximately in the
same proportion as the figures for the conductivities and other powers of
the acids. This is found to be the case, as is shown in the accompanying
figures : oe
Inversion Equivalent conductivity at18° |

Acid coefficient (0.1n except when otherwise noted)
Hydrobromie ............0.see eee ee 1.114 360
Hydrochloric ..............eeeee eens 1.00 351
Nitrie’ .i.ccc 3 pwadine's ie bee stwins de alla b% 1.09 350
Trichloracetic ........... 0.000000 eee 0.754 323 (n/32)
Sulphuric ............ 2. cece eee eee 0.536 225
SOKANIC gen catiwd eww epee eees 0.186 117
Phosphoric .........-.-0 eee eee eee 5 46.8
Monochloracetic . 72.4 (n/32)
Formic ..........-00eee: : 29.3 (n/32)
Acetic: asset oss os Pie sav ove atu nui sa rarest 0.0040 46

 
196 PHYSLOLOGICAL ‘CHEMISTRY

It is a general law that solutions freeze at a lower temperature than
the pure solvent. It has been found by a further study of this phenomenon
that the depression of the freezing point of dilute solutions is propor
tional to the concentration of the dissolved substance, that is to the
number of molecules in a given volume. A solution as concentrated as

 

Fig. 16.—Beckmann freezing-point apparatus. A. tube containing liquid to be frozen:
D, thermometer; H, stirrer; G, side tube for introducing ice crystals, etc.; B, large outer
test tube; O, jar containing freezing mixture; J, stirrer for same.
a one-tenth gram mol. solution, that is a solution which contains 6.06 x 1074
solute molecules in a liter volume, depresses the freezing point of water
0.186°, so that a solution of glucose which contains 18.0 grams of glucose
in one liter will freeze at —0.186° C. A solution half as concentrated
will freeze at —.093° C. In this way by taking the freezing point of a
solution by means of an accurate thermometer measuring to hundredths
or thousandths of a degree, it is possible to tell how many molecules
there are in a liter of any solution. It is found that a 0.205 M solution
of calcium chloride does not depress the freezing point approximately
.370°, as one would expect were there only CaCl, molecules present, but
THE PHYSICAL CHEMISTRY OF PROTOPLASM 197

it depresses it 1.012°. The most probable interpretation of this fact is
that the solution contains more particles than had been supposed. But
to get a larger number of particles it is necessary to split the calcium-
chloride molecules into Ca and Cl particles. About 91 per cent. of the
molecules must have dissociated into Ca and Cl ions. If the number of
such particles is computed from the freezing point, it is found to be about
the same as that which is computed on the ionic theory from the con-
ductivity. As in this case no electricity is used and it is unlikely that
depressing the temperature could cause such a dissociation, this fact
lends support to the view that some substances dissociate into particles
and these particles are the ions, or electrically charged particles, already
mentioned.

There is one circumstance which strongly corroborated the truth of
the ionic theory, namely, that a great number of facts which were for-
merly wholly unexplained were at once explicable; and new facts could
be predicted and found to be true by experiment. It resulted in an
entirely new development of electro-chemistry and quantitative analysis
was put by it on a firm theoretical foundation. For all these reasons we
may repeat what was already said, that no more clarifying, fruitful
theory has appeared in chemistry than the electrolytic dissociation theory.
Inasmuch, however, as there are some who do not yet accept the thcory
as positively established, for reasons into which we cannot go at this
place, it must be accepted provisionally only, as the most probable
explanation of the facts which has yet been proposed. The conception
of the chemical union of solvent and solute may eventually considerably
modify the ionic theory.

When a salt dissolves in water then, as it does in living matter, there
are these reasons for believing that it breaks, in part, into electrically
charged particles which, like so many tiny electrodes, each bearing one
or more electrical charges, float about in the protoplasm and become
thereby capable of doing many things. Living matter contains before
it is stimulated, then, a large number of electrically charged particles.
and it is clear that if in any way an accumulation of positive particles
in one place and of negative in another could be produced, and if the
negative and positive particles had different actions on the vital prov-
esses, momentous changes might thus be brought about in living mat-
ter. This is what happens when an electric current is sent through
protoplasm. Moreover, it is clear that if the nature of these little elec-
trodes is changed so that instead of carrying one charge each carries
two or three, or if they carry them at a different potential, the electrical
equilibrium of the protoplasm might be upset as surely as if a separation
of opposite electricities had occurred. The ionic theory, then, is at
present fundamental to an understanding of the nature of electrical and
198 PHYSIOLOGICAL CHEMISTRY

chemical stimulation and depression of protoplasm; of the action of salts
and drugs on living matter; and it also enables us to see how if by any
reaction taking place in living matter a change in the distribution of
positive and negative ions could be produced something in the nature of
a condenser might be formed which, under suitable conditions, would
discharge. Later on, under the heading of colloids, the relation of these
charges on the ions to the physical state of the protoplasm will be con-
sidered. it may be stated, also, that oxidation in protoplasm is accom-

    

     

  
    

erm ara eae fo

. Fie. 17.—Porous cup and manometer for measuring osmotic pressure as used by
Pfeffer. m, manometer; 2, porous clay cup with ferrocyanide in its pores. In making the
determination this is put into a beaker of water.

panied by such an electrical disturbance which in its turn probably acts
as a stimulus to the surrounding parts of the protoplasm, the stimulus
being propagated in this way.

Another property of salt solutions of great interest is their high
internal pressure. The internal pressure of salt solutions, or even of
water alone, is very high. By the internal pressure is meant the
cohesive pressure due to the attraction of the molecules for each other.
This pressure in such a liquid as ether, which *s very labile and volatile
and of a low internal pressure, is about 2,0(9 kilograms per square
em. at zero degrees; and in water it is certaink~ far greater than this,
THE PHYSICAL CHEMISTRY OF PROTOPLASM 199

being probably between 5,000 and 10,000 atmospheres. The addition of
salt to water increases this pressure still higher, and the more salt there
is added the greater the internal pressure becomes. The internal. pres-
sure being so high, the spaces between the water molecules are very small.
It is this internal pressure which is probably at the basis of osmotic
pressure.

Osmotic pressure —This is another property of solutions of great
importance in vital phenomena, since it is one of the factors controlling
the amount of water in protoplasm and its turgor. It was found by the
British physicist, Graham, that if solutions of two different substances,
or two differently concentrated solutions of the same substance, were
separated by a membrane, either animal or vegetable, the substances in
solution would in some instances pass through the membranes and’ some:
times they would not. Using parchment.paper, or bladder, as the mem-
brane he divided all substances into: two classes: those which passed
through he called crystalloids, and those which did not were called col-
loids. The process of passage of solvent, or solute, through a membrane
is called osmosis or dialysis. It has been found possible to prepare
membranes which are freely permeable to water, but which oppose a
resistance to the passage of the crystalloid solute; such a membrane is
said to be semi-permeable, since only the solvent goes through. The
botanist, Pfeffer, prepared such a membrane by precipitating the
gelatinous copper ferro-cyanide in the pores of a porous clay cup. If
potassium ferrocyanide is put within the.cup of which the pores are
filled with water and the cup is immersed in a 3 per cent. copper sul-
phate solution for 24-48 hours, a gelatinous precipitate of cupric ferro-
cyanide occurs at the junction of the solutions within the porous wall.
This precipitate is permeable to water and some ordinary salts, but it
does not permit cane sugar to pass through it. If a cup thus prepared,
or prepared by the electrolysis method of Morse and Horn, holding a
solution of cane sugar.is immersed in water, sugar cannot go-out, but
water can and does enter. If the cup is closed by a mercury manometer,
water will continue to pass into the cup, expanding the solution and
forcing the mercury of the manometer upward until a certain pressure
is reached, when the manometer becomes stationary and the solution
takes up no more water. This pressure is known as the osmotic pressure
of the sugar solution. Jt is-the pressure which is just sufficient to pre-
vent the solution from increasing in ‘volume when separated from the
solvent. by a _semi-permeable membrane. ‘Before ‘considering - the cause
of this passage ‘of water inward, the relation’ of the amount are
pressure “to thé concentration of the solution may be discussed. 7°” Oe

Pfeffer made an osmometer of the nature of that: just desorbed
(Figure 17) and measured the amount of the osmotic pressure of sugar
200 PHYSIOLOGICAL CHEMISTRY

solutions of various concentrations and at different temperatures. Some
of the results he obtained are given in the following tables. It will be
observed that the osmotic pressure increases with the temperature and

 

— OF, 0 P
pocene renin Gabote prereuce z pemperainre 1% cane sugar
per cent. cms. Hg. c 6.8° 50.5 ems
1 53.5 ems 53.5 13.2 52.1 “
2 101.6 “ 50.8 14.2 53.1 “
4 208.2 “ 52.0 22.0 54.8 “
6 307.5 “ 51.2 36.0 56.7 “

with the concentration; and also that the amount is proportional to the
concentration and is high. Thus a 0.1 molecular solution, 34.2 grams
saccharose in a liter or about 3.1 per cent., has an osmotic pressure of
2.24 atmospheres at 0°; a .05 molecular of 1.12 atmospheres and so on.
This rule only holds for dilute solutions. Concentrated solutions have a
higher pressure than that calculated.

Since it is not always possible to find semi-permeable membranes
with which to measure osmotic pressure directly, recourse must often
be had to indirect methods. The pressure may be determined by taking
the freezing point of the solution. A 0.1 molecular solution depresses the
freezing point of water 0.186°. This has an osmotic pressure of 2.24
atmospheres at zero degrees. If the freezing point is depressed only
half of the foregoing amount, the solution must be .05 molecular and
the osmotic pressure is only 1.12 atmospheres. Ordinarily, therefore,
instead of measuring the osmotic pressure, the freezing point may be
taken, a correction made for the concentration change produced by the
ice which has separated and the osmotic pressure calculated. Of course
the calculation is made on the assumption, which is not always correct,
that the degree of dissociation and association does not markedly change
with the temperature; this is virtually true for most common salts. A
very useful table for calculating the osmotic pressure from the freezing
point is that of Harris and Gortner, on page 201.

The van’t Hoff law of the correspondence of osmotic and gas pressure
only holds for dilute solutions. It does not hold strictly even for a solu-
tion of sugar 0.1 mol. in strength and higher solutions have osmotic pres-
sures greater than that calculated. (Morse; Berkeley and Hartley;
Garrey.) Thus the freezing point of a molecular cane-sugar solution is
not —1.86° C. as calculated from the freezing point of a 0.05 molecular
solution, but it is —2.775°. The osmotic pressure in place of being the
theoretical amount of 22.4 atmospheres at 0° is actually about 33.3 atmos-
pheres. The deviation becomes greater at higher concentrations. It does
not disappear entirely if we calculate the concentration on the basis
of the pressure being that which would be exerted by the gas when caleu-
lated for the volume occupied by the solvent only. The osmotic pressure
THE PHYSICAL CHEMISTRY OF PROTOPLASM 201

of the sea-water at Woods Hole is that of a solution freezing at —1.81° C.
or about that of a 3/4 molecular cane-sugar solution (256.6 grams per
lier. Garrey).

TaBLE OF OsMOTIC PRESSURES TIN ATMOSPHERES FOR DEPRESSION OF THE FREEZING
Pornt To 2.99° C. (Harris and Gortner).

 

Hundredths of degrees, Centigrade

 

>

0 1 2 3 4 5 6 7 8 9

 

0.000} 0.121 | 0.241] 0.362] 0.482] 0.603] 0.724] 0.844) 0.965]! 1.085
1.206} 1.327 1.447 | 1.568] 1.688] 1.809 | 1.930] 2.050} 2.171] 2.291
2.412] 2.532 | 2.652] 2.772] 2.893) 3.014, 3.134] 3.255] 3.375] 3.496
3.616] 3.737 3.857 | 3.978] 4.098} 4.219| 4.339] 4.459] 4.530] 4.700
4.821} 4.941 5.062 | 5.182] 5.302] 5.423} 5.643] 5.664] 5.784] 5.904
6.025) 6.145 6.266 | 6.386] 6.506} 6.627] 6.747] 6.867] 6.988] 7.108
7.229| 7.349 7.469 | 7.590{ 7.710] 7.830! 7.951] 8.071] 8.191} 8.312
8.432) 8.552 | 8.672) 8.793] 8.913] 9.033! 9.154] 9.274] 9.394] 9.514
9.635] 9.755 9.875 | 9.995) 10.12 | 10.24 |10.36 | 10.48 | 10.60 | 10.72
10.84 | 10.96 11,08 | t1z0 [11.382 | 11.44 |11.56 |11.68 | 11.80 | 11.92
12.04 | 12.16 12.28 | 12.40 | 12.52 | 12.64 |12.76 |12.88 | 13.00 | 13.12
13.24 | 13.36 13.48 {13.60 |.13.72 |13.84 {13.96 |14.08 | 14.20 | 14.32
14.44 | 14.56 14.63 {14.80 [14.92 | 15.04 |15.16 |15.28 |15.40 |15.52
15.64 | 15.76 15.88 |16.00 |16.12 |16.24 |16.36 |16.48 | 16.60 |16.72
16.84 | 16.96 17.08 |17.20 [17.32 117.44 {17.56 |17.68 |17.80 | 17.92
18.04 |'8.16 {18.28 |18.40 |18.52 |18.64 |18.76 |18.88 |19.00 | 19.12
19.24 |19.36 |19.48 | 19.60 |19.72 | 19.84 |19.96 |20.08 | 20.20 | 20.32
20.44 | 20.56 |20.68 | 20.80 |20.92 |21.04 [21.16 |/21.28 | 21.40 | 21.52
21.64 | 21.76 {21.88 | 22.00 |22.12 |22.24 |22.36 |22.48 | 22.60 | 22.72
22.84 | 22.96 |23.08 |23.20 |23.32 |23.44 (23.56 |23.68 | 23.80 | 23.92
24.04 [24.16 124.28 |24.40 |24.52 |24.63 |24.75 | 24.87 |24.99 | 25.11
25.23 | 25.385 |25.47 |25.59 (25.71 |25.83 25.95 |26.0; |26.19 | 26.31
26.43 | 26.55 {26.67 |26.79 |26.91 {27.03 |27.15 | 27.27 | 27.29 |27.51
27.63 | 27.75 {27.67 |27.99 |28.11 |28.23 |28.34 |28.46 | 28.53 | 28.70
28.82 [28.94 {294 |29.18 |29.30 |29.42 (29.54 | 29.66 |29.7x | 29.90
30.02 | 30.14 |30.26 | 30.38 {30.50 |30.62 {30.74 /|30.86 | 30.98 | 31.09
31.21 | 31.33 31.45 131.57 [31.69 |31.81 {31.93 /32.05 [32.17 | 32.29
32.41 | 32.53 |32.65 /32.77 |32.89 |33.00 |33.13 [53.25 |33.36 | 33.48
33.60 | 33.72 |33.84 |33.96 |34.08 |34.20 (34.31 [34.43 134.56 | 34.68
34.79 | 34.91 [35.04 |35.16 |35.27 135.39 [85.51 |35.63 [35.75 | 35.87

COHNAMAwWI HS

 

PPNNNNNNNNME EEE EEE EES SS SS909999

CONINAURWNIHOOMDUNRMARwO DMO

 

 

 

 

 

 

 

 

 

 

A convenient form of apparatus for determining the freezing point
of blood, vegetable saps, milk or other animal juices is that shown in
Figure 18, described by Bartley:

“The apparatus consists of a Dewar tube, A, 22 cm. high and 6 em. inside
diameter, set in a wooden base. This is fitted with a rubber stopper having three
holes. Into the large hole is fitted a heavy glass test tube 20 cm. long and 3 cm.
wide passing down to near the bottom of the vessel A. Two other holes are for small
brass or copper tubes, one (C) terminating just below the rubber stopper and the
other (B) passing to the bottom of A and coiled around two or three times. These
coils are perforated with a series of small holes. Inside of the test tube passing
through the rubber stopper is a second test tube of about the same length and 2.5 cm.
in diameter, held in place by a section of rubber tubing drawn over it and separat-
ing the two tubes by « narrow space. In operation, this space is filled with alcohol.
A delicate thermometer (F) with a platinum wire coiled loosely around its lower
end completes the apparatus. In the apparatus as here figured, and as used by
202 PHYSIOLOGICAL CHEMISTRY

the author, the stirrer (E) is operated by a toy motor (D) run by an uray dry
cell. This can be dispensed with, if desired, and the stirrer operated by hand, al-
though this mechanical contrivance makes the apparatus almost automatic.’

To use the apparatus, fill the tube A about one-third full of ether or carbon
disulphide... Insert the rubber stopper tightly, connect the shorter metal tube with
e Richards aspirator pump, attached to the water service. The liquid to be frozen

is placed—in-the-inner test tube. -There should be enough: liquid to cover the mer-
eury bulb of the thermometer, when the latter is lowered to the bottom of the tube.

 

 

 

 

 

Fig. 18.—Bartley freezing-point apparatus.

The wan is then started through.the Richards respirator pump, which draws air
_through the ether in a series of bubbles, causing it to evaporate.

Owing to the well-known principle of the Dewar tube, applied in the. popular
thermos bottle, almost all the heat used to vaporize the ether is derived from the
_ thin layer of alcohol between the two test tubes and from the liquid under examina-
tion... There is no frosting of the outer vessel, the whole system remains clear and
transparent and the thermometer can easily be read at all times.
| When the temperature reaches zero, the stirrer is started. It will be observed
that the temperature steadily sinks to —2° C. o —8° C. before freezing begins, i.e.,

: Bartley: Archives of Diagnosis, 1913.
THE PHYSICAL CHEMISTRY OF PROTOPLASM 203

two or more degrees below the true freezing point of the liquid. Then, suddenly,
freezing occurs and the temperature reading rises to a fixed point and remains there
for some minutes. When this point is reached the water is shut off and an accurate
reading taken. This is the freezing point of the liquid. There is no necessity of
adding ice to start the freezing, as is usually done in other forms of apparatus. The
whole process is automatic and all the observer need do is to regulate the flow of
water running through the pump and read the thermometer. It is advisable, when
the temperature reaches zero, to draw the air through the ether more slowly until
freezing takes place, by partly shutting off the flow of water. For accurate work
the Beckmann adjustable thermometer should be used. The thermometer is the
most important and most expensive part of the apparatus.”

The osmotic pressure may then be defined as that pressure which
is just sufficient to prevent any increase of volume of a solution when
it is separated from its solvent by a truly semi-permeable membrane.

Using the measurements of Pfeffer, van’t Hoff discovered that for:
dilute solutions the osmotic pressures were equal to the pressure which
a true gas would exert if the same number of molecules were contained
in a space as large as that at the disposal of the solute molecules. Thus
a one-tenth gram mol. of sugar in a liter space at 0° exerts an osmotic
pressure of 2.24 atmospheres per square cm. One-fifth of a gram of
hydrogen gas in the same space and at the same temperature would have
the same pressure. Moreover, the temperature coefficient is the same
both for the osmotic pressure and the gas pressure. In the case of a gas
it is known to be 1/273, or .00366 per degree. Pfeffer found for the
osmotic pressure of sugar approximately the same value.

.A 1 per cent. solution of cane sugar contains one gram in 100.6 c.c.
The saine number of molecules of hydrogen in the same space, or .0581
grams per liter at 0° exerts a pressure of .646 atmospheres. Van’t Hoff
gives the following table comparing gas and osmotic pressure:

Temperature Osmotic preseure of Gas pressure of

cane sugar hydrogen gas
6.8° 0.664 0.665 atmosphere
13.7 0.691 0.681
15.5 0.684. 0.686
36.0 0.746 0.735

These facts were all determined empirically, but the explanation has
not yet been given to the satisfaction of all. At first the conceptions of
the molecular kinetic theory of gas pressure were carried over bodily to
explain osmotic pressure. The pressure in the case of a gas is due to
the bombardment of the walls by the rapidly moving molecules of the
gas; the osmotic pressure was ascribed to the bombardment of the semi-
permeable membrane by the dissolved molecules. A more probable
explanation of the pressure is the following: The vapor pressure over
a salt solution is less than over pure water. This is shown either by
direct measure of the vapor pressure or by a boiling-point determina-
204 PHYSIOLOGICAL CHEMISTRY

tion. The boiling point of a solution is that temperature at which the
vapor pressure becomes equal to the external, generally the atmospheric,
pressure. It is found that it is necessary at atmospheric pressure to heat
salt solutions to temperatures higher than 100° C. before they begin
to boil, from which we conclude that their vapor pressures at 100° and
below are less than an atmosphere and lower than that of pure water.
It is also found that the increase in the boiling point is proportional to
the molecular concentration of the dissolved substance for all substances
which vaporize at a temperature higher than does water. Why is the
vapor pressure of a salt solution lower than that of water? Various
reasons may be assigned. One is that the attraction between salt mole-
cules and between water and salt is greater than that of water for water.
Hence the internal pressure of the solution is higher than that of water
alone. Now at the same temperatrres all molecules possess the same
average kinetic energy ; that is, the product of the mass by the square of
the average velocity is a constant for all molecules at any given tem-
perature, the heavier molecules moving more slowly, the lighter faster.
The mean kinetic energy of the water molecules in water and salt solu-
tion is the same. but the cohesive attraction is greater in the salt than
in the water. Only those molecules which have a kinetic energy above
the mean value are able to escape from this cohesive attraction of the
liquid into the vapor. Since the cohesive energy is greater in the salt
solution, there will be, on the average, fewer molecules able to escape this
attraction in unit time. Hence, when equilibrium is reached and just as
many molecules in the vapor are coming into the liquid as escape from
the latter, this equilibrium will be attained when fewer molecules are
in the vapor space in the case of the salt solution and hence the vapor
pressure over the salt solution will be lower than over the water.

If two receptacles are closed except for a glass tube connecting them
and the one is partly full of water, the other partly full of salt solution,
the vapor pressure over the salt solution will be lower than that over the
water. The water will gradually distill over into the salt solution. The
conditions are not different if the two solutions are brought into contact;
for now the attraction, or cohesion, of the salt solution molecules for
water is greater than that of the water molecules for water, and the water
molecules will gradually penetrate the salt solution until equilibrium
is attained, when the solution becomes homogeneous. If we put a semi-
permeable membrane between the solution and the solvent and then
exert a pressure on the salt solution, molecules of solvent may be forced
outward, by filtration, through this membrane. By increasing the pres-
sure, the number of solvent molecules thus leaving the salt solution may
be increased until a point is reached at which the numbers thus forced
out by pressure, added to those which are leaving as vapor, equals the
THE PHYSICAL CHEMISTRY OF PROTOPLASM 205

number leaving pure water when in equilibrium with its vapor. This
pressure will thus just suffice to prevent more water entering the solu-
tion than is leaving and such a pressure is called the osmotic
pressure.

The cause of the osmotic pressure is evidently ultimately the attrac-
tion of a physical or chemical nature between the solvent and the solute
molecules. It is the cohesive or internal pressure of the solution.

Since salt solutions and all things in solution exert osmotic pressure,
protoplasm has a decidedly higher osmotic pressure than water. The
amount of this pressure varies in different cells, but for the mammalian
tissues it is supposed to be about that of a 0.9 per cent. NaCl solution,
since in such a solution the tissue neither gains nor loses weight. This
is about 7.1 atmospheres. For the cells of apples, the juice obtained by
pressing the apples has an osmotic pressure of about 17 atmospheres.
It is partly by means of osmotic pressure that plant and animal cells
preserve their turgor and keep the cell wall stretched; and it is by
changes in turgor that movements are produced in many plants, i.e., the
sensitive plant, and possibly in our own brain cells.

The determination of the osmotic pressure of animal and plant cells
may be directly made by immersing them in solutions of salts or sub-
stances which do not penetrate them and determining whether they shrink
or swell or remain unaltered. That solution in which they neither swell
nor shrink is supposed to have an osmotic pressure equal to that of the
cell contents. This method was used by the botanist, de Vries, to deter-
mine the osmotic pressure of plant cells and also the concentration of
various salts all of which left the size of the cells unaffected. He used
cells of many plants, among others of Tradescantia, the spider lily. Algex
serve as well. Normally the cell contents are under high pressure, due
to turgor which keeps the protoplasm applied to the cellulose wall, but
if the cell is put into a solution of which the solute does not penetrate
the cell, and if the osmotic pressure is high, the protoplasm shrinks away
from the cellulose wall. It is said to be plasmolyzed, and the method is
called the plasmolysis method. By this method the osmotic pressure of
various plant cells was determined. Some vegetable saps have an osmotic
pressure of 14 atmospheres.

This method has several serious sources of error. Tle plant cell is
not a bag of liquid with a semi-permeable wall, but probably a jelly-like
substance. Furthermore, this gel is one of the most unstable substances
known. It is living matter, and the activities of living matter are won-
derfully dependent on different kinds of salts and other substances. It
is not surprising. therefore, that the method has given only approximate
results, although these results have been of great value, since it was from
de Vries’ osmotic measurements made by this method that van ’t Hoff
206 PHYSIOLOGICAL CHEMISTRY

«nd Arrhenius drew part of their material for the laws of osmotic pres.
sure and dissociation.

Animal cells presumably have an osmotic pressure approximately
equal to that of the circulating liquids like the blood, which is some-
what more than seven atmospheres. The freezing point of blood serum
is about —0.6°, which would be an osmotic pressure of 7.2 atmospheres
as shown in the table. The red blood corpuscles of mammals are often
used for osmotic pressure determinations. The concentration of a solu-
tion is determined in which the corpuscles have the same volume (in the
hematokrit, see page 992) that they usually have in the serum. The
osmotic pressure of the serum is hence equal to that of the solution.
For mammalian corpuscles it is about that of a 0.9 per cent. NaCl solu-
uon. Solutions of this osmotic pressure are said to be isosmotic or
isotonic, Stronger solutions which shrink the corpuscles are hypertonic;
weaker, which swell them, are hypotonic. Although these corpuscles have
little chemical activity, they are gels like the plant cells and their use
for determining osmotic pressure is hence very limited.

Surface tension.—Besides the properties of osmotic pressure and
ionization and the physical properties which have been mentioned, salt
solutions, such as occur in protoplasm, or in fact all liquids, possess
certain properties at the surfaces which separate them from other sub-
stances of a gaseous, liquid, or solid nature. Such surfaces are supposed
to and probably do exist in protoplasm between the more solid and the
more liquid parts of the protoplasm ; and the physical properties of such
surfaces of separation become at least worthy of attention in any exami-
nation of the physical properties of protoplasm. It is clear that where
a liquid comes in contact with another substance of a different kind, the
molecules of the periphery of the liquid are no longer under similar
attractions in all directions. It will seldom or never happen that the
attraction between the molecules of the two substances in contact will
be precisely the same as that between the molecules of each substance.
The result of this will be that the molecules in the surface film of the
liquid will be attracted with a different force outward than they are
inward. Their freedom of movement, therefore, will no longer be pre-
cisely the same in all directions, as it is in the interior of the liquid, but
will be restricted in certain directions. The surface of a liquid thus
comes to possess different properties from the interior; and, since the
molecular freedom of movement is restricted in a certain direction, the
surface perpendicular to this direction acquires the property of a solid,
since a solid is a liquid in which the freedom of movement of the mole-
cules is reduced. The surface has a certain resistance to rupture owing
to the inability of the molecules to move freely out of the plane of the
surface; and this resistance to rupture of the surface film is called the
THE PHYSICAL CHEMISTRY OF PROTOPLASM 20?

surface tension. Wherever there are surfaces of separation of liquids,
or of liquids from solids in protoplasm, such surface films will be found;
and their surface tension becomes then a very important matter in the
physiology of the cell.

Method of determining the surface tension.—The surface tension
of a liquid can be determined in several different ways, of which only
a brief outline can be given here. The most accurate is perhaps the
so-called ripple method of Lord Rayleigh, which consists in measuring
the speed of propagation of a series of ripples set up in a pan of the
liquid. There is a relation between the velocity and the surface tension.
This is applicable to pure liquids. Another equally accurate method
is that of measuring directly,, by means of a balance, the tension
of a double surface film of a given length. This method is not
applicable to volatile liquids. There are two methods which are more
convenient, but which are not so accurate. One is the measurement of
the height to which a liquid will rise in a capillary tube of known bore.
From this height the surface tension in dynes per cm. may be calculated
by the formula: Surface tension= vy —%% grh (D,—D,). ris the radius
of the tube in cms.; g, the acceleration due to gravity; h is the height to
which the liquid rises; and D, and D, the densities of liquid and vapor.
The drawback to this formula and this method of the measurement of
the surface tension is that it involves the assumption that the angle of
contact of the liquid with the wall of the tube is zero, so that the cosine
of the angle is unity. While this is very nearly approximated to in
water at low temperatures, it is probably not true at higher temperatures
and particularly for liquids which have a lower tension than water;
hence all determinations of the surface tension by the capillary method
are open to the suspicion of being too low, the error increasing with
the temperature. Another method of determining the tension is the drop
method. The drop weight which any surface film can support is depend-
ent on the surface tension. The number of drops which are formed
from a given volume of liquid is determined by means of a stalagmometer,
Figure 19, and if the density of the liquid is known, the weight of each
drop may be calculated from the weight of the liquid divided by the
number of drops. The surface tension of water being taken as unity,
the surface tension of any other liquid measured in the same stalagmome-
ter may be found. from the formula:

n= rn
y, is the surface tension of the liquid sought; z and z, the number of
drops of equal volumes of water and solution; s, the specific gravity of
the unknown liquid of which y, is the surface tension.

This method, while not so accurate as some others, is nevertheless
208 PHYSIOLOGICAL CHEMISTRY

most applicable for the determination of the surface tension of animal
and plant liquids. It has been refined in the hands of I. Traube and
Morgan. Another accurate method, also avoiding the error of the angle
of contact of liquid with the solid, is that of Eétvs, which is particularly
applicable for the accurate determination of the tension at the junction
of liquid and saturated vapor. It involves only the measure of certain

 

Fie, 19.—Traube stalagmumeters for determining surface tension.

angles determined by reflected light and is carried out in sealed tubes. It
may be used for the determination of the surface tension of condensed
gases. There are also other methods, but these are the more important.
It is found by the use of the ripple method that the surface tension
of pure water is 73.24 (74 by Rayleigh) dynes per em. at 18°. The addi-
tion of any of the common salts increases the tension, as is shown in the
table: \
SurRFACE TENSION OF SODIUM CHLORIDE, PoTassIuM CHLORIDE AND ZnSO, 18°.

Concentration NaCl KCl %ZnS0,
1M 73.42 73.48 73.40
2 73.51 73.60 73.60
3 73.55 73.75 73.75
5 74.10 74.20 74.20
7 74.40 74.50 74.50
1.0 74.80 75.00 75.10

These results are represented by the formula, T,=T,-+kC. The value
of k was for NaCl, 1.53; KCl, 1.71; % Na,Co,, 2.00; % K,Co,, 1.77;
Y% ZnSO,, 1.86. Tw is the tension of water.

It will be noticed that the addition of each salt has a specific effect.
That is, the tension is not increased to the same extent by the same con-
centration of each salt. The surface tension is also a linear function of
the concentration, at least within certain limits. The compressibilities
of the solutions decrease, in homologous salts, as the surface tension
THE PHYSICAL CHEMISTRY OF PROTOPLASM 209

increases, showing that the internal pressure of the solution due to cohe-
sion is also increased by the action of the salt.

Fats and soaps and bile salts decrease the surface tension of water.
The least trace of grease has a marked effect on the surface tension of
water, if the tension is measured by the ordinary methods, where the
surface is not fresh. But if the jet method is used for the determina-
tion of the tension, it is found that the perfectly fresh surface of the
water has its tension changed very little by the addition of soap. It is
only if there has been a chance for the surface to stand for a few moments
that the surface tension is found to fall rapidly. The reason for this
is that the concentration of the soap in the surface film increases up to
a certain point with the time and thereby makes the surface tension
steadily lower. It is a very important fact to remember, in considering
the surface tension of substances which may exist in two or more states,
that the state with the lower surface tension will accumulate in the
wurface.

The surface tension of water may be used to test the presence of oil
in the skin. If camphor is placed on the surface of perfectly pure water,
it darts hither and thither on the surface until by its solution in the
water it has lowered the surface tension of the water a certain amount.
If there is an extremely small amount of grease on the surface, and there
is generally enough grease in the air of an ordinary laboratory to spoil
the surface of water very quickly, the camphor stands still. Now it is
found if a glass rod be touched to the skin beside the nose and then
touched to the water, it makes the camphor still, provided the skin has
a normal amount of oil. A quantitative determination of the oiliness of
the skin in different localities can be made by this method. It has been
found that the ingestion of boric acid in sufficient quantity so completely
prevents the secretion of ofl, causing all the hair of the body to come out,
that the camphor no longer becomes still if the rod is rubbed by the side
of the nose and then touched to the water. Lord Rayleigh has calculated
how thin the film of oil must be to prevent the movement of camphor
on water and he has found that it is about of the order of magnitude of
a single molecular diameter of the oil. In other words, the oil is a layer
only a molecular diameter thick. The reason why the camphor darts
about on the surface is that by the solution of a little of the camphor
under the piece there is a local-lowering of the surface tension so that
the surface yields at this point and is stretched by the superior tension
of the surface elsewhere. This jerks the camphor away with it. It is a
good demonstration of how rapid movements may be produced through
the influence of surface tension. Many believe that the movements of
protoplasm and even muscle contraction are due to surface-tension
changes. But this may be discussed later. When the concentration of
210 PHYSIOLOGICAL CHEMISTRY

‘the soap or oil in the water is sufficient to lower the surface tension to
the point where the addition of camphor can lower it no further, then
the camphor stays still.

A more difficult question is raised if it be asked how it comes that
sodium chloride added to water increases the surface tension and that
soap or fat lowers it. Perhaps it follows from the fact that the cohesion
of salt is greater, and that of fat is less, than that of water. What the
cohesive pressure of soap or oil may be is unknown, but it certainly is
a good deal less than that of water. Water probably has an internal
pressure of about 10,000 kilograms per square em. at 15°. No other
liquid has at this temperature as high an internal pressure as this. The
surface tension of a pure liquid is a function of the internal pressure,
and the surface tension of water is accordingly higher than that of oil.
Acetic acid also lowers the surface tension of water, and here again the
surface tension of the acid is less than that of water. Salt, on the other
hand, has an internal pressure so great that the substance is a solid at
the ordinary temperatures; it is much higher than water. We may say,
then, that those substances with a lower surface tension than water
will move into the surface film and those of a higher surface tension will
move away from the film.

The accumulation of substances in the surface film.—The eminent
American mathematical chemist, Willard Gibbs, drew the conclusion from
that general principle of energetics and thermodynamics which says that
systems always endeavor to take that state, or form, in which their
potential energy is at a minimum, that if any substance lowered surface
tension it would accumulate in the surface film, and that if it raised
surface tension it would be less concentrated in the surface film than
elsewhere. This prediction was experimentally confirmed. It is easy
to see why this should be. If a substance by its presence in the surface
film is going to increase the surface energy it is evident, from the law
of conservation of energy, that this increase of energy can only be
obtained by the doing of work. The substance in order to move into the
film must then do work. This is as if there was an obstacle to its moving
into the film and hence there will be fewer molecules moving into the
film against this pressure than are moving in other directions. Hence
the concentration will be less in the film. Just the contrary will be the
case for substances which by their presence in the surface diminish the
surface tension. For these substances the surface film acts as a trap.
Once in it they find an obstacle to their leaving it, since by their
departure the energy of the surface will be increased, and hence to leave
it they must do work. It is found, as a matter of fact, that this diminu-
tion or increase of concentration in the surface film actually occurs,
although the amount is not usually very great. In sodium oleate solu-
THE PHYSICAL CHEMISTRY OF PROTOPLASM 211
tion, Milner found an excess of 0.4 mg. oleate per square meter in the
surface film.

The difference of concentration between the surface film and the rest
of the solvent may be of considerable importance in protoplasm. Thus
it is suggested that in the surface of contact of protoplasm with water,
lipin substances will accumulate and thus make a kind of intermediate
layer of a lower surface tension and of a fatty nature. But, inasmuch
as the whole substratum of the cell is of a fatty or lipin nature, it is
difficult to see how the surface tension of the junction of fat and water
could be changed by the passage of more lipin into the film; and, as a
matter of fact, there is no good evidence that there is such a layer about
the protoplasm. It is probable that often the protoplasm is not a liquid
at its surface at all but a gel-like solid.

Quite apart from the accumulation of soluble substances in the sur-
face film due to the general principle of maximum stability just men-
tioned, we often find that solid substances will accumulate in the surface.
If finely divided substance be placed in an emulsion and the emulsion
afterwards separates from the liquid, as an oil or ether emulsion may
separate. from water, the material in suspension is carried along with
the emulsion and thus separated from the liquid. This method may
sometimes be used to purify solutions from finely divided precipitates
which filter badly. The accumulation of these solids in the surface is not
due to the principle of Gibbs, just stated. They get into the surface
by movements accidentally carrying them there by the shaking when the
emulsion is made. Once there they are kept there by the surface film
which is like a solid membrane. They are supported at the surface
because they come to lie actually outside the water and between that and
the ether. They are supported there just as flowers of sulphur are sup-
ported at the surface of water and they are mechanically carried up by
the rising oil or ether. In protoplasm substances may get caught in this
same way at the surface boundary of protoplasm and water or possibly
even between boundaries in the cell and thus, perhaps, materials for
the making of shells or membranes may be accumulated (Macallum).
But this process is sometimes confused with that indicated by Gibbs,
whereas it is only remotely related to it.

There can be-no doubt that there is a certain tension of the surface
of the water which touches the protoplasm. The water at least is liquid.
But the same cannot be said of the protoplasm. It was long believed
that the movement of the amceba was due to these surface tension forces
in the protoplasm. The internal protoplasm of the ameba is certainly
at times liquid, for example the protoplasm which rolls out to form a
pseudopod ; but the rest of the protoplasm in the external layer, according
to Kite, is solid and gel-like and it can be cut off and cut into pieces.
212 PHYSIOLOGICAL CHEMISTRY

It is difficult to see how surface-tension changes of the water should cause
the movements in the interior protoplasm. Moreover, the movements
begin, according to Harrington and Leaming, not in the periphery but
in the interior. Jennings, who has very carefully studied the movements
of the ameba, concludes that whatever the cause of these movements may
be they are certainly not due to surface tension. When the pseudopod
moved forward the surface went forward too, not backward as it should
have done if the pseudopod was formed as the result of the lowering
of surface tension at the point of rupture. An examination of the move-
ment of the ameba from the side instead of from the top shows that
the ameeba walks on pseudopods as if they were legs and that the motion
is not surface tension. According to the observations of Kite, the move-
ments seem more probably due to the liquefaction or taking up of water
by the cell protoplasm, this differing in differing regions and causing
the movements in the protoplasm. It is very doubtful, therefore, whether
the movements of an ameba are due to surface tension any more
than those of a fish are due to surface tension. It is very difficult to
apply to such a complex organized half-gel and half-sol substance
such as protoplasm is, the conclusions derived from the study of
pure liquids in the simplest conditions. The application is extremely
hazardous.

When one comes to consider the protoplasm as a whole, it is impos-
sible to say to what extent it is made up of small chambers of capillary
dimensions; to what extent it has a structure of such a kind that capil-
larity should play a large part in it. The granules and droplets of proto-
plasm are many of them solid, not liquid, and they are imbedded not in
a liquid but in a more or less solid gel. It is impossible to say to what
extent surface forces are active in such a semisolid medium. It is,
therefore, at least too early to speak of surface tension as determining
the distribution of substances in the cell, as has been done by some
observers.

Surface tension plays an important physiological réle in its relation
to the absorption of water by the cell colloids. If a colloidal or gel-like
substance such as gelatin, perfectly dry, be put in contact with water
it absorbs a considerable quantity of the latter. This absorption is due
to the chemical affinity of the water for the gel substance. By the pene-
tration of water into the gel there is produced an enormous surface of
eontact of the water and the gel particles. Now, if the gel be acted upon
by any substance which increases its affinity for water, which increases
its power of union with the water molecules, the attraction for the water
is increased and consequently the surface tension of the water at the
surface boundary is lowered and the surface will be increased. In other
words, more water will be absorbed, On the other hand, if we add to the
THE PHYSICAL CHEMISTRY OF PROTOPLASM 213

liquid any substance which increases the surface tension of the water,
the surface tension will be increased and the surface of contact will be
reduced, the gel will lose water. It is because of this fact that the move-
ment of water into and out of the structures of protoplasm becomes pos-
sible. Acids, for example, enormously increase the attraction of proteins
for water, consequently acids will lead to the taking up of water by the
protoplasm, as they are found to do. Salts, on the other hand, may have
an opposite action. The movements of all kinds of living cells are prob-
ably due to this swelling, or dehydration, of the protoplasmic gel; and
we may, therefore, consider it briefly. Since the protoplasmic gel is made
of colloids, we may begin by a study of these substances.

Colloids—The microscopic examination of living matter shows that '
the cell is not alike in all its parts; it is not homogeneous, but it has a
definite structure. This structure is due to the colloids of the cell.
There are numerous coarse granules of various sizes and kinds; a nucleus;
nucleolus; and a clear, more homogeneous matrix in which very fine
granules are revealed under the highest powers of the microscope, and
particularly when photographed by ultra-violet light. The details of
this structure appear somewhat different in different cells, but it has
been suggested by Biitschli, after long investigation both of fixed and
living protoplasm, that, including that part which appears to be homo-
geneous, protoplasm has in reality a foam-like structure, the compart-
ments of the foam being very small and the walls extremely thin. Within
the cavities of the foam a solution is supposed to exist. This conception
is probably not strictly accurate, but there is no doubt of the organiza-
tion and heterogeneity of the cell protoplasm whatever the exact nature
of its finest:structure. The cell is an organized structure; it is not form-
less. If the structure of the cell is destroyed, if the nuclear membrane
or cell membranes are ruptured by mechanical means, as by cutting or
grinding the cell, or by the penetration of ice crystals in freezing and
thawing, or by stirring up the protoplasm so as to bring about a thorough
mixture of its various parts, there is a great outburst of chemical activity -
evidenced by the formation of acid and the liberation of carbon dioxide,
and cell life stops. Organization is, therefore, essential for metabolism.
The different substances of the cell must be kept apart, localized in dif-
ferent regions. If they are mixed, they react with and destroy each
other.

The cell is in fact not a single room in which all the chemical proc-
esses occur in a higglety-pigglety manner, as they occur in a beaker, but
it is rather a well-organized chemical factory with different chemical
processes occurring in different regions and in which substances are being
elaborated as fast as they are required. How their production is regu-
lated will be discussed farther on.
214 PHYSIOLOGICAL CHEMISTRY

.. This division of labor within the cell,-this separation into different
eompartments is due to the fact that protoplasm is not primarily a solu-
tion, or is so only in part. but it is a jelly-like substance or technically a
gel. It is a semisolid substance consisting of solid and water in intimate
admixture or union. This gel structure of protoplasm is due to the fact
that the organic substances of which it is in part composed have very
large molecules, or are large particles, so that they have little velocity of
translation, but cohere together. Such substances are known as colloids,
and it is in virtue of the colloidal nature of the products elaborated
from the foods by the cell’s chemical processes that life is possible.

The colloidal substances in protoplasm contributing to its structure
are the proteins, carbohydrates and lipins. These together form the vital,
organized substratum of the cell, containing in its interstices the water
and substances of simple molecular kind, the extractives, salts and vari-
ous other organic bodies in true solution. As the whole organization of
the cell depends on the colloids and vital activity is so dependent upon
their affinity for the water or solution present, an affinity which is easily
modified by salts, metabolic products, acids, anesthetics and other drugs
and by digestive enzymes, an examination of the general properties of
colloids and the colloidal state and particularly of colloidal proteins is
necessary for the understanding of vital processes.

Properties of colloids. All substances in solution were divided into
two great groups about the middle of the nineteenth century by the
British physicist, Graham; into substances which would diffuse through
parchment paper or other membranes wet by water, substances generally
erystalline in nature, which he named crystalloids; and into substances
which would not diffuse through parchment or other similar membranes,
substances which he called colloid (Gr. kolla, glue; eidos, appearance)
or glue-like bodies, because they behaved like glue in this respect. Among
the colloidal, or glue-like bodies, were albumins, gum arabic, glue itself,
starch and many other animal and plant substances. Besides the prop-
erty of not diffusing through paper, these colloids had several properties
in common. Most of them, but not all, were amorphous, non-crystalline
bodies; they formed viscous solutions which when sufficiently concen-
trated would set, or gel. When in aqueous solution, Graham called them

“hydrosols; when gelled, hydrogels.

It is now known that many colloidal bodies may be crystalline. For
example, the chromoproteins hemoglobin, phycoerythrin and phycocyan
are all readily crystallized; and many other colloidal proteins such as
edestin, excelsin, serum albumin and ovalbumin may be obtained crys-
talline; but nevertheless it is true that in most cases special conditions
are necessary for the crystallization of colloids, and when crystalline
the crystals are small and of microscopic dimensions; and many colloids
THE PHYSICAL CHEMISTRY OF PROTOPLASM 215

have never been crystallized. A great many crystalloids, also, even
substances like common salt, may be obtained in a colloidal form.

It is in virtue of these three properties—non-diffusibility, of forming
viscous solutions and real gels—that the colloids are able to act as true
organizers of the cell’s activity.

The peculiar and distinctive properties of colloidal solutions are due
to the large size of the particles which are dispersed. Owing to this
large size, surface tension phenomena between solute and solvent come
into play at the boundaries of the particles, and these phenomena, which
are lacking in ordinary solutions, give to colloidal solutions properties
which ordinary solutions lack. In order that a substance shall be col-
loidal, the dispersed particles of it must be sufficiently large to separate
the molecules of the solvent beyond the range of their cohesional attrac-
tions. This produces the surface and the surface tension. The range
of molecular attraction is of the order of magnitude of 1<10—* ems., or
1<10-* mms. This is 1p. Dispersed particles must be, therefore, at
least 1 wy in diameter in order that the dispersion be colloidal.

Colloids may be arbitrarily defined as substances of which the par-
ticles in solution have a diameter ranging from 1-100 uu, One pv is the
one thousandth part of a millimeter. They grade into the diffusible crys-
talloids on the one hand, and suspensions on the other.. An idea of the
size of a colloidal particle may be obtained from the fact that a molecule
of ether has a diameter of about 3<10—* ems. or about .000,000,3 mm.
One “ is .000,001 mm. The shortest visible waves of violet light have
‘a wave length of about 400 wy. That the size of colloidal particles
is large is shown not only by their non-diffusibility, but also by the fact
that they may at times be seen in the ultra microscope; that they scatter
light and the light so reflected from their surfaces is polarized (Tyndall
phenomenon) ; and by the fact that they may be centrifugalized out of
solution.

The size of colloidal particles cannot be directly determined by micro-
scopic measurement because of the diffraction halos which surround them
and indeed which make them visible. The particles themselves cannot be
seen. The diameter of a particle of sodium oleate can be calculated
approximately by measuring the thinnest spots of the films of solutions
of sodium oleate. These are found to be about 610 ems. in thickness.
Since the thinnest films are at least three times the diameter of a mole-
cule, a molecule or particle of sodium oleate cannot have a diameter
greater than 210-7 ems. or 210—* mms. The smallest particles which
are visible in the ultra microscope are said to be about,5 wor 5X10—-*
mms. The ultra microscope can show particles, therefore, which have a
diameter little larger than three particles of sodium oleate. Linear
dimensions found for some colloidal particles are: Gold 6-130 my;
silver 50-77 wu; platinum 44 wu,
216 PHYSIOLOGICAL CHEMISTRY

In the ultra microscope light enters the solution from the side instead
of from beneath as in the ordinary microscope. The light strikes the
colloidal particle and is reflected upward to the eye. One sees the col-
loidal particles, when they are sufficiently large, as bright specks on a

   

ee EIR.

Fic. 20.—Lantern and microscope arranged for ultra-microscopic observation. Cardioid
condenser on the microscope.

dark field. These bright points are usually in active Brownian move-

ment. The smallest particles cannot be seen in this way. For example,

. = the particles of casein in. solution are

es colloidal, but they do not appear in

the ultra microscope. When a casein

solution clots, however, the particles

become visible and may be seen to
grow.

When it is remembered that some
forms of living matter exist, sub-
microscopic germs of disease, which
are filterable through a porcelain filter,
but searcely visible in the ultra micro-
scope, it is probable that their dimen-
sions can hardly be larger than a very
few molecules cf a protein colloid.

Fig. 21.—Cardioid condenser for . . 5
Sitsduiteroseaple. slau @he. ways: oF Their organization must, hence, be ex-

aight illuminate the liquid on the glass tremely simple and can hardly be other
than that of a chemical substance.
Colloidal substances readily separate from ervystalloids if brought
into parchment paper immersed in the solvent. The ecrystalloids pass
through; the colloids remain behind. This process of separation is called
dialysis (dia, through; lysis, to loosen). Colloidal solutions may be
purified in this manner.

 
THE PHYSICAL CHEMISTRY OF PROTOPLASM 217

The Tyndall phenomenon. Most colloidal particles are sufficiently
large to show the Tyndall phenomenon. By this is meant that colloidal
solutions have the property of scattering a beam of light passing
through the solution, so that the path of the light rays in the solution
becomes visible, just as in passing through a dusty atmosphere. This
is known as the Tyndall phenomenon, and Tyndall used this method
to determine when the dust particles had subsided out of the air in
his famous experiments on artificial biogenesis. The light which is
thus scattered from the particles, or reflected from their surfaces, is
found to be elliptically polarized like other reflected light. Since the
blue rays are the more easily reflected, colloidal solutions often show
a blue opalescence.

Suspensoids and emulsoids. For convenience, but not because there
is any sharp line of demarcation between them, for on the contrary they
grade one into the other, colloids are divided into two classes: into sus-
pensoids and emulsoids. The colloidal solutions of metals are typical
suspensoids. They are easily precipitated from their solutions by the
action of salts; they do not gel; and they form generally rather dilute
unstable sols; the emulsoids, on the other hand, of which protein colloids,
starch, gum arabic and gelatin are types, have the property of forming
semisolid or solid gels; that is, solid systems containing a great deal of
water. Most of the emulsoids, however, will flock and not gel if the
solutions be sufficiently dilute, so that the distinction is not a funda-
mental one. The colloids in protoplasm are emulsoid colloids.

Suspensoids Emulsoids
Collodia] metals Gum arabic
Kaolin Proteins
Antimony sulphide Starch
Cadmium sulphide Gelatin
Arsenious sulphide Silicie acid

Soap
Agar-agar

Nucleic acid

Colloidal particles are electrically charged. A fundamental fact about
aqueous colloidal solutions is that the particles bear electrical charges,
the charge of opposite sign being in the water contiguous to the colloid.
That the colloids are electrically charged may be shown by placing elec-
trodes connected with a battery in a colloidal solution. The colloidal
particles move with or against the current. Since only electrically charged
particles are thus transported, the colloidal particles must be charged.
The various colloids may be divided into those which move to the anode,
and are, hence, electro-negative; and those which move to the cathode,
and are, accordingly, electro-positive.
218 PHYSIOLOGICAL CHEMISTRY

Electro-negative | Electro-positive
Arsenious sulphide. Ferric hydrate. ;
Antimony sulphide. Basie proteins, histones and protamines.
Gold. Proteins in acid solution.
Platinum. Oxyhemoglobin.
Copper and other metals. Aluminum hydrate.

Most natural proteins in neutral or
slightly alkaline solution.

‘Lecithin and phosphatides.

‘Gum arabic.

Glycogen and starch.

Nucleic acid.

Soaps.

How many charges there are on a single colloidal particle has not beeu
determined, so far as I know. Some writers speak as if there were a
complete electric double layer all about the particle. There is probably bur

 

Fie. 22.—Apparatus for the study’ of cataphoresis of colloids. Non-polarizable elec
trodes are in the top compartments. The culloidal solution is brought into the U tube
below the gelatin plugs. In the figure it may be seen that the colloid is accumulating
below the plug on the anode side and is leaving the cathode chamber. The colloid is
electro-negative.

a single charge on some soap colloids, but the number undoubtedly is
much greater in others. ,

Origin of the electrical charges. The origin of these electrical charges,
of the existence of which there can be no doubt, was at first obscure.
It was originally suggested that the particles owed their charges to the
faster-speed of migration of the hydrogen or hydroxy] ions of water, the
ion which was going faster would presumably strike the colloid first (see
p. 153) and in this way give it a positive charge in acid solutions, where
hydrogen ions predominate, and a negative’ in alkaline, where the
hydroxyl ions are predominant. It is, however, generally recognized that
THE PHYSICAL CHEMISTRY OF PROTOPLASM 219

this explanation is incorrect; and there can be little or no doubt that
they acquire their charges like any other ions by the process of ionic
dissociation. The colloidal particle sends into the water one ion, metal
cr metalloid, and it retains the opposite charge. This process may be
illustrated by glass. Glass in contact with water becomes electrically
uegative and the water positive, the reason being that glass, which is a
silicate, sends potassium or sodium ions into the water, thus making
the glass electro-negative, and the water, containing the ion, positive. It
is quite possible to substitute the sodium ionized from the glass by
another metal. If, for example, a glass bottle contains a solution of
copper sulphate it will be found, if the sulphate is poured out, that some
sodium from the glass has gone into the copper sulphate solution and
some of the copper remains attached to the glass so firmly that it is very
difficult to remove it with water. It is necessary to treat the glass bottle
with acid in order to free it from copper. <A copper silicate has been
formed in place of the sodium silicate on the surface of the glass. It
is for this reason that in trying physiological experiments glass utensils,
which have had mercury or copper salt solutions in them, must be washed
with the greatest care. :

If, instead of using the glass as a bottle, finely pulverized glass, or
glass wool, is placed in a copper sulphate solution, the surface of contact
is so much larger than in the case of the bottle that the power of com-
bining with the metal is greatly increased, so that the effect of the glass

 

Fie. 23.-—Beaker of water showing Fic. 24.—Showing how copper in a
negative charges on the glass and the water copper sulphate solution replaces the sodium
made electro-positive by the sodium ions in oe glass and is absorbed ‘by the beaker
it. wall. ae

in modifying the concentration of the copper in the solution becomes
plainly noticeable. Thus quite large quantities of copper may be sepa-
reted on the glass and removed from the solution. A very interesting
experiment was tried by True and Ogilvie illustrating. this combining
power of glass. They placed sprouted pea seedlings in copper sulphate
solution just concentrated enough to be toxic to ‘the seedlings, as shown
by the wilting of the rootlet. If now some powdered glass or glass wool,
220 PHYSIOLOGICAL CHEMISTRY

or even filter paper, was placed in the bottom of the tube containing the
copper and the seedlings, so much copper was taken out of the solution
by the filter paper or glass that it was no longer toxic and the seedlings
grew.

It is easy in imagination to carry the process of subdivision of the
glass further, until, instead of the pulverized glass, a colloidal silicate
is obtained, the particles becoming so small that their surface compared

 

* A
Go ee
4

Fia. 25.—Colloiual ‘sudium _ silicate Fig. 26.—To illustrate the possible way
showing the silicate particles electro-nega- in which cotton fibers become electro-nega

tive with the positive sodium ions in the tive in water by sending some hydrogen
water close by. ions into the water.

to their bulk is so large that the particles remain suspended in the water.
As the number of particles of silicate in the surface increases by sub-
division, the number of bonds between the water and the silicate increases
also. The action of the glass in detoxicating the copper solution thus
appears to increase proportional to the surface of separation of water
and glass and processes of this kind are often referred to as surface
phenomena, and treated as if they differed in kind from other chemical
reactions. There is, however, no difference in principle between the
condition of the soluble glass as a colloidal silicate in solution in the
water and the solid glass bottle containing the water in it. In each case
the glass is charged by the process of ionization; and the union of metal
and glass is a true chemical union. But it will be seen that if the glass
should be filtered off and analyzed the proportion of copper and glass
would not be found fixed, as in ordinary chemical compounds, but vary-
ing in every degree with the size of the surface of the glass.

Let us suppose, now, that instead of the glass being a solid, so that
it retains its shape and the water is forced by its affinity for the glass
to spread itself over the surface, the glass molecules were freer to move.
suppose the glass were a liquid. It is clear that if the attraction between
the water and glass were sufficiently great, the surface energy would be
most reduced by the most complete and extensive contact possible between
the water and the glass. Hence the potential energy of the system would
be least when the surface of contact was most extensive. The system
would proceed as far as possible in the direction of reducing its surface
THE PHYSICAL CHEMISTRY OF PROTOPLASM 221

energy and the glass would, accordingly, dissolve in the water and the
water in the glass. Ordinarily in a colloidal solution the division
of the colloid proceeds to that point at which the surface energy is a
minimum.

Other colloidal solutions obtain their charges in the same way as the
glass. Thus colloidal silicic acid sends a hydrogen ion into the water.
It is probable that all carbohydrate materials of an insoluble nature ar
of a nature to form colloidal solutions do the same. Figure 26. The
carbohydrates are very weak acids, as discussed in Chapter II. In some

o

 

 

re +t
Oe SS et
— +
Rae A@*
& Ag

Fic. 27.—Showing how various col- Fie. 28.—Showing how a zinc plate in
loidal metals are electro-negative since they water becomes electro-negative while posi-
send a few positive metal ions into solu- tive ions are in the water contiguous to the
tion. Each particle has an electrie double plate.
layer at the surface.

 

 

 

 

 

cases, however, there may be metal ions present in place of the hydro-
gen, as in gum arabic. Filter paper or cellulose or glycogen, like glucose
and cane sugar, probably send hydrogen ions into the water, the insol-
uble residue taking the negative sign. Filter paper will thus combine
with and hold positive colloids filtered through it. Gum arabic is
electro-negative. The positive ion in this case may be calcium, since
this is always present in gum arabic. The colloidal metals are. all
electro-negative. This is due to the fact that all the metals, in the pres-
ence of pure water, send some positive ions of the metal, the number
-varying with the solution tension of the metal, into the solution, and
accordingly the colloidal metal particle is electro-negative. Since the
water is electro-positive, due to the presence in it of the positive metal
ion, there is, along the surface of a plate of metal in water, an electric
double layer. The colloidal solution of ferric hydrate is made by shak-
ing ferric hydrate in ferric chloride solution and then dialyzing out the
ferric chloride. The hydrate is electro-positive because by the affinity of
the iron of the hydrate for the iron of the ferric chloride, the ferric
hydrate is held in solution. The ferric hydrate has thus the charge of
the positive ferric ion and the negative ion in this case is chlorine,
some of which always remains in the colloidal solution, A similar
222 PHYSIOLOGICAL CHEMISTRY

+++ i
1. Fe(OH), + FeCl, = Fe(Fe(OH,) 1+ 3Cl
Colloidal ferric
hydrate.

2. (As,S.), +H,S=H,S(As,S,),— + HS(As,8,) ,

, Colloidal arsenious
sulphide.
phenomenon occurs in the case of arsenious sulphide sol. In this case
the solution is prepared by passing H,S into arsenious acid. The
arsenious sulphide does not precipitate, but remains as a yellowish
red solution. If this solution is dialyzed a long time, to get rid of
the sulphureted hydrogen, and is then evaporated and analyzed, it
is found that there is always more sulphur than is sufficient to form
the sulphide of arsenic. It is clear, then, that the arsenious sulphide, as
it forms, unites with the hydrogen sulphide, probably by the affinity of
the sulphur of the two sulphides for each other. The hydrogen sulphide
ionizes, separating hydrogen as a positive ion and sulphur as a negative,
so that the arsenious sulphide thus is united with the anion and is accord-
ingly electro-negative. Cadmium sulphide and antimony sulphide be-
have in the same way, so that all of these colloids are electro-negative.
In addition cadmium sulphide shows a very interesting phenomenon,
which recalls some of the specific reactions of the precipitins; the cad-
mium sulphide sol is extremely sensitive to cadmium salts and is pre-
cipitated by very small amounts of these salts—by a dilution of 1 :250,000
CdSO,, as compared with 1:20,000 of MnSO,.

Perhaps one of the most interesting and instructive cases of a alt
loidal solution is that of soap in water. In alcohol a soap is quite
normal, the molecules being monomolecular and not colloidal; but in
water there is a true colloidal solution. The explanation of this throws
light on many puzzling colloidal phenomena. In water soap is hydro-
lyzed, that is, some of the soap molecules react with the water to form
sodium hydrate and the free fatty acid, the fatty acids being very weak
acids.

Stearic acid is not by itself soluble in water, but just as atoms unite
readily in a physical or chemical union with other atoms of the same
kind, so stearic acid unites with the stearic ion of that portion of
the soap which has not been hydrolyzed. Every sodium atom has, there-
fore, attached to it one stearate ion, but united with this stearate ion,
in either a physical or a loose chemical union, are two or three molecules
of stearic acid. If now the soap is salted out of the solution, this mixture
of sodium stearate and stearic acid separates in the form of soap. The
soap thus forms an electro-negative colloidal particle consisting of several
molecules of palmitic, or stearic, acid, and this is held in solution because
of the great attraction of the sodium ion for water. The alcoholic soap
THE PHYSICAL CHEMISTRY. OF PROTOPLASM 223

Oo 0

i = Il
1, Na—O—C— ( cH, ) 1e CH, =—— Na+0o—C— (CH,) 10 CH,
Sodium stearate Stearate ion

2, Na—0-—C—(CH,),,—CH, + H,O ———* NaOH + H—O—C—(CH,), «CH,
‘ / Stearic acid

3 No + 0—C—(0H,),,—CH, + 2HO—C—(CH,),,—CH, ==>

I
0

Fs "0—C—(CH,). —CH
ai 2H0—C—(CH.) .—CH,

(

Colloidal soap.
solution is normal, and not colloidal, for the reason that hydrolysis does
not occur in the alcohol and the stearic acid, if formed, has so much
greater an affinity for alcohol than for ‘water that it does not form
molecular complexes. The cleansing power of soap depends upon this same
principle of affinity between the palmitic or stearic acid colloidal. particle
and the fatty acids of the neutral fats. When soap is put on the skin,
the fats of the skin, like the palmitic acid of the soap, adhere to the latter,
and the whole is suspended in water because of the attraction of the
sodium for the water and the electro-static affinity between the sodium
and the palmitate or stearate ion. Very large, loose physico-chemical
aggregates may be built up in this way. Thus vaseline, a hydrocarbon,
does not readily combine with soap, but it does have an affinity for oil
and oil for soap. Thus by rubbing vaseline with oil it is easily removed
by soap, the oil acting as an intermediate body. Probably such unions
as these contribute to the formation of protoplasm ; the union between fat,
phospholipin and cholesterol may be of this nature.

_ These examples will suffice to show how colloidal particles get the
electrical charges.they have in solution and that they are produced by
processes of ionization.

Precipitation of colloids by salts. Many colloids, particularly the
suspensoids, are very easily precipitated from their solutions by ‘salts
of any kind; but all colloids aggregate into larger or separate into
smaller particles, or change their surface of contact with water wihen
they are in the gel state, under the action of salts. This is one of the
fundamental changes which salts can produce in living matter, and since
there is good reason for thinking that the mechanics of living matter
involves this process, perhaps more than any other, a careful study of it
224 PHYSIOLOGICAL CHEMISTRY

has been made. The change in state of colloids by salts is of great
practical importance not only in the industries, in dyeing, in the treat-
ment of sewage, in mining, in chemical technology, etc., but also in
therapeutics and physiology.

The addition of various neutral salts to the solutions of colloids gen-
erally causes their precipitation. But some colloids are dissolved by
small amounts of salts. The amount of the salt necessary to precipitate
varies with the salt and the colloid, but toward all colloids the salts
arrange themselves in about the same order of precipitating efficiency.
The following examples, most of which are taken from the work of Linder
and Picton, who, while not the first to investigate these phenomena, put
their results in a very convenient form, show the minimum amount of
salt necessary to bring about a precipitation of the colloid in a given
time.

Precipitation power of various salts on arsenious sulphide sol (Linder and
Picton) AICI, being taken as unity. The figures represent the relative concentration

of other salts necessary to precipitate. The limiting concentration for precipitation
by AICI, is about .0001 molecular.

Trivalent cations Bivalent cations Monovalent cations
AlCl ‘ 1 SrCl is 20.0 HCl 954
Al, ( 80,) ‘ 0.6 Sr(NO, ) P 20.9 HBr 909
FeCl, 2.2 CaCl, 21.3 HI 933
Fe, (SO,) i. 1.50 CaBr, 21.3 HNO, 933
Cr,(SO,), 1.00 CaSO, 26.0 H,S0, 1,980

ZnSO, 27.3 H,80, 3,640

Bivalent cations ZnCl 5 21.8 H. AsO a 5,100
PbCl, 3.65 FeCl 4 23.1 H FO i 4,430
HgCl, 5.23 FeSO, 31.8 NH,Cl 1,010
CdCl, 16.4 CoCl, 20.9 NH,Br 1,200
CdBr, 15.5 CoSO, 31.9 Kcl 1,590
Cdl : 22.7 NiCl, 24.6 KBr 1,640
Cd80, 15.0 MnSO, 32.8 NaCl 1,680
Cd(NO,) a 14.6 CuSO, 14.8 NaBr 1,770
MgCl ¥ 16.4 BaCl 5 19.1 NaNO, 1,900
MgBr, 21.3 Ba(NO,), 18.6 LiNo, 1,770
MgSO, 34.1 TI,SO, 13

Precipitation of an electro-positive colloid. Albumin from Picea excelsa in 0.1
per cent. HCl (Posternak). The figures indicate the molecular concentration of the
weakest precipitating solutions.

Salt Concentration Salt Concentration Salt Concentration
HCl 0.388 NH,Br 0.230 NaNO, 0.116
NH,Cl 0.385 NaBr 0.200 KNO, 0.136
aCl 0.325 KBr 0.206 %H,SO, 0.0714
Cl 0.380 Nal 0.069 Yy (NH,) 259, 0.0376
Y%MgCl, 0.311 KI 0.098 %Na,SO, 0.0274
¥,SrCl, 0.366 HNO 0.137 %eK,SO, 0.0402

¥,BaCl, 0.414 NH No, 0.135
THE PHYSICAL CHEMISTRY OF PROTOPLASM 225

It will be seen from the examples cited that salts containing monova-
lent metals (cations) require stronger solutions to precipitate electro-
negative colloids than salts containing bivalent metals; and these in turn
require stronger solutions than salts of trivalent metals. The valence of
the ion of the opposite charge to the colloid appears to determine, or to
be a powerful factor in, the precipitation of a colloid. It will be seen
that apparently, at any rate, valence is of more importance than the
chemical nature of the ion. Furthermore, the valence of the salt ion
which is of the same sign as the colloid, the anion in the case of electro-
negative colloids, appears to exert no influence on the precipitation. For
example, while calcium chloride is much more powerful as a precipitating
salt than the chloride of sodium, sodium chloride and sodium sulphate
have about the same precipitating power. These facts have been found
to be very general. Toward electro-positive colloids the valence of the
anion is important.

It is necessary to examine the character of the precipitate formed
if an insight is desired into the mechanism of precipitation by salts.
Does the salt go down with the colloid or not? It has been found in
all cases which are thus examined that the ion of the opposite charge
to the colloid, that is the precipitating ion, always is found in the
precipitate. Thus, when antimony sulphide is precipitated by sodium
chloride, there is always sodium in the precipitate; if by potassium
chloride, there is potassium in the precipitate, and so on. If a protein
is salted out of solution by a sulphate, sulphuric acid is always found
attached to the protein after dialysis of the salt. These general
phenomena have been formulated in the following rules:

1. The precipitating agent is always the ion of the opposite sign to
that of the colloid. That is, if the colloid is negative, the precipitating
ion is always the cation; if: positive, the anion.

2. The precipitating power of the precipitating ion is a function of
its valence. Bivalent ions are much more powerful than monovalent;
polyvalent more powerful than bivalent.

3. Some of the precipitating ion is always precipitated with the
colloid.

4. The valence of the ion of the same sign as the colloid is of no
importance in the precipitation.

5. The ion of the same sign appears to exert an influence antag-
onistic to the precipitating action of the ion of opposite sign.

How does the valence of the ion act? The fact that the precipitation
is a function of the number of valences is of great significance, because
the valences are probably electrical in nature. The electrical state of
the ion thus appears to be of more importance than its chemical nature.
Attempts have been made to explain how the valence might act. There
226 ‘PHYSIOLOGICAL CHEMISTRY

have been two explanations given of the way in which an increase of
valence might increase the precipitating action of a salt. The first is
that of Whetham and Hardy. Since it is the valence, or the number of
electrical. charges on the ion, which is of importance in precipitating,
Hardy suggested, and Whetham computed, that there was a far greater
chance of ‘two charges arriving simultaneously in the neighborhood of a
colloid particle when both charges were on the same ion, than when they
were on separate univalent ions. For a trivalent ion the chances were
very much better that the three charges should arrive simultaneously
if all were on one ion, than if each were on a separate ion. Their idea
was far removed from that of a chemical union between the ion and the
colloid. Hardy supposed that the electrical double layer about the col-
loidal particle was destroyed by the approach of the precipitating ion,
and the solution was in this way made unstable. This interpretation
was rendered very unlikely when it was found that the precipitating ion
went down with the colloid in union with it. Furthermore, the author
has shown that the ion of the same sign as the colloid exerts a dissolving
action, making the colloidal solution more stable, but valence plays no
part in its action. Compare, for example, the sodium and potassium
salts ‘in Posternak’s work. It always takes a higher concentration of a
potassium salt to precipitate than of a sodium salt of the same acid.
If it were simply a question of the opposite action of electrical charges,
the same reasoning should hold for the ions of the same sign; and the
efficiency of a polyvalent ion of the same sign in holding a colloid in
solution should also be greater commensurately than that of a monovalent
ion. Since valence is of importance in one case, that in which the ion
unites with the colloid, but is without importance for the ion which does
‘not unite with the colloid, the writer has suggested that bivalent and
trivalent ions are more effective in precipitating because they unite two
or three or more colloidal aggregates into very large aggregates of the
‘following kind: Ca-colloid-Ca-colloid-Ca-colloid-Ca-colloid. The aggre-
gates are nearly always obviously larger when the precipitation is by
‘a polyvalent ion. Since ions of the same sign do not unite with the
colloid, the number of charges they bear is of no effect.

How does the ton of opposite sign precipitate? This is a very fun-
damental question and one to which no definite answer can as yet be
given. It is essentially the question of solubility. It is not known why
sodium sulphate is soluble and barium sulphate insoluble. There is a
surface of contact between the colloidal particle and the water or. salt
solution. The action of the salt on the surface tension of the water may,
therefore, be considered first. All of these salts raise the surface tension
of the water, as may be seen in the figures cited on page 208.

Any agent which raises the surface tension of the water will, if it have
THE PHYSICAL CHEMISTRY OF PROTOPLASM 227

no other action, cause the system water-colloid to reduce the surface of
contact in order to reduce the potential energy of the surface to a mini-
mum. This factor then will result in the flocking, or the coalescence, of
(he colloidal particles into larger aggregates of smaller surface. An
e.amination of the effects of salts on surface tension shows that they
arrange themselves somewhat in the same order as they do in their pre-
cipitating powers. This does not explain the fact; it simply expresses
another fact. It does not say exactly how this coalescence is brought
about. Another factor may be this: The colloid is rendered stable by
the electric double layer; the effect of this double layer is to reduce the
tension of the surface, because it sets up electro-static stresses across the
surface between colloid and solution. Now, it is generally true that salts
of bivalent metals ionize somewhat less readily than monovalent. Hence,
if a monovalent ion is replaced by a bivalent, the ionization will be
reduced, the electric double layer reduced, the surface tension increased
and hence a reduction of surface will occur, if it can occur. The water
in contact with the uncharged particle has, of course, the highest surface
tension when there is no union or attraction between the water and the
colloid. The consequence is that the undissociated -particle is the least
soluble particle. The ion, or charged colloidal particle, is more soluble,
because the double layer reduces the surface tension.

In some of the colloids, as in the globulins for example, which. are
soluble in dilute salt solutions but not in water, the addition of a little
salt is able to cause the colloid to dissolve. This can only be explained
by supposing that the salt acts on the colloid so as to increase its affinity
for water, so that by this the surface tension is reduced more than it is
raised by the direct action of the salt on the water. This muen be accom-
plished in the following way:

Colloid -— Colloid + a
Ss eee ee

Colloid + H + Na + Cl2—* Colloid + Na + HCl

ColloidNa + HCI—~ColloidHCl + Na
In this case the HCl formed is probably united with the colloid,and may
ionize itself, making the colloid positive at one place and negative at the
other. At any rate, the tension of the surface between NacolloidHCl
and water is reduced below that of the globulin alone, and solubility is
increased. The addition of more salt precipitates. The same thing
may happen in arsenious sulphide, which is also rendered more soluble
by very small amounts of salt, but precipitated by larger. Here normally
the positive ion is hydrogen. By the replacement with sodium in sodium
chloride the ionization will be increased. However, H,S is so weak an
acid that this action will soon cease, then the addition of more NaCl will
228 PHYSIOLOGICAL CHEMISTRY

reduce the ionization and the colloid will be precipitated as the sodium
salt. Addition of the salt pushes back the ionization and the sodium
salt of the colloid is accordingly precipitated.

ee
Na + colloid Nacolloid

Soluble Very little soluble.

According to this explanation the weaker the acid of the sodium salt
used, the larger should be the amount of salt necessary to precipitate.
It should take more sodium iodide than of chloride to precipitate, and
this is found to be the case.

Influence of solution tension. There is another factor in the precipi-
tating power of an ion or salt besides the valence. It is found that hydro.
gen chloride is always more effective in precipitating an electro-negative.
colloid than sodium chloride; and differences exist between the lithium,
rubidium, cesium, potassium and sodium salts, all of which are monova-
lent. Similar differences exist between calcium, magnesium, barium and
strontium, or between aluminum, ferric and chromic salts. These dif-
ferences have been studied by the author and they are illustrated by the
preceding tables. Thus Posternak found that the limiting precipitating
concentrations of NaCl, NaBr and Nal were .325, .200 and .069 M.
respectively. The anions have the same valence presumably, but the
precipitating action of the iodide is greater. In Linder and Picton’s
work, KC! had a precipitating power represented by 1/1590; while HCl
was 1/954.

It is a matter of general experience also that the heavy, and par-
deularly the noble, metals precipitate albumin colloids more effectively
than do the alkaline or alkaline earth metals of the same valence. Cobalt,
cupric and mercuric chlorides are far more powerful precipitants of the
colloids than are the alkaline earths. To explain this difference the
author has pointed out that the metals arrange themselves in the order
of their solution tensions. In other words, that besides the number of
charges carried by the ion, the efficiency of the ion is measured by the
voltage of the ion; that is, by the tendency of the ion to give up its
charge, or by the amount of available energy in the ion. It thus hap.
pens that although silver is monovalent it is a better precipitant of
the colloids than is calcium. There are always two factors in energy,
the volumetric or capacity factor, i., the quantity factor; and the
intensity factor. Just as in an electric current the amount of work
it can do is measured by the amount of the current and the voltage, so
in an ion the work it can do is measured by the number of charges and
by the voltage or intensity factor of the charge. The silver ion holds
its positive charge far less firmly than does sodium. Silver, as an ion,
attempts to get rid of its charge and go over into the metallic state; so
THE PHYSICAL CHEMISTRY OF PROTOPLASM 229

that ionie silver is a good oxidizing agent. When it is reduced a large
amount of energy is set free. In the silver ion there is a large amount
of available potential energy as compared with metallic silver.

It is for this reason also that silver salts are so toxic and poisonous,
whereas the metal is so inert. In the case of sodium the reverse is the
ease. Metallic sodium has more energy in it than the ion. It is this
difference in energy content that makes the properties of ionic sodium
so different from those of the metal, one being a necessary food for the
body, the other a terrible caustic which destroys all living matter with
which it comes in contact. Copper, ferric iron, lead, gold, platinum,
arsenic, bismuth and mercury all resemble silver in the respect that they
have more energy in the ionic than in the metallic form. Of the anions,
chlorine, bromine, fluorine and iodine have very much more available
energy in the atomic than in the ionic form and consequently these sub-
stances are, as elements, strong oxidizing agents. Fluorine with the
most energy is the most caustic and toxic; chlorine, bromine and iodine
following in the order named, iodine being least toxic.

It would take us too far afield, however, to discuss farther in a book
of this character this relationship of the solution tension, or energy con-
tent of the ion, to its precipitating power, and we may now pass on from
a consideration of the effect of salts on colloidal solutions to the properties
and nature of gels.

Structure of gels.—Many colloidal solutions, particularly solutions of
emulsoids, but also some crystalloid solutions have the property of
solidifying as a whole without the separation of the solute and solvent
into visibly distinct zones or phases. A sufficiently concentrated solution
of gelatin will set when cool into a jelly. A gel has the properties of a
solid, in that it holds its shape and resists shearing stresses; it is more
or less elastic. The molecules of which it is composed are not like those
of a solution free to move about. Their motion is in some way restricted
asin asolid. A gel is never homogeneous. It consists always of two dis-
tinct phases or substances, one of these is a liquid and it may generally
be separated from the other by pressure, leaving behind the more solid
phase of the solute. Since protoplasm has the property of changing very
readily from a liquid to a gel state, the study of the structure and physics
of gels becomes very interesting for the physiologist. A gel may be de-
fined as a disperse system of a solid consistence and consistin iqui
and a more solid _phase, or of two liquid phases. It is a solid disperse
system in which the degree of disperson is not to molecular fineness.
Some colloids form gels with great ease. Gelatin is a typical example
of this; agar-agar is another example. A solution of sodium nucleate
gels very easily. These colloids are called hydrophilic colloids, meaning
that they have an attraction for water. On the other hand, some colloids.

     
230 PHYSIOLOGICAL CHEMISTRY

such for example as colloidal metals, do not form gels. They flock out
of solution. Closely allied to the gels are the emulsions. These are sys-
tems of several substances having at times a solid consistence. They
differ from the true colloidal solutions and gels,in that the degree of
dispersion is not so fine. A soap foam is an emulsion of this character ;
cream is another.

Many gels are converted into a sol state by warming or other treat-
ment, and gel again on cooling. These are called reversible gels. Some
gels are not reconvertible into sols. They are irreversible. Gelatin
or agar-agar form reversible gels; blood when it clots forms an irre-
versible gel; a strong solution of coagulable protein forms on heating
an irreversible gel also. It will not redissolve on further heating. Proto-
plasm appears to form reversible gels.

What, then, is the structure of a gel? What has happened when a
colloidal solution gels? How is the water held in the gel? Why do some
colloids flock out of solution and some gelatinize? Since gels are solids,
not liquids, it is clear that in some way or other the freedom of movement
of the solvent molecules is restricted in the gel as compared with the state
in the liquid. How is this loss of freedom produced? To examine the
structure of gels an ultra microscope, that is a dark field microscope, is
best. In this case the light enters from the side instead of from under-
neath and the finest particles are shown as bright spots on a dark field.
The process of gelatinization has been studied with such a microscope. It
has been found that the structures of various gels may be quite different
in details. Some gels are formed of very minute, or rather very thin,
acicular crystals which penetrate the gel in all directions, and which hold
the saturated solution from which the crystals have been deposited
entangled between them. Such a gel made of a crystalloid, not a col-
loid, is very easily obtained by dissolving a good deal of caffeine in water
and allowing it to cool. The caffeine crystallizes out in a mass of very
long, extremely thin, acicular crystals, and the whole makes a gel. so
that the test-tube may be inverted without any liquid escaping. This
experiment shows that crystalloids may form gels as well as colloids.
Tyrosine when quite pure often forms similar gels. To form such a crys-
talline gel it is apparently necessary that the crystals should.come out
in a very minute dimension, at least one or two dimensions must be
minute. The crystals may be very long. Among other examples of col-
loidal substances which gel by the formation of very long, extremely
thin acicular crystals, the clotting of the blood may be mentioned. The
crystals of fibrin are shown in Figure 29. The corpuscles and liquid
are entangled between these crystals. Most gels, however, are not crys-
talline in structure. A typical gel of a non-crystalline form is that of
casein. If rennin is added to a solution of casein under suitable condi-
THE PHYSICAL CHEMISTRY OF PROTOPLASM 231

tions the casein is converted into an insoluble form, paracasein, which
forms a clot or jelly. This. process has been watched under the ultra
microscope and the appearances are reproduced in Figures 30 and 31.
In the solution nothing can be seen. The field is homogeneous and dark.
As the clotting begins there is at first a diffuse, very faint light in the

 

Fie. 29.—The clotting of fibrin showing the long, acicular crystals. A crystalline
gel as seen in the ultra microscope (Stubel).

field, but no visible, distinct points. As the clotting goes on distinct
points, very minute and in active motion, appear first, these grow
larger and gradually cease to move. Finally the gel is seen to consist
of a very large number of small clumps or specks distributed through-
out the gel and having no Brownian movement. The gelatinization
appears in this case to be due to the formation of an insoluble pre-
cipitate which does not flock out of the solution but remains in situ
and which holds the liquid between the particles. The liquid between
the particles will hold other substances in solution and will also neces-
sarily consist of a saturated solution of the substance, in this case para-
casein, which has been in part precipitated. Most gels are of this general
character. Silicic acid, for example, forms a gel of this nature and so
does sodium nucleate. Still another form of gel is sometimes obtained
of the nature of a very fine emulsion. A mixture of gelatin, water
and alcohol will form such a gel as shown by Hardy. If the concentra-
tion of the gelatin was 36 per cent., the gelatin formed solid walls
or alveoli containing a dilute solution of the gelatin in the cavity; if
the gelatin was 13.5 per cent., the more concentrated phase separated
out. as spherical drops surrounded by the more dilute phase. A gel
may be formed of three liquids.

The most probable explanation of the formation of a gel is that the
232 PHYSIOLOGICAL CHEMISTRY

liquid solvent, or more liquid phase, is made solid by surface forces. It
has already been explained that at the surface of boundary of two liquids
or of liquid and solid the attraction of the molecules in the two directions
outward and inward is different, so that the surface layer of liquid mole-

 

Figs. 30 anp 31.—Two stages in the clotting of casein as seen in the ultra microscope.
In Fig. 31 the casein particles have aggregated into coarser clumps (Stubel).

cules have their freedom of movement reduced ; they are really converted
into a solid of two dimensions. In order to make a solid gel it is only
necessary that the amount of liquid in the surfaces shall be large com-
pared to the amount of liquid not under the action of unequal attractions.
In other words, the proportion of liquid in the form of surface film must
be sufficiently high. To accomplish this it is only necessary to have a very
fine state of subdivision of the precipitate, together with a certain con-
THE PHYSICAL CHEMISTRY OF PROTOPLASM 233

centration of the precipitate and an attraction between the precipitate
and the sdlvent. It is a matter of indifference whether the finely divided
precipitate is crystalline or amorphous; all that is necessary is that the
surface of the particles be very large compared to their bulk and that
there be enough of such fine crystals, or other finely divided matter, so
that the amount of free liquid shall not be too great. If the amount of
liquid is too great, the particles, surrounded by their surface layers of
water, will separate out. Emulsions do not differ in principle from gels,
nor do foams. Although a soap foam is composed of a gas and a liquid
soap solution, yet if the liquid be distributed in the form of surface films
it is changed to solid supporting lamelle and the foam is a solid. The
essential thing about a gel is, therefore, that the liquid which is present,
whatever its character, be present for the greater part as surface films.

All surface films, as we have seen, have a contractile action. There is
always a tension in such films. This is shown very clearly in the contrac-
tion of a small soap bubble when one stops blowing it and removes the pipe
from the mouth without stopping the vent. It is this contractile action
which constitutes or measures or is the expression of the surface tension.
It is not surprising, hence, that gels which really owe their solidity to
surface films of liquid generally contract. In this contraction they press
out some of the liquid and this liquid is always, naturally, a saturated
solution of the material of one phase of the gel, generally of the solid
matter, and it often contains other substances in solution such as salts.
This process of contraction of gels with the separation of some liquid of
this character is called ‘‘ Syneresis.’’ It is a true process of secretion,
probably identical in character with the processes of secretion by cells.
The contraction of an agar-agar tube with the formation of the so-called
water of condensation is known to every bacteriologist; and the con-
traction of clotted blood with the formation of serum which is squeezed
out of the jelly is known to many. Most housewives know how annoying
this tendency of contraction of gels is, since the liquid thus expressed is
often a good culture medium for moulds and bacteria.

Protoplasm may be regarded either as a very viscid sol, or as a gel.
Its structure is that of a microscopic emulsion. In other words, it has
the structure of a gel, or when it is more liquid, of a sol. Like other gels
it is able to contract and thus to press out a solution. It can take up
water. It is probable that while a good deal of the solidity of protoplasm
may be due to the formation in the cell of tough fibers or crystals of
protein or other matter, a part of its solidity, and often a large part, is
due to the fact that the liquid in the cell is distributed very largely in
the form of surface films between the granules, microsomes or droplets
of various kinds found in living matter. The liquid between the various
granules is bound, probably, in the form of surface films of a contractile
234 PHYSIOLOGICAL CHEMISTRY

kind. This structure of protoplasm by which it is allied to an emulsion
was particularly studied by Biitschli. Figure 32 shows how closely
the structure resembles that of an emulsion. The real living part of the
protoplasm in its more solid moments is probably a microscopic foam
or emulsion. At other times it may be distinctly fluid. This will happen
when the films are broken, or when the surface tension is diminished, or

 

Fig. 32.—Illustrating the foam-like structure of fixed protoplasm (Hardy).

when there is too much water in the protoplasm in proportion to that
present in the form of surface films. The recent, very important work of
Clowes on emulsions should be read in this connection.

Absorption of water by gels——The absorption of water by gels is
extremely important in physiology, because the protoplasm of the cells
of the higher animals is a gel and many of the physiological activities
appear to be due to the absorption and loss of water by various parts
ot the protoplasm. Thus the contraction of muscle cells is apparently
due to the passage of water into and out of the fibrillar elements, sarco-
styles, as is discussed on page 627; in secretion there is evidently an alter-
nate absorption and loss of water by the cell, the process being so regu-
lated that the loss of water takes place from another part of the cell
than that in which it is taken in; the abstraction of water from the gel of
the nerve fibers causes the development of a nerve impulse; and in plants
THE PHYSICAL CHEMISTRY OF PROTOPLASM 235

the movements of leaves, petals and other parts of the plant are due,
generally, to turgor changes in the different cells. In fact, it appears
that the mechanics, that is the physical part of the protoplasmic activity,
is very largely a matter of the orderly absorption and loss of water by
the cell colloids. In this connection attention may be called to the con-
centration of the chromatin of the nucleus into very dense chromatic
masses during cell division as a probable example of the concentration of
a gel by the loss of water, accompanying a physiological process. The

5 = . ——_4 =,
Na- Na~ Na- Na-
NaOH et rr Acetat| Harta (INSSQ)—Icitrat] Fos
7 7] i | 7 7

j~----| L----| peers a asian

 

 

  

 

 

 

 

 

 

 

 

 

 

   

 

 

 

 

 

 

  

Fig. 32A.—The swelling of fibrin in different salt solutions. qua! amounts of fibrin
in each tube. Fibrin is represented by the punctate masses at the bottom of the tubes

(from Bechold).

 

great importance of a thorough understanding of the physics of the proc-
ess of the absorption and loss of water by gels, of swelling or imbibition
processes in other words, for the understanding of vital actions will be
apparent from this statement; but in addition it may be mentioned
. that from the point of view of pathology and therapeutics the subject
is no less important, since the process undoubtedly plays a very great
role in cedema, in the swelling of the brain after a blow on the head and
in the occasional swelling of organs like the kidneys to such an extent
that the circulation is impeded.

Hofmeister was one of the first to recognize the great importance of
this process of swelling in animal physiology, and he has contributed
much to our knowledge of the conditions of the process. Any protein
gel can be used to illustrate the action of salts, acids, ete., on the water
content, but perhaps gelatin and fibrin are the most easily obtained and
have been most studied. Small muscles may also be used, for in them
the gel is protoplasm itself, but muscles have the drawback that they
are the seat of various chemical processes, which complicate and make
236 PHYSIOLOGICAL CHEMISTRY

obscure the interpretation of the results. The method employed by Hof-
meister, and generally followed: by others, is to make a fairly strong
gel of gelatin by dissolving the latter in hot water. The gelatin is poured
into a flat-bottom vessel in a thin layer, and after hardening it is cut
into cubes of about the same size. These cubes are weighed and then
placed in solutions of salts of various strengths and kinds, or of acids,
or other substances, and then, after varying times, they are removed, the
adhering’ water removed by blotting paper and the cubes weighed. If
the salt or acid has caused the gel to take up more water, the cube will
now be heavier; if loss of water has occurred, it will be lighter.

““ By swelling,’’ Hofmeister says, ‘‘ one understands the taking up of
liquid by a solid body without chemical change.’’ Three different proc-
esses may play a part in this:

1. A porous mass may take up liquid in previously formed capil-
laries and spaces filled with air. This is capillary imbibition and is illus-
trated by the absorption of water by a mass of porous clay.

2. <A porous mass may take up liquid by osmosis into previously built
spaces filled with soluble substances or liquids. This is imbibition by
osmosis. This occurs in all animal and plant cells which have a per-
meable skin.

3. A homogeneous, pore-free mass, like gelatin, absorbs a liquid
undergoing an increase in volume. This is the molecular imbibition of
Fick. This is the process of the swelling of most of the proteins, of
lecithin and other phospholipins and most of the cell structures like the
chromosomes, cellulose fibers, dried peas, etc.

In animal or plant cells all three forms of swelling may coexist.
Thus a muscle brought into a solution takes by capillarity some water
into the spaces between the fibers. A second part is taken by osmosis,
not all substances in the muscle being diffusible through the surface
layer; and a third by molecular imbibition. Of the three forms, the
capillary depends on surface tension; imbibition is a peculiar kind of
process which possibly belongs in the group of adsorption processes and
may also be of a surface tension nature at the bottom.

' Three laws of swelling which have been discovered are these: a. Many
substances capable of swelling take up a definite amount of water, not
an indefinite amount. There is a swelling maximum. b. The volume of
the swollen body is smaller than the combined volumes of the original
substance and the liquid taken up. Swelling is accompanied by a con-
traction in volume (Quincke). c. Swelling is regularly accompanied by
the evolution of heat (Duvernoy ; Wiedemann and Liideking).

Hofmeister used small squares of gelatin or agar not over 0.2 mm.
thick and weighing when dried at 100° C. from .01 to .05 gram. These
were placed for a time in water warmed to a certain temperature and
THE PHYSICAL CHEMISTRY OF PROTOPLASM 237

weighed from time to time to discover the rate at which water was taken
up and the swelling maximum. It was found that the weight of water
absorbed could be represented by the following formula:

If W is the weight of water taken up by one part of the dry substance
in t minutes it was found that

J

14$t
P is the largest amount of water which can be taken up by one unit of
substance, or the swelling maximum; ¢c is a constant computed from thc
experiment; d, the thickness of the plate in the maximum swollen state
measured in mm.; and t, the time in minutes. P was found by leaving
the plates in water for from 1,000 to 2,000 minutes. P depends on the
affinity of the particles for water and the cohesion of the particles for
themselves. The velocity is proportional to P—W.

The following results were obtained with agar plates of a thickness of
0.60 mm. P—5,.62935; c¢/d=.15348.

W = P(I—

 

)

t W found W computed
5 2.44 2.44
10 3.35 3.41
15 3.86 3.92
20 4.31 4.25
30 4,69 4.63
40 4.84 4.84
50 5.07 5.03
60 4.98 5.08
1440 5.52 5.60

It will be seen from these figures that the water is absorbed with
great quickness. The smaller and the thinner the piece, that is the larger
the surface in proportion to the bulk, the more rapid will be the swelling.
A completely dry agar plate 0.206 mm. in thickness in the first minute
absorbed 75 per cent. of the total amount it could take up. If the size
of the plate was that of a red blood cell, i.e., .002 mm., in the first minute
it would have reached the maximum, or at least 98 per cent. of the maxi-
mum absorption. This is a point of very great importance in the inter-
pretation of muscle contraction where the taking up and loss of water
must take place with great speed, if the contraction is to be ascribed, as
it seems that it must be ascribed, to this process. It will be seen, alsc,
that when a celi is placed in water or other solution, the first effect must
be on the delicate membrane surrounding the cell which will almost at
once, because of its great thinness, alter its state of swelling to that of
the solution in which it is placed. It is seen, then, that the first effect
of a solution is thus on the state of swelling of the peripheral membrane
and it is not impossible that this is of importance in determining the ease
238 PHYSIOLOGICAL CITEMISTRY

or difficulty of penetration of substances into cells, and is a matter of
importance in the making of the fertilization membrane of eggs.

The second problem investigated by Hofmeister had to do with the
question, How does the swelling behave when the gelatin is in a salt solu-
tion and not in water? He made cubes of concentrated gelatin and deter-
mined the amount of ash and water in several of them. The gelatin cubes
were then brought into the solution to be tested and on the next or later
days were taken out and after drying were weighed. He at times deter-
mined also the amount of salt taken up. The figures in the table show the
number of times the weight of solution in the plates is that of the dry
substance. At the start the water was 4.42 times the dry substance. The
cubes were placed in solutions of the salts which contained one gram mol.
of the'salt to a thousand grams of water.

After

 

Salt 48 h. 72h. 96h. 11 days 25 days
Na Acetate .... 2.69 2.76 2.99 3.07 3.25
Water ....... 6.39 6.74 8.13 9.37 Putrefaction
HSC a siscadeien acces 6.89 7.49 9.25 10.05 11.86
NaCl ........ 6.82 7.49 9.82 10.81 13.07
NH Cl ....... 9.39 10.33 12.83 14.01 16.47

In solutions of LiCl, MgCl,, CaCl,, NaBr, NaClO,, the gelatin dis-
solved in 24 hours.

In double normal solutions in 48 hours, cubes, which before contained
5.39 parts of water to one part gelatin, contained the following amounts:

AYCOWO]! iicscsiaesd ciseeiesaa ownage 8.70
Cc Hy. Sassicesi soi + Lead eeas vgn uerte oiaiaNe 9.29
Na Acetate: onc sopiuacaenaecascaus 9.83
Sucrose: k20s vaeeuieeseeees ee bed 10.77
Water jaca ceshanticnes chien canoes 12.12
NH,Cl Dis hiiSaod chon e deen tan 15.95
TRC or stsdetitscictountitaewracs meee Sneniee 18.84
NGCD: ssoiinsis sarc samesan aes 22.19

These experiments show that the amount of water taken up by a gel
from a salt solution depends on the nature of the salt present, being
reduced by some salts and increased by others. Most salts, howexer,
inerease the swelling of the gelatin. This is exactly parallel to the solu-
tion of a globulin, which will only dissolve if some salt is present. Hof-
meister interpreted his results to mean that all salts had, as a primary
effect, an increase in the water-absorbing power, but that some had a
greater affinity for water themselves and that there was a strife between
the gelatin and salt particles for the water. It is, however, possible that
the greater power of the ammonium chloride may be due to the presence
of a small amount of acid in its solution and the inhibitory action of the
acetate to the presence of a little hydroxyl in the solution. Acids in small
THE PHYSICAL CHEMISTRY OF PROTOPLASM 239

amounts greatly increase the swelling power of protein colloids. The
amount of salt taken up was in general proportional to the quantity in
the solution, but the absorption of the salt and the water did not run
perfectly parallel. Thus, at first, the salt content increased more rapidly
than the water content. While it is clear that some kind of a chemical
affinity must exist between the swelling body and the liquid, since rubber
swells in ether but not in water and gelatin shows the reverse effect, Hof-
meister concludes nevertheless that the attraction is not that of an ordi-
nary chemical kind, since in the latter a definite proportion of the react-
ing substances is found. The selective absorption of the gelatin for
various solutes was made very obvious by placing the gelatin cubes in
solutions of methyl violet. In dilute solutions most of the color passed
out of the solution into the gelatin. He concludes that the gelatin par-
ticles must have a far greater attraction for the color molecules than for
the water. It is probable, since salts unite with amino-acids, that they
also combine chemically with gelatin.

Similar experiments have been carried out by Pauli and others,
among whom special mention may be made of Fischer for his applica-
tion of the results to the study of the cause and treatment of cedema.
Pauli studied the action of various salts on the fusion points of gels. He
found that the iodides lowered the point of fusion most, then came the
bromides, chlorides, acetates, tartrates and sulphates in the order
named.

While the facts as they stand enable us to forecast the influence of
salts on the colloidal substratum of living matter and thus furnish a
partial explanation of how salts are affecting vital processes, yet it is
instructive to inquire somewhat more deeply into the physics of this
process. Why do salts in dilute solution cause swelling, and in stronger
solution, shrinking? It is probable that the manner of action is the same
as the precipitation of a colloid from solution. If the colloid is sufficiently
dilute, under the influence of the salt it falls out of the water: if it is
too concentrated, as it is in the gel form so that it can no longer precipi-
tate, the salt causes the water to fall out of it. The same result may be
had if a piece of fibrin is placed in water; it takes up some water, but
it takes up far more when there is a little acid present. Some salts and
acids increase the affinity of protein colloids for water and these cause
swelling and dissolve protein colloids; others diminish the affinity, and
these cause a loss of water. Anything which increases this affinity lowers
the tension of the surface of contact of the water and the colloid and
hence this surface is increased, the gelatin swells, water penetrates it;
on the other hand, any salt which increases the surface tension of contact
between colloid and water will cause a diminution of the surface of
contact, and the colloid will lose water. The way in which acids thus
240 PHYSIOLOGICAL CHEMISTRY

act is that they unite with the protein to form a salt which ionizes
readily. This salt thus causes the gelatin particles to be surrounded by
an electrical double layer. By the electro-static affinity across this layer
the affinity of the water and the gelatin is increased, and accordingly the
surface tension of the water in contact with the gelatin is reduced, and
if the gelatin is not too rigid an increase of surface will take place. All
acids, therefore, in more than quite minute amounts, cause proteins to
absorb water. If, however, the amount of acid is very small and the
protein is in a very dilute alkaline solution, the first action will be in
the direction of a loss of water. The production of acid in a tissue
Fischer believes to be the prime cause of edema. The action of salts is
more difficult to understand, but it is probable, as Hofmeister believed,
that the first effect of the salt is to make a weak union with the gelatin,
leading it to take up more water or rendering it more soluble. We may
infer from the fact that iodides cause solution of gelatin that the gelatin
colloid is, in water, chiefly electro-negative, that which direct observa-
tion confirms.

The swelling of solid bodies following or accompanying imbibition
of water is often very great. Thus peas will swell to double their size,
and Irish moss (Chondrus crispus) swells to treble its size when dry. In
all such cases the cohesion of the swollen body is much less than that
of the same body before imbibition. Thus Reinke found that dried
Laminaria absorbed 300 per cent. of water and its ductility increased
sixty times, while the breaking strain fell to one-tenth its value. Solid
substances which take up water in visible pores without swelling undergo
no change in cohesion. Thus a dried brick will absorb a good deal of
water in its pores, but the cohesion of the brick is not altered thereby.
There is probably no difference in principle between the imbibition of
water in visible capillary pores and true swelling where there are no
visible pores and the imbibition is molecular or micellar. The sole dif-
ference is probably that in the case of imbibition without swelling the
attraction between the water and the solid substance is not sufficiently
great to overcome the attraction of the molecules of solid for each other.
The molecules of the solid cannot be separated by the penetrating water
and the body does not swell. The water is able to displace the air from
the capillary spaces and spreads itself accordingly over all free surfaces.
In the case of swelling gelatin, or woody fibers, or liquid crystals, the
attraction between the molecules of the substance and the water mole-
cules is greater than between the molecules of the substance, so that
the latter are separated. Swelling of solid bodies in this way passes
imperceptibly into swelling so considerable that the solid becomes a liquid
solution. Thus gum arabic and peptone at first swell as solids and then
dissolve in the water taken up. Katz has shown that the processes of
THE PHYSICAL CHEMISTRY OF PROTOPLASM 241

molecular imbibition in solids are at bottom identical with the taking up
of water by glycerol, sulphuric acid or other liquids which absorb water
with the liberation of heat.

The heat liberated by these swelling processes may be considerable.
Katz found that dried cellulose liberated the following amounts of heat
on swelling. W is the quantity of heat in gram calories liberated when
1 gram of the dried substance takes up i grams of water. Formula:
W=Ai/(B+i).

Cellulose. A—11.6; B—0.030.

i Wedeter. W cale. A
0 0 0
0.014 3.5 3.7 —0.2
0.041 6.9 6.7 —0.2
0.054 7.6 7.5 —0.1
0.074 9.0 8.3 —0.7
0.261 10.5 104 —0.1

Adsorption by colloids.—Finely divided solids often have the power
of uniting with substances in solution so that if the solid is filtered off
the solute remains very largely in the precipitate of the finely divided
solid. Thus colors filtered through charcoal or diatomaceous earth often
remain in the charcoal or earth and the solution is freed from them. If
permanganate solution is filtered through clean sand, it is said that the
first water which comes through is almost colorless. There are many
other examples known to chemists, and one of them has already been
mentioned: namely, the taking up of color from a solution of methyl
' violet by gelatin, This separation of the solute from a solvent by sub-
stances in suspension is generally said to occur by means of adsorption.
By many observers it is referred to surface forces at the boundary of
liquid and solid. It is probable that many of these so-called adsorptions
are in reality due to true molecular union between the solid and the
substance adsorbed. This is the case, for example, between the filter
paper and copper adsorbed from a solution of copper sulphate. Prob-
ably the majority of adsorption compounds are in reality chemical unions.
They differ, however, from ordinary chemical unions in the fact that the
two combining substances are not united in definite proportions, but the
amount of substance which is adsorbed is more or less proportional to
the amount of surface.

That surface forces may, also, be responsible for some of these
processes of adsorption is, however, possible and on the whole probable.
It was shown by Willard Gibbs that substances which lower surface
tension will accumulate in the surface film. That is, their concentration
will be larger there than in the liquid behind. If there is a very large
amount of surface, it is possible that quite a good deal more substance
will be taken up than by an equal amount of pure solvent not in the
242 PHYSIOLOGICAL CHEMISTRY

surface form. Suppose at the boundary of liquid and more solid por-
tions of protoplasm the surface tension might be lowered by the passage
into the film of some substance present in the protoplasm. In that case
the distribution of the substance might be altered by the presence of the
film. It might be more concentrated immediately about the granules, for
example, than in the protoplasm between the granules. It might be said

 

Fig. 33.—Distribution of potassium ip Acineta. The black granules represent
ee (Macallum). Macallum believes this distribution is determined by surface
to be concentrated by adsorption. Some physiologists believe that this
principle of distribution of substances in protoplasm by means of sur-
face forces of adsorption plays a large part in determining the distribu-
tion of substances in cells. Macallum, for example, has studied the
distribution of potassium in cells from this point of view. He has con-
eluded that probably surface tension forces do determine the distribu-
‘tion of potassium in Acineta. The curious distribution of potassium in
these cells is shown in Figure 33. It is extremely difficult to be sure
in these cases that the substance is really distributed by surface forces
rather than by the operation of the usual chemical means of forming
insoluble precipitates. To what extent adsorption, as distinct from ordi-
nary chemical unions, determines the taking up of substances by living
matter must be left open for the present. There can be little doubt,
however, that many so-called adsorption phenomena are in reality noth-
ing more than chemical unions. As the tension of water in contact with
THE PHYSICAL CHEMISTRY OF PROTOPLASM 243

a solid cannot be measured, it is impossible to say whether any substance
raises or lowers this tension and, hence, whether the accumulation is due
to its lowering tension, or because it unites chemically with the particle.
For potassium to accumulate in the surface in the way mentioned above,
it is necessary to suppose that it unites with the substance which the
liquid bathes and so reduces the tension by increasing the attraction
across the surface; or else that it is present in union with some sub-
stance which reduces the tension and so accumulates in the surface.

Brownian movement.—The fine particles of a suspensoid or emulsoid.
colloid when they are large enough may be seen to be in a state of
violent agitation. The particles dance about a fixed point, first in one
direction and then in another. This movement was studied by Robert
Brown in 1828 and has since been known as the Brownian movement.
The vigor of the movement increases with the temperature and with the
smallness of the particles. It has been variously explained, but it is now
ascribed to the bombardment of the particles by the molecules of liquid
in which they are immersed. It represents the true dance of the mole-
cules. By this bombardment they are knocked first in one direction and
then in another. In protoplasm the particles are not generally in
Brownian movement, but when the protoplasm liquefies the movement
begins at once. It would appear from this that the-protoplasm of many
cells at any rate is rather in a gel than a sol state.

Osmotic pressure of colloidal solutions—As is to be anticipated,
colloidal solutions possess osmotic pressure. They must do so if they: have
any affinity for water. The colloidal particles are, however, so large
that their number is very small even in fairly concentrated solutions
when compared with the number of particles of a non-colloidal substance
in a similar concentration. Indeed, the freezing-point method, or the
boiling-point method of determining osmotic pressure, which depends on
the vapor tension of the solution, is not sufficiently precise to measure the
osmotic pressure accurately, and indeed for some time it was doubted
whether colloids really exerted an osmotic pressure. By directly measur-
ing this pressure in an osmometer, however, there is no doubt of its exist-
ence. The osmotic pressure of hemoglobin, a colloid, is given on page
142. Since osmotic pressure is but the expression of the affinity of par-
ticles and water, it is immaterial whether the particles are in a solid state
or in solution. The swelling pressure of gelatin in water is but the
expression of its osmotic pressure. The protoplasmic colloids, whether
gel or sol, have then an osmotic pressure, but it is customary to call it
osmotie pressure only when the colloid particles are in solution.

It is rather interesting to consider the number of particles which are
necessary to produce a given osmotic pressure. Suppose a gram of a
pure crystalline protein is dissolved in 100 c.c. of solution. The osmotie
244 PHYSIOLOGICAL CHEMISTRY

pressure is measured and found to be 76 mm. of mereury. What is the
molecular weight of the protein and what is the number of particles in this
solution? The number of molecules in a gram mol. of any substance has
been measured in various ways and found to be 6.0610". A gram mol.
of glucose in a liter of solution has a theoretical osmotic pressure of 22.4
atmospheres at 0° C. In each cubic centimeter there must be 6.06 10?°
molecules. The solution just mentioned had an osmotic pressure of 76 mm.
Hg, that is 1/10th of an atmosphere. It must contain in a cc. 6.06
10"°/22.4 particles or 2.710'*. Since the solution contained 0.01 gr.
in a ce., the weight of each molecule is easily calculated. A gram mol.
per liter gives a calculated but not an actual osmotic pressure of 22.4
atmospheres. The actual osmotic pressure is more than this (p. 200).
This solution containing 10 grams per liter gives an osmotic pressure
1/224th of that of a gram mol. Hence 10 is 1/224th of the molecular
weight, and the molecular weight would be 2240. It may be seen from
this that an almost inconceivably great number of molecules may be
present in a solution and yet exert a very small osmotic pressure.

The electrical phenomena of protoplasm.—Every activity of living
matter, whether it is an act of secretion, a muscle contraction such as
the rhythmic contraction of the heart, or a nerve impulse, is accom-
panied by an electrical disturbance in the protoplasm. This electrical
disturbance may be detected by connecting two points in the tissue
which are not equally active with the electrodes coming from an elec-
trometer or galvanometer. In some cases, as for example the electrical
eels, Gymnotus or Malopterurus, or the electrical fishes, such as the
Torpedo, the electrical disturbance may be so heavy as to produce a
strong shock to animals touching or being in the neighborhood of the
discharging animal, and the discharge is of use in stunning enemies or
prey. Every living cell is, therefore, a battery and the study of proto-
plasm as an electrical machine may help elucidate its vital properties.

For the detection of these electrical currents non-polarizable elec-
trodes should be used. These are made usually by immersing a zine wire
or zinc electrode in a strong solution of zinc sulphate, the zine sulphate
is kept from the tissue by interposing between the sulphate solution and
the tissue a solution of inert saline, that is N/6 NaCl solution; a string
from this solution connects with the tissue. Current passing in either
direction through these electrodes does not produce any opposing electro-
motive force. For the detection of the current various devices are used,
such as the oscillograph, the thread galvanometer or the capillary
electrometer, for the operation of which special memoirs may be
consulted.

By the means just mentioned it is found that the part of protoplasm
or tissue which is active is always electro-negative to the part which is
THE PHYSICAL CHEMISTRY OF PROTOPLASM 245

more at rest. Every. excitation of the protoplasm causes a momentary
negativity in the part excited so that a momentary current flows through
the galvanometer from the less active to the more active part; and within
the tissue the current is completed by the current flowing from the more
active to the less active part of the protoplasm. This momentary electric
discharge is called the current of action, or the negative variation, or
by Waller the ‘‘ blaze ’’ current. Waller called it a blaze current to
indicate that it is as if, following the stimulation, the protoplasm sud-
denly blazed up in a conflagration. It is usually of very short duration,
but if the difference of activity is constant the electrical difference is also
constant.

The cause of the electrical phenomena.—The mechanism of the pro-
duction of this current of action, or of the blaze current, is still unknown.
Three or four more or less probable suggestions have been made to
explain it. The first, and in many ways the most important of these
suggestions, is that of DuBois-Reymond, that protoplasm is composed of
polarized electro-motive molecules being electro-negative at the ends and
positive in the center. The current is produced by the rotation of these
molecules. If cohesion is of a magnetic nature, as appears probable, it is
not unlikely that such magnetically polarized molecules really exist in
living matter.

The second view is that of the polarizable membrane. It is supposed
that protoplasm has in it membranes which are permeable for cations
but not for anions and so generates an electro-motive force at the boun-
daries. Excitation lets the anions out, too, and so produces an electrical
disturbance.

The third is that the current is caused by a change in the surface
of contact between colloids and water, as it is in the capillary
electrometer between mercury and acid.

The fourth view is that it is caused by the production of acids set-
ting free hydrogen ions, making a concentration chain effect.

The fifth view is that it is produced by oxidation and reduction, that
it is a burning with the energy set free as electrical energy rather than
heat, as it is in a battery.

It is impossible at present to decide which of these theories is correct,
or whether any one is correct.

The reaction of protoplasm and the preservation of neutrality.—So
far there has been considered the physical chemistry of the structure of
living matter, its properties which are due to its colloids and the surface
tension (and electrical properties) ; those properties, in other words,
have been considered which have to do with the machinery of protoplasm.
We shall now take up briefly the physical chemistry of some of the fun-
damental chemical properties of the cell, dynamical physical chemistry
246 VHYSLOLOGICAL CHEMISTRY

in other words. One of the simplest and most important of these chem.
ical properties is the reaction, whether alkaline or acid, of the protoplasm
and the methods used to preserve neutrality.

Whether the reaction is acid, alkaline or neutral may be determined
with greater or less certainty in a variety of ways, all of which show
that protoplasm is generally very faintly alkaline in reaction, but that
it becomes acid after death and when it works in a medium with too
little oxygen. The reaction of living matter may be determined by the
use of indicators.and in various indirect ways. Many stains are good
indicators: that is, they have a strong color change when they pass from
an alkaline to an acid reaction. Such a stain is neutral red, which is a
deep orange red in an alkaline reaction and a pink in an acid. Neutral
red- penetrates nearly all cells with ease and stains the granules in the
cells and sometimes other structures. It always takes the color it has ©
in an alkaline reaction; moreover, it is found that when protoplasm is
made acid it no longer stains with neutral red. Another stain is
cyanamine, which has been introduced by Bensley and Harvey. This
stain is red in an alkaline solution and blue in an acid solution. It is
found that it stains cells or parts of cells red. Other stains may also be
used. Thus acid fuchsin will not stain protoplasm as long as it is alka-
line, but as soon as it is acid it takes up the stain. By this means it
is found that if acid fuchsin is injected into a frog and then the blood
vessel to one leg is ligated while that to the other leg is intact, and then
if the muscle of the ligated side is made to contract, that muscle will be
found, on taking off the skin, to be a bright red, while the other has
scarcely a tint of red. This proves that acid is formed in muscle during
contraction and accumulates there, if there is not a supply of oxygen.
Ordinarily the protoplasm is neutral or alkaline, since it does not stain.
The same experiment has been tried with the electrical organ of the
Torpedo and with other tissues and the same facts found for the differ-
ence between the reaction of active and passive protoplasm. Miss Green-
wood fed various stains to Stentor, Paramecia and other protozoa and
found the reaction of the protoplasm to be alkaline to litmus and the con-
tractile and digestive vacuoles to be acid.

There are several difficulties in this use of stains for the determina-
tion of reaction. The first is that the stain may itself be poisonous and
by killing the cell bring about the production of acid, since dead proto-
plasm is generally acid in reaction. Another difficulty is that only those
stains will accumulate in protoplasm which combine with some element
in the cell. Not all stains penetrate easily. Moreover, the cell is strongly
reducing so that many stains, like methylene blue, are reduced to a color-
less substance as long as the cell is alive; the color can only be seen in
those parts of the cell where the reducing or oxidizing powers are least;
THE PHYSICAL CHEMISTRY OF PROTOPLASM 247

where, in other words, the vitality of the cell is least. But the most
serious difficulty, and one which is usually overlooked by cytologists,
is that the stain is not, properly speaking, a reagent for acids and alka-
‘lies at all, when used in a heterogeneous system like the cell. In all cases
the difference of color shown by the’stain is between the color of the salt
and the free base or acid. Now, let us suppose that there are in proto-
plasm substances, electro-negative colloids, which will form insoluble
salts with neutral red. If these colloids do not change their sign with a
slight acidity, but remain electro-negative, they will form a true salt
with the stain and give the reaction of the salt, as if the reaction were
acid, even though the reaction is neutral or faintly alkaline. The same
is true of any acid stain. If there are electro-positive colloids anywhere
in the cell, these will react with the acid dyes, forming a true salt and
giving the salt color, which in a true solution would be held to be the
sign of an acid. For example, silk exposed to acid, but thereafter thor-
oughly washed so that it is not acid in reaction, will stain a brilliant
red in acid fuchsin. Stains can be used, then, for the detection of the
acidity of protoplasm only with the greatest caution, but nevertheless
the evidence, as far as it goes, is to the effect that the protoplasm is
usually very faintly alkaline.

Another method of determining the alkalinity of protoplasm is to
study the alkalinity of the liquid in which the cells live. It is not likely
that a cell will have a reaction very different from the medium in which
it lives, although this is at.times possible. The blood may be regarded
as a living tissue. The reaction of the blood plasma and the lymph of
mammals and vertebrates generally is, like that of the sea-water, very
weakly alkaline. The blood is alkaline to litmus, but acid to phenol-
phthalein. The exact reaction of the blood may be determined by the
methods given in Chapter XII. By these methods it is found that the
plasma has a concentration of hydroxyl ions of about 7X10—7 N.

Changes produced by a change in reaction in the cell. The physical
and chemical changes which ensue in protoplasm when its reaction is
rendered less alkaline, or more acid, are extremely important and pro-
found. The first effect of the production of acid in the cell is seen in
a reduction in the rate of oxidation. The respiratory oxidation of the
vell is wonderfully affected by a rise or fall of acidity. By an increase
in acidity it is checked ; by a decrease in acidity it is greatly stimulated.
[t is always found that the appearance of acid, or any reduction of alka-
linity, is accompanied by a failure to oxidize certain cell products which
under normal circumstances are oxidized. Thus acidity in the mammal
is always accompanied by the appearance of acetone bodies in the urine
which may disappear at once, if alkalies, such as sodium bicarbonate, are
given. A second important change is the appearance in the cell of auto-
248 PHYSIOLOGICAL CHEMISTRY

lytic enzymes of various kinds, both proteolytic and carbohydrate split-
ting enzymes. Invertin, amylase and other digestive and deamidizing
enzymes are set free.) The appearance of these enzymes is in the nature
of an adaptive change, the cell by means of them seeking to recover its
metabolic balance. By means of the carbohydrate enzymes it obtains
glucose and levulose from glycogen or cane sugar, or other carbo-
hydrate reserves, and by these it is able to carry on its oxidations
and respiration under conditions of lack of oxygen and acidosis which
it cannot otherwise withstand. Thus in the liver any acidosis is at
once accompanied by the transformation of some glycogen into glucose
for the protection of the respiration of the liver. The proteolytic
enzymes attack the proteins of the cell, setting free. ammonia from them
by means of which the cell acids are neutralized. Thus every acidosis
in the body is at once accompanied by the increase of proteolysis in the
cells and the appearance of ammonia, so that by many ‘physiologists
the appearance of more ammonia in the urine is regarded as charac-
teristic of acidosis. The autolytic digestion of proteins is distinguished
from the digestion by the digestive ferments of the alimentary tract
by the production of vastly more ammonia in the former process.

A third marked change produced by the action of acids in cells is
a slowing down of all the activities of the cell. This is caused by the
period of recovery from activity becoming much prolonged: that is,
fatigue comes on much sooner when there is a reduced alkalinity. It
is for this reason that athletes are able to withstand fatigue longer if
they load the blood with oxygen first. By the action of the muscles in
exercise lactic acid is produced, this is at once oxidized, or otherwise
removed, if plenty of oxygen is present, but it accumulates and produces
fatigue if too little oxygen is present; and by its accumulation it checks
oxidation, having thus an autocatalytic action.

Another physical change produced by the accumulation of acid in
cells in small amounts is an accumulation of water in the cells and
edema. The colloids of protoplasm take up more water in the presence
of acid; this may interfere with the circulation in the case of those organs
which, like the kidneys, are inclosed in a tough inelastic capsule. A
vicious cycle is thus inaugurated, acid causing swelling, the swelling
checking the blood and oxygen supply, thus increasing the production of
acid and increasing swelling. In many cases the ingestion of alkalies
in such conditions produces a sudden and marvelous improvement.

Regulation of the reaction of protoplasm. With such grave conse-
quences following from any permanent derangement of its reaction, it
is not surprising that cells have various methods of preserving that reac-
tion most suitable to their activities. The means employed are in part
organic and in part inorganic. The organic consist in the setting free
THE PHYSICAL CHEMISTRY OF PROTOPLASM 249

of the autolytic enzymes already mentioned by means of which ammonia,
for the neutralization of the acid is set free, and glucose, to enable the
cell to survive the period of depressed oxidation caused by the acid.
There is in addition the power of the protein constituents of the cell to
combine with acid, thus neutralizing it. We have, also, in many cells
another mechanism which may also be brought into relation with this
process, but which is usually held in abeyance as long as carbohydrate
food is present; this is the mechanism of the carboxylase. On the diges-
tion of the proteins amino-acids are set free. By means of these the acids
are in part neutralized by the formation of salts, just as by the ammonia,
but by the action of the carboxylases the carboxyl group of the amino-
acid is split off according to the following equation,

CH,.CHNH,.COOH —-+ CH,.CH,NH, + CO,
Ethyl] amine.

and a base is left with much greater acid-combining powers than the
amino-acid itself. These amines play, also, in the mammalian organism
another important réle, since some of them are strong stimulants of
the heart or vaso-dilators, thus increasing the circulation through the
parts and hastening the removal of the acid.
_ In the simpler organisms, but also playing an important part in the
higher, are the inorganic safeguards against acidosis. These are pri-
marily the carbonates and phosphates. There are considerable amounts
of both these salts in all protoplasm and they have this very important
function among others. They act as buffers, in that they can take up a
good deal of acid with a very slight change in acidity. Sodium acetate
has a similar power. They owe these powers to the fact that both car-
bonic acid and phosphoric acid are very weak acids, so that when acids
such as lactic, or oxybutyric, or almost any other acid, are produced in
the cell, the acid so formed at ance takes the base away from the car-
bonate, making bicarbonate, which is scarcely at all acid. If still more
acid is formed, carbon dioxide is set free, which is a very weak acid and
which escapes from the cell with the greatest ease. The phosphates, also,
play a part, although they are held rather in reserve, as phosphoric acid
is stronger than carbonic. There is in the cell a mixture of disodium
hydrogen phosphate and dihydrogen sodium phosphate. The acid pro-
duced is neutralized by the base taken from the former. The former is
alkaline, the latter slightly acid in reaction. H,NaPO, ionizes very
little into H and HNaPO,, so that the number of hydrogen ions is only
very slightly increased. Moreover, there are some NaHPO, ions from the
Na,HPO, which still further check the ionization. The equilibrium of
this reaction has been studied by Henderson.

To sum up, then, the production of acid in cells: The immediate effect
250 PHYSIOLOGICAL CHEMISTRY

of this acid is to cause the protein gel to absorb water at the point of
formation of the acid and to become more fluid. Possibly the liquefac-
tion of the protoplasm of the ameba at certain regions has this cause.
In other cases, as possibly in fertilization, gelation may result. The
activities of protoplasm are almost certainly due to this action, in part
at any rate. This acid, if it is not oxidized, accumulates. It checks oxi-
dation. It is in part neutralized by the following reactions, which are
written for lactic acid, C,H,O,:

I. c, H 6° 3 + Na, HPO, —~+ NaC_H 0, + NaH, PO,

in é H,9, + Na,CO, —+ NaC,H,0 “+ NaHdo,

Ill. ¢C gts + ProteinNa —— HProtein + NaC, it, 0,

1. Protein + Protease ——> Amino-acids -+- “Protease
IV. | - OHO, + Amino-acid ——~ Amino-acid C,H,O,
Vv. 1. Amino-acid ++ Deamidase —~ NH 3+ Oxyacid + Deamidase
}2. NH, + C,H,0, —- NH,C,H,0, *
VL. } 1. Amino- acid aE Carboxylase ~—— Amine + CO ee Carboxylase
2 C, H ioe + Amine —~ Amine C, H,0

Catalysis —The mechanism of smtapladi certainly involves the
passage of water into and out of the cell or of the cell elements. This
movement of water back and forth is in its turn probably to be ascribed
to a varying affinity of the protoplasmic colloids, whether protein, carbo-
hydrate or lipin, for water and in part to the varying osmotic pressure
of the cell contents. It has been suggested, and the evidence is on the
whole favorable to the view, that this varying affinity of the colloids for
water is due in large measure to the varying reaction of the protoplasm,
often a variation of a strictly local nature, due to the production of acid
in the course of the cell metabolism. This consideration led to the discus-
sion of those chemical processes by which acid is produced and got rid of
and by which the colloids of the cell are made and broken down. These
chemical changes are the source of the energy which moves the water
and animates the machine. While these chemical processes are very
diverse in their nature and may be considered in each tissue in turn,
they are all alike in that they proceed at a rate much superior to that
at which they go on outside of the cell when under similar conditions
of temperature. This superior rate of reactions in protoplasm is due to
the fact that-these reactions are hastened or catalyzed, and it is this
process which we have now to examine.

The word catalysis is from the Greek kata, meaning down, and lysis,
to loosen. Literally a down loosening, it has come to mean the hastening
of a chemical reaction by a third substance, the catalyst, which emerges
at the end of the reaction unchanged in amount, or nearly unchanged,
since all substances are more or less unstable, and which accordingly has
appeared to act only by means of its presence. But while it appears to
have acted by its presence only, there is no doubt that in many, if not
in all cases, it has actually entered into the reaction at some stage or
THE PHYSICAL CHEMISTRY OF PROTOPLASM 251

other, but has become free again. There are a great number of reactions
of this kind known outside of cells. For example, hydrogen and oxygen
gas do not combine at a measurable speed at ordinary temperatures,
although they will combine at higher temperatures. It is, however, to be
inferred that they do unite at ordinary temperature, but at so slow a
rate that it is not measurable in the times so far studied. But if this
mixture of gases is passed over finely divided platinum, union takes place
and at a rate so great that it heats the platinum. In this case the platinum
is the catalyst.. Another example of a catalytic reaction is the union of
sulphur dioxide and oxygen to sulphuric acid in the lead chamber process
of sulphuric acid manufacture. The presence of nitric trioxide, N,O,,
hastens this reaction, the nitric trioxide appearing at the end in
unchanged amount: Water is one of the most important catalytic agents
known. Thus perfectly dry ammonia, NH,, and HCl will not unite with
measurable speed, nor will ammonium chloride dissociate into NH, and
HCl in the absence of water. The presence of a very minute amount of
water catalyzes, or hastens both reactions. Water is necessary for the
rusting of iron, or for the union of chlorine and hydrogen. In fact,
an enormous number of reactions are catalyzed by water.

To understand how catalytic agents may hasten reactions, we must
first consider the factors which determine the velocity of ordinary
reactions.

The velocity of a chemical reaction is directly proportional to the
chemical affinity, and inversely proportional to the chemical resistance.
There is as yet no good means of measuring chemical affinity. It involves
two factors, mass and attraction. Chemical reactions take place in the
direction of doing the maximum of external work. This is simply another
way of saying that the reaction is always in such a direction that the
total potential energy of the system reacting is reduced to a minimum:
in other words, the reaction as a whole, but not necessarily in all its parts,
always goes in the direction of greater stability under the conditions of
the reaction. By the velocity of a chemical reaction is meant the amount
of substance transformed divided by the time. If a gram of cane sugar
is inverted in an hour, the velocity of the reaction is 1/60th of a gram
a minute. The time required for any chemical transformation is evi-
dently the sum of two periods, namely, the time required for the two
or more reacting molecules to come into contact, since chemical actions
only take place between molecules in contact or more probably they only
take place when they are united, that is they only occur within molecules.
It is necessary, then, in order that the reaction shall take place, for the
reacting species of molecules to come into contact and unite into a single
molecule. The second period of the total time taken is for the molecu-
lar rearrangement to take place which constitutes the reaction.
252 PHYSIOLOGICAL CHEMISTRY

These two periods of time may be illustrated by the reaction by which
sulphuric acid is made in the lead chamber process. The first part of
the reaction consists in the time necessary for the formation of the inter-
mediate compound, nitrosyl-sulphurie acid, and the second period the
time required for the decomposition of this compound:

1. 280, 4+H,04+20+N,0, —~ 2(SO,.0H.NO

2. 2(86, OHNO, ) eee 2.80, 1-N.0, 2)

Now the first part of the time, ie., that required for the molecules
to meet, will be the shorter the more molecules there are in the space
in which they are confined ; and evidently the speed of the reaction will
be proportional to the number of molecules of each reacting species in
the space, or in other words to the concentration of each of the reacting
species. This general law of chemical reactions by which the velocity
is proportional to the concentration is known as the law of mass action
and is sometimes called the law of Guldberg and Waage. It may be put
in the form of an equation as follows:

Amt. Transformed
Velocity = V = —-_——_-= K ©, x,
Time Ri
K being the constant of proportion and C, and C,, being the concen-
trations respectively of the two kinds of reacting molecules a and b.
Since in the absence of any special means of keeping the concentration
constant C, and C, must of course diminish as the molecular species
a and 0b are used up in the reaction, it is obvious that the velocity of such
a reaction is not constant but must diminish with the time. If, how-
ever, a very minute interval of time was taken, the velocity would remain
approximately constant during that time interval. If dx is a very minute
amount of substance transformed in the very minute time, dt, then the
velocity at any instant, t, will be dx/dt and this will be proportional to
the amount of substance actually present, and this will be equal to the
amount A at the start of the reaction minus the amount x transformed
during the period, t, or dx/dt—K(A—x) ; this is the differential equa-
tion of the velocity of a reaction in which only a single molecular
species A is undergoing a change in concentration. It applies, for
example, to the hydrolysis of cane sugar, the water, which is the other
molecular species entering the reaction, not materially changing its con-
centration, being present in great excess. Since dx and dt are too minute
for direct measurement, it is necessary to add a great number of these
together to get a time interval and an amount of x which can be meas-
ured. This addition is the process of integration; and if the foregoing
equation be integrated, or added, there is obtained the velocity equation
LogA — Log(A — x) = Kt; or Log(A/(A—x)) = Kt
THE PHYSICAL CHEMISTRY OF PROTOPLASM 253

t being the time from the beginning of the reaction, A the concentration
of the substance at the start and A—x the concentration at the end of
the interval t. :

Temperature coefficient. The velocity of this part of the reaction,
namely the time required for collision of the reacting molecules, is not
only a function of the concentration of the molecules, but also, very
naturally, it is a function of the speed of their movement; the velocity
of the reaction, or rather of this part of it, must hence increase with
the temperature. Most chemical reactions increase in speed with the
temperature, however, at a very much greater rate than can be
accounted for by the increase of velocity of the molecules. For most
chemical reactions at temperatures of from 10-40° C. the rate of the
reaction doubles or trebles with a rise in temperature of 10°, but the
rate of increase is not constant, being greater than twice or thrice at
lower temperatures and less at higher. If only the velocity of the
molecular movement was concerned in this increase in the reaction veloc-
ity, the rate should increase uniformly with the temperature. For
example, the average kinetic energy of all molecules at 20° C. (293° Abs.)
is 2.015 <10—* 298 ergs, and at 30° it is 2.015 X<10—"* 308 ergs. Since
the kinetic energy, 4% MV’, is proportional to the square of the velocity
of movement, the velocity of the reaction, which is proportional to the
speed of molecular movement, at 30° should be to that at 20° as the
square root of 308/293 or 1.017. It is evident from this calculation, as
the actual increase is 2-3 times this amount, that chemical reactions are
increased by a rise of temperature in some other way than exclusively
by the increase of velocity of molecular movement. This brings us to
the second period in a chemical reaction: namely, the time required for
the molecular rearrangement.

Chemical resistance. If it be admitted that rearrangement only takes
place within the molecule, if in other words it is admitted that molecules
really interact only when combined, a longer or shorter period of time
will be necessary for the molecular rearrangement to take place by which
the reaction is consummated and the new molecular species are formed.
Now this intramolecular rearrangement can only spontaneously occur in
the direction of greater molecular stability; that is, of less potential
energy. This brings us to the question of the resistance to chemical
reactions. While very little is known about this, it is not impossible
that it consists, in a measure at any rate, in the stability of the molecular
form of the molecule. The atoms are probably packed very closely
together in a molecule. The resistance to movement, or the internal
molecular friction opposing the molecular rearrangements, may be either
high or low. but it is often high. The length of time union between
molecular species persists before rearrangement takes place, ani the
254 PHYSIOLOGICAL CHEMISTRY

reaction is ended, is extremely variable. In some cases the time is long
and the intermediate substances are hence so stable that they may be
isolated in quantities; in other cases the resistance to rearrangement is
so slight that the reaction takes place almost instantaneously and these
intermediate compounds are so unstable that they cannot be isolated and
often their existence can only be inferred from the character of the
transformation. It is, for example, very difficult often to prove that
molecular oxygen unites with the substance oxidized before the reaction
is consummated, but oxyhemoglobin is such an intermediate substance
which in the absence of either alkaline or acid reaction is fairly stable
and may be isolated. Since a rise in temperature increases the motion
of the atoms in the molecule and thus increases their lability, it shortens
the period of the reaction by shortening the time taken up in the inter-
mediate stage and so hastens the reaction. It accelerates by diminishing
the resistance, but does not so greatly affect the chemical affinities. Heat
having this double action accelerates chemical reactions more than
physical. Nearly all vital reactions or. activities are doubled or trebled
by a rise of temperature of 10° between the limits 10-40° C.

The action of a catalyst may be pictured in very much the same way
as the action of heat, in that the chemical resistance is reduced, so that
the time required for the intermediate stage of the reaction is greatly
shortened and hence the reaction is hastened. For example, it is prob-
able that the reaction in the union of hydrogen and oxygen to form water
involves the intermediate formation of hydrogen peroxide, thus

L, BO, ae EO,
2, 2,0, —~2H,640,
Finely divided platinum has the saeeicaety) of rendering hydrogen perox-
ide so unstable that it decomposes with great speed, so that the total
time required in the reaction is enormously shortened. There can be
very little doubt, also, of the manner in which this hastening is produced.
It is found that any substance which will unite with the platinum, and
thus presumably occupy the bonds of the platinum where the hydrogen
peroxide ordinarily takes hold, will poison or prevent the action of the
platinum. Thus hydrogen sulphide or carbon bisulphide are true poisons
of this catalysis. It is probable, therefore, that in the presence of
platinum there are these reactions:
1. H,+0,—+ H,0,
2. 2H0 _ -Pt— 3H, O,Pt
3, 2H.0,Pt—+2H.040, + Pt
Another reaction of this same type where the action is hastened by the
formation of an intermediate union between the catalyst and an inter-
mediate product of the reaction is that of the formation of ether from
alcohol by the action of sulphuric acid.
THE PHYSICAL CHEMISTRY OF PROTOPLASM 255

Another fundamental property of catalytic agents is that, in many
cases at any rate, they do not change the point of equilibrium of revers-
ible reactions. A great many reactions, possibly all of them, never go
completely to an end. They apparently come to rest, but it is found.that
there is more or less of the unchanged, reacting substance still present
when this happens. Such reactions are said to be reversible. It is char-
acteristic of the common reversible reactions that very little energy
exchange takes place in them. Such a reversible reaction is that gen-
erally cited between acetic acid and ethyl alcohol. If acetic acid and
alcohol are mixed, union takes place between them with the formation of
ethyl acetate. The reaction apparently comes to an end when there are
about 33 molecules each of alechol and acetic acid and 66 of the ethyl
acetate. If ethyl acetate be dissolved in water, it will break up into acetic
acid and alcohol until the three reacting molecules are present in the same
proportion as before. This point is called the point of equilibrium of
the reaction.

C,H,0, + C,H.OH ; oars U,H,.0.CO.CH, +H,0

This reaction goes in the left-handed direction if ethyl acetate is dis-
solved in water, and in the right-handed if a start is made with alcohol
and acid. At the time of equilibrium the reaction has not stopped, but
is going on in such a way that the number of molecules of acetate break-
ing up is just equal to the number being formed in any time interval.

This reaction may be catalyzed by lipase, an enzyme or catalytic agent
found in cells, but it is found that while the point of equilibrium is
reached in a shorter time, it has not changed the relative concentration
of the reacting molecules. The fact that the point of equilibrium of the
reaction is not changed by the catalyst means that the catalyst must
accelerate the reactions in each direction equally, otherwise, in any
interval of time, there would be more molecules. of acid and alcohol unit-
ing than of ethyl acetate breaking up, or vice versa, and the point of
equilibrium would be shifted. On the theory of the catalysis being due
to the formation of an intermediate unstable stage, this behavior is
readily understood, since the reaction has to pass through this stage in
whichever direction it is going. This fact, that the catalysts catalyze
equally both reactions in a reversible reaction, is known as the reversible
action of enzymes. It was first observed in the case of the enzyme
maltase. This catalyzes the union of glucose to form isomaltose and
water, and of maltose and water to form glucose. It has since been shown
also for other reversible reactions and other catalysts. Kastle and Loe-
venhart showed the reversible reaction of lipase. and Taylor reported the
reversible synthesis of protamine by a proteolytic enzyme.

The intermediate body composed of catalyst and reacting molecules
256 PHYSIOLOGICAL CHEMISTRY

is generally unstable, but it is conceivable that when it is formed it
might in some way be rendered more stable. If this were the case, we
should have a complex formed of enzyme and various species of reacting
molecules which might be very complex and stable within narrow limits.
It is possible that the synthesis of amino-acids to make proteins, and
other syntheses, are brought about in this way in protoplasm, the proto-
plasm being essentially composed of the enzymes united with the sub-
stances upon which they act. The substance making the intermediate
stage stable might be called an anti-ferment.

The catalytic agents of cells are known as enzymes, a word meaning
literally in yeast, from the Greek, en, in; zyme, leaven. An enzyme is an
organic catalytic agent found in, or isolated from, living matter. These
catalytic agents are very numerous and it is to them that the activity of
living protoplasm in a chemical sense is due. Some of these enzymes are
easily isolated from cells; they are exocellular; such are the various
digestive enzymes, pepsin, trypsin, invertin, ptyalin, maltase, and the
alcoholic enzyme, zymase. Others, however, have not yet been isolated.
and the more fundamental reactions of living matter, such as the oxida-
tions or the preliminary fragmentations of the molecules, are apparently
due to some enzymes of a very unstable kind which are firmly tied to the
structure of the cell. It may be that for these reactions the simultaneous
presence of two or more contiguous or loosely-bound enzymes may be
necessary, so that by separating them their action is lost. At any rate,
it has so far been impossible to bring about the synthesis produced by
the cell if the structure of the cell is first destroyed. -

There are a great variety of enzymes found in cells; among them are
those which produce hydrolyses, such as the digestive enzymes, and by
whose action the synthetic formation of various colloidal constituents
may be explained ; oxidases; peroxidases ; catalases; and various enzymes
producing fermentations such as zymase. Among the hydrolytic enzymes
may be mentioned invertin, maltase, laccase, amylase, dextrinase, cytase,
emulsin, myrosin, pepsin, trypsin, erepsin, probably rennin, and other
proteases; the esterases, such as various lipases; deamidizing enzymes,
such as adenase, guanase; arginase; nucleases; and glyoxalase. It is
possible that these enzymes form part of the organized protoplast, and
that their hydrolytic activity is checked by the presence of anti-enzymes,
particular conditions of alkalinity and so on.

The physical chemistry of oxidation.—Since the whole of the energy
used in the production of living phenomena comes immediately or sec-
ondarily from the oxidation of the foods, an understanding of the process
of oxidation is necessary before vital processes can be understood; a
short account of the theories of the nature of oxidation may be given at
this place. There are two kinds of oxidations going on in living matter
THE PHYSICAL CHEMISTRY OF PROTOPLASM 257

namely, those taking place at the expense of the oxygen of the air, and
those in which the oxidation is produced by easily reducible food sub-
stances or their metabolic fragments. The first process is called aérobic
respiration ; the second anaérobic. In aérobic respiration the oxidizing
agent is the oxygen of the air.

The term oxidation, which literally means a process of souring, from
the Greek, oxys, acid, includes in chemistry not only processes which
involve the transfer of oxygen, but it is used to signify any process
which results in the increase of the number of positive valences, or the
diminution of negative valences of a compound or element, whether this
is produced by oxygen or some other agent. Thus the reaction between
ferric chloride and potassium iodide by which iodine is set free is called
an oxidation, the iodide being oxidized by the ferric chloride. The re-
action is as follows:

++4+7—>— + = ++ ——— +
fet scl. k+.1—~- fe 43014. K41

It will be seen from the ionic reaction that the oxidation has really
involved the passage of a positive charge of electricity from the ferric
atom, which is the oxidizing reagent, to the iodine atom; or the passage
of a negative electrical charge from the iodine ion to the ferric ion, thus
reducing it. It will be noticed that there cannot be an oxidation without
a corresponding reduction. A similar reaction is that between nitric
acid and silver, leading to the formation of silver nitrate. This may be
written as follows from the ionic point of view:
NO, + OH + Ag — Ag-+0H+NO,
Ag-- OH +H 4NO, —+ Ag+NO, +H,0

In this reaction it will be seen that the oxidizing reagent is the ion, NO,,
which has a positive charge held at a very high potential, and that this
is the cause of the oxidation of the metallic silver to the positively
charged Ag ion. It may at first seem unlikely that nitric acid should
dissociate in this way into NO, and OH, but it is not by any means
impossible that such a dissociation in small amounts takes place. The
weaker the acid is, the more likely it is to dissociate somewhat as a base
as well as an acid. Water, for example, functions both as a base
and an acid. Borie acid is nearly as basic as it is acid. Hypo-
chlorous acid, HCI1O, is also a very weak acid and probably dissociates in

this way in part into a +-OH; the positive chlorine being the active
agent. It will be observed that in all the oxidations of this type a
hydroxyl group combines with the oxidized substance. Another reaction,
an oxidation which does not involve oxygen, is the oxidation of zine by
an acid. In this case there is the reaction

tt —— $4 55
Zn. + 2H + 2Cl —~ Zn +2014 H,
258 PHYSIOLOGICAL CHEMISTRY

In this reaction the hydrogen is the oxidizing agent giving up a positive
charge to the zine which is thus oxidized. Similarly all processes of oxi-
dation, could we trace them out, would be found to involve the transfer
of a negative electron from one element to another, the one which receives
it being reduced and the element losing the negative charge being thus
rendered more positive and being said to be oxidized.

Whether the foregoing picture of the process of oxidation be in all
-particulars right or not, it is beyond question that the oxidation does
involve, in all cases in which the process can Le watched, the transfer
of positive and negative electrical particles or electrons, and that this is
the essence of the process. Moreover, the more easily a substance gives
up a negative charge the more active will it be as a reducing agent; and
similarly the more easily it gives up a positive charge, or acquires a
negative, the more active will it be as an oxidizing agent. Oxygen acts
as an oxidizing agent because it has a great tendency to take away a
negative charge from other substances and go over into the form of an
oxygen ion, or of electro-negative oxygen.

The great importance of this theory from the point of view of physio-
logical chemistry is that it shows at once that every oxidation in proto-
plasm is at the bottom an electrical process involving the transfer of
electrical charges. In other words, an electrical disturbance of some
kind, albeit possibly within molecular dimensions, must occur in every
combustion in the protoplasm. It thus furnishes a point of attack of the
origin of the electrical disturbances which are so characteristic of living
matter of all kinds and enables an understanding of the disappearance
of these currents when the respiration of the protoplasm is prevented.

While ordinarily the transfer of the electrical charge from one atom
to another releases, directly or indirectly, in the form of heat, energy
which had been potential, under suitable conditions this energy does not
take the form of heat, that is of indiscriminate molecular vibrations, but
of a steady migration of the ions, the positive in one direction, the nega-
tive in another, so that we have an electrical current which can do work.
This happens in the particular arrangement which is called a battery. If
a piece of zinc is placed in a solution of sulphuric acid, it dissolves with
the liberation of hydrogen gas and of much heat. In this case the oxidiz-
ing substance is the hydrogen ions of the acid, and the oxidized substance
is the zine which escapes into solution as a positive ion. The heat may
be due to the violent separation of the zine and hydrogen after the trans-
fer of the charge from one to the other. If, however, the zine be placed
in a solution of zine sulphate, and this is in contact through a porous cup
or directly with a solution of copper sulphate in which is a plate of cop-
per, and if the copper and the zinc are connected with a wire, the zine dis-
solves as it did before, and copper is deposited, but there is no appear-
THE PHYSICAL CHEMISTRY OF PROTOPLASM 259

ance of heat or very little, but instead an electrical current is produced
from the zine to the copper in the solution and in the opposite direction
outside. This reaction is a true oxidation-reduction reaction. In this
case the zinc dissolves extremely slowly when the battery is not short-
circuited, that is when the zinc and copper are not connected, for the
reason that there are in a zine sulphate solution so very few hydrogen
ions to oxidize the zinc; but as soon as the connection is made with the
copper, the copper ions in the solution which have a greater oxidizing
potential than the hydrogen are able to give up their charges to the cop-
per plate and these charges are conducted by the wire to the zinc plate,
thus oxidizing the zine so that it can go into solution as a positive ion.
This is, as it were, an oxidation at a distance, the oxidizing and reducing
agents not being in direct union, but indirectly through the copper plate
and wire. There is, of course, some loss of energy as heat produced by
the movement of the ions through the solution and in part by the move-
ment of the electrons through the wire, but the loss is not great. The
important point is that the processes in a battery which give rise to the
electrical phenomena of the battery are oxidation-reduction processes.

It is not inconceivable, although it has not yet been possible to prove
it, that the electrical phenomena of protoplasm might arise directly in
this way from the oxidation-reduction phenomena of protoplasm. They
may, however, have an indirect relation to the oxidation, as has been
pointed out. :

In order that the oxygen of the air shall oxidize it must first go into
solution. It is only in the presence of water that oxygen has the power
of oxidizing rapidly. The first question, then, is the mechanism of the
oxidation by oxygen. What happens to oxygen when it goes into solution
in water? This raises a most fundamental question, to which no definite
answer can at present be given; there have been several answers.

The first view is that of van’t Hoff. According to him, the oxygen
probably ionizes when it goes into solution in water, splitting into a

small number of 0 and O ions. It is the 0 ions which have the oxidizing
power. It has not yet been possible to prove that such an ionization
takes place, although something similar appears to happen in many gases
at high temperatures, dissociation into atoms taking place. It is also sug-
gested by others that all processes of ionization involve a union between
solvent and ionizing substance and this view, while not entirely incom-
patible with the foregoing, will, if Sanevaated cause some modification
of the explanation.

Another view is that of Traube, according to which the oxygen always
unites with the water to form hydrogen peroxide. which is the real oxi-
dizing agent. The reaction might be written as follows:
260 PHYSIOLOGICAL CHEMISTRY

2H,0 + 0, _ 2H,0,
This view leaves unexplained the cause of the oxidizing power of the
hydrogen peroxide.

There is a growing amount of evidence that all true solution is a
process of chemical union between the solvent and solute, possibly
through the extra valences on the molecules of the two kinds. It may
be that the oxidation is similar to that of the oxidation by chlorine or
bromine. When bromine dissolves in water it is known to form hypo-
bromous acid, bromates and bromide. The reactions might be written
as follows on the basis that the interaction of the water and the bromine
can only take place when a chemical union exists between them:

1, 2(Br,) +2H,0 —=+ (2H,0) (Br,),

o. (eH 0) (Br, Y, —- 2HBr + 2HO0Br

3. HBrO ——- H+ OBr; or HBrO —- Br + OH

Or the reaction might be with the water molecules (HO),

l. 2Br ot (H 2)s Se Br, (H 2)

2 H 0, Br, —~ 3HBr + HOBr + H,0,
Since the ion, BrO, has little or no oxidizing power, and the power of
the hydrogen ion is very much less than the solution possesses, the oxi-
dizing agent would be the positive bromine obtained from the bromine
hydrate,

The oxidizing power of oxygen may be represented in the same way:

1. 20, + 3H,0 _ H,0, —- 200H +H,0, +H,0

2, OOH —- 6408

The oxidizing agent would be the oxygen hydrate. The above reaction
would be in case the oxygen is monovalent in the gaseous form. [If it
were bivalent, the reaction would be

20,-4+3H,O ~—+ O(OH), 4 2H,0,

These reactions would account for the general appearance of hydrogen
peroxide during the reaction. They are, of course, in the case of oxygen,
purely hypothetical, since neither O(O0H), nor OOH have been isolated.
There are two facts, however, which are undoubted: one is that the action
of the oxygen is enormously more rapid in the presence of water, and
that hydrogen peroxide is formed accompanying many oxidations. It
would seem, therefore, that a union of some kind between the water and the
oxygen certainly precedes the oxidizing process. The solubility of oxygen
clearly indicates this also, since the solubility is greater than that of a
completely indifferent gas such as hydrogen, or helium. There is no
doubt, either, that in the case of the halogens the acids corresponding to
lypobromous acids are always formed when they dissolve in water; and
it is equally certain that. the oxidizing power of the metals, such as
THE PHYSICAL CHEMISTRY OF PROTOPLASM 261

Cu(OH),, is due to the presence of hydrates in the solutions. The facts
are, then, that the exact behavior of oxygen in water is uncertain, so that
it is impossible to say just what the oxidizing principle really is. It is
worth noting that, if there is in protoplasm a substance which will com-
bine with oxygen in the way supposed for the water, it will form just
such a union with the oxygen. Hemoglobin is such a substance.

Summary.—We may summarize as follows the results of the study
of the physical chemistry of protoplasm made in this chapter. Proto-
plasm, that is the real living protoplast, consists of a gel, or sol, which
is composed of the colloids of an unknown nature which include protein,
lipin and carbohydrate. Whether these colloidal particles consist of one
large colloidal compound in which enzymes, protein, phospholipin and
carbohydrate are united to make a molecule which may be called a
biogen, cannot be definitely stated, but it seems probable that something
of the sort is the case. This colloid exists in the form of a gel. That is,
it always contains a large amount of water and this water has in it salts.
The gel of the protoplasm is not often uniform, but it is differen-
tiated physically and chemically in different parts of the cell. The cell
is not isotropic, as the morphologists say. The movements of the proto-
plasm and so the activity of the cell, the vital activity, appear to be due
to the varying affinity of this gel, or of particular parts of it, for water,
by which water is caused to enter, or leave it. This affinity for water
may be modified in various ways. It may be modified by salts, which
exist usually in loose or more firm chemical union with the colloids. Some
salts if introduced into the protoplasm will cause the protoplasm to take
up more water, others to lose water. It may be modified by a change in
the reaction of the cell, by the production of acid. And, above all, it is
modified by the chemical changes occurring in the colloid itself, for this
colloid is very unstable. The last is the cause undoubtedly of most of
the rhythinic and other activities of protoplasm. The protoplast, which is
probably the continuous phase, undergoes oxidations, and in virtue of the
changes thus produced the affinity for water by the colloid is changed. It
may be simply a local affinity alteration, such as we see in the streaming
ameba, in which the protoplasm suddenly appears to become more liquid
in one region or another of the cell. A stimulus, on this view, is anything
which alters the affinity for water on the part of the protoplast, and as
this affinity is a very delicate adjustment it may be altered by a great
variety of means. Hence stimuli may be either chemical, physical or
mechanical; since by all of these means we may produce chemical changes
which will alter the affinity of the cell for water.

‘Every activity of this protoplast is accompanied by an electrical
disturbance. the blaze current, or current of action. The way in which
this electrical disturbance is produced is still entirely dark; and
262 PHYSIOLOGICAL CHEMISTRY

its significance, or rather its possible function, is equally dark. But
men have imagined three possible ways in which it might be produced:
It might be produced by the change in the surface of contact of the col-
loid and water, as happens in a capillary electrometer when the surface
of contact of mercury and acid is altered; it might be produced by the
appearance of acid as a result of chemical decomposition, the hydrogen
ions in some way setting up a concentration chain effect by their greater
velocity of movement, the colloids assisting by forming semi-permeable
membranes, thus interposing resistances to the passage of the negative
ion; or the electrical disturbance might be the direct result of the oxi-
dation, every oxidation involving a minute current when the positive
charge is passed from the oxidizing to the oxidized body. How such an
effect could be propagated to a distance beyond the molecule has not
been explained. Evidently the explanation of the mechanism of the pro-
duction of this electrical disturbance must be left to the future.

All the chemical processes in the cell, so necessary for the quick
response to a stimulus and to recovery from the effects of a stimulus,
are accelerated by the presence in the cells of accelerators of these re-
actions, and these accelerators are called enzymes. The nature of none
of these is definitely known, and the protoplast itself, or the biogens,
appear to be the most important of these accelerators. The enzymes,
there are reasons for thinking, are not distributed evenly through the
cell, but exist in definite locations, so that the changes in one part differ
from those in another, thus producing a physiological division of labor
and a physiological diversity no less marked than the morphological
diversity.

Finally the structures of cells are so characteristic and definite as
to show that the cell is organized in some way or other. The suggestion
has been made that this organization must in the long run be caused by
the molecules of which the protoplast is composed, just as the form of
a crystal is produced by the molecules of which it is composed. It must
hence be the expression of the molecular form of the biogens.

REFERENCES. Osmotic PREssuRE.

1. Findlay: Osmotic Pressure. Monographs on Inorganic and Physical Chemistry.
Longmans, Green and Co. London, 1913.

Van ’t Hoff: Zeit. physikal, Chem. 1, p. 481, 1887.

Traube: Arch. f. Anat. Physiol. u. wissensch. Med. p. 87, 1867.

Pfeffer: Osmotische Untersuchungen, 1877.

Graham: Phil. Trans. 144, p. 117, 1854.

Kahlenberg: Jour. Physical Chem., 10, p. 141, 1906.

Berkeley and Hartley: Phil. Trans. A, 206, 486, 1906. A. 209, 177 and 319.

Morse: American Chem. Journal, 48, p. 29, 1912. Earlier papers in the same
journal. Osmotic pressure of concentrated solutions.

9. Garrey: Some cryoscopic and osmotic data. Biol. Bull., 28, 77, 1915.

PA AIP eS
10.

11.
12,

13.

14.

10.

11.

12.

13.

14.

15,

16.

li.

18.
19.

20.

21.

22.

23.

THE PHYSICAL CHEMISTRY OF PROTOPLASM 263

Callendar: Pro. Roy Soc. A, 80, 466, 1908.

Stern: Zeit. physikal. Chem. 81, 441, 1912.

I. Traube: Phil. Mag., 8, 704, 1904. Surface tension and osmotic pressure.
Armstrong: Proceedings Roy. Soc. A, 78, 264, 1906.

Taylor: Chemistry of Colloids. Longmans, Green and Co., London, 1915.

PROTOPLASM AS A PHysIcAaL SYSTEM.

Rhumbler: Das Protoplasma als physikalisches System. Ergeb. d. Physiol. 14,
~1914, pp. 475-617.

@Arsonval: Rélations entre la tension superficielle et certains phenoménes
d’origine animale. Arch. de Physiol. (6), 1, p. 460, 1880.

Bechhold: Strukturbildung in Gallerten. Zeits. physikal. Chem., 52, p. 185, 1905.

Bechhold: Die Kolloide in der Biologie und Medizin. Dresden, 1912.

Bernstein: Chemotropische Bewegungen eines Quecksilbertropfens. Ar. ges.
Physiol. 80, p. 628, 1900.

Bernstein: Die Krifte der Bewegung in der lebenden Substanz. Naturwiss.
Rundschau, 16, 1901, pp. 413, 429, 441.

Bernstein: Die Energie des Muskels als Oberflaichenenergie. Ar. ges. Physiol. 85,
p. 271, 1901.

Bernstein: Bemerkung zur Wirkung der Oberflachenspannung im Organismus.
Eine Entgegung. Anatom. Hefte. 27, p. 823.

Berthold: Studien iiber Protoplasmamechanik. Leipzig, 1886.

Bethe: Zellgestalt, Plateausche Fliissigkeitsfigur und Neurofibrille. Anat. Anz.
40, p. 209, 1911.

Biedermann: Vergleichende Physiologie der irritablen Substanzen. Ergeb.
Physiol. 8, p. 26, 1909.

Biedermann: Die Aufnahme, Verarbeitung und Assimilation der Nahrung.
Winterstein’s Handbuch der vergl. Physiol. 2, 1911.

Buglia: Hangt die Resorption von der Oberflichenspannung der resorbierten
Flussigkeit ab? Bioch. Zeits., I, p. 1, 1909.

Biitschli: Untersuchungen iiber mikroskopische Schiume und das Protoplasma.
Leipzig, 1892.

Biitschli: Ueber Strukturen kiinstlicher und natiirlicher quellbarer Substanzen.
Verh. d: naturhist.-mediz. Ver. Heidelberg. N.F. 1895, pp. 460-368.

Biitschli: Untersuchungen iiber die Mikrostruktur kiinstlicher und natiirliche
Kieselsaure Gallerten. Ibid., 6, p. 287, 1900.

Danilewsky: Versuche iiber die elektrische Pseudoirritabilitit toter Sub-
stanzen. Arch. Anat. Physiol. 1906, p. 413.

Dellinger: The cilium as a key to the structure of contractile protoplasm.
Jour. Morph. 20, p. 171, 1909.

Fischer: Fixierung, Firbung u. Bau des Protoplasmas. Jena, 1899.

Fitz-Gerald: On the change of superficial tension of solid-liquid surfaces with
temperature. Scientific writings, p. 307, 1878.

Freundlich: Kapillarchemie. Eine Darstellung der Chemie der Kolloide und
verwandter Gebiete. Leipzig, 1909.

Gaidukow : Dunkelfeldbeleuchtung und Ultramikroskopie in der Biologie und in
der Medizin. Jena, 1910.

Gebhardt: Knochenbildung und Kolloidchemie. Arch. Entwickelungsmechanik,
32, 1911, p. 727.

Gibbs: Equilibrium of heterogeneous substances. Trans. Conn. Acad. Arts
Sci. 3, p. 380, 1878.
264

24.

25.

26.

27.

28.
29.

30.

31.

32.

33.

34,

39.

40.
41.

42.

43.

44,

45.

46.

47.

43,

49.

50.

PHYSIOLOGICAL CHEMISTRY

Gruber: Biologische und experimen. Untersuchungen an Amoeba proteus.
Arch. Protistenkunde, 25, p. 316, 1912.

Hamburger: Physikalisch-chemische Untersuchungen iiber Phagocyten. Ihre
Bedeutung vom allgemein biologischen u. pathologischen Gesichtspunkt.
Wiesbaden, 1912.

Harvey: Studies on the permeability of cells. Jour. Expt. Zool. 10, 1911, p. 507.

Heidenhain: Kine Erklirung betreffend die Protoplasmatheorie. Als Antwort
an J. Bernstein, P. Jensen u. L. Rhumbler. Anat. Hefte, 27, p. 887, 1905.

Héber: Physikalische Chemie der Zelle und der Gewebe. 4th ed. Leipzig.

Héber: Die physikalisch-chemischen Vorgiinge bei der Erregung. Zeits. all.
Physiol. 10, p. 173, 1910.

Hofmeister: Die chemische Organisation der Zelle. Braunschweig, 1901.
Naturw. Rundschau, 16, 1901, pp. 591, 600, 612.

Jennings: Artificial imitations of protoplasmic activities and methods of
demonstrating them. Jour. Applied Micros. 5, p. 1597, 1902.

Jennings: Contributions to the study of the behavior of lower organisms.
Washington, 1904, Carnegie Publications.

Jensen: Ueber den Aggregatzustand des Muskels und der lebendigen Sub-
stanz tiberhaupt. Arch. ges. Physiol. 80, p. 176, 1900.

Jensen: Zur Theorie der Protoplasmabewegung und tiber die Auffassung des
Protoplasmas als chemisches System. Anat. Hefte. 27, p. 831, 1905.

Kite: Studies on the physical properties of protoplasm. <A. Jour. Physiol., 32,
p. 146, 1913.

Kite: Some structural transformations of the blood cells of vertebrates. Jour.
Inf. Diseases, 15, p. 319, 1914.

Hardy: Structure of cell protoplasm. Jour. Physiol. 24, p. 158, 1899.

Chambers : Some physical properties of the cell nucleus. Science, N. S., vol. x],
pp. 824-327, 1914. Lancet-Clinic, March 27, 1915.

Kiister: Ueber Veriinderungen der Plasmaoberfliche bei Plasmolyse. Zeit. f.
Botan. 2, p. 689, 1910.

Kiister: Ueber Zonenbildung in kolloidalen Medien. Jena, 1913.

Leduc: Théorie physico-chimique de la vie et générations spontanées. Paris,
1910.

Leduc: La Biologie synthétique. Paris, 1912.

Lehmann: Fliissige Krystalle sowie Plastizitit von Krystallen im allgemeinen,
molekulare Umlagerungen und Aggregatzustandsveriinderungen. Leipzig,
1904.

Lehmann: Fliissige Krystalle, Myelinformen und Muskelkraft. Miinchen.
(Isaria Ver]. Abteil. Natur u. Kultur). 1910.

Lillie: General conditions of fibrillar contractility. A. Jour. Physiol. 22, p. 75,
1908.

Lillie, R. §.: On the connection between stimulation and changes in the perme-
ability of the plasma membranes of the irritable elements. Science, N. S.
30, p. 245, 1909.

Loeb: The Dynamics of Living Matter. University of Chicago Press, Chicago.

Macallum: On the distribution of potassium in animal and vegetable cells.
Jour. Physiol. 32, p. 95, 1905.

Macallum: Oberflichenspannung und Lebenserscheinungen. Ergeb. Physiol. 11,
1911. :

Michaelis: Dynamik der Oberflichen. Eine Einfiihrung in biologische Ober-
flichenstudien. Dresden, 1909.
52.

53.

54.

55.

56.

57.

58.

59.

60.

61.
62.

63.
64.

65.
66.
67.

68.
69.

70.

71.
72.

73.
74.

75.
76.

77.

THE PHYSICAL CHEMISTRY OF PROTOPLASM 265

Ostwald: Grundriss der Colloidchemie. 1909.

Pauli; Kolloidchemie der Muskelkontraktion. Ueber den, Zusammenhang von
elektrischen, mechanischen und chemischen Vorgiingen in Muskel. Dresden
and Leipzig. 1912.

Prenant: Théories et interpretations physiques de la mitose. Jour. Anat.
Physiol. 46, p. 511, 1910.

Quincke: Ueber periodische Ausbreitung von Fliissigkeitsoberflichen und
dadurch hevorgerufene Bewegungerscheinurigen. Ann. Phys. Chem. N. F. 35,
p- 580, 1888.

Quincke: Bildung von Schaumwinden, Beugungsgittern und Perlmutterfarben
durch Belichtung von Leimchromat, Kieselsiure, Eiweiss, ete. Anna. Phys.
(4) 13, p. 65, 1904. :

Quincke: Doppelbrechung der Gallerte beim Aufquellen und Schrumpfen.
Ann. Phys. (4) 14, p. 848, 1904.

Rhumbler: Physikalische Erklirung der Lebensiiusserungen niederster Lebe-
wesen. Natur, 8, 1912, pp. 129, 176, 214. Other works by this author are
cited in the article in the Ergebnisse, 1914.

Robertson: An outline of a theory of the genesis of protoplasmic motion and
excitation. Transact. Roy. Soc. South Australia, 29, p. 56, 1905.

Robertson: Remarks on the theory of protoplasmic movement and excitation.
Quar. Jour. Exper. Physiol. 2, 1909, p. 303.

Schaefer: On McDougall’s theory of muscle contraction with some remarks on
Hurthle’s observations on muscle structure and the changes which it under-
goes in contraction. Quar. Jour. Expt. Physiol. 3, p. 63, 1910.

Traube: Die osmotische Kraft. Arch. ges. Physiol. 123, p. 419, 1908.

Traube: Die Theorie des Haftdruckes und ihre Bedeutung fiir die Physiologie.
Arch. ges. Physiol., 132, p. 511, 1910.

v. Uexkill: Umwelt u. Innenwelt der Tiere. Berlin, 1909.

Della Valle: Die Morphologie des Zellkernes und die Physik der Kolloide.
Zeits. f. Chemie u. Indus. d. Kolloide, 12, p. 12, 1913.

Verworn: Allgemeine Physiologie. 5 Aufl. 1909.

Verworn: Article on ‘‘ Physiology,” Encyclo. Britannica, 11th ed.

Wilson: On protoplasmic structure in the eggs of Echinoderms and some other
animals. Jour. Morph. Supplement, 15, 1, 1899.

Mathews: The internal pressure of liquids. J. Physical Chem., 17, p. 603, 1913.

Mathews: The nature of chemical stimulation. Amer. Jour. Physiol., 11, p.
455, 1904.

Euler: The general chemistry of enzymes. Translation by Pope. New York,
1912.

Cohnheim: Enzymes. Wiley and Sons, New York, 1912.

Clowes: Protoplasmie Equilibrium. Jour. of Physical Chem., xx, 1916, pp.
407-450.

Clowes: Soap jellies. Proc. Soc. Expt. Biol. and Med., xiii, 1916, pp. ‘114-118.

Clowes: Antagonistic electrolyte effects in physical and biological systems.
Science, N. S., xliii, 1916, pp. 750-757.

McClendon: Physical Chemistry of Vital Phenomena. Princeton University
Press, 1917.

Haldane: Gas laws extended to solids and liquids. Biochem. Jour., xii, 464,
1918. :

Mathews: Molecular Cohesion. Verhandelingen. Amsterdam Academy, Erste
Sectie, XII, 1917.
PART II.

THE MAMMALIAN BODY CONSIDERED AS A MACHINE. ITS
GROWTH, MAINTENANCE, ENERGY TRANSFORMATIONS
AND WASTE SUBSTANCES.

We have now to inquire more in detail concerning the nature of the
processes by which the animal body grows, and maintains itself from
the foods; what the source is of the heat it so constantly produces;
whence comes its power of moving and doing work; what is the nature
of the waste products it forms, and the causes of the variations which
they show in diverse conditions of diet and health. All these processes
are included in the province of the science of nutrition. It is, then, the
nutrition of animals and in particular of mammals which we shall
consider in the following chapters. We shall take up, first, the produc-
tion of heat in the body and then pass on to the study of the processes
mvolved in the maintenance of the animal organization and in its
development and energy transformations.

In order that organisms shall maintain their form unchanged, that
they shall transform into their own substance this mass of material dif-
ferent from themselves on which they subsist,—that they shall make
themselves out of their foods,—it is necessary that there shall be some
kind of an organizing force at work. Organization is at the bottom of
everything living, not only of the material side of our existence, but of
the mental as well. The organism may be but an enlarged, complex
and semifluid crystal. That we shall remember experiences, that we
shall continue to exist as individuals, something of a material kind must
persist, or be reconstituted from moment to moment. What is that some-
thing? What is the nature of the organizing principle of living things?
‘hese are fundamental questions, but they are questions to be solved by
ihe experiments of the future. We cannot answer them at present.

The body resembles a magnet. The body in many particulars
resembles a magnet. A magnet has existence as a magnet only as long
as the materials of which it is composed are organized; only as long as
the molecules are oriented or organized into a definite relation to each
other. The magnet is itself, then, an organism. It has the power, like
the body, of organizing other materials like itself, so that if new iron
is brought to it, it makes the new iron into a part of the magnet by

266
THE MAMMALIAN BODY CONSIDERED AS A MACHINE 267

organizing the ultimate particles of the new iron to correspond to the
organization or orientation of its own molecules. It has the power of
growth. It will pick the right materials, fine iron particles, out of a
mixture of iron and other substances, rejecting the useless and assimilat-
ing the substances like itself. It may be cut in two, like many organisms,
and each part becomes a magnet like that from which it came. If heated
to a point at which its organization is lost; if heated, in other words,
to a point at which the orientation of the molecules in consequence of
their rapid movement is lost, the magnetism disappears. Its property of
magnetism is lost. The magnet dies as an individual. How closely simi-
lar to all this, at least in its superficial aspect, is the life history of the
mammalian organism. Beginning life as a very minute organism, it
assimilates to itself, out of the mixture of foodstuffs brought to it, sub-
stances like itself. It grows like the magnet. Its power of organization
reminds one irresistibly of the magnet’s powers of organization. Like
the magnet, the organism cannot assimilate all kinds of things, but only
those things of a certain special kind depending on the shape and nature
of the molecules. Like the magnet, too, it can only continue to exist below
a certain temperature. If heated to its critical temperature the organ-
ism, like the magnet, ceases to exist as an organism, although composed
of the same elements as before the heating. In the heated magnet,
although it ceases to exist as a magnet, the property of magnetism still
exists concealed from our eyes in the molecules of which it is composed.
Each molecule preserves the property of magnetism. Is 1t not probable
that so in an organism the psychic and other properties of vitalism are
really inherent in and continue to exist in the particles of which the
molecules and atoms are composed, but that they are concealed from our
dull vision? The organism at death may be resolved into a multitude of
psychic elements, of elements possessed of vitalism, but this vitalism, this
psychism, is now in a multitude of small molecular and atomic units and
the organism as an organism ceases to exist. The law of the conservation
of energy, namely, that energy can neither be created nor destroyed,
but may be organized to appear in different forms, is one of the funda
mental laws of physics, chemistry and physiology. The biologist may
some day add its counterpart in the law of the conservation of psychism.
The property of psychism may be a fundamental property of every atom;
nay, of every electron. But it is only when organized that this property
appears to us in the form of an individual, a larger psychic unit.

The magnet does not appear to be undergoing vigorous chemical
change. In this respect it differs from the animal body, which is the
seat of such changes. But this difference is probably but a difference
in degree. The atoms of which the iron magnet is composed, there is
reason now for thinking, are not everlasting and changeless, but they
268 PHYSIOLOGICAL CHEMISTRY

may be having a metabolism of their own, new electrons being absorbed
from the ether, and old electrons being given off to the ether. All is in
flux in nature. Stability is but an appearance. Our brief lives are like
the fraction dt in a differential equation, infinitely brief in the time of
the universe. Things appear constant when observed for so short a time.

We cannot pursue speculations of this nature concerning those great
problems awaiting the future biologists. It is for us to clear the ground.
And we may comfort ourselves with two reflections as we consider how
hopeless the solution of those fundamental problems appears at the
present time: the first is that there is really nothing insignificant in
nature, however small our problems may look to us. The problem most
trivial in appearance, if followed but a little way, brings us to the most
profound mysteries of nature; and the second is that, however small a
fact may appear to be, any discovery made is multiplied by an infinite
factor in the course of time, since it is multiplied by infinite time, so
that every discovery is in reality infinitely valuable.

With these preliminary observations. which are not in many ways
very a propos, we may pass now to the consideration of the immediate
problems of how organisms maintain themselves. what the nature is of
the processes by which the materia] stream which flows through them
is organized into the body itself: whence come from this organization
those properties of life and consciousness which we may include under
the term vitalism ?
CHAPTER VI.
ANIMAL HEAT.

Animal heat.—The mammalian body is generally warmer than its
surroundings. It has a temperature of about 37.5° C., which is kept
nearly constant irrespective of the outer temperature. It produces heat
like a stove; it moves and expends energy in moving. Whence comes
the heat of the body; and what is the source of the energy used in move-
ments, in doing work? What are the natures of the processes by which
heat is liberated? These questions, or their prototypes, were among the
most puzzling which men asked when they began to inquire concern-
ing the nature of things; and very crude were the answers which
were given in the twilight of learning. Men imagined that heat was an
essence, or substance, which found its way into and out of the body ;
or that it was one of the four cardinal elements of which things were
made, earth, air and water being the other three. Men knew nothing con-
cerning the nature of heat. They did not know what was happening
when a piece of wood burned, so that their ideas about animal heat were
‘necessarily crude and generally wide of the mark. Even near the end
of the eighteenth century such brilliant and far-seeing men as Lavoisier
and Laplace still considered heat to be a substance which could be added
to or taken away from bodies; although they also suggested that it might
be a mode of motion of the finer particles of which matter was composed.

History of the discovery of the origin of animal heat.—In the last
part of the seventeenth and first part of the eighteenth century
men began more and more to try experiments to discover something
about nature, for ‘‘ it is only by experiment that we see in the full light
of day.’? Some experiments were tried by Mayow, Boyle and Priestley.
They put small animals into confined spaces and discovered that they
soon died. They found also that if when one animal was dead they
introduced a second into the same jar without renewing the air, the
second died in a shorter time than the first. The air in the jar was no
longer able to support life. Moreover, if a candle was put under a simi-
lar jar, it was extinguished after a time. If, then, a second lighted
candle was introduced without renewing the air, the light at once went
out. These observations showed that animals and candles behaved in
the same way in a closed space. Life and light were extinguished. The
experiment was then tried of seeing whether a mouse would live in the

269
270 PHYSIOLOGICAL CILEMISTRY

air exhausted by a candle and whether a candle would burn in air in
which a mouse had suffocated. In other words, did a candle and a mouse
change the air in the same way? It was found that mice would not live
in air in which candles could not burn. There was evidently something
similar in the burning of a candle and the breathing of an animal.
Black, who studied what had happened to the air, says in 1757: ‘‘ I have
convinced myself that the change produced in healthful air by the act
of respiration consists principally, if not entirely, in converting a part
of it into fixed air [now called carbon dioxide], because I found that in
breathing by means of a tube into water of lime or into a solution of
caustic alkali, I precipitated the lime and caused the alkali to lose its
causticity.’’ Shortly after this Priestley made a kind of air by heating
mercuric oxid2, which he called dephlogisticated air, but which is now
called by Lavoisier’s name of oxygen. He collected this air and put a
lighted candle into it. It burned better than in ordinary air. He then
put 4 mouse into some more of the dephlogisticated air and it lived longer
than in the same quantity of ordinary air. This ‘‘ dephlogisticated air ”’
was beneficial alike to burning candles and mice. Priestley also found
that it changed dark venous blood into red arterial blood, a change which
was known to occur in the lungs.

Lavoisier.—The question now turned on finding out what the candle
did to the air when it burned in it. This problem was solved by the
great French chemist Lavoisier about 1776. He proved by a series of
simple and convincing experiments that Priestley’s ‘‘ dephlogisticated
air ’’ was a gas present in ordinary air and forming about one-fifth of
its volume. When metals were heated in air they became heavier, not
because they lost the light spirit of phlogiston which had been driven
away by the heat, but because they combined with this oxygen. And he
proved also that the so-called ‘‘ fixed air ’’ was carbon dioxide, a com-
pound of carbon and oxygen. The burning of a candle consisted, then,
in the combination of the carbon of the candle with the oxygen of the
air to form carbon dioxide, and in this process heat. was liberated. Since
carbon, or phosphorus, or sulphur burning in this air formed acids, he
called the new or dephlogisticated air, the acid-maker, ‘‘ oxygen ’”’
(Gr. oxys, sour or sharp; gennao, I produce). He then proceeded to find
if animals affected air in the same way as burning candles. He found
that they did. In 1777 he published the observation that animals placed
under a jar until dead changed a portion of the oxygen of the air to
carbon dioxide. To re-establish the air so that it would support life,
it was necessary to absorb the carbon dioxide and restore an equal part
of oxygen. He says: ‘‘ If one augments, or if one diminishes, in a given
quantity of air, the quantity of eminently respirable air (oxygen) that
it contains, one augments or one diminishes in the same proportion the
ANIMAL HEAT 271

quantity of metal that one can calcine in it and up to a certain point
the time that an animal can live in it.’’ Having thus clearly demon-
strated that both burning candles and living mice took oxygen out of
the air and added carbon dioxide to it, he goes on to say, in regard to the
place in the body where the conversion of oxygen to carbon dioxide takes
place, ‘‘ I have been forced to two conclusions equally probable and be-
tween which my experiments do not permit me to decide. Wither the
portion of air eminently respirable (oxygen) contained in atmospheric
air is converted into (carbon dioxide) fixed air in passing through the
lungs; or it makés an exchange in this organ, on the one hand the emi-
nently respirable air is absorbed, and on the other hand the lung returns
for it a portion of carbonic acid almost equal to it in volume.”’

Having thus shown the identity of the chemical changes produced by
combustion and respiration, he at once inferred that, since a candle lib-
erates heat when it burns and animals also produce heat, this animal
heat probably came from the combustion of the body. In his Memoir
to the French Royal Academy in 1777 he says: ‘‘ I have shown that the
pure air after having entered the lungs comes out in part in the state of
fixed air (carbonic acid). The pure air in passing the lungs undergoes
a decomposition analogous to that which takes place in the combustion of
coal. But in the combustion of coal there is a disengagement of heat
(materie de feu), of which there should be a similar disengagement in
the lungs in the interval of inspiration and expiration, and it is this heat,
without doubt, which distributed by the blood throughout the animal _
economy gives rise to the constant temperature of about 32.5° Réaumur.
This idea appears perhaps hazardous at first glance, but before rejecting
or condemning it, I ask that it be considered that it is founded on two
constant and incontestable facts: namely, on the decomposition of air
in the lungs, and on the liberation of the matter of fire (heat) which
accompanies all decomposition of pure air, that is to say, all passage of
pure air to the state of fixed air.’”’ As further proof of this hypothesis
he points to the fact that those animals which are warmest, such as the
birds, produce the most carbon dioxide in a given time in proportion to
their weight.

In order to establish this revolutionary and beautiful hypothesis that
animal heat was due to the combustion of the carbon of the body, he
attempted to measure the amount of heat produced by an animal and
the amount of carbon burned and thus see whether the combustion of this
amount of carbon would account for the heat produced. It was neees-
sary for him to perfect first a means of measuring heat, so with the aid
of his great compatriot Laplace,.one of the greatest: of French physicists,
he perfected the ice calorimeter which had been made by Black. This
calorimeter corisisted of a double-walled can.: as shown in: Figure 34.
272 PHYSIOLOGICAL CHEMISTRY

In the space between the walls cracked ice was placed. This prevented
any heat from reaching the interior of the can from the outside. In
the interior there was placed a cage which would hold a guinea pig, a
rabbit or other small animal, and this cage was packed in ice. The heat
given off by the body of the animal melted some of the ice in the interior
compartment and the water from the melted ice was collected and
weighed. Since the melting of each gram of ice requires an amount
of heat sufficient to raise the temperature of a gram of water from 0°
to 80°, and the amount of heat necessary to raise the temperature of

 

 

Fig. 34.--Ice calorimeter of Lavoisier and Laplace. The animal was placed in the
eage, A, and the heat of its body melted ice in B, the water being collected in @ and
weighed. The outer layer of jce in O prevented heat from entering.

water one degree was taken as the unit amount of heat and called one
calorie, to melt a gram of ice requires 80 calories of heat. Of course,
arrangements had to be made for the ingo and outgo of air for respira-
tion. Before or after being in the calorimeter the guinea pig was placed
m another apparatus in which the carbon dioxide exhaled could be meas-
ured. The result of this experiment was as follows when translated into
modern units: In 10 hours the guinea pig burned 3.33 grams of carbon,
computed from the carbon dioxide exhaled. The heat liberated by burn-
ing this amount of carbon Lavoisier and Laplace determined to be suffi-
cient to melt 326 grams of ice to water at 0°. The guinea pig when
placed in the calorimeter for a given time liberated sufficient heat to
melt in 10 hours 402 grams of ice. This should be a little larger than
the heat generated by the combustion of the carbon, for the pig was at
a lower temperature when emerging from the calorimeter than on enter-
ANIMAL HEAT 273

ing it. Some of the preformed body heat had been lost. Estimating
that the heat thus lost from the body would melt 61 grams of ice, there
remain 341 grams of ice melted, as compared with the 326 grams which
should have been melted by the combustion of the 3.33 grams of carbon
of the body. Thus he arrived at the conclusion that at least 96 per cent.
of the heat of the body was to be accounted for by the combustion of the
carbon. The experiment may be tabulated as follows:

Amount of carbon burned by a guinea pig in 10 hours, 3.33 grams.

Heat liberated by burning this amount of carbon melts 326.5 grams of ice.

‘Pig in calorimeter for 10 hours melted 402.27 grams ice.

Reduction of temperature of body accounts for 61.19 grams ice.

Heat produced by guinea pig in 10 hours hence melted 341.08 grams ice.

Heat produced by burning carbon in per cent. of total = 326/341 — 96 per cent.

Having thus proved that animal heat was due, in large part, to the
union, or combustion, of carbon and oxygen, he proceeded to find out
if the oxygen combined with anything else than the carbon. Measuring
the amount of oxygen consumed and the oxygen in the carbon dioxide,
he found that only 81 per cent. of the oxygen combined with. the carbon;
the other 19 per cent. must combine with something else, namely hydro-
gen, to form water. If the figures just given were corrected by the addi-
tion of the heat formed by the combustion of the hydrogen, there was a
slight excess of heat computed over that found. There was also a source
of error in the experiment. He had measured the heat produced when
the pig was exposed to a temperature of 0°, but the carbon dioxide pro-
duced while the pig was exposed to 18° C. Perhaps the body burned more
carbon and produced more heat when it was cold than it did at room
temperature. This thought led him to other fundamental discoveries.
He undertook to determine whether more oxygen was consumed at low
cemperatures than at high ; and whether more oxygen was consumed and
more heat liberated when work was done. His experiments in this direc-
tion were never published in full, owing to the interruption in the pub-
lication of the Academy Memoirs caused by the French revolution, and
to the death of Lavoisier on the guillotine in 1794, but an abstract
had been published. These conclusions are fundamental for the science
of nutrition. They are as follows:

1. A man in repose and fasting at an external temperature of 32.5°
R. consumed per hour 24.002 liters O,.

2. A man in repose and fasting at an external temperature of 15° R.
consumed per hour 26.66 liters oxygen.

3. A man during digestion consumed per hour 37.689 liters oxygen.

4. A man fasting. doing work necessary to raise in 15 minutes a
weight of 7.343 kilos to a height of 199.776 meters, consumed per hou:
63.477 liters oxygen.
274 PHYSIOLOGICAL CHEMISTRY

5. A man during digestion doing a similar amount of work con-
sumed per hour 91.248 liters oxygen.

Here we have the fundamental observations that the consumption of
oxygen and exeretion of carbon dioxide are increased by cold, diminished
by warmth; and increased by work and by digestion. Furthermore,
the quantitative data are given for man himself.

Lavoisier sums up his work in the following words: ‘‘ Respiration is
only a slow combustion of carbon and hydrogen, which is similar in all
respects to that which takes place in a lamp or lighted candle; and from
this point of view the animals which respire are truly combustible bodies
which burn and consume themselves.’’ ‘‘ In respiration, as in combus-
tion, it is the air which furnishes oxygen and heat, but since in respiration
it is the substance of the animal itself, it is the blood, which furnishes the
combustible, if the animals do not habitually repair by their aliment that
lost by respiration, the oil would be lacking just as in a lamp; the animal
would perish like a lamp extinguished when it lacks nourishment. The
proof of this identity of respiration and combustion is deduced imme-
diately from experiment.’’ ‘‘ In concluding these reflections upon the
results which have preceded, one sees that the animal machine is princi-
pally governed by three regulatory principles: respiration, which con-
sumes hydrogen and carbon and furnishes heat; transpiration, which
augments or diminishes, following the necessity of getting rid of more or
less heat; and digestion, which returns to the blood that which has been
lost by transpiration and respiration.’’

I have dwelt so long on the work of this great scientist because the
discoveries he made and expressed in such clear and beautiful language
are the fundamental discoveries of our science. Of that wonderful group
of brilliant Frenchmen who lived at the end of the eighteenth and the
beginning of the nineteenth century he was one of the greatest. He
terminates his last Memoir by a consolatory reflection wrung from him,
no doubt, by the distracted state of his country and his own threatening
future:

“* We will terminate this memoir by a consolatory reflection. It is
not indispensable, in order that one should merit well from humanity and
pay a tribute to his country, that he should be called to public functions
and aid in the organization and regeneration of empires. The physician,
also, in the silence of his study and laboratory, can exercise his patriotic
functions; he can hope by his work to diminish the mass of evils which
afflict the human species, to augment its happiness and well-being; and
can he contribute by new ways which he discovers to the prolongation of
some years, of some days even, of the average human life, he, also, may
aspire to the glorious title of benefactor of humanity.”’

Where in the body is the heat produced ?—The heat and the energy
ANIMAL HEAT 275

of the body were thus proved by Lavoisier to come from the combustion
of the materials of the body. We may now consider where this burning
takes place by which carbon dioxide and water are formed and heat
set free. Lavoisier left this question undecided. He did not know
whether the combustion took place in the lungs, or whether only an
exchange of gases, of oxygen for carbon dioxide, took place in the lungs,
but the combustion somewhere else. This question was settled by the
work of various men. Lagrange objected if the combustion took place
in the Jungs the amount of heat the body gives off is so great that if all
were liberated there it would injure the lung tissue. Spallanzani showed
that all kinds of animals, whether they had lungs or not, gave off carbon
dioxide and consumed oxygen and liberated heat. He took the lungs
out of a frog and found that the animal still breathed through the skin.
Edwards drew hydrogen gas through blood and found that the blood gave
off carbon dioxide and oxygen to the hydrogen, thus showing that it
already contained both of these gases before it reached the lungs. Finally
Magnus in 1838 received some blood under the receiver of an air pump
and demonstrated that there was a large amount of gas in blood and
that venous blood contained more CO, and less oxygen than arterial
blood. These facts showed that only an exchange between the gases of
the blood and the outside air took place in the lungs; the combustion
took place somewhere else. This conclusion was confirmed by measuring
the temperature of the blood going to the lungs and that of the blood
coming away. The blood coming away was cooler, not hotter: It should
have been warmer if the combustion took place in the lungs. On the
other hand, blood coming from the muscle was hotter, not cooler, than
that going to it. Bertholet showed that muscles became hotter during
contraction. Consequently by 1850 it was believed that the combustion
took place either in the blood of the capillaries in the organs of the body
or in the organs themselves. Finally by the work of Pfliiger and Hoppe-
Seyler, among others, it was shown in 1870 that the blood had little
power of combustion and that combustion took place in the tissues, and
indeed in the living matter of the tissues. The union of oxygen and
living matter, or combustion, takes place and carbon dioxide and heat
are produced, then, in the living cells themselves. The real respiration
occurs here. It is the living matter which is burning, not the food sub-
stances circulating in the blood.

Origin of the heat set free on combustion.— Where is the heat before
it appears as heat? Is it in the foods, or in the oxygen, and in what form
is it? This was long a puzzling question and in some ways it still is
puzzling. It may be answered tentatively and in part as follows: Both
carbon and hydrogen have a great attraction for oxygen. For some
reason, not at present understood, an atom of oxygen and an atom of
276 PHYSIOLOGICAL CHEMISTRY

carbon in certain conditions attract each other so strongly that to sepa-
rate them much work is required. A similar attraction exists, also,
between hydrogen and oxygen. Why the union between hydrogen and
oxygen should be so much firmer than between other elements is not yet
clear; but there is no doubt about the fact. When, therefore, they are
separated, energy is consumed; and this energy is represented by the
separate positions or distance apart of the atoms. It is energy of posi-
tion or potential energy. It is supposed to be a condition of strain in the
ether which fills all space. Light, working in the chlorophyll parts of
plants, is able to bring this separation to pass. The light energy disap-
pears and is represented by the potential energy of the system carbv-
hydrate-oxygen. Now for some reason carbohydrate, which contains both
carbon and hydrogen, does not combine readily with oxygen in spite
of the great attraction between the oxygen and carbon atoms. It is
as if there was some resistance in the way of their union; but under the
conditions prevailing in protoplasm this resistance, whatever its nature,
disappears, and now the carbon and oxygen atoms rush together with
great violence, drawn by their mutual attraction. In the violence of
their impact they rebound and vibrate vigorously back and forth. Some-
times this vibration is so fast and so vigorous as to give rise to light.
This happens in the phosphorescent substances; in other cases the vibra-
tion is communicated to the surrounding molecules and is gradually dis-
sipated in longer molecular vibrations, and this we call heat.

Comparison of the amount of heat liberated with the amount cal-
culated from the oxidation of the carbon and hydrogen. The conserva-
tion of energy.—Having shown that the heat and energy of the body
come from the oxidation of the substances of the body and chiefly the
carbon and hydrogen, for as we shall see nitrogen leaves the body in
an unoxidized form, chiefly as urea, CO(NH,),, we may now consider the
no less important question: Is all the energy of the body due to oxida-
tion? Has the body no other source of energy? Is there an exact balance
of the energy set free by the combustion in the body and the amount
given off from it? Energy may leave the body as heat, or be rendered
latent, that is changed to potential energy, in the evaporation of water
or in the doing of work. Does the law of conservation of energy hold in
living things? Has man no other source of energy than his foods and
oxygen? For there might be some kind of radiant energy penetrating
the universe which is neither light nor heat, nor X-rays, but of a period
of vibration of such a character that it might be absorbed by man’s body
and transformed into intellectual or physical work. Has man any such
source of energy? This, as will be seen, is a very important question
and one which has, perhaps, not yet been absolutely settled.

If there were a kind of ray of radiant energy which could freely
ANIMAL HEAT 277

penetrate the bones of the skull and surrounding tissues, so that these
were transparent to it, but which was absorbed by some of the con-
stituents of the brain cells so as to produce metabolic changes in them,
it might be possible to affect the brains of men, or portions of the brains,
or other organs, directly without the interposition of matter. It might
in this way be possible, if the wave length was such as to influence only
certain specific parts of the brain, to set going processes which might
result in definite ideas in trained minds. There is hardly any question
that each substance has its own absorption spectrum, although that spec-
trum may lie in the ultra-violet or involve wave lengths far too short
for the substances of the retina to perceive. There would seem to be
no theoretical objection to the possibility of such an absorption of radiant
energy on the part of the brain or other tissues of the body; but there
is as yet no evidence for such a source of energy.

Work of Dulong and Depretz.—Lavoisier, in the rough experiments
which had sufficed to lead him-to such correct and brilliant conclusions,
had not been able to strike an exact balance between the income and outgo
of energy. He measured as heat about 104 per cent. of that calculated
from the combustion of the carbon; if he added to the calculated amount
the amount of heat computed from the combustion of the hydrogen, then
he found too little. The result was not satisfactory when left in this
form. Accordingly the French Academy offered a prize for an investi-
gation of this matter. Two investigations were undertaken, one by
Depretz and the other by Dulong. Both of these observers improved on
Lavoisier’s methods in that they measured the heat produced and the
carbon and hydrogen burned at the same time, and the animal was kept
at-or near room temperature. They weighed the carbon dioxide and
water given off while the animal was in the calorimeter. They made use
of. a number of small animals, dogs, rabbits, birds, ete. They made what
has since been called a respiration calorimeter, the heat being absorbed by
water instead of ice. Figure 35 shows Dulong’s calorimeter. The results
obtained by these observers are summed in the table after correcting
their results for more modern values for the heat of combustion of car-
bon and hydrogen.

Dulong: Animals used: dog, rabbit, pigeon, cat, fowl. For every 100
calories registered by the calorimeter that calculated from the respiration
accounted for:

Minimum .. 22... eee cence eee cere cee tenet e eee eee e eens 79.2 cals.

Mai a seiisas sce co Scteesn setae an a vent cca vonhatig lg (G-series 6 wea 99.4 “

Meat: ois. ccusenaeddesns tae Ook Hae ees Ook Bia ker aa AS 90.6
Depretz:

Mini Mini 4 .dcceedatenncine sk sei e eRe ae ew ae ts ini wa as 84.2 “

Mea shinnta tn «hc. dices beac eicn dd radon os sScavarr ois elena ROG aca Rio he w ae uae 1018 “

IMIG@A TN, arr eaeessteageent, aecet eek aes calla ce sta landu-o doevane Roaatneee ia dee ree eect eee 92.3 *
278 PHYSIOLOGICAL CHEMISTRY

As will be seen, they were unable to get a complete agreement between
the heat calculated from the products of respiration and that measured,
but they came sufficiently close to show that certainly 92 per cent. of the
heat of the body was due to the combustion. These observers made one
observation which turned out to be of great importance. They found
that in dogs the volume of carbon dioxide given off was a smaller pro

 
          

PUTT TTP TT

uy

 

 

Fig. 35.—Respiration calorimeter of Dulong. Tbe air entered at D, and after passing
through the coil, S, passed out at D’. The can containing the rabbit is immersed ino
the water of the calorimeter, as shown.

portion of the volume of the oxygen consumed than in rabbits and fowls.
They suggested that this might be due to a difference in the character
of the food. The ratio of the volume of carbon dioxide exhaled to that
of the oxygen consumed (c.c. of CO, exhaled = c.c. O, consumed) is
called the respiratory quotient. This suggestion of Dulong’s has proved
to be correct and has contributed much to the determination of the
character of the substance burning in the body.

Respiratory quotient. Regnault and Reiset.—Dulong’s suggestion,
that the respiratory quotient was dependent on the character of the food,
was substantiated by Regnault and Reiset in what is one of the funda
mental investigations in nutrition. They made use of the apparatus
in Figure 36, which is essentially a closed respiration system in which
oxygen consumption is measured directly. They measured more accu-
rately the ratio of carbon dioxide produced to the oxygen consumed,
and the relation of the respiratory activity to the size of the animal.
ANIMAL HEAT 279

They made two fundamental discoveries. First, that as required by the
theory of Lavoisier, those animals of the smallest size, and consequently
of the largest surface in proportion to their weight so that the radiation
of heat was a maximum, consumed more oxygen per gram per hour and
gave off relatively more carbon dioxide than larger animals of the same

 

 

 

Fig. 36.—Respiration apparatus of Regnault and Reiset. The oxygen was supplied
as consumed from the containers N, WN’ and N” and the COeg was absorbed by the absorption
bulbs, C and CO’, which were alternately moved up and down. The system was closed.

kind. This observation of the relation of respiration to surface was
extended by Rubner many years later to the heat production. Smaller
animals produce more heat per gram than larger. Second, they showed
that there was a relationship between the amount of the oxygen con-
sumed and the amount of carbon dioxide exhaled and the character of
the food. They thus discovered what is now known as the dependence
of the respiratory quotient on diet, a possibility suggested by Dulong.

Regnault and Reiset. CO,/O, ratio and its dependence on diet:

Animal Diet €.¢,CO2/c.c.0,
Rabbit .............. Carrots and vegetables ................ 0.92
DOR os tesa GaN a2 Bread: 2 ccseury cx ceeniia ysacaene ait agers oe ae 0.93
Lean: Meat: iss sch sae oes has a 0.74
Hat: MGAb: iced sacesuwiek Aw Deets vracacece 0.69
Fowl) 2c2ccu co deies Grains .... 0.93
: Bread .... 0.97
Meat 0.68

 

Those foods consisting chiefly of carbohydrate gave a high respiratory
quotient ; those of protein were intermediate; and foods containing much
fat produced the lowest quotient.
280 PHYSIOLOGICAL CHEMISTRY

‘Cause of the variation of the respiratory quotient with diet—The
reason why the respiratory quotient varies with the nature of the mate-
rials being oxidized in the body is the following: Carbohydrates, having
the general formula C,H,,0,, already have in their molecules sufficient
oxygen to oxidize all of the hydrogen to water. This leaves only the
carbon to be oxidized by the oxygen consumed in respiration. Each mole-
cule of oxygen consumed combines then with an atom of carbon to make
one molecule of carbon dioxide. Since both carbon dioxide and oxygen
are gases, they follow Avogadro’s law, according to which the same num.
ber of molecules of every gas occupies the same space at the same tem.
perature and pressure. Accordingly the volume of the carbon dioxide
exhaled equals the volume of oxygen inhaled and the respiratory quotient
for a carbohydrate is unity.

C.H..O —6H,0 ——s Cc, g 6C + 60, —-= 6CO, 3 vol. 6CO, /vol. of 60, Ses

6 12 6
There is not sufficient oxygen in the molecule of fats to oxidize thy
hydrogen of the molecule, consequently a portion of the oxygen consumed
will not reappear in the form of carbon dioxide, but in that of water.
The respiratory quotient will hence be less than unity when fat is burn-
ing in the body.

i Ce 0 0. 6 e.

Tristearine.
2. C ston + 81.5 0, == 57CO, + 49H,0

3. Vol. of 57CO _/vl. 81.5 0, 07

In burning tristearine, then, only 57 volumes of carbon dioxide are
exhaled for each 81.5 volumes of oxygen consumed, the respiratory quo
tient when fat is burning is then 57/81.5 or about 0.7. For triolein
it would be a little higher than this, since there are six less hydroger:
atoms in the molecule.

For protein the respiratory quotient lies between the two, since the
protein molecule contains a larger proportion of oxygen than the fats
and less than the carbohydrates. For a protein the ratio CO,/0O, is
about 0.8.

In the preceding computations it has been assumed that the oxidation
is complete to carbon dioxide and water. In no case, however, does the
oxidation in the body go completely to carbon dioxwde and water. Small
fragments of the molecules partly oxidized are to be found in the urine.
This error is practically negligible in the case of the carbohydrates. In
round numbers the respiratory quotient of a carbohydrate is taken as.
unity; of a fat as .71 and of a protein as .8. If the respiratory quotient.
is high, carbohydrate is supposed to be oxidizing in the body. If it is low,
fat is burning. It is clear that in anaérobic respiration, and all tissues
may have some anaérobic respiration for a short time, carbon dioxide is
liberated, although no atmospheric oxygen is consumed. For short
ANIMAL HEAT 281

periods, therefore, the amount of carbon dioxide exhaled may be greater
than that of oxygen inhaled, giving a respiratory quotient greater than
unity. For other short periods oxygen may be stored, that is combined
without the production of an equivalent part of carbon dioxide, the oxi-
dation not being complete. In experiments extending over 24 hours
or several days, however, these irregularities more or less completely
neutralize each other, since in the long run the oxygen of the CO, comes

 

Fie. 37.—Rubner’s respiration calorimeter for animals.

directly or indirectly from the oxygen of the air, at least in the higher
animals.

Proof of the conservation of energy in the body. Rubner.—It was
still uncertain from these determinations whether the income and outgo
of energy of the body could be balanced. It was necessary, if a complete
energy balance was to be had, to measure not only the energy of the
food taken, but that still left in the feces and urine. The final proof
that such a balance exists and that the law of conservation of energy
holds for the highest animals was not given until 1892. It was the work
of the German physiologist Rubner.

In 1892 Rubner had constructed for him a very accurate respiration
calorimeter. Figure 37. The calorimeter was kept as nearly as possible
at a constant temperature and at the temperature of the room; and the
heat produced by an animal in the calorimeter raised the temperature of
the air passing through the apparatus and could thus be measured.
282 PHYSIOLOGICAL CHEMISTRY

The calorimeter was large enough to receive a dog and permitted the
simultaneous examinution of the water and carbon dioxide produced.
The urine and feces were collected and examined for the amount of
potential or unconsumed energy they contained, and the experiments
were continued cach time for several days. The amount of potential
energy contained in the food was carefully determined. With this
apparatus Rubner was able to get the results a few of which are shown
in the table.’

COMPARISON OF THE Heat ACTUALLY PRODUCED WITH THAT COMPUTED FROM TH

METABOLISM.
Heat calculated Heat directly
Diet Number of from metabolism determined Difference- -

days Cals. Cals. per cent.
St ti 6 1296.3 1305.2 —1.42

arvation ...... 2 1091.2 1056.6
Fat ....... ese 5 1510.1 1498.3 —0.97

8 2492.4 2488.0

Meek and deb, a9 { 12 3985.4 3958.4
Meath xcs cece y 6 2249.8 2276.9 —0.42
} 7 4780.8 4769.3 + 0.43

The amount of heat given off under varying conditions of fasting and
different diets was found to be very close to the amount of heat calcu-
lated as set free by the combustion of the foods. The maximum varia-
tion was some 1.5 per cent. from the calculated and in some cases the
two values agreed almost exactly. By these experiments, then, Lavoi-
sier’s theory of the source of animal energy and heat was demonstrated
to be true. Instead of 92 per cent. of the calculated energy being found,
with the improvement in method and data, 99 per cent. of the energy of
the body was accounted for and the other 1 per cent. lay within the limits
of error of the method.

From these experiments Rubner established the fundamental law of
animal thermodynamics: Energy is neither created nor destroyed in the
animal body. The energy appearing as free energy is exactly equal tu
the energy of the materials burned. Conservation of energy is as true
within the animal body as elsewhere in nature. The animal behaves in
all respects like a machine. But the error of 1 per cent. was still tou
large. It was desired to test the law on man himself and, moreover, tu
carry out on him experiments in nutrition. The ultimate aim of all
physiological inquiry is to enlarge man’s knowledge of himself and to
enable him to control the living world. It might still have been true
that man had some unknown source of energy different from other ani-
mals, although this was unlikely. For the purpose of studying the nutri-
tion of man, calorimeters had to be constructed which should be large
enough to accommodate a man. The best of these calorimeters was first
constructed in this country in Middletown (Conn.) in the laboratories
of Wesleyan University. In the computations which follow the calories
referred to are always kiolgram or large calories.

1 Lusk: Science of Nutrition. 2nd edition, p. 42.
ANIMAL WEAT 283

The Atwater-Rosa-Benedict calorimeter.—The following descrip-
tion of the Atwater-Rosa-Benedict calorimeter is taken from the report
of Benedict and Milner. Figure 38. ‘‘ The respiration apparatus is in
principle essentially the same as that of Regnault and Reiset, that is a
so-called closed circuit apparatus. The same current of air is kept in
circulation through the chamber, the carbon dioxide and water vapor
imparted to it by the subject being removed as it is withdrawn from the

 

Ms |

 

     

RESPIRATION CHAMBER

 

 

 

 

 

 

 

    
 
 

 

 

 

 

 

 
 

   

 

 

SODA Lime * SOOA UME
ABSORBS
f CARBON DIOXID CARBON DOXID I
i
Es 7 7 i me
Fo supmunic 2ciD, sagen 410
FROM SODA UME acts la

 

 

 

 

 

Fig. 38.—Diagram of Atwater- Rosa-Benedict respiration calorimeter for human beings
(Benedict and Milner).

chamber and oxygen restored to it as it is returned to the chamber.”’
The quantities of carbon dioxide and water removed from the air and
of oxygen supplied to it are ascertained as follows: The carbon dioxide
is absorbed by the soda lime tubes C and B in the figure, which are
weighed on an accurate balance: the water is absorbed by the sulphuric
acid absorber just before the soda lime tubes; the amount of oxygen is
determined by weighing the bomb containing compressed oxygen before
and after the period of observation. Of course, corrections have to be
made for the water condensed in the chamber about the water pipes
which carry away the heat; and for the composition of the air left in
the chamber at the end of the experiment. The air is caused to cirecu-

1 Benedict and Milner: Experiments on the Metabolism of Matter and Energy
in the Human Body, 1903-1904. U.S. Dept. Agriculture, Bulletin 175.
284 PHYSIOLOGICAL CHEMISTRY

late by the rotary blower which goes at such a speed that 75 liters of air
a minute are drawn from the chamber. To provide for the expansion or
contraction of the air due to changes of barometric pressure, or of slight
changes of temperature in the apparatus, the pan G. which is provided

 

 

 

 

 

 

 

 

 

a a
£ £
Lia f
{
Lf f
Cc e
d d d

 

Fie. 89.—Vertical cross section of the calorimeter showing wooden outer walls and’
zinc and copper inner walls and air spaces (Atwater and Benedict).

with a rubber disk, which rises or falls as the pressure is greater or less
than that in the outer air, is put into the circuit.

The calorimeter.—‘‘ The device employed in these investigations
combines a respiratory apparatus and a calorimeter in the same con-
struction, and the determination of the heat output of the subject is made
concurrently with the measurements of the respiratory products and
oxygen. Figure 39. The apparatus is so devised and manipulated that
the passage of heat through the walls of the chamber is prevented, and
the heat evolved by the subject cannot escape in any other way than
that provided for carrying it out and measuring it. A small quantity
ANIMAL HEAT 285

leaves the chamber as latent heat of water vapor, but the major portion
is sensible heat absorbed by a current of cold water passing through a
coil of pipe within the chamber.’’ ‘‘ The principle of construction of
the calorimeter as a whole is much like that of an ordinary refrigerator,
namely, a chamber surrounded by a series of confined air spaces. The
walls, ceiling and floor of the calorimeter chamber, which is of course
the respiration chamber, are copper. On all sides of the chamber the
copper is attached to a wooden framework, to the outside of which is
fastened a zinc shell concentric with that of the copper, a dead air space,
about three inches across, separating the two metal shells. About three
inches outside of the zinc is a concentric shell of wood, and an equal
distance from this is the outer wooden structure. The space between the
zine and the inner wooden shell is the ‘inner air space,’ and that
between the two wooden shells is the ‘ outer air space,’ both of which
are thus designated hereafter. The wooden casing surrounding the
chamber on all sides, and especially the air spaces with the devices for
heating and cooling them, afford means for controlling the temperature
of the zine wall and protecting it against fluctuations in temperature of
the air surrounding the outer wooden casing.’’

Gain or loss of heat through the metal walls of the chamber is pre-
vented'by keeping the zinc wall at the same temperature as the copper,
‘in which case there will be no exchange of heat between them. For this
purpose provision is made for heating or cooling the inner air space,
and thus heating or cooling the zinc. The heating is accomplished by
passing a current of electricity through a German silver resistance wire
installed in the space, the amount of heat being controlled by a rheostat
on the observer’s table. For cooling, a current of water is passed through
a small brass pipe in the same space.

Similar provision is made for heating or cooling the outer air space
to aid in protection against changes in temperature of the air of the labo-
ratory. ‘‘ The attempt is made, of course, to keep the temperature of the
laboratory as near as possible to that of the interior of the calorimeter.
In order to know whether to heat or cool the zine wall, it is necessary
to know whether it is hotter or colder than the copper. For this pur- »
pose thermo-electric elements of iron and German silver wire are
installed between the two metal walls in such a way that one end of
each element is in thermal contact with the copper and the other with the
zine wall, and are connected with a delicate galvanometer on the observ-
ers’ table. The difference between the temperature of the copper wall
at one end of the elements and that of the zinc wall at the other end is
indicated by the deflections on the galvanometer, one direction showing
that the zinc is cooler, the other that it is warmer than the copper. and
accordingly whether to heat or cool.”’
286 7 PHYSIOLOGICAL CHEMISTRY

The arrangements are made in such a way that it is possible to heat
the bottom, top or sides separately.
“« In order that the chamber will neither gain nor lose heat by changes

 

 

ip!
ture. The observer controlling the temperature sits at the table in front of the

 

—General view of, the respiration calorimeter in the laboratory. The calorimeter with its window open
he pic

Fie. 40.

stands on the right of t
calorimeter with eho galvanometer before him (Benedict and Milner).

 

in temperature of the air current, the temperature of the ingoing air is
kept the same as that of the air leaving the chamber. A system of thermal
junctions is installed with one end im the ingoing and the other in the
ANIMAL HEAT 287

outgoing air, and provision is made for heating or cooling the ingoing air
as indicated by the galvanometer deflections.’’

Removal of heat from the chamber. A portion of the heat generated
within the chamber is carried out as latent heat of water vapor in the
air current. The major portion, however, is removed by a current of

\

  
  

   
 

 

 

pas

BALANCE

   
 

 

op eh
0 SOSORDERS
r5 oS J

   
 
  
     
     

  

RESPIRATION

wa ‘
CHAMBER Me

PRESSURE
REGULATOR O

 

FI co, ansonptas. 1,
bof Aa

 

 

3 a “k= ditt

BAROMETER

DAYING

cold water which passes through a pipe extending around the chamber
near the ceiling and absorbs the heat. To increase the heat-absorbing
area a large number of copper disks are soldered along the pipe.

It has been stated that an effort is made to extract the heat just as
fast as it is produced, and thus maintain a comparatively constant tem-.
perature in the chamber. To this end it is necessary that the rate of
absorption may be accurately controlled. This is accomplished in three
ways: First, by increasing or diminishing the rate of flow of water
through the pipe; second, by raising or lowering the temperature of the}
288 PHYSIOLOGICAL CHEMISTRY

water entering the heat-absorber; and third, for the finer regulation,
exposure of the absorbing area to the heat may be increased or diminished
by raising or lowering the metal shields which partially surround the
pipe and disks. As the shields are raised they become filled with cold air
and act as insulators; when they are lowered a greater absorption area
is exposed.

Determination of the quantity of the heat evolved. ‘‘ The tempera-
ture of the air within the calorimeter is accurately determined by means
of resistance coils of copper wire placed in different parts of the chamber.
The variations in temperature shown are always small, generally amount-
ing to not over a few hundredths of a degree, since the rate of abstrac-
tion of heat may be regulated so closely in accordance with that at which
it is produced. The heat removed from the chamber as latent heat of
water vapor in the air current is computed from the weight of water
absorbed from the air and the factor for latent heat of vaporization.
According to the best available data, it requires 0.592 Calory (kilogram
ealory) to vaporize one gram of water. This factor is used in these
computations. The amount of heat removed from the chamber by the
cold water passing through the heat-absorbers is computed from the
amount of water that passes through the pipe and its rise in temperature
during its passage. The quantity of water passing through the absorber
is determined by weighing on a special device. The rise in temperature
is determined by observation of carefully calibrated mercury thermome-
ters whose bulbs are immersed, one in the ingoing and the other in the
outgoing water. These are read every two to four minutes, according
to the rate at which the temperature is changing. Corrections in readings
are made for the effect of pressure of water on the bulb of the ther-
mometer. In calculating the quantity of heat removed from the chamber
by the water current, the specific heat of water at 20° C. is taken as
unity, and the results are all expressed as calories at 20°.

‘* The sum of the two quantities of heat determined as just described
comprises practically that evolved within the chamber, though allowance
is made for certain small quantities involved in the change of tempera-
ture of the calorimeter, and in the passage of objects into or out of the
chamber through the food aperture at a temperature different from that
of the chamber.’’

Appointments of the respiration chamber. The respiration chamber
is 7 feet 6 inches long, 4 feet wide and 6 feet 6 inches high, the dimen-
sions being such as to allow a man to stand or lie down at full length,
or even to move about to a limited extent. In one end is an opening
through which a subject enters or leaves the chamber at the beginning
or end of an experiment. During an experiment this is closed by glass,
tightl” sealed in place and serves as a window, admitting ample light
ANIMAL HEAT 289

for reading and writing. In the opposite end of the chamber is a smaller
opening known as the food aperture, through which receptacles for food,
drink, excreta and other articles may be passed in the course of an
experiment. Within the chamber are a chair, a table and a bed, all of
metal and all of which may be folded and put aside when not in use.
For experiments in which muscular work is performed, a device by means
of which the amount of work done may be measured is provided. There
is a telephone for communication with persons on the outside. The
arrangement of the furniture, bed, shelving, etc., in the space available
is such as to make the subject comfortable during an experiment which
may continue for two weeks.

Testing the accuracy of the apparatus. To test the accuracy of the
calorimeter and the respiration apparatus alcohol is burned in the
calorimeter. The results of such a test are given in the following
table:

 

 

 

RESULT oF A TYPICAL ALCOHOL CHECK EXPERIMENT. .
in ees Alcohol Carbon Dioxide Water
se Burned Found |Required eco Found | Required Thee
urs, mip.}| Grams Grams | Grams Grams { Grams
Dee. 16-17 2 30) 50.22 86.02 87.30 98.54 58.47 | 58.16 | 100.53

5 15) 101.76 || 174.27 | 175.84 | 99.11 || 115.73 | 117.15 |. 98.79
11 25 | 212.59 || 369.63 | 369.53 | 100.03 || 248.24 | 246.20 | 100.83
2 05] 40.14 || 70.96 | 69.77 | 101.70 || 46.52 | 46.49 | 100.07

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Total 21 15 | 404.71 || 700.88 | 702.44 99.78 || 468.96 | 468.00 | 100.21
Date Oxygen consumed Heat
Found Required on Found Required Taheeey:
Grams Grams Calories Calories
Dee. 16-17 96.48 95.23 101.31 295.10 288.98 102.11
183.02 191.83 95.41 582.18 582.10 100.00
405.37 403.14 100.55 1223.20 1223.29 100.00
78.27 76.12 102.83 233.13 230.98 100.92
Total 763.14 766.32 99.59 2333.61 2325.35 100.36

 

 

 

 

 

 

What, then, has been the result obtained by this most accurate
calorimeter in answer to the question with which this chapter started -
namely, Does the amount of latent and active heat and external work
done equal the amount of heat which is calculated from the oxidation

_ , of the protein, carbohydrate and fat of the body and that of the food?
The following table taken from Benedict and Milner summarizes all the
experiments which have been tried up to the date of publication of the
bulletin by this calorimeter. It was believed that by taking a large
number of observations the accidental errors would very largely elimi-
nate each other. By the net income is meant the heat calculated from
the combustion of the foods and the material of the body, when there
has been a change of weight, less the heat of -ombustion of the feces and
‘290 PHYSIOLOGICAL CHEMISTRY

urine. By the net outgo is meant the total outgo, including the heat
measured by the calorimeter, the heat made latent by the evaporation of

water and the heat equivalent of the external work done.
ToraL INCOME AND OUTGO oF ENERGY.

 

 

 

 

 

 

 

 

Subjects and eae experiments. Data cane | Net outgo pumerence ae rate of net
Rest EXPERIMENTS Days | Calories | Calories Calories Per cent.
H.F. Carbohydrate 3 5,678 5,712 34 0.6
B. F. D. ee 3 6,441 6,683 242 3.7
A. L. L. m 4 10,458 | 10,301 157 15
Total, 5 rest experiments 10 22,577 22,696 119 0.5
Total, 22 previous rest
experiments 67 151,638 | 152,051 |. 413 0.3
Total all experiments 77 174,215 | 174,747 532 0.3
Work EXPERIMENTS " : ; mi 5
J.C. W. Fat diet 3 15,423 15,852 42y 2.7
J.C. W. Carbohydrate 3 16,378 16,304 74 0.5
BF.D. | @ 1 | 4,463] 4,565 102 2.2
A. L. L. ne 3 14,257 14,464 207 15
A. L. L. Fat diet 3 14,792 14,671 12] 0.8
A.LLL. “ “ severe work 1 7,185 7,137 48 0.7
Total 6 work expts. 14 | 72,4981 72,993 | 495 0.7
Total 23 previous expts. 76 346,167 | 345,641 526 0.2 -
Total all work experiments 90 418,665 | 418,634 31 0.0

 

 

 

 

 

 

The difference between the total Calories computed and those actu-
ally measured in the whole series of experiments, both those reported
here and those previously reported, amounted to only 501 Calories in
a total of 592,880 computed, or a difference of only 0.1 per cent. The
individual experiments show, to be sure, a larger deviation, particularly
when they are for short periods of a day or two, but this deviation is
certainly within the limits of error of the method and probably depends
on the use of factors not exactly correct for the computation of the poten-
tial energy of the foods used in the body; or they come from errors in
the. estimation of the amount of energy in the feces. It is difficult to
determine exactly the feces which correspond to the periods under
examination, when these periods are short.

It may be said, therefore, as the result of these and other experiments,
that the conclusion of Lavoisier is correct and that the combustion of the
materials of the body is the sole source of energy of the human body, as
far at least as our methods permit us at present to determine. The law
of ‘the conservation of energy. aUAS. to the human machine as well as
to: adhe inanimate world.

‘How much energy does the combustion of a gram of various foods yield

the body; do all foods serve equally well for the production of the heat and
energy of the body? Having shown that the source of the energy and heat is the
ANIMAL HEAT 291

oxidation of the materials of the tissues and that there is an equivalence between the
heat and work actually produced and that computed from the carbon dioxide and water
formed by the combustion, we may ask the farther question whether proteins, carbo-
hydrates and fats serve equally well as sources of energy. If we wish to do muscular
work, is the energy for that work obtained with equal ease from the fat, carbohy-
drate, or protein? Have the foods an isodynamic value as heat and energy producers?
This question may in its solution also throw light on the other question of whether
the energy of the muscles comes mainly from carbohydrates, or fats, or proteins.

This question is one which may be attacked and partially solved by the use of
the calorimeter, and in order that the method of attack may be understood I have
presented in some detail the results of a most carefully conducted experiment of this
kind reported by Benedict and Milner. The object of this experiment was to dis-
cover whether fats or carbohydrates serve best as sources of muscular energy. To
solve this question a man entered the calorimeter and, while on a diet in which most
of the energy was in the form of fat, he did a heavy day’s work by riding a sta-
tionary bicycle which was geared to a dynamo so that the amount of the work done
could be measured. A second period of observation followed of three days’ duration
during which he did as nearly as possible the same amount of work, but in this his
main source of energy was in the carbohydrate of the diet.

Many other interesting facts will appear also from this experiment, namely,
the amount of energy in the feces and urine, the proportion of the total energy con-
sumed which appears in the form of work done, in other words the efficiency of the
human machine, the volume of oxygen consumed per day by a man, the weight of
carbon dioxide and water given off. The methods of computation of the results are
no less interesting.

The subject after a preliminary digestion experiment of four days’ duration
entered the calorimeter at 10 p.m. The experiment in the calorimeter began at 7
the next morning and continued for three days. The object of the first experiment was
to test the value of fat as a source of energy as compared with carbohydrate. The
diet for this period was, therefore, a fat diet. He rode the bicycle for six hours
a day. The feces were marked off by taking charcoal before entering the chamber
and at the end of the period. The feces between the passage of the two charcoal
markers corresponded to the period under observation. The daily régime was as
follows: The subject was called at 7 a.m. He took his pulse and temperature, voided
his urine, and weighed himself. He then collected the water from the drip on the
heat-absorbers in dry, weighed bottles and weighed the absorbers to ascertain the
amount of water adherent to them. He had breakfast and began work at 8.15. The
food was carefully analyzed and weighed. All water that he drank was measured.
The feces were collected in a closed can. The perspiration in his clothing was
measured by weighing his underclothing when dry and after exercise. The nitrogen
secreted in the perspiration was also determined by examining the wash water from
the underelothing. It amounted to about 0.5 gram per day. The diet was very
simple and consisted of bread, ginger snaps, shredded wheat, butter, milk and cream
and an infusion of cereal coffee. ,

The diet contained approximately 110.9 grams of pzotein, 450 grams of carbo-
hydrate and 350 grams of fat. The total heat of combustion of the foods eaten was
on the average 5,550 Calories per day. Of this amount the total energy from
the fat, since this was butter fat, was 9.3 Calories per gram or a total of 3,100 to
3,350 Calories or about 60% of the total energy intake. These results more in detail
for the first day are given in the accompanying table.
PHYSIOLOGICAL CHEMISTRY

292

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Gp |8see |09'6 |so'se | Le9F| se Seal je tissetess|leierere nes 91928 | LLer'e | auLiIn [eJOL,
sel |stl jase joeet | ztat| oe Joc ypc) eerie | vese jc * Sep pe,
Ofl jess j9Te jssst| erst] Te Joc yc Pc} geese foese foccctt’ Sep pss
LPL 0693 | 36S [LSI | Leet] Tet} po SSSPI | ysorl | '7'** Aep 4st aul
60 [99°9 | 20% |Os'9s | OST | LG | SSt [Sst fetl | eter | 208s ae Bap oa
126 |G6'6L | Zosl joros | ess | g1s| GLP |Los isse | sels | els eons
66SS |61'963) 94°6L |OO'TeS| C6'8T| rec | asp | @osE]eeLL | LLLOE | vy6e'e | 11. a PE»
GPSS |F6'Z6S| 98'LL |SF'SIS| 898" | O'Ss | LerH | ESrE;Esli | Loses | vourg |. 11177 ABP PS»
SPSS |ET'068| 8S9L Fe 20¢| ¢h'SI| Lz] Ler | srEeleol | sees | cee Aep 381 +1030
H8bS |GP'LS | GB'3e |GL'EIz| Gee. | T'9 | sor |z'ses jose | sor | ozot| (1171771111! P40
68 |0F0F | OS'TI |us'e2 | 009 | og | Oss [OP JOLE | O'8g8 OOO rt Pe ALA
ep 4s_ :peyuewarddng
y
18S |hs'set| So'se |Ssstz] se | o2 | oece |ete [eer | citer] ogett| “°° Foret [eseq [e70L
oy josh (FS jeog | OI 96 VOU S68lL | OOSL| OT aayoo [vata
BLT letes | 26% |¢e'st cy Gp Pons ee ivang
I9T |eg9t | 9s% |gs9t | so | 9 cite |2 lee | ee op Jo yeaya pappolysg
sce |3r'rs | s9e [eres | s¢ rL | oop |e [Te |S 09) oti cts) sdeus sesurp
198 [1S | EL | L1'6s | 60° On jr s'sg {9° 0? Gp pore re: “ JeqQNg
OLZT |6eSIl| ss°ZT |29'ZsI| zr9 | oF | sase |o9 |s'9e | LOLI op ft ans prag
‘sep | ‘emg | sug | ‘sug | ‘say | -suy | ‘sug ‘sary | ‘surg ‘sup “su poo.
3 36 uaZ was sateipcy —
qwaH | wadfxo -orpA moqieg “ON sy oqaug qq [UIe}01g ae wae jeenuneise utes
aUBISqNs aatj IVA

 

 

 

 

 

‘HNTUQ) GNV SHORg ‘G00q dO NOILSOANOD dO LVAY ANY ‘NOILISOdNOD ‘LHDINM ATIVG IVLOL

It will be noticed by inspecting the last column of this table that of the

tained in the food, 300 Calories per day appeared in the feces and

on the average about 138 Calories in the urine, making all told about 8% of the

les con

5,550 Calori

rk
t

total food Calories which were not set free in the body but which were re-excreted.

About 92% of the heat of the food was utilized or was available to the body.

It will be remembered that Lavoisier found that the oxygen consumption of a
man doing work was about 90 liters per hour. The average in the table on page 293

for the whole day was about 41 liters per hour.

i found for a man at rest

isier

Lavo
but digesting about 37 liters per hour. The respiratory quotient of 0.815 is rather

h fat. It is due to the fact that there was a good

ining as muc

high for a diet conta
ANIMAL HEAT 293

deal of carbohydrate also present and the respiratory quotient when carbohydrates
are burned is very nearly 1. When fat alone is burned the whole respiratory
quotient should be 0.71.

WaTER AND CaRBON DIOXIDE GIVEN OFF AND OxyGEN CoNnSUMED. THER OUTGO OF
WaTER AND CARBON DIOXIDE AND THE CONSUMPTION OF OXYGEN.

 

 

 

 

Water of Carbon dioxide Carbon Oxygen consumed |
respiration d a y h eae ates
and perspira- | Grams | Liters | in CO, a Lene || Toten
tion exhaled | dx 509 | ax3/t1 | Stars x07
Ist day 3,702.27 1,775.61 | 903.96 484.25 | 1,545.24] 1,081.67] 0.836
2a “ 4,179.78 1,813.56 | 923.28 494.60 | 1,652.56] 1,156.79) 0.798
3d“ 3,907.14 1,697.41 | 864.15 462.91 1,517.80} 1,062.46] 0.813
Total 11,789.19 5,286.58 | 2691.39 | 1,441.76 | 4,715.60 | 3,300.92] 0.815

 

 

 

 

 

 

 

Amount of heat eliminated——The manner in which the heat is computed for
the various factors involved in the calorimeter is shown in Table C.

TABLE C
SUMMARY OF CALORIMETRIC MEASUREMENTS.

 

 

@ b ¢ c a e J
Heat Change of | Capacity orrection | Heat used Total

 

 

due to tem-
d | t - ot) b: '- | heat deter-
in terme of |ture of cal- of calorim- [PeT@ture Of] ation of | mined
20° cals orimeter | eter bx 60 aches water

: Calories Degrees Calories Calories Calories Calories
Ist day ...... Sgtgire esti at 4,594.50) — 0.01 — 0.60 | — 37.97) 739.17 | 5,295.10
20) oo sawie hones 4,657.59 | + .28 + 16.80 | — 42.33] 801.35 5,433.41
Oe ee casa rineeva ye vesetecasas 4,443.16} —.20 | — 12.00] —44.33| 737.05 | 5,123.88
Total « ccae sess Boifalaesever= 13,695.25) + .07 4.20 |— 124.63 | 2,277.57 |15,852.39

 

 

 

 

 

 

It will be seen from this table that by far the greater part of the heat given off
from the body is measured directly by the outgoing water of the heat-absorbers,
column a. The other principal measurement is that of the water vapor in the air
streaming from the calorimeter. From the amount of water in the air the amount
of heat used in vaporizing it may be computed. These figures are given in column e.
The total output of heat of the subject doing hard work was about 5,300 Calories
per day.

Comparison of the income and outgo of material and energy.--
“From the data given in the preceding tables the amounts of material and energy
received and given off by the body in different ways may be compared, and gain or
loss by the body estimated. Such a comparison is made in the tables which follow.”
“The computation of the amounts of materials gained or lost by the body are given
tor purposes of illustration in the data for the first day of the experiment.”

The difference between the total weight of the income and that of the outgo was
132.06 grams. Tt will be observed on comparing the total income of elements with the
total outgo that there was a gain of 1.69 grams of nitrogen, 20.80 grams of hydrogen,
and 125.75 grams of oxygen. and a loss of 14.68 grams of carbon and 1.50 grams of
ash. “In the lower section of the table are shown the amounts of protein, fat,
carbohydrates, and water gained or lost by the body on the first day of the experi-
ment as computed from the gains and losses of the elements. The gains or losses
 

 

 

 

 

 

 

 

 

 

 

294 PHYSIOLOGICAL CHEMISTRY
Tasre D.
Gains AND Losses oF Bopy Marertat. on First DAY oF METABOLISM EXPERIMENT 56.
alent Nitrogen | Carbon | Hydrogen | Oxygen Ash
a 6 ¢ a e tf
Income Grams Grams Grams Grams Grams Grams
Oxygen from air ....] 1,545.24] .......0.[eccceeeeefeweereeee 1,545.24 |.........
Water in drink ..... 5950.00 8) ocecd- 2.6: csse0 | pee ee crevasse 218.20 | 1,731.80 |.........
Water in food ...... 9935-504) scutes ecees: (| Sisenstersyoe 328.48 | 2,607.02 |.........
Solids in both ...... 914.50 18.45 507.94 76.28 290.13 21.70
Total | 7,345.24 18.45 507.94 622.96 | 6,174.19 21.70
Outgo
Water in feces ..... 191:30)| wsaasce ss ec aeestsie 21.41 168.89 |.........
Solids in feces ...... 48.40 1.80 26.80 4.07 6.63 9.10
Water in urine ..... 1,425.80] --++. --e[eeee. eee] 159.55 | 1,266.25 |,... ....
Solids in urine ..... 69.80 14.31 11.57 2.92 26.90 14.10
Water of respiration si
and perspiration ..) 3,702.27 65* [........ 7 414.21 | 3.287.41 |........ 7
CO, of respiration ..| 1,775.61] ---+----+ 484.25 |... 0.00. 1,291.36 |.........
Total 7,213.18 16.76 | 522.62 602.16 | 6,048.44 23.20
Gain (-+) or loss (—)) | 13996] 4.1.69 | —14.68] + 20.80 | + 125.75] — 1.50
Ash of protein gained 16
Gains and losses
of body material 41014] +£1.69 ee a 4223| 4.16
mee SOA BIN ES asnnipitgd 33.35| +5.17| 45.30 :
Glycogen TEE EET ET |= 120.17} cece ees — 53.36] —7.45 | — 59.36
WiGtor Se PAPO sicaurassn thes hse as + 22.37 | +4 177.56
Wale wdecics eon 450 fice renee dt semene pseeoewiesl eared — 1.50
4132.22; +1.69 | — .+.o.| + 20.80 | + 125.73] — 1.34

 

 

 

 

 

 

 

* Nitrogen of perspiration.
of these different compounds are computed in the following way by means of
formule derived from the following data regarding the elementary composition of

these compounds:

Protein .......
BBUG: sdancagesaes

TABLE E.
-ASSUMED PERCENTAGE OF COMPOSITION oF Bopy MatTERIALs.
Nitrogen Carbon Hydrogen Oxygen Ash
Per cent. Per cent. Per cent. Per cent. Per cent.
eau Silegatians 16.67 52.80 7.00 22.00 1.53
wqeeeiieneuers 76.10 11.80 12.10
re 44.40 6.20 49.40
11.19 88.81

Carbohydrates .

Water ........

In the above table the carbohydrate is assumed to have the composition of glyco-
gen. The ash of the protein may be disregarded and the following equations may be
derived from the above data, letting p= protein; t= fat; r— carbohydrates; and
w= water, and indicating the names of the elements by the initial letter, as is

customary :

0.1119 w + 0.0620 r + 0.1180 t + 0.0700
0.888] w + 0.4940 r + 0.1210 t + 0.2200

0.1667 p
0.4440 r + 0.7610 t + 0.5280 p
P
P
ANIMAL HEAT 295

“The solutions of these equations in terms of nitrogen, carbon, hydrogen and
oxygen give the following formule:
Protein = 6.0 N.
Fat — 0.005 C + 9.693 H — 1.221 0— 2.476N
Carbohydrates — 2.243 C — 16.613 H + 2.093 0 — 2.892 N
Water = 1.248 C + 7.920 H + 0.128 0 + 0.460 N

“Tf the quantity of each element gained or lost by the body as expressed in
grams in Table D is substituted in these equations for the corresponding sign of the
element and the indicated calculations are performed the results obtained will be the
quantities of the different compounds gained or lost. For example, according to
Table D the body gained 1.69 grams of nitrogen on the first day of the experiment.
This value would therefore be substituted for N in the first of the above equations
as follows:

Protein = 6 N= 6 (+ 1.69) = + 10.14.”

The gains and losses of protein, fat, glycogen, and water as thus computed from
the gains and losses of the elements for the first day of experiment 56 are shown
in the lower section of the first column of Table D.

The energy income and outgo in this experiment totaled as follows for the
three days:

Available energy from the foods ............... 15,354 calories
Energy by the combustion of body material lost 69 *¢
Total income of energy ............--..0e0eeee 15,423 ee

“

Total energy outgo, work and heat observed .... 15,852

Excess of heat measured ....................- 429° «

This was the first experiment with the respiration calorimeter in its revised form
and the difference of about 2.7% between the computed and measured heat is much
larger than in other experiments. In making these computations of the energy
equivalent of the materials of the body which had lost in weight the following factors
were used:

Heat of combustion of protein ..... 5.65 calories per gram.
See t fat: .esn2sae 954 “ Sor oes
os * “ carbohydrates 4.19 “ Be at

These are of course the heats as determined in the bomb calorimeter.’ The value
for the protein is that of fat-free muscular tissue from which the nitrogenous non-
proteid compounds have been removed.

Having thus illustrated the method by the detailed experiment just
quoted, the question may now be asked whether it is possible to with-

‘Tt must be noted that if we consider the fats and carbohydrates in the food
alone and not those in the body material catabolized since the absorbability is not
perfect: we do not use the figures given here, but rather the relative efficiency of the
foods are shown in the following table which expresses how many calories of energy
are actually available to the body for each gram of foodstuff eaten:

BEOteIN oes Garena uss aes Career gars Shane 4.0 cals. per gram.
Carbohydrate wscsiescess 5 saasiowe aks oes AQ GE SE ee
Fats: jo backs eguiees abound evans sige 8 anlar 3 BQ

ere ches ses Momnbnbe haute a
296 PHYSIOLOGICAL CHEMISTRY

draw the fat in the diet mentioned and replace it with an equivalent
amount of carbohydrate, equivalent that is in the amount of energy it
contains. For this purpose the subject after a few days’ interval
re-entered the calorimeter and for a period of three days did near!y
the same amount of work, but the cream was now withdrawn from his
diet and cane sugar containing the same amount of energy substituted
for it. On the fat diet the fat furnished 60 per cent. of the total energy ;
on the carbohydrate diet the carbohydrates furnished about 4,000
Calories or nearly 80 per cent. of the total. The total energy outgo was,
as before, 5,500 Calories per day. Of this amount the equivalent of
602 Calories was in the form of external work.

The respiratory quotient while on the carbohydrate diet was 0.92
on the average, on one day being as high as 0.946. Now if the carbo-
hydrate cannot be used as efficiently to produce energy as the fats, then
it would be necessary for the body to call upon its own tissues to supply
the energy, and proteins or fats should be catabolized. As a matter of
fact, the experiment showed that the body lost on the average per day
the following amounts of material : Protein, 1.12 grams; fat, 21.48 grams;
carbohydrates, 26.76 grams, and water was gained to the extent of 199.63
grams.

In the fat experiment there was a loss of body fat, but a gain of
protein and carbohydrate. The change was, however, small. From this
it might appear that the fats were slightly better sources of energy for
the body than the carbohydrates, but as a matter of fact these twa
substances are really nearly equivalent. The slight difference was due
to the energy output being somewhat greater in the second experiment.
From this experiment, then, the conclusion may be drawn that the body
can get its heat and the energy for its external work from either fats or
carbohydrates and that they replace each other in the energy balance of
the body approximately according to their calculated heat equivalents.
They are, in other words, tsodynamic.

We have now to consider the proteins. Are they also equivalent to
the others as energy-producers? Does it make no difference so far as
the body is concerned whether the energy is supplied from protein or
carbohydrate? It is at the outset clear that proteins and fats suffice for
the greater part of the food of many people, namely the Esquimaux.
Further, carnivorous animals generally have very little carbohydrate
in their diet, and yet they are capable of great exertion. Moreover, in
the calculations which have been made in the experiment cited, it is
assumed that the protein is oxidized and produces heat and energy in
proportion to its content. It is not, of course, possible to substitute
the proteins completely by fats or carbohydrates, since the latter con-
tain no nitrogen, but it is possible to supply the whole needs of the body
ANIMAL HEAT 297

for a time at least with protein and a small amount of fat or carbohy-
drate. The proteins can replace the latter as sources of energy as shown
by Rubner. 100 grams of fat are equivalent to 227 grams of protein
or carbohydrate in the food, according to Rubner; or 223 grams, accord-
ing to Atwater.

But, while the foods thus have an isodynamic action in this sense, they
are of course by no means equivalent physiologically. Each has a spe-
cific action in nutrition ; the proteins affect the production of heat directly
apparently by stimulating it; they are necessary to the formation of
many of the internal secretions, and they, or the foods which contain
them, may have a direct action on the nervous system. The digesti-
bility is different. There are also other substances in foods besides the
proteins, fats or carbohydrates which are of great importance, of vital
importance indeed, although present in small amounts. These will be
considered presently.

Growth.—Thus far the foods have been considered only as the source
of energy. That energy was shown to be necessary, first, to supply the
heat necessary to keep the temperature of the body high enough to per-
mit the chemical processes to occur with sufficient speed and to keep the
protoplasm in the necessary condition of viscosity; second, to furnish
the energy which is transformed in doing external and internal work,
for energy is consumed in moving the blood, in secreting the urine, as
well as in all the bodily movements; and third, to furnish the energy
used in the syntheses of specific materials in the body, for not. all the
chemical transformations in the body are exothermic decompositions ;
there are also synthetic, reducing, endothermic reactions by which some
heat is rendered latent and in virtue of which the specific materials of
the body are produced. But foods are needed not only because with
oxygen they supply energy ; they must also construct the machine. They
are the raw materials from which the body is made. It is probable.
although it is not certain, that much of the materials which are oxidized
in the body are built into the real living matter before they are oxidized.
In other words, it is the living matter which is burning and not the foods
as such. Foods may or may not contain energy, but they must have the
raw materials out of which the body is formed. They must, in othe
words, nourish the body. And it is from this point of view that we shall
now consider them.

Summary.—lIn this chapter we have considered the income and outgo
of the energy of the body considered as a whole. The body is warmer
than its surroundings. It is constantly giving off heat. The amount of
heat given off in the course of the day by a man at rest is about 2,500
large calories, and during working days it is greater than this. Energy
leaves the body not only as heat, but some is converted into potential
298 PHYSIOLOGICAL CHEMISTRY

energy of work done and some into the potential energy involved in the
separation of the water molecules to the form of a vapor. The source of
the heat of the body was long a puzzle. It was found by the French
chemist, Lavoisier, that it came from the combustion of the materials of
the body, and in particular from the combustion of the carbon and
hydrogen of the body by the oxygen of the air. Oxygen enters the body
by the lungs and combines with the carbon and hydrogen in the tissues
giving carbon dioxide and water. This combustion does not take place
in large measure in the lungs or in the blood, but chiefly in the tissues
and in the muscles in greater part, since these are the most bulky tissues
of the body.

The measurement of the heat given off is made by means of a respira-
tion calorimeter. The most perfect of these instruments has been con-
structed in this country. By this calorimeter it was shown that the
amount of heat given off in perceptible heat, or in other forms such as
latent heat of vaporization, is equivalent to the amount of heat set free
by the combustion of the carbohydrates, fat and protein burning in the
body. The source of animal heat is, hence, the combustion of these mate-
rials and the amount of heat is that calculated from the combustion. So
far as the measurements go, they indicate that there is a balance between
the income and outgo of energy and that the law of conservation of
energy holds for human beings as well as for other animals. The human
body does not have, so far as can be determined, any other source of
energy than the combustion of its materials. Respiration and heat pro.
duction are thus related.

The amount of heat produced and of carbon dioxide given off per
kilo body weight varies at different ages and is greater in children than
in adults. It is generally larger in small animals than in large
of the same kind. There is, hence, a more rapid respiration and heat
production in the young than in adults, and this is true even for the heat
production per unit of surface area of the body. In other words, the
metabolism of young is higher than that of older people.

Some light may be thrown on the nature of the materials burning
in the body by the study of the respiratory quotient. This is the ratio
between the volume of carbon dioxide exhaled and the volume of oxygen
consumed. The respiratory quotient is unity for the combustion of
carbohydrate, about 0.8 for proteins and 0.71 for fats. It may, for short
spaces of time, be larger than one, or smaller than .7.

By the use of the respiration calorimeter it has been found that the
carbohydrates and fats can very largely replace each other in the
metabolism of the body and that in the absence of much of either of these
the energy required may come from the proteins. The latter, however,
have also a direct action on the body in that they increase the heat pro-
ANIMAL HEAT 299

duction and have in this way a specific action different from the fats and
sugars. The explanation of this action is as yet obscure.

Finally, while we speak of the foods as oxidizing in the body and
while for practical purposes they may be treated as if they were under-
going oxidation in this way, it is more probable that it is not the foods
which are oxidizing, but the living matter; and that the foods only oxi-
dize as they are combined in the living protoplasm. When life ceases,
the oxidation of the food stops. It is impossible thus far to isolate from
dead protoplasm substances, or enzymes, which have the power of oxidiz-
ing the foods apart from living matter. The oxidation of the purine
bases is a possible exception to this statement. The question of the nature
of the combustion is considered more fully farther on. Foods must
above all nourish and reconstitute the ever wasting body.

REFERENCES. Anrmat Hear.

1. Black: Quoted from Gavarret: De la chaleur produite par les étres vivantes,
p- 163. Paris, 1855.
Lawoisier: Experiences sur la respiration des animaux. Histoire et Mémoires de
l’Acad. de Sci. Paris. 1777, p. 187; 1780.
Lavoisier: Sur la combustion en general. Ibid., 1777, p. 599.
Lavoisier et Laplace: Sur la chaleur. Ibid., 1780, p. 355.
Lavoisier: Ibid., 1780, p. 401. .
Lawoisier et Seguin: Premiére mémoire sur la respiration des animaux. Ibid.,
1789.
7. Dulong: Mémoire de Il’Institut, 1844, 18, p. 341; Annales de chimie et de
physique (3), I, 1841.
8. Deprete: Annales de chim. et de phys. (2), vol. 25, p. 337.
9, Regnault et Reiset: Annales de chim. et de phys. (3), 1849, vol. 26, p. 26.
10. Spallanzani: Mémoire sur la Respiration.
11. Rubner: Zeitschrift f. Biologie, 30, p. 73 (1894).
12. Laulanie: Archives de Physologie, 1898, p. 748.
13. Atwater and Benedict: Metabolism of Matter and Energy in the Human Body.
U. S. Dept. Agriculture, O. E. 8S. Bulletin No. 136, 1903.
14. Benedict and Milner: Ibid., Bulletin 175, 1907.
15. Benedict: Carnegie Institution Publications, Washington, No. 42.
16. Atwater: The nutritive value of alcohol. Mem. National Acad. of Science,
vol. 8, 1902.

np

So Sr ee
CHAPTER VII.
THE RAW MATERIALS OR FOODS.

Foods.—The body consists of water, various salts, of pro-
teins, carbohydrates and fats, and various other organic substances
which are formed from the latter in the course of their decom-
position in the body. These organic constituents of the body are
burning or oxidizing as long as life lasts, and it is their combustion
which gives rise to the heat of the body; to the energy appearing
as work done, or transformed into such other forms of energy
as chemical, or electrical. By this combustion of its substances simple
compounds are produced, such as carbon dioxide, water, urea and many
other substances which are constantly leaving the body in the excre-
tions, either through the skin, by the lungs, kidneys or alimentary canal.
The water leaving the body by the skin or kidneys carries out always
some of the salts with it in solution, so that the body is constantly losing
carbon, nitrogen, oxygen, hydrogen, salt and water. The body may be
likened to a lamp which as long as combustion continues continues to
give light and heat. So the body as long as it continues to burn, or, as
it is called in living things, to respire, continues to give off heat and
those various forms of energy found in living things. And just as the
lamp, if the supply of oil runs low, burns its wick, one of its constituent
parts, and is ultimately extinguished ; so in the body, if the supply of food
or raw material for combustion is exhausted, it burns in part its own
substance and then life is extinguished. But, while this analogy with
a lamp or candle is in many ways most apt, it must not be pushed too
far, for the body not only burns the fuel supplied it, but manufactures
or repairs the wasting of the machinery of the body. It is as if the
lamp were able to make new wick material from the oil so as to make
good the loss of wick material which occurs daily.

Food. Definition.—The term food has been variously defined. It
has been said to be a substance containing available potential energy,
that is energy which can be utilized by the body. But by this definition
some poisons are foods, since, like alcohol, morphine or caffeine, they are
partly oxidized in the body and thus setting free their energy contribute
to its work or its store of heat; and other substances such as water, salts
or oxygen which are absolutely necessary to the body are not foods, since

800
THE RAW MATERIALS OR FOODS 301

they carry no energy into the body. Obviously such a definition is of
very little value. A food has one characteristic, it has the power of
repairing waste, or contributing substance necessary for the growth
of the body. The body is not a stove which burns fuel. Foods do not
burn as they do in a stove, being contained in the material but not made
part of it. It is true that some oxidation of this kind may occur, but
not the combustion of the foods. It is the living matter which is burn-
ing, not the foods which are in it. Foods may or may not bring avail-
able energy into the body; but they all repair waste and provide the raw
materials for growth. All substances which have this power are foods;
those which lack it, although they may be burned in the body and set
free energy, are not foods. Thus water, salts, oxygen are true foods.
All things necessary for the construction of the living machine are foods.
Poisons, such as alcohol, caffeine, morphine or other substances which
may be oxidized in the body, but which are incapable of repairing waste,
—these are not foods. All substances which do not nourish living matter
and which are not chemically inert are harmful to it. So a poison may
be defined. as a substance, not a food, which acts chemically on the body.
Too much of one kind of food or too little of another may be harmful,
but such substances do not thereby become poisons. They are still foods.
On the other hand, poisons may change living matter in a manner to
benefit it temporarily, or restore it when it has been disturbed, but such
substances are none the less poisons, although they may be temporarily
used for valuable ends. No material has as yet been found which is able
to improve protoplasm so that it will live longer than it does under its
normal circumstances. All substances not foods shorten life when they
find entrance to protoplasm, provided they are taken in sufficient. amount
to affect it at all.

The food materials must contain, then, the materials out of which the
body is made, and we may at this time examine the constitution of vari-
ous foods as raw materials for the manufacture of the body.

Water.—Human beings normally require 3 to 5 liters of water per
day, although life is possible on less. Some of this is obtained in the
foods; fruit and many vegetables consist of 80 to 90 per cent. of water,
but most is taken in beverages of various kinds. Water that we drink
is never pure. It always contains salts of various kinds in solution
and these salts may have a physiological action. Thus hard and soft
waters may differ in their action on the bowels; in localities using hard
waters goiter is often endemic, though it is very doubtful whether the
hardness of the water due to the lime salts in it has anything to do with
the goiter. It is more probable that the cause of the goiter is an organic
substance or germ and an indirect result of a deficiency in iodine.
Thus in the region of the Great Lakes goiter is very common.
Nearly all the dogs in Chicago are more or less goiterous. In general,
302 PHYSIOLOGICAL CHEMISTRY

also, hard waters are constipating, soft relaxing. But this effect again
is not due to the water, but to the salts. Some waters, too, are radio-
active and at the present time there is a good deal of evidence indicating
that this cireumstance is of importance in the beneficial action of such
waters in many diseased states. How the radio-activity produces any
physiological action is still quite obscure. No one has as yet shown that
living matter is at all radio-active, so that we have no reason for thinking
that radium in minute traces may be a necessary part of our foods. Still
it is not impossible that this may be the case. Minute traces of substances
are frequently of extraordinary importance. Thus it was recently found
by Berthelot that manganese in extremely small amounts was necessary
for the development of the gonidia in Aspergillus. The amount of man-
ganese which was necessary was so small that it was very difficult to
provide water and containers and foods which did not have these minute
traces, so that growth was impossible. This fact makes one very cautious
in concluding that minute traces of substances which occur in living
matter may not have an influence on it. While the favorable action of
various naturally radio-active waters is to be ascribed in part to the
salubrity of the climate, to the more restricted diet generally enjoined on
.those who visit these watering-places for health, to enforced exercise,
to diminished intestinal putrefaction due to the laxative action of the
water or to drinking more water, since many people usually drink too
little, yet some of the action it seems most probable is almost certainly
due to the radio-activity of the water.

Diversity of foods. Importance.—The various food materials of
civilization have enormously multiplied in kind with the improvement of
transportation and the introduction into all countries of the products of
other countries. The temperate zone now obtains the vegetables and
fruits of the tropics ; and America not only has its native vegetables, such
as the potato and maize, but it cultivates also those of nearly all other
lands. The diet of various nations, and in particular of Americans, has
thus become more varied than that of any nation or group of people
previously existing. This is due in part, so far as America is concerned,
to the favorable location of the country, reaching as it does from the
tropics to the north and having no customs duties; and in part it is due
to the efforts of the Department of Agriculture to introduce into the
country all those fruits and cereals which. are cultivated elsewhere and
which custom has shown to be valuable foods. As a varied diet is more
certain to contain all those food and food accessory materials which the
body needs than is a restricted or monotonous diet, the.general result of
this expansion in the kinds of foods consumed is certain to be good; for
what one foodstuff may lack in one particular is certain to be contained
in another.

With the wonderful development of chemistry, also, food substances
THE RAW MATERIALS OR FOODS 303

are now manufactured from materials which were formerly wasted.
Thus while the human body cannot make glucose from cellulose, the
chemist can accomplish this. A great many prepared foods supposedly
more nutritious or partially digested or suitable for special ends, or
having a great many other real or imagined advantages, are being con-
sumed in constantly increasing amounts. Mention may be made of vari-
ous infant foods designed to eke out or replace mother’s milk; break-
fast cereals of all kinds; bran and wheat mixtures for laxative foods;
and so on. This development of the chemical manufacture or chemical
treatment of foods is a matter of the very greatest importance to public
health, since while the end sought is in many ways desirable, the foods
by chemical treatment are often altered in'their chemical composition,
or substances not naturally in the foods may be introduced from the
chemicals used in manufacture. The physiological effects of even small
amounts of certain compounds are so important that it is very necessary
to guard against the admixture of any toxic or harmful substance which
may be present in the chemicals used even in very small amounts. Thus
some years ago sulphuric acid containing arsenic was used in the manu-
facture of glucose from starch. The arsenic contaminated the glucose;
the glucose was used in the manufacture of beer and many people in
England were poisoned, some fatally, by the arsenic. It is important to
remember, also, that in the chemical treatment of foods there is the
possibility of forming in them toxic substances not in the food itself.
Often the effects of such substances in small quantities are not immedi-
ately apparent and it is extremely difficult to trace such injurious effects
as there may be to their true source in the altered food. It is for this
reason that any manufactured or chemically changed food which has not
had years and even generations of tests behind it, as all natural foods
have had, is to be regarded with a certain amount of suspicion, unless
the methods used in its manufacture are such as practically to preclude
injury. Physicians and scientific men have particularly to be on their
guard against hasty indorsements of artificial foods. For example,
physicians and scientific men usually mean by the word glucose the pure
hexose sugar, dextrose, and when the problem of the use of glucose is
presented to them they think only of the pure sugar, which is of course
an admirable food. But the manufacturer uses the term glucose, or
did use it for a long period, for the very impure mixture obtained by
the partial hydrolysis of starch. This substance not only contained pure
glucose, but many other substances such as dextrins, maltose, etc. ; it ocea-
sionally contained some nitrogenous substances, and if made with impure
materials it might contain some deleterious substances such as arsenic.
Before indorsing a food of any kind, a scientific man or physician would
do well to consult the government authorities whose business it is to
304 PHYSIOLOGICAL CHEMISTRY

investigate such foods, their constitution, methods of manufacture, etc.,
and to inform people of their desirability. Food accessory substances
or vitamines are also now known to be vitally important constituents of
the diet.

Amount of food required hy a human adult.—The human adult
requires roughly 60-150 grams of portein a day and sufficient fat and
carbohydrate, or protein, in addition to cover his energy requirements.
All three of these substances are found in all the foods, but in widely
varying proportions. In general a diet for an adult not doing hard work
must contain in addition to mineral matters, salts, and food accessory
substances approximately :

. Calories

100 grams of protein yielding ............ eee eee eee eeeeeee 400
500“ SF CATDODYVATALG? (escssce sai. sntobyaisied wate neie Somers Wilmette oaceved ca buna 2,000
GO SE Bate resonates swt i Sua te Souci Shnsne oie ars gsr Ce asda a ees eons 540
otal - uissy a devas oeeaea nals bas Gee crav aes: G Bie Soe eke 2,940

In addition there is required of phosphorus about 0.6-0.8 gram per day
and of calcium about 0.6 gram. Vegetables contain as a rule a great
excess of carbohydrate, and meat an excess of protein. Many vegetables
contain much water. The following tables give the proportion of food
matters in the principal foodstuffs. Attention is particularly given to
the various meats and such vegetables as potatoes, turnips, squash, peas
and beans, corn, wheat, oatmeal, and the fruits: apples, pears and
bananas.

ComposITION oF SomME Foop MAtTerIats. BuLiteTIn 28. E. O. S. Dept. oF

AGRICULTURE.
Fuel
Foods Refnse Water Protein Fat Totalcar- Ash value per
N x6 2 bohydrates pound
Beef.
Brisket, medium fat as purchased. Cals.

Average: 2s 3i05 ea teen aee 125 23.3 41.6 120 22.3 0.6 1165
Chuck, including shoulder. Medium

fat. As purchased. Average .. 15.2 57.9 166 10.1 0.8 735
Flank. All analyses. Average. As

purchased ...............005-. 5.5 561 186 19.9 0.8 1185
Loin. Medium fat. As purchased. :

AVERAGE: 5st senesa e524 hosed + a ichetand 3.1 582 17.1 111 0.9 785
Round. Fat. As purchased ...... 12.0 54.0 17.5 16.1 0.8 1005
Heart. As purchased ............ 5.9 53.2 14.8 24.7 0.9 1320
Corned beef. Brisket ............ 214 400 144 19.4 45 1085

Veal: Fresh.
Leg, lean. As purchased ........ 9.1 66.8 19.4 3.7 1 520
Leg. Cutlets. As purchased ..... 3.4 68.3 20.1 7.6 1.0 690
Mutton: Fresh.
Leg. Medium fat. As purchased .. 18.4 51.2 15.1 14.7 0.8 900
Loin, without kidney or tallow. As
purchased. .ccsevsvedeerscaotest 14.8 404 13.1 31.5 0.6 1575
Pork: Fresh.
‘Ham. Medium fat. As purchased 10.7 48.0 13.5 25.9 0.8 1345
Loin chops, medium fat ......... 19.5 418 134 24.2 0.8 1270
Tenderloin as purchased .......... 00 66.5 189 13.0 1.0 900
Ham. Smoked ...............55 12.2 35.8 14.5 33.2 4.2 1670
Salt pork, clear fat .............. 00 79 19 86.2 3.9 3670
Bacon, smoked .............00005 87 104 9.5 69.4 4.5

|

2685
Pork sausage

THE RAW MATERIALS OR FOODS

Refuse Water Protein Fat
N x 6.25

SSNS Sais Apes ok 3.3 55.2 18.2 19.7
39.8 13.0 44.2

Chickens. Broilers as purchased .. 41.6 43.7 12.8 1.4
Goose. Edible portion ........... 46.7 16.3 :

a As purchased ........... 17.6 385 13.4 9.8
Fish. Mackerel. Whole. As pur-

Chased. heels wean, ee ose 44.7 40.4 10.2 4.2
Mackerel. Edible portion ....... 73.4 18.7 Ti
Eggs. Uncooked. Edible portion 73.7 14.8 10.5

s s As purchased . 11.2 655 13.9 9.3
Eggs. Boiled whites ............ 86.2 13.0 0.2

= yolks: 6 ssi vewecas 49.5 16.1 33.3
Butter. As purchased ........... 11.0 1.0 85.0
Buttermilk. As purchased ...... 91.0 3.0 0.5
Cheese. American. Pale ........ 31.6 28.8 35.9
(Cheese. Cottage ............... 72.0 20.9. 1.0
Cheese. Dutch .......... acs Rina 35.2 37.1 17.7
Cheese. Roquefort ............. 39.3 22.6 29.5
Milk. Whole. As purchased .... 87.0 3.3 4.0
Milk. Evaporated. Unsweetened 68.2 9.6 9.3
Milk. Condensed. Sweetened ...° 26.9 8.8 8.3
Oleomargarine ................. 9.5 1.2 83.0

Vegetable foods.

Corn meal. Granular .......... 12.5 9.2 19
Corn HOUr esc. gene ete Do4 12.6 7.1 1.3
Oatmeal: «sinc sive cianleeaesvens t0eis 7.3 16.1 7.2
Rolled: Oats: occ cesses eee eee 7.7 16.7 7.3
Oatmeal boiled .............4.. 84.5 2.8 0.5
RiC@) gs gence thieee auatigy’ ae aden Soe as 12.3 8.0 0.3
Wheat flour. Entire wheat ..... 114 13.8 1.9
Wheat flour, Patent roller. Fam-

ily grade. Spring wheat ..... 11.9 10.9 1.1

Wheat flour. Winter wheat .... 13.1 12.3 11
Wheat: Breakfast foods.

Marina. .ingedoe ares sec ea eet 10.9 11.0 14

Shreddedt ssc -s.ssescws ceissamuarce edn oris 8.1 10.5 1.4

MaGatOnt : .0 340s tamara s tee se 10.3 13.4 0.9

ee Cooked ...........4. 78.4 3.0 15
Wheat bread. As _ purchased.

Home made ................55 35.0 9.1 1.6
Wheat bread. Whole wheat .... 38.4 8.7 0.9
Crackers, Oyster crackers ...... 4.8 11.3 10.5

ey Soda: cessceee ax esc es 5.9 9.8 9.1
Apple: pie iia os sguessaese is es 42.5 3.1 9.8
Bey sSnee ecapre ce 18.2 0.4

Vegetables.
‘Asparagus. Fresh ............. 94.0 1.8 0.2
Beans, butter, green. Edible portion 58.9 9.4 0.6

“ “ e As purchased 50.0 29.4 4.7 0.3

“lima. Edible portion .... 68.5 7.1 0.7

“ String. , Cooked ......... 95.3 0.8 1.1
Beets. Cooked ..............-. 88.6 2.3 0.1
Cabbage. Edible; portion. Fresh 91.5 16 860.3
Carrots. Cooked ............. 3.5 7.7 3.6
Corn. Green. Edible portion ... 75.4 3.1 ll
Cucumbers. Edible portion ..... 95.4 0.8 0.2
Egg plant. Edible portion ...... 92.9 1.2 0.3
Lentils. Dried .............66- 8.4 25.7 1.0

Total cur-
bohydrates
0.3(2) 3.8

1.1

0.37

oe
Re Ae Pow
mS wwWoo

75.4
78.4
67.5
66.2
11.5
79.0
71.9

75.6

73.0

76.3
77.9
74.1
15.8

53.3
49.7
70.5
73.1
42.8
81.2

Ney

CAwWSSAIAINB Ses

00
NM EIWHAROORH WD

on

.
PrPOVorvwovMroorre soos

WONNDOBDRNOHRQSON~A “Ot

mOONP On
an OPRNIH DDO

HPs eo

NaOH CWNO WwW

SoePNNYY

FESS SO POS
NAANSoOaoUSS84N

805

Fuel
Ash value ye
poun

1170
2125

295
1830
1505

365
645
720
635
250
1705
3605
165
2055
510
1435
1700
325
780
1520
3525

1655
1645
1860
1850

285
1630
1675

1655
1635

1685
1700
1665

415

1225
1140
1965

© 1925

1270
1520

105
740
370
570

185
146
1790
470
80
130
1620
306 PHYSIOLOGICAL CHEMISTRY

Fuel
Foods Refuse Water Protein Fet Totalcar- Ash value per
N x 6.25 bohydrates po
Peas. Dried .................. 9.5 24.6 10 62.0 2.9 1655
Peas, green. Cooked. Edible
PORHON: ..2 Sukcsacdn erate a ae eeessoe 74.6 7.0 0.5 16.9 1.0 465
Potatoes. Cooked. Boiled ...... 75.5 2.5 0.1 20.9 1.0 440
“ Mashed and
creamed 4.2 tent esa ware cee 75.1 2.6 3.0 17.8 1.5 505
Potatoes. Sweet. Raw. Edible
Portion «,..ceetesewe sees 69.0 1.8 0.7 27.4 11 570
Squash. Edible portion ........ 88.3 1.4 0.5 9.0 0.8 215
Tomatoes. Fresh ........e..00- 94.3 0.9 0.4 3.9 0.5 105
Fruits.
Apples. Edible portion ......... 84.6 0.4 05 14.2 0.3 290
Bananas. “ ee ahs danke 75.3 1.3 0.6 22.0 0.8 460
Blackberries ..............-505 86.3 1.3 0.0 10.9 0.5 270
Oranges. Edible portion ....... 86.9 0.8 0.2 11.6 0.5 240
Pears. Edible portion .......... 84.4 0.6 05 14.1 0.4 295
Nuts.
Almonds. Edible portion ...... 48 210 549 17.3 2.0 3030
Brazil, RWS is scc.j ase aenvinds does 5.3 17.0 66.8 7.0 3.9 3265
Hickory nuts ..........-+..005- 3.7 154 67.4 11.4 2.1 3345
POaN Ut: os cite osc ceceuncor aie eitenehaveisoss 92 25.8 38.6 24.4 2.0 2560

Milk.—Milk is a food for the young and growing. It is a most inter-
esting food, since it represents the answer Nature has given to the ques-
tion as to the best food for developing mammals. After a very long
period of experimentation in the monotremes, marsupials and lower
placental mammals, the milk of the higher placentals was evolved. It
is probable that there are good reasons for the presence in milk of most,
if not all, of its constituents. Milk is the secretion of the mammary
glands and these are modified skin glands. The constituents of the milk
are for the greater part manufactured in the gland itself; some of them,
however, come directly from the blood. The organic substances in the
milk are peculiar to it. They are not found in other tissues of the body.
Human milk is alkaline to litmus; cows’ amphoteric. The specific gravity
is 1.026-1.035.

The composition of the milks of the different mammals varies, but
they are alike in their fundamental qualities. In all, the organic mat-
ter consists for the most part of protein, carbohydrate and lipin, and
each of these is peculiar to milk. The carbohydrate is lactose; the lipin
is peculiar because of the proportion of fatty acids of low molecular
weight it contains and also for its special properties in nutrition; the
protein is largely casein. The composition of human and cows’ milk is as
follows (Meigs) :

Lactose Protein Ash
Per cent. Per cent. Per cent. Per cent.
Human milk ......... 2—4 6—7.5 07-15 . 0.15—0.3
Cow’s milk ........... 2—4 3.5—5 2.5—4 0.66—0.77

Besides the substances mentioned in the foregoing table, both cows’
and human milk contain organic substances which have little or no
nitrogen, which are soluble in ether and alcohcl, as well as water, but
of which the chemical nature is still unknown. The amount of these
substances in human milk at the beginning of lactation is about 1 per
cent., and in the middle period of lactation about 0.5 per cent. Cows’
THE RAW MATERIALS OR FOODS 307

milk at the middle of lactation contains about 0.3 per cent. These sub-
stances are possibly of great importance in nutrition.

The composition of the milk varies at different periods of lactation.
The milk secreted during the first three or four days after delivery is
called colostrum milk. According to Keenig colostrum of cows has the
following composition :

WAteR x ccsse gitadnah eae vee eit 74.67
OIE! ie ald ach seins. ae Gagn ov anbsauiasle ws 25.33
Casein: fiy.cecekw as haoamenewen oa 4.04
Albumin and globulin .......... 13.60
Bat was gravest oevieaaactes suave 3.59
Lactose. itiredsa vgn acca esas 2.67
AICS) aw seaweed Men eM aaah eres 1.56

The colostrum is richer in proteins and total solids than the later milk.
It contains many cells, milk corpuscles, which contain nuclear material,
whereas the later milk is practically free from such material. The fat,
also, is different in that it has a higher melting point, a higher iodine
number and it contains more cholesterol and lecithin. The protein mate-.
rial is different. It contains, probably in the colostrum corpuscles, more
coagulable proteins, so that unlike ordinary milk it coagulates on
heating. These same differences between colostrum and later milk are:
shown also in human milk, but they are not so marked. Meigs gives the
following figures of the human colostrum and later milk, comparing the
total nitrogen, the casein, globulin and albumin, and unknown substances
(figures in per cent. of weight of whole milk) :

Total N Nin caseinand N in albumin Nitrogen not

globulin precip- precipitated by precipitated by
itated by mag- acetic acid and either MgSO.

aoe ‘ sul- heat or heat
phate

Colostrum Human .......... 0.2962 0.1572 0.0514 0.0876
Early milk “ — .......... 0.2186 0.1377 0.0267 0.0542
Cows’ milk (Guernsey) ...... 0.5623 0.4925 0.0482 0.0216

The main differences between human milk and cows’ milk are these:
Human milk contains considerably more lactose than cows’ milk and
more substances of an unknown nature which contain little or no nitro-
gen; it contains very much less protein than cows’ milk. The greater
proportion of lactose in human milk may be correlated with the very
much greater brain development of human beings. There is a very rapid
myelinization of the fibers of the brain occurring shortly after birth
in the first six weeks of life. In myelin there is a large amount of
galactolipins of the nature of phrenosin, or cerebrosides of various
kinds. Galactose is one of the constituents of this material. It may
be that the larger amount of lactose in human milk is to supply this
need. No other place of formation of galactose in the body is known
than the mammary glands and these are of course very rudimentary in
the infant. The replacement of lactose by cane sugar in milk; or in
milk substitutes, would seem to be open to serious criticism on this
account. It must be remembered that the mammary glands have been
evolved primarily to make a food best suited for rapidly developing
human beings; even undiluted cows’ milk cannot be tolerated by very
308 PHYSIOLOGICAL CHEMISTRY

young children, although its composition is so similar to human milk.
Inasmuch as we are quite ignorant of the composition of some constitu-
ents of milk which may be very important, the attempt to find a suitable
substitute for human milk is seen to be very largely a groping in the
dark.

Proteins of milk.—The proteins of milk are casein (English call it
caseinogen) and lactalbumin. These two proteins do not contain all the
nitrogen particularly in the early stages of lactation, when quite a large
percentage of the nitrogen is in the form of substances, extractives, etc.,
which are not precipitated by MgSO, and not by heat after slight acidifi-
cation. They are not precipitated either by Al(OH), and are presum-
ably non-colloidal.

Casein. This is the most abundant and characteristic protein of milk.
It is probable that each kind of milk has its own kind of casein in it. In
cows’ milk there is about 3-4 per cent. of casein; in human milk between
0.5-1.5 per cent. This protein is a phosphoprotein. It is a conjugate,
or compound, protein containing phosphoric acid. It is a strong acid
probably forming three or four series of salts and having 4 or 6 ecar-
boxyls free. Its molecular weight, according to Van Slyke, is about
8,000. Casein has several peculiar properties. It is not coagulated by
heating; it is precipitated by faint acidification with acids, but soluble
in excess; it clots or coagulates when it is treated by a proteolytic
enzyme, and in particular by the enzyme found in calves’ stomachs, or
the stomachs of other young mammals. Its percentage of composition is:
C, 53.00; H, 7.00; N, 15.70; S, 0.8; O, 22.65; P, 0.85. These figures are
for cow’s casein. When hydrolyzed it yields the amino-acids shown in
the table on page 129. It is very unstable and hydrolyzes with great
ease. It will be noticed that it is remarkable for the very small amount
of cystine it contains, for the absence of glycocoll and for the large
amount, reiatively, of tryptophane. Casein in milk is in such a condi- |
tion that it will not pass through a porcelain filter. Among the acids
found in casein, but not found in other proteins, may be mentioned
diamino-trioxydodecanois acid, C,,H,,N,O,, found by Fischer and Ab-
derhalden; dioxydiaminosuberic acid, C,H,,N,O,, found by Skraup:
caseanic acid, C,H,,N,O,, and caseinic acid, C,,H,,N,O,. The nature
of these is uncertain (Skraup). Caseinic acid may. be identical with
dioxyaminododecanoic acid, although it appears to differ from it in its
optical rotation. Another acid recently isolated by Dakin is hydroxy-
glutamic acid. Caseins from various animals differ in the per cent. of
nitrogen and phosphorus. Mares’ and asses’ caseins contain more nitro-
gen (16.44-16.28 per cent.) than cows’. Asses’ casein has more phos-
phorus (Tangl and Csékas). Casein contains calcium. The amount
varies. [@]) =—97.8 to —111.8° in a N/10—N/5 NaOH (Long). In
neutral solution it is lower, i.e., —80°.
THE RAW MATERIALS OR FOODS 309

The preparation of casein is described on page 949. The clotting is
discussed on page 377.

Lactalbumin. This is a crystallizable protein present in small
amounts in milk and resembles seralbumin, but differs from it in its
rotation, (@)p =—87°. It coagulates at about the same temperature as
seralbumin, i.e., 72-80°. Its composition is: C, 52.19; H, 7.18; N, 15.77;
S, 1.73; O, 23.18. The amount of this protein in human milk is about
one-fifth of the casein; and in cows’ milk it is about one-tenth of the
amount of the casein.

Lipins of milk.—The composition of butter fat has already been
given on page 64. It is remarkable and different from the fat of most
other organs in the large proportion of butyric, caproic and caprylic acids
in it; in the proportion, in other words, of the volatile fatty acids. Milk
fat vontains, in addition to the glycerine esters of the usual fatty acids
and those just mentioned, small amounts of both lecithin and cholesterol.
Koch and Woods found in milk 0.036—0.049 per cent. of phosphatide
computed from the phosphoric acid of the alcohol soluble phospho-
lipins as lecithin; and 0.027-0.045 per cent. of a phospholipin computed
as cephalin. This latter is insoluble in alcohol. The total of phospho-
lipins was:0.072-0.086 per cent. For human milk ‘‘ lecithin ’’ was 0.041
per cent.; cephalin, 0.037 per cent., and the total, 0.078 per cent. The
amount of cholesterol has not been determined by the digitonin method
so far as can be found, but Marsh found by the Ritter method 0.021
per cent. The character of the phospholipin should be further studied.

The importance of these lipins in diet has been emphasized by the
work of Stepp, who found that mice could not be nourished on food
waterials which had been carefully extracted by alcohol and ether,
whereas the addition of this extract to the food restored them to
health. The active substance was not fat, cholesterol, lecithin or salts.
Similar observations have been made by Hopkins, who found that the
addition to an incomplete ration of a very small amount of milk powder
enabled mice to thrive when on a diet containing too little of some amino-
acid. Similar observations have been made also by McCollum and
Qsborne and Mendel, who found that the active principle in milk was
contained in the alcohol-ether extract and that similar active substances
were present in the oils of fish livers, such as cod-liver oil. The nature
of the constituent, or constituents, is still quite uncertain. They belong
to the general group of vitamines which are discussed on page 838.
Both Fat Soluble A vitamine and Water Soluble B are obtained by
the mother from her diet. Their presence is absolutely necessary for
growth and health of the offspring in rats and guinea pigs and pre-
sumably also in human beings. Hence the importance of the mother’s
diet during lactation. She must have an unusually large income of
310 PHYSIOLOGICAL CHEMISTRY

calcium and these vitamines at that time. The small amount of lecithin
in milk, which is designed as a food for rapid growth, is in striking
contrast with the large amount oi lecithin of the yolk of hens’ eggs.
This fact indicates that the infant is able to make the necessary phos-
pholipins from the inorganic phosphates. There are other evidences
that the phospholipins are readily formed by most kinds of protoplasm
from inorganic material, fats and amino-acids.

Lactose.—The presence of lactose in milk to the extent of from 4
per cent. in cows to 7 per cent. in human milk is significant for the rea-
sons already stated. The physical properties of this sugar are treated
on page 56; the method of isolating it on page 951. The mammary
glands have the power of making gatactose from glucose.

Other organic constituents of milk.—IMilk contains in small quanti-
ties a considerable number of organic substances. Among these par-
ticular mention may be made of citric acid, which is present in cows’
and human milk to the extent of .05-.1 per cent or even .237 per cent.
Among other constituents may be mentioned one found recently in
human milk by Meigs and Marsh, which crystallizes readily, which
makes a considerable part of the unknown alcohol soluble substances,
present in human milk in the early stages of lactation to about 1 per
cent., and which gives a strong test for unoxidized sulphur. The com-
position of this substance is unknown. Biscaro and Balloni have iso-
lated a substance called orotic acid (Gr. oros, milk), which was precipi-
tated by basic lead acetate. It had the composition C,H,,N.O,.2H,0.
The nature of the acid is unknown. Siegfried has isolated carniferrin,
related to nucleon or phosphocarnic acid, from milk by his iron method.
This yielded on decomposition carnic acid, fermentation lactic acid,
succinic acid and oxylic acid (C,,H.,N,O,). The latter yielded leucine
on hydrolysis with hydrochloric acid. There are also present in milk
small amounts of urea, some purine nitrogen (in 1 liter cows’ milk
0.004-0.006 gram purine N) and various other substances in small
amounts. Some observers have described peptone or proteose bodies in
milk, but it is doubtful whether they occur in perfectly fresh milk.
Cows’ milk as ordinarily obtained contains very large quantities of
bacteria which rapidly change its constitution. To get sterile milk is
difficult and it can be obtained only by passing a sterile catheter past
the sphincter into the ducts. It must be remembered, however, that
bacteria, although they are present in great quantities in ordinary city
milk, often 1,000,000 or more per ¢.c., are not normal constituents, but
have fallen in during the act of milking and that they multiply in the
milk with great rapidity. As Sedgwick has put it: ‘‘ A cow secretes
milk, not bacteria.’’

In 100 ¢.c. milk, both cow and human, there are found in mgs. 20-30
THE RAW MATERIALS OR FOODS 311

total non-protein N; 6-17 urea N; 2.6-7.3 amino.N; 1.0-1.3 creatinine;
2.0-3.4 creatine; 1.4-2.0 uric acid. These amounts are practically those
found in blood plasma. (Denis, Talbot and Minot.)

Inorganic substances in milk.—Milk must contain most of the sub-
stances required for the early development of a child. It must provide
substance for the growth of bone, hence it must and does contain large
amounts of calcium and phosphoric acid. Moreover, all growing organ-
isms need potassium, since potassium in nearly all forms of living matter
is present in larger quantities than sodium. Rapidly growing cells need
potassium. Jron is present in milk in very small amounts. This is in
contrast to the egg where there is an abundant supply of iron in the
yolk. But Bunge has shown that the iron is stored in the liver of the
foetus, this organ at birth having a rich supply which is called upon
during development to make good the lack in the milk. The ash of milk
contrasted with the ash of the new-born pup, showing how closely they
resemble each other, is given by Bunge as follows:

In 1000 parts of ash:

Of dog milk Of new-born dogs
K 29 5.0 fbuseilet odoavadenn a 62s! 149.8 114.2
Na,O ee ee 88.0 106.4
CaO? wsiivincaceate 272.4 295.2
MgO: sasa8 aaass eae 15.4 18.2
Fe,O a rittatsessees 1.2 7.2
P.O, suaualsds a anlenensreseceys 342.2 394.2
Cl aaiicovaceenens 169.0 83.5

In human beings there does not seem to be so close a correspondence
between the ash of milk and of the new-born (Camerer and Soldner).

Zine is found in human milk to the amount of 5.7-13.8 mgs. per kilo
milk; there are 3.6-5.6 mgs. per kilo in that of cows. The amount is
greatest in the early stages of lactation.

Coloring matter——The milk of cows fed on green fodder is more
or less yellow, while the milk of cows fed on dry hay and other forms
of dry fodder is not yellow. Guernsey cows give particularly yellow
milk and their fat is very yellow. This natural coloring matter is
derived entirely from the green fodder and consists of carotin and
xanthophyll.

Hulls of the milk globules.—It has been generally supposed that the
fat globules were surrounded by a very fine sheath of casein kept or
condensed there by the action of surface tension. Recent investigations
indicate that this is incorrect. By allowing the milk globules to rise
through a long column of water they can be separated from the casein
and other constituents of the milk. They are collected, the fat extracted
with ether and the residue, which constitutes the hulls, analyzed. The
result has been to show that the hulls may give no biuret reaction at all.
312 PHYSIULOGICAL CHEMISSRY

When hydrolyzed they yield glycocoll, which is not present either in
casein or lactalbumin. The amount of nitrogen is very low and variable,
4.04-5.70. per cent. The ash varies from 4.57-45.28 per cent., and P
between 0.18 and 0.57 per cent. It is clear that it is not a homogeneous
substance nor is it composed of casein.

Milk glands.—The composition of the mammary glands has been very
little studied. A nucleic acid similar in its general properties to that of
other organs has been isolated from it by Mandel and Levene. This
acid contained pentose, so that it is probable that these glands contain
not only the usual tetranucleotide, but also an acid analogous to, or
identical with, guanylic acid. By treating the glands in the same way
as the pancreas is treated for the extraction of a nucleoproteid by Ham-
marsten (see page 171) a nucleoproteid was isolated by Odenius of the
following composition: C, 46.89-47.15 per cent.; H, 6.04-6.15 per cent.;
N, 17.26-17.29 per cent. ; 8, 0.875-0.904 per cent. ; P, 0.275-0.278 per cent.
and 0.962-0.922 per cent. Mandel and Levene isolated a substance of a
reducing character which contained sulphur, and they called it gluco-
thionice acid. S, 2.65 per cent.; N, 4.388 per cent. There are probably
present substances of the sulpholipin or glycolipin type and these may
yield the reducing substances obtained by several observers on hydroly-
sis of the organic matter. The extractives are about the same as the
other organs so far as they have been studied. The composition of the
substances thus far isolated throws singularly little light on the method
of formation of the milk constituents or on the metabolism, and the gland
should be further investigated.

Metabolism of the glands.—There is very little that can be said about
this. The raw material from which the galactose is made is undoubtedly
glucose. The metabolism undergoes a remarkable rhythmic alteration
dependent upon the sexual organs. The mamme are in the nature of
secondary sexual organs. That is, although they are present in a rudi-
mentary form in the male, they do not develop, but remain rudimentary.
In the female they begin their development with the ripening of the
ovaries and the formation of ova. It is clear that their development must
be stimulated in some way by the ovary. Further research of this very
interesting problem has shown that the corpora lutea have the power
of stimulating the growth and activity of the mammary glands. An
extract of the corpora has a remarkable action on these organs. It is
found, too, that an extract of the uterine wall at the time when it is
undergoing regressive metamorphosis after parturition has the power,
when injected into a female animal of the same kind, of exciting the
activity of the mamme so that the glands become full of milk. On the
other hand, an extract of foetus has an inhibiting action. It will be
noticed, too, when the hypophysis is considered, that the extract of this
THE RAW MATERIALS OR FOODS 313

organ may cause a discharge of milk from a pregnant female. The
development of the gland appears to depend, then, upon the internal
secretion of the ovaries. The development ceases and the gland atrophies
after the discharge of ova from the ovary ceases and there are no more
corpora lutea. On the other hand, the cause of the differentiation of the
skin tissue into milk-gland tissue does not depend upon this internal
secretion, for it occurs in the male also. It would appear that in this,
as in so many other ways, males have in them often many of the poten-
tialities of females, and vice versa, and that whether male or female char-
acters predominate depends upon the development of ovaries or testes.
This is a matter, however, somewhat foreign to the present discussion,
but which will no doubt occupy a much larger space in the biochemistries
of the future.

Enzymes.—The presence of enzymes in the milk has been much dis.
puted, the contradictory results being due possibly to the rich bacterial
flora of most milks examined. Human milk is said to contain small quan-
tities of amylolytic enzyme, and as this is found in the blood it prob-
ably comes from the blood. The presence or absence of lipase is dis-
puted. There is no lipase in the glands (Bradley) and this indicates
that lipase is not the cause of the synthesis of the fats in the gland.
Catalase and peroxydases are present in milk, but catalase is found
almost universally in the organized world. <A proteolytic enzyme of an
ereptic nature has been described (Russell and Babcock) and given the
very inappropriate name of galaciase. This does not appear to be always
present. An enzyme which ferments lactose to lactie acid, alcohol
and CO, has been described by Stoklasa in both human and cows’
milk.

Souring of milk.—This is produced by the action of bitters of vari-
ous kinds which attack the lactose, forming from it lactic acid. The acid
thus set free precipitates the casein and thus clots the milk. Some bac-
teria are able to clot milk in a neutral reaction. These clot because they
secrete a proteolytic enzyme like rennin which forms paracasein from
the casein.

Sterilized milk as a food. —Many very young children do not thrive
when fed exclusively on boiled, diluted cows’ milk, whereas they will
assimilate fresh, diluted cows’ milk. The character of the change pro-
duced in the milk by heating, which is responsible for this malnutrition,
is still unknown. On heating, small amounts of sulphureted hydrogen
are split off from the milk. This is probably one reason for the change
in taste and odor produced by cooking. Whether it has anything to do
with the change in nutritive qualities is uncertain. Cooking destroys
much of the fat soluble and all of the anti-scorbutic vitamine in the milk.
If infants can have a portion of human milk, they generally assimilate
without trouble cooked, diluted cows’ milk to which lactose has been
added.
314 PHYSIOLOGICAL CHEMISTRY

Foreign substances in milk.—Various substances pass directly. from
the blood to the milk. Thus salts of various kinds taken by the mother
may affect the child. Cases are on record of infants developing bromism
from the effects of bromides taken by the mother during nursing. Vari-
ous odoriferous substances pass readily into milk, as everyone knows.
The diet of the mother is hence of importance to the child. It is pos-
sible that various internal secretions from the mother’s body may pass
by means of the milk to the child and may affect it.

Various kinds of milk—Of the various kinds of milk which ar:
available as foods for children, asses’ milk approaches most closely iu
composition to that of human beings. The composition of goats’, asses’
and mares’ milk is as follows:

Water Solide Casein Total Fat

protein  (Lipin) Lactose Ash

Asses’ milk ..........06. 90.12 9.88 0.79 1.85 1.37 6.19 0.47
Mares’ milk ............ 90.58 9.42 pees 2.05 1.14 5.87 0.36
Goats’ milk ..........00. 86.30- 13.70 360 460 400 4.30 0.80

Eggs.—Like milk, eggs represent a food provision for the develop.
ing young. The needs of young birds and mammals are for the greater
part the same. There must be energy foods to supply the energy require-
ments of development and there must be proteins and fats and inor-
ganic substances to provide the requirements of matter. The developing
chick needs iron for its blood and phosphates to make its bones, nucleins
and phospholipins. The manner in which these phosphoric-acid needs are
met is essentially different in the birds and mammals. In milk the phos-
phates required to make nuclein, phospholipin and bone are present for
the larger part as inorganic phosphoric acid; there is in addition phos-
phorie acid tied to organic matter in the casein; there is very little phos-
pholipin present. The casein has attached to it a part of the calcium
required and a part is present in combination with the phosphoric acid.
In eggs the calcium is in part present in the shell which is partly reab-
sorbed during the development ; and in part it is present in both organic
and inorganic form. There is in the yolk of egg a phosphoprotein
analogous to casein, called vitellin; the amount of inorganic phosphate
is less than in milk. But the main difference lies in the fact that the
egg yolk represents a cell and the phosphoric acid is laid down in the
form of lecithin which is digested in the course of development and
inorganic phosphates formed from it. Neither milk nor eggs contain
appreciable quantities of purines and, since these are formed rapidly
in the rapidly developing nuclei of the embryo, both young birds and
young mammals must have the ability to form purines and pyrimidines
at a very rapid rate.

A hen’s egg consists of both white and yolk. The white is a secretion
of the glands in the oviduct; the yolk is a real cell, the egg cell. Th.
contains very little living protoplasm, but an enormous mass of non:
living matter.
TILE RAW MATERIALS OR FOODS 315

Eyg white. The composition of white of the hen’s egg is as fol-
lows (Koto) :

Per cent. Per cent.
Water ...... 87.71 Total N in white 1.75
Solids ...... 12.29 Protein (Nx 6.25) 10.93
Ash wccuends 0.4 Dextrose ........ 0.55
Organicsolids 11.89 = 4.47% of dry matter.
Per cent.
Yolk. Water ....... 49.73
Solids ....... 50.27
Ash ........ 1.44
Organic solids 48.83
Glucose ...... 0.27 of fresh yolk.
Nitrogen .... 249 “ “
Lipins ...... 34.00 “ “ “

The concentration of the glucose in the water of the yolk is about
that of the white.

Egg white.—The reaction of the white of egg when fresh is very
faintly alkaline, but the alkalinity increases on standing, owing to auto-
digestion and the formation of ammonia. As a result of this alkalinity
there is a setting free of sulphur, since the white of egg contains a sub-
stance which has very labile, unoxidized sulphur. The sulphide thus
set free combines with the iron set free from the hematogen to make
sulphide of iron; this colors the white near the yolk a bluish color when
the eggs are cooked.

Proteins of the white. The proteins in egg white are simple pro-
teins for the greater part and consist of ovalbumin and ovoglobuiin. The
latter is precipitated when the white is diluted with water or dialyzed.

Ovoglobulin. This makes about 61% per cent. of the total proteins.
This substance is apparently not a uniform or homogeneous substance,
but a mixture consisting chiefly of ovomucin. It was called a globulin
because of its insolubility in water and solubility in salt solutions, but
it is in reality a compound protein. (See ovomucin.)

Ovalbumin. The composition of this substance has already been
given on pages 129 and 139. Together with conalbumin it forms nearly
90 per cent. of the total coagulable protein of the white. It is an easily
crystallized protein when it is separated from the perfectly fresh eggs.
It coagulates at 64-70° or higher. There is some doubt whether it is
a unitary substance or not, some authorities believing that it is a mix-
ture. The experiments of Abderhalden and Pregl on the glucosamine
content of various successive crystallizations indicate that the substance
is generally impure as it is usually prepared. It usually contains some
absorbed, or otherwise entangled, ovomucoid. They found that the
successive crystallizations yielded respectively on hydrolysis from 7 per
cent. down to 2.5 per cent. of glucosamine. The recent carefully recrys-
tallized and otherwise purified preparation of Osborne and Campbell
316 PHYSIULUGICAL CHEMISIRY

yielded 1.23 per cent. of glucosamine. The amount of amino-acids
recovered from a hydrolysis of ovalbumin totals only about 50 per cent.
of the .nolecule. It is probable, therefore, that there are other products
not yet isolated. Osborne and Campbell suggest, indeed, that ovalbumin
is a conjugate protein and possibly contains some radicle like chondroitic
acid. This would account for the glucosamine and also for some volatile
acid which was obtained when distilled with dilute acid. The acid might
be acetic. Further work on the composition of this protein is much to be
desired.

Conalbumin. This is the second albumin in egg white. It is sepa-
rated from egg white by crystallization. It is the part of the albumin
which is not. erystallizable. It constitutes about one-half of the total
albumin. It resembles ovalbumin in elementary composition, bui wii
not crystallize and coagulates at a lower temperature, i.e., 50-60°. It
contains about 16 per cent. N and 1.7 per cent. of sulphur.

Ovomucin. This is separated in the globulin fraction by precipita.
tion by half saturation with ammonium sulphate (Hichholz). It is a
glycoprotein. It rapidly becomes insoluble in water on dialysis. It
yields about 11 per cent. of glucosamine.

Ovomucoid. This makes about 10 per cent. of the total solids o?
hen’s egg white. It is a typical mucoid substance. It contains 12.65
per cent. nitrogen and 2.20 per cent. of sulphur: According to Seeman,
it contains 34.9 per cent. of glucosamine. It is not precipitated by half
saturation with ammonium sulphate or by complete saturation with
sodium chloride or magnesium sulphate. It does not coagulate on boil-
ing and it may be prepared by this means. All coagulable proteins are
removed by slightly acidifying with acetic acid, coagulating by heat, and
filtering. The concentrated filtrate is precipitated with alcohol. This
behavior and the low content of nitrogen may mean that the substance
is allied to chondroitic acid, or contains a large amount of this sub-
stance. Further work on the composition of this body is needed.

Ash. The ash of the white contains about 30 per cent. of Na,O, the
same amount of K,O, about 29 per cent. of Cl and small amounts of
Ca, Mg, phosphoric acid and other substances.

The yolk.—The yolk is very much more complex than the white.
It contains a very large amount of oil or fat, egg oil, and the protein is
chiefly vitellin. The solids are about 50 per cent., and these consist of
protein, 16 per cent.; fat, 23 per cent.; lecithin, 11 per cent.; cholesterol,
1.5 per cent., and salts 3 per cent. There is some variation in these
figures in different eggs. The lecithin and egg oil make the greater part
of the solids. The phospholipins are not all lecithin; bodies similar to
cephalin and some other myelin-like phospholipins have been isolated
(Thierfelder). The yolk generai!y males about one-third of the whole
THE RAW MATERIALS OR FOODS 317

egy. It weighs, on the average, about 20 grams. There should be be-
tween one and two grams of phospholipins in one egg yolk.

Ovovitellin. This is a typical phosphoprotein, or nucleoalbumin.
According to Osborne and Campbell, ovovitellin as extracted from egg
yolk by salt solution is a mixture of various compounds of lecithin and
vitellin proper. The P content varies from 2.5-4.2 per cent. The amino-
acids it yields are given on page 129, and its composition on page 139.
The ovovitellin is insoluble in water, but soluble in dilute salt solutions
and very dilute alkalies. It behaves much like a globulin, but it differs
from a globulin in that it contains phosphoric acid in some union or
other, and when digested with pepsin HCl it yields an acid rich in
phosphorus, about 8 per cent. P, which was at first supposed to be
nucleic acid, but is not. It yields no purine bases. The vitellin is
extracted from the yolk by shaking the fat out with ether and then treat-
ing the residue with 10 per cent. NaCl and precipitating the filtrate by
diluting with water. The lecithin is separated by boiling alcohol. Pure
vitellin has not been obtained. The nature of the phosphoric acid residue
is unknown and needs further investigation.

Hematogen. Egg yolk contains a substance which on digestion with
pepsin HCl yields a nuclein-like substance which contains iron. This
iron compound is supposed to be the mother substance of the hemoglobin.
It was named hematogen to show this relationship. The composition of
this substance is as follows: C, 43.5; H, 6.9; N, 12.6; P, 8.7;S, trace; Fe,
0.455; Ca, 0.352; Mg, 0.126. The substances analyzed are probably mix-
tures. There is no doubt that egg yolk contains all the necessary ingredi-
ents for the manufacture of hemoglobin, since this substance is formed
during development.

Lipins. Egg oil. This yields a large amount of oleic and palmitic
acid. Liebermann obtained: palmitic acid, 38.0 per cent.; oleic, 40 per
cent., and stearic acid, 15.21 per cent. The composition of this fat is
dependent upon diet. Thus, if chickens are fed fish, the eggs have a very
strong taste of fish; if they are fed on rape seed and the young rape
plants, the boiled yolks are a deep black and have a disagreeable taste.
Fat soluble dyes such as Sudan 3 will be laid down in the yolk if it is
ingested by the birds. The yolk is laid down in rings. Hach ring means
a time interval of 24 hours. The deposition of the yolk is slow at night,
or some reabsorption may occur so that the appearance of the fat is
changed and the yolk is in layers (Riddle). It takes about four days
when a hen is laying rapidly to produce the greater part of the yolk.
The period can be greatly lengthened by cold or poor nutrition. The
phospholipins include lecithin and lipins resembling cephalin and amino-
myelin.

Mineral substances. The following analysis shows the composition
of the mineral constituents in parts per thousand:
318 PHYSIOLOGICAL CHEMISTRY

Na,O 512 65.7 Fe) 11.9- 145
K,O 80.5 89.3 P.O, 638.1 - 667.0
CaO 122.1 132.8 SiO, 5.5- 14.0

Mgo 20.70- 21.1
As in milk so in egg yolk, a small amount of zinc is found.

REFERENCES. Foops.

1. Milk. Meigs and Marsh: The comparative composition of human milk and of
cows’ milk. Jour. Biol. Chem., 16, p. 147, 1913. (Literature cited
in this paper.)

2. Difference between cooked and raw milk. Schardinger reaction. Romer:
Biochem. Zeits.. 40, p. 5. 1912. McCollum and Davis: J. Biol. Chem.,
23, p. 247, 1915.

3. Milk. Value in growth. Stepp: Zeits. f. Biol., 57, p. 135, 1911.

McCollum and Davis: Jour. Biol. Chem., 15, p. 167, 1913.
Osborne and Mendell: Jour. Biol. Chem., 15, p. 311; 16, p. 423, 1914.

4, Lecithin content of milk. Koch. W.: Zeits. f. physiol. Chem., 47, p. 327,
1904.

5. Eggs. Composition of hens’ eggs. Koto: Zeits. f. physiol. Chem., 75, p. 12,
1911.

6. Proteins of the white. Osborne and Campbell: Protein constituents of
egg yolk and egg white. 23d Report Conn. Agri. Expt. Sta, 339 and 348,
1900.

7. Composition of the body. Inaba: Ueber die Zusammensetzung des Tierkérpers.
Archiv f. Physiol., 1911. p. 1. ;

8. Composition of various food matters. U. S. Dept. of Agriculture. O. E. 8.
Bulletin 221, 1909.

9. Effects of different methods of cooking on the digestibility of meat.
Grindley: U. S. Dept Agriculture. O. E. S. Bulletin 193, 1907.

10. Digestibility and nutritive value of bread. Woods and Merrill: U. 8. Dept.
of Agriculture. O. E. S. Bulletin No. 143, 1904. Ibid., Snyder: Bulletin
126, 1903.

ll. Dietary studies in public institutions. Knight. Pratt and Langworthy:
U.S. Dept. of Agriculture. O. E. S. Bulletin 223, 1910.

12. Milk. Composition of cows’, goats’ and human milk. Bosworth and Van
&lyke: Jour. Biol. Chem., 24, p. 187, 1916.
CHAPTER VIII.
SALIVARY DIGESTION.

Digestion in general.—All those chemical processes by which foods
are rendered available to an organism are in a broad sense digestive
processes. Of the various substances used for the nourishment of the
body only a few of the simplest such as oxygen, water, dextrose and inor-
ganic salts are in a condition to be utilized directly by the living matter
of the body for the repair of its waste, for its upbuilding and growth,
or as a source of energy ; all other substances than these must be reduced
to simpler compounds by which their absorbability or availability is
increased. Thus the starches must be changed to simple monosac-
charides, the proteins to amino-acids ; the fats to glycerine and fatty acids.
The object, then, of all these digestive processes is to increase the avail-
ability of the substances as foods.

In this sense all the processes of cooking become processes of diges-
tion ; for by cooking disintegrative chemical changes are brought to pass,
such for example as the transformation of starches into dextrins, sol-
uble starch and even disaccharides; the softening of the hard connective
tissue of meat by the conversion of collagen into gelatin; the changing
of some others of the proteins to the first stages of their decomposition
into acid albumin and albumoses; or the partial splitting of the fats
into glycerol and fatty acids.

For other reasons, too, the processes of cooking are properly included
in digestion, since by cooking the appearance, taste and odor of foods are
improved, in general opinion at any rate, and by its pleasant excitation
of the end organs of the special senses such food leads to a reflex stimu-
lation of the digestive mechanisms which is a marked aid to digestion.
It must not be forgotten that the digestive processes proper in the
body involve a great number of different factors, such as the motility of
the stomach and alimentary canal; the secretion of the digestive juices in
amounts adapted to the food to be digested and at the times when they can
be most efficient; and the formation of a digestive fluid of the proper
activity and chemical composition. In the alimentary canal, too, the
products of digestion are being removed by the processes of absorp-
tion and this very materially modifies the rate of digestion. Digestion
being thus not a simple process occurring in an inanimate beaker, but
a highly complex adaptation of effort to work to be performed, by a
very ingenious and admirable mechanism, everything which affects this

319
320 PHYSIOLOGICAL CHEMISTRY

mechanism becomes capable of directly or indirectly affecting digestion.
So cooking, because of the development of pleasant and appetizing tastes
and smells in foods, aids digestion more than by its direct digestive action
on the foods.

In still a third way cooking may profoundly aid digestion by killing
parasites or bacteria which otherwise would gain a foothold in the ali-
mentary canal and thus change the nature of the digestive processes. The
meat of chickens and pigs is particularly apt to become infected with
bacteria which produce poisonous products, or which become lodged in
the intestine and growing there produce toxines. Many of the head-
aches, attacks of constipation, diarrheas, feelings of malaise, fatigue,
etc., from which we suffer, are due to such infections, which are, as often
as not, so ill defined in their onset, so general in their symptoms and
so gradual in their disappearance that the origin of the ill feeling is often
incorrectly referred to the nervous system, or to any other rather than
to the right source.

Auto-digestive processes occur, too, in many foods. Thus meat and
eggs contain digestive enzymes which come into activity particularly in
meat when its reaction becomes acid. These ferments digest the tissue
and even in the absence of bacteria gradually destroy the structure and
render the constituents soluble. The ripening of fruits; the changes in
tenderness and taste of meat of various kinds, or of glandular organs
used as food; the change in taste of vegetables with age; all these are due
to such auto-digestive processes, which are thus more or less important
adjuncts to the digestive juices of the body.

Saliva. Origin.—The first of the digestive juices formed by the
body with which the food comes in contact is the saliva. This fluid is
secreted by the salivary glands, the parotid, submaxillary and sublingual,
and by the mucous membrane of the mouth.

The saliva is formed by the protoplasm of the cells of these glands
with the admixture of salts, water and some other substances derived
from the blood. The protoplasm of the inner end of the cells of the gland
and sometimes the greater part of the cell body is transformed into
saliva. Saliva is thus nothing else than transformed protoplasm. Dur-
ing the time of active secretion raw material is brought to the gland by
the rapid blood stream, since vasodilation takes place at that time and
the flow of blood to the gland is greatly increased. Out of this raw
material crude protoplasm is formed which afterwards, during the period
of glandular rest, either forms mucin and the other salivary constituents,
or it is itself transformed into these substances by a process of differ-
entiation. The mucin thus formed accumulates in the cells in the form
of granules and is discharged from the cell during the process of
secretion.
SALIVARY DIGESTION 321

The character of the saliva secreted by the different glands varies.
The sublingual secretion is very thick and viscid, due to the presence
in it of a great deal of mucin; the submaxillary, also, generally contains
a great deal of mucin and its saliva is very slimy; the parotid, on the
other hand, has a saliva with less mucin, or none at all. Its secretion is
generally more watery, although it has a good deal of protein in it,
and it is the secretion of this gland, more than the others, which has a
digestive action on starches. Differences exist, also, in the salivas secreted
by the same gland in different species of animals. The parotid saliva
of the horse, rabbit and sheep is generally more fluid than that of the
dog, which indeed may be very viscid.

Nervous control of the secretion.—Each of the salivary glands is
controlled by a double set of nerves, by a nerve which dilates the
arterioles of the gland, a nerve which comes directly from the brain;
and by a nerve which comes from the sympathetic system, and which
when stimulated produces constriction of the arterioles of the gland. It
is by means of the actions of these two nerves that the nutrition of the
gland is controlled and its activities regulated. The quantity of saliva
secreted on stimulation of the cerebral nerves (chorda tympani of the
submaxillary and sublingual, and the auriculo-temporalis branch of the
fifth nerve, which receives fibers from the glossopharyngeal nerve for
the parotid) is much greater and the saliva is more watery than when
the sympathetic nerves are stimulated. By some authors, notably by
Heidenhain, this difference was ascribed to differences in the manner of
action of the nerves on the cells of the gland. Heidenhain suggested that
the cerebral nerve acted upon the gland cells in such a manner as to
cause the secretion proper, that is the discharge of water and salts.
whereas the sympathetic acted upon the cells so as to make the cell
contents soluble, so that the secretion was a great deal more concen-
trated in the latter case. By other physiologists the difference in the
character of the secretion is ascribed to the difference in the state of
the blood vessels accompanying secretion, to the vasodilation when the
watery juice is secreted and to the constriction when the more concen-
trated juice is formed; by still others (Eckhard, Bernard and some
others) the difference in composition of the two kinds of salivas is
ascribed to the fact that in part the nerve acts on the cells of the gland
and the blood vessels, and in part it acts on the basement membrane,
or the basket cells, which are supposed to be contractile and to press
out of the gland some of the stored secretion. The sympathetic causes
its secretion mainly by the latter method, the chorda by stimu-
lating secreting cells, blood vessels and contractile sheath. It is
impossible to go here into a discussion of this question which is still

unsettled.
322 PHYSIOLOGICAL CHEMISTRY

Composition of mixed saliva——The composition of mixed human
saliva was found by Frerichs to be as follows:

Water. .cccinnccseens dete seraeeds peleee eet 99.41%
Solids ........ G felted dane Sielaie: SS -SRO RS SS Rae go 59
Mucin and epithelium ..........ceeee ee eeeeee 213
Soluble organic matter ..........ecceeee cece 142
Inorganic salt ........ cece cence cece eco eeene .219
TRONS © 23 ease Siete erivedseroeein. oaes cd Sas ncaa ne ee .01 - .00

In 1,000 parts of the mineral ash there were found the following
constituents :

Ge BAER ERAG BOERS HS SEE ER Rs ee OR ERR 457.2
ING cinug Sela eA Des CAREER Ulstteny 95.9
CaO and traces of Fe,0, Aecasetteien ek thie Skat 50.11
MeOh ici diuasdidad es eet GeReee eee ee 1.55
SO, Sa ee tacts Bae eee ails ha thts DEM ORS 63.8
P.O, doh itt & sacle CSG Re aera Glow Sa eisben cask 32 188.48
Ol oy eo G ested ee anes tOEG Dea edwaneuwes 183.52

The specific gravity is 1.002-1.008.° The freezing point is A =—0.28 to
—0.43°. The freezing point is higher than that of the blood. The
viscosity of submaxillary-sublingual saliva may be 18-35 times that of
water. It is extremely variable. The great richness of the ash in potas-
sium will be noticed. In the blood, sodium greatly surpasses potassium
in amount. This potassium is not free potassium chloride, but the
greater part is present in organic combination.

The reaction of the saliva of the healthy is generally, if not always,
in its fresh state, alkaline to litmus, but acid to phenol-phthalein. This
would give it a reaction about that-of the blood, or approximately
2X<10‘—®) normal concentration of hydrogen ions. To neutralize it to
litmus there must be added for 10 c.c. saliva 4.5-14.9 c.c. of N/100 acid;
and to make it sufficiently alkaline to redden phenol-phthalein, about
the same amount of N/100 alkali is necessary. It will not infrequently be
found, however, in testing a large number of individuals, that some have
the mixed saliva acid to litmus. This is most often due to an acidity
produced by the action of bacteria on the fragments of food left between
the teeth, particularly carbohydrate food, but it sometimes has another
explanation, for the saliva is found to be acid to litmus when collected
directly from the duct by a cannula. This acidity may be correlated
with age, state of health or diet and should be further studied. The
reaction of the saliva is of considerable importance in the hygiene of the
mouth, for it is a general impression of dentists that the acids thus pro-
duced by the action of the bacteria on the saliva and food remnants
are an important element in the decay and corrosion of teeth, or in the
deposition of the tartar on the teeth. The subject needs 9 thorough
investigation.
SALIVARY DIGESTION 832i

Amount of saliva secreted per day.—It was estimated by Bidder
and Schmidt that about 1,500 c.c. saliva were normally secreted per day,
but the amount is no doubt subject to wide variation, depending on the
amount of water. consumed, the length of time the food is chewed, the
character of the food, its dryness and so on, and the estimate is at best
an approximation only. Since the salivary glands weigh together only
about 66 grams in the adult, it is obvious that the amount of saliva
secreted is many times the weight of the glands. The secretion may be
greatly increased by many poisons, such as pilocarpine, by mercury
saits or by some organic toxins which produce a salivation very similar
to mercury salivation. It is increased by smoking. It was not infre-
quent in the days of mercury treatment of disease to give mercury freely
until three or four liters of saliva, or even more, were secreted daily.
In mercury poisoning one of the symptoms is the copious flow of saliva
and the other manifestations of salivation, such as the sore gums
and lips. ;

Functions of the saliva——In man the saliva has two main functions,
iLe., to aid swallowing and to assist in the digestion of starches. These
functions are subserved by two different constituents of the saliva. The
former by the water and mucin; the latter function by the ptyalin.
These are, therefore, the most important constituents and their chemistry
may now be briefly considered.

Chemistry of mucin.—The saliva is a modified skin secretion, since
the salivary are modified skin, or epidermal glands. It is interesting,
therefore, that they contain mucin, a glycoprotein, which is such a con-
stant constituent of the skins of the amphibia and the fishes and which
occurs even in the skin of mammals in a disease of the thyroid known as
myxedema. Mucin, or mucoid substances, are round not only in the
salivary glands and in the skin, but similar glycoproteins may be iso-
lated from the tendons, connective tissues, from cartilage, from amyloid,
and they occur not infrequently in the invertebrates. The general com-
position of various mucins is shown in the following figures:

Cc TW N S 0 Author
Snail’s mucin... ... 50.32 6.84 13.65 1.75 27.44 Hammarsten
Tendon mucoid...... 48.76 6.53 11.75 2.33 30.63 Chittenden and Gios
Submaxillary mucin.. 48.84 6.80 12.32 0.84 31.30
Salivary mucin...... 48.26 691 10.70  1.38- Miiller
1,45

They contain on the average less carbon and nitrogen and more oxy
gen and sulphur than the simple proteins.

-The mucins are conjugated proteins, they are glycoproteins. That
is, when they are hydrolyzed by acids they yield a carbohydrate radicle.
The exact composition of the mucin of the submaxillary or of any other
324 PHYSIOLOGICAL CHEMISTRY

true slimy mucin has not yet been determined, but extensive studies have
been made by Gies, Levene and others on the mucoid of the tendons.
The relation between the chrondroproteins (mucoids) and muciu is still
uncertain, but the former contains more sulphur than the latter. The
mucins have the general properties of the nucleins, that is they are read-
ily soluble in dilute alkali, are readily precipitated by dilute acid, but
are soluble in stronger acid. They are prepared by extracting tissues
with water, or with dilute alkali and precipitating with acetic acid. Such
preparations generally contain nucleoprotein. They are acid bodies
and exist in the tissues as salts. The mucin of the submaxillary gland
probably exists as the potassium salt. Being acid bodies, like the
nucleins, they form compounds with basic dyes, or at least some of the
mucins thus combine, and one of the principal mucin stains is thionin,
which is such a basic dye. According to Levene, submaxillary mucin
has some of its sulphur as ethereal sulphate.

On decomposition of tendon mucoid by hydrolysis by acids, Levene
found that it split into sulphuric acid, galactose, galactosamine; and by
stronger acid into leucine, tyrosine, levulinie acid and acetic acid. It
contains, therefore, a hexose (shown by the levulinic acid) and sulphuric
acid. It is thus clear that the mucoids must be closely related to the
cartilages. The organic matrix of cartilage consists of two proteins,
one of which is a conjugated protein formed by a simple protein united
with chondroitic acid, that is with cartilage acid, since the word chon-
droitic means cartilage. This cartilage acid, or chrondroitic acid, is
an acid found in small amounts in the urine. When hydrolyzed by acid
it breaks up according to the scheme shown on page 325.

The final products of decomposition of the chrondroitic acid are there-
fore glucosamine, or levulinic acid from it, sulphuric acid, acetic acid, gly-
euronic acid. These are just the splitting products which Levene found
among the decomposition products of tendon mucoid. It seems, there-
fore, that the prosthetic group of the mucoid molecule is chrondroitic
acid, or a closely similar acid. He was not able to isolate the chrondroitic
acid itself. Mucin is related, therefore, to cartilage through the mucoids.
The difference between them is probably to be found in the protein part
of the molecule and the proportion of chondroitic acid. Mucin also
occurs chiefly as the potassium salt, whereas cartilage mucoid is prob-
ably present chiefly as the calcium salt.

The relation of mucin to cartilage is extremely interesting from the
evolutionary standpoint. It shows that mesodctmal and the ectodermal
tissues are possibly not so unlike in their chemical nature. A very inter-
esting fact, also, is the resemblance in composition of mucin, which
occurs in such quantities in the skin of the lower vertebrates. to chitin,
the hard covering of the arthropods. When chitin is hydrolyzed it

.
SALIVARY DIGESTION 325

yields glucosamine and acetic acid. It is supposed to be a polymerized
monoacetyl glucosamine. There is always present, also, some sulphuric
acid, but this is generally regarded as an admixture of inorganic
sulphate. In other words, chitin, which is the main constituent of the
external and internal hard parts of the arthropoda, is chemically related
to the cartilage or the organic matrix of the internal skeleton of the
vertebrates and to mucin, which is such an important constituent of the
skins of vertebrates. These facts lend support to the view that the chitin
of the invertebrate, perhaps by combination: with glycuronic and sul-
phurie acids, formed the matrix of the mucin, mucoid and cartilage of
the vertebrates. These facts are of interest in the light of the theory of
Gaskell and Patten that the arthropods were the ancestors of the
vertebrates.

C,,H,,NSO,, + H,0O=H,80, + C,,H,NO.,
Chondroitic acid. Chrondroitin.

Chrondroitin in its properties resembles gum arabic. It yields acetic
acid and chrondrosin.

C, gH,,0,,N + 3H,0 = 30,10, +C, oH, NO,
Chondroitin. Acetic Chondrosin.

acid.
On further hydrolysis chondrosin, which is a reducing substance,

splits as follows:
C,H, ,NO,,+H,0= C,H 0 + CH (NH,)0.

10° 7,' 6 11

Chondrosin. Glycuronie Gyjc95 ainine
, acid. ;
HC———0-——CH
ae ae ees
OH HCOH | | HOCH OH
| CH, . 2 @ 4 CH,
SO, | HOCH | | HCOH | SO,
I co | i | | co i
0 | HC___'__'|___CH |
|. OHH H NH H { { H HN H OH |
CH,—C—C—Cc—C —C—0O—CH HC—0—C———-C—C—C— —C—CH,
“4! OHH | | H oH lH B
Pact COOH HOOC | ess

Proposed formula for chondroitic acid. (C,H, .N,8,0,,)
(Levene and LaFarge.)

While the composition of the salivary mucin is still uncertain, there
are several facts. which indicate that it is closely related to the mucoids.
Tt has been shown (Miiller), for example, that of the total sulphur in
the compound 35 per cent. is in the form of oxidized sulphur, and
(Levene) that part of it is present as ethereal sulphate. The carbo-
hydrate radicle makes 36.9 per cent. of the salivary mucin, and about
24 per cent. of the submaxillary mucin (Miller). This radicle is prob-
326 PHYSIOLOGICAL CHEMISTRY

ably at least in part glucosamine. Moreover, it is probably a monoacetyl
hexosamine. This relates the mucin at once to chitin, which is a polymer
of an acetylated glucosamine. On cooking with alkali it splits off a
complex carbohydrate, called animal gum by Landwehr, which does not
itself reduce Fehling’s solution but only after hydrolysis. This again
would relate it.to chitin. It appears, then, that the prosthetic group of
mucin, if not chondroitic acid, is at any rate related to it and resem-
bles chitin in many ways. The protein part of the molecule is still not
well investigated. Mucin which has been warmed with alkali gives a
very red color with dimethylaminoazobenzaldehyde (Ehrlich). This
reaction is given also by acetylated glucosamine when it is treated with
alkali. Chitosan from chitin does not give this reaction.

Dry mucin is a white amorphous substance, which swells in water
and dissolves on the addition of a little alkali. As precipitated by acetic
acid its reaction is acid. It gives the protein reactions, but it does not
coagulate on heating a neutral solution. It is completely precipitated by
saturation with ammonium sulphate, but not by magnesium sulphate.

Preparation of mucin.—Mucin is prepared from the submaxillary
gland of the dog by extracting the finely hashed organ with water and
filtering. HCl of 25 per cent. is then added to the filtrate until this
contains 0.15 per cent. of HCl. On the addition of the acid there is at
first a precipitate, but this soon redissolves. The clear solution may be
then diluted with 2-3 volumes of water when the mucin precipitates
out. Tendon mucoid may be prepared from the ox Achilles tendon by
extracting the chopped tendon first with water and then 10 per cent.
NaCl to free it from protein. It is then extracted with Ca(OH,). The
extract is filtered ; the mucoid is precipitated with acetic acid and puri-
fied by repeated solution in dilute alkali and reprecipitation with acid.

B. Digestive action of saliva-——-That human saliva has the power
of converting starch into sugar was discovered by Leuchs in 1831. If
a little saliva is mixed with starch paste, it may be seen that the paste’
very quickly becomes clear like water; if a little of it is then poured
out, it will be found that, whereas it was before very thick, it now pours
easily ; in other words, its viscosity has been greatly reduced ; if a little
of the clear solution is added to some Fehling’s solution in a test-tube
and boiled, it will be found that the Fehling’s solution is reduced, whereas
it was not reduced either by the starch alone, or by the saliva alone; by
the action of the saliva a reducing substance has been formed; finally,
if after a short time one adds to the mixed solution of starch and saliva
a solution of iodine in potassium iodide, it will be found that the iodine
no longer forms the blue color which it formed in the starch solution
before the addition of the saliva, but it either gives no color at all or it
develops a red color. These facts show that saliva changes both the
SALIVARY DIGESTION 327

chemical and physical properties of starch solutions and that the starch
disappears and a reducing substance appears in its place. If the starch
solution thus acted upon by saliva is tasted, it will be found to have
become sweet. From these observations we infer that saliva changes
starch into some kind of reducing sugar.

Nature of the changes involved in the digestion of starch.—The
further study of the changes which have occurred in the starch solution
have not yet succeeded in entirely clearing up the matter. The question
may first be asked, What is the nature of the sugar produced? This is
certainly in greater part the disaccharide, maltose, C,,H,,0,,. There
appears, however, particularly toward the end of the action, to be some
glucose present also. But besides the maltose there are also formed in
the course of the digestion substances of .a very much lower reducing
power, or of no reducing power at all, and substances which give with
iodine a red coloration, or no coloration at all. These substances are
called ‘‘ dextrins,’’ because they are dextro-rotatory. The name was
given by Biot, the father of polariscopic investigations. The dextrin
which gives with iodine a red coloration is called erythrodextrin, or
the red dextrin (Gr. erythros, red). The dextrin which gives no color
with iodine is called achroodextrin, or the colorless dextrin (Gr. achroos,
colorless). These substances are colloidal in nature. While all observ-
ers are agreed that these various substances are formed in the course
of the digestion, there is no agreement as to the exact order in which
they appear and their relation to each other. The difficulty in the way
of a solution of the problem arises in part from the difficulty of sepa-
rating quantitatively the various substances formed, but also because
the substrate, the raw material which is digested, is not a pure substance.
There is certainly in every starch grain some substance of the nature of
cellulose which is far more resistant to the action of the enzyme than
starch proper. Furthermore, the ferment solutions may contain more
than one enzyme. Probably a great deal of experimental work will be
required before this matter is entirely cleared.

The products of the digestion of starch by malt extract, which has a
digestive property similar to, but not in all points identical with, ptyalin,
were found by Musculus and Gruber to be the following:

(a) Soluble starch. This was insoluble in cold water, but soluble at 50°.
Todine colored its solution wine red, but the dry substance blue. The reducing power
was 6% of that of glucose. (a), = -+ 218°.

(b) Erythrodextrin. Soluble in cold water. Both when dry and in solution
gives a red color with iodine. Not a pure substance.

(c) Achroodextrin a. No color with iodine ( a), =+ 210°. Reducing power
12% that of glucose. Less easily changed to sugar than erythrodextrin.

(d) Achroodextrin, 8. (2), =-+ 190. Not digested by malt diastase. Reducing
power 12% that of glucose.
328 PHYSIOLOGICAL CHEMISTRY

(e) Achroodextrin, y. (a), =-+ 150°. Not digested by diastase. Reducing
power 28% that of glucose.

(f) Maltose, C,H, 0... (a), ==+150° Reducing power 66% that of glucose.

It is capable of fermentation oy yeast. It is not attacked by malt diastase.
(g) Glucose, UH U,. (@)p) =+ 56°. Fermentable. Reducing power 100%.

According to Brown and Herron, the simplest formula for soluble starch
is (C,,H,.0.9) 19. This was believed to split off maltose under the action
of malt diastase and to give erythrodextrin (C,,H,.0,,)9. This losing
another molecule of maltose went into a second erythrodextrin, and this
after the loss of another molecule of maltose went into achroodextrin.
The final mixture contained 81 per cent. of maltose and 19 per cent. of
achroodextrin. In their opinion the dextrins had no power of reduction
when they were entirely free from maltose.

Since the maltose comes off almost instantaneously when starch solu-
tion is mixed with saliva, it is generally believed that the course of the
reaction is a gradual etching away of the starch molecule by splitting
off successive molecules of maltose by hydrolysis, as pictured in the
following scheme:

Starch + H. = = Soluble starch + Maltose
Soluble starch + H.0 = Erythrodextrin + Maltose
Erythrodextrin + H,O = Achroodextrin a -+ Maltose
Achroodextrin @ + H,O == Achroodextriu 6 + Maltose
Achroodextrin 8 +H, “O) = = Maltose + Maltose.

We may accept this scheme as the best one possible with our present
knowledge, although there are many grave difficulties in the way of its
acceptance as a real picture of the process. The main fact is, however,
that dextrins and maltose are produced by the action of saliva on starch.
There is also in human saliva some maltase which converts some of the
maltose to glucose.

Ptyalin.—The question may now be asked, What is it in the saliva
which enables it to act thus on starch? A few simple experiments show
that the active substance is destroyed or loses its activity if the saliva
is heated, and that it is precipitated from the saliva by alcohol. It is
then not heat stable, and it is probably an organic substance. Since a
very little of it can convert a very large amount of starch into sugar
by hydrolysis, it evidently belongs in the group of substances known
as catalysts, or enzymes. It is called. ‘‘ ptyalin,’? a name given by
Berzelius to the peculiar organic matter of the saliva, but now confined
to the ferment. The word comes from the Greek, ptyalon, spittle.
Ptyalin is, therefore, the enzyme in saliva which digests starch.. It is
hence an ‘‘ amylase,’’ that is an enzyme which hydrolyzes starch, or
amylum. It is not certain whether there is but a single enzyme in
ptyalin. There may be several,—an amylase converting starch to dex-
trin, and a dextrinase which converts dextrin to maltose. There is hardly

wy
SALIVARY DIGESTION 329

a doubt that in the pancreas the amylase 1s such a mixture, and it is
probable that this is the case in the saliva also. Such amylolytic
enzymes are widespread in nature, being found not only in all vegetable
cells which contain starch, but also widely distributed in animals, being
found in the blood, saliva, pancreatic secretion, in the liver and prob-
ably in the muscle also. They are also very often present in moulds,
yeasts and bacteria, although common brewer’s yeast does not contain
amylase. These amylolytic (literally starch-loosening) enzymes are
sometimes called diastases in English, American and German litera-
ture, but the French use the term diastase as a synonym for enzyme in
general. The French usage is the more correct. The name diastase was
given to the starch-splitting enzyme in malt by Payen and Persoz to
indicate that it was different, or stood in a class by itself. The word
means ‘‘ to stand apart,’’ Gr. dia, apart, or through. With this confu-
sion in its significance it is perhaps better to drop the word entirely
and replace it by the word enzyme as the group name, and amylase as
a specific name of the starch-splitting ferments. Malt amylase was first
discovered by Dubrumfaut in 1823, and isolated and carefully studied
by Payen and Persoz in 1833. It was found by Kraussman and Krauch
in leaves; by Baranetsky in the tubercles of potatoes in repose; by
Duclaux in moulds such as Aspergillus niger. It was found in the saliva
by Leuchs in 1831, and extracted from saliva by Miahle in 1845; the
latter year the amylase of the pancreas was discovered by Bouchardat
and Sandras.

While all these different enzymes have in common a similar action in
producing a soluble sugar, maltose, from starch, they differ among them-
selves so that they constitute not one enzyme, but a group of enzymes.
The amylase of the saliva is much more sensitive to heat than that of
malt. The amylase of the pancreas, amylopsin, converts the starch into
dextrins more rapidly than the dextrins into maltose. And there are
other differences in their actions.

Composition and manner of action of ptyalin—The composition
of ptyalin has not been much studied, owing in large part to the diffi-
culty of procuring sufficient material and of separating the enzyme from
mucin. But the composition of other amylases, such as that from malt,
has been often investigated, as well as that from the pancreas. The best
method for the extraction from malt or the pancreas is to extract the
tissues or material with two to four volumes of dilute alcohol, 20-30
per cent., and then to-precipitate the amylase by the addition of three
volumes of absolute alcohol. This is the method followed by Lintner in
the study of malt diastase. It may also be extracted with water and
precipitated by saturating with magnesium sulphate. Still another
method was followed by Fraenkel and Hamburg. They fermented malt
330 PHYSLOLOGICAL CHEMISTRY

extract by yeast to get rid of any maltose or other fermentable sugar;
filtered from the yeast through a porcelain filter; precipitated the
protein very carefully by basic lead acetate; and fermented the filtrate
with a nitrogen-hungry yeast to free from the amino-acids and peptones:
again filtered through a porcelain filter; and evaporated to dryness
in a vacuum at a low temperature. The per cent. of composition of
various amylases obtained by various observers is as follows:

From Cc H N 8s Ash OandS P
Lintner Malt 44.33 6.98 892 1.07 4.79 34.98
Tegorow 40.24 6.78 4.70 0.70 4.60 4223 1.45
Osborne Hemp seed 52.50 6.72 16.10 1.90 0.66 22.78

Opinions vary widely as to the nature of the active principle. Arm-
strong has suggested that the enzymes are of the nature of the sub-
stances they act on, that glucoside-splitting enzymes, for example, are
glucosides. Lintner thinks that the active principle is a carbohydrate
and the nitrogen present in most preparations is an impurity. On the
other hand, there was no carbohydrate in Osborne’s preparation, so that
the active principle can hardly be a carbohydrate. Fraenkel and Ham-
burg found that their very active preparations gave no protein reac-
tions, except the faintest Millon and xanthoproteic. The carbohydrate
reaction was strong. They concluded with Lintner that it was a carbo-
hydrate. Wroblewski says that he has prepared a very active substance
from malt extract which gave no carbohydrate reactions, but was of the
nature of an albumose. The following facts, however, are admitted by
all investigators: Nearly all preparations, if not all, contain phosphoric
acid which it is very difficult or impossible to eliminate and to keep the
activity of the enzyme. There is a growing consensus of opinion that
in most enzyme decompositions, at least those of the carbohydrates,
phosphoric acid is playing some unexplained réle. There is practical
agreement, also, that generally the enzyme is found to be accompanied
by, or to be part of, a gum, and this gum contains phosphoric acid.
This gum is an araban, a pentose gum. In all organs from which amylase
has been isolated pentoses have been found on hydrolysis. All observers
agree that the active principle is a colloid. It will not diffuse. No
preparation of active amylase has been obtained free from nitrogen and
the protein reactions, whereas some have been obtained free from carbo-
hydrate reactions. In view of these facts we may tentatively conclude
that, while the nature of the enzyme is still quite unknown, the indica-
tions are that the active part of the molecule is a protein, probably
colloidal, and that this active principle is usually combined with a col-
loidal, carbohydrate gum. It is possible that it is only active when united
with the gum, but the probability is, the writer thinks, that the gum acts
wly as a bearer of the active principle, making it more stable, and that
SALIVARY DIGESTION 331

the principle is more active when free from the gum. In other words,
that the compound, gum-active principle, is the proenzyme, or zymogen
as it is called, from which the active principle may be set free.

There is no doubt that the active principle, whatever its nature, com-
bines with starch and other carbohydrates. This is shown by the fact
that it is much more stable in the presence of these bodies. The enzyme
is more resistant to heat, ultra-violet light, alcohol and other poisons when
carbohydrates are present in the solution.

Determination of the activity of amylolytic enzymes.—The activity
of any preparation of amylase may be measured in various ways. As
soon as the starch solution becomes clear, the further course of the
digestion may be followed by the diminution in the rotatory power. In
quantitative determinations by this method it is necessary to correct for
the mutarotation of the maltose by the addition of a little alkali before
polarization. The alkali stops the digestion also. Another method is to
fill glass tubes of an internal diameter of about 2 mm. with a thick
starch paste, cut the tubes in small lengths, place them horizontally in a
small flask with the solution of amylase to be tested and measure the
length of the starch column dissolved at different time intervals.
Another method is to determine the length of time which is required for
the blue iodine reaction of a starch paste of known concentration at a
known temperature and with a known amount of ferment to disappear.
This method is better than measuring the rate of increase of the reducing
power of the solution by Fehling’s solution, for the latter may involve
several enzymes, whereas the iodine reaction probably involves only the
amylase proper. Another method which again involves the dextrinases
as well as the amylases is to measure the rate of change of the viscosity
of the solution by a viscosimeter. In all these measurements, if the
activity of different preparations of the enzyme are to be compared, it
is necessary to be sure that the conditions of activity, such as the acidity,
proportion of inorganic salts, etc., are in all cases at the optimum for
the enzyme being tested.

Conditions of activity of the ptyalin—The optimum temperature for
the action of ptyalin or saliva on starch is 40-45°. The action is per-
manently lost if saliva is heated rapidly to 75°; and more gradually lost
if heated for longer periods at lower temperatures. While the reaction
of the saliva is very faintly alkaline to litmus, the optimum reaction for
the digestion of starch is a very faint acidity. Thus the addition of car-
bonic acid hastens the action, as illustrated by the table on page 332
(Chittenden and Painter). The degree of acidity must, however, be very
slight. The most favorable concentration of hydrogen ions was found
to be: NX10—-5%.

If more acid than this is present, if sufficient is present to turn congo
332 PHYSIOLOGICAL CHEMISTRY

red violet, a hydrogen ion concentration of NX10-+, the activity is
stopped. The digestion will still proceed in faintly alkaline solution, but
it is already partially inhibited if the reaction is alkaline to phenolphtha-
lein and stronger amounts of alkali quickly bring the reaction to an end.
How acids accelerate the ptyalin action, and they act in exactly this same
manner toward another enzyme, invertin, is not yet known. One sug-
gestion is that the acids form the active principle from the inactive
zymogen. Perhaps they set free the active principle from the gum; per-
haps they may act in other ways by combining with the enzyme, or the
starch. This matter has to be left for future investigation.

Starch changed Relative activity
When no gas passed through ........ 24.15% 100
PRAT carats seee capi Os diioec sadandbid vo No cobaves orate 25.02 103.6
oO Pe 27.72 114.7
co Pe 28.82 116.8
A 2° anid IS USS alate ahh oes w vein Oa wale laNeNer OHS 25.23 104.4
D steneste coamseirteiaiacaaaes om 22.86 94.6

The presence of some salt is necessary for the action. Carefully
dialyzed saliva loses much of its activity, and this is restored by the action
of common salt. Phosphates particularly favor some of the amylases.
How the salts act is not clear, but possibly they increase the fineness of
subdivision of the enzyme, just as they help to dissolve globulins, and
thus in effect increase its concentration. Salts of the heavy metals may
be very toxic. Thus uranium salts even in a dilution of .0001 per cent.
retard and 0.008 per cent. of urany! nitrate completely inhibits. Silver
nitrate and mercuric chloride are also highly toxic. The toxic action of
these salts indicates that the active principle is probably a protein. If
the acidity is already beyond the optimum, the addition of amino-acids
greatly improves the rate of digestion, since these substances combine
with the acids and thus help establish the optimum acidity. It will be
observed that the optimum conditions for the action cf saliva are those
present in the medium for which it was designed, if we may use a
teleological expression. When the food is swallowed we have in the
stomach a very faint acidity, some salts and proteins with their decom-
position products.

The products of digestion inhibit the action of ptyalin—As is the
case with most enzymes, the speed of conversion of the substrate is
reduced by the presence of the hydrolytic products of the digestion. It
will be found that if two starch solutions of the same strength and the
same temperature are taken and to one some maltose is added and not
to the other, and if to each the same amount of saliva is added, the one
without the maltose ioses the iodine-starch reaction before the one with
the maltose. The action of the ptyalin on the starch is reduced by the
SALIVARY DIGESTION 333

presence of the maltose. This fact of the retardation of the velocity by
the products of the digestion is generally true for all hydrolytic enzymes.
Now in the stomach and intestines this retarding action is avoided by
the reabsorption of the maltose by the walls of the intestine as rapidly
as it is formed so that it does not remain to vex the ptyalin by its
presence.

The effect of the removal of the maltose in increasing the speed of
digestion of starch is shown very clearly in the following experiment of
Lea, in which the course of the digestion of the starch was followed by
the iodine reaction. In one case the digestion was carried on in a beaker,
and in the other in a dialyzing tube immersed in running water so that
the maltose was dialyzed out.

 

 

 

Time Dialysed starch solution Not dialysed
11.00 Pure blue Pure blue

11.20 Trace of violet o ae

11.40 Red with violet tinge Violet

12.15 Colorless Red with violet
12.00 * Very faint red
1.15 : ¢. Colorless

 

This retarding action of maltose and other sugars on the rate of
digestion of starch by ptyalin was at first supposed to be due to the mass
action of the maltose. The reaction by which the digestion of the starch
is produced was supposed to be a reversible one.

(C,H, 0,,), +H,0 == » C,,H,,0

12 #20 10°n 12° 22°11
Starch. Maltose.

The addition of maltose might be supposed to reverse the reaction and
thus to check the rapidity of the disappearance of the starch. This
explanation is incorrect, or at least insufficient. The fact is that the
maltose combines with the enzyme, thus removing it from acting on the
starch. This can be shown by the addition of sugars other than maltose
to the reacting mixture. All will be found to retard the reaction, but
maltose and glucose retard it most.

Law of action of the ptyalin.—If a dilute solution of starch is used
and the enzyme added in more than a minimum amount, the rate of
digestion will be found to double with a doubling of the amount of
enzyme. In other words, the speed of the reaction goes proportional to
the concentration of the enzyme. Chittenden and Smith, using 100 c.c.
1 per cent. starch solution with varying amounts of saliva which had
been diluted 50 to 100 times, obtained in 30 minutes at 40° C. the follo77-

ing per cent. of sugar:
Saliva .......2-- 0.5 c.c. 1.0 ce. 2.0 c.c.
Sugar per cent. 3.60 7.23 16.92
334 PHYSIOLOGICAL CHEMISTRY

The increase in sugar was nearly proportional to the increase in the
saliva. If, however, tubes of starch paste are used, so that the enzyme
must always act on the starch in the presence of a strong solution of the
products of its activity which diffuse away very slowly, then the rate
goes approximately proportional to the square root of the concentration
of the enzyme. That is, if of two tubes one is placed in an enzyme
solution four times as concentrated as the other, the rate at which the
starch is digested in the two tubes will not be as one is to four, but
approximately as one is to two. The rate will be doubled, not increased
four times. One will digest two millimeters while the other is digesting
one. This is called the law of Schiitz and Borissow. Amount digested

+ by the time = K VCrement Crerment iS the concentration of the ferment.

Time of appearance of the ptyalin in development.—Only the parotid
of the new-born contains amylase; the submaxillary lacks this action.
It appears in the submaxillary and the pancreas only at the end of the
second month of life. At birth the activity of the parotid is little more
than that of many other tissues.

Variation in different types of animals.—Not all salivary glands have
in them ptyalin or an amylase. There is very little or none present, for
example, in the saliva of various carnivores, such as the dog and cat.
The parotid glands of rodents are said to be most active. In the horse
mixed saliva is said to have a very powerful diastatic action, whereas
saliva collected from the parotid ducts is said to be inactive (Ellenberger
and Hofmeister). There is a possibility that a co-ferment or kinase is
necessary for the action of the enzyme and this may be absent in some
glands. The late Dr. G. W. Cook told the writer that if the human
mouth is carefully washed out with a sterile solution of water, or dilute
antiseptic, the saliva collected from the ducts may be inactive, whereas
the saliva which has been in contact with the mucous membrane of the
mouth is very active. It is possible that bacteria are necessary for the
setting free of the amylase, as Ellenberger and Hofmeister suggested for
the horse, or it may be that the mucous membrane contributes a kinase
to assist in the action. This matter should be carefully investigated. It
might clear up some of the contradictory statements in the literature.
Various observers have found that a carbohydrate diet increases the
amylase and maltase content of the saliva of human beings and dogs.
Others have failed to find such an increase.

Other enzymes in the saliva.—Besides the amylolytic enzyme, human
saliva contains a substance, an erepsin, which will split the tripeptide,
1-leucyl-glycyl-d-alanine and probably other peptides; and maltase, an
enzyme which will convert maltose into glucose, the amount of which is,
however, small and its origin unknown. It may be derived from the
maltase of the blood. Saliva also contains catalase, which catalyzes
SALIVARY DIGESTION 335

hydrogen peroxide setting free oxygen; and an oxidase. Saliva blues
guaiac tincture, and develops color with p-phenylendiamin, a-naphthol
and m-toluylendiamin. It is probable that other enzymes in small quan-
tities will be found in it.

Potassium sulphocyanate and other excretory substances.—Potas-
sium sulphocyanate is found in most human saliva in small quantities.
It is generally present in larger amounts in the saliva of smokers shortly
after smoking. It is in the nature of an excretory substance and is not
supposed to have any function. Whenever cyanides are ingested, as
they are in some fruits or in tobacco smoke, they are excreted as sulpho-
cyanates, which are excreted in practically all of the secretions. It is
possible, also, although it has not been proved, that hydrocyanic acid
may be formed in small amounts in the oxidation of protein in the body,
since nitriles appear in the oxidation of proteins by permanganate.
Many other excretory substances may be found in the saliva in larger
or smaller amounts, depending on their concentration in the blood. Thus
urea, uric acid, small amounts of glucose and other excretory sub-
stances have been isolated from saliva. Iodides pass out in the saliva
very quickly and, since nitrites may also be excreted here or be formed
in the mouth by the action of bacteria from nitrates excreted in the
saliva, the iodine of ingested iodides may be set free and produce irri-
tation of the throat and mouth.

Importance of salivary digestion.—The importance of the digestive
action of the saliva on starch was long underestimated, for it was thought
that the action was checked at once on entrance of the food into the
stomach. We now know that this is not the case, but that the food
collects in the fundus of the stomach in a large mass, in the interior
of which the reaction is very faintly acid, the optimum conditions of
ptyalin action prevail and that for a half hour or longer, depending
on the size of the meal, the ptyalin may continue to digest the starches.
Its action is permanently checked as soon as the hydrochloric acid
accumulates sufficiently to give a reaction with congo for free acid. In
another way, also, the saliva may be an important aid to digestion. It
is stated by several competent observers that the food matters when
thoroughly mixed with saliva digest very much faster with gastric
juice than food matters not so mixed, and this is not supposed
to be due entirely to the finer state of division of the food. The pres-
ence of saliva, or its digestive products, may also act as a stimulus
to the secretion of gastric juice. For all these and ether reasons a
thorough mastication of the food undoubtedly conde to good
digestion.

Composition and metabolism of the salivary glands. —The ecompo-
sition of the salivary glands has been very little investigated, and what
336 PHYSIOLOGICAL CHEMISTRY

is known about them throws very little light on their metabolism.
Besides the mucin which the sublingual and submaxillary glands con-
tain in small but undetermined quantities, they contain also a nucleo-
protein and nucleic acid. Xanthine and guanine have been isolated from
a nucleoprotein which is obtained by extracting the mucin first by
water, and then the nucleoprotein by dilute alkali, such as lime water, and
precipitating by acetic acid. The nucleoprotein thus obtained (Holm-
gren) contained 15.11 per cent. of N and some phosphorus. On peptic
digestion it yielded a nuclein containing 2.9 per cent. P. It is said
(Horsley) that after thyroid extirpation in apes mucin is increased in
the glands, and even appears in the parotid gland where it is normally
absent. Ptyalin or a ptyalinogen exists in unknown amounts in the
parotid gland.

The consumption of oxygen by the gland increases when it is in a
state of activity (Barcroft). Like the other glands, it has an unusually
high rate of oxygen consumption per gram of tissue: namely, .028 c.c.
O, per gram per hour. Muscles use .004 c.c. O, per gram per hour.

REFERENCES. Satrivary DIcEstion.

1. Composition of mucin. Levene: Zeit. f. physiol. Chem., 31, 1900, p. 395.
Richard and Gies: American Journal of Physiology, 7, p. 93, 1902.
2. Amylase. Discovery in malt. Payen and Persoz: Mémoir sur la diastase. An-
nales de chimie, 53, 1883.
Discovery in saliva. Leuchs: Kastner’s Archiv f. d. ges. Naturlehre, 1831.
8. Amylase. Composition. Wroblewski: Ber. d. d. chem. Gesell. 31, p. 1128, 1898.
Osborne: Jour. Amer. Chem. Soc., 17, p. 587, 1895.
Fraenkel and Hamburg: Hofmeister’s Beitrige z. chem. Physiol. 8, p. 389, 1906.
4. Digestive decomposition of starch. Musculus and Gruber: Zeits. f. physiol.
Chem., 2, p. 177, 1878.
Brown and Millar: Jour. of the Chem. Soc., 75, 1899.
Moritz and Glendenning: Jour. Chem. Soc., 61, 1892, p. 689.
Nasse: Pfitiger’s Archiv f. d. ges. Physiol., 14, p. 473, 187.
v. Mehring: Zeit. f. physiol. Chem., 5, 1881, p. 185.
Brown and Herron: Liebig’s Annalen, 199, 1880, p. 65.
5. Effects of temperature. Chittenden and Martin: Transactions Connecticut
Acad. Sci. 7, 1885.
6. Effects of acids and other substances on speed of digestion. Chittenden
and Ely: Jour, of Physiol., 3, p. 327, 1882.
Wood: Amer. Chem. Jour., 15, 1893, p. 663; 16, 1894, p. 313.
Cole: Jour. of Physiol., 30, p. 202, 1904.
McGuigan: Amer. Jour. Physiol., 10, p. 444, 1904.
Chittenden ond Smith: Trans. Conn. Acad. Sei., p, p. 348, 1884,
Inhibiting action of digestive products. Lea: Jour. of Physiol., 11, p. 226,
1890.
8. Proteolytic enzyme in saliva. Koelker: Jour. Biol. Chem., 8, p. 145, 1910.
Warfield: J. Hopkins Bulletin 22, 1911, p. 100.
Koelker: Zeit. f. physiol. Chem., 76, 1911, p. 27.

~
SALIVARY DIGESTION 337

9. Amylase in saliva of carnivora. Carlson and Ryan: Amer. J. of Physiol., 22,
1908, p. 1.

10. Excretory substances in saliva. Uric acid. Boucherson: C. R. de Acad. de
Sci., Paris, 100. p. 1308

ll. Activity of mixed saliva .. horses. suwenoerger and Hofmeister: Arch. f
wiss. u. prakt, Thierheiikunde 12, p. 265. 1881.
CHAPTER IX.
DIGESTION IN THE STOMACH.

After a more or less thorough mastication and mixing with the saliva,
the food is swallowed. It passes down the esophagus into the stomach,
where it remains for a period of from one to five hours, undergoing a
process of solution by the action of the juices secreted by this organ. At
first it lies in the stomach in a mass in the left side or fundus of the
stomach. Small portions are gradually separated from this mass by the
contractions of the stomach, partially digested in the pyloric region,
and as soon as they are reduced to a state of fine division or solution
passed on through the pylorus into the intestine for further digestion
and absorption there. We may now proceed to examine in detail the
nature of the processes involved in gastric digestion.

Morphology.—The stomach is an enlargement of the alimentary
canal found in all vertebrates, which functions both as a reservoir and
as a digestive organ. It has an acid secretion and contains hydrochloric
acid. The shape of the stomach differs in different animals, varying
from a slight tubular enlargement of the alimentary canal, such as is
found in the frog, to a many-chambered organ. In most of the verte-
brates, including the mammalia, and particularly in the Carnivora and
Primates, there is only a single cavity, but this may be partially sepa-
rated into two by a muscular constriction, more or less pronounced,
between the fundus and the pyloric portions. The mucous membrane
of these two portions is often plainly different in its macroscopic appear-
ance, and histological examination shows it to be different, also, in its
finer structure. The glands in the pyloric region have in them none of
the so-called parietal cells. There is also a difference in the chemical
nature of the juice they furnish.

The most complex form of stomach is found in ruminants. In cows
and other animals of this class the stomach consists of several chambers.
These divisions are called respectively the paunch, or rumen, which
receives the food as it is first swallowed; the reticulum; the omasum, or
psalterium; and the abomasum. The first two cavities have an alkaline
secretion and here the digestion is carried on by means of bacteria and
saliva. When the bolus of food, after regurgitation from the reticulum,
is chewed and reswallowed, it passes into the second stomach, or psal-
terium, and this corresponds to the fundus portion of the human stomach,

338
DIGESTION IN THE STOMACH " 336

the paunch and reticulum corresponding to an enlargement of the
cesophagus. The psalterium and the abomasum, or rennet sack, have
an acid secretion like the human stomach. The fourth stomach, or
abomasum, corresponds to the pyloric end of the human stomach. In
some of the ruminants there is, in addition, a division of the paunch
for carrying water. This is brought to a high state of perfection in the
2amel,

When empty the human stomach is normally contracted on itself so
that its walls are in contact and the reaction of its mucous membrane is
either very faintly acid or even neutral. When water is swallowed into
an empty stomach it runs rapidly throngh the stomach into the intestine,
where it is absorbed. But when food is swallowed the stomach begins
to relax and it relaxes sufficiently to adapt itself to the amount of food
taken, the powers of relaxation being really remarkable. But while the
stomach is normally contracted and empty between meals, it sometimes
happens that it re-expands, after emptying itself, and becomes filled
with a juice acid in reaction. Such a dilated stomach is a source of much
discomfort, and is pathological.

General Physiology of the Human Stomach.—What actually hap-
pens in the human stomach when food is swallowed and during diges-
tion was first accurately observed by the American army surgeon, Dr.
Beaumont, in the stomach of the Canadian coureur du bois, Alexis
St. Martin. These observations, though ‘old, are still in many respects
the best observations on the human organ, and they have since been
many times confirmed. The nature of the wound in St. Martin and his
robust health enabled a very careful scrutiny of a perfectly healthy
organ. Alexis St. Martin was wounded in the left side by the accidental
discharge of a musket close to him. The charge carried away some of
the lower rib, and a part of the wall of the stomach and abdomen. The
injured lung protruded from the wound. On healing, the wall of the
stomach and the abdominal wall grew together so as to leave a hole or
fistula in the fundus part of the stomach, through which food could be
introduced, or gastric juice extracted, and the inside of the stomach
directly observed. After a while a flap of mucous membrane grew
down over the wound and acted as a valve, preventing the escape of the
contents of the stomach, unless the valve was pushed to one side. The
accident occurred on the island of Mackinac, at the head of Lake Michi-
gan, in 1822. The post surgeon was Dr. Beaumont and he at once seized
this most fortunate opportunity for a study of gastric digestion. He
hired St. Martin to submit to observation and he made out many of
the fundamental facts of our knowledge of stomachal digestion. He
observed that the mucous membrane of the empty stomach was pale
pink and covered by a thin laver of mucus of a neutral or slightly
340 PHYSIOLOGICAL CHEMISTRY

alkaline reaction, but that as soon as the stomach was excited, either by
the introduction of food into it or by mechanical irritation, the mem-
brane became a bright red and appeared to be gorged with blood. Its
temperature rose a degree or so at the same time, and if it was watched
it could be seen that at the openings of the stomach glands clear drops
of colorless juice welled up and, running together, trickled down the
sides of the organ. His description of the organ was as follows:

‘“ The inner coat of the stomach, in its natural and healthy state, is
of a light or pale pink color, varying in its hues according to its full
or empty state. It is of a soft or velvet-like appearance, and is con-
stantly covered with a thin, transparent, viscid mucus, lining the whole
interior of the organ. Immediately beneath the mucous coat, and
apparently incorporated with the villous membrane, appear small
spheroidal- or oval-shaped granular bodies, from which the mucous fluid
appears to be secreted. On the application of aliment the size of the
vessels 1s increased, the color heightened and vermicular movements
excited. The gastric glands begin to discharge a clear, transparent fluid,
which continues rapidly to accumulate as aliment is received for diges-
tion. This fluid is invariably distinctly acid. The mucus of the stomach
is less fluid and more viscid, and sometimes a little saltish, but does not
possess the slightest character of acidity. On applying the tongue to the
mucous coat of the stomach in its empty, unirritated state, no acid taste
can be perceived. When food or other irritant has been applied to the
membrane, the acid taste is immediately perceptible.’’

By irritating the stomach with a feather, a thermometer tube or a
rubber catheter he got severa! ounces of juice secreted and collected for
examination. The human gasirie juice thus obtained was a perfectly
clear, generally colorless solution, like so much water in appearance,
except that it was now and then mixed with some bile. It had in it a
little mucus. It was salty and sour to the taste, turned purple cabbage
red and effervesced with carbonates. Some of the juice was analyzed by
Professor Silliman, of Yale University, who found that it contained a
good deal of hydrochloric acid. Beaumont found that the juice thus
collected had the power of dissolving meat put in it when kept warm
outside the body, and he thus confirmed Spallanzani’s observations of
the: chemical or solvent nature of the juice. The collection of several
ounces of juice was accompanied by no other sensation than that of
faintness and a sinking at the pit of the stomach. Among the other very
interesting properties of this Juice was its power of preventing putre-
faction. Meat in it did not putrefy; it had a preservative action, and
Beaumont found that applied to wounds it kept them clean and fresh,
helping them to heal by first intention and stopping suppuration.

He also examined the time which it took to digest meals: i.e., the
DIGESTION IN THE STOMACH 341

time necessary for the stomach to empty itself after a meal. He found
that pork took longer to digest than beef, and in general fatty food took
longer than lean. Thus after a meal of mutton chops and potatoes the
stomach was empty in two hours, whereas after a hearty meal of pota-
toes, roast pork, vegetables and pudding, it took four or five hours to
empty itself. Among the most valuable of Beaumont’s observations were
those on the optical appearance of the mucous membrane under varying
conditions of health, after drinking ardent spirits and coffee, and during
constipation. He was able to see what effect drugs had on the mucosa
and, above all else, he was able to find out to what extent the condition
of the mucosa could be inferred from external symptoms. Thus he
found that after hard drinking of ardent spirits the mucosa was inflamed,
small ulcers appeared in it and it secreted a large amount of mucus,
but a very small amount of gastric juice, and this almost without
digestive action. The same conditions prevailed in fever, so that gastric
digestion almost ceased. In constipation, too, when the tongue was
coated, the mucosa was bright red, dry and secreted little juice. All
of these symptoms could be changed in the latter case by the use of a
good cathartic, such as calomel, the mucosa recovering its normal condi-
tion. Particularly instructive was the fact that the condition of serious
disturbance of the mucosa could not be inferred from any outward
symptoms.

Beaumont also tried many other experiments. He introduced various
foods, such as vegetables or meat done up in cloth bags, into the stomach,
leaving them there for some time and afterwards withdrawing and exam-
ining them to see what substances would be affected by the gastric diges-
tion. Beaumont did not observe any secretion of gastric juice in St.
Martin purely as the result of smelling food, or tasting it, or from the
.- fact that it was meal time. It secreted only when he ate or when the
stomach was mechanically irritated. He observed no appetite juice, in
other words.

It seems hardly probable that, had there been such an appetite secre-
tion, it would have escaped so acute an observer, but apparently it did
not occur to him‘to try to see whether the mere sight of food would
stimulate the secretion. Pawlow has questioned whether the juice Beau-
mont got was due to mechanical irritation and has suggested that it was
in reality an appetite juice due to nunger, but there is no reason to
suspect that Beaumont’s observations were faulty in this particular.
Pawlow’s criticism was based chiefly on the behavior of the dog’s stomach.
Richet in another case of gastric fistula in a human being later showed
that it was not necessary for the food to enter the stomach, but that the
taking of sugar or other foods into the mouth would start the secretion

of juice.
342 PHYSIOLOGICAL CHEMISTRY

Beaumont also made a suggestion as to the nature of the stimulus
leading to the sensation of hunger. He suggested that the turgidity of
the gastric glands caused this sensetion, which, if it be acute, is often
ascribed to the pit of the stomach. This suggestion remained the most
probable until the recent observations of Boldyreff, and those of Can-
non, and later of Carlson, that a sensation of hunger is accompanied
by, and these observers believe is caused by, the rhythmic contraction
of the empty stomach.

Manner of obtaining gastric juice—a. From fistulas in human
beings. Human gastric juice unmixed with food has been obtained since
Alexis St. Martin from several individuals in whom, owing to a blocking
of the esophagus, a fistula or artificial opening had been made into the
stomach for the purpose of feeding. Among the commonest causes of
such strictures of the esophagus which prevent swallowing have been
the drinking of caustic liquids, such as strong acids or alkalies, either
accidentally cr purposely. It may happen that the health of such per-
sons is not very good, so the composition of the juice obtained from them
may not be altogether normal. An examination of such cases shows,
as with St. Martin, that the stomach when empty is contracted and
contains no juice, but only an alkaline mucous secretion. They can when
hungry often cause the stomach to secrete by chewing food. This
appetite secretion, as it is called, has provided samples of pure gastric
juice of human origin for examination.

b. By stomach tube. Impure juice mixed with saliva and food rem-
nants can be obtained from normal individuals either by causing vomit-
ing or drawing off the stomach contents with a stomach tube after eating
a meal. This is the clinical method for determining whether the secre-
tion is normal in amount and quality. The test meal, as it is called,
is taken in the morning when the stomach is empty, only a light supper |
or none at all having been eaten the evening before. There are several
test meals. That of Ewald consists of a buttered roll and a cup of weak
tea; another such breakfast consists of a roll or a couple of pieces cf
toast, a glass of water, or a cup of tea, and some bacon. This is thor-
oughly chewed. After three-quarters of an hour a stomach tube, a
flexible rubber tube, is swallowed and a portion of the contents are
drawn out. (See page 972 for method.)

e. From animals by artificial fistulas. To obtain juice from animals,
the method usually employed is to make a gastric fistula: i.e., an opening
is made into the stomach, generally at the fundus end, and the walls
sewed into the abdominal walls, leaving an opening into the interior. If
the walls of the stomach are brought out between the muscle fibers, the
pressure of these may act as a valve and prevent the escape of gastric
contents. The gastric juice flows from the cannula introduced into the
DIGESTION IN THE STOMACH 343

fistula when the dog is teased with the sight and smell of food or when
he is fed pieces of meat. Such juice may have saliva mixed with it.
A great step forward in the study of gastric secretion was taken by
Pawlow in his introduction of the cesophageal fistula and his formation
of a small stomach pouch separate from the main stomach. In making an
wsophageal fistula the esophagus is divided and the two ends are then
sutured to the skin. The result is that when a dog eats, the swallowed
food falls out of the upper end of the fistula and neither it nor the
saliva enters the stomach. Meanwhile, from the stomach fistula juice
may be drawn off if it is secreted.

1. Appetite secretion. Richet discovered in human beings that it
was not necessary to swallow food to arouse the stomach to activity.
The taste or smell of food was sufficient to provoke a flow. Pawlow
found the same facts for dogs. Advantage may be taken of this fact
to procure pure juice. It is only necessary for hungry dogs to see or
smell solid food, or even to hear some sound, such as a special note, which
had always been sounded as they were about to be fed and so had become
associated with the idea of feeding, to start the stomach secreting. This
is the appetite secretion and it begins after a very definite latent period
of about five minutes if the dog is hungry. There is no appetite secre-
tion if the dog is not hungry.

2. Juice obtained by sham feeding. Another method of getting the
juice is by means of sham feeding. This provides large amounts of juice.
A dog with an esophageal fistula as well as a gastric fistula is given meat
to eat. Ie swallows it, but it falls out of the opening in the esophagus
A dish put under the cesophageal fistula receives the swallowed food and
the dog will eat it over and over again. By this means a constant stim-
ulus is provided to the taste buds in the mouth and to the gums aad teeth
and the nerves of smell. Under this stimulus the stomach secretes a con-
siderable amount of juice, as may be seen in the experiment, page 344.
By this means pure canine gastric juice was obtained. It is a clear,
limpid liquid, looking like water, but having a doggy smell which is dis-
tasteful to human beings. This smell.is, however, eliminated by aérating
the juice, and such juice, acid and very active, is now obtainable in
Russia for therapeutic purposes. The dogs kept for this purpose are
large and healthy. They are fed two hours a day in this sham feeding
and work cheerfully at producing gastric juice. The flow continues
only during the eating, and grows less as the sham feeding proceeds.

d. The stomach pouch. By the foregoing method it was possible
to obtain pure juice and to study its secretion in the absence of food
in the stomach. It was very desirable to study also the effect of having
food in the stomach on the composition and amount of the juice, and
particularly to see whether the character of the juice remained the same
344 PHYSIOLOGICAL CHEMISTRY

no matter what kind of food was eaten. To do this and yet get juice
free from food admixture, Pawlow devised the small separated stomach

e

   

I rose
1 Muscularis

 
   
   

Mycoses
Pytorus

Plexus gastricu;
anterior vagy

 

Plexus gastricus
posterior vegs.

  

dy 2
Fie. 41.—Manner of making a Pawlow pouch. The incision is made along the line

A—B (1). The complete pouch is shown in (2) ; its cavity, 8, is separated from the general
cavity of the stomach, V, by a double layer of mucous membrane (Pawlow).

or stomach pouch, which in its processes completely mirrors the large
stomach which has the food in it.

SHam FEEDING EXPERIMENT.
Amount of juice collected

Time from stomach

12h 5’ 3.2 cc. Before feeding.
15 3.4
20 3.5
25 4.0
30 3.0
35 2.4
45 1.8 Meat feeding begun.
50
55 10.8

1 h. 00 15.4
05 17.8 Stopped feeding.
10 16.0
15 12.0
20 10.8
25 8.6
30 7.6
35 6.2
40 6.2
45 2.8

The idea of cutting out a part of the stomach and sewing it into a
pouch discharging its secretion to the exterior was not original with
Pawlow. It had been tried by Heidenhain and by Klemenciewicz. They
made the mistake, however, of cutting clear through the walls of the
DIGESTION IN THE STOMACH 345

stomach and thus cutting off all the nerves to the small pouch. The
secretion they obtained was on this account far from normal. Pawlow
made the pouch in another way so as to leave the nerve supply intact.
The method is illustrated in Figure 41.

The nerves of the stomach are the two vagi which come down the
cesophagus and spread out, one on the posterior and one on the anterior
surface of the organ. They run on the surface and then plunge into
the muscular coat to get to the mucosa. There are, also, sympathetic
nerves which come in from the splanchnic nerves from the pylorus end
of the stomach. To make the small pouch, a V-shaped cut is made
completely through all the coats of the stomach in the direction and
location shown in the figure. The mucous membrane and the muscular
wall are now sewed together in such a way that a complete floor is made
for the large stomach, so that food passes normally through it and into
the pylorus. The V-shaped piece is now sewed into a pouch and the
end of it brought to the surface, passing through the muscles of the
abdominal wall, the muscle fibers being separated and not divided. They
thus press the mouth together and act as a sphincter. This little pouch
has its vagus connections intact and it is separated from the large
stomach by a double layer of mucous membrane. If desired, a fistula
can also be made into the large stomach. Various foods can now be
allowed to enter the large stomach, and the amount and character of the
juice secreted by the small stomach under these varying conditions can
be studied. It was found that the secretion in the large and small stom-
achs began and stopped at the same time, and that the introduction of
a large amount of food into the large stomach caused the secretion of
a large amount of juice in the small, and that when the amount of food
was halved, the amount of secretion in the small stomach fell to one-
half. This established the very important fact that the amount of juice
secreted is normally proportioned to the amount of food to be digested
and that the small stomach was a faithful image of the secretion in the
large stomach.

Character of pure gastric juice of the dog.—The pure gastric juice
obtained from dogs or human beings, when unmixed with bile, is a per-
fectly clear, limpid, not viscid, colorless juice. Its specific gravity lies
between 1.002 and 1.0059. It is levo-rotatory. In a decimeter tube
the juice collected by Madame Schumova-Simonowski rotated —0.7-
—0.73° ; that collected by Rosemann, —.109°- —.168°. The freezing point
was -—.490-0.638°. The freezing point of the first juice secreted was
—0.643°, and after two hours —.628°. As the freezing point of the blood
is generally —0.571°, the juice is generally hypertonic, not hypotonic,
to the blood: The secretion of the gastric juice raises the concentration
of the blood when the juice is discharged to the exterior through a fistula.
346 PHYSIOLOGICAL CHEMISTRY

Thus in an experiment of Rosemann the freezing point of the blood
before secretion was —0.589° and after —0.600°. The dry residue is
0.26-0.653 per cent. The ash was 0.133 per cent. (0.105-0.204 per cent.).
The organic matter varied from 0.176-0.484. The ash contained iron,
calcium and phosphoric acid, and the usual salts of blood serum. The
organic substance gives both the Millon and biuret test, although Madame
Schumova-Simonowski stated that it did not give the biuret test when
it was perfectly fresh, but only after standing. Ammonium chloride is
present only in small amounts. There was no lactic acid. The acidity
computed as hydrochloric acid was from 0.46-0.58 per cent. The juice
contained, as a mean, 0.6137 per cent. of chlorine. Of this 0.5322 per
cent. was present in hydrochloric acid; 0.0653 per cent. was in the ash
as chlorides; and the remainder was 0.0162 organically combined. Since
the per cent. of chlorine in dog’s blood is 0.295-0.275 per cent., the
gastric juice is twice as concentrated in chlorine as the blood. During
secretion, therefore, on one side of the mucous membrane the concen-
tration of the chlorine is 0.54-0.64 per cent; on the other, 0.41 per cent.
in the plasma; and in the membrane itself 0.09 per cent.

The hydrogen ion concentration of pure human gastric juice from a
ease of stomach fistula was found by Dr. Menten in the author’s labo-
ratory, using the gas-chain method, to be about equivalent to that of
N/10 HCl, or even a little more acid than this. In the stomach, how-
ever, the juice is generally mixed with food substances so that its acidity
is lower than this. See page 368.

COMPOSITION OF GASTRIC JUICE.

Dog (Rosemann) Human (Bidder and Schmidt)
Water wos siewenaaesas 99.74-99.36 Water ........ ..... 99.44
Dry residue .......... 0.26- 0.64 Organic ............ 32
Organic: sy cssescics wees Oi 0.43 HOD ac cevec eek es 0.20
Inorganic ............ 0.10- 0.21 CaCl, 21S denver vaneeteaynass 0.0061
Hydrochloric acid ..... 0.46- 0.58 NaCl ............... 0.146
Chlorine .............. 0.61 KRG). wescgioee ca tees 0.055
Chlorine in HCl ...... 0.53 NE GL oesccaiveacnee. —
Chlorine in chlorides .. 0.077 Ca, (PO)
Chlorine in organic .... 0.016 Mg, (PO), Sead 0.0125
ABS ccicschse cages 42 ayes 0.127 Fe PO,
Water sol, ash ...... 0.120 Human Appetite Juice (Carlson)
ING) senor Meco costs 0.025 Sp. gravity ..... 1007
TR sees Caagtaca-d Reed 0.030 Total acidity ... 0.45 - 0.50
OD ccsud a aavpnea tears 0.067 Freezing point... 0.580 - 0.530
so, doo intsedantianeecatai ety 0.0012 Total solids .... 0.48 - 0.61%
Insol. ash .......... 0.0023 Organic ...... 0.34 - 0.47
Ca. Saal xs Gree 0.00022 Inorganic .... 0.11 -0.14
Mg SOUS SEES RR Soe 0.00022 Free acidity .... 0.85 - 0.45
BO, verre rece eee 0.0006 Total chlorine... 0.49 - 0.56%

BOs pieises oavilarae iees Trace NES ig Setar: 0.051 - 0.074%
DIGESTION IN THE STOMACH 347

Amount of gastric juice secreted—The amount of juice secreted
is subject to great variations, depending on the amount of food eaten,
the state of health and so on. By means of the small stomach pouch just
described Pawlow found that the amount secreted by the small stomach
was directly proportional to the amount of food eaten. If he doubled
the size of a meal, the amount of juice was doubled. This proportion-
ality is of course true only within limits. It was estimated by Bidder
and Schmidt that in human beings 2-3 liters of gastric juice were secreted
per day, and this is probably not far from the truth. In an experiment
by Rosemann the stomach of a large dog weighing 24,100 grams secreted
during a sham feeding in the first two hours at the rate of 200-300 e.c.
per hour; it then fell to 100 c.c. per hour. It was found by Pawlow
that when food entered the stomach the secretion was larger than when
there was only a sham feeding. We may, perhaps, estimate that in this
dog at least 500 ¢.c. of juice would be secreted for the digestion of each
meal. If a man’s stomach secretes at the same rate, this would give
about 1,500 ¢.c. for the digestion of a meal.

In these fistula dogs the amount of chlorine and water secreted in
the juice may form a very considerable proportion of that contained
in the whole body. Thus in the dog experiment just quoted the total
amount of juice secreted in the sham feeding in 2% hours amounted to
one-half the total volume of the blood in the body. The secretion of this
amount of liquid caused great thirst. In various researches Rosemann
found the total chlorine secreted in 31% hours to be from 4.4-5.5 grams.
In a dog weighing 26 kilos there are in the blood about 5.4 grams of
chlorine, so that as much chlorine was secreted in the gastric juice as
the total amount present in the blood. Such a loss of chlorine would
almost certainly affect the activities of all the cells of the body. The
total chlorine in the body of a dog of this weight was estimated as 20.8
grams, so that approximately one-fourth of it was eliminated by the
gastric juice in three hours.

The gastric juice of human beings contains on the average about 0.3
per cent. of hydrochloric acid; that of dogs about 0.53 per cent. If a
liter or 1,500 ¢.c. of such juice is secreted in each person for the diges-
tion of a meal, this would mean the secretion of from three to five grams
of hydrochloric acid each meal time. Since this acid is poured out
before the secretion of the alkaline bile and pancreatic juice, it causes
a reduction in the acidity of the other juices of the body and an increase
in their alkalinity. It produces, in other words, an alkaline tide
in the body Thus the urine is always reduced in its acidity during
the digestion of a meal and may even become alkaline. It is not
mmpossible that this alkaline tide may be part of the cause of the sen-
sation of well-being which accompanies the eating of a meal. A
348 PHYSIOLOGICAL CHEMISTRY

slight change of alkalinity of the cells very materially affects their
activity.

Variation of the character of the juice with the diet—Among the
interesting facts found by Pawlow and his pupils by the use of the smal]
stomach was that the character of the juice varied with the character of
the diet. The juice was most powerfully proteolytic when the dog had
bread to eat and weakest when fed milk. The figures on page 380 illus-
trate the variation of the juice. It was also found that a sudden increase
in the rate of secretion not only increased the rate but also the concen-
tration of the juice, confirming a fact observed by Heidenhain. The
explanation of the difference in the activity of the juice on these different
diets has not been given. The juice which is secreted at the beginning
of an experiment is usually more active than that after the secretion has
gone on for a time, presumably owing to the fact that during secretion
some of the stored pepsinogen has been exhausted. Some of the varia-
tions noted may be due to the fact that not all the glands of the stomach
secrete at the same time. It is probable that the number entering into
activity may vary with the size of the meal; some may be at rest while
others are secreting. If now by giving meat one arouses those which
have been resting, it is possible that their secretion would be more con-
centrated and more active than the secretion which has been derived
from the glands which had hitherto been in activity and which had been
partially exhausted.

How is the secretion of the juice produced and controlled? Gas-
tric hormones.—There is no doubt that the secretion of the gastric juice
is under the control of nerves, the vagi and the splanchnics. The stimu-
lation of these nerves under proper conditions causes secretion of the
juice. Secretion is greatly reduced in the dog after section of the
nerves. If we ask the further question of how the nerves cause secre-
tion, we ask a question not yet answered. There seems to be evidence
that either owing to the stimulation of the mucosa by the nerves or other
agencies, i.e., the food, there is produced in the stomach cells a substance
which when injected into the blood of another animal or intramuscularly
causes the secretion in that animal of gastric juice. Such a substance
as this is called a hormone, meaning ‘‘ I rouse to activity ’’ (Gr. hormén,
I rouse, or set in motion). As we shall see in other cases, there is some
reason for thinking that such substances are normally produced perhaps
in all cells under the influence of nerves and it is these substances which
directly stimulate the cell to activity. The work on the gastric hormones
is yet incomplete, but the observations of Edkins that such gastric hor-
mones exist have been confirmed in the author’s laboratory (Keeton and
Koch). These hormone substances in the gastric gland are extracted
from the mucosa by hot acid and are hence probably substances of a
DIGESTION IN THE STOMACH 349

basic nature. They are heat stable. The gastric hormone does not cause
secretion in the salivary glands, but only in the stomach. The question
of the relation of gastrin to secretin, the active secretory hormone of
the intestine, is still uncertain. They appear to have many properties
in common. Gastrin injected subcutaneously into dogs having Pawlow
pouches causes a copious secretion of typical gastric juice in the pouch
(Keeton and Koch). (See also Emsmann, Tomazewski and Ehrmann,
who have studied gastrin.)

Digestive actions of gastric juice.—J. Action on proteins. It was
found by Spallanzani that gastric juice exerted a solvent action on
meats. Pieces of meat immersed in the juice gradually dissolve until
only a few fragments of fibers remain undigested. The cause of this
solvent action was early studied. It was at first supposed that it was due
to the hydrochloric acid which had been discovered in the juice by Prout
in 1823, but experiment showed that pieces of meat immersed in solu-
tions of hydrochloric acid as strong as that of the gastric juice did not
dissolve at all, or at most they dissolved at a rate so slow as not to be
comparable to the action of the juice. There must be something else in
the juice to bring about this digestion. It was discovered by Schwann,
one of the founders of the cell theory, that if the juice was boiled first
it lost its digestive action, although its acidity remained. The active
principle, therefore, is destroyed by heat. Since the juice contains a
good deal of organic matter and this is coagulated by heat, it was sup-
posed that the active principle was organic in nature and Schwann pro-
posed that it be called pepsin (Greék, pepsis, digestion). Pepsin is,
hence, the active principle in the juice which digests meat, or, more gen-
erally, proteins, in an acid medium.

2. The clotting of milk—Another property of the gastric juice
which was very early discovered is that it causes milk to coagulate, or
clot, so that it becomes jelly-like. Since acid by itself produces a simi-
lar physical change in milk, as is shown in the souring of milk, it
might be supposed that this power of the gastric juice is due to the acid
it contains; but this is not the case. Carefully neutralized gastric juice,
particularly that of young animals, will still produce clotting if added
to neutral milk. The milk does not change its reaction in the process.
Even an aqueous extract, neutral in reaction, of the calf’s stomach will
clot milk, although the action is faster in a faintly acid medium. Per-
fectly fresh rennet, however, requires to be treated with an acid before
it will clot milk. If the gastric juice or these extracts are boiled, they
lose their power of coagulation. Like pepsin, the active principle is found
stored’ in the mucous membrane. This power of the juice is accord-
ingly due to an active principle or enzyme, and this enzyme is ealled
rennet, or rennin, or chymosin. The mechanism of this clotting and
the question of the identity or difference of pepsin and rennin will be dis-
850 PHYSIOLOGICAL CHEMISTRY

cussed presently. Since this power of clotting milk is most pronounced
in the stomachs of very young animals, it is evidently in the nature of
an adaptation to milk as an article of diet, and the advantages secured
by it are discussed farther on.

3. Action on carbohydrates——Toward carbohydrates gastric juice
has a very unimportant action. Starch and dextrins it does not act
upon, but saccharose or cane sugar, which is the easiest of the disac-
charides to invert, is very slowly split into levulose and glucose by the
hydrogen ions, or the acid of the stomach. There is, however, under
normal circumstances when no regurgitation has taken place from the
intestine, no enzyme in the gastric juice capable of digesting carbo-
hydrates. Lusk found that the inversion of cane sugar was no more
rapid than could be ascribed to the acid of the juice. Since lactose and
maltose are inverted or digested by acid much more slowly than cane
sugar, the action on these sugars is even less important than that on
eane sugar. All three are digested by enzymes found in the intestine.
To what extent cane sugar is inverted in the stomach will depend on
the length of time it remains there after free acid appears. This time
is normally so short that probably little inversion occurs. The concen-
tration of free hydrogen ions at the best is not more than .08 N, and in
a mixed meal is less than this. Such weak acid inverts slowly.

4. Action on fats.—Gastric juice has quite marked powers of
digesting fats which are already emulsified, such as the fats of milk
and of yolk of egg, but it has almost no power on non-emulsified fats such
as those in meat or butter. The power of the juice to digest emulsified
fats was described long ago by Ogata and confirmed by many observers,
but it was for some reason long omitted from the text-books. It has
recently been confirmed by the work of Volhard and his pupils. Thus
the fats of milk and yolk of eggs are split or digested into fatty acid
and glycerine to about 50-80. per cent. in the stomach, whereas non-
emulsified fat is split only to the extent of .5-2 per cent. in the stomach.
The greater action on the fats of milk and yolk of eggs is probably due
to the fact that the area of contact between fat and water is much
greater owing to their emulsion. This power of the gastric juice to split
fats is still under investigation. There is no doubt that the action is
due to a fat-splitting enzyme in the gastric juice, or lipase as it is
called. For some time it was doubtful whether this lipase was secreted
by the stomach, or whether it had regurgitated into the stomach from
the intestine, the juices in the intestine containing much lipase, but
experiment has shown that lipase pre-exists in the stomach mucosa, and
also that the stomach lipase differs in the optimum acidity of its digestive
action from that of the intestine (Davidson).

5. Antiseptic action of the juice—In practically all of the verte-
DIGESTION IN THE STOMACH 351

brates the secretion in the stomach is strongly acid with hydrochloric
acid. The end secured in having an acid rather than an alkaline reac-
tion in this reservoir is presumably to check fermentation. Hydrochloric
acid kills all forms of living things except spores. Many animals swallow
their food alive. Frogs eat each other, or snakes and other living ani
mals. The tissues of these animals live well enough in an alkaline or
neutral digestive medium, but in the strong acid of the stomach they
are attacked, killed and digested. But probably of even greater impor-
tance is the additional protection secured against parasites of all kinds.
Animals are constantly eating with their food bacteria, moulds, protozoa
or other parasites which are killed by the gastric juice. Of the vast
numbers of bacteria we swallow the great majority are killed in the
stomach, and people having a copious and active gastric juice are less
liable to infection by typhoid and cholera than those with less acid juice.
The digested strongly acid material called chyme as it escapes from the
stomach is almost sterile and contains few living bacteria. Any reduc-
tion of the acidity is apt to be followed by a bacterial or yeast fermen-
tation in the stomach which may produce irritating organic acids and .
gas. As long as hydrochloric acid remains in normal amount in the
stomach, one never finds more than traces of lactic, butyric or other
acids which are produced by fermentation, but in the absence of hydro-
chlorie acid these generally appear.

Action of the juice on proteins —1. Pepsin. Iis origin. Pepsinogen.
We may now proceed to consider more in detail the nature and source
of the active principles of peptic digestion, namely the pepsin and the
hydrochloric acid, and the character and conditions of their action on
proteins.

Pepsin is stored in the mucous membrane of the stomach. It was
found by Schwann that if the mucous membrane of the pig’s or dog’s
stomach, which is alkaline or neutral in reaction, is extracted with dilute
(0.4 per cent.) HCl or extracted by water and then the water extract
mixed with acid to make about 0.3 per cent., the extract has digestive
powers similar to that of gastric juice.

This experiment showed that pepsin, or a substance which gave rise
to it, was stored in the mucosa of the stomach, but since the aqueous
extract of the membrane was neutral, it was clear that the hydrochloric
acid was not stored in the mucosa,’ The pepsin was probably made in
the mucosa between meals, but the hydrochloric acid must be made only
at the moment of secretion. Further facts about this pepsin were dis-
covered by Langley. He found that it was extremely sensitive to alka-
lies. If, for example, the juice be neutralized by sodium hydrate or
carbonate and then made acid again, it will be found to have lost much
of its activity. If it be made plainly alkaline, it is entirely inactive
352 PHYSIOLOGICAL CHEMISTRY

when reacidified, although the reacidification is made at once. The
pepsin has been destroyed or rendered inactive by the alkali. It is
necessary, if one wishes to neutralize the juice without destroying the
activity of most of the pepsin, to add CaCO, to it and then a very weak
alkali, such as sodium acetate or milk of lime, very cautiously. Langley
found that if an aqueous extract be made of the mucosa of the stomach,
this extract might be made slightly alkaline for a short time, but would
still be active if made acid again; but if an acid extract be made of
the stomach, this could not be made weakly alkaline without perma-
nently destroying its activity. It appeared, then, that the extract made
with water was more resistant to the action of alkali than the extract
made with acid. Langley interpreted this to mean that the pepsin did
not exist as such in the mucous membrane, but that there was an antece-
dent and more stable substance from which the active pepsin was formed
by the action of acids. This more resistant antecedent substance he
called ‘‘ pepsinogen,’’ since it formed pepsin when acted on by acids.
Another evidence that the substance in the mucous membrane differs
. from pepsin is the fact that if carbonic anhydride gas be passed through
a neutral, aqueous extract of the frog’s esophagus, which contains pep-
sinogen, most of the pepsinogen is destroyed, but if the extract is first
acidified and then neutralized and carbonic anhydride passed through
it, it is not destroyed. Pepsinogen is, then, more sensitive to carbon
dioxide and less sensitive to alkalies than pepsin. It has since been
shown that the loss of activity of the pepsin is not permanent, as Lang-
ley thought, when the juice is carefully neutralized and then made alka-
line. The activity of the pepsin of such juice may be reobtained if acid
is very carefully added nearly to the neutral point and then after
standing twenty hours the reaction made acid. But there is no doubt
of the difference in resistance of pepsin to CO, and alkalies after and
before it has been treated with acid. If his explanation is correct, we
may then assume that pepsin exists in an inactive state in the mucosa as
pepsinogen and is set free by the acid. Since it is quite a common thing
for the enzymes to be in union in the cells with some colloidal matter,
the union being far more stable than the free enzyme, it is probable that
the explanation of Langley is the true one. The bald statement is often
made in the literature that pepsinogen and not pepsin exists in the
mucosa, but it is well to remember that this is as yet an inference and,
while quite probable, should not: be stated as a fact.

The fact that pepsinogen is so very sensitive to carbonic acid gas,
whereas pepsin is not at all sensitive to it, is very interesting and may
throw light on the nature of the change which occurs when pepsinogen
is converted into pepsin. The reaction may be the carbamino reaction
of Siegfried (see page 121). If it is the carbamino reaction, then the
DIGESTION IN THE STOMACH 353

change produced by acid in pepsinogen by which it is converted into
pepsin might be similar to the conversion of creatine to creatinine by
the action of acids. The pepsinogen might be represented thus:

R—CHNH, R—CHNH
\
éa,—coon da—& =0
Pepsinogen. Pepsin.

No importance is to be attached to the relative positions of the carboxyl
and amino groups in the above formula, but only that under the action
of the acid an anhydride may be formed like creatinine. The carbonic
acid in the presence of a calcium salt would combine as follows with the
pepsinogen, but not with the pepsin: 1

R—CHNH—C00O—Ca
cH, —— co—d
Carbamino pepsinogen.

2. What cells of the mucous membrane form the pepsin? The ques-
tion which followed immediately on the discovery of the fact that pepsin
or its antecedent, pepsinogen, existed in the mucous membrane in note-
worthy quantities was that of the location of this substance. A histo-
logical examination of the mucosa of the stomach showed it to contain
a large number of simple tubular glands. There are in these
glands in the fundus end of the stomach in mammals three kinds of cells:
namely, mucous cells near the neck of the gland, chief or adelomorphic
cells, and parietal or delomorphic cells. The latter are also called oxyntic
cells. Oxyntic means acid, and adelomorphic means ‘‘ of uncertain
shape ’’ (Gr. adelos, uncertain; morphe, shape). In the pyloric end of
the stomach the glands have no parietal cells and they differ in some
other particulars from those of the fundus end. The mucous cells secrete
mucin; both the parietal and chief cells contain granules presumably of
a protein nature, but the granules are larger in the chief cells than in
the parietal, and these two kinds of cells differ in their affinities for
certain dyes. The granules are more abundant at certain times than at
others and it is believed, though it has not been demonstratively proved,
from analogy with other glands which can be observed in the living state,
that these granules, elaborated by the cell, are secreted or dissolved and
pass out from the cell when the latter secretes. They are probably pro-
tein in nature, judging from their solubilities and relation to reagents,

+The reaction would probably occur in the absence of calcium, giving simply
the free carbamic acid compound. If ammonia is present a uramido compound might
be formed.
R—CH—NH—COOH R—CH—NH—OO—NH,

|
da —cooH » or CH,—COOH.
364 PHYSIOLOGICAL CHEMISTRY

and it is natural to suppose that they constitute a portion at least of the
organic matter found in the gastric juice. Many have gone farther
than this and called them plainly zymogen granules and assumed that
they represent so much pepsinogen. They are in fact ordinarily called
ferment granules, but it must be remembered that this is an inference.
and whether the pepsin is represented by the granules or by some sub-
stances in solution in the protoplasm it is quite impossible to say at the
present time, although it is possible that some of the granules may con-
tain, or be, pepsin or pepsinogen itself. There is no question from the
work of Harvey and Bensley that the secretion of the parietal cells
is alkaline and protein in character. These observers discovered an
intravital stain, cyanamine, which is blue when acid and red when
alkaline and which is taken up with avidity by the secretion in the
fine canaliculi of the parietal cells. The duct contents stain a clear
red by this stain and the blue is found only close to the neck of
the gland in the lumen and in the cells of the neck and the cells of
the foveole. The secretion of the chief cells is also of an alkaline
nature.

It has been found that pepsin is secreted in frogs by some of the cells
in the lower part of the esophagus, and the opinion is widely held,
although it cannot be said that the evidence is satisfactory, that pepsin
is secreted mainly by the chief cells. Until there is some microchemical
test which will enable a detection of pepsin, it is impossible to say which
kind of cell secretes pepsin. It is of course little likely, but it is by no
means excluded by experiment that some might be secreted in the mucous
cells.

3. The nature of pepsin. Curiosity as to the nature of the digestive
principle, pepsin, is of course very keen and many attempts have been
made to discover something of its chemical composition. It was early
found that a small amount of this substance was able to digest a very
large amount of meat, an amount enormously greater than its own
weight. This at once classified it with the enzymes or catalytic sub-
stances, for this is their main characteristic. But while the amount is
large it is not unlimited. Ifa large piece of meat is put into gastric juice,
the digestion will stop long before the digestion is complete, and it
proceeds if more pepsin is added. Moreover, pepsin is itself unstable,
and gastric juice even when kept in the dark and in the ice box slowly
loses its activity.

Pepsin is colloidal in nature, or if not colloidal itself it is attached
to colloids. If gastric juice is placed in parchment paper tubes and
dialyzed, the pepsin stays in the tube while acids and salts go out; more-
over, it will be found that when dialyzed in this way against water, or
very weak acid, a precipitate appears in the tube and this precipitate
DIGESTION IN THE STOMACH 355

contains most of the pepsin. Under the ultra microscope, also, pepsin
solutions are seen to contain actively-moving, colloidal particles.

Unfortunately very few of the investigators who have attempted to
separate pepsin from the juice have made any quantitative determina-
tions of the activity of the substances they have obtained, but have con-
tented themselves with the statement that the powder was very active,
or digested a piece of fibrin in a certain length of time. In the absence
of decisive, carefully carried-out measurements of the activity of the
powders called pepsin, it is impossible to know whether the powders
described as pepsin contained much or little of the active principle.

Briicke made in the phosphoric acid extract of the mucosa an insoluble
precipitate of Ca,(PO,),. This carried down some pepsin with it. This
was dissolved in hydrochloric acid and then an ether-alcoholic solution
of cholesterol was poured into the solution. The cholesterol was pre-
cipitated and again carried down some of the pepsin. The cholesterol
was removed from the precipitate with ether, leaving the pepsin behind.
The small amount of powder thus obtained was of unknown activity.
It gave no xanthoproteic reaction, nor was it precipitated by tannic
acid or potassium ferrocyanide and acetic acid.

Sundberg, in 1885, extracted the mucosa with saturated sodium-
chloride solution which prevented the solution of much protein. The
solution was very powerful, but contained only a trace of protein. This
solution he precipitated with Ca,(PO,)., redissolved in HCl and dia-
lyzed. He states that the solution was more active than before, but gave
no reactions for protein. It contained ash and nitrogen. Pekelharing
in 1896 and 1897 made an HCl extract of the mucosa and dialyzed it.
A precipitate formed in the tube. This was many times redissolved in
acid and precipitated by dialysis. He finally obtained a precipitate
which had a powerful digestive action. It was a yellow powder giving
the albumin reactions and containing about 1 per cent. P,O;, some of
which was soluble in alcohol. It contained a nucleoprotein. 1/100 mg.
of this powder dissolved in .2 per cent. HCl was able to digest a piece
of fibrin in one hour and a piece of coagulated egg albumin 2 mm. thick
and 1 em. long in a few hours. The substance gave no xanthoproteic
reaction, but was certainly a protein and he thought probably a nucleo-
protein.

Nencki and Sieber examined the natural pepsin precipitated from
natural gastric juice of dogs on standing in the cold. This pepsin con-
tained Cl, .475 per cent.; P, .104 per cent.; Fe, .16 per cent. in the dry
pepsin. After washing with alcohol the ash was .399 per cent.; Fe,
0.115 per cent.; P, 0.059 per cent.; Cl, .188 per cent. of dry pepsin. The
elementary analysis was C, 51.26 per cent.; H, 6.74 per cent.; N, 14.33
per cent.; S, 1.5 por cent. These are the figures of a protein. It lost
356 PHYSIOLOGICAL CHEMISTRY

power on washing in alcohol. Lecithin made about 10 per cent. of the
pepsin. They concluded that the molecule was very complex and labile
and that it contained protein, lecithin, chlorine, iron, phosphorus and
a pentose. These conclusions are, however, quite incorrect for the active
principle. Pekelharing subsequently obtained a pepsin which was active,
but which was free from phosphorus and contained, hence, neither
nucleic acid nor lecithin, but still contained protein. Purified pepsin
contains no chlorine. Pepsin prepared by salting out from commercial
pepsin by (NH,),SO, appears to be a coagulable protein.

It will be clear from this résumé that practically nothing is known
of the nature of the active principle and that the subject is badly in need
of investigation. It seems, on the whole, probable from all its reactions
that the pepsin is either a protein itself or united with a protein. The
fact that it is so easily extracted with acid and that it is so unstable in
alkaline media indicates that it is of a basic nature and hence probably
contains nitrogen. Pepsin is destroyed in a moist state when heated to
57-58°, which is the temperature at which the proteins present coagulate
(Langley).

4. Different pepsins exist. Just as each animal has its own specific
proteins which resemble those of others but which are nevertheless differ-
ent from them, so there appear to be different pepsins. By pepsin, as a
group name, is meant a proteolytic enzyme which acts in a strongly acid,
but not in a neutral or alkaline medium. That there is more than one
kind of pepsin is indicated by the fact that the optimum concentration
of acid is not always the same. For example, that of the dog has an
optimum with a hydrogen ion concentration of .65 N, while human pep-
sin has its optimum at a lower concentration, namely .03 N. They differ
also in their resistance to heat. The pepsin of cold-blooded animals
works as well at 10° as at 40° C., whereas that of warm-blooded animals
works far better at the latter temperature. They show differences, also,
in their speed of digestion of different proteins. Thus the pepsin of the
calf’s stomach digests casein at a rapid rate, but coagulated egg albumin
very much more slowly. The pepsin of the pig’s stomach digests both
proteins rapidly.

5. Pepsin in embryonic development. Pepsin is present in the gas-
tric mucosa of new-born cats and dogs, but this pepsin only begins to attack
voagulated egg albumin toward the end of the third week of life. It
appears at an earlier time in the rabbit. In children pepsin is already
present at birth and casein is changed by it to proteose. Whether this
pepsin is identical with that of adults is not yet certain. Casein is
digested with greater ease than many other proteins. Extract of the
cow’s stomach has digestive power in an acid solution from the third
fetal month. The stomachs of children have less acid in the juice they
secrete than is found in adults and the pepsin in their stomachs works
DIGESTION IN THE STOMACH 357

in more weakly acid media than in adults. Pepsin may, in fact, be
regarded as an enzyme which appears rather late in the course of evo-
lution and also late in the course of embryonic development, and it is
preceded by other enzymes of the nature of erepsin which are active in a
weaker acid. No invertebrate has been found to have a true pepsin
active in strongly acid media and inactive in neutral or alkaline. The
proteolytic enzymes of invertebrates are usually of the tryptic, ereptic or
autolytic class. Some, however, are said to be active in feebly acid media.

6. Conditions of activity of pepsin. The most important of these
conditions is the acidity of the mixture. The most favorable concen-
tration of acid is that which is generally found in the stomach of the
animal of which the pepsin is being examined. For human beings it
is an acidity of about 0.3 per cent. hydrochloric acid, or an hydrogen
ion concentration of about 0.03.N. In dogs the concentration of both
free acid and of hydrogen ions is greater and the optimum is higher:
namely, at about 0.56 per cent. hydrochloric acid, or about 0.05 N
hydrogen ions. Other acids are able to replace hydrochloric acid,
but none are better, and probably none quite so good as the hydrochloric
acid which is normally present. The action of the acid involves not
only the hydrogen ion, but also the anion, since when various acids are
taken with the same hydrogen ion concentration it is not found that
all the acids aid the pepsin equally well. The sulphate ion appears to be
particularly deterrent. Perhaps it acts on the protein to prevent its
ready solution. It appears that pepsin unites with protein which is
being digested and advantage may be taken of this fact to separate pepsin
from a solution. If fibrin or elastin is immersed in a slightly acid solu-
tion containing pepsin, the pepsin will stick to the protein and when
the fibrin or elastin is taken out of the solution pepsin goes with it. If,
- then, the elastin is thoroughly washed with water to get rid of. all
adherent substances and then brought into hydrochloric acid of the right
concentration, the pepsin attacks and digests the protein. The course
of the digestion may be followed very easily by the polariscope, since
the products of proteolytic digestion are optically active and it is only
necessary to filter off some of the solution from time to time and exam-
ine the progressive change in the rotatory power of the solution. The
rate of solution may be followed, also, by means of the ninhydrin, or
other delicate reaction for the presence of amino-acids or other poly-
peptides in the solution. Free acid is not necessary for the action of
the pepsin. If a protein is first soaked in acid so that it combines with
the acid and is then brought into a neutral solution of pepsin, it will
be found that pepsin will digest the protein, although the reaction of
the solution when tested with congo red shows no free acid present. In
fact, it is probably not the concentration of the hydrogen ions which
is important in the digestion, but the combined acid. It is the acid
358 PHYSIOLOGICAL CHEMISTRY

which is combined with the protein and pepsin which enables the pepsin
to act. Neither pepsin nor hydrochloric acid will digest rapidly except
in the presence of the other.

The optimum H ion concentration is difficult to determine. It lies
between 1.7X10-2 N and 3X10—*%. When the concentration of H ions
falls to 10~* N, Michaelis states that the digestion is already bad, and at
NX<10~ it almost ceases. It is probable that the optimum will be found
to differ with different pepsins, since in a baby’s stomach the digestion
goes on with a much weaker avidity than this.

7. Law of peptic activity. The question may now be asked what
relation, if any, exists between the amount of pepsin, the amount of
digestive products and the time of the digestion. What is the relation
between the rate of digestion and the quantity of pepsin? If the quan-
tity of pepsin in unit volume be doubled. will the rate of digestion be
doubled, or will it increase more or less than this? One reason for
finding out any such proportion between rate and amount of pepsin is
that it may enable us to compute the amount of pepsin present, or the
relative amount compared with some standard, by measuring the rate
of digestion. By the rate of digestion is meant the amount of protein
digested divided by the time in minutes or hours during which the
digestion has proceeded. This gives the average rate of digestion per
minute or hour during the period of observation.

It happens that we can only measure differences in the amount of
digestion after an hour or more interval. The rate may not remain
constant during this time. It is, therefore, much more convenient to
take a very short interval of time, an interval so short that the rate
does remain practically constant throughout it. This time which is
relatively very minute we represent ordinarily by the symbol dt. Dur-
ing ‘this interval of time the amount digested, let us say of coagulated
albumin, is a very small amount and it is represented by the symbol dz.
The rate of digestion for this small time interval is, then, the quantity
digested divided by the time, or dz/dt. We can make various guesses
concerning the relation of this velocity of digestion, dx/dt, to the con-
centration of the enzyme. The simplest guess is that the rate is propor-
tional to the amount of the ferment present, or that d2z/dit=—KC,Cs.
Where K is a constant of proportion to be determined by experiment,
and C, and C, are respectively the concentration of the ferment in unit
volume and the concentration of the protein or substrate. If C, remains
constant, the velocity should be constant also, provided of course that
C, does not change. If, however, (, is changing, that is if there is less
and less protein left undigested, then the velocity should of course stead-
ily diminish as C, diminishes. Let us suppose, for example, that this
ic the ease. If a is the concentration of the protein at the start and x
DIGESTION IN THE STOMACH 359

is the amount digested during the interval t, then a—x is the concentra-
tion at the end of the time t. The equation becomes then dx/dt=KC
(a—x). By integrating this expression we sum up all the amounts
digested in the successive intervals which have elapsed since the begin-
ning of the digestion and that enables us to compare the actual digestion
as measured with that computed in this way. Integrating, or summing
the expression, we have, log (a/(a—x))=KC, t. If on the other hand C,
remains constant, we should have x, the amount digested, —KC;t. That
is, the amount digested should be proportioned to the time t.

One of the most useful methods of measuring the rate of digestion is
that of Mett’s tubes, which has already been described (p. 331). The
length of the column of coagulated egg albumin digested away from the
end of the tube is carefully measured. This method is certainly far from
ideal. The cross-section of the protein exposed to be acted upon by the
pepsin remains approximately constant in this method, so that this sub-
strate is constant; but the concentration of the ferment does not remain
constant. The fragments of protein which go into solution are not fully
digested. They combine with and neutralize in this manner some of the
pepsin, so that the concentration of the pepsin immediately at the end
of the cylinder of protein is reduced. They also combine with the acid
and reduce the concentration of the acid at this point, and in both these
ways check the rate to a maximum. Nevertheless, by this method a
certain relation has been found to exist between rate and amount of
ferment: namely, the amount of coagulated protein digested is propor-
tional to the square root of the concentration of enzyme, x=tK VC,
This is known as the law of Schiitz and Borrissow. The time is taken
always as 24 hours. It expresses only a relation within very narrow
and special conditions, but it is a useful method none the less. This
relation between rate of digestion and enzyme concentration is not fol-
lowed if the conditions are changed. For example, Gross has measured
the amount of pepsin by the rate of conversion of casein into caseoses
which are not precipitated by acids. In this method the casein and the
enzyme are both in solution, but the casein is changing its quantity rap-
idly. He measured the time it took various concentrations of pepsin
to convert all the casein into caseoses and he found that if he doubled
the amount of pepsin he halved the time. This corresponds to our first
equation log (a/(a—x))=KC,t. For in this case the concentration of
the casein at the start, a, and the concentration at the time t, or (a—x),
are always taken the same and are constant, so that we have simply log
(a/(a—x) )=Constant=KC,t. That is, the concentration of the fer-
ment multiplied by the time is a constant. If, therefore, the ferment is
doubled, the time will be halved. A very similar law has been found
to hold also for the liquefaction of gelatin by various proteolytic
360 PHYSIOLOGICAL CHEMISTRY

enzymes of bacterial and other origins by Jordan. The time required for
liquefaction is inversely proportional to the concentration of the enzyme
present.

Among other simple but not very accurate methods devised for the
study of the rate of digestion are various colorimetric methods. These
generally involve the setting free of some dye from fibrin by digestion.
Carmine and acid fuchsin are sometimes used. The amount of digestion
is estimated by the color of the solution. Fibrin may be brought into
a slightly acid (acetic acid) solution of acid fuchsin in which it stains
an intense red. The color is fixed more firmly by heating to 80° C. A
small piece of colored fibrin is placed in 5-10 e.c. of the juice to be
examined and the rate of digestion is determined by the deepening of
the color of the solution. For two reasons this method is not accurate.
In the first place, it is impossible to get two pieces of fibrin of exactly
the same surface of contact between it and the enzyme solution. And,
in the second place, the combining power of the fibrin for congo red
or any other color increases as digestion proceeds and more molecules
are set free. One has thus a varying relation between the amount of
color and the amount of fibrin in the solution.

8. Products of the peptic digestion of protemns. The action of the
gastric juice is to dissolve the proteins partially or wholly. To bring
this to pass a chemical change is produced in the protein. If fibrin is
dissolved in gastric juice, the substance in solution is no longer fibrin.
It has been changed to a mixture of derived proteins called meta-protein
and proteoses by the action of the juice. Whether amino-acids and di-
and tri-peptides are produced at the same time or not has been the subject
of much controversy, owing in part to the fact that the digestion has at
times been carried out with pure juice and at times by extracts of the
mucous membrane. In these extracts there are present sometimes other
enzymes than pepsin. The digestion of a protein by pepsin hydrochloric
acid is in its main features as follows: If a mass of fibrin is dissolved
in gastric juice, there remains a small undissolved residue which is sup-
posed to represent nuclein of white blood cells entangled in the fibrin,
or a lecitho-protein (Wooldridge). From this it appears that gastric
juice will not dissolve nuclein or some lecithoproteins. If the clear
filtrate from any such undissolved digestion residue is neutralized care-
fully with NaOH or NaHCO,, there is generally precipitated at or near
the neutral point, if the digestion has not lasted long, a more or less
copious flocculent precipitate which will redissolve if more alkali is
added. This substance (1) is a meta-protein known as syntonin or acid
albumin. The greater part of the digested fibrin remains in solution
even at the neutral point. The solution gives a violet-red biuret test.

If the solution freed from the syntonin is half saturated with
DIGESTION IN THE STOMACH 361

ammonium sulphate, a white, flocculent or sticky precipitate separates
out, which often clings to a glass rod or sticks to the side of the beaker.
This substance (2) is known as primary, or proto-proteose or protal-
bumose. If more ammonium sulphate is added, another precipitate
begins to form and at complete saturation of the warm solution there
separates a second substance, or rather a mixture of substances (3)
which are known as deutero, or secondary proteoses or albumoses. These
two fractions, 2 and 3, make by far the greater part of the weight of the
original fibrin, and the process of digestion evidently consists in con-
verting the protein into acid albumin, proto- and deutero-albumose. It
will be found by the examination of the digested mixture from which
the albumoses have been separated by ammonium sulphate that it still
gives a good biuret test. The separation of these biuret-giving, pro-
teolytic products which are soluble in saturated ammonium sulphate is
a matter of some difficulty. They are all called peptones (4). Whether
some amino-acids or di- and tri-peptides are produced, or not, is still a
matter of dispute, but probably they are not set free by the pepsin, even
though they may appear in the digestive mixtures. It is, perhaps, more
“probable that they are the result of the action of the ereptic protease,
sometimes and perhaps always present. There is a general agreement,
therefore, that by peptic digestion acid albumin, proteoses and peptones
are formed.

We should not leave this part of the subject, however, without a
reference to the very careful work of Zuntz on the extent of peptic
decomposition of proteins, since his results raise many questions of inter-
pretation of which the solution may be of great value. Zuntz found that
protein fragments of small size, precipitated by phosphotungstic acid
and not giving the biuret test, were formed very early in the course of
digestion.

a. The proteoses or albumoses. The proteoses or albumoses are a
group of derived proteins. There are three divisions of the group:
namely, the primary proteoses, including the proto-proteoses and hetero-
proteoses, and the secondary, or deutero-proteoses. The primary pro-
teoses are precipitated by half saturation of their solutions by ammonium
sulphate. Proto-proteose is soluble both in hot and cold water and dilute
salt solution. The hetero-proteoses, while not coagulated by heat, undergo
a precipitation when heated, but the precipitate redissolves readily in
a little acid or alkali. The hetero-proteoses are insoluble in water, but
soluble in dilute salt solutions. The secondary proteoses are only pre-
cipitated by full saturation. It is probable that each of these groups
‘is a mixture of many different fragments of the protein molecule. They
can be further fractioned by ammonium sulphate, but we need not go
into that matter at this place.
362 PHYS1OLOGICAL CHEMISTRY

It is found that the different proteoses differ in the kind and amount of
the amino-acids that they contain. They differ also from each other
depending on the protein from which they come, the deutero-gelatose
from gelatin being different from the deutero-caseose from casein. The
protalbumoses differ from the deutero-albumoses, also, in their reaction
to other precipitating reagents than ammonium sulphate. Thus potas-
sium ferrocyanide and acetic acid precipitate the proto, but not the
deutero-albumose; copper sulphate will precipitate the former, but not
the latter, and other differences have been noted by Kutscher. It is
probable, from his results, that the latter have more of the basic amino-
acids in them than the former and that the protalbumose is more acid
than the deutero-albumose.

9. Character of the linkings in the protein molecule attacked by
pepsin. Although it is uncertain whether some small amounts of amino-
acids are or are not set free by the prolonged action of pepsin hydro-
chloric acid, there is no doubt that the action is incomparably more rapid
in the first stages of digestion by which the protein is split into albumoses
and peptones than in the further decomposition of these substances.
Another very significant fact is that no artificial polypeptides have yet
been made which can be digested by pepsin, although many are digested
by erepsin and trypsin. Moreover, pepsin has no action at all on certain
proteins which are easily digested by trypsin and erepsin. It has no
action on the protamines, salmin and clupein. In this case we have
linkings between the basic amino-acids and proline, serine and valine
which pepsin cannot break. Since it will not act either on any of the
artificial polypeptides, it seems either that pepsin must act only on some
spevial amino-acid linkings, or else that there are in the protein molecule
linkings uniting the albumoses of a different nature than amino-acid
linkings. It may be remarked in this connection, however, that while in
the course of prolonged peptic digestion the number of free amino
groups undoubtedly increases, as is shown presently, yet it is still doubt-
ful whether the early rapid digestion leading to the formation of
albumoses is accompanied by any increase in free amino groups. This
would indicate that if the later digestion is really due to the pepsin and
not to some admixed enzyme of an ereptic nature, like that, for example.
of the saliva, that the pepsin must have acted on some amino-linkings.
The further investigation of this problem will possibly throw light on the
synthesis of albumoses to make complex proteins. In any case the action
of pepsin shows that the possibility that the protein molecule is not a
single long chain of amino-acids, but may be composed of larger groups
of two, three or a larger number of amnio acids, just as starch is com-
posed of a number of maltose groups, cannot be disregarded. The protein
digestion is thus apparently similar to that of the polysaccharides. By
digestion of the polysaccharide, starch, a number of dextrin groups are
DIGESTION IN THE STOMACH 363

first formed, and these are subsequently transformed into monosac-
charides. This is accomplished by two or three ferments: amylase, which
attacks the starch and carries it over into dextrin; dextrinase, which
attacks the dextrin and carries it to maltose; and maltase, converting
maltose to glucose. Amylase, dextrinase and maltase correspond in a
general way with the three proteolytic enzymes pepsin, trypsin and
erepsin. That the pepsin hydrochlroic acid decomposes by hydrolysis is
shown by the investigations of Chittenden and Kiihne. If a protein
is analyzed before and after digestion, it is found that the amount of
hydrogen and oxygen in the split products is much greater than in the
original protein. The total weight, also, of the solid protein left on
evaporation is increased, showing that the elements of water are taken on.

During the course of prolonged peptic digestion there is a breaking
of amino-carboxyl linkings so that the number of free, that is unsubsti-
tuted, amino groups greatly increases. This inference is drawn from
two facts: first, the capacity of the digestive products of combining with
formaldehyde steadily increases, as is shown by the Sorensen titration ;
and, second, coincident with this is a steady increase in the power of
combining with acid, as is shown by the steady fall in the amount of
uneombined or free hydrochloric acid as determined by the Giinzberg
reagent. The following experiments illustrate these facts:

100 c.e 1% gluten in N/10HCl plus 50 ce 1% Parke-Davis pepsin in N/10HC1
in the thermostat at 38°.

a 3 Free HCl Formol
Titration (Giinzberg) titration
ACHONCS: ie aive leads SY grees $8* 6
After 4 days ............ 84 12
After 12 days ............ 17 7

* The figures represent c.c. of N/10 NaOH necessary to neutralize 100 c.c. of the
mixture. 100 c.c., for example, contains 88 c.c. free N/10 HCl. On the addition of
formol the acidity increases equivalent to 6 ¢.c. N/10 acid, prenol-phthalein being
the indicator.

50 c.c. saturated gluten solution in N/J0 HC! plus 50 c.c. 1% Parke-Davis pepsin
in N/10 HCl in thermostat. ‘

Titration
AG ONCE: osc seiea tise ree ees 91 13
After 4 days ............ 84 21

The experiments show that the increase in the formol titration is
almost equal to the decrease in the free acid. This fact is also brought
out in the following experiments which give also the change in the
titration by congo red:

Giinzherg Congo-
acidity Congo Gtinzberg Formol
Titration Free HCl acidity acidity titration
At once 2.0.06 .6 cee wees anaes 12 88 16 18
After 2 hours ........+02eee 64 86 22 25
OL day cece cece ee eee 56 86 30 32
6 GO days .....cee ee eeeeee 29 89 60 58

185 day® ....scceeeeenece 20 88 68 67
364 PHYSIOLOGICAL CHEMISTRY

The congo titration remains practically constant, since this gives both
free and combined acid; and the difference between Giinzberg and congo
acidity represents the amount of hydrochloric acid which has been bound
by the amino groups set free. It will be seen that this number is almost
the same as that of the formol titration which titrates the number of such
amino groups which have been set free in another way.

These experiments show very clearly that whatever the special link-
ings may be which are broken by the pepsin, or other enzymes in the
commercial pepsin, that they involve amino groups. It will be seen,
too, that the free acidity decreases markedly, and since the activity of
the pepsin is dependent on the acidity this factor of diminishing acidity
will probably alter the rate of digestion. In any experiment in which
the activity of the pepsin itself is to be studied the acidity should be kept
constant.

Such a substance as Witte’s peptone, which is in reality largely a
mixture of albumoses, is still capable of partial digestion by pepsin, as
is shown in the following experiment:

50 cc. 4% Witte’s peptone plus 50 ec. 1% Parke-Davis pepsin in N/10 HCl
plus 25 c.c. water at 38° C.
Free acidity Free and combined

Titration (Ginzberg) (Congo red) Formol
AL Once 22s wks vice eancves 15 38 ‘24
After 2 days ...........05 9 37 29
After 9 days ............ —10 29 42

There is a noticeable increase in the free amino groups, as shown by
the rise in the formalin and decrease in the Giinzberg acidity.

If the formation of other products than peptones and albumoses
recorded by Zuntz is really due to pepsin, the following scheme may
perhaps indicate the course of peptic digestion of a simple protein.

Simple protein digested by pepsin HCl.
|

ae |
Syntunin Tripeptide

 

 

 

 

|
| eine
Proto-proteose Tripeptide
|
|
Proto-proteose Triptptide
|
|
Deutero-proteose Tripeptide
|
| |
Deutero-proteose Tripeptide

|
| |
Peptone Tripeptide
DIGESTION IN THE STOMACH 365

10. Energy changes in digestion. Peptic digestion does not involve
a noticeable loss of energy, a matter of great importance for the organ-
ism, since it indicates that neither the synthesis nor the decomposition
of the proteins from the amino-acids involves much energy dissipation.

If the heat produced by burning a certain weight of the protein before
digestion is compared with that produced, by burning the same weight of
the digested products, allowing for the increase of weight due to the
water added by hydrolysis, the difference is negligible. Energy is not
lost, then, by the hydrolytic decomposition of the proteins, or the loss
is extremely little, nor is energy required for their synthesis by
dehydration.

11. Fate of the pepsin. What becomes of the pepsin which is
secreted by the stomach and finds its way into the intestine? Is it
destroyed there by the action of the bacteria and enzymes of the small
and large intestine? or is it reabsorbed? and, if it is reabsorbed, what
becomes of it? Is it picked out from the blood by the stomach mucosa
and used over again or is it taken up and destroyed by the blood or
body cells?

These are questions to which only most imperfect answers can be
given. In fact, it is not known what becomes of most of the pepsin pass-
ing with the food:into the intestine. It was long thought that by the
action of the bile, duodenal and pancreatic secretions, all of which are
alkaline, it would be destroyed and that its action would cease on
elltering the intestine and the pepsin itself be perhaps digested. This is
perhaps the fate of most of it. But it has been found that the reaction
of the chyme in the upper part cf the intestine remains weakly acid
sometimes elear to the large intestine, at other times only a short distance
below the pylorus, but in any case the neutralization gives very little or
no free alkali. Recent experiments by Abderhalden and Meyer have
shown that active pepsin is to be found in the intestinal contents of the
dog's duodenum, jejunum and ileum. By mixing these contents with
elastin to which the enzyme sticks and then placing the elastin in hydro-
chlorie acid solution, the elastin was found to be digested, proving the
presence of active pepsin throughout the canal. There can be little
doubt that the peptic digestion does not cease as soon as the chyme passes
into the intestine. but continues for some time. and that the Pee not
killed at once, at any rate, by trypsin and erepsin.

The further fate of this pepsin is not known. Very little active pep-
sin is found in the feces. There is little doubt that some of it is absorbed.
for the hlood always contains some anti-pepsin, a fact which proves that
pepsin has found entrance to the blood either from the peptie glands
of the stomach or from the alimentary canal. More probably it enters
from the latter. Pepsin is found. also, in small amounts in the urine,
366 PHYSIOLOGICAL CHEMISTRY

which proves that some of it is circulating in the blood. These facts
make it possible that there may be a circulation of pepsin analogous to
the circulation of the bile, the pepsin being in nart reabsorbed, carried
to the stomach and there picked: out and resecreted. The conditions of
this resecretion are of course much less favorable than for the analogous
circulation of the bile, since before reaching the stomach the blood from
the intestine must pass through many other organs, all of which may act
upon the pepsin. How extensive and whether or not there is such
a circulation of the pepsin cannot at present be said and the question
should be investigated. While it is not impossible that a larger propor-
tion of the pepsin which is secreted in the stomach may be reabsorbed
in the intestine than is at present believed, the probability is that most
of the pepsin is destroyed in the intestine either by the other digestive
enzymes or by the bacteria. The whole question is badly in need of
further investigation.

Hydrochloric acid. Methods of its quantitative determination.—
The activity of the pepsin depends on the acidity of the juice. It
becomes, therefore, important from a clinical point of view to determine
what the amount of acid is in the juice and whether it is hydrochloric
or some other acid. Generally the secretions of the hydrochloric acid and
the pepsin run closely parallel to each other, so that if the juice is weak
in pepsin it is also weak in hydrochloric acid. In hyperacidity there
is usually a hyperpeptic activity also. There may, however, be achlor-
hydria, that is an absence of hydrochloric acid, with some peptic action.
The close parallelism of the secretions of these two substances may mean
that they come from the same cells or are even formed together in one
action. The acid is generally lacking in carcinoma of the stomach, and
this is a fact of considerable diagnostic importance. It is desirable to
have, therefore, methods for the easy and accurate determination of
the stomach acid. The examination is usually made in the following
way:

In the morning when no meal, or but a light one, has been eaten the
evening before, and the stomach has, therefore, been resting for 12
hours and should be empty, a test breakfast is eaten of either a roll and
a cup of weak tea, or five Albert or other crackers (biscuits) well chewed.
After 45 minutes the patient either vomits, or a stomach tube is swal-
lowed and a sample of the gastric juice is drawn for examination. It is
wise not to be content with a single sample, but to examine the contents
on more than one occasion, since the acidity of the contents is not always
the same and it may happen that the material withdrawn has not been
thoroughly mixed with the juice. See page 973 for further directions.

A few c.c. of the filtered juice is titrated with N/10 NaOH, using
phenol-phthalein as an indicator. This indicator is so delicate that it
DIGESTION IN THE STOMACH 367

will detect all acids, even very weak organic acids, and that also which
is temporarily combined with the protein. Hence this is the maximum
acidity which can be.present. The number of ¢.c. of N/10 NaOH neces-
sary to neutralize 100 c.c. of the unfiltered contents is called the total
acidity.

If this number turns out to be normal, and human normal gastric
contents should be sufficiently acid so that 100 ¢.c. will require about
74 ee. to 90 c.e., or even 100 cc. at the end of the digestion period, of
the N/10 alkali, the next process is to discover whether the acid is
hydrochloric acid or not, or at least to see whether the hydrochloric
acid is present in a free state. This is best determined by Giinzberg’s
reagent or by Topfer’s. The latter introduced dimethylamino-azo-
benzene as an indicator for free hydrochloric acid, since it only changes
color when the concentration of hydrogen ions is at least NX10—. Any
organic acid occurring in the stomach would need to be present in very
high concentration, 0.5 per cent. or more, to produce this concentration.
This indicator is pink when acid and yellow when alkaline. If now
10 «ec. of filtered juice are taken and a drop or two of the indicator
solution added and then titrated with N/10 NaOH, the result, if the
contents are normal, should give a figure of from 40 to 75 c.c., depending
on the period at which the juice is taken. It is highest just before the
stomach is emptied. The Gtinzberg reagent should also be used as
described on page 368. An accurate determination of the number of
hydrogen ions in the juice, and this is the only true test of its actual
activity or avidity, can best be made at the present time by means of the
gas-chain method explained on page 540. This method is too difficult
for clinical work. A substitute for it may be found in the indicator
method as shown on page 976. A bright green color on the addition of
a drop of gentian violet solution shows a H ion of .06—.1 N or above.
The actual acidity of the juice as determined by this method varies
greatly in different conditions. In the fasting stomach Tang] obtained,
by drawing some of the contents with a sound 10-12 hours after the last
meal at 8 a.m., from 2-25 e.c. All 13 samples, with one exception, were
acid to litmus. Only one was alkaline. The concentration of hydrogen
ions in gram equivalents per liter were: .035; .022; .0001; .00042 ;12x
10-8; .085; .013; .029. The acidity of normal gastric contents should be
about .035. The acidity ran between 10-2 and 7X10-2 N hydrogen ions.
The optimum concentration of hydrogen ions for the digestion of pro-
teins by pepsin was found by Sérensen’s gas-chain method to be 0.020-
0.060 N. Christiansen found the optimum for human juice to be
020-.033 ; for the pig, 0.052; and for the dog, 0.053 N.

While the direct determination of the hydrogen ion concentration
368 PHYSIOLOGICAL CHEMISTRY

is thus too difficult for clinical use, except in a well-equipped laboratory,
a very close approximation may be made by the use of the Giinzberg
reagent. This reagent consists of a mixture of phloroglucin, 2 grams;
vanillin, I gram; alcohol, 100 cc. The reagent should be kept well
protected from the light. One drop of the reagent is placed on a white
porcelain plate, or evaporating dish, and dried cautiously over the flame,
or better on the water bath. Then add one drop of the juice to be
tested to the clear yellow spot left by the evaporation and warm again
carefully. If hydrochloric acid is present in the free state, a red color
develops on heating. If it is heated too much, it will be brown and the
test must be repeated. By diluting the gastric juice a limit will be found
which just gives the reaction. This limit contains 0.0004 NHCIl. The
red color is due to the production of a red erystalline substance,
C.,>H,,03, which is formed by the union of two molecules of phloro-
glucin, C,H,O,, and one molecule of vanillin, C,H,O,, with the elimina-
tion of water. If acid is tested in the cold with this reagent, it will be
found to react only with NHCI1; weaker acid gives no color. A more
convenient method: is given on page 976.

The following table illustrates the findings of a large number of
eases after a test breakfast (Ewald’s). The figures in all except the
first column represent the number of cc. of N/10 NaOH necessary to
neutralize 100 c.c. of the gastric contents when the different indicators
are used. The Giinzberg figures are not determined by direct titration,
but in the manner indicated, i.e., by dilution. They express the amount
of free hydrochloric acid there is in 100 ¢c.c. of juice. The figure 20
under Giinzberg means that 100 e.c. of this juice contained 20 e.c. of
free N/10 NCl. The juice was probably drawn about 45 minutes after
eating.

 

 

 

CHC com-
< ; Pee ee CHC! cor. 3 sien
,, : enol-
exper. CH by gas seagate dieeoelanion Gilnzbere'| “TOpfer red | Litmus | onthalein
chain {as titration
number,
1. .0003 0 |. ON eee Acree 4
2, 033 33 BM! coos ose ay | 6. oe
3. -004 © 4 4 |ieecrnees ae SS ll decrees 47
4, .036 36 BF: | eecaney 62 feel] 88
5. .004 4 4 Trace BE" | Recevers 33
6. .018 18 19 10-15 BS. AM ease 52
itis 022. 22 23 15-20 SS) | cxsiscsees 51
8. 025 25 26 20-25 Ba scunersas Neg eey
9. .056 56 59 42-48 68 82 88
10. -040 40 42 40-45 59 67 78
20. -062 62 67 66 77 83 93
29. .036 36 38 44 59 64 78
32. .0003 0 ; 0 0 13 21 44
39. .042 42 44° 39 59 ‘70 80

 

 

 

 

 

 

 

 

 
DIGESTION IN THE STOMACH 369

It will be seen from the foregoing table that the free HCl as deter-
mined by Giinzberg’s reagent is very close to the actual concentration
of the hydrogen ions as determined by the gas-chain method. Congo red
gives always much more than the free hydrochloric acid and dimethyl-
amino-azo-benzene (Tépfer) somewhat more. If the concentration of
the hydrogen ions is low, however, Ginzberg used in this way gives too
low results. Pure, human appetite juice contains .12 N H ions.

In the case of pure gastric juice, when there is no admixture of
stomach contents, congo red gives results more in harmony with the
hydrogen ion titration. For then there is no admixture of food to bind
some of the acid of the gastric juice. This fact is shown in the follow-
ing determinations of various samples of gastric juice from a ease of
hypersecretion :

 

 

 

c CHC! cor- is ongo benol-
sample | chai | tected for | Ginzberg | On phthatein
1s 035 37 38 0O| 4D 47
2, 035 a 33 1 45 55
3. 037 39 43 | 54 62
4, .022 23 25 | 30 38
5. 10-* 0 0 0 4
6. .035 37 33 44 57
7. .0073 77 11 83 88
8. .056 59 58 64 71

 

 

 

 

 

With pure gastric juice there is not a very great difference between
phenol-phthalein titration and that by Gtinzberg; in other words, there
is very little difference between the free hydrochloric acid and the total
acid, for in such juice when it is normal there is almost no lactic or
organic acid and most of the acid is free and not hound to the organic
matter in the juice. But as the protein admixture in the juice increases,
or when by fermentation organic acids may be formed from the ecarbo-
hydrates, then the difference between the two titrations may be very
large. This fact is brought out if we compare the titrations of the
stomach contents, first, of the pure juice, then of the contents after an
Ewald test breakfast, which contains very little protein, and then after
Bourget’s breakfast which has more meat in it.
370 PHYSIOLOGICAL CHEMISTRY

TITRATION NUMBERS.

Ginzberg’s reagent Congo paper Phenolphthalein
Pure gastric juice ........ 25 30 35
After Ewald’s breakfast .... 25 45 65

After Bourget’s breakfast ... 25 75 125

How much acidity of the juice may vary after various meals and at
different times is shown by the following figures:

Sample drawn

Breakfast hours ater Cy ions Cyc! Ginzberg ee 2. yuan
1. 250 grs. oatmeal soup .. 3% 032 34 36 64 91
50 grs. meat
4 pieces white bread
2. 250 grs. oatmeal soup .. 2 .008 8 8 41 67

100 grs. meat
4 pieces bread
3. 250 grs. soup ........ 3 021
100 grs. meat
2 pieces bread
4, 250 ers. soup .......... 2% 062 67 71 91 117
100 grs. meat
2 pieces bread
5. 250 grs. bouillon ...... 2 .0007 1 —24 30 90
100 grs. meat
2 pieces bread

io}
wo

13 100 190

In the last experiment there was no free hydrochloric acid as shown
by Gunzberg ; indeed, to get a positive Giinzberg test it was necessary to
add hydrochloric acid in considerable amounts, but nevertheless there
was a normal total acidity and a normal acidity to congo paper.

The following table shows the number of c.c. of N/10 NaOH required
to titrate 100 cc. of unfiltered stomach contents after an Ewald test
breakfast when various indicators were used:

Indicator c.c. N/10 NaOH

Tropaeolin ..........-4.. 12-19
Methy! violet ............ 15 - 25 End point indefinite.
Gtinzberg ........ cece eee 25

BO aS) oaitcciwcrvsidian ny Sichuan tunres 25
Dimethyl-amino-azo-benzene 36-38
Methyl orange ........... 41 - 423
Congo paper ..........+-. 43
Alizarin .........00 0.000 49 - 51
Rosolic acid ............. 51-53
Litmus paper ............ 56

Phenol-phthalein ........ 65
DIGESTION IN THE STOMACH 371

Variation of hydrochloric acid in disease.—The determination of
the secretion of hydrochloric acid is of considerable importance in the
diagnosis of stomach disease. Thus in cancer of the stomach particularly,
but also at times when the cancer is in some other part of the body, there
is often a great diminution of the secretion of hydrochloric acid or its
complete suppression. On the other hand, in ulcer of the stomach and
in particular when the ulcer is in the pylorus, or duodenum, there is gen-
erally found hyperacidity. It may happen, however, that when an ulcer
has a cancer grafted on it the secretion may be normal. In various
neuroses the acidity and the pepsin content may be increased above the
normal. In general, carnivorous animals have a more acid secretion than
herbivorous, and a meat diet is supposed to increase the acidity ; although
no very convincing evidence of variations of acidity with diet has been
found.

Theory of titration of stomach contents by indicators,—All the indicators
employed for the titration of acids or alkalies are either acids or bases. Thus
phenol-phthalein and congo red are acids. Dimethyl-amino-azo-benzene is a base.
The color change is due probably to a rearrangement of the molecule to a colored
form, usually a quinonoid, when in the salt form. This rearrangement is probably
due to the dissociation of the molecule, the undissociated molecule not rearranging.
Since the indicators are acids or bases of different avidities, that is since they have
different amounts of dissociation, some are weaker than others. Accordingly some
are able to form salts in sufficient amounts to give a perceptible color in the presence
of more acid than are others which are weaker. Thus congo red is a fairly strong
acid and is able to take some of the base to itself and make a colored salt in the
presence of some free acid, whereas phenol-phthalein is so weak an acid and its salts
dissociate so much hydrolytically that it will only give an alkaline reaction, that
is form enough salt to be seen, when there is quite a good deal of alkali present.
Dimethyl-amino-azo-benzene is so weak a base that enough of the salt is present to
give a red color only in the presence of a strong acid The concentration of hydrogen
ions at which the various indicators change their reactions is shown in the following
table (see also page 546):

Cg ions, at which the

indicator changes.

(Normal divided by 16

raised to the power in-

Indicator dicated hy the following
figures. Cougo red

changes between N/10°
and N/10° H ion)

Tropacolin OO ...... cece eee eee eee 1.4-2.6
Methyl violet: sis ccc cmc cine sede 0.1 - 3.2
Dimethyl-amino-azo-benzene ........... 2.9-4.0
Methyl orange ........-..eee eee eeeee 3.1-4.4
Congo: FEA! sj csicie sss ecag ge Spaces cubed Sree 3-5
Alizarin« 2.2.0... ec ceeee dsRiichwaoaveds 5.5 - 6.8
Litmus paper .....ce ccc ee cece cece eens 7
Neutral red) cas cicuws os anene ceed oe 6.8 - 8.0
Rosoli¢: acid: si-ssccwsav sens eeaawaven 6.9 - 8.0

Phenol-phthalein .................- ..- &3-10.0
372. PHYSIOLOGICAL CHEMISTRY

Giinzberg‘s reagent.—The property of giving a red color under the conditions
of the Giinzberg reaction is not peculiar to hydrochloric acid, since sulphuric, nitric,
phosphoric and boric acid give it also. Of these acids phosphoric and boric are
weak acids, boric being very weak. But boric acid has the property of uniting by
one of its hydroxyls with sugar and becoming thereby a much stronger acid, and it
is possible that phosphoric acid has this same power less developed, since as the
existence of phytin shows it has the property of combining with the hydroxyls of
aromatic alcohols such as phloroglucin. Both these acids probably unite with the
phloroglucin to make esters and their acidity is probably thereby much increased.
Phosphoric acid is, for example, a much weaker acid than formic, which does not
give the reaction. Oxalic, citric and tartaric acids give a positive reaction; succinic,
propionic, lactic, acetic, butyric, benzoic, formic and phthalic are negative. No mono-
carboxylic fatty acid is known which is positive, but if there is more than one
carboxyl present then it may be positive. Hydrochloric acid N/2500 still gives a
noticeable reaction. It is clear that the reaction does not depend upon the number
of hydrogen ions alone. For example, a mixture of glycocoll and hydrochloric acid
having a concentration of hydrogen ions of N x 10-1.96, or roughly .01N, is just
positive; while free hydrochloric acid N/2500 or N x 10-3-4 is positive. Christiansen
concludes that the reaction depends on the nature of the acid, and not on the H ion
concentration. The probability is, however, that when glycocoll and hydrochloric
acid are evaporated more and more of the acid is bound as the concentration in-
creases. Consequently the ion concentration at the end may be far less than that
indicated in the foregoing figures. Gitinzberg’s reagent is certainly the most useful
indicator for the determination of the free hydrochloric acid.

Origin of the hydrochloric acid.—What is the origin of this hydro-
ehloric acid? How shall we picture the processes by which this acid
in a concentration fatal to all animal cells, if once it enters them, is
secreted by living matter from an alkaline fluid like the blood? In what
part of the stomach is it formed and by what glands or cells? These
are questions which are not yet solved. The problem is a difficult one.
The acid, unlike pepsinogen, is not stored in the cells of the stomach
mucosa, for the aqueous extract of the mucosa is neutral, not acid, in
reaction, and the mucosa contains only a small amount of chlorine,
although two or three times as much as most of the other tissues of the
body. Not only is the hydrochloric acid not stored, but neither are the
chlorides stored or an organic chlorine compound, except in small
amounts. The per cent. of chlorine in different tissues computed on the
wet weight was found as follows by Nencki and Schumova-Simonowski:

Panniculus adiposus ....2 00... eee 0.076
Stomach mucosa ........... 0... cee eee eae 0.093
TEI VOR, fis-e seas asus BA cas add o bald iiearbed Wasaoheraedah Raanshote 0.025
BOne@ MarrOW .esecesca vce cecevagsee actiauecnia acess Oe 0.034
Muscle ............. aovabe deface" dnledeafoeieneetvataher 0.033
Kidney FAG: sais vs eetaverurwesnnign warming a agang gs 0.032
BOneS  <yagcissngwtae onsen: eames combs se» 0.033
DIGESTION IN THE STOMACH 373

The chlorine content of the mucosa is, therefore, somewhat greater
than that of other tissues. But, owing to the small weight of the mucosa,
the amount of chlorine stored is but a very small fraction of that
excreted.

The hydrochloric acid is secreted chiefly by the fundus end of the
stomach. The pyloric secretion is certainly less acid than the fundus
secretion. It is found, too, that the fundus mucosa has a little more
chlorine in it than the pyloric mucosa.

Cl Content or Funpus anpD Pyztoric Mucosa. In Perr CENT. oF THE Dry WEIGHT.

 

 

 

 

 

Per cent. chlorine in dry weight of tissue
eaueae / Seach Coney Fundus Pylorns Difference
PIS” |) save Seauneeee wey eaeeecs 0.87 0.67 0.20
Pig Strongly acid ........ 0.62 0.59 0.03
Dog ne Pa Wiles? Aad 0.72 0.68 0.04
Pig Empty. Weak acid .. 0.75 0.44 0.31
Dog |Strongly acid ........ 0.59 0.50 0.09
Pig fe BO edie Gai 0.60 0.50 0.10
Pig Empty. Weak acid .. 0.83 0.62 0.21

 

The fundus has, then, more chlorine than the pylorus. The water con-
tent is about 85 per cent. in each.

The attempt was made by Heidenhain to determine whether the acid
was formed in the pyloric or fundus part of the stomach by making
a pouch of a portion of the stomach in each region. He found that the
pouch at the fundus end was weakly acid and that at the pyloric end
was alkaline. This experiment, however, is open to the criticism that
the nerves were cut and the secretion consequently not normal. The
pouch made by the Pawlow method takes in more of the fundus part of
the stomach and it always secretes a strongly acid secretion. There is
no doubt, therefore, that the secretion of this part of the stomach is cer-
tainly acid. Since in the mammalian stomach there is a marked differ-
ence between the character of the cells in the glands of the two regions,
in that the so-called delomorphie, or parietal, or oxyntic cells are found
in the glands of the fundus part of the stomach but not in the pyloric
portion, the conclusion was drawn that the acid was secreted by these
cells and they were named oxyntic (meaning ‘ acid’) cells in conse-
quence. Many attempts have been made to discover some direct evi-
dence that these cells secrete the acid, but the final result of such attempts
has been to show beyond doubt that they do not secrete acid, but have an
alkaline secretion.

Claude Bernard was one of the first who attempted to get some
direct evidence of the place of formation. of the hydrochloric acid. He
374 PHYSIOLOGICAL CHEMISTRY

injected into one vein of a dog potassium ferrocyanide and into another
the lactate of iron. When these two reagents are brought together
outside of the body, it is only in the presence of acid that they react to
make a blue precipitate of Prussian biue. On killing animals after
such an injection, he found the blue color only in the lumen of the
stomach, and in the necks of the glands, but not in the mucous mem-
brane. He concluded that the mucosa was alkaline in reaction and that
the acid was formed only after excretion. Foster suggested that it was
formed as an organic compound which after secretion decomposed, set-
ting free the acid. All attempts to show the existence of such a com-
pound in the mucosa have so far been fruitless. Fitzgerald recently
repeated the work of Bernard, with the result that she found some of
the parietal cells stained blue by the Prussian blue, but in general the
color was in the lumen of the neck of the gland and in the stomach itself.
Her results were interpreted to mean that the secretion of the parietal
cells was acid. It has recently been shown by Harvey and Bensley that
these conclusions are incorrect. A few of the parietal cells may take
the stain, but the vast majority do not. Moreover, cells of the liver,
and other tissues, may also stain and the blue deposit may be found in
the lymph and blood where there is no possibility of the formation of
acid. It is, therefore, clear that this method gives no reliable indica-
tions of the reaction of a cell or a tissue.

Macallum attempted to follow the matter further by means of a
study of the distribution of the chlorine in the cells. By precipitating
the chlorides with a solution of silver nitrate in dilute nitric acid and
then reducing the silver chloride by exposing the section to the light,
he was able to detect the presence of chlorine in cells. This method is
open to the serious objection that perhaps other substances than chloride
may fix the silver. Nevertheless, the method is better than none at
all and enables a study of the silver-fixing elements of the cells. He
found that both parietal and chief cells gave a strong reaction for
chlorides, but that the parietal cells had the stronger reaction and he
interpreted this finding as favorable to the view that the parietal cells
secreted the acid. <A repetition of this work by Lopez-Suarez gave the
contrary result, that there was more chlorine in the chief cells and that
the parietal cells were practically free from chlorine. That the acid
is not formed by the parietal cells, but only in the neck of the gland
and possibly in the lumen itself, is shown by the work of ‘Harvey and
Bensley. They found that cyanamine. which is blue when acid and red
when alkaline, stains the secretion of the parietal cells an intense red
in the living state. The secretion of these cells is not HCl, but it is
full of organic matter. The secretion of the whole of a fundus gland is
slightly alkaline or neutral until the foveola is reached, The gland con-
DIGESTION IN THE STOMACH 375

tents in the foveola stain a blue color, indicating acid. From these
observations it may be concluded that the acid is not secreted by the
cells of the stomach at all, but is probably formed in the fovea by the
reabsorption of some basic constituent, leaving the acid outside.

We may perhaps picture the formation of the acid of the gastric
juice as follows: It is not formed in the cells, but in the cavity of the
stomach and in the fovee of the glands. The cells lining the mucous
membrane are non-permeable to it. It is possible that the chlorine is
secreted as ammonium chloride, or the chloride of some other weak
base. It might also be secreted as an ester. When in the cavity of the
stomach, perhaps as it passes along the lumen of the gland, or at any
rate in the neck of the gland, a hydrolytic or other dissociation takes
place, setting free hydrochloric acid.

NH,Cl + H,0—— NH OH + HCl
Absorbed.

Either by some kind of selective absorption or adsorption the NH,OH is
removed by the cells of the neck of the gland, leaving the HCl behind.
Such processes of an unknown nature, called selective adsorption, are
believed to occur; but to fall back on this terminology here is in the
nature of a subterfuge, since it simply puts the mystery under a new
name. Possibly by a chemical reaction the ammonia is absorbed and
converted into uramido compounds or carbamic compounds in the cell,
keeping the pressure of the ammonia ion in the cell nearly zero. In
every solution of NH,Cl there are NH, and OH ions, and it is known that
undissociated NH,OH or NH, penetrates cells readily.

It may be urged in favor of this hypothesis that it is supported by
all the direct evidence which we have. A similar formation of hydro-
ehloric acid in just this manner occurs elsewhere in nature. Thus if
the mould, penicillium, is grown in a medium containing ammonium
chloride it absorbs the ammonia, leaving the hydrochloric acid outside.
This is just the mechanism supposed in the foregoing theory for the
stomach. Furthermore, the mucosa of the stomach contains more
ammonia than any other tissue of the body. There are, for example,
40-52 mgs. in a hundred grams of stomach mucosa; whereas arterial
blood contains about 1 mg. per 100 grams. The theory accounts, too,
for the fact that the most acid juice is found in carnivora and in
omnivora on a protein diet, a diet which by the deaminization of amino-
acids sets free large amounts of ammonia. The theory has, theerfore,
much in its favor. The only great difficulty is the small degree of
hydrolysis of ammonium chloride. The theory needs further confirma-
tion before it can be wholly satisfactory. The fact that the mucous
membrane or the cells of the mucosa, as long as they are alive and in
good condition, have a resistance to the entrance of the acid cannot be
376 PHYSIOLOGICAL CHEMISTRY

doubted, since otherwise the acid would be neutralized by reabsorption.
This impermeability is, however, of a very limited kind, and if the cell
is weakened by disease or by partial occlusion of the arteries, or by
great anemia, its resistance may be so lowered that the acid penetrates
and digestion of the stomach wall begins. Particularly hyperacidity
is dangerous, because the limit of resistance of the cells may be sur-
passed. The absorption of the base leaving the acid may be something
similar to the precipitation of an alkaloid by Lloyd’s reagent (hydro-
silicate of alumina). This reagent if added to a solution of strychnine
sulphate or other alkaloidal salt precipitates with itself the alkaloid
leaving the acid in solution.

There have been two or three other suggestions of the nature of the
process of the secretion of the acid which should be noted, although they
are probably incorrect. The first is that of Maly, according to which
by a double decomposition of the acid carbonates and alkaline phos-
phates and the chlorides some hydrochloric acid is formed in the cells
of the stomach and the acid is then, in some unknown way, discharged
in one direction and the alkali in the other. This seems highly improb-
able. In the first place, there is no evidence that acid is formed in the
cells. In the second place, the amount of free acid thus formed would
be extremely small, because the avidity of hydrochloric acid is so much
greater than the very weak carbonic acid. Hydrochloric acid being
stronger would take the sodium from the carbonate, not the other way
around. In the third place, it explains one miracle by supposing a
greater one in the separation of the free acid from an alkaline cell.
Koppe has also made a suggestion that the hydrogen ions of the blood
wander through the membrane and are exchanged for sodium ions
which come in. The membrane is supposed to be non-permeable for
negative ions like chlorine. The difficulty here is also to understand
how such a membrane could be constructed, and the extremely small
numbers of hydrogen ions in the blood. To get the acidity of the gastric
juice as fast as it is secreted would be impossible. The explanation has
the one merit of making the place of formation in the interior of
the stomach rather than in the cells thereof. Otherwise it is no ad-
vance. As a matter of fact, the juice is secreted from the glands
with full acidity and even in the absence of sodium chloride in the
stomach.

One of the results of this acid secretion in the stomach is to render
the whole blood and tissues of the body more alkaline, and this pro-
foundly aifects their metabolism. The sodium of the sodium chloride
which has undergone decomposition finds its way into the blood as the
alkaline phosphate or ‘carbonate. The urine may even become alkaline
during the eating of a meal and always its acidity is reduced. The
DIGESTION IN THE STOMACH 377

change of alkalinity of the tissues increases the oxidation of the tissues
and is responsible in part for the increased metabolism and heat pro-
duction during digestion, observed by Lavoisier. It may very easily
be a factor in the feeling of well-being following eating. Its impor-
tance for the body as a whole is generally overlooked.

Rennin and its action.—Gastric juice, or the aqueous infusion of the
ealf’s stomach or of any other suckling mammal, clots milk. The essen-
tial fact in this clotting is that a soluble protein, casein (caseinogen, as
the English call it), is transformed to an insoluble form called paracasein
(English casein). The paracasein is so concentrated that it does not
flock out of the solution as insoluble precipitates generally do, but it
remains distributed all through the milk, and the water, with the fat, is
held or entangled between the particles of paracasein so that a gel is
formed. The substance in the gastric juice which causes this change in
the casein is the rennin, or chymosin. If a little sodium oxalate or any
other substance which precipitates calcium be added to milk, the milk
will not clot on the subsequent addition of rennin. Calcium as well
as rennin is, therefore, necessary to the clotting of milk. If after the
addition of the oxalate and the rennin one waits for about half an hour
or even a shorter period and then boils the milk so as to kill the rennin,
the milk is still fluid, but if one now adds calcium chloride or some
other calcium salt to the milk so that there is more than enough to com-
bine with the oxalate, then clotting comes on at once. From this experi-
ment it is clear that although in the absence of calcium no clotting has
taken place, yet the rennin must nevertheless have acted on the casein

‘and changed it so that, even in the absence of rennin, as soon as calcium
is added the milk clots. Hammarsten found, also, that when milk clots
and the whey or fluid portion is separated from the clot, a new protein,
an albumose called whey albumose, appears in the whey. When milk

‘clots, therefore, casein disappears and two new proteins appear, whey
albumose and paracasein, the latter of which is found as a calcium
compound in the clot; the former in the whey. Since a pure casein solu-
tion acts in the same manner, we may recapitulate all of these facts in

‘the following explanation: Rennin splits casein into whey albumose ( ?)
and paracasein, the paracasein thus formed is much less soluble than
the casein as is shown by the fact that it is much more easily precipi-
tated by acids, and its colloidal particles are of larger size. The para-
casein forms with calcium an insoluble calcium salt, calcium paracasein-
ate, and this precipitate clots the milk. The molecular weight of casein,
according to Van Slyke, is about 8,500, that of paracasein about 4,500.
He believes that rennin splits casein into two molecules of paracasein.

The action of rennin appears, therefore, to be nothing more than
the early stages of digestion or hydrolysis of the casein molecule. Casein
378 PHYSIOLOGICAL CHEMISTRY

clots when digested, for the reason that one of the early meta-protein
hydrolytic products forms with calcium an insoluble compound. The
action of rennin thus appears to be simply the action of a proteolytic
ferment, and it is an interesting fact that all proteolytic enzymes appear
to act similarly, although they differ considerably in their power. Thus
not only does the gastric juice clot milk, but so will pancreatic juice.
the juice of the intestine, the juice of the pineapple, of the cocoanut, the
secretions of many bacteria and the extracts of some crucifere. In fact.
wherever in nature one finds a proteolytic enzyme there one finds also
a milk-clotting enzyme. It may be concluded from this either that
the enzyme, rennin, is very widespread in nature, constantly accom-
panying proteolytic enzymes; or that the clotting of casein solutions is
an indirect result of the proteolytic cleavage of the molecules of casein.
The latter conclusion seems the more probable.

The foregoing conclusion does not mean, however, that rennin and
pepsin are identical. This is a matter in dispute at the present time
and a brief examination of the facts in the case will be worth while.

If it were possible to obtain a solution which had peptic but no
elotting action, or vice versa. it might be inferred that the two sub-
stances were different. Hammarsten found that it is indeed possible to
prepare solutions which have one but not the other action. By heating
pepsin solutions for several hours at 40° C. they lose their rennin but
not their pepsin action; if precipitated by lead acetate or magnesium
carbonate, pepsin is completely precipitated, but a part at least of the
rennin still remains in the solution. The most. striking fact indicative
of a difference between these two ferments is that peptic digestion takes
place only in acid solution, whereas rennin coagulation will occur in
neutral or amphoteric solution. These facts all look as if the ferments
were two different substances. There is no doubt, however, that pepsin
has a coagulating action on milk. Thus Hammarsten tried the following
experiment: A pepsin solution, which had been heated 48 hours at 40° C.
to make its rennin action very weak. after neutralization coagulated
milk only after 6 hours and 10 minutes when added in the proportion
of 1:5. By the addition of HCl the neutral solution was brought to
an acidity of .1 per cent. HCl and it coagulated now in 12 minutes. A
control showed that this coagulation was not due to the acid. Another
solution of rennin, which according to Hammarsten was pepsin-free,
was diluted with water so that it coagulated milk only after 45 minutes:
it was then made acid to .1 per cent. HCl and it coagulated after 20
minutes. The addition of acid, therefore, accelerated the action of the
solution which contained much pepsin but little rennin far more than
it did the solution containing rennin but no pepsin. The clotting in the
first juice must, therefore, have been due to the pepsin.
DIGESTION IN THE STOMACH 379

Experiments similar to these have been carried out by Schmidt-
Nie!ssen. He heated an acid extract of calves’ mucosa so long at 40° C.
that on neutralizing it with n/10 NaOH the neutral fluid added to
milk in the proportion of 1:5 coagulated the milk only after 4-6 hours
at 38°. This solution he called A. Another portion of the same extract
was not heated, but was diluted with water and neutralized with
n/10 NaOH until it had a rennin action approximately equal to solu-
tion A. These two solutions, A and B, when neutral had, then, the same
coagulating action and presumably had the same content of rennin. If
now there is only one ferment in‘ the juice, then if acid is added to
each of these solutions they ought to be affected to the same degree and
they should have the same action on milk and fibrin. The results were
as follows:

Solution A. Warmed. Solution B. Not warmed.
Time for coagulation Time for coagulation.
1. Neutral milk ............... 370 minutes 355 mirutes
AGId MUNK ios edie doers toes ee 3 * 215 s
Fibrin digestion ............. 3 hours 80 hours
2. Neutral milk ............... 420 minutes 360 minutes
Acid “MINE: i acaiets ts seins x states 18 oe 250 -
Fibrin digestion ............. 3-4 hours 80 hours.

It is clear from these experiments that, although solution B had a
little stronger action in the neutral solution and so presumably con-
tained more rennin, yet the addition of acid affected the two solutions
to very different degrees, A being enormously accelerated by the acid
both in its coagulative and in its digestive power; whereas B was only
slightly accelerated by the acid and’ had a very weak digestive power.
The heating must have destroyed most of the rennin (or changed it to
pepsin) and the coagulation in the acid-solution must hence be due in
chief measure to the pepsin, which is thus shown to have a clotting
action. It has this clotting action only in an acid medium. There seems
to be no escape from Schmidt-Nielssen’s conclusion that the two fer-
ments are not identical, or as he says, ‘‘ that enzyme which coagulates
neutral milk, chymosin, cannot be identical with pepsin.”’

A difference between the ferments is also shown by the time law
of the coagulation. Thus for rennin in neutral solution the product
of the time of coagulation by the ferment concentration is a constant;
if one adds half the quantity of rennin, the time of coagulation is
double, or C, t=K. C, is the concentration of the ferment. Schmidt-
Nielssen found that the coagulation time in acid milk did not follow
this simple proportionality law, as may be seen in the following
experiment:
 

 

 

5 peer Infusion Cooked . j
Milk containing warmed:o? infusion for Concen., of Coag, time— Cer
4 ft
4 per cent. HCl, calves’ atomach dilution eDeyme NUL
10 ee. 2 0 1 9.5 9.5
10 ee. 1 1 0 35 17.5
10 cc. 5 1.5 25 300 75

 

 

An experiment showing the effect of acid in accelerating the clotting
action of the pepsin-rich, rennin-poor solution is the following:

HCl in milk-enzyme mixture— Ccagulation time—
per cent, minutes
0.42 3.5
0.25 10
0.17 42
0.08 280
0.00 Not determined

Other points of difference between the enzymes have been pointed
out by Bang. Furthér evidence that there are at least two different
enzymes is the fact that the infusion of calf’s stomach has a powerful
clotting action, but a weak digestive action on egg white; whereas the
infusion of the pig’s stomach has a powerful digestive but a weaker
clotting action.

This view of the difference of the two enzymes does not stand unop-
posed. Pawlow and his pupils have established many facts which they
have interpreted to mean that the two ferments are the same. Pawlow
at first calls attention to the fact, so extremely significant, that one
always finds a clotting action wherever one finds a proteolytic enzyme.
This is true even in the case of plant enzymes which could never have
been elaborated to act upon milk. This, he maintains, shows that all
proteolytic enzymes coagulate milk and, vice versa, the coagulation of
milk is caused by a proteolytic enzyme, a conclusion which has been
supported by the discovery of the character of the change in the casein
accompanying clotting. They were unable to prepare rennin solutions
which were free from proteolytic power and pepsin solutions which
were free from a rennin action. Pawlow and Parastchuk experimented
with gastric juice of an adult dog obtained by sham feeding. They
found that bread juice has a stronger milk-clotting action as well as a
stronger proteolytic action. One of their experiments consisted in taking
three samples of juice: 1, milk juice; 2, meat; and, 3, bread juice.
These at the start had very different proteolytic actions. By dilution
they were made of equal pepsin content and then they were tested to
see whether they were now equal in clotting power. The experiment was
as follows:

 

 

 

 

Milk juice | Meat juice Bread juice
Digesti ;
aie, eee iy Cvag. time— a sneha Coag. time— D nestive Coag. time—
Mett’s tube. minutes an minutes Poe minutes
24 hours :

1.8 50 3.6 5.25 5.8 OB
1.85 30 4.05 3.0 5.8 0.75
2.45 12 3.8 3.2 6.4 0.7&
2.9 35 4.0 9.0 6.4 5.5
2.5 26 3.6 6.5 6.6 2.5

 

 

 

 

 
DIGESTION IN THE STOMACH 381

It will be noticed that the bread juice coagulates in the shortest time
and is the most powerful digestant, and the milk juice is the weakest.
To show that when they have the same peptic content the coagulation
time is the same, a sample of each kind of juice was taken and the
amount of pepsin made the same in each by dilution, according to the
law of Schiitz and Borissow by which the amount of pepsin is propor-
tional to the square of the egg albumin digested in a Mett’s tube. At
the start the bread juice digested 5.8 mm., the meat 2.8 and the milk
2.0 mm. of egg albumin in the same time. The pepsin in these samples
was then in the proportion of the squares, or 33.64:7.84:4.0. By dilution
with acid they were made equal in pepsin and acid. Equal amounts
of the juices then coagulated milk respectively in 190 minutes, 190
minutes and 195 minutes. It will be observed that this is in an acid
solution. It was found, also, that when the juice was kept in a thermo-
stat it lost both actions at the same rate and both disappeared at the
same time. Moreover, on heating five minutes at varying temperatures
from 15° to 62°, the coagulation power and the pepsin action fell
together, and both disappeared at the same temperature, namely with
five minutes’ heating at 62° C.

Although these experiments were interpreted by their authors as
being in opposition to those of Hammarsten, they are in reality not so.
Hammarsten’s work was done on the neutral infusions of calf’s stomach
and the clotting of neutral milk was studied. What Pawlow and
Parastchuk have really shown is that in dog’s gastric juice pepsin has
a rennin action; and, probably, that in the stomach of an adult dog
gastric juice contains no specific rennin enzyme, the rennin effect being
due to pepsin.

The experiments prove that in the stomachs of calves there is present
in addition to pepsin an enzyme which will clot milk in neutral solution,
and this enzyme was the one originally called rennin or chymosin.

The conclusion seems justified that the coagulation of milk is not
a specific function of any single enzyme, but that it is a general prop-
erty of all proteolytic ferments. The clotting is due to the fact that
one of the first products of the decomposition of the casein forms an
insoluble calcium compound which sets or gels. In the mucosa of the
stomach there may be, and no doubt are, more than one proteolytic
enzyme. There can hardly be a doubt that the mucosa of the stomach
does produce different proteolytic enzymes in different portions of the
stomach. The fundus glands produce pepsin, that is an enzyme active
only in an acid medium and easily destroyed by slight alkalinity. The
pyloric extract, on the other hand, is known to furnish an extract which
is active not only in an acid medium but also in a neutral medium. It
must, therefore, contain proteolytic enzymes more allied to trypsin and
382 PHYSIOLOGICAL CHEMISTRY

erepsin. It is not strange, therefore, that more than a single proteolytic
enzyme should exist in the gastric juice and be present in varying pro-
portions at different ages. It has been observed, indeed, that in the
course of development ereptic enzymes which act on certain native pro-
teins and albumoses appear in the tissues before pepsin, which is the
specific enzyme of the stomach. A -albumosease (erepsin) has been
found in gastric juice.

The facts clearly indicate that the coagulation of milk in a neutral
solution, a power particularly developed in the stomachs of suckling
animals such as the calf, is due to a particular proteolytic enzyme of
the ereptic type active in neutral or very faintly acid solution. This
particular ereptic ferment is rennin (chymosin). But in the adult
stomach, particularly of carnivora, the amount of this enzyme is greatly
reduced, or it may be absent, and the clotting of acid milk by the gastric
juice of these animals is a result chiefly or entirely of the activity of
pepsin. The clotting of milk is, then, no test for the presence of a
specific ferment for casein, and, since other ferments than pepsin clot
milk, the use of the time of clotting as a test for pepsin in gastric con-
tents is entirely unwarranted. It is extremely significant in this con-
nection that erepsin and ereptic ferments, although they do not nor-
mally digest native proteins, but oniy albumoses, do attack and digest
casein, and that casein is one of the most unstable and easily hydrolyzed
proteins known.

The question may be asked whether anything is gained by having
in the stomachs of the new-born an enzyme which will clot and digest
milk in a neutral or very faintly acid medium. The answer to this
question is not difficult, for the advantages are obvious. The mucous
membrane of a child’s stomach is extremely delicate; it is very thin
and so sensitive that it cannot bear even the firm clot of cow’s milk.
It cannot stand much acid. Milk is a liquid and liquids normally pass
guickly through the stomach into the intestine. By having a proteolytic
enzyme acting in a neutral or amphoteric medium on casein the milk is
clotted in the stomach, even when the secretion is only faintly acid. The
fluid portions are passed on, but the curds are held for digestion by the
rennin and, as acidity develops, by the pepsin also.

Salivary and intestinal digestion in the stomach.—The food while
in the stomach is acted upon not only by the juices secreted by this organ.
Saliva swallowed with the food continues to act; and there is, at times,
a regurgitation of digestive juices from the intestine so extensive that
the stomach digestion may partake largely of the type of intestinal
digestion.

The saliva continues to act in the fundus end of the stomach for a
period longer or shorter, depending on the size and character of the
meal and the amount of gastric juice. The center of the mass of food
DIGESTION IN THE STOMACH 383

in the fundus end of the stomach does not become sufficiently acid to
stop ptyalin action for about an hour, on the average, after the food is
eaten (see page 335). During this time ptyalin is active and we now
know that starch digestion can proceed well toward its end in the
stomach. Moreover, we have always the action of the bacteria. These
are killed for the most part when the juice is acid, but in hypochlorhy-
dria, or when the motility of the stomach is depressed, the activity of
the bacteria may be a source of much discomfort. By the fermentation
by bacilli of a lactic acid type, or the butyric acid bacillus, or various
sarcinas, lactic, butyric or acetic acids may be formed, and large quanti-
ties of hydrogen, or carbonic acid gas evolved. A sufficient amount of
gastric juice to produce rapid acidity will prevent this bacterial growth.
Hence, if much hydrochloric acid is formed, there is little lactic acid
produced.

Still another factor enters into the gastric digestion: namely, the
regurgitation of bile, pancreatic and duodenal juice into the stomach.
This ordinarily occurs to a relatively slight extent, but under certain
conditions it may result in the digestion becoming of a real intestinal
nature. This fact, discovered by Boldyreff, has been taken advantage
of by him for obtaining pancreatic juice for clinical examination. The
presence of bile was observed in the stomach of Alexis St. Martin, but
it does not seem to have occurred to physiologists that the pancreatic
juice might come also. Boldyreff found that the presence of fat, and
particularly fat with fatty acid in it, caused this regurgitation. Also, if
the stomach is more than normally acid, there is a pouring-in of large
quantities of intestinal juice to neutralize the hyperacidity. :

Summary of gastric digestion—The empty stomach is normally
contracted on itself and its cavity obliterated so that its walls are in
contact. Under these conditions the mucous membrane is white, or a
pale pink, thrown into folds, or ruge, and the surface is covered with
a thin layer of mucus either alkaline, or neutral, or very faintly acid
in reaction. When one is hungry the smell, or sight, or the taste of
food is sufficient to start the secretion, so that before a meal is eaten
there may be some gastric juice present in the stomach. This juice is
acid in reaction, due to the presence in it of hydrochloric acid, and it
has a strong solvent action on proteins, due to the presence of an enzyme
ealled pepsin. When food is eaten the stomach slowly relaxes as the
food is swallowed and thus adapts itself to the size of the meal which
has been eaten. The food at first accumulates in the fundus end of the
stomach in a large bolus, which retains a very faint acid or neutral reac-
tion in its interior for a considerable period and in this bolus salivary
digestion continues some time.

As soon as food enters the stomach, or even before it entcrs, the
384 PHYSIOLOGICAL CHEMISTRY

4

stomach begins to pour out the strongly acid secretion of the gastric
glands. This juice attacks the exterior of this mass of food and slowly
softens, digests and liquefies the peripheral portions of it. At the same
time movements of the stomach begin and increase gradually in intensity.
These movements are in the nature of peristaltic constrictions, which
appear at about the junction of the pyloric and fundus regions of the
stomach and then travel slowly toward the pylorus. By means of these
movements some of the partially digested, softened portions of the
periphery of the bolus of food are broken from the main portion and
thoroughly mixed with the digestive juices.

At first when the peristaltic wave reaches the pylorus the latter does
not open, but the wave is reflected back again toward the fundus end
of the stomach, thus carrying the portions of the food back with it. By
the combined action of the movements and the solvent action of the
juice, the mass of food is slowly reduced to a state of fine subdivision
and solution in an acid medium and this mixture of foods partly in sus-
pension and partly in solution is called chyme. Gradually, as the free
acidity of the juice increases, the vigor of the movements increases. As
the peristaltic waves reach the pylorus the latter relaxes a little and
some of the more fluid portions of the chyme are thus squirted into the
intestine, where they cause the secretion of the juices of this part of
the canal in the manner shortly to be described. In this manner the
stomach slowly empties itself and the food is passed little by little toward
and through the pylorus. It happens at times that larger masses are
carried along in the peristaltic wave, but when these masses strike the
pylorus the latter closes down upon them and does not let them pass.

The various foodstuffs are acted upon in such a way by the gastric
juice and the saliva that when the contents leave the stomach the proteins
have been reduced for the greater part to the state of acid albumin and
proteoses. Some polypeptides, tri- and di-peptides are also in the chyme.
Some protein particles have not been digested, however, and meat fibers,
as such, may pass the pylorus to be acted upon and digested in the intes-
tine. The nucleins are not digested. The fats, if already saponified, have
been in large part already converted by the lipase of the stomach into
fatty acids and glycerol, but the fats which are not emulsified, such as
the fat of meat, are passed through the pylorus still as neutral fat. The
starches have been in large measure digested by the amylase of the saliva
and changed to dextrins and maltose, so that in the chyme both of these
are found; and some of the cane sugar, if that has been eaten, has been
inverted by the action of the acid of the juice and converted into glucose
and levulose, but most of the carbohydrates are passed on into the intes-
tine only partially digested, to await their final digestion in that organ.

The bacteria and parasites swallowed by the individual have been
DIGESTION IN THE STOMACH 385

for the most part killed by the acid of the juice, so that the strongly
acid chyme is nearly or quite sterile.

The study of the origin of the pepsin and hydrochloric acid, which
are the principal active constituents of the juice, has shown that the
pepsin is formed in the cells of the glands of the stomach and particu-
larly the cells of the fundus region. It exists in the cell presumably in
an inactive form called pepsinogen, and is made active by the action
of the acid of the juice. What cells form the pepsin is still uncertain.
The origin of the hydrochloric acid is still very obscure. It is formed
from the chlorides of the blood and by the fundus part of the stomach.
The probability is that it is formed in the neck of the gastric glands,
and perhaps all along the surface of the mucous membrane, by the reab-
sorption (selective adsorption?) of the basic part of the chloride (which
is possibly ammonia) leaving the acid on the outside. The stomach wall
is impervious to acid. It is certain that the acid is not formed by the
parietal cells, as was at one time believed, but for which there never
was any good, direct evidence. The formation of the acid is a problem
of great importance which must be left to the future to solve.

The acidity of the juice formed is subject to wide variation in disease,
and the determination of the emount and character of the acid is of
diagnostic value in some stomach disorders.. The methods for the inves-
tigation of this acidity have already been described. In carcinoma of
the stomach the acidity is generally below normal or absent; in ulcer
the acidity is generally above normal.

The resistance of the stomach to self-digestion is due to the fact that
the acid is not able to penetrate the living protoplast of the cells of the
membrane; but when they are dead or when their resistance is reduced,
it does so penetrate and then the pepsin digests the dead organ. The
tells contain, also, a substance which checks peptic action, an anti-
ferment.

Not all of the products of digestion pass out into the pylorus. An
unknown proportion, but probably a small proportion, of the food is
absorbed directly by the stomach, according to the most recent
investigation.

REFERENCES. Sromacu Dicestion.

1. General. Beaumont: Physiology of Digestion.
Pawlow: The Work of the Digestive Glands. Translation by Thompson.
Bidder and Schmidt: Die Verdauungssifte.. Leipzig, 1852.
2. Acidity of gastric juice. Christiansen: Untersuchungen iiber freie und gebun.
HCl in Mageninhalt. Biochem. Zeits. 46, 1912, pp. 24, 50, and 287,
Toepfer: Eine Methode zur titrimetrische Bestimmung der hauptsiichlichsten
Factoren der Magenaciditit. Zeit. f. physiol. Chem., 19, 1894, p. 104.
(Previous literature cited here.)
886 PHYSIOLOGICAL CHEMISTRY

8. Secretion in human stomachs. Richet: Comptes rendus de l’Acad. de Sci.
84, Gaz. Hebd. 187, No. 10. Carlson: A. Jour. Physiol. (Various papers,
1912-14.)

4. Formation of HCl. Harvey and Bensley: Biological Bulletin, 23, p. 225, 1912.

Sjéquvist : Skandiniivische Archiv f. Physiol. 6, 1895, p. 255.

Maly: Sitzungsber. d. Wiener Akad. 69. Abth. II and ITI.

Schultz: Die Zerlegung der Chloride durch CO,. Pfltiger’s Archiv 27, p. 454.
Lopez-Suarez: Biochem. Ztschr. 46, 1912, p. 490.

Ritter: Reabsorption of NH, by moulds. Biochem. Zeits. 42, 1913, p. 2.

5. Analysis of gastric contents. Panton and Tidy: Quarterly Journal of Medi-
cine, 4, 1910-11, p. 454. ;

6. Pepsin. Method of determination. Gross: Berl. klin. Woch., 1908, 45, p. 643.

Reicher: Wiener klin. Wochenschr. 48, 1907.

7. Gastric juice. Composition. Rosemann: Pfliiger’s Archiv f. d. ges. Physiol.
118, 1907, p, 467.

8. Passage of duodenal juices into the stomach. Boldyreff:. Pfliiger’s Archiv.
121, 1907, p. 13; 140, 1911, p. 436.

9. Pepsin and rennin. Not influenced by Roentgen rays. Richter and Gerhartz:
Berl, klin. Wochen., 1908, 45.

Action of alkalies. Ticomiroff: Zeit. f. physiol. Chem., 55, 1908.

Law of action, Schiitz: Wiener klin. Wochenschrift, 21, 1908, p. 729.

Various pepsins. Wroblewski: Zeits. f. physiol. Chem. 21, 1895, p. 1.

Identity of rennin and pepsin. Schmidt-Nielssen: Zeits. f. phys. Chem., 48,
p. 92, 1906.

Hammarsten: Zeit. f. physiol. Chem., 92, p. 121, 1914.

Pawlow and Parastehuk: Zeit. f. physiol. Chem., 42, 1904,

10. Pepsin. Colloidal nature. Recherches sur le mode d’action de la pepsine dans
la digestion de l’albumine. Arch. Intern. de Physiol. 13, 1913, p. 316.

11. Pepsin. Composition. Nencki and Sieber: Zeits. f. physiol. Chem., 32, p. 291,
1901.

Pekelharing: Zeits. f. physiol. Chem., 22, 1896.

12. Energy transformations in enzyme reactions. | Lengyl: Pfliiger’s Archiv f. d.
ges. Physio]. 115, p. 7, 1900.

13. Acid control of the pylorus. Cannon: Amer. Jour. of Physiol., 20, 1907, p. 283.

14. Excretion of pepsin in urine. Wilenko: Berlin klin. Wochenschrift 22. 1908.

15. Pepsin in infants. Rosenstern: Berl. klin. Wochenschrift 1908. No. 11

16. Isolation of pepsin by elastin. Abderhalden and Strauch: Zeits, f£. physiol.
Chem. 71, 1911, p. 315.

17. Protection of stomach against digestion. Katzenstein: Berl. klin. Woch. 39,
1908.

18. Other proteolytic enzymes in the stomach, Hirayama: Zeiis. f. physiol.
Chem, 65, 1910, p. 290. :

19. The chemical methods of gastric secretion. Hdkins: Journal of Physiology,
34, 1906, pp. 132-144.

20. Favorable hydrogen ion concentration for gastric digestion. Michaelis:
Die Wasserstoffionen Concentration, p. 70, Berlin, 1914.

21, Gastrin. Moronescu: Inter. Beit. z. Path. u. Therap. Ernihrungsstérunger.,
1, p. 194, 1910. ;

Ehrmann: Ibid., 3, p. 382, 1911-12. Emsmann: Thid.. 3. p. 117, 1912.
Tomazewshi: Cont. f, Physiol. 27, 1913. Keeton and Koch: A, Jour. Physiol.
37, p. 481, 1915.
CHAPTER X.
DIGESTION IN THE INTESTINE.

Duodenal secretion.—The discharge of acid chyme into the intes-
tine causes the secretion of duodenal juice. We have now to inquire how
it causes it and what are the properties and uses of the juice thus poured
out. This brings us to one of the most interesting of the unsolved prob-
lems of physiology: namely, the true functions of the duodenum.

The duodenum (Latin, duodent, twelve each), or the twelve-finger
intestine, as the Germans call it (Zwélffinger Darm), because it is about
eleven inches or twelve finger breadths in length, is that part of the
intestine extending from the pylorus to the jejunum, or the empty intes-
tine, and it receives the two very important secretions of the liver and
the pancreas as well as its own peculiar one. It is lined throughout with
a mucous membrane which contains a large number of small glands, the

.glands of Brunner.

The mechanism of secretion of these glands has not been sufficiently
studied, but they pour into the duodenum a large amount of a strongly
alkaline, albuminous juice. The alkalinity of the juice is due to car-
bonates and it effervesces strongly on the addition of acid. It has by
itself very weak digestive powers. It contains some invertin, so that
it will invert cane sugar; and some erepsin;.but its main function is to
increase the power ci, or to work in conjunction with, pancreatic juice

‘and bile.

The quantity of duodenal juice secreted normally in man cannot be
staied, but from a duodenal fistula of a loop cut off from the intestine
and the stomach an extraordinarily large amount of a clear, colorless,
alzaline (H ion content about 2X10—-* N) strongly albuminous fluid is
discharged. In a dog weighing 5 kilos, 50 ¢.c. were secreted in 120 min-
utes. It does not seem possible, or probable, that the secretion can under
normal circumstances be as large as this.

Enterokinase.—The duodenal juice is remarkable because of a sub-
stance in it called enterokinase, meaning literally the active substance
of the intestine, which has the property, when mixed with pancreatic
juice, of enormously increasing the action of the latter on proteins. If,
for example, three samples are prepared in three test-tubes, one of pure
pancreatic juice, one of pure duodenal juice and one of a mixture of
equal parts of duodenal and pancreatic juice, and a piece of fibrin or

387
388 PHYSIOLOGICAL CHEMISTRY

other protein is introduced into each of the three, the mixture digests
the protein at once, while the pure juice in each case leaves it almost
unaffected.

Enterokinase is found in the mucous membrane of the duodenum and
may be extracted with dilute bicarbonate solution. It is destroyed by
heating. It is found in the intestines of all the vertebrates. In the dog-
fish (Elasmobranch), Mustelus canis, one finds none of it in the stomach
mucosa, but it is found all along the intestine mucosa nearly to the
rectum, but the larger quantities are found in the upper part. It is
supposed to act by converting an inactive trypsinogen'in the pancreatic
juice into active trypsin, but the evidence of this is still uncertain,
although this interpretation is probable. It is claimed by some that
enterokinase, or substances of a similar property, are found in leucocytes
and other cells, but this is denied by Bayliss and Starling. It is cer-
tainly found nowhere else in the dogfish than in the mucous membrane
of the intestine. A small amount of enterokinase is able to increase the
digestive activity of the pancreatic juice on proteins very strongly, and
for this reason it is thought to be a ferment itself.

Other enzymes in duodenal juice.—Besides containing enterokinase,
the duodenal juice is important in digestion on account of its alkaline
reaction, by which it aids in the neutralization of the acid chyme coming
from the stomach, and because of the carbohydrate enzymes invertin,
maltase and lactase it contains, which give it a powerful action on the
disaccharides cane sugar, maltose and lactose, converting these to mono-
saccharides.

Other functions of the duodenum.—There are also certain facts
about the juice which are well deserving of farther investigation. lt
seems to be necessary to life. Dogs live for long periods if the bile and
pancreatic juice are divertéd from the body by fistulas; but if the °
duodenal juice is completely diverted to the outside, they rarely live
more than 48 hours. If, for example, a ligature is placed about the
pylorus, so that duodenal juice cannot go back into the stomach, and
the duodenum is cut away from the intestine about six inches from
the pylorus, a gastro-enterostomy being made so that the food can pass
from the stomach to the intestine, and if the duodenal sac thus made
is drained to the exterior by a fistula, or if the duodenum be taken out
entirely, no serious symptoms show themselves for 24 or 36 hours, but
very shortly afterwards the dogs die. If, however, the duodenum is left
in connection with the pylorus, being cut off at its lower end, so that
the juice can get back into the stomach and so into the intestine: or if,
after tying both ends of the duodenum, a rubber tube is connected with
the duodenum and the jejunum so that the juice can pass along it, no
serious results follow the operation. Indeed, extirpation of the first
DIGESTION IN THE INTESTINE 389

six inches of the dog’s duodenum is almost invariably fatal in the ex-
periments reported. Similar fatal results have been reported in human
beings following resection of the duodenum. The cause of death is still
obscure. Whether it is due to the rapid secretion of some necessary
substance through the duodenum to the exterior; or whether it is due
to the duodenum making something which is necessary to the function-
ing of the gut lower down, or whether it is due to some other cause, is
quite unknown. An experiment has recently been reported in which the
duodenum was removed from a dog after implantation of the gall and
pancreatic ducts into the jejunum and this dog survived. (Moorhead.)

Excretory function of duodenum.—The duodenal juice may have
in it not only its normal constituents, but many substances are excreted
here. Thus sugars of various kinds put directly into the blood are
excreted into the duodenum and reabsorbed farther down. This fact
makes the interpretation of metabolism experiments in which sugars are
injected directly into the blood very difficult, because one cannot easily
tell whether the sugar is used directly, or only indirectly after its excre-
tion and reabsorption. So, also, morphine is excreted here, and potas-
sium ferrocyanide and many metals when they are injected subcutane-
ously, or intravenously ; indeed, this part of the intestine is an important
excretory organ, which functions especially in cases of renal in:
sufficience.

Pancreatic juice——Closely applied to the duodenum, or lying in
the mesentery by its side, is the pancreatic gland, a digestive organ of
the first importance which pours its secretion into the duodenum through
two main ducts, the duct of Wirsung and the duct of Santorini. There
may be, also, subsidiary ducts between these two. The latter of these
ducts opens in human beings about four inches (9-10 cms.) below the
pylorus; the former, in common with the bile duct, about five and three-
fourths inches below the pylorus.

The pancreas, like the other digestive glands, does not secrete con-
stantly, but only intermittently when its secretion is needed. Its strongly
alkaline secretion gushes forth, together with the bile and the duodenal
juice, when the acid chyme is squirted through the pylorus into the
intestine, and it is continued until the acidity of this chyme is
neutralized.

Composition of human pancreatic juice-—Human pancreatic juice
has been obtained from artificial pancreatic fistulas. The juice is alka-
line to litmus, due to the presence in it of sodium carbonate; it is as
clear as water and coagulates on- heating. The pancreatic juice of the
dog obtained on stimulating the vagi or by the action of pilocarpine
may contain so much protein that when heated it forms a solid coagulum.
The freezing point of human pancreatic juice is above that of the blood:
390 PHYSIOLOGICAL CHEMISTRY

i.e.. —0.42° to —0.49°. To neutralize it, using litmus as an indicator,
by titration it requires for 1 ¢.c. of juice 0.1-0.15 ec of N/10 HCl. The
neutralized juice becomes turbid at 47° C. and coagulates between 57°
and 59° (Wohlgemuth). It is precipitated by an equal volume of satu-
rated ammonium sulphate. The following is the composition of the
juice examined by Glaesner and Wohlgemuth :

 

Glaesuer Wohlgemath
Water’ axcevniin textes Pee ee .. 98.72 £8.70
Solids) 2: cs4uaesce's ee 1.27 1.30
Coagulable protein aa 174 0.093
Nitrogen ............... 2 0.0983 0.0813
Alcohol soluble substances ................ 0.508 0.523
Specific gravity ......... cece cece eens 1007.5 1007.13
Ash:
RS areiscsis 1.10% CI. saci 50.75 PO, ferred 1.85
Na .... 36.65 SO, wee 2.05 SO, eaayagels.< 0.34

Traces of Ca, Mg, Fe, sio,. co a 0.11%

This is the secretion from a permanent fistula. The secretion of the
first juice secreted from the dog’s pancreas on opening the duct is far
more concentrated than this. It may contain nearly 10 per cent. of
solids, 9 per cent. being organic matter. The concentration of hydroxy]
ions in dog’s pancreatic juice is equal to N/10,000 (Foa).

Control of the secretion of the pancreas.—How the secretion of the
pancreas is controlled brings us to one of the most interesting of recently
discovered facts concerning the chemical messengers of the body. To
understand what follows we must have some knowledge of the structure,
blood and nerve supply of this vital organ.

It is a relatively small organ weighing in the human adult about
87 grams. It is a tubular gland, the secreting cells being in acini and
they are filled, or more than half filled, with granules which during
secretion disappear from the cells and are replaced by a clear non-
granular protoplasm containing mitochondria. Figure 42. The gland
in the guinea pig and rabbit is very thin and spread out in the mesentery ;
and with a little care a loop of the intestine containing the organ in
the mesentery may be so placed on a microscope stage that the gland may
be watched secreting in the living state. The blood may be seen circu-
lating about the base of the cells and the granules are clearly visible near
the lumen. No structure could be seen by the author in the bases of
these cells, but only a clear protoplasm, and the granules could not be
seen to move in the cell itself. On stimulation of the vagus nerve the
bases of the cells seemed to be a little more indented between the cells,
but no other change was visible. Mitochondria may, however, be seen
in the base of the living cell when appropriate methods are used.

The blood supply is from the pancreatic artery and the blood returns
by the pancreatic vein into the mesenteric and ultimately into the portal
DIGESTION IN THE INTESTINE 391

vein, so that the blood after passing through the pancreas goes through
the liver. There is a double nerve supply, fibers coming both from the
vagi nerves, sometimes from one more than the other, and from the
splanchnics. Stimulation of these nerves under certain conditions causes
secretion from the pancreas. With this preliminary statement we may
approach the problem of how the presence of chyme ir the intestine
causes the pancreas to secrete.

Secretin—Since most organs are controlled by nerves, one naturally
thinks of a nervous reflex. The acid chyme may be supposed either

   

y

Fie. 42.—Section of pancreas of mouse. Osmium bichromate (Mathews).

directly, or indirectly by changes it induces in the mucosa, to stimulate
sensory nerve ends. Everyone knows how acids will stimulate such
endings or such nerves in the skin and they may act in the same manner
in the intestine. Such impulses may ascend the vagi nerves to the brain
and be reflected back over the vagi, or splanchnics, or both, causing a
dilation of blood vessels in the organ and a secretion of its stored juice.
This explanation appeared quite satisfactory until it was discovered
that after all nerves going to the pancreas were cut, putting acid into
the duodenum still caused secretion from the gland. The first interpre-
tation of this unexpected result was that there must be a local reflex
mechanism; that the impulses did not need go to the brain, but were
reflected through a local ganglion and so to the nerves of the organ. It
is very difficult, if not impossible, to prove that this is not the case, but
it was very soon found that certainly another mechanism different from
392 PHYSIOLOGICAL CHEMISTRY

this was called into play. Bayliss and Starling discovered that an acid
infusion of the mucous membrane of the duodenum and the intestine
when neutralized and injected into the blood caused secretion from the
pancreas and liver, whereas acid infusions of other organs of the body
had no such action. There was no doubt that a substance existed in
the mucous membrane of the intestine, duodenum and jejunum, which
could be extracted by boiling dilute acid, which was not protein or
coagulable, which was soluble in alcohol, and which caused, when injected
into the blood, secretion of the pancreas and liver. It caused, also, a
marked fall of blood pressure and a diminution of coagulability of the
blood, although these effects may be due to another substance than that
causing secretion. This substance, stimulating secretion, they called
“secretin” because of-its action on the pancreas. This discovery sug-
gested at once the possibility that the presence of acid chyme in the
intestine sets free secretin in the mucosa; and that some of this secretin
gets into the blood and makes the pancreas secrete.

The fact that such a substance exists in the mucosa does not neces-
sarily mean that the substance is actively engaged in the normal secre-
tion. To prove this it was necessary to prove that blood from animals
actively secreting pancreatic juice really contained enough secretin to
cause secretion. This proof was obtained by making a cross circulation
in two dogs, by anastomosing the arteries with the veins. Cannulas
were put in the pancreatic ducts of both dogs and then acid was placed
in the duodenum of one dog. Under these circumstances both pan-
creases secreted. This experiment shows that secretin actually enters
the blood when acid is put in the duodenum and that the amount is
sufficient to cause the pancreas to secrete. Bayliss and Starling propose
to call such chemical messengers as secretin, which arouse other organs
of the body, ‘‘ hormones,’’ from the Greek, horman, ‘‘ I rouse to activ-
ity.’’ Adrenalin is another hormone which in some particulars resem-
bles secretin. It is probable that this secretin came from the duodenal
mucosa of dog No. 1, but the possibility is not excluded that it arose in
the pancreas of this dog when the latter was acted upon by nervous
stimuli and after causing secretion in this organ passed to the blood and
aroused, then, the second pancreas to secrete.

It has been incorrectly inferred by some authors that this discovery
means that the nervous system is not concerned in the secretion of the
pancreas, but this is of course not true. It is unlikely that the nervous
mechanism plays no part in the secretion. .The existence of this mechan-
ism can be proved by directly stimulating the nerves, causing thereby
a copious secretion. Of course in this case, just as with the supra-renals
and splanchnies (page 778), it is very difficult to prove that the secretion
accompanying vagi stimulation is not due to the nerves acting on the
duodenum or stomach so as to cause secretion of secretin, which then
DIGESTION IN THE INTESTINE 393

indirectly arouses the pancreas. A certain argument from analogy may
be made for this conclusion. For example, it is known that when the
sympathetic is cut and degenerates, organs supplied by this nerve
become more than usually sensitive to the action of adrenalin, so that
after division of the cervical sympathetic on one siae the pupil of the
eye on that side will dilate after amounts of adrenalin have been
injected too small to affect the nornial pupil. The pancreas shows a
somewhat similar action. The vagus does not, in normal circumstances,
cause secretion from the pancreas when it is stimulated, but if it is
cut first and then stimulated after degenerating for two or three days,
it often does cause a secretion on stimulation. It might be, therefore,
that the gland after division of the vagus became hypersensitive to
secretin. But while it is possible that the vagus causes secretion only
indirectly in that it arouses the duodenum to secrete secretin, there is
as yet no evidence that this is the case, and the analogy with adrenalin
would indicate that secretin is probably acting to reinforce a nervous
mechanism rather than to supplant it. There are several differences
between the secretion due to secretin and that due to stimulation of
the vagus. One is that, if the first dose of secretin injected is large, very
little subsequent secretion is produced by subsequent doses. Nothing
of this sort happens on stimulating the vagi. These cause secretion,
under favorable circumstances, as often as they are stimulated and the
secretion is copious. In this respect secretin resembles adrenalin, which
in any but very small doses produces little effect on the second or third
injection. Secretin causes secretion after atropin has paralyzed the
gland so that nerve stimulation no longer causes secretin. The character
of the secretion is different in the two cases also, the secretin secretion
being more dilute with less organic matter than that obtained on nerve
stimulation. In view of these facts, while no definite conclusions can be
drawn without further investigations, it seems more probable that both
chemical and nervous mechanisms are involved in the secretion of the
pancreas and that part of the result of introducing acid chyme into
the duodenum is due to a nervous reflex through the vagi, and part
to the production of secretin by the direct action of the acid on the
mucosa. There is, indeed, a remote possibility which has not been
investigated so far as the author knows, namely, that secretin may pro-
duce its secretion from a different tissue of the gland than that of the
nerve stimulation. There are two tissues in the gland. duct tissue and
secreting acinary tissue. The cells of these tissues are different in their
physical appearance and they react differently when the ducts are
plugged. If a fat with high melting point is injected into the ducts,
thus plugging them, the acinary tissue degenerates, but the duct tissue
remains, so that the pancreas becomes, as Bernard says, like a tree
without its leaves, all the small ducts and ductules standing out with
394 PHYSIOLOGICAL CHEMISTRY

all the acinary cells gone (Figure 63, page 786). It is not known whether
the duct tissue contributes to the secretion and, if so, what constituents.

Secretin; chemical nature.—The chemical nature of secretin is still
unknown. An acid extract of the mucosa contains, according to Dale,
f-imidazolylethyl amine, a substance derived from histidine by split-
ting off carbon dioxide as follows:

a og
CH
don 4 tow 7
|

jie ike

CHNH, CH,NH,

| -imidazolylethyl
COOH 6 o ieninee ea
Histidine. Histamine.

According to some f#-imidazolylethyl amine is identical with vaso-
dilatin. It causes, certainly, a fall of blood pressure and a lowered
coagulation of the blood, but this identity is denied by others. Whether
secretin will act in the absence of any depressor substance in the extract
is still uncertain, so that secretin and vasodilatin or imidazolylethyl amine
are possibly not identical. Secretin is stable in acid solution, but readily
oxidizes in neutral or alkaline solution and is destroyed. These are
resemblances to adrenalin, which is apparently a body of the same kind:
that is, a basic substance possibly derived from some amino-acid by the
splitting off of carboxyl. It is not the only substance causing secretion
of the pancreas. Thus pilocarpine has such an action and so also has
Witte’s peptone and curarin. It is asserted by Popielski that other
tissues than the intestine will also yield extracts, vasodilatin, to acids
which, when neutralized, will cause secretion of the pancreas on injec-
tion. It may be mentioned in this connection that, according to the
author’s observations, adrenalin causes secretion of the salivary glands,
but not of the pancreas. Secretin may be extracted from the mucosa
of the upper part of the intestine, after the mucosa has been hardened in
HgCl,, by boiling, rejecting the filtrate, extracting the residue with
2 per cent. acetic acid containing 1 per cent. of HgCl, and precipitating
the filtrate by the addition of NaOH nearly to the neutral point. The
white flocculent precipitate is treated with H,S, filtered, the filtrate
boiled to free from H,S. The filtrate is very active (Dale and Laidlaw).

Digestive functions of the pancreas. Action on fats.—The greater
part of our knowledge of the fundamental facts of the digestive action
of the pancreas we owe to the great French physiologist, Claude Ber.
nard, whose Mémoire sur le pancreas is one of the classics of physi-
ology. Bernard undertook an investigation into the comparative
physiology of digestion. His own words on the discovery of the actior
of the pancreas are as follows:
DIGESTION IN THE INTESTINE 395

sf During the winter of the year 1846 I studied the digestion of
different alimentary substances in different carnivorous and herbivorous
animals. Having fed fatty substances to dogs and to rabbits, I followed
the physical or chemical changes fitting them for absorption that the
different substances underwent in different parts of the alimentary canal.
I perceived, in opening the intestines of animals, that in dogs the fatty
substances were emulsified and absorbed by the lacteals from the com-
mencement of the intestine and nearly from the pylorus; while in rab-
bits the phenomena became evident only very much farther down at a
distance of 30 to 50 cms. from the pylorus, depending on the size of the
animal. Struck by this difference, I sought with care to see if there
was not some constant, particular, anatomical difference in this region
between the two species. I determined in fact that in the dog the two
pancreatic ducts opened very high up in the intestine, at the commence-
ment of the duodenum, in the neighborhood of the choledochal canal;
while in the rabbit, on the contrary, the principal pancreatic duct opened
much lower than the bile duct and precisely at that point where I had
seen that the emulsification of fatty matters commenced with great
intensity. By this coincidence I was very naturally led to infer that
the pancreatic secretion must have the property of so altering fats as
to make them absorbable.

‘* Tt remained to test this hypothesis by experiments with the secre-
tion of the pancreas. To this end I began experiments to obtain pan-
creatic juice and I was able to determine that the liquid possesses in
truth the special property of emulsifying instantaneously the fats and
of acting upon them chemically in a very remarkable manner.”’

Bernard found that pancreatic juice added to neutral olive oil formed
almost instantaneously a permanent emulsion, the oil not separating as
a layer; and that, if extract of litmus were added, the solution could be
seen to have become distinctly acid. This acidity was due to the fatty
acids which were set free from the neutral fats. The first property then
to be noticed of pancreatic juice is that it splits and emulsifies fats. This
property it loses if boiled first. It is due to an organic substance of an
unknown nature contained in the juice which is called steapsin; and
since it splits fats it is one of the group of fat-splitting enzymes called
lipases, or lipolytic-enzymes. The steapsin of the pancreas is the chief,
cr most important, enzyme in the digestion of fats. By it the neutral
fats are split into glycerol and fatty acid by hydrolysis according to
the following equation:

C,H,0, (C, ,H,,0), + 3H,0 + Steapsin — C,H_0, (C,H, 0) ,.3H,0.St =
Intermediate hypothetical stage.
C,H.O H Steapsin.
clyelrdl  Sledie’ asia.
396 PHYSIOLOGICAL CHEMISTRY

Steapsin. Origin.—The steapsin found in the juice is formed and
stored in the tissue of the gland from which it is secreted. If a little
piece of fresh pancreas is placed in a feebly alkaline NaHCO, emulsion
of olive oil containing blue litmus, it will be seen under the microscope
that very quickly the piece of tissue becomes surrounded by a red
aureole or halo, due to the fatty acid set free from the fat. The piece of
tissue may be put into a fat emulsion in gelatin colored as above. The
lipase causes the gelatin to turn red. Pancreatic tissue differs from all
other tissues in this property. The lipase may be extracted from the
gland by glycerine or by water, but one of the best ways is by the
use of 60 per cent. alcohol. The fresh gland shaken with alcohol of
this concentration gives a maximum yield of lipase. It is probable that
the steapsin is formed in the pancreas itself. It is supposed to be formed
by the acinary cells, but decisive experiments in this regard do not seem
to have been carried out.

Conditions of action of steapsin——Pancreatic juice works usually
in the presence of bile and it is not surprising, therefore, that it has
been found that the addition of bile greatly increases the rate of split-
ting of fats by pancreatic steapsin. The following experiments show
this fact: The figures give the c.c. of N/10 NaOH required to neutralize
the fatty acid formed by the steapsin from the neutral fat with and
without bile. The results also show the relative activity of glycerol
extracts filtered or not filtered, or of the filter residue, and of suspensions
of the gland. The extract was made by allowing glycerol to stand
14 days on pigs’ pancreas. The filtration was through paper.

C.c. of N/10 NaOH required to neutralize equal mixtures of oi] and pancreatic
extracts.

Without bile With bile

 

Gland suspension 10.9 c.c. 21.7 ec.
Ist. filtrate: yuan. 2 secies cain 7.4 18.0
Clear filtrate ...............0. 6.9 14.5
Pilterx residue 055 scence ce scsieed daiadaioene ee 14.2 32.5

_ In the presence of bile the amount of fatty acid formed was nearly
doubled. The steapsin does not easily pass through a porcelain filter,
at least the first filtrates are very poor in lipase. This is called an
adsorption of the lipase by the filter. The filter soon becomes saturated,
however, and thereafter the filtrate has a normal concentration of lipase.
This is shown in the following experiment: A glycerol extract was
made by extracting an alcohol-ether extracted pancreas with glycerol
for 1-3 days. 100 e.c. glycerol was added to 1 gram of dried pancreas.
This was filtered through paper until clear. The acidity was tested by
taking 5 ¢.c. of the glycerol extract. 5 c.c. of a 1 per cent. solution of
Platner’s bile and 5 c.c. of olive oil, which were shaken and left for 20
hours. Then the acidity was determined by titrating with N/10 NaOH
DIGESTION IN THE INTESTINE 397

with phenolphthalein as indicator. The ¢.c. of NaOH required were the
following:

1. Filtered rapidly through paper .............. Suara aeetea etlags 34.1 ec.
2. Ist filtrate through Chamberlain filter .................... 2.0
3. 2d ie fe se BO ian hacen atesia ate waiters gla 11.5
4. 3d ef ss SS eatousa aera a sae 32.5

That the lipase of the pancreas differs from some other lipases is
_shown by the fact (discovered by Rosenheim and Shaw Mackenzie)
that it attacks normal glycerides very much better than the lighter.
normal ethyl butyrate. It is apparently made of two parts. If it be
filtered through paper, it is said that the filtrate is inactive and the
part on the filter paper is inactive. If they are reunited they become
active. The co-enzyme is heat resistant, dialyzable, not soluble in alcoho]
or ether and its activity is lost on incineration. Sodium cholate acti-
vates the inactive enzyme. Glycerylphosphoric acid has also a remark-
able action in activating, and this may account for the results of Hew-
lett, who ascribed such powers to lecithin.

Steapsin is not extracted by glycerine diluted with water from the
pancreas which has previously been extracted by alcohol and ether and
dried.

Inorganic salts do not increase, but instead they always decrease lipase
activity, unless soaps are present in sufficient quantities to inhibit the
steapsin. If this is the case, the addition of salt may appear to stimulate
the action (Hanisek). The action is very dependent on the degree of al-
kalinity ; the digestion goes best in a solution very faintly alkaline with a
hydrogen ion content of N10-*, it is very poor in weak acid or stronger
alkali. Cholesterol diminishes the action and the addition of hemolytic
substances, such as the saponins, hastens the action of the lipase.

The favoring action of bile on the steapsin is not explained. It will
be shown when discussing the bile that it is the bile salts which aid the
lipase. One explanation of the manner of their action is that by the
addition of these salts, which are the salts of weak acids, the change in
acidity is reduced, the bile acting the part of a buffer so called, a sub-
stance like glycocoll which prevents any large change in the concentra-
tion of hydrogen ions. Were this the whole explanation of the action
of bile acids, however, sodium acetate or sodium phosphate or glycocoll
should act as well, since they have the same power of controlling acidity.
But they do not accelerate lipase. Bile also acts by aiding the emulsifi-
cation of the fat in the manner described under Bile, page 412. By this
means the surface of contact between the fat and water is greatly
increased, which has the effect of increasing the concentration of the
fat. Lipase is certainly soluble in water containing glycerol and it
either dissolves in the fat or unites with it, concentrating itself in the
398 PHYSIOLOGICAL CHEMISTRY

layer of separation of fat and water. It probably dissolves in the fat,
since some may be extracted from the pancreas by ethyl butyrate. The
Twitchell process of hydrolysis of fats in soap manufacture makes use
of a sulfuric fatty acid which acts much as an artificial lipose.
Steapsin; its manner of action.—Steapsin, like most or all enzymes,
becomes more resistant to heat, light and other reagents, in tha, presence
of fat, its substrate. It may be inferred from this, and by thé fact that
fat will extract or shake it from water, that steapsin unites with neutral
fat. The most probable point of union is with the double-bonded oxygen
by means of the reserve or extra valences on the oxygen, as the oxygen
of most esters is Rene eet This union protects the steapsin from
&

O = Enzyme
ela ye o CH,

Hod Be (CB,) ,,—CH,
ho Bot Vag
=

Neutral ee “enzyme compound.

toxic influences. It leads to the breaking of the bond between glycerol
and fat and the entrance of water. The action is hence an hydrolysis.
Lipase is very sensitive to acid and if a fresh pancreas is extracted with
water or salt and then treated with acid (Pg=5.3) the lipase is de-
stroyed, while the trypsin is most stable.

Action of pancreatic juice on carbohydrates.—Panereatic juice,
besides its emulsifying and splitting action on fats, has a very powerful
starch-dissolving action. It has an amylolytic power. An aqueous or
salt extract of the fresh pancreas of any vertebrate when added to a
starch solution causes an almost instantaneous clearing of the solution.
Instead of being opalescent, the starch becomes quite transparent; there
is a marked fall in viscosity; and, if the solution is tested by Fehling’s
solution, it is found to have acquired a strong reducing action, due to
the appearance of maltose. This action of the juice is lost if it is boiled;
and a very small amount of the juice is able at ordinary temperatures
to dissolve and digest a large amount of starch. We say, therefore,
that the juice contains a catalytic agent, or enzyme, which converts
starch into maltose. The enzyme is called “ amylopsin.”

Pancreatic juice is thus in most carnivora, in which the saliva has
very little amylolytic power, the most important agent for the digestion
of starch, or of glycogen in their food. The course of the digestion is
much the same as in the ease of ptyalin, but differs from it in certain
particulars. The digestion goes best in both cases in very weak acid
and the optimum temperature is about the same in each. Bile and
DIGESTION IN THE INTESTINE 399

duodenal juice appear to affect the amylolytic action of the juice very
little, or not af all. The starch is converted into various dextrins, and
these finally into maltose. Pancreatic amylopsin differs from ptyalin
in the fact that with amylopsin the conversion of the starch into
dextrin goes at a rate relatively greater than with ptyalin and the reduc-
ing sugar appears more slowly than with ptyalin. In fact, extracts
of the pancreas are sometimes found which convert starch into soluble
starch and dextrin with great speed, but which have little or no sac-
charifying power. For these reasons many observers are convinced that
there are at least two different enzymes in amylopsin and perhaps in
ptyalin, one an amylase which converts starch into dextrin, and one or
more dextrinases which convert dextrin into maltose. These two enzymes
are generally associated, but it may happen that they may exist sepa-
rately or the dextrinase may be destroyed wholly or partly, leaving the
other active. Similar facts are alleged for malt diastase. It is said that
by heating malt diastase with CaSO, solution the dextrinases may be
largely, or completely, destroyed, while the amylases remain, and this
process has been patented for its application to brewing.

The pancreatic amylopsin is apparently the source of most of the
amylolytic enzyme of the blood and urine, being either reabsorbed
directly from the gland itself or from the intestine after secretion into
the latter.

If dialyzed, pancreatic juice, like saliva, entirely loses its amylolytic
action, but the addition of salts such as CaCl,, NaCl, etc., to the juice
restores its action completely. A possible explanation of this fact is
that the active principle is of a globulin-like character insoluble in dis-
tilled water, but soluble in dilute salt solutions. The matter needs further
investigation.

Preparation and properties of amylopsin.—Very active preparations
of pancreatic amylase have been prepared recently by the following
method (Sherman and Schlesinger): Mix thoroughly 20 grams of
pancreatin powder with 200 c.c. 50 per cent. alcohol at 15-20°. Allow
to stand 5-10 minutes, then filter, keeping the temperature below 20°.
[t takes 1-2 hours to filter. Pour the filtrate into 7 times its volume of
a mixture of 1 part alcohol to 4 parts ether. Within 10-15 minutes the
enzyme separates out as an oily solution. Decant supernatant liquid.
Dissolve the precipitate in the smallest amount of pure water at a tem-
perature of 10-15° C. and reprecipitate at once by pouring into 5 vol-
umes of absolute alcohol. Allow to settle, keeping temperature low;
filter, dissolve in 200-250 ¢.c. 50 per cent. alcohol containing 5 grams
of maltose. Pour solution into 500 ¢.c. collodion sack and dialyze against
2,000 c.c. 50 per cent. alcohol at not above 20° C. and preferably not
below 15°. Replace dialyzate twice after 15 hours and a second period
of 8-9 hours with fresh 50 per cent. alcohol. Continue dialysis 40-42
400 PHYSIOLOGICAL CHEMISTRY

hours. Filter. Pour clear filtrate into an equal volume of 1:1 alcohol-
ether mixture. Filter in cold and place at once in a vacuum desiccator.
The elementary analysis of the product on the basis that P was an
integral part of the molecule was: C, 51.9; H, 6.6; N, 15.3; S, 1.0;
P, 0.8; O, and undetermined, 24.4. The substance is evidently a pro-
tein. It coagulates on heating at 68-70°. .000,002,5 gram of the powder
digested 5 c.c. of a 1 per cent. solution of Kahlbaum’s soluble starch to
a pure wine-red reaction with iodine in 30 minutes at 40°. This on
Wohlgemuth’s scale D4 =2,000,000. The weight of the starch digested
was 20,000 times the weight of the enzyme. It later digested 800,000
{umes its own weight of starch in 30 hours to a red color, and in 72
hours to the achromatic point. It formed 455,000 times its own weight
of maltose. In strong solutions it formed glucose as well as maltose.
It acts only in the presence of electrolytes. It loses activity very fast on
standing in pure water at room temperature, 45 per cent. activity being
lost in 20 minutes. This preparation was not pure. It was more active
proteolytically than the original pancreatin. It gave all the typical
protein reactions, biuret, Millon, xanthro-proteic and tryptophane. After
coagulation the coagulum took a violet color in the biuret test, the filtrate
a rose-red color.

The pancreas appears not. only to cause the digestion of starch by
its own action, but it seems from the work of Roger and Simon to reac-
tivate in the intestine the ptyalin which has been rendered inactive but
not destroyed in its passage through the stomach.

Lactase and maltase.—The pancreatic juice acts also in children
and young animals on lactose; it contains a lactase, an enzyme which
splits lactose by the addition of water into galactose and glucose. This
enzyme is absent in adult animals, except those like human beings and
pigs which continue to ingest milk. The attempt to make the enzyme
reappear in the glands of adult herbivorous animals which do not nor-
mally have milk by feeding milk has been thus far a failure. Invertin
has sometimes been found in the juice, but at other times it has been
missed. It appears then not to be a constant constituent.

Maltase, which converts maltose to glucose, is also present.

Action of pancreatic juice on proteins.—Not only does pancreatic
juice act upon fats and carbohydrates in the chyme, but it also, when
mixed with duodenal juice, has a powerful action on proteins, although,
as secreted from the duct, it has very little action on proteins. Unmixed
with succus entericus it will digest casein, but not coagulated proteins.
But when it is mixed with duodenal or jejunal secretion, the mixture
digests nearly all proteins at a very rapid rate and this mixture prac-
tically completes the digestion of the proteins which was begun by
the stomach. Claude Bernard discovered that pancreatic juice would
digest some proteins, but the action was very slow. He found that the
DIGESTION IN THE INTESTINE 401

digestion differed from the peptic digestion in two particulars: first,
it went on best in a feebly alkaline medium and was stopped by acid;
and, second, in a tryptic digestion the digestive mixture in a day or
so acquired the property of giving a violet color when bromine or
chlorine water was added to it. He found no other secretion which
gave this reaction, so that he considered it unique and characteristic
of tryptic digestion. This reaction was called, therefore, the tryptophane
reaction (Gr. trypsis, a rubbing; phanos, bright). It is now known
to be due to the formation of free tryptophane, indol-amino propionic
acid. These three discoveries of Bernard’s were fundamental: first,
that pure pancreatic juice was a weak digestant of proteins; second,
that it worked in an alkaline medium; and, third, that by it other
digestive products were formed than by peptic digestion.

After this work by Bernard further work appeared to show that the
action on proteins was very intense. Thus, if the pancreas be dried,
extracted with ether, and then treated first with salicylic acid solution
for 24 hours at 38° C. and thereafter by Na,CO, solution by Kiihne’s
method, an extract very active on proteins was obtained. It was not
until 1900 that Bernard’s early observations were cleared up. It was
found that the pure juice has indeed a weak action on proteins, as
Bernard observed, but that if it is mixed with only a small amount of
duodenal juice, or with an extract of the intestinal mucosa, the mixture
becomes extremely powerful and digests proteins, even coagulated pro-
teins, rapidly and so completely that monoamino-acids are formed and
very little albumose or peptone remains. It is the enterokinase of the
intestine which acts in conjunction with the pancreatic juice to digest
proteins. The active principle of the pancreatic juice thus acting on
proteins is called “ trypsin” (Gr. trypsis, a rubbing ; referring to method
of preparation). It is probable that it is not a single enzyme, but that
there are at least two, one acting on the complex proteins, for which
the name trypsin may be retained, and the other of an ereptic nature,
acting on the albumoses and some natural proteins like casein, and this
might be called ‘‘ pancreatic erepsin.’’ It is probably this latter which
has the power of clotting milk, or a rennin action, in the absence of
intestinal juice.

Since pancreatic juice is inactive as it is secreted, but when mixed
with intestinal juice digests rapidly, it is believed that trypsin does not
exist as such in the gland, but in an inactive form called trypsinogen.
It is ordinarily stated that the enterokinase acts by activating the
trypsinogen or converting it into trypsin. It is well, however, to bear
in mind the actual facts, rather than any interpretation of them, and
the facts are that a mixture of pancreatic and intestinal juices digests
proteins much more rapidly than either juice alone, and that the active
principle found in the pancreas is called trypsin and that in the intes-
402 PHYSIOLOGICAL CHEMISTRY

tine is called enterokinase. But exactly how they support each the
action of the other, cannot be stated at present and further work is
required. The facts at present known, while not very convincing, indi-
cate that enterokinase may be setting trypsin free from some union
in which it is inactive, but other interpretations than this are possible.
All observations on the digestive power of pancreatic juice made before
the discovery of the réle of enterokinase have to be interpreted with
this fact in mind.

It is generally stated that digestion by the pancreas is most rapid
in the presence of hydrogen ions of a concentration of Py=8.1 or in a
slightly alkaline medium (.3 per cent. Na,CO,). It is a singular fact
that, if this is true, the digestion must not take place in the alimentary
canal under optimum conditions, for the reaction of the chyme is often
feebly acid to litmus for a considerable distance along the intestine. It
is possible that there are two or more enzymes of which the first, trypsin,
working on coagulated protein, may be most active in a feebly alkaline
medium, whereas the action on the albumoses by the erepsin may be
best in a neutral or very faintly acid medium. This is a matter requiring
further work. Rachford states that the bile, and particularly the chyme
as it comes from the stomach, exerts a favorable action on tryptic diges-
tion. He collected pancreatic juice from the pancreatic duct in rabbits.
From his results, which antedated the discovery of enterokinase, it
appears that the digestion is at an optimum under the conditions of the
admixture of bile, duodenal juice and chyme with the pancreatic juice,
such a mixture as exists in the intestine. The experiments made by
Abderhalden and others on the digestion of artificial polypeptides have
been made with juice containing both enzymes. Furthermore, natural,
that is unactivated, pancreatic juice will hydrolyze peptone, casein and
fibrin by means of the erepsin it contains. Besides enterokinase, it is
maintained by Henri that pancreatic juice is activated also by calcium
salts. The results on this are, however, diverse and from recent work
it appears that calcium hastens the action of trypsin after it is
formed and hastens the action of erepsin, but does not activate tryp-
sinogen. The explanation of the action of the salt is, however, still
largely guesswork.

Products of the action of trypsin-enterokinase on proteins.—The
digestion of the natural proteins by the mixture of juices in the duo-
denum passes through much the same stages as peptic digestion, except
that the decomposition of the peptones and albumoses into di-, tri- and
mono-peptides takes place very much more rapidly than in the case of
pepsin. The pancreas has an ereptic as well as a peptic power. As a
result, if the digestive mixture is examined for the primary proteoses,
very little will be found in the case of pancreatic digestion as compared
with peptic. Furthermore, tyrosine, leucine and other amino-acids are
DIGESTION IN THE INTESTINE 40%

produced in great amounts and crystallize out of the digesting mixture;
these products are not formed by peptic digestion. It is uncertain
whether these are produced by trypsin or by erepsin. No method of
separating these enzymes is known.

The tryptic digestion differs, on the other hand, from the autolytic
digestions of organs, that is from their self-digestion, in that little
ammonia is formed in the course of tryptic digestion, whereas much
is produced in autolysis. This, as has been pointed out, has
the teleological explanation that acids set free in cells autolytic
ferments and these form ammonia to neutralize the acid. There
is no doubt that a good deal of ammonia is produced either by
the action of digestive ferments or by the bacteria in the course of
intestinal digestion, for the mucosa of the intestine contains much
ammonia.

The digestion by trypsin-enterokinase does not, however, reduce all
the polypeptides to amino-acids. There still remains in a trypsin diges-
tion mixture a considerable number of carboxyl-amino linkages, since
if a tryptic digestive mixture is titrated with formol in the Sdrensen
method before and after hydrolysis with acid, it is found that the num-
ber of free amino groups is considerably increased by the action of the
acid, showing that some NH-CO linkages had not been split by the
trypsin-enterokinase. This final step in the reduction of the proteins
to the simplest building stones, the amino-acids, is brought about by the
enzyme, erepsin, found in nearly all tissues, but in larger amounts in
the intestinal mucosa and to some extent in the intestinal juice. Trypsin
is, however, able to break many amino-carboxyl linkings, or, at least,
activated pancreatic juice is able to do this, as is shown by the work
of Abderhalden and his colleagues. They found the artificial poly pep-
tides were digested or not digested by this mixture as shown inthe table
on page 404.

Whether the splitting was due to the trypsin-enterokinase or to the
erepsin in the juice could not be positively stated. An examination
of the appended list shows that the property of being digested by this
mixture of enzymes depends on several properties of the molecule. In
the first place (A) it depends on the structure of the molecule. Thus
alanyl-glycine is hydrolyzed, but glycyl-alanine is not hydrolyzed;
d-alanyl-l-leucine is digested, but alanyl-leucine (B), which was probably
Lalanyl-l-leucine plus d-alanyl-d-leucine, was not digested. The first
peptide contains those active amino-acids found in nature. 1-leucyl-l-
leucine, the natural leucine, is digested. while l-leucyl-d-leucine and
d-leucyl-l-leucine are not split. Dakin found, too, recently that casein
and other proteins which have been racemized by dilute alkali are no
longer digested by trypsin.
404 PHYSIOLOGICAL CHEMISTRY

ARTIFICIAL POLYPEPTIDES HyDROLYZED AND NOT HyYDROLYZED BY PURE ACTIVATED
PANCREATIC JUICE (Fischer and Abderhalden).

Hydrolyzed Not hydrolyzed
(r) Alanyl-giycine Glycyl-alanine
(r) Alanyl-alanine Glycyl-glycine
(r) Alanyl-leucine (A) Alanyl-leucine (B)
(r) Leucyl-isuserine (A) Leucyl-alanine
Glycyl-ty rosine Leucy!-glycine
Leuev!]-I-tvrosine Leucyvl-leucine
(r) Alanyl-givevl-glycine Aminobutyryl-glycine
(r) Glycyl-leucy l-alanine : Aminobutyryl-aminobutyric acid (B)
(r) Leueyl-glyevl-glycine Aminobutyryl-aminobutyric acid (A)
Dialany]-eystine Glycy|l-phenylalanine
(r) Alanyl-leucy]-glycine Valyl-glycine
Dileucyl-cystine’ Diglyevl-glyeine
Tetraglycyl-glycine Triglycyl-glveine
Triglycyl-glycine ester (Biuret base) Dileucyl-glycyl-glycine
d-alanyl-d-alanine d-alany]-l-alanine
d-alany]-I-leucine l-alany]-l-alanine
d-alanyl-l-tyrosine 1-leucyl-glycine
1-leuey]-!-1eucine 1-leucy]-d-leucine
l-leueyl-d-glutamie acid d-leucy]-l-leucine

L-leucyl-l-tyrosine
1-proly]-l-phenylalanine
d-alany]-diglycy]-glycine
Peptides marked with an r are the racemic compounds.

In the second place (B) the power of being digested depends on the
number of amino-acids present in the chain. Thus the tetraglycyl-
glycine containing five glycine radicles was digested, while the diglycyl-
glycine containing three was not digested.

Different proteolytic enzymes show quite different powers of hydro-
lyzing the different polypeptides, and the enzymes may be distinguished
from each other in this way. It is besides in the highest degree signifi-
cant that the enzymes found in the same organs in different kinds of
animals show different digestive powers, and wide differences exist
between the different organs of the same animal in its power of hydro-
lyzing these artificial polypeptides. Pepsin will not digest any arti-
ficial polypeptide. Fischer has suggested that this is because it has not
yet been tried on a sufficiently long chain, but it may be due to other
reasons. The various autolytic enzymes, or proteoclastic enzymes as they
are also called, show very marked differences. But in general their
powers of decomposition are greater than those of the pancreatic juice.
Thus glycyl-glycine is hydrolyzed by the liver and muscle juice of the
ox, rabbit, dog and by kidney press juice, but not by the serum or plasma
of the horse. Ox plasma digests glycy}-dl-alanine, but not glycyl-l-
tyrosine. In general, it may be said that when the racemic polypeptides
are digested the naturally-occurring, optically-active, isomers are the
ones digested and not their optical antipodes. Many of these peptides
are split if injected directly into the blood of animals. Sometimes both
DIGESTION IN THE INTESTINE 405

the d and I- forms are utilized, but one better than the other. In such
cases it is possible that racemization is first produced, and the natural
substance then consumed. Often the unnatural antipode is excreted
more or less completely.

Sometimes when a tripeptide is exposed to pancreatic juice or other
enzyme one of the amino-acids is split off very quickly, the other two
being separated very much more slowly; or again, it may happen that
one enzyme breaks the chain in one point while another breaks it at
another, just as happens in the polysaccharides. In the tripep-
tide 1-leucyl-glycyl-d-alanine, the proteases in yeast and saliva split
this into leucine and the dipeptide glycyl-d-alanine; while pancreatic
juice splits off d-alanine, leaving the dipeptide 1-leucyl-glycine.
Other examples have been studied by Koelker and Abderhalden.
By this means differences between different proteases may be
detected.

Law of the rate of tryptic digestion—For the relation between the
rate of proteolysis by trypsin and the amount of the enzyme present the
same facts have been found as have been noted under pepsin. If the
protein is in solution, if for example it be gelatin, there is an inverse
simple proportionality between the amount of enzyme and the time
required to digest to the point of liquefaction. That is, dx/dt=
KC jement Or log (A/(A—X))=KC ferment t- In the gelatin experi-
ment log (A/(A—X)) is constant. If, however, the determination is
made by Mett’s tubes, values are obtained which are fairly in agreement
with the Schiitz-Borissow law, according to which the amount digested in
a certain time is proportional to the square root of the amount of enzyme.
Finally if fibrin colored blue with ‘‘ spritblau ’’ is the substrate, and
the reaction is followed by the depth of color of the solution, the same
law is found as Griitzner found for peptic digestion under similar con-
ditions: namely, that X, the amount digested, =KC7%.,..4. The amount
digested is proportional to the two-thirds power of the concentration of
the ferment (Palladin). Both of the latter relations are purely
empirical.

Pancreatic juice dialyzed against water loses all its tryptic power, but
it retains its ereptic power. It cannot then be activated by the addition
of kinase. Kinase does not, then, hasten the ereptic action of dialyzed
juice, nor will it’ activate trypsinogen in the absence of salts. Possibly
trypsin is rendered insoluble by the dialysis, behaving as if it were a
globulin. These experiments show that the juice contains at least two
enzymes.

Pancreatic nuclease.—Common foodstuffs generally contain nucleic
acid. This is not digested by proteolytic enzymes. There is in the pan-
creatic juice a nuclease which acts on the nucleic acids of the foods and
406 PHYSIOLOGICAL CHEMISTRY

reduces them probably to phosphoric acid, nuclein bases and carbo.
hydrate. From the pig’s pancreas (but the juice was not examined)
Jones extracted a nuclease (tetranucleotidase) which had the property
of splitting guanylic acid from yeast nucleic acid. The investigations
of London show that nucleins are digested after reaching this portion
of the intestine, and it is probable that this enzyme of the pancreas is
the active principle. It is not impossible that the presence of guanylic
acid in the pancreas, and presumably outside the nucleus, may be cor-
related with the presence of this enzyme. It is possible that the nuclease
is in union with the guanylic acid of the gland which thus serves as a
colloidal, non-digestible substrate.

Pancreatic juice. Summary.—The digestive action of the pancreatic
juice may be summed up as follows: When mixed with bile and intes-
tinal juice it very rapidly splits by hydrolysis and emulsifies fats; it
reduces starches, dextrins and glycogen to maltose and glucose; it
inverts cane sugar, at times, and in young animals it has the
power of converting lactose into glucose and galactose; it attacks
the partially digested peptones, albumoses, etc., of the chyme, very
quickly hydrolyzing them into di- and tri-peptides and crystalline
amino-acids which do not give the biuret reaction; it digests nucleic
acid.

Pancreatic juice mixed with bile and intestinal juice often regurgi-
tates into the stomach, as was mentioned when digestion in that organ
was discussed. By stimulating this regurgitation it is possible to obtain
samples for clinical examination. Boldyreff recommends the following
method: The person should have eaten the day before only a small
amount of easily digested food. The following morning 200 c.c. of olive
vil containing 2 per cent. oleic acid is given on an empty stomach. After
1-1% hours the stomach is emptied with a stomach tube. The contents
settle in an oily upper layer and a watery lower layer. The latter is
often alkaline in reaction and slightly brown in color. This can be
tested for its tryptic activity by making slightly alkaline with sodium
bicarbonate and adding fibrin. To see if it has pepsin it may be exam-
ined in acid solution. Digestion in the alkaline solution indicates the
presence of pancreatic juice. The juice may also be investigated for its
other pancreatic ferments.

THE BILE.—The third of the juices co-operating in duodenal diges-
tion is the bile. The bile, or gall, the external secretion of the liver, made
two (black bile and yellow bile) of the four cardinal humors of Hip-
pocrates. It early attracted the attention of physicians and laity alike,
partly because its formation was apparently the only function of one of
the largest organs of the body, the liver; partly because of its green or
golden color and viscidity; partly because of its extremely bitter taste
DIGESTION IN THE INTESTINE 407

and the yellow appearance of the skin and whites of the eyes when its
outflow from the gall bladder was blocked; and partly because of the
formation of gall stones and the extreme pain of their expulsion from
the gall bladder. But while curiosity was very early excited as to the
functions of this liquid, its real function has become known only within
the past half century, and indeed it is still probably very imperfectly
known. Its composition, also, is in many important particulars still
obscure and there can be little doubt that many valuable discoveries
remain to be made concerning its composition, function, formation and
secretion.

The bile in human beings as it flows from a temporary biliary fistula
is generally of a clear, golden yellow or brownish yellow color, but it
is sometimes an olive green; it is very bitter and has a slimy consistence.
The bile taken from the gall bladder after death is generally of a
brownish green color, and is more viscid, containing more solids and
more phosphoprotein (false mucin) than that flowing from a tempo-
rary fistula. The viscidity of the bile is a matter of importance, for the
reason that the bile is secreted under so small a secretory pressure that
the viscous bile may offer such a resistance as to block wholly or partially
its outflow; and under these circumstances reabsorption takes place
through the lymph vessels, leading to the appearance of bile pigment
in the fluids and tissues of the body, and the excretions, and to the con-
dition of icterus (Greek, ikteros, jaundice) or jaundice. The color of
bile’ is generally yellow brown, but it may be green in its fresh state.
If green, it readily changes by reduction, which may be produced by
putrefactive changes or by the walls of the gall bladder, or possibly even
by internal changes in the bile itself, into a reddish or golden brown.
The freezing point of human bile is just about that of the blood, i-e.,
—0.55° to —0.61° C., so that its osmotic pressure is about the same or
possibly a little greater than that of the blood. In this respect the bile
differs from most other secretions which generally have a freezing point
above that of the blood. Its specific gravity is 1.026.

Composition.—The composition of the bile is different in every
species of animal examined, but all, or nearly all, biles contain three
characteristic and very important substances: 1. The bile pigments;
2. the bile salts; and 3, cholesterol. The bile salts vary with the species.
In the elasmobranch fishes they differ widely from those present in
most animals. Most, if not all, biles contain also some phosphatidé
(phospholipin).

Human bile has been obtained for analysis either from biliary fistulas
or from the gall bladder of recently executed criminals or persons dead
by accident. The composition is indicated in the table. Bile is generally
neutral or faintly alkaline in reaction to litmus.
408 PHYSIOLOGICAL CHEMISTRY

COMPOSITION OF THE BILE,
Biapper Bite. Sp. gr. 1.027.

Frerichs y. Gorup - Besanez
Wate isidesnsa sctsecsiesses smatinverd ese me RS 86.00 85.92 82.27 89.81
ONS, i 2 sa recdinle arti cavonnsdrerecoenlavouaysiavelecons.s 14.00 14.08 17.73 10.19
DUE 8A tS oct teseusr ace aecrasie?besedonteese auierd 7.22 9.14 10.79 5,65
Mucin and pigments ..............4. 2.66 2.98 2.21 1.145
Cholesterol ......-..0ccevccceueeceer 0.16 0.26 4.73 3.09
Babi: sresaig segduaneeseaes eee 2s 0.32 0.92
Inorganic: sssacsns caguicaateaass ees 0.65 0.77 1.08 0.62

Fistuta Bite (Hammarsten). Sp. gr. 1.012.

WWREOR siicnese ctuaicadd satan ania nasa 97.48 96.47 97.46
Solids) = a) fis calcd: ne cxcucncuasn teckiheeie aan 2.52 3.53 2.54
BBG; SANCS: 2 acsvosecauencsarscteauand. cssuanante dst censeame 0.93 1.82 0.90
Taurocholate ...........-..-.--e cece 0.30 0.21 0.22
GIVCOChOMACG: sosca are sree neste imonneees 0.63 1.61 0.68
Hearthy Cid 82 asccevsjioe penaviecciirhsiarvauisobis Sots yavanalest 0.12 0.14 0.10
Mucin and pigments ................. 0,53 0.43 0.52
Cholesterol ......... 0.06 0.16 0.15
Lecithin + Fat 0.02 0.096 0.06
Soluble salts .... 0.81 0.68 0.73
Insoluble salts .. 0.025 0.05 0.02

 

The composition of bile flowing from a biliary fistula differs consid-
erably from that of bile taken from the gall bladder. The fistula bile
is less concentrated the more completely the bile has been cut off from
the intestine. Thus bile from a complete fistula in a woman was found
by Pfaff and Balch to contain only 3 per cent. of total solids, and Ham-
marsten found similar figures; whereas bile from the gall bladder con-
tains from 10-20 per cent. of total solids. Moreover, of the total solids
of fistula bile 30-50 per cent. are inorganic, whereas in human bladder
bile only about 6 per cent. are inorganic. The freezing poimt of the
bladder and of the fistula bile does not greatly differ, although that of
the bladder bile is somewhat lower. The reason for this is that some
of the organic constituents of the bladder bile are probably in colloidal
solution, so that they do not much affect the freezing point. The greater
concentration of the bladder bile is generally ascribed to a reabsorption
of water in the gall bladder, but there is little evidence of this. It is
more probably due in part to the fact that some of the organic matters,
mucin, phosphoprotein and possibly some cholesterol, are added during
the stay of the bile in the bladder, but it is chiefly due to the fact that
when. the bile is diverted from the intestine, as it is in most fistulas,
there is no reabsorption of the bile and hence the bile salts are not
re-excreted as they normally are.

Secretion of bile—The bile is poured into the duodenum in man
about 4-5 inches below the pylorus through.the ductus choledochus. This
duct opens very close to or in conjunction with one of the pancreatic
ducts (Wirsung’s), so that these two secretions are intimately mixed
DIGESTION IN THE INTESTINE 409

as they are poured out into the acid chyme coming from the storiach.
They are also mixed with the duodenal juice, and it has been found
that these three secretions mutually interact with each other and with
the chyme and together form a digestive fluid very powerful and capable
of digesting and dissolving all classes of foodstuffs.

The amount, character and time relations of the bile secretion can
be studied by means of a fistula, either temporary or permanent. In a
permanent fistula the gall bladder is opened, brought where possible to
the surface, drained and fastened in the skin in such a way that an
opening persists to the exterior. If it is desired to block completely all
flow of bile into the intestine, it is necessary to resect a portion of the
common bile duct (ductus choledochus), and even then the connection
with the intestine may in time be re-established, so great are the repara-
tive powers of animal tissues. As a rule, however, this connection is
not re-established and the bile passes altogether to the exterior. If no
resection of the duct is practised, some of the bile may continue to find
its way into the intestine, and in human beings and dogs, if no means
are taken to prevent it, the external opening will ultimately close and
normal connections with the intestine will be re-established. The method
usually employed in making such fistulas in dogs is that of Dastre. Such
temporary fistulas are not uncommon in human beings following apete:
tions for gall stones, or for infection of the gall bladder.

The secretion of bile from such a fistula is found to be continuous,
but by no means uniform. It is indeed subject to wide fluctuations in
amount. during the twenty-four hours, and the causes of these fluctua-
tions are still quite obscure. While the formation of the bile and its
flow from the fistula are thus continuous, the flow from the ductus
choledochus into the intestine is intermittent, at least in those animals
provided with a gall bladder, and occurs only when acid chyme is dis-
charged from the pylorus into the intestine. The irregularity of the
secretion of the bile from a fistula is illustrated in the following figures
which give the secretion from a biliary fistula in a woman in the Massa-
chusetts General Hospital, as recorded by Pfaff and Balch:

8am.-2 pm. 149 ec.
2p.m.-8 p.m. 84
8pm.-2 am. 96
2am.-8 am. 136
8 am.-2 p.m. 170
2p.m.-8 pm, 129
8 pm.-2 am, 70
2am.-S a.m. 105

The hourly secretion is subject to a wide diurnal variation, being
least in the hours of the early morning from 2-5 a.m., rising rapidly
after this time and culminating, as a rule, about 1-3 p.m., thereafter
decreasing with some fluctuations. It thus runs a course not very dif-
410 PHYSIOLOGICAL CHEMISTRY

ferent from the body temperature, which is at a minimum about 3-5
A.M, and at a maximum about 4-5 p.m. Other functions of the body also
show such a diurnal or rhythmic variation. Similar variations in the
flow of bile from a human fistula have been reported also by others.
There are, besides the main variations in flow already mentioned, other
variations apparently related in some way, not at present clear, to
digestion. Thus in the case of Pfaff and Balch the great rise in the early
morning coincided with breakfast and the next great rise with dinner,
but sometimes the rise came before and sometimes after dinner, an hour
or more, and the maximum outflow was not coincident with any ascer-
tainable phase of digestion. In another case the flow often showed a
remarkable fall immediately after a meal, particularly at the time of
afternoon tea, and the authors suggested that possibly this sharp fall
might be due to closure of the pylorus following the taking of food, thus
diminishing the discharge of acid into the intestine and so reducing the
stimulation by the secretin formed when acid is discharged. The flow
from a dog’s fistula shows somewhat similar variations.

Amount of bile secreted per day.—The amount of bile secreted per
day by a human being is very difficult to estimate with any accuracy.
Pfaff and Balch report in their case a secretion of 525 c.c. per day, and
similar results have been reported by others. These figures, however.
while they may give us some idea of the amount secreted, are not to be
taken as a certain index of the amount normally produced, since they
are all of them from permanent fistulas. It has been shown that, if
bile is poured into the intestine, the secretion is increased both in con-
centration and amount. In these cases no bile was finding entrance to
the intestine, and hence this would tend to diminish the secretion. On
the other hand, the acid of the chyme acts as a stimulus to the formation
and the discharge of bile from the liver. Now in the absence of the bile
it is possible that the acid chyme remains acid for a longer time than
normal and thus acts as a stimulus for a longer time. The absence of
the bile in this way might act as a stimulus to the formation of more
bile than usual. It is hence impossible to say whether 550 ¢.c. per day
represents the secretion of bile in the human adult or not, but it is
probable that in health and when the bile finds its way into the intestine
the excretion is larger rather than smaller than this.

The amount of the bile formed is dependent also on various other
factors, particularly on diet. There is a general consensus of opinion
of all investigators that the secretion of bile is at a maximum on a
meat diet and at a minimum on a carbohydrate diet. After hemorrhage
the amount and solids of the bile are both reduced. Drinking water
does not apparently influence the secretion. In starvation the bile con-
tinues to be formed, in dogs at least, up to the moment of death, and it
DIGESTION IN THE INTESTINE 411

does not materially change its composition, although it decreases in
amount. The following experiment of Albertoni shows this fact:

Bite Srcrerep oN Successive Days or Fastina sy a Dog. Wr. 21 Kas.

 

 

 

Days of Fresh bile— Dry bile— N Bile dry— N—
fast grams grams per cent. per cent.
1 74,75 3.45 0.115 4.61 0.153
2 40.65 3.34 0.118 8.21 0.290
3 31.25 2.48 0.08 7.90 0.256
5 40.25 2.10 0.123 5.2] 0.305
10 33.25 2.01 0.080 6.04 0.240
17 23.00 2.11 0.079 9.13 0.343
21 25.00 3.05 0.120 12.2 0.480
23 24.05 1.75 0.073 7.20 0.303
27 16.00 1.37 0.260 8.50 1.625 Died.

 

 

Functions of the bile—If the bile is cut off from the intestine anu
drained to the outside by a fistula, very serious disturbances of digestio1:
follow. In dogs and human beings the feces lose their color, they becom:
clay-colored; they increase greatly in amount and become very fatty
if fat is taken in the food; constipation generally occurs; and the odor
of the feces is very strong and offensive, putrefaction evidently being:
very marked. At the same time, although the person or dog may eat
voraciously, there is a constant loss of weight, and dogs, at any rate,
die in extreme emaciation in the course of four or five weeks, if they be
prevented from licking the fistula and thus swallowing their own bile.
Both human beings and dogs acquire a distaste for fats if the bile is cut
off from the intestine and will not willingly eat fatty food.

The changes just described show that the absence of the bile is
followed particularly by a failure to absorb fat, but the reabsorption
of other foodstuffs, proteins as well, is also diminished. The direct
examination of the bile shows that it has itself either no power of diges-
tion at all or but slight action on either fats, starches or proteins. It is
true that human bile is said to have some solvent action on fibrin but not
on coagulated egg white; but on the whole the bile itself does not contain
digestive enzymes. It has, however, very remarkable powers of aiding
the digestive action of the pancreatic and intestinal juices and in helping
the absorption of fats.

The power of the bile to aid the digestion of fats by the pancreas
was observed by Claude Bernard, who clearly recognized the importance
of the co-operation of the bile, pancreatic and duodenal juice in diges-
tion. It was particularly studied by Rachford, who found that the
power of the pancreatic juice of splitting fats was increased two to three
times if bile were present in the digestive mixture, and it has been
recently reinvestigated by von Fiirth and Schiitz, who found that the
addition of 5 ¢.c. of bile to 20 c.c. neutralized olive-oil emulsion contain
412 PHYSIOLOGICAL CHEMISTRY

ing steapsin increased the amount of fatty acid formed from 5-10 times
over that formed by the steapsin alone.

Rachford did not find any accelerating action of the bile on amylase,
but he thought that the digestion of proteins was accelerated. Other
observers failed to find any accelerating action of bile on tryptic diges-
tion, so that the matter has been uncertain, but recently this work has
been repeated with pancreatic juice and bile from permanent fistulas
of dogs. No accelerating action on the protein digestion of pancreatic
juice and intestinal juice was observed, but there was a slight favoring
action on amylopsin.

The substance in the bile which thus accelerates the fat-splitting
action of the pancreas was found by Rachford to be the bile salts,
sodium taurocholate and glycocholate, which were almost as favorable
as the bile itself. Hewlett, who repeated his experiments, attributed the
action of the bile to the lecithin it contained rather than to the bile
salts. Hammarsten has recently shown that human bile has a very
large amount of lecithin, or other phospholipin, in it, so that Hewlett’s
results seemed not unlikely, but von Fiirth and Schiitz in re-examining
the whole matter and using synthetic bile salts, so as to exclude the
possibility of the presence of lecithin as an impurity in the salts, have
shown that the whole power of the bile is due to the bile salts and that
sodium cholate acts as well as the glycocholate or taurocholate.

Circulation of the bile. Role in absorption.—But not only do the
bile salts thus play a very important part in the digestion of fats; they
play a not less important and certainly a very illuminating réle in the
absorption of fats. In the absence of bile the fatty matter in the feces
is found to have been split into fatty acids, but these have not been
absorbed. It might be, of course, that in the absence of the bile this
splitting took place too far down the intestine for absorption to occur
and the bile might by accelerating the splitting cause it to occur so far
up in the gut that absorption could take place; but whether this is true
or not, there can be no doubt that in quite another way bile influences
absorption, if indeed it does not play the major réle in the mechanism
of absorption. When fats are being digested and absorbed, all the lymph
vessels leading from the intestine across the mesentery to the receptacu-
lum chylii and so to the thoracic duct are filled with a white milky lymph
called chyle; and it is by this path mainly, and not by the portal cireula-
tion, that the fats are absorbed. Now, if the bile is cut off from the intes-
tine of a dog and fat is fed the chyliferous vessels no longer are white,
but filled with a lymph not more than opalescent. Evidently in the
absence of bile fat does not pass into the chyle vessels. Furthermore,
it is not absorbed even if it is given in the form of a soap, or as a
fatty acid.
DIGESTION IN THE INTESTINE 413

A further study of the manner in which bile affects the absorption
has led to the discovery that bile is able to dissolve large amounts of
fatty acids. Oleic acid is the more soluble in bile, but palmitic, stearic
as well, acids which are quite insoluble in water, are soluble in bile.
This solvent power of the bile for fatty acid is due at least in large
measure to the bile salts. The probability is that the fatty acids form a
loose union either with their amino groups or in some other way, pos-
sibly by cohesion. One or more molecules of fatty acid are perhaps thus
united to the one molecule chemically bound to the bile salt. The
fatty acids embark as it were on molecules of glycocholate and taurocho-
late and by this means they are ferried across the cell membranes and
into the lymph channels. The bile salts thus reabsorbed are thrown into
the blood, carried to the liver and are there re-excreted. There is thus
a circulation of bile salts from the gall bladder to the intestine and from
the intestine to the blood, by which they are carried to the liver and
re-excreted. Each molecule of bile salt can thus function, theoretically
at least, over and over again.

The circulation of the bile was first particularly emphasized by
Schiff, although its possible occurrence had been suggested before that
by Liebig. Schiff observed that the flow of bile from a biliary fistula
was greatly increased, and in particular the bile solids were increased
when bile was put into the intestine. He was struck by the fact that
when a fistula is first made the bile flowing from it is far more con-
centrated than that coming after six hours or more. This decline in
total solids and amount of bile and the immediate increase of both when
bile was admitted to the intestine led him to conclude that the bile, or
at least the bile salts, were reabsorbed and re-excreted and thus circu-
lated. This fact has been established by subsequent observation, and
particularly by the observations of Stadelmann, who found nearly the
whole of the bile salts put directly into the intestine were re-excreted.
How impossible it is for any process of diffusion to explain secretion
is shown by this excretion of bile salts which are picked out from the
blood, where they exist in the smallest proportions, and carried through
the liver cells and into the bile, where their concentration is many thou-
sand times as great as that in the blood.

Influence of bile on intestinal putrefaction—Bile has a marked
influence in reducing intestinal putrefaction and in stimulating peri-
stalsis. It was at first thought that it must have antiseptic properties, but
bile is itself easily putrescible, at least as long as it contains mucin.
In fact, bile is a fair culture medium and it not infrequently happens
that the gall bladder becomes infected, as in typhoid fever, and con-
tinues to serve as a permanent culture medium, constantly feeding
bacteria into the intestine. The réle of the bile in reducing putrefaction
414 PHYSIOLOGICAL CHEMISTRY

is, hence, probably an indirect one; by stimulating digestion and aiding
absorption it prevents the accumulation of putrescible materials, and
by its peristaltic stimulating powers it sweeps the bacteria out of the
body and prevents constipation. Bile is, in fact, a natural laxative.

Summary of the functions of the bile-—The bile plays a very impor-
tant part in the digestion and absorption of the foods. While of itself
it has little digestive action, it stimulates greatly the digestion of the
fats by steapsin and other lipases, and it holds in solution the fatty acids
set free. By its alkalinity and because the bile salts, the taurocholate
and glycocholate and other similar salts, are salts of weak acids, it helps
neutralize the acidity of chyme and so makes a favorable medium for
the action of trypsin and amylopsin. Even more important is its réle
in absorption, since in its absence absorption is much reduced. The fatty
acids combine chemically or physically with the bile acids and the com-
bination passes into the intestinal epithelial cells with ease. The bile
salts are then freed from the fatty acids and carried to the liver, where
they are re-excreted, thus forming a circulation of the bile. By stimu-
lating peristalsis and absorption, putrefaction is much reduced. Bile
is a natural laxative.

Bile acts also as a channel for excretion of various substances.
Cholesterol is excreted in the bile and bile is the only fluid of the body
which will dissolve this substance. In addition the bile pigments appear
to be purely excretory substances formed by the decomposition of the
blood pigment. So far as known they play no part in the physiology of
the bowel. Other substances may also appear in the bile, such as toxins
or metallic poisons of various kinds.

With this statement of function we may now consider the chemistry
of the various constituents of the bile, the pigments, salts, lipins and
mucin, and the manner and place of their origin.

Chemistry of the bile pigments.—The peculiar color of the bile is
due to several pigments, of which the most important is bilirubin, which
is the mother substance of most of the others. This is the pigment which
gives the golden red color to bile; it is easily converted by oxidation into
a green pigment, biliverdin; and by further oxidation it is converted into
a series of colored substances, of which bilicyanin, a blue pigment, may
be particularly mentioned. By reduction it probably forms urobilinogen,
a pigment of the urine. Bilirubin is derived from the hemoglobin of
the blood, and the amount of bilirubin, or the other pigments derived
from it, in the bile may be increased by any means which causes laking
of the red blood corpuscles and frees hemoglobin in the blood.

Bilirubin.—The chemical composition of bilirubin is still uncertain.
It is a erystalline, organic, iron-free compound, of which the formula is
either C,,H,,N,O, or more probably C,,H,,N,O,, according to Willstaet-
DIGESTION IN THE INTESTINE 415

ter. The last formula makes bilirubin isomeric with hematoporphyrin.
It yields on oxidation hematic acid, C,H,NO,; on reduction with hydri-
odic acid, zine or other reducing agents it yields hemopyrrol. This latter
substance is a mixture of substituted pyrrols from which there have
been isolated methyl, ethyl pyrrol and 2, 3-dimethyl, 4-ethyl pyrrol.

er te fc oe
H—C C—CH CH,—C C—CH,
Dimethyl-ethyl-pyrrol. Phyllopyrrol.

Bilirubin is an acid and in the soluble form is present as the sodium salt.
It probably contains four substituted pyrrol nuclei. The way they are
put together is uncertain. The following formulas have been suggested
for hematoporphyrin, and also of bilirubin, by Willstaetter and Fischer,
although other formulas also have been proposed. This will serve to
indicate the general nature of the compound.

CH, = CH—C — C—CH ‘ CH se — C—CH = CH,
U | Io
Cc C COH
\ /N\<Z
| Mt \ ye Na
oO C=C
| NH / \ NH
CNS oH
M ;
coon—cx,—ca,—d —_ C—CH, cut —C— CH, —CH > C00H
Formula of bilirubin suggested by Fischer and Rése, C Ha N,0, is
CH,—C — CH ‘CH — C—CH = CHOH

Ie <i

CH »OH—CH,—C —C

S G
c———C

é \

HOOC—CH,—CH,—C = C = C—CH,—CH,—COOH
Z
| Sa sé
\
cH,—C = C= C—CH,
|
CH, bar,
Possible formula of nenidladueglineiny C,H, NO.

33-38 4-6
The acid character and the substituted pyrrol nuclei are to be seen
in this formula. In their chemical nature, then, the bile pigments are
clearly oxidation products of hematin, the colored constituent of
hemoglobin. Oxidation products similar to those of bilirubin and
416 PHYSIOLOGICAL CHEMISTRY

hematin are obtained from chlorophyll; all three yield hematic acid, and
the two latter and mesobilirubin yield methyl-ethyl-maleic imide.

COOH—CH,—CH,—C = C—CH, CH,—CH,—C = C—CH,
| I 1k
Oo=C be Oo O=C . =O
\
4 NA
Hematie acid. Methyl-ethy]-maleic-imide.

In every spot of extravasated blood (‘‘ black and blue spot ’’) this
conversion of hematin into pigments analogous to, or identical with, the
bile pigments may be seen. Pyrrol, which thus forms so important a
part of the molecule of the pigments of bile, blood, urine and chlorophyll,
is found in the protein molecule in tryptophane, and in the reduced
form in proline.

Properties and preparation of bilirubin. Amorphous reddish-brown
powder, or reddish-yellow or brown crystals. Crystallizes readily from
chloroform solution by evaporation of the solvent. Long needles. Insolu-
ble in water ; soluble in warm chloroform, but with difficulty in cold ; more
soluble in aleohol; very slightly soluble in benzene, ether, amyl alcohol or
glycerol. Soluble in dilute alkalies. The alkali salts are insoluble in
chloroform. The solutions show no absorption bands. Solutions of
bilirubin in dilute alkali are precipitated by milk of lime. An aqueous
solution of alkali salt of bilirubin treated with ammonia and then with
some zine chloride becomes first deep orange, which changes to brownish
green and finally green. The solution then shows absorption bands close
to the C line and between C and D.

Bilirubin is most easily prepared from gall stones of cattle. The
stones are powdered and extracted successively with ether, boiling water,
10 per cent. acetic acid, alcohol and hot glacial acetic acid. By these
extractions cholesterol, bile salts, mineral constituents, green coloring
matter. choleprasin are removed. The residue is washed with water,
dried and extracted with boiling chloroform. The bilirubin erystal-
lizes out of the chloroform on cooling. It may be recrystallized from
hot chloroform or from hot dimethylaniline.

The reactions by which bilirubin may be detected, namely, those of
Fhrlich, Gmelin, Huppert and others, are described on page 916.

Biliverdin.—Bilirubin in an alkaline solution has the property of
auto-oxidation. If spread out in a thin layer in the air, it changes to
a green pigment which is often present in the bile, known as biliverdin.
The composition of this substance is probably C,,H,,N,O,. It does not
crystallize. By further oxidation with nitric acid, or bromine water,
it develops the series of colors noted for bilirubin. At times, in infants,
‘ especially in certain diarrheas, biliverdin may color the feces a bright
DIGESTION IN THE INTESTINE 417

green. Biliverdin may also be formed from bilirubin by gentle oxida-
tion by sodium peroxide, or by Hiibl’s solution.

Biliverdin was found by MacMunn in the mesoderm of a sea-
anemone (Actinia mesembryanthenum (?)). It is a significant fact
that biliverdin, which contains no iron, combines spontaneously with
oxygen and gives it up again almost as readily as oxyhemoglobin.

Properties. Amorphous. Soluble in alcohol, glacial acetic acid,
dilute alkalies; insoluble in water, ether, chloroform. The alkali salt
is precipitated by heavy metals and alkaline earth salts. By reduction
by putrefaction, or ammonium sulphide, it is converted back to bilirubin.
By reduction with sodium amalgam it is converted into hydrobilirubin.

Urobilin, stercobilin, hemibilirubin and hydrobilirubin.—The bile
pigments do not usually occur in the feces and urine, although they may
at times be found there. The brown color of the feces is due to a reduced
bilirubin called stercobilin or urobilin. These two are probably identical.
Urobilinogen is one of the important coloring matters of the urine. It
disappears from the urine when the bile is prevented from entering the
intestine. It is probably formed from bilirubin by the reducing action
of the bacteria of the gut. It is to be found also in blood serum and in
bile itself. The composition of urobilin, or stercobilin, is still uncertain,
for it is an amorphous body and it is impossible to know whether it has
been prepared in a pure state. It is preceded in the urine by a uro-
bilinogen, a substance which is converted into urobilin by the action of
the oxygen of air. According to Fischer and Meyer-Betz, there are at
least two of these urobilinogens: namely, hemibilirubin, to which they
ascribe the formula C,,H,,N,O,, and an unknown substance. By the
reduction of bilirubin by sodium alagam a series of substances are
obtained which resemble urobilin, but which are less stable. Among
the reduction substances thus formed may be mentioned hydrobilirubin,
which, according to Maly, has the formula C,,H,,N,0,. This formula
calls for 9.8 per cent. of N. According to Garrod and Hopkins, how-
ever, urobilin as isolated by their method contained but 4.11 per cent.
of N. The relation of these bodies is, therefore, still uncertain. Accord-
ing to Thudichum, urobilinogen is but a mixture of decomposition prod-
ucts of urochrome; the hydrobilirubin of Maly he believed to be entirely
different from urobilin. It is possible that what is known as urobilin is
a mixture of various pyrrol derivatives, since all of the unstable pyrrols
show color reactions such as-the Ehrlich reaction and the fluorescence
characteristic of urobilin.

Properties. Urobilin is soluble in water, but may be salted out by
saturating its solution with ammonium sulphate. It is an amorphous,
-brownish substance which in dilute solution is reddish yellow or rose red,
but is nearly colorless if slightly acid. It is distinguished by two proper-
ties: if a solution is made faintly ammoniacal and sufficient ZnCl, is
418 PHYSIOLOGICAL CHEMISTRY

added, the solution acquires a beautiful green fluorescence. In trans-
mitted light it is a reddish color. Even very dilute solutions may be
detected by this property. The other means of distinguishing urobilin
is by the absorption spectrum. In a strong solution the light is ab-
sorbed from the violet end right up to Frauenhofer line b; but in a more
dilute solution there is a well-defined band between b and F. If ZnCl,
is added to the solution, another band appears between b and F, but
nearer b than the former. Since urine has no absorption bands it is
clear that urobilin does not normally occur in it, but that some precursor,
urochrome according to Thudichum, or urobilinogen, is the urinary
pigment. Urobilin has the property of giving the Ehrlich reaction:
that is, a brilliant red color on the addition of a little p-dimethylamino
benzaldehyde to the acid solution. This reaction is given not only by
urobilin, but by hemopyrrol and all pyrrols having an unsubstituted H
atom on one of the carbon atoms of the ring (Fischer and Meyer-Betz).
Urobilin is soluble in CHCI1, and in acid alcohol. The method of sepa-
rating it from the urine is given on page 765. Urobilinogen is said to
give the biuret reaction.

Cholehematin.—Besides the pigments belonging to the bile, other
pigments of an extraneous nature are sometimes found in it. Thus in
herbivorous animals such as the rabbit, ox, hippopotamus, horse or goat,
when fed on green fodder, the bile often contains a red pigment with
very characteristic absorption bands called by its discoverer, MacMunn,
cholehematin. The absorption bands are the following: Band 1, —A638;
band 2, A 604—582; 3, A 570—558; 4,2 530—515. There are two bands
in the ultra violet. This substance is not a true bile pigment, but is
derived from the chlorophyll the animal has eaten. This red pigment,
phylloerythrin, as it is called, is found in the feces of cows. It is derived
in some manner from chlorophyll, presumably by the action of the bac-
teria of the intestine; it is absorbed, carried in the blood to the liver
and there execreted in the bile. If cattle are fed on dry fodder, the color
disappears from the bile and from the feces. This fact illustrates again
that the bile is not only a secretion, but an excretion, and toxins and
poisons absorbed from the intestine may be excreted in it. Cholehematin
is identical with bilipurpurin. It is extracted from the fresh feces of
cattle fed on green grass. The solution in chloroform is cherry red. It
is a weak base, insoluble in alkali. It is changed by HCl to a blue violet.
It would be better to call it phylloerythrin-

Where are the bile pigments made?—Does the liver make the bile
or does it simply secrete it from the blood as the kidney secretes urine?
This question has been answered by seeing whether bile pigment and
bile salts will accumulate in the blood after the liver has been cut out
of the circulation. If the bile duct is tied, bile pigments and other salts
accumulate in the blood of mammals and birds so rapidly that they may
DIGESTION IN THE INTESTINE 419

be easily detected in the blood serum after four to six hours. Now if the
blood vessels, the portal vein and the hepatic artery are tied no such
accumulation occurs, although animals may live 6 to 18 hours after
the operation. Moreover, under such circumstances the bile pigments do
not appear in the urine. In birds the liver may be extirpated, leaving
only very small remnants about the vena cava, since in these animals
a connection exists between the portal and kidney veins, by which the
blood may return from the intestine into the general circulation. In
birds, geese, ducks and hens, Minkowski and Naunyn found that there
was no jaundice, nor did any bile salts appear in the urine, although
the birds lived in some instances in a fairly good state for 12-19 hours
after the operation. It is then clear that normally bile pigments and
salts are formed in the liver itself, although every black and blue spot
in a bruise shows that the bile pigments may be formed from extravasated
blood in almost any location in the body.

The raw material from which the liver makes the bile pigments is
the blood pigment, hemoglobin. Bilirubin and hemato-porphyrin are
isomeric substances. The direct transformation of hematin of blood
hemoglobin into bile pigments, bilirubin and biliverdin, is shown by
the fact that any means which leads to the setting free of hemoglobin
in the blood causes an increase in the bile pigments excreted. Thus
inhalation of arseniureted hydrogen causes a great hemolysis, even
leading to hemoglobinuria ; and injections of toluylendiamine produce the
same result. Bile secretion is greatly increased in animals poisoned by
these substances. Injection of hemoglobin itself has a similar effect.
Moreover, liver pulp rapidly destroys hemoglobin and. although it does
not convert it into bilirubin, it is believed to make it into an antecedent
of bilirubin. Microscopic examination of the liver shows the various
steps in this transformation.

Transformation of hemoglobin into bile pigments—We may now
ask the question of the chemistry and method of the transformation of
the blood into the bile pigments. The red blood corpuscles are con-
stantly being formed in the cells of the red marrow of bones. It is
clear, therefore, that they must somewhere be broken down and done
away with or they would accumulate in the blood. All that is known
indicates that it is in the liver that this destruction takes place, at least
in the birds and amphibia, but that in mammals the spleen also plays
an important, and perhaps the more important, rdle. The destruction of
corpuscles in the liver cannot be demonstrated by the simple and direct
expedient of counting the corpuscles in the portal and hepatic bloods.
Counts of this sort reveal no appreciable differences between the blood
entering and leaving the liver. nor is it to be expected that they would,
since so rapid is the blood flow through this organ and so large is it,
that in any single passage of the blood but a very few corpuscles can
420 PHYSIOLOGICAL CHEMISTRY

possibly succumb. ‘Nevertheless, there is good reason to believe that
they do thus die and degenerate in the liver tissue of both birds and
amphibia, since both normally and when their decomposition is accel-
erated by blood poisons, such as those just mentioned, the process of
decomposition is easily demonstrable by the microscope. Minkowski and
Naunyn have particularly studied these processes in bird’s liver and in
rabbits after poisoning with arseniureted hydrogen, and Keyes has
recently shown the same processes in normal livers of birds and amphibia.
There are many questions which naturally arise in our minds as we
approach this problem. Does the liver secrete into the blood a sub-
stance hemolytic in nature, which is not sufficient in amount to dissclve
all the corpuscles but only the weakest ones? And does this hemolytic
substance cause a discharge of the hemoglobin into the plasma of the
blood from which the liver cells pick it up? or are the corpuscles engulfed
as such by some of the phagocytic cells of the liver which by intracellular
action destroy the corpuscles? If part of the hemoglobin is made into
bilirubin, what becomes of the rest of the corpuscle? There are in the
liver two quite distinct tissues: the endothelium of the blood vessels and
the glandular tissue. Are both of these tissues or only one of them
important factors in the production of bile from blood? While these
questions cannot as yet be fully answered, certain facts have been
discovered.

The endothelium of the blood vessels constitutes a great organ extend-
ing everywhere in the body, for which no function has as yet been found
beyond that of serving as the lining of a tubular conduit of the blood.
Is this its only function? or is it concerned also in the elaboration of
the constituents of the blood; of its proteins, slasma and blood cells? Or
does it play an important part in the regulation of the composition of
the blood and the determination of the viscosity of the blood in the
capillaries by the production of substances which act upon the colloids
of the plasma, affecting frictional resistance? We cannot as yet answer
these questions, for we know neither where the blood proteins arise nor
the functions of the capillary endothelium. One thing, however, is clear
and that is that these capillary cells must certainly play an important
part in the control of lymph formation, to which they stand somewhat
in the same relation as the glomerular cells of the kidney to urine forma-
tion. They are also endowed, in certain regions at least, with phagocytic
powers and, indeed, it has recently been shown that the giant phagocyte
cells found in tissues arise from the capillary endothelium. This phago-
eytic function is shown to a pre-eminent degree by the endothelium of
the liver. Here and there in the liver these cells increase in size and
when large are called Kupfer cells. These endothelial cells engulf
bacteria. They also. according to Keyes, and as may be seen in the
drawings of Minkowski and Naunyn, engulf the red blood corpuscles
DIGESTION IN THE INTESTINE 421

in birds and amphibia. One of these cells may take up many of these
corpuscles which can be distinguished after engulfment by their outline
and by the chromatin material, since the corpuscles of these forms are
nucleated. These engulfed corpuscles go to pieces and may be seen with
out their hemoglobin here and there in the Kupfer cells, and in case
the destruction of the corpuscles is intense, a bright-green pigment.
possibly allied to or identical with biliverdin, may be seen in them. At
the same time the hemoglobin appears in the endothelial cells as masses
of brown pigment. Similar masses appear shortly after in the liver cells
proper and at the same time the cell body, particularly along the bile
capillaries, becomes filled with fine brown granules, which stain a deep
blue in potassium ferrocyanide, or brownish black in ammonium sulphide,
showing that they contain iron. This iron has been set free from the
hematin.

In the mammalian liver this phagocytic réle of the endothelial cells
has not been definitely established, but similar phagocytic cells were
described in the finest capillaries in rabbit’s liver after the injection
of blood poisons by Minkowski and Naunyn. These cells were
present, however, in mammals in much smaller numbers than in the
bird’s liver. There is also an accumulating mass of evidence that in
mammals the spleen probably plays an important part in the destruc-
tion of the corpuscles. Cells like those phagocytic cells of the liver are
found in numbers in the spleen containing engulfed corpuscles, and
recent surgical work indicates that the spleen is active, in some patho-
legical conditions at any rate, in blood destruction. Some kinds of
anemia have been improved by extirpation of the spleen. If, however,
the blood corpuscles are destroyed in the spleen. this cannot be the only
place of their destruction. since the taking out of the spleen does not
check the formation of bile in the normal animal, and the fact that bile
pigments do not accumulate in the blood after an Eck fistula and ligation
of the hepatic artery also shows that, if the spleen is active, only the
first stage of the process can be taking place in that organ. The ques-
tion of the destruction of the red corpuscles in mammals is, thus, in a
very unsatisfactory state and more work must be done before definite
conclusions concerning this very important question can be drawn.

All the evidence. however, points to the conclusion that the hemo-
glohin is not first discharged from the corpuscle in the blood stream
and is then picked out by the liver, but that the corpuscle is engulfed
as a whole and the separation of the hemoglobin occurs intracellularly
in the phagocytic cells. It appears then that red cells in a peculiar
condition, in the condition. possibly, which is produced by the first effect
of a hemolyzing agent before hemolysis occurs, are engulfed by the
phagocytie cells of the liver; these cells in birds and frogs, and possibly
also in mammals, being in part at least the endothelial cells of the liver
422 PHYSIOLOGICAL CHEMISTRY

blood capillaries. Being engulfed, the hemoglobin separates from the
stroma and is passed in some manner not known to the liver cells proper
and there undergoes a transformation of such a kind that iron is split
off and becomes readily demonstrable by microscopic methods; and ulti-
mately the iron-free part of the hematin is further transformed in the
liver cells into the bile pigments, bilirubin and biliverdin. It would be
very interesting to know, in this connection, whether the endothelial
cells of the blood change themselves so that the corpuscles more readily
adhere to them leading to their engulfment, or whether they form a
substance which, acting upon the corpuscles, makes them more easily
engulfed, much as the bacteria are supposed to be acted upon by the
opsonins. These and many other similar queries are among the most
enticing questions of chemical physiology at the present time, since their
solution may throw light on the important subject of the defense of the
organism from bacteria.

It may be mentioned, in this connection, as possibly indicating one
reason for the excretion of the bile pigments, that small amounts of
hematoporphyrin occur normally in the blood. It has recently been
found that hematoporphyrin has a very extraordinary effect in rendering
animals sensitive to the action of light. If hematoporphyrin is injected
into white mice, rats or guinea pigs, or if it is produced in the blood by
the action of poisons, no toxic effects follow from the presence of the
hematoporphyrin as long as the animal remains in the dark or in a dim
light. But, if it is brought into direct sunlight, it presently begins to
scratch vigorously, often rubbing the hair and skin off, it is very rest-
less and it will die if not returned to the dark. Similar effects are pro-
duced in human beings by sunlight and hematoporphyrin, a rash and
skin eruption appearing, followed by a tremendous edema. The effects
may persist for several weeks after taking hematoporphyrin before
the sensitization of the body to light is lost. The mechanism of the
action is not explained, but the observations are of very great interest.
Animals with much pigment in the skin are protected from these
effects. One of the functions of the liver is, then, to pick out the
hematoporphyrin from the blood and to convert it to a harmless bile
pigment.

The close relationship of the bile pigments to the blood pigments is
shown by several facts. Thus if oxyhemoglobin is treated with dilute
acid it splits into globin, a protein, and hematin, C,.H,.N,0,Fe; by
strong acid hematin is converted into hematoporphyrin, and the iron of
the hematin is set free as inorganic iron. Hematoporphyrin is supposed
to have the formula, C,,H,,N,O,, or C,,H,,N,O,, and it is isomeric with
bilirubin, C,,H,,N,O,.1. On oxidation both give rise to hematice acids.

‘The formulas of bilirubin and hematoporphyrin are still uncertain. The older
formula called for 32 atoms of carbon. Kiister gives 34 atoms as more probable,
DIGESTION IN THE INTESTINE 423

C,,H,,N,0,Fe + 2H,0 = Fe + C,,H,,N,O,
Hematin. Hematoporphyrin.
The bile pigments are also closely related to chlorophyll.
The relation of these three pigments may be illustrated by the follow-

ing diagram:

I T III
Hemoglobin Chlorophyll
Globin Hematin : Phyllocyanin
C,,H,N Fel?)
Hematoporphyrin _ Bilirubin Phylloporphyrin
C,H (2) C,H, (?) C,H. .N,0,
Biliverdin
C, HH, oN,
Urobilin
C,H, N,0, ( *)
Hemopyrrols Hemopyrrols Hemopyrrols
C,H, ,N (ete.) C,H, ,N (ete.) C,H, ,N (ete.)

‘

Hematie acids.
0,H,0.,C_H,NO,,C,H,NO,.

The bile salts.—These are the characteristic constituents of the bile
which give to this fiuid its properties of assisting in the digestion and
absorption of fats. In the majority of mammals these salts consist for
the most part of sodium salts of glycocholic and taurocholic acids, but
in addition to these acids there may be present analogous acids such
as taurocholeic and glycocholeic acids. The relative proportion of tauro-
cholic and glycocholic acids differs in various animals; thus in dogs
glycocholic acid may be almost or quite absent, although at other times
it may be present. Its absence or presence may be shown readily by
adding to the bile some neutral acetate of lead which precipitates the
glycocholic acid but not taurocholic. The bile salts are salts of paired
acids; that is, they split on hydrolysis into taurine, or glycocoll, and

whereas Fischer has recently expressed the opinion that there are 33 atoms of ear-
bon and 38 of hydrogen.
424 PHYSIOLOGICAL CHEMISTRY

cholic acid. The taurine and glycocoll are the same in all animals, but
the cholic acid part of the molecule differs in different animals. The
pig or the goose does not have the same cholic acid as the ox or man.

The bile salts appear very early in development, as soon in fact as
the liver has been set apart; in the chick embryo they occur from the
third day of incubation. They are not formed elsewhere in the body than
in the liver, and in fact may be considered as the most characteristic
products of the metabolism of this organ. They give a striking color
reaction when they are mixed with a little sugar or formaldehyde and
the solution placed in contact with concentrated sulphuric acid. A violet
ring develops at the zone of contact. This is known as Pettenkofer’s
reaction. It is the same as Molisch’s reaction for the detection of carbo-
hydrates, with the exception that the bile salts take the place of the
a -naphthol as the chromogenic substance. The reaction depends on the
formation of an aldehyde-like furfural or oxymethylfurfural from the
sugar by the acid and the condensation of this product with the bile
acids, or some decomposition product of the latter, to a colored compound.
The reaction is not at all specific for bile acids. It is given also by
oleic acid, by many alcohols, aromatic substances and other compounds.

Preparation and properties of the bile salts. To make an impure
solution of the bile salts, a simple method is to mix fresh bile with about
1 per cent. by weight of animal charcoal and to evaporate to dryness on
the water bath. The dry residue is powdered and extracted with water
and filtered. The bile salts are extremely soluble in water and go into
solution while the pigments and some other impurities remain in the
charcoal. The solution contains not only the bile salts, but some choles-
terol, mucin, phosphatides and inorganic salts. The bile acids may be
obtained from this solution, if it is sufficiently concentrated, by
acidification. .

Plattner’s bile. Crystallized bile. This is prepared in the same way
as the decolored bile just described, except that the dried residue con-
taining the charcoal is extracted with boiling, absolute alcohol. The
salts are very soluble in alcohol. By this means mucin, pigments and
most of the inorganic salts are left behind. The alcohol after filtration
through a dry filter into a dry flask, using care to prevent the entrance
of water, is placed in a loosely stoppered flask and absolute ether run in
until a precipitate begins to appear. It is then set apart in a cool place
and the bile salts will crystallize out, provided the reagents have had
little water in them. If water is present, they will come out as an oily

liquid, which may later crystallize. The salts thus crystallized are very
- deliquescent and take up water rapidly from the air. They must be kept
in a desiccator. They are known as Plattner’s bile. From this partially
~arified product the acids may be separated. The cholesterol is sepa-
DIGESTION IN THE INTESTINE 425

rated by washing the saits with absolute ether. The salts generally have
a bitter taste. They make a neutral solution. Some of them may be
precipitated by saturating their solutions with ammonium sulphate.

Glycocholic acid.—C,,H,,NO,. a. Properties: White, crystallizing in
needles. Slightly soluble in cold water, more soluble in warm. Very
soluble in strong alcohol, less in dilute. m.p. 193°. Shrinks 133-134° on
rapid heating. Recrystallizes readily from dilute alcohol on cooling
(Alcohol 10-30 per cent.) Slightly soluble in ether (1:1,000), and is
precipitated from alcoholic solution by ether. Practically insoluble in
benzol and chloroform. Rotatory power of alcoholic solution(a)18° =
+32.3° (Letsche). Na glycocholate, (#)13°=+24.8°, dissolved in water ,
-+27.8° in 90° alcohol (Letsche). The alkali and alkali earth salts are
soluble in water; most others are insoluble. All of them are soluble in
alcohol, except lead. Glycocholic acid is precipitated from solution by
neutral acetate of lead. This distinguishes it from taurocholate, which
is precipitated only by basic lead acetate. Its taste is bitter, with a
sweet after taste. The affinity constant K is .0132. The acid is hence
about as strong as lactic acid. Its salts do not make colloidal aqueous
solutions.

b. Decomposition by acids. Cooked with acids it decomposes into
glycocoll and cholic acid as follows:

C,,H,,NO, +H,0=C,H.NO,+C,H, 0..

26 43 £405
Glycocholic acid. Glycocoll. Cholie acid.

By farther heating the cholic acid loses two molecules of water and is
converted inte dyslysine, C,,H,,03.

ce. Preparation. From some biles containing principally glycocholic
acid the glycocholic acid is very easily prepared by acidification after
the addition of some ether. The bile of oxen often contains a good deal
more of glycocholic acid than taurocholic. In some cases it is only neces-
sary to filter the bile through charcoal, to partially or wholly decolorize
it, slightly acidify to remove mucin, filter and then add a mixture of
5 parts HCl and 30 parts of ether to 100 parts of bile to have the glyco-
cholic acid crystallize out. Generally, however, it will be found neces-
sary to make first crystalline bile in the method described. This gets
rid of the mucin. A strong solution of the crystalline bile is made after
first freeing the crystals from cholesterol by anhydrous ether. Some
ether is added to the solution, and then little by little a solution of
sulphurie acid. This is added cautiously at first until crystallization
begins and then more freely. The glycocholic acid crystallizes out, leav-
ing the taurocholic acid in solution. This method does not always yield
crystalline glycocholic acid, particularly if much taurocholate is present.
It may be necessary to separate from taurocholic acid by precipitating
with neutral lead acetate. This precipitates the lead glycocholate. leav-
426 PHYSIOLOGICAL CHEMISTRY

ing the taurocholate for the larger part in solution. The precipitate is
filtered off, washed with water on the filter and dissolved in warm
alcohol. The lead is separated with sulphureted hydrogen, the lead
sulphide filtered off, the filtrate concentrated and the free acid precipi-
tated by the addition of ether. It slowly crystallizes. From the first
filtrate the lead salt of taurocholic acid may be precipitated by basic
lead acetate. .

Taurocholic acid.—C,,H,,NSO,. a. Properties. White, needle-
shaped crystals very deliquescent, and very bitter. Decomposes on cook-
ing with water, but when dry may be heated above 100° without change.
Acids and alkalies decompose it into taurine and cholic acid. In alco-
holic solution (@)p=—+24.5°. Very soluble in water, soluble in alcohol,
insoluble in ether. Dissolves some glycocholic acid. It is a fairly strong
acid, and it decomposes in solution more readily than glycocholic acid.
It is stronger than glycocholic acid. The salts are soluble in water and
alcohol, but insoluble in ether.

b. Preparation. It is most easily prepared from the bile of dogs
which contains often very little glycocholic acid, or from the bile of the
haddock which contains only taurocholic acid. It may be prepared by
precipitation with basic lead acetate in the manner described.

ce. Decomposition. By hydrolysis by acids or alkalies it splits read-
ily into taurine and cholic acid as follows:

C,,H,,NSO,+H,O— C,H,NSO, + C,H,.0,

26 H,, 24° 40
Taurocholic acid. Taurine, Cholic acid.

Taurine is derived from cystine by reduction of the latter to cysteine
and the see of the cysteine followed by a decarboxylization, thus:

O H NH, O H NH,

mee. +0, — ee eee —- aod hls +o,
kok Ju k Suk

Cysteine. Intermediate, Taurine.

Taurine is found very widespread in nature, showing that the
processes by which it is derived from cysteine must be very comnon in
cells. It is found in many tissues of the body and is probably a normal
intermediary product of cystine metabolism.

The determination of the amount of taurocholic acid in various biles
has been generally made by determining the sulphur in the ether insol-
uble, alcohol soluble part of the bile and computing the taurocholic acid
from this on the hypothesis that taurocholic acid is the only sulphur con-
taining substance in the bile having these solubilities. This method of
determination of the amount of taurocholic acid has been shown recently
by Hammarsten to be quite incorrect, owing to the fact that there is in
DIGESTION IN THE INTESTINE 427

many biles some ethereal sulphate and sulpholipin which have the same
solubility in alcohol as taurocholic acid. It is not possible to say posi-
tively how much taurocholic acid there is in bile for this reason. The
following table, taken from Hammarsten, shows the amount of sulphur in
the crystallized bile of various animals:

Amount or SULPHUR IN Various Bites (Hammarsten).

Sulphur in ether in- Snlphur in alcohol

Animal soluble bile salts -— soluble cried bile—

per cent. per cent.

GOGSE ina sities aracveeieres 05 6.34 3.86-4.21

Snake (boa constr.) ..... 6.24 4.11

DOR aie sieisetisgei wey Dhaea reis farce 6.21 re

Snake (python) ......... 6.04

POX: etek e cape es wees cee, 5.96

B@ar™ ecsi-4 sa0de0.8 éAeee Sak oees 5.84

DHCP soos Sioa sree scacere.g wissecn 5.71

ISH inie sd aheiwcea eottesea ase 5.55-5.99

Haddock ............0.-. 5.66 acces

Walrus ....... a iiees veer ae 4.37

Goat: fee swe os vie re soe 5.26 buoys

Wl os beds naan eats 5.08

ROT sss sss asa bie cena 0-5 5e 4.96

GANT eidisc Actes oa are elope sae 4.88 3 as

OR: eatin de ana eentveea secs 3.58 3.43

Musk:0x eines eesti seasons ay sags 2.05-3.05

Kangaroo wee ec iva eec eset 2.47 ah

Hippopotamus ........... 4% 1.84

Orang-utang ............. ee 1.37

Mant peice oke usa yaaa. 0.21-2.67 ae

MAT sites pesesners ceelaeeved,24 18% 0.83-2.99 0.85-1.30

BAB Coir je cstadatal dvi elon Germs 0.33 0.56

Human bile, as will be seen, contains but very little sulphur. In
fact, in one case of a bile fistula Jacobson found none at all. If it be
remembered that taurocholate alone contains 5.95 per cent of 8, it will
be seen that, if the sulphur is contained altogether as taurocholate, some
of these biles can have practically no glycocholic acid. Such is certainly
the case for the haddock and at times in dog’s bile, but in other animals
the sulphur may not be present in the form of taurocholate at all. Thus
in the hagfish, Seymnus borealis, although the alcohol soluble bile solids
contain 5.3 per cent. S, this is all ethereal sulphate and there is no
taurocholic acid, but instead a substance allied to cholic acid, or choles-
terol, called by Hammarsten scymnol, C,,H,,0;, which is paired with
sulphuric acid. Human bile, also, contains these ethereal sulphates to
very differing amounts in different people, and Hammarsten has sug-
gested that they are very likely derived, like the similar bodies in the
urine, from intestinal putrefaction. In the bile of the new-born and
sucklings Jacobowitsch found only taurocholic acid; but Buginsky and
Sommerfeld got also glycocholic. The bile of a ray, Raja batis, also
contained the greater part of the sulphur as ethereal sulphate. The
428 PHYSIOLOGICAL CHEMISTRY

amount of the sulphur as ethereal sulphate in human bile may vary
from 6-17 per cent. of the total sulphur of the alcohol soluble solids.

It is possible by feeding animals cholic acid to increase somewhat the
amount of taurocholic acid in the bile. The body seems to have a supply
et taurine in reserve which it can unite with the cholic acid fed. This
reserve of taurine is, however, soon exhausted and, if more cholic acid
is given than enough to combine with the taurine, it appears in some
other form than as taurocholic acid and chiefly in the form of glyco-
cholic acid. The dog’s body appears to form taurocholic acid by pref-
erence, but in the absence of sufficient taurine glycocoll is used instead.
It thus happens that while taurocholic acid is generally present in large
excess in the dog’s bile, yet more or less glycocholic acid may be found
there also. Indeed, after glycocholic acid is taken by the mouth, it
reappears in the bile as such. But while the body has some reserve
of taurine and a large reserve of glycocoll to pair with any cholic acid
produced, there is no reserve of cholic acid to pair with the taurine. It
has been found that, if cystine is given alone, there is no increase in the
sulphur of the bile, the reason being that there is no reserve of cholic
acid to pair with it; but, if cystine and cholic acid are given together,
taurocholic acid is much increased.

Cholic acid.—C,,H,,0,;. This is also called cholalic acid. It is
formed by hydrolysis from the conjugated acids just described. From
10 liters of ox bile Schryver obtained 225 grams of cholic acid, 75
grams of choleic and 40 grams of deoxycholeic acid in the crude erystal-
line form. Pregl and Buchtala obtained from ox bile 51.2 per cent. of
the total fatty and bile acids as cholic acid.

a. Preparation. The following method of preparation of cholic and
other acids of the bile was employed by Schryver (1912): 2.5 liters of
fresh ox bile were mixed with 170 grams of NaOH dissolved in 300 e.c.
of water and heated 30 hours in an iron digester with a reflux condenser.
The mixture was then diluted with twice its volume of water and, while
still warm, acidified with dilute HCl, vigorously stirring after each addi-
tion of acid. The crude acids separated as a viscid oil, pasty on cool-
ing and sometimes granular. After standing overnight the mass was
filtered, washed free from HCl by kneading in water, dried on the
water bath and powdered. It was dissolved in an excess of dilute
NH,OH8, so dilute that there was in solution finally not more than 5 per
cent. of the ammonium salt, and some pigment was removed by boiling
with animal charcoal. It was filtered, precipitated by dilute HCl, the pre-
cipitate washed with water, and dried in vacuo over CaCl, and soda
lime. From time to time the surface lumps were removed and powdered,
and finally it was a powder, which was recrystallized from hot acetone,
filtered hot. On cooling, the crystals were filtered, using the pump, and
DIGESTION IN THE INTESTINE 429

washed in cold acetone until nearly free from mother liquor. The
filtrates and washings united yielded a second crop of erystals when
concentrated. The process was repeated until only a green mother liquor
was left, from which no more crystals separated. All crystals were
united. The separation of cholic, choleic and deoxycholeice acids was
accomplished by the difference in behavior of the Mg salts. The mag-
nesium salts of choleic and deoxycholeic acids are less soluble than cholic
acid. The crystals are suspended in hot alcohol, a little phenolphthalein
added and, while the alcohol is kept hot, NaOH is added until a faint
alkaline reaction was obtained. The alcohol was then evaporated on
the water bath, the sodium salts taken up in water so that 100 c.c. of
solution corresponded to 1 gram of the crude crystals, filtered and made
neutral to phenolphthalein with acetic acid. The solution was mixed
with one-tenth its volume of 20 per cent. MgCl, and heated on the water
bath. A bulky crystalline precipitate gradually formed. After heating.
1 hour it was allowed to cool. The precipitate consisted of the Mg salts
of choleie acid, deoxycholeic acids, with a little cholic acid. Cholie acid
was obtained by acidifying the mother liquor. Choleic acid was sepa-
rated from deoxycholeic by precipitating it as barium choleate, the
barium salt being insoluble.

b. Properties. White, crystalline, very bitter substance. Almost
insoluble in water, soluble in alcohol, but not very soluble in ether. It
crystallizes from alcohol when water is added to the latter in the form
of rhombic pyramids and tetrahedrons which contain a molecule of water.
m.p. 198° (Bondi and Miiller). Its solutions are dextro-rotatory (@)p—
+85°. It gives Pettenkofer’s reaction. It combines with water, with
the halogen acids and potassium iodide. It is unsaturated. The alkaline
salts and barium salt of cholic acid are very soluble in water, the other
alkaline earths less soluble in water. On oxidation with nitric acid it
forms first dehydrocholic acid, C,,H,,0,, and finally oxalic acid, various
volatile fatty acids and cholesterinic acid, C,H,,0;. Its structural
formula is unknown, but it is related.to cholesterol and the terpenes. A
formula suggested is the following (Pregl, 1910) :

CH,.CH, / CH (CH,OH).CH(CH,OH)\
COOH,CH cnt pe » OH (CH,), )_.CH cH, cH CH,

|
CH,.CH = CH.CHOH

 

Another formula suggested by Panzer is given on page 430. It has
a very characteristic iodine reaction, forming with the latter a blue-
colored compound like starch. If 2 grams of cholic acid and 1 gram
of iodine are dissolved in 40 ¢.c. of alcohol and to this is added 20 e.e.
af a solution of KI containing 1 gram of J, a compound is formed which
m the addition of water with constant stirring precipitates as 2 mass
430 PHYSIOLOGICAL CHEMISTRY

of bluish crystals. These are insoluble in water. They resemble starch
iodide. The formula is supposed to be (C,,H,,0,),KI-+-nH,O. Other
acids, such as deoxycholeic, hyocholiec, bilianic, ete., do not give this reac.
tion. When heated in water to 200° in an autoclave it forms the anhy-
dride, dyslisine.
H = HO 2H
othe ooh “Breltaget °

Alkalies reconvert the dyslisine into cholic acid. The close relation of
cholic acid to cholesterol and its probable derivation from that substance
is shown by the fact that both cholesterol and cholie acid give the
Lifschiitz oxycholesterol reaction after oxidation with benzoylperoxide
(see page 83) and that both yield on oxidation rhizocholic acid, C,H,0,,

which probably has the following composition:
CH—C—COOH

[ pe eee
HOC—C
door
Rhizocholic acid.

The same acid is obtained from camphor and oil of turpentine when
oxidized, which shows that cholic acid and cholesterol are terpenes.

With HCl, cholic acid gives a color reaction (Hammarsten). Pow-
dered cholic acid shaken with 25 per cent. HCl at room temperature
colors the fluid at first yellow to green and then after several hours a
bluish violet, which deepens for the next 24 hours. It shows an absorp-
tion band near D. The change takes place more rapidly on heating. Not
all bile or cholic acids give this reaction.

HH HH HOR HH HE
yy Yr YY
uc’ ‘oa’ “don” Non “on,
Hd cn dncoon da, by du,

x ‘a, Xe
|

|
CH,OH CH,OH
Tentative formula of cholic acid (Panzer).
C,,H,.0
24° 40°65

Glycocholeic acid.—C,,H,,NO,. Besides the ordinary glycocholic
acid found in ox and dog biles, a similar but somewhat different acid
is also found, and it probably occurs elsewhere. It is present in smaller
amounts than the ordinary glycocholic acid. It consists of glycocoll
paired with choleic acid. Glycocholeic acid differs from glycocholic acid
in the following points: It crystallizes in short, thick prisms; it has a
DIGESTION IN THE INTESTINE 431

very bitter taste, with only a weak sweet after-taste; it is soluble with
difficulty in boiling water ; the melting point is higher, namely, 175-176’ ;
its aqueous alkali salt solutions are precipitated by the addition of
barium, calcium or magnesium chlorides. In this way it may be sepa-
rated from glycocholic acid. The sodium salt solution is far more easily
precipitated by the addition of a saturated solution of NaCl than is
the corresponding salt of glycocholic acid; and'the pure solution of the
alkali salt is precipitated by the addition of acetic acid, whereas the
pure alkali salt solution of glycocholic acid is not precipitated by the
addition of acetic acid; a solution of sodium glycocholate is precipitated
by acetic acid, however, if free neutral salts such as NaCl are present
in the solution (Wahlgren, 1902).

Glycocholeic acid may be separated from ox gall by Wahlgren’s
method as follows: The fresh bile is evaporated to a syrup and the
mucin precipitated by the addition of alcohol. The alcohol is evaporated
from the filtrate, the residue dissolved in water and precipitated by
the successive addition of lead acetate, basic acetate and ammoniacal lead
acetate. The first precipitate contains both glycocholate and glyco-
choleate. It is freed from lead by Na.CO,, filtered, the filtrate evapo-
rated to dryness and the residue extracted with alcohol to free from
carbonate. The glycocholate dissolves readily in the alcohol; the glyco-
choleate dissolves with difficulty. It may be separated by precipitating
it with BaCl.,, which precipitates the choleate but not the cholate.

Taurocholeic acid.—This acid has been isolated from dog and ox
bile. It is less abundant than the taurocholic acid. It consists of taurine
and choleic acid. It contains more sulphur than taurocholic acid,
namely, 6.25 per cent: instead of 5.94 per cent. in the sodium salt. It
is separated from the taurocholate by being not so easily precipitated
from its alcoholic solution by the addition of ether, as is the taurocholate,
and when in aqueous solution it is precipitated by the addition of ferric
chloride, whereas taurocholate is not precipitated (Gullbring, 1905). It
is intensely bitter with no sweet after-taste. It has not been obtained in
a erystalline form. Like taurocholic acid it is readily precipitated by
NaCl, as a thick honey-like mass.

Choleic acid—The probable formula for this acid is C,,H,,0,
(Lassar-Cohn). It thus has the same formula as cholic acid, although its
discoverer, Latschinoff, ascribed to it the formula C.,,H,,0,, for the water-
free salt. Choleic acid is very much like cholic acid, but is less soluble
in water, alcohol, and glacial acetic acid. The water-free acid melts
hetween 185-190°; the water-containing acid melts at 135-140° and at
150° forms a homogeneous liquid. After repeated recrystallizations from
glacial acetic acid it melts at 145°. This is probably the acetyl compound.
This is the same melting point as deoxycholic acid, which is probablv
432 PHYSIOLOGICAL CHEMISTRY

identical with choleic acid, according to Gullbring. (See, however,
Schryver). It forms an insoluble barium salt by which it can be sep-
arated from cholic acid.

Other cholic acids——In the. bile of other animals than the ox and
dog, other cholic acids and their conjugates are found. Thus in the
pig hyoglycocholic acid occurs, and in the bile of geese chenotaurocholic
acid. Hyoglycocholic acid has been given the formula C,,H,,NO,.
Chenocholic acid is C,,H,,0,. There is little doubt that a number of
these more or less closely isomeric cholic acids occur in different biles.

Soaps.—Bile contains small amounts of the sodium salts of various
fatty acids (myristic, palmitic, stearic), among which sodium oleate may
be especially mentioned. The presence of these soaps affects the ease of
precipitation of the bile salts by neutral salts, the presence of sodium
oleate particularly interfering with the salting out. From 10 liters of
ox bile Schryver obtained the following amounts of fatty and bile acids:

Cholic acid ........ 225 grams’

Choleic “ ........ 75 grams } 350 grams of crude crystalline acids.
Deoxycholeic acid... 40 grams

_Fatty acids ........ 20 grams : :
Pigment (green) acid 12 grams 65 ae mother liquors of acetone crys
Glassy acid ........ 35 grams 5

Pregl and Buchtala found in the bile of oxen of Gratz 10 per cent.
of the total acids as fatty acids; 51.2 per cent as cholic; 11.9 per cent.
choleic ; 13.5 per cent. deoxycholeic ; 12.6 per cent. non-crystallizable.

Cholesterol in bile—Cholesterol has been found in the bile of all
animals in which it has been looked for, with the exception of the
hippopotamus. While the amount of cholesterol in human bile is
not very great, its importance is increased by the fact that it is one
of the chief constituents of gall stones. The amount in human bile is
given as 1.6 parts per thousand by Frerichs; and as 1.00 per thousand
in ox bile. In venous blood there is in the serum only 0.09 p.m. (Bec-
querel and Rodi), and in the whole blood only about .44-.75 p.m.

* Cholesterol, as its name implies, i.e., solid bile, is one of the commonest
constituents of gall stones of which it may form anywhere from 20-90 per
cent. The general properties of this substance, so common in all cells,
has already been given, page 81, and here only the amount in the bile,
and its origin, funetion, and fate will be considered.

Cholesterol is entirely insoluble in water or in salt solutions whether
neutral, acid or alkaline; its solution by the bile is, therefore, a singular
fact. The bile is in fact able, as Moore and Roaf showed, to dissolve
even more cholesterol than is ordinarily found in it. If solid pieces of
cholesterol are put into bile they dissolve (Naunyn). This power of solu-
tion of the bile depends on the presence of the bile salts and specifically
upon the cholic acid radicle of the salts. The bile salts themselves are
DIGESTION 1N 'THE INTESTINE 433

extremely soluble. The cholic acid part of the salt is probably closely
related chemically to cholesterol. It is probable that cholesterol unites
either physically or chemically, in some way not yet known, with the
cholic acid of the bile salts, and is thus held in solution. It will be re-
called that the bile salts are hemolytic agents and in this respect act
like the saponins and these saponins have been shown by Windaus and
Yogi to form easily dissociable compounds with cholesterol. It is not
improbable, therefore, that cholesterol unites similarly with the hemolytic
group of the bile salts and the compound thus formed is soluble in the
bile.

The origin of the cholesterol of bile is not yet certain. It may be
derived in part from the food and in part from the cholesterol of the
red blood cells, which are destroyed. A part of the cholesterol thus
passing in might be changed to cholic acid, a part might be secreted un-
changed. A part might be made in the liver from fat or sugar by the
metabolism of that organ. Dorée and Gardner have found only traces
of cholesterol in feces after feeding dogs various cooked foods, such as
oatmeal and milk, beef and mutton, horseflesh or other foods. Stercorol
was excreted on a diet of raw sheep’s brains. If cholesterol is given in
food to rabbits some is reabsorbed and finds its way to the blood, causing
there an increase in both the free cholesterol and the cholesterol esters,
as measured by the digitonin method. If phytosterol was fed to rabbits
it, also, was absorbed in part, resulting in an increase of free cholesterol
of the blood; but phytosterol did not appear as such in the blood.
While the reduction of cholesterol to stercorin happens in the body,
cholesterol is not reduced if added to the feces in vitro, possibly owing
to lack of solubility. Dog’s feces contain the unchanged cholesterol
of the bile.

Since bladder bile is much richer in cholesterol than that from a
fistula and the difference is much greater than the increase of con-
centration of the salts, it is generally concluded that cholesterol is added
to the bile largely by the epithelium of the bladder. It is especially
Naunyn who has defended this view and brought it into relation with
the formation of gall stones. It is often the case that the kernel of the
gall stone appears to be bladder epithelial cells and it is necessary to
have some injury to the bladder, an inflammation or traumatism, before
stones form. The experimental introduction of crystals of cholesterol
into the bladder did not cause the formation of concretions unless in-
fection of the gall ducts occurred also. The evidence does not appear to
be at all conclusive that cholesterol is added to the bile in the gall
bladder, and while it may well be that some of the cholesterol finds its
way through the wall of the gall ducts or bladder, it would appear more
probable that it is formed and secreted with the bile salts, to which it
434 PHYSIOLOGICAL CHEMISTRY

is so closely related chemically. It may be that the poor content of
cholesterol in fistula bile, as compared with bladder bile, is due to a
more complete conversion of the cholesterol into the bile salts in the
former case when the bile is unusually poor in bile salts, than happens
normally when there are bile salts being reabsorbed from the intestine.
The presence of cholesterol in fistula bile in considerable quantities is
evidence that certainly much of the cholesterol is secreted by the liver
itself. It is, on the other hand, probable that cells of epithelium escaping
into the bile may become impregnated with cholesterol and serve as the
nucleus for the formation of a stone. Bile of the hippopotamus con-
tains no cholesterol. It would be interesting to know whether the blood
of the hippopotamus contains cholesterol. Perhaps some African
physiologist may make some interesting discoveries by studying the bile
and blood of this animal.

The functions of cholesterol have already been discussed. It has
no function in digestion so far as we know. It is a singular fact that
bile hastens the action of the lipase of the pancreas, whereas the choles-
terol it contains is said to have the power of inhibiting lipolysis. There
is a curious apparent contradiction in these statements. May the liver
by excreting too much cholesterol reduce the power of the blood to with-
stand hemolysis, since cholesterol counteracts hemolysis? Does the liver
make anything more out of cholesterol than the bile salts, if this is the
origin of these substances? Are there any active oxidation products of
the nature of bufonin made here in small quantities and are they active
in the physiology of other parts of the body? These are some of the
questions which must be left to the future for answers.

Whether the cholesterol secreted in bile is reabsorbed with the bile
salts is not yet clear, since the metabolism of cholesterol has not yet been
worked out. Certainly some of that taken in the food, and presumably
some of that found in bile, is reabsorbed. But to what extent it is re-
absorbed is uncertain ; and whether the cholesterol of the body is formed
in the body or absorbed from the food eaten is equally uncertain.
Inasmuch, however, as the sterol of herbivorous animals is cholesterol
and not phytosterol, the sterol of plants, and since cholesterol is found
in all cells, it is probable that the animal body has the power of making
its own cholesterol. The blood of the portal vein contains more choles-
terol than that of the hepatic vein. In the feces of the new-born, the
meconium, cholesterol is found in large quantities, but in ordinary feces
of men no cholesterol is found, but in place of it a reduced cholesterol
called stercorin, by its discoverer. Flint; (from Latin stercus, dung) ;
or coprosterin (Gr. koprosteros, feces). During starvation no stercorin,
but only cholesterol, is to be found in the feces. Stercorin is evidently
formed from cholesterol by the reducing action of putrefaction.
DIGESTION IN THE INTESTINE 435

Stercorin. This is obtained from the dried and powdered feces by
extraction with ether; the ethereal extract is decolorized with charcoal
and evaporated. The residue is extracted with hot alcohol, the alcohol
extract saponified with KOH, mixed with powdered salt and evaporated.
The solid mass is extracted with ether, the ether washed with water until
neutral in reaction, then the ethereal extract filtered and dried. The
residue is extracted with boiling alcohol and on cooling stercorin crystal-
lizes out in long fine needles radiating from centers.

Stercorin (Coprosterin) is soluble in CHCl., and the solution gives
with concentrated H,SO, at first a yellow color, which, by standing,
changes to an orange and then a dark red. In the Liebermann test it
gives at once a blue color, changing to a green. The most probable
formula is C,,H,,0. Unlike cholesterol it does not take up bromine.
The specific rotation (a@)p—= + 24°.

In the feces of horses, cows, sheep and rabbits there is a hippo-
coprosterol, or hippostercorin, C,,H,,0, which is the phytosterol of
grass, which has passed through the intestine unchanged. Gardner and
Dorée have proposed to call it, therefore, chortosterol (Gr. chortos,
grass). This substance melts at 78.5-79.5°. It is optically inactive and
gives no cholesterol color reactions. It is stated by Bondzynski that dog’s
feces do not reduce cholesterol to stercorin.

The following experiment on the influence of diet on the excretion
of stercorin was tried by Bondzynski and Humnicki. In five days on a
normal diet the weight of stercorin in the feces was 4.30 grams. The
next five days one gram of cholesterol was daily added to the food,
making 5.0324 grams in all. In these five days there were excreted 5.835
grams of stercorin, and in the following five, when no cholesterol was
added to the dict, 7.3694 grams, making a total increase of 4.629 grams.
Of cholesterol they found only 0.5326 gram. In the following two days
1.4071 grams stercorin and in the second period of 1 gram cholesterol per
day, for five days, 6.2812 grams stercorin were obtained. So that in
one case only -10 per cent. and in the other only 3 per cent. of the
cholesterol taken were refound in the feces, the rest having been trans-
formed into stercorin. In human bile cholesterol has been found to be
between .06-.6 per cent. It may be said then, roughly speaking, that
about one gram a day of cholesterol may be discharged into the in-
testine from the bile. The feces contain, on the average, about 0.9 gram
of stercorin per day.

Phospholipins in bile—Bile contains also considerable quantities
of lecithin and other phospholipins. In human and dog bile, the lecithin
computed from the phosphorus is generally given as from 1-7 per cent.
of the alcohol-soluble substances, but Hammarsten’s investigations of
human bile show that the amount of phospholipin is much greater than
436 PHYSIOLOGICAL CHEMISTRY

this and is indeed as high as that of the polar bear. The alcohol-soluble
part of the bile contained 1.047 per cent. P, which calculated as lecithin
would be 29.75 per cent. In the bile of the polar bear 23.5 per cent. of
the alcohol-soluble solids were calculated as lecithin. Thudichum states
that thene is no lecithin in ox bile, but in place of it another phospholipin.
in which the relation of P: N is as 1:4. Lecithin is certainly present in
polar bear bile, according to Hammarsten, because a phospholipin was
obtained in which P:N as 1:1 and from which stearic, oleic acids.
glycerol and choline were isolated.

The amount of phosphorus in the alcohol-soluble solids of the bile
of different animals and the amount of lecithin, computing the latter
from the phosphorus on the supposition that all phospholipin is lecithin
and contains oleic, palmitic or stearic acids and so 3.94 per cent. of P,
is given in the following table: *

THe AMOUNT OF LECITHIN IN THE ALCOHOL-SotuBLE SoLtips or Various BILEs.

(Hammarsten. )
P— Lecithin—
per cent. per cent.
Polar bear csewcsrsewsierasamagen segs 0.911 - 1.14 23.12 - 28.96
Man (bladder bile) ...............4.. 0.048 - 1.17 1.33 - 29.75
Man (fistula bile) ...............245- 0.100 - 0.611 2.54 - 15.5
DOB ce so sinsAd ie tithes Beer nae eatin ee -768 19.50
TeAnd-FEAE hack vcthantesd amarhed ee aaweees -502 12.74
aoe UtANY 2.0... eee cece ence ees 420 10.67
DPA 252c2 nae a, Been wiausoand Sfouewsgeseiie, BladeueusasseNts 334 8.47
EVN ODIG | piheehcor ator deaiietal enavedanansvonecanevonsasl vous 332 8.43
Sheep ...... yeild, «fetpansiiia esaseseause 2 davenais 289 7.35
MuskiOxX® weceveccoraictereis ie iuvinens et nasente eraces 272 7.04
Hippopotamus ............0..-006 00 191 4.86
OR” powmaidoneimee Sawaal ES 181 4.60
Seal (Phoea- Gri) sscucsycaeeiess eenayes .168 4.27
Goose: sesxiyeaescsen awd cesses -162 4.10
Walrus: .c¢-vemesscuseue oi sacar oecemos -043 1.08
Séa-Woll . cus ncetca wees rece Vse.8 . 033 0.81
TRC OCKS ica 5c es sconced: dgdetuiicaiey <8 dobcacsvtud oasaene Not determinable

There are probably other phosphatides (phospholipins) in the bile
than lecithin, since one phospholipin had N:P as 8:5 and another
N:P::2:I. A similar phosphatide was obtained from brain by Thu-
dichum and named sphingomyelin.

Nothing is certainly known concerning either the function, origin or
fate of this bile phospholipin. Possibly it is derived, like the cholesterol,
from the red blood corpuscles and it no doubt contributes toward giving
bile its power of dissolving fats and cholesterol. Presumably it is split
and. digested in the intestine by the lipase of the intestinal secretions.
The lecithin content is highest in the polar bear bile and this animal
feeds on seals, which contain large quantities of fat. The great varia-

* Hammarsten. Hreebnisse der Physiologie, 4, 1905, p. 15.
DIGESTION IN THE INTESTINE 437

tiou in the biles of human beings would perhaps point to some variation
in the diet as a cause. The phospholipins of the bile are combined, it
appears, in part at least. with the bile salts, since they are precipitated in
part with them by ether from the alcohol solution, and in part they remain
in the ether and hold some of the bile salts in solution in a liquid in
which, when pure, they are insoluble. It may be, however, we are deal-
ing here with different phosphatides, one of which, like cephalin, may
be insoluble in ether; the other, like lecithin, soluble. The fact that
some of the phospholipin is precipitated with the bile salts by ether
and that some of the bile salts remain in solution with the lecithin makes
all the older analyses of the bile which did not take account of this fact
- unreliable.

Mucin.—Bladder bile is viscid, or slimy, due to its containing a
mucin-like substance secreted by the walls of the gall bladder. Most
of this mucin-like substance is not a true mucin, but a phosphoprotein
(nucleo-albumin), which yields no sugar on hydrolysis and contains
much more nitrogen (16.14 per cent.) than a true mucin. A small
amount of true mucin may, however, be present in human bile, but
according to Hammarsten this is secreted by the gall ducts and is cer-
tainly present in small amounts. This so-called slime or mucin can be
precipitated by alcohol and it yields ordinarily a small amount of a
reducing sugar on hydrolysis. Since many substances besides mucin,
such as phosphatides, glycogen, dextrin, etc., have this same property.
the appearance of a reducing substance under these circumstances is no
proof that mucin is present. Wahlgren has obtained a secretion from
the human gall bladder free from bile and finds it generally colorless
and containing a nucleo-albumin, a little globulin and albumin. The
presence of a phosphoprotein is. therefore, undoubted, but whether true
mucin is present or not cannot be stated with certainty. No function
has been found for the mucin-like bodies in the bile.

BACTERIAL DECOMPOSITION OF FOODS IN THE
INTESTINE.

Both the unabsorbed food and the digestive juices are subjected in
the intestine, and particularly in the large intestine, to the decomposing
action of myriads of bacteria and the products thus formed are of the
greatest importance to the organism. Many of them are toxic, produc-
ing headaches. drowsiness. or, at times. irritability. causing depression
and a general feeling of malaise. By the destruction of red blood cor-
puscles they are one of the factors in anemia. That these products pre-
dispose, also, to various infections. particularly of the skin. but of other
438 PHYSIOLOGICAL CHEMISTRY

parts of the body as well, there can be no doubt. The study of these
decomposition products and the discovery of methods for limiting them
becomes, therefore, of great importance in hygiene. The toxic substances
are derived in large measure from the proteins and are formed by
putrefactive processes. The carbohydrates are decomposed by fermenta-
tion. But there is no essential difference in kind between fermentation
and putrefaction, although, strictly speaking, a fermentation should in-
volve the liberation of a gas.

The bacteria are found all the way from close idliew the pylorus
to the rectum in constantly increasing numbers, but the numbers in the
small intestine are small compared to the large. It is in the large in-
testine, the ascending, transverse and descending colon in which the
intestinal contents, not reabsorbed, are stored for some time that the
main putrefaction occurs. The chyme leaving the stomach is often
sterile, bu‘ as acidity is neutralized a constantly increasing flora is found.
The contents of the small intestine are sometimes weakly acid throughout
and this reaction is unfavorable for the bacteria. The intestinal con-
tents even at the junction of the colon with the ileum are not at all
fecal-like’ They are generally slightly acid in reaction, semifluid in
consistence, and they are nothing more than the undigested remnants of
the foods, cellulose, some starch, some meat fibers, seeds, particles of peas
not fully digested and also, in considerable part, the unreabsorbed secre-
tions of the alimentary canal. It sometimes happens that an artificial
fistula must be made at the end of the small intestine because of
tumor of the large intestine, and by this means knowledge has been
obtained of the character of the material going into the large intestine
and the time it takes to pass through the intestine. It has been observed
that the discharge in such cases is not of a fecal nature. The time re-
quired for the food to traverse the stomach and the small intestine to
such a fistula naturally varies somewhat with the character of the meal
and the idiosyncrasy of the patient, but in round numbers it may be said
that the first discharge from the end of the ileum takes place about
four hours after eating and the discharge persists for about two hours.
The amount discharged is small compared. to the bulk of the food eaten;
practically all water is absorbed and 90 per cent. of the solids of the
foods.

Formation of the feces.—The transformation of the undigested rem-
nants of the food and the secretions of the intestine not reabsorbed inte
the typical dark brown feces occurs in the large intestine, where during
a period varying from ten hours to two days the remnants of the foods
undergo putrefactive changes and fermentative decompositions pro-
duced by myriads of bacteria. Since the absorbing powers of the large
intestine are very great, these products are reabsorbed and, coursing
DIGESTION IN THE INTESTINE 439

throughout the body in the blood, produce in it changes already men-
tioned, finally finding their exit in the urine, the perspiration or in the
breath, which when this decomposition is unusually abundant may have
a very marked fecal odor.

The number of bacteria in the feces is almost incredibly large.
Something between one-half and a fourth of the dry matter of the feces
consists of the bodies of bacteria. In a milligram of feces there are
found by culture methods of living bacteria in normal men on a some-
what restricted diet 26,000,000 bacteria. There are in addition large
numbers of dead bacterial cells, which can be computed by centrifuging
an emulsion of feces so that the coarser fragments are thrown out and
only ‘the bacteria left in suspension, and then counting under the
microscope the number of bacteria in a given volume. By this means the
total number of living and dead bacteria excreted in the feces per day
by healthy young men, on a restricted diet in a metabolism experiment,
was found to be on the average 500101,

The weight of dried feces passed per day is on the average in such
conditions 15-25 grams, and of moist feces 80-120; but the amount may
be much larger, particularly on a diet containing a good deal of in-
digestible substance such as cellulose. Of the 15-25 grams of dry matter
4-5 grams consist of bacterial bodies. Of the total nitrogen in the feces,
which may be on the average about 1.5 grams, one-half is bacterial
nitrogen.

The foregoing figures will give an idea of the enormous numbers of
bacteria in the feces and will make one appreciate the probable
very great importance of the products of their metabolism in human
life.

The kind of bacteria which are present is, of course, of even greater
importance than their numbers, since the character of the putrefactive
and fermentative products formed is dependent on the species of
bacteria. Ordinarily many different kinds of bacteria are found and
the character of the flora varies from time to time. The greater number
-of the bacteria are bacilli of the type of the bacillus coli communis, but
there are many races of this organism, some being much more toxic than
others. To this same group of organisms belong the paratyphoid and
the dysentery organisms and the connection between them and patho-
genic forms, such as the typhoid bacillus, is still uncertain. The bacillus
coli communis is a facultative anaérobe, that is it can grow both in the
presence and in the absence of oxygen. It ferments glucose and lactose,
forming lactic acid, alcohol and carbon dioxide; in anaérobic conditions,
in the absence of sufficient carbohydrate, it forms from the proteins
scatole and indole, the substances in the feces which produce the typical
odor; and it will split off hydrogen sulphide from cysteine and cystine.
440 PHYSIOLOGICAL CHEMISTRY

The study of the bacterial flora and in particular the careful study of
its variation in health and disease and the different strains of the colon
bacillus from different individuals is a matter of the greatest hygienic
importance, in which as yet hardly more than a beginning has been
made.

Bacterial decomposition of the carbohydrates. The bacteria form
from the unreabsorbed carbohydrates and cellulose, carbon dioxide,
butyric, lactic and other acids, alcohol, hydrogen, methane and other
substances. Of these hydrogen, methane and carbon dioxide form a
large part of the gases of the intestine. An analysis of human intes-
tinal gases has shown the following composition in 100 volumes
(Ruge) : :

Vegetable diet .......... CO,, 21-34; H,, 15-4 ; CH,, 44-55; N,, 10-19
Meat diet ............. CO,, 8-13; H,, 0.7-3 ; CH,, 26-37; N), 45-64
Milk diet ............0. CO,, 9-16; H,, 43-54; CH,, 0.9 ; N), 36-38

Lactic acid and alcohol are reabsorbed and burned by the body. That
still other substances are produced from the carbohydrates is undoubted,
but their nature and action are unknown. None of the products of carbo-
hydrate bacterial decomposition is harmful so far as known, although
the gas formation may be distressing.

Bacterial decomposition of the fats. The fermentation of the un-
reabsorbed fats and fatty acids seems to have been but little studied. The
author has not found any data on the subject.

Bacterial decomposition of the protems. It is, however, from the
putrefactive decomposition of the proteins that the most toxic sub-
stances are produced. The amino-acids of the proteins set free by the
intestinal enzymes are physiologically quite inert. They are absorbed
and serve as foods. If they are introduced directly into the blood, they
cause no physiological reaction whatever. The bacteria, however, like
the cells of the body, have the power of tearing these amino-acids to
picces and some of the products are very toxic.

In the first place, it is certain that they have the power of deamidizing
the amino-acids, setting free ammonia and leaving the fatty acid. It is
not yet certain whether in the intestine this process involves the oxida-
tion of the amino-acid to the ketonic acid first, as is the case in the
oxidation of the amino-acids in the tissues of the body (see page 810), or
whether the deamidization may be brought about by a hydrolysis. In
any case, if the oxy-acids are thus formed, they are subsequently re-
duced so that from the amino-acids the fatty acids are produced. This
may be illustrated by the decomposition of tryosine or phenyl] alanine,
which has been most carefully studied:
DIGESTION IN THE INTESTINE 441

C.0H Pe Ze a ye
G Y
AC a Nox He ‘oH HC” Nx ' HC ‘SH He CH
| | b fe (tl ae b de
ee CH HC CH a CH ne , H <
” \S
S "4 SS % 4 \ c WW
| —- | — — —
CH, 2 ra CH,
dann N CH, COOH
| |
COOH COOH
Tyrosine. p-oxy phenyl- p-oxyphenyl Para-cresol. Phenol.
propionic acid. acetic acid.
The putrefaction of tryptophane probably is preceded by a deamini-
zation :
CH CH
a a
HC ( ‘y—c_cH, ,cHNH,.COOH HC 7%: ¢-CH,,.CH, COOH
re ee — WW
Be Cc CH ne
\S
Naf fa Na fa
Tryptophane. Indole propionic acid.
CH oe CH
x:
a ‘oy —c—cH, coon HC” \No—oH HO” No c—cn,
| | — ! | ie "L I |
« Cc CH Cit Cc a ) CH
Not Wa No oa Not Wi
Indole-acetie acid. Indole. Scatole.

A very important change is the splitting off of carbon dioxide by the
action of so-called carboxylase bacteria. This may happen either be-
fore or after deaminization. If it happen before deaminization, amines
of a highly toxic character are produced. Such amines produced by
bacteria from amino-acids are called ptomaines by Gautier. As this
is a process of great importance to the body we may examine it a little
more at length. Thus from histidine there is formed imidazolylethyl
amine; from alanine, ethyl amine; from tyrosine, phenyl-ethyl amine;
from arginine, agmatine, or guanidine-butyl amine. Their formation is
illustrated in the following reactions: ‘

1, CH, .CH(NH,).COOH = co, + CH, CH, (NH,)

Alanine. Ethyl “amine.
2, C H,(OH).CH,.CH(NH, ).COOH = CO, + OH, (OH) .CH,.CH, ‘NH,
“Tyrosine. Tyramine
3. CNH, .CH,.CH (NH,).COOH = co, +C,H.N,. CH,.CH, NH,
"Histidine. *Tmidazolylethyl amine. (Histamine:
4. NH 50 (NH)—NH.CH,. (CH,), .CHNH,.COOH =
” Arginine. oe

co, +NH, .C(NH)-NH. CH,.(CH,),. CH,NH,
Agmatine.
442 PHYSIOLOGICAL CHEMISTRY

Such substances have been called by Kutscher, aporrhegmas (Gr.
apo, from; rhegma, a fracture). Such aporrhegmas are the active
principles of ergot. These substances have, in many instances, a power-
ful effect on the blood pressure and may be of great importance in the
production of high blood pressure and the resulting thickening of the
artery walls. They are very common in the metabolic products of plants
and they are formed also in the metabolism of animals, since the power
of splitting carbon dioxide from the amino-acids appears to be very
general. Imidazolylethyl amine, or histamine, produces vasodilation,
lowers the coagulability of the blood, and has been isolated from Witte’s
peptone and from the mucosa of the intestine. After the injection of
considerable doses of histamine fully half the plasma of the blood escapes
into the tissues, producing a great fall of blood pressure, and great
increase of viscosity. Adrenaline, the active principle of the supra-renal
glands, is a substance of this nature, being a methylated ethyl amine
derivative of the following composition :

C,H, (OH) ,CH (OH) -CH,.NH(CH,)

It can hardly be doubted that similar amines are produced from the
other amino-acids, such as tryptophane, which gives rise to indole ethyl
amine. LHither histamine or its mother substance seems to be formed
in every tissue of the body.

APORRHEGMAS.* ALL THE FRAGMENTS | OF THE AMINO-ACIDS WHICH ARE FORMED
IN A PHYSIOLOGICAL Way DuBING THE LIFE oF PLANTS AND ANIMALS.

Amino-acid Aporrhegma Methylated aporrhegma veuyee
Histidine Imidazolylethyl amine (Histamine)
Arginine Ornithine: agmatine; tetra- Tetramethylputrescine

methylenediamine; 6-aminoval-
erianic acid

Lysine Pentamethylenediamine
(Cadaverine) .
Glutamie acid y-aminobutyric acid (y-Butyro-betaine ( y-trimethyl
a-oxy-beta- amino butyric}

Asparticacid -alanine butyro-betaine)

Glycocoll Methyl amine Sarcosine.
Betaine

Alanine Ethyl amine

Leucine Isoamylamine

Proline Pyrrolidine N-methyl pyrrolidine  Stachydrine

Pheny] alanine Pheny]-ethyl amine

Tyrosine p-oxyphenylethylamine (Tyramine) Hordenine Surinamine

Tryptophane Indole; scatole; Hypaphorine

indolethylamine (trimethyl

tryptophane
betaine. )

*Kutscher and Ackermann: Zeit. f. physiol. Chem., 69, 1910, p. 265.
j It is better to limit the term to the nitrogen-containing products. A. P. M.

The decomposition of cysteine has not been fully worked out, but from
the presence of mercaptans, such as methyl and ethyl mercaptans,
CH,.CH,SH, in the feces, it is probable that there is a preliminary de-
DIGESTION IN THE INTESTINE 443

carboxylation, with the formation of a thio-ethyl amine as an interme-
diate product:
SH.CH,.CHNH,, COOH ——~ co, + SH.CH,. CH,NH, _ SH.CH,.CH, +NH,

Cysteine. Thioethyl amine, or Ethyl mercaptan.
amino-ethy] mercaptan.

If the splitting off of the carbon dioxide occurs after the deamidiza-
tion an amine cannot, of course, be formed, but the next lower
carboxylic acid is produced by way of the aldehyde. Thus from tyrosine
there may first be formed phenyl-pyruvie acid, which may be reduced
to phenyl-lactic acid, reabsorbed and re-excreted in the urine; or the
phenyl-pyruvic acid may be split into phenyl acetaldehyde and carbon
dioxide, and the former be oxidized into phenyl-acetic acid, which is
excreted in the urine. Phenyl-acetic acid is the mother substance of
homogentisie acid and other aromatic compounds of the urine. Phenol
or cresol may be set free.

These deamidized compounds have not as yet been shown to have
any physiological action. But from tryptophane by a similar decom-
position indole and scatole are set free and these bodies are toxic and
give part of the bad odor to the feces. The formation of indole and
scatole is shown in the reactions just given, although the intermediate
products have not been thoroughly worked out.

By the decomposition of cysteine and cystine hydrogen sulphide is
formed. This is reabsorbed readily and produces headaches and depres-
sion even when reabsorbed in small quantities and it is presumably one
of the factors causing solution of the red blood corpuscles and so the
anemia seen in those having chronic constipation. Mercaptans, that is
ethyl or methyl sulphide, may also be formed and these are véry ill-
smelling compounds.

From the conjugated eatains the decomposition of the prosthetic
group, such, for example, as that of the nuclein bases, may give rise to
other products of importance to the body. But this matter has not yet
been sufficiently investigated to permit of any definite statements being
made.

It has been suggested that substances having the property of raising
blood pressure may be produeed from tyrosine in putrefaction and that
these substances are active in causing arteriosclerosis and the ills which
follow from this. Since a man is said to be as old as his arteries it
would appear possible that intestinal putrefaction may be a factor in the
production of the decrepitude of old age, as Metchnikoff has suggested.
To what extent putrefaction produces premature decrepitude cannot be
stated without more investigation.

There can, however, be no question of the importance of these putrs.
factive substances in the general well-being of the individual. We are
all constantly exposed to food poisonings, which, when slight, are gen-
444 PHYSIOLOGICAL CHEMISTRY

erally overlooked as the true cause of inefficiency, depression, sluggish
mental processes, dissatisfaction or abnormal irritability. Particularly
chicken, veal and pork are liable to contamination from some person
handling these meats in the kitchen, who may carry a peculiar rave of
bacteria. Such food poisonings may come from infected meat-choppers,
so that hashed meat is more apt to be a scurce of trouble than unhashed.
If the bacteria are not killed in the subsequent cooking, and the hash
is kept, as it is in restaurants, it may give rise to symptoms of food
poisoning more or less marked. These symptoms generally come on in
1-5 hours after a meal, if they are due to ptomaines or toxic substances
already formed in the meat before eating; but the bacteria themselves
may develop in the intestine and form toxic substances. In such cases the
‘onset of the symptoms is delayed coming 12-48 hours after the meal, vary-
ing with different individuals. If the symptoms are not well marked so
as to lead to cramps, prostration and diarrhea, by which the bacteria are
swept out of the system, «nly feelings of drowsiness, headache, migraine,
lassitude or depression often preceded by mental or sexual excitement
are produced, and the real cause of the trouble is overlooked. The symp-
toms are often extremely baffling and are referred to the nervous
system, the ductless glands or any other rather than the true cause.
Moreover, when once such a bacterium is lodged in the canal it may
persist for a long time and the effects of a single food poisoning in an
experiment on human metabolism were found to be clearly perceptible
for a month or longer. These facts make it desirable, in case of head-
ache, drowsiness or depression, even though there be no symptoms of
deranged digestion or constipation, to try the effects of a good purge.
The effect on-the mentality is often remarkably prompt.

The putrefactive processes in the intestine have also a remarkable
relation to the skin. In nearly all cases of: excessive intestinal putre-
faction the skin, and perhaps the whole organism, has its vital resist-
ance lowered. Pimples, pustules, acnes and boils are constantly forming.
The spotted skin is generally, although not always, a sign of intestinal
putrefaction. This condition is generally referred by the laity to im-
pure blood; and to cure it sulphur, burdock root and other local reme-
dies are recommended as blood purifiers. - Without exception all these
blood purifiers, so called, are intestinal purgatives and their therapeutic
action is due, in large measure at least, to their power of sweeping the
intestine clean. Cold sores on the lips often have a similar origin. The
increased sensitiveness of the skin to infection of the hair follicles is
shown also in many cases of eczema, which may greatly improve if the
diet is limited or if fasting is practised. Many hidden or chronic slight
inflammations are often stimulated to become acute, or more active, by
intestinal putrefaction. Thus colds may develop, catarrh become worse,
chronic nephritis of a mild type become acute, erythemas develop in
DIGESTION IN THE INTESTINE 445

various parts of the body or even old healed wounds with bad circulation
may inflame and break out afresh from this cause; and by attention
to diet, or by the use of good purges, these results may be avoided.

The question is still unsolved whether besides their evil products the
bacteria of the colon are also producers of some essential products. Bac-
teria are certainly not necessary for development, since the alimentary
canal of the new-born is sterile. Chickens have been hatched and raised
under sterile conditions; and guinea pigs removed by Cesarean section
have been found to grow and utilize their food when fed on sterile
diets under aseptic conditions. As yet these bacteria have not been
found to do good; but they certainly cause much evil. And while it
will not do to condemn them completely without farther investigation,
it would appear at the present time the part of wisdom to reduce putre-
faction in the intestine as far as possible.

Methods of reducing putrefaction. The easiest method of reducing
putrefaction is by a strict limitation of the diet, a reduction in the
proportion of meat and a more thorough mastication of that which is
eaten. By the careful use of these two precautions Mr. Horace Fletcher
and others who have adopted his methods have been able to so reduce
putrefaction in the intestine that the feces are entirely without odor.
The general condition of these men has been greatly improved. Men
and women of sedentary habits are very apt, particularly after the
age of forty, to eat too much meat. The exact quantity and the par-
ticular quality of food most conducive to happiness, health, efficiency
and retention of vigor has not yet been determined ; but two facts have
been found, namely, that the retention of vigor late in life, a green old
age, generally goes with an abstemious diet; and in the second place,
many have been greatly improved by a strong reduction in the amount
of food eaten and in particular by a reduction in the amount of protein
food. We cannot do better than to follow the wise advice of Don Quixote
to his squire, Sancho Panza, as the latter was about to depart for the
Island. ‘‘ Eat little at dinner and less at supper; for the health of the
whole body is tempered in the laboratory of the stomach.’’

Another method of reducing putrefaction is by eating carbohydrate
food rather than protein, for the bacteria will not produce these putre-
factive products from the proteins as long as there is carbohydrate food
at their disposal. This peculiarity of the bacteria, which has been
studied by Kendall, we shall have to return to under the chapter: on
metabolism, since it illustrates a general property of all living matter,
namely, that the carbohydrates spare the proteins, and it is only in the
absence of the carbohydrates that the proteins are attacked and torn to
pieces to supply energy. By carbohydrate fermentation, also, acidity is
increased and this also has a part in checking bacterial putrefaction,
446 PHYSIOLOGICAL CHEMISTRY

since many bacteria cannot metabolize in the presence of acid. But
the main action of the carbohydrate is that just stated, that as long as it
is present it is utilized as a source of energy and the protein only to
build the protoplasmic machine.

Summary of digestive changes.—The chief changes undergone by
the foods in the intestinal tract by which they are made available to
the body may be briefly summarized as follows:

The proteins by the action of pepsin hydrochloric acid of the gastric
juice, by the intestinal and pancreatic juices containing enterokinase,
trypsin and erepsin, are resolved into the amino-acids of which they
are composed. The compound proteins, such as the nucleoproteins, are
digested in part by the proteolytic enzymes and in part by the nucleases
of the pancreatic juice, with the formation of phosphoric acid, nuclein
bases, pyrimidin bases,.and presumably carbohydrate, from the nucleic
acid. Hematin is split off from hemoglobin. By the action of the bac-
teria some of these amino-acids are further decomposed, losing ammonia
and being converted into fatty acids, or by decarboxylization being made
into amines, some of which have a very powerful action on the blood
pressure and other functions of the body. Other ill-smelling and harm-
ful substances are produced, such as some of the mercaptans, sulphur-
eted hydrogen, indole and scatole.

The carbohydrates taken as sugars and starches are digested by the
ptyalin of the saliva, the amylopsin, maltase and lactase of the pancreas,
and the invertin of the intestinal juice so that they are all reduced to
the state of monosaccharides. Some of them are further broken up by
the bacteria, with the formation of lactic acid, alcohol, marsh gas, hydro-
gen and butyric acid.

The fats acted upon by the lipase of the stomach and that of the
pancreas and intestine are converted into fatty acids and glycerol, and
by means of the bile salts are carried into the intestinal epithelial cells.

In short, the main object of the whole process of digestion appears to
be to resolve the various food substances into those common building
stones, amino-acids, monosaccharides and fatty acids, which are the com-
mon basis of all proteins, carbohydrates and fats. Thus each organism
ean use these building stones in the proportion and order it needs to
construct its own proteins and organized matter, which in each organism
has an architecture as distinct and characteristic as the form of the or-
ganism itself.

Finally by this decomposition into building stones but very little
energy has been lost, and only a very small amount of heat set free.
The advantage of this to the organism is obvious, for it means not only
that energy has not been lost, but that very little energy will be required
to reconstruct the tissues of the body.
DIGESTION IN THE INTESTINE 447

We may now pass on to the consideration of the changes undergone

by these building stones during absorption, and during their stay in the
blood and the tissues.

f.

oe

18.
19.

20.

REFERENCES. S&cReErTIon or BILE.

Human biliary fistula. Pfaff and Balch: Jour. of Expt. Medicine, 2, p. 49,
1897. :

In fasting. Albertoni: Arch. Ital. de Biol. 20, p. 134, 1890.

In anemia. Korentschewsky: Arch. des scien. Liol. d. St. Petersburg, 16, p.
252, 1911. See also p. 490.

CoMPOSITION OF BILE

Reaction. Quaglianello: Atti R. Acad. dei Lincei, Roma, 20 (11), p. 312, 1911.

Osmotic pressure. Dreser: Archiv f. expt. Path. u. Pharm., 29, 1892. Bon-
nani: Bioch. Centrlblt., 1903.

Toxicity. Bunting and Brown: Jour. of expt. Med. 14, 1912, p. 444.

Iron in bile. Beecari: Archives Ital. de Biol., 28, 1897. p. 206.

Influence of cystine ingestion on taurocholic acid. v. Bergmann: Hof-
meister’s Beitriige 4, 1904, p. 192.

Cholesterol in bile. Doyon and Dufourt: Archives de Physiol. (5) 8, p. 587,
1896.

Amount in human bile. Pierce: Archiv f. klin. Med. 106, 1912.

Conjugated sulphates in bile. Hammarsten: Zeits. f. physiol. Chem. 24, p.
322, 1898.

Stercorin or coprosterol. Flint: Amer. J. of Med. Sci. (N.S.) 44, 1862, p.
306 and following pages. Zeits. f. physiol.. Chem. 23, p. 363, 1897.
Bondzynski and Humnicki: Zeits. f. physiol. Chem. 22, 1896, p. 396.

Chortosterol. Dorée and Gardner: Proceed. Royal Soc. B, 80, pp. 212-26, 1908.

Phosphatides in bile. Hammarsten: Zcits. f. physiol. Chem. 36, 1902. p. 328.

Bile of hippopotamus. Hammarsten: Zeits. f. physiol. Chem. 74, 1911, p. 123,

General review. Article Bile. Dictionnaire de Physiologie, Richet, 2, p. 144,
1898. Hammarsten: Ergebnisse der Physiologie, 1905.

Preparation of unconjugated acids of ox bile. Schryver: Jour. of Physiol.,
44, p. 265, 1912.

Taurocholeic acid of ox bile. Gullbring: Zeit. f. physiol. Chem., 45, p. 448,
1905.

Glycocholeic acid of ox bile. Wahlgren: Zeit. f. physiol. Chem., 36, p. 556,
1902.

Separation of taurocholate and glycocholate from ox bile. Tengstrém:
Zeit. f. physiol. Chem., 41, p. 210, 1904. Bleibtreu: Pfiliger’s Arch., 99.
Letsche: Zeit. f. physiol. Chem., 60, 1909, p. 462.

Choleic acid. Properties. Lassar-Cohn: Zeits. f. physiol. Chem., 17, 1893.
Latschinoff: Ber. d. d. chem. Gesell. 18. p. 3039, 1885. Fischer and Meyer:
Zeit. f. physiol. Chem., 76, pp. 95-8, 1912.

Acids of pig bile. Jolin: Zeits. f. physiol. Chem., 13, p. 205. 1889.

Cholic acid. Relation to cholesterol. Schrétter and Weiteenbéck: Monats-
hefte f. Chem., 29, pp. 395-8, 1908. Lifschiite: Ber. d. d. chem. Gesell. 47,
pp. 1459-60, 1914.

Cholic acid. Color reaction with HCl. Hammarsten: Zeits. f. physiol.
Chem., 61. pp. 495-8, 1910.
448

21.

22,

23.

24,

26.

27.

28.

29.

31.

32.
33.

34.

35.

36.

37.

PHYSIOLOGICAL CHEMISTRY

Phocataurocholic acid and walrus bile. Hammarsten: Zeits. f. physiol.
Chem. 61, p. 454, 1910.

Cholic acid. Iodine reaction. Burger and Field: Jour. Chem. Soc. 101, pp.
1394-1409, 1913.

Cholic acid. Composition. Structure. Pregl: Zeits. f. physiol. Chem. 65
p. 157, 1910.

Synthesis of taurocholic and glycocholic acid. Bondi and Miller: Zeits. f.
physiol. Chem., 47, 1906, p. 499.

Lowering of surface tension by bile salts. Billard et Dieulafé: C. R. de la
Soc. Biol. 54, p. 245, 1902.

Brite PIGMENTS.

Composition of bilirubin. Fischer and Rése: Zeit. f. physiol. Chem., 89, 262,
1914. Some earlier literature cited. Kiister: Zeits. f. physiol. Chem., 59,
1909, p. 63. Lit. before 1909 cited in this paper.

Bile pigments. Properties. Jolles: Arch. f. d. ges. Physiol., 75, p. 446, 1889.

Formation of biliverdin from bilirubin by Na,O,. Hongouneng and Doyon:
Arch. de Physiol. (5) 8, p. 525, 1896.

Absorption spectra of bile pigments. MacMunn: Jour. of Physiol., 6, p. 22,
1885.

Cholohematin, bilipurpurin and phylloerythrin. Marchlewski : Zeit. f. physiol.
Chem.. 45, p. 466, 1905.

Urobilin. Spectrum. Lewin and Stenger: Arch. f. d. ges. Physiol., 144, 1912,
p. 279.

Urobilin. Origin, Fromholt: Zeit. f. expt. Pathol., 20, 1911, p. 268.

Bile pigments in invertebrates. Molluscs, Schulz: Zeits. f. all. Physiol. 3,
1904, p. 91.

Origin in liver. Naunyn and Minkowski: Archiv f. expt. Path. u. Pharm., 21,
1886, p. 1. Fiessinger and Lyon-Caen: Jour. de Physiol. et Path. gen., 12,
p- 958, 1910. Weill: Arch. Internat. de physiol., 1913, 13. p. 181.

Urobilin. Urobilinogen. Fischer and Meyer-Betz: Zeit. f. physiol. Chem., 75,
1911, p. 232. Garrod and Hopkins: Jour. of Physiol., 20, 1896, p. 112; 22,
1898, p. 451. ,

Chlorophyll composition and relation to bile and blood pigments. Will-
stdtter: Jour. Amer. Chem. Soc., 37, p. 323, 1915.

Viscosity of bile. Burton-Opitz: Biochem. Bull., 3, p. 351, 1914.

INFLUENCE OF BILE ON DIGESTION AND ABSORPTION.

Influence of the bile on pancreatic digestion. Rachford: Jour. of Physiology,
12, p. 72, 1891. Ibid., Jour. of Physiol., 25, 1899, p. 165. Martin and Wil-
liams: Proceed. Roy. Soc., 48, p. 160 Tschermak: Cent. f. Physiol., 16,
p. 329, 1902. Bruno: Arch. des Sci. Biol., St. Petersburg. 7, 1899. v. Fiirth
and Schiite: Hofmeister’s Beitriige, 9, p. 28, 1907. (Earlier literature
cited here.) Hewlett: Effect of bile on ester splitting action of pancreatic
juice. Johns Hopkins U. Bull., 16, p. 166. 1906.

Effect of synthetic bile acids on pancreatic fat digestion. Magnus: Zeits. f.
physiol. Chem., 48, 1906, p. 376.

Influence on absorption. Pfliiger: Arch. f. d. ges. Physiol. 88, p. 431, 1901
Moore and Rockwood: Pro. Royal Society, 60, p. 439. Jour. Physiol., 21, p.
373, 1897.
_

We

8.

DIGESTION IN THE INTESTINE 449

Influence on peptic digestion. Schiff: Arch. f. d. ges. Physiol., 3, 1870, p. 200.
Ibid., p. 613.

Fat absorption. Bidder and Schmidt: Verdauungsifte, p. 223. Dastre: Arch.
de Physiol., 1890.

Reabsorption cf bile. Schiff: Arch. f. d. ges. Physiol., 3, p. 598, 1870.

PANCREAS.

’ General. Pawlow: Work of the Digestive Glands.

Discovery of function in digestion. Bernard: Mémoire sur le pancreas. Paris,
1856.

Composition of human pancreatic juice. Wohlgemuth: Biochem. Zeit., 39,
p- 321, 1912. (Other recent literature cited.)

Clinical method of obtaining pancreatic juice from the stomach. Balagrens
Archiv f. d. ges. Physiol., 121, 1907, p. 13; 140, 1911, p. 436. ~

Secretin. Discovery. Bayliss and Starling: Journal of Physiology, 29 and 31,
1902. Carlson et al.: Jour. Amer. Med. Assn., 66, p. 178, 1916. Bibli-
ography.

Discharge of HCl into duodenum causes secretion of pancreas. Popielski:
Cent. f. Physiol., 10, 1896, p. 405-9.

Secretion of pancreas due to both humoral and nerve action. Popielski:
Zeit. f. physiol. Chem., 71, 1911, p. 186. Bylina: Archiv f. d. ges. Physiol.,
143, 1911, p. 531. Popielski: Ibid., 121, 1907-8, p. 239. Mazurkiewicz:
Ibid., 121, 1907-8, p. 75. Good bibliography in Bylina’s paper, p. 565.

Secretin appears in blood when acid put in duodenum. Hnriquez et Hallion:
C. Ren. de la Soc. Biol., 55, 1903, p. 233.

Secretin causes bile secretion. v. Henry and Portier: C. Ren. de la Soc. de
Biol., 54, 1902, p. 620.

Secretin inactivated by pancreatic extracts. Lalon: Jour. de Physiol. et de
Path., 12, 1911, p. 343.

Secretory action of curarin. Czubalski: Archiv f. d. ges. Physiol., 133, 1910, p.
225.

Method of extraction of secretin from intestinal mucosa. Dale and Laidlaw:
Pro. Physiol. Soc. Jour. of Physiol., 44, 1912, p. xi.

Fate of secretin in pancreatic diabetes. Hvans: Jour. of Physiol., 4, 1912,
p. 461.

Existence in mesenteric ganglia. Delezenne and Frouin: C. Rend de la Soc.
Biol., 54, p. 896, 1902.

Enterokinase. Discovery. Chepowalnikow: Thesis, St. Petersburg, 1899.
Bayliss and Starling: Jour. of Physiol., 30, 1903, p. 61.

Supposed activation of trypsin by Ca salts. Hekma: Pro. Amsterdam Acad-
emy of Sciences, 18, (2), p. 1002. Delezenne: C. Rendu de la Soe. Biol.
1905, pp. 476, 523 and 612; 1906, p. 1070; 1907, p. 274. Zinz: Annal. de
ia Soe. Roy. des Sci. Med. et Nat. Bruxelles, 16, 1907.

Conversion of trypsinogen to trypsin. Mellanby and Wooley: Jour. of
Physiol., 45, p. 370-88, 1913.

Time of appearance in fetal life. Ibrahim: Biochem. Zeits., 22, pp. 24-32, 1909.

Erepsin of pancreatic juice. Schaeffer and Terroine: Jour. de Path. et.
Physiol., 12, 1910, pp. 884 and 905.

Amylase of pancreas. Composition and methed of obtaining. Sherman
and Schlesinger: Jour. Amer. Chem. Soc., 34, 1912, p. 1104.
450 PHYSIOLOGICAL CHEMISTRY

9. Lipase of pancreas. Influence of dialysis on lipolytic activity. Bierry and
Giaja: C. Ren. de la Soe. Biol., 62, p. 432, 1907. Slosse et Lambosch:
Biochem. Cen. 7, 1908, p. 510. Hanisek: Zeit. f. physiol. Chem., 71, 1911,
p. 239. Rosenheim and MacKenzie: Jour. of Physiol., 40, 1910, pp. viii, xii,
xiv.

10. Lipase action on various esters. Morel et Terroine: Jour. de Path. et
Physiol., 14, 1912, p. 72.

1l. Lipase sensitive to heat. Visco: Arch. Ital. de Biol., 54, 1911, p. 243.

12. Alkalinity of pancreatic juice. Foa:C. Ren. de la Soc. Biol., 59, 1905, p. 867.

13. Trypsin and trypsinogen. Mellanby and Wooley: J. of Physiol., 47, pp.
339-60, 1914. Vernon: J. of Physiol., 47, pp. 325-38, 1914.

14. Erepsin. Conditions of action. Rona and Arnheim ; Biochem. Zeits., 57, 1894,
p. 84.

15. Intestinal Lipase. Falk: J. Amer. Chem. Soc., 36, 1914, p. 1047.

16. Action of pepsin and trypsin on each other. Hdie: Biochem. Jour., 8, 1914,
p. 193.

17. Optimum concentration of hydrogen ions for liquefaction of gelatine by
trypsin. Palitesch and Walbwm: Biochem. Zeits., 47, p. 1, 1914.

18. Quantitative determination of trypsin. Wahlschmidt: Arch. f. d. ges.
Physiol., 143, pp. 189-229, 1911. Various methods are described.

19. Ferments of Pancreas. Mellanby and Woolley: Jour. Physiol. 49, p. 246,
1915

20. Pancreatic Insufficizncy. Spriggs and Leigh: Quart. J. of Med., 9, p. 11, 1915.

BACTERIA.

1. Fecal bacteria of healthy men. MacNeal et al.: Jour. Infectious Diseases, 6,
pp. 123-169, 571-609, 1909.

2. Method of estimation of fecal bacteria gravimetrically. Strasburger: Zeit.
klin. Med., 46, 413, 1902.

3. Method by microscopic counting. Winterberg: Arch. f. Hygiene. 59, p. 283,
1906. Klein: Centrlblt. f. Bakt. Abth. 1. Orig.. 27, p. 834, 1900.

4. General treatise on the feces. Schmidt and Strasburger: Die Faeces des
Menschen, Berlin, 1905.

5. Bacteria and chemistry of feces of healthy nen. Studies in Nutrition by
H. S. Grindley and W. J. MacNeal. Vols. 3, 4 and 5, University of
Illinois, 1912,

6. Histamine shock. Dale and Laidlaw, Jour. of Physiol., 52, p. 376, 1919.
CHAPTER XI.
ABSORPTION

Absorption.—The foods in the alimentary canal are not yet, strictly
speaking, in the body, since the cavity of the canal is in communication
with the external world. How do the digested foods get into the body? Is
it by a simple physical process of diffusion or osmosis occurring through
the mucous membrane of the intestine, between the blood on the one side
and the intestinal contents on the other? Or is it by the vital activity of
the epithelial cells? The foods move in as though there was an attractive
force exerted upon them by the hungry body cells. We do not believe
any such force exists, but how is their entrance to be explained? Does
anything happen to them during the process of absorption?

Absorption takes place throughout the intestine, which is a tube
some 27 feet long, without counting the six feet of large intestine. It
is slight in the stomach; most of the absorption is in the small intestine.
Only a small part of the food remains to be absorbed in the colon.
The food in the small intestine is spread out as a thin layer over the
surface of the gut. This surface is enlarged by the presence of finger-
like processes, the villi, which dip down into the cavity of the intestine.
Each villus is supplied with artery, capillaries and veins. It has in the
center a lymph space called a lacteal, opening into the chyle vessels of
the mesentery. Each villus has both longitudinal and circular muscular
fibers so that it can expand and contract, taking up food like a sponge
and being squeezed by the contraction. The mucous membrane, which
lies between the food and the lacteal, is thin, only about 0.012 mm. in
depth. It seems probable that a good deal of the absorption is due to
purely physical processes of diffusion or osmosis of the amino-acids and
monsaccharides from the intestine, where they are present in quantities,
into the blood in which they are present in very small amounts; but the
problem is not so simple as this. This membrane of epithelial cells,
although thin, is alive. The food matters must pass through the living
protoplasm that is constantly altering its state. Such alterations in
activity constantly change the rate of absorption; they change the
permeability of the cells. Now substances do not pass through mem-
branes by simply shooting through the holes. There is some kind of a
union between the solvent and the solute and between the solvent and
the membrane. Do the substances go through the cells because they dis-

solve in the water which penetrates the cell? Or because they unite with
451
452 PHYSIOLOGICAL CHEMISTRY

the organic basis of the cell? One thing is probable, namely, that the
activity of the cell in some way controls this process.

Absorption of fats——That absorption is not due, primarily at any
rate, to osmotic pressure is shown by the absorption of fat. Fat is en-
tirely hydrolyzed in the intestine. The fatty acids thus set free are held
in colloidal solution by the bile salts, probably by the union already dis-
eussed. The colloids have almost no motion of translation and very little
osmotic pressure and yet the absorption of fats takes place with speed.
The absorption is greatly aided by the bile. The bile salts are reabsorbed
with the fatty acids. Glycerol also passes into the cells. The glycerol
and fatty acid thus brought together in the living cell are resynthesized
into neutral fat, which can be seen scattered about in the cell. Just
how this resynthesis is produced it is at present impossible to say. Some
have referred it to the reverse action of the lipase of the intestinal
mucosa. But while it is true that lipase added to oleic acid and glycerol .
causes some resynthesis and this resynthesis is hastened by the bile, yet
when water is present, as it is in the cells, the lipase produces only a
very small amount of synthesis. Moreover lipase is not found precisely
in those cells in which the synthesis is going on most rapidly, as in the
cells of the mammary glands. All of these synthses are dependent on
a supply of oxygen and are inhibited by ether, which does not check
lipase activity. The resynthesis is hence, probably, one of the synthetic
powers of the cell correlated with cell respiration.

However the resynthesis of the neutral fats is brought about, it cer-
tainly occurs. The very finely emulsified fat is discharged in some way,
in the larger proportion (60 per cent. or more) into the lacteal from
which it is pressed along into the lymphatics of the mesentery in the
form of a milk-white emulsion called chyle, into the receptaculum chyli
and from here it passes by the thoracie duct, which lies just behind the
pleura, not far from the median line posteriorly, up to the left shoulder,
where it is poured into the blood at the junction of the jugular and sub-
clavian vein. The chyle is forced up this duct partly by the negative
pressure of the thorax, partly by the pressure due to inspiration on the
abdominal viscera and partly by the pressure of the new chyle or lymph
behind it, coming from the liver and other abdominal organs. Not all the
reabsorbed fat is to be found in the chyle thus discharged into the jugular
vein. A portion (some 40 per cent.) has disappeared, perhaps finding its
way into the blood and so to the liver; or has been catabolized during its
passage through the mucous epithelium; the fate of this remnant is not
yet clear. Why the fat should thus be passed into the blood by going
around the liver through the thoracic duct is not yet clear. But there is very
little doubt that some good reason exists for this peculiar arrangement.

The chyle thus poured into the blood gives to the blood serum or
plasma during the absorption of a fatty meal a milky appearance, and
ABSORPTION 453

the amount of the fat in the blood may be so increased that it will rise on
the plasma like so much cream. By the blood it is carried about the
body, each tissue taking what it needs and any excess being picked out
and stored in the fat depots of the body, the fat cells of the adipose tissue
of the mesentery, the panniculus adiposus, or about the internal organs.
How the blood fat finds its exit from the blood; how it gets through
the vascular wall, whether by being split into soaps and glycerine or as
neutral fat; and how it is accumulated in fat tissues, these are problems
not as yet solved. There is a lipase in the red blood corpuscles and
perhaps all the blood fat is rehydrolyzed before leaving the blood.

Absorption of carbohydrates.—What if anything happens to the
carbohydrates during their passage through the mucuous membrane is
unknown. They are nearly all absorbed in the jejunum and ileum.
They are believed to pass directly into the blood capillaries as levulose,
glucose or galactose. Sometimes maltose, lactose and cane sugar are
absorbed as such and can be detected in the blood and urine, since the
two last named sugars are not used if they enter the blood, but pass out
in the urine. If sugars are injected into the blood they are in part
excreted into the duodenum and presumably then reabsorbed. The
sugars thus absorbed are found in the portal blood to the extent of
0.2-0.4 per cent. They pass to the liver, where a portion may be taken
out and stored in that organ as glycogen; and in part they circulate to
the other organs of the body, each of which removes from the blood what
it needs. The sugar in the blood is generally glucose and it is in a form
which may be dialyzed out of the blood by vivi-diffusion. Page 470.
The ingestion of starch or sugar is always followed by a rise in the
blood sugar which in normal individuals reaches a maximum value of
about .15 per cent. about 2-3 hours after a meal.

If sugar in a concentrated solution is ingested it may cause vomiting,
and in any case it is diluted in the bowel by the passage outward of water
through the mucous epithelium. Ultimately, if the mucous membrane
remains in a living, active form the sugar and water are reabsorbed.

Absorption of proteins.—The proteins are absorbed probably for
the most part, if not altogether, in the small intestine in the form of
amino-acide. It is certain that in the cephalopods, in which the condi-
tions for studying this problem are very good, owing to the simple com-
position of the blood, they are absorbed in the form of amino-acids.
There is now good evidence that they are absorbed in that form in the
mammals also, since small amounts of the amino-acids are found in the
blood. (Page 472.) The quantity of non-protein and amino nitrogen
inereases in the blood during the absorption of the proteins, the increase
being particularly shown in the amino N of the red blood corpuscles
which rises 4-5 hours after a meal from 7-8 mgs. amino N per 100 ce. of
corpuscles, to 20 mgs. or more. The presence of erepsin in the wall of
454 PHYSIOLOGICAL CHEMISTRY

the intestine and the elaborate mechanism to secure the reduction of the
proteins to the form of amino-acids is strong a priori evidence that they
are absorbed in this form. This view is strengthened by the fact that the
albumoses, when brought directly into the blood stream, act like foreign
bodies, lowering the blood pressure and reducing the coagulability of the
blood, whereas the amino-acids are quite inert. There is no doubt that at
times complex polypeptides are reabsorbed, since some individuals are
found who have an idiosynerasy toward some one protein, reacting by
anaphylactic shock to the ingestion of this particular protein. Sometimes it
is the protein of milk, sometimes of egg or some other protein of the food.
This reaction is proof that at some time this protein in an unchanged
form secured entrance to the blood. The very fact that this reaction is
rare shows how unusual it is for the undigested protein to be reabsorbed.

There is no question that deamidization, that is the splitting off of
amnio groups, occurs to some extent during their passage through the
wall or during their bacterial decomposition, for the intestinal mucosa
contains more ammonia than any other tissue of the body with the ex-
ception of the stomach mucosa; and the blood of the mesenteric veins
coming from the intestine carries from 6-10 times as much ammonia as
any other blood in the body. A part of this ammonia is probably
derived from the amide nitrogen split off by tryptic digestion, but a
portion also comes, no doubt, from the amino groups. What proportion
of amino-acids are thus deamidized it is impossible to say, but presuma-
bly not more than a third and it may be less than this.

The amino-acids which get past the intestinal mucosa are found in
the blood rather than in the chyle. Their discovery here has been re-
cently made. They do not accumulate, for they are removed from the
blood by the tissues about as rapidly as they enter it. At any instant of
time there are, then, only minimal amounts present.

For a long time it was believed that the amino-acids were resynthe-
sized during their passage through the mucosa into some of the blood
proteins and probably into serum albumin. This was supposed to be the
food of the various tissues. This view, for which there never was any
convincing or even strong evidence, has now been rendered very un-
likely by the discovery of the constitution of the proteins and the
presence of amino-acids in the blood.

Réle of the white blood corpuscles in absorption. During the absorp-
tion of food and particularly of protein food, white blood corpuscles ac-
cumulate in the mucous membrane of the gut. They are to be found not
only beneath the epithelial cells, but crawling between them and even out
into the lumen of the gut. They may be seen also coming back into the
chyle vessels. The rédle they are playing in absorption is still quite ob-
scure. After the absorption of a meal they are found in the blood in
unusually large numbers. There is, as it is said, a digestive leucocytosis.
ABSORPTION \ 455

That fat may be found in the epithelial cells of the intestine indicates
that most of the fat absorbed goes through these cells rather than through
the leucocytes. The leucocytosis is most pronounced during the digestion
of proteins, and since the leucocytes show themselves to be positively
chemotactic toward any decomposing tissue, or toward the products of
digestion of proteins when the latter are in capillary tubes and placed in
various locations in the body, it has been suggested by Hofmeister that
most of the proteins are absorbed by uniting with the leucocyte body. By
the decomposition of the whole or a part of the body of the cell they are
then supposed to be set free. This view does not seem very probable in

 

 

 

 

 

 

 

 

 

 

 

 

 

Fig. 43.—Polyfistula dog of London. The dog has three fistulas at different levels of
the intestine. One is at & and the two others are discharging into the vessels d and e.

the light of the recent work, indicating that the proteins are absorbed as
amino-acids and get into the blood in that form. It is objected also
that the absorption is too rapid and too large to be accounted for by the
relatively small weight of leucocytes found in the intestine during the
absorption. The leucocytes of dog’s blood cannot weigh altogether
more than 2 grams. Still there are many indications that the blood pro-
teins may be formed by the leucocytes, and perhaps they are active in
securing sufficient protein to supply the needs of the blood tissue alone.
Further work is needed before any positive conclusion can be drawn
concerning the réle of the leucocytes during absorption.

The amount of absorption in different parts of the tract. By making
fistule at various levels of the alimentary tract, London and his co-
workers have studied the rapidity of absorption during the passage of
456 PHYSIOLOGICAL CHEMISTRY

food down the tract of dogs. They have found that the greater part of
the absorption takes place in the upper parts of the intestine and that at
the end of the small intestine only a small fraction of the total food
remains to pass into the colon. At least three-quarters of the food is
absorbed into the small intestine. Water passes very rapidly through
the stomach and is absorbed in the intestine.

Conclusion. The absorption of the products of digestion is in part
a physical process, but in a much larger degree it is a physiological
process dependent upon’ the physiological activity of the cells of the
intestinal epithelium. These cells have the power not only of reabsorb-
ing food products, but of secreting into the bowel a good deal of water,.
salts and other substances. Moreover the solution of the problem. of
the nature of the processes involved in absorption is greatly complicated
by the great sensitiveness of these cells to various conditions. It would
be thought fairly easy to study absorption by means of perfusion experi-
ments with defibrinated blood, but such experiments have hitherto shat-
tered on the impossibility of keeping the mucous membrane in a normal
condition. Whether it is that the epithelium is abnormally sensitive to
lack of oxygen, or whether the defibrinated blood acts as a poisonous sub-
stance, or for some other reason the epithelium undergoes very readily
a kind of autolysis in such experiments and the perfused blood escapes
into the lumen of the intestine. Certain it is that the absorption
normally oceurs in large measure in the upper part of the small intestine
and that relatively little remains to be reabsorbed in the colon.

The changes undergone by the food matters during absorption are
still badly known, but the fats are apparently resynthesized to neutral
fat, the carbohydrate is absorbed unchanged and the amino-acids are in
part deaminized, but in larger part they pass as such into the blood.

REFERENCES. Prorern ABSORPTION.

1. Abderhalden: Zur Frage des Albumosengehaltes des Blutes und speziell des
Plasmas. Biochem. Zeit., 8, 1908, p. 360.

2, Abderhalden uw. Rona: Fiitterungsversuche mit durch Pankreatin, durch Pepsiu-
salzsiiure Pankreatin und durch Siure hydrolyzierten Casein. Zeit physiol.
Chem., 42, p. 528, 1904. Also 44, p. 198, 1905.

3. Abderhalden and Oppler: Weiterer Beitrag zur Frage nach der Verwertung von
tief abgebauten Eiweiss im Organismus des Hundes. Zeit. physiol. Chem.,
51, p. 226, 1907.

4. Abderhalden u. Rona: Same title. Zeit. physiol. Chem., 52, p. 507, 1907.

5. Abderhalden and London: Same title except investigation on an Eck fistula dog.
Zeit. physiol. Chem., 54, p. 80, 1907.

6. Abderhalden and Ollinger: Same title as 4. Zeit. physiol. Chem., 57, p. 74, 1908.

7. Abderhalden and London: Weiterer Beitrag zur Frage nach. dem Ab- und Aufbau
der Proteine im tierischen Organismus. Zeit. physiol. Chem., 65, 1910, p. 251.
ABSORPTION 457

8. Abderhalden and Suwa: Same title as 4. Zeit. physiol. Chem., 68, p. 416, 1910.

10.
11.

12.
13.

14,
15.
16.
17.
18.
19.
20.
21.
22.
23.

24,

25.
26.

27.

28.
29.

30.
31.

32.
33.

34.
35.

36.

Magendarmkana! eingeftihrten Monaminosiiuren. Zeit. physiol. Chem., 53,

_-p. 826, 1907.

Ascoli and Vigano: Zur Kenntnis der Resorption der Eiweisskérper. Zeit.
physiol. Chem., 39, p. 283, 1903.

Bergmann: Notiz tiber den Befund von Verbindungen im Blute, die mit Naph-
thalinsulfochlorid reagiren. Beitr. chem. Physiol. Pathol., 6, p. 40, 1905.

Bostock: On deamidization. Biochem. Jour., 6, p. 48, 1911.

Brasch: Weitere Untersuchungen iiber den Bakteriellen Abbau primarer
Eiweissspaltprodukte. Biochem. Zeit., 22, p. 403, 1909.

Cathcart and Leathes: On the absorption of proteins from the intestine.
Jour. Physiol., 33, p. 462, 1905.

Cohnheim: Die Umwandlung des Eiweisses durch die Darmwand. Zeit.
physiol. Chem., 33, p. 451, 1901.

Cohnheim: Weitere Mittheilungen tiber Hiweissresorptionsversuche an Octopoden.
Zeit. physiol. Chem., 35, p. 396, 1902.

Cohnheim: Versuche iiber Eiweissresorption. Zeit. physiol. Chem., 59, p. 239,
1909. See also 61, p. 189, 1909.

Cramer and Pringle: Assimilation of protein introduced enterally. Jour.
Physiol., 37, p. 158, 1908.

Embden and Knoop: Ueber das Verhalten der Albumosen in der Darmwand, etc.
Beitr. chem. Physiol. Pathol. 3, p. 120, 1903.

Halliburton: The absorption of proteins. Lancet, I, 1909.

Hedin: Trypsin and antitrypsin. Biochem. Jour., 1, p. 474, 1906.

Henriques: Die Eiweisssynthese im tierischen Organismus. Zeit. physiol.
Chem., 54, p. 406, 1907.

Hofmeister: Das Verhalten des Peptons in der Magenschleimhaut. Zeit.
physiol. Chem., 6, p. 69, 1882.

Hopkins : The utilization of proteins in the animal. Science Progress, 1, p. 156,
1906.

Howell: Amino-acids in blood and lymph. A. Jour. Physiol., 17, p. 278, 1907.

Kiihne: Ueber die Verdauung der Eiweissstoffe durch den Pankreassaft. Arch.
f. path. Anat. u. Physiol., 39, p. 130, 1867.

Lang: Deamidization in the animal body. Beitr. chem. Physiol. Pathol., 5, p.
821, 1904.

Levene and Van Slyke: Ueber Plastein. II. Bioch., Ztschr., 16, p. 203, 1909.

London: Various papers on digestion and absorption in the Zeit. physiol. Chem.
from 1907 on. Especially 61, 69, 1909; and 60, 191 and 267, 1909.

MacFadyen, Nencki and Sieber: Die chemische Vorginge im menschlichen
Diinndarm. Arch. f. expt. Pathol. u. Pharm., 28, p. 310, 1902.

Omi: Resorptionsversuche an Hunden mit Diinndarmfisteln. Arch. f. d. ges.
Physiol., 126, p. 428, 1909.

Paton and Goodall: Digestion leucocytosis.. Jour. Physiol., 33, 20, 1905.

Robertson: Synthesis of paranuclein through the agency of pepsin. Jour. Biol.

_ Chem., 5, p. 493, 1908. :

Salaskin: Hiweissresorption im Magen des Hundes. Zeit. physiol. Chem., 51,
p- 167, 1907.

Taylor: On the synthesis of protein through the action of trypsin. Jour.
Biol. Chem., 3, p. 87, 1907. Ibid., 5, p. 381, 1908.

Reid: Intestinal absorption. Phil. Trans. Roy Soc., 192, p. 240, 1900.
CHAPTER XII.
THE BLOOD. THE CIRCULATING TISSUE.

Since each cell of the body needs food and oxygen and is injured
by the accumulation of its own waste products, it is necessary, if a
multicellular organism of a large size is to exist, that there be some
means of getting food and oxygen to the cells in the interior of the
mass, and some means of removing the waste matters. This problem was
solved by one of the tissues, the blood, becoming liquid so that it could
easily penetrate all the crevices of the body and come into intimate
contact with every cell, and thus nourish it. There was finally developed,
also, a system of tubes to contain the blood and a pumping organ, the
heart, which drove it all over the body. Every cell of the body comes
then into close contact with the circulating fluid and exchanges food
and waste products with it. In some organisms this fluid bathes each cell
directly, so that all cells of the body really live in the blood. This is the
case, for example, in invertebrates and in the cortical parts of the supra-
renal capsules of vertebrates where there are no capillaries. In these
eases the blood passes directly from the ends of the arterioles into the
tissue spaces and is collected from these spaces into the veins. But
usually in vertebrate organs the blood is confined to the blood vessels. It
has evidently been found on the whole to be better, for one reason or
another, to confine the blood in this manner. The finer blood vessels,
the capillaries, have walls so thin and so permeable, or are physiologically
so constituted, that oxygen and other gases and water, salts and food
matters pass easily through them, so that the tissue cells, while not
directly in the blood stream, are in the lymph, which comes from the
blood. The principal advantage of a closed vascular system is that
the circulation is probably more easily controlled with such an arrange-
ment than when the blood flows from the arteries into lacunar spaces
in the tissues. By this device it is possible to drive the blood faster past
the tissues and to provide more food and particularly more oxygen. The
main difference in the metabolism of the higher and lower vertebrates
is the much more intense oxidation in the higher. It is the nervous sys-
tem which especially requires a very large supply of oxygen and it was
probably primarily to supply this tissue, which has the keenest metab-
olism of all, that the many devices for improving the circulation have

458
THE BLOOD. THE CIRCULATING TISSUE 459

been evolved. It must be remembered that it is the nervous system
which has been, on the whole, consistently selected in vertebrate and
also in invertebrate evolution. The rest of the body really exists for it.
Each cell of the body, then, even of the higher vertebrates, really lives
in an aqueous medium just as do unicellular organisms, which live in
water itself. And for this reason the blood has been called the internal
medium of the body.

Lymph.—The spaces of the tissues are filled with a liquid called
lymph, derived from the blood by secretion through the capillary walls.
Lymph resembles, in many ways, the plasma of the blood, and contains
some white corpuscles or cells in suspension in it. These are called
lymphocytes. Some of this lymph, which is the immediate liquid en-
vironment of the tissue cells, passes back to the blood by absorption
through the capillaries, but part of it is collected into thin-walled
vessels called lymphatics, which resemble veins in structure, and after
passing through lymph glands the liquid finally finds its way back to the
blood by the thoracic duct and other lymphatics opening into the blood
at the junction of the internal jugular and left subclavian vein. Food
substances pass from the blood to the lymph and the cells take them
from the lymph. The waste products pass in the opposite direction.
One object of a separate lymphatic system may be to guard against infec-
tions, since when bacteria find access to the tissues they are carried by
the lymph stream to the lymph glands, where they are generally digested
and the infection is thus confined. There may be other advantages also
in this separate lymphatic system.

Functions of the blood.—The blood has four great functions: 1. It
earries food from the intestine to the tissues; and gaseous food, or
oxygen, from the lungs to the tissues. 2. It removes waste products
from the tissues and carries them to the kidneys, lungs, intestine, and
skin, the excretory organs of the body. 3. It provides for the metabolic
co-ordination of the body, in that it distributes the internal secretions
from each organ to other organs which utilize them. It thus keeps
tissues, which may be far separated, in metabolic co-ordination or ex-
change with each other; it is the internal medium of exchange. 4. It
plays a very important part in the defense of the organism against the
invasion of parasites.

Composition of the blood.—The blood is generally considered to be
a liquid tissue lying within a system of tubes, the blood vessels, and the
latter are not considered as part of the blood itself. More correctly,
however, both blood and the endothelium of the blood vessels make part
of a single system and it is impossible to alter one without at the same
time altering the other. The liquid part of the blood, the plasma, is
regarded as corresponding to the lifeless intercellular substance of con-
460 PHYSIOLOGICAL CHEMISTRY

nective tissue and cartilage; the cells or living part of this tissue being
the red and white corpuscles. From this point of view the liquid part
of the blood is a lifeless carrier of waste materials and food substances
to the various other tissues of the body and only the cells of the blood
are living. One can look at the blood, however, in a different way, as
suggested by Wooldridge, and from this point of view it gains wonder-
fully both in interest and instructiveness. The blood as a whole, includ-
ing the endothelial cells of the blood vessels, may be considered to be
living matter, distinguished from most other living matter by its greater
fluidity. There are,-however, other kinds of living matter of a liquid
kind. The protoplasm of the amceba and many plant cells is so liquid
that it flows readily and is in reality a circulating liquid. The blood
plasma may be regarded as a very liquid protoplasm formed essentially
by the cells of the vascular endothelium, by the blood cells and the
hematoblasts. The blood platelets and the red blood corpuscles may be
regarded, from this point of view, as homologous with the granular
inclusions of many cells. Blood separated from the endothelial cells
dies and clots; in just the same way a peripheral nerve dies if severed
from its nutritive center. We shall in this chapter adopt this point
of view of Wooldridge and consider the whole blood, the more liquid
portions together with the corpuscles both white and red, the platelets,
and the cells lining the blood vessels, as consisting of a great mass of
living protoplasm. The processes which occur in the blood are then in
many important particulars probably identical with those occurring in
living matter generally. Thus the clotting of the blood is not to be
regarded as a separate and independent property peculiar to this tissue,
but as typical-.of similar processes occurring in every form of living mat-
ter. For all living matter like the blood has the property of forming a
gel or clotting. Blood, too, respires, like living matter. When the
amino-acids, carbohydrates, etc., enter the blood by diffusion, or by secre-
tion of the endothelial wall, they are not then entering an inert fluid,
but they are entering real living matter and their subsequent fate in
this fluid becomes extraordinarily interesting as showing the probable
fate of similar substances in the body cells generally. The proteins of
the blood plasma are not to be considered as inert simple proteins, as
they are ordinarily considered, as so much globulin, albumin and
fibrinogen in solution, but they are probably united in the blood plasma,
at least to some extent, with phospholipins, just as they are in cells; and
they probably make part of a complex substance, in reality an organized
and very unstable substance, the blood plasma. The alkalinity of the
blood is about the same as that of the tissues generally and the methods
of maintaining its alkalinity are those employed by all forms of living
matter. A study of the alkalinity of the blood, its variation both physio-
THE BLOOD. THE CIRCULATING TISSUE 461

logical and pathological and the means employed to hold it constant,
throws light on the alkalinity of every cell.

We probably know more about the composition and the chemical and
physical changes taking place in the blood than of any other tissue, but
that our knowledge is still extremely fragmentary will appear in the
pages which follow. The study of the blood is in many ways easier
than that of any other tissue. Being liquid, it is readily obtained in
large amounts for chemical and physical investigation, and by centrifu-
galization we are able to separate it, without inducing serious chemical
changes, into portions of lighter and heavier specific gravity. Moreover,
we can get such quantities of it that, we are able to experiment with it
more than with almost any other tissue of the body.

General composition of mammalian blood—Mammalian blood as it
circulates within the blood vessels consists of a more fluid part, the blood
plasma, and this contains a large number of cells and organized struc-
tures in suspension. The cells are the leucocytes, or white blood cells;
the special organized structures are the red blood corpuscles, or erythro-
cytes, and the blood platelets. The other cells belonging to this system,
the endothelium of the blood vessels, are fixed, not circulating, cells and
we shall for the present neglect them; but more than any other cells of
the blood they probably control its composition and metabolism. The
general composition of mammalian blood is as follows: (see also
page 468.)

1. Blood (mammalian) consists of:

A. Plasma ..... 60-70% by volume; or about 55% by weight.
B. Corpuseles .. 40-30% .“ e « & 45% “
A. Plasma.
(Specific gravity — 1.0237-1.0276 at 25°)
Bie WATER cislack iw agasace is wade eieln ravayade 90 - 92%
Dir SOW AS secs ceaseless etait srsions 10- 8
Organic
Lipoproteins ............ 5.5 -8.4
Carbohydrates .......... 0.1 -0.2
Cholesterol ............. 0.09-0.14
Fat and fat acids ....... 0.3 -0.6
Extractives ............. 9.05-0.2
Inorganic ..............6. 1-2
3B. Red corpuscles (Abderhalden).
(Specific gravity — 1.088)
Bs Water sosaicn tareied neers od at 68.7-59.2
bs ‘SOlCS. cues ss eautee wees cae 31.3-40.8
Organle: <s.666as as nares x00 30.4-39.9
Hemoglobin ............ 31.7
Stroma
Phospholipin ......... 0.37-0.39
Cholesterol ........... 0.14-0.17
(Grigant et Huillier)
Protein. ssacce se exageass 6.7 -6.4

Inorganic (excluding Fe) -- 0.9
462 PHYSIOLOGICAL CHEMISTRY
The following analysis of human blood was made by Carl Schmidt: *

BLOOD OF A MAN TWENTY-FIVE YEARS OF AGE

ONE THOUSAND GRAMS OF BLOOD CONTAIN

513.02 BLOOD CORPUSCLES

 

 

 

Water cchicccia cogs eeees. Bee 349.69
Substances not vaporizing at 120° 163.33
FREIWNGENN: - getiean tesnescecutashcue we Sapecécea 7.70
(including 0.512 iron)
‘Blood easein,” etc. .........-. 151.89
Inorganic constituents ......... 3.74
(excluding iron) ‘i
FRE sinc midcod aeons roa 0.898 ; :
Bidpnuiie AO i sucieddampncieese wees 0.031 ! Potassium chloride ............. 1.887
Phosphoric acid ........-.....-- 0.695 Potassium sulphate ...........-. 0.068
Potassium aot aece ote eaten eon newien 1.586 Potassium phosphate ........... 1.202
RCN TINT cee ret tadcesbeanoese heceledataseneiad 0.241 f = 1 Sodium phosphate ............. 0.325
Phosphate of lime ............-. 0.048 Soda... cece cece ee eee cence ee 0.175
Phosphate of magnesium ..... .. 0.031 Calcium phosphate ............. 0.048
OXV2eN) cess is eeeesrrewsae teres 0.206 Magnesium phosphate ......... 0.031
PEOUA, | ee sigcectis tc opeseeee arevepevers 3.736
486.98 PLASMA
Watel> codsiiataadtnomenn a arucinns oe 439.02
Substances not evaporated at 120° 47.96
Ribyiny cgcueuewepens sauweeae Aes 3.93
Albumin, ‘etes 25 hen sacamea saree 39.89
Inorganic constituents .......... 4.14
ChIOTING: coc. scaeesacawansy meee 1.722 Potassium chloride ............. 0.175
Sulphuric acid .............2005 0.063 Potassium sulphate ............. 0.137
Phosphoric acid ................ 0.071 Sodium chloride ............... 2.701
Potassium .......-.. 0. eee ee eee 0.153 | _ | Sodium phosphate .............. 0.132
SOdiUM: | sis sce sqisend a. rato toes oeeve L6G) WSOd a: ais Mace cvigscom eitveoncacaninne.aitosnentcs 0.746
Calcium phosphate ............. 0.145 Calcium phosphate ............. 0.145
Magnesium phosphate .........- 0.106 | Magnesium phosphate .......... 0.106
OXYGEN csc eaiadessenaarns pee 0.221 |
Total). cccay sstgossesamee se 4.142

SPECIFIC GRAVITY = 1.0599

1000 GRAMS OF BLOOD CORPUSCLES

WATE ssicspidenin Sedpl ay iearesasouesare Gey say 681.63
Substances not volatile at 120° .. 318.37

Hematin: ceiev scaeaseewaaiend ses 15.02
(including 0.998 iron)

“Blood casein,” etc. .......000. 296.07
Inorganic constituents ......... 7.28

(excluding iron)

*C. Schmidt, “ Charakteristik der epidemischen Cholera,” pp. 29, 32: Leipzig
and Mitau, 1850. Quoted from Bunge, Physiologic and Pathologic Chemistry, 2d
edition, p. 212-213, Philadelphia, 1902.
THE BLOOD. THE CIRCULATING TISSUE 463

PWIOFING: fore sige oy eddy reas cea ey 1.750)  { Potassium sulphate ............. 0.132
Suiphurie acid ..............04. 0.061 | Potassium chloride ............. 3.679
Phosphorie acid ................ 1.335 | Potassium phosphate ........... 2.343
Potassium ............. cece eee 3.091; _ | Sodium phosphate .............. 0.633
SOMO 22 Sen wana edenanuh naar O70 FF Boda: cane tae oicaexdanienvaneene 0.341
Calcium phosphate ............. 0.094 + Calcium phosphate ............. 0.094
Magnesium phosphate ........... 0.060 Magnesium phosphate .......... 0.060
ORV BEN: - ose aie cnc Beem ye eats 0.401 ; ——

Total inorganic constituents (excluding iron) .. 7.282

SPECIFIC GRAVITY = 1.0886

1000 GRAMS OF PLASMA CONTAIN

 

Water .aiiccccsasiwennacadiacs a 901.51

Solids non-volatile at 120° ..... 98.49

IBTIT eee: hes £5 Saale hay Shee 8.06

Albumin, ete. .................. 81.92

Inorganic constituents .......... 8.51 .

Chlorine™ ass.:6sseners 4 oes on 3.536 | — { Potassium sulphate ............. 0.281
Sulphuric acid ...............5. 0.129 Potassium chloride ............. 0.359
Phosphoric acid ................ 0.145 Sodium chloride ................ 5.546
Potassium ............. eee s eee 0.314; _ } Sodium phosphate ............. 0.271
SOGRUM: g4 cihicne ins ods eae nutans Serewwere 3.410 | TP Sodas -esrcimiig diace cheers acs Seeeers CRA ea 1.532
Calcium phosphate ............. 0.298 Caleium phosphate ............. 0.298
Magnesium phosphate .......... 0.218 Magnesium phosphate .......... 0.218
Oxygen 3 estou a ees eh gears 0.455 t woo

Total of inorganic constituents .. 8.505

SPECIFIC GRAVITY = 1.0312

1000 GRAMS OF SERUM CONTAIN

 

Water iscsi saadires svineses.eg 908.84
Solids not volatile at 120° ..... 91.16
Albumin, ete. ................. 82.59
Inorganic constituents ......... 8.57
Chlorine wisi ice assay cacerdie eis deers 3.565 f Potassium sulphate ............. 0.283
Sulphuric acid ................. 0.130 | Potassium chloride ............. 0.362
Phosphoric acid ................ 0.146 Sodium chloride ........... eee. 5DOL
Potassium: ssicwsscsueus ctor ees 0.317 | _ | Sodium phosphate ............. 0.273
Sodium .............2-2--0--. 3.438 1 = Soda. 2s. guctiveeaccataaacias “.. 1.545
Caleium phosphate ............. 0.300 | Caleium phosphate ............. 0.300
Magnesium phosphate .......... 0.220 Magnesium phosphate .......... 0.220
OXY PEN: 22 kadeditiiin eae eee gue 0.458 | i

Total inorganic constituents ..... 8.574

SPECIFIC GRAVITY — 1.0292

Corpuscles of the blood. White.—The white corpuscles are true
cells. They have a nucleus; they are capable of spontaneous movement
and reproduction. They are colorless, amceboid cells which have the
power of phagocytosis; that is, of eating bacteria and other solid matters
which enter the blood. Their specific gravity is less than that of the red
blood corpuscles, so that they collect as a layer above the reds when
the blood is centrifugalized. They have an active metabolism, and their
composition is that of typical cells consisting of true nucleins, phospho-
464 PHYSIOLOGICAL CHEMISTRY

lipins, globulins and albumins, which are at least in part in union with
phospholipin. They have the extractives usually found in cells. They
are variable in size, but they are somewhat larger than the red blood
“corpuscles. In mammals’ blood their diameter is about 4-13 yw. Their
number is very variable. Normally there is about one leucocyte to 350-
500 red cells in human blood, or about 7,000-15,000 per c.mm. of blood.
The number is relatively larger in the young, and it is increased in many
infectious diseases, by the injection of nuclein, by suppuration, and
by a meat diet. In the pathological condition of leucemia, or leuco-
cythemia, the number may be greatly increased so that the blood has a
creamy appearance, as if pus were mixed with it. The numbers may rise
to 500,000 per c.mm. of blood. This disease may be analogous to a
neoplasm involving the’ white cells or the tissue which produces them.
The white corpuscles are of various kinds: a, polymorphonuclear; b,
lymphocytes; ¢, eosinophile; d, basophile, etc. They are formed in the
bone marrow, or in the lymph glands, and possibly in part in the
spleen.

Motility of white cells. The white (and also the red) corpuscles
when placed in a thin film of blood or Ringer’s solution, and possibly
under other circumstances as well, undergo very remarkable changes
of form: not only are the white corpuscles amceboid, but they shoot out
from themselves long, whip-like processes which extend many cell
diameters and have an active movement. These processes move back
and forth and they are very sticky, so that bacteria stick to them like
flies on sticky fly-paper. The processes may be very quickly withdrawn
within the cell and the latter resume its ameboid form (Kite). These
processes may be seen with particular clearness with the dark field illu-
mination. They seem .to be due to some surface tension action. Very
similar processes are found also on the red cells under certain conditions.
By means of their motility the white cells can crawl out of the blood
vessels into the tissues, and their main function appears to be to remove
old, worn-out or diseased tissues and cells, and to digest or otherwise -
overcome parasites of all kinds attacking the body. Probably they help
also to form the proteins of the blood plasma.

Red corpuscles. Erythrocytes.—The number of red corpuscles in
mammalian blood is very great. In human blood it is normally between
5,000,000 and 6,000,000 per c.mm. of blood, but in leucemia and in
anemias of various kinds it may be reduced to half this number or even
less. These corpuscles have normally in human blood the form of
biconcave disks and they contain no nuclei. But in mammalian embryos;
or in adults after hemorrhage when the blood is regenerating, larger,
nucleated red cells may be present; and in all the vertebrates below
mammals the corpuscles are red nucleated cells, oval in outline and
THE BLOOD. THE CIRCULATING TISSUE 465

-biconvex. The mammalian red blood corpuscles, as they lack nuclei,
are not complete cells. Their diameter in human blood is usually about
7-8 uv, but there may be some as small as 5 » and larger ones up to 12 yu
in diameter. Their numbers are increased by living at high altitudes,
or by exposing an animal to a lowered pressure of oxygen. An animal
responds to either of these conditions by the production of more cor-
puscles and more hemoglobin, so that the blood can carry more oxygen
at a lower partial pressure of the oxygen.

The red corpuscles contain hemoglobin and their chief function is
to carry oxygen to the tissues from the lungs. Besides the hemoglobin,
they contain chiefly lipoprotein material. Their composition will be
taken up presently. They are formed in the red marrow of the bones
by cells called hematoblasts. How long they live is not known. Their
form, while quite characteristic and apparently fixed in most prepara-
tions so that it is apt to give the notion of considerable rigidity, is in
reality not so. They are soft, and their peripheral layer at least is
apparently made of a sticky, fluid matter. Under certain conditions,
when they are mounted on a glass slide at least, they undergo extraor-
dinary changes of form. At times in a moment they all become covered
with sharp projections like burs; they are then said to be crenated, and
under high powers of the microscope, particularly in the dark field, it
may be seen that fine protoplasmic processes may extend over a cell’s
diameter in length from the ends of the sharp processes. These processes
may be in active movement. The processes may be withdrawn as rap-
idly as they are extruded and the cell round up to the typical shape. If
stimulated by touching the corpuscle, the processes may be suddenly
shot out, or as suddenly withdrawn, or by a slightly stronger stimulus
the erythocyte may withdraw them, suddenly swell and undergo solu-
tion. In all these respects the erythocytes behave like many other cells,
like the white blood cells, a great many kinds of egg cells and tissue
cells of the lower animals, such as sponges. They resemble, too, some
of the mitochondria (Kite; Oliver). These changes in form indicate
very clearly that the corpuscles are irritable and at least the exterior
substance of the cell. is liquid, since only liquids have the power of
molecular movement necessary for these rapid and extraordinary
changes. The changes in shape have been variously interpreted. Some
have regarded them as the beginning of death changes; but to the author,
as to many who have watched them, they appear exactly analogous to
the vital responses of all kinds of living matter. They show that the
property of movement is not lost to the red cells, and that they are real
living’ cells. albeit of a special kind, and not rigid. or semirigid, quiescent,
dead structures. They lack nuclei and with this they have lost to a
large extent, but not entirely, the power of respiration and all power
466 PHYSIOLOGICAL CHEMISTRY

of growth, or synthetic metabolism, but they are still reactive, although
far less so than the white cells. They probably live for a certain time
only and grow old like other cell structures. These are not the only
cells without nuclei in the animal kingdom. In certain molluses there
are found in the seminal fluid gigantic anomalous spermatozoa in which
the nucleus has totally degenerated and disappeared, and yet the cells
continue to exist for a long time, and these cells without nuclei are capa-
ble of movement.

It is because of the presence of the corpuscles of the blood that even
in thin layers the blood is not transparent, but opaque. A good deal
of light is reflected from the surfaces of the corpuscles. If the corpuscles
are dissolved, the blood takes a darker tint and becomes transparent. It
is then said to be ‘‘ laked.’’ The aarker tint is due to the fact that in
laked blood there is a smaller admixture of white light reflected from
the corpuscles.

Because of their small size, flat disk shape, and enormous numbers,
the total surface of the red corpuscles in a human adult amounts to
from 3,000-4,000 square meters. The blood makes about one-twelfth
of the total weight of the body. In a man of 72 kilos weight there would
be some 6 kilos of blood or something over 5.5 liters, since the specific
gravity of the blood is about 1.055. The total number of red blood cor-
puscles in the body would be about 30,000,000,000,000. The surface area
of a corpuscle is about 0.000,1 sq. mm. The large surface area of a
corpuscle in comparison with its bulk facilitates the exit and entrance
of oxygen.

Blood platelets.—The blood platelets are spherical, or disk-shaped,
bodies found in the shed blood of mammals, but not in the blood of birds
or cther vertebrates lower than the mammals. The platelets are of
somewhat irregular size, the diameter varying from 1.5-3m. Their
numbers are also very variable, and it is a very suggestive fact that the
more care that is used to keep the blood in a living state the fewer plate-
lets there are. Injury to the wall of the blood vessels greatly increases
their numbers. They are refractive bodies and apparently nearly or
quite homogeneous. There is no evidence that they contain nuclei. The
number of these bodies in shed blood, as has been said, is very
variable, but they may be present in very large numbers: namely, from
300,000-800,000 per c.mm. of blood. Sometimes they contain hemo-
globin, but they are generally colorless. The origin and nature of these
bodies has been much disputed, but the following conclusions are those
generally accepted. They are almost certainly derived from the white
blood corpuscles, and in part at least from the red. In their chemical
composition they appear to be identical with the stroma of the red
corpuscles or the protoplasm of the leucocytes, and like undifferentiated
THE BLOOD. THE CIRCULATING TISSUE 467

protoplasm generally. By some they have been supposed to be com-
posed of nuclein, but this is almost certainly incorrect and is due to the
fact that they show some of the properties of nucleins, such, for example,
as solubility and the property of yielding, when digested by pepsin
hydrochloric acid, an insoluble residue rich in phosphoric acid. In
reality the platelets consist of a phospholipin-protein compound. The
phosphoric acid is in a phospholipin. The platelets consist of what
Wooldridge calls A-fibrinogen. As we shall see when we come to the
clotting of the blood, they play a very important réle in this process and
they yield fibrin, and also a substance called thrombin or fibrin ferment,
and a proteolytic ferment.

It is very difficult to decide whether the platelets are preformed
in the living, unchanged blood or whether they appear there with great
ease when blood is disturbed. We now know that the chromosomes of
the nucleus, which are certainly organized structural constituents, may
appear with great rapidity when the nucleus is stimulated in any way
(Chambers). It may be the same with the platelets. Although they
have been seen in the living capillaries, yet it is significant that there
are fewer the more care is taken to avoid injury to the capillaries
and stasis of the blood. It is impossible to examine the blood without
causing some injury to it. The platelets form the first beginnings of
the thrombus in intravascular clotting and they appear in such numbers
that it is impossible that they should have been preformed in the blood.
If, for example, one ties off a portion of the carotid artery of the dog
with its contained blood and then, the dog lying on its back, one injures
the wall of the carotid on the upper (ventral) surface with silver nitrate
or heat, a thrombus forms in the vessel at the injured point. It is found
that the thrombus consists chiefly of platelets and there are vastly greater
numbers than could have been present in the small amount of blood
caught between the ligatures. Injury to the endothelial wall appears
to eall them forth. This observation is very strong confirmation of the
view of Wooldridge that the platelets, in part at least, are in solution in
the plasma in a supersaturated form and when disturbed they crystal-
lize out. He regarded them as in the nature of imperfectly crystalline
proteins. They appear to the author to resemble what are known as
fluid crystals. Moreover, substances of this chemical nature are par-
ticularly apt to form fluid crystals. Schwalbe, who tried the experiment
on thrombosis just cited, concluded that they must come from the red
corpuscles, because some of the red corpuscles were found in various
unusual forms as if they were decomposing. It is hard to see how with-
out motion of the blood the platelets could accumulate at the upper part
of the artery where they were found. since they are heavier than the
blood plasma and would naturally sink. It is hence more probable that
468: PHYSIOLOGICAL CHEMISTRY

they are formed in the method described by Wooldridge by a process of
crystallization from the plasma, the crystallization taking place first
at the point of injury of the artery wall. It is probable that some are
preformed in the blood as it circulates, but this would not at all militate
against this view. Many authorities, however, are of the opinion that
the platelets are almost wholly preformed, living elements (see Dietjen).

The platelets may be prepared by receiving blood in an equal volume
of 0.2 per cent. sodium oxalate solution, or sodium metaphosphate or
fluoride, so as to prevent coagulation and the dissolution of the platelets,
and then by centrifugalization at a relatively slow speed to separate out
the corpuscles. The plasma with the platelets is then poured off from
the red and white corpuscles and centriftiged very fast for some time.
The platelets are thus thrown out and collect as a grayish-white layer
at the bottom of the tube. They are quite soluble in the plasma on
warming when they are first separated (Wooldridge), but on standing
lose their solubility. Their numbers are increased by cooling the plasma
before centrifugalizing it. At first they dissolve readily also in salt
solution containing a little alkali, but on standing rapidly change, becom-
ing more insoluble and like fibrin. A suspension or solution of the
washed, perfectly fresh, unchanged plates has the property of forming a
typical clot, yielding fibrin. (Wooldridge; Schittenhelm and Bodong.)

Blood plasma.—Horse blood contains about 34.5 per cent. by weight
of corpuscles; and 65.5 per cent. of plasma. In other mammals the pro-
portions are not very different from this. In human blood the corpuscles
make 35-40 per cent. of the blood by volume and nearly 50 per cent. by
weight, since their specific gravity is greater than that of the plasma.
The plasma contains about 10 per cent. by weight of solids, of which
about 7-9 per cent. are proteins; 1 per cent. salts. and the rest lipin and
various other substances, such as urea, creatine, amino-acids, dextrose,
ete., present in small amounts. 100'c.c. plasma contain 7.8-11.8 mgs. Ca.
The composition of the serum is given in the following table:

Ox Buioop (Abderhalden, Bunge’s textbook).

WEP cue eba beer baw 4 Soe ks ee Be ee ee 913.64

SOUQSS 5 gues bre es CES RS SSG HT RSH Oar eee 86.36
WOROLCIO) -saieconss a Bra eset see eRe oO dae hese Ae eteeeices, BGs 72.5
SULLA cies Gla. a tages dole hudshte Bis yestncqaca 8 auetenann 2/0 paver co 1.05
PUOUSIOTGL, 25 icawleweeamis woman Reames ake dears 1,238
QCD | dey sek apetati tare anand a a hatrindes a avehel eh dewey ae Sc 1.675
Rats <2, Beas. _y aoe wl eres osname ens Anterior anon ds 0.926
Phosphoric acid as nuclein ......... cece cece eee 0.0133
SOd2 5 eae daw-nges tad Se hsb Garg away eee tas 4.312
Potash weswsacares mre sedeas wy eeeeks saw ieee 0.255
Tron Ox1d@: «aus eee suecn es eased oe 6 Sees ea ane oh es & Gnuck
Lini@. qa shh cece ss ghee ss Seed in 4 aloe eNews ees 0.1194
MNGTGSIGE. “tty acess O ouaie BRE RiGee RAINS Be chepss eases is xs 0.0446
CUTIE, 5 sin ys reat ss ed ene sane actepades dReayiair ne auhense ed. hs 3.69
PHOS PNG RIO MCT a. eccics  5 0 sas ga evapsvauel € huararneel diag Aodes 0.244

Tnorganic phosphori¢ acid ....... cece eee eee eee 0.0847
THE BLOOD. THE CIRCULATING TISSUE 469

The proteins which may be separated from it are 1, fibrinogen; 2,
serum globulin; and 3, serum albumin. It is possible that the globulin is
a mixture of two or more proteins. Another protein is present in the
blood serum, but not in the plasma, called serum fibrinogen by its dis-
coverer, Wooldridge, but more commonly fibrinoglobulin. The com-
position of the blood proteins will be taken up later on page 551. They
are in part at least, as they exist in the plasma, in combination with
phospholipins and possibly with cholesterol. The plasma is generally

 

 

 

 

 

oN

 

1g. 44.—Abel’s apparatus for the vivi-diffusion of the blood.

colored a light yellow, although in carnivorous animals it is at ‘times
almost colorless. The coloring matter in herbivorous animals is in part
carrotin and.is derived from green fodder, but in part it is due to
urobilin. Plasma has a reaction alkaline to litmus, but acid to phenol-
phthalein, so that its hydrogen ion concentration is about 210-3
normal. It contains both carbonates and phosphates as well as chlorides.
The greater part of the salt is sodium chloride.

With this brief statement of the structure and general composition
of the blood: we may proceed to discuss its functions.

1. The blood as a carrier of food from the intestine to the tissues.
—The blood is to carry to the tissues the digested and absorbed foods.
Water and salts; amino-acids and ammonia formed from the digestion
470 PHYSIOLOGICAL CHEMISTRY

of the proteins ; glucose and levulose from the digestion of carbohydrates ;
fats which have been split and then resynthesized during the process
of absorption; all of these must find their way into the blood in order
that they may be distributed to the tissues which need them. And all
of them are to be found in the blood. Some hours after the ingestion
of a fatty meal the blood contains so much of finely divided fat that
the plasma may have a milky appearance and be no longer clear as it is

 

Fic. 45.—McGuigan and von Hess apparatus for vivi-diffusion. The blood enters and
leaves at @ and c, passing through coilodion tubes inclosed in the diffusiun jacket. By
raising or lowering 0b diffusion is accelerated, and the pressure may be increased or
diminished on the outside of the diffusion tubes through g.

in fasting animals. There may be so much fat present that it will rise
on the serum separated from the defibrinated blood like so much cream.
The amount of fat and fatty acid in the blood serum is normally 0.1-0.3
per cent., but after a fatty meal it may be from 0.6-1 per cent.

Dextrose is always present in blood in varying amounts. Normally
the blood contains about 0.08 per cent. of dextrose, but it may in health
rise to 0.15-0.2. per cent.; and in venous blood it may fall to 0.06 per
cent. The dextrose is for the greater part in solution in the blood plasma
in a dialyzable form. It may be dialyzed out of the blood by the method
known as vivi-diffusion and, since this method is of the greatest impor-
tance in the investigation of the composition of the blood, it will be well
to describe it at this point.

Vivi-diffusion method. The gist of the method consists in sending
the blood from an artery through a collodion tube which is surrounded
with physiological salt solution. Substances which are diffiisible through
collodion tubes will pass from the blood into the outer liquid and accu-
THE BLOOD. THE CIRCULATING TISSUE 471

mulate there until the concentration in the dialyzate is the same as that
in the blood. After passing through the dialyzer, the blood returns to
a vein. The dialyzer is thus a kind of artificial organ of excretion. The
apparatus as described by Abel is shown in Figure 44. In this
apparatus it is necessary to add hirudin or some other anticoagulant to
the blood to prevent clotting.. This introduced an element of complica-
tion in the interpretation of the results. The apparatus has been sim-
plified and improved by McGuigan and von Hess, as shown in Figure 45.
By avoiding all roughness in the glass and by keeping up a. pulsation in
the dialyzer they were able to prevent the blood from clotting without
the use of any anticoagulant and greatly to hasten dialysis. The results
obtained by the use of this method are very interesting, and the diffusion
of dextrose is illustrated in the accompanying table (McGuigan and von
Hess) : ,

 

 

 

 

Per cent. sugar in
sheatneton used Dialysis
Plasma Dialysate Plasma H,O | Dialysate H,O
Morphine ......... 3.0. hrs. 0.135 0.139 0.147 0.141
Morphine ......... 5.0 “ 0.125 0.133 0.136 0.135
Morphine ......... 6.0 “ 0.088 0.099 0.096 0.100
Ether’ \2¢ssnaw ess 45 “ 0.083 0.087 0.090 0.088
Morphine and ether] 5.0 “ 0.073 0.082 0.080 0.083
Urethane ......... 2.0 “ 0.151 0.168 0.164 0.169
Urethane and ether 3.5 “ | 0.163 0.175 0.177 0.177
Urethane and ether 45 “ 0.153 0.169 0.167 0.171
Urethane and ether 10 “ 0.157 0.173 0.170 0.175

 

 

 

 

 

 

It is clear from the foregoing results that the sugar in the circulating
blood is practically altogether in solution in the plasma and is not in
union with a colloid, as had been suggested. In rabbits and man some
glucose is found, also, in red cells (.05-.2 per cent.). Maltose also has been
found in the blood plasma; and at times, during lactation or at the end
of pregnancy, lactose in small amounts may be found there. It is sur-
prising that the amount of dextrose in the blood does not increase more
than it does during the absorption of a large amount of the products/of
starch digestion from the intestine. The reason is, probably, that the
‘tissues take the dextrose out of the blood as rapidly as it enters, so that
it does not accumulate in the blood. Liver and muscles have the power
of storing a Jarge amount of glucose as glycogen.

Water and salts are taken up by the blood from the intestine at a
very rapid rate, but as the kidneys have the function of keeping the
osmotic pressure of the blood approximately constant, when the tissues
have taken out what they need any excess is rapidly eliminated in the
urine. Nevertheless, foreign salts or the salts taken with the food can
472 PHYSIOLOGICAL CHEMISTRY

be shown to be present in blood. They are for the most part in solution
in the-plasma.

The end products of protein digestion are also found in the blood,
but in very small amounts. After the injection of glycocoll into a loop
of the intestine of a cat, there is an increase in the non-protein nitrogen,
other. than urea and ammonia, in the blood (Folin and Denis). A
solution of glycocoll was injected into the ligated small intestine of a cat
which had been fed 24 hours previously. The amount of non-protein
.. nitrogen in the mesenteric and carotid blood before and after the injec-
tion was as follows:

Total non-protein N in 100 c.c. portal blood before injection .. 30 mgs.
ae ef “« « « “mesenteric vein 45 minutes after

Njection, 22 .ceaes is teeeaase 85
= * SB ie ee carotid blood before injection.. 30 “
os e Se 6 minutes after in-

JOCUION 5 it.2 2 erecrinnann pices crepe oe 34 “
sig = «  « « earotid blood 45 minutes after

injection ............-.056- 57“
es se grams muscle before injection .. 250 “
re © oO “ at end of experi-

MeN sees we aweg pee se che 346 “
“ Urea N eee grams muscle before injection .. 27 “
* s SS at end of experi-

TONG seo oes sa pets na SA EOS 37.

While the presence of amino- acids in the blood was made probable by
these observations, by the discovery of their presence in invertebrate
blood, and by the presence of these acids in the urine, their actual isola-
tion from vertebrate blood has only recently been accomplished. They
are present in very small amounts. A very large amount of blood was
received from the slaughter-house (Abderhalden) and each liter poured
into 15 liters of boiling water. After 15 minutes’ boiling, 1 per cent.
acetic acid was added, little by little, until the coagulation was complete:
and the solution became clear. It was filtered, the filtrate concentrated,
-and by the use of mercuric acetate and sodium carbonate the amino-acids
precipitated. The precipitate contained proline, leucine, valine, alanine,
glycocoll, aspartic acid, glutaminic acid, tryptophane, lysine, arginine
and histidine. There is no doubt, therefore, that the amino-acids in
extremely small amounts are to be found in solution in the blood. Their
presence may also be shown in the blood plasma by the vivi-diffusion
‘method. They are diffused out of the circulating blood and may be
detected in the dialyzate by the ninhydrin reaction and in other ways.
Most’ of these amino-acids are found in the red blood corpuscles, but
some’ are in the plasma. Thus in fasting dog’s blood there may be in
the whole blood (Gyérgy and Zunz) 4-5 mg. amino N per 100 ce. In
the plasma 1.8-3.9 mgs.; and in the corpuscles, 7.2-8 mgs. per 100 e.c.
Four hours after a meal of meat the amount in the carotid blood rose to
from 10 to 12 mgs. per 100 e.c., the amino-acid N in the corpuscles
THE BLOOD. THE CIRCULATING TISSUE 473

increasing over 100 per cent. to 20.6 mgs. per 100 c.c. of corpuscles, and
the plasma rose to from 7-8 mgs. The amount in venous blood was 54-76
per cent. of that of the carotid.

In the blood of a fasting dog Van Slyke and Meyer found in the
femoral artery blood 4.4 mgs. of amino N per 100 cc. blood; in the
carotid artery 5.4-3.1 mgs., and in the mesenteric veins 3.9 mgs. per
100 ¢.¢ blood.

While a very large amount of protein may be absorbed in the form
of amino-acids by the blood, it is so rapidly removed therefrom by the
tissues, or metabolized by the blood itself, that there is very little
accumulation, but still there is some. From the figures just quoted from
Folin and Denis it appears that amino-acids accumulate to some extent
in the muscles after protein ingestion. The possibility exists that the
amino-acids taken into the blood are in part synthesized into the proteins
of this tissue, and by the digestion of these proteins again set free
(Nolf) ; so that the amino-acids found in the blood need not be those.
immediately reabsorbed from the intestine. There is no good evidence,
however, for any considerable transformation into blood proteins as an
intermediary step.

_2. The blood as the carrier of oxygen from the lungs to the
tissues.—a. The amount of different gases in the blood. Venous blood’
as it leaves the tissues is purple in color. It returns to the right side
of the heart and is driven through the pulmonary artery to the lungs.
It there changes its color to a bright scarlet, and this scarlet, arterial
blood is pumped by the heart to the tissues. The change in color of
the blood in passing through the lungs is due to the fact that in these
organs it takes on oxygen and loses carbon dioxide, so that arterial blood
contains more oxygen and less carbon dioxide than venous; while in
the tissues it loses oxygen and picks up carbon dioxide.- The blood is
then constantly carrying oxygen from the lungs to the tissues and carbon -
dioxide from the tissues to the lungs.

That arterial blood contains more oxygen and less carbon dioxide than
venous blood may be shown by exposing the blood to a vacuum. The
gases are given up to a vacuum and may be collected and examined. - The
blood yields all its oxygen to a vacuum and nearly all the carbon dioxide,
but to get the last traces of carbon dioxide it is necessary to add to
the blood some acid. This last portion is some of that. combined as
carbonate. In this and other ways it has been found that 100. c.c.. of.
average human blood is able, if fully saturated, to take up from air or
to.give off to a vacuum between 18 and 19 c.c.-of oxygen measured at
0° and 760 mm. pressure. Since arterial blood is usually only 96 per
cent. saturated, the actual amount of oxygen recovered from blood under
usual conditions is a little over 18 c.c. O, from 100 c.z. of blood. Venous
blood yields generally about two-thirds as much oxygen as arterial, so
474 PHYSIOLOGICAL CHEMISTRY

that blood returns to the heart with about 12 per cent. by volume of
oxygen still in it. In passing through the lungs 6 volumes per cent. of
oxygen are restored to it.

The amount of carbon dioxide which can be pumped out of blood
after the addition of acid, that is the total carbon dioxide, is about
40 ec. from 100 cc. of arterial human blood and about 48 ec. from
venous human blood. So that about 8 volumes per cent. are lost in pass-
ing the lungs.

The composition of cat’s blood gases has been recently determined
by Buckmaster and Gardner as follows, the oxygen capacity being some-
what less than that of humans:

ARTERIAL BLOoD. VOLUME IN C.C. aT 0° C. AND 760 MM. Pressuge From 100 C.C.

 

 

 

BLoop.
Total gas COg Og No

CaTOtid) iscsces eh erdiani iia 39.34 25.81 12.70 0.83

Ser axnealenesn dd eaonetecd ayeavenene 28.59 15.79 11.97 0.83

EE Lailecasdhncand os Sraentite eae 33.73 18.54 14.18 1.00

So” . . desutahiestlelateieoetatdealaraoreeNasa 38.41 22.11 15.13 1.17

Femoral i sicicisie sssinas veers 50.07 34.52 14.50 1.05

CO” eer inaesd eran a eteareS 47.91 33.68 13.10 1.14
Mean: ssscscxiorscnees evans 39.68 25.07 13.60 1,00°

 

Venous BiLoop. ANESTHETICS AND HIURDIN.

 

 

 

Total gas CO Op, Ng

Right auricle .............. 56.66 44.24 11.31 1.12
* Of eee acaanere Des 46.39 37.42 8.54 0.42
MCalte se ocso cece cose Se eet tas 51.53 40.83 9.93 0.77

 

 

 

 

 

Besides carbon dioxide and oxygen, blood also contains small quan-
tities of nitrogen. Nitrogen composes about four-fifths of the atmos-
phere. It is taken up by the blood in the lungs and exists in solution in
all the tissues and fluids of the body. The amount of nitrogen gas given
off by animals appears to be slightly greater than that inhaled, which
would indicate the production of a small amount of gaseous nitrogen in
the metabolism of the body. This result is not accepted by most observ-
ers, but it seems not unlikely, since by the action of bacteria in the
alimentary tract nitrates are reduced to nitrites, some nitrites are con-
stantly taken in the food:and ammonium nitrite decomposes spontane-
ously, setting free nitrogen gas. The amount of nitrogen so formed is.
however, very small. Nitrogen is very inert and exists simply in solu-
tion in the blood. :

The amount of nitrogen taken up by the blood will depend on the
pressure of nitrogen in the lungs. The amount absorbed is considerable
in men working under compressed air in caissons, and as this nitrogen
THE BLOOD. THE CIRCULATING TISSUE 475

is released as a gas when the compression is suddenly removed it may
collect as bubbles of gas in the blood vessels and by forming gas emboli
be one of the causes of caisson disease. The amount of nitrogen in the
blood of dogs, arterial and venous, for there is no difference usually
between the amount in arterial and venous blood, was determined by
Bohr to be 1.2 c.c. in 100 c.c. of blood. Recent determinations in the
blood of cats shows only 1.06 c.c. per 100 c.c. of blood. The air in the
lungs, alveolar air, contains about 83.67 per cent. by volume of nitrogen.
If this nitrogen pressure is reduced, the blood will lose N in passing the
lungs and the venous blood may temporarily have more nitrogen than
the arterial blood, due to the washing out of nitrogen from the tissues
of the body. Nitrogen dissolves readily in fat, and at normal tempera-
tures fat dissolves at least five times as much nitrogen as blood. The
nitrogen in the blood and tissues is inert and probably plays no part
in metabolism.

The amount of oxygen simply dissolved in the blood is small. It
may be directly determined for the plasma and serum, but for the whole
blood it is determined indirectly by finding the solubility of some inert
gas like hydrogen, which does not combine with the blood corpuscles,
and then multiplying the result thus found with the ratio of the solu-
bility in water of hydrogen to that of oxygen.

The solubility of oxygen in ox serum at 29.7° is 2.47 per cent. by
volume (0°, 760 mm.) or 94 per cent. that of its solubility in water.
The solubility of hydrogen in ox serum is 1.56 per cent. by volume, or
95.5 per cent. of its solubility in water.

The difference in solubility of these gases in water and serum is due
to the salts in the serum. A salt solution dissolves always less gas than
an equal volume of water. Indeed. by the addition of salt, gases can
be salted out of their solutions just as the proteins can be. Horse plasma
dissolved 94 per cent. of the oxygen dissolved by an equal volume of
water. The whole blood dissolved 91 per cent. as much hydrogen as an
equal volume of water. The difference is due in part to the salts
and in part to the volume of the dissolved protein and the solid
matter of the corpuscles. It is evident, since the corpuscles make
as a rule about 40 per cent. by volume of the blood, that hydrogen
must dissolve in the water of the corpuscles as well as in the blood
plasma.

b. How are the gases carried in the blood? Oxygen. Human arte-
rial blood contains in 100 ¢.c. such an amount of gas that it will yield
to a vacuum 18-19 e.c. of oxygen, 40 ¢c.c. of carbon dioxide and about
1 @e. of nitrogen, argon and other gases, all of these measured at 0°
and 760 mm. pressure. Such an amount of gas is vastly more than can
be dissolved in 100 c.c. of water, or in 100 ¢.c. of blood plasma. 100 e.e.
of water at body temperature and the usual pressure of oxygen, that is
- 476 PIYSIOLOGICAL CHEMISTRY

-a pressure of about one-fifth of an atmosphere, will absorb only 0.4 c.c.
of oxygen, and serum will absorb about 94 per cent. of this amount.
It is clear that oxygen must be combined chemically or physically with
something in the blood so that its solubility is increased. It is, as a
matter of fact, in greater part in union with the red coloring matter of
the blood, hemoglobin, with which it forms oxyhemoglobin. 0.744 gm.
Hb combines with 1 ¢.c. of oxygen, or 1 gm. of Hb combines with 1.34 ee.
of O,.

Carbon dioxide. Similarly 100 ¢.c. of water will absorb of carbonic
‘anhydride at the temperature of the body and under the pressure of one-
tenth to one-twelfth of an atmosphere, which is the pressure of CO, in
the tissues, only about 10 c.c. Carbon dioxide is in part combined with
the proteins of the blood plasma, and in part it is present in the plasma
as the carbonate and bicarbonate of soda. There is in the blood plasma
disodium hydrogen phosphate and sodium carbonate. When carbon
dioxide comes into a solution of these salts, it combines with some of the
‘sodium to make bicarbonate of sodium and is carried in the blood in
‘large measure in this form. The proteins, such as the globulins, are
also present in the blood as sodium salts. This sodium is removed from
the globulin by the carbon dioxide. The corpuscles, too, act in the
same manner as the globulins. They have sodium and potassium in them
in organic union, and when carbon dioxide is given to the blood in the
capillaries as it passes through the tissues, some alkali leaves the cor-
pusclés to saturate the carbon dioxide so that the plasma has its total
alkali increased by the action of carbonic acid, since the carbonates and
bicarbonates have an alkaline reaction, or at any rate are titratable like
alkalies. These various reactions by which the carbonates are formed
may be written as follows:

Na,HPO, + CO, + H,0 —~ NaH_PO, + NaHCO,
is Na. 200, +-C0, +H, o—— 2NaHCO,
Na. globulinate + CO, +H,0 NaliCO q+ Globulin.

1 Na lecithinate, etc., in the blood corpuscles + CO, +H,0 —~ NaHCO, + Organic
aii : compounds in a more acid state.

 

"+" All these changes take place when the blood is passing through the
capillaries in the tissues. By this means it will be seen that the acidity
“of the corpuscles of the blood -is increased, since they have lost alkali,
Na and K, to carbonic acid. The result of this increase of acidity is that
‘the power of the hemoglobin in the corpuscles to take up oxygen is
greatly reduced, as we shall see presently, and the entrance of carbonic
anhydride into the blood thus helps to turn the oxygen out of the blood
and into the tissues. This factor is of great importance in cold-blooded
» animals where the affinity of hemoglobin for oxygen is so great, owing
to the low temperature. that the pressure of the oxygen in the capil-
laries would be small. In the lungs, on the other hand, the opposite
THE BLOOD. THE CIRCULATING TISSUE 477

change takes place. With the passage of carbon dioxide outward alkali
is set free again, is taken up by the corpuscles of the blood and their
affinity for oxygen is so increased thereby that the blood saturates itself
with oxygen very quickly in its passage through the lungs. The oxy-
hemoglobin thus formed is presumably a stronger acid than hemoglobin
and thus helps turn carbon dioxide out of its union with sodium and so
out of the blood. This probably explains the well-known acid action of
the red blood corpuscles. This. change is associated with a change of
volume of the corpuscles. The volume in venous blood is larger than in
arterial blood. Water passes into and out of the corpuscles.

Carbon dioxide is probably also carried in the blood in union with
the proteins. When carbonic anhydride enters a solution of a protein
which has free amino groups, the acid unites with these groups to make
carbamino compounds (carbamino reaction of Siegfried). This reaction
is the following:

NH, NH—COOH
| |
R—CH +H,CO, —+ R—CH +H,0
2 oe
boon cooH

Carbamino compound

Protein. of the protein.

These compounds are dissociable and the carbonic acid is easily
recovered from this union. In all these ways, then, is the carbon dioxide
carried back to the lungs. For the most part it is in solution in the
plasma as carbonate, bicarbonate and protein compound, but some of
it also is united with the corpuscles, presumably with the proteins of
these structures (hemoglobin). A part of the carbon dioxide is dis-
solved as such in the blood. All these forms of carbon dioxide are in
equilibrium with each other and with free carbonic anhydride, so that
if the latter escapes more is set free to take its place, being dissociated
from some of these unstable compounds. In the tissues the pressure of
earbon dioxide is higher, 11 per cent. of an atmosphere, than in the
blood, so that carbonic anhydride enters the blood until it is under
equilibrium with this pressure of the gas. But when the blood reaches
the lungs the pressure of carbon dioxide in the air in the lungs is low
and carbon dioxide escapes from its solution and into the alveolar air.
As soon as some of it escapes, more is set free from its union with
alkali and protein in the blood. The pressure of carbon dioxide in the
alveolar air in the lungs is about'5-6 per cent. of an atmosphere, whereas
in the blood as it leaves. the tissues it is about 8-10 per cent. Conse-
quently in passing the lungs carbon dioxide is given up. The equilib
rium may be represented as follows:
478 PHYSIOLOGICAL CHEMISTRY

Tissues CO = 10% of an atmosphere pressure. Alveolar air of lungs CO, =
5-6% atmosphere.

£0, +H 0S h.e0.
H,CO, ++ Na,CO,>—2NaHCO,
H,CO, + Na,HPo, =—= NaHCO, + NaH,PO,
H,CO, + NaProteinate ——" NaHCO, + H proteinate.
HCO, + Protein —— Protein COOH + H,0

Carbamino compound.
The reactions go in the right-hand direction in the tissues and in the
left-hand direction in the lungs. Since the corpuscles and proteins have
the power of combining with the sodium hydrate set free when the car-
bonie anhydride escapes in the lungs, they act as acids. It is for this
reason that it is possible to pump nearly the whole of the carbon dioxide
out of blood by means of a vacuum, whereas it is not possible to pump
carbon dioxide to the same extent from a solution of sodium bicarbonate.
From a solution of bicarbonate of sodium one can pump the carbon
dioxide until sodium carbonate is formed. Thereafter the decomposi-
tion is almost immeasurably slow. A change occurs in the volume of
the corpuscles when CO, enters them. They swell. This change is very
significant, since it shows how small a change in acidity is required to
cause swelling changes in vital structures. It is possible that it is the
same process which is at the bottom of the contraction of muscle, as we
shall later see. It is besides a true process of secretion of water into
and out of the corpuscle, and is also a rhythmic process. When CO,
increases in the corpuscle water enters it; when CO, is diminished water
leaves, the sodium ion re-enters and the corpuscle shrinks. There is an
exchange of sodium ions and hydrogen ions back and forth between the
corpuscle and the plasma.

ce. The mechanism of the entrance of oxygen into the blood and the
passage of CO, outward. The exchange in the lungs. We may now ask
the question cf the manner in which oxygen passes through the alveolar
membrane into the blood. Is it by a simple physical process of diffusion,
or is it by the active secretion of the lung tissue; or do both these
processes occur ?

The answer to this question, so important in medicine, cannot yet
be given with certainty.

‘The walls of the alveoli of the lungs are extremely thin. They are
composed of flattened plates. There is no doubt that in the lower forms,
such as the amphibia, these plates are true cells and composed of living
tissue; but there is a difference of opinion whether the plates in the
mammalia are living or dead, and whether or not they have nuclei.
Besides the very thin layer of alveolar plates, the gases must pass also
the endothelium of the capillaries, which is also very thin, but certainly
alive. The oxygen might enter either by diffusion, or one or both of

op Y wy P
THE BLOOD. THE CIRCULATING TISSUE 479

these membranes might intervene actively in the process. If they do
so intervene, they would probably be controlled by the nervous system.

The solution of this question of the method of entrance of the oxygen
was approached in the following form: if the pressure of oxygen in the
arterial blood as it comes from the lung is always lower than the pres-
sure of oxygen in the alveolar air, then the process is probably one of
diffusion and the membranes presumably do not actively intervene in
it; if, however, the pressure of oxygen in the blood is ever higher than
that of the alveolar air, then the exchange cannot be a simple physical
process of diffusion. The first requisite for the solution of this problem
was to find a method of estimating the oxygen tension of the blood in
the arteries and of the air in the alveoli.

This tension is measured in two ways, a direct and an indirect. The
direct method has been most frequently employed. The aérotonometer
is an instrument designed for this purpose. The essential principle of
the aérotonometer is the following: The blood is introduced directly from
the blood vessels into an atmosphere of nitrogen, carbon dioxide and
oxygen and allowed to remain in contact with the gas in a thin layer so
that equilibrium is attained. The gas is then analyzed.

The principle of the method used by Krogh is to shake a small air
bubble in a very small amount of the blood to be examined. As little
as 1 ¢.c. blood may be used. The gases in the air bubble come quickly
into equilibrum with those of the blood. The amount of oxygen is meas-
ured by the decrease in volume when the oxygen is absorbed by alkaline
pyrogallate or some other oxygen-absorbing fluid, such as acid hypo-
sulphite.

All measurements which were made with this instrument resulted
uniformly in showing that the pressure of oxygen in the alveoli was
slightly greater than the pressure in the arterial blood.

The matter thus seemed settled, but in 1899 Bohr? got the first defi-
nite evidence that the process was not one of simple diffusion. He
observed in a few cases that the oxygen pressure in the arteries was
higher than that in the alveoli. His results obtained by the aérotonom-
eter method were so irregular as to suggest errors of manipulation and
they have been seriously criticised by Krogh and by Haldane and Doug-
las. The aérotonometer is very sensitive to a change of temperature, and
an accidental variation in this might have accounted for his results.
Moreover, in one or two cases, as Haldane and Douglas point out, the
results are so improbable as to indicate error very plainly. In spite
of these defects, Bohr’s paper served the end of reopening the ques-
tion. The possibility of a definite secretion of oxygen by the lungs also
gained in probability by the discovery of the high oxygen content of the

air bladder of fishes, the organ from which the lungs were evolved. In
* Bohr: Skan. Archiv f. Physiol., 2, 1890, p. 236.
480 PHYSIOLOGICAL CHEMISTRY

1907 Bohr afforded other evidence of the secretory activity of the lungs.
He found that when pure air was breathed by one lung and air con-
taining 8.8 per cent. by volume of CO, by the other, CO, was still given
off from the lung breathing the CO, mixture, although the pressure of
CO, in the venous blood from the right side of the heart was that of an
atmosphere containing only 5 per cent. of volume.

It will be noticed that the pressure of CO, was determined in the
heart blood, and the pressure in the lungs was supposed to be equal to
this. However probable this assumption is, it weakens the proof.

Recently Haldane and Smith also got some evidence of the existence
of a secretory activity of the lungs, but their work contained so many
assumptions and possibilities of errors of fact and interpretation that
not much weight can be given it. A more recent paper will be con-
sidered presently.

Krogh ' has recently re-examined the whole question. He and Mrs.
Krogh measured with great care by means of the micro-aérotonometer
the pressures of oxygen and CO, in the lungs and the arterial blood.
They found always that the pressures of CO, in the arteries and in the
alveolar air were equal, the result which the diffusion theory demands.
The oxygen pressure in arterial blood was always slightly less than the
pressure in the alveoli, a result also in accord with the diffusion theory.
These experiments give no evidence of a secretory activity on the part
of the lung.

The matter is not yet settled, however, for Douglas and Haldane *
have made a very complete study of the matter recently and obtained
very interesting results.

Their method of measuring the arterial oxygen pressure was an
indirect one. It consisted in partially saturating the blood with CO
gas. When blood or hemoglobin is exposed to a mixture of O, and CO,
the hemoglobin takes up some of each and the relative amount depends
on the partial pressures of the two gases. But always far more CO than
O, is held by the hemoglobin at the same pressures. If a person is
made to breathe air containing a small per cent. of CO, the blood ulti-
mately, after 30 minutes about, has taken up all the CO it will. If now
the per cent. of saturation of the Hb by CO can be determined, then the
amount of oxygen in the arterial blood can also be determined, since
from the per cent. of saturation of hemoglobin by carbon monoxide the
tension of oxygen necessary to prevent total saturation by carbon
monoxide and to permit only the per cent. of saturation actually
observed can be calculated. This calculated oxygen tension is assumed
to be present in the blood. To determine the per cent. of saturation of

Krogh: Shan. Archiv f. Physiol. xxiii, 1910, p. 274.
2 Douglas and Haldane: Journal of Physiology, 44, 1912, p. 305.
THE BLOOD, THE CIRCULATING TISSUE 481

the Hb by CO a sample of the blood is drawn and its tint, when diluted,
is compared with a carmine solution which has previously been standard-
ized against blood completely saturated with carbon monoxide. From the
amount of dilution of the carmine solution the per cent. of saturation of
the Hb can be caleulated, Column 3, and from that the arterial pressure
of oxygen can be computed, Column 5.

The results obtained by Douglas and Haldane are illustrated in the
following protocol. Mice breathed air mixed with varying amounts of
CO. They were then drowned and two drops of blood taken from the
heart for analysis. The inspired air contained on the average 19.79 per
cent. of O, and 0.29 per cent. CO,; the alveolar air contained 14.06 per
cent. O, and 5.64 per cent. CO,.

 

 

1. 2. 3. 4. 5.
Per cent. saturation of Hb i joni
Per cent. of CO Duration of with co arene Og tension ja
in inspired expt. per cent. of an atmos-
air minutes phere calculated
in vivo in vitro from 3
0.016 60 26.2 17.2 12.2
0.018 45 26 18.5 13.5
0.046 40 29.1 22.7 15.0
0.053 : 40 37.7 30.2 16.2
0.100 32 45 43.0 19.3
0.129 31 56.4 56.3 20.8
0.213 13 59.1 75.5 44.7 (Animal died)
0.244 12 67.3 71.7 25.7 (Animal died)
0.262 20 66.4 73.7 28.2
0.275 25 66.5 76.9 35.9

 

 

 

 

 

The experiment shows that as long as hemoglobin was not more than 30
per cent. saturated with carbon monoxide, the pressure of oxygen in
arterial blood (12-15 per cent.) was less than that in the alveoli; but
when the per cent. of saturation of the hemoglobin with carbon monoxide
was more than this, the calculated arterial tension was always higher
(16-36 per cent.) and might be over 100 per cent. greater than that of
alveolar air. The per cent. of saturation of the Hb by CO was less in
vivo than in vitro.

A similar result was obtained for human beings when breathing air
containing varying amounts of oxygen. The experiment was tried on a
man breathing in a closed system. While resting and breathing air con-
taining normal amounts of oxygen, the tension of oxygen. in the alveolar
air being from 12-15 per cent. and that of CO, 5.6 per cent., the arterial
blood had an oxygen tension varying in different experiments from 91.6-
104.4 per cent. of the tension in the alveolar air. In other words, it was
only once found to be higher than the tension in the alveoli, but was
generally lower as the diffusion theory demanded. The same result was
obtained when the subject was resting and breathing air containing more
than the normal amount of oxygen. When, however, the amount of
482 PHYSIOLOGICAL CHEMISTRY

oxygen was reduced so that the per cent. of oxygen in the alveolar air
was lower than normal, the pressure of oxygen in the blood was larger
than the tension in the alveoli; the difference was particularly large
when work was done.

 

 

wes eee eee : Oy tension CO, tension
arterial Op tension in per cent. of aly eolar alveolar
alveolar oxygen tension

115.4 : 5.98 4.93

121.6 6.99 4.78

128.1 6.40 3.48

112.1 5.53 4.74
Moderate work. One arm

124.8 12.82 5.39
Severe work. One arm

131.7 15.11 5.33

135.0 15.49 5.69

128.0 11.64 5.22

128.0 19.38 4.43
Moderate work. One arm

147.6 10.20 3.30

 

 

 

There is no doubt, therefore, that in normal circumstances during rest
oxygen enters by a process of diffusion; or at least there is no evidence
of any secretory activity by the alveolar endothelium. During work,
however, or when there is a deficiency of O,, the pressure of O, in the
arteries rises far above, to 185 per cent., that in the alveolar air. It
appears from these experiments, then, that the lungs may, when there
is necessity, actively secrete oxygen into the blood. This discovery, if it
be sustained, is evidently a very important one. The way in which the
lungs are aroused to activity when the tissues need oxygen is still obscure.
It may be either by way of the nervous system or by some metabolic
products of the tissue activity. The authors state that it is certainly not
by means of CO,, or lactic acid, since these leave the process practically
unaffected.

Conclusive though these experiments seem, they are not completely
so, and the whole question must still be regarded as open. This is
owing to the fact that in any indirect method of determining the pres-
sure there are always many assumptions, some of which cannot easily
be tested. In this method the following assumptions are made: First,
that the colorimetric method of estimating the degree of saturation of
the hemoglobin is reliable. A better method has been devised by Hart-
ridge. But, even if the degree of saturation is correctly determined,
the inference that the rest of the hemoglobin is combined with oxygen,
or uncombined, is not proved. It is possible that hemoglobin unites with
many other substances than gases. If so, these substances may be present
in the blood in larger amounts than usual under the conditions of the
experiment when there is partial asphyxia.
THE BLOOD. THE CIRCULATING TISSUE 483

Another possible source of error in this indirect method of deter-
‘mining the oxygen tension of the blood is this. What is actually deter-
mined is the amount of carbon monoxide hemoglobin in the blood. From
this figure one calculates how large the tension of oxygen must be in
order to prevent the hemoglobin from taking up more carbon monoxide
than it does. The assumption that is made in this is that the avidity
of the oxygen and hemoglobin undergoes no change in the course of the
experiment, but remains the same as in the experiments in vitro. This
assumption may not be correct. In the experiment oxygen and carbon
monoxide are quarreling for the hemoglobin. The power of the oxygen
is measured by its success in the struggle under certain conditions. But
let it be supposed that in times of stress, as in partial asphyxia, the body
has the power of strengthening the hands of the oxygen; it might then
wage a very much more successful struggle for the hemoglobin than
before and displace more of the carbon monoxide. There are reasons
for thinking that the body does possess just this power, because it
forms oxidases which hasten oxidation when it needs oxygen. The
formation of oxyhemoglobin is a process of oxidation. It is possible,
therefore, that the smaller saturation of the hemoglobin by carbon
monoxide in partial asphyxia is not due to the fact that the tension of
the oxygen has been increased, but that the efficiency of that actually
present has been increased in its oxidizing power by the oxidases. Per-
haps the proportion of active oxygen molecules is increased. It would
seem unlikely that oxidases should play no part in the oxidation of such
an important substance as hemoglobin. This possibility should be
investigated. That there is something in its favor is shown by the fact
that a person exposed to a lew oxygen pressure, if the pressure is not
too low, shows a betterment of condition when slight work is done. The
asphyxia seems somewhat relieved by the work. Perhaps by the activity
of the tissues more of the oxidase is produced.

d. Nature of the union of hemoglobin with oxygen. There is little
doubt that the union between oxygen and hemoglobin is chemical in
nature. This opinion was almost universally held until W. Ostwald sug-
gested that the union was one of adsorption. If the union is chemical.
then if the per cent. of saturation of the hemoglobin is plotted along
the ordinate and the tension of oxygen along the abscissa, as is done in
Figure 46, the curve of saturation of the hemoglobin by oxygen should
be a rectangular hyperbola. Bohr did not find this to be the case. The
cause of the discrepancy was investigated by Barcroft, who found that
if the hemoglobin solution was thoroughly dialyzed so as to rid it com-
pletely from salts, then the curve was a rectangular hyperbola, as the
theory demanded.

That the union is chemical is shown also by Barcroft and Hill. The
rate of reduction of HbO, by nitrogen was strongly influenced by tem-
484 PHYSIOLOGICAL CHEMISTRY

 

Fig. 46A.—Curve of dissociation of oxyhemoglobin showing the effects of various ealtz. I. 0.7% NaCi
II, NaHCO,; Ill, Na,HPO, Bicarbonate and Na,HPO, concentration equivalent to NaCl. Ordi
Tate : % saturation of Hb. Abscisea: tension of Oz in mms. Hg.

   

B

Fie. 46B.—Dissociation curver of sheep blood at various tensions of COs, V at 5;IV at 10; IIL at
20; 11 at 40; and 1 at 80 mm. Hg. tension. Numbers below curves show actual (Os tensions ob-
served Temperature 87-38° C. Ordinate: % saturation of oxyhemoglobin. Abscissa: tension of O2 in
mms. Hg (Barcroft and Camis).
THE BLOOD. THE CIRCULATING TISSUE 485

perature, going on at a much more rapid rate at a higher than at a
lower temperature and the temperature coefficient between 38° and 18°
is 3.7 for 10°. This indicates strongly a chemical union, for asa rule
physical processes have much lower temperature coefficients than this,
which is that of a chemical reaction. They also determined the heat set
free when one gram of Hb was oxidized to HbO,. They found for 1
gram Hb 1.85 calories. From this they calculated the molecular weight
of the hemoglobin as 15,200, which is about that found by Hiiffner, if
one molecule of oxygen combines with one molecule of hemoglobin and
if there is one atom of iron in the molecule. The process is evidently a
limited, or partially consummated, oxidation process. One molecule of
hemoglobin combines with one molecule of oxygen and a‘certain amount
of heat is liberated in this process. We see, therefore, that to this extent
at least Lavoisier was right and that some combustion really takes place
in the lungs. There is some heat liberated there.

We have already considered on page 256 the physical chemistry of the process
of oxidation, but we may at this point consider the application of those principles to
the oxidation of hemoglobin. The velocity of the oxidation is then proportional to
the concentration of the active oxidizing agent, to the concentration of the active
reducing agent, and to the time required for the passing over of the positive charge
from the oxidizing (O,) to the reducing (Hb) body. This last factor, which varies
so enormously in different oxidations, is in the case of hemoglobin in ordinary cir-
cumstances very long. It is much longer than oxyhemoglobin ordinarily exists before
the reaction is reversed in the tissues. Hence HbO, is stable for a considerable
period. This molecule has quite a long span of life. Nevertheless it is stable only
under very narrow conditions and a change of alkalinity sufficiently great causes
the consummation of the oxidation and the formation of methemoglobin, smal]
amounts of which are present in normal blood and which under pathological con-
ditions when nitrites, chlorates, aniline and many drugs are taken, is formed in.
large amounts. Hemoglobin is a substance combining easily with oxygen, but in
which the oxidation does not go to a conclusion.

The velocity may be written in the form of an equation:

d(HbO,) /dt = KO%x 0,

C* is the concentration of the active oxygen, that is oxygen in a condition tc
9 : zi : ; aot
unite; and Co is the concentration of the active hemoglobin. The reaction is

1. Hb2—Hb”
2. 0,-—=0,”

a Bb 04 == Hp,

The point of equilibrium and the velocity of the reaction will be determined not
by the total concentration of the Hb and the O,, but by the concentration of the
active molecules present. Now most oxidations have these two peculiarities: They
are accelerated by light, particularly by ultra-violet light, and they are all de-
pendent upon water. In light, therefore, the per cent. of saturation of the hemo-
globin at a given pressure of oxygen should be higher than in darkness and the
velocity of the oxidation should be greater.

A second important fact in oxidation is the cule of water. Substances do not
oxidize in the dry state. The probable explanation of this fact, or at least one
486 PHYSIOLOGICAL CHEMISTRY

explanation, is that given in the case of bromine oxidations (p. 260). It is almost
certain that when bromine oxidizes the active agent is not bromine itself, but a
positive bromine ion, which is formed by the interaction of bromine and’ water as
follows:

1. Br, + HOH-—HBr + HOBr

+ -
2. HOBr=—-Br + OH

The Br+ set free is a powerful oxidizing agent and the speed of the reaction is
probably proportional to its concentration. The oxidation by copper and oxygen is
probably very similar as already discussed on page 260. We probably have the
reactions:

3. Hb” + O++(OH) ——"HbO, + H,0

Heat is liberated in the last reaction.

The condition of the hemoglobin must also be a great factor in the speed of the
exidation. All the evidence we have shows that reducing bodies are not always in
a state to receive the oxidizing body and as a rule the condition of ionization of the

100
90
80
70
60
50
40
30

20

          

° 20 30 40 50 400 110120

Fic. 47.—Effects of temperature on rate of reduction of sheep's blood by hydrogen.
(Oinuma). Ordinate: per cent of saturation with oxygen; abscissa: time in minutes.

reducing body is of great importance. Now Hb is probably a salt of a metal, sodium
or potassium, and the condition of-the iron, with which the oxygen is combined, will
probably be found to be a function of the particular metal in combination with the
hemoglobin.

It will be seen then from the foregoing equations that the speed of oxidation
and the per cent. of saturation of the hemoglobin with O, will depend in the first
. instance on the alkalinity, or number of hydroxyl ions. Hence an increase in the
alkalinity of the blood will cause Hb to take up O, faster and hold a larger propor-
tion of it since this increases the active mass of the oxygen; and acidity will have
an opposite effect, causing the HbO, to give up O,, hastening the reduction and
lowering the point of equilibrium when saturation is reached. The effect of tem-
perature, since the reaction is exothermic, will be to increase the dissociation as
the temperature rises. Alkalies may also affect the active mass of the Hb. These
theoretical conclusions are borne out by experiment.
THE BLOOD. THE CIRCULATING TISSUE 487

e. Factors influencing the dissociation of O, from HbO,. Bohr
opened this subject by his discovery that carbon dioxide strongly influ-
enced the dissociation curve of HbO,. Hemoglobin takes up less O,
as the tension of CO, increases; and HbO, gives off O, much quicker in
the presence of CO,. This is a matter of great importance in the body.

 

10 20 30 40 50 60 70 80 90 1¢0

Fig. 48.—Curves illustrating the dissociation of oxyhemoglobin of sheep blood on the
addition of various amounts of lactic acid (Barcroft and Orbeli). The upper curve repre-
sents the per cent. of saturation of normal blood when the oxygen tension in mm. is that
be pena along the abscissa. The two lower curves show the effect of the addition of
lactic acid.

The studies of Barcroft and his associates have shown the infiuence of
alkalies, acids, salts, temperature and light on the dissociation of this
oxide.

1. Effect of temperature on dissociation. The per cent. of satura-
tion of hemoglobin in air and at differerit temperatures was determined
by Barcroft and Hill as follows:

Dog’s hemoglobin.
Saturation (%) .....cces esses ee eee eee 96 89 77 52
Temperature (°) ............ sie abacus reves 16 25 32 38

2. Acids and alkalies. Alkalies increase the speed and the per cent.
of saturation under a given oxygen pressure and acids have the oppo-
site effect. This is shown in the following figures from Barcroft and
Camis and in figure 48 and 46A:
488 PHYSIOLOGICAL CHEMISTRY

Per cent. saturation of hemoglobin in water ........ 29 40 60 77.5 90.5
Tension 0, AMM +509 sees oss eunis e's Gere has BAe ese Ue 12.5 15.5 31 45 72

Per cent. saturation Hb alkaline with (NH,) 29, .. 785 79 92.5 97 99
Tension O, in MM, ....... 1. esses eeeeee cere eee erees 20.5 21 305 37 59.5

It will be seen that in water at a tension of 50 mm. Hg hemoglobin is
less than 80 per cent. saturated. In ammonium carbonate solution it is
98 per cent. saturated. The effect of carbon dioxide in reducing the
saturation of the hemoglobin is shown in the figures and curves in
Figure 46B:

Washed dog’s corpuscles in Ringer’s solution.

Tension of co, AT MAMAS 9 Sis cesdatys.a Sia diadt-ae eases eaceeras 2 5 76 69
Tension of 0, AT MIM ie Geese gte wasn saecenes Ps MeN es ae 18 19 17 18
Per cent. of saturation of Hb .............-eee eens 55 57 6 9

Sheep blood at varying CO, tension.

Tension of 0, in mm....... 10 15 20 30 40 50 60 70 80 100
Per ) 5 mm. tension co, 28 35 47 585 89 95 96 98 99 99.5
cent. 10 “ se “11 26 38.5 63 83 91.5 94.5 96.5 97.5 98.5
satura- es x ee e 0 10 25 53.5 845 90 93 95 97.5
tion of | 40 “ e oe 0 O11 425 77 = 83.5 88.5 93 95.5 .
Hb. ) 80 “ ss a 0 0 1 33 69.5 77 83 87.5 92.5

3. The effect of salts on dissociation. The action of salts is also
very important. Human blood corpuscles contain more potassium salts
than sodium, whereas dog’s corpuscles contain more sodium. Potassium
salts are particularly efficient in increasing the per cent. of saturation of
the Hb. The salts in the corpuscles, or the nature of the base in union
with the Hb, is, therefore, of considerable importance in this exchange.

At a tension of 50 mm. of oxygen the hemoglobin in solution in
0.7 per cent. NaCl is 85.5 per cent.; in 0.9 per cent. KCl it is 95 per cent.
saturated ; and in Na,HPO, it is more than this. It is probable that this
difference in salt content explains the difference in saturation of differ-
ent bloods when exposed to the same tension of Q,.

4. Other factors possibly wmfluencing the dissociation of HbQ,.
There is, in addition to the factors already mentioned as controlling
the union of Hb with O,, one other which has been so far neglected, but
which was mentioned on page 483. It may be that there are in the plasma
or corpuscles substances which may facilitate the union of hemoglobin
with oxygen. It would be interesting to see what influence small traces
of iron might have on this process. It is said that small amounts of iron
are constantly getting free from the Hb, particularly from reduced
hemoglobin. Indeed, Bohr suggested that the iron was alternately set
free and reunited with the Hb, but such is probably not the case. The
matter should be further studied.
THE BLOOD. THE CIRCULATING TISSUE 489

f. Biological significance of factors influencing HbO, dissociation.
The general biological significance of the facts thus described for blood
is very great. We may indeed take hemoglobin as a type of a sub-
stance uniting with oxygen. Substances having the power of uniting
with oxygen are found in all cells of the body, and it is probable, since
the dissociation of the cell from oxygen is a matter of a good deal more
difficulty than the dissociation of HbO., that these substances hold their
oxygen a good deal more firmly than the oxygen is held by Hb. They
do not easily give up their oxygen to a vacuum. The tension of oxygen
in the tissues is very low. There are many reasons for thinking, how-
ever, that oxygen storage may occur there. The protoplasm is made up
of reducing substances. We may be certain, in any case, that the oxida-
tion of a cell, like that of hemoglobin, will be profoundly affected by
sunlight, temperature, alkalinity and acidity and by salts. And indeed
all our facts prove this to be the case. Oxidation is facilitated in the
light ; by fevers or high temperatures ; by slight alkalinity ; and by vari-
ous salts.

The acidity produced by carbon dioxide and lactic acid is very impor-
tant in turning oxygen out of its union with hemoglobin when the blood
reaches the tissues. This must play a great part in cold-blooded animals,
where at low temperatures the oxygen-hemoglobin compound dissociates
very little. Carbon dioxide does not in the frog find its way out through
the lungs, but through the skin. Perhaps it is kept in the body for this
purpose.

g. The exchange in the tissues. While the exchange in the lungs
has been supposed by some to involve secretory activity on the part of
the plates of the lung endothelium, the exchange in the tissues is believed
to be due only to processes of diffusion. The pressure of oxygen in the
tissues is less than that in the capillaries and the pressure of carbon
dioxide in the tissues is greater than that in the blood. So that there
is no reason for supposing that any other factors than those of diffusion
piay a part in this exchange.

The clood as it leaves the tissues is still rich in oxygen. It is never,
under ordinary circumstances, completely reduced. Indeed, venous
blood still contains a large proportion of its oxygen. Analyses of blood
coming from different organs show differences in this respect as might
be anticipated, but the following table illustrates the composition of the
gases of the venous blood from various organs; the figures are c.c. for
100 c.c. of blood:

Organ Blood co, 0, Observer
Submaxillary Arterial 53.1 15.2 Chauveau and Kaufmann
gland resting Venous 55.2 11.4 (Barcroft: Ergebnisse 7)

Leg of dog { Arterial 21.92 14.4 Zunst
muscle resting { Venous 36.32 1.2
490 PHYSIOLOGICAL CHEMISTRY

Organ Blood co, 0, Observer
Supra-renal Arterial 21.79 Chassevaut and Langlois
gland ( Bareroft)
Brain Arterial-carotid 40.86 16.82 Hill and Nabarre
Venous (Mean values)

(Torcula Heroph.) 44.74 13.39
The following table from Barcroft shows the consumption of oxygen
in ¢.cm. pro gram per minute by various tissues:

OxyGEN CONSUMPTION PRO GRAM PER MINUTE BY RESTING TISSUE

Name of tissue Oxygen consumption Animal Observer
Skeletal muscle .0037 c.c. Horse Chauveau and Kaufmann
Heart muscle 010 Dog Bareroft and Dixon
Salivary glands 028 Dog Barcroft and Dixon

023 Cat Barcroft

Pancreas -03-0.05 Dog Barcroft and Starling
Intestinal canal 023 Dog Brodie, Halliburton and Vogt
Kidneys 026 Dog Barcroft and Brodie

h. Is the respiratory pigment as tt exists in the blood itself hemo-
globin? Does hemoglobin as it exists in the blood differ in its properties
of oxygen absorption from isolated hemoglobin? Bohr thought that it
did. He supposed that there were distinct differences between hemo-
globin and the blood pigment, which he called hemochrome. The reason
for this opinion was that solutions of hemoglobin in water were found
by him to have a different curve of dissociation of the HbO, compound
than the curve for the dissociation in blood itself. But Bohr overlooked
the fact that the dissociation curve depends on the amount and character
of the salts present. Barcroft and Camis showed that a solution of dog’s
hemoglobin, to which had been added the salts found in dog’s corpuscles,
gave a curve of dissociation like that of dog’s blood; and that if to dog's
hemoglobin salts, like those in human red corpuscles, were added a curve
of dissociation was obtained like that of human blood. It appears, there.
fore, that there is no reason to suppose that the pigment as it exists
in the blood is different from hemoglobin. There is no evidence, in
other words, that the shape of the corpuscle, its wall or other properties
play a part in the process of the union of hemoglobin and oxygen. Blood,
in virtue of its alkali salts, has, however, a great advantage over a simple
solution of hemoglobin in water. Thus at 30 mm. oxygen pressure
hemoglobin in aqueous solution is only 62 per cent. saturated; whereas
blood at the same pressure saturates itself to 69 per cent., owimg to the
potassium salts in the corpuscles. Hemoglobin in the corpuscles is, how-
ever, almost certainly in chemical union with the stroma, so that the
oxygen-carrying substance in the blood is in reality stroma-hemoglobin
compound and not free hemoglobin.

i. Respiration of the blood itself. Does the blood, then, not respire
itself? Does it consume no oxygen and give off no CO,? While the
great bulk of the oxidation occurs in the tissues, there can be no doubt
that a certain amount occurs in blood itself. Blood is a living tissue.
THE BLOOD. THE CIRCULATING TISSUE 491

The white cells in it certainly respire, and the red corpuscles probably do
also to a limited extent, since they contain in their stroma oxidizable
substances. But the rate of their respiration is undoubtedly small. Not
so, however, with the white or nucleated cells both white and red. Par-
ticularly after hemorrhage the blood shows a considerable power of oxi-
dation of itself, and also during asphyxia, or whenever the tissue decom-
position is greater than the tissue oxidation can burn, these substances
get free in blood and in part burn there. Thus Bohr found that the
ratio of O, to the iron of the blood underwent marked changes after
hemorrhage, so that he suggested that there was more than one kind of
hemoglobin in the blood. But it has since been shown (Douglas) that
the oxygen capacity of the blood after hemorrhage is exactly propor-
tional to its hemoglobin content, so that there is no change in the char-
acter of the hemoglobin. It has been found, too, that the blood of rab-
bits made anemic (Morawitz and Pratt) by repeated injections of phenyl
hydrazine or repeated hemorrhage has a remarkable power of absorption
of oxygen and production of CO,. The following figures show the con-
sumption of oxygen by rabbit blood after repeated hemorrhage:

0, Per cent. co, Per cent.
After: a@ration: 26-6. eo eee saad vaewed 8.7 30.8
Incubated % hour .............-.00- 6.0 34.8
& DY BE eg ecadutes: cis essyannans Beenie 2.8 36.0
“ Ds A A ad eerie 0.3 39.1

Ae IES acta Pernt sane cath 39.9
Respiratory quotient CO,/0, = .91.

The main factors in this consumption are the white and the nucleated red
blood corpuscles. That the oxygen-consuming power is found chiefly
in the white corpuscles can easily be made evident by centrifuging
defibrinated blood. It will generally be observed that the red color
immediately beneath the layer of white corpuscles which rests upon the
red corpuscles is that of reduced hemoglobin. In the blood of inverte-
brates the corpuscles are in many cases altogether white corpuscles. In
Limulus, the king crab, there is a blood pigment which 1s blue when
oxidized and colorless when reduced. This pigment corresponds to
hemoglobin, but contains copper in place of iron. It is called hemo-
eyanin. It will be observed in this blood if it is allowed to clot that the
blood is white or reduced, except in the upper layers of the clot, where
it comes in contact with the air.

j. Evolution of hemoglobin. The evolution of hemoglobin is of
interest. Iron is found in all forms of living matter and in all it plays
perhaps a predominant réle in oxidation. In the course of evolution an
iron compound was evolved which, while permitting the iron to take
up oxygen, was not itself oxidized by it. It remained. therefore, an
easily reduced, but otherwise fairly stable, oxide. This substance is
492 PHYSIOLOGICAL CHEMISTRY

hemoglobin. It is found very low in the animal kingdom in annelids.
nemertines and mollusks. In the lowest forms and in its primitive con-
dition it is a constituent of the muscles, just as it, or a closely allied
substance, is found in many vertebrate muscles to which it gives a red
coloration. Here it plays its primitive réle of a storer of oxygen. The
next step consisted in having it free in the circulating fluid as it occurs
in the nemertines, so that it could obtain oxygen at the surface of the
body and bring it back to the tissues. Finally we have it inclosed in
corpuscles, where it may be surrounded by salts, which are pariicularly
useful for its functions, but which, if at large in the blood stream, would
be harmful to the organism. It is possible, also, that the concentration
of hemoglobin in the blood can be increased by this means above that
which is possible by simple solution; and finally it may be that the wall
of the corpuscle has been particularly evolved to make a membrane
which, like the gills of the fish, will let gases through readily. but will
prevent the entrance of many substances which might combine with
hemoglobin. It is possible that there are in the tissues other colorless
protein or other compounds to which oxygen is more firmly attached
than it is in the hemoglobin and which serve the purpose of storing
oxygen in the tissues. The existence of such compounds can hardly be
doubted ; some have been described (Griffiths) ; and they are supposed
by many physiologists to play a great réle in anaérobic respiration.

k. Other compounds of hemoglobin with gases. Hemoglobin com-
bines with many other substances than oxygen, and perhaps one advan-
tage of placing it within corpuscles may be to protect it from such other
substances. They may find difficulty in entering the blood cell. Indeed,
it may be that the envelop of the red blood corpuscle has been devised
to permit the easy passage of CO, and oxygen through it, but to resist
most of the other food and metabolic products of the body. Among
the substances readily penetrating the red blood corpuscles is carbon
monoxidé, CO, which is found in illuminating gas. This substance is
either far more reactive than oxygen or else it forms a firmer compound
with the hemoglobin. Probably the latter is the case. The HbCO com-
pound, carbonyl-hemoglobin, dissociates less readily than oxyhemoglobin,
or else the active mass of the CO, that is the proportion of molecules in
a condition to unite with hemoglobin, is greater in it than in oxygen.
When blood is exposed to a mixture of carbon monoxide and oxygen or
air hemoglobin takes up by preference the carbon monoxide so that even
though there is little carbon monoxide present, as compared with oxygen,
blood saturates itself to a considerable extent with carbon monoxide. If
hemoglobin becomes 50 per cent. saturated with CO, the life of an animal
is endangered. Tn human blood 50 per cent. saturation of the hemoglobin
with carbon monoxide occurs at room temperature in the presence of
THE BLOOD. THE CIRCULATING TISSUE 493°

air containing about 0.05 per cent. by volume of CO gas. It is for this
reason that the presence of even small amounts of carbon monoxide in

a

   

 

A Bot

Fig. 49.

EXPLANATION: Spectra 1, 2, 3 and 4, Oxyhemoglobin of various degrees of coxcentra-
tion; Spectrum 5, Hemoglobin; Spectrum 6, CO-Hemoglobin; Spectra 7 and 8, Hematin
in alkaline solution of different degrees of concentration: Spectrum 9, Hemochromogen
(Stokes’ reduced hematin); Spectrum 10, Methemoglobin: Spectrum 11, Acid hematin
(blood treated with acetic acid) ; Spectrum 12, Acid hematin in ethereal solution; Spec-
trum 13, Acid hematoporphyrin; Spectrum 14, Alkaline hematoporphyrin.

the air of houses is so detrimental to health. The bloods of different
animals show different powers of saturation with carbon monoxide when
they are exposed to the same mixture of air and carbon monoxide. and
494 PHYSIOLOGICAL CHEMISTRY

there is a variation also in different species of the same animal. This
fact has not yet been explained. It may be due to the fact that the
saturation of the blood by oxygen is dependent upon various factors
which do not affect the carbon monoxide. Thus the percentage of satura-
tion of the blood by oxygen is dependent upon carbon dioxide, alkalinity
and lactic acid, whereas the dissociation of carbon monoxide hemoglobin
does not appear to be much if at all affected by the presence of these
substances. It may be, therefore, that the salts of the blood corpuscles
being different in different animals determines that the per cent. of
saturation of the hemoglobin with oxygen shall be different, and accord-
ingly leave more or fess hemoglobin free for carbon monoxide to unite
with. Mouse blood, for example, is one-third less saturated with carbon
monoxide than human blood when both are exposed to the same mixtures
of O, and CO gases (Hartridge). The firmness of the union of carbon
monoxide with hemoglobin makes it very difficult to replace it with
oxygen and so to resuscitate a person poisoned by illuminating gas.
Inasmuch as alkali does not affect the firmness of the union of the hemo-
globin with carbon monoxide, but does increase the power of hemo-
globin to unite with oxygen, it would appear wise, in cases of poisoning
by coal gas, to give large amounts of sodium bicarbonate.

The spectrum of COHb differs slightly from that of oxyhemoglobin,
Figure 49, the two absorption bands being shifted slightly to the blue
end in the carbonyl hemoglobin. From this a very good method has
been devised by Hartridge for estimating the per cent. of carbonyl
hemoglobin in blood. The absorption bands of CO Hb are in an M/5000
solution 10 mm. deep: I, 582.1-560; IT, 548.5-522.9. The bands of HbO,
under similar conditions are: I, 585-567; II, 550.7-527.

Carbon monoxide hemoglobin is more stable than oxyhemoglobin. It
eoagulates at a higher temperature and at a slower rate than HbO,;
earbonmonoxyhemoglobin is not attacked by ferricyanide of potassium in
the dark to make methemoglobin, but is in the light. This shows that
carbonmonoxyhemoglobin, or carbonyl hemoglobin, as it may be called,
is dissociated by light, at least when in the presence of oxygen. If blood
be exposed to the same mixture of oxygen and carbon monoxide in the
dark and in the light the relative amounts of oxy- and carbonyl
hemoglobin will be different in the two cases. It is probable that reduced
hemoglobin is less stable than oxyhemoglobin and the reason that car-
bonyl hemoglobin coagulates more slowly and at a higher temperature
than oxyhemoglobin is that there is less of the reduced hemoglobin
formed by dissociation. Carbon monoxide probably unites like oxygen
with the iron atom of the hemoglobin molecule. The union may be of
the nature of a carbonyl of iron, since this gas has the property of
forming such carbonyl derivatives with the metals of the iron group.
THE BLOOD. THE CIRCULATING TISSUE 495

Haldane and his co-workers have generally assumed that carbon
monoxide unites only with hemoglobin and that it owes its toxicity
solely to the fact that it thus interferes with the oxygen-carrying power
of the hemoglobin. It is very doubtful whether this is the case. It is
more probable that it unites with other oxygen receptors, as well as
those of hemoglobin, and it may thus act directly on cells. It is found
to be somewhat more toxic than an equivalent asphyxia for mammals,
which would bear out this view. It is toxic toward some animals and
plants which have no hemoglobin, but is not toxie for others.

Tluminating gas has in it another substance, ethylene, CH,:CH,,
which toward many plants is vastly more toxic than carbon monoxide.
Although ethylene is present in the gas in very small quantities it is the
most toxic element of the gas for trees and various seedlings. The action
of this gas on animals should be carefully investigated. It is possible
that a part of the beneficial action of sleeping out of doors may be due
to escaping the poisonous action of small amounts of illuminating gas,
which penetrate from leaking pipes, joints and cocks into all dwellings.
It has recently been stated that ethylene is the anesthetic element
present in ether, and there seems little question but that the addition
of ethylene to ether improves its anesthetic power. The anesthetics
generally unite with hemoglobin.

Nitrous oxide hemoglobin. Nitrous oxide, or laughing gas, also
forms a loose combination with hemoglobin. It is very suggestive that
this mild and typical anesthetic is thus found to unite with an oxygen
receptor in an easily dissociable union. It suggests that anesthesia may
be produced by the saturation of the oxygen receptors of the protoplasm
by anesthetics.

Nitric oxide (NO) hemoglobin is a firmer compound, and with this
gas hemoglobin is easily oxidized to methemoglobin.

Hydrocyanic acid hemoglobin. Hydrocyanic acid, HNC, also unites
with hemoglobin probably by means of its bivalent carbon atom H-N=C.
There, again, is another typical respiratory poison and anesthetic occu-
pying an oxygen receptor. It probably unites in the same manner in
the cell and thus prevents union with oxygen. The union is in both
cases dissociable.

Carbon dioxide unites with some part of the hemoglobin molecule,
but it is more probable that it unites with the protein part of the
molecule than with the iron.

Sulphureted hydrogen, H,S, forms the compound HbH,S. The
union is probably with the extra valences on the sulphur, H,S. Pre-
sumably, also sulphur, 8,, will unite with hemoglobin to give sulphur
hemoglobin, but this compound does not seem to have been dis-
covered.
496 PHYSIOLOGICAL CHEMISTRY

To what extent hemoglobin unites with other substances has hardly
been studied, but it will probably be found that many other substances
will unite with it. For example, it is known that the anesthetics, such as
ether and chloroform, when in blood, unite chiefly with the red blood
corpuscles. It is believed by most observers that they form a loose union
with the lecithin or other lipin of the corpuscles. Solutions of lecithin
and cholesterin have the power of dissolving more anesthetic than water
alone; but there may be in addition a union with the hemoglobin, which
will retard its oxygen-carrying capacity, and thus play a part in
anesthesia. Particularly chloroform from its greater chemical activity
may be supposed to act in this way. The observations of Buckmaster
and Gardener show that anesthetics in some way or other do lower the
oxygen-carrying capacity of the blood.

1. Summary of the oxygen-carrying capacity of the blood. We may
now briefly summarize the discussion in the previous pages. The blood
contains in the red blood corpuscles a red pigment, hemoglobin, which
is probably in union with the stroma. Normal human blood contains
in 100 ec. about 14 grams of Hb. Hemoglobin has the property of
uniting with molecular oxygen and giving it off again in a molecular
form. In virtue of this property the blood is able to unite with con-
siderable quantities of oxygen in the lungs to form oxyhemoglobin, which
is of a scarlet color, and to carry oxygen to the tissues, which take the
oxygen away in virtue of their reducing powers. In the tissues the
pressure of the oxygen is extremely low, and in virtue of this fact oxygen
dissociates from oxyhemoglobin and enters the tissues. The oxy-
hemoglobin is thus partially reduced and the blood changes to the purple
color of venous blood, due to the presence in it of hemoglobin. The
amount of oxygen in the arterial blood, as it leaves the lungs, is different
in different individuals and in different animals, and it depends in the
first instance on the amount of hemoglobin there is in one cubic eenti-
meter of blood. But in general there can be extracted from 100 ee.
human arterial blood about 19-20 e.c. of oxygen gas, measured at 0° and
760 mm. of Hg pressure. One gram of Hb combines with approximately
1.34 ¢.. of O, gas. From venous blood less oxygen can be extracted,
the average amount being in human beings about 15 c.c. of oxygen,
although it may under circumstances be less. The amount of oxygen
taken up by the blood depends, in part, upon the partial pressure of the
oxygen, but even when this partial pressure is reduced to only 13 per
cent, of an atmosphere instead of the usual 20 per cent., the blood is
still 93 per cent. saturated.

The per cent. of saturation of the blood by oxygen depends upon
several factors; upon temperature, alkalinity or acidity of the blood,
upon light and upon the presence of salts and of certain specific salts.
THE BLOOD. THE CIRCULATING TISSUE 497

Since these factors vary in different animals, the per cent. of saturation
of their blood by oxygen when exposed to the same gas mixture varies
also.

Blood also carries carbon dioxide from the tissues to the lungs, where
it is given up. This carbon dioxide is in part dissolved as such in the
water of the blood and the corpuscles, but in large part it is present
combined with other substances in solution in the plasma. In part it is
there in sodium bicarbonate and in part in union with the proteins of
the blood plasma. It is also carried in the red blood corpuscles, presum-
ably united with the hemoglobin, but not united with the iron of the
hemoglobin. The passage of carbon dioxide into blood from the tissues
renders the blood in the capillaries more acid, or rather less alkaline,
so that carbon dioxide in this way helps to turn the oxygen out of its
union with hemoglobin and so make it available to the tissues. And
when the lungs are reached the passage of carbon dioxide outward into
the alveoli sets free the alkali to which the carbon dioxide had been
attached. This facilitates the taking up of oxygen in the lungs. Other
acids act in the same manner as carbon dioxide, so that the products of
oxidation in the tissues, the organic acids, may thus assist in providing
the tissues with oxygen by which these products may be oxidized.

The passage of oxygen into the blood and carbon dioxide out of
the blood in the lungs is generally supposed to be due to processes of
diffusion and to be thus a physical process. The pressure of oxygen in
arterial blood is always, under ordinary conditions, lower than that in
alveolar air; and the pressure of carbon dioxide is higher than that of
the alveolar carbon dioxide. A few observations exist, however, which
appear to indicate that in time of stress the lung epithelium may
actively intervene in the process and actually secrete oxygen inward.
so that the pressure in the arteries may be higher than that in alveolar
air. The observations, however, upon which this conclusion of the
activity of the lung or capillary endothelium depends are still open 1o
other interpretations. They do not conclusively show that the oxygen is
thus secreted. It is also unlikely that such very thin plates as the
alveolar epithelium should have a secretory function, although the
capillary endothelium might. It is better at present, therefore, to
conclude that certainly diffusion is the principal factor concerned in
the entrance of oxygen into the blood, but that possibly at times an active
secretory process may also assist. Further work on this matter must be
done before a definite conclusion can be drawn.

Hemoglobin unites not only with oxygen, but with many. other sub-
stances, such as coal gas, or CO. The latter union is firmer than the
oxygen union with hemoglobin, and a part of the toxicity, probably the
chief, and by some thought to be the total, action of the gas is to
498 PHYSIOLOGICAL CHEMISTRY

asphyxiate through its power of union with hemoglobin, so that the
blood can no longer carry oxygen to the tissues.

Laking of the blood.—Blood may be laked, that is hemoglobin may
be caused to pass out of the corpuscles into the plasma, by various agents.
Many toxins lake the blood, particularly some of those of snakes and
bacteria. Corpuscles are laked also by small amounts of alkali; by the
addition of water to the blood, by the action of anesthetics, such as ether
or chloroform, by bile salts and soaps. Blood may be laked also, by freez-
ing and then thawing it. The explanation of this laking or hemolysis
is still a matter of dispute. Some, perhaps the majority of observers,
consider that hemoglobin is held in the corpuscles by the wall of the
latter. This is not permeable to hemoglobin. The corpuscles are con-
sidered to be bags, or little cells, containing a concentrated solution of
hemoglobin. When the membrane of the corpuscle changes its state it
may happen that it becomes more permeable to the hemoglobin, which
now diffuses out of the corpuscle. All these various laking agencies are
said to act by affecting the permeability of the corpuscular membrane.

There are many reasons for doubting whether this explanation is
correct. Hemoglobin may be held in the corpuscle by union with the
stroma. It is true for all other cells, and probably it is true for the
corpuscles, that they are not bags filled with fluid, but they are or-
ganized jellies. The corpuscles behave in many ways as if they were
such jellies also. Hemoglobin does not escape, as one would expect it
would if it were in solution, when the corpuscle is punctured or cut
* across, but it stays in the divided corpuscle. Moreover, when hemo-
globin is set free in the corpuscle by some of these methods, particularly
in the very large cells of Necturus, a tailed amphibian, the hemoglobin
may crystallize in the corpuscle itself, which shows that it must be pre-
vented in some way from crystallizing in the normal cell. Moreover,
the concentration of hemoglobin in the mammalian corpuscle is greater
than the solubility of oxyhemoglobin in an equal bulk of water. For
these and other reasons some observers are of the opinion that hemo-
globin is held in some kind of a loose chemical or physical union, pre-
sumably the former, with the stroma of the corpuscle and that the
various hemolytic agents break this union. It is-not at all impossible
that the union is with certain reserve valences of the hemoglobin and the
stroma and such unions are very unstable and easily broken. Stroma
freed from its hemoglobin behaves as a poison, causing intravascular
coagulation. The hemoglobin-stroma compound as it exists in the cor-
puscle is inert in this respect. Carbon dioxide protects the corpuscles
from the hemolytic action of various hemolytic sera (Sawtschenko).

Whatever may be the explanation of hemolysis the process itself is
of very great interest from a physiological point of view. It may be
THE BLOOD. THE CIRCULATING TISSUE 499

taken as a type of the processes which are occurring in all protoplasm.
It is particularly instructive if the view be adopted that Hb and stroma
are in union, for it indicates that similar loose unions may occur in
protoplasm between other substances, for example between the fats and
the proteins, or between lipins and proteins, and that the instability of
protoplasm and its power of responding to stimuli of all kinds may
depend in part upon this fact. ‘It is well known that the presence of a
certain amount of salt is necessary for the preservation of many cells
and that if the salt is reduced in quantity the activity of the cell is
profoundly affected. We see from the foregoing that the composition of
the corpuscle, the hypothetical union of hemoglobin and stroma, de-
pends on the presence of a certain amount of salt in the plasma, since
diluting the plasma increases the tendency to hemolysis.

Composition of the red corpuscles.—The red corpuscles consist of
hemoglobin and stroma. The latter contains a considerable proportion
of phospholipin and cholesterol. 1,000 parts of erythrocytes contain,
according to Abderhalden, the following amounts of lipins:

 

Bull |Horse 1/Horse 2) Rabbit}. Pig | Dog1]) Dog2 |Sheep 1/Sheep 2) Ox

 

Cholesterol......... 1.824 } 0.3888 | 0.661 | 0.720 | 0.489.] 2.155 | 1.255 | 2.860 | 3.593 | 3.379
Phospholipin. .....]| 2.850 | 3.978 | 4.855 | 4.627 | 3.456 | 2.568 | 2.296 | 3.379 | 4.163 | 8.748
Total lipin......... 4.674 | 4,361 | 5.536 | 5.847 | 3.945 | 4.723 | 8.451 | 5745 | 7.756 | 7.127
Phospholipin

 

 

 

 

 

 

 

 

+cholesterol. ...| 1.563 | 10.240 | 7.845 | 6.426 | 7.067 | 1.192 | 1.829 | 1.428 | 1.159 | 1.109
H

 

The total lipins make, therefore, from 0.34-0.77 per cent. of the weight of the
corpuscles.

1. Hemoglobin. Chemisiry—a. Occurrence. Hemoglobin is a
pigment which is widespread in the animal kingdom and which is allied
to chlorophyll of plants and to the pigments phycocyan and phycoery-
thryn found in alge. It, or an allied pigment, occurs in many of the fixed
tissues of animals, as well as in the blood, as, for example, in the striated
muscle of most vertebrates, in heart muscle, in the pharyngeal muscles of
many mollusks, such as Paludina, and in the pharyngeal muscle and
ganglia of the polychete annelid, Aphrodite. Similar pigments, having
the same power of combining with oxygen and giving the spectra of
‘hematin and hemochromogen, have been found by MacMunn in the
cells of sponges and echinoderms and he has called these pigments histo-
hematins. While not identical with hemoglobin, they closely resemble it.
The function of this pigment is apparently to serve as a storehouse of
oxygen, and MacMunn has suggested that this was the original function
of the pigment and that it was later developed into a means of trans-
porting oxygen from the exterior to the tissues. Besides being in the
tissues, hemoglobin is found in the blood or body fluids of a great variety
of invertebrates and in all vertebrates with one or two exceptions. It
occurs in these fluids either in solution, or confined to certain small
500 PHYSIOLOGICAL CHEMISTRY

bodies called erythrocytes, literally meaning ‘‘ small red bodies.’’ In
all the vertebrates and in certain lamellibranch mollusks, i.e., Arca
tetragona, Pecteniculus, ete.; in the polychete, Terebella; in various
holothurians, i.e, Cucumaria Planci, ete.; in the worm, Thalassema
erythrogrammon ; in the polychete, Capitella, it is found in erythrocytes.
Tt occurs in solution in the blood or body fluids of various Cheetopods,
crustacea, insects, leeches, and even in the echinoderm, Ophiactis virens.
It is clear from this list that the power of forming hemoglobin must be
very widespread in nature. Since the“essential part of the molecule con-
sists of various pyrrol nuclei, the power of making such pyrrol nuclei
and uniting them to form hematin or chlorophyll must be a very general
possession of protoplasm. Hemoglobin, or similar iron-containing pig-
ments, are not the only oxygen-carrying proteins found in blood. In
the blood of Limulus, and various crustacea and mollusks, there is found
a copper-containing protein with a similar function, called hemocyanin.
This is a blue pigment and these animals are the truly blue-blooded
animals of the sea. Hemocyanin contains copper in place of iron. The
composition of this pigment has not been investigated with the thorough-
ness of that of hemoglobin, but it resembles hemoglobin in its high histi-
dine content and in some other properties. It is not so efficient an
oxygen-carrier as hemoglobin and cannot carry nearly as much oxygen
per gram of pigment as hemoglobin.

The development of hemoglobin in the blood has gone on pari passu
with the development of the central nervous system. This system has
a very great need of oxygen. It is more dependent on oxygen than any
other tissue of the body, and its consumption of oxygen per gram of tissue
appears to be larger. In the course of evolution the nervous system under-
went a progressive development, presumably because animals have been
selected chiefly for brain power. Hence as this system developed there
developed the need of carrying large amounts of oxygen to it. The
hemoglobin content of the blood increases more or less parallel with this
growth of the nervous system. Thus man, with the largest nervous
system, has the largest amount of hemoglobin in his blood. In human
beings there is normally in the blood 14-15 per cent.; in dogs there is
less; horses and sheep have still less, and in fishes and the lowest verte-
brates the quantity is further reduced. The amount of hemoglobin in
human blood corpuscles is larger than could be held in solution in them.
This large amount is made possible by placing the hemoglobin in the
erythrocytes.

b. Crystalline form. There is not a single hemoglobin, but a whole
series of hemoglobins, each animal probably having a kind differing
from that of every other species. They all resemble each other in their
main features, but they differ slightly in their composition, and above
all, they differ in their crystalline form.
THE BLOOD, ‘THE CIRCULATING TISSUE 501

 

g
a

Fic. 50, Varions forms of oxyhemoglobin crystals of different animals. a. Necturns
maculatus: b. Trumpeter swan, Olor buccinator: ec. Guinea fowl; d, Goose; e, Tasmanian
wolf; f. Fox squirrel: ¢. Ground squirrel (Reichert and Brown}.
502 PHYSIOLOGICAL CHEMISTRY

ce. Method of crystallization. Oxyhemoglobin erystallizes with very
great ease. In some animals in which hemoglobin is relatively little
soluble, it is only necessary to lake the blood under the microscope to
produce erystals. The crystals may even form in the corpuscles them-
selves, as in Necturus. Horse blood, guinea pig blood and squirrel blood
crystallize most readily; ox blood with more difficulty. But all hemo-
yiobins may be crystallized by the use of special methods. To obtain
large amounts of crystals in dog’s blood it is only necessary to lake the
blood corpuscles by shaking them with toluene and pleeing | in the ice-box.
The following methods are, however, better.

Hoppe-Seyler’s method. The defibrinated dog or horse blood is di-
luted with 10 volumes of 3 per cent. salt solution, and the corpuscles
allowed to settle. The supernatant liquid is poured off, the corpuscles
washed twice with cold salt solution and allowed to settle in a cool place.
The salt solution is then poured off and the mass of corpuscles is mixed
with its own volume of ether. This lakes the corpuscles. After laking
the ether is separated by rapid filtration, the filtrate cooled to 0° and
diluted with a %4th volume of absolute alcohol also cooled to 0°. It is
kept at —5° or —10° until crystallized. The crystals separated by centri-
fuge or filtration are washed with cold, 25 per cent. alcohol, dried by
pressure and recrystallized by dissolving them in water heated to 54°,
cooling and adding 4th volume of alcohol as before. Hiifner has short-
ened the method by using the centrifuge and laking pig’s blood by the
addition of distilled water.

Reichert and Brown have made a very careful study of the
crystalline form of the hemoglobins from a great number of animals.
Some of their figures are reproduced in Figure’ 50. They found
that each species of animal had its peculiar kind of hemoglobin.
The crystals of related animals were generally similar so that it was
possible, the authors thought, to use the crystalline form of oxyhemo-
globin as a means of aiding in the classification of animals and in dis-
covering relationships. It often happens that one kind of animal may
have more than one crystalline form of its hemoglobin. In such ease
it is possible that the crystals may differ in the amount of water of
erystallization that they contain. This amount is sometimes as much as
1] per cent.; but it may be half this quantity. The crystals of oxyhemo-
globin do not keep well, but even in a vacuum or when dry they are
slowly converted in part to methemoglobin, become less soluble and have
a brownish color. Nearly all the crystals belong to the rhombie system.
The crystals when examined in polarized light are pleochroic. That is
some of the crystals appear a brilliant scarlet; others have an orange
color. This is due to the fact that in some of the crystals the light is
coming through one axis of the crystal, whereas other crystals are so
placed that light passes through the crystal in another direction and
THE BLOOD. THE CIRCULATING TISSUE 503

the refraction and dispersion of the light is a little different.in the vari-
ous axes. The erystals placed between an analyzer, Nicol prism, and a
polarizer, with the interposition of a gypsum plate, show various colors
when the Nicol is rotated. The crystals show the same absorption
spectrum as that of the solution, except that the distance between the
two absorption bands is a little greater when the light comes through
one axis of the crystal than when it passes through another axis. Re-
duced hemoglobin crystallizes with greater difficulty than oxyhemoglobin.

d. Properties of oxyhemoglobin. The solubility is increased by the
addition of very small amounts of alkali. It is not precipitated by
NaCl or MgSO, added to saturation; it is precipitated by (NH,),SO,
beginning to precipitate when about two-thirds saturated and continuing
until saturation is reached. It resembles in this property the albumins.
It is soluble in distilled water, but not soluble in alcohol or ether.
Alcohol renders it insoluble and makes methemoglobin. It is a weak
acid, goes to the anode on passing an electric current through the solu-
tion, and the isoelectric point, that is the point of minimum dissociation,
is at a concentration of H ions of 1.8<X10—". . Acids, even when dilute,
and alkalies decompose it into hematin and globin. It is not precipitated
from solution when neutral by CuSO,, FeSO,, AgNO,, HgCl,, or by
lead acetate. Methemoglobin is precipitated by lead acetate. Oxyhemo-
globin is precipitated by CHCl, at 55° and the precipitate is insoluble
in water. When heated oxyhemoglobin coagulates at 64° and decomposes,
setting free hematin. An alkaline solution heated does not coagulate
but begins to decompose at 54°. Oxyhemoglobin is dextro-rotatory,
(a)c=+104 (Gamgee). The globin component is levo-rotatory
(@)o =—54.2° Reduced hemoglobin is extremely resistant to putre-
faction, remaining unchanged in putrefying mixture for long periods.

Oxyhemoglobin may be freed from all other proteins of the plasma,
stroma, etc., by precipitating the latter with alumina cream [freshly
pptd. Al(OH,)]. Oxyhemoglobin is not precipitated by this reagent.

e. Absorption spectrum. The spectrum depends on the concentra-
tion. Strong solutions absorb from the violet end clear to the red;
weaker solutions show two absorption bands between D and E. The
centers of these bands are for @ at 576 mus; 8 at 587 “pu; there is also
an absorption band in the ultra-violet, y, at 414 mu. The relation of
the width of the absorption band to the concentration is shown in
Figure 51, from Rollet, in which the spectrum is plotted on the abscissa
and the concentration on the ordinate. The depth of solution looked
through is in each case I em. Reduced Hb has a single absorption
band between D and E.

It is the absorption of green and blue light which makes hemoglobin
look red. It is possible that this absorption of blue light is of service to
504 PIYSIOLOGICAL CHEMISTRY

the organism in that it helps to protect the tissues from the more active
blue light.

f. Quantitative determination of the amount of hemoglobin and oxy-
hemoglobin. Various clinical methods for the determination of the
amount of hemoglobin in the blood are given in the practical part (page
993), and need not be repeated here. For the accurate determination
of the amount of oxyhemoglobin present, even in the presence of other
pigments which do not have the same absorption spectrum, the best
method is probably that of the spectrophotometer. This, as its name
implies, is a spectroscope so arranged as to measure the intensity of the
absorption of any light ray desired, but a description may be omitted in
a work of this character. The intensity of the absorption of the light is a
simple function of the concentration of the hemoglobin. The product of
the concentration by the depth of solution through which the light has
to pass in order to produce the absorption of a given proportion of the
incident light, say nine-tenths is'a constant. If c is the concentration
of the hemoglobin, ¢ the reciprocal of the depth expressed in centi-
meters necessary for the absorption of nine-tenths of the light, ¢ is
called the coefficient of extinction, then c/é=a constant A. A being
once determnied if c or eis known the other may be found. It may also
be determined accurately by the refractometer. (Howard.)

g. Composition. Although the hemoglobins differ among themselves
they are all alike in their general composition. They are all conjugated
proteins, that is they break easily into a simple protein, called globin,
and an iron containing radicle, which is hematin if oxyhemoglobin is
broken up, or hemochromogen, if hemoglobin is broken up. Hemo-
chromogen is reduced hematin. This decomposition is easily brought
to pass either by the action of dilute acids or alkalies, or by boiling
hemoglobin or by the action of digestive juices. The following table
shows some of the results of the elementary analysis of hemoglobin by
various observers :

 

 

 

 

 

 

 

 

Beno u cule oak ‘Te 0 N s | Fe | 0 P Observer
Dog | 3.4 | 53.85 | 7.382 | 16.17) 0.43 | 0.39 | 21.84] 0 Hoppe-seyler
11.39 | 54.57 | 7.22 | 16.38 | 0.568 | 0.336 | 20.93 0 Jaquet
1 54.15 | 7.18 | 16.33] 0.67 | 0.43 | 21.24 0 C. Schmidt
Horse 5 54.87 | 6.97 | 17.31}0.65 | 0.47 | 19.73] 0 Kossel
51.15 | 6.76 | 17.94] 0.39 | 0.335) 23.43] 0 Zinofisky
54.81 | 7.01 | 17.06] 0.6 | 0.468] 19.80; 0 Nencki
Beef 9.52 | 54.66 | 7.25 | 17.70 | 0.447 | 0.40 | 19.54] 0 Hiifner
Pig 8.04 | 54.71] 7.88 | 17.43) 0.48 | 0.399) 19.60} 0 Hiifner
Fowl 9.33 | 52.47 | 7.19 | 16.45) 0.858 | 0.335 | 22.5 |0.197 | Jaquet
Goose 54.11 | 6.83 | 16.58] 0.65 | 0.51 0.0059) Abderhalden and
i Medigreceanu.

 
x

 

eCB D Eb F a «CB D Eb
B

THE BLOOD. THE CIRCULATING -:TISSUE 505

It will be noticed in the foregoing table that in dog’s hemoglobin
there are three sulphur atoms to one iron atom; while im horse
hemoglobin there are two sulphurs to one iron. The sulphur of
oxyhemoglobin, or of globin, is not split off by the action of alkali.
Jaquet states that neither hen, horse nor dog hemoglobin contains
sulphur in a form which can be split off by the action of alkali and
lead acetate. If an alkaline oxyhemoglobin solution is cooked after the
addition of a few drops of MgCl, solution the hematin split off is
precipitated with the Mg (OH),, leaving an almost colorless solution.
This alkaline solution of globin contains no sulphur precipitable by
lead acetate as sulphide. The small amount of phosphorus in the
hemoglobin of bird’s blood is due to an impurity of nucleic acid, and is
not part of the hemoglobin molecule.

h. Composition of globin. If the formula of hematin, namely, C,,H,,
N,0,Fe, be subtracted from the formula of hemoglobin, C;,,H,23Ni955;
FeO,,,, there is left CrosHiig9N9:830213, a8 the formula of globin. It
might be that there were three molecules, each with one atom of sulphur,

   

 

 

 

 

 

 

 

F G oh

  

  

A

ic. 51.—Absorption spectra of hemoglobin (A) and oxyhemoglobin (B). The
abscissa shows the Frauenhofer lines of the spectrum; the ordinate represents the concen-
tration when the depth is 1 cm. (Rollet).

attached to the hematin molecule in place of one very large molecule
like the above. Globin is a histone-like. body containing rather more
nitrogen than most proteins, about 17 per cent., and somewhat more
carbon. It is like histone in the fact that it is not dissolved by ammonia
in the presence of ammonium chloride, and in the particular that it
yields a very large amount of the basic amino-acids, namely, about 11
per cent. of histidine, 5.4 per cent. of arginine and 4.3 por cent. of lysine.
506 PHYSIOLOGICAL CHEMISTRY

For the preparation of histidine, blood corpuscles are most commonly
used. Globin is insoluble in water, but readily soluble in alkalies and
acids; it is coagulated by heat, but the coagulum is readily dissolved by
acids (Schulz). It is interesting that the analogous blood pigment,
hemocyanin, also yields a large amount of histidine on hydrolysis. The
other amino-acids are those ordinarily found in proteins, as may be
seen in the table on page 129, the percentage of leucine, namely 29 per
cent., being rather larger than usual. It is rather interesting that, al-
though globin is so strongly basic itself, yet it forms, when united with
hematin, an electro-negative colloid. This is due to the fact that
hematin is an acid. Concerning the nature of the union between
hematin and globin nothing is known, but the great ease with which
the bond is split both by acids, alkalies and by boiling water suggests
that the union is simply that of a base and acid. It resembles, in this
respect, such nucleo-proteins as protamine nucleate, which is also a salt
union, and which forms an electro-negative colloid. Hemoglobin, being
an acid, is usually combined with some base and sodium or potassium is
ordinarily found in it. It is more soluble in very dilute alkali than
in water for this reason. The acidity of methemoglobin is particularly
strong.

i. Hematin and hemin. Hematin, the colored constituent of oxy-
hemoglobin, is a brown, amorphous substance the constitution of which is
not yet accurately known. It is an acid which is insoluble in water,
but is soluble in alkalies and in acid alcohol. It is formed when oxy-
hemoglobin is heated, or treated with acid, alkalies or various digestive
enzymes, and it gives the brown appearance to cooked meat. The prob-
-able formula of hematin is C,,H,,N,FeO,. Hematin has not been ob-.
tained crystalline, but when it is treated with hydrochloric acid it is
converted into hemin, which forms small brown erystals. Hemin is
hematin chloride and has the formula, when obtained by the action of
alcohol, sulphuric acid and sodium chloride by the method of Moérner,
of C,,H,,N,FeO,Cl. This hemin probably contains a molecule of hydro-
chloric acid and one molecule of alcohol. If this is subtracted this would
make C,,H,,N,FeO,, if two molecules of water had been eliminated in
the process. Kiister has proposed the formula C,,H,,N,0,FeCl for
hemin, which by treatment with KOH would give for hematin the
formula, C,,H,,N,O,Fe. This formula was that suggested by Hoppe-
Seyler and is the most probable one. Hematin, when reduced, forms
hemochromogen. Heated with strong acid it loses iron and is converted
into hematoporphyrin, a possible graphic formula of which has been
given on = o
NO peo erage FeCl, +cC,H. NO

i H,, 4 84 88 4 6
Hematin. * Hematoporphyrin.
THE BLOOD. THE CIRCULATING TISSUE 507

j. Hematoporphyrin. C,,H,,N,O,: The Willstetter formula is
Cy3H,,N,O,. This, the iron-free derivative of hemochromogen or hematin,
is isomeric with bilirubin. It can be made by. the method of Nencki and
Zaleski, which consists in adding hemin, little by little, to 75 ¢.c. of acid
and glacial acetic acid, which has been saturated with HBr at 10°, and
stirring constantly. One adds 5 grams of hemin to 75 cc. of acid and
allows the mixture to stand at ordinary temperature for 3 to 4 days.
The hemin having dissolved, the solution is diluted with water (1-2
liters) and filtered after some hours. The hematoporphyrin is then
precipitated by the addition of just sufficient sodium hydrate to neu-
tralize the hydrobromic acid. The precipitate is filtered, redissolved on
the water bath by the addition of sufficient sodium carbonate and
reprecipitated by addition of acetic acid. The precipitate is filtered,
washed, sufficient water is added to make a thick suspension and HCl
is added in small amounts until the hematoporphyrin has redissolved,
the solution filtered and HCl added in excess, any resinous precipitate
being filtered off. The filtered liquid is concentrated in vacuo until it
erystallizes. The crystals are filtered, washed with 10 per cent. HCl and
once recrystallized. These crystals are hematoporphyrin chloride. The
free hematoporphyrin may be obtained by dissolving in water and
neutralizing the acid with sodium acetate. It is a brown powder. The
probable formula of this reaction is the following:

Caatash O4FeCh + 2HBr + 2H,0 — C, H..N,O, + FeBr, + HCl.

emin. Hematoporphyrin.

Properties. Hematoporphyrin is a dark, violet-colored powder,
having a green tint in transmitted light. It was discovered and given
the name cruentine by Thudichum. It is insoluble in water and very
slightly soluble in CHCl,, amyl alcohol or ether; it is soluble in ethyl
aleohol, and in water containing sodium hydrate or other alkali or dilute,
strong acid. . It is probably an amphorteric electrolyte. It is not very
soluble in weak organic acids. The alcoholic solution, when neutral, is
red, which becomes red-orange on the addition of a little acid, the color
‘changing to a purple or violet by the addition of more acid. The solu-
tions are very fluorescent and in the light have a strong physiological
action. (See page’422.) Alkaline aqueous ‘solutions are also red and
have a different spectrum from acid solutions. (See the practical part,
page 1003.) Hematoporphyrin is an acid and forms definite salts, of
which the sodium salt has been erystallized. The alcoholic and alkaline
solutions have four absorption bands: in the red between C and D
621-610 wu), two between D and E (the first at D, 590-572 ju, the
second near EH, 555-528 uu), and the fourth from b to F (514-498 yy),
For the spectrum, see page 493. The position of the bands depends
508 PHYSIOLOGICAL CHEMISTRY

somewhat on the concentration of the alkali, and their width on the
concentration of hematoporphyrin. According to Schulz the third band
can be resolved into three constituents. The spectrum of acid hematopor-
phyrin is different. There are two bands on each side of D: the first from
597-587 MM, the second at 541 uy. In both acid and alkaline solutions
there is an absorption band in the ultra-violet beween h and H (Gam-
gee). The acid salt dissolved in alcohol has the five-banded spectrum of
alkaline hematoporphyrin, but by the addition of more HCl, the spectrum
changes to the acid hematoporphyrin spectrum.. Hematoporphyrin
strongly resembles phylloporphyrin obtained from chlorophyll, but it
contains two atoms of oxygen more than the latter. Heated with zine
and distilled both yield pyrrols. It unites with metals in excess of
alkali or in acetic acid. The synthetic iron compound is almost iden-
tical with hematin but the absorption bands are slightly different.
(J. A. Milroy.)

Composition. When decomposed with hydriodic acid in glacial acetic
acid, hematoporphyrin yields various hemopyrrols and hematic acids.
The composition of some of these bodies has already been indicated on
page 416. It is the general opinion that the molecule of hematoporphyrin
contains four substituted pyrrol nuclei, but the exact manner in which
these are arranged is still uncertain. There is still some uncertainty
about the molecular size, but the probability is that the molecule con-
tains only four pyrrol nuclei. It is both an acid and a base, forming
true salts with acids and bases. It is closely related to bilirubin in
structure and it is the mother substance of the bile pigments. MacMunn
and Thudichum showed that hematoporphyrin is one of the pigments
found in the shell of birds’ eggs and that it is the pigment of Uraster
rubens; of various mollusks, such as Limax and Arion; and of Lum-
bricus and Actinia. As a copper compound it is found as a pigment in
the feathers (Laidlaw) of birds of the genus Mucophaga. It occurs in
small amounts in the urine and in larger quantities after sulphonal
ingestion and in some pathological conditions.

k. Reduced hematin. (Also called hemochromogen.) It is formed
by the action of acids or alkalis on hemoglobin, or it may be formed by
the reduction of hematin. It oxidizes spontaneously in the air and forms
hematin. It loses its iron more easily than hematin and forms hems-
toporphyrin. Like hemoglobin, one molecule unites with one molecule
of CO and probably one of HCN. The union of reduced hematin with
oxygen is apparently more stable than that with carbon monoxide, since
oxygen will readily turn the latter out of its union with hemochromogen,
but not with hemoglobin. This shows that the firmness of the union be-
tween the gas and the chromogenic radicle is affected by the presence of
the globin radicle. There is still some question as to the amount of

By
THE BLOOD THE CIRCULATING TISSUE 509

oxygen combined with reduced hematin to make hematin. It was
observed by Ham and Balean that when hematin is formed from oxy-
hemoglobin by the action of strong acid some oxygen is set free, and
the amount was about equal to one-half that which was set free from
oxyhemoglobin by the action of ferricyanide of potassium. Oxygen,
moreover, cannot be pumed out of the hematin solution to form reduced
hematin. In this particular the state of the oxygen in hematin would
appear to be more nearly that in methemoglobin. If carbin monoxide
hemoglobin is acted upon by acid a carbonyl-hemochromogen is ob-
tained. The union is one molecule of CO to one molecule of hematin.
The carbon monoxide can be displaced by a current of hydrogen. Nitric
oxide also forms a molecular and firm, red-colored compound with re-
duced hematin. (Linossier.)

Reduced hematin may be easily prepared from blood or hemoglobin by
adding to solutions of the latter some solution of hydrazine hydrate, 5 per
cent., in 10 per cent. sodium hydrate. The spectrum of hemochromogen
appears. On shaking with air that of hematin temporarily appears,
but is quickly reduced. The alkaline solution of reduced hematin
is cherry red. It has absorption bands in the green between D and
IX nearer D (567-547 wu mean at 559 wy) and one less well-defined
from E to b (532-518 uu ; mean at 525 wu). In the ultra-violet there
is one between h and g at 420 wu (Gamgee). In air solutions oxidize
readily and take the brown color of hematin. It differs from hematin
particularly in its sensitivity to acids. Even dilute acids transform it
into hematoporphyrin, whereas hematin is decidedly resistant to acids.

3. The blood as the carrier of waste substances.—This is the third
great function of the blood. All kinds of waste materials are found in
the blood in solution in the plasma. The blood carries them from the
tissues to the kidneys and other excretory organs. Some of these sub-
stances contribute to the composition of the medium in which the cells
are accustomed to work and they are, therefore, important factors in
their environments. Thus while urea and carbon dioxide are waste
materials and generally regarded only as excretory substances some, and
possibly all of the cells of the body, work better when they are present
in the blood in the usual amounts. As they thus condition the activities
of cells they may be regarded, also, as hormones. Thus carbon dioxide
helps to rouse and regulate the respiratory center; and the heart beats
with greater vigor, in some of the lower vertebrates at any rate, when
there is a small amount of urea present. The amount and character of
various waste products found in the blood is shown in the adjoining
table. There are many which are not mentioned in this table, since
nearly all the numerous substances excreted in the urine have been in
the blood.
510 PHYSIOLOGICAL CHEMISTRY

ACCUMULATION oF NrrrRogENouS WaAsTE Propucts IN Bioop UNpdER Various
ConpDITIONS.*

’

Mes. Per 100 Grams Buioop.

 

 

on- A Creatin-
Condition of diet and health pratein Uree ae 3s ee 4 rae ‘ne and
Normal, purine-free, ‘high N diet.

Urinary N 24 grams ............ 34 16 | 0.1 2.5 1.1 9.5
Normal(?) Urinary N 17 grams ... 37 18 | 0.1 3.0 1.2 8.5
Normal. Low N. Urinary N 6.2

VANS. oe ek bie eee nes etl es 4 32 15 | 0.14] 2.0 1.3 6.5
Normal. Urinary N 4.5 grams .... 24 11 |] 0.11 | 2.0 1.4 6.9
Toxemia 3 weeks after delivery ..... 26 12 | 0.1 4.4 0.9 6.0
Typhoid fever. 104°. Second week .. 38 19 | 0.08 | 2.0 1.4 | 10.0
UCM cc ceresmae eae anetet on eae 288 222 9.5 | 31.0
Urem ia: 0 vcgeihvors gyaaceamety ven’ 284 228 | 0.66) 6.6 | 26.0 | 46.0
Uremia, convulsions ............26: 200 160 | 1.0 8.0 | 10.0 | 27.0
Chronic nephritis ..... ey eee 200 140 7.5 | 13.0
Pneumonia (before crisis) ......... 72 44 5.0 1.5 | 20.0

 

 

 

 

 

 

 

* Folin and Denis: Jour. Biol. Chem., 17, p. 488, 1914.

4. Blood as a distributor of internal secretions. Its co-ordinating
function.—The. blood plays a very important part in the metabolic co-
ordination of the body. It carries from one organ substances elaborated
there and which are necessary for the growth and normal life of tissues
in other parts of the body. One of the first of these internal secretions
(internal because secreted into the blood) to be recognized was that of
the sexual glands. The metabolic products of these glands in some way
or other determine the development, or aid in the control of, the so-called
secondary sexual characters. Thus oxen are proverbially less fierce than
bulls, their bodies have a different shape, their horns are larger. Many
other organs besides the sexual glands produce internal secretions, which
are necessary for the development of other organs. Thus the develop-
ment of the long, silky hair and smooth skin of human beings, the de-
velopment of the skull and intelligence, depend upon the thyroid gland.
The metabolic products of this gland, whatever their nature, are sup-
posed to find their way into the blood and to be distributed by that
tissue to the tissues which need them. In this way the blood plays the
part of a middleman, or a system of transportation in human society.
The blood, after the nervous system, is the most important co-ordinating
agency in the body. These internal secretions are probably present in
the blood in very small quantities. They are probably in solution in the
liquid part of the blood. Their nature will be considered in the chap-
ter which deals with the glands which produce them. (Cryptorrhetic
tissues. Chapter XVI.)

5. Physico-chemical factors of the blood important in its circula-
tion. Its viscosity ; coagulation.—-Under this heading will be considered
THE BLOOD. THE CIRCULATING TISSUE 511

the physical chemical features of the blood, which are important in its
functioning as a circulating fluid. The most important of these char-
acters is its viscosity. The blood must be a liquid in order to penetrate
freely all parts of the body. In order that the blood should be driven
in a constant stream through the capillaries of the tissue it must be
kept at a high pressure in the arteries. The pressure of the blood in the
arteries is maintained by the contraction of the heart, which constantly
pumps blood into the arterial system, and by the peripheral resistance
in the arterioles and capillaries which prevents the blood running too
rapidly through the capillaries. This peripheral resistance may be
diminished or increased by widening or narrowing the path of the blood
by dilating or contracting the arterioles; but it may also be changed
by variations in its viscosity.

Viscosity of the blood. By the viscosity of a liquid is meant stick-
iness, or the resistance it opposes to flowing or changing its shape. A
perfect gas has no viscosity. The molecules are so far apart that their
size and mutual attractions in no way affect the freedom of their move-
ments. In liquids the size of the molecules relative to the space be-
tween them is so large that the molecules get in each other’s way and
limit their freedom of movement. This resistance to freedom of move-
ment due to cohesion and the space occupied by the molecules is known
as the viscosity. In determining the viscosity of a pure liquid, or of a
liquid containing molecules in solution, molecular size and cohesion are
the determining factors. When the liquid contains substances in sus-
pension in it if the amount of this material is small compared with the
total space of the liquid there will be little change in the viscosity
from that of a true solution, but when the amount ef added material
becomes large enough to compare with the bulk of the liquid then the
viscosity is increased. Water containing colloidal metals or clay in sus-
pension has its viscosity very little changed until the amount of clay
is large. If, however, the substances in suspension have a marked affinity
for water so that they are united chemically or physically with several
molecules of water, thus making what Naegeli called micellae, or what
we may call hydrophile or emulsoid colloids, then the viscosity is more
or less increased.

In blood the viscosity is ‘affected by the corpuscles which are in sus-
pension and by the proteins which are in solution. Blood consists to
nearly half its volume of corpuscles, so that its viscosity is much greater
than that of a salt solution, owing to this fact. And in the second place
the proteins are markedly soluble. They are hydrophyl colloids. They
may change their state so as to bind more or less water. There is about
8 per cent. of protein and this is sufficient to make the viscosity of the
plasma greater than that of water.

Measurement of viscosity. The viscosity of a liquid is measured
512 PHYSIOLOGICAL CHEMISTRY

by an instrument called a viscosimeter. There are various forms of this
instrument. The method consists essentially in measuring the time
required for a given bulk of a liquid to flow through a small opening
or capillary tube under a constant temperature. One can use so simple
a plan as to determine the time required for a liquid to flow from a
pipette and compare the time with that taken by water under similar
conditions, the viscosity of water being taken as unity. If it takes twice
as long for plasma to pass as for water, the viscosity of the plasma is
said to be 2.

Viscosity of the blood.—The viscosity of mammalian blood is about
4.4-5.5. The viscosity of plasma or serum is much greater than that of
water. At 38° it is 1.78-2.09 (Bence. Burton-Opitz). The viscosity
diminishes with a rise of temperature until coagulation is reached. This
high viscosity of the plasma and serum is due to the proteins in it. A
dilute salt solution has about the same viscosity as that of water.

The viscosity of blood is much greater than that of serum, owing to
the corpuscles in the blood and is much influenced by their tendency
to clump. The viscosity is a function of the number of corpuscles, as
may be seen in the following figures (du Pre’ Demming and Watson) :

No, of blood Viscosity of sernm
corpuscles + corpuscles
0 1.9
3.2 x 10° 3.3
6.3 x 10° 4.9
12.6 x 10° 15.6

The figures are for horse’s blood at 32.2°. The corpuscle numbers are
for cubic millimeters. The high viscosity of the last number is probably
due to a partial blocking of the small capillary opening by the clumped
corpuscles. The viscosity of the blood appears for this reason, unlike
that of water or a true solution, to increase with the diminution of the
size of the capillary. A capillary of 2 mm. diameter, a wide capillary.
gives the more reliable figures. It is clear, however, that in the living
body a narrowing of the capillary path will have the effect of appar-
ently increasing the viscosity of the blood for the reason just stated.
The effect of temperature on the viscosity of the blood is marked.
At 387° the viscosity is about 16 per cent. less than at 17° so that varia-
tions of temperature of 5°, which is within the limits of fever, may
change the viscosity of the blood about 4 per cent. The viscosity of
human blood in the mean is about 5.1 (Beech and Hirsch). It may vary
between 4.73 and 5.89 at 38°. Since carbon dioxide causes an imbibition
of water by the corpuscles and an increase in their size the viscosity is
increased by carbon dioxide and diminished by oxygen: Hence the
viscosity of venous blood is greater than that of arterial blood.
THE BLOOD. THE CIRCULATING TISSUE 513

Viscosity of O, saturated blood ............seseeeeeeees 5.4
. after H, passed through it ...............0+. 5.88
. “co, “ ADS 6 hand oa liens tuk Bee a ee 3.8 6.57

In dyspnea the viscosity is increased. In dogs, hunger diminishes the
viscosity (Burton-Opitz), a meat diet increases it most; a carbohydrate
and fat diet produces a medium effect.

The blood contains a special mechanism for affecting its viscosity
quite apart from those factors thus far mentioned. This is its power
of clotting, or coagulation. By clotting the viscosity of the blood be-
comes so great that fluid'y is lost and the blood is converted into a
jelly-like solid. This power of clotting is of great service to the body in
preventing hemorrhage, but it is not impossible that a partial coagulation
takes place in certain regions of the circulation, causing a great increase
in the viscosity and peripheral resistance in these regions. This would
cause a very high intracapillary pressure and might contribute to a
resulting edema. The tendency to clotting is always present. Even a
sluggish movement of the blood in the veins may result, in certain special
circumstances, in a premature permanent clotting, or thrombosis. This
happens sometimes after anesthesia, which increases coagulability, or
after parturition while the patient is lying flat in bed. Clotting is due
to the conversion of one of the soluble proteins of the blood plasma, the
fibrinogen, into an insoluble form of fibrin. This fibrin comes out in
the form of a net which entangles the corpuscles and water and makes a
jelly-like’ clot. Without considering at this point the nature of the
change in the fibrinogen and the cause of the change, we may briefly
examine the reasons for thinking that this change may perhaps nor-
mally occur to a slight extent and thus change the viscosity of the blood.
The chief reasons are the following: ;

An extremely small amount of formation of fibrin from fibrinogen is
sufficient to change enormously the viscosity of the blood. The con-
version of .001 per cent. of fibrinogen into fibrin is able to change blood to
a condition where it will hardly flow, but behaves as a gel. Even
less than this will greatly affec. its viscosity. If this happened in a
certain part of the kidney, for example, the effect might be greatly to
increase the pressure in the capillaries of the glomeruli and thus to in-
crease the filtration. There is in the arterial blood entering the kidney
generally about 0.1 per cent.-0.2 per cent. of fibrinogen. In the blood
of the kidney vein there are always a few hundredths, or thousandths
of a per cent., less than in the arteries. What becomes of this fibrinogen
which thus disappears has never been entirely explained, although Nolf
suggested that it deposited on the endothelial cells. It is not now be-
lieved that it is used as food, since the tissues are probably nourished
by the amino-acids rather than by the blood proteins. It is possible that
514 PHYSIOLOGICAL CHEMISTRY

it has been converted (Nolf) into fibrin and possibly afterwards re.
dissolved or partially digested, but not converted back to fibrinogen. A
similar loss of fibrinogen occurs in nearly all the organs in the body, the
kidneys perhaps being the most important of the organs destroying
fibrinogen. The question may be raised whether the conversion of
minute amounts of fibrinogen to fibrin may not take place in the periph-
eral capillaries, thus leading to an increased resistance and to a loss of
fibrinogen. It may be that this was the original purpose of the fibrino-
gen. These are questions which cannot be answered positively without
experiment. But the presence of this peculiar protein which has the
function of enormously altering viscosity suggests that it may be a very
important factor in the normal regulation of blood viscosity.

The great importance of the viscosity of the blood, and of the pro-
teins of the plasma in determining it, is shown by the circumstance that
if the blood is withdrawn from a dog’s body and the corpuscles removed
by centrifugalization and then the corpuscles suspended in a salt solu-
tion of the proper strength, such as Ringer’s solution, and reinjected,
the animal will not live. But it will live if gum arabic is added to the
Ringer’s solution in sufficient quantity to restore the viscosity to its
normal amount.

Clotting of blood.—When mammalian blood escapes from the blood
vessels into the tissues or when it is collected in a vessel, it changes in
the course of two to ten minutes from a liquid to a jelly-like solid,
which has the same volume as the blood in the liquid form. It clots. No
heat is disengaged in this process of clotting. If the clotting takes place
in a beaker or other vessel, the clot after a longer or shorter time,
generally in the course of half an hour, begins to shrink and the clotted
blood gradually separates into two portions, a clear, slightly straw-
colored liquid, called the serum; and the solid, contracted clot consisting
of the corpuscles, the greater part of the serum, and of an insoluble pro-
tein substance called fibrin. This process of contraction of the clot
with the pressing out of the liquid serum is not peculiar to blood, but
is a common property of many gels. It is called syneresis. The con-
traction of the blood clot is, however, more extensive than that of most
other gels. The particular object secured by this process of conversion
of the liquid blood to a solid clot is to stop bleeding and so to prevent
fatal hemorrhage. While clotting is common to all forms of blood from
that of the echinoderms to man, the process reaches its highest degree
of perfection in the mammalia and birds, where the blood pressure is
highest and the danger of fatal hemorrhage is correspondingly increased.
We may now inquire more at length into the nature of this process of
clotting. It may be stated at the outset that while no physiological
phenomenon has been more extensively studied and for a longer time, yet
THE BLOOD. TIIE CIRCULATING TISSUE 51E

we are still quite ignorant of the explanation of many of the steps of
the process.

Clotting not due to contact with air. That clotting is not due to
contact of the blood with air may be shown by the fact that the entrance
of air into the veins does not cause clotting. In caisson disease, when
men are too suddenly transferred from a compressed air to normal
atmospheric pressure, there often occurs a disengagement of bubbles of
nitrogen gas in the blood vessels of the body. These bubbles, while they
may cause death by air embolism, do not cause any clotting about them
in the blood. Moreover, if blood is received from an artery or vein
into an evacuated vessel, or if it be collected over mercury without con-
tact with air, clotting occurs as usual. Contact with the air is not, then,
the cause of the clotting of blood as one might at first think.

Stopping of circulation not the cause of clotting. It might also be
thought that the cessation of movement of the blood, its lack of agitation
ensuing after its discharge from the blood vessels, is the cause of clotting,
but this again can be shown not to be a sufficient explanation of the
facts. If, for example, two ligatures be placed about a portion of the
jugular vein of a horse so that some blood is imprisoned between the
ligatures, it will be found on opening this vein after some time that
the blood in it has remained quite fluid. It will, however, ultimately
clot in the vein in these conditions, but its clotting takes very much
longer. If the fluid blood is poured out of the vein into a glass vessel
the blood clots at once. Moreover, if an attempt is made to keep blood in
circulation through glass pipes at body temperature, it will be found
that it clots in the pipes and fully as rapidly as if it remained at rest.
Whipping the blood with glass rods increases the speed of clotting.
The quick clotting of shed blood cannot then be due to the stoppage of
the flowing motion of the blood, although this may, at times, in the
blood vessels be a predisposing cause of clotting. Clotting is not due
either to a lowering of temperature of the shed blood, since cooling
usually retards the process of clotting, and a high temperature accel-
erates it.

Contact with the tissues greatly accelerates clotting. When blood is
shed into the tissues, or when in the process of collecting it, it passes
over the wounded surfaces of the tissues, its clotting is greatly acceler-
ated. In fact, if blood of vertebrates lower in the animal scale than
mammals, for example the blood of birds, reptiles, fishes and amphibia, be
carefully collected by means of a cannula introduced into an artery, the
greatest care being taken to avoid any contact with a wounded piece of tis-
sue, the blood thus drawn will remain fluid for a long period of time, and
if it is collected into vaselined glass vessels so that the blood does not touch
the glass walls it will-remain fluid almost indefinitely., [f to such blood
516 PHYSIOLOGICAL CHEMISTRY

a small amount of the normal salt extract of any tissue of the body is
added; clotting occurs very quickly. It is evident from this that there
must be something in the wounded tissues which accelerates clotting of
blood. In mammalian blood the conditions are essentially the same.
except that clotting will occur without contact with the wounded tissue.
Nevertheless such contact greatly accelerates clotting here also, and the
addition of extracts of the tissues hastens the clotting of mammalian
blood just as it does that of bird’s blood. Among the substances of the
tissues which have this power of accelerating clotting, phospholipins of
the nature of cephalin, or unknown substances which accompany this
fraction of phospholipin when the latter is separated from the tissues,
have the power of acting in the same manner and are presumably the
active substances in the tissues. It may be said here that these ac-
celerating substances have a certain specificity of action in that the tissue
of one kind of fish or other animal accelerates the clotting of its own
kind of blood far more than it does that of blood of other species
(Loeb, Nolf). The specificity is not, however, absolute, but is relative.
The substances in the tissue extracts thus accelerating clotting are
sometimes called thromboplastic substances, thrombokinase, tissue fibrin-
ogens, or coagulins, as different writers have made different pictures of
their method of acting.

Contact with foreign bodies greatly accelerates clotting. It has been
found that not only will tissues hasten the clotting of blood. Any finely
divided foreign substance, which can be wet by the blood, hastens clot-
ting. Thus finely divided glass, glass wool, clay filters, feathers, or
absorbent cotton, hasten clotting. It is not necessary for the blood to
be shed in order to produce clotting by these means. If, for example, a
needle be passed through the walls of an artery or vein it becomes
covered with a coating of fibrin ; and if the walls of a vein be injured, as
by the action of an inflammatory process, by a hot needle or by caustics
(AgNO,), clotting more or less limited to the place of injury will be
observed. Sometimes if the blood has been rendered abnormally easy
to clot, as it is by anesthetics after an operation, or after hemorrhage,
slight injury to a vein by an inflammatory process or mechanical process
may lead to clotting in the vein and thrombosis. This is especially apt
to happen, for example, after parturition. After any hemorrhage. in-
cluding the more or less extensive hemorrhage of parturition. hlood is
more easily clotted, the circulation in the veins of the leg is slow and
by tight bandages about the abdomen may be rendered slower than usual.
Conditions somewhat similar,.except for the hemorrhage. prevail after
operations for appendicitis and clotting in the veins of the legs. par.
ticularly in the right leg after appendicitis operations. is not unusual.
Tf these clots get loose in the circulation they may lodge in the pul-
THE BLOOD. THE CIRCULATING TISSUE 517

“monary circulation and produce serious trouble or even death. If they

remain fixed they disturb, in a painful way, the circulation of the leg.
Milk leg is a result. Such clots are in part reabsorbed and in part are
incorporated into the vein walls. While contact with many forms of
matter produces clotting, yet blood received under paraffine oil and
into smooth glass receptacles which have been oiled with paraffine oil
has its clotting much delayed. Oftentimes the first signs of clotting in
such blood occurs in the surface where dust particles may be present.
Examination generally shows that the foci of such clots are some ex-
traneous matters. Contact with foreign substances which may be wet
with the blood is, hence, one of the determining factors of the process of
clotting. Many inert solid substances thus have a thromboplastic action.

Physiological variations in tendency to clotting. It is observed that
the tendency of blood to clot undergoes quite wide variations in different
individuals. As has just been said, previous hemorrhage always greatly
accelerates the tendency to clot. This is of course in the nature of an
adaptive change to stop any recurrence of the hemorrhage. Certain indi-
viduals have blood which clots with much greater difficulty than normal.
If this tendency toward delayed clotting is very pronounced, it may
constitute a positive danger to life. Such people are known as bleeders;
they have the disease known as hemophilia. This trouble is inherited
and generally runs in families. In pneumonia and some fevers the clot-
ting of the blood is somewhat slower than usual, and the settling of the
corpuscles being rather more rapid, when the shed clotted blood of such
individuals is examined it often happens that the upper parts of the
clot may be found to be composed of plasma with few or no corpuscles,
the corpuscles having settled before clotting occurred. The blood has
a clear coat above. This is called the crusta inflammatoria. Horse’s
blood usually acts in this manner, the corpuscles settling very fast, par-
ticularly if the blood be cooled as soon as it is shed.

Ways in which the clotting of blood may be caused or prevented.
Action of albumose. Tf a strong solution, 5 to 10 per cent., of albumose
(Witte’s peptone) in 0.9 per cent. NaCl be injected rapidly into the
jugular vein of a dog in an amount of 0.2-0.3 gram albumose per kilo
body weight, it is found that blood drawn from'a few minutes to a
couple of hours later has a greatly prolonged time of clotting. By the
use of larger amounts of albumose the blood drawn 15 minutes after
injection does not clot at all. Such blood is known as peptone or pro-
peptone blood. This injection has no such pronounced action in rabbits,
the injection of amounts of peptone which the animals will survive is
followed by very little change in the coagulability of the blood. It has
been found that, if the injection of peptone is made in a fasting dog,
the results are different from those when the dog is in full digestion
518 PHYSIOLOGICAL CHEMISTRY

(Wooldridge). The very first effect of the injection is a very brief
period of quickened coagulation. Furthermore, if a small amount of
peptone is injected first, so that the coagulability of the blood is only
slightly depressed, then the subsequent injection of a large amount of
albumose solution has no effect at all in changing the speed of clotting.
The first injection appears to have made the dog immune to the albumose.
What it is in the albumose which has this action is not known. It is
perhaps significant that albumose prepared by the digestion of fibrin
is most efficient in this action, but albumose from meat is also efficient,
though to a less degree. Albumose added to blood outside the body
does not have the effect of retarding coagulation. It somewhat acceler-
ates the clotting. The effect of preventing clotting is, hence, not a
direct action of the albumose on the blood, but on the tissues of the
body. Some have thought that it is particularly acting on the leuco-
cytes; others that it is acting on the liver. We shall come back to this
presently.

Leech extract. If an aqueous extract be made of the head of the
medicinal leech, it is found to have quite remarkable powers of delaying
coagulation when injected into the body, or when added to the blood
outside of the body. The active principle is called hirudin. It appears
to be a deutero-albumose. The blood will not clot after leéch extract has
been added to it. Hirudin is an anticoagulant.

Tissue extracts. These have a most interesting action. If a normal
salt extract be prepared of the thymus gland, spleen, testicle, brain,
lymph glands, or other tissues, something goes into solution which is
precipitated with dilute acetic acid. Redissolved in salt solution made
slightly alkaline by sodium carbonate and injected into the vein of a dog,
it will cause death if sufficient is injected. On post-mortem examina-
tion it will be found that intravascular clotting has occurred in the portal
region. Sometimes the clot is confined to the portal vein and its branches
entering the liver, but in digesting dogs it will be found that the clot
may extend into the right heart and lungs. In rabbits by such tissue
extract injections the whole circulation may be suddenly clotted. In
dogs, however, it is found that a clot forms preferably in the portal circu-
lation. If smaller amounts of the tissue extract are injected, it is
observed that at first, immediately after the injection, there is an
increased coagulability of the blood, and that there is a temporary ces-
sation of the respiration and a sudden fall of blood pressure, but that
in a few moments the dog recovers. Larger quantities of the tissue
extract may now be injected without any obvious ill effects, but if the
blood be drawn, it is found that it has lost wholly or partly its powers
of spontaneous clotting. The addition, however, to this non-clotting
biood of more of the same extract which had just been injected causes
THE BLOOD. THE CIRCULATING TISSUE 519

clotting at once outside the body, provided too much of it has not been
injected, although it is ineffective within the body. If a dog which has
received a dose of tissue extract not sufficient to kill him be killed in
some other manner, it will generally be found on post-mortem examina-
tion that there are clots in the portal vein. These clots are already
white, owing to the blood corpuscles having been washed out, and they
ultimately undergo digestion and disappear. It is, hence, a very singular
fact that these tissue extracts, which act in so many ways like peptone
or albumose, produce within the body first a positive tendency to coagu-
lation, followed by a negative reaction, whereas when added to blood
outside the body they produce only the positive phase. This fact indi-
cates that the negative phase of clotting, that is the tendency of the
blood to remain fluid, is due to a reaction on the part of some tissues of
the body to this dangerous substance, and not to a reaction on the part
of the fluid blood itself.

The substance in the tissues which is responsible for the clotting
and which was called ‘‘ tissue fibrinogen ’’ by its discoverer, may be
purified by precipitation by half saturation with ammonium sulphate,
or half saturation with sodium chloride, or by the addition of
acid in small quantities or by carbon dioxide. It is insoluble in dis-
tilled water, but readily soluble in dilute salt solutions. It is found
in the greatest amount in the lungs, then in the brain and in
the kidney. Muscles contain relatively little. It was called tissue
fibrinogen for the reason that in all its properties it strongly resem-
bles the fibrinogen of the blood. Like the latter it is precipitated
by mixing its solution with an equal quantity of saturated sodium
chloride, and by CO, and by acids, although it is precipitated at a little
different point of acidity than blood fibrinogen. It coagulates at the
same temperature as blood fibrinogen, i.e., 56°. It is electro-positive in
solution and goes to the cathode. Like fibrinogen also it is composed of
a phospholipin united with a protein. In fact this substance has been
repeatedly extracted from tissues and called fibrinogen on account of
these properties and confused with the fibrinogen of the blood. When
examined it was found to contain 10.7 per cent. of N and 1.53 per cent.
of P. It yields approximately 36 per cent. of its weight as a phospholipin.
These facts have been discovered by Mills, but some of them are but the
confirmation of earlier studies by Wooldridge. Unlike fibrinogen it does
not clot, but if it is added to a solution of fibrinogen it increases the
weight of the fibrin obtained from the latter. A small amount of this
tissue fibrinogen is found also in the blood.

When tissue fibrinogen is injected, so that the blood becomes abso-
lutely incoaguable, it is found that all of the blood fibrinogen has dis-
appeared from the blood. It is unknown what has become of it. Such
520 PHYSIOLOGICAL CHEMISTRY

blood, although it will not clot itself, will hasten clotting in other bloods.

This tissue fibrinogen thus obtained is the most powerful of known
styptics and it is the natural styptic of the tissues. The activity appears
to be dependent upon a union of the phospholipin, or some substance
associated with it, with the protein since if the phospholipin is removed
by treatment with benzene, an emulsion of the phospholipin is much
less active than the original substance and the remaining protein is
almost inactive. Union of the two solutions restores the activity. More-
over the addition to the tissue extract of an emulsion of the phospholipin,
which by itself is almost inactive, renders the tissue extract far more
active. (Mills.)

Action of oxalates, fluorides and citrates. If blood is received into
a solution of sodium or ammonium oxalate, fluoride or citrate so that
it contains about 0.1-0.2 per cent. of the first two of these salts, or
if 100 c.c. blood is received into 5-10 c.c. Na, citrate 2.5 per cent. in
NaCl 0.9 per cent., it will remain indefinitely liquid. If, however,
sufficient ‘calcium chloride is afterwards added to such oxalate, or
citrate blood so that there is an excess of calcium in the blood, clotting
occurs normally. Fluoride blood does not clot spontaneously by the
addition of an excess of calcium. Since these substances, except
the citrate, have the power of precipitating calcium, and since the
addition of calcium causes the blood to clot, it is concluded that
the presence of calcium salts is necessary for the clotting of blood.
While the citrate does not precipitate calcium, it is believed to unite
with it and hold it in an un-ionized form so that its action also
is supposed to be that of a decalcifier. The injection of calcium
chloride solutions into the blood increases somewhat its tendency to
clot, but does not in itself cause clotting, nor does it do so outside the
body. Both strontium salts and barium salts will enable decalcified
blood to clot, although the action is not so good as that of calcium,
By collecting blood in oxalate solution and centrifuging the corpuscley
out there is obtained a clear plasma, sometimes slightly colored by
hemoglobin, which will not clot spontaneously, and this is called oxalate
plasma.

Action of strong solutions of magnesium sulphate and sodium
chloride. The clotting of the blood may be prevented in vitro by receiv-
ing the blood in an equal volume of 10 per cent. NaCl, so that the mix-
ture contains 5 per cent. of NaCl. Such blood remains fluid for a long
period. If the corpuscles are centrifugalized off, the clear plasma is
ealled sodium chloride plasma. Such plasma will clot spontaneously
if it be diluted with four volumes of water. Similarly, if bood be
received into a saturated solution of MgSO, so that there will be threw
THE BLOOD. THE CIRCULATING TISSUE 521

volumes of blood to one of the solution, the blood will not clot. If the
corpuscles be centrifugalized off, then the addition of water to the
magnesium sulphate plasma does not cause spontaneous clotting, but
the diluted plasma will clot on the addition of some of the serum from
clotted blood.

Explanation of the facts just cited. The facts which have just been
stated about the clotting of blood, and some other facts which we shall
now discuss, have been known most of them for a very long period,
namely from 20-40 years, but the satisfactory explanation of these facts
is still impossible. With these experimentally ascertained facts in mind
we may now proceed to examine the nature of the processes involved
in clotting. It is very important for the student to hold the facts
themselves clearly in mind, rather than any explanation of them, for
there are about as many explanations offered as there are investigators,
and none of these explanations is as yet satisfactory. The great diffi-
culty in the explanation is due to the complexity of the blood and our
ignorance of the fundamental properties of solutions and the processes
of crystallization, for the clotting of the blood, as we shall see in a
moment, is at the bottom the crystallization of a supersaturated solution,
the crystalline substance which separates being called fibrin.

Fibrin. Whatever may be the exact nature of the processes involved,
all are agreed that the conversion of the blood from a liquid to a solid
clot is due to the fact that a substance of an insoluble nature appears
in it. This substance is called fibrin. It is a protein substance. It is
deposited first as very fine acicular crystals lying between the blood
corpuscles, and radiating from the blood plates or other finely divided
foreign particles. (Figure 53.) These crystals shortly coalesce, or stick
together, and form a network in the interstices of which corpuscles, both
white and red, and the blood plasma are held so that the whole is in
the nature of a solid clot. If blood as it is drawn be whipped with a
glass rod, the fibrin as fast as it separates sticks to the rod in the form
of tough strings in which some corpuscles are entangled. This is fibrin.
The blood from which this fibrin has been thoroughly removed will no
longer clot. It consists of a mixture of serum and corpuscles and is
called defibrinated blood. ,

The question is, then, what is the origin of this fibrin? Does it
pre-exist in the blood or does it appear only at clotting? Is it in the
corpuscles or plasma and why does it appear when blood is shed? Some
of these questions are not difficult; others of them are very hard to
answer.

Fibrinogen. Origin of fibrm. The first question which may be asked
is this: Does the fibrin come from the corpuscles or from the plasma?
This question may fortunately be answered with certainty. The greater
522 PHYSIOLOGICAL CHEMISTRY

part of the fibrin comes from the plasma, not from the corpuscles, or
at least not directly from the corpuscles. If blood is prevented from
clotting by the action of any one of the anticoagulants just mentioned,
such for example as strong NaCl solution, or leech extract, it is possible
to remove all the corpuscles, both red and white, by centrifugalization.
The clear plasma thus obtained, if it be peptone plasma or salt plasma,
will clot if diluted with water. Fibrin appears in it. If oxalate
plasma is obtained, it is necessary to add some serum or an extract
from the alcohol-coagulated serum, or a calcium salt to make it clot,
but then it clots and yields a typical fibrin. On the other hand, the
blood corpuscles washed free from plasma by suspending them in
physiological salt solution and recentrifugalizing will not clot and form
fibrin under similar circumstances. This experiment shows clearly that
the fibrin has come from the blood plasma. It is found that the amount
of fibrin which a dog’s blood will yield is about 0.1-0.3 per cent., but after
suppuration or inflammation it may rise to double or treble this amount,
ie., to 1 per cent. The plasma yields naturally a larger proportion, since
the corpuscles which make 30-40 per cent. by volume of the whole blood
contain none. Plasma generally yields from 0.2-0.6 per cent. of fibrin.
Since the fibrin once formed is, as such, quite insoluble or nearly insolu-
ble in blood plasma, it is believed that the fibrin does not exist as fibrin
in the plasma, but as a substance which gives rise to fibrin. This pre-
existing substance is called fibrinogen. The essential act of clotting,
therefore, appears to be the conversion of the soluble fibrinogen into
fibrin. It may be said here, however, that besides that in solution in the
plasma fibrinogen is also contained in the blood plates.

The optical phenomena of clotting. Before taking up the nature of
the relation between fibrinogen and fibrin, we may for the moment turn
aside to the microscopic examination of the clotting blood. The process
of clotting of plasma or of blood itself can be best studied by means of
the ultra microscope, that is the dark field microscope. If plasma or
blood be watched as it clots, it will be found that fibrin comes out in the
form of very fine, long, acicular crystals shown in Figure 58. These
crystals are probably not solid crystals, but liquid crystals, since liquid
crystals not infrequently take this form. Moreover, they have another
property of liquid crystals, that of coalescing or sticking to things; it is
because of this property that they will stick together to make thick, long
strands of fibrin of which the crystalline nature could not be inferred
in any way by microscopical examination except from their double
refraction. That indeed shows that their molecules are oriented. The
crystals appear first, as a rule, about little specks of dust, or, if blood
platelets are present, they adhere to them and grow out from them. We
shall later come back to the discussion of the relation between the plate-
THE BLOOD. THE CIRCULATING TISSUE 523

lets and coagulation. The fact is, however, that we have in fibrin forma-
tion a process of crystallization. The clotting of the blood is a crystal-
lization of fibrin. This fact was clearly recognized by Wooldridge many
years ago. The problem we have to answer is then this: Why does crys
tallization occur when blood is shed and why does it not occur in the
blood vessels ?

The relation of fibrin to fibrinogen. The first question which we wish
to ask is then this: Is the fibrin preformed but held in solution in the
plasma until clotting occurs; or is fibrin formed only at the moment of
clotting from the fibrinogen which is itself soluble. If the latter
hypothesis is true, namely, that fibrinogen is converted into an insoluble
protein, fibrin, which crystallizes out, then the problem is essentially

 

Fic. 538.—The clotting of fibrin showing the long, acicular crystals. A crystalline
gel as seen in the ultra microscope (Stubel).

to learn the nature of the difference between fibrinogen and fibrin. The
conversion of fibrinogen into fibrin is the essential thing. If, on the
other hand, the fibrin is simply held in solution by union with some
substance in the blood plasm and crystallizes out, when the influ-
ence of this substance is withdrawn, then the problem is quite a dif-
ferent one. The first question, then, to be discussed is the relation
between fibrinogen and fibrin. What is the nature of fibrin? Now it
unfortunately happens that we do not know what is the composition of
fibrin. It is known that it is a protein. It is generally stated that
it is a simple protein. The composition of it and of fibrinogen have been
given by various authors. The figures of Hammarsten are as follows:
Cc H N oO Ss

Fibrinogen ..........seeeeeees 52.93 6.90 16.66 22.26 1.25
Fibrin... oe cece cece eee eeeeee 52.68 6,83 16.91 22.48 1.10
524 PHYSIOLOGICAL CHEMISTRY

It will be noticed that the analyses show no difference in composition
between fibrinogen and fibrin. But, while these figures appear so clear-
cut and decisive, their reliability is in fact an illusion. The substance
which is analyzed as fibrin and called fibrin in the foregoing analysis
is not the substance as it appears in the blood; and the substance which
is analyzed as fibrinogen is not the substance which is obtained from
blood by salting out, but each is a modified substance. Each of these
substances before analysis has been extracted with alcohol and ether to
remove the fat and lipoid. The substance remaining is a protein, of
which the composition has just been given. Now it may be just in the
lipin part of the molecule that the difference lies, and indeed Wooldridge
maintains that this is precisely the case. It is true of all the proteins
of the plasma that when they are precipitated from plasma by the
addition of salt, the precipitate which is obtained always contains a
considerable proportion of a phospholipin. Wooldridge showed that
fibrin might contain as much as 14 per cent. of its dry weight as phospho-
lipin. This phospholipin, or phosphatide, is often regarded as an impur-
ity, but the probabilities are that it is a compound with the protein. The
amount of phospholipin in the fibrin is less, Wooldridge thought, than
that in fibrinogen, and this author, one of the ablest and keenest-sighted
of all who have worked on this problem of coagulation, believed that
the principal difference between these proteins consisted in the propor-
tion of lecithin in the two cases. It may be mentioned in this connection
that we have a very similar state of affairs in the case of hemoglobin and
the red biood corpuscles. The amount of hemoglobin contained in the
blood of most, or at least of many, mammals is very much more than
can be dissolved in the liquid of the corpuscles when the oxyhemoglobin
is free. It is held in solution by union with the phospholipin and protein
of the stroma of the red blood corpuscles. When this union is broken
hemoglobin crystallizes out, just as the fibrin crystallizes out. There are
so many points of identity between laking blood and clotting that we
may with some confidence believe that the processes are at bottom essen-
tially of the same kind. There is at least one difference between fibrin-
ogen and fibrin, which was emphasized by Wooldridge, but which seems
to have been overlooked by all subsequent workers. If fibrinogen is ob-
tained by Hammarsten’s method of salting out by means of a saturated
sodium-chloride solution, Wooldridge found that this fibrinogen when
digested by pepsin hydrochloric acid left a large residue. This residue is
by some authors (Pekelharing) called,a nucleoprotein, or nuclein residue,
owing to the fact that it is soluble in alkalies and contains a great deal
of phosphoric acid; but it is incorrectly so regarded. The phosphoric
acid of the residue is soluble in aleohol. It is not, therefore, a nuclein,
since nucleins are insoluble in alcohol. Nearly the whole of the
THE BLOOD. THE CIRCULATING TISSUL 525

phosphoric acid is extractable with alcohol. This shows that fibrinogen
as it exists in the plasma is a lecithoprotein, or at any rate that it con-
tains a good deal of a phospholipin. Now Wooldridge found that fibrin
did not vield nearly as abundant a residue of phospholipin when digested
by pepsin-hydrochlorie acid. It dissolved in the digestion mixture almost
perfectly. The small residue it yielded Wooldridge believed to be due
for the most part to some unchanged fibrinogen. These observations
appear to indicate that fibrinogen and fibrin are not identical substances.
but differ in the proportion of phospholipin in them, fibrin containing
less.

Fibrinogen. The protein usually called fibrinogen, but which we
may call Hammarsten’s fibrinogen, may be obtained from centrifugalized
oxalate plasma by adding to the plasma an equal volume of concentrated
NaCl. None of the other proteins of the blood plasma precipitate with
this treatment. The precipitate redissolves in dilute salt solution. It
is insoluble in water and acts in this respect like a globulin. It may be
redissolved in salt solution and reprecipitated several times so as to
get it as free from the other proteins as possible. Thus obtained it
is always contaminated with, or joined to, a phospholipin of some kind.
It has a tendency to become insoluble and to be converted into a fibrinous,
insoluble substance, apparently fibrin. If left long in the salt solution
from which it is precipitated, it undergoes this change. Wooldridge
called this a true conversion into fibrin. It is hard to know whether
it is or not. Dog’s fibrinogen particularly easily undergoes this change;
horse’s changes less readily. If we disregard the phospholipin and con-
sider this only an impurity, the fibrinogen appears to be a simple pro-
tein, coagulating at 56° C. The kinds of amino-acids it has is shown on’
page 144. It has the property of clotting when in solution on the addi-
tion of thrombin or of serum. It is optically active.

We may sum up this discussion, then, by the statement that it is
at present impossible to say whether fibrin pre-exists in the blood plasma
being held in solution by its union with other proteins, or other sub-
stances, until the time of clotting; or whether an insoluble protein is
formed from a soluble one, fibrinogen, the fibrin then crystallizing out.
The latter. view is the one at present held by nearly all physiologists, but
it was strongly criticised and condemned by Wooldridge, and his objec-
tions, in the writer’s opinion, have never been answered.

The role of the structural elements of the blood in clotting. When
blood clots, then, we observe that the clotting is due to a protein substance
which had been in solution in the blood plasma, crystallizing out of solu-
tion; while the exact nature of the changes involved in this protein
which determines that it becomes insoluble or capable of crystallization
is still unknown, we may proceed to examine the causes which are
526 PHYSIOLOGICAL CHEMISTRY

playing a part in the transformation. The first question which we
shall ask is whether the red, the white corpuscles, or those more
unknown elements, the blood plates, play any part in the process.
This question is generally answered in the affirmative, namely, that
both the white blood corpuscles and the platelets play a very
important part in the. initiation of the clotting process, but, as
we shall see, the evidence that the white corpuscles are concerned in
the process is at the best very dubious. On the other hand, there is
no doubt that the elements called blood platelets are a very important
factor in the clotting of the blood. Schmidt, who worked for many
years on the clotting of the blood, and to whom most of the essential, but
possibly erroneous, conceptions of the process are due, was of the opinion
that the white corpuscles played a very important part. According
to him, when blood is shed the white corpuscles break down in part and
liberate a substance which he called prothrombin. This substance does
not act, according to him, by itself, but in certain circumstances, i.e., in
the presence of calcium salts, it is converted into a third substance,
thrombin, or fibrin ferment, as he called it. This substance he believed
to be a ferment, and by its action on the fibrinogen of the blood, with
the co-operation of the paraglobulin, it formed fibrin. This view of
Schmidt as regards the réle of the leucocytes, the furnishing of pro-
thrombin and thrombin and its action on fibrinogen, is accepted by the
majority of workers on this subject. There is a doubt, however, whether
it is essentially correct and well-founded. The evidence that Schmidt
adduced to prove that the white corpuscles cause coagulation was this:
If horse’s blood is received directly from the artery or vein into a
cooled glass vessel and the blood immediately cooled in a salt-water-ice
mixture and kept cooled to about 0°, the clotting is so prolonged that
time is given for the corpuscles to settle. The red corpuscles being more
dense settle faster than the whites, and accordingly there is at the bot-
tom of the tube a layer of red corpuscles, above this is a thin layer of
white corpuscles and above this is the plasma, which is somewhat turbid,
due to the admixture of some platelets and leucocytes. Now coagulation
ultimately ensues in this blood and it is observed that the clotting begins
always in the white layer, where the white corpuscles are most abundant,
and that the plasma at the top and the red corpuscles underneath may
not be coagulated, whereas the plasma just above and including the layer
of the whites is coagulated. On examining this white layer under the
microscope, Schmidt observed that there was among the white corpuscles
a large amount of a granular precipitate looking like the débris of
corpuscles which had gone to pieces. Schmidt interpreted the débris
as being due to disintegrated leucocytes, although there was no evidence
that it was so derived, and we now know that it has in part at least
another origin. Schmidt concluded from these observations that whcr
THE BLOOD. THE CIRCULATING TISSUE 527

blood is shed many of the corpuscles disintegrate and set free a sub-
‘stance which causes clotting. This substance he called fibrin ferment.
To support this conclusion Schmidt tried the experiment of injecting
leucocytes derived from lymph glands, or débris of the latter, into the
circulation and he believed that the intravascular clotting sometimes
obtained was due to this fibrin ferment from the lymph glands. Now
this conclusion of Schmidt, although it has persisted in the literature as
a fact, is incorrect. Its incorrectness was shown by Wooldridge. So
far as is known, the leucocytes of mammalian blood do not disintegrate
to any considerable extent in shed blood. They persist and the débris
which Schmidt observed did not come from them, but consisted of the
substance now known as platelets, and the origin and nature of which
we shall soon consider. All the evidence which Schmidt accumulated
really applied to the blood plates, and it is these indeed which really
inaugurate the process of clotting. That the leucocytes do not cause the
clotting and are not necessary for it may be shown in several ways.
Blood plasma quite freed from leucocytes and red blood cells by centrifu-
galization, such for example as salt plasma, or peptone plasma, will
coagulate if simply diluted with water, or if a current of CO, is passed
through the plasma. No corpuscles of any kind are necessary. More-
over, it can be shown that the leucocytes are quite inert. Wooldridge
found that if lymph corpuscles are carefully washed with salt solution,
they will cause clotting if added to peptone plasma, or if added to salt
plasma, provided the substance of the platelets, which he called
A-fibrinogen, is also present. But these same leucocytes do not clot the
lymph in which they are. Exudates exist containing many leucocytes
and fibrinogen, but these do not clot. Moreover, if the leucocytes which
have been once in blood are collected by centrifugalization, washed and
suspended in peptone plasma, they do not cause clotting of the plasma,
although Jeucocytes of lymph glands, which have never yet been in the
blood, will cause clotting. It is clear from this extraordinarily instruct-
ive and important observation that the leucocytes of the blood have not
a power of inaugurating clotting. Wooldridge tried another very sug-
gestive experiment. If very strong peptone plasma is used, a consid-
erable amount of lymphocytes may be added without producing clotting.
If now these added lymphocytes are centrifuged from the plasma and
washed carefully in salt solution, so as to free them from adherent
plasma, and are now introduced into a peptone plasma not so strong
but one which will clot spontarreously in time, they do not clot this
plasma, whereas a similar quantity of the same lot of lymphocytes which
had not first been in the peptone plasma will clot it at once. This fact
shows, according to Wooldridge, that leucocytes which have once been
in the blood become inert toward it; whereas those which have not been
528 PHYSIOLOGICAL CHEMISTRY

in the plasma act like foreign bodies and hasten the clotting. Wool-
dridge was able to show, also, that it was the substance of the blood
platelets which was the important substance in clotting. It is, however,
possible to extract from the leucocytes a substance which hastens blood
clotting. That the red corpuscles are not primarily concerned in the
clotting is also clearly shown by the fact that the plasma has all the
powers of clotting in the absence of the reds. But here again, just as
with the whites, it is possible to isolate from the red corpuscles a sub-
stance which acts exactly like the substance from the whites and causes
clotting. The fact, then, that it is possible to isolate from the white
corpuscles a substance which hastens coagulation is no argument that
the white corpuscles play a part in normal clotting, since exactly the
same argument applies also to the reds, which are universally admitted
to play no part in the process. It is a very interesting fact that while
the intact red corpuscles are quite inert toward the clotting process, yet
if the hemoglobin be separated from the stroma it is found that the
stroma is very toxic, its injection produces intravascular clotting and
when added to shed blood it greatly accelerates clotting. There is no
good evidence, then, that either the reds or the whites are concerned
in the normal clotting of blood, and we may now turn to the blood plates,
the other morphological constituent of the plasma. Practically all
authorities are of the opinion that these play a vital part in the clotting,
since the addition of these to blood enormously accelerates the clotting
process.

Réle of blood plates in clotting. We now enter one of the most dis-
puted fields of the morphology of the blood, for there is no unanimity
of opinion as to the nature and origin of the blood plates, or indeed
whether they pre-exist in the blood in the vessels. But whether they
pre-exist or appear only after shedding, they play a part in clotting,
and in fact their appearance is the first visible sign of the changes of
the plasma resulting in clotting. The platelets are generally disk- or
spindle-shaped bodies without color, about 1.5-3 4 in diameter. In other
words, they are about one-third to one-seventh the diameter of a red blood
corpuscle. They may be present in enormous numbers, as many as
800,000 per c.mm. of blood in dog, or human blood, or they may be
quite absent, as in bird’s blood and the blood of the lower verte-
brates. Normal human blood contains about 300,000 per cu. mm.
They may be fixed by osmic acid. When first separated from oxalated
blood by centrifugalization they redissolve readily in dilute alkaline
salt solutions, but after. a short separation they lose this power of
dissolving and become converted into a fibrinous mass. It is very diffi-
cult to decide whether they are to be considered as morphological cells,
as they are at present generally considered, or whether they are to
be regarded as liquid crystals. It is well known that supersaturated
THE BLOOD. THE CIRCULATING TISSUE 529

liquids form crystalline deposits with the greatest ease, so that it is quite
possible that we have in the blood plasma normally something in the
nature of a supersaturated solution of the materials from which the
plates are formed and that this deposits the plates very quickly on
contact with foreign substances. Something of a very similar kind
appears to happen in the cell nucleus and to result in the sudden
appearance of the nuclear chromosomes. It has recently been shown by
Chambers that the chromosomes may appear verv suddenly in the clear
homogeneous nuclei of the sperm mother cells of the testis of grass-
hoppers. The chromosomes appear whenever the cell is irritated, and
their appearance inaugurates the phenomena of cell division just as
the appearance of the plates inaugurates the process of clotting. The
chromosomes are also organized substances of definite shape and, like
the plates, are often disk-shaped. The phenomena of the appearance
of the plates from the plasma has been particularly studied by Wool-
dridge. If horse’s blood is cooled, the corpuscles separate and the
plasma may be removed. The same result is had with dog’s peptone
blood. The corpuscles are separated by centrifugalization. The plasma
which results is perfectly clear at the temperature of the body, but if
it be cooled to about zero degrees there comes out of it a precipitate of
platelets. This precipitate is not an amorphous precipitate, but is made
up of clear, granular refractive disks, which look like imperfect protein
crystals. On warming the blood plasma the platelets swell and redis-
solve and may be reseparated by cooling. The little plates thus sepa-
rated from the plasma look exactly like very small colorless red blood
cells and when slightly warmed they coalesce and may form larger
disk-shaped, colorless structures of the shape and size of a red blood
corpuscle. If after separating them in the cold, the precipitate with
some plasma is warmed slightly, the disks lose their crystalline appear-
ance and rounded or spindle shape and stick together in rows and clumps
of granules which form fibrinous strings and become insoluble. They
become changed to typical fibrin (Wooldridge, Schittenhelm and
Bodong). The disks when they first appear look like the incompletely
crystalline stage of proteins. Their behavior reminds one of the liquid
crystals described by Lehman. Wooldridge concluded that they were
in reality crystalline. By many authors they are supposed to be alive,
because they show ameeboid changes of form. But this again is a char-
acteristic of some liquid crystals. Such crystals also have the power
of changing their shapes and to make refractive myelin-like forms resem-
bling protoplasm. It is possible, of course, that in some bloods they are
present in such amount that they may be in part precipitated in the
circulating blood. The plates have been. collected by Wooldridge and
their chemical composition seems to be identical with that of the stroma
530 PHYSIOLOGICAL CHEMISTRY

of the red blood cells and like the protoplasm of the whites. They resem-
ble this stroma, also, in their physiological action, as we shall see. They
contain a protein, fibrin and various lipins, such as a phospholipin,
thrombin and a proteolytic ferment. They closely resemble the stroma
of the red corpuscles of birds’ blood, as the stroma also has the power of
clotting and yielding fibrinogen.

It was believed by Lillienfeld and Pekelharing that these plates-were
composed of nuclein. This belief was based on the fact that they were
soluble in dilute alkalies, precipitated by dilute acids, were soluble in
strong NaCl, contained phosphorus, stained with methyl green and basic
dyes, and that they left a phosphoric acid containing residue when
digested with pepsin HCl. Small amounts of nuclein bases may be
obtained from them. But all these characteristics, except the last, are
possessed by the lecithoproteins as well as the nucleins, and Wooldridge
showed that the phosphoric acid in the residue was soluble in great part
in alcohol. We know, besides, that particularly the phospholipins have
this property of forming liquid crystals. While, then, the origin, and
indeed the proper interpretation of the blood plates, is thus still in
many points obscure, we may consider their relation to clotting. It may
be said, in passing, that the interpretation of the clotting of the blood
really rests on the determination of the origin and significance of the
plates.

If any foreign substance wet by the blood is put into the blood
stream, such as a needle or a thread put through a vein or artery, it is
found that the foreign substance becomes covered with a deposit of
plates and that this deposit becomes covered by, or is partially con-
verted into, fibrin. Moreover, when platelets are added to plasma and
the latter clots, it will be observed that the platelets are the centers from
which the fibrin formation starts. The relation of the plates to clotting
was also shown by Wooldridge as follows: Dog’s blood, which is inco-
agulable from the injection of peptone, becomes coagulable if a stream
of CO, passes through it. The same is true of the peptone plasma, pro-
vided the dose of peptone has not been too large. Peptone plasma will
clot spontaneously if CO, is passed through it, or if acetic acid is added
to the plasma, or if the plasma be diluted with two or three volumes of
water, but it will not clot spontaneously if diluted with two or three
volumes of 1 per cent. NaCl. Wooldridge found that if such coagulable
plasma was cooled to a low temperature and the platelet stuff centrifu-
galized out of the blood and the plasma then filtered through several
thicknesses of filter paper so that the plasma was perfectly clear and
free from plates (A-fibrinogen, as he called it), the plasma had lost its
power of clotting when diluted with water or when CO, was passed
through it, but it reobtained its power if the platelets either suspended
in a little plasma or redissolved in very dilute alkaline salt solution were
THE BLOOD. THE CIRCULATING TISSUE 531

added to it. Moreover, this plate material separated and dissolved in
the manner indicated had the property when injected into the circula-
tion of an animal, of causing intravascular clotting, in this respect
resembling the stroma of the blood corpuscles or extracts of the various
tissues. There can be no doubt, then, from these facts that the stuff
known as blood platelets stands in some very important relation to clot-
ting. They seem to resemble chemically and in action the substances
(fibrinogens) obtained from the tissues and from the wounded cells and
tissues. A further very important fact is that the amount of fibrin
recovered from plasma by the addition of plates to it increases with the
amount of platelet stuff added, so that Wooldridge believed that the
platelet stuff actually contributed to the substance of the clot, as well as
acting as a starter of the process of clotting. This view has now been
substantiated. Exactly the same fact holds for tissue fibrinogen.
Prothrombin and thrombin. So far, we have seen that the plasmz
contains all the elements necessary for the clotting as long as the plasma
contains platelets or the substance A-fibrinogen, which gives rise to the
platelets. There remains, however, another factor to be considered. It
has been found that, after plasma has clotted and the serum separated.
the serum always, or nearly always, contains a substance which was not
in the plasma and which has a remarkable action on the clotting of
Hammarsten’s fibrinogen. This substance is known as thrombin, or
fibrin ferment. It is better to call it thrombin, since there is no evidence
that it is a ferment, although it may be one. If blood serum is pre-
cipitated by adding to it four volumes of strong alcohol and the pre-
cipitate allowed to stand under alcohol for two to four weeks, all the
proteins of the precipitate are coagulated by the alcohol. If now this
coagulum be extracted with water, or dilute sodium chloride solution,
an organic substance goes into solution and this solution added to a
solution of Hammarsten’s fibrinogen causes it to clot in a typical fashion.
Furthermore, although its action is somewhat quicker in the presence of
calcium salts and in the absence of oxalate, yet it will clot the fibrinogen
solution in the presence of oxalate. The substance in the solution which
has this property of clotting fibrinogen is destroyed by continued heat-
ing to 60°. It was called fibrin ferment by Schmidt, its discoverer, but
is now generally called thrombin. Nothing is known about the nature
of this substance. Lillienfeld and Pekelharing believed it to be a
nucleoprotein, but it is probably not a nucleoprotein. There are rea-
sons for thinking that it originates from the platelets, and these are
almost certainly not nucleoprotein and contain none. Its nature is
unknown. Now thrombin does not pre-exist in the blood. Like fibrin,
it appears only if the clotting has occurred. It is an accompaniment of
the clotting process, and. with as much justification as fibrin, is to be
regarded as the result of the clotting process, but not its cause. It is,
532 PHYSIOLOGICAL CHEMISTRY

however, generally believed by most physiologists from Schmidt’s time
to be the cause of clotting. Both Wooldridge and Nolf have urged grave
reasons for disbelieving that it is the appearance of this body which
inaugurates the process of clotting normally. There is one reason which
appears to the writer to be practically conclusive that the appear-
ance of this substance is not the cause of normal clotting: that is the
astonishing fact that enormous amounts of it may be injected into the
circulation without causing any intravascular clotting. Now, if it be
assumed that there is some anti-clotting substance normally present
which is counteracting its action and keeping blood fluid, there cannot
be the quantity required to hold back the action of so much of this sub-
stance. A very small amount of thrombin will cause clotting of a
fibrinogen solution in the test-tube, but one can run in nearly as much
thrombin as there is of fibrinogen in the blood without causing any
intravascular clotting and without producing any change, or more than
a slight change, in the clotting power of the blood. Now it is very aston-
ishing, if thrombin is the cause of clotting and the formation of minute
amounts of this substance in the shed blood causes its clotting, that
the injection of large amounts of thrombin into the blood, which con-
tains already fibrinogen and calcium salts, has no effect at all. The
result is the more astonishing since the injection of very little tissue
extract, or blood platelet material, causes clotting both within and with-
out the body with the greatest ease. Moreover, these same substances
will clot peptone plasma outside the body, whereas thrombin does not
clot it. These facts do not seem to have been properly appreciated by
most writers who have accepted Schmidt’s view. They constitute, in
the writer’s opinion, an insurmountable objection to the present view,
that the first thing that happens in clotting is the formation of thrombin,
and that this latter causes clotting by making fibrin out of fibrinogen.
Wooldridge, because of these facts and others, held thrombin to be a
result of clotting, not to be a ferment, and maintained that the fact
that thrombin would not clot blood intravascularly showed that blood:
did not contain the substance known as Hammarsten’s fibrinogen, but
that the latter had been formed from the real fibrinogen of the blood,
which he called B-fibrinogen, by the actions necessary to separate it.
Since thrombin did not exist in the plasma before clotting, but
appeared after clotting, Schmidt and his pupils concluded that there
must be an antecedent substance there which was called prothrombin,
but which has also been named thrombogen, to indicate that it gave rise
to thrombin. According to Schmidt, thrombogen was not present in the
plasma, but was set free from the leucocytes when the latter disinte-
grated. This is the generally accepted belief at the present time, but it
is not well grounded. There is no sufficient evidence that the leucocytes
go to pieces in shed blood; Schmidt’s evidence that the substance came
THE BLOOD. THE CIRCULATING TISSUE 533

from the leucocytes was vitiated by the fact that he neglected the plate-
lets; it is the platelets, not the leucocytes, which added to peptone blood
cause clotting, and it is from the platelets, not the leucocytes, that the
prothrombin appears to be derived.

The réle of calcium salts. There is no doubt that calcium salts play
an important role in clotting (Green, Arthus). They are not necessary
for the clotting of fibrinogen solutions by thrombin, but such solutions
will not clot on the addition of platelets unless calcium salts are present,
and the calcium must be in an ionized form. The salts are supposed
to act, hence, by converting thrombogen, or prothrombin, into thrombin.

Explanation of the clotting process by Morawttz and Fuld and Spiro.
According to the work of Morawitz and Fuld and Spiro, the leucocytes
contain proferment, or thrombogen. This thrombogen in the presence
of calcium salts is converted by tissue extract, which they call throm-
bokinase, into an active thrombin. The thrombin, in some unknown way,
converts the fibrinogen of the blood into fibrin. Blood remains fluid
in the vessels of the body, because there is no thrombokinase present.
This is supplied by the blood coming in contact with the tissues of the
body. These authors, then, make the clotting of the blood analogous -
to the digestion of fibrin by pancreatic juice. The latter is supposed to
have its trypsinogen converted to trypsin by the action of the entero-
kinase of the intestine in the presence of calcium salts. Thrombin,
according to this view, is really an enzyme, and it is the thrombin of
the blood which causes clotting. This view encounters two or three main
objections. First, there is no evidence that the thrombin is a ferment,
but on the contrary it can be shown that it unites with the fibrin; and
the amount of fibrin formed is pretty strictly proportional, when the
conditions are kept constant, to the amount of thrombin added. In the
second place, the thrombokinase, which they supposed to be killed: by
heat, is in reality heat resistant, and Wooldridge showed that it was a
phospholipin, but distinct from the usual lecithin of eggs. Howell has
shown that it belongs to the cephalin group of phospholipins. There is
no evidence that the conversion of the fibrinogen into fibrin is of the
nature of a proteolytic digestion, although there is no evidence against
this view, either. They cannot explain how it happens that the addition
of very little of cephalin, or extract containing it, clots the blood far
better than the addition of thrombin itself.

View of Nolf and Howell. To account for the fact that the blood
remains fluid in the body, although it contains all the materials for
clotting, the simplest explanation is that there is present some antago-
nistic substance which prevents clotting. This view has been suggested
from varicus sources from Wooldridge down and lias recently been
advocated by Nolf and Howell, According to Nolf, the blood contains
534 PHYSIOLOGICAL CHEMISTRY

a substance called hepatothrombin to indicate that it is formed by the
liver. This hepatothrombin unites with leucothrombin (the thrombogen
of other authors) to form thrombin. Leucothrombin is supposed to come
from the leucocytes, hence its name. According to Nolf, hepatothrombin
by itself unites with fibrin and holds it in solution so that it cannot be
precipitated. There is normally a slight excess of hepatothrombin in
the blood so that it keeps the blood fluid; but when blood is shed there
is a discharge of leucothrombin from the leucocytes. The leucothrombin
then unites with the hepatothrombin, and the two together with calcium
make thrombin; this then unites with the fibrinogen, and the three
make fibrin. It happens that there is a slight excess of thrombin in
the blood so that some remains in solution in the serum after the clot-
ting has taken place. Nolf’s hepatothrombin corresponds in a general
way with Wooldridge’s A-fibrinogen and with other tissue thrombins
or fibrinogens. The hepatothrombin is what others have called anti-
thrombin. In later work Nolf modified this view somewhat. According
to Howell, the process is a little different. The blood remains fiuid in the
body because there is an excess of antithrombin there. This anti-
thrombin is probably formed by the liver. The coagulative action of
tissue extracts is due to the fact that they contain a cephalin-like body,
as Wooldridge thought, but this cephalin-like body causes the clotting by
uniting with the antithrombin and thus getting it out of the way. In
the absence of antithrombin the thrombogen is converted into thrombin
by the action of the calcium and unites with the fibrinogen to make
fibrin. This view is in many ways like Nolf’s, although the terminology
is somewhat different. Both recall the work of Wooldridge.
Wooldridge’s view. It will be obvious from this brief consideration
bow obscure the process is. Nearly all observers have neglected a pos-
sible change in the fibrinogen and have concentrated their attention on
the thrombin and the changes it undergoes. Hammarsten’s fibrinogen
may not exist in the blood as such. The mere fact that there is a pro-
tein often present which will coagulate at 56°, the temperature of coagu-
lation of fibrinogen, and that fibrinogen can be isolated from the blood,
is not conclusive evidence that the fibrinogen exists there in an uncom-
bined form as it does in the fibrinogen solution. It must be remembered
that so-called tissue fibrinogens not clotting with fibrin ferment, but
coagulable at 56°, can be isolated from a great variety of tissues of the
body. In these circumstances of great confusion it is better, in my
opinion, to go back to the work of Wooldridge, whose writings are the
clearest and freest from preconceived ideas of any who have worked on
this subject. A word may not be out of place here about this talented
man, who was far ahead of his time in this work. His work has suffered
a most unmerited neglect and his observations have been rediscovered
since by those who were ignorant of his precedence. Wooldridge started
with the Schmidt view of the importance of leucocytes in clotting, but
THE BLOOD. THE CIRCULATING TISSUE 535

he clearly showed in 1883-88 the untenability of this view. He suuwed

the predominant place of the platelets in clotting and proved that these

bodies were, in part at least, in solution in the blood and that they were

to be considered as crystals rather than as morphological elements. It

was Wooldridge who discevered the intravascular clotting by tissue
extracts and that the acceleration of the clotting by these extracts and

the plate material was due to the alcohol-soluble lecithin-like body in

them. This body was not lecithin itself, since the lecithin from eggs
had no such influence. It was he again who maintained at that time
that the clotting of the blood was in the nature of the crystallization of
some supersaturated liquid, and that view has now, within the past

few years, been demonstrated to be correct. Wooldridge, a young
man, was opposed to the views on coagulation of Schmidt and his school.

views which were at that time, as at present, the dominant views, although

he showed their weakness. This opposition secured for him the enmity

or opposition of his older colleagues in England, and the Royal Society
refused to publish the Croonian Lecture which he delivered before them, -
but buried it in their archives, and it was only published after his death.

There is no doubt that that lecture was far ahead of any treatise at that

time on the coagulation of the blood. This lecture, together with that

entitled ‘‘ Blood Plasma and Protoplasma,’’ and his reports to the
Grocers’ Committee are among the most lucid and able treatises on

coagulation and should be read by anyone who wishes to know the
coagulation of the blood. The great difficulty in the way of the problem

of coagulation at the present day arises from the attempt to harmonize
the facts of coagulation with the facts of immunity and with those of
the action of digestive enzymes. All of these subjects are in the greatest

confusion from the complexity of the problems. It is probable that they

all have the same basis. In all the same elements are found: namely.

phospholipins, an enzyme-like substance, calcium salts, carbon dioxide
and a protein. In nearly all cases the substance coagulates at about 56°.

Study of the coagulation of the blood will no doubt elucidate these

other processes, also.

Wooldridge very carefully avoided giving any names to his sub-
stances which might indicate a preconceived view of the réle they were
playing. According to his view, blood plasma contained two fibrinogens,
A-fibrinogen and B-fibrinogen. C-fibrinogen was the ordinary fibrinogen
of Hammarsten, but it did not pre-exist in the blood in more than very
small amounts. A-fibrinogen gave rise to B-fibrinogen, and this in turn
~ to C-fibrinogen, and this to fibrin. A-fibrinogen was the substance which
constituted the blood plates. This was in solution in the plasma. Blood
plasma was like protoplasma and was indeed nothing else than a dilute
protoplasma. Now, in consequence of various actions when blood was
shed, there was a change in this very unstable form of material. The
first change was the precipitation of some of the A-fibrinogen. When-
536 PHYSIOLOGICAL CHEMISTRY

ever this was precipitated the equilibrium of the plasma was upset in
some way and chemical changes were set up. The A-fibrinogen sep-
arated from the plasma became changed into fibrin, and one of the
products of the change was thrombin. The action of calcium salts in
hastening this change was unknown to Wooldridge. The main difference
between the various forms of fibrinogen and fibrin was supposed to be a
difference in the proportion of phospholipin they contained united with
the protein. This view is, of course, inadequate, but it has the greal
advantage of being, in a sense, a physico-chemical explanation and i
free from the preconceptions of complement, amboceptors and antibodies
which enshroud most modern views in an impenetrable cloud of misun-
derstanding and confusion.

Summary of the clotting of the blood.—To unravel this tangled skein
and to solve the mystery of the coagulation of the blood we must adopt
the methods of those immortal detectives, Lecocq and Sherlock Holmes.
We must put ourselves in the place of the criminal, in this case Mother
Nature, and by seeing clearly the problem she had to solve form a clear
idea of the probable methods employed. The problem was in its essen-
tials this: The great object or trend of evolution has been to develop
consciousness and reason. These properties depend in some way or
other which we do not understand upon a certain structure, such as
exists in the brain, and upon a most irritable and unstable form of
living matter. This living matter requires an unusual amount of
oxygen. It is conscious only when the flames of its burning are leaping
high. Permit the fire to die down and consciousness, reason, all the
higher traits of the mind are lost. It was necessary then not only to
provide a brain, but above all the nervous system must be supplied with
an abundance of oxygen, that the fires might burn. The solving of ths
problem of supplying oxygen was not difficult. Hemoglobin was in.
vented, it was packed into thin corpuscles, so that more could be carried
under the most favorable conditions, lungs were made and the blood
was confined to a closed vascular system and driven at a high rate of
speed from lungs to brain by a clever pump, the heart. As the demand
for more oxygen increased as the nervous system developed, more
hemoglobin was put in the blood, and the blood was driven faster and
faster, under a greater and greater pressure. Now as a result of this
great pressure danger arose of the rupture of the blood vessels and
hemorrhage from wounds. To safeguard from this danger the clotting
of the blood was devised. The problem was this: how can the blood be .
made to clot instantly when it is shed from the ruptured blood vessels,
but yet be in no danger of clotting in the blood vessels themselves. This
problem was solved in the very simple manner of putting some of the
clotting factors in the blood and another highly important one in the
tissues. In the blood was placed fibrinogen and blood platelets; but
these do not interact. In the tissues was placed the other factor, tissue
THE BLOOD. THE CIRCULATING TISSUE 537

fibrinogen, so that as soon as the blood should escape from the blood
vessel it would clot. A further step was taken when the tissue fibrinogen
was placed in greatest amounts in those tissues in which the danger to
life following hemorrhage was greatest, namely, first and foremost in
the lungs, second in the brain, third in the kidney. In birds’ blood there
is practically no tissue fibrinogen in the blood but in mammalian blood
there is a very little in the blood itself, so that clotting of the latter may
occur without tissue admixture, although the latter enormously ac-
celerates and is the usual cause of the clotting. The exact details of the
method by which the tissue fibrinogen acts on the blood to cause clotting
are still obscure. But with this fundamental conception of the separa-
tion of the two elements which cause clotting into those in the blood
and those in the tissues, the general problem appears in a far clearer
light.

This conception of Nature as acting in an intelligent manner to
secure definite ends will no doubt be repugnant to many, but I am
convinced that its general adoption as an aid to investigation, though
not as a tenet of belief, would save science from many of the grievous
errors into which a failure to realize the cardinal facts of adaptation
have led in the past few years.

The clotting of the blood is essentially the crystallization of a protein
substance, fibrin, in the form of liquid crystals which coalesce to form
fibrin strings. Whether the crystalization of this substance is due to the
fact that the fibrin has been held in solution by its union with some
substance which is dissociated from it at the time of clotting, or whether
it may be that the fibrin does not pre-exist in the blood as such but in the
form of a soluble precursor, fibrinogen, cannot be said with certainty.
When blood is shed, and particularly when it.comes in contact with
wounded tissue, this crystallization occurs. Tissues contain substances
which hasten the clotting. There is in blood plasma a substance which
may appear in the form of the structural elements known as blood plates.
This substance has the property of hastening clotting. Calcium salts
are also necessary to clotting. There appear in the blood as the result
of clotting two new substances; one of these, called thrombin, has the
property of clotting fibrinogen solutions but not circulating blood. The
other is known as serum fibrinogen. Its relation to the clotting is not
known. Clotting is very much hastened by and generally dependent
upon contact with some foreign substance. Such substances appear to
act as the points of deposition of the crystals of the blood plates, and
from this separation the fibrin crystallizes out. The process of clotting -
is greatly accelerated by the intravascular injection of tissue extracts.
How these various facts are interacting is still uncertain, but various
theories have been given to explain them. None of these explanations
are satisfactory. The process has many points of resemblance with the
hemolysis of the red blood corpuscles. . There, as in the blood, a crystal-
538 PHYSIOLOGICAL CHEMISTRY

line substance comes out when its union with a lecithoprotein is broken.
It is suggested that the lecithoprotein holding the fibrinogen in solution
is the serum fibrinoglobulin which appears in the serum when the fibrin
erystallizes out. The origin of the fibrinogens is still obscure, but the
general resemblance in chemical composition of the plates and the stroma
of red and white corpuscles would suggest the derivation of one from
the other. The réle of the liver in the formation of the fibrinogen would
thus be explained, also. It is clear that the whole subject of the clot-
ting of the blood is a very attractive field for study from the modern
point of view of colloid chemistry and liquid crystals.

The following view is suggested very tentatively as harmonizing many
of the facts of coagulation: Blood plasma is a very unstable, supersatu-
rated solution corresponding to a very dilute protoplasm. Its insta-
bility may be upset in a variety of ways, these ways being generally
those which we recognize as acting as stimulants of cells. Among the
substances in solution in the plasma is a complex of enzymes, calcium,
phospholipin and protein, a complex which probably corresponds pretty
closely in nature to the clear, homogeneous substratum in cells. The
blood plasma is supersaturated with this substance and under various
conditions, particularly when removed from the blood vessels and brought
in contact with foreign matter which it wets, it crystallizes out. These
crystals are liquid crystals and, like many liquid crystals, they readily
change their shape. They are known as blood platelets. When in solu-
tion they constitute the substance called by Wooldridge A-fibrinogen,
and the part remaining in solution may be called B-fibrinogen. The
composition of this material may possibly be of the following nature:
Protease-fibrin-phospholipin-thrombin-calcium-antithrombin. This anti-
thrombin is partially dissociable. The stability of the whole complex
depends upon it. If a little cephalin, or tissue fibrinogen from injured
tissues which contains cephalin (thrombokinase), is added to this mix-
ture it combines with and removes some of the antithrombin or unites
with this complex. What remains is unstable and breaks apart between
the phospholipin and the thrombin. The protease and fibrin crystallize
out as fibrin, taking with them some of the phospholipin. The thrombin
remains in solution. The protease-fibrin-phospholipin fragment may be
what is called Hammarsten’s fibrinogen, or C-fibrinogen of Wooldridge.
The action of tissue fibrinogen in offsetting the action of antithrombin
would be explicable in the way suggested by Howell and others. The
addition of thrombin to C-fibrinogen causes it to clot, but even in the
absence of thrombin the dissociation of some of the phospholipin would
lead to a slow conversion to fibrin. The addition of thrombin to blood
would have no effect, since C-fibrinogen does not exist free in blood itself.
The rapid digestion of the fibrin by the protease under appropriate con-
ditions would be explained, also. It may be that the instability of the
protease-fibrin-phospholipin-thrombin complex is much greater when this
THE BLOOD. THE CIRCULATING TISSUE 539

is present as the calcium salt, and perhaps greatest of all when carbon
dioxide is added to the other bond of the calcium. That there are
difficulties in the way of this view is self-evident and it is at the best but
tentative. The advantage of it is that it agrees, on the whole, better
with what we know of the general structure of protoplasm than the
others suggested. But the further development of this subject cannot be
taken up here.

Alkalinity of the blood.—Both the reaction of the whole blood, as
well as that of the blood plasma, is alkaline toward litmus, but acid
toward phenolphthalein. This means that there are very few hydroxyl
ions present in the blood. But, while it is true that blood is thus almost
neutral so far as its hydrogen ion content is concerned, it nevertheless
has the power of neutralizing quite a large amount of strong acid before
its reaction turns acid to litmus. The coincidence of these two properties
means that blood has in it a good deal of some very weak alkali, i.e.,
NaHCO,.

Total alkalinity. The power of the blood of neutralizing acid added
to it without itself becoming acid to litmus is called the total alkalinity.
It is the total titratable alkali. The amount of this titratable alkali
varies with the amount of protein present and under other circumstances.
In man it has been found to be equivalent to a sodium carbonate solution
of 0.34 to 0.59 per cent. strength. In other mammals it generally lies
between these extremes. In the dog it is 0.49, and in the horse 0.44 per
cent. Na,CO,. It is somewhat reduced after violent exercise, in partial
asphyxia and in diabetes. The alkalinity is said to diminish rapidly
outside the body, perhaps owing to the production of acid in the blood
itself, or perhaps for some other reason.

This total alkalinity of the blood is due to the presence in the blood
serum or plasma and in the corpuseles of sodium bicarbonate, sodium
carbonate and of a little Na,HPO,; and to the presence of the alkali
salts of the proteins of both the corpuscles and the plasma. When a
strong acid is added to blood it takes away the sodium and potassium
from these weak acids, the carbonic, disodium hydrogen phosphate and
the proteins, to make the neutral salts of the strong acid. All of these
acids are very weak, and accordingly it takes a large amount of them be-
fore they furnish enough hydrogen ions to turn litmus red. The proteins
of the blood will also, in another way, combine with the acid by means of
their amino groups, so that in two ways proteins neutralize acids pro-
duced in the tissues and which find their way to the blood. This acid-
combining power of the blood is of very great importance, for it is in
virtue of it that the blood is able to pick up the carbon dioxide from
the tissues and carry it to the lungs. It is also of very great value in
caring for an excessive pathological acid production in the tissues, such
as occurs in diabetes. In this disease large amounts of hydroxybutyric
540 PHYSIOLOGICAL CHEMISTRY

acid and aceto-acetic acid are set free in the tissues and the blood must
carry them to the kidneys to get rid of them.

If, for example, HCl is added to blood, the strong acid takes first
the sodium of Na.CO,, setting free the very weak acid H,CO, and mak-
ing NaCl. Similarly it dispossesses Na,HPO, of its Na to form NaCl
and NaH,PO,, and the latter is also a very weak acid. Finally the
proteins exist both in the corpuscles and blood serum as salts, still weaker
than these others. Na globulin and albumin at first give up their
metals, leaving the free globulin, hemoglobin and albumin, and then,
as more acid is added, they unite with the HCl to form globulin
and albumin HCl, which dissociate few hydrogen ions. Moreover, the
blood, like most tissues, possesses a power of ammonia manufac-
ture by which small amounts of amino groups are split off from the
proteins. This power of the blood of combining with acid is called the
alkali reserve. It is due principally to the bicarbonate present and it is
discussed with methods of determining it on page 1042.

Hydrogen ion content of the blood. Blood, as so frequently empha-
sized, is a living tissue. The processes which occur in it, the processes
of clotting and respiration, are processes probably like those taking part
in all living tissues. Like the other tissues of the body, the blood to work
at its best must be nearly neutral in reaction. It is normally very faintly
alkaline, but when great quantities of carbon dioxide and other acid are
poured into it by the tissues it may probably become nearly, if not quite,
neutral. Normally, however, when tissues are thus pouring acid into
the blood the circulation to that organ is so increased that sometimes
the blood comes away from such a tissue less acid and less charged with
carbon dioxide than when the tissue is at rest. The blood often issues
from the vein of an organ doing work in an obviously arterial state. It
has not been reduced.

The concentration of the hydrogen ions in the blood may be deter-
mined most accurately by means of the gas-chain method, but it may
also be determined in blood plasma by means of indicators.

The gas-chain method. In this method a platinum electrode saturated
with hydrogen gas is partially immersed in the blood, and another elec-
trode also saturated with hydrogen gas is partially immersed in a weak
solution of hydrochloric or some other acid of which the concentration
of the hydrogen ions is known. The two electrodes are now connected
through an electrometer, and the solution of acid and the blood are
connected by putting in between them a saturated solution of KCl. The
arrangement is shown in Figure 54. The hydrogen of the electrodes
tends to go into solution as a positive ion, leaving the electrode in each
case negative. There is thus set up at each electrode a difference of
potential between the electrode and the blood or the solution in which
the electrode is placed. The amount of the potential difference between
THE BLOOD. THE CIRCULATING TISSUE 541

the electrode and the solution is a function of the number of hydrogen
ions in the solution. It is proportional to the logarithm of the ratio of
the pressure P of the hydrogen in the electrode to the pressure of the
hydrogen ions in the solution, zy, or the electromotive force E=K log
P/7q. If 7, is the pressure of the hydrogen ions in the blood and zy’

 

 

 

 

 

 

  

me

ae Sa +

inecarevest La Blew nein meme ee ie ee ee ce ee ee et

Akumuutor.

Fic. 54.—Arrangement of apparatus for the measurement of the hydrogen ion content
of any fluid (Sérensen). 4 contains the fluid to be examined and the thin platinum
electrode which barely touches the liquid and is surrounded with hydrogen gas; B contains
a saturated solution of KCl; C is a 0.1 normal calomel electrode, voltage against N acid
0.3377; D is the normal element which enables a determination of the potential of the
accumulator FE; F is a Wheatstone bridge; H is a capillary electrometer; J a key permitting
short-circuiting of the capillary electrometer and the throwing into it_either the current
from the normal element or the gas chain. 4 is connected either with 5 or 6 at will by a
stiff copper wire. The electrode A is shown somewhat enlarged in Fig. 55. This electrode
is particularly designed for the investigation of the blood, as fresh blood is easily introduced
without permitting air to mix with the hydrogen, and equilibrium between it and the
CO, content of the hydrogen atmosphere above it is thus readily obtained.

that in the acid, then the potential difference between the blood and the
electrode immersed in it would be

E = K log(P/7,)
and that between the acid and the other electrode would be
EB’ = K log(P/m,')

and the difference between the two electrodes, or the electromotive force
of the circuit when the electrodes and the blood and acid are connected
in the manner indicated, would be

e—E— E—K (log P/7, = log P/z,,') = K (log =,,’— log7,,) = Klog (7,'/tg)

In this equation the difference of potential between the boundaries of
the solutions is disregarded, but this can be neglected when a strong
connecting solution of KCl is used, since the two ions of this salt move
with the same speed approximately and so no difference of potential is
set up. Since the pressures of the hydrogen ions are proportional te
542 PHYSIOLOGICAL CHEMISTRY

the concentrations of the ions in the two solutions, we can put C,, and
C,,’ in place of 7, and 7,’ and we have then:
e=K log (C'g/Cq)

The value of K is known to be .0577 volts when Briggs’ logarithms are
used and the temperature 18° C. Hence, if we measure the electro-
motive force between the two electrodes and C,’ is known, that is the
concentration of hydrogen ions in the acid solution of known strength,
a simple calculation gives the value of ae the concentration of the
hydrogen ions in the blood. Instead of using a second hydrogen elec-
trode in an acid of known strength, a calomel mercury electrode of
known electromotive force when balanced against a N hydrochloric acid
solution may be used instead. The current, or e, is measured by balanc-

 

Fig. 55.—Enlarged electrode vessel for the examination of the hydrogen fon content
of blood or other liquids containing CO2. Capacity 15 ¢.c.; liquid 7 c.c. Metal spring
clips hold the top on firmly.
ing a current of known strength through the electrometer against the
eurrent between the electrodes. If the 0.1 N calomel electrode is used:
e = 0.3377 + .0577 p,,, Py having the significance of page 543. Further
discussion of this method appears on page 1049.

The indicator method of determining the hydrogen tons. The gas-
chain method requires some experience and apparatus. A very useful
substitute for it is the indicator method by which an approximation can
be made to the actual concentration of the hydrogen ions. This method
depends on the fact that various colored substances change their colors
when they are made alkaline or acid, and the point at which the color
changes in the different substances is at a different degree of alkalinity.
If, then, it is known at what hydrogen ion concentration a color change
of any substance occurs and we find that a solution just produces this
color change, it must ‘have at least this concentration of hydrogen ions.
If another indicator can be found which does not change its color at
THE BLOOD. THE CIRCULATING TISSUE 543

this concentration of hydrogen ions, but requires a higher concentration
of hydrogen ions, then, if the blood can change one indicator but not
another, the hydrogen ion concentration of the blood must lie between
these two concentrations. Two such indicators are litmus and phenol.
phthalein. Litmus changes to a blue violet when the concentration of
the hydrogen ions is equivalent to a N/107 concentration. In concen-
trations of hydrogen ions greater than this litmus is red; in concentra-
tions less than this it is blue violet or blue. Phenolphthalein is still
colorless at this concentration of hydrogen ions. It only becomes pink
when the concentration of hydrogen is less than N/10®. Hence, as the
blood is alkaline to litmus and acid to phenolphthalein, its hydrogen ion
concentration must be between N/107 and N/10*. By finding indicators
which change between these two points and at other points, it is possible
to find in this way the approximate hydrogen ion concentration of a
liquid. The method may be still further refined by the comparison of
the depth of color produced and the tint. For this purpose a set of
tubes are made up of which the concentrations of hydrogen ions are
known, having been determined by the gas-chain method or in other
ways. These tubes may have different mixtures of Na,HPO, and
NaH,PO,. A drop of the indicator is added to these tubes, and then
the indicator is added to the solution of unknown acidity and the
unknown is matched against the colors of the known tubes until one
is found to match. The concentration of the hydrogen ions in the
unknown tube will then be equal to that in the known mixture, or it
will lie between two of the standards. Of course, this method cannot
be used with an already highly colored liquid like the blood, but it may
be used with the urine or lymph, blood plasma or gastric juice or other
body fluids little colored.

The following table shows the concentration of H ions at which the
different indicators undergo their color changes. The figures under the
heading p,, are the exponents of the powers of 10 by which a normal
concentration (1 gram per liter) of H ions must be divided to give the
required concentration. If P,, is 2, it means that the concentration of
hydrogen ions is .01 N, i.e., N/10?; if Py is 3, it means N/10*, or .001 N.

1. Hydrogen ion concentration of standard solutions of acids (Michaelis).

 

 

 

 

 

H | at 180 Acetic acid at 180
Concentration | Concentration Pp {| Concentration Concentration p
x normal H ions x N A x H ionsx N 'H
1.0 0.78 x 10-° 0.11 1.0 0.42 x 10-7 2.38
0.1 0.91 x 10" 1.04 0.1 0.13 x 10-7 2.89
0.01 0.96 x 10-7 2.02 0.01 0.42 x 10-* 3.38
0.001 0.98 x 10-* 3.01 0001 0.13 x 10-* 3.89

 

 

 
544 PHYSIOLOGICAL CHEMISTRY

2. Hydrogen ion concentrations of different salt mixtures for use in the indi-
eator method.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Acetic acid ga Sa acetate Primary and Secomtnny: Na phosphate
Acetic acid
a Cone Acetate | Concentration Pu alae a 4 a a Concentration Py
taining1 | dilution | H ions x m/3 m/3 H ions x N
gram mol) dilution +| dilution
1 32 5.76 x 10-* 3.24 1 32 0.64 x 10° 5.19
1 16 2.88 x 10-* 3.54 1 16 0.32 x 10-° 5.49
1 8 1.44x 10-* 3.84 1 8 0.16 x 10-° 5.80
1 4 0.72 x 10-* 4.4 1 0.80 x 10-° 6.10
1 2 0.36 x 10-* 4.44 1 2 0.40 x 10-* 6.40
1 1 1.80 x 10-° 4.74 1 1 0.20 x 10-* 6.70
2 1 0.90 x 10-* 5.05 2 1 1.0 x10? 7.00
4 1 0.45 x 10°* 5.35 4 1 0.50 x 10-7 7.30
8 1 9.22 x 10°° 5.66 8 1 0.25 x 107 7.60
16 1 0.11 x 10-° 5.96 16 1 0.12 x 10-7 7.92
32 1 0.56 x 10-° 6.25 32 1 0.61 x 10-° 8.21
64 1 | 0.28 x 10-* 6.55
Primary and acco ary sodium phosphate
fo
NaHyPO, Na, HPO,
m/3 m/3 Concentration H ions x N Py
dilution dilution
1 32 0.77 x 10° 5.11
1 16 0.38 x 10-5 5.42
1 8 0.19 x 10 5.72
1 4 0.96 x 10-° 6.02
1 2 0.48 x 10-* 6.32
1 1 0.24 x 10-* 6.62
2 1 1.20 x 10-7 6.92
4 1 0.60 x 10-7 7.22
8 1 0.30 x 10-7 7.52
16 1 0.15 x 10-7 7.82
32 1 0.75 x 10* 8.12
NH,OH 180 NH,O8 and NH,C! at 180
Concentration | Concentration P NH,OH NH,Cl Soncentration Py
Nx H ions H dilution | dilution H ions
1.0 1.8 x10? 11.74 32 1 1.02x 10" | 7.99
0.1 0.57 x 10-*? 11.24 16 1 0.51 x 10-* 8.29
0.01 1.8 x10-° 10.74 8 1 0.26x 10" | 8.59
0.001 0.57 x 107° 10.24 4 1 0.11x10-°| 8.88
2 1 0.64 x 10-° 9.19
1 1 0.32 x 10-° 9.49
1 2 0.16 x 10-° 9.80
1 4 0.80 x 10°) 10.10

 

 

 

 

 

*The figures of these tables except NH OH mean liters of solution containing
1 gram mol or equivalent. Thus in the last’ table, where NH, OH is 32 and NH cl
1, this means that 1 liter of NH, Cl solution containing I gram mol of NH cl and
1/32 gram mol of NH OH has a concert=~#!-— SE ES eae ee cece
EWS

PHYSIOLOGICAL CHEMISTRY—MATH

 

(,cdUNqON ,, wotf paonposdag

‘ajodjuy, wort payrpoyr)

‘VLVd NOILVYLNAONOO NOI NAIDONGAN JO LUVAD “VY ALVIT

*pise d1)Apooes ‘ayzejApooed wnipos ‘pice 913998 'a]k799e wnipos N0zZ:0
yuk SUOIINJOS YayIO AY ‘HORN NOLO 23 TU! ploe d1J0q “ByOb-Z1 ‘HORN N OZ-0 JO asi Tu! prise 919319 6g00-12 *3u2!! 43d ¥Od 7HY'6B0-6
‘guj! Jad O2HZ**OdH ZeN 6928 TI : 8471 vad “1D eN “Bge-g +1109004)5 BSOS-Z ‘HOPN NOT-O ‘19H NOLO ‘e4e SuO!{NjOS UasUesOG ay

“INIMINISNOD ,alDy SS37,, YO ,INIIVWIy JYOW , JO JUNIXIW JO NZ] NI SLUVd

 

 

 

 

 

 

 

 

     

 

 

 

 

 

 

8-2¢-2:1_ 3N78 IOWAHL :

  

          

 

0 ¥—6-2 INSZNIG-OZV-ONIaY-TAHTSNIG |

O-b —6-2 JONVYO TAHIIW
9 >-8-2 3aNiG IONIHd-ONONE
0-9-0-S NMOL3IVN3Hd
€9-2-b Gay TAHIIW]
Fidyhd 10S34d-OWOYE

         

  

  
   

  

Ga- 26

9:2-0-9 3N1d TOWAHL-OW0Ud

£:8-£:2 NIZWHLHd-TOHLHdYN

   

 

 

 

 

 

 

 

 

 

 

 

 

   

>[ S-01 — £6 NIFIWHIHd TOWAHL

 

0 0. 9 b
96E 1
Lo
| an
spl | | 2
doo2h! Ry
ae
Le 2 ols ZI ¢
LO sof] | b
¥ —F
He by PN Lt
| any oy HApY
On fener] 79]
oe Lm /
22 A sn | ae ee 9
BH,
: 2 | ep ean Nn]
(:sosviwHt 140] o1nty Su 7ONra W533} 3 base vga HY
2 os ,
5 ee a vw oF WLRIN TL TOSEV)
= 2
D-a)Af€ NUVIUINYE WOH} 8 is | y, (ara -
g dat sy g J
we *
. 6 or BIZ KD 6
| Be Se
2 roe Le
1)} 01 s6f* oer] bad
oA
" " us i
a 2 re zi
fpo.nda1q aware}
|_—T—_ NJ jute ae
SGINTI JO SNOLLOVINY S) SINTOd OTULITTAOSI ¢ —— good

 

 

 

Zo
THE BLOOD. THE CIRCULATING TISSUE 545

 

 

NaOH at 180
Coneeaaae Concentration H ions PH
1.0 0.754 x 10-?* 14.12
0.1 0.65 x 10-75 13.19
0.01 0.61 x 10-7 12,21
0.001 0.59 x 10-1? 11.23

 

The mean value of the hydrogen ion content of defibrinated mam-
malian blood at room temperature, i.e., about 18-20° C., has been found
to be between 6X10~8 and 2X10-® (p,=7.2—7.7). If the dissociation
constant of water at this temperature is 0.72<10—*, the OH ion con-
centration of the blodd would be 1.2X10—‘ to 3.6X10—*. The blood is,
therefore, a very weakly alkaline fluid. The concentration of the hydro-
gen ions depends, however, on the amount of carbon dioxide in the blood.
Thus at 38.5° C. for defibrinated ox blood Hasselbalch and Lundsgaard
got the following values: At this temperature the dissociation constant of

water, that is the product of the hydrogen and hydroxyl ion concentra-
tion, is 2.710—“, so that p,, of water is 6.78.

CO, tension Py(mean value)
30 mm. 7.45
40 “ 7.36
50 “ 7.31

An increase in the CO, increases the hydrogen ion content.

The suspension of blood corpuscles has a higher concentration of
hydrogen ions, the serum a lower, than the whole blood when under
the same pressure of CO,. Sérensen gives the following figures:

CO, tension Py serum Py corpuscle P gw hole blood
M.m Hg. suspension

13.4 7.88

19.7 7.55
29.5 7.68

30.0 7.42
41.0 7.03

41.7 7.63 7.31

53.5 6.96

54.0 7.60 7.28

The meaning of this is that most of the acid neutralizing substances
are in the serum. Venous blood contains at least twice as many hydro-
gen ions as arterial blood. Henderson showed that the concentration
of the hydrogen ions is only slightly altered in passing from 18° to
88.5°. Since the value of the dissociation constant of water increases
from 0.72—2.7<10—" within these same temperature limits, it is clear
that the hydroxyl ion concentration of the blood must enormously
inerease in passing from 18° to 38°. The product of the concentration
of the hydrogen and the hydroxy] ions is a constant. The concentration
of the hydroxyl ions at 35° is at least twice or thrice that at 18°, and
it increases 15-20 per cent. in rising from 38° to 42°. It is obvious, since
PHYSIOLOGICAL CHEMISTRY

546

pal.

asoy asueig ***°
pat

AOT[AA

 

per
asueiC

whieiovee penaane|

 

MoTyad
ystuaeiy

rrr i er ee ey inset wie

qui} pez

MoTat
SMOTTOA cect tsetse orl?

BSOY corr ssses ss sete

qaqora poy --**** (iad eg ee ae esate

weer e te ees oe

yaora

MO[[PA

qejora ong ****"
ang  ssa]10Jo)

unndind
-oz09q

eaezueq
-omq UII,

o mo
-edoly,

~

aduwig ttt

aBueig ---

were ween ete coe ee een n es Heer eee ees
Ce ey
ean eee seen:

ystuaery *

er a eves

68810] 00
4pyomb

pea poy

aniq
ystuaety «°

PIA

ee

oelry

Seer re ee

 

UWse1r) ped

 

 

asoy |”

889] 10[0F) i

 

 

asoyy
UMOIG
qysrys

pea

Soares: grate S EOE Gye teres elec

uUMOlg | | yelteog

ee ee

enews wee

 

uMOIg
ST

on[d

en[d

SsaT10[oQ =». MOT JOA

wees
AOTPA

ayeuoydyns
-jousqd UpVZIyTe

va wnIpos

-(wy%q) SHOLVOIGN] 40 SHBNVHD YOTOD 7

moypa£
uMolg

auazueg urereqqyd

pros
toqiqden

o1osoy

pel
osu0p

a ce echursianaaehs | qelOLA

re

PLOLA

ant g
uaels
anid

 

uaad?)

 

AMOLLPA

 

er

oe eereeee

cane etaeee

 

 

oAnRyL

99
”

MOTIPA

esueiCQ

pez
asuevioQ

93u8I0
Yen

a

or OT
sr-OT
sr-OL
trOT
or OL

e01

«01
+01
+01
+01
01
+01
«01
01

OL
0%
Nx

suo!
“1900; +
THE BLOOD. THE CIRCULATING TISSUE 547

oxidation is greatly dependent on the concentration of the hydroxyl
ions, that combustion or respiration must be far more intense in fever
than at normal temperature. Blood is, moreover, but a type of a
tissue. The elements controlling the alkalinity of the blood are the
same as those which control the alkalinity of the cells. A rise of
temperature in fever probably increases the hydroxyl ion concen-
tration of the body cells in the same manner as that of the blood.
The hydrogen ion concentration of the blood is not very different from
that of sea-water, but as a rule sea-water is a little more alkaline, par-
ticularly in the south.

The number of H ions in the blood keeps remarkably constant. This
is due to the co-operation of three factors: namely, the presence in the
plasma of the salts of three weak acids—carbonic, phosphoric and pro-
tein. The first of these is present in the largest number of molecules
and is by far the most important. When acid enters blood it reacts
with the carbonates and phosphates to form carbonic acid and acid phos-
phates. The carbonic acid is removed through the lungs very quickly,
and the kidneys pump out the acid phosphates, restoring the blood to its
proper alkalinity. Any rise of H ions in the blood at once stimulates
the respiratory center and leads to the elimination of carbon dioxide.
It must not be forgotten, however, that a slight variation in P, means
a very great variation in H ion concentration, for P, is an exponent
of 10. Thus if P, of blood changes from 7.4 to 7.3 it means an increase
in H ion concentration of 25 per cent. The actual number of H ions in
1 ce. of blood plasma having a Py 7.4 is 2.41 X 10"; and in blood of P y
7.3 is 3.04 « 10°,

Osmotic pressure of the blood.—Since the relative osmotic pressure
of the blood and the tissues helps determine whether liquid shall pass
from the blood to the tissue, or vice versa, and since the activity of every
body cell is dependent upon the amount of water in it, any change in
the water content at once altering its activity, the osmotic pressure of
the blood is of great importance in its functioning. It is obviously
desirable that the osmotic pressure of the blood shall be kept as nearly
uniform as possible, in spite of the considerable quantities of water
leaving the body in the lungs, urine and through the skin, and the con-
siderable income of water from foods and drink and from the oxidation
of the hydrogen of the foods. It is one function of the kidneys to keep
the osmotic pressure of the blood as constant as possible. The osmotic
pressure of the plasma or the whole blood is determined by the freezing-
point method, which has already been described (page 201). The deter-
mination may be made with only a few c.c. of blood by the Wilson modi-
fication of this method. The freezing point of the blood of various
mammals is as follows:
548 PHYSIOLOGICAL CITEMISTRY

Freezing point

Mammal an
Mani) cacsccnvsauiasw inn aaicieenas soe — 0.526° (Varies .482-.605)
OX! ee be eats beeen y eaten oa meee 0.585 ( “« ,543-.662 )
HOrge: .4o.ccee ee Bed VRE ees 0.564
TRADDIE: 2.4. vsyccnsarainuceener 03-2 aueeasacreietnorican ne 0.592
DHECD: ca lecyenctuien besa nieromeemninie a exerenenaoret 0.619
PUB etsy cone ia ds grap dvi are rancho ose adeenada tee 0.615
DOG uh secgurateeiie keane semiaass 0.571
Cat ncsirssmas sanios emer eaae 2s 0.638

The freezing point of mammalian blood is, hence, about —0.6° C.
This depression of the freezing point would mean an osmotic pressure of
0.6/1.85 22.4 atmospheres. or 7.3 atmospheres. This is about equal to
a one-third molecular sugar solution. This osmotic pressure is subject
to some variation even in the same individual. Thus Koeppe found in
himself the freezing point to be as follows:

Freezing point Freezing point
Aa Aa
QOS vse eegiesersne seen Ds — .535° + Morning fasting 9 a.m. ......—0.581°
V2: Me esi WS tc ceiidewntas anges Bavsunse aa 558 Dg, SRM sp 3s. pantera acaba, BeSesayeus 0.512
1% p.m. (After dinner) ....... 585 LE PIM Goes ese Sums eitoniisd ules 0.551
D8, MEME: .. pale cid enseaion £4 mibeeheanes 528 2 p.m. (After dinner) ........ 0.617

The secretion of the gastric and intestinal juices thus increases the
osmotic pressure of the blood. Ina fistula dog this increase may be quite
marked. (See page 346.)

The arterial blood has generally a slightly lower osmotic pressure
than the venous blood. The difference is not marked (Nolf):

Carotid Jugular

(Uncoayulated blood)
A A
— .574° — .589°
.580 .587
572 .576
595 597
591 593
.567 565
The portal vein has a lower osmotic pressure than the hepatic vein
(Fano and Botazzi) : :
Dog’s blood
Portal vein Hepatic vein
A A
.692 .722
617 .667
.602 .633

The osmotic pressure of the blood is due chiefly, but not exclusively,
to the crystalloids it contains, to the salts, sugar, urea, etc., but the
proteins also contribute somewhat to it.

In cholera, or in very hot, dry regions, the blood may have its osmotic
pressure markedly increased. Its viscosity increases at the same time.
hence the necessity of diluting it either with water or salt solutions.

The tissue fluids of invertebrates and some of the lower vertebrates
have the freezing points shown in the accompanying table.
THE BLOOD. THE CIRCULATING TISSUE 549

The great difference between the cartilaginous (Selachians) and bony
fishes is seen in the table. The former have very little control over
the osmotic pressure of their body fluids. Their blood is about the same
freezing point as that of the sea-water. In teleosts partial control is
attained, so that the osmotic pressure of their body fluids is lower than
that of sea-water in the sea-fishes, and higher than that of fresh-water
in the fresh-water fishes. This independence of the medium is in some
fishes so complete that, like the salmon, they can pass from sea-water
to fresh-water. It would seem that the covering of the gills of fishes
inust be of such a nature that it permits gases to pass but not water. In
Selachians a considerable part of the osmotic pressure is due to the
urea in the blood, which may be present to the extent of 1.5 per cent.
The osmotic pressure due to the urea would be about 5.5 atinospheres.
The total pressure is about 27.8 atmospheres. The osmotic pressure of

 

 

human urine is about that of sea-water. A —=—1.3-2.3°. ’
FREEZING PoINTs oF THE FLuips oF SOME INVERTEBRATES AND VERTEBRATES
(Botazzi).
Aa
Coelenterates: Aleyonium palmatum ...............6-4. — 2.196
Echinoderms: Astropecten aurantiacus .........0e cesses 2.312
Worms: Sipunculus nudus 2.31
Crustacea: Maja squinado ........ 2.36
Homarus vulgaris 2.29
Cephalopoda: Octopus macropus 2.24
Selachians: Torpedo marmorata ..... cc ceee cence cnenes 2.26
Mustelus vulgaris ..... 0... cc eee cece eee 2.36
Trygon violacea .........ceceesseececeee 2.44
Teleosts: Charax: punt?220) 6.002 scares dase sss caresses 1.04
Coma; PIZas sss cewessscins nasser eeewwss 1.035
Crenilabrus pavO ......... cece ence eeeeee 0.74-0.76
Box: Sapa asec i-iens, tcaydie ssene dig wieieions o:6 epee 0.82-0.88
Reptilia: Thalassochelys carelta ..............0000- 0.61
Fresh-water forms (Fredericq, etc.)
Crustacea: Astacus fluviatilis 0.80
Teleosts: Anguilla vulgaris 0.58-0.69
Barbus fluviatilis eke 0.475-0.558
Lenciscus lobula ........... 2 cece cece eee 0.45
Perea fluviatilis .......... eee e cece scene 0.512
Amphibia: Rana esculenta .........seeseeeeeereeeee 0.465
Salamandra maculosa ...........eeee8 secs 0.479
Reptilia: Emys europea ............. hererasaa'e eisiaieue’ 0.474

Conductivity of the blood.—The conductivity of the blood is of
interest mainly because it enables a computation of the volume of the
corpuscles in the blood. The conductivity is due to the salts in the
plasma. The corpuscles occupy a certain amount of space in the plasma,
but have almost no conductivity, so that the conductivity of the plasma
is greater than that of the blood. If the conductivity of the plasma is
determined on the one hand and that of the blood on the other, the
volume of the corpuscles may be calculated by the formula (Stewart) :

Vi;= mi (180—A, — VA5)
550 PHYSIOLOGICAL CHEMISTRY

V, is the volume of the corpuscles in the blood volume of 100; A, the
conductivity of the blood, and 4, the conductivity of the serum.

Enzymes of the blood.—The blood plasma contains many different
enzymes. Indeed, the blood plasma may be regarded as a very dilute,
liquid, nor organized cell protoplasm, having in it many of the cell
substances and showing many of the processes of cell metabolism. The
blood plasma, like the plasma of cells, contains enzymes, and among
these bodies are some proteolytic enzymes of which the importance in
immunity and to the body is fundamental.

1. Amylase. There is always present a very small quantity of an
enzyme which converts glycogen or starch to a reducing sugar. This
enzyme is present in very small amounts. It is increased if dextrins,
or starch or glycogen, are injected into the blood, or if these are fed
in large amounts. It is increased very much if the pancreas ducts be
ligatured. It is believed that this enzyme comes, at least in part, from
the pancreas, but it is not impossible, since such enzymes are present in
many other tissues such as the liver, the salivary glands, the white blood
cells, that the enzyme is derived, in part, from other sources.

2. Invertin. This is also present in small quantities, but increases
when large amounts of cane sugar are fed, or when cane sugar is injected
directly into the blood. The amount present, however, is extremely
small, the inverting power of the serum being very slight. Slight acidifi-
cation greatly increases the activity of this enzyme.

3. Glycolytic enzyme. The blood plasma always has some power of
destruction of glucose. This is ascribed to the presence of a glycolytic
enzyme. What is made of the glucose, whether it is converted into
isomaltose, or whether alcohol, lactic acid or other substances are formed
from it, is uncertain. The amount of the glycolytic power is said by
Lepine, who has principally studied this question, and by Slosse to be
reduced in diabetes.

4. Ltpases are also present. These have their origin perhaps in the
pancreas.

5. Proteolytic enzymes. These are found normally in the plasma,
together with their antibodies: that is, substances which prevent or
inhibit their action. Thus there is always present in serum or plasma
an anti-pepsin, anti-rennin and anti-trypsin. These digestive enzymes
are thus rendered inactive. Where they come from, whether they are
reabsorbed from the intestine in digestion or from the glands in which
they are formed is still uncertain. Blood platelets contain or yield a
good deal of proteolytic enzyme (Abderhalden and Deetjen). A very
fundamental observation has recently been made by Abderhalden,
an observation which may go far toward clearing up some obscure facts
of immunity. He has found that the injection into the blood of strange
proteins of any kind leads to the appearance in the blood within 24
THE BLOOD. THE CIRCULATING TISSUE 551

hours of enzymes which will digest the albumoses, formed by acid hydrol-
ysis from those proteins injected, and they will not split other albu-
moses. This fact he has applied to the detection of pregnancy. It was
found that the blood serum of a pregnant woman, or other mammal,
has the property of digesting the albumose-peptone mixture made by
hydrolyzing the placental tissue of that mammal with sulphuric acid.
The albumose is prepared in the following way: The tissue or protein to
be tested, in this case placeatal tissue, is ground fine in a meat-chopper
and then allowed to stand 48 hours in 50 per cent. sulphuric acid at room
temperature. The material is diluted, neutralized, filtered, boiled, fil-
tered, and the albumoses precipitated by saturating with ammonium
sulphate. It is freed from sulphate by dialysis. One c.c. is then mixed
with 1 ¢.c. of blood serum, diluted to fill the tube of a polariscope and
placed at 35° C. Any digestion occurring is shown by the change in
rotatory power. This change is very slight and the observation must be
carefully controlled, but there appears to be no doubt of its existence.

Another method consists in allowing the digestion to take place in a
collodion tube. The products of the digestion are dialyzed out, the
dialyzate is concentrated and the presence of amino-acids or proteins
shown by the ninhydrin reaction.

By the optical method it has been possible to differentiate pregnancy
from various tumors,-and the method promises to be of value in diag-
nosis as well as of great theoretical interest. It has been found that
any kind of protein, when injected, produces an enzyme in the blood
which splits the albumose from that protein, but not from others.

What then is the explanation of this extraordinary power of the
body to make a special enzyme which will digest the albumoses of the
kind of protein which calls it into existence, but no others? How is it
possible for the body to know at once how to make an enzyme which
fits the particular protein injected, but no other, and to do this at the
first attempt for a protein it and its ancestors have probably never met
before? Possibly the enzymes are in each case formed from the proteins
themselves, and hence resemble the proteins from which they came so
closely that they fit them best.

6. Cholesterases. These split the cholesterin esters. They are pres-
ent in the blood corpuscles.

7. Peroxidase and catalase. Blood contains also peroxidase and a
catalase that decomposes hydrozen peroxide.

Proteins of the blood plasma.—The plasma of mammalian blood is
obtained by centrifugalizing blood which has been rendered non-
coagulable by potassium oxalate, sodium fluoride, hirudin or other means.
Such mammalian blood plasma contains normally 5-8 per cent. of
coagulable proteins. These proteins are serum albumin, or seralbumin as
it is called; serum globulin, or serglobulin; and fibrinogen. They are
552 PHYSIOLOGICAL CHEMISTRY

separated from each other by their varying ease of precipitation with
acids or neutral salts. The relative amounts of these three substances
vary under different conditions, but are approximately as follows:

Fibrinogen: 3.4003 sche hace eee etios were he wae 0 15-0.6
SeHUiMi: ClOHV A si, sieve AOE aM Lee RES 3.8
Serum albumin .......... cece cece reece recente eceees 2.5

The fibrinogen is subject to the widest variation, since whenever there
is prolonged leucocytosis or suppuration anywhere in the body, the
fibrinogen increases and may double or quintuple or even increase to
eight times its normal amount, reaching as much as 0.9 per cent. of the
whole blood or approximately 1.6 per cent. of the plasma. (Author’s
observations.) It is the impression of the author, although no definite
study of this matter has been made, that young animals generally have
more fibrinogen in their blood than old animals. On the other hand,
the total protein of the blood plasma in white rats increases from early
to adult life.

The relative proportions of serum globulin and serum albumin are
reported to be different in different animals and in the same animal
under different conditions, but there is no method which permits a
sharp separation of the two bodies, hence all observations of their
relative amounts are open to serious question.

Fibrinogen. Fibrinogen is the least soluble of the three proteiné.
It is almost completely precipitated by saturating the plasma with
sodium chloride; or by the addition of a very small amount of acetic
acid. It is also easily precipitated by water. It coagulates, also, at the
lowest temperature, becoming insoluble at 56°-60° C. under the usual
conditions of the plasma. It is rendered insoluble and converted into
fibrin, undergoing some change as yet unknown, by various agents gen-
erally supposed to be catalytic agents, or enzymes, and found in all
cells. These agents, whatever their nature, are called fibrin ferment,
or thrombin. It has not yet been shown that they are enzymes, and
strong reasons have been given by Howell for doubting that they actu-
ally are.

Serum globulin. Although the globulins are ordinarily defined as
being insoluble in water and precipitated from their salt solutions by
dialysis, it is not possible to separate the globulin of the blood from
the albumin in this way. Only a small fraction of the globulin is
separated by dialysis of the serum. This small fraction is sometimes
treated as a separate protein and called eu-globulin (eu meaning well) ;
the name signifying that it is a typical globulin. A much larger amount
of globulin is precipitated by diluting the serum several times with
water and then mixing it with an equal volume of saturated solution of
ammonium sulphate. This fraction, which is not precipitated by water,

“but is by half saturation by ammonium sulphate, is called pseudo-
globulin. It is still uncertain whether they are distinct proteins, or
THE BLOOD. THE CIRCULATING TISSUE 553

whether the pseudo-globulin fraction continues to give small quantities
of eu-globulin. The ease with which the proteins change their solu-
bilities makes it very difficult to settle a point of this nature. The con-
tent of amino-acids in the two fractions is approximately the same. The
globulin may be protected from precipitation by dialysis by the presence
of some other colloid. The precipitate obtained by salting out the
globulin is always strongly impregnated with a phospholipin 8-10 per
cent. (Hardy). To separate this it is necessary to extract it with alcohol.

Serum globulin is a white, coagulable protein, coagulating in
the plasma or in 3 per cent. salt solution at 75°. It is soluble in
salt solutions, but is partially precipitated by a small quantity of car-
bonie or acetic acid. It is electro-negative for the most part, and prob-
ably exists in the plasma as the sodium salt. Its name comes from its
supposititious origin from the white blood globules (A. Schmidt).

The difference in the composition of the globulin and albumin may
be seen by comparing their basic amino-acids. The albumin contains far
more of the basic amino-acids than the globulin.

In 100 Grams AsH-FREe ProTetn (Lock and Thomas).

Serum albumin Serum Serum Fibrin
(average) globulin 1 globulin2
Histidine 202 sterawnsardag ass 3.48 1.45 1.74 2.85
ATRINING (65:5 shoei se wtdies ne ese 4.67 4.51 4.07 5.52
Lysine? issisvsaseiacsdeawsas aes 11.08 6.75 6.72 7.40
ROCA <ov5, dec eee i aceceaiaya seve Meeks 19.23 12.71 12.53 15.77

Serum albumin. This simple protein resembles serum globulin
closely, it coagulates at about the same temperature, but it is not pre-
cipitated from the serum by saturation of the latter with magnesium
sulphate, or by half saturation with ammonium sulphate, but it pre-
cipitates if the filtrate from the globulin, which is half saturated with
ammonium sulphate, is acidified with acetic acid. The precipitate con-
sists of a sulphate of the albumin.

Origin and function of the plasma proteins.—The function of the
blood proteins is still a matter of investigation. They were formerly
supposed to be the protein food of the tissues, but this does not seem
so probable since the presence of amino-acids in the blood has been
shown and the radical differences between blood and tissue proteins
have become clear. There never has been any evidence of the consump-
tion of these proteins by the tissue cells. The whole matter of their
nutritive value must be left to the future to determine. One function
they undoubtedly have: they give to the plasma one of its most im-
portant properties, its viscosity. This is the function of the proteins
in the other tissues of the body, for it is the proteins which give the
structure, the jelly nature, to protoplasm; in other words, it is the pro-
teins which determine the viscosity of the protoplasm. Similarly in the
554 PHYSIOLOGICAL CHEMISTRY

blood, which is a living tissue, the protcins contribute to the viscosity,
a matter of much importance in determining the peripheal resistance.
The proteins of the plasma resemble the proteins of the tissues of the
body in other ways. As they occur in the blood, they are in union with
phospholipins, and there is reason for thinking that the proteins of the
body cells generally and of all forms of protoplasma are similarly joined
to lipin. Just as the blood changes its viscosity with the greatest ease,
at one time becoming more fluid and at another more solid, so do we
see the same propertié¢s in the cell. The cell protoplasm clots like the
blood. The blood, when shed, becomes more acid, just as the proto-
plasm when it dies becomes more acid. Moreover, there is a marked
resemblance between the character of the proteins in the two cases. All
tissues of the mammalian organism, or at least the majority of the
tissues, contain a protein which resembles, but is probably not identical
with, fibrinogen, a protein which coagulates at 56°. Many of them, also,
contain globulins and albumins, which resemble, but are not identical
with, the proteins of the plasma. Another important function of these
proteins is in regulating the alkalinity of the blood. They neutralize in
the ways described on page 545 the acids formed by the tissue cells, in
part by yielding up to the acids the alkali metals they carry, and in
part by uniting directly with the molecules of acid. Both these func-
tions are in common with the proteins of the cell protoplasm. In fact we
shall probably not go far wrong if we consider the blood plasma as a
very dilute protoplasm. The processes which occur in it are probably
the mirror of the processes which occur in all forms of living matter.

The origin of the plasma proteins is still uncertain. Schmidt, who
worked for many years on this problem, believed that they came from
the white blood corpuscles on the disintegration of the latter. Similar
but not identical proteins are found in a wide variety of cells. Probably
more work has been done upon the origin of fibrinogen than upon any
other member of the group. This substance, because of its peculiar
property of clotting and the ease with which it can be removed from
the blood, is most easy to study, but the conclusions arrived at by various
observers are nearly as diverse as the number of investigators. Some
have concluded that the fibrinogen is formed in the liver, others from
the white blood corpuscles, others from the bone marrow.

The problem has been attacked in the following way: If a dog or
cat be anesthetized, a cannula put in the femoral artery and another in
the femoral vein, between one-third and one-half of the blood may be
withdrawn from the artery without killing the animal, if the blood is
quickly reinjected. The whole blood is estimated at about one-thirteenth
the body weight. This blood is kept warm and defibrinated by whipping,
and after filtering through a cloth is reinjected slowly into the body. The
injection must be slow or intravascular clotting may occur. If it does
THE BLOOD. THE CIRCULATING TISSUE 555

occur the clots generally appear in the heart, being attached to the valves
and corde. After allowing 5 minutes for the reinjected blood to mix
well with the blood in the body and the circulation to recover itself, the
same amount of blood is again withdrawn and whipped and reinjected.
By repeating this process 5-6 times the whole time of defibrination taking
from 11-24% hours, about nine-tenths of the fibrin of the blood is removed
and so little remains, i.e., .002-.004 per cent., that the blood either will not
clot at all, or at the most gives but a very weak jelly. The amount of
fibrin thus recovered is generally a little less than, about nine-tenths of,
the amount which was calculated to be present by a quantitative de-
termination in a sample of blood taken at the outset of the defibrination.
This fact shows that neither any pronounced destruction nor reforma-
tion has taken place during the process of defibrination, otherwise, unless
both processes occurred equally, the amount recovered would be less
or more than that found. The defibrinated animal reforms the fibrin and
in the course of 24-48 hours the normal fibrinogen content of the blood
is restored. This reformation of fibrinogen takes place equally well
whether the animal is fed or is fasting, and it is clear that it does not
come from the proteins of the food. Whence then does it come? Ex-
periments have shown that the reformation takes place with normal,
or even increased, speed after extirpation of the kidneys, spleen, pan-
creas, or after ligating off the brain and most of the nervous system. It
also occurs normally after making an Eck fistula which cuts off all the
portal blood flow through the liver, but leaves the hepatic artery open.
Neither spleen, pancreas, kidneys, reproductive organs, lymph glands of
the mesentery, nor nervous system is then necessary for the reformation.
Perfusion experiments by which defibrinated blood is perfused through
the legs, intestine and kidneys have shown no formation of fibrinogen in
these organs. Perfusion through the liver is also negative in the experi-
ments of some, and positive in others. The fibrinogen is reformed nor-
mally if the liver and intestine are intact, but the reformation is greatly
reduced if the intestine is absent. An examination vf the fibrinogen con-
tent of the blood in the arteries and veins in different parts of the body
shows that in all regions of the body but the intestine the venous blood
has less fibrin than the arterial. The portal or mesenteric veins alone
show generally, but not always, a larger amount of fibrinogen than the
arterial blood. From these observations it would appear that fibrinogen
is taken out of the blood in its passage through most of the organs of the
body and in particular in the kidneys, and that it is added to the blood
during its passage through the vessels of the intestine or the liver, since
some observations indicate that the amount of fibrinogen in the hepatic
blood is greater than in the portal. While none of the methods of the
quantitative determination of fibrinogen are quite satisfactory the
results are so uniform as to indicate that the differences obtained between
556 PHYSIOLOGICAL CHEMISTRY

the arterial and venous blood really represent actual differences. The
fibrinogen appears, then, to be added to the blood in the portal area
and chiefly in the intestine. Whether the liver is active or not in the
process is undecided. It was not supposed that the intestine formed the
fibrinogen from its own peculiar tissues, except, perhaps, the lymph
tissues, but that the fibrinogen came probably from the white blood cells
and that the place of their destruction was probably the intestinal area.

The only method known to increase the amount of fibrinogen in the
blood is by suppuration. Any prolonged suppuration anywhere in the
body, and however produced, is accompanied by a great increase.in the
fibrinogen content of the blood. This increase may be enormous, more than
eight times the normal amount being present. The reason for this great
increase in fibrinogen has never been explained. It is possible that it is
part of the defensive mechanism of the body against infections and
fever, the viscosity of the blood being increased thereby. The blood in
all such cases nearly always shows that the red corpuscles agglomerate
and sink much more rapidly than normal so that the blood may have a
coat of serum or plasma above the clot free from corpuscles, the so-called
buffy coat, or crusta inflammatoria, which had been observed by the
physicians when bleeding was common. This process of agglutination of
the corpuscles must also increase the viscosity of the blood and would
tend to reduce the speed of blood flow and perhaps make conditions
favorable for the passage of the white blood cells out into the tissues
where the infection is. Inasmuch as any great or prolonged leucocytosis.
as in infections, is accompanied by an increase in fibrinogen and it is also
accompanied by an increase in the decomposition of the leucocytes, the
author drew the conclusion that probably the fibrinogen, and the other
blood proteins as well, originated in the white blood cells.

Opposition to this view has, however, been fairly widespread. Both
Nolf and Doyon believe that the liver forms the fibrinogen. The reason
for this conclusion is that the liver, like many other organs, contains a
protein which may be extracted and which coagulates at the same tem-
perature as fibrinogen. It has never been shown, however, that this
protein is fibrinogen and that it has the property of clotting on adding
fibrin ferment. Another fact supporting the view that the liver forms
fibrinogen is that when one poisons the liver with phosphorus or chloro-
form, so that there is extensive degeneration of this organ, then the
amount of fibrinogen in the blood is reduced. These latter observations,
however, do not show that the fibrinogen is formed in the liver. It is
not to he supposed that the liver is the only organ affected in phosphorus
or chloroform poisoning. The disappearance of the fibrinogen might be
due to the fact that the consumption rose above the power of produc-
tion. or that directly or indirectly other tissues which produced the
fibrinogen were affected. It may be that the liver forms an enzyme,
THE BLOOD. THE CIRCULATING TISSUE 557

which may digest the fibrinogen and that on its destruction this enzyme
gets loose in the blood. .

There is still another possibility of the origin of the fibrinogen. It
may be formed in the bone marrow. It is said, indeed, that the amount
of activity of this tissue is greater after defibrination. And that is not
improbable. It is said also that fibrinogen can be isolated from the
bone marrow. Since this is the blood-forming tissue in the adult body it
is most probable that the proteins are derived from the bone marrow
or from the blood cells. The blood is a living tissue. Every living tissue
has proteins peculiar to that tissue. Were the proteins the same, the
chemical processes in different tissues would be the same. But we know
that they are different. Every tissue forms its own peculiar proteins.
It is probable, therefore, that the blood is formed by itself. It makes its
own proteins. The place where it is made is either in the bone
marrow, which is the blood-forming organ in the body, or by the dis-
integration, that is the further differentiation, of the blood corpuscles,
and in particular the white cells. It is certain that the white cells, and
possibly the reds as well, are constantly giving off substance to the blood.
They disintegrate. Their surfaces, as Kite has shown, are sending out
constantly great streamers of protoplasm into the plasma. It is probable,
then, that they are constantly contributing to the protein constitution
of this tissue and that they keep its constitution fairly constant. It is
possible that the endothelial cells of the blood vessels also play a part
in this process. This possibility should be carefully investigated, but
the fact that the reformation of fibrinogen has never been described in
the great number of perfusion experiments of living organs with de-
fibrinated blood indicates that more than one tissue is concerned in
this process. It may be that defibrinated blood does not reform its
proteins on perfusion, because the raw materials, the leucocytes, are
lacking. A circulation through the bone marrow and then through the
intestinal area would possibly be more successful. The origin of fibrino-
gen and other proteins is then still obscure and must be left for future
investigations, but the evidence favors its origin from the blood cells, the
white blood corpuscles, and possibly the blood-forming organs, the bone
marrow.

Experiment has shown that when the fibrinogen is reformed after
defibrination, there is no increase in the per cent. of the other proteins
of the blood. That is, the reformation of the fibrinogen does not in-
volve a simultaneous increase in the globulin and albumin, as would be
expected if all three were formed simultaneously by the decomposition
of some cells such as the leucocytes. However, this fact is not conclusive
evidence against the common origin of all three of the proteins, since
some regulatory mechanism might remove these proteins as rapidly as
they were formed. There is often a slight decrease in the other blood
558 PHYSIOLOGICAL CHEMISTRY

proteins coinciding with the reformation of the fibrinogen, from which
it might be inferred that the fibrinogen was derived from the globulin,
as Schmidt thought. But against this conclusion is the fact that the
decrease takes place during the defibrination and not during the reforma-
tion. It looks rather as if some of the serglobulins contributed to the for-
mation of the fibrin and this is not impossible. Schmidt found that the
weight of the fibrin recovered from serum was increased by the addition
of paraglobulin to the serum. The relation of the fibrinogen to the other
proteins is still, therefore, a matter to be investigated.

The source of the other blood proteins is still more obscure than that
of fibrinogen. The same method may be used in their study. The
defibrinated blood may be centrifugalized, the corpuscles suspended in
Ringer’s solution and reinjected. It is necessary to add to the Ringer’s
solution some gum arabic to make the viscosity right. After several
drawings and reinjections the blood is nearly freed from the proteins.
The course of their reappearance can then be watched. The method is
tedious and time-consuming. It is found that the paraglobulin and
albumin will be reformed in about the same time as is required for the
reformation of the fibrinogen. Nothing is known of the origin of these
proteins.

Function of the endothelium of the blood vessels.—It is the pur-
pose of this book to raise questions and if possible to raise more, by
far, than it answers. The question must occur to every student of
physiology what is the function of the endothelium of the blood vessels?
These are generally thought of as passive tubes for the transportation
of the blood, but we must now consider them as living things. They
have a good nerve supply. What is the function of these nerve fibers
distributed to the capillaries of the body? The vascular and lymphatic
endothelium is a great organ, a living tissue, penetrating all the organs
and parts of the body. It is probably not passive since it certainly plays
a part in the coagulation of the blood. It must be constantly interacting
with the blood, changing its composition, possibly affecting its viscosity,
controlling the secretion of lymph, and possibly contributing hormones
of importance to the body. Histamine has a powerful influence upon it.
What substances does it require for its nutrition? Does it, like the
corpuscles of the blood, send out into the stream fine processes? Does
it take fibrinogen out of the blood and put it in? If it controls the
viscosity, as its relation to the clotting implies, how does it do it? Is it
by shedding fibrin ferment? By secreting enzymes specifically suited
to the blood proteins? Is it in these cells that the strange proteins
injected into the blood are imprisoned and do they form the specific
enzymes which appear in the blood when strange proteins are injected?
Do they form the precipitins and immune bodies? Is their activity
controlled by the nervous system? Are they actively phagocytic? Do
THE BLOOD. THE CIRCULATING TISSUE 559

they change their adhesiveness as the corpuscles do, so that bacteria and
other cells will stick to them? Here is an organ coextensive with the
body of which we know very little. Who knows but that some great
gaps in our knowledge of the most fundamental questions may not be
solved by its study. In the text-books of the future it may be that more
than a chapter must be given to this tissue of whose chemistry,
metabolism and function we are so profoundly ignorant.

REFERENCES. Coaeutation oF BLoop.

1. Wooldridge: Chemistry of blood. (Collected Papers.) Report to the Scientific
Committee of the Grocers’ Assn. I, pp. 201-249; II, p. 266 et seq. Croonian
Lecture, p. 141. Reply to Halliburton, Jour. of Physiol., 10, 1889, p. 329;
Protoplasma and blood plasma; Arris and Gale Lecture, Chemistry of
Blood, pp. 174 et seq. Various papers in the Proceedings of the Royal
Society, 36, 1884, p. 417, and succeeding years to 1889.

2. Nolf: Archives internat. de Physiol., 9, 1910, pp. 407-459; 204-261. References
here to earlier papers on coagulation by the same author.

3. Archard and Ayrand: Action des anticoagulants sur les globules. C. R. Soe. de
Biol., 1908, 1, pp. 898-900; 2, pp. 724-725.

4. Zak: Relation of plasma lipoids to coagulation. Archiv f. expt. Path. u. Pharm.,
70, p. 274, 1912.

5. Gengou: Adhesion moléculaire et phénoménes biologiques. Arch. internat. de
Physiol., 7, pp. 1-87; 115-210, 1908-9.

6. Fuld and Spiro: Influence of coagulation inhibitors on bird plasma. Beitriige z.
chem. Physiol. u. Path., 5, p. 171, 1903-4.

7. Morawitz: Beitrige zur Kenntnis der Blutgerinnung. Peitriige z. chem. Physiol.
u. Path., 5, p. 133, 1903-4; 4, p. 381, 1903. See also Morawitz: in Oppen-
heimer’s Handbuch der Biochemie.

8. Schittenhelm u. Bodong: Coagulation and especially the action of hirudin.
Archiv f. allg. Path. u. Pharm., 54, pp. 217-244, 1906.

9. Schmidt: Ueber die Beziehung der Faserstoffgerinnung zu den farblosen Ele-
menten des Blutes. Arch. ges. Physiol., 11, p. 515, 187.

10. Popielski: Importance of the loss of coagulability of the blood for the activity
of the digestive glands. Archiv f. d. ges. Physiol., 144, pp. 135-151. Bull.
internat. de Vacad. de Sci. de Cracovie, 1912, pp. 755-681; 1137-1196.
Maly’s Jahresber., 42, 178, 1912.

ll. Collingwood and MacMahon: Anticoagulant substance in blood serum. Jour. of
Physiol., 45, p. 119, 1911-12.

12. Hayerafi: Action of a secretion from the medicinal leech on coagulation.
Pro. Royal Soc., 36, p. 478, 1884.

13. Wooldridge: Relation of blood coagulation to the vascular wall. “On auto-
infection in cardiac, disease.” Pro. Royal Soc. 1889, 45, p. 309. Du Bois’
Archiv f. Physiol., 1888, p. 174.

14. Bruce: On the disappearance of leucocytes from blood after peptone injection.
Pro. Roy. Soc., 55, p. 295, 1894.

15. Wright: Pro. Roy. Soc., 52, 1892-3, p. 364.

16. Franz: The nature of hirudin. Arch. f. expt. Pharm. u. Path., 49, pp. 342-
366, 1903.

17. Green: On certain points connected with the coagulation of the blood. Jour.
Physiol., 8, 354, 1887.
18.

19.

20.

21.

22.

23.

24,

25.

26.

27.

28.

29.

30.

31.

32.
33.

34.
36.

36.
37.

PHYSIOLOGICAL CHEMISTRY

Ringer and Sainsbury: The influence of certain salts upon the act of clotting.
Jour. Physiol., 11, p. 369, 1890.

Arthus et Pages: Nouvelle théorie chimique de la coagulation du sang.
Archives de Physiol., 1890.

Wright: A study of intravascular clotting. Pro. Royal Irish Acad. 3d series.
2, p. 117, 1891-3.

8. B. Jones: Presence of prothrombin in blood platelets. Amer. Jour. Physiol..
30, p. 74, 1912.

Davis: Intravenous injection of thrombin. Amer. Jour. Physiol., 29, p. 160.
1911.

Howell: Réle of antithrombin and thromboplastin in the coagulation of blood.
Amer. Jour. Physiol., 29, p. 187, 1911.

Howell: Thrombin. Ibid. 1910, 25, p. 453. Nature and action of thrombo-
plastic (zymoplastic) substances of the tissues. Amer. Jour. Physiol., 31,
1, 1912.

Howell: Prothrombin. Ibid., 35, 1914, p. 474.

Retger: Mechanical factors in clotting. Ibid. 24, p. 429, 1909.

Howell: Optical phenomena of coagulation. Ibid.

Modrakowski: Action of vasodilatin on coagulation. Archiv f. d. ges. Physiol.,
133, p. 301, 1910.

Burker: Apparatus for determining time of coagulation. Arch. f. d. ges.
Physiol., 118, p. 452, 1907.

Nias: Relation of salts given by the mouth to the coagulation of blood.
Lancet, 174, p. 96, I, 1908.

Fox: Coagulability of blood in pregnancy and puerperal state. Lancet, 1908,
p. 99, 1908.

Lesourd et Pagniez: Jour. de Physiol. et Pathol. gen., 1909.

Wright: Influence of CO, and O, on coagulation of blood in vivo. Pro. Royal
Soc., 55, p. 279, 1894.

Tait: Types of Crustacean Blood Coagulation. Journal of Mar. Biol. Assn.,
Plymouth, 9, pp. 191-198, 1911. .

Hardy: Journal of Physiol., 13, 1892, p. 165.

Fredericq: Arch. de Zool. Expt. 2d, 1, p. 413, 1883.

Bordet et Gengou: Coagulation of blood and the genesis of thrombin. Annales
de Institut Pasteur, 26, p. 657, 1912.

Bioop PLATELETS.

Wooldridge: Chemistry of Blood. Report to the Scientific Committee of the
Grocers’ Association.

Solwalbe: Thrombose u. Gerinnung. Ergebnisse der allg. Path. u. Path. Anat.,
11, 2, 1907, pp. 901-927. (Literature.)

Schwalbe: Bau u. Entstehung der Blutplittchen. Ibid., 8, 1902, p. 192.

Biker: Blood platelets and coagulation. Arch. f. d. ges. Physiol., 102, pp. 36-94,
1904.

- Pasoucci: Composition of platelets. Beitriige z. chem. Physiol. et Pathol., 6,

pp. 540-552, 1905.

Hayem: Récherches sur |’évolution des hématies dans le sang de homme et des
vertébrés. Archives de Physiologie, 1878-9.

Meves: Thrombocytes of Salamander blood and their relation to coagulation.
Archiv. f. Mik. Anat., 68, pp. 311-358, 1906.
AS

THE BLOOD. THE CIRCULATING. TISSUE 561

Kreidl u. Newmann: Ueber das Vorkommen von Ultramikroskopischen Teilchen
im fétalen Blute. Zent. f. Physiol., 24, 54. 1910.

ProTErns oF BLoop PLASMA.

Wooldridge: Chemistry of Blood. (Collected papers.)

Iscovesco: Etude sur les constituents colloides du sang. Le fibrinogéne. C. R.
Soc. Biol., 60, 1906, pp. 923-5.

Hardy and Mrs. Gardiner: Jour. of Physiol., 40, 1910, p. 68. Pro. Physiol. Soc.

Freund and Joachim: Nucleoproteid of blood serum. Zeits. f. physiol.. Chem.,
36, 407.

Lock and Thomas: Content of blood proteins in basic amino-acids. Zeits. f.
physiol. Chem., 86, 1913, p. 74.

Huiskamp: Fibrinoglobulin question. Zeits. f. Physiol. Chem., 44, pp. 182-197,
1905.

Schaefer: Proteids of chyle and the transference of food materials. Proc. Roy.
Soc., 38, p. 89, 1885.

Suaars rn Boop.

Héber: Distribution of sugar between corpuscles and plasma. Biochem. Zeits.,
45, p. 207, 1912.

Spallita: Sur la nature du sucre du sang. Arch. Ital. de Biol., 53, 1910, p. 356.

Scott: The content of sugar in the blood under common laboratory conditions.
Amer. Jour. Physiol., 34, p. 271, 1914. Literature cited here.

Von Hess and McGuigan: Sugar content determined by vividiffusion. Jour.
Pharm. and Expt. Ther., 6, 1914.

Fat in BLoop.

Hermann and Neumann: Ueber den Lipoidgehalt des Blutes normaler u.
schwangere Frauen sowie neugeborene Kinder. Biochem. Zeits., 43, p. 47,
1912.

VIVIDIFFUSION MetHop or Stupy1ne BLoop.

Abel: On the removal of diffusible substances from the circulating blood of living
animals by dialysis. II. Jour. Pharm. and Expt. Ther., 4, 1914, p. 611.
Von Hess and McGuigan: See above, Sugars in blood, 4.

FERMENTS IN BLoop.

Moechel and Post: Diastase in different animals. Review of literature. Zeits.
f. physiol. Chem., 67, 1910, p. 433.

Gould and Carlson: Relation of pancreas to serum and lymph diastases. Amer.
Jour. Physiol., 29, p. 174, 1911.

Cytronberg: Cholesterase of blood corpuscles. Biochem. Zeits., 45, p. 281, 1912.

Haberlandt: Existence of a leucocyte diastatic ferment. Archiv f. . ges.
Physiol., 132, p. 175, 1910.

Bial: Diastatiec ferment of lymph and serum. Archiv f. d. ges. Physiol., 52, p.
137, 1892.

Tchereskoff: Récherches sur le ferment amylolytique du sang. Archives d
Physiol. Path. et Norm., 1895, pp. 629-635.

Bainbridge and Beddard: The diastatic ferment in the tissues of diabetes mel-
litus. Biochemical Journal, 2, p. 89.
562

8.
9.
10.

11.
12.

13.
14,
15.
16.
V7.

18.

anprwbd

iy

~

29

PHYSIOLOGICAL CHEMISTRY

Carlson and Luckhardt: Amer. Jour. Physiol., 23, p. 148, 1908.
Arthus: Sur la ferment glycolytique. C. R. Soc. Biol., 43, p. 60, 1891.
Lépine: Le diabate et les lésions du Pancréas. Revue de Médec., 12, 1892, I, p.
402; II, p. 481.
Lépine et Bonal: C. R. Soc. Biol., 44, p. 271, 1891.
Abderhalden u. Kopfberger: Invertin in blood serum. Zeit. f. physiol Chem., 69,
p. 23, 1910.
Abderhalden u. Rathsmann : Serological studies with the aid of optical methods.
Zeits. f. physiol. Chem., 71, p. 367, 1911.
Abderhalden u. Schilling: Ibid., 71, p. 385, 1911.
Abderhalden u. Kampf: Ibid., 71, p. 421, 1911.
Abderhalden u. Fodor: Studien tiber die Spezifizitat der Zellfermenten mittels
der optischen Methode. Ibid., 87 and 88, 1913.
Abderhalden u. Schiff: The speed of appearance of protective ferments after re-
pented action of substrates foreign to blood plasma. Tbid., 87, p. 225, 1910.
Abderhalden : Abwehrfermente, 1914. Vam Slyke et al.: J. Biol. Chem., 23,
p. 377, 1915.

LrucocyTes AND RED CELLS.
Patella; Origin in endothelium of blood vessels. Arch. Ital. de Biol., 54, p.
213, 1911.
Morawite and Pratt: Miinchener med. Wochen., 55, p. 1817, 1908.
Morawitz: Arch. f. expt. Path. u. Pharm., 60, p. 298, 1909.
Lohner: Erythrocyte membrane question. Arch. f. Mik. Anat., 71, p. 129, 1908.
Deetjen: Arch. f. Path. Anat. u. Physiol., 165, p. 280.

Meltzer: Effects of shaking on red blood corpuscles. J. H. U. Hospital Reports,
9. p. 135.
Oliver: Science, N. S., 40, p. 645, 1914.

_Kite: Journal Infectious Diseases, 15, p. 319, 1914.

Jaeger: Destruction of red blood corpuscles. Arch. f. d. ges. Physiol., 94, p. 65,
1903.

INORGANIC CONSTITUENTS.

Bertrand et Medigreceanu: Sur le manganese du sang. C. R. Acad. Sci., Paris,
154, p. 941, 1911.

Henze: Vanadium compounds of blood cells. Zeits. f. physiol. Chem., 72, p. 494,
1911.

ALKALINITY OF BLOoop.

Friedenthal: Reaction of blood serum. Criticism of vital stains as tests for
reaction. Zeits. f. allg. Physiol., 4, p. 60-61, 1904.

Lundsgaard: Reaction of blood. Biochem. Zeits., 41, p. 247, 1911.

Peters: Combined tonometer and electrode cell for measuring the H ion concen-
tration of reduced blood at a given tension of CO,. Jour. Physiol., 48, Pro.
Physio]. Soc. VII, 1914.

Hasselbalch: Biochem. Zeits., 49, pp. 449, 457, 1913; 30, p. 317, 1910; 46, p.
403, 1912.

Kormkoff : Ibid., 51, p. 202, 1913.

Svrensen: Ergeb. d. Physiol., 12, 1912, p. 527. Literature. The measurement
and significance of the H ion concentration for biol. processes.

Héber: Physical chemistry of blood. Oppenheimer’s Handbuch der Biochemie, II,
2, 1914. Literature.
10.

11.

12,

13.

14.

16.

16.

17.

THE BLOOD. THE CIRCULATING I1SSUE 563

Michaelis: Die Wasserstoffionenkonzentration. Ihre Bedeutung ftir die Biologie
und die Methoden ihrer Messung. Berlin, 1914.

ConpbuctTIvity oF BLoop.
Bugarsky and Tangl: Archiv f. d. ges. Physiol., 72, 1898, p. 531.

OsMoTic PRESSURE.
Botazzi: La Pression osmotique du sang des Animaux marins. Archiv. Ital. de
Biol., 28, p. 61, 1897.
Héber: Arch. f. d. ges. Physiol., 70, 1898, p. 630.

RESPIRATORY FUNCTION oF BLOOD.

Douglas and Haldane: Jour. of Physiol., 44, p. 338, 1912.

Peters: Chemical nature of specific oxygen capacity of blood. Jour. Physiol.,
44, p. 131, 1912.

Buckmaster and Gardmer: Gases of arterial and venous blood of cat. Jour.
Physiol., 41, p. 60, *910.

Barcroft and Hill: Jour. of Physiology, 1912.

Krogh: The diffusion of gases through the lungs of man. Jour. Phys., 49, p.

271, 1915.
Barcroft: Effect of altitude on dissociation curve of blood. Jour. Physiol., 42.
p. 44, 1911.

Vernon: Respiration of tortoise heart in relation to functional activity. Jour.
Physiol., 40, p. 295, 1910.

Vernon: Conditions of tissue respiration. Action of poisons. Jour. Physiol., 39,
p- 149, 1909.

Hill: Effects of aggregation of Hb on its dissociation curve. Jour. Physiol., 40,
p. IV, 1911.

Douglas: Determination of total oxygen capacity of the blood at different alti-
tudes by the CO method. Jour. Physiol., 40, p. 472, 1910.

Hiifner: Archiv f. Physiol.. 1894, p. 130; 1904, Supp. 387.

French, Pembrey and Riffle: Compensatory increase in Hb and red corpuscles
of blood in congenital heart disease. Jour. Physiol., 39, 1909. Pro.
Physiol. Soc., p. ix.

Firket : Tension des Gaz du Sang arteriel. Arch. internat. de Physiol., 9, 1910,
p. 291.

Hiifner : Composition of hemoglobin. Beitrige zur Physiol. Ludwig’s Festgabe
70 Geburtstage, 1887.

Willstatter: Chlorophyll. This article contains much also on the composition
of hemoglobin and references to literature. Jour. Amer. Chem. Soe., 37,
p. 323, 1915.

Milroy: Metallic compounds of hematoporphyrin. Biochem. Jour., xii, p. 318,
1918.

Henderson, L. J.: The equilibrium between O, and CO, in blood. Jour. Biol.
Chem., 41, p. 401, 1920.

VoLUME.

Dreyer and Ray: Blood volume of mammals as determined by experiments on
rabbits, guinea pigs and mice and its relationship to the body weight and
to the surface area expressed in a formula. Philosophical Transactions B,
vol. 201, 1911, p. 133.
564

_

co

PHYSIOLOGICAL CHEMISTRY

VISCOSITY OF BLOop.

Chick: Viscosity of Blood protein solutions Biochem Journal, 8, p. 261. 1914.

Burton-Opitz: Vicosity of blood. Amer. Jour. Physiol., 35, p. 51, 1914. Arch.
ges. Physiol., 82, p. 447, 1900. Jour. Physiol., 32, p. 8, 1904.

Dupré-Demming and Watson:

DuBois-Reymond, Brodie and Miiller: Influence of viscosity on the circulation.
Archiv f. Physiol. Supp., 1907, p. 38. :

Albutt: Viscosity of the blood. A review. Quarterly Journal of Medicine, 4,
p. 342, 1910-1911.

White: A new viscosimeter and its application to the blood and blood serum.
Biochem. Zeits., 37, p. 489, 1911.

HEMOLYSIS.

Sawtschenko: Action inhibitrice de l’acide carbonique sur hemolyse et la bac-
teriolyse. Annales de I’Instit. Pasteur. 26. pp. 1032-1035, 1912.

Meyer: Mechanism of saponin hemolysis. Hofmeister’s Beitriige z. chem. Physiol.,
II, p. 357, 1907-8. Previous literature cited.

AMINO-ACIDS AND EXTRACTIVES.
Abderhalden: Der Nachweis von freien Aminosauren im Blute, etc. Zeits. f.
physiol. Chem., 88, p. 480-1, 1913.
Van Slyke and Meyer: The amino-acid nitrogen of the blood. Preliminary ex-
periments on protein assimilation, Jour. Biol. Chem., 12, p. 399, 1912.
Gyérgy and Zunz: A contribution to the amino-acid content of blood. Jour.
Biol. Chem., 21, p. 511, 1915.

Bock and Benedict: Jour. Biol. Chem., 20, p. 47, 1915; Greenwald: Ibid., 21,
p. 61, 1915.

Myers and Fine: Comparative distribution of urea, creatine, uric acid and
sugar in the blood and spinal fluid. Jour. Biol. Chem., 37, pp. 239-244,
1919.
CHAPTER XIII.

THE MASTER TISSUE OF THE BODY.

THE BRAIN AND NERVOUS SYSTEM.

CHEMISTRY AND METABOLISM OF THE MASTER TISSUE OF THE BODY.

The brain and nervous system control, either directly by nerve im-
pulses or indirectly through the blood stream, the metabolism and ac-
tivity of all the other tissues of the body. They are, hence, the master
tissue of the body. While the greater part of the metabolism of the
body is muscular metabolism, the muscles by their bulk dominating
the character of the metabolism of the body as a whole, the superior
reactivity or irritability of the nervous tissue enables it to control or
to set the pace for all the other tissues.

The chemistry and metabolism of the nervous tissue is from almost
every point of view the most absorbing and interesting of the problems
of physiological chemistry. In the evolution of the vertebrates it seems
to have been this tissue more than any other upon which the attacks of
natural selection have been directed. Between the marsupials and the
placental mammals the chief difference is not so much a change of form
or structure of the body as it has been a change in capacity of the skull;
a change in brain power. And the marsupials were, undoubtedly, in this
respect, superior to the monotremes. It is brains which have won sur-
vival in the struggle for existence. Evolution. since the appearance of
the vertebrates at any rate, is especially characterized by the steady
development of the nervous system, its increase in amount and com-
plexity. Concomitant with this perfecting has come the development of
memory, self-consciousness and reason. It is, indeed, astonishing how
extraordinarily small was the amount of nervous tissue in the gigantic
Dinosaurs, reptiles which once must have dominated creation. But they
were undoubtedly supplanted by their more clever though smaller rela-
tives. The vertebrates differ from the invertebrates, also, as profoundly
in the amount of nervous tissue they contain as in any other way. The
circumesophageal ring of nervous matter of the invertebrate is re-
‘placed. by the great ganglia of the vertebrate brain. In fact the whole

of evolution is characterized by the steady development of the nervous
565
566 PHYSIOLOGICAL CHEMISTRY

system and by the steady development of no other tissue. The power
of adapting the organism to a changing environment and thus to
secure liberty from environmental trammels has been the problem
nature has had to solve. It solved it by the development of a tissue
of the body which should be most irritable, which should control the
other tissues and which, having memory, could profit by experience.
Adaptability of organisms was the end sought and this was obtained by
the selection of the function of irritability. The nervous tissue and it
alone shows, hence, a fairly steady progress from the lowest to the
highest animals. It is by means of his nervous system and in that re-
spect only that man stands at the summit of the animal world.

Not only has the nervous system been the point of attack of natural
selection and so has played a predominant part in evolution, but it is
also of the highest importance in embryological development. Since it
is to control, directly or indirectly, the metabolism of all the other tissues
of the body, it is almost or quite the first of the tissnes to be set apart
in embryogenic development. Its development appears, according to
Child, to set the pace for the development of the other tissues which
follow it. It is probable that, like the growing bud of the plant, the
metabolism in the embryonic nervous system is higher than in anv other
tissue of the body. In the vertebrate it is the rudiment of the nervous
system, the nervous folds of the cord and brain and the eye vesicles
which appear earliest in development.

The function of the fore-brain and particularly of the cerebral
hemispheres is memory and reason: This organ is, as it were, an epitome
of the whole body, for it is brought into relation with every part by
means of nerve fibers. No doubt this centralization of the body in the
brain plays a part in the development and perfecting of self-conscious-
ness and memory. Reasoning and all our psychic life are dependent, in
some unknown way, upon this master tissue. For these reasons the study
of the chemical composition and chemical transformations of the brain
possesses a fascination above that of the metabolism of any other part of
the body whatever.

Structure.—The nervous system is partly condensed into great
ganglia constituting the brain and spinal cord, which are found in the
cavities of the skull and vertebre; and in part it is distributed through
the body in the form of ganglia or nerve fibers or isolated nerve cells.
Among these groups of ganglia, which are outside of the spinal canal.
the ganglia of the sympathetic system found ventral to the vertebre and
in the abdomen are the most important. We shall treat, in this chapter,
almost exclusively, the composition of the brain and cord, since these
have been most studied.

The brain is composed chiefly of nerve cells with their processes, the
dendrites and nerve fibers. There is also present, however, a small
THE MASTER TISSUE OF THE BODY 567

amount of connective tissue supporting the blood vessels; and a kind of
supporting tissue composed of peculiar branching cells called neuroglia
cells. These neuroglia cells appear to support the nerve cells. Nothing
is more striking or significant in considering the metabolism of this
tissue than the arrangements which insure a very large blood supply.
The brain is supplied with blood from the external carotids, the internal
carotids and the vertebral arteries; these pour their blood into a com.
mon series of great vessels about the base of the brain called the circle
of Willis and from this the blood vessels are given off penetrating every
portion of the brain substance. It thus happens that even if both ex-
ternal carotids are compressed, or otherwise rendered incapable of carry-
ing blood, the other arteries are able to supply the needs of the brain.
This remarkably copious blood supply, when taken in connection with
the sudden change in activity of the brain when that supply is deficient,
indicates, very clearly, that the brain must have a very intense me-
tabolism.

The brain cells have usually two kinds of processes, the axon, so
called, and the dendrites; the former generally constitutes the nerve
fiber and in the vertebrates is generally surrounded by a special sheath,
the medullary sheath. By means of these fibers the cells are brought
into connection with other nerve cells at a distance, or with muscular
and other tissues. One of these processes, the principal one, as has been
said, is often surrounded by a peculiar cylindrical sheath of a glistening,
white, fatty matter of a peculiar chemical nature. Nothing is definitely
known of the function of this sheath, but it gives to nerves their glisten-
ing white appearance; and in the brain the nervous matter may be
separated into white and gray matter, the difference between these de-
pending on the relative amounts of medullated nerve fibers and cell
bodies. The parts of the nervous tissue which consist chiefly of cell
bodies and dendrites are gray ; the fibers are white. The corpus callosum,
the broad thick band of medullated fibers connecting the two cerebral
hemispheres, is purely white matter; the great ganglia of the corpora
quadrigemina and striata, and the cortex of the cerebrum are largely
gray matter. These different parts of the brain, the white and gray
_ matter, have different functions and different chemical compositions.
The gray matter is, on the whole, more automatic, in that nerve impulses
originate in it; the white matter is more purely a conducting tissue.

Chemistry.—The chemical composition of the brain can be best
studied in the human brain, for not only is man’s brain the largest, but
the peculiar psychical processes correlated with, or dependent upon,
the nervous system have in him reached their highest development.
His brain is the most highly differentiated and appears to be the most
perfect. We should expect to find in it, in the largest amounts, and in
56S PHYSIOLOGICAL CHEMISTRY

the purest unmixed state, the peculiar substances upon which the psychic
processes depend. It is, moreover, relatively easy to obtain human brain
material for analysis; the large number of accidents by which men in
the prime of life are killed making available brain material, unaltered by
disease.

The most striking point of difference between the chemistry of the
brain and that of other tissues is the very large quantity of alcohol-
ether soluble substances it contains; that is the very large proportion of
lipins in it. No other tissue has anything like such a proportion, with
the exception of fat tissue itself. The lipins of the brain, however, are
almost entirely free from neutral fat. There is practically no neutral
fat in the human brain; the lipins found there contain large amounts
of phosphoric acid, or at least many of them do; they are phospho-
lipins, glycolipins and cholesterol. That these lipins have a very im-
portant function in the physiology of the brain there can be no doubt.
since they are present in very much smaller amounts in the embryonic
nervous system and they develop pari passu with the development of
the functions of the brain. Most of these lipins are in the white matter
of the brain; but the proportion is also high in the gray. The proteins
are far less prominent among the total solids than in muscle. In both
white and gray substance, however, water makes by far the greater
proportion of the weight. Thus, in the gray matter, it is 85.27 per cent.;
in the white matter of the adult human brain it is 70.23 per cent. The
proportion of water varies with the age, being largest in the youngest
brains.

Our knowledge of the chemical composition of the brain is owing
largely to Thudichum, a man of extraordinary care, accuracy, insight
and industry, whose abilities were much underrated during his life.
For, owing to various causes, he alienated many of his colleagues, espe-
cially those in Germany who ignored and neglected his work. There
is now, however, no question that he was far in advance of all others in
this difficult field and his book, published in 1884, entitled, A treatise
on the chemical composition of the brain, based throughout on original
researches, and republished in 1901 in German, is a monument to his
ability and insight. A German by birth, he lived most of his life in
England. He died in 1902.

Chemical Examination of the Brain. Separation of the Phosopho-
lipins.—The method adopted by Thudichum for the chemical examina-
tion of the brain consisted in first freeing the brain from its membranes,
the pia mater, ete., and the blood vessels, then drying and extracting it
thoroughly with alcohol and ether. To get rid of the water in it the
brain can either be ground, or cut into small pieces and dried in a cur-
rent of air; or the pieces may be placed in 90 per cent. alcohol, at least
three volumes of alcohol to each volume of brain. The latter method

 
THE MASTER TISSUE OF THE BODY 569

is the one described here as it is, on the whole, the better. The cold
alcoholic extraction should be once repeated. This treatment coagulates
all the proteins and takes in the alcohol most salts and so-called ex-
tractives. The hardened brain substance is then ground still finer and
suspended in a small amount of fresh alcohol and put through a wire
sieve of 144 meshes to the inch, by means of a stiff brush. This reduces
the material to a fine purée, which can be thoroughly extracted. The
material is then heated to 70°, with a large amount of 85 per cent alcohol,
filtered hot through a cloth first and then a filter paper, and the residue
reheated with fresh alcohol five times. To exhaust it completely it
must be boiled at least 15 times more with 85 per cent. alcohol or abso-
lute alcohol; even then some substances slightly soluble in alcohol, prob-
ably anhydrides of the lipins, remain behind in the proteins. By this
repeated boiling with alcohol of 85 per cent., followed by absolute al-
echol, practically all of the brain lipins are removed and the proteins
left in a coagulated form. These united, hot alcohol extracts, if allowed
to stand cool for 12 to 24 hours, separate out a large quantity of a white
precipitate, which appears crystalline under the microscope, but is not
homogeneous. This material may be called ‘‘ white substance.’’ It is
collected on a cloth, the mother liquor pressed out and serves for further
fractioning into its constituents. It is sometimes called crude protagon.
It contains nearly all the glycolipins (cerebrosides), cerebric acids,
much cholesterol, cephalin (kephalin), various myelins and, if the alco-
hol solution was concentrated, some lecithin, amino-lipotides (amino-
lipins) and small amounts of other substances. If the alcoholic filtrate
from the white substance is concentrated by further evaporation until a
test portion settles out a precipitate on cooling and is then allowed to
cool, a precipitate comes out having, when pressed in a cloth, a soft
buttery consistence and a yellow color. This precipitate, which may be
called the ‘‘ buttery substance,’’ is separated by filtration. It consists
of much cholesterol, most of the lecithin and other phospholipins (phos-
phatides) ; the amino-lipins; but only traces of cerebrosides and cerebric
acids.

The alcohol filtrate from the ‘‘ buttery substance ’’ is still further
evaporated ‘by distillation as long as alcohol, capable of burning, comes
over. It is then evaporated further on the water bath in a porcelain
dish. As soon as the last traces of alcohol are gone, there form, on
the surface of the remaining water, oily drops uniting to masses, which
hang to the sides of the dish. This forms the “‘ oily material or sub-
stance.’’ It is separated hot, because if allowed to cool 1 mixes again to
an emulsion with the water. The ‘‘ oily substance’ contains some
cholesterol, but consists chiefly of amino-lipins (amino lipotides)
(bregenin) and. phosphatides (kephalin}.
570 PHYSIOLOGICAL CHEMISTRY

The aqueous solution, which is left, contains the extractives, namely,
inosite, lactic acid, salts, succinie acid, hypoxanthine, alkaloids, amino-
acids and inorganic salts. This extract may be united with the extract
obtained by evaporating the cold alcohol, which was used for dehydra-
tion. The latter has in it, also, some phospholipin. This may be re-
moved most easily by shaking it up with water to make an emulsion,
then adding some chloroform in small amount, shaking again and mak-
ing it acid by the addition of hydrochloric acid. The lipins come wut as
an emulsion, which may be filtered from the water which contains the
extractives.

The partition of substances sketched above may be summarized in the
following table (Thudichum, Gehirn, p. 79):

1. First extractive substances in the cold alcohol for hardening and dehydrating.
2. Insoluble protein and tissue residue containing neuroplastin, protein, nuclein,
phosphoproteins.
3. White substance containing:
Kephalin with varieties and compounds.
Lecithin with varieties and compounds.
Paramyelin with varieties and compounds.
Myelin with varieties and compounds.
Amino-myelin with varieties. and compounds.
Cholesterol and Phrenosterol.
Cerebrin mixture, mixture of cerebrosides (glycolipins), cerebric acids,
‘ pecehito: -sulphatides and amino-lipotides with sphingomyelin and assurin.
4. Buttery substance containing:
Kephaloidins with varieties and compounds.
Lecithin.
Paramyelin.
Myelin.
Amino-myelin.
Sphingo-myelin, and assurin (small amount).
Cholesterol and Phrenosterol.
Phrenosin and other cerebrosides.
i. Amino-lipins (Amino-lipotides).
5. Oily substance containing:
a. Lecithin; b. Paramyelin; ¢. Oily liquid material of amino-lipotides.
6. Last aqueous brain extract containing:
a. Alkaloids (hypoxanthine, etc.). b. Amino-acids. c. Inosite. d. Organic
acids and salts. e. Inorganic acids and salts.

Rmrops oe

Pam Sao op

Fractioning the white substance. Both the white and buttery sub-
stances may be fractioned. or separated into their constituents by the
methods given in Thudichum. By extracting these substances with cold
ether all the sterols and most of the phospholipins go into solution, leav-
ing a white ‘substance. cundissolved. This white substance consists chiefly
of cerebrin and. cerebrosides, cerebric acid and sulphatides. The kephalin
is separated from the lecithin and some other phospholipins in the ether
THE MASTER TISSUE OF THE BODY 571

extract of the white and buttery substances by the addition of three
volumes of absolute alcohol. This precipitates an impure kephalin.
It may be purified by repeated solution and precipitation and finally by
precipitation with cadmium chloride. Further details are given in
Thudichum. We may now consider these various products, or educts, in
detail.

Lecithin.—This is a phospholipin, or phosphatide, as it is called by
Thudichum. The term phosphatide lays chief stress upon the phosphoric
acid of the molecule; while phospholipin emphasizes its fatty character.
Lecithin is found in the white substance in part, but in largest amount
in the buttery substance. It is separated from the white or buttery sub-
stance by extracting these with cold ether. The cephalin is separated
from the lecithin by the addition of three volumes of absolute alcohol to
the ether. This precipitates nearly all of the cephalin, but leaves the
lecithin in-solution. The filtrate is precipitated by the addition of ai-
coholic ammoniacal lead acetate to get rid of the rest of the cephalin,
cephalin being precipitated by this reagent, but lecithin not. The mye-
lins, ete., are also precipitated ; the filtrate is distilled to remove ammonia
and ether until fairly pure cholesterol begins to come out. If some salve-
like lecithin comes out, this is redissolved in 85 per cent. warm alcohol.
To this warm solution a warm saturated solution of CdCl, in 85 per cent.
alcohol is added, little by little, as long as a precipitate forms and then
about as much more CdCl, as had already been added is poured in.
The lecithin CdCl, compound erystallizes out as a white precipitate.
This is washed by decantation with 85 per cent. alcohol. This precipitate
is dried and freed from ether-soluble substances by prolonged extraction
with boiling ether. Krinosin is the main impurity removed. The pre-
cipitate is next extracted with cold, water-free benzol to remove traces
of the cephalin-CdCL, and finally is extracted with hot benzol, which dis-
solves the lecithin cadmium chloride, but leaves behind the para-and
amido-myeliz cadmium chlorides, which remain insoluble. The lecithin
cadmium chloride compound is precipitated from the benzol by the
addition of absolute alcohol and is recrystallized from hot alcohol.

There is thus obtained lecithin-CdCl,, which crystallizes in spheres
and stars of microscopic crystals. The purest lecithin cadmium chloride
compound of ox brain had the composition: C,,H,,NPO,CdCl,. Lecithin
may be freed from the cadmium chloride by suspending it in 85 per
cent. alcohol and passing in H,S. It is filtered through a hot funnel.
On cooling a felt work of fine, needle-shaped crystals of lecithin
chloride separate out. These crystals are microscopic plates, often hexag-
onal and very thin, so that they may be bent over to look like needles,
They dry in vacuo to a white, easily-powdered mass of the composition:

C,H, .NPO.Cl; or C,H, ,NPO,Cl.. HCl: N: P:: 1: 1.03: 1.09.
572 PHYSIOLOGICAL CHEMISTRY

Pure lecithin. This is a white crystalline body, the crystals being
thin plates, which, when pressed together, have a waxy consistence, but
it is stickier than wax. It is very soluble in 85 per cent. alcohol and
more soluble still in absolute alcohol. It is soluble in ether and chloro.
form, but does not crystallize from them on evaporaticn. It is not
precipitated from its alcoholic solution by ammonia and lead acetate.
When put in concentrated sulphuric acid it dissolves with a yellow color
and if to this sugar solution is added, a deep purple red color develops
(Pettenkofer’s reaction). This is due to the oleyl group in the lecithin.
The purple coloring matter is soluble in glacial acetic acid and shows
absorption bands between D and E and in the blue. If lecithin cadmium
chloride is suspended in water and dialyzed the lecithin remains in the
tube; the cadmium chloride is dialyzed away.

The chief properties of lecithin are due, according to Thudichum, to
the oleic-acid group it contains, but it is obvious that all of its constitu-
ents, and particularly the phosphoric acid, contribute to its properties.
The oleic-acid radicle is somewhat more easily separated than the others.
Thus it comes off readily when the PtCl,HCl lecithin compound is made.
By decomposition with acids, or barium hydrate, lecithin yields neurine
(choline according to most observers), oleic acid, palmitic acid or stearic
and glyceryl-phosphoric acid. The formula for lecithin, given on
page 30, is that of Diakonow, derived from the study of egg yolk lecithin,
but it is uncertain what the formula really is, since Diakonow at no time
probably had pure lecithin in his hands for analysis. How the various
radicles are united to make lecithin is still uncertain except that gly-
cerol is united with the phosphoric acid to make glyceryl-phosphorie
acid. The chief lecithin of the brain is oleylpalmityl-glyceryl-neuryl-
phosphatide. The other lecithins, containing steary! in place of palmityl,
are present only in small amounts. To indicate his belief that phosphoric
acid is at the basis of the molecule, whence the name phosphatide, Thudi-
chum represents the molecule as follows:

C_.H,.O

18 83 2

Oo=P CoH 1% = C,H, .NPO
are C,H,0, — “42°” 82 8

CH N

6 11

It is probable that the formula is really that given by Diakonow with
the fatty acids substituted in the glycerol; and the choline, if present.
united through the hydroxyl of the carbon, rather than that of the
nitrogen, to the phosphoric acid. Thudichum states that he has verv
carefully examined the base of brain lecithin and that it is neurine and
not choline. Unless the lecithin is suspended in water and shaken with
hydrochloric acid it is impossible, he says, to free it from potassium,
THE MASTER TISSUE OF THE BODY 573

which on hydrolysis precipitates with the platinum chloride-neurine com-
pound, so that most of the cholines examined have been impure. If
neurin pre-exists in the molecule it is very difficult to see how it can be
attached, since the formation of the chloride of lecithin indicates, very
clearly, that the hydroxyl of the nitrogen is free. It seems to the
author more probable that the base in lecithin is choline, from which
neurine is formed on decomposition. There do not seem to be determina-
tions of the molecular weight of lecithin. It is certainly colloidal in
aqueous solution. It is possible that the formula is more complex than
appears. Possibly the phosphoric acids may be joined much as they
are in nucleic acid to give poly-phosphatides. The whole matter of the
composition of the lecithin molecule is in need of investigation. Lecithin
is often considered to be unstable, but Thudichum states that lecithin, in
a dry state, or as the CdCl, compound. is so stable that it may be kept
for years without change; and even as the hydrate when suspended in
water it does not easily change. It is wet by water, does not float like
fats, but sinks to the bottom, and swells to form an emulsion if it is
‘present in less than 1 part to 100 of water. It does not dialyze. A very
interesting fact is that, as ordinarily prepared, it contains some potas-
sium. It probably exists in the cells in part as a potassium salt. It has
the property of making myelin forms which are liquid crystals.

Kephalin.—Another mono-amino-mono-phosphatide is kephalin (Gr.
kephalos, brain), or cephalin if the Latin spelling is used. This differs
from lecithin, according to Thudichum, in that it contains another acid,
cephalinic acid, which is an unsaturated acid of the linolinic acid series,
.in place of oleic acid. It differs, also, as we now know, in the character
of the base it contains; it contains no choline, but in place of it amino-
ethyl alcohol, or oxy-ethyl amine. This was isolated from it by Thudi-
chum. Possibly other bases are also present, i.e., @-hydroxy-a-amino
butyric acid (McArthur).

Preparation. It is isolated from the ether extract of the ‘‘ white
substance ”’ or by extracting the dry brain with ether. The extracts are
concentrated and freed from cholesterol by precipitation with acetone.
The precipitate is redissolved in ether, if not clear, allowed to stand
until any white matter has separated out, and the decanted clear solu-
tion precipitated by the addition of absolute alcohol as long as a pre-
cipitate forms. Cephalin is precipitated. After standing 24 hours in
the cold the liquid is poured off from the cephalin precipitate. This
precipitate is redissolved in ether and reprecipitated several times with
aleohol to remove cholesterol and lecithin. It is then emulsified with
100 parts of water, allowed to stand, separated from any precipitate
which may form by decantation and precipitated by the addition of
hydrochloric acid just sufficient to precipitate it. The precipitate rises
574 PHYSIOLOGICAL CHEMISTRY

to the top and is lifted off and washed with water until by the separa-
tion of HCl it begins to get slimy. Then it is freed from water by
alcohol, dissolved in ether, precipitated by alcohol, dried in vacuo and
it is ready for analysis. The emulsification and precipitation of the
cephalin, by acid, is necessary to free it from bases, Ca, K and Na, and
phosphoric acid, which stick to it. There is always some ammonia
separated in the water, and Thudichum states that the aqueous solu-
tion from the emulsification contains copper, giving, on evaporation, a
deep blue solution with ammonia. This point should be reinvestigated
to see whether this is in reality a normal constituent of all brains, or
present only in human brains which happened to come for analysis. It
is possible that the human brains he examined might have come from
brass-workers or others exposed to copper poisoning. The calcium and
potassium are attached, in part, directly to the cephalin molecule. These
salts predominate over the others in the cephalin just as they do in the
cell. It is not impossible that the greater predominance of potassium
over sodium in the cell may be due to this firm union between cephalin
and these bases. Similar salts are recovered from most phosphatides,
particularly from myelin. Their possible rdle in the nerve impulse has
been discussed by Pike.

Properties. The cephalin thus isolated is quite possibly still impure.
It is at first a light yellow or white color, but in ether solution it changes
rapidly to a red. It unites with water, forming an emulsion, the soluble
cephalin becoming insoluble by heating the aqueous solution. <A part of
the cephalin, after precipitation with CdCl,, when freed from CdCl,
by dialysis in the manner described for lecithin, will diffuse through
the paper. It makes a finer emulsion with water than does lecithin.
It is soluble in water saturated with ether. 100 parts of boiling absolute
alcohol. dissolve 9 parts of cephalin; 2 parts come out on cooling, 7 parts
remain in solution. It is much less soluble in alcohol containing water.
Heated in water to 90-100° it melts to a thick, dark red oil. It forms a
chloride with hydrochloric acid. This chloride is soluble in ether and is
not precipitated by alcohol. It is precipitated from an ether-aleohol
solution completely as cephalin PtCl,HCl by PtCl,. It is precipitated
also by Ba (OH),, and Ca (OH), and ZnCl,. The affinity of CdCl, for
cephalin is less than that for lecithin. The Pettenkofer reaction is never
so good as that of lecithin. In cephalin the nitrogen base splits off first
on hydrolysis, like the base in sphingomyelin. Thudichum obtained by
hydrolysis neurine, a second base probably amino-ethyl alcohol, which
may be a decomposition product of neurine; a third base, of unknown
nature; cephalinic acid, which is apparently an unsaturated partially
oxidized palmitic or stearic acid; and glycerol. He isolated cephalyl-
phosphoric acid, which is cephalin minus the neurine. More recent de-
THE MASTER TISSUE OF THE BODY 575

terminations of the composition of cephalin indicate that it contains no
neurine. Koch found that when heated with hydriodic acid it gave rise
to less isopropyl or methyl iodide than did lecithin, so that the base is
certainly not neurine or a methylated base. Miss Foster has found that
the iodide obtained by Koch was isopropyl iodide from the glycerol.
No:methyl iodide is formed. It is probable that the preparation of
Thudichum was still not pure. There is no doubt that the chief base
present is oxyethyl amine. Baumann obtained, like Thudichum, amino-
ethyl aleohol and believes that this is the only base present; on the other
hand, MeArthur has isolated amino-oxy-butyrie acid, serine, ethoxy
amine and ammonia. How far these are decomposition products and how
far they are preformed in the molecule is uncertain. No reliance can be
placed on the results of the study of the decomposition products, unless
the cephalin has been carefully separated from lecithin, myelin and
other phosphatides, and emulsified and treated with acids to free it from
extractives and salts. Neurine breaks up readily in alkaline solution,
so hydrolysis in an alkaline solution yields results which are at the best
difficult to interpret, but it is stable in acid. Wither there are a number
of cephalins with different nitrogen bases or products in various stages
of decomposition have been analyzed. The recent work of Levine indi-
cates the latter as probable. There is also unexplained the number of
oxygen atoms which are not accounted for by the decomposition products
isolated.

The composition of cephalinic acid is still uncertain, but it appears to
be either C,,H,,0, or C,,H,,03. It is probably a mixture of very un-
saturated acids of the linoleic or linolinie acid type and their partially
oxidized derivatives. The constitution of cephalin was represented as
follows by Thudichum, but the neuryl radicle should be replaced by
amino-ethyl alcohol.

Kephalyl C_H.O

1824-3 |
Stearyl CH, O
O=—P 1835 2 =
: Glyceryl C,H,O, CrHeoNPO,

Neuryl C,H, ,ON

The manner in which these are united is unknown, as is also the
molecular weight.

The extraordinary reducing powers of cephalin due to the unsatu-
rated acids are extremely suggestive and interesting. In cephalin we
have a body greatly more reactive than lecithin, capable of auto-
oxidation and hence of respiration, and unstable. These phenomena,
as pointed out on page 590, are possibly related to the phenomena of
respiration and memory shown by the nervous system. This insta-
bility is one of the difficulties in the way of obtaining pure cephalin.
Another possibility exists also: namely, the molecule may be formed
576 PHYSIOLOGICAL CHEMISTRY

of several phosphoric-acid groups to which various radicles are united.
The further investigation of cephalin will probably yield results of great
value.

The further study of the acids present indicates that they are stearic,
as found by Thudichum, linolic, linolenic and carnaubic (McArthur).

Paramyelin.—This is another mono-amino-mono-phosphatide which is
present in the white substance. It is precipitated by CdCl,, but is sepa-
rated from cephalin CdCl, by the solubility of the latter in cold, water-
free benzol ; and from lecithin CdCl, by its solubility in hot benzol. The
paramyelin CdCl, is insoluble in hot benzol. The CdCl, salt is finally
dissolved in boiling 85 per cent. alcohol, from which it crystallizes on
standing. The CdCl, compound dissolved in hot alcohol, decomposed
by H,S and filtered hot, crystallizes out in white crystals as the chloride.
When this ‘is recrystallized from alcohol it loses HCl and crystallizes
in rhombic and hexagonal plates like lecithin containing 4.31 per cent.
of P and 2.06 per cent. of N. The computed molecular weight was 721.
The formula: C,,H,,NPO,.CdCl,. On decomposition with Ba (OH), it
yields glyceryl-phosphoric acid, neurine, an acid giving Pettenkofer’s re-
action and another acid. It is a white, solid, crystalline substance, erys-
tallizing from hot alcohol in plates and needles. It is a weaker base
than lecithin or kephalin, easily losing the HCl. It is found both in
the white and butter substance. This substance requires further exami-
nation. The character of the base is particularly doubtful.

Myelin.—This mono-amino-mono-phosphatide is found in the brain
only in small amounts. It is sharply distinguished from paramyelin by
its lead compound.

Preparation. If the white substance is extracted with cold alcohol
and allowed to stand in the cold, myelin separates out mixed with
sphingomyelin. The precipitate redissolved in hot alcohol is precipi-
tated by alcoholic lead acetate; the lead compounds are extracted with
boiling alcohol which dissolves the sphingomyelin. The lead compound
is also insoluble in benzol. If it is suspended in hot alcohol, decomposed
by H,S§, filtered hot, white myelin crystallizes out on standing. When
recrystallized from alcohol it tends to form insoluble anhydrides. It is
not precipitated in alcoholic solution either by CdCl, or by PtCl,.

Properties. It crystallizes out of ether or alcohol on cooling the satu-
rated solution either in spheres or very small microscopic crystals. In
masses it is white like bleached ivory and when powdered it is entirely
white. It dissolves in concentrated H,SO, without color, but if sugar
solution is added it gives a deep purple substance soluble in CHCl,. The
myelin lead compound is insoluble in ether; that of cephalin is soluble.
Lecithin, paramyelin, amidomyelin and sphingomyelin give no lead
compound. The analysis gave: C, 63.41 per cent.; H, 9.83 per cent.: N,
THE MASTER TISSUE OF THE BODY 577

1.79 per cent.; P, 4.087 per cent.; O, 20.874 per cent., which corresponds
to C,,H,;NPO,,. The decomposition products are unknown.

Diamino-mono-phosphatides.—There are also phosphatides with the
nitrogen and phosphorus in the proportion of 2:1. These are di-amino.
mono-phosphatides.

Amidomyelin. This is a diamino-mono-phosphatide of the formula,
C,,H,,N,PO,,, which is isolated as a CdCl, salt from the buttery sub-
stance. It crystallizes as the salt C,,H,,N,PO,,2CdCl,. This phos.
phatide has a very curious property: namely, it is completely soluble in
cold water when freed from CdCl, by dialysis, but the solution gels on
heating. The gel is not reversible, it does not redissolve on cooling. This
property differentiates it from the other phosphatides. The free amido-
myelin forms snow-white, microscopic needles and plates mostly arranged
in stars. Over sulphuric acid it drys to a perfectly white powder. It
gives Pettenkofer’s reaction very quickly and intensely. Nothing is
known about its decomposition products.

Sphingomyelin. This is the chief, but not the only, phosphatide left
in the ‘‘ white substance ’’ when this is extracted with ether. The
method of its separation is long and involved and will not be given here.
It erystallizes out of alcohol in thick masses of needles, stars and six-
sided plates. It is almost insoluble in ether even when HCl is added.
It is thus separated from lecithin. It appears to combine with the
cerebroside, kerasin, as a base in a weak union. It is precipitated by
CdCl, from 85 per cent. alcohol solution. It swells in water and makes
an emulsion. The CdCl, salt loses its CdCl, on dialysis. - The analyses
gave C, 65.37 per cent.; H, 11.29 per cent.; N, 2.96 per cent.; P, 3.24
per cent.;.O, 17.14 per cent., which corresponds to the formula,
C52H,),.N,PO,H,O. On hydrolysis it yields no glycerol. After 5 hours’
heating with Ba(OH), only neurine is given off. The amount of neurine
found as the platinum salt was 1.1 grams, where 1.3 grams was the
theoretical. There remains sphingomyelinic acid, C,,H,,NPO,,. N:P::
1:1. This yields sphingol, an alcohol, C,H,,0, or C,,H,,0,, soluble in
alcohol and ether; sphingosin, a base, C,,H,,NO,, and sphingo-stearic
acid, C,,H,,0,, probably an isomer of stearic acid melting at 57°. The
CdCl, salts are beautifully crystalline and white. Sphingosin is appar-
ently an unsaturated, mono-amino-dihydroxy alcohol. The structure is
still unknown. The sulphate melts at 233-234° (uncor.) and the rotation
is (q@)i}° =—13.12 (+0.00) in a solution of .5304 gr. of sphingosin in
5 ec. CHCl, and 1 ec. glacial acetic acid. The recent analyses of
sphingomyelin by Levene showed the composition, C, 64 per cent.; H,
11; N, 3.40; P, 3.60: inorganic bases, 3 per cent. N:P::2:1. It con-
tains no free amitio nitrogen. One nitrogen atom is in the form of
choline; the nature of the other base is unknown; the acids split off on
578 PHYSIOLOGICAL CHEMISTRY

hydrolysis are lignoceric acid, C,,H,,0,, and cerebronic acid, C,,H,.0,;
(a hydroxy pentacosanic acid), and sphingosin. The composition of
sphingosin is still unknown, but its composition has been suggested by
Levene to be C,,H,,CH—=CH.CHOH.CHOH.CH,NH,.

Diamino-diphosphatides.—These contain two atoms of phosphorus
and two of nitrogen to the molecule. They are present in small amounts
only.

Assurin. This is found in the alcoholic extract of the cerebroside
mixture after the separation of the myelin, sphingomyelin and kerasin.
It is crystallized as the PtCl, salt, which is insoluble in boiling alcohol
or ether and is either 2(C,,H,,N,P,0O,HCl)+PtCl,; or 2(C,,H,o,N,
P,O,HCl)+PtCl,. There is apparently united with this body an amino
lipotide phosphorus free, C,,H,,NO,. It may be identical with bregenin.

The cerebrosides or galactosides.—In addition to the phospholipins,
the brain is remarkable for the group of bodies called the cerebrosides,
or glycolipins. These are not found in the embryonic brain, but develop
as medullation comes on and are to be found chiefly in the medullary
sheaths in the white matter of the brain. The most important of the
cerebrosides are Phrenosin and Kerasin, or the cerebron of Thierfelder.
The cerebrosides contain no phosphorus, but they all contain nitrogen,
a sugar, first named cerebrose, but later shown to be for the most part
galactose, and a complex fatty acid.

Phrenosin and kerasin. These two cerebrosides make the greater
part of the white substance left behind on extraction of the phosphatides
with ether. They are quite insoluble in ether, but are soluble in alcohol,
particularly in warm alcohol. They crystallize very readily, although
it is not so easy to get them quite clean. Phrenosin has also been named
cerebron, by Thierfelder, but it is better to keep the original name.
They are mixed in the white substance with another cerebroside, cerebric
acids, from which they may be separated by precipitating the latter
with lead oxide. They are prepared by exhausting the white substance
by extracting with ether. If now they are redissolved and recrystallized
from hot alcohol, the phosphorus content falls to about .8 per cent., but
it is hard to get it lower. To purify further the mass is rubbed in a
mortar with an alcoholic solution of lead acetate containing a little
ammonia, and then, constantly stirring, it is poured into hot 85 per cent.
alcohol. When all is transferred add an alcoholic solution of lead
acetate and ammonia as long as a precipitate forms; filter hot; extract
the precipitate repeatedly with hot 85 per cent. alcohol and collect
the precipitates from all the hot alcohol extracts on cooling. Recrystal-
lize from absolute alcohol and filter after 24 hours. The precipitate
is a mixture of phrenosin and kerasin. On longer standing kerasin
erystallizes out. The separation of these two is made by dissolving them
THE MASTER TISSUE OF THE BODY 579

in alcohol so that they are not too concentrated and allowing them to
cool. Phrenosin comes out above 28°. Kerasin, which is more soluble,
comes out below 28°.. The sphingomyelin has to be separated by CdCl..
Recrystallizing from glacial acetic acid also helps to eliminate the last
traces of the phosphatide. ,

Phrenosin. Properties. The name is from the Greek, phren, brain.
It is a white crystalline substance, which when warmed in water becomes
neither doughy nor slimy, but floats in loose flocculi through the liquid.
Rubbed with concentrated H,SO,, it apparently completely dissolves,
then gradually develops a purple red color which is attached to particles
in the acid. These are soluble in CHCl, or glacial acetic and show spe-
cific absorption spectra. The color reaction belongs to the sphingosin.
No sugar need be added, since galactose is already there. Phrenosin has
the composition C,,H,,NO, (Thudichum), but more probably C,,H,,NO,
according to Levene. When boiled with acids it hydrolyzes and galactose
is set free, the solution acquiring a strong reducing power in conse-
quence. It yields sphingosin, C,,H,,NO,, already described as a
decomposition product of sphingomyelin; galactose; and a new acid,
which Thudichum thought to be an isomeric stearic acid, but which is
now known to be C,;H,,0,, phrenosinic, or cerebronic, acid (a hydroxy
pentacosainic acid). On decomposition of phrenosin, the cerebronic
acid breaks off first and there remains a compound of sphingosin
and galactose of a basic nature named psychosin, C,,H,,NO,. Out of
hydrated phrenosin sulphuric acid splits off galactose, leaving esthesin,
C,;He.NO,(?), which is probably a compound of sphingosin and cere-
bronic acid. Sphingosin has a bitter taste and causes a burning sensa-
tion in the throat and a feeling of illness. Its pharmacology would
perhaps be of interest. The composition of phrenosin may be repre-
sented as-follows:

heenneint ;
Phrenosinie acid, C,H. 00g

Phrenosin | (a hydroxy pentacosanic)
C,H, N 0, Galactose, C eet %
Sphingosin, C__H,,NO,

Kerasin resembles phrenosin in most particulars, except it is optically
different and it is more soluble in alcohol. If not more than one part
is present in 321 parts of alcohol, it does not crystallize out above 28°.
100 ¢.c. of acetone dissolve 0.1576 gram of kerasin at 15°. It does not
give the Pettenkofer reaction so easily as phrenosin. The differences
between these two bodies have been shown to be due to the presence
in phrenosin of cerebronic acid m.p. 84°; while in kerasin it is lignoceric
acid, C,,H,,0,. The cerebron of Thierfelder appears to be a mixture
of both these bodies. Thudichum thought that the difference probably
was that the cerebronic acid of the kerasin had about one carbon atom
580 PHYSIOLOGICAL CHEMISTRY

more than that of phrenosin. Kerasin is levo-rotatory. [a],——2° in
pyridine. The composition of kerasin may be the following:

Lignoceric acid, C,H, ,0,
Kerasin Galactose, C,H, 9,
C,H, NO, Sphingosin, C,,H,.NO,

The phrenosin and kerasin spherocrystals are readily distinguished
by microscopic examination with polarized light and a selenite plate
(Rosenheim).

It is an interesting fact that the medullary sheath contains so large
a proportion of galactose. It is possible that it is to supply this raw
material to the brain at a time when medullation is proceeding at a
rapid pace that the sugar of the mammary glands is lactose and con-
tains, therefore, galactose.

Protagon. It was for a long time believed that the crystalline mate-
rial falling out of the alcoholic extract of the brain constituted, when
purified, a single substance composed of a compound of cerebrin and
lecithin or cephalin. This was called ‘‘ protagon.’’ Since Thudichum
succeeded in isolating 14 different substances of a complex nature from
this mass, it is probable that protagon is a mixture and not a chemical
individual. Nevertheless, it is not impossible that some of the compounds
isolated may have been in weak chemical union. It is known, for exam-
ple, that cholesterol forms a molecular, crystalline compound of definite
composition with digitonin; but if the compound is extracted with ether
it is decomposed, the cholesterol dissolving. The similar compound of
cholesterol and saponin is stable in ether, but breaks up in hot alcohol.

The sulphur of the brain. Cerebro-sulphatides. Sulpholipins.—The
eerebrosides while impure always contain some oxidized sulphur and,
according to Thudichum, some unoxidized sulphur. The oxidized sul-
phur body was believed by Koch to be a union of sulphuric acid, a
cerebroside and a phosphatide as follows:

Oo

I!
Cerebroside—O—S—O—Phosphatide

1

Oo

Its nature is still entirely unknown. Taurine is found in the brain, but
the unoxidized sulphur fraction of the lipins noted by Thudichum is
still uninvestigated. It is probable that further study will show that
sulphur is as important in the metabolism of the brain as phosphorus.
The phospholipins from other cells not infrequently, also, vontain
sulphuric acid not extracted with water, but split off on hydrolysis with
acid; and also a carbohydrate group. The sulphatide isolated by Thu-
dichum from the brain contained 4 per cent. of sulphur.
Amido-lipotides. Amino-lipins.—These are found chiefly in the oily
THE MASTER TISSUE OF THE BODY 581

matter separating out at the end of the evaporation of the alcohol. They
contain no phosphorus, but an amino group. When heated they collect
as a thick oily mass on the surface of the water, or cling to the sides of
the beaker, but on cooling they take up water again and swell to make
a gelatinous mass. None of them are well characterized and they are
present in relatively small amounts. Whether they are preformed or
represent partially digested phospholipins cannot be said. Among these
bodies two were isolated by Thudichum: Krinosin, C,,H,,NO,, and
Bregenin, C,,H,,NO;. (From the Platt Deutsch word, bregin, brain.) If
II,PO, is added to this formula, we get the formula of the lowest lecithin.
Nothing is known of the composition of these bodies. Their physical
properties are so interesting that they should be carefully studied. It
is possible that they might throw light on the composition of the phos-
phatides. It is very hard to separate them completely from cholesterol.
Cholesterol esters, also, have the property of taking up large amounts of
water. See page 87.

Sterols.—The brain contains extraordinarily large amounts of choles-
terol (1.6 per cent. of the wet weight of the brain), which appears to be
for the greater part free cholesterol. This sterol is chiefly cholesterol
melting at 145°; but there is also present another sterol melting at 137°,
which is the melting point of iso-cholesterol. It was called by Thudi-
chum phrenosterin, or as we should call it now, phrenosterol, to indicate
its origin in the brain. The composition and characteristics of choles-
terol have been discussed on page 82.

AMOUNT OF CHOLESTEROL IN DIFFERENT Brains (Rosenheim).

Water—per cent. Cholesterol—per Cholesterol—per

cent. wet weight cent. dry weight
Man ....... ee eer 78.86 1.95 9.22
Child 3 months ............... 85.80 0.69 4.89
Foetus 36 weeks ............4-. 90.29 0.39 4.07
Child'S. days: . ss ceies seer dec ee 89.99 0.53 5.29
Dog sieve swans since arene es 76.18 2.76 11.59
Cate a ccoxarsacayen gs Spiess nema ove aucnoes Beasmeane 76.53 2.35 9.99
DOK” Siac seca tthenameng tye Biases dusco:lebdnaguevaseiierd 78.83 2.39 11.28
atid 2.13 10.37
2.12 9.57
1.45 7.40
1.92 12.02

 

The extractives—When the lipins have been removed from the
evaporated alcoholic extracts, cold and hot, there remain a group of
water soluble substances, organic and inorganic, of which only a few
have been isolated and identified. These are called the brain extractives.
The solution is generally brown in color, with the smell of meat extract
and a bitter, salty taste. Some of the organic extractives contain nitro-
gen and are precipitated by the alkaloidal precipitating agents like
phosphotungstic acid. They are alkaloidal in nature; others are of a
582 PHYSIOLOGICAL CHEMISTRY

carbohydrate nature; others are acids. It is very difficult to know
whether these substances are normal constituents of the living brain,
or whether they are formed by the hydrolytic decompositions which
begin as soon as the circulation of the brain is interrupted. It is prob-
able, however, that they pre-exist in the brain and that their accumula-
tion there after death is due to the persistence of the vital processes
in the absence of blood supply to remove the waste products. This view
is supported by the relatively slight autolytic decomposition which brain
material shows.

Alkaloid extractives. Of these hypoxanthine is the most important,
but Thudichum isolated also another of unknown character having the
formula for its gold salt C,,H,,N,O,HClAuCl,, and another having a
strong odor of human sperm. Hypoxanthine is discussed in connection
with the occurrence of it in muscle. It is impossible to say whether it is
formed from the nucleic acid of the brain by a partial hydrolysis, or
whether it is found in the cell in a free state. Its function is unknown.
It is interesting to recall that when caffeine is taken it accumulates in the
brain tissue to a far greater degree than in any other tissue of the body.

Amino-acids. Among the amino-acids in the extractives Thudichum
isolated tyrosine and a new leucine, a sweet leucine which he called glyco-
leucine, and correctly inferred it to be the normal caproic acid leucine
having the formula:

H HH H NG,
n—¢_b_b_bb_coou

hha hn

It has been recently isolated also by Abderhalden, who proposes to call
it caprime. It is curious that the brain proteins should contain this
normal leucine, whereas most other proteins have the iso-butyl-amino-
acetic leucine. Caprine is less soluble in cold water and also the copper
compound is less soluble than the ordinary leucine. Besides leucine and
tyrosine, creatine and taurine have been found in the extractives.
Creatine is methyl guanidine acetic acid, NH,—C—NH.N(CH,)—CH,—
COOH. The significance of its occurrence in the brain is unknown.

Organic acids. The principal organic acids found in the extractives
are lactic, C,H,O,, succinic, C,H,O,, and also formic acid, CHOOH.
These same acids are also the principal acid extractives of muscle. The
lactic acid is the para, or meat, lactic acid.

Carbohydrates. In this group inosite, C,H,,0,, an optically inactive,
sweet-tasting alcohol, not reducing Fehling’s solution, is present in con-
siderable quantities. This again is found in quantity in the muscles.
Inosite is believed to be hexahydroxyhexamethylene. Its significance is

unknown.
THE MASTER TISSUE OF THE BUDY 583
H OH
*

L
H © 4
SW SA <
HO H
e| [3
»e Cc
HO Xe OH
\

H” OH

Inorganic salts. The salts of the extractives are phosphates, chlorides,
sulphates of calcium, potassium, sodium, magnesium and iron.

Distribution of substances between gray and white matter.—We
may now take up the question of the distribution of these substances
in the gray and white matter of the brain and their functions. While
the methods of separation employed by Thudichum were far from quan-
titative, they give at least some idea of the distribution of many of the

substances. Some analyses are given also by the improved methods of
W. Koch.

Gray MatTrer or THE CorTEX. Driep First. {Thudichum.)

Water ats 95 cnoiie os cre aecenty lane eh edie ede bud eased cena as 85.27%
Solids: sacec ees oe ieee te Gitte ee siGidres eee ee wing Voc alamie aleve 14.73
Neuroplastin:. 2 sig iseseoseiss pa riaceae yi ner ete mines ee mene & 7.608
Ether extract (kephalin, lecithin, cholesterol) .................. 1.950
Cerebrosides, cerebric acid and myelin ..................0..0000- 0.424
Lecithin, kephalin, myelin from last oily matter ................. 0.780
TnOSite> c.stnsius sagen coe msee hte paeccuint eo ctvenesdlee amacedodw vende 0.193
Dactioraeld) ovc.cA cds we oa ea enaeitiei ed aloe edeee Saad ate 0.102
Alkaloids: esc saauie to i Seas een dae Sa Geee NOES eGR east | Aaa
H,SO, in neuroplastin ..........-. see eee cece ence eee ence eee ees 0.06
< extract: sei sks yaw ates teieees 16 wees tietaame ee asee Pas). wean
H 39, spotted Leda Maas ee Guala sate e h Vauatia ns eee es we a dod a 0.017
ee a hoshncs alas eos G48 edie 5 OC Atnie Ge eee a des Aes Nee es Mente Re hes 0.025
SIN Eh ob8 asad cut nbd’ 0 cahanais. ses ¥duancaabanede3 fevsenld a couasosa avoir slice tua suateseuinel a, a¥e ateunr 0.092
Water extract) isciu <rapaen. dst: atelebacena Betigaane Seielelaned-acd Shavelas g ataeuetele ste 0.500

Loss by operations about 25% of the total solids.

CoMPOSITION OF THE WHITE MATTER OF THE CoRPUS CALLOSUM.

Water (dried at-95°)) aces aiisacdcn Sag taseead scene Foose Bere 70.23%
NetPro plastini« <3) ¢.sa5 wpessecses 9 ahseecd 5,2-5icuetand, & gutsaon Welesemanccard euneiguern Sociche 8.63
Ether extract (kephalin, lecithin, cholesterol) .................. 11.497
Cerebrosides and myelin .......... 0. ccc cece ee cece eee eee enes 6.91
Water ‘extract. xi niinuee yaa etine cee dos Di Gucahepele Wiaatenbence wing 1.403
Dbaeticiacid: 2cahuciet snag 24 seme atearGday envrr tan meee ee wars see 0.0456
Wnesité cece sagovisniodenw signe: cetdb ee eee Swen sas eies see ae 0.2171

Alkaliées' or carbonates....¢.. :4ka ssc akw oe ecanesaee sy saakeas ee as 0.1717
584 PHYSIOLOGICAL CHEMISTRY

In contrasting these two tables it will be noticed that the white mat-
ter contains more solids and less water than the gray, and these solids
are chiefly present in the ether extract and include the cerebrosides.
Thus the cerebrosides make roughly 7 per cent. of the white matter and
only 0.4 per cent. of the gray; and the cephalin, lecithin and cholesterol
are 11.5 per cent. of the white and only 1.95 per cent. of the gray. The
per cent. of protein is also higher in the white than in the gray. These
figures indicate very clearly that most, if not all, the cerebrosides are
in the medullary sheaths and that the greater proportion of phosphatides,
myelins, etc., are there also. On the other hard, the proportion of these
substances is very high even in the gray matter, the proteins making
only about 50 per cent. of the total solids, the other 50 per cent. being
extractives and ether-soluble material.

COMPOSITION OF THE SOLIDS OF THE HuMAN BRaIN.

Figures are per cent. of dry matter.*
Whole brain Whole brain

(child) (adult) Corpus callosum
BrOten an: ise: alse tes cevens eee ecesane a ecw aaa a 46.6 37.1 27.1
Extractives .......ccscceccescnsees 12.0 6.7 3.9
TAISIE » 2 osha Socctutealaet wiaedare eters Ga 8.3 4.2 2.4
Phosphatide ..........ccecsecceeee 24.2 27.3 31.0
Cerebrosides: . sisg.62:.ndeae o% aaweess 6.9 13.6 18.0
Lipoid sulphur ...........-..00ees 0.1 0.3 0.5
Cholesterol: escaiss escees seed ess 1.8 10.9 17.1

* Koch: Die Bedeutung der Phosphatide (Lecithane) fiir die lebende Zelle. II.
Mittheilung. Zeitschrift f. physiol. Chemie, 63, p. 432, 1909.

Of the extractives, inosite forming 0.193 per cent. of the gray mat-
ter and 0.217 per cent. of the white appears to be present in larger
amounts in the latter. Lactic acid, on the other hand, is more abundant
in the gray matter.

The specific gravity of the gray matter, 1.041, is somewhat greater
that that of the white, 1.032; the specific gravity of the whole human
brain is about 1.037, but there is some uncertainty about these figures.
Computing from the specific gravity of the whole brain and the specific
gravities of white and gray matter, the relative amounts of these two
kinds of tissues were estimated as about 55 per cent. of white and 45 per
cent. of gray matter. Tables giving a summary of the composition of
the human brain have been published by Thudichum. More accurate
analyses have been made by Koch. The adult human brain weighs
about 1,200-2,000 grams; it contains approximately 78.9 per cent. of
water and 100-120 grams cf dry protein matter after all ether-soluble
and alcohol-soluble matters have been extracted.

Proteins of the brain and nerve tissues.—There is nothing particu-
larly striking or novel about the proteins of the brain which has been
discovered so far, except the presence in the brain proteins, or some
THE MASTER TISSUE OF THE BODY 585

of them, of the normal amino-caproice acid. This is a matter probably
of not very great significance and it is unlikely that this acid is only
found in these proteins. The nervous tissues of frogs contain a globulin
which coagulates at a very low temperature, 35°, and in the brains of
mammals a globulin is present, Halliburton says, which is coagulated: at
about 42°. It is possible that the coagulation of this protein may be of
importance in the pathology of heat stroke. A similar protein is found
in the muscles of frogs, according to von Fiirth.

Nucleic acid. The brain contains relatively little nucleic acid. This
acid, isolated by Levene, had the usual structure of a typical animal
nucleic acid. It yielded phosphoric acid, adenine, guanine or the oxi-
dation products of these bases, hypoxanthine and xanthine. It yielded,
ulso, levulinie acid and hence contained a hexose in the molecule like
the thymus nucleic acid. Nothing is known of the. character of the
protein in the nuclei of the brain cells. The Nissl substance is sup-
posed by some to be a nucleoprotein or a nucleoalbumin, but, since
there is no certain way of distinguishing these bodies microchemically, it
is impossible to be certain that the Nissl substance really contains phos-
phorie acid. The amount of purines isolated from the brain by Burian
and Schur was small in amount, as was to be expected from the rela-
tively small amount of nuclear matter.

Physical consistence of the protoplasm of the nerve cell.—lIt is
very important to determine the physical consistence of the protoplasm
of the ganglion cells. The experiments of Harrison, who cultivated
embryonic nervous tissue under the microscope, showed that the fine ends
of the growing axis cylinders were capable of spontaneous movement.
This observation would indicate that the contents of the tips of the axis
cylinder, at any rate, were liquid in nature and not jelly-like. It was
found, also, by Heger and others in his Institute at Brussels that the fine
terminations of the dendrites in the brain were movable and capable
of retraction. It was suggested as the result of this observation that
the making and breaking of the connections by means of these processes
accounted for the co-ordination in the brain and the psychical disor-
ganization which sometimes occurs. On the other hand, Mott says that
the substance of most ganglion cells is of a jelly-like consistence and
that in the living cell there is no trace of the Nissl substance to be seen.
It is possible that the cell bodies are jelly-like and the tips liquid. The
observations of Kite certainly show that most tissue cells of the higher
animals have a jelly consistence. Mitochondria, probably of a phospho-
lipin nature, occur in nerve cells (Cowdrey).

Cerebro-spinal fluid.—This fluid was collected by Halliburton from
a young woman in whom, owing to some malformation, there was a con-
nection between one nostril and the ventricles of the brain so that the
liquid dropped constantly from one nostril. The amount formed was
586 PHYSIOLOGICAL CHEMISTRY

2.378 ¢.c. in 10 minutes when sitting quietly, but during straining or
when the abdomen was compressed it might rise to 3 and 4 ec. per
10 minutes. Analysis of the fluid showed that it was alkaline in reac-
tion and of the composition:

Waters ais t2.dcclan! ofa Saale mamta pease otihdede 99.004%
Solids.» scaccieaieice scalp avaceye's eieaia ap anes gia wednre 0.966
Organic: ‘solids es63ssscae sc saeeg es aes eee ee 0.118
Inorganic solids .......... cc cece cece eee ees 0.878

This is a true secretion, as is shown. by its low specific gravity, 1.004-
1.007; it contains only a trace of proteid, of a globulin character.
Fibrinogen and albumin are absent; it contains a reducing, non-
fermentable substance which Halliburton thought to be allied to pyro-
eatechin. It is hypertonic to lymph. It is probably formed by the
secretory cells covering the choroid plexus. Its function is unknown.
It is probable that the secretion of the pituitary passes into the cerebro-
spinal fluid.

Developments of the method for obtaining cerebro-spinal fluid and
its growing importance in the diagnosis of disease, particularly of the
nervous system, has led to a considerable increase in our knowledge of
this liquid in the past few years. In normal individuals it is a per-
fectly clear, colorless liquid containing not more than 3 to 5 white cells
perc.mm. When first drawn it is acid to phenol phthalein, like the blood
plasma, but on standing it loses carbon dioxide and becomes alkaline
to that indicator. Its alkalinity is altered in disease and a method of
diagnosis of tuberculous meningitis has been based upon this fact. It
contains always about 0.08 per cent. dextrose, and about 0.02 per cent.
protein. Its composition as determined from the analyses of Mestrezat
is given in the following table. The total amount present in the brain
and spinal cord was estimated at 60 c.c. (Magendie). The pressure is
about 45 to 90 mm. water in children; and in adults, in a sitting position,
200 to 250 (recumbent 130 to 150 mm. water). The specific gravity
varies from 1.001 to 1.006.

COMPOSITION OF CEREBRO-SPINAL FLUID. (Mestrezat.)

Pts. per 1000 Pts. per 1000
Water: casas seas siceoe 996.7 ASH soise'v ca niente ss eee es 8.80
Solids: z.sgce-es2daee-s Ses 10.93 Na,O ia) Bieta eeca Sib eae ts Re 4.35
Organic matter ........ 2.13 KO eee eee eee 0.25
Mineral « wccewedeones 8.80 CBO) sive 3 mane 4 Reked een dye 0.095
Protein sip eccneeiaae 0.18 MgO srncsineaniacg never enn 0.050
Dextrose ...........05- 0.52 Fe,0, datintts we aise a ercanarh to As 0.002
Organic acids ........ 0.30 PLO cece cee e eee ee ees 0.030
Amino acids ......... 0.01 80, (total) sisssgedediraliubsncr desta usa 0.056
Wee: ssaennanruee es eiages 0.06 co, (Of BSH): as 5 diene caiewes 0.550
Ammonia .............. ? ai" eAdasradirecvecteind hiatal den weeare  ahasere 4.448

Chlorid as NaCl........ 7.32 TotalN: x siiass ota veces 0.0196
THE MASTER TISSUE OF THE BODY 587

P,, when first drawn, 7.4 increasing on standing to 8.3. (Tashiro and Levinson)

Alkali reserve 58 to 63. (McClendon)

Non-protein N 17 to 26 mgs. per 100 c.c. (Leopold and Bernhard)
Creatinine 0.7 to 1.5 mgs. per 100 c.c. i
Urea N 7 to 13.5 a =
Dextrose 0.07 to 0.1 per cent. “ *

Viscosity 1.0424-1.0489 (Levinson).

Surface tension 72.0 dynes at 20° C (Polanyi). Freezing point —.56°.
Refractometric index: 1.33516 (Polanyi). It contains no fibrin ferment or fibrinogen;
it has no amylolytic power.

It is clear from these figures that the cerebrospinal fluid is probably
to be considered as a dialysate from the blood (Mestrezat), or as an
ultrafiltrate resembling in its properties the aqueous humor of the eye.
It contains, as will be noticed, very little protein as long as the fluid is
normal, but about the same amount of salt, urea, dextrose, and creatinine
as the blood plasma.

In pathological cases these properties change. The fluid may be
greatly increased in amount and under high pressure. It may contain
many cells. Its protein content increases. By taking advantage of its
change in alkalinity and rate of change of alkalinity on standing Tashiro
and his pupil, Levinson, devised a method for differentiating tuberculous
meningitis from epidemic meningitis. If to 1 ¢.¢c. cerebrospinal fluid
there is added 1 ec. 3 per cent. sulphosalicylic acid; and to another
1 «ec. of fluid a like amount of 1 per cent. HgCl, then in tuberculous
meningitis the protein which settles down on standing after 24 hours
is more voluminous in the mercury tube, whereas in epidemic meningitis
it is more voluminous in the sulfosalicylic tube.

Trimethyl amine is normally present in cerebro-spinal fluid (Dorée
and Golla).

Physiological interpretation of the chemical composition.—In the
brain three fundamental properties of living matter are brought to their
highest perfection: i.e., conduction, irritability and memory. One fune-
tion, on the other hand, is practically in abeyance from an early age:
that is, the function of growth. The brain early reaches its full devel-
opment and thereafter division of the nerve cells does not occur, although
there may be some increase in complexity and growth of the dendritic
processes. We have before us, therefore, in the brain, m as pure a form
as we can find it, a tissue specially built for the functions just men-
tioned, and we may infer from a study of its composition and a com-
parison with those tissues which are specialized for growth, or move-
ment, what kind of protoplasm, or what substances in protoplasm, are
particularly concerned in irritability.

The fact which at once strikes us when we undertake such a study
is that the nerve fibers, which are pre-eminently conducting, are sur-
588 PHYSIOLOGICAL CHEMISTRY

rounded by medullary sheaths which consist chiefly of phospholipins,
glycolipins (cerebrosides) and sulphatides. While it is undoubtedly true
that conduction takes place in the axis cylinder, which is of the nature
of gray matter, yet the gray matter also is unusually rich in these same
constituents, or some of them, such as the phosphatides and cholesterol.
The composition and function of the medullary sheaths. The medul-
lary sheaths consist for the most part of glycolipins (cerebrosides), phos-
pholipins of various kinds and cholesterol. What the function of the
medullary sheath may be is still uncertain. It has been suggested that
it is in the nature of an insulating material which serves to prevent the
spreading of the impulse from one fiber to the other; and, on the other
hand, a nutritive function has been assigned to it. It is probable that
the latter is the true explanation of its function. It may serve as a
source of nutriment for the axis cylinder. The length of the latter is
often so great that it would seem to be very difficult to control its nutri-
tion from the cell body from which it is derived. Another fact which
points in this same direction is that the medullated nerve fibers are
almost indefatigable, that is they recuperate at once from fatigue, and
the medullated nerves are less fatiguable than the non-medullated. The
nerve fibers have been shown by Tashiro to have a very active metabolism,
one of the most active of any tissue in the body, and the fact that they
are not fatigued would indicate that they have a good source of supply
of raw material. In the medullary sheath there is a large amount of
inosite, galactose, fatty acids, various nitrogen bodies, phosphoric acid,
sulphuric acid and of potassium, calcium and sodium. We find these
same materials, or materials of a similar kind, in locations in which a
very active metabolism is about to take place. They seem to constitute
a peculiarly favorable material for the production of some of the active
bodies of the cell. Thus they are found in the yolk of birds’ eggs, or in
the eggs of lower forms of life, like the starfish. In the egg after fer-
tilization there is a sudden outburst of metabolism at the expense of its
stored materials. The composition, then, of the medullary sheath would
lend some support to the view that the function of the sheath is nutritive.
This view of its function has been greatly strengthened by Tashiro’s
recent discovery of the active metabolism of the nerve fiber, particularly
of the non-medullated fiber. The axis cylinder appears to have a
metabolism but little, if at all, less intense than that of the cell body
from which it is derived. There are facts which he has found which
indicate, also, that this keenness of metabolism is correlated with the
rapidity of transmission of the impulse, being more intense in the fibers
which transmit the impulse most rapidly, as in mammalian nerve. It is
obvious that the power of immediate recovery from a stimulus, the
short latent period and rapid transmission are very important in the
functioning of the nervous system. If rapid transmission implies, as
THE MASTER TISSUE OF THE BODY 589

seems probable, a rapid metabolism also, a reserve supply of food readily
assimilable by the fiber to replace at once that used up in the transmis-
sion of the impulse would seem to be necessary. Some mechanism simi-
lar to that of the medullary sheath would seem the most simple and
direct way of solving the problem. The medullary cells on this view
function as a kind of nurse cells, similar to the nurse cells of the eggs
of many invertebrates. The laying down of this reserve substance within
the living matter of the axis cylinder would obviously have many disad-
vantages and would probably reduce the-rate of its metabolism and the
rate of conduction, since that appears to be the result generally of an
accumulation of foreign or lifeless matter in the living protoplast. The
device adopted by the animal economy of having long nerve fibers, very
thin, so that the surface is large in proportion to the bulk, and sur-
rounded by a large number of special nourishing cells, would appear to
be a most satisfactory solution of a very difficult problem which the
organism had to face. If such rapid. conduction were not needed, the
problem might be solved by introducing relays of cells so that the length
of any single axis cylinder was not beyond the powers of nourishment
from the cell body ; but this contrivance is not suitable where very rapid
action is required, as in the brain and in those skeletal muscles by whic
an animal escapes death or secures its food by superior agility; for
at every relay, or sinapse, there is an unavoidable delay in the
rate of transmission of the impulse. In the internal organs where
speed and agility, or indefatigability, are not the prime requisites,
the nerves are still non-medullated for the most part and relays are
used. ‘

A second very striking and interesting fact in the chemistry of the
brain, and one which may perhaps be correlated with the provision of
reserve food in the medullary sheath, is the absence of reserve carbo-
hydrate food in the brain, and thé entire absence of neutral fat. Glyco-
gen, which is found in so many tissues and which occurs in the young,
undifferentiated nervous system, is absent in the adult brain. When
medullation appears it disappears and no other carbohydrate is present
in the adult brain, except inosite and galactose. The function of
inosite is unknown, but it readily combines with phosphoric acid to
make phytin and similar esters. Perhaps it plays the réle of an inter-
mediary substance. Inosite is‘readily destroyed when taken by the
mouth. The absence of neutral fat while the phospholipins and other
compound lipins are so abundant is indeed remarkable.

If the medullary sheath has this function of serving as a nutritive
material, the question at once arises as to the constitution of the real
living matter, the matter which has the properties of conduction, irri-
tability and memory par excellence. How does it differ, for example,
from living matter which is chiefly secretory, or metabolic, or con-
590 PHYSIOLOGICAL CHEMISTRY

tractile? The only facts to guide us in this dark region are analyses
of the gray matter, for although it is impossible to get gray matter
éntirely free from medullary matter, the admixture in some of the
regions of the brain is small. The main facts which may be gathered
are these: The proportion of protein in the gray matter is high. The
proteins make in the gray matter a larger proportion of total solids
than in the white; the proportion of phospholipins is also high. Irri-
table nervous living matter has a larger proportion of water, 85
per cent., than contractible; it contains relatively less protein, though it
is still protein which makes the main part of its solids; it contains a
very large proportion of phospholipins and cholesterol, but no fat or
carbohydrate. The irritable, conducting protoplasm is, therefore, decid-
edly aqueous and the colloids are protein-phospholipin colloids. These
few facts tell us very little about this irritable matter, but so far as
they go they indicate the importance in nervous activity of the chemical
substances just mentioned. It may be significant that this substratum
is of a kind which so readily forms liquid crystals: i.e., a kind whose
molecules are readily oriented.

A third fundamental property of the human cerebrum is memory.
It is possible to make some kind of an impression on the human brain
of so persistent a nature that when made at from 4-6 years of age it
may persist for from forty to eighty years. Of the physical-chemical
basis of memory very little is known. When an impulse strikes into
the brain something remains there, some trace, perhaps an enzyme.
Something happens to the brain with every impulse fed into it. We may
perhaps picture the process as follows in a tentative way, although the
evidence is extremely meager that this picture at all represents the real
process. There is a growing number ,of facts which indicate that when
nerve impulses impinge on cells they do not at once arouse the peculiar
activity of that kind of cell. There is always a latent period. It seems
that under the influence of the nerve impulse a substance is produced
which in its turn acts as the excitant of the cells’ activity. This would
explain the latent period of muscle and other cells. We may regard
the gastric hormone or the secretin of the cells of the intestinal mucosa
as such a substance. In muscle possibly lactic acid is the hormone. By
a hormone is meant a substance which rouses to activity. Now in the
nerve cells it may be that such memory hormones are produced and
accumulate, or persist in the cells. There are only two facts of the
chemistry of the brain, so far as the author sees, which may be brought
into relation with this extraordinary power of memory. One of these
facts is the great reduction of autolytic power of the brain tissue as
compared with that of any other tissue of the body. If the stomach,
the liver, the spleen, the muscles are finely hashed and kept sterile in
water at 37° C., their proteins undergo rapid changes of autolytic diges-
THE MASTER TISSUE OF THE BODY 591

tion. The brain under similar conditions undergoés very little autolysis.
In fact, the autolysis was at first overlooked, it is so slight, a very little
alkali apparently checks it completely. What autolyis there is seems
to occur in the first few hours after death. This great reduction ‘of
the brain’s autolytic power points toward a stability of the brain’s pro-
teins, of an absence of wear and tear in the brain, which possibly, but
only possibly, for in reality the matter is guesswork, but which invol-
untarily recalls the stability of impressions and memories in the brain.
When autolysis does occur, or when nerve cells are destroyed in injury
or by toxins, then memories disappear also, showing that there is some
substantial basis of the memories. The stability of the nerve cells, the
absence of autolysis, of cell division, all would appear to favor the
stability of impressions of whatever kind which are made on the brain
by the events about us. It is possible, also, that this absence of autoly-
sis is correlated with the fact that the brain does not lose weight
in starvation, but lives on those organs which autolyze the most
readily.
’ The second fact of interest, although it may be simply a parallelism
and not fundamentally connected ‘with the process of memory, is the
presence in the cephalin of the brain of very unsaturated acids of the
type of linolinic acid. It has already been pointed out on page 67 that
in the spontaneous oxidation of linolinic acid phenomena closely parallel-
ing memory and learning occur. These are autocatalytic processes. In
the case of linseed oil the memory, if it may be so called, consists in
the persistence in the oil of an intermediary, catalytic oxidation product.
If this substance could in any way be made stable, so that it would per-
sist for a long period, linseed oil once illuminated would remember that
illumination forever and would always respond differently, when
exposed with oxygen to the light, from linseed oil which had not had
the experience of a previous illumination. If it were possible that
an impulse coming into certain cells caused in those cells the formation
of a persistent autocatalytic, intermediary oxidation product, a physical
basis of memory might be given. Our ignorance is so profound,
however, that at present we can hardly do more than guess at this
interesting problem and we must be on our guard against assuming
that the basis of memory is in reality that sketched above. All experi-
ence shows how often we may be misled by seductive parallelisms of
this kind into the conclusion that Nature is working in the manner
we imagine her to be. To quote an old and homely, but true, saying:
““ Nature knows more than one way to kill a cat.’’

Having now considered the general composition of the nervous tis-
sue and the connection between that composition and function, we may
pass to the consideration of the metabolism of the brain. Is the respira-

’
592 PHYSIOLOGICAL CHEMISTRY

tion of the brain keen, or very low? What substances does it use as
foods? What are its waste products?

The first question, that of the respiration or rate of metabolism of
the brain, is very important for the light it will throw on the nature
of the nervous mechanism. If those processes consist, as many have
believed, in the simple dislocation of inorganic ions in the periphery
of the nerve fiber, a dislocation which may be accompanied by a change
of state in the nerve colloids,—if they are, in other words, physical
processes,—one would expect the metabolism of the brain to be small,
very small, for no material would be used up in this process, a temporary
dislocation of the ions alone occurring. The only energy needed is the
jar, or slight explosion, which starts it going, and if this took place in
the sense-organs no energy would be required to be expended in the
brain. If, on the other hand, the nerve impulse is in the nature of a
chemical change taking place with great ease and swiftness, and if the
velocity of conduction is proportional to the respiration, then the
metabolism of the brain, as measured by its respiration, should be
extremely high, higher than that of any other tissue of the body. The
lack of cell division, the stability of the nerve cells, the large amount
of neuroglia, the absence of autolysis, all point to a low formative
metabolism, that is a low protein or growth metabolism after the adult
condition is reached; but, on the other hand, the respiratory metabolism
should be very high, if this is correlated at all with irritability. What,
then, is known of the metabolism of the brain?

A few general facts may at once be noted. First, the blood supply
to the brain is unusually large and every provision is made to supply
the brain with a copious amount of blood. Not only are there the large
direct carotids, external and internal, but there are also the indirect
anastomoses through the vertebrals, which, in dogs at least, are able
alone to maintain the life of the brain. This copious blood supply points
unmistakably to a very vigorous metabolism of some kind which involves
oxygen.

A second well-known fact is that a supply of oxygenated blood is
absolutely necessary to the maintenance of consciousness in human
beings. Consciousness cannot persist more than a few moments with-
out it. Even though the tissues are saturated to their full extent with
oxygen by inhalation of the latter and forced respiration, the supply
cannot maintain consciousness for more than five minutes and, if the
supply of oxygen is low, loss of consciousness comes on easily. Thus
with a low blood pressure,.as in fainting or syncope, the higher centers
of the brain appear to pass out of action at once. Similarly swimmers
under water holding their breath to the utmost sometimes become uncon-
scious, and that it is the oxygen of the blood which is important is shown
by the fact that if that oxygen be replaced in the blood by carbon
THE MASTER TISSUE OF THE BODY 593

monoxide, as in poisoning by illuminating gas, consciousness is lost.
There is no question from this observation that it is only the oxygenated
brain tissue which is irritable and conscious. In the absence of oxygen
the brain loses its function more quickly than any other tissue of the
body. These facts show that brain tissue either has a higher rate of
consumption of oxygen, so that it exhausts its supplies more quickly;
or that the nervous processes involve more oxygen than others; or that
the brain has a smaller supply of stored oxygen than any other tissue
of the body. The great dependence of the brain on atmospheric oxygen
may be correlated, also, with the fact that there is no stored carbo-
hydrate. The presence of stored carbohydrate like glycogen appears
to increase the powers of tissues tc live without atmospheric oxygen.

A third general fact which points unmistakably in the same direc-
tion is the great sensitiveness of the brain to cyanides and other drugs.
Cyanides certainly check respiration and undoubtedly combine with
the cell protoplasm. The brain is most sensitive to cyanides, these drugs
abolishing consciousness very quickly, and particularly the activity of
the highest centers. Since it can be shown that many drugs accumulate
in the nervous system, it must be that the latter has compounds with
chemical bonds in a reactive or open form with which they can unite.
It has been shown, too, that those tissues which are most active in
respiration or have the highest rate of respiratory metabolism are the
most easily affected by drugs like the ecyanides. Hence, the sensitiveness
of the brain to drugs also indicates clearly a higher rate of metabolism
in the brain than in other tissues.

The persistence of the weight of the brain during starvation means
either that the rate of formation of its substances must be very high or
the rate of consumption extraordinarily low, since it is always the
case that the more rapidly metabolizing tissue draws for its raw mate-
rial upon the tissue having the less intense metabolism.

The blood coming from the brain also indicates metabolic changes
in that organ. It is venous blood. Oxygen is consumed there and in
large amount. That means that substances in the brain have been oxi-
dized or burned, hence they must be constantly replenished or they
would be used up. The blood coming from the brain has less sugar
in it than in the arterial blood going to the brain. Evidently the brain
consumes sugar. The blood of the jugular vein also contains less
fibrinogen than the arterial blood of the carotid artery. But the inter-
pretation of this fact is uncertain.

Direct determinations of the rate of respiration of the nerve fibers
have now been made by Tashiro, who found indeed that the amount
of carbon dioxide given off from the nerve fiber per gram of substance
in 10 minutes was very high, higher than that of any other tissue exam-
ined. Thus the resting non-medullated claw nerve of the spider crab
c04 PHYSIOLOGICAL CHEMISTRY

at 19° gave 6.7X10-7 grams CO, per eg. per 10 minutes; the frog’s
sciatic nerve, a medullated nerve, gave 5.5X10—* grams CO, per cg.
per 10 minutes, and the rate was about doubled when the nerves were
stimulated by a weak electrical current. This rate of metabolism was
probably somewhat larger in the cut than in the uninjured nerve, owing
to the effect of the injury, but the amount is very high, higher than for
the frog as a whole when equal weights are compared. The examina-
tion of the various parts of the nerve shows, also, that there is a gradient
in its metabolism, the part of the nerve fiber nearer the source of the
normal stimulus having a higher rate of metabolism than that farther
from this source. Ganglia, too, seem to have a somewhat higher rate
of metabolism than the nerve fibers, but the difference is not very great.
Evidently the nucleus of the cell is not so important in respiration as
had been thought. There is no doubt from these observations that nerve
cells and nerve fibers produce carbon dioxide at a very rapid rate. If
equal weights of nervous and other tissues are compared, the nervous
tissues are found to give off more carbon dioxide per gram of tissue
than any other tissues.

The examination of the oxygen consumption of the brain has been
made by the method of Barcroft, which consists in estimating the amount
of oxygen in the blood going to and coming away from the brain. The
results of this study were as follows (Alexander and Cserna) :

By comparing the oxygen content in the arterial blood with that of
the venous blood of the sulcus longitudinalis and measuring the velocity
of flow, it was found that in the waking animal the brain consumed
0.360 ¢c.c. oxygen per gram per minute. This amount is enormously
greater than that of skeletal muscle of 0.004 e.c. and of the salivary
glands of 0.028 ¢.c. per gram per minute. In narcosis by ether the
consumption sank in the mean 77 per cent.; the CO, output 59 per
cent. In morphine narcosis the oxygen consumption sank 57 per cent.;
the CO, output 61 per cent. Under ether the O, consumption could be
reduced still farther. The first effect of the anesthetic is, however, to
increase the consumption of oxygen. The initial action might increase
the oxygen consumption 150 per cent.; the CO, production 359 per cent.
This increased consumption of oxygen corresponds to the preliminary
stimulation which is the first effect of the anesthetic. Tashiro and Adams
found in the case of the nerve fiber and other cells that the first effect of
other anesthetics was to increase the CO, production concomitant with the
excitatory action, and that the second effect was to reduce the CO,
output. By injection of CaCl,, which aroused the brain from anesthesia
by MgSO,, the metabolism rose again to normal amounts. It is clear,
then, that there is in the brain, as in the nerve fiber, a complete or wide-
reaching parallelism between metabolism and the state of irritability.
‘hese results completely confirm the view of the chemical nature of
THE MASTER TISSUE OF THE BODY 595

anesthesia. They indicate clearly the conclusion that conduction and
irritability are at bottom chemical processes, rather than physical, as
they are sometimes pictured.

The rate of consumption of oxygen by nerve fibers has not yet been
made so accurately and directly as the rate of carbon dioxide produc-
tion, as the methods are badly in need of improvement. But there is no
doubt from indirect evidence that the irritability of the nerve fiber is
dependent upon a supply of oxygen and that it fatigues more rapidly
in the absence of oxygen than when it is present. Thus, if a frog’s
sciatic nerve is stimulated in hydrogen or nitrogen, it becomes non-
irritable more quickly than in oxygen and it recovers if returned to
oxygen. The oxidavion appears, however, to be in part independent
of the oxygen of the atmosphere, or else there is some sort of storage
of oxygen in the nerve, as the irritability of frogs’ nerves will persist
for some hours, although diminished in amount, in the absence of
oxygen. The carbon-dioxide production does not appear to be due to '
a direct oxidation by the oxygen of the air. It may be that the oxygen
in an available form is stored in some kind of a loose compound in the
medullary shvath. Whether the nerve stores oxygen or not must be left
for further investigation to decide. The carbon dioxide might be formed
in various ways, either by carboxylase from the proteins or amino-acids,
or by fermentation from the carbohydrates, or by indirect oxidation of
the fatty acids, or by the decomposition of an unstable peroxide.

The attempt has also been made to get some direct measure of the
metabolism of the brain in human beings by the use of the calorimeter,
but so far without results. The brain in human adults weighs between
1,200 and 2,000 grams; it makes, therefore, between 2 per cent. and 8
per cent. of the weight of the body. On the other hand, the muscles
make 42 per cent. of the body weight. If the brain could be brought
into a condition of complete rest, the maximum change of metabolism
of the body produced thereby would be only two per cent. But it is
impossible to make the brain rest. The centers are always more or less
active even during sleep, so that the difference in the metabolism to be
expected would only be a per cent. or so of the total. Impulses are
always coming into the brain over the sensory nerves from the skin
or sense-organs or from the tendons and muscles. No difference in heat
output, or in respiration, or metabolism could be detected by Benedict
as a result of active intellectual work. There is, however, one clear and
unmistakable evidence that intellectual work involves metabolism, pre-
sumably respiratory activity of the brain tissue: namely, keen intel-
lectual work always causes a turgidity of the head and brain; blood
increases in the head when one is working, as is evidenced by the flushed
face, and if it does not increase keen intellectual work is impossible.
In its absence one becomes sleepy, pale, and thoughts are sluggish, rela-
596 PHYSIOLOGICAL CHEMISTRY

tions are not pcrceived. No change can be detected, so far, in the com-
position of the urine as a result of brain work. There is, in fact, no
product of brain metabolism, that is, no substance whose presence indi-
cates brain metabolism, which has been found as yet in the urine.
Changes in the metabolism of the master tissue of the body cannot, there-
fore, as yet, be determined by any examination of the excretions of the
body. It is, hence, impossible to say whether any drug, preservative or
food substance has acted harmfully or beneficially on the brain or nerv-
ous system by an examination of the urine or excretions.

One of the most striking facts about the chemistry of the brain is
the very large amount of phosphoric acid it contains. This fact was
discovered very early, since in the ashing of the brain substance some
of the acid is reduced and phosphorus formed. It has become so much
a matter of common knowledge as almost to lose its significance. This
phosphoric acid is in part free, as the salts of phosphoric acid; in part

_ 15 is in combination in the nucleic acid and in the phosphoproteins; and
in larger part it is present in the phospholipins. Liebreich crystallized
the impression of the importance of phosphorus in the metabolism of
the brain in the saying: ‘‘ Ohne Phosphor keine Gedanken ’’ (Without
phosphorus no thoughts). It seems that phosphoric acid plays a highly
important, though still obscure, réle in the metabolism of all living mat-
ter. Not only do we find compounds of phosphorus most common in
the protoplasm of the brain, and its importance was emphasized by
‘Thudichum by the selection of the word phosphatide to indicate that
the other radicles were grouped around it, but phosphorus occurs in
large amounts in the nucleus and in phytin; it helps regulate the cell
reaction and, combined with calcium and some organic radicles, it forms
the basis of many structures like the bones; it has, besides, some peculiar
relation to the alcoholic fermentation of sugars and other fermentations.
Calcium metaphosphate may not be at the basis of life, but the impor-
tance of phosphoric acid is not to be denied. |

Summary.—We may summarize the contents of this chapter very
briefly, as follows:

In its chemical composition the human adult brain is characterize:
by the very large amount of alcohol-ether-soluble material in its solids:
by its vigorous respiration; by its low rate of autolysis; by its lack of
formative or growth metabolism. Of the solids the alcohol-ether-soluble
material consists of a variety of phosphatides, or phospholipins, such as
lecithin, kephalin, myelin, sphingomyelin, amino-myelin, paramyelin; of
glycolipins or cerebrosides, such as phrenosin, kerasin, cerebron, homo-
cerebrin, cerebric acids; of sulphatides; of cholesterol and phrenosterol,
and of nitrogenous fats or amino-lipins (amino-lipotides). The brain
contains no neutral fat. These substances are found chiefly, but not
THE MASTER TISSUE OF THE BODY 597

exclusively, in the medullary sheaths of the nerve fibers. Their propor-
tion in the gray matter is also high. The protein material, or neuro-
plastin, contains only a small proportion of nucleic acid, but differs from
other proteins in that it contains a normal amino-caproic acid or glyco-
leucine. The proteins of the brain have not in other respects been
shown to be different from those of other tissues, although they are no
doubt specific to the tissue. It has not yet been possible to examine the
neurokeratin separately from the other proteins of the brain. There are
also present extractives, among them inosite, lactic, succinic and formic
acids, creatine, tyrosine, glyco-leucine, hypoxanthine and other ni-
trogenous bases of an unknown nature. On the whole, the brain ex-
tractives are very similar to those of muscle. Of the functions of these
bodies nothing is known. Inorganic salts are also present.

The function of the medullary sheath is probably nutritive. The
really active protoplasm of the nerve cell and body is characterized by
its large content of water, at least 83 per cent. and probably more, by
the absence of stored glycogen and neutral fat, and by the presence of
large amounts of phospholipins and cholesterol. While the growth or
formative metabolism of the brain is low, it has a very keen respiration
and oxidative metabolism, the keenest of all tissues of the body, and
the metabolism of the nerve fiber does not differ very much from that
of the nerve ganglia in its intensity. The activity of the brain and
nerve tissues in general is very dependent upon oxygen and they pro-
duce large amounts of carbon dioxide, the amount of which runs in
general parallel to the state of activity of the tissue. It is probably its
rapid and keen metabolism, which means that it has many reactive bonds
in it, which makes the nervous tissue so very sensitive to drugs of all
kinds.

We may infer tentatively from these facts that the lipins probably
play a very important part in cell irritability without at this time ven-
turing to define more in particular just the réle they are playing. The
phenomena of conduction appear to be closely correlated with the
respiration of the tissue and are, hence, probably chemical in nature.
More work along these lines is, however, necessary before any definite
conclusion as to the nature of the nerve impulse can be drawn. At the
present time it appears possible that it may be in the nature of an
explosive decomposition of an organic oxide or peroxide which is propa-
gated along the fiber. There are difficulties in the way of this view,
one of them being the fact that the nerve seems to liberate no heat
at the time it conducts. The chemical composition has so far thrown
very little light on the chemical basis of memory, but it is suggested
that possibly the presence in the brain of highly unsaturated acids of
the linolinic-acid type may possibly be in some relation to this phe-
nomenon, since in its oxidation this acid shows some of the phenomeng
598 PHYSIOLOGICAL CHEMISTRY

we are accustomed to call memory, teaching and forgetting when they
occur in living things.

We may close this chapter in no better way than in opening the
question of the origin of the psychic qualities which are so related to
the nervous system. Do these qualities arise de novo in the nervous
system? Are they not found in their faintest form way down the slope
of animal life? Do we not indeed see the beginnings of psychic life
among the plants? And is it possible to start with the plants? Do not
the foods every minute change into living matter in our bodies? Are
not the atoms the same in the foods and living matter, and is it pos-
sible that they have different properties in the living and lifeless form?
The atoms we now know are composed of electricity and the valences, or
chemical bonds, are probably also electrical in nature. Are our thoughts
also at the bottom electrical? Whenever a nerve impulse sweeps over
a nerve it is accompanied by an electrical disturbance, and this dis-
turbance is the surest sign of life. When the nerve impulses play back
and forth over the commissures of the brain they are accompanied by this
pale lightning of the negative variation. Is that pale lightning what we
recognize as consciousness in ourselves? It would seem that there must
be some psychic element in every electron if the atoms are made of
electrons. There must be some psychic disturbance in every union of
hydrogen and oxygen to make water and in every wave of the wireless
telegraph. When an electron moves it generates a magnetic field; does
it also generate a psychic field? How shall we escape the conclusion that
there must be a psychic element in all matter both living and lifeless,
since that matter is the same in the two forms? May it not be that just
as magnetism, which is probably an attribute of all molecules, becomes
most evident under certain conditions in certain substances, so the
psychic life common to all matter shows its true character plainly only
when organized as it is in the brain during its life? A magnet when
heated loses its magnetism as surely as an organism when heated loses
its vitality and its psychic life. In the case of magnetism all that has
happened by the heating is that the orientation of the molecules has been
changed so that the magnet is no longer an individual; in the case of the
organism a similar loss of individuality results.

REFERENCES. Braln.

1. Thudichum: The chemical constitution of the brain based throughout on
original researches. London, Balliere, 1884. German translation, 1901.
Die chemische Constitution des Gehirns des Menschen u. der Tiere.
Tiibingen, 1901.

2. Rosenheim: Purification of kerasin and phrenosin. Biochemical Journal, 8, 1914,
p: 110.

3. Dorée and Golla: Trimethyl amine in cerebro-spinal fiuid. Biochem. Jour., 5
1911, p. 306.

4, Koch: Zur Kenntnis der Schwefelverbindung des Nervensystems. Zeits. f. phys.
Chem., 53, p. 486, 1907.
10.

11.
12.

13.
14,
15.
16.
17.
18.
19.

20.
21.
22,
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.

35.
36.

THE MASTER TISSUE OF THE BODY 509

Simon: Autolysis of brain. Zeits. f. physiol. Chem., 72, p. 463, 1911.
Kutscher and Lohmann: Autolysis of brain. Ibid., 39, p. 313, 1903.
Pighim: Indophenol oxidase in central nervous system. Biochem. Zeitschrift,
42, 1912, p. 124.
Barbieri: Catalase in cerebro-spinal fluid. Biochem. Zeits., 42, 1912, p. 138.
Nizzi: Absence of esterase and lecithase in nervous tissues. Biochem. Zeits., 42,
1912, p. 145.
Masuda: Cholesterol and fatty acid content of different parts of brain
Biochem. Zcits., 29, 1910, p. 161.
Alexander: Respiration of brain. Biochem. Zeits., 44, p. 127, 1912.
Loeming and Thierfelder: Cerebrosides. Zeits. f. physiol. Chem., 74, p. 282.
1911.
Batelli and Stern: Peroxidase of brain. Biochem. Zeits., 13, p. 44, 1908.
Levene and Jacobs: Sphingosine. Jour. Biol. Chem., 11, p. 545, 1912.
Baumann: Cephalin; nature of base in. Biochem. Zeits., 54. 1913, p. 30.
MacArthur: Cephalin. Constitution. Jour. Amer. Chem. Soc., 1914.
Abderhalden: Normal leucine in brain. Zeits. f. physiol. Chem., 84, 1913, p. 39.
Frank: Cephalin in liver. Biochem. Zeits., 50, 1913, p. 273.
Schreiber and Lenard: Oxycholesterol in brain. Biochem. Zeits., 49, p. 458,
1913.
Kaufmann: No free choline in ox brain. Zeits. f. physiol. Chem., 74, 1911, p.
175.
Hannemann: Inhibitory influence of the brain on body metabolism. Biochem.
Ztschr., 53, p. 80, 1913.
Abderhalden and Weil: Amino-acids by hydrolysis of gray and white matter ot
brain. Zeits. f. physiol. Chem., 83, p. 425, 1913.
Levene and Jacobs : Cerebrosides of brain. Jour. Biol. Chem., 12, 1912. p. 389.
Levene and Jacobs: Cerebroniec acid. Jour. Biol. Chem., 12, p. 381, 1912.
Takaki: Tetanus binding constituents of brain. Hofmeister’s Beitrage, 11.
p. 288, 1907-8.
Halliburton and Dixson: Choroid plexus and secretion of cerebro-spinal fluid.
Jour. Physiol., 40, 1910, p. xxx, Pro. Phys. Soc.
Koch and Koch: Chemical differentiation of the brain. Jour. Biol. Chem., 15.
p. 423, 1913.
Koch, M.: Brain of the albino rat at birth compared with that of fetal pig.
Jour. Biol. Chem., 14, p. 267, 1913.
Tashiro and Levinson: Journal of Infectious Diseases, XXI, 1917, p. 571.
Levinson: Cerebrospinal fluid. St. Louis, 1919.
Mestrezat: Bull. soc. chim., 9, pp. 683-8, 1911.
Mestrezat: Journal de physiol., 14, p. 504, 1913.
Buitendijk: Consumption of oxygen and action of anesthetics. Proc. Am
sterdam Acad., 13, p. 577, 1913.
McClendon: Cerebrospinal fluid. Jour. Am. Med. Assn., 70, 1918, p. 977.
Polényi: Hydrocephalus fluid. Biochem. Zeitschrift, 34, 1911, p. 205.
Leopold and Bernhard: Amer. Jour. Diseases of Children, 13, p. 34, 1917.
CHAPTER XIV.
THE CONTRACTILE TISSUES. MUSCLE.

Amount.—Muscle forms the greater proportion of the living tissues
of the body. In an adult the weight of the muscle is about 42 per cent
of the body weight. Of the whole metabolism during muscular rest
about 50 per cent. is the metabolism of muscle; and during muscular
activity probably nearly three-fourths of the chemical changes occur-
ring are taking place in the muscle. These figures will make it clear
that the muscles dominate, as it were, the metabolism of the body and
their composition and the nature of the chemical changes taking place
in them become of the first importance.

By far the greater proportion of the muscle is under the control
of the will and is called voluntary muscle; but a smaller portion is not
so controlled and is found in the heart, along the arteries, in the
walls of the alimentary canal, in the uterus, bladder and other organs.
This latter is involuntary muscle. These two kinds of muscle differ
in their physiological properties. Thus the voluntary has a short latent
period, it contracts and relaxes very quickly, whereas the involuntary
muscle contracts much more slowly. They differ also in their optical
appearance, in their structure and in their chemical composition. Heart
muscle in all its properties takes an intermediate position between the
other two. Thus voluntary muscle appears optically to be composed
of alternate lighter and darker bands of matter. It is said to be cross-
striated; its nuclear material is relatively small in proportion to the
whole bulk; it contains a very high proportion of proteins and extract-
ives and relatively little water. Involuntary muscle has relatively more
nuclear material and it is not cross-striated, so that it is called smooth
muscle. It is very faintly striated longitudinally in fixed preparations.
All muscle begins development as smooth muscle. Cross-striated muscle
may be regarded as smooth muscle which has been differentiated into
a special structure, probably securing thereby greater speed of con-
traction. Having less nuclear material, it is possible that the striated
material has a lower rate of nitrogen metabolism than the unstriated.

Structure.—A striated muscle consists of bundles of muscle fibers
united or bound together by connective tissue. The amount of this
connective tissue varies in different muscles and the more there is of
it the tougher is the muscle. The connective tissue often contains

600
THE CONTRACTILE TISSUES. MUSCLE 601

deposits of fat. The blood vessels running in the connective tissues carry
a large supply of blood to the muscles, the capillaries branching about
the muscle fibers in such a way that they are in intimate contact with
them. This arrangement brings directly to the muscle fibers a copious
supply of oxygen and food and provides for the rapid elimination of
wastes. During activity the arterioles dilate, owing in part to the
action of chemical substances formed by the muscle, and a more than
usually copious supply of blood is provided thereby. The nerves of
muscle are both motor and sensory. The sensory are distributed at least
in part to the tendon, but the motor fibers branch freely and a branch
penetrates each muscle fiber, piercing the sarcolemma (the membrane
surrounding the fiber) and ending in a swelling consisting of nuclei and
granular protoplasm called the muscle plate and situated about the
middle of the fiber. While the nerve fiber thus enters into a very inti-
mate connection with the muscle substance so that nerve impulses
pass freely to the latter and profoundly influence its metabolism,
the nerve fibers are none the less nourished from the cells which
originate them and they degenerate if they are cut off from these
cells.

‘The muscle fiber itself is the real living, contractile part of the muscle.
Such a fiber is an elongated, spindle-shaped cell. The structure differs
in different kinds of voluntary muscle, appearing to have reached the
highest perfection and differentiation in the wing muscles of insects in
which the power of rapid contraction and relaxation is most developed,
the cross-striation and fibrillar arrangement of the contents being most
marked ; in mammalian muscle, also, it is highly differentiated, but in
the amphibia, and in particular in the very low vertebrate, Ammocetes,
the fibers are of a more primitive structure and their cellular character
is more plainly seen, the differentiation not being complete. In these
latter the nuclei are arranged down the center of the fiber, surrounded
by a larger or smaller part of undifferentiated protoplasm; but in mam-
malian and insect muscle the nuclei, of which there are several, are
distributed through the contractile protoplasm and many lie close under
the sarcolemma.

The exact structure of the differentiated contractile substance is still
uncertain. It certainly consists of alternate disks or bands of different
optical and staining properties. One of these disks is doubly refracting
and more dense than the other and it stains more deeply in various stains
like iron hematoxylin. In many muscles, and in particular in the insect
muscle, these alternate disks appear to be arranged in fibrils which are
called sarcostyles. There seems to be no doubt that in these muscles,
at any rate, such a fibrillar arrangement really exists. Their exact
structure in living muscle is still a matter of controversy. There are,
602 PHYSIOLOGICAL CHEMISTRY

besides, in the living muscle granules, some of which may be glycogen.
It is generally supposed that there is also about the fibrils a more liquid
portion called the sarcoplasm, but the existence of this is very uncer-
tain. Some observers believe that there are networks of an elastic
material extending in a regular pattern through the muscle and con-
tributing to its relaxation. Such networks can be made out in the fixed
muscle, but their existence in the living muscle has been disputed. Kite
found that the resistance to the movement of a needle in the muscle of
Necturus was about the same in all-directions, and Kiihne and Eberth
observed in certain muscle fibers of frogs a minute parasitic nematode
worm which moved through the content of the muscle fiber as if it were
fluid.

The physical consistence of the contents of muscle fibers has been
earefully examined by Kite by his very ingenious method of micro-
scopical dissection. By means of very fine glass needles carried in a
mechanical holder he cut the large muscle fibers of Necturus into pieces.
He found the inside of the fiber, the protoplasm, the most ‘‘ viscous,
elastic and cohesive of the living gels examined.’’ ‘‘ The muscle sub-
stance sticks to a glass needle and can be drawn out into extraordinarily
long threads, which when released almost regain their previous shape.
The absorptive powers and turgidity of this substance are compara-
tively high.’’ ‘‘ When the whole or a piece of 2 muscle cell is stretched
the striations become faint or disappear, only to reappear when the ten-
sion is removed.’’ ‘‘ If the point of a needle be pushed into a muscle
cell, it can be moved in one direction about as easily as another.’’ He
goes on to say, ‘‘ The optical appearance of a striped muscle is very
misleading. Dissection has shown that the dark bands seen in living
muscle are produced by concentrated areas of muscle substance, which
absorb enough transmitted light of low intensity to appear as dark
bands in the optical image. No fibrils could be dissected out. The sub-
stance lying between the concentrated regions and appearing as light
bands is a highly viscous elastic gel, and has no physical properties that
serve to distinguish it from the surrounding protoplasmic gel.’’ ‘‘ Hence
absorption, diffraction, refraction and dispersion are involved in the for-
mation of the optical image of striped muscle.’? The sarcolemma is ex-
tremely elastic. It is evident from this description of Kite that the muscle
substance consists at least in part of alternate disks of more and less con-
centrated gel. It is, however, unlikely that this gel would split into
the very regular and characteristic fibrils of the muscle of the water
beetle as described by Schaefer if there was not in these latter, at any
rate, a well-marked fibrillar arrangement into sarcostyles. The muscles
of Necturus are possibly less differentiated than those of the insects.
There is no doubt, however, that in all these cases the substance is
THE CONTRACTILE TISSUES. MUSCLE 603

highly elastic, and of a jelly consistence; it is probably not fluid as it
was once supposed to be.

General composition.—Both striated and unstriated, mammalian
muscle contains, in the adult, from 72-78 per cent. of water, and 22-23
per cent. of solids. In youth and in the fetus the per cent. of water is
higher. The solids are very largely protein in nature, and in this re-
spect the muscle shows a striking contrast to the nervous system in
which the lipins hold so prominent a place. The total ether extract,
lipin of all kinds, of the dog’s heart is between 2.86-3.73 per cent.
(Lederer and Stotte). The glycogen of the dog’s heart is between 0.709
and 0.576 per cent. In the skeletal muscle it may be a good deal less
than this, namely, 0.15 per cent. -0.3 per cent; 18-20 per cent. by weight
of the muscle substance is, therefore, protein. The composition of ox
meat, after extraction of the glycogen and fat, was found by Argutinsky
to be as follows: C, 49.6 per cent. ; H, 6.9 per cent.; N, 15.3 per cent.; Ash,
5.2 per cent.; O and S, 23.0 per cent. The extractives of muscle are also
important. The proteins may be first considered. They are generally
divided into two groups, the proteins of the muscle plasma and the pro-
teins of the stroma. Part of the proteins are soluble in ammonium
chloride solution. This protein is sometimes called myosin. In human
muscle Danilewski found of 21.48 per cent. total solids, 3.68 per cent.
extracted as myosin and 11.90 per cent. remaining insoluble as protein
of the stroma. In most animals a somewhat larger per cent. of the
protein may be extracted by ammonium chloride than this. In calves’
muscle 15.6 per cent. was insoluble protein, 3.6 per cent. soluble. The
composition of the muscle is strikingly different from that of the elec-
trical organ which is formed from muscle. In the torpedo the electrical
organ contains only about 8 per cent. of protein and over 90 per cent. of
water. The development of a large amount of protein substance in mus-
cle appears to be correlated, therefore, with the function of contraction.

Proteins of muscle.—The proteins of muscle are generally divided
into two groups, those of the muscle plasma and of the muscle stroma.
If a muscle is ground or hashed and then pressed in a hydraulic press,
with very high pressures, 250 to 300 atmospheres, there is squeezed out
a thick fluid, the plasma, which has the property of clotting spontane-
ously on standing. The amount of the plasma, which may thus be
obtained from most muscles when they are living and subjected to the
high pressures just mentioned, is about 60 per cent. of the weight of the
muscle. The plasma has often a faint red color. After it clots the
clot contracts somewhat just as does the blood clot, though not to the
same degree, and the liquid which is thus expressed from the clot is
called the serum. Muscle plasma may also be obtained by a method
used first by the physiologist Ktihne. The muscle is frozen, shaved
604 PHYSIOLOGICAL CHEMISTRY

fine in a knife machine or microtome, rubbed with sand while still cold
and then pressed under high pressure. The part which remains behind
in the press is called the stroma. Of course the proteins may be ob-
tained directly from the muscle without this preliminary expression of
the muscle juice or plasma, by the use of appropriate solvents. Thus
either 5 per cent. sodium chloride or 5 per cent. ammonium chloride or
magnesium sulphate may be applied directly to the ground-up fresh
muscle. By this means a portion of the proteins go into solution and
these are supposed to come from the muscle plasma. The stroma, or
tho insoluble portion of the muscle, is insoluble in all neutral salt solu-
tions and can only be dissolved by the action of strong acids and alkalies.
It behaves like coagulated protein.

Proteins of the muscle plasma.—There is a great deal of uncer-
tainty and contradiction in the literature in the account of the
muscle proteins and much confusion.in nomenclature. In these cir-
cumstances we shall follow the account of von Fiirth, who was given a
great deal of study to this subject. It should be stated at the outset,
however, that the conclusions of von Fiirth are not accepted by all and
have been contradicted in many important points recently. The diffi-
culty arises from the uncertainty whether the change of a soluble to an
insoluble protein involves a change in chemical composition, or is only
a change in the state of aggregation. According to von Fiirth there
are three coagulable proteins, albumins or globulins in the narrow sense,
in muscle plasma; these three are myogen, myosin and soluble myogen
fibrin.

Myosin. This is the musculin of the older observers, or para-
myosinogen of Halliburton. It is a globulin-like substance coagulating
on quick heating between 44 and 50°, and it is characterized by its
power of spontaneously coagulating on long standing, when it passes
into an insoluble protein called myosin fibrin. Myosin is soluble in
neutral salt solutions, is precipitated by dialysis and by water; is easily
salted out of solution by one-half saturation with (NH,),S0O,, which
permits its separation from myogen; and it is easily precipitated by
acids. Botk in salt solution and when standing in water it becomes
insoluble (myosin fibrin). Its per cent. of composition is as follows:
(Kiihne and Chittenden).

C, 52.79; H, 7.12; N, 16.86; S, 1.26; O, 22.97.

Myogen. This is another protein constituent of muscle plasma.
It was formerly called, by Kiihne, myosinogen, but this name von Fiirth
thinks should be dropped, since it implies.that it gives rise to myosin
and this is incorrect. There is about three to four times as much myogen
in muscle plasma as myosin. Unlike myosin it is not precipitated by
THE CONTRACTILE TISSUES. MUSCLE 605

dialysis with water, nor by one-half saturation with ammonium sulphate.
Thus it belongs to the class of albumins rather than to the globulins.
It is precipitated by dilute acids like a globulin. The peculiar property
which differentiates it from other proteins except myosin is its power
of spontaneous coagulation. "When in solution it passes first into a
soluble metastable form called soluble myogen fibrin, and then into
myogen fibrin. Some consider this simply a change in the state of ag-
gregation of the same protein, the protein in solution passing slowly
into an aggregated state. According to von Fiirth, then, the spon
taneous coagulation of muscle plasma takes place according to the fol-
lowing scheme:

_ Muscle plasma
Myosin Myogen

| Soluble myogen fibrin

Myosin fibrin Myogen fibrin.

It is not impossible that this slow coagulation is due to the gradual
hydrolytic decomposition of a salt of the protein, forming an insoluble
free protein, but of the nature of the change involved nothing is cer-
tainly known. Halliburton believed that it was caused by an enzyme
analogous to the fibrin ferment, but this view has not been accepted by
von Fiirth. The myosin fibrin and myogen fibrin are insoluble proteins
behaving like coagulated proteins. They cannot be turned back into
myosin and myogen. The change is not reversible.

Soluble myogen fibrin. This substance is easily detectable, either
in solution or when present in muscle itself, by its extremely low tem-
perature of coagulation. It coagulates at 30-40°, so that when soluble
myogen fibrin is formed from myogen there is a fall in the temperature
of coagulation of the solution from 55-60° to 30-40°. According to Bo-
tazzi, this apparent coagulation of the myogen is due to the heating has-
tening the spontaneous change of myogen to myogen fibrin, or in other
words it is not a true coagulation, but the hastening of the process of
aggregation already going on. The difficulty in this explanation is that
it does not explain the change in the state of aggregation. According to
von Fiirth soluble myogen fibrin behaves like a globulin. It is precipitated
by half saturation with ammonium sulphate or by dialysis, and is in-
soluble in water. It differs from myosin in its lower coagulation tem-
perature. Stewart and Sollman believed that myosin and soluble myo.
gen fibrin were identical, but von Fiirth says that this is not so, for it is
impossible to change a pure solution of myosin to soluble myogen
fibrin coagulating 10° lower. Furthermore in frog’s muscle both soluble
myogen fibrin and myosin co-exist even in the living state. but in warm.
606 PHYSIOLOGICAL CHEMISTRY

blooded animals no soluble myogen fibrin exists in the muscle, for did
it exist, it would be coagulated by the heat of the body. The ease of
transformation of myogen into soluble myogen fibrin is thus described:
‘* Tf one adds to a pure freshly prepared myogen solution some per
cent. of a neutral salt and leaves it standing overnight, in the morning,
although it shows no optical change to the eye, yet already a large
portion of the myogen has been changed to soluble myogen fibrin.’’

Myoproteid. This is a non-coagulable protein found in the muscle
plasma of the fish. It is not present in the muscle plasma of mammals.
Its nature is still undetermined, but it is not an albumose.

Of these various proteins, myosin and myogen are found in all ver-
tebrate voluntary muscles. Myogen has not been found in invertebrate
muscle. Soluble myogen fibrin is found preformed only in the muscles
of fishes and amphibia; while in reptiles, birds and mammals it occurs
there only as a secondary decomposition product of the myogen. Myo-
proteid is found only in the muscles of fish in large amount, in the
amphibia it is present only in traces and in reptiles, birds and mammals
it is absent. In heart muscle there is twice as much myogen as myosin.

Proteins of the stroma.—The greater part of the proteins of the
muscle are insoluble in ammonium chloride solution. These are the pro-
teins of the sarcolemma and the stroma. It is stated that the structure
of the muscle is not changed by the extraction of the myosin; the
stroma is still doubly refracting. The amount of stroma insoluble in
ammonium chloride is very hard to determine with any accuracy, owing
to the tendency of the muscle proteins to become insoluble. From per-
fectly fresh living mammalian skeletal muscle it is said (Saxl) that
84-90 per cent. may be extracted by NH,Cl, if rigor is prevented;
whereas, when the muscle has stood for a short time only, 2-3 per cent.
is extractable. According to Salkowski, of the nitrogen of flesh 77.4 per
cent. was in the form of insoluble coagulable protein and 10.08 per
cent. in the soluble coagulable form. 12.52 per cent. was non-protein
nitrogen.

The stroma contains phosphorus. It contains a small amount of
nucleoprotein. Two grams were obtained from 543 grams of dog
muscle and this nucleoprotein contained 0.7 per cent. of phosphorus.
Corresponding with this small amount of nuclear matter, it was found
that the amount of purine bodies obtained by the hydrolysis of muscle
was very much less than from the thymus and other organs. The result
clearly indicates that the nucleus contains the nucleoproteins and that
these are confined to the nucleus. It is possible that a nucleoprotein con-
taining inosinie acid may be found in the cytoplasm, but the source of
inosinie acid in the cell is uncertain.

The stroma contains also phospholipins (phosphatides) apparently
THE CONTRACTILE TISSUES. MUSCLE 607

in union with the proteins, although this cannot be said to be definitely
proved. The phospholipins, which are present, are discussed on
page 611. The protein matter of the stroma partakes of the character of
coagulated or insoluble protein, myosin fibrin and myogen fibrin or
albuminoid. It is either an albuminoid or metaprotein. It is partially
digestible in pepsin hydrochloric acid and, if so treated and the extract
neutralized with sodium carbonate, a precipitate called myostromin,
containing phosphorus and probably a nucleoprotein or lecithoprotein,
was obtained.

From the fact that the stroma retains its optical and structural
properties after the extraction of some of the myosin Danielewski sup-
posed that the stroma consisted of a doubly refracting network or
spongy substance in the interstices of which the myosin was in solution.
It seems probable from the general phenomena of the squeezing out
from gels on contraction of the latter by the process called syneresis, of
a solution containing the same matter as the gel, but in different con-
centration, that the plasma may, in reality, contain the same proteins
as the stroma, but in more dilute form. The myosin is itself, when in
strong solution or partially dried, doubly refracting like the stroma
(liquid erystals). The fact of double refraction would indicate that
the molecules of protein in the muscle are not arranged haphazard, but
they must be oriented in some way similar to their orientation in a
erystal whether liquid or solid. Otherwise there could be no definite
double refraction. As a matter of fact there is no double refraction
as long as the myosin is in solution; in other words, as long as the
molecules are so far apart that they can take any position freely and
have their axes directed in all directions. If the myosin is in the form of
a gel, the gel as it exists in the muscle must be so concentrated that the
molecules are oriented and have their freedom of movement somewhat
restricted as they do in a solid. Furthermore the tenacity of the pieces
of muscle cut out by Kite and the remarkable elasticity which the sub-
stance showed indicate that the muscle substance has rather the
properties of an elastic solid, hydrated perhaps, but no less a solid,
than the properties of a gel. If muscle is a gel it is certainly not a
homogeneous gel, but one which in some way is definitely organized so
as to give the remarkable structural pictures of the muscle. It is
more comparable to a liquid crystal than a gel.

The protein of the stroma substance appears. then, to be made of
an albuminous, insoluble material possibly united with phospholipins
to make lipo- or lecitho-proteins. This union, however, is not very firm,
it is dissociable and some of the protein remains free. The molecules
are definitely oriented. It is also quite possible that in the living muscle
some of the extractives, such as creatine, may be united with this com-
608 PHYSIOLOGICAL CHEMISTRY

plex and that carbohydrate, either glycogen or glucose, is also joined to
it to make a very complex, unstable protein molecule.

The proteins of the different kinds of muscles do not differ widely
in the composition of the amino-acids that they yield by hydrolysis, as
may be seen in the following table:

Perhaps the interesting fact here is the relative large amount of
the basic amino-acids, since these occur also among the extractives.

AMINO-ACID CONTENT oF VARIoUS Musctes (Osborne and Heyl).

  

Fish—Halibut Chicken Ox Scallop
GUY COCON iii oa tcoicesa aed wleletsiee see 0.00 0.68 2.06 0.00
AV AMING: scpiesie eco stanttete rs sting a5 erets 2 2.28 3.72 diese
Mane: <6 onuoe so eases shaw be 0.79 2 0.81 weeded
Leteine: i. spars iss airews oe nares 10.33 11.19 11.65 8.78
Proline: ssw s oneaw ss eemeen ex 3.17 4.74 5.82 2.28
Phenyl alanine ................ 3.04 3.53 3.15 4.90
Aspartic acid ................. 2.73 3.21 4.51 3.47
Glutaminie acid . -- 10.13 16.48 15.49 14.88
Serine ........... : ? ? ? ?
Tyrosine ........ -. 2.39 2.16 2.20 1.95
Arginine .... a a schatarords 3 0.as3 6.34 6.50 7.47 7.38
Histidine: vices is aes oad cares 2.55 2.47 2.66 2.02
HAY SIMO: cece os Soaseishuare 5 eeReRCe a & tes 7.45 7.24 7.59 5.77
NH, SS Joadcsuie) OSS sSualones des Sepalnadedsiveniacsuese 1.33 1.67 1.07 1.08
Tryptophane ............0.2.00- Present Present Present Present

Proteins of smooth muscle—Smooth muscle of mammals or birds
extracted with 0.7 per cent. NaCl also yields a muscle plasma,
which spontaneously coagulates at room temperature. By dialysis a
globulin separates, which coagulates at 54-60°, and in the filtrate from
the globulin an albumin was found which coagulated at 45-50°. This
latter substance was myosin(?). Swale Vincent and Lewis got from the
smooth muscle of the sheep’s stomach by extracting with .7 per cent.
NaCl a plasma coagulating at 47°, but no other protein was present.
If the extraction was made with 5 per cent. MgSO,, the coagulation tem-
perature of the extract was 56°. The coagulation temperature of pro-
teins varies within several degrees, depending on the salt content, nature
of the salts, and reaction of the solution. 56° is the temperature of
coagulation of the fibrinogen of the blood and also of similar globulins
found in liver and other organs. Smooth muscle contains a larger
proportion of nuclear material than striated and the nucleoproteid is
five times the proportion of that of striated muscle. From these ob-
servations it appears that smooth muscle contains myosin and nucleo-
protein, but no myogen.

The extractives.—-The extractive substances in muscle are those
organic substances which may be extracted with boiling water. The
great interest attaching to these substances is due to the circumstance
that they represent products of muscular metabolism and thus throw
THE CONTRACTILE TISSUES. MUSCLE 609

some light on the nature of the chemical changes going on in it. Further-
more, since the muscles form nearly half the weight of the body these
substances probably represent the precursors of some of the substances
excreted in the urine. The total amount of extractives, including both
inorganic salts and organic matter, obtained from perfectly fresh living
muscle by boiling it with water amounts to about 2 per cent. of the
weight of the muscle. Of this amount .7 per cent. is organic and 1.3
per cent. inorganic.

The nitrogenous extractives. In this group are found creatine,
methyl guanidine, carnosine, inosine, carnitine, sarcosine, taurine,
glycocoll, urea and hypoxanthine.

Creatine. Creatine, or methyl guanidine acetic acid, C,H,N,0,
NH, — C( = NH) —N (CH,) —CH,— COOH, is described on page
702. The amount of creatine in voluntary muscle varies from 0.3-0.45
per cent. of the weight of the fresh muscle. Most of the creatine may
be extracted by soaking the muscle in cold water, but a somewhat better
yield is obtained by making a boiled extract. Creatinine is found only
in small amounts in perfectly fresh muscle and it is doubtful whether
it exists in the living muscle.

The creatine in muscle is probably in union with the colloids of the
muscle, since it does not readily dissolve out of the muscle. Thus muscle
contains in 100 grams 400 mgs. of creatine, whereas the blood contains
only 2-3 mgs. per 100 grams. Creatine is given off only in small
amounts to the blood by the normal beating heart or by the living muscle,
and while this might be due to the fact that the sarcolemma was almost
impermeable to creatine it is more probably due to the creatine being in
a loose chemical or molecular union with the muscle substance.

The origin of the creatine in muscle has been much debated and no
satisfactory source has yet been established. It might be derived from
arginine, which is guanidine-amino-valerianic acid, or from some of the
phospholipins, or by synthesis from urea and sarcosine. The matter
is still. under investigation and no definite conclusions have yet been
reached. It appears probable that the muscle is able to methylate
guanidine acetic acid or glycocyamine. Whether the creatine of muscle is
inereased or diminished by stimulation has been much disputed. The
older statements in this regard are all open to question because of the
methods of determining creatine, which were not quantitative; by
the method now used of determining it, by conversion into creatinine,
a more accurate determination is made, but one also open to many
errors. If the muscle is stimulated in situ with its normal blood supply
several observers have found that the creatine is diminished instead of
inereased by stimulation. Liebig reported that the muscles of a hunted
fox contained several times the normal amount of creatine. According
610 PHYSIOLOGICAL CHEMISTRY

to recent work, contraction of muscle does not change its creatine
content, but any means which diminishes the tonicity of the muscle
does diminish the creatine content.

Iso-creatinine. This has been isolated from fresh fish muscle. Its
nature is uncertain, but it differs from creatinine in the greater solu-
bility of the base and picrate in water and by the fact that oxidation by
potassium permanganate does not produce methyl guanidine, but am-
monia. It may be that the nitrogen is bound to different carbon atoms
or that it is a tautomeric form of creatinine.

Carnosine (Ignotine) C,H,,N,0,, is a basic extractive precipitated ©
by phosphotungstic acid and by silver and barium hydrate, according
to Kossel’s histidine method. On hydrolysis it yields histidine, C,H,
N,0,, and what is believed to be @-alanine. If this is so carnosine is
the first natural dipeptide which has been found to have in it a
68-amino-acid. It has been suggested that it may arise from histidyl-
aspartic acid by the action of a carboxylase, splitting off the last
carboxyl group of the aspartic acid.

Carnosine has the following properties:

100 grams water at 24.9-25° dissolve 31 grams carnosine. It is precipitated from
water by alcohol. (aye? = + 20.77. The nitrate melts at 222° (Thiele’s block).
Colorless needle-shaped crystals, m.p. 219° with decomposition. ‘Carnosine tastes
insipid and reacts strongly alkaline. It is not precipitated by K,FeCy,, lead acetate,
acid or basic, nor by HgKI,. Saturated picric acid does not precipitate it but tannic
and phosphotungstic acids do. Gold hydrochloride gives a precipitate. It has been
found in the extract of horse, ox and calf muscle, and of fish, crabs and oysters, but
only in the muscle. It is not found in kidneys or in liver. In the hind leg of a fresh-
killed horse from 6.4 kilos of muscle, 0.58 gram of free purines, 2.0 grams of
carnosine and about 10 grams of methyl guanidine were obtained. The accom-
panying table shows the amounts of various extractives contained in one kilo of
various muscles. The amounts are expressed in grams.

AMOUNT OF EXTRACTIVES IN GRAMS IN ONE KILO MUSCLE.
Wild

Horse Ox Salmon Maguro Calf rabbit Pig
Creatine ..........eee. . 0.58 3.2 3.0 2.0
URIS? soiree cig vateece donee Sincere 0.09-.07 0.04
Carnosine .......... sere, 182: 1.30 0.55 2.0 1.76 2.23 1.95
Methyl guanidine ....... .083-.1 0.22
Carnitine ...........00. 2-17 0.19 0.3

Carnitine (Novain?). This has been found in horse, calf and pig
muscle. It is a base precipitated by phosphotungstic acid, giving with
corrosive sublimate a double salt, m.p., 196°: C,H,,NO,.2HgCl,. Carni-
tine is probably y-trimethyl-@-oxybutyrobetain, the hydroxyl probably
being in the f position (or @).

co

0
(CH,) NC |
CH,—CHOH—CH .
THE CONTRACTILE TISSUES. MUSCLE 611

It would seem not impossible that this base might be the mother sub-
stance of choline. It is conceivably formed from glutamic acid by split-
ting off carbon dioxide and methylation. This base is said, by Krim-
berg, to be identical with the Novain isolated from meat extract by
Kutscher, but according to the latter it is rather an isomer of Novain.
The oblitin of Kutscher is the diethyl ester of carnitine.

Taurine. Taurine is found only in small quantities in mammalian
muscle, but it is the chief nitrogenous extractive in the muscles of vari-
ous invertebrates. The fresh muscle of the cephalopod, Octopus. con-
fains at least 0.5 per cent. of taurine, but no creatine or creatinine.
This muscle contains about .03 per cent. of hypoxanthine and no glyco-
coll. Taurine is

i
N H,.CH = CH,—S—OH

g

Purimes of muscle. The fact that muscle contains far less purines
than other tissues was discovered by Kossel. There is less nuclear sub-
stance in muscle than in most other tissues. In the cells of the thymus
glands, for example, the nuclei nearly fill the cells and these cells con-
tain seven to eight times as much purine nitrogen as the muscles, in
which the greater part of the cell is cytoplasm. The pancreas is inter-
mediate in the amount of nuclear material and purine bases it contains.
This fact indicates that probably the purine bases are confined to the
nucleus. The amount of purines in various tissues is shown in the follow-
ing table in which the purine nitrogen is expressed in per cent. of the
weight of the fresh organ:

ToraL PuRINE NITROGEN IN PER CENT. OF THE WET WEIGHT OF THE ORGAN
(Burian and Hall).

Organ Per cent.

Horse meat ......... .055

Ox meat ..........56. .062

Veal ...... Sateatioacss 071

Thymus ............ 482 (by quick work)
ST isndaua tata toxtaes .429 (by slow work)

Pig’s pancreas ....... 123

Ox pancreas ........ 183

The hypoxanthine of muscle increases during muscular work, whereas
if the perfused muscle is stimulated the purine base nitrogen falls from
06 to .04 per cent. Muscles give off purine bases to the blood stream
when they do work; they also give off some uric acid. The purine base
and uric acid in the blood perfused through muscle behave as follows:

Total purine Uric acid N -Uric acid Purine base

3 N mgs. mgs. mgs. N mgs.
JefOPe: PETIUSION: 20.000. ssc d wise tea ards 0

after 1 h. at rest—1000 c.c. blood perfused .. 52 1
2d period muscle at work—1000 c.c. blood .. 8.5 2.
3d period muscle at rest 1 h.—1000 cc. blood .. 10.5 5
612 PHYSIOLOGICAL CHEMISTRY

Glycocoll and Urea. Large amounts of glycocoll are found in the
muscle of pecten irradians, 0.39-0.71 per cent. being present. Urea is
present in only small amounts in the muscle of mammals, .04-.08 per
cent., but about 1-2 per cent. is present in the muscles of elasmobranchs,
such as the sharks and rays. If elasmobranch muscle dries, some of the
urea decomposes yielding ammonium carbonate, so that the muscle ap-
pears to have urease in it.

Inosine (Carnine). C,,H,,N,0,. This substance is a decomposi-
tion product of inosinie acid and consists of a compound of hypoxan-
thine and d-ribose, a pentose. Similar substances (vernin) have been
found in plant cells by Schulze. Of its function or significance nothing
is known.

Inosinic acid. C,,H,,N,PO;. This was discovered in muscle by
Liebig, who quite overlooked its phosphorus content, but found that it
yielded sarkine (hypoxanthine) and a carbohydrate. It has since been
shown to be a compound of hypoxanthine, d-ribose and phosphoric acid
and to have the probable formula: _

0 = C—NH
Pore

CH bi
mi Hci

About .11 per cent. of inosinic acid is present in the muscles of fowls,
whereas none is present in the muscles of pigeons. None was found by
Schlossberger in human muscle. Inosinie acid is obtained from the
alcohol-extracted and powdered meat by extracting one kilo of the dry
residue with 2-3 liters of warm water. The phosphates are carefully
precipitated with Ba(OH),, the solution neutralized with HNO, and
the inosinic acid precipitated with AgNO,. The precipitate is decom-
posed with H,S and the filtrate evaporated to a syrup, which becomes
powdery with alcohol. The free acid is insoluble in alcohol and ether.
1,000 parts of water at 16° dissolve 2.5 parts of the salt. Inosinic acid
is a mono-nucleotide. Whether it is found in the nuclei or cytoplasm
cannot be said. Its relation to the nucleic acid of the nucleus is un-
known.

i-Inosite. C,H,,0,; CsH,(OH),. Hexahydroxyhexamethylene. Ino.
site is found in nearly all organs of the animal body, in the heart,
skeletal muscle, liver, pancreas, spleen, kidneys, supra-renals, testes,
spinal cord, brain and lungs of mammals. It does not occur in the
normal urine of man or rabbits, but it has been found in the urine in
diabetes insipidus and mellitus (Kulz). It is not constantly present in
the urine in these diseases, for in eight cases of diabetes insipidus it was
found only once. It is sometimes present in quantities in the urine.

 
THE CONTRACTILE TISSUES. MUSCLE 613

One case of inosite-uria has been described in which 18-20 grams of
inosite were excreted daily (Vohl). Inosite is also widely spread in
the plant world and it occurs in especially large quantities in green
beans. It appears to be oxidized, or destroyed, in the body, since Kulz
gave a man 30-50 grams of inosite and recovered in the urine only
0.2-0.5 gram. The wide occurrence of inosite in all forms of living
cells indicates its connection with some fundamental process in the
living protoplasm. It is found united with phosphoric acid as phytin in
bran. Phytin is probably
H OPO(OH)s

H H
(HO),OPO o ‘e OPO(OH),
(HO),0PO fs C OPO(OH)

\Z

H OPO(OH).

Phytin.
Phytin has a marked laxative action on the bowels and inosite also,
when taken by the mouth, produces diarrhea.

There are in nature three forms of inosite: i-inosite, found in mus-
cle, which is the meso-inosite and inactive; d-inosite and l-inosite. It
crystallizes in large, rhombohedric crystals; it is very soluble in hot
water, insoluble in absolute alcohol and ether; its solutions have a sweet
taste, but it does not reduce Fehling’s solution.

It is generally thought to occur free in the muscle, but some think
that it is in union with some other substance. The amount in muscle
is small, only about .003 per cent. being present. From the brain 10
grams were obtained from 50 pounds. Of its function nothing is known.
Presumably it is formed from carbohydrates.

MytiliteThis is a body related to inosite which is found in the aqueous extract
of the muscle closing the shell of Mytilus edulis. It appears to be a penta alcohol,
having five groups capable of acetylation. It contains a hexa carbon ring and gives
Scherer’s reaction. It is isomeric, or stereoisomeric, with quercite and isoquercite.

It resembles an anhydride of inosite. Its formula is C,H,,0,.2H,O. Among the
other extractives of this muscle are betaine (Brieger; Jensen), taurine and 1.5% of

glycogen.

Xylose. Xylose was found by Henze to make from 0.4-0.9 per cent.
of the dry weight of the muscles of Octopus.

Carnic and phosphocarnic acid. This is a substance found in the
extractives of muscle and obtained as a copper salt. Carnic acid is
probably a dipeptide of the formula C,,H,,N,0,. This substance binds
hydrochloric acid so firmly that it forms no precipitate with silver
nitrate except on boiling. It gives the biuret reaction and is identical
with anti-peptone. It is said to yield lysine and some ammonia on
614 PHYSIOLOGICAL CHEMISTRY

hydrolysis. Carnic acid exists also in combination with phosphoric
acid, according to Siegfried, to form phosphocarnic acid, to which im-
portant functions were originally assigned. From further work it seems
doubtful whether phosphocarnic acid is a pure substance. There may
be a series of similar compounds of an analogous nature. It need not be
considered more at length until further work has been done upon it.

Succinic acid. C,H,O, COOH. CH,—CH,.COOH. This acid is
one of the more abundant, non-nitrogenous extractives of muscle. It is
found in the fresh muscle as well as in meat extract. 1.5 kilos of
dog’s muscle two hours after death yielded 0.24 gram of succinic acid;
from 1.8 kilos of beef muscle, 48 hours after slaughter, 0.133 gram
were obtained; and seven days later 1.75 kilos of the same beef yielded
0.122 gram. The amount is so large that it cannot possibly all be de-
rived from phosphocarnic acid, even if it be admitted that the latter is
an individual. Succinic acid possibly plays a réle in oxidation, since
it was found by Thunberg to influence oxidation of the muscle in quite
a special manner. This acid is also one of the constituents of the
oxidizing ferment laccase, which may be of interest in connection with
the results of Thunberg. Whether muscle can oxidize or destroy this
acid is unknown. It is doubtful whether it is oxidized to malic acid.
The réle it is playing in muscle, the conditions of its formation, and its
fate, if normally formed there, require further investigation. It is
found also in the brain extractives.

Lipins of muscle.—These consist of neutral fat, cholesterol and phos-
pholipins. The latter are very little known at the present time. The
neutral fat probably comes, for the greater part, from the connective
tissue; the phospholipins, however, are derived from the muscle itself.
Of the skeletal muscle the phospholipin makes about 30 per cent.,
usually, of the total lipins, but in the heart the proportion of phospho-
lipin may be 60-70 per cent. of the total lipin. The cholesterol is about
0.2 per cent. of the dry substance. The phospholipins of the heart and
skeletal muscle have been investigated recently by Erlandsen. In the
heart there is found a peculiar lipin like cephalin, having highly un-
saturated fatty acids, which has already been described on page 95.
There are also present other lipins more of the nature of lecithin. Since
these bodies have probably not been obtained as yet in a pure state
and have been but little investigated, we need not discuss them further
here. The total lipin content of the muscle is between 2 and 8 per cent.
of the wet weight of the muscle. Figure 56.

Inorganic constituents of muscle.—The principal points of interest
in the inorganic constituents of muscle are the relatively large proportion
of potassium as compared with sodium and the considerable quantity of
phosphoric acid present. The phosphorus is mainly in the inorganic
THE CONTRACTILE TISSUES. MUSCLE 615

form. The total amount in muscle is about 0.2 per cent. Of this
amount in ox flesh, Constantino found 81 per cent. as inorganic and 19
per cent. as organic phosphorus. Grindley got 93 per cent. in the form
of inorganic phosphorus in the beef he examined. Of the organic phos-
phorus, 16 per cent. is present as phosphatide and 3 per cent as other
organic phosphorus compounds, nucleins, etc. In heart muscle, the inor-
ganic is only 40 per cent. of the total and the organic 60 per cent. (Con-
stantino). In smooth muscle 50-70 per cent. of the total phosphorus is
organic, but the non-phosphatide, organic phosphorus is larger, probably

  

A.

Fig. 56.—Distribution of _lipin in muscle cells (Noll). 4A, Lipin stained with osmic
acid in pectoralis of pigeon. B, Osmic acid preparation of rabbit smooth muscle.

owing to the larger amount of nuclear material present. Thus in the
heart there were 42 per cent. of phosphatide phosphorus and 20 per
cent. of non-phosphatide, organically bound phosphorus. In smooth
muscle of the 50 per cent. organic phosphorus, 27-42 per cent. were in
the form of non-phosphatide phosphorus organically combined.

In striated muscle there are from 2-3 times as many atoms of potas-
sium as of sodium present. The atomic ratio K: Na is 2.60—8.34: 1.
In smooth muscle the ratio is variable. In the retractor penis it is very
nearly 1:1. But in the stomach it is about the same as in striated mus-
ele. In striated muscle of mammals there is about 0.32-0.42 per cent. of
potassium and 0.04-0.07 per cent. of sodium computed on the wet
weight; of smooth muscle that of the stomach has about the same propor-
tion of sodium and potassium as striated, but the retractor muscle of the
penis and smooth muscle of the uterus have about 0.26 per cent. of potas-
sium and 0.15 per cent. of sodium (Constantino. See also Fahr and
Meigs).

The internal secretion of muscle.—Like all other tissues of the
body, the muscles are constantly giving off to the blood substances
which are to be eliminated, or which affect more or less profoundly
other organs. Knowledge of the substances thus given off is still un-
certain, but the finding in the urine in small quantities of most of the
extractives of muscle (Kutscher) shows that these substances are prob-
ably given to the blood by muscle. It has been found by perfusion
experiments that carbon dioxide, creatine, methyl guanidine, hypo-
616 PHYSIOLOGICAL CHEMISTRY

xanthine, lactic acid and acetone are, sometimes at any rate, given off
from contracting muscle. Some of these substances are of importance
in stimulating the respiratory center. This is true of the carbon dioxide
and the lactic acid; others produce dilation of the arterioles and in
this way serve the muscle; the acids, also, have the very important func-
tion, particularly in amphibia and cold-blooded animals, of helping to
dissociate oxygen from oxyhemoglobin, thus turning the oxygen out
of the blood so that it can be utilized by muscle. (See page 487.)
Blood of tetanized dogs is said to be toxic and on transfusion to other
dogs causes in them disturbances both of the circulation and respiration.
(Compare parathyroid tetany, page 655.) The substances so acting
are said-to be soluble in alcohol. There is no doubt that much remains
to be done in this branch of the subject. Methyl guanidine, which is
found in some muscles, is decidedly toxic, as is also oblitine. These toxic
bases have been found in the urine of dogs after parathyroid extirpation.

The metabolism of muscle.—It is extremely difficult, from the
account which has just been given of its composition, to form any
probable picture of the nature of the metabolism of muscle and the
changes occurring in it, particularly during contraction or muscle rigor.
By many authors an attempt has been made to distinguish between that
part of the metabolism which is concerned with the formation of the
muscle cell itself, its formative metabolism so called, and the metabolism
which is involved in its activity during contraction, or its energy
metabolism. It is doubtful to what extent this separation of the two
phases is justified, but as it is not unlikely that the character of the
chemical processes involved in forming the protein machinery of the
muscle are different from those involved in furnishing the energy for
contraction, the total metabolism may be discussed under these two
headings. .

The formative metabolism of muscle.—The formative metabolism of
muscle, like that of all other cells, is that metabolism in virtue of which
the substance of the muscle is produced. Its general features are those
common to all cells. The proteins of the muscle are characteristic and,
indeed, the muscle of each species of animal has its own specific protein.
Probably were our methods fine enough, we should find the proteins
of -each individual of the same species to be different from the cor-
responding proteins in other individuals. These proteins are prob-
ably made from the amino-acids brought to the muscle from the digestive
tract by the blood. The synthesis of the amino-acids into the specific
proteins is, hence, a part of this formative metabolism. It is unknown
how each tissue picks out or uses just those amino-acids in the right
proportion to form its own specific proteins. Possibly it depends, in
part, on a slightly different rate of destruction of the different amino-
THE CONTRACTILE TISSUES. MUSCLE 617

“acids in different muscle cells. But not only has muscle the power
of forming proteins from amino-acids; it is also able to destroy these
proteins and reduce them to amino-acids. It has the property, in other
words, of auto- or self-digestion. This happens in starvation; in wast-
‘ing diseases, when fever exists; and it probably happens, also, in the
wasting of a muscle from disuse. The fundamental protein metabolism
of muscle is not especially keen. On the contrary, there are many indi-
cations that muscles are not remarkable for their power of growth and
repair. The fundamental formative metabolism goes on at a faster or
slower rate, depending on circumstances, such as temperature; and
possibly upon the number and character of the nerve impulses reaching
it; or upon age, being more rapid in youth than later in life; or upon
health or sickness, a rapid catabolism taking place under conditions
of starvation, etc.

But muscle is more than a simple protoplasmic cell. Its living
toaterial has either been transformed in large part into a machinery
capable of producing changes in shape of the muscle at a rapid rate, or
the living material has fashioned in itself a machinery, possibly not a
truly living machinery, which is the machinery of contractility. It is
impossible to say at present whether the contractile machinery is truly
self-perpetuating or not; whether, in other words, it is living or not,
or whether it is not to be regarded as a piece of lifeless protein ma-
chinery more like connective tissue, or the catgut fibers of Engle-
mann’s artificial muscle, which works under the influence of the chemi- .
cal changes occurring in the living material which has formed and
which surrounds it. Whatever may be the truth of this matter it seems
possible that the muscle has quite a special metabolism, distinct from its
formative metabolism, and that this second metabolism, the energy
metabolism, is concerned with its functions as a motor, or contracting
engine. This second metabolism, that of contraction, is thought to be, as
it were, superimposed upon, or separate from, the fundamental metab-
olism of the muscle cell proper. It is a destructive metabolism, or catab-
olism, by which heat and energy are set free sufficient to move the
machinery and produce contraction.

The nitrogen output of muscle is probably not so keen during rest as
that of many other tissues of the body. There is a general law which
prevails throughout the whole animal and plant kingdom that that
organ which has the most powerful formative metabolism always draws
upon and reduces the metabolism of other organs of a less intense
metabolism. Thus the apical buds of plants inhibit the development of
buds farther down the branch. So in times of fasting or starvation the
muscle protein is torn to pieces and converted into amino-acids, which,
passing from the muscle to the blood, are carried by that internal
618 PHYSIOLOGICAL CHEMISTRY

medium to those organs of which the metabolism is keener, to the brain
and nervous tissues and the heart, and serve to nourish those organs at
the expense of the muscles. Thus during fasting the voluntary muscles
waste away, but the brain and the heart keep their weight almost un-
changed, and those muscles which are of the most importance to the
animal and are the most exercised draw upon those of less importance.
This conversion of the muscle proteins, or self-digestion, is accomplished
by means of autolytic enzymes; a plentiful supply of food and oxygen
normally holds these enzymes constantly in check. Hence a rapidly
contracting muscle like the heart is able to maintain its material intact,
whereas a resting muscle gradually wastes away. Acids are particularly
prone to set free the autolytic enzymes of cells.

The formative metabolism of muscle is also dependent directly,
or indirectly through the blood supply, on the innervation. Muscle of
which the nerve has been cut degenerates, the cross-striations break
down, fat staining in osmic acid appears and there is a marked reduc-
tion in the bulk of the muscle. This degeneration may be the result
either of the falling away of the nerve impulses of a heat-producing or
tonic kind, which may normally be constantly impinging on the muscle,
impulses which do not cause muscle contraction but keep it in a state of
tone; or it may be due to the lack of control of the blood supply of the
muscle. Ordinarily the metabolic demands of the muscle are very care-
fully met by the vascular system; but in the absence of this control,
_ no regulation of supply and demand any longer exists. Which of these
mechanisms determines metabolic control cannot be decided. It would
seem possible, also, that the nerve impulses play an important part in
the organization of the muscle substance. The re-establishment of the
connection of the nerve with the muscle, after the nerve has been cut
and degenerated, leads to the reorganization of the muscle substance
and its restoration to a normal state. It would seem possible that this
organization proceeds outward from the nerve terminals and is pro-
duced by the nerve impulses, just as the brain material is organized by
the impulses coming into it. It is not possible to say whether the growth
of a muscle accompanying its use is due to a stimulation of the form-
ative metabolism coincident with a stimulation of the energy metab-
olism, or whether it is an indirect result of the stimulation of the latter.
It is, on the whole, probable that it is a direct result of the energy stimu-
lation and that a nerve impulse striking the muscle, not only causes the
great catabolism which liberates energy and produces contraction,
but also directly stimulates the other. chemical changes of a form-
ative nature. Something similar occurs in nerve tissue, in which it
has been shown that the formative metabolism, which causes the develop-
ment of the centers of the brain, is thus influenced by nerve impulses.
THE CONTRACTILE TISSUES. MUSCLE 619

For example, if the eyelids of a puppy are sewed together at birth, so
that they cannot be opened and no stimulation of the retina occurs.
it has been found that the cells of the brain ganglia concerned with
vision do not develop as they normally do, but remain in an infantile
state. In the case of muscle it seems probable, as has already been
stated, that a constant stimulation of the muscle through the nerve
occurs. This stimulation does not cause muscle contraction, because it is
not sufficiently intense, but it is sufficient to stimulate the formative
metabolism and perhaps to maintain a certain tonus in the muscle.

In the course of the formative metabolism of muscle creatine, among
other things, is produced. Nothing definite is as yet known of the real
function of creatine, but according to the work of van Hoogenhuyze
and Verploegh creatine is produced in the course of that metabolism
which is concerned with tonus. These authors have shown that tonic
contraction of muscle increases the creatine output in the urine and
increases the creatine content of muscle. Some authors have reported
an increase in the creatine content of muscle as a result of nerve stimu-
lation. While this is not improbable, the evidence is as yet unsatis-
factory.. That muscle has in it the precursors of creatine is probable
from the fact that on autolysis an increase of the total creatine and
creatinine of the muscle occurs. On the other hand, Mellanby has
failed to get any such increase when bacteria were absent. But blood
certainly contains such a precursor. It is stated that the addition of
gelatin to autolyzing muscle increases the creatine formed on autolysis.
A study of the creatine content of muscle during disease also indicates
the connection of creatine with the formative metabolism. Thus in dis-
eases involving the wasting of muscle abnormally large amounts of
creatine appear in the urine (Shaffer), if the methods used for detecting
creatine are reliable, which seems somewhat doubtful.

Nucleins also play a part in the formative metabolism of muscle.
The uric acid excretion undergoes a remarkable rise following muscular
work. This rise lasts only for a couple of hours and is then followed
by a fall so that the total excretion is not changed by work. Muscular
work appears, in the first place, to increase the hypoxanthine content,
and this is then oxidized to uric acid. It has been suggested that this
oxidation happens only as the hypoxanthine is about to leave the mus-
cle and is due to the fact that the hypoxanthine oxidase is found only
in the periphery of the muscle. It is probable, since muscle is formed
in young children when on a purine-free diet, that muscle has the
power of making purines from non-purine precursors.

The formative metabolism includes the power of making glycogen
and possibly of making carbohydrate from non-carbohydrate material,
such as the proteins. Glucose brought to muscle is, in part, stored as
620 PHYSIOLOGICAL CHEMISTRY

glycogen. The origin of the d-ribose in inosinic acid is unknown, nor
can anything be said concerning the origin of the phospholipins.

On the whole, then, the formative metabolism of muscle presents
just those problems which all other tissues present; problems of which
the solution remains for the future.

The energy metabolism of muscle.—The energy metabolism of mus-
cle is, as it were, superimposed upon the formative metabolism. This
metabolism is peculiar in that it does not directly involve any increase
in protein catabolism. Muscular work does not increase the nitrogen
output of the body or of muscle. This metabolism involves primarily
the carbohydrates and we may now consider the changes in composi-
tion of muscle produced by muscular work.

1. Change in glycogen content. That muscles contain glycogen
in considerable quantities was discovered shortly after Bernard found
glycogen. The amount of glycogen in different muscles and in the cor-
responding muscles of different animals is variable. Thus horse muscle
contains an unusually large quantity of glycogen, 1-2 per cent., and
since glycogen can be detected in muscle microscopically by the brown
reaction it gives with iodine, it is possible in this way to detect the
presence of horse meat in sausage, or other food products in which it
has been used. Another muscle having a large amount of glycogen is
that of the scallop, or the shell-closing muscle of Mytilus edulis, which
contains also about 1.5 per cent. Ox flesh and other forms of muscle
contain less. Thus it has been found that perfectly fresh ox muscle
contains, on the average, only 0.3 per cent. of glycogen. The glycogen
content of muscle is subject to variation with the diet, but the variation
is less than that of liver glycogen. Thus, in fasting, glycogen disappears
more rapidly from the liver than from muscle. A large intake of glu-
cose or other carbohydrate increases the glycogen of the liver more than
that of the muscle, but still glycogen increases also in muscle in such
cases. The heart muscle normally contains fully as large an amount
of glycogen as the skeletal muscle, but glycogen disappears after death
more rapidly from the heart than from skeletal muscle, so that many
observers have found less glycogen in the heart than in other muscles.

The localization of glycogen in the muscle has been repeatedly in-
vestigated. It is found in the form of granules in the living matter
itself (Arnold), in the sarcoplasm, but not in the fibrils. According to
Arnold glycogen is best fixed by corrosive sublimate containing some
glucose.

The glycogen of muscle undergoes a rapid decomposition after death,
and in beef which has hung for some time the amount of glycogen is
much reduced. In hen’s muscle 30 to 60 minutes after death, 25-58 per
cent. of the glycogen is lost and rabbit’s muscle in 4 hours at 40° may
THE CONTRACTILE TISSUES. MUSCLE 621

lose 88.8 per cent. of its glycogen (Boruttau). This behavior is similar
to that, of the liver and, like the liver, it is due to the presence in
muscle of an enzyme, an amylase or glycogenase, which rapidly converts
glycogen into glucose when the reaction is slightly acid. There may also
be found some iso-maltose in the muscle extract, which probably also
comes from glycogen, but there can be no doubt that most of the sugar
formed from the glycogen is glucose.

QUANTITY oF GLYCOGEN IN Various Doe’s Muscies, SHOWING THE QUANTITY OF
GLycoGEN IN CORRESPONDING MUSCLES ON Opposite SIDES OF THE Doc’s Bony,
THE EFFECT OF FasTING AND oF AGE (Maignon).

Dog Bnace ane

Very old. Fat. Semi-tendinosus Right 0.146

Left 0.229

Biceps femoralis Left 0.235

"= = 0.550

0.516

0.446

0.669

0.365

0.527

0.417

0.077

0.109

0.660

0.559

4 years old. Fat.
Semi-tendinosus
“c “

Triceps brachialis
3 years old. Biceps femoralis

“ce “

1 year old.

oe “

1 year old.

Perna

Fasting

Old dog. Temporal. 0.134

0.161

0.441

0.544

0.679

0.708

0.221

0.200

0.220

0.255

0.280

0.255
Besides having the power of converting glycogen into glucose, liv-

ing muscle has a very remarkable power of destroying glucose, and

even autolyzing or hashed muscle has this power to some degree, al-

though it is soon lost after death. This glycolytic power, as it is called.

it is needless to say is the most important problem in the metabolism

of the carbohydrates and it has been keenly investigated of recent years.

We will leave it for the moment to consider the change in the amount

of glycogen, which accompanies muscular contraction and work.
Glycogen is much reduced when muscles do work. Thus the follow-

ing results were obtained when dogs were anesthetized with ether, mor-

phine and chloroform, and after the ligation of both the external iliac

arteries and compression of the dorsal aorta the crural nerve on one

side was stimulated as long as the muscle would contract. The stimu-

4 years old. Semi-tendinosus
Semi-membranosus

Old dog. 5 days fasting. Semi-tendinosus

Old, fat dog. 14 days fasting. Biceps femoralis

HHP rR PRCA

2 years old. 5 days fasting. “ “
622 PHYSIOLOGICAL CHEMISTRY

lated muscle was then stimulated directly as long as it would respond.
The two corresponding muscles of the two sides, stimulated and un-
stimulated, were then removed and their glycogen determined by the
not very accurate Briicke process. The unstimulated muscle contained
from .532-.684 per cent. of glycogen; while the active contained 0.116-
0.194 per cent. Irritability of a muscle is lost long before its glycogen
is exhausted. On a less-extended stimulation there was found in the
unstimulated 0.716-0.560 per cent. and in the stimulated 0.440-0.112 per
cent. On the other hand, division of a motor nerve increases the glyco-
gen content of that muscle, as compared with the corresponding muscle
of which the nerve has been left intact. In frogs the increase
may be 20-30 per cent. of the original amount; in cats and rabbits the
change is not so marked. Tetanus of frog’s legs caused a diminution
of 24-50 per cent. of the glycogen of the stimulated, as compared with
the unstimulated side. In prolonged stimulation and tetanus by strych-
nine, glycogen undergoes a great diminution.

From the foregoing results there is no doubt that during muscular
work there is a great diminution in the glycogen content of muscle.
Glycogen is used up, presumably converted first to glucose and then
oxidized. The energy uséd in muscular work may come, therefore, from
this combustion of the glycogen. The question arises whether this is the
sole source of the energy. Is there enough glycogen in the body to do
the whole work which the muscle does? This question is very difficult
to answer. A comparison of the amount of energy used in the work
done with the amount which would be set free from the combustion of
the glycogen which had disappeared during the working period has
given a very bad agreement between the two values. Seegen found
that the work done by an extirpated muscle represented 2-11 per cent.
of the energy presumably available by the burning of the glycogen.
From this factor he calculated that there was not enough glycogen in
the body to yield the energy of muscular work, but Schenck has very
justly criticised these conclusions because the muscle was working under
unfavorable conditions, because in the body glycogen can be rapidly
reformed, and because there was no proof that the disappeared glycogen
had actually been completely burned. There is no doubt from the
persistence of the power of muscular contraction in severe diabetes,
where the power of combustion of carbohydrates has, presumably, been
completely lost, that certainly muscle has the power of burning other
foods, fats for example, as a source of its energy.

The fasting heart holds its glycogen longer than the body muscles,
for it draws on the voluntary muscle for its nourishment during starva-
tion. The heart has: the power of nourishing itself even from a very
greatly impoverished blood. In rabbits after six days of fasting the
THE CONTRACTILE TISSUES. MUSCLE 623

muscles still contain 0.04-0.06 per cent. of glycogen ; cats retain 0.05-0.07
per cent. after 12-14 days of fasting; the muscles of doves after 2-8 days
of fasting contain from 0.07-0.32 per cent. The most surprising result
is found in horse muscle, which still contains from 0.98-2.48 per cent.
of glycogen after 9 days of fasting (Aldehoff).

The glycogen in muscle, like that in the liver, is under the control
of the internal secretions of the body, and particularly under that of
the supra-renal gland. The observation was made nearly forty years
ago that if cats were simply tied down on an operating board they showed
after half an hour a very marked glycosuria, and that if they remained
there the animals died after 36 hours, with a great fall of blood
pressure and body temperature. The muscles of these animals proved
to be completely free from carbohydrates and the liver lost its glycogen
before the muscles. This disappearance of glycogen is probably due to

‘the discharge of adrenaline from the supra-renal glands into the blood;
a discharge which accompanies emotions such as anger and fear and
the object of which may possibly be to promote the liberation of the
carbohydrate so that it may be in a condition for rapid burning. Thus
the chances of escape of an animal might be facilitated (Cannon). By
causing the transformation of glycogen into glucose there are put at the
disposal of muscle large stores of fuel, thus making possible a supreme
effort. In this case the skeletal muscles are affected by nerve impulses
to the supra-renals, these nerve impulses acting, as it were, at a distance,
that is indirectly on muscle. The impulses impinging on the supra-renal
glands cause these to set free substances which profoundly affect the
metabolism of the muscle; may it not be possible that the effect of the
nerve impulse to the muscle itself is to set free substances which cause
the contraction of muscle? The nerve impulse may not be a direct, but
an indirect, cause of the contraction. The latent period may have this
explanation. The muscles have much less glycogen in pancreatic and
phlorizin diabetes, but it does not completely disappear. Accompany-
ing this loss of glycogen there is a marked weakening of muscular pow-
ers. Arsenic, also, causes a disappearance of muscle glycogen in cats.
As adrenaline causes a discharge of glycogen from muscles so there may
also be substances which cause a retardation of the transformation of
glycogen to glucose, and it is not impossible that certain antipyretics,
such as acetanilide or antipyrin, may have some such action. This pos-
sibility should be investigated.

From these observations it appears that glycogen is contained in
muscle in considerable quantities. In a man’s body, if his muscles con-
tain 0.3 per cent. of glycogen and weigh 30 kilos, there would be roughly
90-100 grams of glycogen. This is partly consumed during muscular
work, the transformation into glucose being caused by an enzyme simi-
624 PHYSIOLOGICAL CHEMISTRY

lar to the glycogenase of the liver. The glycogen of muscle is less
affected by diet than that of the liver, but still it is affected somewhat.
In starvation it diminishes, and on carbohydrate food it increases.
After death it disappears with greater or less speed, and under the
influence of adrenaline or phlorizin it is greatly diminished. We have
now to inquire concerning the further decomposition of the.glucose set
free from the glycogen.

The glycolytic power of muscle. Most of the sugar which is burned
in the body is burned in muscle. It is a veritable conflagration which
is there going on, a conflagration which warms, animates and invigorates
us, but a conflagration giving a light too subtile to be seen. What is
the nature of this conflagration, of this wonderful combustion in an
aqueous medium, which causes heat and electricity but so seldom pro-
duces light? The true solution of this problem still eludes physiolo-
gists. Do the sugar molecules burn as such, or are they first torn to
pieces by chemical union with some catalytic substance? It may be
imagined that they unite with some of the substances in living matter
and when the union is made the sugar molecule is no longer stable but
breaks into fragments. When a piece of wood burns it is not the wood
which burns, but the pieces of the molecules of wood which have been
dissociated by the heat and which have such an affinity for oxygen of the
air that they inflame spontaneously. And it is probably so in muscle,
also, only it is not heat which fragments the sugar molecule in muscle,
but a substance which unites with the sugar. Protoplasm is itself the
torch.

The property of thus fragmenting the sugar molecule, is it a prop-
erty of the living protoplasm only, or is it due to a substance which
may be isolated from the protoplasm and which will have the same
properties outside the cell as in it? Many men have sought for such
a substance. The property of thus fragmenting the sugar molecule
seems to be a property of living muscle only. Dead muscle no longer
has this power. It is true that ground muscle has, also, some glyco-
lytic power, but this power is soon lost; it inheres only in fragments of
the living matter itself; it is never very great. Extracts of muscle do
not have the power of glycolysis. It is impossible to say whether this
power is due to some substance which is very unstable and thus hard
to isolate, or which is destroyed by the action of other ferments such
as the digestive ferments of muscle, or whether it is only the organized
biogenic molecules which have the power. The course of development
of the science in the past leads one to hope that the isolation of this
substance will ultimately be made, but if it is made it will be one of
the most fundamental discoveries in the history of science, for every-
thing indicates that this fragmentation of the foods is a preliminary
THE CONTRACTILE TISSUES. MUSCLE 625

to their whole metabolism and not alone to their oxidation. It is a
problem to be actively prosecuted by every means. Lepine and the
French and Belgian physiological chemists with characteristic genius
have most keenly attacked this fundamental problem. They have found
that not only muscle but blood and other tissues have the power of
making glucose disappear, but they have not shown that the disappeared
substance has been oxidized or destroyed. It may have been resynthe-
sized into maltose or some other substance having a lower reducing
power than glucose. From yeast a substance has been isolated which
does fragment the sugar molecule into carbon dioxide and alcohol.
This substance is zymase. Possibly a similar enzyme fragmenting the
sugar molecule in the same or slightly different way may exist in muscle.
Definite staterhents cannot yet be made, but the presence of small
amounts of alcohol in muscle tissue is held by some to be significant.
What, then, is the character of the fragmentation of the muscle
sugar? This question cannot be answered. By some it is suggested
that the glucose is fragmented by a process analogous to or identical
with the fermentation by yeast into alcohol and carbon dioxide and that
the alcohol is then oxidized to CO, and H,O. But, while small amounts
of ethyl alcohol have been recovered from muscle and alcohol is readily
burned in the body, it is not probable that the whole metabolism goes
in this direction. Lactic and succinic acid are produced during mus-
cular work and acetone in large amounts when work is done during
fasting. The fact that muscle has the power of methylating many sub-
stances, such as glycocyamine, would indicate that some formaldehyde
was formed during the process of fragmentation. The ready synthesis
of imidazole from pyruvic aldehyde and ammonia according to the reac-
tion on page 185 and the presence of considerable amounts of histidine
in muscle might suggest the formation of pyruvic aldehyde as an inter-
mediate substance. Nothing is certainly known of the character of the
first products of the fragmentation of the sugar molecule. The solution
of this problem is of the highest importance for understanding muscu-
lar contraction and for the treatment of diabetes mellitus. While it is
generally assumed that the fragmentation of the glucose molecule pre-
cedes its oxidation, it is not impossible that this view is incorrect and
that primarily an oxidation to an osone or other ketone occurs and that
this compound is then fragmented by some constituent of the muscle.
These partially oxidized sugar molecules would probably be far less
stable than the normal molecule, just as the partially oxidized fatty
acid molecules are less stable than the saturated, unoxidized molecules.
According to the view of Hermann. glucose is built into the nitrogen-
containing complex molecules of the cell; this is then oxidized, the carbo-
hydrate burned and the inogen muiecule, so called, is reconstituted, tho
626 PITYSIOLOGICAL CIIEMISTRY

nitrogen part being retained in the cell, so that the total nitrogen
excreted is not increased.

Lactic acid. Du Bois-Reymond found that muscle is normally alka-
line in reaction to litmus, but becomes acid when it works. The acidity
is very slight and not demonstrable if the muscle is well supplied with
oxygen and blood, but it becomes noticeable if the circulation is inter-
rupted and the muscle stimulated. The acid formed is in part the dextro-
rotary lactic acid, or sarco-lactic acid, CH,.CHOH.COOH. It is still
undecided whether lactic acid is formed exclusively from carbohydrate
or not, but the best evidence is that most of it is derived from ‘glucose.
It is usually removed, if oxygen is present, as rapidly as it is formed. It
was formerly thought to be oxidized, but Hill thinks it is resynthesized
into muscle substance. Any accumulation is thus prevented, unless the
entrance of oxygen is restricted. The development of acidity in muscle
can be made very strikingly evident by an experiment devised by
Dreser. If acid fuchsin is injected under the skin of a frog, it is car-
ried by the blood all over the body, but it will not stain the tissues and
muscles while these are alkaline. At best only a little superficial pink-
ness appears in parts of the connective tissue. After giving time for
this distribution, the blood supply of both legs is interrupted by ligating
the blood vessels and the sciatic nerve going to one leg is tetanically
stimulated for some minutes. If, then, the skin is removed from the
hind legs, it will be found that the muscle which has been stimulated
is intensely red, while the resting leg is at most a faint pink. If the
blood supply remains intact, stimulation of the muscle does not have
this effect, for the acid is neutralized and removed as rapidly as it is
formed.

The acidity of muscle may also be shown by immersing two gas-
trocnemius muscles in sodium chlorate, n/8 solution, and stimulating
one. Sodium chlorate in neutral, or slightly alkaline, solutions is not
toxic, but in the presence of only a little acid it produces rigor of the
muscle. The tetanized muscle will be found to go into rigor while the
other remains living.

Various mechanisms exist in muscle for the maintenance of its
neutrality. These mechanisms have already been discussed on page 247.
They are (1) the oxidation of the acid to very weak acid, such as CO,,
which will easily escape from muscle or combine with the amino groups
of the proteins. (2) The neutralization of the organic or mineral acid
produced either by sodium carbonate or sodium phosphate. By this
the neutral salt of the strong acid is formed and carbonic and NaH,PO,
being very weak acids are but little ionized; (3) there is a deamidiza-
tion of some amino-acid setting free ammonia which neutralizes the acid
and then the carbon part of the amino-acid is oxidized to CO, and H,0.
THE CONTRACTILE TISSUES. MUSCLE 627

Every acidosis is accompanied by a larger or smaller increase in ammonia.
It is reported that a working perfused muscle adds a little ammonia to
the perfused blood. This is probably greater when the respiration is in
any way reduced. (4) It is possible that other bases may be formed
also. Muscle has the power of forming various diamines such as
putrescine. These are formed by the splitting off of carboxyl from
the amino-acid. Such bases occur in muscle and they may be formed
in small quantities as part of the neutrality mechanism. They are cer-
tainly less important, however, than any of the other neutrality factors
mentioned. (5) Anotier mechanism which might have some importance
is the conversion of creatine to creatinine. The latter being the stronger
base has much greater powers of neutralizing acid than has creatine.
To what extent creatine thus plays a part in regulating the acidity of
muscle cannot be said. If it does play any considerable part in this
way, it would be an interesting example of how the balance of living
matter is preserved or, as Hering puts it, how the need produces the
satisfying of the need; of how, in other words, living matter shows its
wonderful powers of adaptation to circumstances. The acid creates the
base which saturates the acid.

Does the nucleus play a part in contraction? A very interesting but
entirely unsolved problem is the possible involvement of the nucleus
in the processes of contraction and energy metabolism. On stimulation
of the muscles of amphibia it is said that there is a fall in the total
purine nitrogen of the muscle of about 9-17 per cent. of that present
at the start (Scaffadi). Burian states that on stimulation the hypoxan-
thine at first increases. These facts, scanty as they are, indicate the
possibility that the nuclei are not the passive spectators of muscular con-
traction that they are often imagined to be. Perhaps they actively inter-
vene. Histological studies of the great cells of Necturus might throw
light on this problem.

The mechanism of the contraction—During contraction of muscle
carbohydrates disappear, heat is liberated, carbon dioxide is given off
and lactic acid appears. No change in the bodies containing nitrogen
is known to occur, except a slight increase in the ammonia and the
changes in the purines which have been described. "Work does not
increase the nitrogen output in the urine. We may now ask the ques-
tion: How is the contraction produced? Like all the other fundamental
questions of physiology. this question cannot be completely answered
at the present time. Indeed, we can do but little more than conjecture.
‘The suggestions which have been made are the following:

The machinery or contractile engine in the muscle is certainly pro-
tein in nature and doubly refracting. The muscle sarcostyles consist
of a verv dense or concentrated gel and these gel particles are in very
, 628 PHYSIOLOGICAL CHEMISTRY

small units, so that their surface is very large compared to the bulk.
Protein gels have the peculiarity of swelling greatly and very rapidiy
in the presence of acid. It has been suggested that the gel particles in
protoplasm are arranged mechanically in very fine fibrils and that a
more fluid gel or a liquid, the sarcoplasm, is about them. When the
nerve impulse sweeps over the muscle it causes in some unknown way
the combustion of the carbohydrate, which in part at least is probably
built into the machinery, and this combustion produces acids such as
lactic acid and succinic acid. Heat is liberated at the same time.
Under the influence of the heat and the acidity these gel particles at
once absorb water from their surroundings and swell. Being confined
in tubes, or the molecules being oriented in a certain way, as in a crystal,
the swelling does not take place uniformly in all directions, but they
broaden and shorten in length. They swell in a direction transverse
to the long axis of the muscle and thus cause a shortening in the other
direction. This produces the contraction. But almost at once the
lactic acid-is burned or recombined and the carbon dioxide escapes from
the muscle; the acidity is quickly reduced; the affinity of the con-
tractile elements for water returns to the normal; and water passes out
of the sarcostyles, which thus return to their normal size. The elastic
properties of the muscle, which Kite has described, help it to regain
its normal condition. On this view, then, the essential cause of the
shortening is the production of acid. Everything follows as a result of
this. Of course, the heat liberated will also assist in the process. There
are various additional circumstances which may be urged in support
of this view. If it is correct, then if the oxidation or removal of the
‘lactic acid could be prevented the muscle should remain permanently
shortened. Just this is what happens, according to many observers in
“‘ rigor mortis.’? Muscles after death go into a condition of extreme
contracture and hardness. They become rigid and this rigor persists
for some time. A muscle which has been stimulated for some time just
before death becomes rigid at death much quicker than one which has
been at rest. Certain poisons of the picric acid group, which cause
great fever in mammals, cause rigor to appear immediately after death.
If a little acid is added to a fluid which is being perfused through muscle,
its rigor comes on much earlier than normal; whereas, if alkali is added,
rigor is delayed or may be prevented entirely. The beginning of rigor
can be checked or entirely prevented by alkaline Ringer’s solution.
Furthermore, since oxygen in the presence of the muscle destroys lactic
acid or causes it to recombine, oxygen should prevent rigor, and such
is the case. If muscles are placed in atmospheres of compressed oxygen
so that a plentiful supply of oxygen is present all the time, they take
much longer to become rigid or they may not go into rigor at all. On
THE CONTRACTILE TISSUES. MUSCLE 629

the other hand, the muscles of frogs placed in hydrogen become rigid
unusually soon. All of these facts agree with the hypothesis that acid
is the direct cause of the muscle contraction and that in contraction we
are dealing with a swelling or imbibition phenomenon of protein col-
loids. Another fact which points in the same direction is that muscles
placed in salt solutions containing smali amounts of acid take up more
water and increase in weight much faster than if the muscle is in a neu-
tral salt solution.

On the other hand, there are certain objections which may be urged
to this view. The first objection is the very great speed of the con-
traction and relaxation of the wing muscles of insects. These may con
tract (bees) as many as 400 times per second. .It has seemed to many
physiologists that it is extremely unlikely that any such rapid imbibi-
tion and syneresis could occur. They have suggested as an alternative
view that the change can only be one which affects the surface of the
fibril, like a change in surface tension, so that the change, whatever its
nature, need not penetrate any distance. The insurmountable objection
to this view, or at least it seems insurmountable at the present time, is
that the interior of the muscle where these changes are taking place is
probably not a liquid, but is a gel and elastic and tough. (Perhaps more
of the nature of a liquid crystal.) It is impossible to suppose that the
phenomena of rapid change of form under the influence of surface ten-
sion can occur in such a solid material where the molecules are not free
to move readily. It is only in mobile liquids that surface tension changes
of form can occur. The objection that the rapidity of the process is
too great in insect wing muscles to permit of this explanation may be
met as Hofmeister met it, when he tried his first experiments in this
direction, by the reply that, if the elements are small enough, the imbi-
bition can take place very rapidly. The velocity is a function of the
amount of surface. Now it is just in these insect muscles that the
structure of the muscle has reached its highest perfection and the sar-
costyle elements are extremely small. On the other hand, histological
research has not yet been able to demonstrate clearly and in a convincing
manner that the necessary machinery really exists in muscle which is
required by this explanation.

Another suggestion was made by Englemann, who laid stress on
the fact that double refraction and contractility go together. He sug-
gested that the muscle fibrils absorbed water because of the heat of the
combustion. It is possible to make an artificial muscle by taking a cat-
gut-string, which is also doubly refracting, passing a wire about it
and suspending it in water or salt solution. Ifa current is sent through
the wire so that the water is warmed about the string, it at once takes
up water and contracts. On cooling it expands again. The phenomena
630 PHYSIOLOGICAL CHEMISTRY

of the contraction resembled in many particulars that of muscle. The
difficulty in this theory in the minds of many is the improbability of
the differences in temperature within the muscle in minute dimensions
being sufficiently great to account for the whole process, but undoubt-
edly the heat of the contraction does facilitate the contraction. There
is no contradiction between this view and that of imbibition due to acid.
Very great differences in temperature within minute distances may
momentarily occur, since temperature is only molecular kinetic energy.

It seems that the imbibition theory is the most probable theory of
muscle contraction which we have. It is very suggestive that the myosin
which is obtained from muscle is extremely sensitive to very minute
amounts of acid. Thus one of the best solvents of it is ammonium
chloride, which by hydrolytic dissociation produces small amounts of
atid. One reason why this explanation of muscle contraction is satis-
factory is that other living phenomena, such as those of secretion, may
be explained in the same manner. In secretion we have also, probably,
a rhythmical imbibition and syneresis on the part of the secreting cell,
only in this case the cell acts as a whole and when the water leaves the
protoplasmic gel it leaves it in a different direction from that in which
it entered it. There is thus a regular pumping of water through the
cell, such as occurs, for example, in the absorption of water from the
intestine, or in the secretion of liquid from the salivary and other glands.
In this case, too, acid may be at the bottom of the process. One could
picture the process of secretion as follows: Let us suppose that a nerve
impulse first impinges on the base of the cell and passes through it to
the lumen end. As it passes down the cell it causes an explosive wave
of oxidation by which acid is set free. This acid at once directly, or
indirectly, increases the affinity for water on the part of the cell colloids.
The protoplasm at the base of the cell first takes the water from the
capillary and the lymph space about it. It swells. The oxidation of
the acid in this part of the cell causes it to lose its affinity for water,
but the next succeeding segment of the cell is now acid and has a great
affinity for water so that it imbibes that which is lost from the base. The
water is thus handed on from one part of the cell to the next until it
reaches the lumen end of the cell, where it escapes more or less loaded
with soluble substances into the gland lumen.

Not only are secretion and muscle contraction shown to be identical
in principle upon this hypothesis, but other processes, such as ciliary
movement and even amceboid movement, can be explained in the same
way. By the production of acid at some point in the interior of an
amceba water is absorbed here, the protoplasm becomes more mobile or
fluid, the internal or osmotic pressure is increased and the firm ectosarc
yielding at some point it bursts and the fluid contents stream in this
THE CONTRACTILE TISSUES. MUSCLE 631

direction. Thus the ameba actually appears to flow through itself.
There are facts which indicate that the movements are not due to sur.
face tension, as they were once supposed to be, but that the beginning
of the process is in the interior. And if in a cilium either in the basal
granule or along the cilium there was a rhythmic absorption by one side
of the granule or cilium its bending might be understood. There is no
doubt that in red blood corpuscles the slight change of acidity produced
by the entrance of so weak an acid as carbonic acid is sufficient to cause
the corpuscle to swell. On losing carbonic acid it shrinks again. Per-
haps the sarcostyles act in the same manner.

But while so many facts find on this hypothesis a natural, simple
explanation, so far without serious contradiction, yet experience teaches
us to be cautious and the theory cannot be regarded as proved. It is
not proved that the change in acidity is sufficient to account for the
change in imbibition. It is rather at present to be looked upon as a
guiding hypothesis helping us to see and experiment. It must not be
forgotten that while acids do much, enzymes do more. Hydrochloric
acid alone will cause the swelling and solution of fibrin, but hydro-
chloric acid plus pepsin causes a much more powerful effect. It is, hence,
not improbable or unlikely that if the theory prove true in its essentials,
namely, that the contraction is of the nature of an imbibition of liquid
by an organized protein substance, it may be found that the imbibition
is not induced directly by the acid, but that this only indirectly, by set-
ting free enzymes which may cause a reversible change in the proteins,
affects the imbibition. Or it might be that the organized living matter
itself may in some other way have its affinity for water directly changed.
It might be, for example, that living matter itself was composed of
large colloidal aggregates, or biogens which were really the irritable sub-
stances. These biogens may be considered to consist of protein, oxygen,
carbohydrate and possibly lipin material, and to have a certain affinity
for water with which they are in union. On stimulation it might
happen that the oxygen combines with and burns the carbohydrate, and
by this process the biogen itself at once, by its change in composition,
might acquire a greater affinity for water and absorb more of the latter.
The biogen molecule at once reconstitutes itself, uniting with more oxy-
gen and carbohydrate, thus changing to its former water-holding power.
The irritable complex is re-established and relaxation occurs. Accord-
ing to this view, the essential explanation of the imbibition as the cause
of the contraction remains unaltered, but acid no longer plays the part
of the sole or chief cause. This view is in its essentials that of Hermann.
who proposed it many years ago; the hypothetical substance here called
a biogen being called by him ‘‘ inogen.’’ By the former hypothesis
the contractile substance is in the nature of a lifeless protein similar
632 PHYSIOLOGICAL CHEMISTRY

to so many connective tissue fibrils which absorb and lose water under
the influence of acid; by the latter view the contractile matter, while
still colloidal and having the properties of colloids, is the living
matter itself. The former view has a certain naiveté which is at
the same time its charm and its weakness. In it the toiling spirit of
life weaves the contractile web, remaining itself outside of and unaf-
fected by the fate of the web; while, according to the latter view, the
web is itself the organized living, self-perpetuating material which con-
tracts because of its physical properties. Ignorance does not permit, as
yet, a choice between these divergent views.

REFERENCES. Muscigs.

1. Lederer and Stotte: Composition of human and dog hearts. Biochem. Ztschr., 35,
pp. 108-112, 1910.

2. Argutinsky: Ueber die elementare Zusammensetzung des Ochsenfleisches. Archiv
f. d. ges. Physiol., 55, pp. 345-365, 1894.

3. v. Firth: General summary on the composition of muscle. Literature. Ergeb-
nisse der Physiologie, 1, Abth.. I, p. 110, 1902; Ibid., 2, I, pp. 575-606, 1903.

4. v. Firth: Archiv f. expt. Path. u. Pharm., 37, 1896, p. 389; Hofmeister’s
Beitriige, 3.

5. Stewart and Sollman: Proteins of muscle. Journal of Physiol., 24, 1899.

6. Winterstein: Physiological nature of rigor mortis. Literature. Archiv f. d. ges.
Physiol., 120, p. 225, 1907.

7. Fletcher: Relation of oxygen to the survival metabolism of muscle. Journal
Physiol., 28, p. 474, 1902.

8. Baglioni: Comparative chemical study of the muscle, the electrical organ and
blood serum of Torpedo ocellata. Beitrige z. chem. Physiol. u. Path., 8,
1906, p. 456-472.

9. Weyl: Physiology and chemical composition of electrical organ of Torpedo.
Zeits. f. physiol. Chem., 7, p. 541, 1883.

10. Buglia and Constantino: Urease in muscle of Scyllum catulus. Zeits. f. physiol.
Chem., 86, 1913, p. 137.

11. Gautier and Landi: Gas exchange of sterile muscle. Anaerobic respiration.
Maly’s Jahresbericht, 24, p. 410, 1895.

12. Wilson: Partition of water-soluble non-protein nitrogen. Jour. Biol. Chem., 17,
1914, p. 385.

13. Meyers and Fine: Creatine in muscle. Jour. Biol. Chem., 1913.

14, Hill: Oxidative removal of lactic acid. Jour. of Physiol., 48, 1914, p. x, Pro.
Phys. Soe.

15. Peters: Lactic acid and heat. Jour. Physiol., 47, p. 20, 1913.

16. Hill; Total energy available in isolated muscle kept in oxygen. Jour. Physiol.,
48, 1914. Pro. Physiol. Soc., xi.

17. Botazei and Guagriello: Constitution physique et les propriétés chimico-
physiques du suc des muscles lissés et des muscles striés. Archives internat.
de Physiol., 12, p. 409, 1912.

18. Galeotti: Condensation of amino-acids with formaldehyde. (Methylation in
muscle.) Biochem. Ztschr., 53, pp. 474-492, 1913.

19. Gulewitsch: Constitution of carnosin. Zeits. f. physiol, Chem., 73, 1911, p. 43.
20.

21.

22.

23.
24.

25.

26.

27.
28,
29,
30.
31.
32.
33.
"34.

35.
36.

37.
38.

39.

40.
41.
42.

43.
44,

45,
46.

47.

THE CONNECTIVE, OR SUPPORTING, TISSUES 633

Thunberg: Muscle respiration. Skan. Archiv f. Physiol., 25, 1911, p. 37.
Previous communications cited in this paper.

Jona: d-alanyl-d-alanine anhydride isolated from Liebig’s extract. Zeits.
physiol. Chem., 83, 1913, p. 458.

Locke and Rosenheim: Consumption of glucose by the mammalian heart. Jour-
nal of Physiol., 31, 1904.

Mosso: Die Ermtidung, Leipzig, 1891.

Contardi: Gazetta chimica Italiana, 42, pp. 408-412, 1912. Synthesis of phytin
from inosite and phosphoric acid.

Cabella: Creatine content of different kinds of muscles. Zeits. f. physiol. Chem.,
84, p. 29, 1913.

Embech: Succinie acid in meat extract and fresh muscle. Zeits. f. physiol.
Chem., 87, 1913, p. 145.

Jansen: Mytilite in muscle of mytilus. Zeits. f. physiol. Chem., 85, p. 231.

Siegfried: Carnie acid. DuBois Archiv f. Physiol. 1904, pp. 401-418.

Roaf : Lactic acid theory of contraction. Pro. Roy. Soc. London (B), 88, pp. 139-
150, 1914.

Scaffadi: Relation of purine bases of muscle during work. Biochem. Ztschr.,
30, pp. 473-480; 33, pp. 247-251, 1911.

Pekelharing: Nucleo-proteid of muscle. Zeits. f. physiol. Chem., 22, p. 245,
1896-7.

Buriam: Action of musele work on purine excretion. Zeits. f. physiol. Chem.,
43, p. 532, 1905.

Buglia and Constantino: Total amino nitrogen titrateable with formol in vari-
ous kinds of muscle. Zeits. physiol. Chem., 81, pp. 109-112, 1912.

Gulewitsch: Extractive substances. Carnosin. Zeits. physiol. Chem., 87, 1913,
p. I.

Arnold: Position of glycogen in muscle. Arch. f. Mik. Anat., 73, 1908-9, p. 265.

Maignon: Influence of hunger on glycoger in muscle. Journal de physiol., 10.
pp. 203-221, 1908.

Preti: Formation of acetone in muscle. Biochem. Ztschr., 32, p. 231, 1911.

Widmark: Chemical conditions necessary for preservation of normal structure
of cells. Skan. Archiv f. Physiol., 24, 1911, p. 339.

v. Fiirth and Leuch: Rigor mortis an acid swelling and not a coagulation.
Biochem. Ztschr., 33, 1911, p. 341.

Jensen and Fischer: State of water in living and dead muscle. Zeits. f. all.
Physiol., 11, 1910, p. 23.

Fletcher: Lactie acid formation in autolysis. Journal of Physiol., 43, 1911, p.
286.

Feldman and Hill: Influence of oxygen on lactic acid formation. Jour. of
Physiol., 42, 1911, p. 439.

Krimberg: Carnitine. Zeits. f. physiol. Chem., 53, p. 514, 1907.

Mendel and Leavenworth: Composition of embryonic muscle and nerve. Amer.
Jour. Physiol., 21, p. 9, 1908.

Henderson and Brink: Compressibility of muscle. Amer. Jour. Physiol., 21,
1908, p. 248.

Fischer: Analogy between absorption of water by fibrin and by muscle. Archiv
f. d. ges. Physiol., 124, p. 69-99, 1908.

Fahr: Sodium content of skeletal muscle of frogs. Zeits. f. Biol., 52, p. 72, 1909.

Constantino: Potassium, sodium, chlorine. etc., in muscle. Biochem. Ztschr., 37,
1911, p. 52; 43, p. 165, 1912.
CHAPTER XV.

THE CONNECTIVE, OR SUPPORTING, TISSUES. THE BONES.
CARTILAGE. TEETH. CONNECTIVE TISSUE.

The chemistry and metabolism of the supporting tissues of the body
present several points of general interest. In plants thtse tissues are
carbohydrate in nature. In the invertebrates the harder parts of the
supporting tissues are chitin, which is a polymerized acetylated glu-
cosamine and the composition of which has already been discussed on
page 825, It contains deposited in it large amounts of calcium
phosphate and carbonate, thus giving it its rigidity. In the verte-
brates these tissues are composed of an organic matrix which is
always of a protein nature and in it is often deposited more or less
inorganic material of the nature of phosphates and carbonates of
calcium and magnesium. The connective tissues consist of both
living and lifeless matter. These tissues are true tissues consisting
of cells, but these cells have formed a large amount of intercellular, .
protein, lifeless matter which is generally of a fibrous nature and always
tough and resistant to most reagents. There are several kinds of con-
nective tissue proper and we may distinguish the yellow connective
tissue, which occurs, for example, in the ligamentum nuche of the ox;
white connective tissue, of which the tendon of Achilles is a good exam-
ple; and reticular connective tissue found in lymphatic glands and else-
where in the body. The yellow elastic tissue contains elastic, tough,
yellowish fibers. It occurs not only in the tendons, but in the walls
of the blood vessels, in the lungs and elsewhere in the body. It consists
largely of a protein called elastin. The white fibrous tissue is also found
generally throughout the body in the connective tissues as well as in
the tendons of muscle. Bone also contains fibers of white tissue. Bone
is indeed connective tissue in which mineral salts have been deposited.
The character of the cells, and of course the character of the ground-
work in which the fibers are embedded, differ in the various connective
tissues. Cartilage is closely related to the white connective tissues. Our
knowledge of the composition of these structures is largely owing to the
work of Gies and his collaborators in this country and to Mérner.

1. Composition of white connective tissue. Tendo Achillis.—
The tendo Achillis consists chiefly of white connective tissue and its
composition is given by Buerger and Gies as follows:

634
THE CONNECTIVE, OR SUPPORTING, TISSUES 635

ComposiTIon oF TENDO ACHILLIS OF THE Ox.

 

 

 

 

Fresh tissue Dry tissue Ash
Constituents
Calf Ox Calf Ox Ox
Wee 4G6 Fee ie eee Chee ees Seeks 67.51% |62.870%
Solids: 33 sonanmeednds ees ev ears Se eee 32.49 |37.130
Inorganic matter ....... 6. c eee e eee e ees 0.61 0.470 1.88 | 1.266
So, Sa ik ko Sone WARE IS Se aa ae eee 0.031 |..... 0.084] 6.65
P,0, iUaseu ah tage suavetnstraueacuentose: Wass ore tensioner) Soares ao ee 0.039 |..... 0.106| 8.34
Oh din ch Modano wien te ueee s Sede ss Sad 0.147 |..... 0.397| 31.37
Organic matter .......... cc cece tenner eeee 31.88 |36.660 |98.12 |98.734
Fat (ether-sol. matter) ............220-- deat 1.040 |..... 2.801
Albumin, globin ...............ee ee eee eee ee 0.220 | .... | 0.593
Mucoid) ¢.ccisia'ed cee cea peeney rans ao aan eae 1.283 wee | 3.455
Bastin’ scincac oarcein ba vnaran Renee ees sce 2.633 ... | 4.898
Collagen (gelatin) ...........0..e.e0-ee- ---. [31.588 | .... [85.074
Extractives and undetermined ............. Batis 0.896 |..... 2.413

 

 

 

 

 

 

2. Composition of yellow connective tissue. Ligamentum nuchae.
—The ligamentum nuche of the ox consists for the most part of yellow
connective tissue and its composition was found by Vandegrift and Gies
to be as follows:

CoMPOSITION oF LIGAMENTUM NUCH orf THE Ox. IN PER CENT.

 

 

 

 

Fresh ligament | Dry ligament Ash
Constituents
Calf Ox Calf Ox Ox
Water chine engine eee ee aes se nee eee 65.10 |57.570)
Solids) .so.i0o2.5 Ssieae Gas iat le Ae oes 34.90: | 42.430
Inorganic matter .........c eee e sence eee eeee 0.66 | 0.470) 1.90 | 1.10u
So, beeen eee teen een enn e ete teneeeee tele nees 0.026].... | 0.062] 3.64
P,0, satan ava cern aera sania a oe aad arcane veces | 0.035].... | 0.081] 7.39
Ol ng ee sate beans beatae PAS Seana lone 0.136).... | 0.318/28.95
Organic matter ........ 6c cece eee eee eee 34.24 |41.960/98.10 |98.900
Fat (ether-sol. matter) ..........0e.ceeeeeee[eceeee 1.120)..... 2.640
Albumin, globulin .......... 0. cece eect cece leeeeee 0.616]..... 1.452
MAU COTG: 5.5. Siees ore neeid.c emer Aabana eon & bativere d4. ates alens Gore 0.525}..... 1.237
Las Cine ce disses cce verso ace Geena se tse ah ee Gudoniavel ig Sassee fates a2 .|31.670]..... 74.641
Collagen (gelatin) ........... cece eee cece eleneeee 7.230]..... 17.04C
Extractives and undetermined ............0ccee}eceees 0.799]..... 1.883

 

 

 

 

 

By an inspection of the foregoing tables it will be seen that the
greater part of the dry matter of the tendons consists of protein material
and that the per cent. of inorganic matter is low. The dry matter in
each case consists of from 1-3 per cent. of mucoid. In white elastic tissue
85 per cent. of the dry matter consists of collagen which yields gelatin
636 PHYSIOLOGICAL CHEMISTRY

on heating with water; in the yellow elastic tissue, on the other hand.
there is only some 17 per cent. of collagen in the dry matter, but
the greater part of the dry matter, namely 74.64 per cent., consists of
elastin.

The composition of the mucoid from the tendons has already bee::
discussed on page 324. This mucoid is obtained by cutting the tendon
or ligament into small parts and then boiling with half-saturate:!
Ca(OH), and precipitating the mucoid by acidification with acetic acid.
This mucoid differs from true mucin apparently in several properties.
It does not form the very stringy, sticky masses characteristic of mucin;
it is less soluble in 0.1 per cent. HCl; and it contains about twice as
much sulphur. The per cent. of sulphur in tendon mucoid is about
2.3 per cent., whereas in submaxillary mucin it is a little less than 1 per
cent. Moreover, the mucoid of tendon contains chondroitie acid which
relates it to the constitution of cartilage. Mucin and mucoid resemble
each other in the fact that both yield a reducing sugar, either glu-
cosamine or galactosamine, on hydrolysis. Both are glycoproteins. It
would, perhaps, be well to call these mucoids chondroproteins, as Ham-
marsten does, to indicate that they contain a conjugated chondre‘‘ic
acid. This acid will be discussed presently.

The composition of the gelatin, or collagen from which it is derived,
has already been given in the table on page 129 and on page 110. It is
remarkable, first, for its powers of gelatinization. It is a protein which
contains a very large amount of glycocoll and which lacks tyrosine and
tryptophane. Collagen yields with tannic acid an insoluble resistant
substance, which does not putrefy readily, and this peculiarity is the
basis of the tanning of leather.

Elastin is a very resistant, elastic protein, of which the composition
is given on page 129. It consists of at least 25 per cent. of glycocoll and
differs from collagen in the very much larger proportion of leucine in
it, namely 21.38 per cent., as contrasted with 2.1 per cent. The three
simple amino-acids, glycine, alanine and leucine, make over 50 per cent.
of the molecule. Elastin is easily digested by pepsin. It contains 0.1-
0.3 per cent. 8, but none which is split off by alkali.

Elastin is readily prepared from the ligamentum nuche. It is first
thoroughly extracted with 5 per cent. NaCl under thymol for 3-4 days
to remove albumins and globulins, and then repeatedly boiled with water
to convert all the collagen into gelatin and remove it, so that the filtrates
give only a very slight turbidity with tannic acid. The undissolved
residue is thoroughly washed with water, extracted with alcohol and
ether and dried at 110°.

Both of these tissues yielded some extractive matter. Creatine and
purines were recognized qualitatively in the extractives.
THE CONNECTIVE, OR SUPPORTING, TISSUES 637

The general composition of several connective tissues is given in the
following table compiled by Buerger and Gies:

 

 

 

 

Tendon Ligament r
Constituents ———| “ous” | Saris | with [“ttebne.
Calf Ox | Cale Ox |humor| lage |marrow| kidney
Fresh tissue
Water inetsssentsescanes 67.51-| 62.87 | 65.10] 57.57 | 98.64 | 67.67 | 50.00 | 4.30
SOlidS) oasis essed cogeioes 31.88 | 37.13 | 34.90] 42.43] 1.36 | 32.33 | 50.00 | 95.70
Organic .........00.000% 0.61 | 36.66 | 34.24] 41.96] 0.48 | 30.13 | 28.15 | 95.51
Inorganic ...........055 32.49} 0.47| 0.66] 0.47] 0.88] 2.20| 21.85) 0.19
Dry tissue
Organic ........eeseeeee 98.12 | 98.71 | 98.10 | 98.90 | 35.29 | 93.20 | 56.30 | 99.80
Inorganic .............. 1.88} 1.29] 1.90] 1.10] 64.71] 6.80 | 43.70] 0.20

 

 

 

 

II. Composition of cartilage.——Cartilage forms the internal skele-
ton of the lowest or cartilaginous fishes and in the higher forms it is
generally laid down first and is later changed into bony tissue. In the
cartilages of the trachea, and in particular of the larynx, it never under-
goes transformation to bone, but remains cartilage throughout life. Car-
tilage is not found in any of the invertebrates, except in the cephalopods
and the arthropods. The cartilage of these forms is, however, related
to chitin rather than to true cartilage.

Histology. Cartilage consists of cells which are embedded in a
homogeneous, or slightly fibrous, matrix. This matrix has in it some
blood vessels for the nourishment of the cells and channels for the pene-
tration of food materials to the cells. But the amount of blood supply
must be extremely little and the penetration of the lymph would appear
to be very slight. Most of the food materials find access to the cells pos-
sibly by soaking through the intercellular substance. This intercellular
substance is secreted or formed by the cartilage cells. The cartilage cells
arise from the mesodermal layer in embryonic development.

Chemistry. If cartilage is cut into small pieces and boiled in water
for a long time, or, better, heated in water under pressure, the organic
matrix of the cartilage slowly ‘dissolves and there is left a mass of cell
bodies and connective tissue fibers and blood vessels. The cell bodies
consist, for the most part, of coagulable protein so that they are resistant
to this treatment. By a careful study of the organic matrix MGrner
found that it consisted, or rather that it yielded on decomposition, the
following substances:

1. Chondromucoid.

2. Collagen.

3. Albuminoid.

4. Chondroitic acid.

5. Inorganic salts.

The chondromucoid is in all essential respects like that isolated by
638 PHYSIOLOGICAL CHEMISTRY

Gies and Buerger from tendons and it has already been described.
Chondromucoid has the composition C, 47.30; H, 6.42; N, 12.58; S, 2.42;
O, 31.28. It contains sulphur both in an oxidized and an unoxidized
form. A part is split off as sulphide when boiled with alkali, a part as
sulphate. This chondromucoid yields chondroitic acid. It is a white,
acid-reacting substance insoluble in water, but soluble in very dilute
alkali and precipitated by acetic acid. It is precipitated by iron alum.
lead acetate and ferric chloride, but it is not precipitated by tannic acid.
It gives the usual reactions of the proteins.

2. Chondroitic acid, or cartilage acid, is sometimes called chondroit-
sulphuric acid, but as chondroitic acid is shorter and was the name first
given, it should be adopted and the awkward name of chondroit-sulphuric
acid dropped. This acid has already been described in part. It will
be recalled that it splits on hydrolysis into sulphuric acid, a hexosamine
(chondrosamine), acetic acid and glycuronic acid. It is a paired sul-
phate. Some of it is apparently free in the cartilage or attached through
bases to the protein, since it may be extracted easily from the cartilage
by Morner’s method by extracting with alkali and removing the proteins
by neutralizing and precipitating the peptone by tannic acid. The salts
of chondroitic acid are nearly all soluble. The composition of chondroitic
acid is not yet definitely settled, the statements in the literature still
being somewhat contradictory. (Fraenkel, Annal. d. Chem. u. Pharm.,
351.) It will be recalled that the composition of this acid is of consid-
erable interest, since the presence in it of an acetylated hexosamine allies
it at once with chitin. It is either identical with, or very similar to, the
glucothionic acid isolated from tendomucoid by Levene. A_ possible
graphic formula is given on page 325.

3. Albuminoid.

Chondroalbuminoid is very closely similar to, but not identical with,
the osseoalbuminoid of bones. It is the albuminous substance which
remains after extraction of the osseoalbuminoid and the nucleoproteins,
and the prolonged treatment of the cartilage with hot water. It is sol-
uble in boiling 0.1 per cent. KOH and also in boiling 5 per cent. KOH.
By the former it is hydrolyzed into various derived products. This sub-
stance is present in the cartilage in very small amounts. It is believed
to form the lining of the tubules in the cartilage and bones. By some
authors it was supposed to resemble keratin, but it is more closely related
to elastin. It was prepared in the following way, according to Hawk
and Gies:

The cartilaginous portions of the nasal septum of the ox were used.
Several pounds of these were hashed in a machine after removal of the
outer membranes; the hash washed in running water; mucoid, nucleo-
proteins, etc., were extracted by several treatments with dilute alkali
THE CONNECTIVE, OR SUPPORTING, TISSUES 639

after a preliminary treatment with 0.1-0.2 per cent. HCl; the alkali
washed out and the residue hydrated in boiling water for several days.
The final product was extracted first with 0.1 per cent. sodium carbonate
and then 0.5 per cent. HCl in which it was insoluble. The albuminoid
remains undissolved. The composition of this substance and that
derived from bone, or osseoalbuminoid, was as follows:

Cc H N Ss oO
Chondroalbuminoid ...............004. 50.46 7.05 14.95 1.86 26.86
Osseoalbuminoid ........... 0c cece eee 50.16 7.03 16.17 1.18 25.46

The greater proportion of the organic matrix of the cartilage is collagen,
but I have been unable to find any quantitative determination of the
amount present.

III. The bones.—The bones also consist of cells and intercellular
substance, but as in cartilage the intercellular substance is far in excess
and determines the character of the tissue. The intercellular substance
is formed by the cellular. It consists of two parts: of an organic basis
of an albuminous nature in which a great amount of inorganic matter is
deposited. The cells, it will be recalled, have to be nourished and the
nourishment is arranged for by a series of channels, the cells being
grouped in concentric rings about the blood vessels and having fine
branching channels penetrating the bone in all directions.

The organic intercellular substance. This resembles strongly the
organic matrix of cartilage. It consists of osseomucoid, the composition
of which has just been given ; and of osseoalbuminoid, which is present in
only small amounts, and which is almost identical with the similar
substance in cartilage and is supposed to be the lining of the Haversian
canals. The greater part of the organic matter here as in cartilage and

-in white connective tissue is collagen, which yields gelatin on cooking
with water.

The bones are many of them hollow and contain marrow in their
centers. This marrow is of two kinds, being in part yellow in color
and consisting of a large proportion of fat; and in part it is red mar-
row, the red color being due to the presence of a large number of red
corpuscles in the erythroblasts.

The composition of the osseomucoid and some other mucoids is given
in the following table taken from Cutter and Gies:

Cc H N s 0

Chondromucoid (Mérner) .............. 47.30 642 12.58 2.42 31.28 .
Tendomucoid (Chittenden and Gies) ..... 48.76 653 11.75 2.33 30.63
Tendomucoid (Cutter and Gies) ........ 47.47 6.68 12.58 2.20 31.07
Osseomucoid "(Hawk and Gies) ......... 47.07 6.69 11.98 2.41 31.85

The composition of the gelatin from bone was found by Fremy to be
C, 50.0 per cent.; H, 6.5 per cent.; N, 17.5 per cent.; O and S, 26.0 per
cent. The following table is given by Richards and Gies:
640 PHYSIOLOGICAL CHEMISTRY

: Cc H N 8 ce)
Ligament gelatin (Richards and Gies) .. 50.49 6.71 17.90 0.57 24.33
Tendon gelatin (Van Name) ........... 50.11 6.56 17.81 0.26 25.24
Commercial gelatin (Chittenden and Solly) 49.38 6.81 17.97 0.71 25.13

The inorganic constituents of bone. These make about 40 per cent.
of the dry residue of the bone, 60 per cent. being organic. In bone with-
out the marrow the relations are just about reversed. The inorganic
material is chiefly calcium phosphate and carbonate, but there is 4
little magnesium and there is also present a trace of fluoride and chloride.
The composition of the inorganic materials is about as follows:

Calcium phosphate ......... ccc cece eee ene eeeeee 85%
Magnesim phosphate ......... 0: ccc eee ee ee tent re ceeece 1.5
Calcium: fluoride ..2). cneae sac ccude vs case sees sae ees 0.3
Caleium chloride ......... 0... cece eee eee ener 2 acer 2, wis eters 6.2
Caleium carbonate .......... cece c eee cee eee eee neees 10.0
Alcala Ball tis? gacatiie cae asenalibenn ah cavtecrend a iactanties soe ualaba lace dleyarece’ (6 2

Hoppe-Seyler pointed out that the relation of Ca to phosphoric acid is
about the same as in apatite, namely, 10 Ca:6PO,. There are about
three molecules of tricalcium phosphate to one molecule calcium car-
bonate. The relationships remain the same, although the bone may be
gaining or losing its inorganic compounds.

Practically nothing is known concerning the nature of the processes
by which the salts are deposited in the organic basis of bone. It seems not
impossible that the conditions are such as to cause the precipitation of
this double phosphate and carbonate here,—or else that either the phos-
phoric acid or the calcium is united by one bond to the organic matrix,
while with the other they unite with phosphoric acid or calcium brought
from the blood so as to precipitate and so hold it. The attempt has been
made to explain the deposition on the basis of an adsorption, but the
relations are so regular, quantitatively, that it seems perhaps as probable
that the union is a true chemical union. It is possible that the deposi-
tion of calcium phosphate and carbonate in bone is due to ammonia
liberated in that tissue. If this is the case the deposition of these salts
would appear to be due to the same processes as are responsible for their
deposition in the sea, geologically. In the lowest forms of life and in
plants the skeletal hard parts are generally calcium silicates; above these
in the invertebrates, both in shells and internal skeleton, we have mainly
carbonate with a little phosphate; in the higher forms we have princi-
pally phosphate and only a portion of carbonate. These salts are in
equilibrium with the calcium carbonate and phosphate of the blood. Too
small an intake of calcium or an acidosis leads to the impoverish-
ment of the liquids of their calcium and the salts are redissolved
from the bones. The bones are not dead, inert structures, but
living plastic material which is moulded by its position, stresses and
environment.
THE CONNECTIVE, OR SUPPORTING, TISSUES 641

The growth of the bones is in some way dependent upon the internal
secretion of both the thyroids and the hypophysis. In case the hypophy-
sis is extirpated in puppies, the bones do not grow normally, they remain
in the state of youth and the epiphyses remain open. Normal growth
of the bone is absent if the thyroids do not develop properly. The bones
are also closely related to the reproductive organs. Castration of young
animals nearly always causes the production of large-boned animals;
and in osteomalacia of women it is not uncommon to remove the ovaries,
the condition of the bones changing at that time. Changes occur in the
bones and teeth in pregnancy. In fact, the bones are by no means a
fixed tissue. They are alive with their own metabolism and the calcium
may be laid down or again removed. We cannot for lack of time enter
here into the work which has been done on the pathological changes
which occur in bones and the attempts which have been made to produce
rickets or other bone diseases artificially. That the proper formation
of both bones and teeth is dependent on a proper supply of calcium in
the diet is perhaps self-evident. How largely the composition of the
hones may be affected by a change in the amount of calcium in the diet
is shown in the following figures from Rohloff:

In growing dogs fed one on calcium rich, the other on calcium poor
food, the composition of the shoulder blade was as follows, the amounts
is shown in the following figures from Rohloff:

Diet Dry substance Ash CaO Phosphoric acid
Ca poor. Shoulder blade ...... 2.678 1.178 0.638 0.499
Ca rich. sf Se Cadets ens 9.021 5.240 2.861 2.211

Similar and more extensive figures are given by Aron and Sebauer.

Teeth.—Teeth are composed of several parts: namely, enamel, den-
tine and cement. The cement is identical with bone in its structure
and composition. The dentine makes the greater part of the tooth and
in its chemical composition is practically identical with bone, although
its structure is somewhat different. The enamel, however, is the product
of an epithelial tissue. It is the hardest substance in the body and con-
tains the smallest per cent. of water. It contains about 5 per cent. of
water. The composition of the enamel of human and calves’ teeth is
as follows (Bertz) :

Human Calf
Organic substance . 16.56
CaO 44.24
MgO . 0.96
PO, a 37.02

 

91 per cent. consists, therefore, of calcium phosphate. It is extraordi-
nary to what widely different uses this interesting and common mate-
rial, calcium phosphate, is put in the body. Here we find it called on
to make the hardest and most resistant structure of the body, while it
642 PHYSIOLOGICAL CHEMISTRY

was but a short time ago that we found it assisting in the coagulation of
the blood and in activating the enzymes, invertin and trypsinogen.

REFERENCES. Connective TISSUES.

1. Oswald: Ueber Myxomucin. Zeit. physiol. Chem., 92, p. 146, 1914.

2. Levene: Preliminary communication on the chemistry of mucin. Jour. Amer.
Chem. Soc., 22, p. 80, 1900.

3. Hawk and Gies: Chemical studies of osseomucoid, with determination of the
heat of combustion of some connective tissue glucoproteids. Amer. Jour.
Physiol., 5, p. 388, 1901.

4, Vandergrift and Gies: The composition of yellow connective tissue. Amer. Jour.
Physiol., 5, p. 288, 1901.

5. Richards and Gies: Chemical studies of elastin. mucoid, and other proteids in
elastic tissue, with some notes on ligament extractives. Amer. Jour. Physiol,
7, p. 93, 1903.

6. Richards and Gies: Methods of preparing elastin, with some facts regarding
ligament mucin. Amer. Jour. Physiol., 5, 1901, p. xi.

7. Gies and Cutter: The composition of tendon mucoid. Amer. Jour. of Physiol., 6,

p. 155, 1901.

Hawk and Gies: On the composition and chemical properties of osseoalbuminoid,
with a comparative study of the albuminoid of cartilage. Amer. Jour.
Physiol., 7, p. 340, 1902.

9. Posner and Gies: Do the mucoids combine with other proteins? Amer. Jour. of

Physiol., 11, p. 340, 1904

lv. Seifert and Gies: On the distribution of osseomucoid. Amer. Jour. of Physiol.,
10, p. 146. 1903.

ll. Buerger and Gies: The chemical constituents of tendinous tissue. Amer. Jour
Physiol., 6, p. 219, 1901.

nN
CHAPTER XVI.

THE CRYPTORRHETIC TISSUES. THE THYROID. PARATHY-
ROLD. SUPRA-RENAL. HYPOPHYSIS. REPRODUCTIVE
GLANDS.

The cryptorrhetic tissues and organs.—There are quite a number of
organs in the body which do not play a part in movement and, while
they are more or less glandular in nature, they either have no ducts for
the discharge of their secretions to the exterior or, if they have such
ducts, there is abundant evidence that they form a hidden secretion
which is passed back to the blood. Of course all organs form such
substances which come back to the blood, but in the cryptorrhetic organs
there are ‘reasons for thinking that the formation of such substances is
their main function. They are sometimes called glands of internal
secretion or endocrine glands (G. endon, within; krino, separate). I
have given them the name ‘‘ Cryptorrhetic organs,’’ from two Greek
words—kryptos, concealed; and rhoia, flow. They are the tissues of hid-
den flowing. Among these organs the thyroid, parathyroids, ovaries and
testes, pancreas, hypophysis and supra-renals are the most important.

THE HYPOPHYSIS.

Among the organs more particularly designated as those of internal
secretion, the hypophysis is certainly among the most interesting and
important.

Structure.—In human beings this organ is about as large as a pea,
lying at the base of the brain with the optic chiasma just in front
of it. The hypophysis cerebri (G. hypo, below) is also often designated
as the pituitary body. It is connected with the third ventricle of the
brain in the thalamus by a narrow stalk called the infundibulum, which
is continuous with the epithelium lining the ventricles. The hypophysis
proper is easily separated into three main parts: an anterior lobe, com-
posed of glandular tissue and which is the larger part; a posterior lobe.
composed of nervous tissue, and a pars intermedia, which resembles
the infundibulum (?). These different parts have quite different struc-
tures, compositions and functions.

Auteriorly, the hypophysis is closely adherent to the tuber cinereum
of the brain, and its separation from this in operations is a matter of
great difficulty. This fact greatly complicates operations on the hypophy-
sis, since any injury to this part of the brain is very serious.

643
644 PHYSIOLOGICAL CHEMISTRY

By means of the infundibulum the organ is in direct connection with
the brain ventricles, its secretions, if any, may be discharged into them,
and when cut the ventricular fluid may escape from it. The hypophysis
is thus essentially a downgrowth from the central nervous system toward
the palate.

Situated as it is in the oldest part of the nervous system, it is not
surprising that this organ, both phylogenetically and embryologically,

 

Fie, 57.—Under surface of the dog's brain showing the hypopbysis, Hyp. V., jusi
behind the optic chiasma (Aschner).
is found to be very old. It is found in all the vertebrates without excep-
tion, except possibly in amphioxus, in which indeed the glandula sub-
neuralis is supposed by some to be homologous with it. Even in the
invertebrates a somewhat similar tissue has been described in worms.
mollusks, and even in Echinoderms, although whether this tissue is
homologous in nature and analogous in function is quite unknown.

Embryologically the organ is derived in part (posterior lobe) from
the nervous system by a downgrowth of the floor of the third ventricle,
and in part from the alimentary canal (anterior lobe) by a proliferation
of cells from the dorsal side of the buccal cavity. It arises very early
in embryological history. From all these facts it is evident that we have
to do with a very old organ and one probably correlated with the most
fundamental processes of the body.

The phylogenetic explanation of this organ generally accepted is
that formerly the neural canal connected at this point with the alimen-
THE CRYPTORRHETIC TISSUES 645

tary canal. A probable and almost the only explanation. of this, though
an explanation almost universally rejected by zodlogists, is that of Gas-
kell, who has maintained that the vertebrate alimentary canal is a new
structure, and that the old invertebrate alimentary canal is the present
neural canal. The infundibulum, on this view, would correspond to the
old invertebrate cesophagus, the ventricle of the thalamus to the inverte-
brate stomach, and the canal originally connected posteriorly with the
anus. The anterior lobe of the pituitary body could then correspond to
some glandular adjunct of the invertebrate canal, and the nervous part
to a portion of the original circumesophageal nervous ring of the inverte-
brates.

The hypophysis has always, even from the time of Aristotle, attracted
attention and many guesses were early made as to its nature, some of
them very near the truth. Thus its connecticn with the third ventricle
led Diemerbroeck in 1686 to the conclusion that it made a secretion
which was poured into the third ventricle, a view on the whole sub-
stantiated by modern work; another view was that it was the source of
the cerebro-spinal fluid; another that it secreted the cerebro-spinal fluid
into the blood. Engel called attention in 1839 to the pathological cor-
relations between the hypophysis, pineal gland and the thyroid and to
the commonness of pathological changes in the sexual glands when these
glands were diseased. He called the hypophysis a secreting gland. The
American-French physiologist, Brown-Séquard, in 1869 definitely placed
it among the organs of internal secretion analogous to the thyroid. This
he did because of its structural resemblance (presence of colloid) to the
thyroid, a resemblance now known to be physiological as well as
anatomical,

The modern view of the function of the pituitary dates mainly, how-
ever, from the work of Pierre Marie and Marie and Marinesco in 1886-
1889. Marie, studying the disease known as acromegaly, a form of
gigantism of the extremities (Gr. akron, extremity ; megas, large), called
attention to the invariably pathological state of the hypophysis in this
disease and in true gigantism, and concluded that the abnormal hypophy-
sis was the primary cause of the growth changes involved in acromegaly.
A very large amount of work has since then been done by pathologists
which has confirmed Marie’s findings; though opinions are not unani-
mous whether the connection between the glands and the bones and other
growing tissues is direct, or indirect through its influence on the
thyroids, thymus and sexual organs. Pathologists have also shown that
in several true dwarfs the gland is rudimentary or almost lacking. The
relation to the sexual organs is very close. Thus during pregnancy the
hypophysis undergoes a characteristic metamorphosis, in common with
the thyroid and some other organs.
646 PHYSIOLOGICAL CHEMISTRY

The pathological correlation between the hypophysis and disturb-
ances of growth, aud in the sexual organs (dysplasia adiposo genitalis)
in the deposition of fat, many years ago led to the inference that the
hypophysis was concerned in the regulation of the growth and
metabolism of the body, but it was impossible to say whether it inter-
vened through the nervous system, or directly by an internal secretion.
The general conclusion was the latter and that it was the anterior lobe
which particularly intervened in growth. The exact function of the
organ could only be determined by experiment.

Such experiments were early undertaken, but, owing to the great
difficulties of operating, the evidence obtained has been, until very
recently, contradictory and confusing. The experimental difficulties
arise from the inaccessibility of the organ; from the great difficulty of
freeing the gland from the brain without injury to the tuber cinereum
and surrounding parts, since a very slight injury here may be fatal;
to the contraction of scar tissue putting tension on these parts which may
result in killing the animal after several months; in part to the danger
of too great loss of spinal fluid and fatal results from opening the third
ventricle; and in part also to the danger of infection. The resistance
to infection, particularly to skin infections, is said to be somewhat re-
duced and animals not infrequently die from these infections or from
pneumonia.

Many of the symptoms which were obtained in the early experiments
were undoubtedly due to injury to the brain, rather than to absence
of the hypophysis. In nearly all of the early experiments extirpation of
the organ was followed by the death of the animal within two to three
days or weeks; and death was accompanied by pronounced nervous symp-
toms: cramps, coma, etc. A few animals lived without any apparent
change for several months; but in these instances it was impossible to
know whether the survival was due to some tissue being left in. Thus
in Vasale and Sacchi’s experiments the gland was destroyed either by
thermocautery or acid. The animals, cats or dogs, always died in a
few days, or hours, showing psychic depression, apathy, motor disturb-
ances, fall in body temperature, polyuria, cramps, polydipsia, anorexia
and loss of weight. Paulesco and Horsley got practically the same
results. These symptoms are now known to be due to injury to the
brain tissue, not to the hypophysis. On the other hand, Kreidel and
Biedel in 1898 and Friedman and Mass conclude that animals can live
a long time without an hypophysis. The American surgeon, Cushing,
was one of the first to prove that definite metabolic changes occur after
the partial extirpation of the anterior lobe of the hypophysis in dogs,
particularly in young dogs. There was a great increase in fat
in the body everywhere, fatty degenerations of the internal organs,
THE .CRYPTORRHETIC TISSUES 647

and, most instructive, in young animals there was a persistence
of the thymus and a rudimentary condition of the sexual organs,
a persistent infantilism. These results of Cushing’s have been
confirmed by a very careful recent study of Aschner, who has so
perfected the method that most of his animals survive for long
periods. His results are briefly as follows and they are illustrated in
Figure 58.

Aschner found that in adult dogs extirpation of the gland was not
followed by any marked disturbances of health. The genital glands
showed more fat than normal (dysplasia adiposo genitalis) ; there was
a decided tendency to fatness; the vital resistance was reduced. Most
animals died of pneumonia or buccal infections. There was no marked
atrophy of the sexual organs, such as Cushing described in his dogs.
If the hypophysis was extirpated during pregnancy, abortion always fol-
lowed after 2-3 days. The only marked change noticed in metabolism
was a decrease in the nitrogen excreted per kilogram body weight on
the third to fifth day of fasting as compared with the normal animal,
and a marked change in the reaction to adrenaline. The whole sympa-
thetic nervous system seemed depressed. There was no diuresis, very
little glycosuria and no mydriasis or other reaction to adrenaline, such as
occurs in normal dogs. This is illustrated in the following experiment
which is taken from Aschner’s work. There was always a decrease in
oxygen consumed per hour. Normal dogs of 4-5 kilos on the third or
fourth fasting day give off 0.4-0.45 gram of nitrogen per kilo per 24
hours; whereas dogs with the hypophysis out excreted only 0.22-0.27
gram per kilo, a reduction of fully one-third in the nitrogen metabolism.
The amount of sugar excreted after the injection of 2-3 c.c. of adrenaline
subcutaneously in a dog of 4-6 kilos on the third or fourth hunger day
is about 3 grams per day; whereas after hypophysis extirpation adrena-
line caused only traces of sugar to appear in the urine. The following
experiment illustrates this:

 

 

 

 

 

 

 

 

 

 

 

 

u N in 24 hours
ene lowe. Temp. bear Dextrose
e In grms.
Total Hourly per kilo f

Ist period 15 5.5 39.6° | 127 ¢.c.| 2.891 | 0.193
2d “ 10 ee asia 98 1.336 0.134
3d“ 23.5 5.45 38.7 116 2.482 0.106 aoa,
4th “ 24.5 5.3 38.9 92 1.888 0.080 0.362
5th “ 24* 5.25 38.5 63 1.953 0.081 0.372 waves
6th “ 14 aye ive 121 1,126 0.080 0.364 3.184
7th “ 9 5.3 39.7 55 0.620 0.069 0.312 0.196

 

 

* At end inject 4.c.c, adrenaline,
648 PHYSIOLOGICAL CHEMISTRY

5 days later extirpated the hypophysis. Observations begin 9 days after extirpation.

 

 

 

N in % hours
aoe | Wt. Temp. ae haem Dextrose
Total Hourly per kilo.

1st period 14 5.4 39.1 106 2.017 0.144 oer ee
2a 9 ais oe 64 0.964 0.107 weed sities
3d“ 25 5.3 38.7 88 1.722 0.069 brecie selec
4th “ 24 5.25 38.9 84 1.114 | 0.047 | 0.214 | ....
5th “ 23* 5.1 38.5 57 1.018 0.044 0.208
6th “ 14 ois eavda |) 65) 0.678 0.048 0.232 Traces
7th “ 13 5.0 39.7 60 0.568 0.044 0.210 ee

 

 

 

 

 

 

*40c.c. adrenaline at end.

The temperature of the dogs was always about one degree lower than
the normal. The longest period the dogs were kept alive was 16
months.

The most interesting changes were in young animals. If the anterior
lobe alone, or the whole organ, is extirpated in young dogs from 6-10

 

 

Fie. 58.—Two dogs of the same litter about ten months old, the smaller having had
the hypophysis extirpated eight months earlier (Aschner).

weeks old, a remarkable cessation of growth takes place, as shown in
Figure 58, which represents two dogs from the same litter, the smaller
one having no hypophysis. The dogs do not grow except in fat; they
keep their puppy hair and milk teeth, although a second set develops very
late; the genital organs, testes, ovaries, uteri, prostates, etc., remain in
the infantile condition or in a condition many months behind the normal.
See Figure 59. The thymus persists much longer; the intelligence also
remains subnormal. Toward adrenaline they show diminished power of
THE CRYPTORRHETIC TISSUES 649

response. If the operation takes place after 10 weeks of age, the re-
tardation of growth is still marked, but not so extraordinary as when
performed on younger dogs.

It seems, therefore, from Cushing’s and Aschner’s experiments and
the pathological evidence, that the hypophysis, like the thyroid, is nec-

 

Fie. 59.—Section of the testis of normal dog at ten months of age. Notice the
aumerous sperm (Aschner).

essary for the growth and development of animals. The body weight, the
development of the sexual organs, bones, etc., may be called secondary
characters of the anterior lobe of the hypophysis.

The experiments of feeding or injecting extracts, or preparations,
of the anterior lobe of the gland (Pituitrinum glandule) confirm these
results. Schaefer has obtained a slight increase of growth by feeding
such tablets to normal puppies; Magnus Levy and Solomon reported an
increased gas exchange after taking hypophysis tablets; Falta found that
an injection of the extract caused an increase of protein decomposition
and a greatly inereased glycosuria and increase of other reactions fol-
lowing adrenaline.

We may sum up the facts just presented as follows: In the absence
of the anterior lobe in young animals growth is checked, an increase
650 PHYSIOLOGICAL CHEMISTRY

of fatty deposit occurs and a persistent infantilism, such as infantile
sexual organs, persistent thymus, long retention of puppy hair and milk
teeth ; the epiphyses of the bones remain open; there is a marked diminu-
tion in susceptibility to adrenaline; lack of brain development and intel-
ligence; a lowered body temperature; a lowered respiratory exchange,

 

Fie. 69A.—Section of the testis of the brother of the dog whose testis is shown In
69. This dog had the hbypophysis extirpated early in life. Note the absence of
spermatozoa. These two dogs were the same age.

a lowered nitrogen decomposition of the tissues. In adults there is no
marked change, except an increase in fat; a lowering of temperature;
lowered nitrogen output; and lowered sensitiveness to adrenaline. There
is also an increase in the eosinophile cells of the blood and a diminution
of vital resistance, particularly on the part of the skin. Feeding tablets
of the hypophysis brings about the reverse change. These conclusions
are supported by pathology. Hyperfunction of the anterior lobe of the
hypophysis is accompanied by gigantism, true or acromegalous. Hypo-
function is accompanied by persistent infantilism.

From these experiments we may conclude as probable that the ante-
rior lobe furnishes an internal secretion necessary for normal growth
and metabolism and which acts much as the thyroid acts. How this
THE CRYPTORRHETIC TISSUES 651

internal secretion acts, and where; whether by direct action on the tis-
sues, or indirectly by trophic changes of the brain, thymus, sexual
organs or in some other manner, is still an open question. Castration of
the young generally leads to the production of an abnormally large
animal. Castrated animals deprived of the hypophysis are less retarded
in growth_than those deprived of the hypophysis alone. The nature and
point of attack of the secretion, whether it goes into the blood, lymph
or cerebro-spinal fluid, are questions needing further examination.

The posterior lobe is quite distinct anatomically, phylogenetically,
embryologically and physiologically. No disturbance of growth takes
place when it alone is taken out. The sole change noted was one by
Cushing: i.e., the increase of sexual desire. This, however. was not
observed by Aschner in any of his experiments and may possibly have
been due to slight injury to the brain parts immediately adjacent to the
hypophysis. Indeed, it must be again emphasized that these parts are
extremely important, injury to them being followed by glycosuria,
polyuria, and changes in the sexual organs; and stimulation of them by
contraction of the intestines, bladder, etc. The connection with the
vagus is very close. Experiments by 8. A. Matthews show that in very
eareful extirpation of the posterior lobe by Aschner’s method there is
often an enormous polyuria, or diabetes insipidus. A dog of 4 kilos
may secrete as much as 3 liters per day. This diabetes insipidus is not
accompanied by any lowering of the sugar tolerance, nor is the operation
followed or accompanied by glycosuria.

The posterior lobe contains a substance, pituitrine, which raises the
blood pressure like adrenaline, but its effect is not so transient as adren-
aline, the blood pressure remains high for a considerable time; it may
cause polyuria; and, most remarkable of all, it is one of the few sub-
stances which causes a flow of milk from the mammary glands. This
last observation of the American physiologists, Ott and Scott, is extremely
interesting, since only one or two other substances have such an action:
namely, the extract of the reabsorbing uterine wall and the extract of
the corpus luteum. If extract of the hypophysis is injected into a preg-
nant or parturient cat, a continuous stream of milk may often be
expressed from the lacteal glands. The total quantity of milk is not
increased. The substance raising the blood pressure has not yet been
chemically isolated and identified. Its presence allies this lobe, per-
haps, to the chromaffine tissues of the body. In other respects it acts like
adrenaline. Its preparation should provide a valuable remedy to medi-
cine. The presence of pituitrine in the body and the resemblance of its
actions to those of adrenaline makes it always somewhat doubtful
whether such a substance in the blood has enme from the hypophysis or
the supra-renals.
652 PHYSIOLOGICAL CHEMISTRY

Among its other physiological actions may be mentioned its action on
the uterus and bladder. It causes in each prolonged tonic contractions.
It seems to act directly on smooth muscle regardless of the innervation,
in this respect differing from adrenaline.

The composition of the active principle of the gland, or pituitrine,
is not yet known. It is apparently a base and is precipitated by phospho-
tungstic acid (Engeland and Kutscher). The solutions of the active
principle give a very strong histidine reaction with p-diazobenzene sul-
phonic acid (Fuhner; Aldrich). It would appear from this to be pos-
sibly a histidine derivative and in this connection it is of interest that
f-imidazolylethylamine also causes contraction of the uterus like pitui-
trine. The action of histamine on the lungs is, however, quite different
from the action of pituitrine. Histamine causes an intense spasm of the
bronchioles, but pituitrine does not do this. It is destroyed by trypsin,
but slowly by pepsin, from which it would appear to be a polypeptide.
It is very unstable in alkali and liberates a volatile amine. Aldrich
obtained a crystalline picrate.

Pituitrine, whatever its nature, is apparently allied in its action
to the digitalis group. A substance of somewhat similar properties has
been found in the skin glands of toads. In the latter case the substance
is an oxycholesterol. There is also a pressor substance in the anterior
lobe, but it is in small amounts and its presence is masked by a depressor
substance which may be separated by its solubility in ethyl alcohol.

The facts that are here presented show that in its actions on general
metabolism the anterior lobe of the hypophysis strongly resembles the
thyroid. Its action on metabolism is less pronounced. It is clear, from
its relation to glycosuria, the chromaffine tissue and the sexual organs,
that the hypophysis, thyroids, sexual glands, adrenals and thymus arc
a closely related series of organs which mutually influence each other’s
growth. The attempt has been made to bring these into a general
scheme among themselves by Eppinger, Falta and Rudinger and later
by Aschner. Such a scheme is that below.

Thyroid. Hypophysis-

Pancreas, ovaries,parathyroids Chromaffine system

 

Inhibition

Thus the thyroid and hypophysis stimulate the chromaffine tissue, the
latter inhibits the pancreas, ovaries and parathyroids; the latter inhibit,
or are inhibited by, the thyroids and hypophysis. Such a scheme,
while useful, is too restricted. It must be remembered that the meta-
THE CRYPTORRHETIC TISSUES 653

bolie co-ordination or correlation of the body embraces every organ in it.
The body is an organic whole and these so-called eryptorrhetic organs,
organs of internal secretion, are not unique, but the bones, muscles,
skin, brain and every part of the body are furnishing internal secretions
necessary to the development and proper functioning of all the other
organs of the body. Such a scheme, to be complete, must embrace every
organ; only the barest beginning has been made in this study so im-
portant, so necessary for the understanding of development and inheri-
tance. Problems of development and inheritance cannot be solved until
these physiological questions are answered.
The following table expresses the action of some of these organs:

On protein metabolism.

Stimulating Inhibiting
Thyroid Pancreas
Hypophysis Parathyroids
Chromaffine system

Ovaries, Testes.
On calcium retention:

Favorable to Inhibit
Hypophysis Sexual glands
Thyroids
Parathyroids.

The internal secretions appear to the author to constitute strong
evidence against the existence of such things as unit characters and
inheritance by means of structural units in the germ, which represent
different characters. We see in the internal secretions that every char-
acter in the body involves a large number of factors. The shape and
size of the body; the coarseness of the hair; the persistence of the
teeth; tendency toward fatness, may easily depend on the hypophysis,
on the thyroid and the sexual organs; and these, in their turn, are but
the expression of other influences played upon them by their surround-
ings and their own constitution. An accurate examination shows the
untrustworthiness of any such simple or naive view as that of unit

characters.
REFERENCES. Hypopuysis.

1. Aschner: Ueber die Funktion der Hypophyse. Full bibliography. Archiv f. d.
ges. Physiol., 146, p. 1-126, 1912.

2. Cushing: Hypophysis cerebri. Jour. Amer. Med. Assn., 53, pp. 249-255, 1909.
Johns Hopkins Hospital Bull., 1909, p. 105.

3. Sotti et Soteschi: Sur un cas d’agénésie du systéme hypophysaire accessoire
avec hypophyse cérébrale intégre et gigantisme acromégalique, avec in-
fantilisme sexuel. Archives Ital. de Biol., 57, p. 22, 1912.

4, Miller and Lewis: The effect on blood pressure of extracts made from the various
anatomical components of the hypophysis. Transactions Chicago Patho-
logical Soc., 8, pp. 133-134, 1911. _

5. Wells: Presence of iodine in human pituitary glands. Jour. Biol. Chem., 7,
1910, pp. 259-261.
654 PHYSIOLOGICAL CHEMISTRY

6. Aldrich: On feeding young pups the anterior lobe of the pituitary gland Amer.
Jour. Physiol., 30, p. 352, 1912.

7. Schaefer: Pro. Roy. Soc., 81, p. 442, 1909.

8. Engelund and Kutscher: Ucber einige physiologische wichtige substanzen
A. Die physiologisch wirksamen Extraktivstoffe der Hypophyse. Zeit. £.
Biol., 57, p. 527, 1911.

9. Ott und Scott: The action of animal extracts on the secretion of the mammary
gland. ‘Therapeutic Gaz., 35, pp. 689-691, 1911.

10. Schaefer: On the effect of pituitary and corpus luteum extracts on the mam-
mary gland in the human subject. Quarterly Jour. Expt. Physiol., 6,
pp. 17-19, 1913.

11. Schaefer and Herring: Action of pituitary extracts upon the kidney. Phil.
Trans. Roy. Soc., 199, B, pp.. 1-29, 1906.

12. Hammond: The effect of the pituitary extract on the secretion of milk. Quar-
terly Jour. Expt. Physiol., 6, pp. 311-338, 1913.

13. Guggenheim: Proteinogene amine. Peptamine: Glycyl-p-oxyphenylethylamine,
ete., Biochem. Ztschr., 51, pp. 369-387, 1913.

14. Fiithner: Pharmakologische Urtersuchungen tiber die wirksame Bestandteile der
Hypophyse. Zeits. f. d. ges. expt. Medizin. 1, pp. 397-443, 1913.

15. Fihner: Das Pituitrin und seine wirksame Bestandteile. Miinch. med. Wochen-.
schr., 59, pp. 825-853, 1912.

16. Dale and Laidlaw: A method of standardizing pituitary extracts. Jour. Pharm.
and Expt. Ther., 4, pp. 75-95, 1912.

17. Barger: The simpler natural bases. Active principle of the pituitary body.
p. 108. Monographs of Biochemistry. Longmans, Green and Co., 1914.

18. Klotz: Experimentelle Studien u. die blutdrucksteigende Wirkung des Pitui-
trins. Arch. f. expt. Pathol. u. Pharm., 65. p. 348, 1912.
19. Schaefer: Die Funktionen des Gehirnanhanges. Zent. Physiol.. 15. p. 427, 1911.
20. Hamburger: The action of extracts of the anterior lobe of the pituitary gland
upon the blood pressure. Amer. Jour. Physiol., 26. p. 178. 1911.
21, Pankow: Ueber Wirkungen des Pituitrin auf Kreislauf u. Atmung. Archiv f. d.
ges. Physiol., 147, p. 89, 1912.

22. Simpson and Hill: Effects of repeated injections of pituitrine on milk secretion.
Amer. Jour. Physiol., 36, p. 347, 1915.

23. Weed and Cushing: Effects of pituitary extract upon secretion of cerebro-
spinal fluid. Amer. Jour. Physiol., 36, p. 77, 1915.

PARATHYROIDS.

The parathyroids, or epithelial bodies, as they have been also rather
badly named, are some small, pale, glandular masses either embedded
in the thyroids, or lying close to them or attached to the thymus gland.
There are in many cases four of these bodies, two within the thyroid
ealled the internal parathyroids, and two without, or external parathv-
roids ; but particularly in herbivora there are often accessory parathyroids.

For a long time these bodies were confused with the thyroids and
results of their extirpation were attributed to the thyroids. Their rela-
tion to the thyroid is not yet clear, but it is certain that the tetany
and death after 9-10 days originally ascribed to thyroidectomy are in
reality due to the extirpation of the parathyroids.
THE CRYPTORRHETIC TISSUES 655

Complete extirpation of the parathyroids is generally followed by a
remarkable set of symptoms. Morel has described these very well:
A dog 2-3 years of age which has undergone parathyroidectomy at first
presents no appreciable symptoms. The symptoms begin on the second
day. The animal then becomes sad and restless; it moans and moves
about. It eats no more, but drinks abundantly. From the second to the
third day it seems stiff in its movements; a fleeting twitching of its
muscles analogous to that of a horse bitten by a fly may be observed.
There is fibrillary twitching of the tongue. On the 4th day it is worse.
The animal makes a miserable appearance. It retires to a dark corner;
it seems to suffer much and cries out at the least touch. Stiffness or
rigor increases so as to interfere with its movements; the tremblings
become general, more ample and less discontinuous. Convulsions throw
the dog on the floor, with feet in extensor tetanus and head bent back;
at times disordered movements come on, the feet beating the air and the
face grimacing. At the same time respiration is quickened, the heart
accelerated and the temperature increases. The attack is prolonged
several minutes; then the dog comes to itself, gets on its feet and stag-
gers about. The attack reappears after some hours, either spontaneously
or under some stimulus such as a light, touch or noise. The attacks
increase in number and intensity and tend to become continuous. Re-
fusing all food, or vomiting the little it has taken, the animal rapidly
becomes cachexie and gives out a putrid odor. Toward the 8th day it
enters on the terminal phase of the malady, the convulsive seizures
are repeated, but their violence diminished. Thin and without
strength, the dog lies on one side. He asphyxiates little by little
and the temperature falls. The respirations become few, deep and
irregular. Then the animal dies. Death comes on the 9th or 10th
day.

But while death generally follows, certain cases have been described in
cats and other animals, in which recovery took place and no symptoms
followed parathyroidectomy ; and on autopsy no accessory parathyroids
could be found. Such cases are most common in cats. Whether they
are to be explained by undiscovered accessory parathyroids, by the
getting into finction of some other organ usually working with the
thyroids, for example the hypophysis, or whether there is spontaneous
cure by immunity, cannot be positively stated.

The tetany can be entirely done away with or greatly reduced by
the giving of sodium bicarbonate or alkalies, or by bone injury, or by
grafting parathyroid into a bone, or by calcium salts, but the length of
life is little if at all prolonged thereby, provided all the parathyroids
have been taken out. If, however, a small amount has remained in, but
not enough to keep the animal alive and free from tetany, then the
injection of calcium salts is said to permanently save the animals. It
656 PHYSIOLOGICAL CHEMISTRY

appears that the tetany is not the cause of death, but an indirect result
of some profound change underlying it.

With the parathyroids, as with all other organs of internal secretion,
we have to ask the question whether they are acting by an internal secre-
tion, or by a process of detoxication. Of course both processes may
coexist. In this case there is no doubt that the tetany is due to a
poison of some kind circulating in the blood of the parathyroidectomized
animals. It has been shown that bleeding and the injection of fresh
blood or salt solution relieve the tetany. Furthermore the injection of
the serum of animals in tetany may produce similar symptoms in othe:
animals. The suggestion has been offered by some that the normal func
tion of the gland is to remove this substance from the blood. Such a
conclusion, however, is not justified. It may very easily be that this
poison is not normally found in the body when the parathyroids are
there. It may be formed as the result of their falling out of function
Methyl guanidine, a poison, has been isolated from the urine of dogs
dead of tetany. Both guanidine and methyl guanidine produce tetany.

The symptoms of this tetany are similar to those which sometimes
follow an Eck fistula when a dog is fed on meat. A bread and milk
diet relieves them; meat predisposes to them. Substances appear to be
formed in the digestive tract, either as the result of bacterial decomposi-
tion of meat or of digestion, or to pre-exist in the meat, which produce
tetany and which the liver ordinarily removes. Substances of unknown
nature and unknown origin of similar action appear also during para.
thyropriva and it has accordingly been suggested by Morel that the
parathyroids are necessary for the liver to function normally; in their
absence the liver is no longer able to detoxicate the body. Others hava
attempted to show that the liver has lost the power of holding back
ammonia and that the tetany is an ammonia tetany, but their methody
have generally been too inexact and their conclusions accordingly not
dependable. It is not sure, also, whether the substances causing the
tetany are the cause of death or an indirect result of the morbific
process. It has also been suggested that an acidosis follows parathyro.
priva. Among the other suggestions made, again badly founded, was
that in parathyropriva there was a hypersecretion of calcium and tetany
as the result of this. This, however, is not the case, although there is
no doubt that the calcium content of the blood is reduced and that by
calcium salts, as by several other ways the tetany may be prevented,
The way in which the parathyroids act is, therefore, still obscure. It is
certain that there are serious disturbances of digestion.

One very interesting result of parathyropriva is the action on the
dentine of the teeth and on the bones. Neither bones nor teeth calcify
properly, so that in rats conditions analogous to rachitis and osteo
malacia are produced.
THE CRYPTORRHETIC TISSUES 657

THYROID.

The thyroid gland is a small, highly vascular, ductless gland, lying
in the neck close to the larynx. It gets its name from the large carti-
lage of the larynx, the thyroid, or thyreoid cartilage, so called from
its shield-like shape. (Gr. thyreos, shield.) In most of the higher
vertebrates the gland is composed of three parts, two lobes lying
close to the larynx one on each side and a little to the front of the
trachea, and an isthmus of connecting glandular tissue which extends
across the trachea in front. The organ is largest relative to the body
weight in the mammalia and smallest in the fishes. The gland is found
in all classes of vertebrates, but in the fishes and cyclostomes it is rep-
resented only by some small diffuse glandular patches little larger than
the heads of pins, which extend along the aorta on the ventral side and
out a little way along each gill arch. The gland is larger and fairly
compact in amphibia and reptiles, but still small. The histological
structure in all forms from the fishes up is the same and quite character-
istic. In amphioxus, one of the lowest of the vertebrates, the gland is
said to be represented by the neural groove, but there is some doubt
about this homology. The only organ of the invertebrate which has been
homologized with it is a gland of scorpions and arachnids. This gland
has a structure very similar to that of the thyroid of the cyclostomes
(Gaskell). It is interesting to notice that if this homology is correct,
and few if any zodlogists would admit its correctness, the vertebrate
thyroid represents an accessory sexual organ of the invertebrate. This
is of interest on account of the close connection physiologically between
the sexual organs and the thyroids of vertebrates of which we shall
presently speak.

The thyroid is a very vascular gland. Relative to its weight a very
large amount of blood passes through it, and in goiter this vascularity
becomes still greater and the carotid arteries supplying the gland are
generally enlarged, sometimes to double their normal size. So great is
the blood supply that dilation of the arterioles of this gland alone may
lower the general blood pressure many millimeters of mercury.

The nerve supply of the thyroid is from the superior and inferior
laryngeal nerves. These nerves carry vasomotor and probably afferent
and secretory fibers for the gland.

The thyroid is, therefore, pre-eminently a vertebrate gland. It has
developed side by side with the vertebrate characteristics of a dry,
hairy skin instead of a moist, mucus-bearing or chitinous skin; with a
great development of the brain and intelligence; with an internal bony
skeleton and a large skull. It may almost be called the vertebrate gland
par excellence, for as we shall presently see it is intimately connected
with these three fundamental vertebrate characteristics. Indeed, it ap-
658 PHYSIOLOGICAL CHEMISTRY

pears to be related in a causal way with these organs, so that these typi-
cally vertebrate characters might almost be called secondary thyroid
characters, just as the horns of cattle may be secondary sexual char-
acters. Injury to the thyroid, particularly in growing animals, is asso-
ciated with profound retrogressive changes or cessation of development
in skin, internal skeleton and nervous system.

Embryology. The thyroid gland, although ductless in its adult state,
arises as an outgrowth of the ventral side of the embryonic esophagus.

 

Fie. 60.—Section of the normal thyroid of a cat (Vincent and Joly). Note the low
epithelium with the colloid material in the center of the alveolus.

It is hence hypoblastic in origin. Another such outgrowth is the lungs.
It corresponds on the ventral side to the anterior lobe of the hypophysis,
which grows out of the dorsal side, and there is some connection and
similarity between the functions of these two organs. Very soon, how-
ever, connection of the thyroid with the cesophagus is lost; the gland is
encapsulated with connective tissue and has no duct for the discharge of
its secretion.

Histology. Histologically the gland consists of closed alveoli con-
taining a single layer of epithelial cells and with the center of the alveolus
(Figure 60), consisting of colloid material, which stains strongly in
eosin. This colloid is believed to be the active principle of the gland. A
similar colloid is found in the hypophysis formed by the pars intermedia
and discharged into the third ventricle of the brain by means of the
infundibulum. How the colloid escapes from the thyroid, whether it
goes into the lymph or into the blood, or whether it is dissolved by some
ferment which splits it into a soluble substance, osmosable and passing
into the blood, is still unknown. It is, perhaps, most likely that such an
enzyme exists in the gland and the colloid does not, as such, find its
exit from the gland.

Function. The function of this gland was until recently obscure and
even now not all the details of its action are known. The gland was
classed as an organ of internal secretion by the great physiologist,
Brown-Séquard. Knowledge of its function was derived first from
THE CRYPTORRHETIC TISSUES 659

pathology and then from surgery. It was found by surgeons that extir-
pation of the gland was followed by very grave symptoms in human
beings ; and the pathologists observed its connection with certain peculiar
changes in the skin and skeleton. ,

(a.) Cretinism. If the gland fails to develop or atrophies or de-
velops insufficiently in embryonic life, a cretin is the result. Growth
is retarded, and a dwarf is produced, with a protuberant abdomen, low
intelligence, or imbecility ; the hair is coarse and scanty; the skin thick
and dry with mucin in it. These cretins, or dwarfs, are found generally
in regions where goiter is endemic, as in Switzerland and Styria. If,
while very young, they are given sheep’s thyroids, or extracts of thyroids,
growth begins again and: the abnormal symptoms more or less completely
disappear. The thyroids must be taken constantly, however, or myxe-
dematous symptoms recur. It is then clear that arrested mental and
physical development is a result of thyroid insufficiency. The proper
development of the brain and skull and various other structures of the
body depend upon the secretions of this gland.

(b.) Myxadema. Extirpation or atrophy of the gland in adults is
followed by the symptoms of myxcedema, so called from the deposit
of mucin-yielding material in the thickened skin. (Gr. myza, mucin;
oidema, swelling.) The skin becomes thick, dry, the connective tissue
is increased and yields mucin when extracted with alkali. The hair
becomes scanty and coarse; there is a marked falling off of the sexual
function ; intellectual apathy and disinclination to exertion of any kind
result. A general obesity may also develop. The body temperature falls
about 1° below normal and the oxygen consumption and nitrogen metab-
olism are reduced. (Compare with results of extirpating anterior lobe
of hypophysis.) Patients with complete atrophy of the thyroid have a
basal metabolism only 60 per cent. of the normal.

(c.) Basedow’s disease. On the other hand, if the thyroid is too
active, exophthalmic goiter, so called from the protuberance of the eyes
which commonly occurs, or Basedow’s disease, results. In this case the
symptoms are generally just the reverse of those in thyreopriva, or
diminished function. The heart is very fast and often irregular (tachy-
cardia) ; instead of a low temperature the temperature is high, one or
two degrees above the normal; there is great nervous irritability in place
of nervous sluggishness; nitrogen metabolism and oxygen consumption
are increased, instead of diminished; and patients are thin instead of
fat. Moreover, the symptoms are ameliorated by reducing the activity
of the gland by tying some of its arteries or partial extirpation. The
gland may or may not be larger than the normal, although it is generally
somewhat enlarged, but it is generally found to contain but little colloid
and more of the secretory epithelium.

From this pathological evidence it is clear that this small organ is
660 PHYSIOLOGICAL CHEMISTRY

enormously important in the proper development of bone, teeth, skull,
brain and skin of the body; that its absence is attended by retrogressive
changes in these tissues and organs; and that when it overfunctions it
stimulates metabolism and nervous activity. The gland obviously has
a very close relation to the nervous and the vascular systems throughout
the vertebrate phylum.

(d.) Extirpation of the gland in animals. The results of extirpa.
tion of the gland differ in different animals. This is owing to the fact
that the thyroid contains often in it the parathyroids, for example in
dogs and cats; whereas in rabbits and the herbivora generally there
are external parathyroids lying outside of the thyroids. The results of
taking out the parathyroids are very different from those of taking out
the thyroids alone. Since, in the beginning, the importance of the
parathyroids was not recognized, many apparent contradictions resulted
in the effects of the operation. Here only the effects of taking out the
thyroids proper will be considered.

If the thyroids alone are taken out, or if the thyroids and internal
parathyroids only are extirpated, leaving the external parathyroids
intact, no very obvious symptoms follow at once. But in adults dis-
turbances in metabolism shortly begin to appear and the symptoms of
myxcedema develop. The animals live for months or years (dogs, cats,
rabbits). The hair falls out. They are apt to become mangy; their
nitrogen metabolism diminishes ; they do not so easily develop adrenaline
glycosuria when adrenaline is injected; the excitability of the vagus,
depressor and other nerves is diminished ; animals become apathetic and
tend toward obesity, somewhat as they do after extirpation of the
hypophysis. Their sexual life is also less intense. If the parathyroids
are also removed tetany and death in 9-10 days nearly always result.

In young animals the consequences of extirpation of the thyroids
are far more serious. Growth is checked; myxcedematous symptoms de-
velop and intelligence remains very low.

(e.) Effect of extirpation on the excitability of nerves. It was men.
tioned that one of the results of extirpation of the gland is that the sym.
pathetic nerves of all kinds, and in general what are known as the
autonomic nerves, namely, those which regulate the visceral as distinct
from the voluntary system of the body, are less active on stimulation
than they were before. Now, in the case of the supra-renals there is no
doubt that the action of the glands is due to an internal secretion, a
substance of known composition, adrenaline, which comes from the gland
to the blood. It is, hence, not unlikely that the thyroids also have an
mternal secretion. Physiologists have, accordingly, sought for evidence
that such an internal secretion is produced by the gland. Such experi-
ments were tried by von Cyon many years ago. He proved that stimu-
THE CRYPTORRHETIC TISSUES 661

lation of the thyroid branches coming from the superior and inferior
laryngeal nerves caused a great: dilation of the blood vessels of the
gland, and that as a result of this stimulation, or of the injection of
thyroid extracts, a remarkable change took place in the irritability of
the vagus nerve and the depressor nerves. Stimulation of the vagus
was far more effective in stopping the heart; and the depressor effect
was far more sensitive after stimulation of the gland nerves or after
the injection of the extract of the gland than before. On the other
hand, when the thyroid was reduced in activity, as in some goiters, or
when it was extirpated, there was a remarkable diminution in excita-
bility of the vagus and depressor mechanisms. Both of these nerves
are inhibitory nerves and are acting in an antagonistic manner to the
sympathetics. These results have been confirmed by several observers.
They appear to be well substantiated. It is also stated that the power
of the splanchnics to raise blood pressure on stimulation by weak cur-
rents is also greater when the thyroid nerves are stimulated at the same
time. This experiment is of importance, since the short time necessary
to produce an effect, i.e., ninety seconds, would indicate that the thyroid
secretion did not find its way into the lymph, but directly into the
blood. A direct action of the thyroid is thus shown to be upon the vaso-
motor apparatus and sympathetic system, including the internal muscles
of the eye. This effect is presumably partly peripheral and in part
flirect. This is of great importance in interpreting the symptoms
of exophthalmic goiter, where such hypersensitiveness is marked.
In thus increasing the excitability of the autonomic system the in-
ternal secretion of the thyroids appears to act somewhat like that of
the supra-renals. But the thyroid extract does not itself cause a rise
of blood pressure; it is much less powerful than that of the latter
organs.

(£.) Action of extracts of the thyroid. Blood pressure and vascular
system. While nearly all are agreed that extracts of the thyroid, in-
cluding thyreoglobulin, which is obtained from the colloid matter,
and iodothyrin, formed from the globulin by hydrolysis, influence
the excitability of the vasomotor apparatus and the sympathetic system,
very contradictory effects on blood pressure have been obtained as a
‘direct result of the injection of extracts of the gland. These contradic-
tory results appear to be due (a) to the use of anesthetics, curare and
morphine in some cases but not others; and (b) to different methods of
making the extract. Drugs often cause very different actions on the cir-
culations of anesthetized and non-anesthetized animals. Thyroidec-
tomized animals generally have, after a time, a low blood pressure. The
serum of such animals is also hypotensic. Jeandelize and Perisot ob-
tained the following results: Rabbits were operated on when 8 days
662 PHYSIOLOGICAL CHEMISTRY

old and tested after several months. The blood pressure in the controls
and the thyroidectomized animals compared as follows:

 

 

Blood pressure in cms. Hg.

 

Time from operation

 

 

Controls Thyroidectomized
2 months 18 days ...............000.. 11.5 9.6
Gs ie Bae lla ves ulate ll 6.5

 

 

The effect of injecting extracts of the gland is not uniform. Von
Cyon and Asher got no direct action of extracts of the gland on blood
pressure. Nearly all observers, however, have obtained a fall in blood
pressure following the injection of aqueous, glycerol or acid extracts
of the gland. It was suggested that this was due to choline. Popielski
showed that if the depressor substance was first removed from the gland
extract, a rise in blood-pressure resulted on the subsequent injection of
the extract. The amount of choline in the extracts is said to be too
small to produce the result. The depressor substance in the acid extract
of the gland is vasodilatin, according to Popielski. This extract causes
all the typical symptoms produced by this substance, namely, secretion
of the pancreas, fall in blood pressure, abolition of the coagulability of
the blood. This substance would appear to be either imidazolylethye-
amine or some similar amine. The recent work of Hunt on the great
activity of some of the esters of choline suggests that possibly some of
these may be present.

As regards the nature of the active principle which produces the
change in excitability of the vagus and sympathetic nerves, experiments
indicate that it is either the colloid, namely the iodine containing
thyreoglobulin, or some derivative of this, such as iodothyrin, or some
proteose from the globulin. The experiments of Popielski indicate the
probability that there are also in the gland basic substances which
may act directly on the blood pressure. The whole matter is, how-
ever, in such an uncertain state that more work will have to be done
before the relation of the gland to the vascular system, a relation which
no one doubts, is cleared up.

(g.) Effect of feeding thyroids or extracts of thyroids on metabolism.
The relation of the thyroid to metabolism is shown very clearly by the
cessation of growth and proper development on its removal. Moreover,
feeding experiments give very positive results. If human beings or
carnivora (the effects on herbivora are doubtful) eat thyroids or take
extracts of the gland, there results a stimulation of their nitrogen
metabolism. It is as if the protein oxidation had been stimulated. The
temperature of the body is raised, just as it is by the ingestion of
THE CRYPTORRHETIC TISSUES 663

protein. The protein is rapidly oxidized. There is an increased con-
sumption of oxygen and production of CO, and nitrogen excretion is
mereased. A diminution of the fat in the body results, so that thyroid
tablets are recommended or used often for reducing fatness. The
nervous and heart symptoms produced are, however, unpleasant. Very
similar effects are produced on metabolism by the ingestion of very
large amounts of protein. In amphibia feeding thyroid greatly hastens
the transformation from the larval to the adult form. Tadpoles change
into frogs long before the usual period if thyroid is eaten.

The nature of the active principle of the gland, which is thus active,
-is considered below. It appears to be correlated with the colloid matter
in the cells. It may be, however, that the nervous symptoms and the
metabolic symptoms are due to different substances. The way in which
the active principle acts is still quite obscure; it is uncertain whether
it acts directly on the cells of the body, or indirectly by its action on
the nerves. Further work in this direction will be needed. The indica-
tions are, however, from the work which follows, that it is the iodine-
containing substance which is active. .

(h.) Nitrile reaction. A very remarkable and curious reaction,
making an almost incredibly delicate biological test for thyroid tissue, is
the nitrile test of Reid Hunt. If so little as 0.1 mg. of dried thyroid
tissue, or an equivalent amount of the thyroglobulin, per gram of body
weight is fed to a white mouse on a cracker diet each day for 10 con-
secutive days, no visible change occurs except a loss of weight; but it
is found that such a mouse will survive as much as 10 times the amount
of acetonitrile given subcutaneously, which would have killed his brother
or sister under conditions identical with the foregoing, except the thyroid
feeding. White rats, on the other hand, are rendered far more sensi-
tive to acetonitrile by this treatment. This test is the most delicate test
for thyroid tissue which we have. All of this activity of the gland
is found in the thyroglobulin of the gland and also in the metaprotein
derived from it by acid hydrolysis; also in the iodine containing protal-
bumose formed from the metaprotein by acid hydrolysis. The amount
of activity computed on the iodine present in these preparations remains
what it was in gland substance containing the same amount of iodine.
By further hydrolysis iodothyrin is formed, and while this is active, it is
not so active per mg. of iodine as the original substance. The extreme
sensitiveness of this reaction and the fact that the nitriles generally kill
by their action on the respiratory center strongly indicate that the
thyroid extract produces, in mice and rats, a change in this center, in the
one case causing an increase, in the other a decrease in its sensitiveness
to acetonitrile.

(i.) Nature of the internal secretion of the thyroid. This problem
is now solved. The thyroid is peculiar among all the glands of the
664 PHYSIOLOGICAL CHEMISTRY

body because it normally contains iodine. No other tissue normally con-
tains iodine in more than traces, except possibly the pituitary, and here
the evidence is doubtful. The fact that the thyroids contain iodine
naturally leads to the conclusion that the peculiarity of the gland’s
action is due in some way to this fact and that the active principle is
also an iodine compound. There are strong reasons for believing this to
be a fact, and some reasons for doubting it. The connection of iodine
with the thyroid was long known empirically. It was found that iodide
taken internally, or iodine painted on the outside. was beneficial in
goiter, and reduced the goiter. It was early stated that the gland con-
tained iodine and that regions in which goiter was endemic were marked
by less than the normal amount of iodine in the water. These results, *
however, were not generally accepted until Baumann, in 1895, showed
that iodine was present in the gland.

The amount of iodine in the gland is extremely variable and is much
increased by various forms of medication by iodine, iodides or iodine-
containing materials. The gland has the power of picking out iodine
from the blood and storing it in organic union. Iodine is found in
combination with a globulin which occurs in the colloid of the gland
and is called thyreoglobulin. All of the iodine present is in this sub-
stance and certainly the effect of the gland on the nitrile susceptibility
of mice is due exclusively to this substance. Glands which contain no
colloid contain no iodine either; those which have much colloid usually
contain also much iodine. As a rule the thyroids of persons living in
goiterous regions contain, per gram of dry substance, less iodine than is
common in other localities. The rule has, however, many exceptions.
In a lot of human thyroids from the Stiermark (Styria), a goiterous
region, von Rositzky found that the fresh weight of the glands of adults
varied from 29 to 231 grams, and the dry substance from 5.2-67 grams.
In 1 gram of dry substance the amount of iodine varied from 0.077 to
3.85 mgs. of iodine. The last figure was obtained in a case of tubercu-
losis. The total amount of iodine in the whole gland varied from 0.99
tu 54.67 mgs. In children from birth to 1% years of age, Baumann
found from 0.09 to 0.842 mg. of iodine in the whole glands. In animal
thyroids the amount of iodine usually runs from 1-1.5 mgs. of iodine in
a gram of dry substance, but pigs may have less. In sheep there appears
to be a seasonal variation in the amount of iodine, perhaps correlated
with the character of the food. In Sweden in human adults the average
amount found by Jolin was 11.20 mgs. of iodine in the whole glands.
It is clear from these figures that the iodine content of human thyroids
is subject to very wide fluctuations and these may be due in part to
medication, disease, diet and other unknown causes. In exopthalmic
goiter, when the colloid may be much reduced in amount, the iodine is
reduced also. Some have interpreted these facts to mean that the gland
THE CRYPTORRHETIC TISSUES 665

simply stores iodine to get rid of it, as some organs store copper and
arsenic. But this view is not borne out by the studies of Kocher, Os-
wald, Hunt and F. C. Koch.

Thyreoglobulin. This is the iodine-containing constituent of . the
gland and is the principal constituent of the colloid matter. It may be
extracted by carefully drying the ground glands at room temperature,
after previously separating them as far as possible from connective
tissue, extracting the dried glands with gasoline to remove the lipins,
removing the gasoline by evaporation at room temperature, and grind-
ing the lipin-free material in a mill and passing the powder through a
fine sieve. The globulin is then extracted from the fine powder with
dilute salt solution. The globulin is purified by the usual methods of
precipitating with ammonium sulphate, resolution, and reprecipitation
by dialysis. It is a white, amorphous powder. The per cent. of iodine is
variable, generally ranging from 0.34-1 per cent. Oswald found 1.6
per cent. The activity is proportional to the iodine content. It is not
impossible that some of the symptoms of exophthalmic goiter may be due
to some other constituent of the gland. The iodine-free globulin is in-
active on the blood pressure, and does not affect the irritability of the
vasomotor apparatus and sympathetic systems. The iodine-containing
globulin is active in these particulars and Koch found that all the
activity of the gland in the nitrile susceptibility was due to this con-
stituent. There is then no doubt that thyreoglobulin is one of the active
substances of the gland. Whether, however, it is the only active sub-
stance and whether it gets into the blood as such is doubtful. Koch
found that full activity persisted in some of the first digestive products
of the globulin. It is, therefore, possible that there is in the gland an
enzyme, which, under nerve influence or other stimulation, digests some
of the protein, setting free in the blood, not thyreoglobulin, but an
iodine containing digestion product and this produces the action on the
nervous system and possibly on the other cells of the body. It has not
yet been shown, however, that any such enzyme exists in the gland and
is active in this way and it is not known in what form the active prin-
ciple is discharged from the gland. If such a process as that just men-
tioned occurs the gland would resemble the liver, which stores glycogen ;
the latter is not poured into the blood as such, but, by digestion, glucose
is formed from it. Basedow’s disease might, on this supposition, be
due to the too great conversion of thyreoglobulin into its absorbable
digested products and as a result the gland strives to manufacture more,
just as the body makes glucose in diabetes. It is as if a brake had been
removed. The small content of colloid, the activity of the gland, the
thyroemia all point in this direction. The condition would be analogous
to diabetes, only it is a protein diabetes, not a carbohydrate,
666 PHYSIOLOGICAL CHEMISTRY

The active principle of the gland has been isolated and identified by
synthesis by Kendall. He has given it the name ‘‘thyroxin.’’ This sub-
stance is trihydro-triiodo-oxyindole-propionic acid. Its formula is as
follows:

IH
c
HH.” \ H H O
oe
Hf G0
NWN 7
cH ‘NH

This substance easily opens the ring by hydrolysis and goes over into
the open ring form in which Kendall believes it to be present in the
gland:

IH
Cc \
BON H H O
te = C—C—C— con
Hd l OOH
a
cH NH,

Thyroxin, open ring form.

Thyroxin has all the powers of increasing metabolism possessed by
thyroid extracts. Its activity is presumably due to its power of opening
or closing the ring. If this is prevented by acetylization, the compound is
inactive. Kendall suggests that the iodine in some way influences its
power of closing the ring and that its influence is thus indirect.

The great importance of iodine in sufficient amounts in the diet is
now being generally recognized. It appears that throughout the central
portions of the United States, as for example in the state of Ohio, goiter,
particularly in young girls, is so common as to be almost the normal con-
dition. It has been found by the work of Marine that if iodine is given
in small quantities to school children at the ages of about six and again
at about twelve years they do not develop goiter. This has now been
tried on a large scale in the cities of Cleveland and Akron. The results
have been remarkable. The change in the mentality of the pupils, and
the sudden increase in growth shown is striking. The unexpected result
also appears probable that if women have sufficient iodine during
pregnancy, the children do not develop adenoids and other lymphatic
derangements which are so common at the present time.

There is no doubt that much can be done for the population by the
general application of similar efforts.
THE CRYPTORRHETIC TISSUES 667

Marine also quotes a very interesting case of sheep raising in Michi-
gan. This was on the point of being abandoned owing to the fact that
the ewes produced very few young, many of these dying shortly after
birth, and that growth and wool were both very poor. By the substitu-
tion of salt containing some iodine all these conditions were remedied.
This is a striking illustration of the importance of extremely small
amounts of necessary constituents of the foods. Kendall reports that
the administration of 10 mgs. of thyroxin to myxedematous patients
raises the basal metabolism 30 per cent.; their mentality becomes normal ;
_ their hair ceases to fall and becomes soft; the voice changes its char-
acter; the skin becomes moist and the edema disappears. It is remarka-
ble that so small a quantity of this substance should produce so great,
so diverse and such permanent changes.

The other constituents of the gland are those of cells in general, such
as lipins, nucleic acid, probably guanylie acid, cholesterol, choline, albu-
mins and extractives. The arsenic formerly reported to be present in
traces in the normal gland, Gautier has shown really to have come from
impurities in the reagents. The gland contains no arsenic.

Iodothyrin. By acid hydrolysis of the gland Baumann and Ross
isolated a brownish, amorphous substance containing from 2-14 per cent.
of iodine. This iodothyrin gives no protein reaction. It is soluble in
dilute alkali but not in weak acids, and, indeed, looks and behaves
somewhat like melanin. It has been found that this substance has an
action on goiters, cretins, myxedematous patients and in modifying the
irritability of nerves like the action of the gland itself but weaker than
the latter, if the amount of action is compared with the amount of
iodine in the iodothyrin and in the dried-gland substance. That is an
amount of iodothyrin containing a milligram of iodine is less active than
an amount of the dried gland containing an equal amount of iodine.
Computing its activity in comparison with the iodine content it is only
about one-tenth as strong as thyreoglobulin in rendering mice resistant
to nitrile poisoning (Koch).

SUPRA-RENAL CAPSULES.

Anatomy.—The supra-renal capsules are two small glandular organs
lying like caps on the upper poles of the kidneys, whence their name.
They are richly supplied with blood vessels and nerves and together
they weigh in an average adult about 6-7 grams. In youth and fetal
life, in common with most of the glandular organs of the body, they are
relatively larger. They are supplied with blood from two small arteries
from the dorsal aorta and wide, short veins empty directly into the
inferior vena cava to which the organs are closely attached. The amount
of blood passing through them is very large compared to their size. The
nerves which govern both the blood vessels and the tissue of the gland
668 PHYSIOLOGICAL CHEMISTRY

itself, being both secretory and vasoconstrictor, come from the two
splanchniecs, the left splanchnic supplying only the left gland, but the
right sending a branch to the left as well as to the right.

Histology.—The gland is composed of two different kinds of tissue,
making respectively the cortex and the medulla. The boundary between
these layers is not sharply delimited, strands of cortex tissue penetrating
here and there down into the medulla, particularly along the sympa-
thetic nerves. These two tissues differ in their histological, chemical and
physiological characters and in their embryonic origin.

Cortex. The cortex is composed of three regions, an external zone.
a reticular and a glomerular zone, the differences between them being
not sharp. The cells are arranged in a reticulum with blood spaces
between. There are no endothelial cells on these cell strings and the
circulation is of that extra capillary kind described by Minot in the
liver and characteristic for most invertebrates. Hence the blood comes
directly in contact with the cells of the tissue. The cortex does not
stain in bichromate of potash and the cells are simply protoplasmic cells,
containing some vacuoles and granules. During pregnancy and par-
ticularly just after parturition, this layer undergoes, in the guinea pig,
a rapid cell proliferation and degeneration, the significance of which is
not yet understood.

Medulla. The medulla is composed, in part, of nerve cells, but in
large measure of granular protoplasmic cells, which are distinguished
by their staining yellow when the gland is fixed in a solution of bichro-
mate of potash and formol. This yellow stain is taken by some other
cells also and tissue so staining is called chromaffine tissue. The stain-
ing power appears to be dependent upon, or correlated with, the presence
of adrenaline, the active principle of the organ, since the content of
adrenaline and the depth of stain rise and fall together. The stain is
not, however, a peculiar characteristic of adrenaline, for other aromatic
reducing bodies will probably be found to give the reaction also. How-
ever adrenaline content and chromaffine reaction often go together.
Thus such tissue is found in the poisonous skin glands of toads and
these contain an unusually large amount of adrenaline. The supra-
renals have no ducts. If they be called glands their secretion is dis-
charged (cryptorrhesis) into the blood stream, so that they are truly
organs of hidden flowing.

Comparative anatomy.—The supra-renals are found in all classes
of vertebrates and besides the organs themselves chromaffine tissue is
found outside the glands. In all the mammals the glands are well-
developed and in monkeys and human beings they are surrounded with
strong capsules. In fishes, in which the pronephros or head kidney
persists, chromaffine tissue resembling that of supra-renals is found
THE CRYPTORRHETIC TISSUES 669

in the head end of the kidney. In the frog, in which the mesonephros
persists, the supra-renals are represented by small masses of tissue just
above the kidneys. In mammals, besides the supra-renals, little accessory
masses of chromaffine tissue are found along the trunk of the great
sympathetic, sometimes in the kidneys also, and one large mass in par-
ticular is called Zucherkandl’s paraganglion. Nothing definite is known
of the function of these accessory masses, but they are believed to act
as accessory medullary substance. Whether chromaffine tissue exists
in the so-called carotid gland is doubtful. The quite general occur-
rence of chromaffine tissue outside the supra-renals would not be sur-
prising if it be true that many cells normally form small amounts of
adrenaline when the nerve impulses strike them, as has been suggested
by Sherrington.

Embryology.—The cortex and the medulla have different sources in
the embryo. The cortex is derived from the mesoderm which gives rise
to the kidney; the medullary portion contains many sympathetic cells
and appears to be a modified mass of sympathetic nerve cells.

Functions.—The functions of these small organs have only been par-
tially discovered and that within the past few years. As was the case
in so many of the cryptorhetic tissues, attention to them was directed by

' Brown-Séquard, who classed them as organs of internal secretion many
years ago and stated that they were essential to life. Pathology first in-
dicated their function. It was found that in Addison’s disease the
autopsy constantly showed lesions of the supra-renal capsules. These
lesions are generally tubercular in nature. The symptoms of this dis-
ease are a peculiar bronze coloration of the skin; a marked lassitude;
lowered blood pressure; and emaciation. In no other disease is this pig-
mentation found, except in connection with the supra-renals. By this
observation a connection was supposed to exist between skin pigmen-
tation, the general tone of the body and the supra-renal capsules.

Extirpation. The consequences of extirpating the glands differ in
different animals. In man the quick death following lesions, hemor-
rhages, pus formation, etc., in the glands leads us to believe that their
absence could not be long survived. They have been extirpated in
monkeys. The following protocol of one of Kahn’s experiments will
show the general course of events after adrenalectomy. The extirpation
is easier in monkeys than in most animals because, in monkeys, the
glands do not lie so near the vena cava and they are encapsulated. A
female Macacus monkey, weight about 2,000 grams and fed on fruit.
Right supra-renal removed under ether November 9, 1911: The wound
heals quickly. On the 4th December, 25 days after the operation, when
the weight was 1,850 grams, took out the left supra-renal. On the 5th
the animal is very well and eats heartily. On the 6th she eats with
670 PHYSIOLOGICAL CHEMISTRY

normal appetite. Is active. There is a little edema at the edges of the
wound. On the 7th normal. On the 8th normal, but appetite a little
less. On the 9th at 9 a.m., is fairly weak, lies stretched out on the bot-
tom of the cage; no appetite; wound in best state. At 9:12 a.m., great
increase in prostration. Electrical stimulation of the points of exit of
the sciatics produces no effect. Direct stimulation of muscle effective.
Apathetic. The eyes are open and look about. At 1:45 p.m., being
nearly moribund. was killed with chloroform and the liver examined
for glycogen, only a trace being found. The animal lived 5 days. For
4 days it could not be told from a normal animal. The sudden onset
of the symptoms of extreme depression has the appearance of an in-
toxication.

Rabbits have an accessory paraganglion on the aorta and they sur-
vive total extirpation for at least a year without any symptoms. Rats
also will survive for about three weeks, but dogs and cats rarely live
more than 24 hours, the symptoms being again those of intense depres-
sion. It will be noticed that the carnivorous animals die most rapidly,
and that the herbivora die with the same symptoms when their supplies
of glycogen are exhausted and the animal must presumably call upon its
protein for food.

The cause of this sudden death is by no means clear. The survival
of monkeys for 4 days quite normally would seem to indicate that death
was not due to the sudden withdrawal of something necessary for the
body. The indications are, rather, that in the absence of the supra.
renals, poisons, possibly protein degradation products, are formed or
not destroyed. But whatever the explanation, there is no doubt of the
fact that the organs are essential to life in most animals. The symptoms
following adrenalectomy are little, if at all, alleviated by the injection
of extracts of supra-renals. The extirpation is followed by the almost
complete disappearance of glycogen in rats, dogs and monkeys from
both liver and muscles. This is a point of some interest in connection
with diabetes.

The glands have a particular relation to blood pressure. Oliver
and Schaefer discovered that an extract of these glands made with 0.7
per cent. NaCl, when injected intravenously into cats or other mammals.
causes a very quick, intense rise in blood pressure, the effect passing off
rapidly. The substance producing this action was found to be in the
medulla and not in the cortex. It was isolated by Abel as a benzoyl
compound, which he called epinephrine, by extracting the glands with
weak acid and benzoylating the extract. Unfortunately the crystalline
substance Abe] obtained was not the active principle itself, but the
- benzoylated active principle. A benzoyl group remained attached to it.
Aldrich and Takamine, who followed Abel, obtained the free base which
THE CRYPTORRHETIC TISSUES 671

was named adrenaline. Adrenaline may be readily prepared by the
following method (Abel) :

The ground organs are extracted with an equal amount of 3.5% trichloracetic
acid in absolute alcohol, shaken and filtered. The concentrated filtrate forms a micro-
crystalline precipitate of adrenaline on the addition of ammonia. The precipitate
contains about 10% of ash. ,To purify it the crystals in a little water are treated
with some oxalic acid and then alcohol-ether mixture added. The oxalate remains
undissolved. It is converted into the acetate, taken up in alcohol-ether and pre-
cipitated with ammonia. v. Fiirth has another method.

Chemical nature and properties of adrenaline. Adrenaline or
suprarenin or epinephrine, as it is also called, is dihydroxyphenyl-
hydroxyethyl-methyl amine. It is a derivative of pyrocatechin, with the
following formula:

HOC = CH H H
Hoc€ Yo—-b—b_wa cH,

|
HH OH H

It is seen to be related on the one hand to choline, or oxyethyl amine,
and on the other hand to tyrosine, of which it is possibly a derivative.
The natural adrenaline is levo-rotatory and is far more toxic and active
than the dextro-synthetic product. It has been synthesized. (a)}°—
—50.72° (Abderhalden and Guggenheim) ; —51.30 (Abel and Macht).
It gives an emerald green in an acid solution with ferric chloride, and
carmine red in an alkaline solution. It is crystalline and precipitated
from solution by ammonia.

Adrenaline is an unstable, weak base, decomposing rapidly when in
solution in the free form, but being fairly stable as the dry free base and
as the hydrochloride, in which form it is generally sold. Its decomposi-
tion is hastened by alkalies and light. It is a reducing substance and
oxidizes spontaneously, if left in an alkaline solution exposed to oxygen.
The oxidation products are physiologically inactive. The d-adrenaline is
more stable. By oxidation with iodine, persulphate, mercuric chloride
and other oxidizing agents, a red color is formed. It is distinguished
readily by the green coloration it gives with ferric chloride. It is prob-
ably the substance which gives the yellow color to the medullary sub-
stance when the organs are fixed with bichromate. It gives a red color
with I,KI and Hg(l,. It is thermostable in an acid solution. It dis-
solves in strong alkali, NaOH, and probably forms a sodium salt.

Quantitative estimation. The amount of adrenaline in the glands
may be estimated chemically by colorimetric and by physiological
methods. The latter methods are the more delicate and extremely small
amounts may be detected. For the colorimetric method, see page 1010,
experiment 224. For the physiological methods, i.e., the frog eye, blood-
672 PHYSIOLOGICAL CHEMISTRY

pressure, uterus strip methods, reference must be made to other works.
The free base is so active that as little as .000002 gram intravenously
will produce a noticeable effect on blood pressure.

Amount of adrenaline in the glands. Bullock’s supra-renals contain
about 0.3 per cent. (Abel) or 0.25 per cent. (Hunt) of adrenaline.
Folin, Cannon and Denis determined the amount in the adrenals of cats
to be about 0.15 per cent.; in cattle and rabbits, 0.3 per cent.; in dogs
and monkeys, 0.2-0.25 per cent. From human adults suddenly killed
the supra-renals contained. about 0.1 per cent.

Functions of adrenaline. The discovery of the active principle of
the gland has given to the physician one of the most valuable of drugs.
It is one of the most powerful local styptics. It causes contraction of
the arterioles and it has proved invaluable in diminishing inflammation
when a weak solution is dropped in the eye; in reducing hemorrhages,
as in operations on the nose; in checking oozing hemorrhages in the
mucosa of the stomach and bowels; in cases of shock with low blood
pressure it stimulates the heart and raises blood pressure both by its
central action on the central nervous system and by its peripheral
action on the blood vessels. When subcutaneously injected in human
beings it has been found of great value in asthma. Here it causes re-
laxation of the muscles of the bronchioles, upon which it has a remark-
able action just the reverse of histamine. It has besides many other
actions. Injected intravenously, and sometimes when injected sub-
cutaneously, it causes glycosuria; in very minute doses it checks the
rhythmic peristalsis of the intestine. It causes contraction of the preg-
nant cat’s uterus, but not of that of the virgin cat. In large doses in
cats it causes the erection of the hair, relaxation of the bladder, dilation
of the iris and all the symptoms which ordinarily accompany fright or
anger. It will cause secretion of saliva and tears, but not of the pancreas
or stomach. In fact we may at once say that it acts pre-eminently on
all the functions innervated by the sympathetic system. Its action on
any organ is always the same as is produced by stimulation of the sym-
pathetic nerves to that organ and in different animals the effect may
vary, since in some stimulation of a sympathetic nerve may cause activity,
while in others it causes inhibition of activity.

But while adrenaline thus acts on all the sympathetic end organs,
its action is not limited to these. It is, as a matter of fact, a general
protoplasmic stimulant and in large doses it acts on all cells of all kinds
of animals. Thus it checks the development of eggs of all kinds and in
smaller doses produces monsters; it stops ciliary movement; and may
poison the heart. It may make processes of repair slow. But, never-
theless, while thus acting, its main effect in small doses is, as stated, on
the organs innervated by the sympathetic nerves. It has, however, an
THE CRYPTORRHETIC TISSUES 673

action also on skeletal muscle, helping it to recover from fouieue and in
the perfused heart it strengthens the beat.

One of the most interesting of the effects of adrenaline is its power
of producing hyperglycemia, which is nearly always accompanied by
glycosuria. This is the case in nearly all mammals. At first it was
believed that the action was on the pancreas, a gland most intimately
connected with the carbohydrate metabolism of the body. Herter re-
ported that painting adrenaline on the pancreas produced glycosuria
very readily and he and Richards described changes in the islands of
Langerhans as a result of this application. He and Wakeman found
also that the glucosuria following pancreatectomy disappeared after
extirpation of the adrenals. The general result of all the work has been,
however, to indicate clearly that the substance acts by producing a
stimulation of the liver and other cells analogous to that produced by
stimulation of the sympathetic nerves (Figure 61). Claude Bernard

 

Fia. 61.—Section of a lobule of mammalian liver showing central necrosis of the
lobule produced by repeated injections of adrenaline (Drummond).

discovered long ago that puncture of the floor of the fourth ventricle of
the brain, the so-called sugar puncture, was followed by an immediate
glycosuria, particularly apparent in rabbits; and shortly afterwards,
Eckhard proved that this only happened if the splanchnie nerves were
intact and if there was glycogen in the liver. It has been found by
MacLeod that stimulating the splanchnic nerves increases the per. cent.
674 PHYSIOLOGICAL CHEMISTRY

of sugar in the blood. It was not for a long time imagtned that the
sugar puncture could involve the supra-renals, but it was recently
suggested that possibly the splanchnics caused glycosuria, not by
direct action on the liver, but indirectly through the action of the nerves
on the supra-renals, causing them to discharge more adrenaline into
the blood and so to produce an adrenaline glucosuria. Such, how-
ever, is probably not the case. The amount of adrenaline in the blood

is too small to produce such a glucosuria. Furthermore, MacLeod
found that stimulation of the hepatic branches of the splanchnics still
caused hyperglycemia, whereas when the plexus about the veins was
destroyed, stimulation of the splanchnics no longer had this effect. It
appears from these experiments that two factors enter into this hyper-
glycemia: stimulation of the splanchnics acting directly upon the liver
and causing glycogenolysis; and, in the second place, setting free adrena-
line. Adrenaline appears to be necessary for stimulation of the liver
tissues by the nerves, either because it acts upon the sympathetic nerves
themselves, or their terminations, increasing their excitability or their
effectiveness ; or because by direct action on the gland cells the latter are
rendered so much more sensitive that they will respond to nerve stimula-
tion ; or because in some other way the nerve is modified. It is not im-
possible, as suggested elsewhere, that normally the nerve excites its
terminal organ by causing the formation in that organ of either
adrenaline or some similar hormone and that the amount formed by
direct action of the nerve is not quite effective without the addition of
that derived from the adrenals. MacLeod suggests that the adrenaline
acts in the same way as the nerve stimulation.

One of the most interesting facts bearing upon this problem of the
manner in which adrenaline stimulates is that tissues supplied by the
sympathetic system and which have been denervated by cutting the
nerves, or extirpating the ganglia, become hypersensitive to the action
of the hormone. This is the case, for example, in the pupil of the eye
and is the explanation of the so-called ‘‘ paradoxical ’’ dilation of the
pupil. Stimulation of the cervical sympathetic nerve causes dilation of
the pupil by stimulation of the dilator or radiating fibers of the iris. If
the superior cervical ganglion is removed on one side, say the left, the
pupil on that side at once contracts, owing to the tonic action of the
sphincter muscle. After a few days, however, if a little adrenaline is
injected subcutaneously, or intravenously, it will be found that the
pupil of the operated side suddenly dilates, whereas that of the normal
side does not. Furthermore, if the cat is excited, or frightened, or
etherized, dilation of the pupil on the operated side occurs before that
of the other side. This experiment was at first supposed to mean that
the sympathetic carried inhibitory as well as stimulatory fibers to the
iris, but was recognized by Langley as due to hyperexcitability and
THE CRYPTORRHETIC TISSUES 675

has been carefully studied by Anderson. The hyperexcitability of
denervated tissue is not confined to the iris, but all denervated tissue
ordinarily supplied by the sympathetic becomes hypersensitive to
adrenaline after degeneration of its nerves. This fact has not yet been
explained, but it may possibly be due to lack of function rendering the
cell temporarily more unstable. There is perhaps an accumulation in
the cell of substances with which the adrenaline usually unites to
produce its action, and hence the cell is more than usually sensitive.
Possibly the so-called paralytic secretions which occur after division of
gland nerves may have a similar explanation.

This fact of the hypersensitiveness of denervated tissue may be made
use of in detecting the presence of minute quantities of adrenaline in
the blood. Thus, during anesthesia of a cat, the pupil of the side
on which the cervical ganglion has been previously taken out dilates
widely, whereas the normal one does not. This indicates the presence
of adrenaline in small amounts in the blood at the beginning of anes-
thesia, a deduction confirmed by other experiments of which we shall
shortly speak. Section of the splanchnics a few days before renders
rabbits more easily glycosuric on adrenaline injection. Similarly the
difference in behavior of the virgin and pregnant uterus may also be
thus explained. Adrenaline added to blood intensifies the absorption
bands of oxyhemoglobin. (Menten.)

The parathyroid glands have functions in many ways complementary
to the supra-renals. The absence of the parathyroids renders the or-
ganism very resistant to adrenaline. No longer will glycosuria appear
on its injection and the various symptoms of adrenaline poisoning re-
quire much heavier doses for their production.

That the supra-renal glands are constantly discharging adrenaline
into the blood stream is now hardly doubtful. Thus Dreyer first showed
that stimulation of the splanchnics made the blood from the supra-renal
veins more active in raising blood pressure than it was before; this
observation has now been confirmed both by direct and indirect experi-
ment. During anesthesia the glands are very largely drained of their
adrenaline, as is shown by making extracts of the gland at the end of
anesthesia and comparing the amount of adrenaline with that found in
similar normal non-anesthetized glands, and it is also shown by the loss
of the chromaffine reaction in the anesthetized gland. This discharge
of the adrenaline, which is physiologically so important, is due to a
central stimulation, since after section of the splanchnic nerves it no
longer takes place. During anger or fright, in cats at any rate, a sub-
stance having the properties of adrenaline is discharged into the blood
in more than normal amounts and may be detected in the vena cava
blood at the level of the supra-renal veins. The dilation of the pupil,
erection of the hairs, etc., are all produced by adrenaline. The dis-
676 PHYSIOLOGICAL CHEMISTRY

charge may be so pronounced that a glucosuria may be produced, a
glucosuria which does not occur if the glands are extirpated, or if one
gland is extirpated and the veins of the other temporarily compressed,
so that the secretion cannot get into the general blood stream. These
observations are of the greatest interest as indicating the remarkable
participation of the glands in the emotions. It must not be incorrectly
inferred, however, that the emotion of fright depends on the supra-
renal glands, or that the manifestation of this emotion necessarily so
depends. The fact is probably quite otherwise. The central nervous
system is responsible both for emotion and for the stimulation of the
sympathetic system, only the sympathetic stimulates the supra-renals
to secrete adrenaline, which in its turn makes the sympathetic innerva-
tion more efficacious. It is like a process of autocatalysis ; the sympathetic
system, as it is stimulated, automatically raising the efficacy of its own
stimulation. Similar processes probably obtain in the nervous system
itself, and are concerned in the matter of learning and habit formation,
processes which also resemble autocatalysis.

The amount of adrenaline in the supra-renals varies at different
ages and in different conditions of health. It is found in large amounts
in those young animals born in an advanced age, such as lambs, colts
and calves. These animals are able to run about when born and their
adrenals contain normal quantities of adrenaline. In human beings this
is not the case. Some observers have not found adrenaline in children’s
adrenals except in very small amounts up to one year of age. But it
must be remembered that disease may, in these cases, have caused a
discharge, or diminution, before birth or before death. A man and a
horse are not at the same physiological age at birth. In comparison
with their total span of life a horse is as old at birth as a human being
at two years. And a child at birth is as old physiologically as a horse
fetus at four months.

The amount of adrenaline in the glands is greatly reduced in poison-
ing by phosphorus, arsenic, and mercury.

Conclusion.—The supra-renal glands then have the very important
function of manufacturing and secreting into the blood stream the active
principle adrenaline. This substance is 1, 2, dihydroxyphenyl, 4,
hydroxyethyl methyl amine.

OH
aN
Hoc” CH
ll
HC C—CHOH—CH ,—NH (CH 3)

Ng
H
THE CRYPTORRHETIC TISSUES 677

This substance is presumably formed from tyrosine, or phenyl! alanine,
though there is as yet no direct preof of this. A 3.4 dioxyphenyl
alanine has recently been isolated by Guggenheim from Vicia faba.
Substances of a similar nature are found elsewhere in the animal king-
dom than in the supra-renal glands. Chromaffine tissue is found in the
skin glands (parotid gland) of poisonous toads and much adrenaline is
found there also (Abel and Macht in Bufo agua). In ergot one of the
active principles is hydroxyphenylethylamine or tyramine:

OH
Cc

Neg

HC
nt Ue
Nf on » -CH,—NH,

This would appear to be an intermediate product in the formation of
adrenaline. This substance is formed from tyrosine by carboxylase
splitting off carbonic acid. By oxidation and methylation this substance
may be made into adrenaline. Similar substances, called by Kutscher
by the group name aporrhegmas, are formed from other organs of the
body and many of them are physiologically very active. It has been
suggested that adrenaline, or related substances, may be formed in all
cells on the advent of the nerve impulse and that it is by means of these
active, or hormone, substances tzat the cell is roused to activity. Adren-
aline is an unstable, easily oxidized substance, passing readily into a
reddish and brown pigment matter. It is secreted into the blood stream
from the glands and its function appears to be to reinforce the action
of the sympathetic nervous system, thus increasing vasomotor tone, sym-
pathetic tone, the tone of the digestive system, the bladder and uterus,
and al) other structures innervated by the sympathetic. It may also
act on tissues not. innervated by the sympathetic, but in small amounts
its action is confined chiefly to that system. It also affects the central
nervous system and it may play a part in the saturation of oxyhemo-
globin. Its powerful action in dilating the bronchioles and the fact that
it is discharged into the inferior vena cava and in greatest amounts
during anesthesia, fright, exertion, etc., when the need for oxygen is
greatest, indicates that its main physiological a¢tion is upon the right
heart and lungs.

The action of the supra-renals cannot, however, be said to be com-
pletely elucidated by these striking facts. We have as yet no satisfac-
tory explanation of the sudden death of carnivorous animals following
their extirpation and the later death of other animals. These have all
the symptoms of an intoxication, and the injection of adrenaline has
678. PHYSIOLOGICAL CHEMISTRY

very little action in prolonging their life. It is not probable that death
is the result of the lack of production of adrenaline. The complete loss
of general irritability, skeletal as well as that of the sympathetic sys-
tem, is too great to be thus explained. The result might be indirect. It
is certainly possible that other factors come into play; the glands may
have a detoxicating action. Further work on the cause of death follow-
ing adrenalectomy is needed; nor is there any explanation of the pigmen-
tation of the skin in Addison’s disease.

The functions of the cortex of the gland are still entirely obscure.
It has only a slight action on blood pressure, extracts causing a light
fall. In fetal life the cortex is most developed and in the guinea pig it
undergoes marked hypertrophy during pregnancy, followed by rapid
retrogression after birth. It has been suggested that it elaborates the
early stages of adrenaline, but this is not substantiated by any evidence.
Nothing, in fact, is known of its function. It is, of course, not impossible
that the adrenaline is made in the cortex and stored in the medullary
cells. These medullary cells are modified sympathetic nerve cells and
they may have been elaborated to act simply as storehouses for adrena-
line, this substance having always a marked predilection for sympathetic
tissue. Something analogous to this appears to be the case in the
hypophysis. Pituitrine is found chiefly in the posterior lobe, which is
nervous tissue, but there are some reasons for thinking that it may be
made in the anterior lobe, which is glandular in nature. This matter,
however, will need further investigation. Pituitrine resembles epine-
phrine closely in its action, but appears in many ways more nearly like
d-adrenaline than the physiological l-adrenaline.

Besides producing adrenaline the supra-renals are remarkable chem-
ically because of their very large lipin content. They contain more
phospholipins, and other lipins of the general nature of those found in
the central nervous system, than any other non-nervous tissue of the
body. They have also a very intense metabolism.

THE SEXUAL GLANDS.

That the ovaries and the testes have a very marked’ effect on the
growth and metabolism of the body has long been known. Indeed, these
were the first tissues of which the eryptorrhetic powers were generally
recognized. Castration has been practised for a very long time with
the purpose of modifying the body form and temperament. That the
effects were not due simply to cutting the nerves was easily shown.
The reproductive organs give off either from the germ or interstitial cells
substances which, circulating in the blood, change the growth of the horns
and bones in cattle, the development of the hair and mammary glands
and genitals in human beings, and profoundly modify the general psycho-
THE CRYPTORRHETIC TISSUES 679

logical properties. There are a large number of so-called secondary sexual
characters which depend upon the internal secretions of these organs.

The nature of the secretions thus formed is unknown, and this book
is already so long that we can give no more here than a brief notice
of some of the recent interesting work in connection with the corpus
luteum. It will be remembered that when an ovum is discharged from
the Graafian follicle of the ovary there is formed what is known as the
corpus luteum, a yellow, glandular structure which after a time
atrophies. It has been found that the extract of this corpus luteum
determines the growth of the mammary glands which occurs during
pregnancy. In some animals the ova are only discharged and corpora
lutea formed after copulation. If copulation is performed by a sterilized
male, so that pregnancy does not follow, the corpora lutea are formed
as usual. The other symptoms of pregnancy follow, although there
is no pregnancy. Thus dogs and cats will in such cases develop milk
in the mammary glands, often they will proceed to prepare a nest
for the litter. Marsupials, in similar conditions, show some of the same
phenomena; they have been observed to clean out the pouch at the time
when the young should normally appear. If, on the other hand, the
corpora lutea be taken out, then these symptoms do not follow.

It seems probable that the corpora lutea play a very important part
also in preparing the uterus for the fixation of the ovum. It is pos-
sible that extracts of the corpus luteum might be of value in checking
abortion, or in aiding the implantation of the ovum. It has no relation
to estrus.

That the ovary has also a very close relationship to the skeletal
system is indicated by the healing effects of ovariotomy in osteomalacia,
and by the large size of the bones of castrated cattle.

Owing to the uncertainty of the interpretation of many of the facts,
however, a further discussion of this extraordinarily important subject
will not be taken up here. It is not impossible that the further study
of this subject may confirm or demolish some of the present theories
of inheritance.

THYMUS GLANDS.

These glands also are ecryptorrhetic organs, but their function is
still unknown, or at least there is little that is clear-cut and definite
to say about them. By many observers they have been brought
into relation with the growth of the bones and the calcium excretion,
but the evidence is contradictory. From a chemical point of view they
are remarkable for their richness in nuclear material. The chemistry
of the nucleic acid and histone found in them has already been dis-
eussed elsewhere. That they have an important function in the young
animal can hardly be doubted.
oxo PHYSIOLOGICAL CHEMISTRY

PINEAL GLAND.

This is also a rudimentary organ in the brain corresponding in a
general way on the upper surface to the hypophysis on the lower.
The function of this organ is still unknown, but it appears to be closely
related to the sexual organs. Tumors involving the pineal body are
often accompanied by an extraordinarily early development of sexual
maturity with all the secondary sexual characters. Somewhat similar
observations have been made for tumors involving the cortical parts
of the supra-renals. Feeding pineal glands to young chicks or guinea
pigs accelerates their development (McCord).

REFERENCES. Sexuar Oreans.

1. Steinach: Willktirliche Umwandlung von Siugetier-Minnchen in Tiere mit aus-
geprigt weiblichen Geschlechtskarakteren und weiblichen Psyche. (Eine
Untersuchung iiber die Funktion und Bedeutung der Puberti&tsdriisen.
Archiv f. d. ges. Physiol., 144, p. 71, 1912.

2. Bouin and Ancel: Récherches sur la signification physiologique de la glande
interstitielle du testicule des mammiféres. Jour. de Physiol. et Pathol. gen.,
6, p. 1012, 1904.

3. Lederer and Pribram: Experimenteller Beitrag zur Frage tiber die Beziehung
zwischen Placenta und Brustdriisenfunktion. Archiv f. d. ges. Physiol., 134,
p. 531, 1910.

4. Ribbert: Ueber Transplantation von Ovarium, Hoden und Mamma. Archiv f.
Entwickelungsmechanik, 7, p. 688, 1898.

5. Clayron and Starling: Pro. Roy. Soc., 77, p. 505, 1906.

6. Marshall: Phil. Trans. B 198, 1905.

7. Meyns: Ueber Froschhodentransplantation. Archiv f. d. ges. Physiol., 132, p.
433, 1910.

8. Gudernatsch: Fiitterungsversuche an Amphibienlarven. Zent. f. Physiol., 26,
p. 323, 1912.

9. Oliver: On the question of an internal secretion from the human ovary. Jour.
Physiol., 44, p. 355, 1912.

10. Paton: The thymus and sexual organs. III. Their relationship to the growth
of the animal. Jour. Physiol., 42, p. 267, 1911.

ll. Marshall: The ovarian factor concerned in the recurrence of estrus. Jour.
Physiol., 43, p. xxi, 1911.

12. Marshall: Physiology of Sex. (Literature.)

13. Marshall: A note on the chemistry of the vesicular fluid of the hedgehog.
Jour. Physiol., 43, p. 259, 1911.

14. ‘Marshall: The male generative cycle in the hedgehog; with experiments on

ue the functional correlation between the essential and accessory sexual

i:! - organs. Jour.’ Physiol., 43, p. 247, 1911.

15. Scherbok: Versuche tiber innere Sekretion der Brustdrtise. Wiener klin.

a. Woch., 25, p. 199, 1911.

16. Steinach: Geschlechtsbetrieb und echt sekundare Geschlechtsmerkmale als
folge der innersekretorischen Funktion der Keimdrtisen. Zent. f. Physiol.,
24, p. 552, 1910.
17.

18.

10.

11.

12,

a

THE CRYPTORRHETIC TISSUES 681

Marshall and Runciman: On the ovarian factor concerned in the recurrence
of the estrus cycle. Jour. Physiol, 49, p. 17, 1914.

Sand, K.: Experimental hemaphroditism. Jour. of Physiol., 53, pp. 257-263,
1920.

SUPRA-RENALS.

General.

Barger: The Simpler Natural Bases, pp. 81-105, 1914. Monographs on Bio-
chemistry.

Bayer: Die normale u. pathologische Physiologie des chromaffinen Gewebes der

Nebennieren. Ergebnisse der pathol. Anat., 14, 1910.

Biedel: Innere Sekretion, ihre physiol. Bedeutung f. d. Pathologie. Berlin, 2d
ed., pp. 313-521, 1913.

Rolleston: Suprarenal bodies. Goulstonian Lecture. British Med. Journal, I.,
pp. 629-634; 687,691; 745-748, 1895. Earlier literature cited.

Vincent: Innere Sekretion u. Driisen ohne Ausfiihrungsgang. Ergebnisse dir
Physiol., 9, pp. 509-586, 1910. Literature.

Pharmacy of adrenaline.
Beckwith: The pharmacy of adrenaline. Journal of American Pharmaceutical
Assn., vol. III, pp. 1547-1554, 1914.

Physiology of glands.

Elliott: The control of the suprarenal glands by the splanchnic nerves. Jour.
of Physiol., 44, p. 374, 1912.

Cannon and Hoskins: The effect ‘of asphyxia and sensory stimulation on adrenal
secretion. Amer. Jour. Physiol., 29, p. 274, 1911.

Cannon and de la Paze: Emotional] stimulation of adrenal secretion. Amer.
Jour. Physiol., 28, p. 69, 1911.

Cannon, Sholl and Wright: Emotional glycosuria. Amer. Jour. Physiol., 29,
p. 280, 1911.

Wertheimer and Battez: Sur les nerfs glyco-sécréteurs. Archives internat. d.
Physiol., 9, p. 363, 1910.

Mayer: Sur le mode d’action de la piqfre diabétique. Rédle des capsules sur-
rénales. C. R. Soc. Biol., 60, p. 1123, 1906; 219, 1908.

Dreyer : On secretory nerves to the supra-renal capsules, Amer. Jour. Physiol., 2,
p. 203, 1899.

Joseph and Meltzer; Effect of stimulation of the peripheral end of the splanchnic
nerves upon the pupil. Amer. Jour. Physiol., 29, p. xxxiv, 1912.

Paton and Watson: The action of pituitrin, adrenaline and barium on the cir-
culation of the bird. Jour. Physiol., 44, p. 418, 1912.

Oliver and Schaefer: The physiological effects of extracts of the suprarenal
capsules. J. Physiol., 18, pp. 230-276, 1895.

MacLeod: The relation of the adrenal glands to sugar production by the liver.
VIII, Amer, Jour. Physiol., 29, 1912, p. 419.

Kahn: Archiv f. d. ges. Physiol., 144, p. 396, 1912; 146, p. 578, 1912.

Adrenaline. Chemistry and physiology.

Abel: Further observations on the chemical structure of the active principle of
the suprarenal capsules. Johns Hopkins Hospital Bulletin, 9, pp. 215-218,
1898.

Abel: Further observations on epinephrine. Johns Hopkins Hospital Bulletin,
12, pp. 80-84, 1901.
682

10.

11.

12,

13.

14,

15.

16.

17.

18.

19.

20.

21.

22.

23.

24,

25.

26,

PHYSIOLOGICAL CHEMISTRY

Abel: On epinephrine and its compounds with especial reference to epinephrine
hydrate. Amer. Jour. Pharm, 75, pp. 301-325, 1903.

Abel and Macht: The poison of the tropical toad, Bufo agua. Jour. Amer. Med.
Assn., 56, 1911, pp. 1531-1536.
Abel and Macht: Two crystalline pharmacological agents obtained from the
tropical toad. Jour. Pharm. and Expt. Therapeut., 3, 1912, pp. 319-377.
Aldrich: A preliminary report on the active principle of the ee gland.
Amer. Jour. Physiol., 5, p. 457, 1901.

Aldrich: Adrenalin the active principle of the suprarenal gland. Jour. Amer.
Chem. Soc., 27, pp. 1074-1091, 1905.

Takamine: Adrenalin the active principle of the suprarenal glands and its mode
of separation. Amer. Jour. Pharm., 73, 1901, pp. 523-531.

Takamine: The isolation of the active principle of the suprarenal gland. Jour.
Physiol., 27, p. xxix, 1901.

Schultz: Quantitative pharmacological studies: Adrenalin and adrenalin-like
bodies. Bulletin Hygienic Laboratory, 55, Washington, 1909.

Schultz: Quantitative pharmacological studies: Relative physiological activity
of some commercial solutions of adrenalin. Bulletin 61, 1910, Hygienic
Laboratory, Washington.

Stewart: The alleged existence of adrenalin in pathologica] sera. Jour. Expt.
Med., 15, pp. 547-569, 1912.

Folin, Cannon and Denis: A new colorimetric method for the determination of
epinephrin. Jour. Biol. Chem., 13, pp. 477-483, 1913.

Friedmann: Die Konstitution des Adrenalins. Beitriige z. chem. Physiol., 8,
pp. 95-120, 1906.

Ewins: Some color reactions of adrenine and allied bases. Jour. Physiol., 40,
pp. 317-326, 1910.

Ewins and Laidlaw: Alleged formation of adrenine from tyrosine. Jour.
Physiol., 40, 275-278, 1910.

Fenger: On the presence of active principles in the thyroid and surprarena]
glands before and after birth. Jour. Biol. Chem., 11, 1912, pp. 489-492; 12,
pp. 55-59.

Barger and Jowett: The synthesis of substances allied to epinephrine. Jour.
Chem. Soc., 87, pp. 867-974, 1905.

Blum: Ueber Nebennierendiabetes, Deut. Archiv f. klin. Med., 71, 146-167, 1901.

Abderhalden and Thiess: Weitere Studien tiber das physiologische Verhalten
von 1, dl and d, Suprarenin. Zeit. physiol. Chem., 59, pp. 22-28, 1909.
Earlier papers on subject cited here.

Abderhalden and Guggenheim: The action of tyrosinase from Russula delica on
tyrosin containing polypeptides and suprarenin. Zeits. physiol. Chem., 57,
1908, pp. 329-331.

Dakin: On the physiological activity of substances indirectly related to ad-
renalin. Pro. Roy. Soc., B, 76, pp. 498-503, 1905.

Gottlieb and O’Connor: Ueber den Nachwies u. die Bestimmung des Adrenalins
im Blute. Handbuch biochem. Arbeitsmethoden, VI, pp. 585-603. 1912.
Guggenheim: Dioxyphenylalanin, eine neue Aminosiure aus Vicia Faba. Zeits.

physiol. Chem., 88, pp. 276-284, 1913.

Kolmer : Relation of adrenals'to the sexual function. Archiv f. d. ges. Physiol.,
144, p. 361, 1912.

Elliott: Some results of excision of the adrenal glands. Jour. Physiol., 49
p. 38, 1914,
10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21,

22.

23.

THE CRYPTORRETIC TISSUES 683

‘THYROID.

Thompson: On the thyroid and parathyroids throughout vertebrates, with ob-
servations on some other closely related structures. Phil. Trans. Roy. Soe.
B, 201, p. 91, 1911.

Koch, F. C.: On the nature of the iodine-containing complex in thyreoglobulin.
Jour. Biol. Chem., 14, p. 165, 1913.

Oswald: Neue Beitrage zur Kenntnis der Bindung des Jods im Jodthyreoglobulin
nebst einigen Bemerkungen tiber das Jodothyrin. Arch. f. expt. Path. u.
Pharm., 60, p. 115, 1908.

Hunt: The influence of thyroid feeding upon poisoning by acetonitrile. Jour.
Biol. Chem.. 1, p. 33, 1905.

Hunt and Seidell: Bulletins 48 (1908) and 69 (1910). Hygienic Laboratory,
Washington.

Baumann: Ueber das normale Vorkommen von Jod in Thierkérper. Zeits. f.
physiol. Chem., 21, p. 487, 1896; 22, p. 1, 1896.

von Cyon and Oswald: Ueber die physiologischen Wirkungen einiger aus der
Schilddriise gewonnener Producte. Archiv f. d. ges. Physiol., 83, p. 1901.

von Cyon: Metholodische Aufklarungen zur Physiologie der Schilddriise. Archiv
f. d. ges. Physiol., 138, p. 575, 1911.

Olaude and Blanchetiere: Sur la teneur en iode de la glande thyroid dan ses
rapports avec la constitution anatomique de l’organe. J. de Physiol. et Path.
gen. 12, p. 563, 1910.

Oswald: Ueber den Jodgehalt der Schilddriisen. Zeits. f. physiol. Chem., 23,
1897. Eiweisskérper der Schilddriise. Ibid., 27, p. 14, 1899.

Krause and Cramer: On the effects of thyroid feeding on nitrogen and carbo-
hydrate metabolism. Jour. Physiol., 44, p. xxiii, 1912.

Gley: Récherches sur la pathogenese du goitre exoptalmique. Jour. d. Physiol.
et Pathol., 13, p. 955, 1911.

Jeandelize et Parisot: La pression artérielle aprés la thyroidectomie chez le
lapin. J. de Physiol. et Pathol. gen., 12, p. 331, 1910.

Rossi: Sur les effets de la thyreo-parathyreoidectomie chez les animaux de la
race ovine. Archives Ital. de Biol., 55, p. 91, 1911.

Vincent: Innere Sekretion u. Driisen ohne Ausfiihrungsgang. Ergebnisse der
Physiol., 9, 1910. Literature.

von Firth u. Schwartz: Ueber die Einwirkung des Jodothyrins auf den Zirku-
lationsapparat. Arch. f. d. ges. Physiol., 124, p. 113, 1908.

Modrakowski: Ueber die Identitét des blutdrucksenkenden Kérpers der Glandula
thyroidea mit dem Vasodilatin. Arch. f. d. ges. Physiol., 133, p. 291, 1910.

Bircher: Weitere Beitrige zur experimentellen Erzeugung des Kropfes. Zeits.
f. expt. Pathol. u. Ther., 9, 1911.

Justchenko: Die Schilddriise u. die Fermentativen Prozesse. Zeit. f. physiol.
Chem., 75, p. 141, 1911.

Fenger: On the presence of active principles in thyroid and suprarenal before
and after birth. Jour. Biol. Chem., 12, p. 55, 1912.

Koch: On the occurrence of methyl-guanidine in the urine of parathyroidec-
tomized animals. Jour. Biol. Chem., 12, p. 313, 1912.

Greenwald: Further experiments upon parathyroidectomized dogs. Jour. Biol.
Chem., 14, p. 363, 1913; 14, p. 369, 1913.

Fenger: On the iodine and phosphorus contents, size and physiological activity
of fetal thyroid gland. Jour. Biol. Chem., 14, p. 397, 1913.
684

ol

10.

11.

12.

13.

14.

15.

PHYSIOLOGICAL CHEMISTRY

Pinzat GLAND.

Vincent: Interna] secretion and ductless glands. 1912.

Baily and Jelliffe: Tumors of the pineal body. Archives of Internal Medicine, 8,
p. 851, 1911.

McCord: The pineal gland in relation to somatic, sexual and mental develop-
ment. 65th Session Amer. Med Assn., June, 1914.

Dana and Berkely: Medical Record, 83, p. 835, 1913.

Foa: Hypertrophie des testicules et de la eréte apres l’extirpation de la glande
pinéale chez le coq. Arch. Ital. de Biol., 57, p. 233, 1912.

Berkely: Med. Record, New York, 85, p. 513, 1914.

PaRATHYROD.

Carlson: Condition of the digestive tract in parathyroid tetany in its and dogs.
Amer. Jour. Physiol., 30, p. 309, 1912.

Cooke: Changes in nitrogenous metabolism after parathyroidectomy. Jour.
Expt. Med., 13, p. 439, 1911.

Greenwald: The effect of parathyroidectomy upon metabolism. Amer. Jour.
Physiol., 28, p. 103, 1911.

Ver Hecke: Etude de Vinfluence de la sécrétion interne du corps thyroid sur les
échanges organiques. Archives internat. de Pharmacodyn., 4, p. 81, 1898.

MacCallum and Voegtlin: On the relation of tetany to the parathyroid glands
and to the calcium metabolism. Jour. Expt. Med., 11, 1909, p. 118. Jour.
Pharm. and Expt. Ther., 2, 1911, p. 421.

Berkely and Beebe: A contribution to the physiology and chemistry of the para-
thyroid gland. Jour. Med. Research, 20, p. 149, 1909.

Morel: Les parathyroides dans l’ontogenase. C. R. de la Soc. Biol., 67, 1910.

Gley: De Y’exopthalmie consécutive 4 la Thyroidectomie. C. R. Soc., Biol., 68, p.
858, 1910.

Falta and Kahn: Klinische Studien iiber Tetanie mit besonderer Beriicksichtigung
des vegetativen Nervensystems. Zeits. f. klin. Med., 124, p. 1.

Wiener: Ueber die Art der Funktion der Epithelkérperchen. Archiv f. d. ges.
Physiol., 136, p. 107, 1910.

Vincent: Some observations on the functions of the thyroid and parathyroid
glands. Jour. Physiol., 32, p. 65, 1905.

Arthus and Schapermann: Parathyroidectomy et sels de chaux chez le lapin.
Jour. de Physiol. et Pathol. gen., 12, p. 177, 1910.

Morel: L’Acidose Parathyroprive. Jour. de Physiol. et Path. gen., 13, 1911, p.
542. :

Meltzer ana Joseph: The inhibitory action of sodium chloride upon the phe-
nomena following the removal of the parathyroids in dogs. Jour. Pharm.
and Expt. Therapeut., 2, p. 361, 1911.

Paton, Findlay and Burns: On guanidin or methyl guanidin as a toxic agent
in the tetany following parathyroidectomy. Jour. Physiol., 49, Proce.
Physiol. Soc., p. xvii, 1915.
CHAPTER XVII.
THE EXCRETIONS OF THE BODY.
THE URINE.

It will appear from the account which has been given of the chem-
istry of the different tissues of the body how fragmentary is our
knowledge of the chemical transformations undergone by the food mate-
rials after their absorption. For example, the simple proteins of the
foods are reduced by digestion to the amino-acids. Some of these amino-
acids are deamidized by the action of bacteria, or the enzymes of the
alimentary muccus membrane. The amino-acids enter the blood of the
portal system partly as amino-acids, partly as ammonia and ketonic
acids, and to some extent possibly in other decomposition products not
yet recognized. In the blood they circulate dissolved in the plasma and
as they pass the various tissves each tissue removes, it is believed, those
amino-acids in the quantity which it needs for its own metabolism ;.and
it builds up its own protein from them. It remains for physiological
chemistry to follow the subsequent fate of these amino-acids through
the complex chemistry of each organ of the body, discovering what
proportion is synthesized into the cell proteins; how rapidly they are
broken up and what decomposition products arise from them; and fol-
lowing, also, the fate of the amino-acids not synthesized into protein,
but broken up directly by the cell into products of great importance to
the cell or its neighbors. This task, however, is beyond our powers at
present and is indeed a vastly difficult task, since each organ has a dif-
ferent metabolism and must be studied by itself. We know scarcely
anything, for example, about the chemical composition of many of these
organs and particularly little about the master tissue of the body, the
nervous system, so that we cannot proceed directly to each organ and
describe just what chemical changes occur in it. We must of necessity
take a roundabout course and follow the path which the science has
actually followed in its development. Having traced the foods into
the blood, the internal medium of the body, we turn to see in what form
the various substances, nitrogen, carbon, hydrogen and oxygen, leave the
body; and by working backward from the excretions gradually narrow
down the problem to the metabolism of the various organs. We shall

turn now to study the composition of the various excretions of the body.
685
686 PHYSIOLOGICAL CHEMISTRY

These excretions are those of the kidneys, the skin, the lungs and the
alimentary canal. :
The total amount of the excretory material varies with the amount
of food and drink consumed and the amount of work done. By refer-
ence to the metabolism experiment on page 292 it will be seen that a man
doing pretty hard muscular work, so that he expends 5,500 calories of
energy per day, excreted about 1,000 c.c. of water per day in his urine,
about 240 c.c. per day in the feces, and about 4,000 grams per day in
the respiration and perspiration. The total output of water amounted,
therefore, to about 5,240 grams per day. For a person not working the
amount is less than this and about 3,000 c.c. The relative loss through
the lungs and skin can be determined by the very ingenious method of
Lombard. He placed a man naked on one pan of a delicate balance, the
opposite pan of which had a lever attached to it so that it traced a curve
on a moving drum covered with smoked paper. Under these circum-
stances, of course, since the man is constantly losing weight by the
evaporation of water from the skin and by the exhalation of water and
carbon dioxide by the lungs, he grows constantly lighter and the pointer
traces a fairly uniform falling line on the revolving drum. The man
visibly evaporates before one’s eyes. The loss in this case is bath by
the skin and lungs. The slope of the curve tells how rapidly the change
of weight is occurring. Now while this curve is being taken, if the man
simply holds his breath there is no longer any loss through the lungs,
but there is a continuous loss through the skin. The slope of the curve
for this period shows how rapid the loss is through the skin alone. By
this means and by other experiments it has been found that by respira-
tion there is lost about 30 per cent. of the total water, by the skin 17 per
cent. and by the urine about 50 per cent. These figures are subject to
wide variation. In the experiment just quoted the loss through the skin
and lungs was at least double that through the urine, and it may be a
still larger proportion during exercise on a hot day. In the lungs there
is lost, also, about 1,700 grams of carbon dioxide per day. Water and
carbon dioxide pass out, then, in large measure through the lungs. The
skin carries out chiefly the water in the perspiration; but there are also
present some salts and some nitrogen substances, such as urea. The
amount of nitrogen lost through the skin is variously estimated. It is
generally determined by collecting the perspiration in some form of
cotton underclothing, washing this out and determining the amount
of nitrogen in the wash water. The method is not exact. The nitrogen
loss through the skin is undoubtedly larger in hot, dry climates than in
moist, cold ones. It may be estimated as about one-half gram of nitro-
gen daily, but it may be nearly a gram a day. The feces have been con-
sidered already. They contain on the average about 1-2 grams of nitro-
THE EXCRETIONS OF THE BODY 687

gen per day. All the rest of the nitrogen excreted, except small por-
tions lost in hair and the wear of the skin, passes through the kidneys
into the urine. It is this fact which makes the urine of such great
interest. The urine also contains various carbon compounds free from
nitrogen and, in small quantities, a great number of substances which
have escaped from the metabolism of the body. The composition of these
substances is often of great interest because of the light they throw on
the intermediate metabolism of the body.

The urine.—The urine is secreted by the kidneys. In man and mam-
malia these are two organs, more or less ellipsoidal in shape, weighing
in male adults about 130 grams each, situated on each side of the spinal
column, behind the peritoneum at about the level of the first three
lumbar vertebre. They are of mesodermic origin and develop in em-
bryonic life from the genito-urinary ridge, a ridge of mesoblastic tissue
extending from head to tail. They are very long organs in the lowest
fishes, extending nearly the length of the body cavity from far forward
in the head; and are called pronephros or head kidneys. In amphibia
the middle region of the ridge gives rise to the kidneys, which are called
the mesonephros, and in mammalia only the posterior region or caudal
portion thus develops into a more compact organ, the metanephros or
true kidneys. In the invertebrates somewhat similar excretory organs
are the segmental nephridia of the annelids and arthropods. The kid-
neys lie very close to the dorsal aorta, receiving an unusually large
amount of blood by very large, short, straight arteries which deliver the
blood under high pressure into a number of capsules of capillaries, each
capsule inclosed in a very fine endothelial membrane. The resistance
to the flow in the capillaries must be high. The glomerulus is supposed
to function as a filtering apparatus (see, however, the recent work of
Brodie) and from it a large amount of liquid containing salts and other
soluble constituents, and at times protein, is believed to be filtered into
the head of the kidney tubule. From these glomeruli the filtrate passes
along contorted tubules, the kidney tubules, making up the greater part
of the kidney substance and which are lined by epithelial cells, which
are supposed to secrete the various nitrogenous and other constituents
of the urine and, perhaps, to reabsorb some of the water. It cannot be
said, however, that the mechanism of secretion of the urine is as yet
in many particulars clear. The secretion is under the control of the
blood flow; a more rapid blood flow, a higher pressure in the glomeruli,
other conditions remaining normal, is accompanied by a higher secretion
of urine.

Amount. The amount of urine secreted per day is extremely vari-
able for different individuals, depending on diet, external temperature,
amount of water drunk and on the constitution of the individual. Thus
688 PHYSIOLOGICAL CHEMISTRY

in warm weather, or when a restricted diet is being taken or little water
drunk, it may be as little as 600-700 cc. per day. In cold weather it is
always larger and also on a heavy diet. An average abstemious Ameri
can student in the Northern States secretes about 1,200-1,500 ¢.c. per
day. The drinking of tea, coffee or beer increases the amount in part
because of the fluid they contain, and in part because of the presence
of diuretics in these beverages. Some individuals, generally of a nerv-
ous temperament, secrete larger amounts, from 2,000-3,000 cc. The
cause of this mild polyuria (diabetes insipidus) is still obscure, but it
may be produced artificially by some injury to the part of the brain
about the infundibular body. It may be due to slight pressure in this
region.

Specific gravity. The specific gravity varies usually inversely with
the amount secreted from 1.008-1.080. The color is generally a darker
yellow, or brown the more concentrated the urine. In polyuria, hys-
teria and some nervous affections the urine is often almost colorless.

Reaction. Normal human urine is usually acid to litmus, though it
may at times be alkaline. It is most acid on a meat diet; on a vegetable
diet it may be neutral or alkaline. Its acidity is greatly reduced during
the secretion of the gastric juice in the stomach during digestion, so
that shortly after a meal an alkaline reaction may be had. There is at
that time an alkaline tide in the body due to the separation of the
hydrochloric acid (page 376). The urine of carnivorous animals is
usually acid; that of herbivorous, such as cows and horses, is alkaline
except during fasting, when it may be acid. The reason why the urine
is acid when on a meat diet and alkaline on a herbivorous is that in
protein there occur sulphur in an unoxidized condition, and esters of
phosphoric acid in nucleic acid, phosphoproteins and in lecithin. Dur-
ing metabolism these are oxidized to sulphuric acid or set free as phos-
phoric acid. It is this acidity which accounts for the acidity of the
urine. Nitrogen from the proteins is eliminated in the unoxidized form
as urea, for the most part, but this is a very weak base, practically neu-
tral in reaction, and hence not capable of neutralizing the acid pro-
duced. There are also some organic acids formed from the oxidation
of parts of the protein, such as phenyl acetic acid, benzoic acid, uric
acid, urocanic acid and so on, which also contribute to the urine acidity.

The alkaline reaction of the urine of herbivorous animals is owing to
the fact that vegetables and fruits contain acid salts of dibasic acids,
or polybasic acids, or other carboxylic acids, such as acid potassium
malate, citrate, acetate, tartrate and so on. On oxidation in the body
these are burned to carbonates. Some of the carbonic acid finds its
exit through the lungs, leaving the associated base, generally sodium
or potassium, with the very weak acid, carbonic acid, to find its way into
THE EXCRETIONS OF THE BODY 689

the urine. The carbonates have an alkaline reaction owing to their form-
ing some free alkali by hydrolysis. It seems paradoxical at first sight
that by drinking an acid citrate the urine should be made alkaline, but
it is explained in the way just stated.

The amount of acid which can be titrated in the urine by alkalies
with phenolphthalein as an indicator amounts in a day to the equiva-
lent of from 150-400 c.c. of N/10 acid. It is about equivalent to N/40th
acid by titration. It varies greatly and is often less than this.

The real acidity of the urine, that is the number of the hydrogen
ions it contains, can be determined either by the gas-chain method
described on page 539 or by the indicator method. <A recent extensive
investigation of the acidity by the latter method by Henderson has
shown that the acidity is about equal to NX10—* hydrogen ion. It may,
for short periods, rise to about NX10—®! or become as alkaline as
NxX<10—*4. As a rule the total acidity as determined by titration and
the concentration of the hydrogen ions go parallel. This may be seen,
for example, in the following case:

Amount c.c. H’ Reide'e. NH, ¢.c. Total acidity Acidity

Case No. hiring (Pq) N/10 Nyto ec ne ae :
3 1000 6.0 222 360 582 0.62
820 5.5 410 600 1010 0.68

1100 6.3 195 390 585 0.50

1500 5. 315 535 850 0.59

1140 6.0 150 290 440 0.52

It will be observed that when the hydrogen ion concentration is great-
est and NX10-*5 the total acidity is highest, i.e., 1010.

There are no characteristic variations of the acidity in disease thus
found, except that in cardio-renal disease the mean acidity is somewhat
higher than normal, H ion=NX10—*-%, but this is not higher than may
occur in normal urine. It appears to be easier to develop high acidity
in disease than greater alkalinity. This may be due to the fact that
in serious disease the diet is generally restricted and the patient may
become more purely carnivorous, the body calling on its own reserves.
While all the acids of the urine contribute to this acidity, phosphoric
acid, ov account of its greater quantity, predominates in producing the
effect. It is a weak acid and such strong acids as sulphuric will satisfy
themselves at the expense of any alkali metal bound to the phosphoric
acid.

In case the total acidity is increased, the ammonia generally rises,
but not always proportional to the amount of acid produced. The
amount of protein eaten, or other variations in the diet, such as the
amount of fruit and so forth, determine whether the acid produced will
be neutralized more by ammonia or by the fixed alkalies.

By the ingestion of acid and bicarbonates the acidity of the urine
690 PHYSIOLOGICAL CHEMISTRY

may be either increased or diminished. Four hours after the ingestion
of 10 grams of monosodium phosphate the acidity of the urine had risen
from NX10—*7 to NX10—-*3 in an experiment by Henderson and
Palmer. In one experiment 12 grams of sodium bicarbonate were
ingested at 10.00 a.m. when the acidity of the urine was NX10~5°°, At
12 o’clock the acidity had fallen to NX10-*". The urine was
then strongly alkaline. It remained at this alkalinity for several
hours.

Osmotic pressure of the urine. The osmotic pressure of the urine is
much higher than that of the blood. This means that the kidney must
do work in the secretion of the urine and the amount of work done can
be determined from the difference of osmotic pressure. It is evident
that the secretion is not a simple filtration, but that the vital activity
of the cells enables them to do this work. The work is done either in the
reabsorption of water by the tubules, thus concentrating the filtrate from
the glomeruli, or more probably in the secretion of the urea and other
substances into the urine. The osmotic pressure is generally deter-
mined by the freezing-point method described on page 201. The freezing
point of the urine varies from about —0.65° to --2.71°. The latter rep-
resents an osmotic pressure (see page 201) of 32.4 atmospheres. The
freezing point of the blood is about —0.6°, which is an osmotic pressure
of only 7.23 atmospheres. If the difference in osmotic pressure of the
blood and the urine amounts to 25 atmospheres and there is secrete
1 liter of urine per day, the kidneys would have to do 25 liter atmos.
pheres of work. 1 liter atmosphere is equivalent to 1.0132 10° ergs, or
2.421110 small calories (15°). In a day, therefore, the kidneys would
do 25 liter atmospheres or 605 small calories of work. This would be
0.605 large calories. As the total output of energy of the body per day
is about 2,500 to 3,000 large calories, the work of the kidney represents
a very small proportion of it. The burning of something less than 0.7
gram of glucose would yield this amount of energy. The osmotic pres-
sure of the urine approximates more closely to that of the blood the
more rapid the secretion; during exertion on very hot, dry days the
freezing point may be still lower than —2.71° and may go below —3.0°.
Bugarsky has found a formula of fairly general applicability express-
ing the relation between the osmotic pressure (freezing point or A)
and the specific gravity: A—=75(s—l), where s is the specific gravity.
In certain circumstances the urine may have a freezing point of only
—0.2°, as after drinking large quantities of beer.

General composition of the urine.—The following table illustrates
the average composition of human urine on an average diet containing
420 grams of protein per day. The amount of solids and water are of
course widely variable. A small quantity of both zine and copper are
found generally present in human urine.
THE EXCRETIONS OF THE BODY 691

ToraL URINE PER Day 1500 c.c. Tora Sonips 61 Grams.

 

 

Organic solids ........... 38.2 grams. Inorganic solids ......... 22.7 grams.
Urea: singin Seyeein aes dae 32 Sodium chloride ....... 14 grams.
Uric acid ....... geese 0.7 HPO, 0. .eeeeee eee ees 2.0
Creatinine ............. 1.8 HSO, ...-- sees renee 2.6
Ammonia ............. 0.7 KO... ee eee eee eee 3.0
Hippuric acid ......... 0.8 MgO and CaO ......... 0.9
Residual organic ....... 2.2 Residual inorganic ..... 0.2

38.2 22.7

Nitrogenous constituents of the urine.—The nitrogenous constitu-
ents found in the urine are urea, uric acid, ammonia, creatinine and
creatine, amino-acids, allantoine, hippuric acid and a great number of
basic and other nitrogenous substances found in very small quantities.
But while present in small quantities many of these are of very great
interest, since they undoubtedly represent intermediate products in the
course of the transformations of the amino-acids in the body. In addi-
tion there may be found in pathological conditions proteins, or derived
proteins, of various kinds. In human urine urea makes by far the
larger proportion of the nitrogenous substances. Normally from 85-92
per cent. of the nitrogen in the urine is in the form of urea nitrogen;
but on a diet containing a minimum quantity. of protein food the pro-
portion of urea nitrogen falls, so that it may be not more than 68-80
per cent. of the total urinary nitrogen.

In a group of about 25 normal young men in the course of an
extended metabolism experiment, the average excretion of nitrogen per
24 hours was as follows:

Body- Ingested Total Urea, "NH: Creati- Uric Undeter- Total
weight nitrogen urinary N N nine acid mined urinary
—kilos —grams N N N . N N as per cent.

g. g. g. g. g. g. of ingested.

71.0 13.43 11.30 9.30 0.49 0.659 0.198 0.65 84.12
Per cent. of total Nin urine 82.29 4.35 5.87 1.76 5.73

Urea. Chemistry.—Urea is the diamide of carbonic acid, CO(NH,),:

ig
Oo=—C
|
NH,
Urea.
It may be regarded as formed from carbonic acid as follows
OH NH
2

| |
o=C + NH, —~0=C + H,0
| |
OH OH
Carbamic acidy
692 PHYSIOLOGICAL CHEMISTRY

NH NH.
i i
Oo=C + NH, 0=C + H,0

| |
OH NH

Carbamic acid. Urea.

According to this formula urea should be a very faintly basic sub-
stance, and acids should have some power of union with the amino
groups, as they do have. If a urea solution is heated, however, it sets
free some hydrogen ions and behaves as an acid. This is probably due
to a rearrangement of the molecule in the direction of the imide form,
and some molecules having the imide constitution probably exist in all
solutions of urea. The imide form is the following:

NH NH NH NH
| * i I ie
o=C0 HO—C Cc 2 €
| : I| II II
NH, NH +H,0 N
Urea. Imide form. Carbodiimide. Cyanamide.

Urea may be synthesized by heating ammonium cyanate which under-
goes a rearrangement into urea:

NH,—0O—C=N =~ NH,—CO—NH,
Ammonium cyanate. Urea,

Urea has no taste. It is colorless and odorless and crystallizes in
long prisms, m.p. 132-1383°. It may be purified by recrystallization
from amyl alcohol. It is extremely soluble in water and in alcohol, but
with difficulty in cold acetone. It is insoluble in ether. It forms salts
with acids, and the oxalate and nitrate, CH,N.O.HNO,, are much less
soluble in water than urea itself. By heating with acids or alkalies it
is split into ammonia and carbonic acid and the same change occurs on
heating with water. Dry urea heated forms biuret, cyanuric acid and
the amide of cyanuric acid, or ammelide:

NH NH NH.
\
2 2 2:
sl chest ! i
_ oe ao 2. 23 i —_— 3NH, — Hoc fou
NH, NH—CO—NH, NH,
Urea. Biuret. Cyanuric acid.

Oxidized by hypobromite or nitrous acid, it is decomposed into
carbon dioxide, nitrogen gas, some carbon monoxide and nitric oxide.
The greater part of the decomposition, however, leads to the formation
of nitrogen gas and carbon dioxide. The latter reaction is used for the
clinical estimation of urea.
THE EXCRETIONS OF THE BODY 693

co (NH 2HNO, = CO,+2N +3H.0
co (MES 3NaOBr = CO! t aNaBr + $11,04.N,
Urea will also, like the amino-acids, form molecular compounds with
salts. Thus, if a solution is evaporated with sodium chloride, prisms
of CO.(NH,),.NaCl.H,O erystallize out. Urea is decomposed into
ammonium carbonate by an enzyme, urease, found in the Soya bean, in
several bacteria and in other plant and animal tissues. Urea is found in
the excretions of nearly all classes of invertebrates.

Amount.—The amount of urea secreted by the kidneys per day
is very variable and depends upon the amount of protein in the food.
For a person eating an average diet containing about 120 grams of
protein per day, the urea excretion will be in the neighborhood of 30
grams; but on a low protein diet the amount of urea is greatly reduced.
With a diet containing 50 grams of protein per day, or 8 grams of
nitrogen, the urea will be only about 8 to 10 grams a day. No con-
stituent of the urine is more variable than the urea. Ordinarily, when
the protein intake is high, about 90 per cent. of the nitrogen of the
urine is in the form of urea nitrogen; but on a low protein diet the
proportion of urea nitrogen falls in the urine to about 60 per cent. of
the total. This is shown in the table on page 750 taken from one of
Folin’s experiments.

The explanation of this variation of urea with the diet is that when
more protein is eaten than is necessary to replace that decomposed in
the vital processes in the body, the body does not store the excess, since
there is no provision for the storage of an excess of protein, except in
relatively small quantities. Instead of storing the excess, the nitrogen
is split off from the amino-acids, converted into urea and excreted,
while most of the carbonaceous part of the amino-acid molecule is con-
verted into glucose, or fat, and stored in that form. Hence, when a
very heavy protein diet is consumed, the amount of urea increases
enormously and proportional to the protein consumption.

The fact that there is only a very limited storage of protein in the
body is of very great significance. It indicates that the proteins play
a different réle from the fats and carbohydrates, probably because the
proteins make part of the living matter of the cells. It is impossible
to increase very much this vital matter without at the same time increas-
ing the surface of the body, increasing its supply of oxygen and in
other ways providing for its needs. Lifeless protein for storage evi-
dently does not exist in the body, except in limited amounts: for
example, in the connective tissue fibers, possibly the proteins of the
blood, and some other supporting tissues.

Origin of the urea in mammals.—What is the origin of the urea
found in the urine? In what organ is it formed? One turns first to
the kidneys. Do the kidneys form the urea or do they only excrete it?
694. PHYSIOLOGICAL CHEMISTRY

A definite answer to this question is obtained by extirpating the kid-
neys and examining the urea content of the blood before and after
extirpation. If the kidneys are the chief or sole producers of the urea,
then there will be no accumulation of urea in the blood after the extir-
pation of these organs; if, on the other hand, they simply excrete the
urea which is formed elsewhere in the body and brought by the blood
to them, then urea will accumulate in the blood, if there is no other
organ which can take over the excretion. These experiments were tried
by von Schroeder and some other experimenters. They showed that
there was always an accumulation of urea in the blood in eats, dogs
and other animals after kidney extirpation. Mammals survive loss of
the kidneys for about three days. The cause of death which ensues is
not yet certain. Von Schroeder and others got the following results:

 

 

 

Per cent. of urea in the blood
Observer Animal
Before kidney extirpation After extirpation
v. Schroeder Dog 0.045 0.208
Prevost and Dumas Dog Saas 0.830
a st Cat ease 1.040
0.083 (24 hours)
Hammond Dog 0.026 0.093 (48 “ )
0.097 (61 “ )
Voit Rabbit eae 0.388 (60 “ )

 

 

These experiments showed that in the absence of the kidneys urea
accumulates rapidly in the blood, and, up to a certain point, the longer
the animal lives the greater the accumulation becomes. A similar
accumulation has been observed in human beings who have had anuria
following or accompanying nephritis. When the kidneys fail to elimi-
nate urea and it accumulates in the blood, the amounts in the other
secretions greatly increase. Thus it may crystallize out on the skin on
the evaporation of perspiration; urea goes also into the saliva, and
particularly into the secretions of the duodenum and the intestine.
since the intestine is one of the most important excretory organs of
the body. In prolonged diarrhea or vomiting in nephrectomized ani-
mals the amount of urea in the blood may be considerably reduced.

From these observations we may conclude that the kidneys are not
the chief organs for the production of urea and that it must be formed
elsewhere in the body.

It would seem at first glance easy to solve a problem of this sort,
since it would appear only necessary to examine the amount of urea
in the blood before and after passing an organ in order to tell whether
urea was produced there. If the blood coming away from the organ
THE EXCRETIONS OF THE BODY 695

has more urea than that going to it, it must be formed in it. Actually,
however, this method is seldom feasible, since the blood goes so rapidly
through an organ as to take up at any one passage a very minute amount
of the substance sought, an amount so small as to lie within the limits
of experimental error. But while the amount taken away from any
organ at any one circuit may be small, the total for the day may be
large. Nor can we solve the problem by simply analyzing the organs
and determining the amount of urea they contain, concluding that the
organ with the highest urea content probably is the source of the sub-
stance. In the first place, the determination with accuracy of minute
amounts of substances in such an albuminous fluid as the blood, or in
any organ, is beset with many difficulties. Moreover, with such a soluble
substance as urea which passes in and out of cells with ease, and which
does not apparently form a union with the colloids of the cell, there is
little accumulation, the stuff being excreted as rapidly as it is produced.
But could we send the blood repeatedly through a single organ, the
blood passing through the organ again and again, we might finally secure
a considerable accumulation of the metabolic products of that organ
in the blood. This we may do in the following way by perfusion: By
taking the still living organ out of the body and establishing through
it an artificial circulation of defibrinated, warm, arterial blood, the same
blood passing through the organ again and again, we may finally secure
so great an accumulation in the blood of any metabolic substances the
organ may form that their nature may be determined. For such obser-
vations the organ to be examined is placed in a warm, moist chamber
and as quickly as possible after removal from the body an artificial
circulation of defibrinated blood, which has been arterialized by shaking
with air, is sent through it. The blood is collected from the vein, shaken
with air, warmed and injected over and over again, and this is repeated
for many hours. We can, if desired, add certain substances to -the
blood and see how they are affected in passing through the organ and
thus get an idea of the chemical powers of the organ.

This method, which looks so simple, is by no means without its
drawbacks and difficulties. In the first place, many organs withstand
very badly deprivation of their normal »lood supply even for so short
a time as twenty minutes. The intestines appear to be particularly
sensitive in this regard, and the brain is also. A far more serious
trouble comes from the fact that the organs are cut off from the nerve
impulses which normally constantly impinge upon its cells and adjust
the amount of blood coming to each part of the organ to the needs of
that part. For no organ functions as a whole. Every gland has
usually some alveoli in activity; some at rest. The circulation is
adjusted to the needs of each alveolus. The same is true of smooth
696 PHYSIOLOGICAL CHEMISTRY

muscle which is constantly active, such as that of the intestine. The
fibers take turns working and resting, so that at any instant of time
some are at rest, others at work. Now in an organ taken out of the
body, the blood vessels, as a rule, dilate and there is no control of the
blood supply. The gland or other organ swells; it is apt to become
cedematous ; the circulation becomes smaller and smaller and finally stops
entirely, or there may be extravasations of blood into the tissue. It
has been found advantageous, also, to cause a rhythmic motion in the
perfused blood to imitate as nearly as possible the natural conditions.
Then, too, defibrinated blood is abnormal blood. It is not impossible
that one of the main functions of the fibrin of the blood may be to
regulate the viscosity of the blood in its passage through an organ, and
this regulation will be lacking in the blood when it is defibrinated. For
all these reasons the perfusion method, which at first glance promises so
much for the study of the metabolism of individual organs, is subject
to serious drawbacks and requires to be perfected.

With all its drawbacks, however, the method has thrown some light
on the chemical possibilities of many organs of the body and will no
doubt do far more when it is made a method of as great precision as
it is capable of being made. Perfusion experiments have been carried
out by von Schroeder, Salomon and others for the purpose of deter-
mining the origin of the urea. The following experiment illustrates
the results obtained :

Bioop Prr CENT. oF UREA.

Before perfusion After perfusion
Kidneys ............ J... 0.0402 0.039
Musclesi 24 siscsescoataneede 0.014 0.0137
Liver ..... cc cece eens 0,045 0.0812
0.0538 0.1177 (3 hours perfusion)
, 0.1253 (5 hours a
LAVE?* eae eg anaes ca ies 0.0193 0.0236 ( No(NH,) CO, added)
0.0599 ( (NH,),CO, added)

From these figures it is seen that neither the kidneys nor the muscles
added urea to the blood during perfusion; but the liver did, and par-
ticularly when ammonium carbonate had been added to the blood before
perfusion. The blood after passing the liver is uniformly richer in
urea than when it entered it. This experiment shows very clearly that
the liver has the power of forming urea and that it is capable of trans-
forming ammonium carbonate into urea..

One cannot conclude from this experiment that the other organs
have not the power of making urea. A positive result is convincing,
but a negative result may mean many things. It might be, for example,
that the liver happened to have a larger amount of precursors of urea
THE EXCRETIONS OF THE BODY 697

in it than the other organs and that, if the proper forerunners of urea
had been added to the blood, the other organs also would be found to
have this power of urea formation. As a matter of fact, there is
reason for believing, as we shall see in a moment, that other organs than
the liver can make urea.

A second method of attacking a problem of this kind, and a very
important method in physiological chemical research, consists in leaving
the organ in the body, but in some way shunting the blood about it so
that very little or no blood enters the organ and then examining the
subsequent changes in the composition of the blood, or the excretions
of the body. For the liver such experiments are difficult to perform.
If, for example, the liver is taken out of the body of a mammal it dies
in the course of a few hours. The cause of the death is still unknown
and would probably well repay study. The liver has a double blood
supply, getting arterial blood from the hepatic artery and venous blood
from the portal vein, which has gathered the blood from all the intestine
and its glandular annexes. About one-third of the blood from the liver
is estimated to go through the hepatic artery, and the other two-thirds
through the portal system. The first question which we will ask our-
selves is: What will be the effect on the urea excretion of cutting off
the blood supply coming through the portal vein? This supply cannot
be cut off in mammals by simple ligature of the vessels and have the
animals live for more than three or four hours. The blood accumulates
in the intestinal area, there being no way for it to get back to the heart.
It is necessary to so arrange the blood vessels as to send the blood around
the liver and back into the circulation. Such a result can be accom-
plished by the so-called Hck fistula.

Eck fistula. The portal vein and the inferior vena cava run side
by side before the former enters the liver. If one makes a slit in the
adjoining sides of the veins and sews together the edges of the slits,
the two veins are united and the blood can pass from one to the other
through the hole, or fistula. This is called the Eck fistula, from its
inventor. With modern technique it is not a very difficult operation
to perform in dogs. After the fistula is made the portal vein beyond
the fistula is ligatured and now the blood from the intestinal region
no longer passes through the liver, but crosses through the opening into
the inferior vena cava and back to the heart through that vessel. Ani-
mals so operated upon may live for months or years. The liver still
has its circulation through the hepatic artery. The results of this opera-
tion on the liver cells is striking, since they shrink in size and look
quite abnormal under the microscope.

As a result of this operation the blood from the intestine no longer
is subjected to the action of the liver, but passes directly to the body
698 PHYSIOLOGICAL CHEMISTRY

cells and to the kidneys. The urine should show some change in com-
position. If the urea is formed in the liver and in the liver alone from
raw materials brought from the intestine, marked changes in the excre-
tions should result. Pawlow and Nencki and Hahn, who early studied
this question, reported a marked change in the proportion of urea
and ammonia nitrogen in the urine as the result of making an Eck
fistula. They recorded the following ratios of ammonia and urea nitro-
gen in various experiments:

Ratio or NH, To Unga In Doa’s URINE.
Before the Eck fistula After the Eck fistula
1:73 1:33
: 24.5
: 16.1
: 7.6
: 9.9

et oe

There was, in other words, a great decrease in the urea nitrogen
and an increase in the ammonia nitrogen of the urine. They found
in the blood and urine of their Eck dogs ammonium carbamate, NH,—
O—CO—NH,. There seemed, in other words, te be a failure of the
body to convert ammonium compounds and carbamic acid into urea.
In some of the experiments the changes were not so pronounced as
those given in the foregoing table. The operated dogs when fed meat
seemed to be poisoned. They became excitable, attempted to climb up
the sides of the cages and showed symptoms like those of ammonia
poisoning. The following experiment quoted from these observers will
illustrate this:

An Eck fistula was made in a bitch on the 23d of January, 1892.
She weighed 23.674 kilos. On March 11 her weight was 14.169 kilos, a
loss of 9 kilos. She had been on a diet of bread and milk. On March
1 she was given 1,200 c.c. of milk and 200 grams meat powder. A
stage of excitation ensued on the evening of this day; she turned con-
stantly in the cage and tried to climb up the wall and bit objects near
her. She seemed to be blind. The next day she received no meat. The
weakness of the muscular system continued and the uncertain and
irregular gait. The animal appeared to feel no pain. In three days
on a bread and milk diet the pathological symptoms disappeared, but
on repeating the experiment the animal died. The urine was very
alkaline.

The authors go on to say: ‘‘ We have observed, therefore, an
undoubted and very characteristic fact: dogs in whom the blood has
been shunted around the liver by means of the Eck fistula, so that it
flows directly from the intestine without going through the liver, can-
not stand meat without showing serious disturbance of the nervous
THE EXCRETIONS OF THE BODY 699

system which may often result in death. It appeared from these
experiments that one function of the liver was to detoxicate the blood;
to take out of it ammonia which if it passed the liver would be extremely
harmful.’’ These experiments also indicated, like those of von
Schroeder, that the liver was the chief source of the urea of the urine,
since, when the blood supply was reduced, urea diminished and its
place was taken by the carbamate of ammonium. Eck fistula dogs do
not always show these symptoms. Sometimes they live hearty and well
for months, particularly if care is taken to provide them with bones.
A diet of bread and milk is not normal for a dog; they develop diarrhea
on it. Dogs may live normally without loss of weight, or only slight
loss after the Eck fistula if fed bread, meat and bones. Adhesions
always form in these operations, and in the adhesions small blood vessels
may grow into the liver from the pancreas or intestine, and it is pos-
sible that the nerves may at times be involved in the adhesions so that
the interpretation of results is not always easy. As far as they go, how-
ever, the experiments cited bear out the conclusion that the liver forms
a good part of the urea of the body.

Urea formation after corrosion of the liver and disease. If the liver
cells could be injured, the metabolism might be changed in such a way
as to throw some light on the question whether the liver is the sole
source of the urea. The liver cells may be thus damaged in various
ways either by disease or by the injection of corrosive or poisonous sub-
stances into the bile ducts. In acute yellow atrophy of the liver,
interstitial hepatitis and cirrhosis of the liver, there is a very extensive
degeneration of the liver cells. In all of these cases there is a reduction
of the amount of urea in the urine, and an increase in the ammonia con-
tent. The change, however, is not so great as one would expect were
the liver the sole source of the urea. In phosphorus poisoning one
may have a great increase in the ammonia and a decrease in the urea.
But in all these cases it is difficult to distinguish cause and effect. For
whenever there is a production of acid in the body, there is always an
increase in the ammonia of the urine, even though the liver cells be
intact. In phosphorus poisoning there is such an acidosis. In acute
yellow atrophy the nitrogen of the ammonia may amount to as much
as 70 per cent. of that of the urea, whereas normally it is not more.
than 7 per cent. These experiments also indicate, then, that the liver
is an important source of urea.

Experiments have also been tried of giving ammonium carbonate
or citrate to patients suffering from liver disease, but the results have
not been concordant. It seems, however, that except in the last hours
of life in such cases the body is still able to convert the ammonia thus
mgested into urea.
700 PITYSIOLOGICAL CHEMISTRY

Experiments in the corrosion of the liver by the injection of sul-
phurie acid into the bile ducts of dogs have been tried by Pick. The
liver cells are injured, but the circulation persists. Dogs thus treated
remain apparently normal for 24 hours; they then become comatose and
die in some six hours more. An analysis of the urine of docs thus
treated gave the following results:

 

 

Before corrosion After corrosion
Urinary N per cent.as HN, N Per cent. as urea N Per cent. as NH, N Per cent. as urea N
1.51 98.23, 3.93 82.6
5.21 81.88
2.17 75,42 3.62 84,12
5.01 76.91 5.4 76.65
4.59 81 9.37 60.29
5.6 3.36
2.95 1.51

 

 

As a rule, the per cent. of urinary nitrogen as ammonia increased after
the corrosion, but the difference was not marked.

Extirpation of birds’ livers. Now, although in mammals the liver
cannot be removed without causing speedy death, nor can the liver cells
be killed with sufficient accuracy to yield sharp and certain results,
it is possible in birds and reptiles to remove the liver without leading
to the immediate death of the animal. It happens that in birds there
is a connection between the portal and the renal circulation so that
blood can go back to the circulation from the intestine after removal
of the liver. Geese thus operated vpon recover very quickly. They
set at once to smooth their feathers and eat readily. After fifteen to
twenty hours, however, or even earlier, symptoms of serious trouble set
in. The animals stagger, become comatose and soon die. They live long
enough, however, to answer the question as to what happens to the
metabolism when the liver is gone. In birds and reptiles the urea of
the urine is replaced by uric acid. There is a very small amount of
urea excreted, but the urine consists of damp masses of crystallized
urates. It has been shown by other observers that the uric acid of birds
is formed from the same substances as the urea of the mammal. Thus,
if urea is fed to birds, it is excreted in the form of uric acid. Ammonium
compounds also are formed into uric acid. The uric acid of birds is,
then, fairly commensurate in metabolism with the urea of the mammal,
and presumably it is formed by the same organs of the body. Minkowski
extirpated the livers of geese and studied the changes produced in the

urine with the following result:
Per cent, of total N as

Uric Acid N Ammonia N
Before: 5.2 siaesaus 33s eines 9:5 GS a RS ES oa ew 60-70 10-18
ALLER? os 4 tend 5.4 Git sh 6 deste 8s BHRWWE acct eae 3-6 45-60
Before ticeceseasanes iS Susbagstcvee erernereies arise Sata Brigid 50
THE EXCRETIONS OF THE BODY 701

From the results of these experiments it is seen that after removal
of the liver from birds, uric acid disappears from the urine to a small
remnant and is replaced by ammonia. The small amount of uric acid
which appears after the extirpation may be the residue which had
accumulated in the body and is slowly excreted. There is no doubt,
from this experiment, that uric acid is formed in the bird’s liver and
not elsewhere in the body in any amount. Since the uric acid of birds
clearly corresponds to the urea of mammals, this is an additional indi-
cation of urea formation by the liver.

Other sources of urea. The liver is not the only source of the urea,
however, for dogs having an Eck fistula and with the hepatic artery
ligated still have the power of increasing their urea output when amino-
acids are injected under the skin (S. A. Matthews). Some of the other
organs, perhaps all of them, certainly have the power in these animals
of forming urea. Nevertheless, the evidence is that the liver is the main
source of the urea of the urine.

The precursors of urea—From what substances, then, is urea
formed? In the first instance it is undoubtedly formed from ammonia.
Not only does ammonia appear in the urine in larger amounts than
normal in the total or partial absence of the liver, but direct deter-
mination of the ammonia of the blood before and after passing the liver
shows that this substance is removed from the blood by this organ. It
will be remembered that in the course of protein absorption and diges-
tion some ammonia is split from the proteins. A part of this ammonia
is split from the amide linkings of the proteins during digestion and
by the action of the bacteria in the intestine; a part is formed from
the partial deamidization of the amino-acids during the process of
absorption ; still another portion may arise in the liver itself by the
action of thé deamidizing enzymes which probably occur there. The
blood of the portal vein is rich in ammonia. This ammonia comes in
part from the intestine and in part from the pancreas, which also has
a very large amount of ammonia in it. The relative amounts of
ammonia in blood of the portal and hepatic veins are shown in the
following table:

Mes. NH, rex 100 Grams Bioop. (Pawlow, Nencki, Sieber.)

Portal vein Hepatic vein
5 1.5
8.4 fee
5.6 11
12.0 1.9
4.0 1.8
3.5 19

The amount of ammonia in the portal blood is very much more than

that in the hepatic blood, showing that the liver removes ammonia from
the blood.
702 PHYSIOLOGICAL CHEMISTRY

Various tissues had the following amounts of ammonia in mgs. per
100 grams of fresh tissue:

Intestine Stomach Intestinal Stomach
Lungs Muscle Liver mucosa mucosa contents contents Pancreas
1.1 9.2 22.8 23 37.1 42.6 16.4 8.8
19.4 21.2 48.9 43.2 22.4 9.9 7.9
41.7 52.8 40.2 24.3 16

The very high figures for the mucosa of the stomach will be noticed.
It always contains more than the contents. The intestinal contents gen.
erally have more ammonia than the mucosa. Blood from various parts
of the body had the following amounts of ammonia:

Mes. AMMONIA IN 100 Grams oF BLoop OB ORGAN.

Hepatic vein Portal vein Pancreatic vein Liver Stomach mucosa
1.8 4.0 8.2 12.2 44.9
1.5 3.5 0.25 13.7 31.8

The carotid artery blood of a dog on a meat diet contains 1.5 mgs
of ammonia per 100 grams of blood. On fasting it may fall to 0.38
mgs.

Nencki, Sieber and Pawlow conclude their article with the following
words: ‘‘ The liver is, therefore, the true guardian of the organism,
which changes the poisonous substances coming from the alimentary
canal into harmless substances; since that which is true of ammonia
may be assumed to be true also for the substituted ammonias, such
as various plant alkaloids, bacterial poisons, etc.’’

From the foregoing it is apparent that all the organs contain more
ammonia than the blood. A part of the nitrogen probably escapes from
them in this form to the blood. Experiments show that when the
ammonium salts of organic acids are fed to animals they increase the
urea excretion. But ammonia is not the only substance which can
serve as a precursor of urea. The same is true of any amino-acid and
many other substances which contain amino nitrogen. The following
results were obtained by von Knierem in the excretion of uric acid in
hens when they were on a constant diet and fed an additional ration
of various substances shown in the table:

 

 

 

Uric acid excreted in grams
Substance added to the diet a
Before After adding to diet
ASPATAQINE © 2... cece cece eee e eee ee eee ees 0.7601 2.4382
Aspartic acid .. ccc cece cece cence ee ceceerenncs 0.9673 1.5596
GIy COCOLE: tee 26 i aceuscecs a aeiaate alae Ate, oe a Ries a SeereN + 1.3912 1.8910
TQ CGN TAG 555-5: casa Sesion ccd ocasaise bia era vatelldrele acduare- eserayacenscel’ 1.001 1.6282
NH,Cl Wiaiiova! Siw eaiecere 0G weyars: estore) Sererrrrre nr eeeee 1.8999 1.9F28
(NH, } BO, ccsnsaretenass savanxeaomuevarcase 1.140 1.19%

 

 
THE EXCRETIONS OF THE BODY 703

Very closely similar results were recorded by Schulzen and Nencki
after feeding dogs glycocoll or leucine. The urea excreted rose from
3.8 grams per day to 8.3 grams per day after glycocoll was fed; a similar
rise occurred with tyrosine and ammonium carbonate and formate.

Another precursor of urea in the mammal is arginine. This amino-
acid may be split by an enzyme, arginase, found in the liver and intes-
tine, but not in muscle, into urea and ornithine.

N H,—C—NH—CH,—CH, —CH,—CHN. H,—COOH + H,O —~

I
NH
Arginine.
NH,—CO + NH,CH,—CH,—CH,—CHNH,—COOH

|
NH

Urea. g Ornithine.

In some animals, too, although it is doubtful whether it occurs in man,
there is an oxidation and hydrolysis of uric acid to urea. This is
probably, if it occurs, an unimportant source of human urea.

Summary. In the process of digestion and absorption of protein
food large amounts of ammonia are set free. This ammonia is liberated
in part as the result of the action of the enzymes of the digestive juices;
in part by the action of the bacteria in the intestine, and in part, during
the process of absorption, from the amino-acids of the proteins of the
food. The ammonia thus liberated accumulates in the mucous mem-
brane of the stomach and intestine, whence it is gradually removed by
the blood and carried to the liver in the portal blood. The liver con-
verts at least part of it into urea and the kidney eliminates it from
the body. Another portion of ammonia is set free in the liver itself
from the decomposition of the amino-acids brought to that organ in
the portal blood, and the carbon moieties of those molecules are converted
in part into glucose and glycogen. A relatively large fraction of the
nitrogen of the food thus never becomes part of the living protein of the
body at all, but is converted into urea and thrown out of the body. On
a meat diet the amount of nitrogen thus converted into urea, so called
superfluous nitrogen, is relatively large. It is not certain just how
large it is, but it probably is at least a third of all the nitrogen in the
urea, since on fasting the urea drops at once to about two-thirds its
former value. Certainly with an output of 35 grams of urea per day
this digestive nitrogen will represent at least ten grams of urea.

But this is not the only origin of the urea. The tissues are also
capable of tearing their protein to pieces and deamidizing the amino-
acids. For example, if a muscle has not a sufficient amount of carbo-
hydrate to supply its energy, it will tear its protein to pieces to get
704. PHYSIOLOGICAL CHEMISTRY

the energy. This is always accompanied by a process of oxidation which
forms a ketonic acid from the amino-acid and ammonia is set free in the
way mentioned on page 123. Lactic acid is formed in muscles during
their work; some ammonia probably escapes from the muscles in the
form of ammonium lactate and this is in part made into urea. The
endogenous urea has, therefore, a very varied origin, coming from the
different organs in the form of a variety of nitrogenous substances which
are somewhere, possibly in the liver, made in part into urea. Even on
the minimum protein diet of only three or four grams of nitrogen intake
per day the urea still makes 60 per cent. or more of the total nitrogen
in the urine.

It is a very interesting observation that when large amounts of benzoic
acid are fed to animals the excretion of urea is reduced and in place
of the urea there appears the glycocoll of hippuric acid. Now the fact
that under these circumstances glycocoll takes the place of urea has
Ted some to think that possibly ammonia is synthesized into glycocoll
normally before it goes into urea. This is not at all improbable.
Glyoxal, COH.COH, and glycolaldehyde, CH,OH.COH, are probably
formed in the decomposition of the carbohydrates. With the former
ammonia will condense as follows to form glycocoll:

H—C—0O H—C = NH
| + NH — | ; +0 —
H—C=—0 H—C—0
Glyoxal. H
H—C=— NH je
H—C—NH.
68 on os (eee
: COOH
Glycocoll.

Other processes which may lead to the formation of the urea have,
however, been suggested. Drechsel suggested that it is formed in part
from ammonium carbamate by an alternate oxidation and reduction
and he synthesized it in this manner by means of a very rapidly alter-
nating electrical current.

O

I|
1, NH 4 0—C—NH, + Oo— NH,—O—CO.NH,
2. NH,O—CO.NH, + H os ee
Urea.

Salkowski has suggested that it is formed by the transformation of
eyanamide:
NH,—C=N + H,0 —~ NH—CONH,
Cyanamide. Urea.
THE EXCRETIONS OF THE BODY 705

It is probable that it originates in a number of different ways, and
in other tissues as well as the liver.

Physiological action of urea. Urea has several quite important
physiological actions. In the first place, it is a natural diuretic and
whenever there is an increase in the urea excretion there is always, other
things being equal, an increase in the amount of urine. A person on a
heavy protein diet will excrete per day between 1,000 and 2,000 cc.
of urine, except in very hot weather, when there is a great loss.of
water through the skin. On the other hand, a person having a small
intake of protein, and so a small excretion of urea, will secrete not more
than 300-500 c.c. per day. When one observes in a patient a small
secretion of urine, it is always desirable to inquire into the nature of
the diet before concluding that the kidneys need stimulating and giving
a diuretic. Many people secrete this small amount of urine for years
without any indication of uremia or other symptoms indicative of
abnormal secretion. Whether there is any advantage in a low or a
high secretion of urine has not yet been determined. It is possible
that by stimulating the secretion of the urine the drinking of more
water results and a kind of catharsis of the cells of the body might be
produced which might be either advantageous or disadvantageous to
them.

Urea has also a very definite function in the cells of the elasmobranch
fishes and possibly in the mammalia also. It is one of the normal con-
stituents of the cell and of the blood and other fluids of the body, and
since these cells have for long years of time been selected to work with
the highest degree of efficiency in this urea-containing medium, it is
fcund that the addition of a little urea to artificial perfusion solutions,
when one is perfusing the heart or other organs, is as a rule advan-
tageous. The effect of such an addition to the salt solutions used to
sustain the heart-beat of fishes, both teleosts and elasmobranchs, is very
marked. Tissues live much longer in the presence of some urea than
in its absence. The effect on mammalia is much less marked, since the
amount of urea in the blood of mammals is very small, .02-.04 per cent.
The effect produced by the addition of urea to the artificial salt solu-
tions in the elasmobranchs is that of a stimulation and the same effect
may be produced by the addition of small amounts of ammonium car-
bonate. It is possible that in these animals there may be some conversion
of urea into ammonium carbonate, or vice versa, and the effect may be
due to the action of the ammonium carbonate in neutralizing acids.
Urea is not, then, entirely inert.

Creatine and creatinine in the urine.—One of the most interesting
of the nitrogenous substances found in urine is creatine and its anhy-
dride. creatinine, creatine being methyl-guanidine acetic acid. In the
706 PHYSIOLOGICAL CHEMISTRY

urine of the average human adult there is present between 0.8 and 2
grams of creatinine a day. Creatine occurs in the urine of healthy men
only in very small amounts, or it may be entirely absent if no creatine
is taken in the food, but it is found in larger quantities in the urine of
women and children even when they are taking no creatine in the food,
and in men’s urine the amount is increased by fasting and in disease.
Both creatine and creatinine occur in the urine of other mammals.
The amount of creatinine excreted daily by human beings varies with
the weight,. muscular development, state of health, sex and age, and
slightly with the creatinine and creatine intake, but ts almost or quite
independent of the protein wmtake of the body. These facts show that
creatine and creatinine have a very different significance from urea
and that they stand in a very special relation to the fundamental
metabolism of the body.

Chemistry. Creatine. Creatine (Greek kreas, fiesh) is methyl-
guanidine-acetic acid and has the formula C,H,N,O, or:

NH oe —N —CH 9 C—O
| lI
NH CH, 0
Creatinine is the anhydride of this: i.e., C,H,N,O, or

NH—CO

te
NH=C |

Se.
CH,N —-CH,

Creatine is thus related on the one hand to arginine, which is
6 -guanidine-w-amino-valerianic acid; from which it might possibly be
derived by oxidation and methylation ; and on the other hand creatinine
may be regarded as an imidazole derivative. It is thus allied to histidine
and to the purines, both of which contain imidazole rings. Creatine may
also be considered as a ureide of methyl glycocoll (amino-acetic acid),
or sarcosine, which relates it to the betaines, methylated glycocoll
derivatives found in many plants and represented in an extreme form
of methylation by choline in animals. Creatine is also methyl-glyco-
cyamine, glycocyamine being guanidine-acetic acid. It may be syn-
thesized with great ease by the direct union of cyanamide, NH,.CN,
with methyl-amino-acetic acid, or sarcosine. If a strong aqueous solu-
tion of amino-acetic acid and cyanamide is made slightly ammoniacal
at room temperature, glycocyamine crystallizes out. The reaction is as
follows:

NH..CN + HN.CH—COOH —- NH —C(—NH)—NH—CH —COOH

Cyanamide. : Glycocoll. Glycocyamine, i
THE EXCRETIONS OF THE BODY 107

The same reaction carried out with methyl-amino-acetic acid gives
creatine.

NH,.ON + HN (CH, )—CH,—COOH——~

NH,—C (= NH)—N(CH,)—CH,—COOH

Creatine.
It will be noticed that cyanamide is an anhydride urea and can be
prepared from urea by the action of sodium; and cyanamide yields urea
when treated with 50 per cent. sulphuric acid. It would seem possible
that urea might give a similar synthesis with amino-acetiec acid:
1. NH,—CO—NH, — ae

OH
2. NH,—C=NH + NH(CH,)—CH,—COOH —-
| Sarcosine.
OH
Urea.
NH,—C (= NH)—N(CH,)—-CH,—COOH + HO

Creatine.

It is by no means impossible that small quantities of cyanamide may
be present in urea solutions and this synthesis is one which might pos-
sibly occur in the body where both urea and sarcosine are to be found.

Creatine is a colorless, bitter, biting substance crystallizing in
rhombie prisms with one molecule of water, which is lost by heating
to 100°, the crystals then becoming white and opaque. It dissolves in
74.4 parts of water at 18°, but is much more soluble in hot water, almost
insoluble in alcohol and insoluble in ether. Its aqueous solutions are
neutral in reaction. It occurs not only in the urine, but in most organs
and especially in the voluntary and involuntary muscle of vertebrates
and some invertebrates. It has been isolated also from the blood, brain,
liver, testis, transudates and the amniotic fluid. It has a stimulating
action on the central nervous system, and methyl-guanidine, one of its
decomposition products, is very toxic.

By hydrolysis with boiling barium hydrate creatine yields methyl-
hydantoic acid, urea, methyl-amino-acetic acid and carbon dioxide.
Probably it forms first by hydrolysis of an amino group the intermediary
substance, HO—C(=NH)—N(CH,)—CH,—COOH, which by molecu-
lar rearrangement is transformed into methyl-hydantoic acid, NH,—
CO—N(CH,)—-CH,—COOH. By oxidation by permanganate or hydro-
gen peroxide it forms urea and methyl-amino-acetic acid, or sarcosine.
Arginase does not decompose it. By boiling with mercuric oxide it
reduces the latter to metallic mercury and the creatine is oxidized to
methyl-guanidine and oxalic acid. Creatine has, therefore, some reduc-
ing action, but it is far less marked than that of creatinine. It is pre-
708 PHYSIOLOGICAL CHEMISTRY

cipitated in neutral solution by mercuric nitrate. It is not precipitated
by cadmium chloride, phosphotungstie acid, or by lead acetate. For-
maldehyde converts creatine to dioxymethylene creatinine.

Creatinine. Creatine goes over spontaneously into its anhydride
form of creatinine, the reaction being similar to the transformation of
glycocyamine to glycocyamidine or of uramido acids to hydantoins.

NH. 2 C (=NH)—N( CH, )—CH,—COOH —. HN—CO

|
NH =

c |

CH tba

Creatinine. P
This reaction is hastened by various tissue extracts, but it occurs if
an aqueous solution of the creatine is boiled, or by the action of acid.
A solution of creatine in half-normal hydrochloric acid heated in an
autoclave for 30 minutes to 117° goes over almost quantitatively into
creatinine (Benedict). The synthesis of creatinine from creatine is a
synthesis of an amino and carboxyl group similar to the synthesis of a
polypeptide from the amino-acids. The power of making such syntheses
is an attribute of all living matter without exception. This synthesis is
also instructive as illustrating the formation of a ring compound from
an aliphatic. A similar synthesis, that of proline from glutamic acid,
has already been discussed on page 124. In the latter case there is the
formation of the pyrrolidone ring from glutamic acid by a union of a
carboxyl and amino group, followed by the reduction of the ring which
thus is rendered stable. The proline ring, like that of glycocyamidine,
is also often methylated to form alkaloids of the type of stachydrine,

CH, _ CH,

|
CH da—co

KF i
Na
eae Na

Stvabydsine.

which occur in plants. Whether it is possible to reduce the creatinine
ring in the body to form the imidazole ring is still unknown. The
creatinine ring being thus an oxidized ring is unstable and creatinine
in an alkaline solution is less stable than creatine from which it is
derived.

Creatinine crystallizes in colorless, monoclinic prismatic crystals.
It is readily soluble in water and alcohol. It forms with zine chloride
a good crystallizing, not very soluble compound by which it may be
identified, (C,H,N,O),ZnCl,. It is a strong base, the aqueous solution
being alkaline; stable in acid solution, but not in alkaline. It is a
reducing substance, reducing alkaline solutions of copper, silver and

+ H,0
THE EXCRETIONS OF THE BODY 709

mercuric salts, but not reducing bismuth oxide. It reduces picric acid to
the reddish picramie acid in alkaline solution, and it is determined quan-
titatively colorimetrically by means of this reaction. See page 1093.
Origin and significance of the creatine and creatinine of the urine.
The origin and significance of the one or two grams of creatinine and
the few milligrams of creatine secreted daily have been keenly inves-
tigated of recent years, but have not yet been brought to a complete
solution. Creatine is found in a great many organs of the body. It
was discovered by Liebig as a constant constituent of voluntary muscle.
It and creatinine are found in Liebig’s beef extract, there being usually
about twice as much creatine in it as of creatinine. Recent analyses by

Beker have yielded the figures shown in the table.

AMOUNTS OF CREATINE AND CREATININE FouND IN VaRIOUS ANIMALS.

 

 

, Mgs. creatine and creatinine computed as
Animal Organ creatinine per 100 grams of the organ
OX: easiest Liver 25.3-37.24
Calf sics dssnad 26.54
Rabbit ...... Ks 18,9-21.2
Cats nec iakres s 20.6
Goat ........ sf 11.2
PIG! ic iciawnad e 15.72-17.46
OR 4 ewes sashes Pancreas 12.51-19.62
DOR essisiergsctenr e 12.9-16.13
Pigs cocunues se 10.69-14.2
OX siccenete.. Thyroid 11.4
OX sec wee Kidneys 12.26-17.6
DOP sce aed “ce 10.28-16.34
PYG cece iowenwdsins ae 15.2
ORS Sie ca eatin Spleen 14.67
DOS sua carcass fe 13.28-19.5
Calf ....... Thymus 9.76
Buil ...... Testis 76.4-97.2; 181, dog testis, Janney and
Blatherwick.
OX! cee useaad Brain 51.4-63; 112, dog brain, Janney and
Blatherwick.
White matter 47.9-56.2 ,
Cerebellum 64,2-71.3
DOB: sie a wiv, es Brain 54.6-57.5
ORs, coredreiets Voluntary muscle 403 (Average of 20 determinations)
Dog xcs “4 314 pee, 56 =
Pig seals ae 338 Se EES =
Rabbit ..... White muscle 451 Cm %
e Red muscle 326
Cat. cseccno ids ai 354
Goat ....... ca 316
OR: sssieie aes Heart 220
DOP ees ya ses <e 228-257.4
Rabbit ..... Intestinal muscle
oe T.arge intestine 32.5
Small intestine 23.4
OX AS evicroaty Uterus 29.9-43
Pig? sewaeese as 29.9-31.2
OX ccgcagees Blood 1,93-2.68
Doge cscie ess Be 1.86-2.44
PI ones ee 2.00-2.08

 

 

 
710 PHYSIOLOGICAL CHEMISTRY

A fundamental and significant fdct concerning the excretion of
creatine und creatinine is that the amount of these excreted in the urine
ner day is independent of the amount of protein taken in the food and
is wonderfully constant for each individual. This fundamental observa-
tion by Folin has been the starting point of the modern investigation
of these substances. It shows clearly that creatinine has a different
significance in metabolism from urea, of which the excretion varies
directly with the protein intake. A second fundamental fact is that
the exeretion of creatine and creatinine in adult men is almost inde-
pendent of the creatine intake, and that creatinine taken in the food
appears in large measure in the urine as such. The third fact is that
age, sex, state of health and starvation profoundly affect the excretion
of these bodies, and the amount of creatinine excreted varies directly
with the weight of the body—at least within certain limitations this is
true. The significance of this latter fact will be apparent if it be
remembered that nothing of the sort is true of urea, since a very small
person, if he eats much meat, may excrete more urea than a large one
on a more restricted diet. These facts show that creatine and creatinine
are products of the endogenous metabolism of the body.

That the excretion of creatinine in male adults is independent of the
amount of the protein ingested is shown by the following observations
by Folin. The figures represent the excretion of an individual on two
occasions, once when on an ordinary protein diet containing about 120
grams of protein per day; and the other time on a restricted protein
diet when only about 20 grams of protein are ingested.

Usual protein intake Low protein intake

Vol. Of Urine. :544 css scaeise seine 1170 385 ae.
COCR) ING 2g si Siva co: fo sioner d dupsavesonsg a. 16.8 3.60 gr.
Urea iN) aicdc ca ek amaanteecavonee nae 14.7 2.20
Creatinine N ...... cc. cece eee eee 0.58 (3.60%) 0.60 (17.2%)

The creatinine excretion remained practically the same, although the
protein was reduced so enormously. The contrast with the urea excre-
ticn is profound. The creatinine nitrogen made in the one case 3.6
per cent. of total nitrogen ; and 17.2 per cent. in the other.

But, while the total amount of creatinine secreted is independent of
the protein intake, it is closely dependent on the bodyweight and chiefly
upon the muscular development of the body, although other factors
affect it also. The average creatinine excretion of Dr. H., weighing
87 kilos, was 1.6 gram; Dr. A., weighing 56 ks., eliminated 1.15 gram;
and F., who weighed 70 kg.. but is rather short and corpulent, excreted
14 gram. The analytical data indicate that moderately corpulent per-
sons (adults) eliminate per 24 hours about 20 mgs. creatinine per kilo
bodyweight: while lean persons yield about 25 mgs. per kilo. The num-
ber of milligrams of creatinine excreted in 24 hours per kilo bodyweight
THE EXCRETIONS OF THE BODY 711

is called the creatinine coefficient. Sometimes the term is applied to
the milligrams of creatinine nitrogen per kilo bodyweight. Shaffer
found that the creatinine coefficient in the latter sense had a value of
between 8.1-11; for children it usually lies between 3.3 and 6.5.

But while the creatinine excretion is independent of the protein of the
food it is affected slightly by the intake of creatine and greatly by that of
creatinine. The ingestion of creatinine is followed by the reappearance
of most of it in the urine. The following figures from Shaffer show both
how constant the creatinine excretion is from hour to hour and how it is
inereased by the ingestion of creatinine. The urine was passed each
two hours. The average excretion in grams of creatinine per hour in
successive two-hour periods was as follows: .066; .062; .057; .065; .068.
From 6.30 to 12 at night the average was .053; from 12 to 9 a.m., while
sleeping, it was .056; from 9 to 11.15 a.m. it was .062. The amount,
therefore, is less at night than in the daytime. Creatinine does not
then undergo the wide hourly variation which uric acid does. At 10.40
A.M., while his hourly excretion was .067 gr. per hour, he took 250
c.c. of water containing 0.70 gram of pure creatinine. From 10.15-
11.15 the hourly excretion of creatinine rose to .084; 11.15-12.15 it was
174; 12.15-2.15, .188; 2.15-5.15, .101; 5.15-12.06 a.m., .084; 12.06-7.20
A.M. it was .073. In this experiment 76 per cent. of the ingested
creatinine had been excreted in 21 hours.

The ingestion of creatine leads to a very small increase in the
creatinine and is generally, but not always, followed by the excretion
of a small amount of creatine. This point has been the subject of a
good deal of controversy. Folin made the extraordinary observation
that ingested creatine did not increase either the creatinine, creatine
or total nitrogen of the urine. The creatine disappeared and he believed
it to be metabolized in the body and retained there. It was afterwards
suggested by Mellanby that it had been retained by the bacteria of the
intestine and he isolated from the intestine a bacterium which would
metabolize the creatine of a solution. It is, however, not yet certain
what has become of the ingested creatine. Further experiment has
shown that there is a marked difference between children and women,
on the one hand, and men on the other, as regards their power of
metabolizing ingested creatine. Men usually destroy nearly all, or all
of that ingested, so that no increase of the creatine, or creatinine,
occurs. In some cases, however, there is a very small increase in the
creatinine. This is perhaps the rule, and occasionally some creatine
appears in the urine unchanged. Women and children excrete more or
less of it unchanged.

Amount of creatine excreted under various conditions. Any discus-
sion of the excretion of creatine in the urine must be prefaced by the
statement that the creatine is not determined directly, but by difference.
712 PHYSIOLOGICAL CHEMISTRY

The preformed creatinine is determined by Folin’s colorimetrical method,
and then the creatine present, if any, is converted into creatinine by
heating the urine with acid, and the creatinine is then redetermined.
This is called the total creatinine. It is sometimes larger than the
figures first obtained and the difference is supposed to be due to the
conversion of some creatine to creatinine. The difference is hence called
creatine. It is obvious, however, that there is a considerable uncer-
tainty about determinations made in this way, and it involves the
assumption that the heating with acid has not in any way changed the
urine, so that it will affect the creatinine determination, except by
the conversion of creatine to creatinine. When it is remembered that the
quantitative determination of the creatinine itself is made by measuring
its reducing action, a property which is not peculiar to it, it is clear
that inferences as to the presence or absence of creatine in the urine
must be very cautiously drawn. As a matter of fact it has recently
been alleged that the appearance of the presence of creatine is really
due to the presence of small amounts of acetoacetic acid, which in the
unheated urine make the creatinine determination somewhat lower than
it should be. This acid is destroyed by heating the urine with acid,
so that the second determination of creatinine is larger. It is just
this difference which is usually called creatine. It is a very suspicious
circumstance that most of the methods which are supposed to increase
creatine excretion are just those which are known to increase acetoacetic
acid excretion. As it is at present controverted whether the acetoacetic
acid present will account for the whole of the so-called creatine or not,
the excretion of creatine is treated here as if it certainly occurred. It
may be that the so-called creatine is really creatine.
EXCRETION OF CREATINE IN HEALTHY CHILDREN. Grams Per Day.

Sex Age Urine c.c. Total N. Creatinine N. Creatine N,
Boy’ hers giao: 5 620 6.10 112 .025
BOY? oie eis chia cert eet 8 710 7.20 163 0
BOY. Hiss ccusnttacgen hss ay ici 15 1150 11.97 378 0
Boy Seana hase acseameed 11 460 5.31 157 0
Boy: serie ae ays sth 12 680 4.19 -086 0
Boy sie orb thers eae ise es 2 300 3.00 -025 023
BOY: costs 2s 4 ede Be eee Set 3 580 5.48 -057 022
BOY. sus tie stead needs eae 16 625 8.10 -268 0
1120 11.70 374 0
GIT age ak seth guevelel dees 5 500 4.20 -069 -005
GED cass) octneralew enone, Ss 6 500 2.33 .032 -003
Girl i cxegtec ns sties ned 7 830 9.75 -157 .066
Gith: 3.5 cate te te ene ek 10 825 8.50 147 .020
GUE, osc ihecine Rarq trance 12 510 8.68 201 011
1000 6.70 224 042

Creatine is found in the urine of most if not all mammals. In
human beings it occurs in large amounts, relative to creatinine, in the
urine of children, but in small amounts only in the urine of adults,
except under special conditions of sickness, or diet, when the amount
THE EXCRETIONS OF THE BODY 713

of creatine may be at least half of the total creatinine and creatine
together. The results included in the table on page 708 show the
excretion of creatine and ene in healthy children (Krause) when
on a creatine-free diet.

From this table it is seen that on a creatine-free diet boys stop secret-
ing creatine at about seven years of age, and that before this age the
amount of creatine secreted, which in the first years of life has been about
equal to the creatinine, becomes relatively and absolutely smaller until
it disappears. Girls, on the other hand, continue to secrete creatine
until puberty, and with women it reappears in the urine at each monthly
sexual cycle. It will be noticed, furthermore, that the absolute amount
of creatinine secreted increases with the age of the child and there
is also an increase in the amount of creatinine nitrogen in mgs. per
kilo bodyweight. This last factor, the ‘‘ creatinine coefficient,’’ in adults
on a creatine-free diet is 8.1 to about 11. In the experiments just quoted
the creatinine coefficient is higher in children of 12 than in those of 5.
The coefficient at ages of 13 to 16 is only 3.6 to 4.1. In infants, Amberg
and Morrill have found creatinine coefficients still lower. At 7 to 14
days old the coefficient was 1.8 to 3.8. The increase in the creatinine
coefficient thus goes pari passu with the relative development of the
musculature. In infants the brain and liver and other organs make
relatively a larger proportion of the body weight than later in life. Not
only does creatine appear in the urine of children, but it has been shown
(Krause) that their power of metabolizing creatine is less than that
of adults. By feeding creatine to children on a creatine-free diet it was
found that the younger the child the less ingested creatine was retained.

Per cent. ingested creatine

Sex Age excreted as creatine
GD eset baie ee aasclgls seieaiere eeisinceut S-sieyetessieieraalonsl ater e Guaiete 6 56
By « ssisaite:gacetecaarn tra eter diei'e 0 ve fetesses “9:0 avgyn date sietevoloeaa.e “ers 8 43
Git), ua Syssiag hen saeads saua teeieee cea eel 8S Seis Il 31

In the case of women creatine is often absent from the urine, but reap-
pears during menstruation, and during pregnancy and after delivery
it occurs there for several days. Benedict and Meyers found consid-
erable quantities of creatine in the urine of insane women when on a
milk diet, or a creatinine and creatine-free diet. The ages of the
patients ranged from 19 to 95 years and the amount of creatine
excreted per day ranged from 70-200 mgs., while the creatinine was
from 292 to 700 mgs. per day.

The ingestion of creatine causes in children and women always an
increase in the creatine of the urine and a slight increase in the
creatinine. In men, however, large quantities of creatine may be
taken by the mouth without causing any increase in the creatine
im the urine or indeed in the total nitrogen excreted. This is particu-
714 PHYSIOLOGICAL CHEMISTRY

larly noticeable when the amount of protein in the food is low. This
fact was discovered by Folin, who on successive days took several grams
of creatine with no change in the creatine or creatinine in the urine.

Krause in various experiments on two male adults of 26 and 36
on a creatine-free diet and with a total nitrogen in the urine of from
3.27-13.44 grams, gave .052-.126 grs. creatine nitrogen per day without any
creatine appearing in the urine. The creatinine was only very slightly
inereased, and in some cases not at all increased. The total nitrogen
in the urine not only did not increase, but seemed, on the contrary,
to decrease. Thus it fell in three of the four experiments from 3.27
to 3.14, from 18.44-12.55 and from 13.30-12.07. Similar results were
obtained in the case of children, except that in them a part of the
creatine ingested reappeared as such in the urine.

‘Weber ingested meat extract and found a greater increase in the
creatinine than was taken in the extract, so that some of the creatine
had gone over into creatinine. Van Hoogenhuyze and Verploegh, Plum-
mer, Dick and Lieb always got a small part of the creatine as creatinine.
Pekelharing and van Hoogenhuyze found that, if they gave dogs and
rabbits creatine subcutaneously in small amounts, it always increased
both the creatine and creatinine, but if they gave it all at once they
got an increase only in the creatine. Towles and Voegtlin also found
a transformation of some of the creatine to creatinine. The impor-
tance of these observations. arises from the fact that they show that
creatine is turned into creatinine in the body and hence indicate that
the creatine of the body is probably the source of the creatinine.

Fasting causes a decrease in the creatinine, but an increase in the
creatine, so that the total is very little affected. Even in men who
do not normally excrete creatine in the urine it appears there if they
fast. In women the amount normally present is increased. A very
interesting case of this sort was reported by Benedict and Diefendorf.
The patient was an elderly woman having a religious mania so that she
fasted periodically. One such period is that contained in the following
table:

 

Total creatinine Preformed Preformed
—er. creatinine—gr. creatine—gr.
0.50 0.50 0.00
0.66 0.61 0.05
0.63 0.60 0.03
0.67 0.61 0.06
0.68 6.05 0.03
Begins to fast. First three days no water.
DO le SENG ear a 4.19 0.65 0.61 0.04
DES vekvevecaieneeigeanedy 6.05 0.66 0.57 0.09
0 - Gad Ss alleen au te i 6.38 0.61 0.54 0.07
2G ev eres aoe res ea 6.93 0.50 0.44 0.06
DT se es Misamis a 6.16 0.65 0.49 0.16

2B? <a Ghouai aie @ ees 4.4] 0.49 0,34 0.15
THE EXCRETIONS OF THE BODY 715

End of fast.
Total creatinine Preformed Preformed
Date Total N —gr. creatinine—gr. creatine—gr.
Nove 29. wiascavcances (3.04 ) (0.34) (0.23 ) (.11) Some loss
OO)! 2s ysiepiredyetersae 3 4.87 0.67 0.56 -ll No food
DOC Pose scssutavs, Seanceabens 3.17 0.55 0.50 0.05
Dm "ic tenisocelatasd nadecetlce 5.09 v.64 0.61 0.03
Bs hinet tna tise ere 6.05 0.61 0.60 0.01

The great increase in the creatine on the 27th and 28th will be
notived as well as the wonderful constancy of the total of creatinine and
creatine. The falling off in the creatinine is illustrated also in the
following results on the professional fasting woman, Flora Tosca (van
Hoogenliuyze and Verploegh) -

Before the fast Fasting continued
Total N Creatinine per day TotalN Creatinine per day
in grums in grams

P00 Reale Me ede hee 1.087 6-70 reese rece ceereenenececee -689
UBOOE SeiacBe ousniars again aloes aaa aaatone 715
Fasting BBO scieroce avverd\aie ctaalestoreyecalenele-<4 08 602
S976) au cyanea LA task 0.904 G4 ciisdes 3G dae eee ieee d 453
QBS: hir.aa kee 9. abc nha raphe he poets 577 G07 ac acske eet aad s eae ose 566
LOTS 2 dese sa ceeds Ce araeunns tetas 581 G2 wee eee eee eee ee eee eee 548
QA Qn ac tee PIs Sut ene KAS 634 BOB. sae bstiaceisenretsseceee teecets ats 426
iB Le “spss visited dag Atalaiow yonaieiades 6633 HBB eee cee eee eee eee ee -715

Mich DO sheial ere-pyatene age anerers ardueeses este 590 Broke fast
Gal. iceeugaae cess cece ed .469 1.028

(In this case the creatine was not determined.)

The reappearance of creatine in the urine of fasting men and
the corresponding decrease in the creatinine seems to be an interesting
casting back of their metabolism to the type of the juvenile metabolism.
Regarded from this point of view it would appear that the metabolism
is rejuvenated by fasting. It would be interesting to see if other signs
‘of rejuvenescence are to be found; to see, for example, if allantoine
would reappear in the urine of fasting men. Fasting rabbits also
increase the creatine at the expense of the creatinine. The creatine
quite disappears if the animals are given carbohydrate food, but not if
fed fat or protein. It may be that the change in metabolism has affected
chiefly the liver and muscles and particularly the former and that the
exhaustion of the carbohydrate of the liver has affected its power to
change creatine into creatinine. By some authors the increase in the
creatine is supposed to indicate an unusual protein catabolism, but the
small amount of the total nitrogen excreted and the slight decrease in
the total creatine and creatinine is against such an interpretation.

Influence of carbohydrate food on creatine excretion. It has been
found that a deficiency of carbohydrate food in the diet produces
creatinuria. Various suggestions have been made to account for this
fact. Thus it is supposed by some that creatine appears as the result
of the abnormal catabolism of flesh in the body consequent on the
withdrawal of carbohydrate; or owing to an impairment of the func-
716 PHYSIOLOGICAI, CHEMISTRY

tion of the liver. Thus creatinuria often accompanies diabetes both
natural and phlorizin. The amount of creatine and creatinine together
in these cases, however, is not larger than normal, so that creatine
appears as the result of the loss of power of the body to change creatine
to creatinine. As was stated at the outset, however, the reported creatine
may not have been creatine, but acetoacetic acid.

Influence of the thyroid on creatine excretion. The thyroid has a
marked but unexplained influence on the metabolism of the body.
Injection of thyroid extract causes a stimulation of the protein catabo-
lism of the body. It is interesting that it also causes an increase in
the creatine excretion (acetoacetic acid?). Just how this result is pro-
duced it is impossible to say. It might be that the thyroid extract
caused directly or indirectly the discharge of creatine from the muscles
so that the excretion is increased. It may be that the creatine acts as
a brake on the protein catabolism and its removal results in an abnormal
catabolism of proteins.

Origin of creatinine. The foregoing facts show that the creatine and
the creatinine of the urine have the same origin, and that creatinine is
derived from the creatine of the tissues of the body. There is in the
body of an average adult something more than 120 grams of creatine.
That this creatine gives rise to the creatinine of the urine is indi-
cated not only by the facts cited, but also by the following observa-
tions of Meyers and Fine:

 

 

 

 

 

Creatine— Creatinine— | Ratio— Ratio—
muscle per cent. coefficient muscle creatine coefficients
Rabbit........... 0.52 14.3 14 17
MaDe: cneincpsioee 0.39 9.0 1.05 1.07
Doge vcesen es 0.27 8.4 1.0 1.0

 

Origin of creatine. From what substances then does the body form
the creatine found in the tissue? Where is the creatine changed into
creatinine? How much of the creatine and creatinine is destroyed?

There are several possible sources of creatine. It might be formed
from arginine, which is guanidine-amino-valerianic acid. By oxidation
this might be turned to guanidine-amino-acetic acid and by methylation
this changed to creatine. An ingestion of arginine does not increase
the output of creatinine. But Inouye found in autolysis of the liver
to which he added some arginine that there was an increase in the
creatine and creatinine. The change was, however, small. Jaffé showed
that the ingestion of guanidine acetic acid is followed by a slight in-
crease in the excretion of creatine and this has been confirmed by others.
He found. also, that the ingestion was followed by an increase in the

.
THE EXCRETIONS OF THE BODY 717

creatine content of muscle. The greater part of the guanidine acetic
acid was excreted unchanged, only 5-8 per cent. having been changed
over. The ingestion of guanidine caproic acid and guanidine butyric
gave no better results. It has not been possible to find glycocyamine
in the metabolism of the body and it is accordingly very doubtful
if it is an intermediate product in the formation of creatine. Another
possibility is that creatine is formed from choline or some similar product
of the phospholipins. Koch’s experiments gave no increase in creatinine
after the ingestion of large amounts of lecithin or choline. Another
possibility is that the synthesis might be made with methyl guanidine as
the intermediate body. Methyl guanidine has been found both in muscles
and in the urine (Kutscher). Experiment has shown, however, that this
is a very toxic substance and Jaffé and others have got no increase in the
creatinine after injecting it. The synthesis might be made using urea
and methyl amino-acetic acid. There is no evidence of this beyond the
fact that the addition of urea to autolyzing liver completely prevents the
destruction of creatine by the liver and the same is true of other organs.
It would appear that in some way urea is concerned in the metabolism
of creatine. The ultimate origin of creatine is thus uncertain. The
presence of sarcosin and glycocoll so frequently in the muscles of in-
vertebrates may be significant.

The transformation of creatine into creatinine is probably carried
out in many different tissues. For example, the creatine of the blood
slowly changes to creatinine, and the liver, kidney, muscle, spleen and
lungs have the same power, due possibly to an enzyme they contain. It
is even possible that this enzyme occurs in the urine since creatine turns
to creatinine in the urine more rapidly than the number of hydrogen
ions present would lead one to expect. Perfusion experiments also show
clearly that the liver and muscles have the power of destroying and
of making creatinine out of creatine. Creatine is destroyed also in many
tissues, but it is not known what becomes of it. It has been suggested
that it is first turned into creatinine and then destroyed, but this is not
certain. If one extirpates the kidneys the amount of creatine in the blood
increases from about 2 mgs. per hundred grams to 8 in the course of 48
hours. In six hours there is no noticeable change, a fact that indicates
that the power of the body to destroy creatine is very great. That
one place of transformation of creatine to creatinine is in the liver is
indicated by the fact that if the liver is poisoned by phosphorus or
hydrazine, creatine replaces creatinine in the urine. An Eck fistula,
however, does not cause creatine to appear in the urine. Even heated
blood serum is more powerful in transforming creatine to creatinine
than is water. Blood contains the following amounts of creatine and
creatinine in 100 grams:
718 PHYSIOLOGICAL CHEMISTRY

Carotid defibrinated blood a epee
Cat (young female) ......cccccereseeeeeereeenee 0.85 2.44
Dog (young female) ......... Sedevaria eva dubien ealarh Gidvecdies 0.72 1.54
Adult (male dog) ............e secs ee cece e cece 0.38 2.92
Human in 100 ceo... i.e ee cece eee 1.2-1.8 3-5

Methylation. Creatine is a methylated amino-acid. This is a point
of much interest, since none of the amino-acids of the proteins are
known to be methylated. The question at once arises as to the mechan-
ism of methylation, the place where it occurs, its significance and what
substances may be methylated in the body. This general problem of
methylation may very well be treated here in connection with creatine
and ereatinine which are the most important methylated substances in
the urine.

The power of forming methyl-amino derivatives is very widespread
in nature and many of such derivatives are of very great physiological
and pharmacological importance. Choline and its more active rela-
tives, nevurine and muscarine, are methyl derivatives, the former being
the trimethyl-oxyethyl ammonium hydroxide; neurine, trimethyl-
vinyl-ammonium-hydroxide and muscarine is supposed to be the alde-
hyde of choline. Neurine and choline occur in the animal organism.
Adrenaline is a methylated amine, being dioxyphenyl-oxyethyl-methy]-
amine. In plants the betaines and similar compounds are common, and
trigonellin, and stachydrin are examples of this class of bodies.

It has been found that human and other mammalian organisms
have the power of introducing methyl zroups into various substances
ingested. The methyl derivatives are found in the urine. Thus ingested
pyridine is excreted by dogs as methyl pyridine (His) ; tellurium salts
are excreted as methyl tellurides (Hofmeister) ; and naphthalene also
appears as the methyl derivative (Cohn). But rabbits do not methylate
pyridine but excrete it unchanged, the carnivora and herbivora showing
important differences in this respect.

The exact manner in which this methylation is produced is not clear,
hut it has been suggested that, in plants at any rate, it comes from the
union of formaldehyde with amino groups. It will be recalled, from
the discussion of the Sérensen titration method, that formaldehyde unites
with amino groups to form methylene derivatives. By reduction these
methylene amines can be changed to methyl amines. This seems the
probable method of their formation. It has not been shown that for-
maldehyde is an intermediary product of metabolism in animals, but in
the decomposition of the carbohydrates it is not impossible that small
quantities may arise and that this formaldehyde isthe source of the
methylation. This view, however, can have no solid basis until the
presence of formaldehyde in animal tissues is unequivocally established.

Concerning the place in which such formation of formaldehyde
THE EXCRETIONS OF THE BODY 719

might occur one turns naturally to the two organs of the body most
concerned in carbohydrate metabolism, the liver, where the carbohy-
drates may be formed from proteins and stored, and the muscles, the
tissues in which carbohydrate is torn to pieces and the fragments
burned for the production of muscle energy. It is, perhaps, more
likely that it is in the muscles, where decomposition of carbohydrate
occurs, that small amounts of formaldehyde may be produced and there
the synthesis of methyl glycocoll take place. Certainly in some of the
invertebrates glycocoll is found in large amounts, not methylated, in
the muscles. Formaldehyde is itself very toxic and possibly glyco-
coll, which serves in other instances as a means of making toxic sub-
stances harmless, may also thus function in the muscle, thus forming
sarcosine.

Relation of creatine and creatinine excretion to muscular metabolism.
But while the muscles are the organs in which creatine is found in the
largest amounts, and while the muscles are able to form creatine, it is
probable that the creatine metabolism does not play a part in muscular
contraction. While the evidence is somewhat contradictory on the be-
havior of muscle creatine in tetanus or during muscle contraction, it is
certain that doing muscular work does not increase creatinine excretion.
On the other hand, it is true that creatinine excretion is larger during
the day than at night when the muscles are relaxed. It has been sug-
gested that creatine is concerned in the tonic contraction of muscles.
Thus standing in a rigid military position is said to increase creatinine
excretion. It is doubtful, however, whether there is this fundamental
distinction in kind between what is known as tonic contraction and the
ordinary contraction of muscle. On the other hand, it has been sug-
gested that creatine is evolved in the course of the formative metabolism
of muscle which involves the making of muscle substance and its ma-
chinery. It represents rather the wear and tear of the machine than
consumption for purposes of obtaining energy. This is, perhaps, the
most reasonable view of creatine and it explains why all organs have and
produce creatine. It is evidently not a substance having to do with a
particular function, like that of contraction, but some function, like
growth, common to all cells.

It is probable that creatine of muscle is not free, although it is so
easily extracted with water, but that it is combined with the colloids of
the muscle. Otherwise it would most probably escape from the cells.
How much of it is actually given off from the muscles in the course of
the day cannot be said. Safe conclusions cannot be drawn from the
fact that it is given off from the muscles to perfusion liquids, for the
condition of perfused tissues is generally far from normal. Most ob-
servers are of the opinion that the greater part of the creatine is de.
720 PHYSIOLOGICAL CHEMISTRY

stroyed in the body and that only a small portion escapes in the form
of creatinine.

Summary. The principal results and conclusions of the chapter
may be summarized as follows:

There is excreted in the urine of human male and female adults
about 1 to 2 grams of creatinine a day. The amount is entirely independ-
ent of the protein intake, but is increased by the ingestion of creatinine,
most of which reappears unchanged in the urine. The ingestion of
creatine increases creatinine only very slightly and it may not increase
it at all; it may increase somewhat the creatine excretion. What be-
comes of the greater part of the creatine thus ingested is unknown.
Creatine is found only occasionally and in small quantities in the urine
of adult men, a few mgs. per day, but it occurs, or is supposed to occur,
in larger amounts, 60-100 mgs. per day, in the urine of women during
menstruation, in the first few days after childbirth, and during preg-
nancy. It is usually present in children’s urine, particularly in girls’,
and may make from one-tenth to one-third the amount of the creatinine.

The amount of creatinine excreted in adults both male and female
is roughly proportional to the body weight; about 7-11 mgs. of creatinine
nitrogen per kilo body weight being excreted in 24 hours. This figure is
called the creatinine co-efficient. The combined creatine and creatinine
undergoes little change during fasting, but the amount of creatinine
diminishes and that of creatine increases, if the method of determina-
tion of creatine is correct. The same result occurs in carbohydrate
starvation, in diabetes, both natural and phlorizin, and in the early
stages of wasting diseases such as fevers, muscle-atrophy, etc. These
facts all show that creatinine has an endogenous origin and that unlike
urea it is unaffected by the intake of protein.

There is found in the voluntary muscles about 0.35-.48 per cent. of
creatine and large amounts are found also in the liver, the involuntary
and heart muscle, in the brain, testes and other organs. The total
amount of creatine in the body is thus about 120 grams. This creatine
of the body is probably the origin of the creatine and creatinine of
the urine. The muscles have the power of forming creatine from
glycocyamine and they, and other organs, can turn creatine to‘creatinine.
Extracts of the organs have this same power which is probably to be as-
cribed to a ferment. Probably the kidneys are active in changing the
creatine arriving by the blood to creatinine, but it is certain that they
are not the only organs having this property. The tissues are also
able to destroy creatine, but what is formed from it is unknown.

The increase of creatine in the urine in diabetes and carbohydrete
starvation, or after the injection of thyroid extract, is ascribed by Folin
Shaffer and others to the increased catabolism of the tissues, but it is
THE EXCRETIONS OF THE BODY 721

also possible that it is due to the setting free of the creatine from the
combination in which it is in the muscles. Nothing definite is known
of the function of creatine in the muscles and the other organs; whether
it is primarily a waste product, or whether, by its presence, it affects
the metabolism of the tissues. Further investigations are very neces-
sary on this point. While all the really important questions about the
origin and significance of creatine and creatinine still remain unan-
swered it appears that these substances hold a very important place in
metabolism and the constancy of the excretion of creatinine indicates
that it is, as Folin suggested, an index of the real catabolism of the
vital machinery of the body proper, in distinction from that catabolism
which increases the free energy. The accumulation of creatinine in the
blood only occurs after long-continued, serious impairment of the kid-
neys. An amount of 5 mgs. or more in 100 e.c. blood indicates an
irremediable condition of the kidney and death within three months.
(Meyers and Killian.) (See page 1017.)

Purine bodies and allantoine in the urine-—The most important
purine found in human urine is uric acid, but there is also present about
30-50 mgs. of purine bases, xanthine, hypoxanthine, guanine and
adenine. The first two are the more abundant. Allantoine is present in
adult human urine in extremely small amounts, not more than 14 mgs.
per day; but in other mammalia it may be present in very much larger
amounts, 25-30 grams a day being secreted by cows. While allantoine
is not, strictly speaking, a purine, it represents in many mammalia the
end product of purine metabolism and must, therefore, be considered
with the purines. For methods, see page 1097.

Uric acid.—Probably no nitrogenous substance in the urine has been
more studied than uric acid. It is present in human urine only in small
quantities, the excretion varying from 0.3-1.2 gram per day in the
human adult, the average amount being about 0.6 gram. The amount
varies with the diet, state of health and individual idiosyncrasy. Of
the total nitrogen of the urine between 5 and 10 per cent. is excreted as
urie acid nitrogen. But though present in small quantities, this acid has
long attracted the attention of physicians and physiologists because of
the problems involved in its origin, its significant variation in disease,
its insolubility, which causes it to form part of the sediment of the
urine, and above all because of its deposition in the joints in the form
of erystalline insoluble salts in arthritis and gout.

While in the mammalia, amphibia and fishes the quantity of uric
acid excreted in the urine is small and is but a small proportion of the
total nitrogen outgo, in birds and reptiles this. substance takes the place
of urea and in these animals most of the nitrogen excretion is in this
form. In the invertebrates, also, in the arthropods and particularly in
722 PHYSIOLOGICAL CHEMISTRY

the mollusks, the excretion of nitrogen, to a considerable extent at any
rate, is in the form of the purine bases and uric acid.

There can be little doubt that the substitution of uric acid for
urea as the form of nitrogen waste in reptiles and birds is an adaptation
fitting them to a dry climate, the particular object of the change being
the conservation of the water of the body. Uric acid has very little
affinity for water and is almost insoluble; and the acid salts are also
insoluble ; hence uric acid is not excreted in bird’s urine in solution, but
in the form of masses of crystals, very little water being excreted at the
same time. Urea, on the other hand, is a very soluble substance; it has
a very great affinity for water and takes a good deal of it out of the body
when it is excreted. It is a good diuretic. The birds were evolved from
the reptiles and the reptiles from the amphibia which have urea as the
nitrogen end product. The reptiles were probably formed or evolved in
some arid region, since in all the particulars of their bodies they show this
same adaptation. Thus they have replaced the moist skin of th>
amphibia by the scaly, hard, dry covering of the reptile, which
prevents loss of water through the skin. Possibly if their lungs
were examined they would be found to allow of less water pass-
ing through than is the case with the amphibian or mammalian

lung.
Chemical nature. Uric acid is 2, 6, 8 tri-oxy purine.
ania oe 0 N—C—OH
o=d done (2) HO—C bona
eal | os Oo | | o-OH (8)
ee —— ae
= acid. Lactam form. Lactim form.

The formula is usually written in the form first given, but another
form is possible and accounts for the fact that it has an acid nature
and that it forms two series of salts. This second form, the lactim
form, is probably in equilibrium with the ordinary or ketone form.
According to the latter formula the acid should be a tribasic acid,
but only two series of salts are known. It is probable that the third
hydrogen ion dissociation would be very weak. Intermediate forms
between the lactam and the lactim probably exist in which only one
hydroxyl is present. The lactam form is the less stable. The hydrogen
in the 2 and 8 positions may be substituted, making possible two series of
salts. Of these salts the acid salts are the less soluble and particularly
the free acid and the mono-ammonium salts are very insoluble. The
solubility of the free acid in water is one part in 39,480 parts at 18°
and 1 in 15,505 parts of water at 87° (Gudzent).
THE EXCRETIONS OF THE BODY 723

It is a white, tasteless powder or crystalline substance, composed
of rhombic prisms or plates (Figure 62). As it comes down in the
urine it is combined with or associated with a red coloring matter,
uroerythrine, and the crystals are colored red, or brown. The forms are
very various, the so-called whetstone shape being common. Dumb-bell
and other shapes occur. The acid urates also are, for the most part,
very little soluble, but the dibasic salts of the alkali metals are more
soluble. The acid may precipitate in the urine in the form of the
acid sodium or ammonium salt in balls of needle-like crystals, of a
brownish red color, or having irregular shapes. The solubility of the
monobasic sodium salt is 0.8328 gram of the salt in a liter of water at
18°, and 0.4141 gram of the ammonium salt. At 37° it is respectively

‘;
By 8

\

Fie, 62.—Crystals of urie acid.

"&

Oe

1.5043 and 0.74138 grams per liter. According to Gudzent these solu-
bilities are only true of the fresh solution, since the solubility gradually
diminishes due to the transposition of the lactam to the less soluble
lactim form. One part of the normal sodium salt dissolves in 77 of
water at 18°. The normal potassium salt dissolves in 44 parts of cold
water ; the normal calcium salt in 1500 parts and the acid lithium salt in
60 parts of water (Ralfe). The acid piperazine salt is much more soluble
than the alkali salts. It dissolves in 50 parts of water at 17° C.

CH,—CH,
eo S
C,H.N,0, NHC DNE
CH,—CH,

The methylglyoxalidine salt dissolves in 6 parts of water (Ladenburg.
Ber. 27, 2952).

In acid solution uric acid is very stable. It may be dissolved in
concentrated sulphuric acid without being destroyed, and from this
solution it may be precipitated by addition of water. In alkaline solu-
tion, on the other hand, it is very unstable, breaking up rather rapidly
and in the presence of oxygen oxidizing itself. Folin and Denis
724 PHYSIOLOGICAL CHEMISTRY

state that 0.5 per cent. Na,CO, solution boiled three minutes with
10 mgs. of uric acid in 20 cc. destroyed 12 per cent. of the acid
present. In alkalies it probably breaks into dialurie acid and
urea.

 

HN—CO HN—CO

oc bwa + 2 H0 eG) HOR + HN
Le IL

HN—C—NH HN—CO HN
Uric acid. Dialuric acid. Urea.

Uric acid, being auto-oxidizable in alkaline solution, is a reducing
substance and reduces Fehling’s solution, ammoniacal silver nitrate,
phosphotungstic acid and other oxidizing substances. It is readily
oxidized also by permanganate and it can be titrated and its amount
quantitatively determined in this way. See page 1097. When oxidized
it forms various substances, such as alloxan, or oxaluric acid, urea,

oxalic acid, carbonic acid, tartronie acid, allantoine and uroxanic
acid, C,H,N,O,.

NH—C — 0
NH—C=0 NH—C=—0 | ue
| |
oz dao o=C CHOH o= C—NH
| |
vu-d—o NH—C=0 | Sexo
Alloxan (Mesoxalyl urea). Dialurie acid. H—C—NH
(Tartronyl urea.) %
OH
O—C—0OH Intermediate form.
|
H—C—OH
|
O—C—OH

Tartronie acid.

Reactions of uric acid. Murexide reaction. The crystals moistened
with nitric acid and evaporated to dryness on the water bath on a
porcelain plate at first dissolve and are partially oxidized, a red
residue being finally obtained. On moistening this, after cooling, with
very dilute ammonia a purple red develops, due to the formation of
ammonium purpurate, or murexide. It is called the murexide test be-
cause ammonium purpurate resembles the scarlet substance in the dye
obtained from the sea-snail, murex. The coloring matter of murex, the
purple of the ancients, is, however, di bromo-indigo blue. If caustic soda
is used in place of ammonia a deeper blue is obtained, and the color
disappears quickly on warming. Some other purines give this reaction.
With xanthine and guanine the color does not disappear on heating.
The formation of purpurate of ammonia is probably as follows:

By the hydrolysis and oxidation of uric acid, dialuric acid and
alloxan are formed. These condense to form alloxantin which, in the
THE EXCRETIONS OF THE BODY 725

presence of ammonia, forms ammonium purpurate or murexide. The
reactions are as follows!:
HN—CO HN—CO HN—CO ' OC—NH

tg He dle |
/ pe
oc C—OH + OC CO —+ 0c C—o0-—C CO

es ea abd \_.

HN—CO HN—CO HN—CO OC—NH
Dialuric acid. Alloxan. Alloxantin.
HN—CO OC—NH
eect
Alloxantin + NH, oc C——-NH—C CO
ie. lah I |
HN—CO OC—NH

Purpuric acid.

Origin of uric acid. It was long believed that uric acid in mam-
malian urine was an intermediary product of protein metabolism,
which was usually almost completely oxidized by the body to urea. The
occurrence of more than the normal amount of uric acid in the urine
was, hence, supposed to mean that the oxidative powers of the body
were impaired in some way. While this view has certain elements of
truth in it, it was in its essentials quite erroneous, as we now know.
Uric acid does not come from the ordinary protein metabolism. The
end product of that metabolism is urea; but it comes from the metab-
olism of the nucleins, both of those of the food and those of the tissues.
The nucleins, it will be remembered, contain nucleic acid, and nucleic
acid contains purines, which, when oxidized, form uric acid. Uric acid
is, hence, of very particular interest because of its relation to nuclear
metabolism.

That uric acid came from the nucleins either of the food or the
tissues followed directly from Kossel’s discovery that the nucleins were
the mother substances of the nuclein, or xanthine, bases as they were
called, adenine, guanine, xanthine and hypoxanthine. These bodies are
now called purines following the suggestion of Emil Fischer, being re-
garded as all derived from purine. Uric acid was known to belong to
the same group of substances and to be simply oxidized xanthine. Kossel
suggested, therefore, that uric acid in the mammalian urine did not come
from the proteins in general, but only from the nucleins. When nucleic
acid was discovered by Altmann, this theory was made still more precise,
the uric acid coming from this constituent of the nucleins.

The fact that uric acid comes from the metabolism of nucleins was
shown in the first instance by feeding experiments. If one determines

+The formulas of alloxantin and purpuric acid are still uncertain,
726 PHYSIOLOGICAL CHEMISTRY

the amount of uric acid in the urine it is found that the quantity in-
creases when there is an increase in the nucleic acid ingested, but it does
not increase nearly as much if there is an increased intake of proteins
which do not contain nucleic acid. Glandular organs generally contain
a good deal of nucleic acid, so that a diet of such organs in place of meat
means an increase in the nuclein intake. In all such diets the excretion
of uric acid is increased. This is shown in the following protocols

(Jerome) :
Uric acid excreted per day—grms.

Usual diet oc. cicisccecusiecaenier ee es 0.554
ee BO Sas eta retevtecdy ian datakelave- ie ubabeunltec sss 0.480
es GA _ Pag cn aussie dene abate ot FOS ap ai overt 0.590

Nuclein diet. Testicles of herring.... 0.740

oe “ ity “ce “ a anes 1.010
« iy ec “ce “ Fe te Nae 0.754

After period. Usual diet ............ 0.452
ey ee a PO" 3 Yale ua.teparnlesstentus 0.462

Nuclein diet. Pancreas period ........ 0.606

s a a BE dec tate as 0.820
ee ff * SA ieee etecea ee 0.612

After period. Usual diet ............ 0.446
g s # OS aces ok teloaei oe he 0.474

Nuelein diet. Thymus period ......... 1.546

e s Se caress tailereia 0.740

Diminished nuclein period ............ 0.398

The usual diet was a fairly hearty diet. The breakfast consisted of
two eggs, bread and butter, porridge, malt coffee, milk and saccharine.
The dinner consisted of meat, potatoes, vegetables, milk or rice pudding,
bread and butter, fruit, and two dessertspoonfuls of whisky, or a pint
of champagne; lunch of fish, bread and butter, apples.

It will be observed that whenever glandular organs rich in nucleins
were ingested then the excretion of uric acid was increased. On a
nuclein-free diet, a starch and cream and egg diet, for example, the
excretion fell to a minimum of about 0.4 gram per day. The same re-
sult has been obtained by many observers. There is no doubt that the
ingestion of nucleins increases the excretion of uric acid; and the
elimination of nucleins from the diet decreases the excretion to a cer-
tain minimum, but does not abolish it entirely. For a nuclein-free diet
eges, milk, sugar, starch and cream furnish an admirable diet almost
purine free. On a diet of cream and starch Folin reduced his daily
output of uric acid to about 0.3 gram and others under his direction did
the same.

The increase in the uric acid excreted after the ingestion of nucleins
might be either a direct or indirect result. That is the nucleins of the
diet might directly in the course of their decomposition give rise to the
uric acid of the urine, or indirectly they might stimulate uric acid
production. It is believed that they act in the first manner and we
accordingly say that some of the uric acid of the urine has an exogenous
THE EXCRETIONS OF THE BODY 727

source, meaning that it comes from outside, from the nucleins of the
food, which have not been incorporated in the nucleins of the body, but
are decomposed and part of the molecule split off and excreted as uric
acid. The details of this process, however, are very badly known. It
is known that the nucleins of the food are digested by the juices of
the intestine and its accompanying glands, such as the pancreas, and the
purine bases are set free. In most tissues, taken as foods, the bases in
the nucleins have already been partially oxidized, while still in the
nuclein molecule, by the action of the auto- digestive and oxidizing
enzymes of the tissue, so that they get free in the intestine in a partially
oxidized form; in the form of hypoxanthine and xanthine, as well, prob-
ably, as guanine and adenine. These bodies are at least in part ab-
sorbed. Their absorption is followed by the appearance of some uric
acid in the urine, but if it be asked where the oxidation of these sub-
stances occurs, whether in the intestinal mucosa, or whether in the
liver or some other tissue, and whether they have, or have not, been
part of the living matter when they were oxidized, or whether they were
only dissolved in the cell sap and never incorporated in the living mat-
ter of the cell; and whether, indeed, they may not have acted by dis-
placing some of the bases already in the cell,—to these questions very
imperfect answers can be given, or none at all.

It is, however, worthy of note that of the total purine ingested, but
a small part reappears in the urine as uric acid. Some of the re-
mainder is probably destroyed in the intestine by the action of the
bacteria, but some is probably destroyed in the tissues or retained there
for the time being. The per cent. of purine nitrogen in the thymus
gland, according to Burian, is 0.482. This would correspond to about
1.2 grams of purine in 100 grams of the fresh tissue. If one eats two
hundred grams of sweetbreads it should, therefore, increase the urie-
acid exeretion by 2.4 grams, making in all nearly 3 grams per day of
uric acid. The actual increase of uric acid in the urine is, however,
not more than half this required amount, showing that the purines
either had not been absorbed, or that they had been retained, or that
they had been destroyed. This fact of the destruction of uric acid in
the human body is well illustrated in the following experiment of Tay-
jor and Rose. For three days the subject was on a purine-free diet, con-
sisting of milk, eggs, starch and sugar; then for three days a part of the
total nitrogen, 10 grams per day, was substituted in the form of sweet-
breads, so that of the 10 grams, 7 grams were in egg and milk and 3
grams in the sweetbreads; for the next four days, six grams of nitrogen
of the eggs and milk were replaced by sweetbread nitrogen; and for
the next four days the purine-free diet, containing 10 grams of nitro-
gen, was restored.
728 PHYSIOLOGICAL CHEMISTRY

 

 

i 2 4th period
Pavietnes ate end period 3rd period Purine free diet

Total urinary N ....... 8.9 8.7 9.1 8.8

Urea N and NH, athens 7.3 7.1 7.1 7.05
Creatinine N .......... 0.58 0.55 0.56 0.47
Purine N (Total) ...... 0.11 0.17 0.26 0.10
Urie acid N ............ 0.09 0.14 0.24 0.07
Remainder N .......... 0.91 0.88 1.18 1.18

 

 

 

 

 

The intake of purine N in the 2d period was 0.17 and in the 3d, 0.34
grams per day. The increase in uric acid excreted accounted for less
than half of that ingested. The purine base N in the urine remained
constant. That much the larger portion of the purine nitrogen is
either not absorbed, or else is retained or destroyed, is shown by
Weintraud, who calculated that the amount of uric acid which was
excreted after a nuclein diet was not sufficient to cover more than
one-fifth of the amount computed that there should be from the
increase in the phosphoric acid excretion.

Time of excretion. The study of the output of uric acid from hour
to hour has led to the discovery of some very curious and unexpected
facts. Hopkins and Hope found that taking food, even when it was free
from nuclein, led in an hour or two to a great increase in uric acid
excretion, which was at a maximum 3-4 hours after the meal, while the
urea maximum was about 6-7 hours after eating. After fasting 6
hours a meal of bread and potatoes was eaten at 1:30 and the urea and
uric acid measured in the urine each hour.

Time Urea—grams Uric acid—mgs. Amount of urine—c.c
10—11 1.07 26 175
11—12 » 1.13 27 “ 118
12—1 P.M. 1.07 24 164

1—2 (meal) 0.64 21 60

2—3 1.12 22 43

3—4 1.16 38 41

4—5 0.84 40 53

5—6 1.16 56 59

6—7 1.20 39 56

7—8 1.37 30 95

8—9 1.47 33 183

9—10 1.33 24 155
10—11 1.33 23 180

These results have been confirmed by Smetanka, who showed that
eating purine-free meat and even carbohydrates causes, 2-3 hours later,
a marked increase in uric acid excretion. This increase is not due to the
daily variation in the uric acid excretion which occurs even in fasting,
the morning excretion being always greater than the afternoon, as in
the experiment following from Smetanka:
THE EXCRETIONS OF THE BODY 729

Hourty Excretion or Uric Acip 1n Mes.

When at 9P.m.
Time When fasting 830 grams of cusein eaten Increase
6—7 P.M. 9.6 10.
7—8 117 9.7
8—9 12.2 9.6
9—10 11.6 9.7
10—11 10.7 17.9 7.2
11—12 10.5 19.7 9.2
12—1 A.M. 10.6 19.7 8.6
1—2 11.4 19.2 7.8
2—3 11.7 ° 19.2 7.5
3—4 11.7 17.5 5.8
4—5 13.5 17.5 4.0
5—6 12.9 17.5 4.6

—_—

Total 54.7 mgs.

What is the cause of this increase? It cannot come from the diet.
It might come from synthesized uric acid, but Smetanka believes that
it comes, as Marec thought in 1887, from the work of the gastric and
intestinal glands. It would seem not impossible that it might be due
to a reabsorption of uric acid precursors from the intestine, due to the
decomposition of the bacteria there and the increased blood supply,
causing an increased reabsorption when digestion begins.

Endogenous uric acid. But by cutting the nucleins completely out
of the food, or by starving, it is not possible to suppress the uric acid
excretion entirely. There is still excreted about .3-.5 gram uric acid
per day. The amount varies in different individuals. This residual
uric acid evidently must have its origin either in the body tissues or
else in the bodies of the bacteria of the alimentary canal. Since there
is reason for thinking that the nuclei of the body cells are undergoing
metabolism, it is generally believed that this uric acid takes its origin,
largely at least, from the nucleins of the tissue nuclei, and the bacteria
of the intestine are not supposed to play any important part in its
formation. At the same time the increase in uric acid excretion, which
accompanies the activity of the intestine, an increase just noted, would
make a careful investigation of this possible source of uric acid desirable.
The uric acid which is still produced in the body after the nucleins
have been cut out of the foods is called endogenous uric acid, meaning
formed, or generated, within.

Variation with disease. The endogenous uric acid will be, then, an
indication of the nuclear metabolism of the body and may be expected
to increase when that catabolism increases, and to decrease when it
decreases. Thus it would be expected that the uric acid would increase
during embryonic development, or during growth, when much nuclear
material is being formed. That this is the case is shown by the exere-
tion of uric acid per gram body weight from pregnant women, and also
from children at different ages. The per cent. of uric acid nitrogen in
730 PHYSIOLOGICAL CHEMISTRY

the urine, computed on the total nitrogen, is also greater at this time,
showing that the nuclear metabolism is greater relative to other metab-
olisms.

In diseases involving tissue decomposition uric acid excretion is also
increased. Thus after the crisis in pneumonia, when the exudate of
the lungs containing a large amount of leucocytes is being digested by
autolysis and reabsorbed, there is a great increase in uric acid excretion.
A similar increase is seen in leucemia (leucocythemia), a disease in
which the number of white blood cells is much increased above the nor-
mal and their decomposition is probably greater than the norma!
amount. After extensive burns of the skin causing marked destruction
of tissues and their reabsorption, there is, similarly, an increase of
uric acid.

Source of the endogenous acid. We have now to inquire what are
the steps, what the processes and in what organs the endogenous acid
is produced. The first steps in the solution of this problem were taken
by Mareg¢ and Horbaczewski. The latter first succeeded in deriving uric
acid from a mammalian tissue by autolysis. By grinding dog’s spleen
in a meat chopper and then with sand, mixing the spleen pulp with
blood well aérated and kept at body temperature, he was able to show
that uric acid was formed from some elements of the tissue or from
nucleic acid added to the pulp.

From these experiments Horbaczewski concluded that uric acid was
produced by the oxidative decomposition of the body nucleins. Of the
various cells of the body undergoing decomposition the leucocytes or
white cells of the blood were those most obviously disintegrating. Hor-
baczewski suggested that most of the uric acid, namely, that following
digestion, pneumonia, leucocythemia, came from the nucleins of these
cells and the uric acid excretion was an index mainly, but not exclu-
sively, of leucocytic decomposition. He suggested, also, that the rise in
uric acid following nuclein ingestion was not due to the direct trans-
formation of nucleins of the food into uric acid, but was an indirect
result of the digestive leucocytosis and decomposition. Subsequent re-
search has shown this view not to be strictly true. Leucocytosis may
occur for a short period without an increase in uric acid, but it is none
the less true that a long continued leucocytosis, involving as it does an
increased decomposition of leucocytes, always increases uric acid. The
parallelism is between the amount of leucocytic decomposition and uric
acid excretion, not between the number of leucocytes in the blood and
uric acid excretion.

Further investigation of the nature of the decomposition of nucleic
acid in the body has led to a more precise knowledge of the various
steps in its formation in mammals. There are in nearly all cells, and
THE EXCRETIONS OF THE BODY 731

possibly in all cells, autolytic or endocellular enzymes, or nucleases,
which decompose nucleic acid into its various constituents. The decom-
position of the nucleic acid may occur in various ways. 1. There may
be a cleavage into mono-nucleotides, such as guanylic acid. This acid
consists of guanine, d-ribose and ortho-phosphoric acid. Such nucleases
exist probably in yeast where guanylic acid and adenosine have been
found. They occur also in the pancreas of the pig (Jones). The
enzyme which thus splits nucleic acid into horizontal slices, as it were, is
called polynucleotidase. 2. A second nuclease, phosphonuclease, splits
off phosphoric acid either from the mono- or polynucleic acids, leaving
the nuclein base and the carbohydrate united as they are in guanosine
and adenosine. 3. Still another cleavage separates the nuclein bases
from the molecule, leaving the phosphoric acid joined to the carbo-
hydrate radicle.

These various cleavages appear in the autolysis of different cells
and are believed to be due to different nucleases. At any rate the
purine bases are set free in most autolyses. Before being set free, how-
ever, they may be oxidized or deamidized and then split free from the
sugar. Thus in some organs during autolysis guanine is not set free as
such, but as xanthine by an hydrolysis as follows:

HN—C —O HN—C —0
Lc bd
NH=C C—-NH + H,O + (Guanase.) ———- O= NH + NH,
cH | | | CH
a er HNC
Guanine. Xanthine.

Adenine may be converted into hypoxanthine by hydrolysis by adenase
and then by oxidation to xanthine. The xanthine may then, if oxygen is

present, be converted by oxidation through the agency of the ferment
xanthineoxidase to uric acid. .

N=C—NH, HN—C — NH HN—C=—0O

nd | — ul b_na + H,0 + (Adenase.) —~ Hd down + NH,

ns (l=

Adenine. Imide form. Hypoxanthine,
HN—C=—0O HN—C=—O0O
ud Pe +O4 (ie eaten ot bs
cH S cH
> i>
{I _l|_?

Hypoxanthine. Xanthine.
732 PHYSIOLOGICAL CHEMISTRY

HN—C=0 HN—C—0
| I
o¢ t —NH + O + (Xanthinoxidase.) = OC C—NH
| || Sox | | c=0
YA
HN —C— € HN —C — NH
Xanthine. Urie acid.

The following list of enzymes concerned in nucleic acid decomposi-
tion has been given by Jones and Amberg:

1. Phosphonuclease,
2. Purine nuclease.
3. Guanosine desamidase. Inosine hydrolase.
4, Adenosine desamidase. 9. Xanthinoxidase.

5. Adenase. 10. Uricase.

The phosphonuclease splits off phosphoric acid from nucleic acid; while
the purine nuclease splits off the purines, leaving the phosphoric acid and
sugar group united. This is quite similar to the splitting of raffinose by
the two enzymes emulsin and invertin. Raffinose is fructose-glucose-
galactose. Invertin splits off fructose, leaving melibiose, or glucose-
galactose. Emulsin splits off galactose, leaving saccharose. By the
action of these various enzymes uric acid will be formed from the catab-
olized nucleic acid.

The distribution of these various enzymes in different organs differs
in different animals. They are found, however, for the most part, in
the liver, the spleen, the pancreas and thymus. It is, on the whole,
probable that some members of the group of enzymes are found in all
tissues of the body, since the partial destruction of nucleic acid and the
conversion of guanine and adenine in their nucleic acids to hypoxan-
thine and xanthine on autolysis appears to be a very common, if not a
universal phenomenon. The determination of the presence or absence
of these various enzymes in different organs in vitro is subject to
various sources of error. Thus there may be inhibitory substances
present, or the enzymes may be present at times but not at others, or
diet may play a part in their appearance. The statements in the litera-
ture are, therefore, in part contradictory. Negative evidence is not
worth a great deal. Wells has compiled the results and from his state-
ment the following excerpt has been made:

1. Nuclease. Present in all cells investigated.

2. Adenase. Present in all cells and tissues examined, including
bacteria, except human spleen, liver, pancreas, kidney and lung, the
human fetus of three months, tissues of the fetal dog until birth.

3. Guanase. Present in tissues and cells investigated, except human
spleen, spleen and liver of the pig and the pancreas of the dog.

4, Xanthine-oxidase. Present in the spleens of dogs, ox, horse, but
absent from the spleen of man and the pig; present in the liver of
men, cows, pig, rabbit, and possibly the dog; present in bovine muscle,

Guanase.
Xanthosin hydrolase.

CSAS
oD
as

THE EXCRETIONS OF THE BODY

‘lot ‘d (uryeq)

 

 

 

 

Se woryeprxo oe
oo'H'N so stsfjorpéy *o'n’H’O oaeorig ‘o'n'a'o
wn < eulozUe] Ty a ; plow orn
@sEplxo UlyUEX |
/ aseprxo-ourgquexodégy
O'N’H'O _s ‘o'n’n’a
SS en:
“*o's’o0'N"Ho *o'H'0"0'N' HO
eulsouy oursoqqueyX
aseuepy aszueny
quemaosider “EN quemeoetdes “Ey
“ANN'HO sisiorpfg —°0°g*o (“NO ‘OH DON HO sicorpég — (*ER)-0'N' HS
auruep _ — puisoueny) => __ ouraeng
es $
*sasearan Ny
[lve a
(un)’n’H') “o'n"o0—a=o

r
Cawo'n’n'o “o’n’o0— 0
HO
“(abyeq) AawowioaANIg sv qaLNassaany
GOV oEMOON TVOIdkL, ‘COV OLFIOAN 10 NOILVGIxQ GNV SISATONCA GEL ONTLNGSIudaY WVEDVIGE
734 PHYSIOLOGICAL CHEMISTRY

intestine and lung. but not in the thymus and blood of cattle, nor in
the lungs, pig’s pancreas, dog’s pancreas and human placenta. It is
lacking in the chief human tissues except the liver.

Destruction of uric acid. Uricolysis. Uric acid is an easily oxidized
and hydrolyzed substance. It is not surprising, therefore, that what
appears in the urine is only that portion which has escaped destruction.
By oxidation allantoine is easily formed and this is certainly one of the
substances formed in the bodies of most mammalia as a result of uri-
colysis. The human body alone and that of the chimpanzee appears to
have lost the power of destroying uric acid. If uric acid is given by
the mouth only a small portion of it reappears in the urine as such.
For a long time it was thought that the remainder had been destroyed
by the tissues, but it now seems more probable either that it has been
destroyed by the bacteria in the intestine or not absorbed. Its fate is
unknown. In most of the mammalia the purines are excreted chiefly in
the form of allantoine, uric acid is the next most important substance
and the bases are excreted in very small amount; but in other mammals
the bases may, at times, surpass the uric acid excretion. In human
beings, as has been said, all observers except Croftan have failed
to find any uricolysis by the extracts of human organs and allantoine is
present in such small amounts in human urine that the opinion is
generally accepted that human tissues have lost the power of destroying
uric acid. On the other hand allantoine, taken by human beings by the
mouth, does not appear as such in the urine (Minkowski).

This destruction of uric acid is brought to pass chiefly in the liver
or kidneys, which contain in mammals except man and the primates a
uric acid-destroying enzyme or uricolytic enzyme, called uricase.

The first thorough study of the destruction of uric acid was made
by Croftan, who found that uric acid is destroyed chiefly in the liver by
carnivora, in the kidney by herbivora, and by both these organs in
omnivorous animals, men and pigs. He succeeded in isolating a sub-
stance from these organs, an albumose-like body and a nuclein, which
were inert when separate, but which were actively uricolytic when
united. His results have been criticised by various workers to the effect
that he did not sufficiently guard against the decomposition of uric acid
by alkali and air alone, but in view of his controls the criticism appears
to the author to be unfounded and subsequent investigations have con-
firmed nearly all of his findings. According to Schittenhelm and
Wells, human liver does not contain uricase. It has been found in mon-
key’s liver, but not in that of the chimpanzee. Croftan’s positive find-
ing of uricase in human liver remains, as yet, unexplained. It is pos-
sible that under different conditions of disease, or possibly of diet, the
uricase may vary in amount.
THE EXCRETIONS OF THE BODY 735

The following are some of the results of Croftan in the destruction of
uric acid by the ground-up dried organs after they had been extracted
with alcohol and ether. The uric acid was dissolved in weak sodium
carbonate and the organ powders were suspended in this and a stream
of air passed through.

 

 

| Flask 1. _ Flask 2. Flask 3. 48 honrs
sine | Orme atid ateeente| Mra at | ate pron
Dog Liver 0.327 0.325 0.225 31.1
Kidney 0.319 0.319 0.312 2.4
Muscle 0.330 0.326 0.303 8.2
Blood 0.321 0.317 0.313 2.5
Spleen 0.327 0.320 0.319 2.4

 

 

 

Similar results have been obtained by Schittenhelm, Wiechowski and
other observers.

Chemistry of the destruction of uric acid. Allantoine. In most
mammals allantoine is formed by the oxidation of uric acid. Certainly
in the dog all of the uric acid appears to go into allantoine, but whether
this is always the case in other mammals or not is very doubtful. In
most experiments in which uric acid has been ingested or injected only
a portion of the uric acid thus ingested has been recovered as allantoine.
What becomes of the rest is unknown. In human beings the allantoine is
not increased by uric acid ingestion or injection, but the uric acid is in
part excreted as such. In fowls it appears from the work of Ascoli that
certainly a portion of uric acid is hydrolyzed in the liver to form
dialuric acid, as shown on page 740. Perhaps this happens in other
animals. This question must be left for further investigation.

According to Sundwik, the oxidation to allantoine by permanganate
probably goes through uroxanic acid as follows:

NH—CO NH—coO
| I
Oc C—NH + HOH + O— + 0° bon) a —> + NaOH ~~
| | Sco | co
Ph ge oe ! | 7.
NH—C—NH NH—C(OH) NH
Urie acid. Intermediate form.
NH, COONa NH,
ae |
oc C(OH)NH -—— by acid —- “ _
: | Nco to co + ©O,
NH—C (0H) NH NH—CH—NH
Uroxanie acid Allantoine,

(sodium salt).
736 PHYSIOLOGICAL CHEMISTRY

The oxidation in dogs and other mammals may follow a similar
course,

Distribution of nitrogen between different purine bodies in different
mammalia. In most of the mammalia 79-98 per cent. of the uric acid
formed in the body is converted into allantoine. In man 80-100 per
cent. appears to escape destruction. The proportion of uric acid and
bases also varies widely. The following table, taken from Hunter and
Givens’ work, illustrates this variability in different mammalia. The
figures under total purine nitrogen are simply average figures added
from their other tables to give some idea of the total nitrogen appear-
ing per day in the form of purines, including allantoine. This total is
very variable and depends in part on diet, since many vegetables con-
tain allantoine.

 

 

 

etal ousiie Per cent. of purine-allantoine nitrogen - : ?
Orders and species ni froven Bees eon
grms. Allantoine Uric acid Bases
Marsupialia
Opossum..... 0.04 786.0 19.0 6.0 79 4.1
Rodentia
Rabbit....... 95 26.0
Guinea pig... 91.0 6.0 3.0 94 27.0
Rat. sciacce eee 93.7 3.7 2.7 96 37.0
Ungulata
Sheep........ 0.2-0.6 64.0 16.0 20.0 80 8.0
Goat......... 1.0 81.0 7.0 12.0 92 17.0
Cow......... 8.0 92.1 7.3 0.7 93 18.0
Horse........ 1.6 88.0 12.0 0.5 88 3.7
PAR es cueeet 0.3 92.3 1.8 5.8 98 12.0
Carnivora
Raccoon...... 92.6 5.4 2.0 95 16.0
Badger....... 0.25 96.9 1.9 1.2 98 28.0
DOP iii eeeens 0.1-0.3 97.1 1.9 1.3 98 29.0
Coyote....... 0.15 95.6 2.6 1.8 97 23.0
Primates
Monkey...... 0.045 66.0 8.0 26.0 89 4.5
Chimpanzee. . 0
Man......... 0.2 2.0 90.0 8.0 2 2.5

 

 

 

 

 

 

 

The foregoing table shows, at a glance, the exceptional nature of the
purine metabolism of man and the chimpanzee. Attention is called to
the fact that in the monkey and some other mammalia the proportion of
purine base nitrogen may be larger than that of uric acid. The fact that
in man uric acid is so much greater than the allantoine has led to the
conclusion that man has no power of destroying uric acid, and this is
in harmony with the fact of the absence of uricase from his tissues.
But, on the other hand, the very small purine coefficient arouses the sus-
picion that some of his purine catabolism is represented in other forms
of nitrogen, and the question whether man does or does not destroy uric
THE EXCRETIONS OF THE BODY 737

acid or other purines cannot be said to be definitely settled. The purine
coefficient in the above table represents the milligrams of purine-
allantoine nitrogen secreted per day per kilo body weight;
the uricolytic index is the ratio of allantoine nitrogen to the sum
of allantoine and uric acid nitrogen only. ‘‘It is taken as the
measure of the animal’s capacity to oxidize uric acid arising interme-
diarily.’’

With the discovery of the origin of uric acid we have not, by any
means, exhausted the subject. Uric acid is but one of the purines,
purines are important constituents of the most important constituent of
living matter, namely, the chromatin of the cell nuclei. The very preg-
nant question remains behind, namely, can the animal organism make its
purines from other nitrogenous material of a non-protein kind, or must
it depend entirely on purine materials in the food? If it does make
purine from amino-acids, why may not some of the uric acid have this
origin? Why must it be formed altogether from the nucleins of the food
or from those of the body cells? The question thus raised is susceptible
of but partial answer at this time. A recent. observation of Taylor and
Rose, in which a man on a purine-free, egg, starch and sugar diet in-
creased the nitrogen intake from about 6 to 40 grams of nitrogen per
day with an accompanying increase of uric acid excretion from
about 0.3 to 0.82 gram per day would indicate that the uric acid of
the urine may be synthesized in part from non-purine precursors.
On the other hand, this rise may be due to a stimulated nuclein
catabolism.

Synthesis of uric acid in birds and reptiles. There is no doubt that
the sauropsida, the birds and reptiles, are able to form purines and
uric acid from non-purine forerunners. Thus, if they be fed on proteins
poor in nuclein, the greater portion of the nitrogen appears in the
urine in the form of uric acid. Similarly, if their livers be perfused
with blood containing various amino-acids, uric acid is formed. They
convert ammonium lactate into uric acid. Purine synthesis is for them
very easy of accomplishment. Moreover all the invertebrates, so far as
they have been examined, are found to secrete their nitrogen largely
in the form of purine nitrogen. They must manufacture their uric acid
also from non-purines. All plants have this power. Assuredly so
general a property of living matter is not lacking in the mammals. All
mammals live during the first months of life chiefly on milk, which is
almost free from purines, and at this very time they are manufacturing
and catabolizing nucleins at a very rapid rate. In the developing bird’s
egg the purine-free proteins of the yolk and white are in part con-
verted rapidly into nucleic acid.

The power of synthesizing purines from non-purine precursors ap-
738 PHYSIOLOGICAL CHEMISTRY

pears, therefore, to be a universal attribute of living cells. It is not at
all probable that this power is lost in the adult mammalia, for no evi-
dence has thus far been obtained that purines are necessary in the food
to make good purine waste. With the mammalian organism having this
power of synthesis of purines which are converted so readily by the
purine oxidases into uric acid, it would appear surprising if some of
the uric acid was not formed directly from purine thus produced, be-
fore it has been incorporated into the nucleic acid. We have no evi-
dence, however, that it is so produced, and it might be that the synthesis
took place only in the nucleus of which the membrane might permit the
passage inward of raw materials, but prevent the passage outward of
the synthesized purines before they were incorporated in the chromatin
or nuclein. The fact that the nuclear wall is, in many cases, derived
from or composed; in part at least, of chromatin, through which the
purine must pass before escaping to the cytoplasm, might be a device
responsible for the failure of uric acid to be set free from these purines.
The purine oxidases, perhaps, are in the cytoplasm, rather than in the
nucleus, and so act only: on those purines which escape from the
nucleus.

Various attempts have been made to discover what the raw ma-
terials are from which pyrimidine and purine might be formed. This
matter has been discussed already on page 183. The presence in the
sperm head of such large amounts of the basic amino-acids, histidine,
lysine, and arginine, draws attention to these substances as the possible
precursors. Moreover, arginine has guanidine already in its molecule.

Not only does the dog and ox liver have the power of destroying
uric acid, but it will also resynthesize it if the conditions are changed
(Ascoli and Izar). The same fact is true of the livers of birds, although
their behavior in this respect appears to vary with the diet. Thus hens
fed in the laboratory resynthesized uric acid without difficulty (Izar),
and behaved both in regard to uricolytic powers and resyntheses like
dog’s liver, whereas little resynthesis was obtained from the livers of
hens bought in the market. Brunton and Bokenham showed long since
that the power of resynthesis was lacking in livers of dogs which have
fasted 72-192 hours; such dogs have also a much-reduced power of
uricolysis, a fact of importance in understanding the contradictory
results obtained with human livers. If blood was added which had been
taken from a dog fed shortly before, the power of synthesis returned.
The following protocol illustrates this.
THE EXCRETIONS OF THE BODY 739

XPERIMENT ILLUSTRATING THE DEPENDENCE oN Drier or Uric Acip DrsTRucriun
AND SynTuESIS ny Doa’s LIvER:

160 grams of sieved liver pulp of « dog 5 days fasting plus 1400 e.c. 0.85% NaCl
plus 948.0 mgs. uric acid in 200 c.e. 1i,CO, (1:90) solution. 3 days autolyzed with
air drawn through and then divided into 4 equa! parts.

Uric acid recovered

A. Coagulated immediately .......... 0. ccc cc cee cece eee eens ... 149.5 mgs.
B. Added 100 c.c. NaCl solution ........ ime 147.3
C. “ * © defibrinated blood of soaace. ||
dog fasting 72 hours ........... { . sa iter Bail iced 164.3
D. Added 100 e.c. defibrinated blood of | ms fe te
dog fed 12 hours before ......... { haem oe eeg 226.22

It will be seen that under CO, the autolysis for 72 hours caused no
reformation of urie acid except when blood had been added from a fed
dog. The uric acid increased in this case 77 mgs.

URICOLYSIS AND REGENERATION or Uric ACID IN THE LIVERS OF Hens, GEESE AND

 

 

 

TURKEYS.
10 per cent. Uric acid found Uric acid found
Liver of sieved liver Hours Added uric after 72 hours after a further 72
pulp fasting acid—mgs. autolysis under . hours autolysis
c. c. : air under CO,

Hen 1 180 3 441 60.2 322.8
“ 2 160 48 s 223.4 242.4
“e 3 190 72 £ 210.3 334.9
se 4 290 6 391.2 30.2 314.7
% 5 220 48 417.0 254.6 309.0
i 6 350 3 790 256.4 635.8
Turkey } 1250 10 640.7 67.8 556.1
o 2 1010 120 817.4 752.6 740.2
ne OSD 1140 4 767.2 136.6 741.8
ce 1380 10 970.1 188.0 814.2
“65 1170 96 832.1 654.2 708.2
“« 6 1240 48 847.4 550.2 622.8
Goose 1 1430 10 811.1 34,2 738.8
sf 2 1000 10 817.4 168.6 784.2
te 3 1750 48 1014.0 966 982.2
“ 4 1730 96 890 830.0 804.6
es 5 1540 48 1034.0 802.0 879.4
ee 6 1150 102 913 876.0 868.0

 

 

 

 

 

These experiments show, first, that birds’ livers have great powers
of uricolysis and this power is enormously reduced by previous fasting ;
and, second, that the uric acid reappears if autolysis is continued for
72 hours under CO,. If the uric acid is really destroyed, it would
appear that when oxygen is present uric acid is destroyed or hydrolyzed
and resynthesized by reduction or when CO, is abundant. The quantity
reappearing is in all cases proportional to that which disappears. The
resynthesis depends here, also, on the presence in the blood of a thermo-
labile enzyme, and an alcohol-soluble, heat-stable component in the
liver. but this coferment is not present in the kidney. Hen’s blood alone
740 PHYSIOLOGICAL CHEMISTRY

destroys uric acid very fast. The ferment is not specific, that is the
ferment in dog’s blood will act in the case of the hen’s liver. A
further investigation showed that in the presence of CO, liver forms
uric acid out of dialuric acid and urea. On the other hand lactic,
paralactiec, tartronic, acrylic, oxalic, mesoxalic acids or their salts caused
no uric acid formation. Allantoine had no effect. Izar was unable to
isolate the intermediary substance. It would seem probable from these
observations that the liver decomposed uric acid to dialurie acid and
urea and resynthesized them under conditions of reduction and possibly
of a change in reaction due to the CO,. Recent work makes these results
doubtful.

NH—C —=0 aan ess 0
o= Leror NH . — o=
| | + oo (IL > co + HO
NH—C = 0 NH,
Dialurie acid. Urea. a a

Allantoine.—C,H,N,O,. Allantoine is the diureide of glyoxylic acid.
This acid will unite with urea through each of its hydroxyl groups to
form a diureide according to the following equation:

NH, HO—CH—OH HN NH—CH—NH—CO—NH,
| | | fw

CO 4 | - ch = 2S6 | + 3H,0
| | | 4

NH, HO—C —0 HN NH—C—O0

Urea. Glyoxylie acid. Urea. Allantoine.

Boiling with alkalies decomposes it by hydrolysis into urea and
glyoxylic acid.

Allantoine is easily produced by the oxidation of uric acid with
lead peroxide and the allantoine of the urine arises certainly in part
from the oxidative decomposition of the purines as already discussed.
Carbon dioxide is produced at the same time.

By boiling water allantoine is hydrolyzed into urea and allanturic
acid as follows:

NH—CH—NH—CO—NH, NH—CH—OH

f | |

oc | + H,0 — oc | + H,N—CO—NH,
I | i. |

NH—CO NH—cCO

Allantoine. Allanturie acid. Urea.

If allanturic acid is reduced it yields hydantoine, C,H,N,O,, which
when hydrolyzed goes over into hydantoic acid (glycol uric acid) and
finally to glycocoll and ammonium carbonate.
THE EXCRETIONS OF THE BODY 741

NH—CH—0H NH—CH,
| by | |
oc | + 2H —- oc | + H,0
Lf | |
NH—CO NH—CO
Allanturice acid. Hydantoine.
NH—CH
2
od | + H,0 —~ NH,—CO—NH—CH,—COOH —~
ee NH,—CH,—COOH + NH, + CO,
Hydantoine. Hydantoic acid. Glycocoll.

It will be noticed that the ring in hydantoine, except for the double
bonds, is the imidazole ring. Allantoine is thus related to the base
histidine. It is also related to cyanuric acid, C,H,N,O;. Allantoine
crystallizes from urine in sheafs of plate-like crystals, but when pure in
clumps of prisms, m.p. 231-282.

Allantoine has been found in the urine of herbivorous and carnivo-
rous animals; in calves’, cows’ and sheep urine and in that of the dog, cat
and monkey. It was first found in the allantoic fluid of the calf, whence
its name. It is not found in the urine of human adults, except that it
has been, at times, found in the urine of pregnant and nursing women.
It is said to occur, however, in the urine of children in the first week of
life. Human adults are said to be able to destroy allantoine taken by
the mouth, but the lower animals, such as monkeys and the carnivora,
are not able to do so, nor can a young child. It is doubtful whether
human adults really have the power of destroying the substance.

In the lower animals and monkeys allantoine appears to be a ter-
minal product of the purine metabolism and allantoine, given by the
mouth, is excreted unchanged in the urine. In man, however, allantoine
taken by the mouth does not appear as such in the urine (Minkowski).
It is apparently oxidized but the products of its oxidation have not been
found. It is possible that the destruction occurs in the intestine. Allan-
toine may be prepared from the urine of cows which secrete 20-30 grams
a day. The method for its quantitative separation from the urine is
complicated and described on page 1100.

Since purines are found in plants as well as animals it is interesting
to note, as showing how closely similar the chemical processes are in the
two divisions of the living kingdom, that allantoine is also found there.
It occurs in sprouting wheat seedlings and is a constituent of the bruise-
wort or slippery root, Symphytium officinale.

Hippuric acid.—This acid is found in the urine of herbivorous ani-
mals, such as horses or cows, in large amount, but only about .7 gram
per day occurs in human urine. It is of especial interest because, unlike
742 PHYSIOLOGICAL CHEMISTRY

all the substances discussed hitherto, it represents one of the chemical
methods of defense of the organism against toxic substances and is
formed in the kidney itself, or at least part of it is. From both these
points of view it will well repay a careful study. The name connects it
with horse urine. (Gr, hippos, horse; and ouron, urine.)

Chemistry. Hippuric acid is benzoyl-glycin, C,H,NO,,

O
Cc _UL_wH—cH—coon
0
He * CH

Hippuric acid.

It is easily decomposed by alkalies, by bacterial action, by boiling
acids, or by an enzyme of the kidney (histozyme) into glycocoll and
benzoic acid. It crystallizes in long, fragile, rhombic, four-sided prisms.
which dissolve in 600 parts of cold water, more easily in hot, readily
in alcohol, slightly in ether, but readily in acetic ester; but which are
not soluble in benzene, petroleum ether or carbon bisulphide. The
melting point is 190.2° (187.5°?) and by farther heating the crystals
form a red mass, which decomposes with the formation of an odor of
hay and then of hydrocyanic acid and benzoic acid. Hippuric acid may
be recognized by the odor of nitro-benzene obtained on evaporating it
with nitric acid and heating the residue, a reaction given also by benzoic
acid. It is differentiated from benzoic acid by the insolubility of hip-
puric acid in petroleum ether. The alkali and alkaline earth salts are
soluble in water; the silver salt is less readily soluble.

Occurrence. Hippuric acid occurs, not only in the urine of cows,
horses, pachyderms, carnivora and man; it has been found in the sweat
after heavy doses of benzoic acid; in the urine of turtles and some
insects; but not in bird’s urine. In the urine of birds one finds in place
of hippuric acid ornithuric acid, a compound of benzoic acid with
diamino-valerianic acid, or ornithine, C,JI,.N,0.,, in place of glycocoll.
It is said by Baumann that hippuric acid completely disappears from
dog’s urine if there is no putrefaction in the intestine.

Amount. The amount contained in the urine varies with the diet.
On a diet containing much fruit or vegetables the excretion by human
beings may rise to two grams a day. Herhivorous animals excrete a
great deal more than carnivorous. Its variation in disease has not yet
been studied.

Origin. The wide variation of the excretion with the diet indicates
at once that part at least of the hippuric acid must be derived from the
THE EXCRETIONS OF THE BODY 743

food and this has been confirmed by experiment. An increase in the
intake of benzoie acid, or of substances which can form benzoic acid in
the body, causes a marked increase in the hippuric acid excreted. An
increase in glycocoll intake, however, produces no change in the output
of hippuric acid. Glycocoll is supplied by the body, benzoic acid
mainly from the foods. Since benzoic acid is more toxic than hippuric
acid, the conversion is evidently a process of detoxication, a means of
defense of the organism against poisons. We find, indeed, that not only
does the organism defend itself against benzoic acid by pairing it with
glycocoll, but also against other aromatic substances, such as phenyl
acetic acid, cresole and phenols, the same means of defense is used. The
formation of glycocholic acid in the bile may be a similar process, since
cholic acid is decidedly toxic. It may be mentioned also, in this con-
nection, that other substances than glycocoll may be used for pairing
purposes, namely, sulphuric and glycuronic acids.

Toxic substances owe their toxicity to two peculiarities: first, they
possess a large amount of available potential energy, being generally
unstable compounds which liberate energy on decomposing ; and, second,
they are abnormal substances not used in the normal metabolism of
the cells in question. The organism protects itself against such sub-
stances in several different ways. It may oxidize them and thus make
them more stable; or by making them unite with other compounds
which are stable and inert they are rendered indifferent to the body. It
may happen that oxidation renders substances unstable rather than
more stable and an organism may in this way increase the toxicity of
substances. Benzene, for example, which is not very toxic, is converted
by oxidation into the toxic phenol; and some nitriles, like the propio-
nitrile, may be oxidized to the lacto-nitrile, which is much more re-
active and poisonous. :

We may take advantage of this property of the body of pairing some
of its metabolic substances with unstable toxic substances to studv
the intermediate metabolism of the body, as will be shown in discussing
cysteine (page 819.) By giving a toxic substance there may be com-
bined with it, and thus brought into the urine, an intermediate sub-
stance not normally found there or found in very small amount. Gly-
curonic acid and cysteine are substances of this kind.

Place of origin of hippuric acid. The formation of hippurie acid
from benzoic acid and glycocoll is of the general type of amino-acid con-
densations, like that of creatinine, and the power of making such conden-
sations is an attribute of all living matter. The work of Bunge and
Schmiedeberg proves that in the dog the perfused kidney can bring
about this synthesis, but in the rabbit, and probably other animals as
well, other tissues may do it also (Salomon). The synthesis depends.
744 PHYSIOLOGICAL CHEMISTRY

like all such syntheses, upon a plentiful supply of oxygen and Drechsel
considered it to be probably an oxidation-reduction synthesis.

Source of the benzoic acid. We not only take benzoic acid itself in
small amounts in fruits and berries, particularly in cranberries, but
grass and vegetables often contain other aromatic compounds, such as
quinic acid, which by digestion, fermentation by bacteria or by oxi-
dation yield benzoic acid. It is for this reason that a fruit or vegetable
diet increases the hippuric acid secretion. Benzoic acid or benzoates are
often used, also, as preservatives in canned fruit, catsup, or other food
products, and even in milk, so that the consumption of such preserved
foods leads to the consumption of benzoic acid. But the proteins them-
selves may also, by bacterial decomposition, give rise to benzoic acid.
Thus phenyl alanine by oxidation goes over into phenyl pyruvic acid,
C,H,.CH,.CO.COOH, which by further oxidation is converted into
phenyl acetic acid and this into phenyl carbonic or benzoic acid. It is
probable from Baumann’s observations that this latter process only
occurs with the intermediation of the putrefactive bacteria in the in-
testine.

Source of the glycocoll. The question naturally arises how much
glycocoll the body has at its disposal to neutralize toxic matters like
benzoic acid and what is the origin of this glycocoll. Does it come from
the protein or is it synthesized in the body? Concerning the first ques-
tion of the amount of glycocoll which the body can supply for purposes
of detoxication, experiment has shown that the herbivora have quite
remarkable powers in this respect. Thus Ringer found that a goat
might take 25 grams of benzoic acid a day and excrete it as hippuric
acid. Magnus-Levy found in sheep and rabbits that after ingesting ben-
zoic acid 27.8 per cent. of the urinary nitrogen might appear in the form
of hippuric acid nitrogen; and Wiechowski that 50 per cent. might thus
appear. There is only a slight diminution in the other nitrogen con-
stituents of the urine, except possibly a diminution in the uric acid
(Weiss and Levin). This would indicate that the glycocoll nitrogen
was supplied in addition to that which would normally have been
eliminated. In carnivora, and probably-in man, the conditions appear
to be different. No more glycocoll is in them available for pairing than
can be accounted for by the decomposition of their body or food pro-
tein. Abderhalden, Gigon and Strauss found that in carnivora, herbiv-
ora and hens the entire amount of glycocoll in the whole body, exclusive
of fat, feathers and intestinal contents, was only 2.33-3.34 per cent. of
the total proteins. This amount is far too little to admit of the explana-
tion that the glycocoll in herbivora is derived simply from the body pro-
teins. It might come in small part from the purines which may yield
glycocoll on certain decompositions and it will be remembered that
THE EXCRETIONS OF THE BODY 745

glycocoll is present in considerable amounts in the muscle of pecten and
other mollusks. It would seem probable either that glycocoll is syn-
thesized in the body from ammonia and glyoxylic acid by reduction, or
else the hippuric acid may be formed in part by the benzoic acid pairing
with other amino-acids of a longer carbon chain, as happens in birds in
ornithurie acid, and these longer chains are afterwards partially oxi-
dized to amino-acetic acid. The former explanation is, perhaps, the
more probable, since glyoxylic acid, or its aldehyde, is easily derived
from the carbohydrates and the synthesis of ammonia and the aldehyde
to glycocoll very probably occurs in the body, although demonstrative
proof of this has not yet been found. It is not impossible that glycocoll
may be formed in this way normally as one of the precursors of urea
in the transformation of ammonia to urea. Whether the use of glycocoll
to detoxicate benzoic acid reduces the amount of glycocholice acid in
the bile should be investigated.

No definite answer can, however, be given as yet to the question of
the origin of the large quantities of the glycocoll in herbivora until the
matter has been more extensively studied.

Method of isolation. Hippuric acid is readily obtained from fresh
horse or cow urine by first boiling it with milk of lime, filtering off
the phosphates, evaporating to about half its original volume, cooling
and adding strong hydrochloric acid to a plainly acid reaction. The
hippuric acid crystallizes out. The crystals are separated by suction,
pressed as dry as possible in filter paper, redissolved in milk of lime
and recrystallized by the addition of acid. They may then be recrystal-
lized from hot water, being decolorized if necessary by charcoal.

Quantitative determination. The method formerly used is that of
Bunge and Schmiedeberg, which is very cumbersome and by no means
exact. The urine, very slightly alkaline with sodium carbonate, is
evaporated nearly to dryness. The residue is extracted thoroughly with
strong alcohol, the alcohol evaporated on the water bath, the dry
residue dissolved in water, transferred to a separatory funnel, the solu-
tion acidified with sulphuric acid and extracted by thorough agitation
repeatedly (five or more times) with acetic ether. The acetic ether
solution of hippuric acid is now shaken repeatedly with water in a
separatory funnel, the acetic ether evaporated and the residue
extracted with petroleum ether to remove benzoic acid, fats, oxy acids,
phenols, etc. The hippuric acid remains undissolved. The residue is
now dissolved in a little warm water and evaporated at 50-60° to erys-
tallization. The crystals are weighed in a small weighed filter. The
mother liquor is extracted with acetic ether, the ether evaporated and
the weight of the residue added to that of the crystals. The better
method of Folin and Flanders is given on page 968.
746 PHYSIOLOGICAL CHEMISTRY

Ammonia.—Blood contains small amounts of ammonia and some of
this is excreted in passing through the kidneys and appears in the urine.
The amount of ammonia in the urine is greatly increased in any condi-
tion in which larger than normal amounts of acid are produced.
Ammonia is one substance which is used to neutralize the acid formed in
the course of cell metabolism, as has already been discussed on page 248.
The amount of ammonia normally present in the urine of human adults
is about 0.7 gram per day. The amount and the relative proportion it
makes of the total nitrogen of the urine may be increased by the inges-
tion of mineral acids. In diabetes or in fasting, where there is an
abnormal formation of acetoacetic acid, ammonia is also increased. On
a high and a low protein diet, page 710. Folin found the total amount
not much changed, but the relative proportion of ammonia was greatly
increased on low protein. Directions for the determination of the
ammonia are given on page 1093.

Other nitrogenous substances present in small quantities.—A mino-
acids and peptides. Normal urine contains smal! amounts of these sub-
stances and under pathological conditions the amount of amino-acids
may increase. Thus after phosphorus poisoning, or in cirrhosis of. the
liver and in some other conditions, amino-acids such as tyrosine, leucine,
glycocoll, etc., have been isolated from the urine. Normally, however,
these substances are present in very small amounts indeed. A number
of substances have been isolated, however, which are probably peptides
or partially oxidized fragments of the protein molecules which have
escaped the metabolism of the body. Such bodies are the oxyproteic acid
of Bondyzynski and Dombrowski ; antoxyproteic acid of the same authors
and alloxyproteic acid; and uroferric acid of Thiele. The total amount
of N in these substances found by Ginsberg and Gawinski in the urine
of a man on a mixed diet amounted to 3-6.8 per cent. of the total
nitrogen of the urine. This, it will be observed, is just about the amount
of N of unknown nature in the urine of an average adult. The relative
amount is greatly increased in phosphorus poisoning and in various
conditions when body protein is being decomposed and the intake of
protein is low.

Cc H N 0 Reactions
Oxyproteic acid .... 39.62 5.64 18.08 35.54 1. 12 No Ehrlich, biuret, or xan-
thoproteic.

Antoxyproteicacid .. 43.21 4.91 244 26.33 0.61 Ehrlich diazo _pnsitive.
Others negative.

Alloxyproteic acid .. 41.33 5.70 13.55 37.23 2.19 Biuret and Ehrlich negative.

Uroferric acid ..... 3.46 Biuret, Millon, Adamkiewicz
negative.

It is doubtful whether these substances are unitary substances.
Basic substances. Small quantities of basic substances correspond.
THE EXCRETIONS OF THE BODY 747

ing in a general way to those found in meat extract have been isolated
from the urine by Kutscher and his colleagues and by other observers,
particularly by French physiological chemists. Among these are tri-
methyl amine, methyl guanidine; novain; reductonovain; dimethyl-
guanidine; gynesin, C,,H,,N,0,, from female urine; mingin, C,,H,,
N.O.; vitiatin, a homologue of choline; histidine; imidazole-acetic acid;
and methyl-pyridine chloride. This last is probably derived from
tobacco or coffee and is not a natural product of the body metabolism.
In addition putrescine and cadaverine, the former tetramethylendiamine
and the latter pentamethylendiamine, were isolated from the urine by
Baumann and von Udransky. Gr*fiths and Bouchard have particularly
studied the ptomaines of urine. They generally isolate them by making
the urine alkaline and shaking it out with ether. The bodies thus iso-
lated have not been, for the most part, identified. They are said to be
toxic to animals, and Bouchard and other French observers have stand-
ardized the toxicity of the urine by injecting it into rabbits. The
urotoxic coefficient Bouchard defines as the weight of rabbit in kilos
which is killed by the amount of urine secreted by 1 kilo of the body
weight of the individual whose urine is being investigated. A part
of the toxicity of human urine is generally ascribed to the potassium
salts it contains, but probably not all of it can be thus accounted
for.

A very interesting urinary constituent is that of urocanic acid found
in the urine of dogs but not thus far isolated from human urine. although
other imidazole substances have been found there. Urocanic acid has
recently been found to be imidazolyl-acrylic acid and is. therefore, a
decomposition product of histidine. It was found by Hunter in the
digestive products of casein when digested for a long time by a pancreas
mixture. The. conditions of its appearance in such mixtures have not
been determined. The formula is as follows:

CH—NH

e CH

COOH
Urocanice acid.

Urocanic acid nitrate forms very curious, sickle-shaped crystals.
Aromatic oxy acids of the urine.—These include phenol, indoxyl,
scatoxyl, and phenyl acetic, paraoxyphenyl propionic, oxymandelic and
748 PHYSIOLOGICAL CHEMISTRY

homogentisic acids. They are all derived either from tyrcsine, trypto-
phane, phenylalanine or other unknown pheny] derivatives of the pro-
teins. The first group includes the ethereal sulphates or conjugated
sulphates.

Ethereal sulphates. By the decomposition of the aromatic amino-acids
of the proteins, phenols and indoles are produced. They are chiefly
formed in the putrefactive decomposition of the proteins in the intes-
tine. In their passage through the body they are oxidized to indoxyl,
scatoxyl or hydroxyphenol, and then are paired with, or conjugated
with, sulphuric acid to form what are known as the ethereal or con-
jugated sulphates. They are excreted in the urine for the most part
as the potassium or sodium salts of these bodies. The amount excreted
per day varies from 0.1-0.6 gram sulphuric acid. Urinary indican,
that is the potassium salt of indoxyl-sulphuric acid, is such a substance.
The place where pairing with sulphuric acid occurs is supposed to be
the liver.

Phenol, C,H,OH, and cresol, methyl phenol, C,H,(CH,)OH. The
mother substance of phenol and cresol is tyrosine and phenylalanine,
and possibly other aromatic amino-acids if any exist, other than trypto-
phane. The greater part of the phenol and cresol is excreted as con-
jugated sulphate, but a small portion is free in the urine, and a part
is conjugated with glycuronic acid. If phenol is ingested, only a
portion reappears in the urine. A part is evidently destroyed in the
body. Probably like benzene some is destroyed by the rupture of the
ring. The source of the phenol and cresol of the urine is believed to
be the putrefactive decomposition of tyrosine and phenylalanine in
the alimentary canal. The reason for this view is that the amount in
the urine is much increased by excessive intestinal putrefaction and
may be reduced to a minimum by a milk diet and by the ingestion of
carbohydrates, a procedure which reduces putrefaction; or by the use
of cathartics, and particularly such as have an antiseptic action such
as calomel. These bodies are supposed, then, not to come from the
metabolism of the tyrosine in the tissues, but entirely to be derived
from the putrefactive decomposition in the intestine. For this rea-
son the determination of the conjugated sulphates is supposed to
give an indication, albeit a very uncertain one, of the amount of
intestinal putrefaction. It is much easier, however, to determine
putrefaction by means of the indican test to be spoken of in
a moment.

The exact manner in which the tyrosine and phenylalanine are
decomposed in putrefaction is not certainly known. But the transfor-
mation is believed to be as follows, all of these steps occurring in the
intestine :
THE EXCRETIONS OF THE BODY 749

Pe CoH COH COH
@ O
HC 7 ‘on HO” CH HCO” Nox Ho” ‘on
Hd bes He ar Ho bes Hb Mas
Ss. 7 \7 SZ Se
CH, bn ' bn, du,
bua, bu . COOH.
boon boon
Tyrosine. p-Hydroxyphenyl Hydroxyphenyl p-Cresol.
propionic acid. acetic,
eee COH Pe
Ho’ ‘cH Hc” Yon HO” cH
Hb x Was Hd thes re ss
NG f \ oft \ '
| le
Phenol. Pyrocatechol. |
HCHNH,

p-Hydroxyphenylethylamine.
It is possible, though perhaps not very probable, that p-hydroxyphenyl-
ethylamine is first formed which is then oxidized to hydroxypheny] acetic
acid. After the administration of phenol the urine may become dark
colored, owing to the formation of hydrochinon, p-dioxybenzene and
pyrocatechol. These in the air undergo spontaneous oxidation with the
formation of dark coloring matters. They are reducing bodies, reducing
Fehling’s solution and other metallic oxides. If pyrocatechol is added to
a very dilute ammoniacal solution of ferric chloride containing tartaric
acid, the solution is colored a violet, or cherry-red color which is changed
to a green on the addition of sufficient acetic acid. This same reaction
is given by adrenaline. The oxyphenols are fairly stable in acid reac-
tion, but very unstable in alkaliue.
Indoxyl-sulphuric acid. Indican of the urine. This is found in the
urine as the potassium salt. Its formula is as follows:
CH

A :
HC tb \No—c-o-—so OK

| i ol
HO C CH

“oa wf
Indican.

indole is formed by the putrefaction of tryptophane in the intestine.
The indole thus formed is oxidized to indoxy] during its passage through
750 PHYSIOLOGICAL CHEMISTRY

the body and is paired for the most part with sulphuric acid, but in part
also with glycuronic acid, presumably in the liver.

CH CH CH
HO” ‘y—c_cu,—cHNH,—co0H uo” ‘Noon Hc” ‘es —con
ne & Ua — ab | Ur ad U &
Nog Sa NG iN Xt
Tryptophane. Indole. Indoxyl.
CH CH
HG 7s co oc— co” Nou
a a
Nf fn wi of

Indigo blue.

The steps in the transformation of tryptophane to indole are not
entirely certain. It is probable that deamidization happens at first and
then the carbon side chain is oxidized off, indole-propionic, indole-acetic
acids and scatole being intermediate products (p. 441); or that by
decarboxylization the indolethylamine is first formed, which is later
converted into indole by oxidation. The important fact is, however, that
the formation of indole does not occur in the course of the metabolism
of tryptophane in the body, or if it does the indole so formed is destroyed.
The presence of indican in the urine shows, therefore, that indole is
being formed in the intestine. If more than the normal amount of it
is present, it indicates the occurrence of an abnormal amount of intes-
tinal putrefaction, or of putrefaction elsewhere in the body, as for
example in decomposing abscesses.

The test for indican in the urine is very simple and is described
in the practical exercises. It consists essentially in oxidizing the indoxyl
in an acid solution by means of hypochlorite or ferric chloride to indigo
blue and shaking out the indigo blue in chloroform. If the chloroform
is more than a light blue, it means an abnormally large putrefaction.
A satisfactory method for the determination of the amount of indican
in the urine is that of Jolles. It was suggested by Folin that the
color be compared with a standard of Fehling’s solution in a colorimeter.
The method is not very satisfactory. The total conjugated sulphuric
acid may be estimated accurately by the gravimetric method, but this
is too difficult a method for clinical use. A very good idea of the relative
amount of putrefaction can usually be obtained by a little practice in
making the test for indigo blue, so that an abnormally large putrefaction
can easily be detected.
THE EXCRETIONS OF THE BODY 751

The amount of the putrefaction is greater on a heavy meat diet. It
may be so great that the chloroform becomes almost black. On the
other hand, it is often extremely faint. It increases after obstruction
of the small intestine.

It must not be supposed that the indican test, if it is negative, means
necessarily that intestinal processes are normal. It might be that the
intestinal products contained little tryptophane, or that the bacteria
present did not form indole, or that the indole absorbed was in part
destroyed,. or that the absorption was defective. But when the test is
positive the conclusion is justified generally that there is abnormal
putrefaction in the intestine, or rather an abnormally large absorption
of putrefactive products, due possibly to constipation. According to
Jaffé, the amount of indigo in the urine of a healthy man is between
» and 20 milligrams in 24 hours.

Scatoxyl-sulphuric acid. Secatole (Gr. skor, excrement) is a crys-
talline, fecal-smelling substance, C,H,N, formed by the putrefaction of
tryptophane by certain kinds of bacteria. It occurs in the feces. Some
is absorbed from the large intestine and when passing through the body
it is oxidized to scatoxyl and paired like indole, presumably in the
liver, with sulphuric acid and excreted as the salt of this substance. In
constipation so much of this substance may be absorbed as to give to
the breath and exhalations of the body a very distinct fecal odor. Its
presence in the urine, therefore, is an indication of intestinal putre-
faction.

@
HC Nie c—CcH,

a ae
No WA

Scatole.

CH CH
as

II II
Cc  COH

SNA

Scatoxyl.

4G

HC C—— C—CH,
|

HC

CH

a
AC a Sea:

HC Cc C—O—S0,.0 Na
\ Z
S CH

Sodium scatoxyl sulphate.

The constitution of the scatole of the urine is unknown, and also
that of scatoxyl. The ingestion of scatole produces in the urine a
chromogen which gives a bright red color when the urine is made
strongly acid with hydrochloric acid. This color is sometimes referred to
as urorosein. It is probable that urorosein is in reality indole acetic acid.
752 PHYSIOLOGICAL CHEMISTRY

Seatole is probably formed from indolacetic acid, and may be one stage
in the formation of indole from tryptophane.

Parahydroxyphenylacetic, parahydroxyphenylpropionic and para-
hydroxyphenylglycolic acid (oxymandelic acid). These occur in small
quantities in the urine, the last, particularly in acute yellow atrophy of
the liver. They all represent intermediary products of the oxidation of
tyrosine and possibly of phenylalanine.

COH COH COH COH

a @ @ G
HC” \ cn HO” \ on ac? Nox HC i
ud a nbn ub on nb on

\ \ S

\ ae \ ne \ ra ra

|
CH, bs, da, aon)
| | |
CHNH, CH, COOH boon
| |
COOH COOH
p-hydroxyphenyl- p-hydroxyphenyl- p-hydroxyphenyl-
aminopropionic propionic acetic acid. p-hydroxyphenylgly-
acid. acid. colic acid.

These acids all give the Millon reaction. They are soluble in water
and in ether in the free state. The melting point of p-oxyphenyl-
propionic acid is 128° and that of paraoxyphenylacetic acid is 148°.
The oxymandelic acid crystals melt at 162° C.

Homogentisic acid. Dioxyphenylacetic acid. C,H,(OH).CH,
COOH. Hydrochinone acetic. This acid is the peculiar acid found in
aleapton urine. It oxidizes spontaneously in the air and the urine turns
black, at first in its upper layers where the oxygen has entrance. This
acid is hydrochinone acetic acid.

OH

HO” Nou

Hb b_cx.—coon
Y
bx

Homogentisic acid.

This substance is formed from tyrosine and phenylalanine. The amount
formed in some cases may be as large as 16 grams a day, usually, how-
ever, it is less than this, ie., 3-5 grams. This substance is probably
formed by a disturbed tyrosine metabolism, as discussed on page 813.
The cause of alkaptonuria is still quite obscure. It appears to run in
THE EXCRETIONS OF THE BODY 753

families. Baumann thought it was due to an abnormal intestinal flora,
but that opinion is now abandoned. According to Garrod, it occurs
most commonly where there is blood relationship of the parents. It 1s
more common in males than in females. It is still doubtful whether the
normal course of metabolism of tyrosine carries the latter through
homogentisic acid, alkaptonuria being due to more of the intermediate
substance escaping in the urine; or whether it is due to an abnormal
manufacture of homogentisic acid in certain individuals. The work of
Dakin indicates the latter.

Homogentisic acid is a strong reducing agent, reducing Fehling’s
solution, alkaline silver solutions and other metals. It does not reduce
bismuth subnitrate. It crystallizes in large clear prisms. m.p. 146.5-
147° C. It is soluble in alcohol, water and ether, but only slightly in ben-
zene. It gives with ferric chloride a transient blue color, a reaction not
specific but given by many other reducing substances such as cysteine,
other phenols, ete. It is inactive. (For its preparation from the urine
see Orton and Garrod, J. of Physiology, Vol. 27.)

Sulphur of the urine—From 75 to 80 per cent. of the total sulphur
of the urine is in the form of inorganic sulphate, when a person is on
an ordinary diet of about 14-16 grams of nitrogen per day. The
remainder of 20-25 per cent. is in organic union; a part as ethereal or
conjugated sulphates, a small portion as taurine, a portion in the oxy-
proteic acids of the urine, some of this sulphur being unoxidized sulphur ;
and a part is present as cystine or cysteine.

Amount. The total amount of sulphur excreted per day depends
upon the sulphur intake, rising and falling with this. Sulphur enters
the body chiefly in the unoxidized form of protein sulphur and the
total sulphur of the urine excreted per day is about 0.7-0.85 gr. The
amount present as inorganic sulphate is about 0.5-0.6 gram per day.
The remainder of 0.1-0.2 gram is organically bound. The methods of
determining these different fractions in the urine are given in the
practical part, page 1106. The inorganic sulphates, the conjugated or
ethereal sulphates and the neutral or unoxidized sulphur, are generally
determined. The last group is a very heterogeneous group and has
been very little studied. It is determined by difference.

Effect of diet on the distribution of sulphur. Sulphur is ingested
chiefly as protein sulphur. There are, however, small amounts of inor-
ganic sulphate, of sulphuric acid paired with phenols, or present as

. Sulpholipins, and of taurine in all tissues, so that many different sul-
phur compounds are ingested. The ingested protein sulphur is for the
most part excreted as inorganic sulphate, since protein ingested is not
stored, but is burned in the body. Hence on a high protein diet not
only does the total sulphur increase, but also the proportion of inorganic
sulphur as well. On the other hand, on a low protein diet the total
154 PHYSIOLOGICAL CHEMISTRY

amount of sulphur is much reduced and the proportion present as
neutral sulphur is increased. Since on a low protein diet the putre-
faction in the intestine is reduced, there is also a reduction of the
conjugated or ethereal sulphate. The following figures from a meta-
bolism experiment by Folin will show the change in the distribution
of urinary sulphur when one changes from a normal to a reduced pro-
tein diet.

Normal protein diet Reduced protein diet
July 13 July 20

Volume of urine ...........eeeeeeeeees 1170 cc 385 ce.
Total nitrogen ...........ecee eee eees 16.8 gr. 3.60 gr.
Urea. nitrogen: vce secais sec siealale ceamimes 14.70 “ = 87.5% 2.20 * = 61.7%
Ammonia nitrogen ...........0e esse eens 0.49 “ = 3.0 0.42 “ = 11.3
Uric acid nitrogen ............0 ee eens 0.18 “= 1.1 0.09 “* = 2.5
Creatinine nitrogen ...............505. 0.58 “ = 3.6 0.60 “ = 17.2
Undetermined nitrogen ................ 0.85 “ = 49 0.27 “ = 7.3
Ota SO be. eiescosiacesevtvapeqadetaveyeseuestverd ace iosesabes 3.64 “ 0.76 “
Inorganic SO, ...... eee e cece cece ee eee 3.27 “ = 90.0 0.46 “ — 60.5
Ethereal sO, sibahttants fabsgne ee ote Sueaele esi cguochiers 0.19 “ = 5.2 0.10 “ —13.2
Neutral 80, eu ile od ay Seah ahat ah “ereilagii cee 0.18 “= 48 0.20 “ = 26.3

In these results it may be observed that the proportion of ethereal
sulphate rose on the low protein from 5.2 per cent. to 13.2 per cent.,
although the absolute amount excreted diminished. But the greater
relative rise was in the neutral sulphur, which rose from a proportion
of 4.8 per cent on the normal protein diet to 26.3 per cent. on the
reduced protein. It is very suggestive, also, that the absolute amount
of the neutral sulphur excreted did not diminish at all on the low pro-
tein diet, but on the contrary rose slightly, the neutral SO, on the
normal protein diet being 0.18 gram and on the low protein diet being
0.20 gram. It is probable from these facts that the neutral sulphur
corresponds to the endogenous wear and tear, whereas the other two
fractions are largely derived from the protein of the food torn to pieces
by the oxidation of the body.

Variation of the sulphur under other circumstances. ‘Vhs yer eent.
of neutral sulphur in the urine is also dependent upon various drugs.
Thus cyanides and nitriles when ingested ieave the body in mammals
largely in the form of sulphuocyanate in the urine. Hence, when any
substance is ingested which can form hydrocyanie acid in the body, it
will increase the neutral sulphur. There is, also, an increase of neutral
sulphur under the influence cf chloroform and other anesthetics. In
cystinuria also the proportion of this sulphur increases.

Phosphorus in the urine.—Form. Phosphorus is found in the urine
wholly in the oxidized form of orthophosphorie acid. It is present as
the disodium, and monosodium phosphate and free phosphoric acic,

1Wolin: American Jour, Physiol. 13, 0, 118 1005.
THE EXCRETIONS OF THE BODY 755

the relative amounts of these substances present depending on the char-
acter of the diet. On a heavy protein diet the urine is acid, due in a
large measure to the sulphuric and other acids formed from the meat;
under such circumstances there will be more free phosphoric acid and
of the monosodium salt; on a vegetable diet, or when salts of acids
which are burned to carbonic acid in the body are ingested, the propor-
tion of disodium phosphate is larger. The bases of the urine distribute
themselves among the various acids in proportion to the strength of the
acids, the stronger acids taking the stronger bases. Phosphorus in an
unoxidized form is toxic, and not present in any food. The phosphorus
of the foods is present always as phosphoric acid, which is either inor-
ganic or it may be in an ester form, as it is in nucleic acid, in phospho-
lipins, phytin, ete.

The amount in the urine. The amount of phosphorus excreted per
day depends on the amount ingested in part, but it is still more depend-
ent upon the condition of the bowels. In other words, it depends upon
the absorption of phosphates. The total amount of phosphorus ingested
per day in the foods is between 1.2 grams and 2.0 grams in an adult
on an ordinary diet. The proportion of this which goes out in the urine
is very variable. In human beings from 50-65 per cent. of the income
is found in the urine, and 30 per cent. to 50 per cent. in the feces. In
constipation the proportion in the urine increases; in diarrhea the pro-
portion in the feces increases and that in the urine diminishes. An
examination of the phosphorus excretion in the urine alone in a metab-
olism study is quite worthless, since so large a proportion of the phos-
phorus is passing out through the feces. Both these excretions must be .
examined in any metabolic experiments on phosphorus metabolism.
The total amount of phosphorus in the urine of human adults is between
0.5 and 1.2 gram per day.

‘Variation in disease, and under various conditions. The excretion of
phosphoric acid is increased during the catabolism of nucleins in the
body, as for example in leucemia, and during the reabsorption of pneu-
monie exudates. It is increased by the ingestion of nucleins. During
starvation the bones are drawn upon for fuel and there is an increase
over the amount of phosphorus excreted when the food contains suffi-
vient fuel matter, but little phosphate. There appears to be a dis-
turbance in the phosphate excretion after parathyroidectomy. There
is an inerease of phosphoric acid in the urine of dogs after feed-
ing thyroid glands (Roos. Cauter). Wer Eecke found after complete
thyroid and parathyroidectomy in rabbits that the secretion of urine
was diminished by 30 per cent., the urea diminished 33.7 per cent., but
the P,O, of the urine was diminished by 61.7 per cent., while the NaCl
augmented 60-8 per cent. In another experiment the P,O, excretion
diminished 72 per cent., while the NaCl augmented 164 per cent. Green-
756 PITYSIOLOGICAL CIIEMISTRY

wald has confirmed these findings after parathyroidectomy in dogs on a
carefully controlled diet. The urinary P as PO, fell on the day follow-
ing the operation from 0.257 to 0.029 gram per day. It kept at this
level two days and then increased as tetany came on to the normal of
0.254 gram. There was no corresponding increase in the phosphorus
of the feces. It would appear, then, that in some way extirpation of
the parathyroids caused phosphorus retention. There is as yet no
explanation of this conservation. It may be correlated with the great
changes in digestion which follow this operation.

The dependence of the phosphorus excretion on the diet is well illus-
trated in the experiments of Folin. On a diet containing 16 grams of
nitrogen per day the excretion in the urine was 4.1 grams P,O, in 24
hours. <A week later on a diet of cream and starch containing 3.9 grams
total nitrogen, the urinary P,O, was 1.1 gram. The relation of the
phosphoric acid retention to calcium intake and to bone deposition is
not yet understood.

Chlorides——The amount of chlorides, chiefly sodium chloride, ex-
creted per day is dependent upon the food chlorides. The amount is very
variable, but generally lies between 10-15 grams. Some people ingest
very large amounts of salt with their food. This salt is absorbed and
passes rapidly through the kidneys into the urine.

The chlorides may, under certain circumstances, almost completely
disappear from the urine. This is the case, for example, in pneumonia
during the formation of the exudate in the lungs. During its reabsorp-
tion, on the other hand, the chlorine reappears. It is not yet known
how this failure to excrete chlorides is to be explained. A part
of it is no doubt due to the great diminution in the intake, but
this is not the whole explanation. The chlorine is in such cases
held more firmly in the tissues. It is not to be explained by the
formation of so much exudate. Further work on this problem is
necessary.

It is a singular fact that no organic chlorine compounds other than
chlorides are known to occur in the body. Whether there are any such
in the urine is still disputed, but it is possible. In the tissues there is
in lecithin or similar phospholipins a place for the attachment of
chlorine to the choline radicle where it may replace hydroxyl. It is
possible that under the conditions of disease this or some similar union
becomes firmer, the chlorine is not dissociated as before and is so
retained. It is not probable that all the chlorine of the tissues is in
the form of inorganic chlorides.

The method for the determination of the chlorides of the urine is
given on page 1107.

Calcium and magnesium.—These occur in the urine in small amounts
as phosphates, about 1 gram of these earthy phosphates being secreted
THE EXCRETIONS OF THE BODY 757

per day. A great deal of work has been done on the calcium metab-
olism of the body, particularly from the point of view of explaining
the defective formation of bone and other hard parts of the body.
Calcium leaves the body largely in the feces and to a less extent in the
urine. About 0.7 gram of calcium are needed per day by an adult. As
these studies have not yet led to definite conclusions, further discussion
of the subject will be omitted.

Pathological constituents of the urine.—Protein. Various forms of
protein may appear in the urine under pathological conditions, but
normally the urine is free from any protein matter. The forms which
may appear are: 1. Coagulable protein, generally derived from the
blood and being either serum globulin, albumin or fibrinogen. 2. Pro-
teoses, formed from some hydrolysis in the tissues, as in pus. 3. Special
proteins, such as Bence-Jones’ protein, a kind of hetero-proteose found
in disease of the bone marrow, myelomas and possibly osteomalacia.
4, Proteins derived from the urinary passages or glands such as mucin,
from the bladder, or spermatozoa. The significance of these is of
course very different. It is the coagulable or blood proteins which are
the commonest forms and these occur in kidney inflammation, or
nephritis, both acute and chronic. Small amounts of these proteins may
be present without nephritis, but as a result of serious disturbances of
the circulation, such as a dilated heart and a low blood pressure. Thus
a certain number of individuals whose urine is free from protein nor-
mally may develop an albuminuria of slight degree following some very
great exertion. During a Marathon race of about 23 miles on a cold
day against a damp head wind nearly all the runners, whose hearts and -
urines had been perfectly normal before the race, developed albuminuria
with heart murmurs and dilated hearts at the end of the race. Similar
_observations have been made on soldiers. after exhausting marches. In
all ordinary circumstances the appearance of appreciable amounts of
coagulable protein in the urine is abnormal and should arouse suspicion
of nephritis.

The presence of proteoses in the urine is not rare during the absorp-
tion of exudate in pneumonia or any other absorption of partially
digested pus.

The Bence-Jones protein, described on page 1069, is found during
myelomas and possibly osteomalacia. It is apparently due to a decom-
position of the bone marrow.

Carbohydrates.—Deztrose. Very minute amounts of dextrose are
present in normal urine, .01—.07 per cent. About 1 gram total sugar is
excreted in the urine normally per day. Of this about one-half is
dextrose. The kidney is not a perfect filter and small amounts of glucose
ean penetrate it. The appearance of enough dextrose to give a precipi-
tate of cuprous oxide in the FMehling test is abnormal. Dextrose may
758 PHYSIOLOGICAL CHEMISTRY

uppear in the urine under several different circumstances. We accora-
:ugly distinguish the glucosurias as follows:

1. Alimentary glucosuria. Due to the ingestion of more dextrose
than the body can store for the time being. Different individuals have
different powers of utilizing carbohydrates and the power of the body
may be increased by previous reduction in the amount of dextrose taken
in the food. Tolerance of normal individuals is about 50 grams of
dextrose when: taken on an empty stomach. More than this causes a
glycosuria.

2. Diabetes mellitus. This is a disease in which large amounts up to
200 grams of dextrose a day may be excreted in the urine. The word
diabetes signifies an increased flow of urine; mellitus (from mel, honey)
that sugar accompanies the polyuria. The urine is increased in quantity
so that it may be four or even 10 liters per day. The urine is generally
very light colored, but its specific gravity is about normal, due to the
dextrose in it. There are all grades of the trouble, from a slight excre-
tion of dextrose when carbohydrate food is ingested to a complete failure
to burn any of the ingested carbohydrate, or that formed in the body.
It is believed to be correlated often with disease of the pancreas.

3. Emotional glucosuria. <A slight glucosuria appears in some indi-
viduals as a result of strong emotions such as anxiety. This glucosuria
is believed to be due to the secretion of more than a normal amount of
adrenaline by the supra-renal glands.

4. Glucosuria due to drugs. Dextrose may appear in the urine after
the ingestion of certain drugs, of which phlorhizin is an example. This
glucosuria is accompanied by a hypoglycemia. It is generally attributed
to an impairment of the kidney which lets more than the normal amount
of dextrose pass into the urine.

These various forms of glucosuria and the interpretation of them
are discussed in Chapter XVIII on the carbohydrate metabolism of the
body. Methods of identifying and estimating the glucose are given in
the practical part, pages 940 and 984.

Lactose. Lactose has been obtained from the urine of women shortly
before or immediately after childbirth. It is derived from lactose
reabsorbed from the mammary glands. Its significance is of course
totally different from that of dextrose. Lactose reduces Fehling’s solu-
tion just as dextrose does, so that if such a reduction is obtained under
circumstances in which lactose might be formed it is necessary to
identify the sugar more carefully. This can be done most readily by
the fermentation test with yeast. Dextrose ferments with ordinary yeast,
but lactose does not. Lactose reduces bismuth in the Nylander test. It
is also dexro-rotatory as is glucose. For tests, see page 1070.

Pentoses. Pentoses may occur in the urine in the condition which
THE EXCRETIONS OF THE BODY 759

is called pentosuria. There is still some doubt as to the nature of the
urinary pentose. Neuberg isolated i-arabinose; Luzzatto, l-arabinose.
Pentoses may occur in the urine after the eating of fruits and fruit
juices. They are also supposed to be diagnostic of pancreatic disease.
The pancreas contains a pentose in its guanylic acid which is d-ribose.
It is probable that it also contains in the amylolytic ferment, or the
gum to which it is attached, an arabinose. Pentoses were first isolated
from the urine of a person addicted to the morphine habit. They are
identified by the orcine or phloroglucine tests, or by making their
ozazones which melt at about 156-160° C. They reduce Fehling’s solu-
tion, but do not ferment with yeast. The tests are described on page 1075.

Glycuronic acid. This acid, see page 1075, occurs in the urine in small
amounts in the paired or conjugated form. It is levo-rotatory in the con-
jugated, but dextro-rotatory in the free state. It is evidently a normal
intermediary product of metabolism, but unless it is united with some-
thing else it is burned in the body. The ingestion of various drugs
increases the quantity in the urine. Thus camphor, chloral hydrate,
many aromatic alcohols or phenols, and morphine appear in the urine
conjugated with this acid. They are reducing substances and may be
confused with dextrose or the pentoses. They may be distinguished
from dextrose in that the latter is dextro-rotatory, whereas the paired
glycuronic acids are levo-rotatory. The free glycuronic acid gives the
orcine test and phloroglucine tests like the pentoses, but the conjugated
acid does not give the orcine test. It may be distinguished from the
pentoses best by the bromphenylhydrazine compound. This when dis-
solved in a mixture of alcohol and pyridine is very strongly levo-rotatory
and may be distinguished in this way.

The origin of the glycuronic acid is still uncertain. It is clear from
the fact that the aldehyde group is not oxidized that probably the oxida-
tion of the glucose to the acid took place when the glucose was in a gluco-
side union, so that the aldehyde group was protected. After oxidation
the acid was split off by hydrolysis.

Acetone and diacetic acid.—These substances appear in the urine
together with hydroxybutyric acid in severe diabetes mellitus, or after
the prolonged administration of phlorizin to dogs, or during various
diseases when there is a deficient nourishment of the body.

CH, CH, CH,

| |
HCOH -— do == 0

bn — bs,

cooH boon co

Bllydroxybutyriec Acetoacetic Acetone +
acid. acid. Carbon dioxide.
760 PHYSIOLOGICAL CHEMISTRY

‘The amount of these bodies in the urine may be as large as 250
grams a day. They produce a veritable acidosis in severe forms of
diabetes.

The acetone is formed from diacetic acid by splitting off of carbon
dioxide. Usually very little of this transformation occurs, but in dia-
betes a considerable amount of dissociation of this sort takes place so
that the urine, may have the odor of acetone. Acetone, or some simi-
lar smelling substance, is in the urine and excretions in quantities in
the disease of milk sickness, contracted from milk-sick cattle. There
do not appear to have been any determinations of the amount of
acetone given off in this disease.

The origin of the acetoacetic acid is in part from the fats and in
part from the proteins. Several of the amino-acids, as for example
leucine, tyrosine and histidine, give rise to acetoacetic acid when per-
fused through a dog’s liver or when administered to dogs. This origin
of acetoacetic acid is discussed on page 815. A more important source
of acetoacetic acid is from the fats. By the oxidation of butyric acid,
acetoacetic acid is formed in the body. The administration of fats con-
taining butyric acid particularly increases the secretion of this substance
in diabetics; but acetoacetic acid is also formed as the last term of the
oxidation of the other fats, since it is probable that in all of them the
fatty acids are oxidized in the @ carbon, two carbon atoms at a time,
leaving at last butyric acid, which then goes over into acetoacetic acid.
It appears either that the power to oxidize this substance is reduced
in diabetics or else that the amount formed is increased beyond the
power of the body to destroy it. It will be recalled that in diabetics
a large proportion of the energy requirement of the body must be cov-
ered either from fats or from proteins, both of which lead to this
substance.

The source of the oxybutyric acid is not now doubtful. It is formed
by the reduction of the acetoacetic acid. It was for a time thought that
the oxybutyric acid was formed first and that the acetoacetic acid was
derived from this. But it is impossible to incfease the excretion of
acetoacetie acid by giving oxybutyric acid. On the other hand, the
excretion of oxybutyric acid is increased by giving acetoacetic acid,
from which it is inferred that the ketonic acid is first produced and the
alcoholic acid by reduction from this.

It is probable that in the normal body, and perhaps in the diabetic
as well, the oxidation usually goes to the next step in the oxidation of
the acetoacetic acid to two molecules of acetic acid, both of which are
then burned to carbonic acid. This decomposition appears to fail
largely, or completely, in diabetics.
THE EXCRETIONS OF THE BODY 762

The methods for the detection and the quantitative estimation of
these acetone bodies are given on pages 1073 and 1117.

The appearance of these substances in the urine means always that
an acidosis is taking place. They accumulate in the blood. They com-
bine with the alkali of the blood, thus setting free carbon dioxide and
greatly diminishing the amount of this substance in the blood. The
result of this is that the elimination of carbon dioxide is interfered
with. In such cases the administration of sodium carbonate or bicar-
bonate is often very beneficial.

Metabolism of various substances not foods.—A great deal of
information concerning the probable course of transformations of the
foods, and concerning the chemical powers of the body, has been obtained
by giving to animals substances of known chemical composition and
then by an examination of the urine determining whether they have been
affected at all in their passage through the body, and if they have been
affected what the body has been able to do to them. A very large
number of experiments of this kind have been tried and it is not pos-
sible in a book of this character to deal with all of them. Nor indeed
would it be desirable, since it would burden the mind to no good end.
But it is worth while to consider the general principles which have
been worked out by experiments of this nature. Many different ani-
mals have been used for these purposes, often the substances have been
ingested by the experimenters themselves when it is certain, or fairly
certain, that they are not toxic, but as a rule the experiments have been
tried on dogs, cats or rabbits. The chemical powers of different kinds
of animals of course vary, else there would not be various species of
animals.

A great many substances which are not foods, and many of them
substances which the organism has had no experience in handling
before, are burned wholly or partially, or otherwise affected by the
cells of the body. This fact is of itself very interesting, for it indi-
cates very strongly that at least one way of burning substances is while
they are in solution in the water of the protoplasm and not when they
are in the living protoplast itself, if there be such a thing. It is hardly
likely that such materials as brombenzene are first built up into a living
molecule before being oxidized. It is far more probable that they are, as
it were, burned in the liquid of the cell while in solution there. This
point, it will be recognized, is a matter of very great interest from a
theoretical point of view, and we shall have occasion to come back to
it in considering the combustion of the excess protein and amino-acids.
It is in general harmony with the view that there are two kinds of com-
bustion going on in a living cell: first, the oxidation of the living matter
itself ; and, second, the oxidation of substances not strictly a part of the
762 PHYSIOLOGICAL CHEMISTRY

living matter, but dissolved in the water which penetrates it. In most,
if not all, oxidations where there is a spontaneous oxidation there is,
as has been pointed out already in discussing the physical chemistry
of oxidation, a formation of hydrogen peroxide accompanying the
main oxidation. Now it is a peculiarity of many of the oxidations of
living matter that they may be closely simulated outside the cell by
oxidation with hydrogen peroxide at ordinary temperatures. This point
has been particularly established by Dakin, who has made many beau.
tiful discoveries concerning the nature of the oxidations in the body
by following out this idea. Hydrogen peroxide is probably formed in
the course of the vital reactions or oxidations. There is in practically
all cells catalase for the purpose of destroying the peroxide. It would
be strange if this ferment was so generally present and if it had nothing
ta do. It is probable, then, that there is produced alongside of the vital
oxidation, or respiration, a secondary oxidation of a purely chemical
kind of the materials in solution in the water of the protoplasm by the
hydrogen peroxide formed. It is possibly this oxidation which gets
hold of the substances as they enter the cells and partially or wholly
oxidizes them. This oxidation is not at all specific and it may happen,
then, that the cell has, in general, powers of oxidizing things it has never
met with before. There is, however, another oxidation which is specific, ©
as has been pointed out.

Substances which appear im the urine paired with glycuronic acid.
One of the first general principles which was found was that the sub-
stance ingested, after undergoing a partial oxidation so that it obtained
a hydroxyl group at some point, combined with glycuronic acid in an
ester union and was excreted in the urine as the glycuronic acid ester.
A very large number of different kinds of substances are thus excreted.
Not always does all of the compound ingested reappear. Generally some,
or most of it, is destroyed beyond recognition, but some of it by uniting
with glycuronic acid becomes so stable that it is no longer oxidized or
otherwise metabolized and it appears in the urine united with it. Just
where it finds the glycuronic acid, in what organ, whether the liver or
the muscles or somewhere else, is not definitely known; but it is
probably one of these two organs, for it is probably an organ which
has a supply of carbohydrate on hand from which the glycuronic acid
can be formed. Among the substances which thus appear in the urine
conjugated with glycuronic acid are a great number of aromatic sub-
stances which contain the phenol group, such as phenol, cresol, camphor,
cyclic terpenes such as borneol, menthol, thymol, naphthalene, antipy-
rine, oil of turpentine, oxyquinolines. orthonitrotoluene and many other
substances of this class. In addition many aliphatic alcohols also com-
Line with glycuronic acid and appear in the urine in that form. Small
THE EXCRETIONS OF THE BODY 763

quantities of the glycuronic ester appear after the ingestion of isopropyl
aleohol, amyl alcohol and many ketones and aldehydes which are
probably first reduced to aleohols in the body. Chloral hydrate ap-
pears in the urine, after its ingestion, as trichlorethylglycuronie acid,
C,Cl,H,.C,H,O,.

Substances which appear in the urine paired with glycine. Glycocoll,
or glycine, is another substance which is very often united with ingested
substances and when so united protects them from further decomposi-
tion. Hippuric acid is a glycine conjugate, but there are many others.
Thus in the bile we have glycocholie acid, a conjugate of cholic acid
and glycine. The body is apparently able to form very large amounts
of glycine and thus to protect itself against many different foreign
substances which by their decomposition might give rise to waste or
active products different from those in whose presence the protoplasm
is accustomed to work. The aromatic substances which by oxidation
give rise to benzoic acid in the body appear in the urine, in part, as
hippuric acid, although they are in part often oxidized further. In the
table on page 814 it will be seen that phenyl-amino-acetic acid, pheny]-
propionic, phenylcinnamic acids, phenylserine, phenylglyceric and
phenyl-b-hydroxypropionic acids undergo oxidation in the body to ben-
zoic acid and leave the body in part as hippurie acid, being paired with
glycine. Phenylethyl alcohol and phenylacetaldehyde are oxidized to
phenylacetic acid and paired with glycine to form phenaceturic acid in
dogs but not in man. In man they are paired with glutamine and appear
as phenylacetic glutamine. In small part, too, they pair with glycuronic
acid. Xylene is oxidized to toluic acid, C,H,(CH,).COOH, and paired
with glycine is excreted as toluric acid; mesitylene, C,H,(CH,),, is oxi
dized to mesitylenic acid, C,H,(CH,),.COOH, and after pairing with gly-
cocoll is excreted as mesitylurie acid, C,H,(CH,),.CO.NH.CH,.COOH;
eymene, (CH,),CH.C,H,.CH,, is oxidized into cumie acid, p-isopropyl
benzoic acid, and excreted as the glycocoll conjugate, cuminuric acid,
(CH,),.CH.C,H,:CO.NH.CH,.COOH.

Substances which appear in the urine conjugated with sulphuric
acid. We have already considered the principal substances of this
group. They are for the- most part aromatic alcohols, such as phenol,
indoxyl, seatoxyl, pyrocatechol and the substituted members of the
group. The formation of these conjugates is still essentially unexplained.
It is not impossible that they may pair with taurine and the organic
part of the molecule be split off. They may, however, be formed from
sulphuric acid itself. The amount is in any case not very large.

Substances which are paired with ornithine. In the bird’s body there
does not appear to be on hand a stock of glycine for pairing purposes.
Substances which in the mammals leave the body paired with glycocoll
appear in birds’ urine paired with ornithine to make ornithuric acid.
764 PHYSIOLOGICAL CHEMISTRY

This is not the only difference between the. birds’ metabolism and that
of mammals. Nitriles which leave the mammal’s body paired with
sulphur, as sulphocyanate, do not appear in this form in birds’ excre-
tions. Ornithine is diamino-valerianic acid, NH,.CH,.CH,.CH,.CHNH,.
COOH.

Uramido acids. Attention has already been called to the fact that
some substances leave the body conjugated with carbamic acid,
NH,.CO.OH. The formation of substances of this sort indicates that
Siegfried’s carbamic reaction occurs in living matter. Perhaps there
first occurs a union of carbonic acid with the amino group, followed by
its subsequent union with ammonia through a second hydroxyl of the
CO,. Thus in rabbits m-amino-benzoic acid appears in part in the urine
as uramino-benzoie acid, NH,.CO.NH.C,H,.COOH. Reactions similar
to these may be at the bottom of the synthesis of uric acid, or of allan-
toine. Uramido acids may undergo dehydration and appear in the urine
as substituted hydantoines. Hydantoine is

NH—CO

at

Hydantoine.

Nitriles and cyanides. These appear in the urine of mammals as
the sulphocyanates, having somewhere in the body picked up an unoxi-
dized sulphur atom. In birds this union does not occur, or if it does
the substances do not appear in the urine in this form. It has not been
determined, so far as I can find, what becomes in birds’ bodies of in-
gested cyanides and nitriles.

Processes of reduction in the body. Whenever they enter protoplasm
substances are exposed both to reductions and oxidations. Probably the
oxidations occur chiefly in the part of the cell where the oxygen is most
plentiful: namely, that near the blood vessel. It probably happens,
therefore, that the first exposure is to strong oxidation, but on pene-
trating into the interior of cells where processes of strong reduction
occur it is not surprising that many substances are reduced as well
as oxidized. This happens, for example, to the foods, since from the
oxidized carbohydrates the reduced fats are formed. But many sub-
stances besides the carbohydrates are reduced in the body. Thus alde-
hydes and ketones are not infrequently reduced in part to alcohols,
while a part is oxidized to acids. This happens, for example, to
acetoacetic acid, which is reduced in part to #-hydroxybutyric acid.
m-Nitrobenzaldehyde is in part carried over into m-acetylaminobenzoic
acid. This is a reaction which involves an acetylation which is discussed
later. (See page 824.)

Methylation and demethylation. Some substances such as pyridine
THE EXCRETIONS OF THE BODY 765

and tellurium compounds are methylated in passing through the body,
and some other substances lose their methyl groups perhaps by oxida-
tion. The matter of methylation has been considered already under
creatinine. The methylated xanthines, such as caffeine and theobro-
mine, are in large measure demethylated somewhere in the body, since
they appear in the urine in small part as the dimethyl or monomethyl]
purines.

Conclusion. The general conclusion from this work, besides the
establishment of the general character of the decompositions of various
compounds, both foods and non-foods, is that in their passage into the
various cells of the body the ingested molecules of all kinds come into
contact with a great variety of other molecules. Some of these molecules
are of such a character that union takes place. The subsequent fate
of the union depends upon its stability. If it is more easily oxidized than
the substance which found entrance to the cell, decomposition and oxi-
dation occur and we say that that particular cell contains an oxidase
which is capable of hastening the oxidation of the ingested matter; if,
on the other hand, the union is less easily oxidized, or otherwise decom-
posed, than the original substance, the compound thus formed takes
its exit from the cell and eventually appears in the urine in the form
of a paired substance. In such case we say that conjugation, or pairing,
has preserved the substance and perhaps protected the body from the
action of a toxic substance. For every substance which is thus ren-
dered inert by the compound being more stable it is possible, however,
that there are other substances which by union are rendered less stable
and made more toxic. Thus some nitriles, such as benzonitrile, may be
rendered far more toxic by partial oxidation, for the oxidized nitriles
set free hydrocyanic acid more easily.

Urinary pigments.—Normal human urine has a yellow color, which
may deepen on exposure to air and light. This yellow color is due to
certain pigments. The first of these to be discovered and studied was
urochrome, which is the principal pigment of the urine. It is precipi-
tated from the urine by phosphotungstic acid or basic lead acetate. It
was discovered and named by Thudichum. He has described its proper-
ties, but its composition is still unknown. It gives rise according to him
to various decomposition products called uropittine, omicholine, etc.
This pigment is badly in need of further investigation. Thudichum’s
work on the pigments of the urine was neglected with the same com-
pleteness as his work on the brain, his work on the bile pigments and
his work on the blood pigments. The whole subject of the relation of
the urinary to the bile pigments and the nature of both should be eare-
fully re-examined with the exact work of Thudichum made the point of
departure. There are many points in the chemistry of these compounds
which cannot be reconciled with the prevailing opinions of their nature.
766 PHYSIOLOGICAL CHEMISTRY

The other pigment of the urine is urobilinogen. Thudichum main-
tained that this substance was but an impure mixture of decomposition
products of his urochrome and gave excellent reasons to support that
view. It is still badly characterized and the analyses do not agree with
the calculations. Urine when made strongly acid becomes a dark red
color, due to the development of a pigment called uroerythrine. This red
color is probably derived from the scatole of the urine. The composi-
tion of urobilinogen and urochrome is still uncertain. They are prob-
ably derived from the bile pigment, bilirubin, and urobilin appears to
be identical with stercobilin, a reduced bilirubin found in the feces. It
is quite possible that this reduction of bilirubin to urobilin can take
place elsewhere than in the alimentary canal, since urobilin is found in
the bile and blood. Both urochrome and urobilin yield pyrrol deriva-
tives. They are probably derived in the long run from the hematin of
the blood as discussed under the bile. As the chemistry and origin of
these compounds is still so uncertain, further discussion may be post-
poned until methods of purifying them, particularly the urochrome, shall
be perfected. Clinically dimethylamino benzaldehyde (red reaction,
Ehrlich’s) is used to detect urobilin in the urine, feces and bile.

REFERENCES. Ursa.

1. von Schroeder: Ueber die Bildungsstitte des Harnstoffes. Arch. f. expt. Path. u.
Pharm., 15, p. 364, 1882.

2. Gottlieb: Ueber die quantitative Bestimmung des Harnstoffes in den Geweben
und den Harnstoffgehalt der Leber. Arch. expt. Path. u. Pharm., 42, 1899.
p. 239,

3, Baglioni et Frederico: L’azione fisiologica dell urea sul cuore dei vertebrate.
Zeit. f. allg. Physiol., 6. p. 482, 1907.

4. Baglioni: Der Einfluss der chemischen Lebensbedingungen auf die Tiitigkeit des
Selachierherzen. Zeit. f. allg. Physiol., 6. pp. 71-91, 1907; 6. pp. 213-216.

5. Schultzen and Nencki: Die Vorstufen des Harnstoffes im tierischen Organismus.
Zeits. f. Biol., 8, p. 124, 1872.

6. Macleod and Haskins: Contributions to our knowledge of the chemistry of
carbamates. Jour. Biol. Chem., 1, p. 319, 1905-6.

7. v, Knierem: Beitriige zur Kenntnis der Bildung des Harnstoffes im tierischen
Organismus. Zeit. Biol.. 10, p. 263. 1874.

8. Salaskin: Ueber die Bildung von Harnstoff in der Leber der Siiugethiere aus
Amidosiiuren der Fettreihe. Zeits. physiol. Chem., 25, p. 128; 25, p. 449,
1898.

9. Richet: Compt. rend. Acad., 118, 1894; Compt. rend. Soe. Biol., 49.

10. Stolte: Ueber das Schicksal der Monoaminosiiuren im Tierkiérper nach Einfthr-
ung in die Blutbahn, Hofmeister’s Beitriige, 5, p. 15, 1903.

11. Nencki, Pawlow and Zaleski: Sur la richesse du sang et des organes en am-
moniaque et sur la formation de l’urée chez les mammiféres. Arch. d. sci.
biol. de St. Petersburg, 4, p. 197, 1896.

12. Kossel and Dakin: Ueber die Arginase. Zeit. physiol. Chem., 41, p. 321; 42,
p. 181, 1904.
13.

14,

16.
16.

17.

18.

19.

oe

10.
11.

12.

13.

14.

15.

16.
17.

THE EXCRETIONS OF THE BODY 167

v. Jaksch: Weitere Mittheilungen iiber dic Vertheilung der stickslulfhaltigen
Substanzen im Harne des kranken Menschen.. Zeit. klin. Med., 50, p. 167,
1903.

Folin: Laws governing the chemical composition of the urine. Amer. Jour.
Physiol., 13, p. 66, 1905.

Osterberg and Wolff: Day and night urines. Jour. Biol. Chem., 3, p. 165, 1907.

v. Schroeder: Ueber die Harnstoffbildung der Haifische. Zeit. physiol. Chem.,
14, p. 576, 1890.

Nencki and Hahn: La fistule d’Eck de la veine cave inférieure et de la veine
porte, etc. Arch. sci. Biol. de St. Petersburg, 1, p. 447, 1892. Massen et
Pawlow: Ibid., p. 401.

Abel and Muirhead: Uebér das Vorkommen der Carbaminsiure im Menschen-
und Hundeharn nach reichlichem Genuss von Kalkhydrat. Arch. expt. Path.
u. Pharm., 32, p. 467, 1893; 31, p. 15.

Salaskin u. Zaleski: Ueber den Einfluss der Leberexstirpation auf den Stoff-
wechsel bei Hunden. Zeits. physiol. Chem., 29, p. 517, 1900.

Uric Acb.

Dakin: Oxidations and reductions in the animal body. Monographs on Bio-
chemistry. Longmans, Green and Co., London. :

Jones: Nucleic acids. Their chemical properties and physiological conduct.
Monographs on Biochemistry. London. s

lzar; Beitriige z. Kenntnis der Harnsiiurezerstérung u. Bildung. Zeit. physiol.
Chem., 73, p. 319, 1911.

Horbaczewski: Monatshefte f. Chemie, 10 p. 614, 1889; 12, p. 221, 1891.

Hopkins and Hope: Uric acid and diet. Jour. Physiol., 23, p. 271, 1898-9.

Jerome: The formation of uric acid in man and the influence vf dict on its
daily output. Jour. Physiol., 22, p. 146, 1897.

Minkowski: Untersuchungen zur Physiologie und Pathologie der Harnsiure bei
Siugetieren. Archiv f. expt. Path. u. Pharm., 41, p. 375, 1898.

Loewi: Beitriige zur Kenntnis der Nucleinstoffwechsel. Archiv f. expt. Path. u.
Pharm., 44, p. 5, 1900; 45, p. 157, 1901.

Smetanka: Zur Herkunft der Harnséure beim Menschen. Archiv f. d. ges.
Physiol., 149, p. 275, 1912-13.

Wendt: Skan. Archiv Physiol., 17, p. 231, 1905.

Arnold and Larrabee: .The purin content of common articles of food. Jour.
Amer. Med. Assn., 58, p. 18, 1912.

Mares: Sind die endogenen Purinkérper Produkte der Titigkeit der Ver-
dauungsdriisen? Eine Antwort auf die Frage Sivens. Archiv f. d. ges.
Physiol., 149, p. 275, 1912-13.

Siven: Ueber den Purinstoffwechsel des Menschen. II. Sind die endogenen
Purinkérper Produkte der Verdauungsdriisen? Archiv ges. Physiol., 146, pp.
499-516, 1912.

Mares: Zur Theorie der Harnsiiure Bildung im Siugethierorganismus. Sitz. d.
Kais. Akad. Wien, p. 101, 1892.

Mares: Der physiologische Protoplasma stoffwechsel u. die Purinbildung.
Archiv ges. Physiol., 134, pp. 59-102, 1910.

Brunton and Bokerham: Zent. f. Physiol.

Ascoli and Izar: Quantitative Riickbildung zugesetzter Harnsiure in Leberex-
trakten nach vorausgegangener Zerstérung. Zeit. physiol. Chem., 58, p. 529,
1909.
768

18.

19.

20.

21.

1 i]
nh

23.

24,

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

PHYSIOLOGICAL CHEMISTRY

Oroftan: Synopsis of experiments on the transformation of circulating urie
acid in the organism of man and animals. Medical Record, N. Y., p. 6,
1903.

Oroftan: Zur Kenntnis der Harnsiure Umwexdlung im Tier u. Menschen-
kérper. Archiv ges. Physiol., 121, p. 377, 1907-8.

Milroy and Malcolm: Metabolism of the nucleins. Jour. Physiol., 25, p. 105,
1899.

Amberg and Jones: Ueber die bei der Spaltung der Nucleine in Betracht kom-
menden Fermente mit besonderer Beriicksichtigung der Bildung vom Hypo-
xanthin in der Abwesenheit von Adenase. Zeit. physiol. Chem., 73, p. 407,
1911.

Jones: Ueber die Beziehung der aus wiisserigen Organextrakten gewonnenen
Nucleinfermente zu den physiologischen Vorgingen im lebenden Organismus.
Zeit. physiol. Chem., 65, p. 383, 1910. (References to earlier literature by
same author. For general summary of work on this subject sce Jones,
Nucleic acids.) ,

Voegtlin and Jones: Ueber Adenase u. ihre Beziehung zu der Entstehung von
Hypoxanthin im Organismus. Zeit. physiol. Chem., 66, 1910, p. 250.

Taylor and Rose: Uricolysis in the human subject. Jour. Biol. Chem., 14, p.
419, 1913.

Schittenhelm: Ueber den Nucleinstoffwechsel des Schweines. Zeit. physiol.
Chem., 66, p. 53, 1910. (Earlier papers cited here.)

Tollens: Gicht und Schrumpfniere. Ausscheidung von Harnsdiure u. Purin-
basen im Urine u. im Kote des Gichtkranken bei Nierenstérungen. Zeits.
physiol. Chem., 53, p. 164, 1907. (Historical account.)

Van Noorden: Handbuch d. Pathol. d. Stoffwechsels. II. Die Gicht.

Block: Die Herkunft der Harnsiiure im Blute bei Gicht. Zeits. physiol. Chem.,
51, p. 472, 1907.

Brugsch: Zur Stoffwechselpathologie der Gicht. Zeits. f. expt. Path. u.
Therapie 6, 1909, p. 278.

Miller and Jones: Ueber die Fermente des Nucleinstoffwechsels bei der Gicht.
Zeit. physiol. Chem., 61, p. 395, 1909.

Biberfeld u. Schmidt: Ueber den Resorptionsweg der Purinkérper. Zeit. physiol.
Chem., 60, p. 292, 1910.

Mendell and Mitchell: The enzymes involved in purine metabolism in the em-
bryo. Amer. Jour. Physiol., 20, p. 97, 1907.

Preti: Beitrage zur Kenntnis der Harnsiiurebildung. IV. Zeit. physiol. Chem.,
62, p. 354, 1909.

Schittenhelm: Ueber die Umsetzung verfiitteter Nucleinsiure beim Hunde unter
normalen u. pathol. Bedingungen. Zeit. physiol Chem., 62, p. 80, 1909.

Bezzola, Izar and Preti: Wiederbildung zerstérter Harnsiiure in der kiinstlich
durchbluteten Leber. Zeit. physiol. Chem., 62, p. 229, 1909.

Ascoli and Izar: Harnsiiure Bildung in Leberextrakten nach Zusatz von Dialur-
siure u. Harnstoff. Zeits. physiol. Chem., 62, p. 347, 1909.

Frank and Schittenhelm: Ueber die Umsetzung verfiitterten Nucleinsiiure
beim normalen Menschen. Zeit. physiol. Chem., 63, p. 269, 1909.

Wiechowski: Ueber die Zersetzlichkeit der Harnstiure im menschlichen Or-
ganismus. Archiv expt. Pathol. u. Pharm., 60, p. 185, 1909.

Schittenhelm u. Weiner: Ueber das Vorkommen u. die Bedeutung von Allan-
toin im menschlichen Urine. Zeit. physiol. Chem., 63, p. 283-88, 1910.
Wiechowski: Das Vorhandensein von Allantoin im normalen Menschenharn u.
seine Bedeutung fiir die Beurtheilung des menschen Harnsiiurestoffwechsels

Biochem. Zeits., 19, p. 368, 1909.
41.

42.

43.

44,

45.

46.

48.

49.

50.

61.

52.

53.

THE METABOLISM OF THE BODY 760

Schmid: Der Abbau methylierte Xanthine. Zeits. physiol. Chem., 67, p. 154,
1910.

Schittenhelm: Die Nucleinbasen der Feces unter dem Einfluss anhaltender
Faulniss. Zeit. physiol. Chem., 39, p. 199, 1903.

Levinthal: Zum Abbau des Xanthins u. Caffeins im Organismus des Menschen.

‘Zeit. physiol, Chem., 77, p. 259, 1912.

Kriiger u. Schittenhelm : Die Menge u. Herkunft der Purinkérper in der mensch-
lichen Feces. Zeit. physiol. Chem., 45, p. 14, 1905. Method.

Kriiger u. Schmidt: Zur Bestimmung der Harnsiure u. Purinbasen im mensch-
lichen Harn. Zeits. physiol. Chem., 45, p. 1, 1905.

Bennett: The purines of muscle. Jour. Biol. Chem., 11, p. 221, 1912.

Burian: Die Herkunft der endogenen Harnpurine bei Mensch u. Saugethier.
Zeits. physiol Chem., 43, p. 532, 1904-5.

Sachs: Weber die Nuclease. Zeits. physiol. Chem., 46, p. 337, 1905.

Kojo: Ueber Unterschiede im Harnbefunde beim Gesunden u. Carcinomatosen.
Zeits. physiol. Chem., 63. p. 416, 1911.

Ackroyd: The presence of allantoin in certain foods. Bioch. Jour., 5. p. 400,
1911.

Wiechowski: Das Schicksal intermediarer Harnsiiure beim Menschen und der
Allantoingehalt des menschlichen Harns; nebst Bemerkungen tiber Nachweis
und Zersetzlichkeit des Allantoins. Biochem. Zeits., 25, p. 431, 1910.

Jones und Rohde: The purin ferments of rats. Jour. Biol. Chem., 7, p. 23%,
1910.

Fine and Chace: The influence of salicylates upon the uric acid concentration
of the blood. J. Biol. Chem., 21, p. 371, 1915.

CREATININE AND CREATINE.

Jaffé: Untersuchungen tiber die Entstehung des Kreatins im Organismus. Zeit.
physiol. Chem., 48, p. 430, 1906. :

Dorner: Bildung von Kreatin u. Kreatinin im Organismus besonders Kaninchen.
Zeits. physiol. Chem., 52, p. 225, 1907.

Folin: Laws governing the chemical composition of the urine. Amer. Jour.
Physiol., 13, p. 66, 1905.

von Hoogenhuyze u. Verploegh: Beobachtungen iiber die Kreatininausscheidung
beim Menschen. Zeit. physiol. Chem., 46, p. 415, 1906; 57, p. 161; 59, p. 101.

Pekelharing and Harkink: The excretion of creatinin in man under the influence
of muscular tonus. Proc. Amsterdam Acad., 14, p. 310, 1910.

His: Ueber das Stoffwechselprodukt des Pyridins. Arch expt. Path. u. Pharm.,
22, p. 253, 1887.

Cohn: Ueber das Verhalten einiger Pyridin u. Naphthalinderivaten im Tierischen
Stoffwechsel. Zeit. physiol. Chem., 16, p. 112, 1894.

Hofmeister: Ueber Methylierung im Tierkérper. Arch. expt. Path. u. Pharm.,
33, p. 198, 1894.

Abderhalden, Brahm u. Schittenhelm: Vergleichende Studien tiber den Stoff-
wechsel verschiedener Tierarten. Zeit. f. physiol. Chem., 59, 1909, p. 32.
770

10.
11.
12.
13.
14.
15.

16.

17.

18.

19.

20.
21.

8.

9.

10.

11.

PHYSIOLOGICAL CHEMISTRY

Inouye: Ueber die Entstehung des Kreatins im Tierkérper. Zeit. physiol.
Chem., 81, p. 79, 1912.

Folin: Hammarsten’s Festschrift, 1906.

Mellanby: Creatine and creatinine. Jour. Physiol., 36, p. 447, 1907-8.

v. Klercker: Beitrag zur Kenntnis des Kreatins u. Kreatinins im Stoffwechsel
des Menschen. Biochem. Zeits.. 3, p. 45, 1907.

Benedict and Meyers: Determination of creatine and creatinine. Amer. Jour.
Physiol. Chem., 18, p. 397, 1907; 18, p. 377; 18, p. 408.

Cathcart: Ueber die Zusammensetzung des Hungerharns. Biochem. Zeits., 6,
p. 109, 1907.

Jaffé: Ueber den Niederschlag welchen Pikrinséure in normalen Harn erzeugt
und tiber eine neue Reaction des Kreatinins. Zeit. physiol. Chem., 20, p.
391, 1886.

Achelis : Ueber das Vorkommen von Methylguanidin im normalen Menschenharn.
Zent. Physiol., 20, p. 455, 1906.

Kutscher and Lohmann: Der Nachweis toxischer Basen in Harn. III. Zeit.
physiol. Chem., 49, p. 81, 1906.

Eingeland: Ueber den Nachweis organischer Basen im Harn. Zeit. physiol.
Chem., 57, p. 49, 1908.

Meyers and Fine: Jour. Biol. Chem., 21, 1915. Various papers.

Traut and Mellanby: Jour. Physiol., 44, p. 43, 1913.

Hrepvuric Acm.

Baumann: Zur Kenntnis der arom. Substanzen d. Tierkérper. Zeits. physiol.
Chem., 7, p. 282, 1883.

Baas: Ueber das Verhalten des Tyrosins zur Hippursiiurebildung. Zeits. physiol.
Chem., 11, p. 485, 1887.

Salkowski: Ber. deutsch. Chem. Gesell. 11.

Wiechowski: Die Gesetze der Hippursiiuresynthese. Ein Beitrag zur Frage der
Stellung des Glykokolls im Stoffwechsel. Hofmeister’s Beitriige, 7, p. 204,
1906.

Lewinski: Ueber die Grenzen der Hippursiiurebildung beim Menschen. Zugleich
ein Beitrag zur Glykokollfrage. Arch. expt. Path. u. Pharm., 58, p. 397,
1907-8.

Abderhalden, Gigon and Strauss: Studien tiber den Vorrat an einigen Amino-
siuren im Organismus bei verschiedenen Tierarten. Zeits. physiol. Chem.,
51, p. 311, 1907.

Magnus-Levy: Ueber die Neubildung von Glykokoll. Bioch. Zeits., 6, p. 523,
1907.

Schmiedeberg and Bunge: Ueber die Bildung der Hippursiiure. Arch. expt. Path.
u. Pharm., 6, p. 233, 1876.

Schmiedeberg: Ueber Oxydationen und Synthesen im Tierkérper. Arch. expt.
Path. u. Pharm. 14, p. 288, 1881.

Folin and Flanders: A new method for the determination of hippuric acid in
urine. Jour. Biol. Chem., 11, p. 257, 1912.

Kingsbury and Bell: The synthesis of hippuric acid in nephrectomized dogs.
Jour. Biol. Chem., 21, p. 297, 1915.

InpIcan,

Jolles: Ueber eine neue Methode zur quantitativen Bestimmung des Indikans im

Harne. Zeit. physiol. Chem., 94, p. 79, 1915.
CHAPTER XVIII.

THE METABOLISM OF THE BODY CONSIDERED AS A WHOLE,
CARBOHYDRATES.

In the immediately preceding chapters there have been considered the
chemistry and metabolism, so far as they are known, of the different
organs and tissues of the body. Since each of these organs is normally
working in conjunction with others, being co-ordinated either by the nerv-
ous system or the blood in the manner described, their normal metabolism
can only be studied while they are in situ. The possibility of studying
each organ separately is greatly limited by this fact. Much, however,
can be learned by studying the metabolism of the body as a whole, with-
out inquiring at first just where the various steps of that metabolism
occur. In this method of study the organs are all working in their
normal relations and at their maximum efficiency. Moreover, the funda-
mental processes of metabolism probably do not differ very widely in
different organs, although, of course, the metabolism differs in par-
ticulars in each, so that the constitution of each is specific. The general
course of the change which the carbohydrates, proteins and fats undergo
in the course of their passage through the body, from the time of their
absorption to their excretion in the form of various fragments, may now
be examined.

The metabolism of sugar—The carbohydrates are absorbed chiefly
as glucose, levulose and galactose and about 500 grams of carbohydrate
are normally consumed per day. We have now to follow, so far as
we are able, the sugar thus absorbed in its course through the body.
From the intestine it passes to the blood, where it is found in the blood
plasma in a concentration varying from .08-.15 per cent. of the whole
blood. The glucose thus present in the plasma is dialyzable and the
greater part of it is free and not united to any colloidal matter, as was
at one time suggested (McGuigan and von Hess).

In taking up this subject of the carbohydrate metabolism it is
perhaps most interesting and instructive to follow the historical method.
About the middle of the nineteenth century it was known from the work
of Lavoisier that the carbohydrates, like other foods, were burned or
oxidized in the body in large measure to carbon dioxide and water;
that. by means of this combustion heat was liberated to just the same
amount as would have been produced by burning so much sugar out-

771
172 PHYSIOLOGICAL CHEMISTRY

side the body; that of this heat part was consumed, or rendered latent,
in the evaporation of water, or in doing work, and the greater part
supplied the heat which the body was constantly giving off as if it were
a stove. But where this combustion took place, how it could take place
in an aqueous medium, and whether the sugar underwent intermediary
transformations before oxidizing, as was indicated by the appearance of
fat after carbohydrate ingestion, these were matters quite obscure.

A great step forward in this dark field was taken in 1843 by the
French physiologist, Claude Bernard, a man whose name should be
remembered for his striking discoveries, ingenious and skillful experi-
ments, his clear thoughts, lofty imagination and the beautiful, simple
and luminous style in which his books and papers were written. Since
it was impossible to follow the course of the sugar directly after its
entrance into the blood, Bernard turned his attention to those patho-
logical cases in which sugar appears in the urine. Disease tries many
experiments which we cannot as yet with our clumsy vivisectional
methods hope to imitate, and for the wise man who can read the experi-
ments aright pathology reveals many secrets of metabolism. It was
known to Bernard that at times large amounts of glucose may appear
in the urine of human beings, although it normally is present only in
amounts ot about .04 per cent. The urine is then greatly increased in
amount, it is often sweet to the taste, very light colored, having a high
specific gravity and often a sweetish or aromatic odor. Such pathological
urine excretion is called glycosuria, or glucosuria, or diabetes mellitus
(dia, through, and mel, honey; literally, honey diabetes). In severe
forms of this disease with a fatal ending in coma, 60-500 grams of
glucose might be eliminated per day. Nothing was known of the cause
of the disease so grave in its prognosis; or of the origin of the glucose
which appeared in the urine.

Bernard tried to produce the disease artificially. He found that
injury to the floor of the fourth ventricle of the medulla oblongata in
dogs and rabbits produced almost invariably a discharge of glucose
in the urine. This puncture is known as the sugar puncture. This
glucosuria was, however, temporary and never had a fatal ending. It
was found on examining the matter farther that the puncture only pro-
duced glucosuria if the animals had been fed on carbohydrate, or were
well fed. Starving animals produced little or no sugar. It did not
oceur either if the splanchnics, two sympathetic nerves supplying the
abdominal viscera, had been divided before the puncture (Eckhard).
Bernard did not succeed in producing artificially a severe glycosuria
with the accompanying symptoms of the human disease, nor were physi-
ologists more successful until about 1890, when von Mering and Min-
kowski discovered that extirpation of the pancreas would produce it.
THE METABOLISM OF THE BODY 773

Bervard attempted to find where the sugar came from which
appeared in the urine after the sugar puncture. He examined the glu-
cose content of the blood in different regions of the body. He found
that the blood of the jugular and femoral veins contained less glucose
than the corresponding arterial blood :

Ves JUBWIATIS: sscesccs se eeereahs 08% Femoral artery ............. 0.12%
Carotid artery ............ 12 Femoral vein ..............- 0.08

Evidently glucose was taken from the blood in passing through the
tissues supplied by these blood vessels. Next, by means of a sound
passed into the jugular vein, he drew a sample of blood from the right
auricle. This contained as much glucose as the arterial blood. It was
clear that somewhere in the body sugar must be added to the blood to
make good the loss of that taken out by the tissues. A sound passed
through the heart and down into the inferior vena cava to a level just
above the kidneys showed that the blood of that vein contained 0.08 per
cent. of sugar. The sound was now drawn carefully back to the level
of the opening of the hepatic vein. The blood from here contained
0.14 per cent. or more of glucose, or more than the arterial blood. Evi-
dently, then, somewhere in the portal circulation glucose was added
to the blood. An examination of the blood of the portal vein showed
generally that the portal vein blood contained less glucose than the
hepatic blood. This proved that sugar was added to-the blood during
its passage through the liver and an examination of this organ resulted in
the discovery of the following facts: If the liver was taken as rapidly as
possible after decapitation out of the body of a rabbit and, excluding the
gall bladder, was thrown into boiling water and cut up in it while boiling,
only very small amounts of a reducing sugar could be extracted from
it. Evidently the liver did not contain more sugar than might be
uttributed to the blood in it. But there went into solution in the water
a substance which made an opalescent solution and which when boiled
with acid or acted upon by saliva or malt diastase quickly set free a
reducing sugar. Moreover, if the liver was left in the body after death,
then it was subsequently found to contain considerable quantities of
glucose and maltose. The living active liver, then, contained little or
no glucose, but it contained a considerable quantity of a material from
which glucose could be and was made, and this substance was called by
Bernard for this reason glycogen, the glucose-maker.

Thus was made one of the first or fundamental discoveries in our
knowledge of sugar metabolism and it placed the liver in an entirely
new light. It had before that been known only as a digestive gland;
it was now seen that it piayed another and possibly far more important
réle in the carbohydrate metabolism of the body and was one of its
most important chemical factories.
774 PHYSIOLOGICAL CHEMISTRY

Further study by Bernard showed that the glycogen which he had
discovered was very generally found in the animal kingdom, where it
played the réle that starch plays in the plant world. He showed that
it was present in muscle cells, in pus cells, in embryonic tissues, in fly
larve, in earthworms and in other invertebrates as well as vertebrates,
and it was present, though in varying amounts, in the livers of nearly
all vertebrates. It became at once one of the fundamental constituents
of animal cells.

Glycogen. (C,H,,0;),. The glycogen thus discovered is a col-
loidal polysaccharide which is digested by ptyalin into maltose and by
the liver tissue and muscle tissue into dextrose and maltose. When
inverted by acid it yields dextrose. It is entirely resistant to 30 per
cent. KOH and its quantitative determination depends upon this fact.
It is prepared by boiling the tissue with 30 per cent. KOH until it dis-
solves; filtering through asbestos or glass wool and precipitating with
alcohol. All protein and reducing sugars are destroyed by the strong
alkali. It is snow-white in color, soluble in water to an opalescent solu-
tion. It is dextro-rotatory: (@)p=+196.63°. It does not reduce Feh-
ling’s solution; with iodine-potassium iodide it gives in the cold, if salt
is present, a pert-wine color and this is one of the means of its detection.
By this reaction it may be detected in sections, and it can be seen in
the liver tissue often deposited in radiating masses or granules about
the nucleus. Some of the mitochondria described by histologists are
possibly glycogen. It is, as will be seen from its resistance to alkali,
an inert substance. It is colloidal and it is plainly a reserve material
of the cell.

Origin of glycogen. The question of the origin of the glycogen of
the liver was at once attacked. Bernard found that it not only ap-
peared in large quantities when carbohydrate food was taken and hence
was made presumably from the carbohydrate of the food, but it ap-
peared also when a purely meat diet was consumed. Putrefying muscle
contains almost no carbohydrate, but the larve of flies which have fed
upon this muscle and on nothing else contain large amounts of glycogen
in their bodies. Evidently they must be able to make glycogen from
protein and since dogs fed exclusively on lean meat also have livers
containing large amounts of glycogen they too must be able to make
carbohydrate from protein. A second fundamental discovery in the
metabolism of the sugars was thus made, namely, that animals can
make carbohydrate from protein. This showed that animals had powers
of synthesis which it had been believed were peculiar to plants.

To try these experiments properly it is necessary to get the liver
practically glycogen free at the start of the feeding. This can, perhaps,
be done best by Lusk’s method of the injection of phlorhizin of which
THE METABOLISM OF THE BODY 175

we shall presently speak, but before the properties of this drug were
known recourse was had to other methods. Animals were made io
fast for considerable periods, forced to exercise by being put in a re-
volving cage, exposed to cold, which causes consumption of glucose, and
finally given small doses of strychnine to produce mild tetanus. By
these means the liver may be made almost glycogen or sugar-free and the
amount in the muscles is reduced to a minimum.

Origin of glycogen from protein and carbohydrates. That the glyco-
gen is formed from protein and carbohydrate food is illustrated by the
following experiments by Kiilz and C. Voit. Kiilz fed hens casein,
serum albumin and egg albumin, control hens being sacrificed at the
start of the feeding to determine the amount of glycogen in the liver.

 

 

 

 

Glycogen content of the liver—grms.

Substance fed Before | After feeding
Cadein suntv es vans se ounexeeeea eeeaeae se 1.013 (1.85
Berti AIDWMIN ok. ose oo one bs eeceees bees 0.917 1.56
Egg albumin ..............,0. 000s eee, . 1.016 1.78

 

C. Voit starved hens and rabbits for five days which reduced the
glycogen to a minimum. He then fed various carbohydrates and killed
the animals after six hours.

Sugar fed - Animal. Amount fed Glycogen in liver
Coek 50 grams 15.34%
1. Dextrose ....} Rabbit 80° 16.88” (9.269 grams in liver;
8.972 grams in muscle)
2. Sucrose ..... Hen 100 c.c. 50% solution. 3.75% (1.215 grams)
8. Levulose .... Cock 54.89 grams 10.50% (3.99 grs.; 3.562 grs. in

rest of body)

Rabbit 130 ¢.c. 54.8 grams 9.08% (5.26 grams)
4. Maltose ..... } tek 60 grams 10.43 (4 grams)
Rabbit 60 “ 9.822
Cock 55 grams 1.29% (0.6716 gr.)
5. Galactoze .... | Rabbit 68.2 grams 13 (0.871 gr.)
Hen 16 grams 0.19%
6. Lactose ...... Rabbit 16 ° “ 1.70
f 50 grams 0.69 before; 3.61% after.

From these results it appears that feeding glucose, levulose, sucrose
or maltose leads to a loading of the liver with glycogen. Lactose and
galactose are utilized very little, if at all, by rabbits or hens. They are,
however, utilized by dogs, human beings and other animals.

As regards the origin from carbohydrates Voit (Otto), after four
days’ fasting, gave a rabbit 80 grams of glucose in solid form and killed
it after 8% hours. The liver weighed 55 grams; he obtained 9.269 grams
of glycogen or 16.85 per cent. In the rest of the body there were 8.972
grams of glycogen. The urine contained during this period 0.816 grams
-of nitrogen equivalent to 5.10 grams of albumin which at a maximum

‘
776 PHYSIOLOGICAL CHEMISTRY

could only have yielded 2.42 grams of glycogen. There is, then, no doubt
of the origin of glycogen from carbohydrate of the food.

Origin of glycogen from fat. While glycogen can thus be readily
formed from protein and other carbohydrate very little is formed from
fat. It is found by feeding glycerol to animals rendered artificially
diabetic, or to diabetic human beings, that the glycerol of the fat mole-
cule, if it get free, may be turned over.into carbohydrate. But if fats
are fed to diabetic animals or men they do not materially change the
excretion of dextrose. It appears from this that the fatty acids do
not go over into carbohydrate in the body, although the reverse formation,
the change of carbohydrate to fat, goes with the greatest ease, and may
very possibly be a part of every anaérobic respiration. It seems prob-
able that the dextrose molecule in its decomposition may furnish oxygen
to the living matter and be itself reduced to fat or other less oxidized
substances. In this way in the absence of sufficient oxygen it aids in the
respiration of the cell and it is itself stored as fat to be used when
the supply of oxygen is more plentiful. The oxidation of the fatty
acids has already been briefly discussed on page 75, and it will be
recalled that they split into acetic acid and the fatty acid poorer in
carbon by two carbon atoms. Acetic acid does not go over into glycogen
but is oxidized to carbonic acid.

Origin of glycogen from other substances. Further study has shown
that the liver can convert many other substances into glycogen, some
of them very simple substances. Thus it can convert lactic acid, pyruvic
acid, glycerol, propyl alcohol, glycerol aldehyde, aspartic acid, alanine,
and some other amino-acids to carbohydrate. The power of the body to
convert other substances into carbohydrate is most conveniently studied
by Lusk’s method of using phlorhizinized dogs.

Fasting dogs, when injected subcutaneously with 1 gram of finely
powdered phlorhizin suspended in 7 e.c. olive oil develop a severe
glycosuria lasting for several days. The ratio of dextrose to nitrogen,
D:N, in the urine after a day or so reaches the value of 3.5-3.7. If now
these dogs are fed or injected with substances which are converted into
glucose in the body, the extra glucose thus formed is excreted in the
urine and the amount may be determined. In this way it may be
found whether animals have the power of converting any given substance
into glucose. The following table has been compiled from Lusk’s work;
the table showing that the first four amino-acids are clearly capable
of being turned either wholly or in part into glucose in the dog’s body.
The amount of these acids in meat will account for about 50 per cent.
of the glucose which is formed when meat is fed phlorhizinized dogs.
Probably serine, arginine, lysine, and valine also go into glucose in whole
or in part. Leucine probably does not form glucose. Since these amino-
THE METABOLISM OF THE BODY 777

acids thus go into glucose in the phlorhizinized dogs it is probable that
they have this power in the normal animal and thus contribute to the
formation of glycogen.

Theoretical if all Per cent. of
Extra glucose amino-acid con- amino-acid

| Amount— excreted— verted into glu- made into
Amino-acid fed grams grams cose—grams glucose
Glycecoll  vsckwaane can waas 20 14.8-15.8 16 100
Alanine sia. ses.ew cnx 20 18.76 20.2 100
Aspartic acid ............. 20 13.4-14.9 13.52 100
(If 3 C atoms into glucose.)
Glutamic acid ............ 20 13.16 12.24 100

(If 3 C atoms to glucose. )

PyTrosine: cus /aicaleesiteseen 0 0

The place of the transformation of amino-acids to glucose is cer-
tainly in part in the liver. Glucose and glycogen can be made by the
liver from protein. It will be recalled when discussing digestion that
some deaminization of the amino-acids took place in the canal wall or
before absorption. The amino-acids are carried also as such to the liver
and this organ has the power of deaminizing some of them. This
process has been studied by Knoop and Neuberg. Alanine, for example,
when perfused through the liver loses its amino group and passes into
pyruvic acid; glycocoll goes into glyoxylic acid: °

CH,.CHNH,.COOH + 0 —~ CH,.CO.COOH + NH,

Alanine. | Pyruvic acid.
CH,.NH,.COOH + O —~+ CHO.COOH +NH,
Glycocoll. Glyoxylic acid.

Not all of the amino-acids which reach the liver are thus converted
into glucose. Some, and probably a large part of them, escape the
gauntlet and circulating in the blood reach and nourish the tissues of
the body. The power of oxidizing them in the way just indicated is
probably an attribute of all living matter, although possibly especially
developed in the liver. Folin and Denis have questioned whether the
liver really deaminizes most of the amino-acids at all. Perfusion experi-
ments appear, however, to be positive that some deaminization occurs
there.

Conversion of glycogen into glucose. The circumstances and exact
control of the conversion of glycogen into glucose by the liver are still
a matter of investigation and they cannot be said to be clear in all
their details. The great importance of this subject arises in part from
the fact that the process is typical of what goes on in cells in general,
for stored foods are not uncommon. It may be said at the outset that
the interruption of the blood flow through the liver, or depriving it of
oxygen, always leads to the conversion, more or less complete, of glycogen
into sugar. The liver can only form glycogen if it be supplied with
sufficient oxygen. During digestion, when vasodilation occurs in all the
778 PHYSIOLOGICAL CHEMISTRY

intestinal organs, the liver receives blood by the portal vein which is only
slightly poorer in oxygen than arterial blood. At this time, therefore,
glycogen is formed in abundance. But during digestive rest the liver re-
ceives blood by the portal vein which is already strongly venous; and the
supply of arterial blood is relatively small by way of the hepatic artery,
so that at this period the liver must be on the verge of asphyxiation.
Under these circumstances glycogen will be converted into glucose.
Sugar puncture leads to the disappearance of glycogen from the
liver; and the conversion of glycogen into glucose can be hastened by
the stimulation of the splanchnics, or of nerves accompanying the blood
vessels into the liver. Carbon monoxide poisoning leads to the dis-
charge of the glycogen; curare injection has a similar result because,
owing to the paralysis of the skeletal muscles, the respiration is reduced
and partial asphyxia results. Chloroform and some other anesthetics
easily produce glycosuria in rabbits and in dogs and human beings, also,
if the body be chilled during the anesthesia. A very important discovery
was that the injection of adrenaline, the internal secretion of the supra-
renals, greatly increased the blood sugar, provided the liver contained
glycogen, and hence produced hyperglycemia and glycosuria. So that
the supra-renal glands were thus brought into relationship with the
glycogen conversion. The question has now arisen whether the conver-
sion of glycogen to glucose by stimulation of the splanchnics, or by
sugar puncture, is due to the direct effect of the stimulus on the cells
of the liver, or whether it is the indirect effect of stimulation of the
supra-renal capsules, since these nerves supply these organs also.
Further examination of this problem has shown that if the supra-
renals be extirpated, and rabbits will live for months without these
organs, stimulation of the splanchnics no longer causes a conversion of
glycogen into glucose. On the other hand, the amount of adrenaline dis-
charged into the blood is not sufficient in itself to cause glycogenolysis.
MacLeod is probably right in his opinion that stimulation of the
splanchnics does two things: it directly stimulates the liver cells to
convert glycogen into sugar; and it indirectly facilitates this result by
setting. free adrenaline, which carried to the liver greatly increases or
re-enforces the effect of the stimulation, just as it does that of other sym-
pathetic nerves. Strong emotion, either of fright or anxiety, causes
hyperglycemia and in some human beings glucosuria. By some authors
this result is supposed to be due to the indirect action of adrenaline,
which is known to be discharged into the blood in large amounts in
emotional excitement. It is, however, more probable that we are dealing
here with a double action, the stimuli coming from the brain under
emotional strain pass over the splanchnic nerves in part directly to the
liver, arousing the cells to glycogenolysis, and in part indirectly they
THE METABOLISM OF THE BODY 779

increase the efficiency of this stimulation by action on the supra-renals,
causing a discharge of adrenaline.

The transformation cf glycogen into glucose in the liver is then, in
a condition of health, the product of at least two, and probably more,
factors. In the first place there is the effect of nerve stimulation which
in some way accelerates this transformation; and in the second place this
stimulation is rendered perhaps more powerful by the increase in the
adrenaline content of the blood by the nervous impulses impinging on
the adrenals. By the co-operation of these two actions glycogen is
changed over into glucose and hyperglycemia is produced. Indirectly.
also, either as the result of the hyperglycemia alone, or because at the
same time the nerves going to the kidney influence the state of that
organ, there may result an emotional glycosuria.

The hyperglycemia thus produced is undoubtedly in the nature of
an adaptation. The object of the mechanism is to supply to the muscles,
which under normal circumstances in nature may be ealled upon to
make great exertions as the animal fices or attacks or defends itself, an
abundant supply of glucose from which they can sustain their energy.
Experiments have clearly shown that in the normal animal maximal
contractions are made possible by an increase in the sugar of the blood.
The heart, also, nourishes itself from dextrose and in its presence

Just as the emotions may stimulate glycogenolysis, so also does ex-
posure to the cold. If rabbits are simply cooled, or if dogs are cooled
and anesthetized, or if human beings are similarly treated, hyper-
glycemia and discharge of glucose from the liver results. This again
is in the nature of an adaptive change since the muscles, the great
thermogenic tissues of the body, require the glucose for fuel.

The fact that emotions or cooling have this effect explains the nega-
tive results of several observers who have attempted to increase the
glycogen content of the liver by the direct injection of glucose into the
mesenteric or other veins. Thus Croftan got quite a negative result in
such experiments unless glucose was given by the intestine, whereas
positive results were obtained provided care was taken to prevent the
access of stimuli from the nervous system to the liver.

It has been remarked that stimulation of the splanchnic nerve causes
a change of glycogen to glucose. How is this produced? Does the nerve
stimulation in some way set free a glycogenolytic enzyme in the tissue;
or does it in some other way, for example by stimulating the active dis-
charge of glucose from the cells, upset the equilibrium between glucose
and glycogen and thus cause more glycogen to change to glucose? Is
there in fact such an equilibrium? The same question arises in con-
sidering the physiology of muscle where also following nerve stimulation
780 PHYSIOLOGICAL CHEMISTRY

contraction takes place, glucose is destroyed and glycogen consumed.
Just how does the nerve impulse act? This question cannot at present
be answered definitely. MacLeod has especially studied it. There are
many possibilities besides those just mentioned. Thus it might be that
always a small amount of free glycogenase exists in the cell, but it is
prevented by colloidal membranes from reaching the glycogen. It might
be that the nerve impulse altered the state of these membranes so that
the enzyme might get access to the glycogen. Another possibility is that
the enzyme is anchored to some of the colloids of the cell, and the union
is broken on stimulation. Or by the nerve impulse an oxidative change
might result forming acid, this acid then might set free the enzyme from
its substrate and enable it to attack the glycogen. Or perhaps the
glycogen is being constantly attacked by the glycogenase and digested,
the glycogenase being always present in the cell, but usually the sugar
thus set free is at once resynthesized by the vital activity of the cell.
The nerve stimulus might act by inhibiting in some way this synthetic
activity, so that the action of the enzyme proceeds unchecked; or it
might be, as already suggested, that the nerves simply stimulated the
cells to discharge or secrete their glucose and automatically the cells
produced more to make good the loss. MacLeod, in studying this ques-
tion, found that no increase in glycogenase could be detected either in
the tissue or in the vein as a result of nerve stimulation. On the other
hand, an increased alkalinity of the tissue prevented the nerve stimula-
tion from being effective.

The transformation of glycogen to glucose goes on rapidly in the
liver after death, at least it goes rapidly in the first few hours. There-
after it goes more slowly. It acts as if it might be a vital transforma-
tion. It is natural to suppose that this transformation is due to an
endocellular enzyme, of the nature of amylase, which becomes active
under the post-mortem acidity of the tissue. Extracts of liver tissue,
however, have little more amylolytic power than those of the blood
(Eves; Dastre); and Dastre thought the conversion not due to an
enzyme. The conversion is greatly accelerated by chloroform. Paton
gives the figures on the next page illustrating the speed of this conver-
sion of glycogen to glucose (dog’s liver).

The figures show that the conversion is very rapid in the first half
hour and thereafter goes on more slowly. It is also clear that there is
practically no post-mortem destruction of carbohydrate, or’ glycolysis.
If the liver is thoroughly ground in a mortar, so as to destroy the cells
as completely as possible, the conversion goes on much more slowly, but
is not completely stopped. Chloroform has practically no influence on
such a finely ground liver. This transformation is greatly retarded or
stopped by an alkaline reaction and accelerated -by small amounts of
THE METABOLISM OF THE BODY 781

acid. Perhaps the slowing of the reaction is due to the destruction of
the enzyme, or to the slowing of the reaction by the development of
alkalinity, owing to the formation of ammonia by autolysis.

 

 

 

Time after death t...........ceeeeeenseeeee Two minutes 24 hours
GI CO MEN: <a sess) genes cacti aaacan erase mrot 5.88 grams 0.55 grams
GLUCOSE cya ieeraic cena eunaiee eed adunte even Trace 5.29
Total carbohydrates ......... bediehe 5.88 grams 5.84
45 minutes 315 minutes
j Without 4 i
Time after death:....| Two minutes With CHCl, CHOly With CHCl, CHa
Glycogen ........ 2. 7.09 grams| 5.68 grams | 6.23 grams | 4.60 grams/| 5.42 grams
Glucose ..........- 0.23 1.39 0.98 = 2.53 1.88
Total carbohydrates .|. 7.32 7.07 7.21 7.18 7.20

 

 

The influence of the pancreas. Still another factor in the control of
this transformation of glucose to glycogen and of glycogen to glucose has
been discovered and it is one of the most important factors in the
sugar control. It is the pancreatic gland. That this gland plays a
very important part in this as well as other aspects of the control of
the body carbohydrate metabolism was discovered by von Mering an:l
Minkowski in 1889, a discovery not less pregnant and important than
the discovery of glycogen itself. These authors found that if the
pancreas be completely extirpated, hyperglycemia, glucosuria, and the
complete loss of all power to burn glucose or other carbohydrate resulted.
The animals showed all the symptoms of the severe form of human
diabetes mellitus. They emaciated, were very thirsty and voracious,
their muscles were weak, they passed urine containing not only glucose
but also acetone, acetoacetic acid and (-hydroxybutyrie acid, and they
died in coma invariably in four to six weeks.

CH
d =O CH ,(CO.CH, .COOH CH,.CHOH.CH,.COOH
I
CH,
Acetone. Diacetic acid. B-hydroxybutyric acid.

Another very interesting and significant fact was that their vital
resistance to infection was enormously reduced so that it was extremely
difficult to avoid infection in the operation, or afterwards, and wounds
healed slowly. A similar susceptibility to blood poisoning, boils, etc.,
in other words to the attacks of various streptococci, is shown by human
beings with a diabetic trait. The possible significance of this fact in
considering the cause of diabetes should not be overlooked.

Results similar to these have been obtained in all classes of verte-
brates; in frogs, selachians (dogfish), birds, snakes and mammals.
782 PHYSIOLOGICAL CHEMISTRY

The total extirpation of the pancreas is always followed by death and
usually by a loss of power of burning carbohydrates.

By this discovery the problem set by Claude Bernard which he
failed to solve, namely, the experimental production of the symptoms
of diabetes mellitus, was solved.

The results which follow pancreas extirpation are not due, as was
at first thought, to the extensive division of the nerves and traumatism
of the sympathetic ganglia involved in the operation, but must be
ascribed primarily to the actual taking out of the pancreas tissue. This
was proved by Hedon, who made the following experiment: The abdomen
of a dog was opened, a portion of the pancreas was extirpated and the
branch of th» gland, which extends into the mesentery, was brought
to the surface and fastened into a pouch made underneath the skin
of the abdomen. The blood vessels and nerves of this portion were
left intact. After several weeks, during which no glycosuria appeared,
the blood vessels of the skin enter into union with the vessels of the
gland graft. Then another operation was performed and the grafted
piece of pancreas was cut loose from the stalk connecting it with the
abdomen. If the operation had been successful, if the blood connection
in the skin had been established, no symptoms of diabetes resulted.
The juice secreted by this grafted piece might discharge outwardly, or
make a tumor, but there was no glucosuria as long as the graft remained.
Some time later the graft was either removed, or at times it degenerated,
and when this happened glycosuria began and continued until shortly
before death.

These experiments of Hedon’s were not very numerous but they were
decisive. They showed clearly and conclusively that the. nervous lesions,
or cireulatory disturbances, of this operation were not the cause of the
glycosuria. The presence of the grafted tissue even under the skin was
sufficient to prevent hyperglycemia and to enable the body to carry on
its ordinarv carbohydrate metabolism. There were, or seemed to be,
but two possible explanations, namely, either the pancreas adds some-
thing to the blood or lymph, which is necessary for normal carbohydrate
metabolism. or it takes something out, which if not removed causes
glycosuria. Most investigators preferred the first of these possibilities
and believed the pancreas must have an internal secretion even more
important. than its external and vitally necessary for the carbohydrate
metabolism of the body. But the second or detoxication theory has alse
liad its advocates.

This discovery of von Mering and Minkowski and Hedon led to an
enormous amount of work to prove the existence of this internal secre-
tion. This work extended over twenty years before a decisive result
was obtained. It was found, however, that neither feeding the gland
THE METABOLISM OF THE BODY 783

itself nor injecting the gland extracts caused any definite and clear-
cut change in the course of the discase or of pancreatic diabetes. There
were, it is true, many apparently successful experiments indicating that
pancreatic extracts might have some temporary or trifling action in
reducing the glycosuria; but other observers either failed to get any
action at all, or the glycosuria might even be increased by feeding pan-
creatic tissue to depancreatized dogs or to human beings with the severe
form of the disease. The ease with which a temporary relaxation of
glycosuria might be produced was a special cause of error. In many
instances, indeed, feeding pancreas after pancreatectomy so far from
diminishing seemed really to increase the glycosuria. It appeared in
1910-1911 that if the pancreas had any internal secretion it did not
accumulate, or was not stored in the gland as are all the other internal
secretions known, and investigators began to turn to the study of other
possibilities such as the detoxication theory, believing that the internal-
secretion theory must be wrong. Within the past few years, however,
more favorable results have been obtained. The most decisive experi-
ments are those of Starling and Knowlton made with an acid extract of
the pancreas. Their results in the following figures show that the addi-
tion of a neutralized acid decoction of the pancreas to diabetic blood
which is being perfused through the heart taken from a depancreatized
dog causes the heart to beat much stronger and to consume glucose
added to the blood. The consumption of the glucose before the addition
of the pancreas extract had been reduced to the vanishing point.

Diasetic Heart Fep witu DIABetic BLoop.

(Sugar consumption in mg. per gram heart muscle per hour.)
First hour with : Second hour after

blood alone addition of pancreatic extract
1.5 4.3
0.5 _ 3.0
0.5 2.8
0.5 3.6

The normal heart of the dog when fed on normal blood under these
circumstances consumes per hour per gram of heart muscle 3.5-5 mgs.
of dextrose. It is clear from these experiments that the diabetic heart
fed with diabetic blood has lost its power of burning glucose to a very
considerable degree, and that the addition of the pancreas decoction
has restored this power.

More recent experiments have indicated that the respiratory quotient
of the diabetic heart is not usually changed by the addition of pan-
creatic extract as one would expect if the heart really had acquired
the power of decomposing glucose. Moreover both alkalies and adren-
aline are said to have an equally beneficial effect. It was not shown in :
the experiments that the glucose had been actually burned. It might
784 PHYSIOLOGICAL CHEMISTRY

have been converted to glycogen. In view of these considerations it is
still doubtful whether the results obtained really show the existence of
an internal secretion.

These experiments and others indicate, however, that normal blood
has in it a very small amount of a substance which enables glucose to
be burned ; this substance is derived from the pancreas. It is its internal
secretion. If to an extract of muscle juice pancreatic extract is added,
Cohnheim found the glycolytic power increased, but McGuigan and
others have been unable to confirm these observations and Cohnheim
himself in repeating them generally had negative results. Evidently
some accidental circumstance in a few cases caused a greater disappear-
ance of the sugar. The manner in which the extract acts is quite ob-
scure. It may be that in the experiments cited the muscle, instead of
destroying the sugar, may have converted it into maltose. The internal
secretion might act by changing the permeability of the muscle mem-
branes or by combining with glucose, acting as a between or intermediate
body by which it is anchored to the protoplasm; or it might be the raw
material from which the glycolytic substance of the muscle is made.
Since a decomposition of the sugar almost certainly precedes its com-
bustion, the muscle has probably lost not the power of burning the
decomposition products, but rather the power of decomposition itself.

The internal secretion of the pancreas, like that of the supra-renals,
is either rapidly eliminated or destroyed in the blood. It may possibly
be a basic body very unstable in a free form, but more stable as a salt.
At any rate blood contains no great amount of it and it quickly dis-
appears when the pancreas is eliminated. It is not impossible that it
is normally destroyed or eliminated by the kidney, as is adrenaline, and
one of the causes of human diabetes may be that the destruction or
elimination of this body may at times be greater than its rate of forma-
tion, so that’ a partial or total loss of combustive power ensues.

This internal secretion of the pancreas plays a part also in the con-
trol of the glycogenic function of the liver. In the absence of the pan-
ereas, although the blood contains two to three times its normal supply
of glucose, the liver and muscles form very little glycogen. If, however,
the liver be perfused with blood containing glucose, or if glucose is
injected directly into the circulation, it stores more glycogen if at the
same time there is added to the blood an extract of the pancreas. How
this extract acts, however, is still quite uncertain. It may be acting on
the ends of the sympathetic nerves reducing or counterbalancing the
action of adrenaline, or it may be acting directly on the liver cells them-
selves. It will, no doubt, seem peculiar, at first glance, that the body

“should still be able to form small amounts of glycogen even when the
further oxidation of glucose is impossible. The probable reason for this
THE METABOLISM OF THE BODY 785

is suggested by the experiments of Lobry de Bruyn and also of Nef.
For the complete oxidation of the glucose molecule a more extensive
decomposition of the molecule is necessary than for the synthesis of the
monosaccharides to the disaccharides. For the latter it is only necessary
to produce a rearrangement of the last two carbon atoms of the chain;
for the decomposition it is necessary to break the chain also. If, for
example, sugars are put into very weak alkali, such as milk of lime,
synthesis into disaccharides and mutual transformations of one sugar
into another occurs, but the molecules do not break into fragments; it is
only in the presence of stronger alkali that the decomposition of the
molecule takes place (see page 31). The diabetic body, then, seems able
to produce the easiest transformation of the carbohydrate, namely, that
involved in the transformation of one kind of sugar into another, levulose
into glucose, for example, or the synthesis of the monosaccharides in part
to glycogen, although it has lost the power of breaking the molecule far
enough to oxidize it. It has lost also most of the glycogen-forming
power. The result of this may be that all dextrose taken in the food
or made in the body, is at once eliminated and hence cannot be held.
It may also be that dextrose must be polymerized before it can be burned.

Recently some results have been obtained by Dakin which may
ultimately throw light on the relation of the pancreas to sugar metab-
olism. Dakin has found that nearly all tissues of the body, i.e., liver,
thymus, thyroid, supra-renal, pituitary, kidney, spleen, heart muscle,
skeletal muscle, lung, brain, etc., contain an enzyme known as ‘‘ glyoxa-
lase.’’? This enzyme has the property of converting a simple or substi-
tuted glyoxaldehyde into the corresponding glycollic acid.

R. CO. CHO -++H,0 —~ R. CHOH.COOH
Glyoxal. d Glycollie acid.

It is a very suggestive fact that the pancreas is the only organ, except
the lymph glands, which lacks this enzyme, and in the pancreas alone
is found a thermolabile substance, which antagonizes the glyoxalase, an
antiglyoxalase. This antiglyoxalase is not found in the blood, it occurs
in small amounts in the external secretion of the pancreas and probably,
Dakin thinks, may -be secreted internally. Whether this be the case or
no, the formation of antiglyoxalase appears to be a specific function
of the pancreas. (Another very interesting fact is that these glyoxals
will be transformed into amino, as well as hydroxy-acids, when perfused
through the liver (Dakin and Dudley)). While the relation of these
results to the carbohydrate metabolism is still obscure, we have at any
rate for the first time a specific chemical property of the pancreas ob-
viously closely related to carbohydrate metabolism.

Internal secretion. Where produced in the pancreas. There are at
least two distinct kinds of cells in nearly all glands, namely, the secret-
786 PHYSIOLOGICAL CHEMISTRY

ing cells of the acini and the cells of the ducts. These cells evidently
differ in their chemical nature and their physiological function, but
practically nothing is known in any gland of the functions of the duct
tissue beyond its function of conducting the secretion. We do not
know in the pancreas whether all the digestive enzymes are secreted by
the acinary cells or whether some are secreted by the duct cells. It is
known that the secretion obtained by the injection of acid in the duo-
denum often differs markedly in composition from that produced by
secretin ; but whether these two come from the same or different tissues

 

Fie. 63.—Islets of Langerhans in the guinea-pig pancreas. The Islets are the dark
cell masses attached to the ductules which grow out of the larger ducts. The acinary
tissue has degenerated as a result of blocking the ducts (Bensley).

of the gland is unknown. There have also been described in the pancreas
small clumps, or groups of cells which differ in their staining reactions
and appearance from the acinary cells, but are in connection with the
ducts. These are known as the Islets of Langerhans. Until very re-
cently no methods were known for the certain identification of islet
tissue, but not long ago Bensley found that in the guinea pig they
stain more readily in neutral red, injected intra vitam, than do the
acinary cells. Here and there, however, outside the islets proper, similar
cells are to be seen. According to Bensley ligation of the duct of the
pancreas in the guinea pig leads to the destruction of the acinary tissue,
while the much branching duct tissue with the islets as masses of duct
cells at the extremities of the outgrowths of the ducts remain. See
Figure 63.

It was Bernard who first succeeded in showing the difference in
THE METABOLISM OF THE BODY 787

reaction and function of the duct and the acinary cells. He found in
dogs that injection of the ducts with a fat of high melting point, solid
at the temperature of the body, produced a complete atrophy of the
acinary cells, whereas the ducts remained intact, ‘‘ like a tree which had
lost its leaves.’ If such dogs developed diabetes Bernard did not
observe it. If they did not, this experiment shows very clearly thai duct
tissue alone is sufficient to preserve the life of the animal in the absence
of the acinary cells.

Some years ago Opie reported that in many cases of diabetes lesions
were apparent only in the islands of Langerhans. He, accordingly, sug-
gested that the internal secretion of the pancreas was due to the islets
and not to the acinary cells. Unfortunately for this theory many cases
of diabetes show lesions in neither tissue. The opinion has, however,
become widespread that the internal secretion is due exclusively to the
islets. The evidence for this is still very doubtful. Both in Bensley’s
guinea pigs and in Bernard’s dogs there is no suggestion of diabetes.
This does not show that the acinary cells form no internal secretion, but
only that the duct tissue, including the islets, is alone sufficient to main-
tain life. Possibly both tissues form the secretion. The contrary ex-
periment of the destruction of the duct and the survival of the acinary
tissue has not been, and seemingly cannot be, tried. We cannot at
present say, therefore, whether the internal secretion of the pancreas is
due to the duct cells and the islet tissue alone, or whether the acinary
tissue also contributes to it. The categorical statement often seen in
text-books and papers that the internal secretion is supplied by the
cells of Langerhans is entirely unjustified.

Cause of diabetes. Finally what is the cause of diabetes in human
beings? Nothing is known of this whatsoever. The facts just stated
lead pathologists generally to attribute the disease often to a lesion in
the islets of Langerhans in the pancreas. Whether all diabetes have this
origin is not certain. Nothing is known of the cause of the lesion if any
such exists. Whether it is due to the absorption of a poison from the
intestine, as some have thought, or whether it is due to a bacterium or
other parasite, has been very little investigated and so far without result.
Attention has been focused entirely on the symptoms of disturbed metab-
olism which accompany this disease, rather than on a search for the
real cause. The fact that diabetics generally show such a lowered resist-
ance to the attacks of the streptococci of blood poisoning arouses the sus-
picion that the trouble might be due to an infection of the islets by
this organism which thus produces diabetes, just as it produces inflam-
mation of the valves of the heart when it strikes them, or of the kid-
neys when it is located in those organs. Perhaps a strain of this pro-
tean organism will be found which will act on, or show a specific affinity
for, the islets. At any rate the search for the cause of this disease,
788 PHYSIOLOGICAL CHEMISTRY

rather than a search for a method of alleviating its symptoms. would
appear to be the part of wisdom.

Summary of the réle of the liver in carbohydrate metabolism. We
may now summarize the réle of the liver in carbohydrate metabolism of
the body. Carbohydrate food after digestion finds its way into the
blood, where it is to be found in small amounts: 0.1-0.3 per cent. in the
blood of the portal vein. The sugar thus circulating in the blood is free.
It is diffusible and may be removed from the blood by the process of
vividiffusion. The portal blood at the same time carries, also, any
metabolic products or internal secretions of the spleen, pancreas, and
intestine. Brought thus to the liver, this organ during digestion, when
it is supplied with much oxygen, picks out from the blood some of the
glucose, levulose and galactose passing through it and converts these
into a colloidal polysaccharide, glycogen, which is laid down in such
quantities in the liver tissue that on a rich carbohydrate diet the liver
may contain 12-15 per cent. of glycogen.

The liver acts, thus, in the first instance, as a storehouse, or reserve
depot of carbohydrates, in which surplus carbohydrate is stored until
it is needed by the other tissues. It functions, then, in this particular
like the fat tissue which stores superfluous fat and like the muscles
which store glycogen.

Between meals, on the other hand, when no glucose is arriving from
the intestine, this stored glycogen is called upon and more or less com-
pletely consumed; the liver of a fasting animal containing much less
glycogen than that of a well-fed animal.

This conversion of glucose into glycogen and of glycogen into glu-
cose depends on various factors. If the glucose in the blood surpasses
0.1 per cent., if the liver is well supplied with oxygenated blood, and
with the internal secretion of the pancreas, the conversion of glucose
to glycogen is at least as rapid as that of glycogen to glucose and no
diminution of glycogen occurs. If, however, the content of glucose in
the blood coming to the liver falls below 0.1 per cent., or if oxygen
supply is reduced; or if impulses come into the liver over the splanchnic
nerves; and particularly if there is at the same time an abundance of
adrenaline in the blood due to the excitation of the supra-renal glands
by the splanchnic nerves following emotional or other excitement, then
glycogen is converted into glucose and this will take place even though
the amount of glucose in the blood is already above the normal and
as much as 0.2-0.3 per cent. It is uncertain how this glycogen is
converted into glucose, but it is probably due to an endocellular
glycogenase. Whether stimulation of the nerve causes an increase of
glycogenolysis, or a decrease of conversion of glucose to glycogen, it is
as yet impossible to say, but it is possible that normally nerve stimula-
tion is re-enforced by the concomitant stimulation of the supra-renals,

a
THE METABOLISM OF THE BODY 789

causing them to throw into the blood an increased amount of adrenaline.
There is no evidence of the setting free of glycogenase during the stimu-
lation. The experiments of Stewart have also aroused doubt that
adrenaline is playing a réle in glycogenolysis.

The liver is not the only storehouse of glycogen. The muscles con-
tain an amount little, if any, less than that of the liver, and even in
the absence of the glycogenic function of the liver, as, for example,
when an Eck fistula is made, the eating of carbohydrate food seems no
more liable than usual to produce glucosuria, nor does there seem to
be any reduction in the sugar tolerance of the organism. This fact
has not yet been explained, but it is probably to be ascribed to two or
three circumstances. One is the storage power of the muscles; another
is that possibly the liver, after the Eck fistula is made when its cells
are greatly altered in their appearance and size and probably in func-
tion, is no longer able to form glucose from protein so that the pro-
duction of glucose in the animal itself is reduced; and finally the nutri-
tion of animals with the Eck fistula is often poor and possibly there is
less complete absorption of carbohydrate and other foods.

But the liver is not only a magazine of carbohydrate; it is also a
factory where proteins, amino-acids, glycerol, lactic and pyruvic acids,
dioxyacetone and other substances are converted into dextrose. Many,
if not all, of these syntheses involve oxidations.and for their accom-
plishment the liver must have a supply of oxygenated blood. In some
of these syntheses ammonia is split off and replaced by oxygen. This
ammonia serves a double purpose: in part by its alkalinity it reduces
acidity and hence checks glycogenolysis; in part it is changed to urea, a
substance of importance in the maintenance of the activity of other
organs; ie., the heart. By this synthetic power of the liver not only is
the body able to form dextrose from protein and other foods, being able
indeed to live on protein alone, but many of the partially oxidized
metabolic products of other tissues, such as lactic acid, are saved and
resynthesized into glucose to be used over again. The liver in this re-
spect works over waste products.

Finally the liver is not only a producer and storer of dextrose; it is
also a consumer. Like all cells and tissues of the body it has the power
of consuming carbohydrate. It respires and burns some of this car-
bohydrate, though how much it is impossible to say. Not all pyruvic
aldehyde or acid is converted into dextrose, but a part at least is oxi-
dized, burned to carbon dioxide and water; a part may, by reduction, be
reunited with ammonia to form some of the simpler amino-acids, so
that the reverse process of protein synthesis can also occur. In the
bird’s liver some of the products of carbohydrate destruction are used
in synthesizing uric acid; and in the mammalian liver we have a forma-
tion of glycuronic acid so important as a means of neutralizing poisons.
790 PHYSIOLOGICAL CHEMISTRY

No doubt, also, a ready transformation to fat occurs. But we have as
yet a most imperfect picture of the chemical transformations occurring
in this most important glandular organ.

But while we picture the liver as acting thus as a storehouse for
glycogen with which it parts when glycogen is needed by the other
tissues, there are reasons which make it probable that glycogen is stored
in the liver primarily for the benefit of the liver itself. Glucose has a
very particular relationship to anaérobic respiration. It is already par-
tially oxidized and it can furnish energy for various decompositions and
activities of the body, being itself reduced to alcohols and fatty acids
at the same time. All tissues live very much longer without oxygen, if
they are supplied with glucose. The liver is peculiar in that it must
carry on its activities while its blood supply is very largely venous in
character. It must be always nearer asphyxia than most of the tissues
of the body. There can hardly be a doubt that the primary object of
this stored supply of glycogen in the liver is to enable the liver to func-
tion properly in the presence of very little oxygen. It is found, indeed,
that the liver in the absence of glycogen is far more liable to necrosis
than when it is present. Particularly in chloroform anesthesia such
necrosis is apt to occur and it shows itself first in the center of the
lobules where, presumably, the oxygen need is greatest (Graham).
This necrosis is very much less apt to occur if an animal has been well
fed on carbohydrate before being anesthetized. It is possible that the
giving to patients about to undergo operation considerable amounts of
carbohydrate so as to load the carbohydrate reservoirs of the body would
be a wise precaution, provided other unlooked-for results do not de-
velop. If glycogen is discharged from the liver by adrenaline, central
necrosis also occurs. (Figure 61, page 673.) The liver then stores
glycogen not only that that glycogen may later be available for the
other tissues of the body, but probably because glycogen is very neces-
sary to its own safety in time of stress and oxygen want.

The further fate of glucose. The liver supplies glucose to the blood.
What then is the ultimate fate of this glucose? The tissues which con-
sume most of the carbohydrates are undoubtedly the muscles; because of
their bulk; if for no other reason, this would be the case. They make
about 50 per cent. of the weight of the body. They are always in
activity and they produce much heat. The muscles contain a consider-
able quantity of glycogen. Indeed the glycogen may persist in the
muscles after it has disappeared from the liver. That glycogen disap-
pears from a muscle, or is consumed during muscle contraction, is shown
by the determination of the glycogen content of muscle before and
after prolonged tetanus, as shown by the figures on page 622.

Moreover that some ron-nitrogenons substance furnishes the energy
THE METABOLISM OF THE BODY 794

for muscular work is shown by the fact that physical work does not
increase the nitrogen outgo of the body, but only that of carbon dioxide
and water. We may, therefore, conclude that the sugar thus circulating
in the blood is picked out by the muscles, which store it as glycogen,
depositing it in their tissue, and that it is oxidized to carbon dioxide and
water or to lactic acid during muscle work. The muscles are the great
carbohydrate-consuming tissues of the body.

The question now arises concerning the consumption of dextrose by
the muscle. How is the sugar consumed? Is it oxidized directly as
glucose? Or is it decomposed first into simpler substances which then
undergo oxidation? Or must it be synthesized into a more complex
carbohydrate? Do the muscles burn glucose by themselves or do
they need the assistance of other tissues or glandular organs? Does this
power of destruction depend on the vitality, structural integrity of the
muscle, or does it occur also in hashed muscle? Is it due to an enzyme
which is found in the muscle, or is it a vital process, that is one involving
the structural integrity of the cell? Is the glucose burned in the muscle
sap or must it first be broken and then built into the living tissue itself?
How are we to explain the explosive decomposition which occurs when
a nerve impulse sets up a muscular contraction? These and many other
questions press at once for solution, but to only a few of them can
we give a complete answer.

Conditions of sugar burning in muscle. The same factors which are
active in the sugar metabolism of the liver play a réle here in the
metabolism of muscle. We may consider first the question whether
the muscle is able by itself to burn sugar brought to it from the exterior,
or only when it is assisted by the co-operation of other organs. To this
question a definite answer may be given. The muscle cannot utilize
extrinsic glucose as food and as a source of energy except in the pres-
ence of the pancreas. If the pancreas be completely extirpated in any
vertebrate so far studied, the power of consuming glucose is com-
pletely lost by all the tissues of the body. If a portion of the gland be
left in the body, the power of sugar consumption persists more or
less completely. If glucose or glucose-producing food is given to a de-
panereatized animal it appears in the urine practically quantitatively.

But after pancreatectomy not only is ingested glucose completely
eliminated, but also all that glucose normally produced in the body from
protein is excreted also. It thus happens that even on a carbohydrate-
free diet the excretion of glucose continues. It is as if the body were
turning to sugar.

There are two aspects of this failure of the muscles and other tissues
of the body to burn carbohydrate in the absence of the pancreas, which
are truly remarkable. The power of burning carbohydrate and in
particular the power of burning or fermenting glucose is practically
792 PHYSIOLOGICAL CHEMISTRY

universal. All kinds of plants, probably all plants, including the bac-
teria, have this power. Glucose is found in all the invertebrates in one
form or another. Now it certainly is an almost incredible fact that this
fundamental property of living matter should, in the vertebrate organ-
ism, be so completely lost, that no glucose at all should be burned in the
absence of the pancreas. That the organs should have come to lean on
the assistance of the pancreas would not be surprising, but that they
should have totally lost one of the most fundamental properties of
living matter so that they can only exist in the presence of the pancreas
is so astounding as to arouse the greatest suspicion of the truth of
the conclusion. It appears from the facts, however, that there is no
escape from this conclusion, however unlikely it may appear.

The other aspect of the affair which is of great interest is this.
Sugar is constantly being made in the diabetic organism. Indeed, the
power of sugar formation is certainly not diminished and many have
thought that it is stimulated in the diabetic organism. This glucose may
be made out of all the substances from which it is made in the normal
organism, from alanine and other amino-acids, from glycolaldehyde and
other very simple substances. Now if the failure to burn dextrose is
due to the fact that the molecule can no longer be fragmented, as is
assumed by some investigators, then we should expect the fragments
to burn as well as ever if these fragments were fed. No one has been
able to discover any such fragments of the dextrose molecule, which
can be burned by the diabetic organism when the power of burning
glucose is lost. Of course there may be fragments which are normally
burned and which will be found in the future. But it certainly is sin-
gular that all the search for such combustible fragments is still unsuc-
cessful in spite of the considerable number which have been tried.
Furthermore it is clear that if the hypothesis is correct that the failure
of the tissues to burn the molecule is due to the fact that the pre-
liminary fragmentation is wanting, these combustible fragments cannot
be any of those from which the carbohydrates are formed. In other
words, the molecule must break down in its fragmentation into sub-
stances of another kind than are used in the synthesis. For example,
since the diabetic organism will carry lactic acid into sugar and not
burn it, it is clear that the combustive decomposition of the carbohy-
drates is not through lactic acid, if the theory as to the reason of
non-combustion is correct. It might perhaps be worth while to re-
examine with great care the evidence upon which the conclusion is based
that no sugar is burned in the diabetic organism to see whether this
view is correct. It might be that the sugar formation was stimulated
to such an extent that all that could be burned was being burned. Any
excess accordingly appears in the urine. In other words, perhaps the
THE METABOLISM OF THE BODY 793

sugar-burning powers of the body of the diabetic are already at a
maximum without the introduction of glucogenetic foods from the ex-
terior. The power of glucose storage is certainly greatly reduced, and
perhaps this stimulates glucose production.

D:N ratio. If the carbohydrate is withdrawn as completely as
possible from a depancreatized animal or from a diabetic individual,
dextrose continues to be secreted in the urine. This dextrose, since
it has not come from carbohydrate food, must have been made in the
body itself, and since its amount rises with the protein and not with a
rise in the fat intake, it must be derived from protein food. How ex-
tensive this manufacture of glucose from protein may be is illustrated by
experiments by Minkowski and Lusk. If the protein was split quanti-
tatively into glucose and urea there should be obtained, since 100 grams
of protein contains 50 grams of carbon and 16 of nitrogen, about 175
grams of glucose. Not all of the protein, however, is converted into
glucose. Some of it appears as acetone, diacetic acid, and other decom-
position products. Some is burned to carbon dioxide and water so that
actually the maximum amount which can be made into glucose in a
dog’s body in a condition of complete glucose intolerance is 60 grams.
We have, then, since 100 grams of protein yield roughly 16 grams of

“nitrogen in the urine, a ratio of D: N, dextrose to nitrogen, of 60:16=

3.7. Higher ratios than this may easily be obtained on feeding food
containing carbohydrates, but on a strictly protein and fat diet in a
condition where glucose cannot be utilized at all, this ratio has been
obtained by von Mering and Minkowski in depancreatized dogs, in
diabetic human beings, and in dogs made completely sugar-intolerant by
phlorhizin by Lusk. In an experiment by Reilly, Nolan and Lusk a
fasting phlorhizinized dog was given 500 grams of meat without altering
the D:N ratio. The figures are for the urine of 12-hour periods.

Dextrose— Nitrogen— D:N
grams grams
Fasting: caicaceeise cose. vege ntesues 23.87 7.0 3.41
500 grams meat « 49.59 14.0 3.54
Pasting: ~...s54 gee Seats Oe ee a 25.36 7.1 3.56

 

This D: N ratio of 3.3-3.7 Lusk calls the fatal ratio, since its appearance
in human diabetics when on a strictly carbohydrate-free diet means
that none of the sugar can be consumed.

From the foregoing experiments it appears that at a maximum 60
grams of glucose may be made from 100 grams of protein. Several
questions at once arise concerning this point. In the combustion of
proteins in the body does it always happen that 60 per cent. are con-
verted into glucose and are burned in that form? Or are we to assume
that in the condition of carbohydrate starvation prevailing in dia-
betes, the process of manufacture of glucose from proteins is greatly
794 PHYSIOLOGICAL CHEMISTRY

stimulated in an endeavor to adjust the disturbed metabolism of the
body and to refill the depleted carbohydrate reservoirs? There is not
at present any way of deciding this question, but many facts indicate,
as we shall see in studying protein metabolism, that certainly a very
considerable part of the protein is normally made into dextrose.
Decomposition of ‘sugar im the muscle. Fermentation. One of the
most important questions in the combustion of sugar in the muscle is
whether the dextrose is burned as such or is first decomposed into
various splitting products which then are oxidized. Some light may
be thrown on this question by a study of the spontaneous oxidation of
glucose. In discussing the chemistry of the carbohydrates it was shown
that oxidation did not occur directly but in large measure indirectly,
the glucose molecule breaking up in alkaline solution and the particles
thus produced then either oxidizing themselves spontaneously, or if suffi-
cient oxygen was not present, the particles oxidized and reduced each
other, or else condensed to form various other products. There are
many indications that dextrose is not burned as such in the body, but
by the action of the protoplasm decomposes, much as it does in alkali, to
form various decomposition products which either oxidize if oxygen is
present, or are reduced or condense to form fats or other metabolic
products. The processes may very possibly be similar to the alcoholic
fermentation of glucose. This decomposition tekes place in the absence
of oxygen, very little energy being set free; by the subsequent oxidation
of alcohol energy is obtained. The exact nature of this alcoholic de-
composition is not clear, but it may pass through glyceric aldehyde.

CH,OH CH,OH CH,OH
| | | + 30,—~2C0, + 3H,0
CHOH CHOH _ CH,
| |
CHOH —_ COH co,
bao CoH co,

|
buon CHOH —_— CH, ‘

3 0, —-> 2CO 3H,O
box bis on daon oa on oe
Glyceric aldehyde.

As a matter of fact muscle and other tissues are able to burn alcohol
readily and alcohol is found in small amounts in normal tissue; it is,
however, very unlikely that in the combustion of glucose in animals
alcohol is an intermediate product, since its toxic actions are too intense.
The exact course of the destruction of the glucose in muscle is still
entirely unknown.

The liver and the muscles are not the only tissnes which need and
consume dextrose, although they are hy far the largest. The heart,
THE METABOLISM OF THE BODY 795

the intestine, both support their movements by burning dextrose. Thus
the addition of glucose to an artificial circulating fluid like that of Locke
or Ringer or Tyrode restores or quickens their contractions.

Altogether aside from muscle tissue, however, there can be no doubt
that the metabolism of other organs also requires dextrose and in its
absence other sources of raw materials for energy and substance must be
found. Particularly the relation of the kidneys to sugar metabolism
needs careful investigation. There can be little doubt that the kidneys
must have some sort of an affinity for glucose to enable them to secrete
it from the blood to the urine, where at times it is in a much higher
concentration. This power is greatly stimulated by phlorhizin; it seems
also to be reduced in pancreatic and human diabetes, since the kidneys
are no longer able to prevent an accumulation of sugar in the blood and
a hyperglycemia. It is, of course, possible that the kidney is only
able under the best of circumstances to secrete a certain amount of glu-
cose, and that the hyperglycemia means that the kidneys are overwhelmed
and unable to reduce the blood sugar to its normal level.

Phlorhizin diabetes.—Hitherto we have considered two experimental
methods for producing glycosuria and more or less serious disturbances
of carbohydrate metabolism. A third method was discovered by von
Mering and has been particularly developed by Lusk. This method
consists in the injection of the drug phlorhizin. Phlorhizin, a glucoside
derived from the bark of the roots of the plum, apple, cherry and pear
trees, as the name signifies (Gr. phloios, bark; rhiza, root), has the re-
markable property of inducing glycosuria which is not accompanied by
a hyperglycemia, but rather by a hypoglycemia. The method of
employing the drug as developed by Lusk is by subcutaneous injection
of 2 grams per day, one gram twice a day dissolved in a little Na,CO,.
Another method of injecting 1 gram suspended in 7 c.c. olive oil is also
used. The drug thus given. or when given in large doses by the mouth,
causes a very great glycosuria, and if the injections are continued the
usual symptoms of severe mellituria follow, namely, besides the
polyuria, muscular weakness, acidosis, acetonuria and death in coma.
If, however, the injections are stopped the animal recovers. The glyco-
suria is accompanied by a hypoglycemia. The dextrose content of the
blood falls from 0.12-0.08 per cent. Nevertheless the kidneys continue
to excrete glucose. The action of the drug appears to be, therefore,
primarily on the kidney, causing it to secrete glucose more rapidly than
usual and hence to keep the level of the dextrose in the body below
the normal. As a result of this impoverishment of the blood, the liver
and the muscles not in activity give up glucose to the blood in order
to supply that organ (the kidney) whose consumption has thus re-
duced the whole supply. But as this happens to be the kidney, the
796 PHYSIOLOGICAL CHEMISTRY

result is simply like pouring water into a sieve. The dextrose is
drained out of the body.

That phlorhizin thus acts on the kidney cells primarily, though not
exclusively, is shown by an experiment of Levene. An anesthetized dog
had cannulas in each ureter. Into the kidney artery of one side was then
injected a small dose of phlorhizin. This kidney secreted glucose at
once and the first appearance of glucose in the other kidney did not
take place for two minutes later.

The drug causes also marked degenerative changes in the kidney
epithelium leading ultimately to its complete destruction. It is some-
times stated that phlorhizin increases the permeability of the kidney
epithelium to sugar, as if the kidney acted as a filter which normally
held back the glucose and by the action of the drug was made more
permeable so that glucose went through. While this may be the means
of its action it seems more probable that the secretion of glucose is an
active process and that this process is in some way stimulated by the
phlorhizin. It is indeed probable that not only does phlorhizin in-
crease the secretion of glucose by the kidney, but under its action glu-
cose appears also in the bile. The secretion of urea is also increased
directly or indirectly by the phlorhizin. The nitrogen output of fasting
dogs is increased 3-5 times by a dose of phlorhizin. It has also been sug-
gested that the glucose is in combination with some of the colloids of
the blood and that the kidney under the action of the drug is able to
make the glucose free, which now escapes and is excreted. The evi-
dence for this is, however, extremely meager. It has been shown that
the sugar in the blood is capable of diffusing by the method of vividiffu-
sion and that its concentration in the dialysate is about that calculated
to be in the blood. It is extremely hard to see why even if glucose is set
free from such a hypothetical colloidal union, it should diffuse from a
region where it is present in only 0.08 per cent. to one in which it is
present to the extent of 2 per cent. Any such a concentration as this by
a reabsorption of water by the contorted tubes of the kidney would be
impossible. In any case the secretion of the glucose must be an active
process quite analogous to that of the secretion of the bile salts from
the blood by the liver cells. Phlorhizin produces microscopic changes in
the pancreas. The fact that all the symptoms of the most severe
form of diabetes can be produced by simply emptying and keeping
empty the glycogen storehouses of the body, by drawing the sugar
out through the kidneys, lends some support to the old view
that the primary cause of the usual diabetes is not a loss of power
of burning sugar, but a loss of the power of filling these reservoirs with
glycogen.
PROTEIN METABOLISM OF THE BODY 197

REFERENCES. CARBOHYDRATE METABOLISM.

The literature of this subject is so enormous that no attempt will be made to

give more than a few of the recent and some of the classical papers on this
subject. A list of some 1,200 references to the literature will be found in Allen:
Glycosuria and Diabetes, Boston, 1913.

1.

3.

=

9

10.

11.

12.

13.
14,

15.
16.

17.

Books.

Allen: Glycosuria and Diabetes, Boston, 1913. 1180 pages. This book has a
fairly complete and exhaustive exaniination of our knowledge of diabetes
mellitus and insipidus and various glycosurias, together with a very large
number of original experimental observations.

Lusk: The Science of Nutrition, 2d edition, 1912. The writer considers this book
to be the best general treatise on the subject of nutrition and a large amount
of space is given in it to carbohydrate metabolism. References are given, the
most important facts are cited in an interesting way and the book is not
swamped by details.

Pfliiger: Glykogen. Archiv ges. Physiol., 96, pp. 1-398, 1903. This has a very
complete critical summary of the work done up to 1903.

von Noorden: Metabolism and Practical Medicine. Chicago, 1907.

REFERENCES.

Bernard: Lecons sur la diabéte et la glycogenése animale, Paris, 1877.
Bernard: Lecons sur la physiologie et la pathologie du systéme nerveux. Vols.
land 2. Paris, 1858.
Bernard: Comptes-rendus de l’Acad., Paris, p. 884, 1859.
v. Mering: Ueber diabetes mellitus. Verhandl. d. Kong. f. inn. Med., 6, pp. 349-
358, 1887.
v. Mering: Ueber diabetes mellitus. Zeits. klin. Med., 16, pp. 431-446, 1889.
v. Mering and Minkowski: Diabetes mellitus nach Pancreasextirpation. Arch.
expt. Path. u. Pharm., 26, p. 371, 1889-90.
Hedon: Arch. de Physiol., 1894, p. 269.
Kiilz: Beitriige zu der Lehre von der Glykogenbildung in der Leber. Arch. ges,
Physiol., 24, p. 1, 1881.
Kile: Beitriige zur Kenntnis des Glykogens. (Beitriige zur Physiologie. Lud-
wig.) Marburg, 1891, pp. 69-121.
Voit, C.: Ueber die Glykogenbildung nach Aufnahme verschiedener Zuckerarten.
Zeit. f. Biol., 28, p. 245, 1891.
Minkowski: Untersuchungen iiber den Diabetes mellitus nach Extirpation des
Pankreas. Leipzig, 1893.
Minkowski: Ueber die Zuckerbildung im Organismus beim Pankreasdiabetes.
Arch. ges. Physiol., 111, p. 13, 1906.
v. Mering: Zeit. f. klin. Med., 16, p. 437, 1889.
Lusk: The influence of cold and mechanical exercise on the sugar excretion in
phlorizin glycosuria. Amer. Jour. Physiol., 22, p. 163, 1908.
Lusk: Ueber phloridzin diabetes. Zeit. Biol., 42, p. 31, 1904.
Lusk: Metabolism after the ingestion of dextrose and fat, including the be-
havior of water, urea and sodium chloride solutions. Jour. Biol. Chem., 13,
p. 27, 1912.
Lusk and Stiles: On the formation of dextrose in metabolism from the end
products of « pancreatic digest of meat. Amer. Jour. Physiol., 9, p. 380,
1903. :
798

18.

19.

20.

21,

22,

23.

24,

25.

PHYSIOLOGICAL CHEMISTRY

Ringer and Lusk: Ueber die Entstehung von Dextrose aus Aminosiiuren bei
Phlorhizinglykosurie. Zeit. physiol. Chem., 66, pp. 106, 1910.

Ringer: Protein metabolism in experimental diabetes. Jour. Biol. Chem., 12,
1912, p. 431.

Opie and Alford: The influence of diet on hepatic necrosis and toxicity of
chloroform Jour. Amer. Med. Assn., 62, p. $95, 1914.

Schéndorff : Ueber den Maximalwerth des Gesammtglykogengehalts von Hunden.
Arch. f. d. ges. Physiol., 99, p. 191, 1903

Whipple: Insusceptibility of pups to chloroform poisoning during the first
three weeks of life. Jour. Expt. Med., 15, p. 259, 1912.

Graham: The resistance of pups to late chloroform poisoning in its relation to
liver glycogen. Jour. Expt. Med., 21, p. 185, 1915.

Starling and Evans: The respiratory exchanges of the heart in the diabetic
animal. Jour Physiol., 49, p. 67, 1914.

Dakin and Dudley: Glyoxalase. Part IV. Jour. Biol. Chem., lv, 1913-14,
p. 505.
CHAPTER XIX.
PROTEIN METABOLISM OF THE BODY.

In the chapters on digestion and absorption, the course of the protein
taken in the food was traced through the processes of digestion and into
the blood. The simple proteins, it will be recalled, find entrance to the
blood, in large measure at least, in the form of amino-acids, the primitive
building stones of which the protein material of the body is to be con-
structed. Whether some of these amino-acids are synthesized to protein
in passing through the wall of the intestine cannot be positively denied,
but certainly the evidence that any such synthesis occurs, except for the
building up of the proteins of the epithelial cells, is extremely unsatis-
factory. Some of the amino-acids have been destroyed by the action of
the bacteria of the tract and some have lost amino groups and been
changed into ammonia and a carbon residue, possibly ketonic acids like
pyruvic acid, during absorption. It is probable, however, that most of the
amino-acids get into the blood as such. In the blood itself they are found
in very minute amounts, but most of the important amino-acids have been
found there in small quantities. So rapid is the circulation of the blood
and so admirable are the mechanisms for maintaining its composition
that the amino-acids are removed from the blood almost as rapidly as
they find entrance to it; there is not, under normal circumstances, any
accumulation of amino-acids in the blood. It has been shown, however,
by Folin and Denis ‘hat there is always some increase, and not an insig-
nificant increase, in the non-protein nitrogen of the blood after the
ingestion of protein foods. The non-protein nitrogen includes amino-
acids, urea and ammonia, among other constituents. We may now ask
ourselves the question concerning the farther fate of these amino-acids.

We have already at various times touched on these questions and
have considered at length certain aspects of protein metabolism when
dealing with the origin of the nitrogenous substances in the urine. Thus
we have already discussed the purine metabolism, the origin of urea,
the formation of ammonia, the transformation of amino-acids to sugars,
the significance of the creatine and creatinine excretion, and the origin
of various other urinary constituents which arise from the proteins.
Something has been said, too, about the influence of the thyroid gland
on protein catabolism. In this chapter we shall only touch briefly on
certain general questions of protein metabolism: What quantity of

* 799
800 PHYSIOLOGICAL CHEMISTRY

amino-acids are destroyed per day? ‘What is the course of the trans-
formations they undergo when they are decomposed and destroyed in
the body? Whether they are also synthesized in animals as they are
in plants. Has the body any power of storing protein when more pro-
tein is ingested than the organism needs at the time? How much pro-
tein per day must a person take and what are the consequences of taking
more or less than enough?

Amount of protein needed per day by a human adult.—Few ques-
tions of recent times have been more debated than this: How much pro-
tein food must we eat a day in order to keep in the highest state of
efficiency. This is a question of the highest importance in human nutri-
tion. The proteins are the most expensive foods that we consume. It
is nitrogen that is expensive. We may say at the outset that the quan-
tity of protein needed will not be independent of the character of the
protein, since the amino-acids are the substances which are really needed,
rather than protein as such, and those proteins which have all the amino-
acids in about the same proportions as they are found in the body as
a whole will probably be more efficient than those which have an excess
of one kind or another. We shall come back to this question presently.
The minimum amount of protein required by the average human adult
was stated a few years ago by Voit to be about 120 grams of protein per
day. This amount was arrived at by measuring the amount which peo-
ple in moderate circumstances consumed. The idea was that the human
race had been for generations experimenting in order to arrive at this
minimum. Proteins are expensive and difficult to get. In the struggle
for existence which presses so hard on human beings as upon all ani-
mals, it is to be supposed that this amount of food which was so hard
to get would be the minimum upon which a high state of efficiency,
sufficient to conquer in the struggle for existence, could be maintained.
If carbohydrates and fats, which are much easier to get and much
cheaper, could give a more efficient individual, the persons who ate
more of them than of proteins would, in the course of generations, have
survived and supplanted their less sensible brothers. As a matter of
fact, it was found that the races which used less protein, and above all
less dairy products, which werc chiefly vegetarian races, were on the
whole less active, vigorous and progressive. They were the Bengalis of
India and races generally regarded as somewhat inferior and retro-
gressive. It seemed, then, that the point of view of Voit was well taken
and that the minimum requirement for efficiency was about 120 grams
of protein per day for an adult.

This idea was seriously attacked by an American, Mr. Horace
Fletcher, some dozen years or more ago and his work gave rise to a dis-
cussion of the whole matter which has done much to clarify our views
of the réle of protein in the animal economy. Mr. Fletcher being past
PROTEIN METABOLISM OF THE BODY 801

middle life, and being refused life insurance on account of his poor con-
dition, went seriously to work to regulate his diet so as to improve his
condition. In this he was imitating the similar conduct of Louis
Cornaro, an Italian of the fifteenth century, who in similar circum-
stances acted in the same way. After some experimentation both cut
down their diets until they were eating far less food than before, and
Mr. Fletcher particularly cut down his protein consumption. Cornaro
took about 12 ounces of food per day. The physical condition of both
Cornaro and Fletcher greatly improved. A bad catarrh and liability
to catch cold which had troubled Mr. Fletcher quite disappeared. His
general condition was so greatly improved that he became an extremely
active man and was able to do exercises of a physical kind which only
young men in good physical training can do without great fatigue and
lameness. These results were so remarkable that he has devoted himself
since then to teaching the great value of a restricted diet, particularly
for men over forty years of age. The results in Cornaro’s case were
no less remarkable. He lived to be 102 years of age and at 82 and again
at 94 he wrote treatises on the art of living long. The sum and sub-
stance of his prescription was temperance in all things. Neither Mr.
Fletcher nor Cornaro restricted their diet to one kind of food. Cornaro
is not very specific as to his exact diet, but apparently he partook of
the ordinary foods, except fish and some things that did not agree. with
him; he drank wine temperately ; and certainly Mr. Fletcher took what-
ever he felt a desire for. In each case there was a great diminution in
the quantity of food taken.

The results were of such a nature that a careful investigation was
undertaken in this country by Chittenden, a squad of soldiers volun-
teering to serve for the experiment to see what the effect would be of
limiting protein consumption. Mendel, Folin and many others have
contributed to this study.

The general result of this work has been to show that it is possible
to live for a considerable period, at any rate, and apparently in a state
of good health and without loss of weight, on far less protein than the
Voit standard demanded. 120 grams of protein requires a nitrogen
outgo of some 19 grams of nitrogen per day. Most of this of course
will go in the urine, but some will be in the feces. The amount of
nitrogen in Fletcher’s urine was about 6 grams per day; in the soldiers’
in Chittenden’s experiments it ranged from 6-10 grams per day, and
in himself and some of his colleagues and students it fell to a similar
figure. Van Sommeren, a son-in-law of Fletcher, lived on an amount of
protein food so small that his urinary nitrogen was only 4-6 grams per
day and presumably it remained there for a long period, although he
was actually under observation for a short time. Folin, by eating a
302 PHYSIOLOGICAL CHEMISTRY

diet containing chiefly starch, cream and sugar, reduced his nitrogen in
the urine to about 6 grams a day for several days; and Thomas, in
Rubner’s laboratory, reduced his urinary nitrogen on a starch and cream
diet, when a large amount was taken so as completely to cover the energy
requirement, for various short periods during two years, to as little as
2.2 grams per day. This amount of nitrogen corresponds to a protein
intake of only 15-20 grams per day.

It is clear from these experiments that it is possible to maintain the
weight of the body and carry out ordinary exertions, to establish nitro-
gen equilibrium, at a far lower level than the Voit standard required.
So perfect is the mechanism of the body that the utilization of the pro-
tein taken in the food is at a maximum under these conditions. It is
almost completely absorbed and utilized, putrefaction in the intestine
being reduced to a minimum. Furthermore, the general health in many
of these individuals was better than it would have been under their
former régime. It is, therefore, clear that the total nitrogen waste of
the body may be reduced to a very low figure, and it must be concluded
either that the proteins in the body are being torn to pieces very little,
if at all, in metabolism, or else that the pieces into which they are torn
are carefully saved and used over again. Which of these points of view
is correct it is very hard to say, but perhaps modern work has emphasized
the latter possibility rather than the former.

In order to reduce the amount of protein intake to a minimum
while nitrogeneous equilibrium is maintained, that is while the outgo
and income of nitrogen balance each other, it is necessary to cover the
energy requirements of the body by eating carbohydrates and fats, for
if sufficient energy-yielding food is not eaten, then the body tears its
own tissues to pieces to secure the fuel necessary. Furthermore, the
quantity of non-protein food eaten must be more than sufficient to
cover the energy requirement, since there are reasons for believing
that the carbohydrates in particular have the additional virtue of
enabling a partial synthesis of at least some, and perhaps of many, of
the amino-acids in the body from carbohydrate decomposition products
and ammonia or other nitrogen derivatives of protein catabolism. For
this reason they assist in keeping the nitrogen in the body. The total
effect of the ingestion of carbohydrate is, therefore, to save the proteins
ot the body and they and fats are said to have a protein-sparing function
in metabolism. The explanation of this action is not certainly known,
but it may be in part that they are so much more easily oxidized that
they protect the proteins from oxidation in this way; or they may, in
the manner just cited, make possible the resynthesis of amino-acids from
ammonia and other decomposition products of protein metabolism; or
they may be important aids in the anaérobic respiration of cells which
PROTEIN METABOLISM OF THE BODY 803

presumably occurs about the nucleus. Thomas found that in order to
keep his nitrogen outgo down to 2.2-4.63 grams per day large amounts
of carbohydrate had to be eaten. If fat were substituted for carbohy-
drate, the amount of nitrogen in the urine was somewhat increased.
Perhaps this was due in part to the slight acidosis which generally occurs
in the metabolism of large amounts of fats. The fats do not burn so
easily and completely as the carbohydrates to carbon dioxide and water,
but fragments of their molecules, such as acetoacetic acid, are apt to
escape unburned in the urine. This acid is neutralized in part with
ammonia and when it appears it carries out some ammonia in the urine,
thus increasing somewhat the nitrogen outgo.

Since it is the amino-acids which are used to synthesize the proteins
of the body it is necessary, if a real physiological minimum is desired,
that just the right amount of each particular kind of amino-acid shall
be eaten. Since the different proteins contain quite different propor-
tions of the aming-acids, it makes a great difference to the body which
protein is eaten. Dog fiesh nourishes dogs with less waste than any
other kind of protein. Some of the proteins lack completely certain
amino-acids, and if the animal organism is incapable of manufacturing
these acids in sufficient amounts to cover its needs, it will be impossible
to maintain nitrogen equilibrium when that particular protein is used
asafood. For example, gelatin lacks both tyrosine and tryptophane and
it has been found impossible to nourish completely any mammal when
gelatin is the sole protein food in the diet. However much gelatin may
be taken, and however much carbohydrate be added to it, there is a slow
Joss of nitrogen to the body resulting eventually, if the diet is not
changed, in death. Evidently it is impossible for the animal body to
manufacture the lacking amino-acids from the food supplied in amounts
sufficient to cover its requirements. Thomas found a considerable differ-
ence in the power of the different proteins to supply in the most efficient
manner the nitrogen needs of the body. Meat and milk protein could
replace the protein consumed with the greatest efficiency. It was neces-
sary to eat least of these in order to supply the 2.2 grams of nitrogen
which was the minimum outgo. Some of the vegetable proteins were
far less efficient. If the protein minimum was covered by them, it was
necessary to take far more of the protein. Indeed, of the total nitrogen
taken in the form of vegetable protein, sometimes 60 per cent. was
wasted: that is, that proportion of nitrogen was not in a form to cover
the nitrogen minimum of the body. Potato protein was better for the
physiological minimum than either peas or beans or wheat. McCollum
states that bean protein is about one half as good as that of wheat and
corn; and the latter about one-half as good as milk and egg protein.

Recent work on the necessity of other constituents than protein in
the foods (see page 837) makes the interpretation of these experiments,
804 PHYSIOLOGICAL CHEMISTRY

somewhat obscure, since it is possible that the greater efficiency of milk
and meat might be due to the presence in them of some non-protein
constituent necessary to the body but not found in the vegetables con-
sumed. But there is no doubt of the fact that the body can get along
for a considerable time and often with advantage on less protein than
is usually consumed. Rubner in 1883 expressed the opinion that not
more than 5 per cent. of the energy requirement of the body had to be
in the form of protein. The 2.2 grams of nitrogen in the urine of
Thomas, Rubner suggests, came from the bacteria of the intestinal tract
and from the blood decomposition. When doing very hard, muscular
work while on this diet Thomas raised his nitrogen to 2.6 grams per
day. This shows that the muscle substance does not wear out rapidly.
The machinery does not wear out. Rubner thinks that there is a mini-
mum decomposition of 2.0-3.0 grams of protein per day. Since the
whole amount of protein in a man’s body is about 2,000 grams, only 0.1
per cent. goes to pieces daily. If the loss were equally distributed, this
would mean that the protein was renewed once in 5 years. The actual
necessary wear and tear, he thinks, is less than this.

It appears, then, that the amount, of nitrogen wear and tear of the
body is not necessarily very great. This may mean one of two things.
First, that the protein is metabolizing at a very slow rate indeed; fhat
the protein makes actually a machinery of a very stable kind which
moves and organizes the cell, but which does not itself burn, or metab-
olize at a rapid rate, but is moved by the energy set free from the
combustion of the carbohydrates and fats; or, second, it may mean that
the body is able to save and use over again the waste products of the
protein metabolism so that it saves its ammonia. The first possibility is
of considerable interest, since it may be that the stability of the brain
proteins makes possible the stability of the memories of the body. This
possibility is discussed under the chapter on the brain. The second pos-
sibility, however, has much in its favor. It is now certain that the body
has the power of manufacturing some amino-acids from some of the
products of carbohydrate metabolism and ammonia, and it is possible,
hence, that the nitrogen is thus saved to the body and remade into amino.
acids which are used to make good the protein wear and tear.

Is minimum protein desirable? It is possible to live for several years
on less protein than is ordinarily consumed. Is it desirable that the bulk
of the population should reduce their protein consumption so as tc
approach the minimum? Most physiologists are of the opinion that it
is undesirable and that it is safer to provide for a certain excess above
the minimum requirement. In the first place, it is certain that growth
is very dependent upon protein food. For the proper development of
the body it is necessary that protein should be eaten in considerable
PROTEIN METABOLISM OF THE BODY 805°

quantities. There is hardly a doubt that the increase in the average
stature of the population of this country is due, in part at least, to
better nourishment of the children. Growth is stunted by too little
food. For growth protein is needed. Nature probably had to solve
this problem by blind experimentation and the food provided for the
rapidly-growing young is always protein food. Milk contains as much
protein as it does carbohydrate or fat; young birds are fed on worms,
larvee and insects, even though the adults may be graminivorous. But
there are reasons even in adults for the excess of protein consumption
above the minimum. While there is no protein storage in a narrow
sense, there is certainly a reserve power which a well-fed person has
and which an ill-fed one lacks. The muscles and cells of the body
full of living matter have certainly a greater vitality and a greater
resistance to disease than when they are depleted. Experiments have
shown that the resistance of rats and other animals to snake venom’
is greater when they have been fed protein than when they have not
been fed protein. It may be that the difference is due not to the pro-
tein, but to other constituents of the diet, but in our ignorance of what
those constituents are it would appear wiser to eat the food which contains
them. The whole matter is, hence, in an unsettled state. We are con-
fronted, on the one hand, with the fact that long life is usually accom-
panied by a temperate disposition, and temperance in eating and drink-
ing; and that many people, particularly those past middle life,'are bene-
fited by reducing their protein; on the other hand, peoples of great
vigor are generally heavy protein consumers, and for the young, cer-
tainly, a plentiful protein diet of a special kind seems to have been that
elaborated by nature after many experiments.

Will the body store protein? The human body has the power of
storing both carbohydrate and fat. If one eats more carbohydrate food
than is necessary to cover the energy requirements of the body, it is
not at once completely burned and got rid ‘of, but up to a certain point
it is stored either as glycogen in the liver, muscles and some other tissues,
or it is converted into fat, and deposited as such in the great fat reser-.:
voirs of the body, which lie under the skin or about the internal organs.
With proteins the matter is quite different. It is true that many plants
have the power of storing proteins in their seeds and in other tissues.
Nuts generally contain stored protein. The proteins in these cases are
dead, reserve proteins. They are more stable than the usual proteins
and they are often laid down in crystalline deposits in. the cells. Some
animals also store a small amount of protein in eggs to serve as food
for the developing embryo. But it is always found that the cells which .
thus store protein have a metabolism slower than usual. Hither they
store nrotein because their metabolism is small, or else the accumulation
806 PHYSIOLOGICAL CHEMISTRY

of this mass of inactive protein checks their metabolism. In the adult
human being there is certainly a very limited storage of protein. If one
doubles the amount of protein necessary to replace the wear and tear
of the protein of the body, in a well-fed person the sole result is to
increase the output of nitrogen. If we eat 200 grams of protein per
‘lay, the body does not retain this, but it is at once oxidized and got
rid of. The nitrogen is increased in the urine to the same extent as
it has been increased in the food. It is not retained in the body. It is
only after a long fast, or particularly after a prolonged low protein
diet, when the body has been covering its needs with carbohydrates and
saving its proteins, that there is a retention of nitrogen in the body.
And this power of retention is not very great. It is. for example, after
wasting diseases when there has been a great loss of muscle substance,
or after hemorrhage when there must be a rapid reformation of blood,
that protein storage occurs.

In fact, so far is it from being the case that eating protein leads to
protein storage, that the reverse is true. A large protein diet far in
excess of the protein requirements leads to a consumption of fat, so
that the body is thin and may actually lose weight. Proteins have a
certain specific action. They stimulate heat production. If one eats
more protein, it is not as it is with the fats that the excess is stored.
but it is burned at once, so that a large protein diet means an increased
output of heat. The heat production is at a minimum on a low protein
diet. In rest it may then sink to 2.000 calories per day; whereas on a
high protein diet even at rest it rises to 3,000-3.500 calories per day.
It is as if the fats burned in the heat of the proteins, for one way of
getting thin is to eat large amounts of protein (Banting cure).

The explanation of this peculiarity of the proteins when contrasted
with the fats and carbohydrates has not yet been given in its entirety.
but it is not impossible that it has the following teleological explanation.
The proteins make part of the real living matter. It is impossible to
increase the living matter of the cell, or the living matter of the body
as a whole, beyond the powers of the blood to supply oxygen to keep
it alive. If more living matter is formed than can be supplied with
oxygen, hydrolytic or autodigestive processes are set at work which
digest the protein and thus tear down that which has been formed. The
amount of the living matter is evidently limited by the ratio of bulk
to surface and to the possibility of supplying oxygen. This may be the
way in which the amount of living matter is limited in the body. Pro-
tein cannot be laid down in the protoplasm in the form of dead or
reserve material without seriously checking the metabolism of the cell.
Stable, inert proteins are found only in those cells where the metabolism
is not very intense. If it be asked how it happens that protein is not
PROTEIN METABOLISM OF THE BODY 807

deposited in cells in spite of the possibly deleterious result of such a
deposition, we have to confess that we know very little about it. The
experiments of Ascoli and others on uricase and its variation in
the liver of fasting and well-fed individuals appear to be full of sig-
nificance. It will be recalled that in bird’s and dog’s liver the enzyme
to destroy uric acid disappears when the diet is restricted and reappears
when the diet is plentiful. This is evidently in the nature of an
adaptive metabolic change. When the diet is restricted, or during
fasting, it is possible that the uric acid is needed to replace the waste
of the nuclein material. The activity of the uricase disappears under
these circumstances. Feed the body well and destruction of uric acid
results. The simplest explanation suggesting itself is that some of the
food decomposition products give rise to the enzyme which destroys uric
acid. But whether this explanation is correct cannot be said. Perhaps
it is the same with the amino-acids. After fasting we know that the
destruction of amino-acids is greatly restricted and the organism builds
them over into protein to replace that which has been used up. It may
be that the enzymes which destroy or hydrolyze the proteins, or oxi-
dize the amino-acids, are reduced in quantity, so that the speed of the
destruction of the amino-acids is reduced. On the other hand, when
there is a luxus consumption of the amino-acids, perhaps some of their
decomposition products are converted into catalytic agents which hasten
the decomposition of the amino-acids. Consequently the oxidative
decomposition is greatly increased and the heat of the body increased.
Whatever may be the exact mechanism by which a storage of protein
is prevented, there is no doubt of the fact that very little storage occurs,
but that excess protein is torn to pieces, and the nitrogen eliminated as
urea. Heat production is at the same time increased, and there appears
to be a stimulated decomposition of the fats. Of the non-nitrogenous
part of the protein molecule a portion at least is converted into glycogen,
as has already been discussed on page 774, and may be stored as
glycogen.

Catabolism of proteins.—The question we have now to ask is a very
fundamental one and, like most fundamental questions, we cannot
answer it. The question is this: What is the course of the metabolic
decomposition of the proteins of the cells of the body? These proteins
are complex, conjugated, colloidal proteins. Do they undergo oxidation
or deaminization while they are in this form; or is the first step in their
catabolism a digestive process which results in setting free the amino-
acids and other constituents? And are these fragments then oxidized,
or first fragmented by fermentation and the fragments oxidized? It
will be recalled at the outset that the proteins with which we are deal-
ing, that is the real, organized, protein basis of living matter, is not
808 PHYSIOLOGICAL CHEMISTRY

a simple protein, but it contains in its molecule certainly phospholipin,
possibly various enzymes and carbohydrate and other material, some of
it inorganic. Its composition is probably illustrated by the composition
of the blood platelets or the stroma of the red blood corpuscles, or we
might even say of the red blood cells as a whole, while they still have
in them hemoglobin. Presumably in the red blood cells the composi-
tion of the protein is represented by a compound of hemoglobin-phos-
pholipin-protein-lipase-cholesterol-potassium. This compound, if indeed
it be a chemical compound, is known to be very unstable and a great
variety of agents cause it to decompose. On the whole, the evidence
is favorable to the view that something similar happens in all cells and
that the first step in the catabolism of the proteins is a decomposition
of this complex, and the digestion of its constituents. Thus it has been
found that in all cells there ensues on death a digestion of the protein
material with the appearance of the splitting products of the simple and
conjugated proteins. This digestion is known as autolysis. Thus in all
tissues, as soon as they die, there is a digestion of the purine bases,
adenine and guanine, ammonia is set free and the deaminized bases,
hypoxanthine and xanthine, are formed; the bases may also be split
free from their union with the carbohydrate or phosphoric-acid group.
Lipases also become active and a digestion more or less extensive of
the fats and phospholipins occurs; the simple proteins are digested with
the appearance of albumoses and amino-acids and other peptides. We
have, then, in cells after death the appearance of digestive enzymes
which deaminize and decompose, or hydrolyze, a great many of the cell
constituents and among them the proteins. Nearly all cells yield pro-
teolytic enzymes of the erepsin type and of the deaminizing type.
Nearly ‘all of these digestive actions occur best in a very faintly acid
medium and they are checked or prevented by the addition of a little
bicarbonate of soda. This is particularly true of the proteases. It has
been suggested, and seems on the whole very probable, that these enzymes
do not begin their work only at the moment of death when the reaction
of the cell has become acid, but that they are more or less active all the
time, but that normally their activity is reduced either by the presence
of antibodies, or else by the reaction, or else that the synthetic power
of the cell is so great that in spite of their action the cell is not
destroyed. It would seem probable that the first step in catabolism was,
then, a hydrolysis of the proteins and that the oxidation or fermentation
of the products set free succeeded this. This view, however, has not
been universally accepted. It is a very singular fact that it is impos-
sible to isolate these digestive enzymes before autolysis has begun. It
is the same difficulty which was noted in the case of the decomposition
of the glycogen in the liver; the enzyme appears only in very small
PROTEIN METABOLISM OF THE BODY 809

amounts and after digestion has begun. A potato, although it under-
goes hydrolysis of its starch very easily after lying for a time, seems
to have no diastase in it when the potato is green. In other words, the
diastase appears when the digestion begins. A quite similar fact is
noted in the clotting of the blood: no thrombin can be isolated from
the plasma, but only from the serum after clotting has occurred. The
question in the clotting of blood is the same as in the digestion of the
proteins, Is the enzyme which appears a result or a cause of the clot-
ting or digesting process? No doubt it appears most probable that
the enzyme is present and active even during life, but its activity is
checked in some of the ways stated. Tissues waste away in starvation,
even though they live, and this is probably due to a partial hydrolysis.
There are not wanting those who maintain, however, that the enzyme
is not the primary cause of the catabolism in the living tissue. The
reason why the enzyme is not to be found in living active cells may be
illustrated by the phenomena of the clotting of the blood. The blood
platelets, according to Wooldridge, are of the nature of crystalline
products. Now, if they are examined fresh, no fibrin ferment can be
extracted from them, but if they are allowed to clot first then they
yield fibrin and thrombin. They also yield an hydrolytic enzyme which
has the pewer of digesting the fibrin and producing fibrinolysis. The
probability seems to be that the enzymes in cells are in union with the
substances upon which they act, they are in union with their substrates
and so cannot be extracted ; under certain conditions this union is stable
and the substrate is not affected by the enzyme, but under certain other
conditions, and a very slight reduction in alkalinity appears to be one
of them, the reaction is consummated, the compound is hydrolyzed and
both the hydrolytic product and the enzyme appear free at the same
moment, just as fibrin and thrombin appear simultaneously. If this
view is correct, the protoplasmic proteins already have combined with
them the various enzymes which under different circumstances decom-
pose them by autolysis. Perhaps under certain conditions these same
enzymes have been responsible for the synthesis of the proteins which
they. digest under other conditions.

By this autolysis of the proteins amino-acids are produced. These.
amino-acids are either fermented, carbon dioxide and amines being
formed from them; or they are oxidized with the formation of certain
substances, among them ketonic acids, ammonia and aldehydes. UIti-
mately the nitrogen residues escape from the body chiefly as urea;
while the carbon residues are in part at least changed to glucose and
glycogen in the manner indicated in the previous chapter.

Course of the oxidation of various amino-acids in the body. The
course of oxidation of various amino-acids in the body has been studied
810 PHYSIOLOGICAL CHEMISTRY

by Neubauer, Knoop, Embden, Dakin and others by perfusing the liver
with blood containing the amino-acids, by obtaining their decomposition
products from the urine and by oxidation with hydrogen peroxide.

The general course of the oxidation of the simple amino-acids is
first to form by oxidation the ketonic acid and ammonia. A subsequent
oxidation converts them into the acid of the next lower series by the loss
of carbonic acid. According to Dakin the aldehyde is formed as an in-
termediate product. The course of the oxidation of the simpler acids
is shown by the following reactions. In not all cases has it been actually
shown that the decomposition follows this rule, but it has in many of
them and it is probable for the others.

NH,CH,—COOH +0 —~ CHO—COOH + NH,
Glycocoll. Glyoxylic acid.

The reaction probably goes in two stages (Knoop and Neubauer), the
oxyamino-acid forming first:

NH = CH—COOH + H,0

we Imino-acid.
NH,CH,—COOH + 0 —~+ NH,CHOH—COOH
Glycocoll. Hydrated imino, O = CH—COOH -- NH3

intermediate stage. Glyoxylic acid.

The hydroxy amino compounds may be regarded as hydrated imino.
acids. It will be remembered that oxygen and NH are very similar in
many of their properties and mutually replace each other in com-
pounds.
Alanine on oxidation forms pyruvic acid, thus:
H —CHNH,— HL —~+ CH,—
ee gig ae

On further decomposition this acid yields by oxidation acetic acid
and carbon dioxide:

CH,—CO—COOH —_+ CH,—COH + CO,; CH,—COH + 0 —-~ CH,—COOH
Pyruvic acid. Acetic aldehyde. Acetic acid.

Relation to hydroxy acids. The amino-acids are converted often
into hydroxy acids. Thus in perfusing the liver with blood containing
alanine, lactic acid was obtained. Similar hydroxy acids have been ob-
tained also from other acids. The hydroxy acid is not formed by a
direct replacement of the amino group by hydroxyl, but by the reduction
of the ketonic acid which is formed by the oxidation of the amino-acid
and the subsequent elimination of ammonia. From alanine pyruvic acid
is formed in the way described and this is by reduction converted into
lactic acid.
PROTEIN METABOLISM OF THE BODY 811

CH ,--CO—COOH + 2H ——> CH, —CHOH—COO0H
Pyruvie acid. Lactic acid.

Lactic acid may, therefore, arise from the proteins as well as from the
carbohydrates.

The evidence that lactic acid is not first formed and then oxidized
to pyruvic acid is the fact that a substituted lactic acid, such for ex-
ample as p-hydroxyphenyl-lactic acid, does not yield homogentisic
acid when administered to an alcaptonuric, whereas p-hydroxyphenyl-
pyruvic acid does yield it. This observation shows that the hydroxy acid
cannot be in the normal course of oxidation of tyrosine; and that
p-hydroxyphenyl pyruvic acid is probably in the chain of normal oxida-
tion. The relation between the hydroxy acids, the ketone and amino
acids may be represented as follows:

CH .- CHOH—COOH
“ol fr
CH,—CHNH,—cooH o CH,—CO—COOH + NH,
2 O
CH,—COOH +- CO,

[asl

It is not impossible, however, that the exact course of the transforma-
tion may not be correctly represented by the foregoing scheme. It may
be that an unsaturated acid is formed at the outset of the reaction by a
deaminization and that this unsaturated azid is subsequently oxidized.
to the hydroxy or oxy acid. An amino group behaves in general like a
hydroxyl group. Hydroxy acids, like lactic acid, easily lose water and are
transformed into the unsaturated acids like acrylic acid. However the
f-hydroxy acids undergo this transformation more readily than the
a-acids. The reaction would be as follows:

CH,—CH.NH,—COOH —~- CH, =CH—COOH + NH,
Amino propionic acid. Acrylic acid.

By subsequent hydration or oxidation acrylic acid might be converted
into the hydroxy or the ketonic acid:

1. CH, = = CH—COOH + HOH—~CH ,--CHOH—COOH

Acrylic acid. Lactic acid.
CH, = CH—COOH +0 ——+ CH 3" CO—COOH
Acrylic acid. Pyruvie acid.

Such an unsaturated amino-acid has been obtained recently by Hunter
from dog’s urine, namely, urocanic acid. It has also been obtained
from a pancreatic digest. It is derived from histidine by deaminization:
812 PHYSIOLOGICAL CHEMISTRY !

CH—NH\

A a
be -
Ue CH

|
COOH
boon is Dest oats
Urocaric acid. Cinnamic acid. \
(Imidazolylacrylic acid.)

The corresponding derivative trom phenyl alanine, called cinnamic acid,
is found in oil of cinnamon, balsam of Tolu and elsewhere. An isomeric
cinnamiec acid, the alpha-phenyl acrylic acid, atropic acid, is obtained
from the alkaloid, atropine. The evidence at present, however, favors
the Knoop view of a preliminary oxidation before the desaturation.

Tyrosine and phenyl alanine. By oxidation tyrosine is converted
probably in the first place to para-hydroxyphenyl pyruvic acid, and
phenyl alanine to phenyl pyruvic acid. .The isolation of these acids
has not been accomplished from the urine after ingesting tyrosine,
but indirect evidence shows their formation. Their further fate is very
interesting. Under certain not well understood conditions a peculiar
acid, homogentisic acid, appears in the urine. This acid is a dihydroxy-
phenyl acetic acid and has the property of turning the urine dark on
standing by the formation of melanin by the spontaneous oxidation
of the acid. That this substance is derived from tyrosine is shown by
the fact that the administration of tyrosine to alcaptone patients
increases the amount of homogentisic acid. This acid differs from
tyrosine in that the hydroxy group and the acetic acid radicle are not
in the para positions to each other, so that to understand the formation
of homogentisic acid from tyrosine it must be surmised that a rear-
rangement of the hydroxy groups takes place. This will be obvious from
the following formule:

C—CH,\-CHNH » COOH C—CH,—COO0H C—CH,—CHNH,—COOH

war @ a
= AL HC \ con HC Me Nox
| I| |

a y CH HOC CH HC ber

\ \ \ ZF

CH s of : Ca
Tyrosine. Homogentisie acid. Phenyl alanine.
3, hydroxyphenyl- 2.5, dihydroxyphenyl acetic

propionic acid. acid.

A similar transformation has, however, been observed by Bamberger
and ‘others, showing that such rearrangements are not uncommon and
that they are due, probably, to the intermediate appearance of quino-
noid derivatives formed by oxidation. Neubauer’s explanation of the
PROTEIN METABOLISM OF THE BODY 813

formation of homogentisic acid includes this explanation and is illus-
trated as follows:

OH
C—CH,—CHNH,—COO0H C—CH,—CO—COOH | ;
ON ON C—CH,—Co—COOH
HC CH HC CH JOS
| | es it i! —= Bc CH
HC CH Hi CH \| \|
SZ. 7 HC CH
COH COH \
para-hydroxyphenyl pyruvic c=O
Tyrosine. acid. Quinonoid form.
ie i
Cc Cc
oN ON

HC C—CH,—CO—COOH HC C—CH,—COOH + CO,

| |
HC be ae ub be

\ Con \ Zon

Homogentisie acid.
2.5 dihydroxyphenyl pyruvic 2.5 dihydroxyphenyl acetic
acid. acid.

Homogentisic acid is possibly a normal product of tyrosine oxidation,
which is usually further oxidized. On the other hand, it is possible that
it is usually formed only in small amounts, and that norrially most of
the oxidation goes either directly from the quinonoid to the further de-
composition of the tyrosine and relatively little is first carried over to
_ homogentisic acid, or it does'not go through the quinonoid form at all.
' Dakin found that when para-methyl-phenyl alanine and para-methoxy-
~phenyl alanine were administered to aleaptonurics they did not go over
into homogentisic-acid derivatives, but were completely oxidized. These
-substances cannot form the quinonoid intermediate products. From this
it would appear more probable that the quinonoid form was not usually
- gone through in the oxidation of tyrosine, but that most of the oxidation
went directly from the dihydroxy acid.

Formation of acetoacetic acid. Acetoacetic acid, .CH,.CO.CH,.
COOH, is formed by the oxidation of butyric acid and is nearly the final
; ;Stage in the oxidation of the fatty acids. It is a substance of very great
‘interest because of its appearance in the urine in diabetes and under
‘various other circumstances. It is also a very reactive substance and
acetoacetic esters form the starting point of some of the most funda-
mental syntheses of organic chemistry. It is extremely interesting that
' -acetoacetie acid is produced, also, from the proteins and from several
amino-acids. It has been shown by Dakin to be formed by the oxida-
tion of tyrosine and phenyl alanine. In this case two of the carbons
PHYSIOLOGICAL CHEMISTRY

814

Up Pozesoxa seouvzsqng)

‘0D Y udseixy ‘suvmBuoT ‘Ty ‘d ‘Zier

(-uxr0y peuquicoun 9} Ul paplooas ox OUTOLTS 41M TOT} BUIQUIOD UL MISIUeBI0 OTF

“Apoq [eullus oy} UL sUOLNpet PUB BUOT}EPIXO * ULC »

 

 

oP ” ” reess"*"* 000'00 “HO *(HQ)"H ‘pyoe oyansAd[Auayddxorphyi ‘e°% 1B
F ” gf) RA “""* 7000°00" HO ( OF "HO ‘poe opansddiAuayddxoyyour-d ‘9g
+ i ze Beene meee ee eee meee e nn neeene tererersoess pie oranssd[Auayqd£yjeu-d eS
afi. | © = . ; a is Fe ee meee eer een eee rete rer eeee sploe oranidd [Aueyd 4xoipéy-w “9 &d Ss
+ + uOLeprxo aqozduno:) eee ers dete e aden ner enenreeeneres H000'00"'HO"H’) ‘prow oransdd [Auayg ‘gz
H000'(HO) ay? gftttt O56: 8) 88 Rorvera ge a GUMTREE s.6 Ghee 5 kw eRe spree aruordosdAuayd fxoapAy- -d ‘-m “o BS
HOOD" *H’) acca gueMerees wee ie 89S RNS eS ae Sees bee ha poe oruordoid4xo1p4y-q-[Aueyg "IZ
+ UOL}eprxo aqatdui0g PER mem eee meee ee rere ener erences pre o1jouj-v-[AuayddxorpAqip eZ 02
9
— HO00"H’) Peas olay Ste AG Uaa Se We Reece Se 6 euereye! < HOOO'HOHO'HOHO’ HO ‘o11a04|3-[Aueqg “61
a es 3 Ce eee ere meme eer ere errr ene trees ploe o1ovy-e- -[Auayddxo1pAy-01 pue £0 ‘SI
= ger ee daapep ll Rn Raa An Gop pase te pa a a Sp oe a Lee
Tayyqnog { atiaMin MaBEpIED . proe oryovy-e-[Auoyddxoipéy-d 9 “LT
+ + uorqeprxo ye{dmog a eae eee Tete ett eeeee eee eerees BOQD HOHO" “0° “AO ‘pyoe anoer-e-[Aueyg ‘OT
HO0O0"H’9)|" 2 SOC S Ae 8 ee ee ness "HOOD! “HNHO'HOHQ” H'0 ‘ures [Auaya “CL
A[rpeat peziprxo 4ON ae he a ee OOO’ "HO" “INHO" “A 85 ‘aulueye- -g- [Auaqg FL
+. a i 3 (RES Breer cene BS er ge caepeegeinet aie deaiceniel ww p lenanieyiel dep Seceuiere aurueye,AueydAxoyjzom-d ‘eL
uolyepixo
+ { anes t 5 ss o- teeter eee e en eeee Fs NC Ea Sie ees cece ee eee ee ‘ZI
= ” ay RAS PONE SL ESSE”! FAOOD *HNHO* “HO (Ho)* TH 0 ‘oursox4oporp ‘oe “Il
+ + “ * eas oh ee ewe neee ene 2 2.5 Gay se ae HOOO0" “HNHO" HO “(Ho)” HH” 9 ‘gursoi J, ‘OL
+ oF woryeprxo aqordmo) rng Be oe gies ae eer ee * HOOD’ “HNHO "HQ" HO ‘ouruvyepAueyg ‘6
9
a BODE) et ci ENA sem mean eaent ees “<* HOOQHO = HO’ yo ‘pion ormreuatg °g
= = HOO ge HOOO’ "HO? HO" H’ D (bree ormorderd|énong “y
ae a, HOOO' HO rea eee rere ee reeee HOQO" HNHO" *y? 9 ‘oryaovourmeAueyg ‘9
+ + Woryeprxo ayojduiog} "st “9 O00" HO" (HO) "H°O ‘PPE osyUaBoMoH “¢
% ay [eater Ss Prttttersterseeees spre onjaoeiduayddxosphy-d pue “mu “0 “F
2a peziprxo gon] F24 quaite-s Sa phdy bas SNe os SeUNINE 45 H009" “HO, “y, ®%5 ‘poe orjaoe [Auayg ‘e
9
1000" HO" sa.) capers 2 6 ies LAS toe Seecten, 8 Se eierosye ss 900" *HO" “HO ‘apAypprejeoetAueyg °Z
eee HOOD’ HO" H ) aneia; 8 Ea wen meee ewe wees ce eRe HO HO’ HO H oO ‘qoyoore[ Ayo, Auoqg 'T
*IOAL[ SUL
TATAIUS UIQ}! «olny deuys
Pesnjied | 02 Waals GAYM | gg raeF10 [UNION Uy IB aoueysqug
way uo} | TonsUIIOJ pos
“BULIOJ plow] oIsIynesoUO
9199080129

 

 

 

 

“( » UIyeq) Sal0y OLLVNOAY ATHLO GNV ANINVIVIANGHG ‘ANISOHAT, JO NOILVGIXQ
PROTEIN METABOLISM OF THE BODY 815

of the acetoacetic acid are derived from the benzene nucleus of the
aromatic acids. The researches of Jaffé show that when benzene is
given to dogs small amounts of muconic acid may be isolated from the
urine, thus showing that the benzene ring is broken in the course of
metabolism. The reaction is as follows:

CH COOH
HC ‘ CH HOOC CH
ue Was ut . thea
NoG \
Benzene. Muconic acid.

Dakin gives the following scheme for the possible formation of
acetoacetic acid from pheny)] alanine:

COOH COOH COOH
| | |
CHNH, co co COOH COOH
| | | | |
ie a ale’ ie
Cc — C — c= —-+ C= —~ CO
a TNS | | |
my a i 1 CH cH cH,
eee ia eaoey:
HC CH CH . s
f Z CH CH Acetoacetie acid.
\& No | |
Phenylalanine. Phenylpyruvic acid. CH cH co,
I! II
CH CH H,0
| |
HC = HC=
Open chain
phenylpyruvic |
acid.

The formation of acetoacetic acid from histidine has already been
mentioned.

Decomposition of arginine. The easiest and most direct decomposi-
tion of arginine is the splitting off of urea from it by the action of the
enzyme, arginase, found in the liver and various other tissues by Kossel
and Dakin:

NH,—C—NH—CH,—CH ,— CH, --CHNH,—COOH ++ H,0—
I Arginine.
NH
co (NH,), --+ NH 3 CH,—CH — CH,—CHNH,—COOH
Urea. i Ornithine.
816 PHYSIOLOGICAL CHEMISTRY

The further fate of the ornithine and the arginine is unknown. The
possibility that creatine may result from the intermediate formation
of guanidine butyric acid has already been discussed on page 716
in connection with the origin of creatine. There is no satisfactory evi-
dence that arginine forms creatine in this way in the body, although
it is not improbable. The further fate of this most interesting sub-
stance should be studied. Its close relation to the cell nucleus in
protamine makes its fate of particular interest. In the urine of some
people having an abnormal secretion of cystine, in cystinuria, Bau-
mann found unusual quantities of the ptomaines, cadaverine and
putrescine. These bodies almost certainly come from the amino-acids
lysine, arginine and ornithine by a process of decarboxylization:

NH,,.CH,.CH,,. CH,.CH,.CHNH,.COOH —-~ NH,CH,.CH,.CH,.CH,.CH, NH 2 + CO,

Lysine. Pentamethylenediamine
(Cadaverine).
NH,CH,.CH,. CH,.CH NH,.COOH —~> NH,.CH,.CH,. CH,.CH,NH, + co,
Ornithine. Tetramethylenediamine
(Putrescine).

It is unlikely that more than a small part of the arginine or lysine decom-
position normally takes this direction.

Sulphur metabolism of the body.—Decomposition of cystine. While
the attention of physiologists and physiological chemists has been directed
in the main to nitrogen in connection with protein metabolism, the
possible réle of sulphur which makes a part, albeit but a small part,
of the protein molecule has of recent years come more into the fore-
ground. Nearly all the proteins of the body, both the living proteins
and those of the circulating liquids, contain a small amount of sulphur
and this sulphur is in an unoxidized form. It is in the form of sulphide
sulphur. Most proteins contain from 1-2 per cent. of this sulphur. It
occurs in the protein molecule either in cystine or cysteine and possibly
in some other similar acids, although no others have been positively
identified.

The sulphur income of the body is almost altogether in the form
of the sulphur of the proteins. Some of the foods, indeed the majority
of them, contain small amounts of sulphates, but these sulphates do
not, so far as we know, enter into the living matter of the body, although
in small amounts they are to be found there. The per cent. of sulphate’
in the blood and tissues is small. It is not certain that sulphur taken
in the form of sulphate is reduced in the body. It is more probable that
it remains oxidized and while it may contribute something to the forma-
tion of the paired sulphates of the urine, even this is uncertain. There
is taken on the average about 110 grams of protein per day. In
PROTEIN METABOLISM OF THE BODY 817

{lis protein, sulphur makes about 1.2 per cent. This would mean
1.32 grams of sulphur income per day in the form of unoxidized
sulphur.

Sulphur leaves the body for the most part in the form of oxidized
sulphur. The amount of sulphuric acid excreted per day is about 2.5
grams. This is excreted as the sodium or potassium salt, principally the
first. About two-thirds of the sulphur leaves in this form. The other
one-third is excreted for the most part as so-called neutral sulphur in the
form of conjugated sulphates, being united with indole, scatole, cresol,
or phenol, as the case may be. There is also present a small amount
of unoxidized sulphur, consisting of cystine, or polypeptides containing
cystine, or some other sulphur compound.

In the intermediary metabolism of the body, that is the metabolism
of the tissue, sulphur probably plays a very important réle. This is
shown not only by the fact that it is absolutely necessary for the con-
tinued existence of the body, as necessary as nitrogen or any of the
other elements, but also by the fact that it is one of the most labile ele-
ments of the protein molecule. No other element is split off from the
proteins with greater ease than this. It is, indeed, the labile element
par excellence. Moreover cysteine, which is one of the amino-acids,
readily oxidizes itself. It is a reducing body. It oxidizes spontane-
ously and there are many points in its oxidation which strongly
resemble the processes of respiration. Thus the most favorable concen-
tration of hydrogen ions for the oxidation of cysteine is the same as that
in protoplasm; both cysteine and protoplasm are poisoned by many of
the same substances, such as the nitriles, the cyanides, acids, and the
heavy metals; their oxidations are catalyzed or hastened in the same
manner by iron, arsenic and some other agents. For these reasons it has
been suggested by Hefter and the author that there is more than a
superficial connection between the oxidation of cysteine and the respi-
ration of the cell.

If we try to follow the course of the absorbed protein sulphur
through the body, we find that the fate of the cystine set free from
the proteins by the digestive action of trypsin and erepsin is not known
in all its details. Some is decomposed by the bacteria of the alimentary
tract, forming hydrogen sulphide, a toxic and ill-smelling gas, which,
when absorbed in quantities, dissolves red blood corpuscles and con-
tributes, no doubt, to anemia; some cystine is probably absorbed as
such. In the liver there is a quantity of taurine in taurocholie acid.
Taurine is produced from cystine, or rather from cysteine, by a car-
boxylic decomposition. Perhaps thioethyl amine may be formed first
and then the sulphur oxidized to sulphuric acid, or oxidation may occur
first and decarboxilation second.
PHYSIOLOGICAL CHEMISTRY

HS—CH,—CHNH,—COOH _— HS—CH,—CH,NH, + co,

Cysteine. Thioethy!] amine.
HS—CH,—CHNH,—COOH + 30 —~ HO,S—CH,—CHNH,—COOH
Cysteine.
HO,S—CH,—CHNH ,—-COOH——~ HO,S—CH,—CH,NH, + ©O,
Taurine.

A portion of the cysteine probably passes the liver and is picked out
by the tissues of the body. It must circulate in the blood as cystine,
since it would at once be converted to cystine by the oxygen of the blood.
When it enters the cells of the tissues it is probably, in part, built up
into the proteins of the tissue: in part it is probably carried over into
taurine since taurine is found in a great variety of tissues. Taurine is
an abundant constituent of the extractives of molluscan muscle, particu-
larly of the muscles of cephalopods; it is found also in the mammalian
brain and indeed there are few tissues without some. Whether all the
decomposition of cystine takes place through taurine is unknown.
Possibly a deaminization may occur, leading to a ketonie acid, but no
such compound has as yet been found. When cyanides or nitriles are
given to mammals they appear in the urine in the form of sulpho-
cyanates, from which it may be inferred that they unite in the cell
with the unoxidized sulphur, which in some way they cause to be de-
tached. This union with sulphur does not seem to occur in birds. From
the fact that the cyanides have such a remarkable inhibiting effect on
respiration it has been inferred by some that sulphur must be very
important in respiration.

It occasionally happens that some individuals excrete more cystine
than normal. They have a cystinuria. This cystine comes from the
tissues of the body, since cystine given in the food does not increase the
amount excreted under such conditions. The cause of this metabolic
anomaly is still completely unknown, but it may be due to a great
hydrolysis of some of the proteins of the body, since there often are
found in such cases more than the normal amounts of the other amino-
acids, such as tyrosine and leucine, and since the bases, cadaverine and
putrescine, may occur in the urine at that time.

Cystine was first discovered, as its name implies (kystis, bladder) in
bladder stones, which often contain cystine or are composed of it. The
meaning of the excretion of this substance was long obscure and it is
worth while to examine how information was obtained that cystine was
a normal product of the body metabolism, since the method employed
for the solution of the problem was a general one and often used for
the solution of similar problems. The question was whether the cystine
which appeared at times in the urine was a normal constituent of the
body metabolism, usually present in very small amounts, but now in-
PROTEIN METABOLISM OF THE BODY 819

creased, or whether it represented a wholly new and strange metabolism.
This problem was settled by Baumann, who at the same time introduced
a very valuable method for the study of the intermediary metabolism
of the body.

In the course of metabolism many substances of a very unstable
nature are produced. They have a very temporary existence, since,
being unstable, they are normally quickly oxidized and we can only
guess at their existence, or infer from general principles that they
may be formed. Now it is exactly these intermediary substances which
are of the greatest importance in metabolism. From one such substance
it is often possible by taking slightly different courses to go to several
different end products; and we must know what these substances are
in order to understand the nature of the metabolism of any single sub-
stance. One way of finding out what these substances are is by com-
bining them with something so as to make them stable and thus cause
them to escape, or to pass unscathed through the fire of metabolism,
coming like Shadrach, Meshach and Abednego to testify to the truth or
falsity of our faith. Baumann discovered that cysteine was such an
intermediary metabolic product. He found that when bromo- or chloro-
benzene was given to dogs there appeared in the urine a very remark-
able complex, namely, bromo-phenyl-mercapturic-glycuronic acid. In
this complex was cysteine. The composition of this mercapturic acid

was as follows:
BrH ,C,—_S—CH,—CH—CO0H

bu—co—cu,

Bromophenylmercapturic acid
(Bromophenylacetylcysteine) .

The brompheny] had united with the sulphur of a sulphur complex
which was acetylated and appeared then conjugated with glycuronic
acid. Here we have, in this complex, three substances of intermediary
metabolism: cysteine, acetic acid and glycuronic acid. The acetylation
has already been discussed. It may come from the union of am-
monia with a molecule of pyruvic acid and possibly a molecule of the
ketone acid, corresponding to cysteine, namely:

HS—CH,—CO—COOH
Thiopyruvic acid.

The reaction might take this course:

HS—CH,—CO—COOH HS—CH,—CH—COOH
“of |
NH, = NH + CO,+H,0

+ |
CH,cO—CO00H CO—CH,
820 PHYSIOLOGICAL CHEMISTRY

This discovery proved that cysteine, which did not appear at all in dog’s
urine, or was not recognized at that time, but which did appear as cystine
in certain cases, was a normal intermediary product of metabolism.

We do not know the method of the decomposition of cystine in
the metabolism of the various tissues. Is the sulphur of cysteine
oxidized while the latter is still a component of the protein molecule,
giving a sulphuric-acid group attached to the protein? Can it be for
this reason that so small a proportion of the total sulphur of most pro-
teins is to be recovered in the form of cysteine? (See page 151.) Is the
sulphur split off as sulphureted hydrogen, which is later oxidized to
sulphuric acid, the cysteine persisting as serine? Or is the cysteine split
out of the protein molecule by the action of autolytic enzymes, then
losing carbon dioxide and with the sulphur oxidized to sulphuric acid,
giving taurine? These questions are not yet answered. One thing at
least is certain, namely, that in human beings most of the sulphur is
oxidized to sulphuric acid before its excretion, four-fifths of it at least
leaving the body in the oxidized form. It is, in fact, in large measure
owing to the sulphuric acid formed in the course of protein oxidation that
the urine of carnivorous animals is acid in reaction. Moreover, this oxi-
dation takes place anywhere in the tissues.

The excretion of sulphuric acid is largest on a meat diet and
the proportion of oxidized sulphur is also largest. The excretion of
sulphur on a cream and starch diet is much reduced and its dis-
tribution in the urine is changed, a larger proportion appearing as
unoxidized sulphur. This would indicate that cysteine or cystine of
the diet is either largely decomposed in the alimentary canal,
or that a great proportion of it is changed in the liver to taurine or
sulphuric acid; whereas that set free in the tissues is relatively less
oxidized and hence more of it escapes in the urine.

The sulphur compounds are often ill-smelling. The active principle
of the odoriferous gland of the skunk is n-butyl mercaptane, C,H,SH.
Ethyl sulphide is found in dog’s urine. Its origin is unknown. Neu-
bauer observed that on feeding ethyl sulphide to dogs it was methylated
and excreted as diethylmethyl-sulphonium hydrate, CH,—_SOH=
(C,H,),. Allyl sulphide and other unoxidized sulphur-containing com-
pounds are found in mustard oil and in many other plants, i.e., oil of
garlic. Ethyl mercaptane, C,H,SH, smells very bad, but the diethyl
sulphide, when pure, has an ethereal odor. When impure its odor is
disagreeable. The source of these compounds in animal metabolism is
still uncertain. Presumably they are derivatives of cysteine. "When
sulphur is thus combined with alkyl radicles it has a strongly basic
character, so that diethylmethyl-sulphonium hydrate, as mentioned, is
a strong base.
PROTEIN METABOLISM OF THE BODY 821

HAHAH
oA, oy a
8 H—C—C—C—C—S8H
0H, Pevee
Ethyl sulphide. HH H H

N-butyl mercaptane.
In dogs a great part of the sulphur fed as cystine appears in the
urine as sulphuric acid, but a part also, as thiosulphate, which indicates

that thiosulphuric acid, HS—S—OH, is an intermediate product.
H\
Oo O

This is confirmed by the fact that in rabbits taurine, taken by the
mouth, appears. for the most part, as thiosulphate in the urine. In
human beings taurine is excreted in part as taurocarbamic acid.

OH

oe
NH,—CO—NH—CH,—CH,—S — 0

I|
0

Taurocarbamie acid.

This is another example of the carbamino reaction in the body. The
formation of thiosulphuric acid would require a reduction. It is not
improbable that it is this thio acid which unites with the cyanides and
nitriles in mammals to form sulphocyanates. Many bacteria, the so-
ealled sulphur bacteria, have the power of reducing sulphates to sul-
phides. Nothing appears to be known about the sulphur metabolism
of birds.

Ethereal sulphates. The quantity of ethereal sulphate in the urine of
men in 24 hours is widely variable. v. d. Welden gives the limits of
0.094-0.620 gram. Sulphuric acid is used by the organism to pair with,
and thus render less toxic, various aromatic decomposition products,
most of them intestinal putrefactive products of the proteins. These
compounds are called ethereal, or conjugated sulphates. They are in
reality esters of phenol, cresol, paracresol, scatole and indole. All of
these are the products of the putrefactive decomposition of tyrosine,
tryptophane and phenyl alanine, and since this putrefaction takes place
generally in the intestine the amount of the conjugated sulphates, or the
amount of ethereal sulphuric acid, is an indication, although not a very
good one, of the degree of intestinal putrefaction. The putrefaction
may, however, take place elsewhere in the body, and putrefying and
decomposing pus in old abscesses will also cause an increase in these
bodies in the urine.

On the other hand, the elimination of ethereal sulphate can be
greatly reduced by giving calomel or by reducing the protein diet. or by
any other method which reduces intestinal putrefaction. Some proteins,
for example casein, contain a great deal more tryptophane than others,
822 PHYSIOLOGICAL CHEMISTRY

so that the amount of ethereal sulphate will depend, too, on the char-
acter of the protein of the food, as well as upon its amount.

Benzene is itself a nearly inert substance, but by oxidation in the
body it is converted into the reactive, unstable, toxic, convulsant phenol
or carbolic acid. Fortunately phenol pairs very readily with sulphuric
acid. In the human organism it appears to encounter most frequently
sulphuric and glycuronic acids, with which it unites to form a non-
toxic stable compound eliminated in the urine. The place in which this
pairing occurs is probably the liver.

Synthesis of amino-acids in the animal body.—All plants have
the power of synthesizing the amino-acids from ammonia and the
products of carbohydrate fermentation. Have animal cells the same
power or must they be fed on amino-acids already formed? This is
obviously a very important question. A few years ago it was be-
lieved that animal cells differed from plant primarily in their powers
of synthesis, animal cells being chiefly catabolic and plant cells anabolic.
Further experience has served to correct that view. We now know that
animal cells are able to synthesize carbohydrates from very simple sub-
stances, possibly even from formaldehyde, and almost certainly from
glycolaldehyde; they can make fats from carbohydrates; nucleic acid
from non-nuclein material; and in fact bring about a great many other
syntheses so that animal cells certainly lack little of the powers of syn-
thesis possessed by plants. Nevertheless the great fact remains that
plants are able to nourish themselves from very simple sources of nitro-
gen, such as ammonia and nitrates or asparagine, whereas animals
require, or at any rate eat, ready-made proteins. It may be observed,
in passing, that the synthetic power of plants does not depend directly
upon chlorophyll or light, since moulds and bacteria are able to construct
their own particular kinds of proteins, which possess all the various
sorts of amino-acids, from a single source of nitrogen, such as asparagine,
if they at the same time have carbohydrate food. These plants contain
no chlorophyll. The problem of how far animals have the power of
making amino-acids has been attacked in various ways. Experiment
has shown that at least glycocoll can be synthesized in large amounts
in the animal body. If benzoic acid is fed mammals it leaves the body
chiefly in the form of hippuric acid, having united with glycocoll some-
where in the body. It has been found by giving large amounts of benzoic
acid that herbivora have the power of supplying glycocoll in far larger
amount than is present in the proteins of the body. The solution of the
question whether the other amino-acids can be formed has been sought
by feeding proteins which lack some specific amino-acid. These experi-
ments have generally been tried on young rats, since these animals grow
very rapidly and are easy to keep. It was found by Hopkins, and Men-
PROTEIN METABOLISM OF THE BODY 823

del and Osborne, that young rats would grow and develop normally when
fed on a ration of butter fat, lard, some carbohydrate, such salts as are
present in milk, and with a single pure protein added, provided that pro-
tein had in it the various amino-acids found in the body. Thus casein,
edestin, serum albumin, were each sufficient to supply the protein needs.
But if zein was the only protein in the diet, then the rats would not ccn-
tinue to grow, or indeed to live for long. Zein contains little or no lysine
and lacks tryptophane. If these were added to the zein, then the diet
became sufficient. We have already seen that gelatin is not in itself
able to supply the whole of the protein requirement of the human body,
and gelatin lacks both tyrosine and tryptophane. It was found also
in the course of the experimentation that the amount of protein which
it was necessary to add to the diet for the purposes of maintenance of
the body weight or growth differed in different proteins. The minimum
amount necessary appeared to be determined by the amount of some
amino-acid which was required by the body and which was present in
small amounts. In some proteins it was cystine which set the minimum;
it was necessary to supply a certain amount of cystine per day and
enough protein had to be fed to supply this amount. of cystine. This
emphasizes the point, made some time ago, that it is not protein as such
but amino-acids which the body requires. It appears from these experi-
ments that these animals, at any rate, cannot make sufficient tryptophane,
tyrosine, lysine and cystine to supply their needs, but that these amino-
acids must be present in the diet. This important field of investigation
has just been opened and much more work must be done before we
shall know how far different animals can synthesize different amino-
acids. It is possible that the bacteria in the intestine may be playing a
very important part in this process; they can synthesize many different
amino-acids from a single one. By digestion these amino-acids might be
set free and made available to the body, and thus the bacteria might be
of considerable use to us. Perhaps the preliminary transformation of
some of the amino-acids to amines by the intestinal bacteria may also be
of value in the formation of certain hormones.

The formation of amino-acids from ketonic acids. A very funda-
mental fact was discovered by Knoop. Not only have animals the
power of converting amino-acids into ketonice acids, and some of these
latter into carbohydrates and indirectly into fats, but the reverse process
is also possible. Out of carbohydrates ketonic acids are formed and
the ammonia salts of these acids may, in some instances at any rate, be
converted into amino-acids by reduction. Thus from ammonia and
carbohydrate the body may have the power of making its proteins. It
is not by any means possible, however, to cover all its protein needs by
this reaction and it is doubtful how extensive this process may be in
824 PHYSIOLOGICAL CHEMISTRY

the animal body. There is no doubt that the liver, and possibly other
tissues as well, may carry out this synthesis which occurs probably in
nearly all plant tissues.

The reaction is the reverse of that for the formation of ketonic acids
and probably goes through the imino stage:

CH,—CO—COOH + NH, —- CH,—C—COOH + H,0 CH,—CHNH,—COOH
I +H,
NH
Pyruvie acid. Iminopropionic.

This reaction being a reduction reaction consumes energy, it is endo-
thermic. It may occur by the utilization of some of the energy set free
by the oxidation of the system of which it is part. Another way in which
the reaction might go is by means of acetylation. It will be recalled
that when brombenzol is given dogs it is excreted in part as an acety-
lated derivative of cysteine and glycuronic acid. Acetylation of amino-
acids is not unusual in the animal body. Knoop found that the phenyl-
a-ketobutyric acid was excreted as the acetylated-amino-butyric acid. It
has been suggested (Knoop) that the acetylation is brought about by a
process analogous to the Canizzaro reaction. If pyruvic acid and
ammonium carbonate interact there is a rise of temperature, the whole
reaction is exothermic and acetylalanine and CO, are produced.

CH,—Co— COOH CH,—CH—COOH
a! = |
CH,—CO—COoH NH + C0,4H,0
Pyruvie acid. |
CH,—COo

Acety] alanine.

It is not improbable that the acetylations in the body are thus produced
from pyruvic acid and ammonia. In this case it will be noticed that one
molecule of pyruvic acid is oxidized by the other. The total energy of
the system is reduced, but the energy of one molecule of alanine is
greater than of one molecule of pyruvic acid. It is clear from this
that the amount of energy set free by the splitting off by oxidation of
one carboxyl from the chain of carbon atoms is greater than the amount
set free by the formation of the ketonic acid by oxidation from the
amino-acid.

REFERENCES. Boogs.

Cathcart : The Physiology of Protein Metabolism. Monographs on Biochemistry, 1912.

Dakin: Oxidations and Reductions in the Animal Body. Monographs on Bio-
chemistry. Londen, 1912.

Leathes: Problems in Animal Metabolism. Philadelphia, 1906.

Lusk: Science of Nutrition. 2d edition.

Fletcher: The A, B, Z of Digestion.
PROTEIN METABOLISM OF THE BODY 825

Cornaro: The Art of Living Long. Translation. Milwaukee.

Chittenden: Physiological Economy in Nutrition. 1905.

Voit: Hermann’s Handbuch der Physiologie, VII, Leipzig; 1881.

Rubner: Die Gesetze des Energieverbrauchs bei der Ernihrung. Berlin, 1902.

Mendel: Theorien des Hiweissstoffwechsels nebst einigen praktischen Konsequenzen
derselben. Ergeb. d. Physiol., 11, pp. 418-525, 1911.

‘1, Abderhalden and Steinbeck: Weitere Untersuchungen tiber die Verwandbarkeit

der Seidenpeptone zum Nachweis peptolytische Fermente. Zeit. physiol.
' Chem., 68, p. 312, 1910.

2. Abderhalden, Hinbeck and Schmidt: Studien tiber den Abbau des Histidine im
Organismus des Hundes. Zeit. physiol. Chem., 68, p. 395, 1910.

3. Abderhalden and Suwa: Weiterer Beitrag zur Frage nach der Verwertung von
tief abgebauten Hiweiss im tierischen Organismus. Zeit. physiol. Chem.,
68, p. 416, 1910.

4, Abderhalden and Pincussohn: Serologische Studien mit Hilfe der optischen
Methode. Zeit. physiol. Chem., 64, p. 100, 1910; ibid., 66, p. 88, 1910.

5. Abderhalden and Frank: Weiterer Beitrag zur Frage n. d. Verwertung v. tief
abgebauten Hiweiss im tierischen Organismus. Zeit. physiol. Chem., 64,
1910, p. 158.

6. Abderhalden and Rona: Ibid., Zeit. physiol. Chem., 67, p. 405, 1910.

‘7, Abderhalden and Massuri: Ueber das Verhalten von mono-palmytyl-l-tyrosin,
disteary]-1-tyrosin und p-amino tyrosin im Organismus des Alkaptonurikers.
Zeit. physiol. Chem., 66, p. 138, 1910.

8. Abderhalden et al.: Weitere Studien tiber die Verwertung verschiedener Amino-
siiuren im Organismus des Hundes unter verschiedener Bedingungen. Zeit.
physiol. Chem., 74, p. 481, 1911.

9. Abderhalden and Lampé: Weiterer Beitrag zur Frage nach der Vertretbarkeit
‘von Eiweiss resp. einem vollwertigen Aminosiuregemische durch Gelatine u.
Ammonsalze. Zeit. physiol. Chem., 80, p. 160, 1912.

10. Abderhalden: Anaphylactic reaction with a synthetic polypeptide. Zeit.
physiol. Chem., 81, p. 322, 1912.

ll. Abderhalden and Hirsch: Fortgesetzte Versuche den Eiweiss bedarf des
Hundes durch Ammonsalze u. ferner durch einzelne Aminosiiuren ganz oder

2 * teilweise zu decken. Zeit. physiol. Chem., 80, 1912, p. 136.

12. Abderhalden: Weiterer Beitrag zur Frage nach der Verwertung von tief abge-
-bauten Eiweiss im tierischen Organismus. Zeit. physiol. Chem., 57, p. 348,
1908; same title, ibid., 61, p. 194, 1909; 44, 1905, p. 198; 51, p. 226, 1907; 52,
-p. 507, 1907; with London: ibid. 54, p. 80, 1907; with Messner and
Windrath: ibid., 59, p. 35, 1909; with Frank and Schittenhelm: ibid., 63,
“p. 215, 1909.

13. Abderhalden: Zur Frage des Albumosengehaltes des Blutes und speziell des
Plasmas. Biochem. Ztschrft., 8, 1908, p. 360. See also Zeit. physiol. Chem.,
42, 1904, p. 155.

14. Abderhalden and Rona: Die Zusammensetzung des “ Eweiss” von Aspergillus
niger bei verschiedener Stickstoffquelle. Zeit. physiol. Chem., 46, p. 179,
1905.

15. Abderhalden and Schittenhelm: Studien tiber den Abbau racemischer Amino-
sduren im Organismus des Hundes unter verschiedener Bedingungen. Zeit
physiol. Chem., 51, p. 323, 1907.

16. Abderhalden and Block: Protein metabolism of an Alkaptonuriker. Zeit.
physiol. Chem., 53, p. 464, 1907. :
826

17.

18.

19.

20."
21.
22.
23.

24.

26.
27.

28.

29.
30.
31.
32.
33.
34.
35.
36.
37,
38,

39.

PHYSIOLOGICAL CHEMISTRY

Abderhalden, Bergell and Dorpinghaus: Verhalten des Kérpereiweisses im
Hunger. Zeit. physiol. Chem., 41, p. 153, 1904.

Abderhalden, Funk and London: Weiterer Beitrag zur Frage nach der Assimila-
tion des Nahrungseiweisses im tierischen Organismus. Zeit. physiol. Chem..
51, p. 629, 1907.

Abderhalden, London and Reemlin: Weitere Studien tiber die normale Ver-
dauung der Eiweisskirper im Magendarmkanal des Hundes. Zeit. physiol.
Chem., 58. p. 432. 1908.

Ackermann and Kutscher: Ueber die Aporrhegma. Zeit. physiol. Chem., 69.
p. 265, 1910.

Ueber ein neues auf bakteriellem Wege gewinnbares Aporrhegma. Zeit. physiol.
Chem., 69, p. 273, 1910.

Argutinsky: Muskelarbeit und Stickstoffumsatz. Arch. f. d. ges. Physiol., 46,
p. 652. 1890.

Ascoli and Vigano: Zur Kenntnis der Resorption der Eiweisskirper. Zeit.
physiol. Chem., 39, p. 283, 1903. :

Benedict: The influence of inanition on metabolism. Carnegie Institution Re-
port. Washington. 1907. See also Bulletin 203, 1914.

Benedict: The nutritive requirements of the body. Amer. Jour. Physiol., 16,
p. 409. 1906.

Benedict and Diefendorjf: The analysis of urine in a starving woman. Amer.
Jour. Physiol., 18, p. 364, 1907.

Bergell and Brugsch: Ueber Verbindungen von Aminosiiuren und Ammoniak.
Zeit. physiol. Chem., 67, p. 97, 1910.

Bergmann and Tangstein: Ueber die Bedeutung des Reststickstoffs des Blutes
fiir den Fiweissstoffwechsel unter physiologischen und pathologischen Beding-
ungen. Zeit. chem. phvsiol. Pathol.. 6. p. 27. 1905.

Bickel u. Pawlow: Untersuchungen iiber pharmakol. Wirkung des p-oxy-
phenylethylamins. Biochem. Zeits., 47, p. 345. 1912.

Biedel and Winterberg: Beitriige zur Lchre von der Ammoniak-entgiftenden
Function der Leber. Arch. f. ges. Physiol., 88, p. 140, 1902.

Billard: Sur la valeur nutritive des albumines étrangéres et spécifiques. C. R.
Soc. Biol., 68, p. 1103. 1910.

Blum: Ueber das -Verhalten des p-aminophenylalarins beim Al]kaptonuriker.
Zeit. physiol. Chem., 67, p. 192, 1910.

Borchardt: Ueber das Vorkommen von Nahrungsalbumosen im Blut und im
Urin. Zeit. physiol. Chem., 57, p. 305, 1908.

Boehm ; Ueber den feineren Bau der Leberzellen bei verschiedenen Ernihrungs-
zustiinde. Zeit. Biol., 51. p. 409, 1908.

Brown and Catheart: The effect of work on the creatine content of muscle.
Biochem. Jour.. 4, p. 420, 1909,

Brugsch: Ueber die Rolle des Glykokolls im intermediaren Eiweissstoffwechsel
beim Menschen. Zent. ges. Physiol. Pathol. d. Stoffw., 8, p. 529, 1907.

Brugsch and Hirsch: Gesammtstickstoff und Aminosiiureausscheidung im
Hunger. Zeit. f. expt. Path. u. Ther., 3, p. 638, 1906.

Burckhardt: Beitriige zur Chemie u. Physiologie des Blutserums. Arch. f.
expt. Path. u. Phar.. 16, p. 322, 1883.

Busquet: Contribution a V’étude de Ja valeur nutritive comparée d’une al-
bumine spécifique et. d’albumineg étrangéres chez la grenouille. C. R. Soc.
Biol., 65, p. 652, 1908,
40.
41.
42.
43.
44.
45.
46.
AT.

48.

49.

50.

51.

52.

PROTEIN METABOLISM OF THE BODY 827

Caspari: Physiologische Studien tiber Vegetarismus. Arch. ges. Physiol.. 109.
p. 473, 1905.

Cathcart: The Physiology of Protein Metabolism. Monographs on Biochem-
istry. London. 1912. Extensive literature cited in this.

Cathcart: The influence of carbohydrates and fats on protein metabolism.
Jour. Physiol., 39. p. 311, 1909.

Cathcart and Leathes: On the absorption of proteins from the intestine. Jour.
Physiol., 33, p. 462, 1905.

Chittenden: Physiological Economy in Nutrition. 1905.

Chittenden: The Nutrition of Man, 1905.

Cohn: Zur Frage der Glykokollbildung im tierischen Organismus. Arch. f.
expt. Path. u. Pharm.. 53. p. 435. 1905.

Cohnheim: Die Umwandlung des Eiweisses durch die Darmwand. Zeit.
physiol. Chem., 33. p. 451, 1901.

Cohnheim: Weitere Mittheilungen tiber Eiweissresorptionsversuche an Octo-
poden. Zeit. physiol. Chem., 35, p. 396, 1902.

Cohnheim and Makita: Zur Frage der Eiweissresorption. Zeit. physiol. Chem.,
61, p. 189, 1909.

Cramer: On the assimilation of proteins introduced parenterally. Jour.
Physiol., 37, p. 146, 1908.

Cramer and Pringle: The assimilation of protein introduced enterally. Jour.
Physiol., 37, p. 158, 1908.

Cremer and Henderson: Ein experimenteller Beitrag zur Lehre vom physio-
logischen Eiweissminimum. Zeit. Biol., 42, p. 612, 1901.

53-54, Dalin: The products of the proteolytic action of an enzyme contained in the

55.

56.

57.

58.

59.

60.

61.

62.

63.

64.
85.

cells of the kidney. Jour. Physiol., 30, p. 84, 1904.

Dakin and Dudley: Conversion of a-amino acids to a-ketonic aldehydes and
their relation to hydroxy acids. Proceed. Chem. Soc., 29. p. 192, 1913.
Dakin and Wakeman: The catabolism of histidine. Jour. Biol. Chem., 10, p.

499. 1912.

Dakin: Racemization of proteins and their derivatives. Jour. Biol. Chem.,
13, p. 357, 1912.

Ehrlich: Ueber die Entstehung der Bernsteinsiure bei der aleoholischen Giirung.
Bioe. Zeit., 18, p. 391, 1909.

Ellinger and Kotake: Synthese der p-oxymandelsiiure und ihre angebliche
Vorkommen im Harn bei akuter gelber Leberatrophie. Zeit. physiol.
Chem., 65, p. 402, 1910. /

Engeland and Kutscher: Ueber ein methyliertes Aporrhegma des Tierkiirpers.
Zeit. physiol. Chem, 69. p. 282, 1910.

Embden and Almagia: Ueber das Auftreten einer fliichtigen jodoformbildenden
Substanz bei der Durchblutung der Leber. Beit. chem. physiol. Path., 6,
p. 59, 1905.

Embden and Knoop: Ueber das Verhalten der Albumosen in der Darmwand
und iiber das Vorkommen von Albumosen im Blute. Beit. chem. Physiol.
Path., 3, p. 120. 1903.

Embden and Schmitz: Ueber synthetische Bildung von Aminosiure in der
Leber. Biochem. Zeits., 29, p. 423. 1910.

Folin: A theory of protein metabolism. Amer. Jour. Physiol., 13. p. 117. 1905.

Epstein and Bookman: Formation of glycocoll in the animal body. Jou.
Biol. Chem., 13, p. 117, 1912.
828

66.

67.

68.

69.

70.

71.

72.

73.

74,

75.

76.

77.
78.

79.

80.

81.

82,
83.

84.

85.

86.

87.

88.

89.

90.

PHYSIOLOGICAL CHEMISTRY

Frank and Schittenhelm: Beitrag zur Kenntnis des Eiweissstoffwechsels.
Zeit. physiol. Chem., 70, p. 98, 1910.

Frank and Trommsdorf: Der Ablauf der Eiweisszersetzung nach Fiitterung von
abundanten Kiweissmengen. Zeit. Biol., 43, p. 117, 1902.

Fraser: Rendering animals immune against the venom of cobra and on the
antidotal properties of the blood serum. Brit. Med. Jour., I, p. 1309, 1898.
II, p. 416; IJ, 1896, p. 910.

Funk: The nitrogenous constituents of lime juice. Biochem. Jour., 7, p. 81,
1913.

Frentzel: Ein Beitrag zur Frage nach der Quelle der Muskelkraft. Arch. ges
Physiol., 68, p. 212. 1897.

Freund and Popper: Ueber das Schicksal von intravenés einverleibten Eiweiss-
abbauprodukten. Biochem. Zeit., 15, p. 272, 1909.

Freund and Toepfer: Ueber den Abbau des Nahrungseiweisses in der Leber.
Zeit. f. expt. Path. Ther., 3, p. 633. 1906.

Friedlander: Ueber die Resorption gelister Eiweissstoffe im Diinndarm. Zeit.
Biol., 33, p. 64, 1896. ;

Glaessner: Ueber die Umwandlung der Albumosen durch die Magenschleimhaut.
Beit. chem. physiol. Pathol., 1, p. 228, 1901. /

Grafe: Beitriige zur Kenntnis des Stoffwechsels im protrahierten Hungerzu-
stande. Zeit. physiol. Chem., 65, p. 21, 1910. ,

Halliburton: The absorption of proteins. Lancet, I, 1909.

Halpern: Beitrag zum Hungerstoffwechsel. Bioc. Zeit., 14, p. 134, 1908.

Hopkins and Willcock: The importance of individual amino-acids in metabo
lism. Observations on the effect of adding tryptophane to a dietary in
which zein is the sole nitrogenous constituent. Jour. Physiol., 35, p. 88,
1907.

Haskins: The effect of transfusion of blood on the nitrogenous metabolism
of dogs. Jour. Biol. Chem., 3, p. 321, 1907.

Hamill and Schryver: Nitrogenous metabolism in normal individuals. Jour.
Physiol., 34, 1906, p. x.

Hawk, Howe and Mattill: Fasting Studies. III. Jour. Amer. Chem. Soce., 33,
p. 568, 1911,

Hedin: Trypsin and antitrypsin. Biochem. Jour., 1, p. 474, 1906.

Hedin: Investigations on the proteolytic enzymes of the spleen of the ox.
Jour. of Physiol., 30, p. 155, 1904.

Heidenhain: Neue Versuche iiber die Aufsaugung im Diinndarm. Arch. ges.
Physiol., 56, p. 584, 1894.

Heilner: Ueber die Wirkung grosser Menge artfremden Blutserums im Tier-
kérper nach zufuhr per os und subcutan. Zeit. Biol., 50, p. 26, 1908.

Henderson and Dean: On the question of protein synthesis in the animal body.
Amer. Jour. Physiol., 9, p. 386, 1907.

Henriques: Die Hiweisssynthese im tierischen Organismus. Zeit. physiol.
Chem., 54, p. 406, 1907.

Henriques: Lisst sich durch Fiitterung mit Zein oder Gliadin als einziger
stickstoffhaltiger Substanz das Stickstoffgleichgewicht herstellen? Zeit.
physiol. Chem., 60, p. 105, 1909.

Henriques and Hansen: Weitere Untersuchungen iiber Eiweisssynthese im
Tierkiérper. Zeit. physiol. Chem., 49, p. 113, 1906.

Hirschfeld: Ueber den Einfluss erhéhter Muskeltiitigkeit auf dem Eiweise-
91.

92.

93.

94,

95.

96.

97.

98.

99.

100.

101.

102.

103.

104.

105.

106.

107.

108.

109.

110.

111.

112,

113.

PROTEIN METABOLISM OF THE BODY 829

stoffwechsel des Menschen. Arch. f. Path. Ant. u. Physiol., 114, p. 301,
1889.

Hofmeister: Das Verhalten des peptons in der Magenschleimhaut. Zeit.
physiol. Chem., 6, p. 69, 1882.

Hopkins: The utilization of proteids in the animal. Science Progress, I, 1906,
p. 159.

Hoogenhuyze and Verploegh: Creatinine excretion in man. Zeit. physiol. Chem.,
46, p. 415, 1905.

Hooven and Soliman: A study of metabolism during fasting in hypnotic sleep.
Jour. Expt. Med., 2, p. 405, 1897.

Howell: Presence of amino acids in blood and lymph. Amer. Jour. Physiol.,
17, p. 273, 1906.

Inaba: Ueber die Zusammensetzung des Tierkérpers. Arch. f. Physiol.
1911, p. 1.

Kauffman: Ueber den Ersatz von Eiweiss durch Leim im Stoffwechsel. Arch.
f. ges. Physiol., 109, p. 440, 1905. z

Kayser: Ueber die eiweisssparende Kraft des Fettes verglichen mit derjenigen
des Kohlenhydrats. Archiv f. Physiol., p. 371, 1893.

Knoop: Ueber den physiol. Abbau der Siuren und die Synthese einer Amino-
siuren im Tierkérper. Zeit. physiol. Chem., 67, 1910, p. 489.

Knoop and Kertess: Das Verhalten von a-Aminosiuren und a-Kentonsiuren
im Tierkérper. Zeit. physiol. Chem., 71, p. 252, 1911.

Kotake: Ueber l]-oxyphenylmilchsiure und ihr Vorkommen im Harn bei Phos-
phorvergiftung. Zeit. physiol. Chem., 65, p. 397, 1910.

Kovalensky and Markewicz: Ueber das Schicksal des Ammoniaks im Or-
ganismus des Hundes bei intravenoser Injektion von kohlensiuren Am-
moniak. Biochem. Zeit., 4, p. 196, 1907.

Kiesel: Ueber den fermentativen Abbau des Arginins in Pflanzen. Zeit. physiol.
Chem., 75, pp. 169-197, 1911.

Krogh: Ueber die Bildung freien Stickstoffes bei der Darmgihrung. Zeit. f.
physiol. Chem., 50, 289, 1907.

Lang: Ueber Desamidierung im Tierkérper. Beitr. chem. physiol. Path., 5,
p. 321, 1904.

Leathes and Cathcart: On the relation between the output of urie acid and
the rate of heat production in the body. Proc. Roy. Soc., 79, p. 543, 1907.

Lehmann, Muller, Munk, Senator and Zuntz: Untersuchungen an zwei hun-
gernden Menschen. Arch. f. Path., Anat. u. Physiol., 131, Suppl. 1, 1893.

Levene and Meyer: The elimination of total nitrogen, urea and ammonia
following the administration of some amino acids. Amer. Jour. Physiol, 25,
p. 214, 1909.

Loewi: Eiweisssynthese im Tierkérper. Arch. f. expt. Path. u. Pharm., 48, p.
3038, 1902.

Lusk and Ringer: Ueber die Entstehung von Dextrose aus Aminosiiuren.
Zeit. physiol. Chem., 66, p. 106, 1910.

Luthje: Zur Frage der Eiweisssynthese im tierischen Kérper. Arch. f ges.
Physiol., 113, p. 547, 1906.

Lusk: The fate of the amino acids in the organism. Jour. Amer. Chem. Soc.,
82, p. 671, 1910.

McCay: Standards of the constituents of the urine and blood and the bear-
ing of the metabolism of Bengalis on the problems of nutrition. Scientific
Memoirs, Govt. India, 34, 1908.
830

114.
115.

116.
117.
118.
119.
120.
121.
122.
123.
124,
125.
126.
127.
128.
129.
130.
131.
132.
133.
134.

135.

136.
137,

138.
139.

140,
141.

142.

PHYSIOLOGICAL CHEMISTRY

Lusk: The Science of Nutrition, 2d edition.

McCollum: Nuclein synthesis in the animal body. Amer. Jour. Physiol., 25,
p. 120, 1909.

Magnus-Levy: Ueber die Neubildung von Glykokoll. Bioe. Zeit., 6, p. 523, 1907.

Mendell and Rockwood: On the absorption and utilization of proteins with
out the intervention of the alimentary digestive processes. Amer. Jour
Physiol., 12, p. 336, 1905.

Michaud: Beitrag zur Kenntnis des physiologischen Eiweissminimum. Zeit.
physiol. Chem., 59, p. 405, 1909.

Morawitz: Beobachtungen tiber den Widerersatz der Bluteiweisskérper. Beit.
chem. physiol. Path., 7, p. 153, 1906.

Medwedew: Ueber Desamidierungsvorginge im Blute normaler u. Schild-
driisenloser Tiere. Zeit. f. physiol Chem., 72, p. 410, 1911.

Mérner: Zur Chemie des Alkaptonharns bezw. der Homogentisinsiure (nebst
einigen ihrer Verwandten). Zeit. physiol. Chem., 69, 1910, p. 330.

Murlin: The Nutritive Value of Gelatin. Amer. Jour. Physiol., 19, p. 285,
1907.

Neubauer: Ueber den Abbau der Aminosiiuren im gesunden u. kranken Or-
ganismus. Deut. Arch. f. klin. Med., 95, p. 211, 1908.

Neubauer and Gross: Zur Kenntnis der Tyrosinsabbaus in der kiinstlich
durchbluteten Leber. Zeit. physiol. Chem., 67, p. 219, 1910.

Neubauer and Fischer: Beitrage zur Kenntnis der Leberfunktionen. Zeit.
physiol. Chem., 67, p. 230, 1910.

Neuberg and Langstein: Ein Fall von Desaminierung im Tierkérper. Arch. f.
Physiol., 1903, Supp., 514.

Oppenheimer: Ueber die Anteilnahme des elementaren Stickstoffes am Stoff-
wechsel der Tiere. Bioc. Zeits., 4, p. 328, 1907.

Romburgh: Hypaphorine and the relation of this substance to tryptophane.
Pro. Amsterdam Acad., 13 (2), p. 1177, 1911.

Paton: On Folin’s theory of protein metabolism. Jour. Physiol., 33, p. 1, 1905.

Paton and Goodall: Digestion leucocytosis. 33, p. 20, 1905.

Paton and others: On the influence of muscular exercise, sweating and
massage on the metabolism. Jour. Physiol., 22, p. 68, 1897.

Pekelharing and v. Hoogenhuyze: Die Bildung des Kreatins im Muskel beim
Tonus und bei der Starre. Zeit. physiol. Chem., 64, p. 262, 1909.

Pfliiger: Die Quelle der Muskelkraft. Arch. ges. Physiol., 50, p. 98. 1891.

Pfliiger: Glykogen. Arch. ges. Physiol., 96, p. 381, 1903.

Robertson: Synthesis of paranuclein through the agency of pepsin. Jour.
Biol., Chem., 5, p. 493, 1908.

Réhmann: Ueber kiinstliche Erniihrung. Klin. ther. Woch., 1, 1902.

Rubner: Theorie der Ernahrung nach Vollendung des Wachstums. Arch. f.
Hyg., 66, p. 1, 1908.

Rubner: Verluste und Wiederneuerung im Lebensprozess. Archiv f. Physiol.
p. 39, 1911.

Rubner: Die Beziehungen zwischen dem EHiweissbestand des Kérpers und der
Eiweissmenge der Nahrung. Archiv f. Physiol., p. 61, 1911.

Rubner: Ueber den Eiweissansatz. Archiv f. Physiol., p. 67, 1911.

Salaskin ; Ueber das Ammoniak in physiologischer und pathologischer Hinsicht
und die Rolle der Leber im Stoffwechsel stickstoffhaltiger Substanzen.
Zeit. physiol. Chem., 25, p. 449, 1898.

Salkowgki: Ueber Autodigestion der Organe, Zeit, f, klin. Med., 17, p. 77, 1890
143.

144,

145,

146.

147.

148.

149,

150.

151.

152.

153.

154,

155.

156.

157.

158.

159.

160.

161.

162.

163.

164.

165.

186.
167.

METABOLISM UNDER VARIOUS CONDITIONS g71

Schryver: Chemical dynamics of animal nutrition. Biochem. Jour. 1, p. 123,
1906.

Schulze and Winterstein: Studien tiber die Proteinbildung im reifenden
Pflanzensamen. Zeit. physiol. Chem., 65, p. 431, 1910.

Schware: Ueber Verbindungen der LEiweisskérper mit Aldehyden. Zeit.
physiol. Chem., 31, p. 460, 1901.

Seite: Die Leber als Vorrathskammer ftir Eiweissstoffe. Arch. ges. Physiol.,
111, p. 309, 1906.

Shaffer: Diminished muscular activity and protein metabolism. Amer. Jour.
Physiol., 22, p. 445, 1908.

Shaffer and Coleman: Protein metabolism in typhoid fever. Arch. of Int. Med.,
4, p. 538, 1909.

Siven: Zur Kenntnis des Stoffwechsels beim erwachsenen Menschen mit be-
sonderer Beriicksichtigung des Eiweissbedarfs. Skend. Arch. f. Physiol., 11,
1901, p. 308. ‘

Starling: Die Resorption vom Verdauungskanal. Handbuch der Biochemie.
(Oppenheimer) ITT, p. 240, 1909.

Stepp: Versuche iiber Fiitterung mit lipoidfreier Nahrung. Biochem. Zeit.
22, p. 452, 1909.

Stolte: Ueber das Schicksal der Monaminosiuren im Tierkirper nach Hin-
fiihrung in die Blutbahn. Beit. chem. physiol. Pathol., 5, p. 15, 1904.

Straub: Ueber den Einfluss des Kochsalzes auf die Eiweisszersetzung. Zeit.
f. Biol., 37, p. 527, 1899.

Tallqvist: Zur Frage des Hinflusses von Fett und Kohlenhydrate auf den
Hiweissumsatz des Menschen. Arch. f. Hygiene. 41, p. 177, 1902.

Taylor: On the synthesis of protamine through ferment action. Jour. Biol.
Chem., 5, p. 381, 1908.

Taylor and Cathcart: The influence of carbohydrates and fats on protein
metabolism. The effect of phlorizin glycosuria. Jour. Physiol., 41, 1910,
p. 276.

Stein: Ueber die Bildung von Milchsiiure bei der antiseptischen Autolyse der
Leber. Biochem. Zeits., 40, p. 486, 1912.

Thomas: Ueber die Zusammensetzung von Hund u. Katze wihrend der ersten
Verdoppelungsperioden des Geburtsgewichts. Arch. f. Physiol., 1911, p. 9.

Thomas: Ueber das physiologische Stickstoffminimum. Arch. f. Physiol.
1911, Supp. 249.

Totani: Ueber das Verhalten der Phenylessigsiiure im Organismus des Huhns,
Zeit. f. physiol. Chem., 68, 1910, p. 75.

Thompson: The nutritive effects of beef extract. Proceedings British Assn..
1, 1910.

Toepfer: Ueber den Abbau der Eiweisskérper in der Leber. Zeit. f. expt. Path.
u. Ther., 3, p. 45, 1906.

Tsuchiga: Ueber den Umfang der Hippursdiure synthese beim Menschen. Zeit.
f. expt. Path. u. Ther., 5, p. 737, 1909.

Veraguth: The effect of « meal on the excretion of nitrogen in the urine.
Jour. Physiol., 21, p. 112, 1897.

Vernon: The rate of tissue disintegration and its relation to the chemical
constitution of protoplasm. Zeit. f. allg. Physiol., 6, 1907.

Voit: Die Grésse des Eiweisszerfalls im Hunger. Zeit. f. Biol., 41, p. 167, 1901.

Voit and Korkunoff: Ueber die geringste zur Erhaltung des Stickstoffgleich-
gewichtes nothige Menge von Eiweiss. Zeit. f. Biol., 32, p. 58, 1895,
832

168.

169.

170.

171.

172.

173.

174.

175.

PHYSIOLOGICAL CHEMISTRY

Voit and Zisterer: Bedingt die verschiedene Zusammensetzung der: Eiweiss-
kérper auch einen Unterschied in ihrem Nahrwert. Zeit. f. Biol., 53, p.
457, 1910.

Weinland: Ueber die Ausscheidung von Ammoniak durch die Larven von
Calliphora und iiber eine Beziehung dieser Tatsache zu dem Entwickelungs-
stadium dieser Tiere. Zeit. f. Biol., 47, p. 232, 1906.

Weiss: Untersuchung iiber die Bildung des Lachsprotamin. Zeit. f. physiol.
Chem., 52, p. 107, 1907.

White, Hale and Spriggs: On metabolism in forced feeding. Jour. Physiol.,
26, p. 151, 1901.

Wolf and Osterberg: Protein metabolism in phlorizin diabetes. Amer. Jour.
Physiol., 28, 71, 1911.

Zisterer: Bedingt die verschiedene Zusammensetzung der Eiweisskérper auch
einen Unterschied in ihrem Nahrwert? Zeit. Biol., 53, p, 157, 1910.

Zuntz: Ueber die Bedeutung der verschiedenen Nahrstoffe als Erzeuger der
Muskelkraft. Arch. f. Physiol., p. 541, 1894.

Winterstein and Kung: Ueber das Auftreten von p-oxyphenylethylamine im
Emmenthaler Kise. Zeit. f. physiol. Chem., 59, p. 138, 1909.
CHAPTER XX.

METABOLISM UNDER VARIOUS CONDITIONS. RESPIRATION.
VITAMINES. CONCLUSION.

Metabolism during starvation and under various conditions.—
When the body is starving or supplied with insufficient nourishment to
cover its wear and tear and energy requirements, the tissues are
themselves consumed, although to a very different degree in the various
organs. A condition of temporary starvation must have been one
of the commonest vicissitudes of life among our progenitors, who
probably lived very much as the Patagonian savages described by
Darwin. These savages are able to go for long periods without food,
and then when they find a stranded whale or some other abundant
source of food, they gorge themselves for days in succession. It is only
after agriculture was cultivated that man began to be superior to these
accidents and to have a regular and even food supply. These periods
of fasting having probably been common, it is not surprising that we
find that the body has a mechanism to provide for them. When abun-
dant food is eaten it is stored in part, and during fasting these stores
are drawn upon. Foods are stored pre-eminently as fat, and princi-
‘pally as the saturated fats in the great fat reservoirs of the body.
These reservoirs are the fat tissues under the skin, the panniculus
adiposus, the fat about the intestines and internal organs and in the
connective tissue about the heart and muscles and even in the muscle
cells themselves. The brain alone of all the organs appears to be free
from fat. A second reserve of food, but one of far less importance and
.weight, is the glycogen which is stored in the liver and muscles. For
the protein there is also a certain amount of reserve, since the muscles
contain a small amount of amino-acid nitrogen and the proteins may
be increased in the body up to a certain point, but nothing comparable
to that of the fats or carbohydrates.

The first effect of starvation, or fasting, is to set free these reserve
foods. The fats are first called upon to supply the needs of the body
for energy or fuel. In fasting animals the adipose tissue almost
entirely disappears, 93-97 per cent. of it being consumed. Glycogen
is also greatly reduced, but a little remains in the liver and muscles
even in a very advanced state of starvation. But the fats and
carbohydrates cannot supply the protein decomposition of the body, and

833
834 PHYSIOLOGICAL CHEMISTRY

since there is very little protein reserve it is necessary for the body to
conserve this in every way possible. The mechanisms by which the
nitrogen is conserved in the body are still somewhat obscure, but we
know now that the body has some power at least of resynthesizing the
ammonia, which may have been set free, into some amino-acids which
can be used for the resynthesis of the proteins. When fats or carbohy-
drates are eaten or drawn from the body’s stores, not so much protein
is decomposed as when they are absent. We say accordingly that these
substances have a protein-sparing action. Possibly they act by furnish-
ing the energy for the resynthesis and so aid the retention of the amino-
acids; perhaps when they are oxidizing the activity of autolytic enzymes,
which may attack the protein, is reduced; or it may be that in some
other way they check the excretion of nitrogen and the destruction of
protein. Certain it is that they do thus act. Whatever the explana-
tion, it is found that in starvation the nitrogen output is reduced to
a minimum. The lowest figures reported are those of Cetti, a profes-
sional faster, whose N output on the 25th day of fasting had fallen to
a little more than 2 grams, and Thomas reduced his on a starch and
cream diet to 2.2 grams. The normal excretion is approximately 12-16
grams. But while the nitrogen loss is thus reduced to a minimum,
‘the total calories given off by the body per kilo body weight are not
reduced in anything like the same proportion. The normal output of
heat from a human adult, when very little active muscular work is done,
is about 35 calories per kilo; it falls on fastir.g to about 30-32 calories
per kilo.

The fact that the nitrogen excretion is so reduced in fasting has
led several to the conclusion that this represents the necessary wear
and tear of the tissues and the normal output, which is usually so far in
excess of this, is due to a luxus consumption of protein. But this mini-
mum may rather be regarded as the maximum reduction of nitrogen
waste of which the body ‘is capable. It is probable that this is the best
which can be done under the most favorable conditions. It is quite pos-
sible that the amount of protein catabolized is far greater than this, but
that the nitrogen is saved and resynthesized and used over again, the
energy for the resynthesis coming from the carbohydrates or fats. It
would be quite erroneous to conclude, as is sometimes done, that of the
proteins of the body only an amount equivalent to this 2.5 grams are
being catabolized per day. It is at least possible that this figure repre-
sents only the amount of net catabolism. How large the real catabolism
and anabolism may actually be we have at present no means of
knowing.

During fasting or starvation the various organs of the body lose
weight. Human beings may fast so that their body weight is reduced
METABOLISM UNDER VARIOUS CONDITIONS 835

to about two-fifths of the average weight and fully recover from the
fast. Children, however, have a higher metabolism than adults, owing
in part to their greater heat loss, as the surface of the body is larger
relative to the body weight in them than in adults, and in part to the
fact that their protoplasm is also younger and catabolizes faster. They
can starve for much shorter periods without dying. The amount of loss
of weight of the different organs of the body in fasting is given by Voit
for a male cat as follows:

Adipose tissue ... 97% Kidneys ......... 26% Bones .......... 14%
Spleen .......... 67 BIN. jc acianes 21 Heart .......... 3
Liver ...seses eon 54 Intestine ........ 18 : Nervous system .. 3
Testicles ........ 40 Lungs) gies sence 18

Muscles ......... 31 Pancreas ........ 17

The heart and nervous system resist starvation better than any other
organs. They lose but a small per cent. of their weight. The liver,
which stores glycogen and fat; the spleen, which has to do with the
blood destruction and formation; the testicles and the muscles, these
besides the fat are most reduced. It is their material which is being
used for the metabolism of the other tissues. It is not to be supposed
that because the heart and the nervous system lose least that their
metabolism is less than the other tissues. The direct contrary to this
is probably the fact. It is those tissues of the most intense metabolism
which preserve themselves best. We know that the heart has such an
intense metabolism. It must continue at work whatever happens; and
so must the nervous system. It is probable that the preservation of
these tissues is brought about in the following way. By the autolysis
of the other tissues a certain amount of amino-acids and other substances
are set free. The metabctism in the heart and nervous system is so
intense that in these organs the amino-acids and other products are in
part catabolized and in part built up into the tissue substance. To
maintain the equilibrium new food substances pass into these organs
from the blood to make good the loss of the catabolized products; and
from the other tissues amino-acids and other products pass to the blood
to make good the loss from that tissue. Thus that organ with the high-
est rate of metabolism will call upon the other tissues of the body which
have the lowest rate of metabolism ; they will waste away at its expense.
It is thus probably that a cancer impoverishes the other tissues.

The preservation of the brain and heart in this way at the expense
of other less vital tissues of the body is evidently a measure of adapta-
tion. These are the vital or master tissues and it is absolutely necessary
that they be preserved. From its earliest origin the nervous system
appears to have the most active metabolism and to dominate that of the
rest of the hody in the way so conclusively shown by Child.

Effects of fasting. The general effects of fasting are extremely inter-
836 PHYSIOLOGICAL CHEMISTRY

esting. There is no doubt that in many cases fasting is very beneficial
to the general health. Chronic diseases, such as catarrhs and pimples,
boils, ete., are said often to have been permanently cured by this simple
expedient. The first few days of the fast may be, and generally are,
trying; there is not infrequently considerable nausea for a few days;
but thereafter the deprivation of food does not appear to cause any
very painful sensations beyond great hunger. The total effect on the
body is to sweep out of the protoplasm all the deposited waste or reserve
material. At least half of the muscle substance has to be regenerated
and, according to Child, fasting is, in its essence, of the nature of a
regeneration or rejuvenation. This is certainly the case in some of
the lower animals, in particular in the flat worms, Planaria, and other
animals upon which he worked. In these forms it is possible to show
that the fasting animals are in reality rejuvenated and emerge from
the fast with all the characteristics of young animals, including a stimu-
lated metabolism, heightened respiration and so on. Whether the human
being is capable of a certain degree of rejuvenation by this same process
is not yet certain, but there are some indications that some reju-
venescence is possible. It seems generally true that the deposition of
colloidal matter in protoplasm is one of the conditions of senescence. It
is possible that such depositions increase the difficulty of passage of
certain substances into and out of cells, or they interpose barriers in the
way of a free exchange of material between the different parts of cells
and so ultimately break down the co-ordination of cell metabolism. If
this explanation of senescence, which has been proposed by Child, is
true, then fasting would appear to be a means of combating the process
to some degree.

There are other changes in the excretions accompanying fasting.
Thus the proportion of urea nitrogen in the urine instead of being about
85 per cent. falls to 55 per cent. or 60 per cent. of the total nitrogen (see
page 754). The quantity of ammonia increases slightly, due to the slight
acidosis produced by the burning of fat and protein; acetone and diacetic
acid appear ; and there is a relative increase in the neutral sulphur and a
corresponding decrease in the inorganic sulphates of the urine. These
are the same changes observed by Folin and others in low protein diets.

The blood holds its composition fairly uniform in fasting, although
there is some sinking in the per cent. of protein present in the plasma.
This falls from 6 per cent. to about 4-5 per cent. of the weight. There
is a considerable reduction in the amount of blood in the body, and the
per cent. of fat in the plasma may in the early days be somewhat higher
than the average, as the fat reserves are mobilized.

The number of cells of the body does not decrease so much as the
size of the cells. The general course of a fasting experiment lasting
*q10de1 om Jo Burpear fhyero & ut pesoddnsesd st pred ETA} OF BOTIOIOEA
AUBAEIOD B 4ST} SNOLACO EI 41 “4x04 O43 UT sUOELIedUICD AUT 10} SEAIRO OST} 0} OPBUT st aoUEIEJoI OYTOeds OG
Wnoyyy "10908; pearasqo Alfeieuad erour Jo yuuzrodur ysour ey} ATO ory -poqoaT[09 EATY oa COUETT
“aoljoadsur quetuaAdod epniserd 04 se ozIs 4Bd13 YONs Jo aq pom yoafqns SsIq}, UO pamMsvour 610,08} OG} JO
Tie Joy s9AmMo Zuturequoo yreqo YW ‘*seA.MO Jo seLies B Aq pamnoes 489q ST UNST[OQEJOUL JO 810}98} SNOLIVA 04}
‘wsem4aq diqstorser oy} Jo TOMEATTENsTA 8 “yoolqns sty jo UASTOquyeuT oy} Jo SJUSUIOINSEOUT FUATEHTP 94} UT:

PeAfOAUr s[epleyvUT Jo seryTWENb oy} Jo uorsserdxe [wor}eUTAyCUE Jouxe oy} SAIS O[quy ON} YSNOTUY ea ne
“syasnyopssy py “uopsoge ‘ftoz0.40 oe ToNNyNSTy erdou.e:
women ly 91g) 20 wot 919 yroySnosg 7 yotqns oe peeeoens eof anes qaotu 4 fo pany wssoqnya yy —" Eg "OT £06 ‘ON UoHeoyqng wor

le 0€ 62 G2 22 92 Sz be Ee 22 IZ ooze si zi OI Siti el 2iroré @ 29 Sve z

 

oz

09

oo! O01

ovt O02 oF
O81 COE OY
O22 OoOv OF
092 SSG Be *SaIt0S Wl0L
ooe oo!
Ove oz
ose Oz OF!
Ozy Of O91!
oor Ov Of

00s 0s" 002
T—$—— SWVud ‘N-VINONWY
Ors o

90
tT wivud (S) uNHE TINS “VLOL
oss 00°

ozo sz OP

099 OS = 09

nnn 99 ALICION TWLOL

seo sf 08

oso 001 001

SLO" $2") 021

OOF OST ORL scayo ‘guiun go AOUaNS
‘ ‘SN

ser szi 00s an 2

osi 00% 009

         

Ser Szz 004 ia teas aden
ve w

or ]o coe ‘ ‘ el ‘
WHO 'N-aiov o1y

oz 02 006 Hees

of oy ood!

ovo 09 oOOll
tied (009) WHIDTVS
00° O8 02!
Missing Page
METABOLISM UNDER VARIOUS CONDITIONS 337

31 days recently carried out by Benedict is shown in the accompanying
chart.

Lack of water and mineral substances. The body can go without
food for 20-30 days, but not without water. Water is constantly escap
ing from the body in the urine, in the sensible and insensible perspira-
tion and in the lungs. And the amount of water which is formed in
the body by the combustion of the hydrogen is by no means sufficient
to make- good this loss. The loss of water makes the viscosity of the
blood greater, the resistance +o its flow increases; water passes from
the tissues to the blood to make good the loss from the latter and this
reduces the amount of water in the tissues, giving rise to excessive
thirst which is almost intolerable. As the physical activities of the
tissues are dependent upon the viscosity of the protoplasm, and the
chemical activities on the water present, both the physical and chemical
processes in the cells suffer. There is at first a reduction in the metab-
olism, which may be followed by an increased catabolism. An adult
cannot live more than three or four days without water, the time depend-
ing naturally on the external temperature, amount of water lost by
evaporation and so on. Death is the result probably of the increased
viscosity of the blood.

Mineral substances are also necessary to life, and the result of keep-
ing them out of the food is disastrous. In the perspiration and urine
salts of various kinds are constantly being lost. 100 ¢.c. blood plasma
contain 7.6-11.8 mgs. Ca; 100 ¢.c. corpuscles contain about 3 mgs. Ca.
The Ca content of various foods is given in the following table: (Rose:
Jour. Biol. Chem., 41, p. 349, 1920).

%o %
Brad s..j2scii8 Saisinsetaeaue s 0.032 Apple eicadecvacneee scac 0.010-0.0024
Beef, lean .............4. 0.018-0.004 Tomato juice ........... 0.015
Milk sas svesisonracereiae os 0.116 RCC _ironiseiotmnegue sen .. 0.012
Honey (strained) ....... 0.004 Coffee infusion .......... 0.003
Butter: gus sateen eeetanet 0.014-0.010 Carrots) 2 cheese esr ee ts 0.044-0.052
POA CheOSsccsiis adcancudsaceveuana 9 0.006 Soda cracker ............ 0.025

In urine there leaves about 0.07 gram of calcium per day and in feces
about 0.25 gram. The calcium from carrots and other vegetables is
well utilized. Food as free as possible from mineral substances pro-
duced disturbances in the muscular system in Taylor’s experiments on
himself; disturbances of the nervous system have also been noted by
Forster. A sufficient supply of phosphates and calcium are essential to
the development of the bones and teeth. Herbivorous animals constantly
have a diet poor in sodium and relatively rich in potassium. Such
animals require from time to time some sodium chloride added to their
ration. Carnivorous animals require no salt, since the salts in their prey
are about those of their own bodies.
838 PHYSIOLOGICAL CHEMISTRY

Vitamines. Accessory food substances of an unknown nature
necessary for the nourishment of the body.—As has been so often
remarked, nearly all of the really fundamental facts in nutrition remain
still to be determined. This is illustrated in no more striking fashion
than by the discovery in the past few years of the specific action of
foods in nourishing the body quite apart from their protein, carbo-
hydrate and fat content. A great field has thus been opened which
promises to yield many valuable discoveries. It seemed a few years
ago as if with the discovery of the fuel value of a food, of how many
calories of energy it contained which were available to the body, and
with the estimation of the grams of fat, carbohydrate and protein in
it, all that was necessary to determine its food value was known. How
far that was from being the truth will appear from what follows. We
now know that the character of the fat, protein and carbohydrate is of
as great importance as the amount. It is by no means the same whether
one eats cane sugar or lactose, although they resemble each other so
closely in calories and composition. It has been found, for example,
that there is in the brain a large amount of the sugar, galactose. This
occurs in the cerebrosides in the medullary sheaths of the nerves. We
do not know whether the body, particularly in youth, has the power
of making galactose from other carbohydrates. We see, in fact, that
Nature, which has had charge of the rearing of young for millions of
years, has provided in the mammary glands an organ for the manu-
facture of galactose, so that the child during the period of the medulla-
tion of the nerves of the cerebrum, when cerebrosides may be produced
in great abundance, that is in the third to sixth month, may have a
nourishment which contains quantities of galactose. The sugar of milk
is not cane sugar; it is not maltose; it is not dextrose or levulose, or
ribose; but lactose, a sugar containing half its weight of galactose, that
important sugar of the brain. It may almost be stated as a truism that
had it been more advantageous to have dextrose in milk than lactose,
the sugar found there would have been dextrose. The suggestion, there-
fore, advocated by some physicians, to substitute for the lactose of the
milk either cane sugar or dextrose, in artificial feeding of children, can
only be regarded with misgivings. It is wiser to accept the conclusions
of nature which has tried, no doubt, many thousands of experiments of
which we are ignorant, and which has provided lactose in the food of
infants only after a prolonged research.

Neither is the fat consumed a matter of indifference, if only it be
fat. It is not enough that the fat shall burn and liberate a certain
amount of energy in the body. The nature of the fatty acid is impor-
tant. We know indeed that the character of the fat laid down in the
cells is somewhat dependent upon the character of the fat eaten. In
the fat of dogs, an abnormal quantity of mutton fat appears when after
METABOLISM UNDER VARIOUS CONDITIONS 839

fasting a dog is fed on large amounts of these fats. It was found, too,
by Herter that the character of the fat laid down in the tissue of growing
pigs was altered when the pigs were fed large amounts of acetic acid.
The power of making the necessary fats peculiar to the body of the
animal in which they are found, and indeed peculiar to the tissue, is
so great that the fats may easily be formed from the carbohydrates.
Nevertheless the melting point, and probably the ease of oxidation of
the fats in the cells, is not entirely independent of the character of the
fat fed.

Above all, the character of the protein is not a matter of indiffer-
ence. Hitherto the protein of the food has been calculated by deter-
mining the nitrogen and multiplying by the factor 6.25 to give the
amount of protein in the food. Sometimes only the coagulated extracted
food has its nitrogen thus measured, sometimes it is raw food, and lipin
nitrogen and nuclein nitrogen have been included. This method it is
recognized is very crude. Not all nitrogen is protein nitrogen. There
is the nitrogen in the phospholipins, in the amino lipins and in nucleic
acid and other nitrogen-containing substances. Furthermore, it makes
a difference what amino-acids are present in the proteins, and the vari-
ous amino-acids we now know have specific functions which cannot be
replaced by other amino-acids. This point has already been considered.

But beyond all these qualities of the food, evidence is accumulating
that the foods must contain other bodies not protein or carbohydrate
or fats or minerals, but of an organic nature and without which the
organism will surely perish. These bodies are required apparently in
very small quantities, but they are of vital importance. They have
been named, therefore, vitamines by Funk, who has worked a good deai
upon them, to indicate his opinion that they are necessary and that they
contain basic nitrogen. While the whole matter is still under investiga-
tion and it is too soon as yet to draw any positive conclusions, the
results obtained have been so extraordinary and so interesting that they -
should surely be considered here, even though opinion is not unanimous
as to their nature.

So far.there appear to be at least three of these accessory food sub-
stances or vitamines. These three vitamines have been named ‘ Fai
soluble A’; ‘ Water soluble B’; and ‘ Water soluble C.’ The first, as
its name implies, accompanies the fat portion of the extracts of celis
and tissues; it is the substance necessary for growth and it appears to
be also the substance the absence of which from the food produces the
diseases of rickets and xeropthalmia, a disease of the eyes (Gr. zeros,
dry; + ophthalmos, eye) ; it is found in greatest amounts in milk, eggs,
cod liver oil, and in green leaves. The second, or ‘ Water soluble B,’
is the substance which protects against neuritis. In its absence the
disease of beri-beri results. It is found in large amounts in yeast and
840. PHYSIOLOGICAL CHEMISTRY

in grains and cereals. The third, or‘ Water soluble C,’ is the constituent
of the foods which prevents scurvy. It is the anti-scorbutic vitamine
and it is found in largest amounts in orange juice, lemon and lime
juice; in onions, cabbage and other green and living vegetables which
have long enjoyed a reputation of usefulness as antiscorbutics. There
may in addition to these be other accessory food substances. In fact
each of these may be a group of different substances.

The first of these to be considered is that first. discovered and called
now ‘ Water soluble B.’ This is the anti beri-beri vitamine.

Bert-bert. There is a curious metabolic disease, called beri-beri,
found among Eastern peoples such as the Filipinos, the Japanese and
East Indians, peoples who have a very restricted diet, of which rice is
the main staple. This disease is characterized as follows:

It begins with a feeling of lassitude accompanied by numbness, stiff-
ness or cramps in the legs. There is edema of the ankles and face.
In its further progress the patient loses the power of walking, there
is partial paralysis of the leg muscles and other muscles, accompanied
by anesthesia in the affected areas, and often by pains and tingling sen-
sations in the feet; the edema becomes more general and breathlessness
and palpitation may come on. There are neither fever nor brain symp
toms. The symptoms are those of a peripheral neuritis which may
involve the pneumogastric and phrenic nerves, but which generally
begins in the regions farthest from the nerve centers. There is degen-
eration of the muscles. The mortality may be as high as 50 per cent.
This disease has been variously explained in the past, some consider-
ing it due to spoiled rice, others to an infection of some kind. It has
become possible to study it through the discovery of a similar disease
in fowls which may be produced artificially, this is the polyneuritis of
birds.

If fowls are fed exclusively on polished rice, that is the white rice,
the reddish exterior having been polished away, no changes are apparent
for several weeks, but suddenly the symptoms appear and in the course
of a couple of days or more they go rapidly to a fatal ending. The
fowls become unable to walk about, then they become weaker, lie over
on their sides and will surely die if the diet is not changed. Figures 64
and 65. If, however, the fowls are fed rice to which a little of the bran
has been added, or if they are fed unpolished rice, they are not subject
to the disease. They recover even when very ill by the injection of the
extract of bran. It seems, therefore, to be clear that there is some-
thing in the bran of rice, or in the outer layer of the kernels, which is
absolutely necessary for the nourishment of the body of the fowl when
it is on a rice diet. This substance is present in very small amonnts.
An amount of ‘solid extract weighing a few mgs. is sufficient to cure
METABOLISM UNDER VARIOUS CONDITIONS 841

 

 

 

 

 

 

*Fics. 64 anp 65.—Early and late stages of volyneuritis in fowls after eating
ecclusively polished rice (Funk).

a fowl when very ill. Concerning the nature of this substance, it may
be said that it is not protein, it is soluble in alcohol, it probably con-
tains nitrogen, it is organic in nature. Funk thought that it was allied
to the pyrimidins, but the evidence is very unconvincing. Whatever
842 PHYSIOLOGICAL CHEMISTRY

its nature, it would seem that it must be present in the foods. Fat,
protein, carbohydrate, mineral matters and energy are insufficient to
nourish the body in its absence. Its lack appears to affect the peripheral
nerves first, leading to their degeneration. Figure 66.

 

 

 

 

Fig. 66.—Degeneration in the peripheral nerves of fowls with polyneuritis (Funk).

Inasmuch as human beings who take wnpolished rice seem to be free
from beri-beri, the conclusion has been drawn that beri-beri is also due
to the lack of this vitamine which is found in the bran of rice, of
wheat and in many other foods. It must be remembered, however,
that peripheral neuritis may be produced in many different ways. It
occurs in arsenical and lead poisoning, also in pellagra and in alcoholics.
It is perhaps not entirely certain that beri-beri is caused directly by
polished rice, although that seems at present most probable.

Further studies into the nature of the curative substance have shown
that it is extractable from a great variety of tissues, both plants and
animals, by alcohol. It follows the lipin fraction, but is probably not
itself a lipin. Lecithin, cephalin, cerebrin, protagon, cholesterol, choline,
nicotinic acid, guanidine and other substances isolated from the lipoid
fraction are without any curative action. The substance contains no
phosphorus. It probably contains nitrogen, since it is precipitated by
phosphotungstic acid. It is precipitated from its alcoholic solution by
ether. It is insoluble in acetone, benzene, chloroform and ether. It is
precipitated from its aqueous solution by Lloyd’s alkaloidal reagent,
a hydrosilicate of alumina (fuller’s earth) which precipitates such
alkaloids as strychnine, atropine, emetine, etc., from solutions of their
salts. When so precipitated adenine may be recovered from the pre-
cipitate, but adenine itself has no curative action. It has been suggested
METABOLISM UNDER VARIOUS CONDITIONS 843

that it is a tautomer of adenine, but of this there is no proof. It may
be separated by Lloyd’s reagent from the anti-scorbutic, Water soluble
C, which is not precipitated. It is soluble in water and is destroyed
by boiling for some time, and by alkalies, even weak alkalies like am-
monia. It is more stable in acids. It loses its activity on standing in
a desiccator. Solutions which have a curative action generally, if not
always, give a blue color with phosphotungstic acid, both the uric acid
reagent and the polyphenol reagent of Folin and Denis. It is hence
apparently a reducing substance. The substance is found not only in
bran, but in milk, in muscle of all kinds, in the alcoholic extract of the
brain, in autolyzing yeast and in other locations. It is apparently very
unstable, particularly in light and in the presence of oxygen. It is
probable that the results obtained by Stepp, who found that food sub-
stances thoroughly extracted with alcohol would no longer permit of
growth and properly nourish animals, were due to the absence of these
vitamines and not to the absence of the lipoid, as he supposed. The
relation of vitamines to growth will be discussed in a moment.

Further careful investigation has shown that Water soluble B is
found in rich amounts in seeds, tubers, such as carrots, turnips, leaves,
yeast, milk, eggs, and in fact it is widespread in nature. It is found in
cotton seed, millet seed, flaxseed, kafir corn, hempseed, cabbage, alfalfa,
clover, timothy, spinach, potato, carrots, onion, turnips, leaves, stem and
root of beet, and tomatoes. There is very little in patent flour. It sticks
to edestin, in the purification of the latter, as if it were an acid.

The presence of B may be proved by yeast. Williams has shown
that yeast will not grow in its absence.

It appears, then, from these experiments that birds, and human
beings as well, require in their food certain unknown substances of an
organic nature which are absolutely necessary to life. The evidence
points, on the whole, to Funk’s conclusion that they are pyrimidin
derivatives. Possibly they are allied to alloxan or alloxantin, both of
which are unstable. The discovery of the nature of these substances, or
of this substance, is a very important matter. Nicotinic acid is found in
the partially purified product, but nicotinic acid is itself inactive. Funk
has suggested that possibly a mother substance of nicotinic acid is the
active principle. All of the purified substances extracted from bran
have been found to be inactive.

As to its nature Williams states that a hydroxy pyridine has a
curative action on polyneuritis and that it is the pseudobetain tautomer
which is active. He concludes that the curative form of nicotinic acid
is similar. But this result has been questioned by Harden. Dutcher
reported that pilocarpine, thyroxin and tethelin had some curative action
on beri-beri, but assuredly the action of these substances is far weaker
844 PHYSIOLOGICAL CHEMISTRY

than the vitamine itself. All that one gets is a slight prolongment of life.

Pellagra. Another disease with some points of resemblance to beri-
beri is pellagra. The name means rough skin, the skin, particularly on
the backs of the hands and about the neck, being thickened and rough
Neuritis occurs here also and the symptoms of disturbance of the central
nervous system are more pronounced. This disease was long ascribed
by the Italian investigators to spoiled maize. It is common in the
Southern States and in Italy. Other investigators have recently ascribed
it to an infection carried by a fly or insect-carrier. Whatever may be
the explanation of the cause of this disease, there can be no doubt about
the fact of the impairment of the nutrition of the nervous system; but
whether this is due to a poison elaborated by a parasite of some kind
either in the body or in maize, or whether it is due to the lack of some
substance in the diet, cannot be positively stated. It occurs generally
among those having a very restricted diet with several possible de-
ficiencies, but other conditions of an unsanitary nature have usually
been present, making the determination of the etiology difficult.

Fat soluble A. The growth vitamine. In addition to the anti beri-
beri, water soluble B vitamine, there is necessary for growth of young
animals, and probably for the nourishment of adults as well, another
different vitamine or food accessory substance which is usually found
in the lipin fraction of the extract of the food and is accordingly called
fat soluble A. It was first clearly recognized and distinguished from
the other by McCollum and Davis, although the necessity of some acces-
sory substance for growth of the young was first shown by Hopkins in
England and by Mendel and Osborne in this country. At first, how-
ever, the two vitamines were confused.

One would naturally look for such substances in milk and possibly
in eggs, since these two foods have been provided especially to serve
the needs of the rapidly-growing organism. If any substances stimu-
latory of growth are to be found anywhere, one would naturally look
first in those foods which we know to be particularly good for growing
animals and particularly good for the rapidly-growing nervous system.
They ought to be found in human milk, since this food has to meet the
requirements of a very rapidly-growing nervous system. Such sub-
stances have been found in milk. Hopkins, McCollum and Davis, Hop-
kins and Nevill, and Osborne and Mendel found, in testing the efficacy
of various pure proteins and inorganic saits in promoting the growth of
young white rats, that artificial diets containing some protein, such as
edestin, albumin or casein, some inorganic salts like those of milk, some
starch, and lard nourished the animals for a time, but that sooner or later
they ceased to grow, so that they rarely attained more than two-thirds of
the weight normal for rats of their age. If at this time some butter
METABOLISM UNDER VARIOUS CONDITIONS 845

fat was substituted for a portion of the lard, the remainder of the diet
being the same and the total energy not- changing, the rats began to
grow again and very rapidly reached their normal weight. Further-
more, milk itself has in it all the substances necessary for the growth
and maintenance of rats. These experiments are illustrated in the

 

Fic. 67.—Curves of body weights of rats which have ceased to grow and have declined
on foods containing the natural “ protein-free milk’ and have recovered when 18 per cent.
unsalted butter replaced the same quantity of lard in the diet. as indicated by the inter-
rupted lines (—o—o—o—). Rats 1204, 1281, 1292 had casein; rats 1268, 1276 had
‘ovalbumin as the sole protein. Ordinates represent grams body weight; abscissas 20-day
intervals. The diet was: Purified protein, 18 per cent.; starch, 26 per cent.; protein-
‘free milk, 28 per cent.; lard, 10 per cent.; butter, 18 per cent.
curves in Figure 67. Sterilization of the milk did not in any way
interfere with the value of the fat as a growth stimulant.

The experiments of Hopkins and Nevill were as follows: Twenty-
four rats from various sources weighing from 50-60 grams each were
placed on a diet containing protein and starch, which had been care-
fully extracted with alcohol; lactose, which had been repeatedly erys-
tallized and extracted with alcohol; and a salt mixture similar to that
used by Mendel and Osborne. Every rat, although eating well and

taking sufficient food to cover its energy needs, quickly ceased to grow,
846 PHYSIOLOGICAL CHEMISTRY

some on the sixth day, some on the ninth and all before the fifteenth day.
A short period followed when they kept their weight, and to this suc-
ceeded a period of gradual decline in weight. The diet was kept the
same for 18 of them up to death. Fourteen of them died about the
fortieth day. In the case of six of the same set of rats after the decline
had begun, 2 c.c. of milk per diem were added to the ration. An imme-

 

Fic. 67A.—Suie as illustrated in Figure 67 except that the rats were all males and
18 per cent. of butter fat, in place of butter, as in Figure 67, was adde. to the diet, after
decline had set in, in place of an equal amount of lard which was discontinued. Rats
1224, 1235 had casein; 1391, edestin; 1616, zein and casein.
diate betterment of the general condition was observed, growth was re-
established and health was maintained. Another lot of six rats were
given a little milk in addition to the ration of alcohol-extracted foods
and these grew normally.

The active substances in the butter fat are still undetermined. Cod-
liver oil will act like the butter in promoting growth. Olive oil does
not. Cholesterol is ineffective. The butter fat used by Osborne and
Mendel contained neither ash, phosphoric acid nor nitrogen. The active
substance for growth, they conclude, is probably not a glyceride of
the ordinary fatty acids, nor a phospholipin, nor a vitamine in the sense
of Funk. What it is must be determined by experiment.

Some experiments by Carrel may also be mentioned in this
connection. Carrel has been growing tissues taken from the living

a

ee ae ee

piace gi ie aa es A te |
METABOLISM UNDER VARIOUS CONDITIONS 847

organism in artificial culture media by Harrison’s method of tissue
culture. He found that if he placed these growing tissues in blood
serum taken from adults, they continued to live for a long time, but
they did not grow. The cells did not reproduce. If, however, the
culture was made in the blood serum of young and growing animals,
then the growth took place vigorously. It seems that there is some
substance in the blood of young and growing animals which is not pres-
ent in that of adults. What the nature of this substance is, is still
unknown. If this experiment shall prove to be generally successful with
many tissues and with the young and old sera, it would seem that we
might at last be on the track of the substances of youth.

Fat soluble A has been found in butter fat, egg yolk and fat, cod-
liver oil, beef oil, oleomargarine, cod testicles, pig kidneys, pig liver and
liver oil, whale oil, fat fish, seal oil, fish oils, dried and unsweetened
condensed milk; in corn, wheat germ, rye and oats, in leaves of plants,
cotton seed flour and oil, olive oil in small amounts, flaxseed, millet
and hemp seed, soy beans, peas and bananas. It is absent or present in
very small amounts in lard, pig heart, almond oil, sunflower oil, linseed
oil, corn oil, wheat, soy beans, cottonseed oil, nut margarine, hydro-
genated vegetable and animal oils (destroyed by the temperature), vege-
table margarine, white beans, barley, potato, pancreas, thymus and
suprarenal.

In experiments on rats a diet free from fat soluble A is generally

chosen for a basal diet somewhat of the following kind:
Basal. Mixture: sits sas bs dee cee eae ee enas 12 grams.
Starch 75 per cent.
Casein 20 per cent.
Salts 5 per cent.!

Antiscorbutic (equiv. to lemon juice) ...... % 5 ce.
Autolysed yeast (to give B)................-. 5 cc.
OVE Oth asso sg aries dicta ae anyon she Seed a adheleye 0.75 c.c.

Fat soluble A has also another function. It appears to be the substance
which protects against rickets. Rickets is a disease of the following
characteristics: Softening of the bones, defective teeth or retardation
of development, soreness and tenderness of body, enlargement of liver

and spleen and malnutrition. This disease, or a disease which appears
+The McCollum salt mixture is as follows:

NaG@h: c4se02. yeheet bones 1.73 grams.

MgSO, ..---.sseeeeeeeee 2.66 “ (anhydrous) -
NaH,PO,H,O ........++. 3.47

K HPO, .. eee sees seca ee 9.54 “

CaH (PO, Ja tttttreteseee 540 “

Ca lactate ........ece eee 13.0 “
848 PHYSIOLOGICAL CHEMISTRY

in its main features to be identical with the human disease may be
induced very easily in pups by the following diet (Mellanby) :

Separated milk ............... 175-350 e.c.
70 per cent. wheaten bread..... Ad lib.
Linseed oil ................... 5-10 ee.
Weast: i icausincnamiene.<a Siete ths 5-10 grams.
Orange or lemon juice......... 3 ce

INGOT, euicturs yoxscape scans aa etavenaveenes 2 gms

This diet is deficient in calcium possibly, and in fat soluble A. This
will produce a rapid development of rickets easily recognizable by
X-ray photographs within six weeks of the beginning of the diet. It
comes on more rapidly the more rapidly the pup is growing. 10 grams
of added butter or cod-liver oil completely prevents the disease. Cotton-
seed oil, olive oil or babassu oil does not prevent it when substituted for
the butter. Hydrogenated fats act like linseed oil and do not prevent
rickets. It appears that fat soluble A or something associated with it is
particularly concerned in the calcification processes of bones and teeth.
McCollum thinks that several factors co-operate in the production of
rickets, namely deficiency of calcium and of fat soluble A and incom-
plete proteins. Still another function of fat soluble A is to protect
against a disease of the eyes called xeropthalmia. Rats which are on
a diet deficient in A develop this disease and if the diet is not changed
they become blind. The disease is marked by ulceration of the cornea
and dryness of the eyes. A similar disease occurs among children. The
cause of the disease, whether bacterial or not, is not known. It may be
that the deficiency simply lowers the vital resistance. At any rate this
disease seems a fairly sure indication that the animals are not receiving
their requirement of A. The addition of a little butter, of fat from fresh
leaves, cod-liver oil, or other substance to the diet which contains A leads
to a prompt recovery if the disease has not gone too far.

The anti-scorbutic vitamine. Water soluble C. Scurvy is a disease
which was very common in armies and in crews of sailing vessels in the
past when fresh and canned vegetables were seldom eaten and the diet
consisted largely of flour and salt or preserved meat. It was early
recognized that certain articles of diet such as lemons, oranges or fresh
vegetables were preventives of the disease and that they had also a
remarkable curative effect on scurvy victims. Scurvy is not at all con-
fined to sailors and camps. It is very common among children and its
milder forms, or incipient stages, were until recently generally over-
looked. Many eases of teething troubles, so called, in children are in
reality incipient scurvy.

The symptoms of scurvy are as follows: Soreness, sponginess and
bleeding of the gums, loosening of the teeth; hemorrhages under the
METABOLISM UNDER VARIOUS CONDITIONS 849

periosteum of the bones and in almost every part of the body; weakness
and almost complete powerlessness of the legs or hind limbs, pseudo-
paralyses, the result apparently of hemorrhages in the hip and knee
joints. The bones become fragile and there is enlargement and dis-
ruption of the rib junctions. In the advanced stage of the disease the
whole body and limbs are tender. In infants in the incipient stages the
children are generally anemic and growth is retarded. They become
fretful and their gums sore. These are the first signs to be observed.
Infants later cease to gain and begin to lose weight. An interesting
ease of this sort occurred in the Hebrew Infant Asylum in New York
(Hess and Fish). In this institution it had been the custom to pasteurize
the milk at 63° C.. (145° F.) for 30 minutes and it was also the custom
to give a certain amount of orange juice to the children. Under this
diet there was no scurvy. But owing to the report of the American
Medical Milk Commission that pasteurized milk was in every way the
equivalent of raw milk the use of the orange juice was discontinued.
The result was that the cessation of the orange juice was followed by the
outbreak of an epidemic of mild scurvy about 2 to 4 months later. The
children were fretful, showed marked pallor, loss of appetite, and some
degree of anemia; they ceased to gain in weight and length. In a few
cases there were distinct hemorrhages in the mouth and elsewhere and
in others the blue line of induration about the margins of the teeth.
All were more than 6 months old. The scorbutie nature of the epidemic
was proved by substitution of raw milk for the heated and by the addi-
tion of an extra antiscorbutic to the diet when the symptoms disap-
peared.

The foregoing case shows that heated milk is not so good an anti-
seorbutic as raw milk. Further investigation has shown that milk is
at the best but a poor antiscorbutic. Large quantities of milk must be
taken to prevent scurvy. Flor example, in guinea pigs Barnes and
Hume report that while 1.5 to 10 grams daily of many raw vegetables
and fruits will protect from scurvy, 100 to 150 cc. of cows’ milk was
necessary to accomplish the same end. Heated milk is not nearly so

_good as unheated, and dried milk powder, although in the process em-
ployed it is exposed to high temperature for a few seconds only, yet
its antiscorbutic powers are largely destroyed. Raw vegetables, raw
fruits are necessary. Even drying vegetables at a low temperature
destroys much or all of their antiscorbutic powers. The armies in the
late war tried dried vegetables as an antiscorbutic, although they had
been tried many times before with the same negative result. Whatever
the nature of the vitamine may be it is clearly of a very unstable nature
under the conditions in which it naturally occurs.

Among the means found of rendering some foods poor in water
850 PHYSIOLOGICAL CHEMISTRY

soluble C sufficiently rich to be valuable is by germination. Seeds such
as barley and wheat, or peas, beans or lentils, have no antiscorbutic
properties. But if they are sprouted, then they acquired antiscorbutic
properties. This fact again was discovered by uninstructed people
ignorant of the science of nutrition, and it has been confirmed by scien-
tific investigation.

Concerning the nature of the anti-scorbutic vitamine C little can
be said. It is soluble in water; it is very unstable; it is not adsorbed
by fuller’s earth.

The great value of green vegetables, salads, etc., consists in the fact
that they act as anti-scorbutics. The food value of cabbage, from the
point of view of calories, or energy, is very little, but as an anti-scorbutie
it is very great. Lettuce, fresh tomatoes, fresh berries of all kinds,
fruits—these are the food articles which by common experience have the
reputation of being in some way healthful. Science has only confirmed
the discovery of our ancestors. It may in the future show what the
accessory foods are. The general result of many years’ investigation
may be summed up as being the discovery that milk and eggs are very
good foods for children, and that no artificial food mixture has been
found so good for children as their mother’s milk.

Tissue respiration.—Combustion is the source of all the energy of
the human body in whatever form that energy may show itself. This
combustion occurs in the living matter; it is the process of respiration.
‘We cannot do better than to close this account of the principles of
chemical biology by a brief examination of this most fundamental
process. It is the breathing brain which is conscious. When oxygen
unites with carbon and hydrogen in the brain under certain conditions,
it produces or is accompanied by the psychic processes. The nature of
respiration appears then to be the problem of all others of a chemical
nature which it is desirable should be solved. Unfortunately we are
stil! far from understanding the nature of this process, and there are
in fact two main views as to its nature, and these two views are on
the whole very antagonistic. They have been expressed by various
observers under various guises, for the problem remains the same,
although its outward form may change. The problem is this: Do the
substances which enter the living protoplasm become part of the living
protoplast before they burn, or not? What conception shall we have
of the living matter with which the chapters of this book have dealt?
There are two possibilities: one is that the cell is a kind of factory,
the walls separating the rooms of which are made of colloidal matter
which is relatively inert. The chemical changes which occur in the
cell are due to the changes which are occurring in these separate rooms.
This may be called the compartment theory. These compartments may
METABOLISM UNDER VARIOUS CONDITIONS 851

be very small. Each may be imagined to have one or several enzymes
or catalytic agents in it. These produce chemical decompositions, by
purely chemical processes, in the amino-acids, sugars, or other substances
which penetrate the compartments. They may undergo a process of
fermentation by which CO, is produced. They may be oxidized, or
hydrolyzed or otherwise changed. Or they may be combined with the
walls of the compartment, either physically by adsorption, or by chemical
union, and thus contribute to the chemical constitution of the colloids.
According to this view, the oxidations in the cell are in all respects
like those outside the cell. They are produced by active agents, oxidases
of various kinds, which can be isolated from the cell and found outside
to carry on the activities of protoplasm. By the oxidations of these
substances acids or other compounds are produced, or heat is set free,
and these compounds, or this heat, cause the change in the amount of
water in the cell and so the physical changes of the lifeless colloidal
material of the cell. These find their expression in the various forms
of vital actions. This view was advocated by Hofmeister, among
others, and it has been defended by Hopkins. According to this view,
there is no vital matter in the cell; and the chemical transformations
do not involve any large biogene molecule, but only such transforma-
tions as those with which we are all familiar, of relatively simple com-
pounds in solution., The evidence in favor of this view is the fact that we
are constantly succeeding in isolating from cells catalytic agents which
cause in aqueous solution, in the beaker, oxidations, fermentations and
hydrolyses identical in their main features with those in living matter.
There is no denying the fact that there are many powerful evidences for
the truth of this view. But it cannot be denied that there are also grave
difficulties in its way. According to this view, the cell is not a unit;
it is a large collection of separate enzymes, confined to separate parts,
or compartments, of the cell. It gives no explanation of the apparent
unity of the cell and the organism. The shapes of organisms are as
characteristic as those of erystdls. In crystals the matter is organized.
In living matter, also, the matter is certainly organized. What is the
organizing force in living matter? How shall we explain irritability and
the phenomena of narcosis or anesthesia on the basis of the view just
stated? The hypothesis that these drugs are acting by altering the
permeability of the walls of the compartments has not yet been sub-
stantiated by any evidence which is really conclusive. It is certain that
they influence the chemical processes profoundly since growth and
respiration are annihilated by them; but anesthetics do not materially
alter most of the oxidations outside the cell.

For these reasons another view has been proposed which has certain
merits of its own. This view has been expressed by Pfliiger, Verworn
852 PHYSIOLOGICAL CHEMISTRY

and other physiologists, often in slightly different forms, but in its essen-
tials the same. The essential basis of this view is that the organizing
property of the cell is made the point of departure. Living matter
organizes the food that it receives and keeps what it needs; it makes
always the right kind of living matter. Now there are two ways in
which we may picture this organization force. The cell may be pictured
as a kind of a kitchen in which there is a maid of all work, as Du Bois
Raymond puts it. She receives the provisions, cooks the meals, throws
out the wastes and keeps all in order. This maid of all work hac
received various names. Sometimes she is called entelechy, or vital
force, but whatever her name her functions and activities are pictured
by all who would employ her in their scheme of cell physiology, as
being essentially the same. As long as the cell lives she is there; when
she departs, things go at sixes and sevens and the machinery no longer
works and the cell dies. The only escape from this conception, which
recently has been again strongly advocated by Sir Oliver Lodge in his
Presidential address before the British Association for the Advancement
of Science, is to ascribe the organizing forces of the cell to the molecules
of which it is composed. There are only two possibilities: either the
molecules organize themselves as they do in‘a crystal or else something
else organizes them, for that they are organized into a definite and
characteristic form there is no doubt. Accordingly it is assumed that it
is the molecular forces in the biogenes or large molecules of the cell.
These large molecules are themselves the living, respiring units. It is the
living matter, the organized matter itself, which is primarily burning
in the protoplasm. But this is only part of the combustion. "When
combustion occurs in an auto-oxidizable matter, such as a biogene, there
is formed, according to nearly all observers, a portion of hydrogen
peroxide. That hydrogen peroxide is also formed in the course of living
oxidations is indicated by the fact that there is present in all forms of
living matter a special enzyme, catalase, which has the property of decom-
posing it and setting free oxygen. It is hardly probable that this
catalase would be present in all forms of living matter without excep-
tion, unless it had some function there. We may assume that this
hydrogen peroxide burns some of the molecules in solution in the cell.
This is the other part of the combustion. Respiration consists on
this view of two processes, the physiological or auto-oxidation of the
real living protoplast or biogenes, and the oxidation, by means of the
hydrogen peroxide, which is a secondary product of the primary respi-
ration, of amino-acids and other fragments present in the solution. It
is this oxidation which Hopkins has described. But this oxidation on
this view is not the essential and primary oxidation. It is certainly a
suggestive fact that the oxidations of this nature, of the amino-acids in
METABOLISM UNDER VARIOUS CONDITIONS 853

cells and various fatty acids and other substances, are identical in char-
acter with those which are produced by hydrogen peroxide, as Dakin
has shown.

We may, therefore, make the following picture of the fate of sub-
stances, such as amino-acids for example, on entering protoplasm. The
amino-acid may enter the cell in solution, or it may ultimately find itself
in solution in the. water in the cell. Before it is combined with the
protoplast or otherwise made over into the colloidal material of the
cell, it has to run the gauntlet, of the hydrogen peroxide and the oxidases.
A portion of the amino-acid is decomposed by this means and con-
verted into ketonic acid and ammonia. The ketonie acid may be subse-
quently burned or it may be synthesized into the colloidal substratum of
the cell. A portion of the amino-acid is caught up as such and syn-
thesized into the protoplast. Oxygen when it enters the cell combines
tn the first instance with water and the living protoplast to make the
irritable substratum of the cell. It is this substratum, a molecular union
of oxygen, water and protoplast, which conducts, respires, contracts
and is anesthetized by ether. The protoplast in its turn consists of
unions of protein, phospholipin and various enzymes.

Whether this picture of the process is correct or not, there is no
doubt that the respiration of the cell involves in its totality some oxidases,
hydrogen peroxide, catalase, iron or manganese, and substances, pre-
sumably the living protoplast, which have the power of uniting with or
decomposing the food matters and thus increasing their power of
combustion.

This brief summary will serve to show how meager, as yet, is our
knowledge, how impotent our attempts, to give any explanation of the
great and fundamental problems of physiology and physiological chem-
istry: the nature of consciousness, of animal and plant forms and of
the fundamental properties of respiration, irritability and growth.

REFERENCES. ViraMINes AND GRowTH SUBSTANCES.

General.

1. Funk: Ueber die physiologische Bedeutung gewisser bisher unbekannter Nahrungs
bestandteile, der Vitamine. Ergeb. d. Physiol., 13, pp. 125-205, 1913. Good
literature lists for beri-beri, scurvy, pellagra, rickets and growth sub-
stances,

Special.
1. nie and Casimir Funk: Experiments on the causation of beri-beri. Lancet,
p. 1266, 1911.
2. Funk: On the chemical nature of the substance which cures polyneuritis in
birds induced by a diet of polished rice. Jour. Physiol., 43, p. 395, 1911.
Funk: The preparation from yeast and certain foodstuffs of the substance the

£8
854

10.
11.
12.

13.

14.
15.

16.

17.

18.
19.

20.

21.

23.
24,
25,
26.

27.

PHYSIOLOGICAL CHEMISTRY

deficiency of which in diet occasions polyneuritis in birds. Jour. Physiol.,
45, p. 75, 1912.

Funk: An attempt to estimate the vitamine fraction in milk. Biochem. Jour., 7,
p. 211, 1913.

Drummond and Funk: The chemical investigation of the phosphotungstate pre-
cipitate from rice polishings. Biochem. Jour., 8, p. 598, 1914.

Funk and Macallum: On the chemical nature of substances from alcoholic ex-
tracts of various foodstuffs which give a color reaction with phosphotungstic
and phosphomolybdie acid. Biochem. Jour., 7, p. 356, 1913.

Barger: The simpler natural bases. Monographs of Biochemistry. Tondon, 1914.

Cooper: The preparation from animal tissues of a substance which cures poly-
neuritis in birds induced by diets of polished rice. Bioch. Jour., 7, p. 268,
1913.

Oooper: Curative action of autolyzed yeast against avian polyneuritis. Bioch.
Jour., 8, p. 250, 1914.

Cooper: The relation of vitamine to lipoids. Bioch. Jour., 8, p. 347, 1914.

MacLean: Purification of phosphatides. Bioch. Jour., 6, p. 355, 1912.

Funk: Is polished rice plus vitamine « complete food? Jour. Physiol., 48, p.
228, 1914.

Edie, Evans, Moore, Simpson and Webster: The antineuritic bases of vege-
table origin in relationship to beri-beri, with a method of isolation of torulin,
the antineuritic base of yeast. Bioch. Jour., 6, p. 234, 1912.

Suzuki, Shimamura and Odake: Ueber Oryzanin ein Bestandteil der Reiskleie
und seine physiologische Bedeutung. Bioch. Ztschr., 43, p. 89, 1912.
Funk: The effect of a diet of polished rice on the nitrogen and phosphorus of the

brain. Jour. Physiol., 44, p. 51, 1912.

Chamberlain, Bloombergh and Kilbourne: A study of the influence of rice diet
and of inanition on the production of multiple neuritis of fowls and the
bearing thereof on the etiology of beri-beri. Philipp Jour. Sci., 6, p. 177,
1911.

Holst and Fréhlich: Experimental studies relating to ship beri-beri and scurvy.
Jour. Hyg., 7, p. 634, 1907.

Funk: Nitrogenous constituents of lime juice. Bioch. Jour., 7, p. 81, 1913.

Lane-Claypon: Value of boiled milk as a food for infants and young animals.
Report to the Local Govt. Board, N. &., p. 63, 1912.

Brachi and Carr: Infantile scurvy in a child fed on sterilized milk. Lancet,
662, 1911.

Lane-Claypon: Observations on the influence of heating upon the nutritive value
of milk. Jour. Hyg., 9, p. 233, 1909.

Stepp: Weitere Untersuchungen tiber die Unentbehrlichkeit der Lipoide fir
das Leben. Ueber die Hitzezerstirbarkeit lebenswichtiger Lipoide der
Nahrung. Zeit. f. Biol., 59, p. 336, 1912.

Stepp: Experimentelle Untersuchungen tiber die Bedeutung der Lipoide fiir
die Ernihrung. Biochem. Ztscher., 22, p. 452, 1909.

Osborne, Mendel and Ferry: Feeding experiments with isolated food sub-
stances. Carnegie Instit., Washington. Nr. 156.

Osborne, Mendel and Ferry: Feeding experiments with fat-free food mixtures.
Jour. Biol., Chem., 12, p. 81, 1912.

Hopkins: Feeding experiments illustrating the importance of accessory factors
in normal dietaries. Jour. Physiol., 44, p. 425, 1912.

Hopkins and Neville: A note concerning the influence of diets upon growth.
Bioch. Jour., 7, p. 97, 1913.
METABOLISM UNDER VARIOUS CONDITIONS 855

28. Osborne, Mendel and Ferry: Maintenance experiments with isolated proteins.
Jour. Biol. Chem., 13, p. 233, 1912.

29. Macallister: A new cell proliferant: its clinical application in the treatment of
ulcers. British Med. Jour., Jan., 1912.

30. Macallister: The action of Symphytum officinale and allantoin. British Med.
Jour., Sept., 1912.

31. Coppin: The effect of purin derivatives and other organic compounds on growth
and cell division in plants. Bioch. Jour., 6, p. 416, 1912.

32. Oarrel: On the permanent life of tissues outside of the organism. Jour. Expt.
Med., 15, p. 516, 1912.

33. Carrel: Artificial stimulation and inhibition of the growth of normal and
sarcomatous tissues. Jour. Amer. Med. Assn.. 55, p. 32, 1911.

34. Umnus: Die photobiologische Sensibilierungstheorie in der Pellagrafrage.
Zeits. f. Immunitiitsf., 13, p. 461, 1912.

35. Hunter: The sand-fly and pellagra. Jour. Amer. Med. Assn.. 58, p. 547. 1912.

36. Aldrich: The feeding of white rats on the pituitarv body. ‘Amer. Jour. Phys.,
31, p. 94, 1912.

37. Orgler: Die Kalkstoffwechsel des gesunden und des rachitischen Kindes. Erg.
d. inn. Med. u. Kinderheil., 8, p. 142, 1912.

38.. Mohr: v. Noorden’s Handbuch der Pathol. d. Stoffwechsels, 2. p. 865.

39. McCollum and Davis: The necessity of certain lipins in the dict during growth.
Jour. Biol., Chem., 15, p. 167, 1913.

40. Osborne and Mendel: The relation of growth to the chemical constituents of
the diet. Jour. Biol. Chem., 15, p. 311, 1913; 16, p. 423. 1914; 17. p. 401.

41. Benedict: Experiment on a man fasting 31 days. Publication 203, Carnegie
Institution, Washington, 1915.

42. Funk and Macallwm: Die chemischen Determinanten des Wachstums. Zcit.
physiol. Chem., 92, p. 13, 1914. Funk: Ibid.. 88, p. 352. 1913.

43. Folin: On starvation and obesity with special reference to acidosis. Jour.
Biol. Chem., 21, p. 183, 1915.

44. McCollum and Davis: The influence of the composition and amount of the
mineral content of the ration on growth and reproduction. Jour. Biol.
Chem., 21, p. 615, 1915; The influence of certain vegetable fats on growth.
Ibid., 21, p. 179, 1915; Dietary deficiencies of rice. Tbid., 23, p. 181, 1915;
The essential factors in growth. Ibid., 23, p. 231, 1915.

45. Funk and Macallum: On the probable nature of the substance promoting growth
in young animals. Jour. Biol. Chem., 23, p. 413, 1915.

46. Osborne and Mendel: The resumption of growth after long continued failure to
grow. Jour. Biol. Chem., 23, p. 439, 1915.

47. Hunter, Givens and Lewis: Preliminary observations on metabolism in Pei-
lagra. Hygienic Lab. Bull. No. 102, U. 8. P. H. Service, 1916.

Mellanby, Edward: A further demonstration of the part played by accessory food
factors in the etiology of rickets. Jour. of Physiol., 52, p. liii (Proc.,
Physiol. Soc.), 1919.

McCollum, Simmons and Parsons: Study of rickets. Jour. of Biol. Chem., 41, p.
lxxxi (Proc., Biochem. Soc.), 1920. —

Chick, Hume and Skelton: Antiscorbutic value of cow’s milk. Biochem. Jour., 12,
pp. 131-153, 1918.

McCollum, Simmonds and Parsons: A biological analysis of pellagra-producing
diets. VI. Observations on the faults of certain diets comparable to those
856 PHYSIOLOGICAL CHEMISTRY

employed by man in pellagrous districts. Jour. of Biol. Chem., 38, pp.
113-145, 1919.

McCollum and Simonds: The nursing mother as a factor of safety in the nutrition
of the young. Amer. Jour. of Physiol., 46, p. 275, 1918.

Osborne and Mendel: Further observation on the distribution of water soluble
vitamine. Jour. of Biol. Chem., 41, p. 451, 1920.

Osborne and Mendel: Nutritive value of the proteins of the barley, oat, rye and
wheat kernels. Jour.'of Biol. Chem., 41, pp. 275-306, 1920.

Harden and Zilva: The differential behavior of the antineuritic and antiscorbutic
factors towards adsorbents. Biochem. Jour., 12, p. 93, 1918.

Drummond, J. C.: Study of the water-soluble accessory growth promoting sub-
stance. IJ. Its influence upon the nutrition and nitrogen metabolism of
the rat. Biochem. Jour., 12, pp. 25-41, 1918.

Zilva, 8. 8.: The action of ultra-violet rays on the accessory food factors. Biochem.
Jour., 13, p. 164, 1919.
Drummond, J. C.: Researches on the fat soluble accessory substance. Observation
on its nature and properties. Biochem. Jour., 13, pp. 81-94, 1919.
Drummond, J. C.: Researches on the fat soluble accessory substance. II. Observa-
tions on its réle in nutrition and influence on fat metabolism. Biochem.
Jour., 13, pp. 95-102, 1919.

Delf and Skelton: Antiscorbutic value of dryed cabbage. The effect of drying on
the antiscorbutic and growth-promoting properties of cabbage. Biochem.
Jour., 12, pp. 448-463, 1918.

Delf and Tozer: The antiscorbutic and growth-promoting properties of raw and
heated cabbage. Biochem. Jour., 12, pp. 416-445, 1918.

Barnes and Hume: Relative antiscorbutic value of fresh, dried and heated cow’s
milk. Biochem. Jour., 13, p. 306, 1919.

Ohick, H., and Delf, EH. M.: The antiscorbutic value of dry and germinated seeds.
Biochem. Jour., 13, p. 199, 1919.

Bulley, E. C.: Note on xerophthalmia in rats. Biochem. Jour., 13, pp. 103-106, 1919.

Drummond, J. C.: Note on the rate of the antiscorbutic factor in nutrition. Bio-
chem. Jour., 13, p. 77, 1919.

Osborne and Mendel: The nutritive value of yeast protein. Jour. of Biol. Chem.,
38, p. 223, 1919.

Falk, McGuire and Blount: The oxidase, peroxidase, catalase and amylase of fresh
and dehydrated vegetables. Jour. of Biol. Chem., 38, pp. 229-244, 1919.

Emmet and Luros: The stability of lactalbumin towards heat. Jour. of Biol. Chem.,
38, p. 257, 1919.

Hess and Unger: The scurvy of guinea pigs. III. The effect of age, heat and
reaction on antiscorbutic foods. Jour. of Biol. Chem., 38, pp. 293-304,
1919.

Emmet and Allen: Vitamines necessary (B especially but A also) for annual
growth and development of tadpoles. Jour. of Biol. Chem., 38, pp. 325-343,
1919.

Palmer and Kempster: Relation of plant carotinoids to growth, fecundity and repro-
duction of fowls. Jour. of Biol. Chem., 39, pp. 299-313, 1919.

Funk: Action of substances influencing the carbohydrate metabolism in experi-
mental beri-beri. Jour. of Physiol., 53, p. 247, 1919.

Leggate, A. R.: Beri-beri among the Chinese in France. Edinburg Med. Jour., 24,
pp. 32-36, 1920.

Osborne and Mendel: The distribution of water soluble vitamine. Jour. of Biol.
Chem., 39, pp. 29-34, 1919.
METABOLISM UNDER VARIOUS CONDITIONS 857

Osborne, Wakeman and Ferry: Preparation of protein free from water soluble
vitamine. Jour. of Biol. Chem., 39, p. 35, 1919.

Chick and Hume: Note on the importance of accurate and quantitative measurements
in experimental work on nutrition and accessory food factors. Jour. of
Biol. Chem., 39, p. 203, 1919.

Emmet, A. D., and Luros: The absence of fat soluble A vitamine in certain ductless
glands. Jour. of Biol. Chem., 38, p. 441, 1919.

Dutcher, R. A.: Antineuritic properties of certain physiological stimulants. Jour.
of Biol. Chem., 39, pp. 63-69, 1919.

Zilwa, S. 8.: Influence of deficient nutrition on the production of agglutinins, com-
plement and amboceptor. Biochem. Jour., 13, pp. 172-194, 1919.

Harden and Zilva: Accessory factors in the nutrition of the rat. Biochem. Jour.,
12, p. 408, 1918.

Steenbock and Boutwell: Thermostability of the fat soluble vitamine in plant ma-
terials. Jour. of Biol. Chem., 41, p. 163, 1920.

Steenbock, Gross and Sell: The fat soluble vitamine content of green plant tissues
together with some observations on their water soluble vitamine content.
Jour. of Biol. Chem., 41, p. 149, 1920.

Steenbock and Gross: Fat soluble vitamine. Jour. of Biol. Chem., 40, pp. 500-529,
1919.

Willaman, J. J.: The function of vitamines in the metabolism of sclerotinia cinerea.
Jour. of Amer. Chem. Soc., 42, pp. 549-585, 1920.

Sugiura and Benedict: The action of radium emanation on the vitamines of yeast.

; Jour. of Biol. Chem., 39, pp. 421-435, 1919.

Hart and Steenbock: Maintenance and reproduction with grains and grain products

as sole dietary. Jour. of Biol. Chem., 39, pp. 209-234, 1919.
PART III.
PRACTICAL WORK AND METHODS

CHAPTER XXI.

Equipment of the laboratory.

a. Desk reagents.

Strength Strength
1, Hydrochloric acid. Cone... 37% 11. Ammonium oxalate ........ 2%
2. Hydrochloric acid. Dilute.. 10 12. Copper sulphate ........... 2
3. Sulphurie acid. Cone. ..... 95 13. Ferrie chloride ............ 2
4. Sulphuric acid. Dilute. .... 10 14. Potassium ferrocyanide .... 1
5. Nitric acid. Cone. ......... 70 15. Millon’s reagent.
6. Acetic acid. Dilute. ...... 10 16. Magnesium sulphate ....... Solid
7. Lead acetate ............4. 2 17. Ammonium sulphate ....... Solia
8. Sodium hydrate ........... 10 18. Litmus paper. Red.
9. Sodium carbonate ........ 10 19. . Litmus paper. Blue.
10. Ammonium hydrate ........ 10

A Word to the Student.—Laboratory work is of value only when it is done
with a thorough understanding of what one is trying to do; otherwise it is of no
more value to a student than any other form of mechanical exercise. An experi-
ment should never be begun until the whole experiment has been carefully read
through and « clear conception had of the object of the experiment. If you do not
understand the object of the experiment and cannot discover it from reading the
directions, ask the instructor. If he cannot tell you the object do not try the
experiment, or if you do, try to find out from it what the object was. The experi-
ments in this book all have a particular object in being there. Most of them are
to enable you to see for yourselves what is described in the text; but some are also
put in to show you methods of solving problems in chemical physiology or in
clinical diagnosis. One generally remembers better what one sees than what one
simply hears about. The experiments should be done while the text bearing on the
matter is clearly in mind. Read over the work to be done and the text bearing on the
general problems before coming to the laboratory. This will make the laboratory
work far more valuable, and also remove from it much of the drudgery. In the
quantitative work, if you cannot get a result you expect, or which you think is
right, tell the instructor frankly about it and ask his advice. Perhaps your
expectation may be wrong and your results are really right; or it may be that you
have made some error in technique for which he can suggest a remedy. It is no
disgrace to fail in getting an accurate result the first time a new quantitative
method is tried. Even skilled chemists often fail in this same way. But by
repeating the work one will become skillful. It is better to do one thing well than
a dozen things badly. Remember that you are working for your own benefit, and
that honesty is the first requisite for success in all walks of life, but above all it
is the foundation stone of science.

858
ona

13.

14,
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31,
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.

. Acetic Acid
. Acetic Acid
. Alcoholic Alpha Uaphthol.
. Alizarin Red
. Ammonium Hydrate (0.90)

Oak wre

. Ammonium Sulphate :
. Ammonium Sulphide
. Aniline Acetate
10.
11.
12.

PRACTICAL WORK AND METHODS

2. Side-shelf reagents.

Ammonium Molybdate in
HNO, (5% in mixt. —
pts. HO and HNO,).

Barfoed’s Reagent
Barium Chloride
Benedict’s Qualitative So-
lution
Benedict’s Quantitative So-
lution
Bismuth Subnitrate
Boas’ Reagent
Borax Solution
Borie Acid
Bromine Water
Calcium Chloride
Calcium Hypochloride ....
Casein
Charcoal (animal)
Chloroform
Congo Red
Cotton (absorbent)
Dextrin
Dimethylamidoazobenzol ..
Egg White
Esbach’s Reagent
Fehling’s Solution No. 1...
Fehling’s Solution No. 2...
Ferric Ammonium Sulphate
Folin-Shaffer Reagent ....
Formaldehyde
Gelatin
Glucose
Glycerol
Glyoxylic Acid
Guaiacum Tincture
Gum Arabic
Gtinzberg’s Reagent
Haines’ Solution
Hubl’s Iodine Solution ...
Hydrochloric Acid
Iodine in KT
Lactic Acid

Strength
Glacial
N

5%

5%

 

 

Sat.

 

50%

 

5%

 

Solid

 

2%
Sat.
Sat.
5%

 

Solid

 

 

Solid
0.5%
Dried

 

 

47.
48.
49.
50.
61.
52.
53.
54.
55.
56.

57.

58.

59.

60.
61.

62.
63.
64.
65.
66.
67.
68.
69.
70.
71.
72.
73.
74.
75.
76.
77.
78.
79.
80.
81.
82.
83.
84.
85.
86.

87.
88.
89.
90.
91.
92.
93.
94.

 

 

 

 

 

859
Strengto
Lactose: sccicvnas eesecae. Solid
Lead Acetate, basic ......
Levulose .............4.- Solid
Magnesia Mixture ........
Magnesium Chloride ...... 1%
Maltose ................. Solid
Mercuric Chloride ........ 1%
Methyl Orange ........:.
Nitrie Acid .............. N
OL Ve: OW eisicsei de saude a vera
ORG ies hsb den Gat 50% Sat.
Oxalic Acid ............. N
Para -dimethyl - amido - ben- —
zaldehyde .............
Paratin. execs ctnedinsnae ou Solid
Peptone® ...0 ceten eaters Solid
Phenol .................. 2%
Phenolphthalein ......... 0.5%
Phenylhydrazine HCl ..... Solid
Phloroglucin in alcohol ... 2%
Phosphomolybdie Acid .... 2%
Phosphotungstic Acid .... 2%
Picrie Acid in Water ..... Sat.
Potassium Bichromate .... Com’l
Potassium Bichromate .... N/2
Potassium Bisulphate .... Solid
Potassium Chromate ...... Sat.
Potassium Iodide ........ 1%
Potassium Mercurie Iodide. Sat.
Potassium Oxalate ....... Solid
Potassium Sulphocyanate. . 2%
Resorcin in Alcohol ...... 0.5%
Saccharose .............. Solid
Silver Nitrate ........... 1%
Soap Solution ........... 1%
Sodium Acetate .......... Solid
Sodium Alcoholate ....... 10%
Sodium Carbonate ........ N
Sodium Chloride ......... Solid
Sodium Chloride ......... N
Sodium Hydrate at gms. in
LOOM: ssa ts wise. ca eens
Sodium Nitrite .......... 5%
Sodium Nitroprusside .... Solid
Sodium cay euitey 1%
EATEN. os mcittr acter hectv ew wets Solid
Sulphanilic Acid ......... 5%
Talcum Powder .......... Solid
Tannic Acid in Alcohol ... 20%,
Thymol in Alcohol ....... 10%
860 PHYSIOLOGICAL CHEMISTRY

 

Strength Strength
95. Toluene ................- 100% 28. Vaseline ........... sete
96. Tropacolin .............. 0.5% 99. Zine Sulphate ........... Solid
97. Turpentine .............. 100. Zine Sulphate ........... Sat

 

3. Special apparatus for general use.

a. Blast of air and special apparatus carrying aération tubes for ammonia deter-
minations by aération. Each apparatus has places for 12 simultaneous
aérations. See Figure 72.

Kjeldahl! digestion apparatus.

Block tin condenser still with 12-20 places for ammonia distillations.

2 oi] baths with tubes for use in Benedict’s urea determination. Figure 71.

Long copper lined trough with running water for dialysis experiments, 8” x 8”
x 15 ft.

Burettes connected with large bottles containing N/2 and N/10 H,SO, and NaOH
for quantitative titrations; also with saturated Ba(OH), solution.

g. 40% solution of soda lye containing Na,S for Kjeldah) determinations. To
10 Ibs. NaOH (Commercial powder; soda lye) add while stirring 11, 320 ec.
water and then 250 grams powdered K,S or 180 grams NaS. Stir until all
is dissolved. Allow to settle out for 2-3 days and siphon off the clear
supernatant liquid. For use in Kjeldahl-Gunning nitrogen determination.
Take 100 cc of this solution to neutralize the 20 c.c. concentrated H,SO
used in the digestion. This soda lye solution is best kept in a large bottle
sitting on a large, enameled-ware plate to protect from the drip, and pro-
vided with a bicycle pump or compressed-air connection by which air pres-
sure can be made on the solution so as to drive some over from the delivery
tube. The cork has two holes, one connecting with the compressed air and
provided with a side tube to remove the air pressure when the required
amount of liquid is discharged; the other carrying the delivery tube which
opens near the bottom of the soda lye bottle.

h. The laboratory must of course have other common pieces of apparatus such
as thermostats, polariscope, spectroscope, colorimeters, balances, etc.

i. Woolen blanket, pail of sand, pail of water, five extinguishers.

pases

La

4. Desk outfit for each student.

One of the blanks and the outfit’ is in the desk when the student enters the
course. He checks off the apparatus, signs the blank and returns blank to the store-
room. The blank is as follows:

PHYSIOLOGICAL CHEMISTRY OUTFIT.

This list must be checked, signed, and returned to the storeroom before the
student can receive materials from the storeroom.

Not returnable goods in the drawer :—

Filter paper, 3 sheets. 1 Test-tube brush.
Matches (safety), 1 box. 1 Towel.
Soap, 1 cake. 1 Wire gauze (4x4 in.).

1 Sponge.
PRACTICAL WORK AND METHODS

Returnable goods in the drawer :—

861

1 Burette with pinch clamp. 1 Mortar and Pestle, porcelain.
1 Cylinder, graduated, 5 c.c. 1 Pipette, 10 cc.
1 Cylinder, graduated, 100 c.c. 1 Pipette, 25 c.c.
1 File, triangular. 1 Pipette, graduated, 5 c.c.
2. Flasks, Erlenmeyer, 150 c.c.. 1 Test-tube holder.
2 Flasks, Erlenmeyer, 300 c.c. 1 Thermometer, 150° C.
1 Flask, Erlenmeyer, 500 c.c. 3 Tubes, fermentation. .
2 Flasks, Florence, 250 c.c. and 500 e.c. 1 Spatula, horn.
1 Funnel, Buchner, with rubber stopper 2 Watch glasses, 2-inch.
No. 8. ‘ 1 Watch glass, 3-inch.
2 Glass rods, 8-inch.
Returnable goods in the cupboard :—
3 Beakers, low, with lip, 75 c.c. 1 Flask, filter, 800 c.c.
2 Beakers, low, with lip, 250 c.c. 2 Funnels, 5 cm. and 10 cm.
1 Beaker, low, with lip, 1000 ce. 3 Rings, iron.
3 Bottles, narrow mouth, 300 c.c. 1 Ring stand, iron.
1 Burner, Bunsen, with tubing. 12 Test tubes, 6-inch.
1 Clamp, iron and attachment. 1 Test tube rack.
1 Disiccator. 1 Tripod, iron.
4 Evaporating dishes, 7 em., 10 cm., 15 1 Wash bottle, 800 c.c.

em. and 21 cm.

1 Wire basket.

Received these articles in good condition and 1 key for desk.

Signed

University Address ...

Home Address
Checked by

Dm eae mw e ww en errr nese er envaee eee

Pee eer emma wren mercer tween tance eeee

(The reverse side of the blank carries the breakage account.)

B. General directions for work.—The general aim should be to
make as much of the work accurate and quantitative as possible. A
quantitative experiment teaches all that a qualitative experiment teaches,
and much besides; in addition it requires careful and accurate manipu-
lation and cleanliness, qualities so necessary to a physician or chemist.
In the experiments which are described it is essential when quantities
of certain materials are mentioned that they be accurately measured
and all the conditions of the experiments carefully noted and fulfilled.
The results of many of the experiments are instructive only when they
have been accurately performed, under given conditions and with suit-
able controls.

The first requisites to proper laboratory manipulation of any kind,
whether chemical, physical or surgical, are neatness and order. Neat.
workers only can be accurate manipulators. Orderly workers usually
make fewer mistakes in laboratory manipulation than do those who go
over the work hurriedly and without system or plan.

Apparatus found in the desks should first be cleaned, rinsed with
distilled water and allowed to drain. The apparatus should be cleaned
862 PHYSIOLOGICAL CHEMISTRY :

each time immediately after using and kept ready for use. Most of the
apparatus soiled in this work is best cleaned by rinsing well with warm
water, then washing with soap and water and brush or cloth, then again
rinsing well with warm water and finally rinsing with distilled water.
In every case friction must be used by rubbing with a brush. Never
dry the inside of a beaker or other chemical vessel with a towel. [If it
must be dried quickly, rinse with alcohol and ether and dry with a cur-
rent of air; or dry by gently warming after rinsing in distilled water.

Test-tubes should never be rinsed only, but should be washed by
means of a brush and then should be well rinsed. In most cases clean-
ing mixture is not necessary, especially as usually employed by the inex-
perienced. Use judgment in cleaning your apparatus; for instance, do
not try to remove fats by rinsing with cleaning mixture and do not
try to remove barium salts by means of cleaning mixture. Always use
the appropriate solvent first, then remove the excess of the solvent and
continue with the washing as above. Pipettes, after the preliminary
cleaning with the appropriate solvents and rinsing as above indicated,
often require further treatment with hot cleaning mixture. Finally,
rinse well with warm water and distilled water.

In measuring reagents, do not insert a pipette into the reagent
bottle, but, pour off about the amount desired into a dry vessel and then
measure therefrom by the pipette. Never return solid or liquid reagents
to the stock bottles. Never suck up strong acid or alkali into a re
but measure these from a measuring glass or burette.

In taking samples or reagents with a pipette be sure to have your
pipette clean and dry, or rinsed with a part of the solution at least
twice before measuring it off. After rinsing with a part of the solution,
hold the pipette between the thumb and second finger, draw up the
liquid, place the index finger on the mouthpiece and by turning the
pipette between the thumb and second finger allow the lower part of
the meniscus to come on a level with the mark. Then observe that no
drops adhere to the outside of the pipette and transfer the liquid to the
proper vessel. Allow to drain by touching the side of the vessel with
the tip of the pipette, but never blow into the pipette except with the
Ostwald pipettes.

Above are given two lists of reagents arranged in the order they are
found on the shelves. Do not insert pipettes, glass rods or other utensils
into these reagents. Do not lay the stoppers on the desk or other sur-
faces; learn to hold the stopper in your hand while pouring from the
bottle. Do not moisten litmus paper by means of the stoppers. PLEASE
RETURN EACH BOTTLE TO THE PROPER PLACE IMMEDIATELY AFTER USE.

Your note-book must contain for each experiment the following data:
1. The object of the experiment; the question to be answered by the

Spo ue
PRACTICAL WORK AND METHODS 863

experiment. Do not begin the experiment until you know this. 2. A
brief but accurate statement of the methods employed for its solution.
3. All results of weighings, readings of burette, measurements, etc., at
the time they are made. All calculations should be given. 4. A concise
critical statement of the conclusion.

Filtering. Nearly all slimy or flocculent precipitates are best filtered
by folded or creased filters without the use of suction. Crystalline or
granular precipitates may be filtered to advantage with suction. If it
is desired to save a precipitate, select the size of the filter with regard
to the size of the precipitate, not to the size of the filtrate. Albuminous
precipitates which stick to the filter are best taken from the filter paper
while they are still somewhat moist. Use filter plate and small filter
paper where possible.

General principles. Avoid using excess of reagents; keep the num-°
ber of different kinds of substances in a solution as small as possible.

CAUTION.—Acetone, alcohol, ether, benzene, glacial acetic acid are
inflammable. Never heat any of them over the free flame. Heat them
on the steam or water or electric bath. Have no lights near when
pouring ether, acetone or benzene from one vessel to another. In case
of fire, if in a dish or flask, cover it with a wet towel, thus suffocating it.
Water may be added to an alcohol or acetone fire. Blankets are kept in
the laboratory for use if clothing catches fire. If strong ammonia or
alkaline liquids spurt into the eye, wash out at once with water and
follow as quickly as possible with concentrated boric acid solution
(No. 17). If acids enter the eye wash with water at once and then with
borax solution (2%) (No. 16).
CHAPTER XXII.
QUANTITATIVE SOLUTIONS AND ANALYSIS.
Exercise I. ACIDIMETRY AND ALKALIMETRY.

In this exercise there are to be prepared for subsequent use standard
solutions of acid and alkalies.

Experiment 1. The preparation of a standard N/1oth acid, from
benzoic acid.—Prepare first a small beaker so that it is perfectly clean.
This is to be used to weigh into. To clean chemical glassware, wash
first in hot water and soap, rinse in hot water, then in distilled water,
then with acetone. The outside of the beaker can be dried with a towel
or linen or silk clean handkerchief, but the inside must always be rinsed
in pure water or alcohol or acetone or ether and allowed to dry. There
must be no lint on the beaker when it is weighed. Place the clean
beaker in your dessicator after it is dried. Do not handle it with your
fingers. In lifting it from the dessicator use a forceps or a clean dry
linen or silk handkerchief.

To weigh out the acid. First test the balance to see that when set
in motion by momentarily placing the rider on the right-hand arm, it
swings equally to each side of the center. With the arms of the balance
supported by the beam support place the dry beaker on the left-hand
scale pan. With the beam support supporting the pans now place a
weight on the right-hand side. Never handle the weights with fingers
or with anything else than the forceps, preferably ivory tipped, fur-
nished with the weights. Never handle anything else than the weights
with these forceps. If the first weight put on is too heavy, turn up the
beam support which lifts the knife edges off from the agate plate,
remove this weight and put on the next lighter. Lower the beam
always very gently so as not to injure the knife edges. Proceed in this
manner until a weight is on the right-hand scale pan which is lighter
than the beaker, but not enough lighter to permit the left-hand pan to
be depressed when it is supported by the pan arrest. The weight will
then usually be within one gram of the required amount. Now put on
the 0.5 gr. weight, release the pan arrest for a moment to see if this is
too heavy or too light. If it is too light add the next heavier weight,
and proceed in this manner until the beaker is balanced almost exactly.

The final balancing is done by means of the rider. Having accurately
864
PRACTICAL WORK AND METHODS 865

balanced the beaker, record the weight accurately in your book. Then
add to the right-hand scale pan weights of exactly 1.22 grams.

Benzoic acid, C,H,COOH, mol. wt. 122, comes in the trade in a very
pure state, it may be resublimed to practical purity if desired but that
will not be necessary for your work. It has no water of crystallization
nor does it condense water on its surface. For all these reasons it is
chosen for a standard. Phthalic anhydride, or the monopotassium
phthalate is an equally good standard substance.

Now carefully add little by little, without dropping any, air-dried
benzoic acid to the beaker on the left-hand scale pan. By removing a
little on the end of a knife blade the weight may be made exact. Having
added so much benzoic acid that an exact balance is had, raise the beam
support. Then carefully add up the weights on the scale pan without
disturbing them and put down the figures in your note book above the
figures of the weight of the beaker. Having done this check this reading
by taking off the weights, beginning with the heaviest and proceeding
steadily to the lightest, and as each weight is taken off check the cor-
responding figure in your note book. Having taken off the gram weights
take off the decigrams and check that figure, then the centigrams, and
finally the milligrams. It is necessary to do this, as mistakes at reading
the weights are easily made. :

To bring the benzoic acid into solution add to the beaker a few e.c.
of 95 per cent. alcohol, enough to dissolve the acid. Now take a 100 c.c.
volumetric flask which is clean, but need not be dry. Place in the neck
a small funnel. Then without losing any pour the benzoic acid solution
into the funnel and while holding the beaker still suspended over the
funnel with the left hand pick up your wash bottle with the right and
carefully wash out every portion of the inside of the beaker with a
stream of water, catching every bit of the water in the funnel. Wash
out the beaker again and again so that no particle of the benzoic acid shall
escape going into the flask. Now put the beaker down, but before doing
so turn it around and wash off the outside of the lip into the funnel. Put
the beaker down and wash out carefully the inside of the funnel with
distilled water. Then lift the funnel from the flask. If any of the
benzoic acid comes out of solution add a little more alcohol. Enough to
dissolve it. Now add water carefully from the wash bottle, giving the
flask a whirl from time to time until the bottom of the meniscus is
exactly at the mark in the stem. Stopper tightly with a glass or rubber
stopper and shake thoroughly so that the contents are thoroughly mixed.
Turn upside down several times.

The standard is now made. It consists of 100 c.c. of solution con-
taining 1.22 grams of benzoic acid. 1.22 grams is exactly one one-
hundredth of the molecular weight. A liter of the solution of the same
866 PHYSIOLOGICAL CHEMISTRY

.. Strength would contain 12.2 grams or 1/10th of the molecular weight.
This solution is hence a 1/10th molecular solution. That is, a liter would
contain 1/10th of a molecular weight expressed in grams. It is usually
written M/10.

Since benzoic acid has but one hydrogen atom in a molecule which
can be replaced by a metal, there is 1/10th of a gram of replaceable
hydrogen in a liter of the solution, the atomic weight of H being unity,
so that the solution is 1/10th normal, or N/10, a normal solution of an
acid being a solution which contains in one liter one gram equivalent
of hydrogen. A normal solution always contains a gram equivalent of
the substance, toward which its normality is reckoned, in a liter of
solution.

Experiment 2. The preparation of a liter of N/1oth NaOH free
from carbonate.—Having obtained the standard benzoic acid solution,
proceed to make a liter of standard N/10th NaOH. To do this procure
a 1 liter volumetric flask and clean it. It need not be dry. The standard
NaOH is to be made from a saturated NaOH solution, a large amount of
which is prepared several days beforehand and which has settled until it
is clear. It is kept in a large bottle provided with air pressure for
delivery, as explained on page 860. Such a solution crystallizes out the
carbonate and leaves the hydrate in solution. When saturated it is
approximately 50 per cent. by weight and its specific gravity is 1.530.
A 1/10th N NaOH solution will contain in a liter one-tenth of the
molecular weight. The molecular weight of NaOH is 40. A 1/10th N
solution will contain 4.0 grams NaOH in a liter.

To make it calculate how much you will need of the saturated solu-
tion. 100 cc. of this solution at 15° will weigh 153 grams. It contains
50 per cent. of NaOH or 76.5 grams. You wish only 4.0 grams of NaOH.
You will need 8 grams of solution, or 8/1.53 e.c. or 5.23 ec. Measure
out this amount from the burette attached to the bottle, into a 1 liter
flask. Now add 450 cc. of distilled water and thoroughly mix the solu-
tions by whirling the flask.

The next step is to find out how much water must be added to make
this solution exactly N/10. To do this it is necessary to compare the
strength of the alkali against the standard benzoic acid.

Clean two 50 c.c. burettes which have been provided with rubber or
glass tips and a pinch cock. To clean them wash out first with soap
and water if they are greasy. If not rinse out with cleaning fluid
(sulphuric acid and bichromate) then with tap water and finally with
distilled water and wipe off the outside with a towel. Allow to drain so
that they are nearly free from water. Into one pour a little of the
benzoic acid solution, a few ec. (2 or 3) and with thumb over end
invert several times and then drain through the pinch cock. Do this
PRACTICAL WORK AND METHODS 867

again. This washes out the burette. It is not necessary if the burette
is clean and dry to begin with. Now fasten in the lamp stand and fill
to a little above the zero mark. Allow the liquid to run out by. the tip
so that the bottom of the meniscus is at the zero point. To read it put
the eye on a level with the mark and hold a little black paper around the
tube just below the meniscus. This makes the meniscus black and sharp.

Rinse the other burette with the sodium hydrate solution from the
liter flask twice and then fill it in a similar manner.

If we were doing really very accurate work it would be necessary to
calibrate the burettes.

Now into a clean 100 or 200 c.c. Erlenmeyer flask draw about 25 c.c.
of the benzoic acid solution, add to it 2 drops of phenolphthalein solu-
tion in alcohol (2 per cent.) to serve as an indicator. Now read accu-
rately and note in your notebook the reading of the NaOH burette to
hundredths of a c.c. When this is done run in from the NaOH burette
into the benzoic acid. The tip of the burette should be a little below
the mouth of the flask so that none may be lost. About 7 cc. may be
run in quickly. It will be noticed that the red color appears but fades
with each addition of the alkali. Now go more slowly, adding about
one-fourth of a ¢c.c. at a time and shaking between each addition so as
to mix the contents. Give the flask a whirl to mix the contents. The
red color disappears less and less rapidly. Finally a few drops give the
flask a permanent pink. When this happens remove from the NaOu
burette, with a wash bottle wash down the sides of the flask, since some
drops may have gone on the side, then add drop by drop from the
benzoic acid until the red color just disappears. Now add one drop of
the alkali so that it is barely pink. This is the end point. Now read
both burettes carefully. Compute now how much of the NaOH solution
must be taken in order that when diluted to 1000 ¢.c. it will be N/10,
or very nearly. Measure out the amount needed into a 1 liter vol. flask,
tnake up to 1 liter with water, mix and titrate for exact value against
N/10 benzoic acid. e.c. NaOH needed to make N/10=c.c. benzoic/c.c.
NaOH. Transfer to a liter, rubber-stoppered bottle.

Exercise IT.

Experiment 3. Preparation of standard acid. N/1o HCl.—Con-
centrated HCl of sp. gr. 1.160 is approximately 10N. To make a N/10
solution it must hence be diluted 100 times. Introduce into a clean
1 liter vol. flask by means of a 10 c.c. volumetric pipette, 10 cc. of con-
centrated HCl. Dilute to one liter.

Now standardize this against the N/10 NaOH and mark exactly
what 1 c.e. of it is equal to in terms of the benzoic acid. Dilute if neces-
sary to make it equal to N/10.
868 PHYSIOLOGICAL CHEMISTRY

Exercise IIT.

Experiment 4. Indicators—Indicators are substances which
change their color when they change from alkaline to acid solutions.
There are many such substances and they are used for particular pur-
poses. There is no one indicator which can be used everywhere. Some
are weak acids and some are weak bases. One of these has been used
already, phenolphthalein. This changes from a pink to colorless or vice
versa at a hydrogen ion concentration of N/10%°. It has become the
custom for shortness simply to write the exponent in the foregoing
fraction. This exponent is called P,. Pin this case is 8.3. The H
has reference to the hydrogen ion concentration. In water Pyis 7, that
is, the concentration of H ions in water is N/10*. That is, water is as
acid as a one ten-millionth normal solution of hydrochloric acid.

 

Changes at se Use in titrating Do not use
Phenolphthalein 8.3 Weak acids for weak bases
Litmus 7.0 Weak acids or bases
Congo red 5.0 Strong acids or bases for weak acids
Methyl red
Methyl orange 5.0 Strong acids or bases for weak acids

 

 

 

To show this use of indicators, make an approximately N/10th solu-
tion of acetic acid and titrate it with N/10 NaOH, using in the one case
phenol phthalein, and in the other congo red and methyl orange. Keep
a record for the instructor of your titrations.

Repeat, using a solution of about N/10 NH,OH made by yourself.
The solution need not be accurately made. Titrate this with N/10 HCl,
using different indicators. Explain the results.

EXeErcise IV.

Experiment 5. Buffer solutions.—Buffer solutions are solutions of
salts so chosen that there is little change of acidity or alkalinity pro-
duced by the addition of a strong acid or base. Protoplasm is such a
buffer solution and the blood is another. Large amounts of acid may
be added to blood without greatly changing its hydrogen ion concentra-
tion. Buffers are made of strong acids and weak bases, such as am-
monium chloride, for example, or of strong bases and weak acids, as in
sodium bicarbonate, acetate, or phosphate.

Such solutions are of great value in keeping the nydrogen ion con-
stant in experiments in which acids or bases are formed as in the course
of digestion. They are addea to enzyme solutions in order that they
may work under the most favorable conditions. Good buffers are made
PRACTICAL WORK AND METHODS 869

of standard concentration of H ions from mixtures of the di- and mono-
sodium. phosphates, of bicarbonates and of mixtures of sodium acetate
and acetic acid. If HCl is added to a solution of sodium acetate, sodium
chloride is formed and acetie acid. Acetic acid in the presence of acetate
ions is almost undissociated so that it forms very few. hydrogen ions.

From the tables, pp. 544 and 545, prepare buffer solutions 100 c.c.
of each of P,, 8.0, 8.3, 7.4, 6.5, 6.0, 5.38 and 5.0. Determine the H ion of
each one by comparison with the standard furnished.

To show how they act as buffers take 5 c.c. of the solution of Py 8.3.
This solution will just be alkaline to phenolphthalein. Now determine
by running into it from a burette or pipette N/20 HCl how much of this
is required to make the solution have a H ion concentration of P, 7.
Observe and record how much more is necessary than to change a NaOH
solution just barely pink to phenolphthalein and of the same P, as the
buffer. Acids are constantly being poured into the blood from the oxi-
dations in the tissues, but these acids do not greatly change the H ion
concentration of the blood because in the blood is a buffer solution of
NaHCO, and Na,HPO,.

EXERCISE VI.

Experiment 6. Determination of nitrogen in uric acid.—To in-
culcate habits of accuracy and to show a standard method of analysis
which involves acidimetry and alkalimetry, the determination of nitrogen
in uric acid by the Gunning-Arnold modification of the Kjeldahl method
is put in here.

Obtain from the store-room a weighing tube containing about 0.25
gram of uric acid. (If uric acid is not on hand some other crystalline,
nitrogen containing, organic substance of known composition may be
substituted for it.) Wipe off the tube carefully with a clean, dry linen
or silk cloth. Remove the stopper and dry to constant weight in the
oven at about 105° C. Remove at the end of one hour, restopper loosely,
place in dessicator until cool, and weigh on the analytical balance to
1/10th of a mg. Replace in the oven and heat another hour, cool and
reweigh. Repeat until the weight remains constant. Do not handle the
tube with the fingers but always with a clean, dry cloth, or forceps. In
weighing follow the directions already given on page 864. Record all
weights in your note book. Do not record them on loose pieces of paper
to be afterwards transferred. When weight is constant, transfer the
uric acid without loss to a 500 or 800 ¢.c. Kjeldahl flask. Stopper and
reweigh the tube. The difference between this weight and the weight
while the uric acid was still in the tube gives the weight of uric acid
taken. From now on proceed in the manner described for the determina-
tion of N in protein on page 931,
CHAPTER XXIII.
THE CARBOHYDRATES.

ExercisE VII. REDUCING ACTION OF CARBOHYDRATES. TESTS FOR
DETECTION.

The experiments of this exercise are designed to show a very im-
portant property of carbohydrates, namely, their reducing powers. It
is by means of this property that they may be easily detected in the
blood, urine and other liquids, and the following experiments are tests
for their presence. The reduction is made evident to the eye by a change
in color of the substance reduced. (Read pages 256-261 on the chemistry
of oxidation; and pages 30 to 41 on the dissociation and oxidation of
carbohydrates.) The word oxidation means, literally, a souring or
acidification and in all of the following experiments the carbohydrate is
oxidized to various acids.

Experiment 7. ‘The reduction of cupric compounds to cuprous.
(Trommer’s test.)—The copper atom in the cupric state carries two
positive charges of electricity and is blue in aqueous solution. It gains
a negative charge with great ease, whence its oxidizing powers, and goes
over to the cuprous state, in which it has a single positive charge, and
it is then either colorless, or when united with oxygen to make cuprous
oxide, Cu,O, either yellow or red, depending on whether it comes out in
a very finely divided state or a coarser state.

Place in a test tube 1 ¢.c. of M/5 glucose and add two drops of cupric
sulphate, CuSO,, 2 per cent. Then add 2 drops of NaOH, 10 per cent.
Observe that the addition of NaOH causes no precipitate of gelatinous
blue Cu(OH),, as it does in the absence of the carbohydrate. Instead
a deep blue solution is formed. The carbohydrate has now combined
with the Cu(OH), and holds it in solution. It acts in this respect
exactly like the sodium potassium tartrate in Fehling’s solution. (See
page 39.) The first step in the oxidation has now taken place. The
oxidizing agent, the Cu(OH), has combined with the reducing reagent,
the glucose, but the negative charge or electron has not vet passed from
a carbon atom of the glucose to the cupric atom. Now heat the tube
gently in a flame. The blue color disappears and the solution becomes
filled with a fine, yellow, colloidal precipitate. This precipitate is very

finely divided cuprous oxide, Cu,0. The glucose has been oxidized to
870
PRACTICAL WORK AND METHODS 871

‘various acids. This production of a red or yellow precipitate of Cu,O
when a substance is heated with an alkaline cupric salt solution is an
indication of the presence of a carbohydrate, but is not conclusive evi-
dence, as‘many other reducing substances, such as uric acid, phloro-
glucinol, creatinine, etc., have a similar reducing power, and some carbo-
hydrates, such as the glucosides, cane sugar, starch and the polysac-
charides generally will not reduce unless they are hydrolysed first. This
is interpreted to mean that in these non-reducing carbohydrates the
aldehyde or ketone group has been substituted and rendered more stable
so that the carbon atom of that group is not able to receive the positive
charge and to be oxidized as easily as when these groups are free. (See
formulas on pages 52 and 55.)

Experiment 8.—In the same manner as in experiment 7 test the
reducing powers of M/5 solutions of maltose, levulose, galactose, lactose,
cane sugar, starch, and, if it can be obtained, some pentose, such as
arabinose or xylose, and record your observations. Starch, cane sugar
and any glucosides you may test should not reduce, but the others
should reduce.

Experiment 9. The action of the alkali in experiment 7.—To make
clear the action of the NaOH used in experiment 7 repeat the experi-
ment without using the alkali.

To 1 c.c. of M/5 glucose solution add two drops of 2 per cent. CuSO,
and heat to boiling. It will be observed that no reduction occurs, al-
though if the boiling is continued long enough, i.e., about 1 hour, reduc-
tion will slowly occur and erystalline metallic copper may thus be
obtained. If after heating and noting that no reduction has taken place,
2 drops of 10 per cent. NaOH be added and the heating repeated, reduc-
tion takes place at once. The addition of alkali is necessary for a rapid
reduction. It enormously accelerates the reduction. How does it do
this? To settle this question the action of the alkali on the sugar alone
may be tried. Take 1 ¢.c. of M/5 glucose and add 2 drops of 10 per cent.
NaOH and heat to boiling. It will be observed that the solution becomes
first yellow and then brown and has a distinct caramel odor. This brown
eolor, when a substance is heated with NaOH, is known as Moore’s test
for a carbohydrate. As soon as it becomes light brown stop heating and
cool under the tap or by immersing in a beaker of water until it is of
room temperature. Label this tube, A. Into another tube (B) put 1 ce.
of M/5 glucose and 2 drops of 10 per cent. NaOH. This tube is a control
tube. We have now in the first tube (A) glucose which has been heated
with NaOH and thus altered, and in the second tube (B) we have the
same materials but the tube has not been heated. Add to (A) and (B)
2 drops of 2 per cent. CuSO, and allow both tubes to stand at room
temperature. After a few minutes tube A changes to a yellow color
872 PHYSIOLOGICAL CHEMISTRY

and becomes filled with a colloidal precipitate of yellow Cu,O whereas
tube B remains blue for many minutes. In other words, we have shown
that the alkali has greatly increased the reducing power of the glucose
so that after heating it reduces the copper at a very much more rapid
rate than it did before. The alkali, therefore, is added to all these
reducing tests primarily for the purpose of increasing the reducing
power of the sugar.

Experiment ro. Character of the change in the carbohydrate pro-
duced by the alkali. Production of enols-—What has the alkali done
to the carbohydrate? Its first action has been to produce enols, p. 32,
and the subsequent action has been a fragmentation and condensation of
the enols thus formed. Enols have the property of forming with ferric
chloride a deep Bordeaux red (wine color). To show this take 1 cc. of
M/5 glucose or maltose in a test tube, add two drops of 10 per cent.
_ NaOH and heat until yellow. Then cool as before to room temperature.
Now add I drop of 2 per cent. FeCl, solution. The solution turns a
deep red, which is the enol reaction. This test is used for the detection
of acetoacetic acid in the urine under the name of Gerhardt’s reaction.
(Page 1074.) If after heating in alkaline solution the solution be made
just perceptibly acid with sulphuric acid it will turn blue on the addition
of a drop of FeCl,. This is probably due to an intermediate stage
(between ferric and ferrous) of reduction of the ferric iron, since
cysteine and many other reducing agents show a similar reaction. Among
the substances giving a green color with ferric chloride are adrenaline
and hydrochinone. With ferrous salts no color is produced.

Experiment 11. Fehling’s test for sugars. (Pages 38-39.)—If the
sugar is present in only small amounts there is not enough of it to hold
the Cu(OH), which is formed by the addition of the NaOH to CuSO,
in solution. It precipitates as blue Cu(OH), and this changes to black,
or dark brown CuO on heating. To show this put in a test tube 1 ec.
of distilled water and then 5 drops of 2 per cent. CuSO,. Now add 2
drops of 10 per cent. NaOH. Observe that a blue precipitate, Cu(OH),,
is formed. Heat the tube. The blue precipitate changes to a dark
muddy brown due to finely divided CuO. This precipitate would mask
the yellow Cu,O were the latter present in small amounts. To obviate
the possibility of the formation of this masking brown precipitate one
may add to the solution something which will hold the Cu(OH), in
solution and prevent its conversion to CuO. This is done in Fehling’s
solution by adding Rochelle salts or sodium potassium tartrate,
NaKC,H,0,, and in Haines’ solution by the addition of glycerol. Both
tartaric acid and glycerol are oxidized by the cuprie hydrate at a very
slow rate. In Fehling’s solution the tartrate and hydrate are in one
solution (B) and the cupric sulphate in (A). To use take equal parts

e
PRACTICAL WORK AND METHODS 873

of A and B, mix, and add to them an equal volume of the solution to
be tested for a reducing sugar, and heat.

Prepare 20 cc. of Fehling’s solution by mixing 10 ec. each of A
and B. Put 2 ¢.. of the mixture into each of eight test tubes. To each
tube add 2 ec. of the sugar solution to be tested, namely, M/250 solu-
tions of arabinose, maltose, glucose, levulose, lactose, galactose and
saccharose. Mix well and immerse all tubes at the same time in a beaker
of boiling water. Observe the time at which in each tube the red precipi-
tate of Cu,O appears. It will be observed that all of these reduce except
saccharose.

This power of polyhydrie alcohols to hold in solution Cu(OH), ap-
pears in reverse form in Schweitzer’s reagent (page 883), in which
euprie hydrate dissolves cellulose.

Experiment 12. Benedict’s qualitative test—Fehling’s solution has
been modified by Benedict so as to increase its sensitivity. The al-
kalinity of the solution is decreased by substituting Na,CO, for NaOH,
and Na, citrate for Rochelle salts. Benedict’s reagent is the following:
85 grams of sodium citrate, Na,C,H,O,11H,0, and 50 grams anhydrous
sodium carbonate are dissolved in 400 c.c. water. <A solution of cupric
sulphate of 8.5 grams dissolved in 50 ¢c.c. of hot water are now poured
slowly with stirring into the carbonate-citrate solution. Filter if
necessary.

To make the test for sugar in a solution, such as urine, take 5 c.c. of
the reagent in a test-tube, heat to boiling (a pebble may be added to
prevent bumping), add about 8 drops of the urine or sugar solution and
boil for 2 minutes. In the presence of more than 0.2 to 0.3 per cent.
dextrose in urine or equivalent amounts of other reducing sugars, the
tube will be filled with a colloidal, greenish yellow or reddish precipitate.
Smaller amounts of sugar make the precipitate appear only on standing
and cooling. With pure sugar solutions the sugar may be detected in
greater dilution than this.

Experiment 13. The reduction of picric acid to picramic acid pro-
duces a deep reddish brown color and this color change has been used to
detect the presence of glucose in blood and other fluids of the body, and
is the basis of the colorimetric method for the determination of creatinine
and sugar in blood.

Into a test tube measure 1 ¢.c. of M/5th solution of maltose, glucose,
lactose, or other reducing sugar, add 3 drops of 10 per cent. NaOH and
1 @e. of a saturated aqueous solution of picric acid. Heat to boiling.
On heating the light yellow color of the picric acid changes to the deep
brownish red of the picramic acid. As the depth of color produced
depends on the amount of sugar present, by estimating the color the
amount of sugar can be determined. (See experiment 204, page 1028.)
874 PHYSIOLOGICAL CHEMISTRY

Reaction:
C,H,.OH. (NO,), + 3H, segs C,H,.OH. (NO, ) oN, + 2H,0
Picrie acid Picramie acid

Note: Creatinine gives this test but the color comes in the cold promptly with-
out heating, and if heated the color due to creatinine fades very promptly, leaving
the color due to the sugar alone. Even in the cold the color due to creatinine will
fade in the course of fifteen to twenty minutes.

Experiment 14.—The action of the alkali in this reduction test is,
as before, to increase the reducing power of the sugar. This fact is
illustrated in this experiment as well as in experiment 9.

Into each of twe test tubes measure accurately with a 1 e.c. volu-
metric pipette 1 c.c. of maltose, glucose, or other reducing sugar solution
of M/5th strength. Then add to each, 1 c.c. of N/2 NaOH. One of
these tubes (A) set to one side; the other (B) heat to boiling. It
becomes yellow.. When this happens cool this tube by holding under
the faucet until it has the same temperature as (A). Now add to each
tube, (A) and (B), I «ce. of saturated picric acid solution, and after
mixing let each tube stand at room temperature. It will be observed
that tube (B), the one which has been heated, becomes a deep red, due
to the reduction of the picric acid to picramic acid. The other, (A),
which had not been heated, remains yellow and changes to an orange
very slowly. In other words, heating the sugar in alkali has greatly
increased its reducing power, so that it now reduces the picric acid
very rapidly.

Experiment 15. Sensitivity of reduction of picric to picramic
acid.—To show how sensitive this test is take 1 e.c. M/2000 glucose solu-
tion, add 1 drop of saturated aqueous picric acid solution, 3 drops of
10’ per cent. NaOH and heat to boiling for a few seconds. Make a
control tube with 1 ¢.c. of water in place of the sugar solution. The
glucose tube becomes the darker of the two. The limit of concentration
in which glucose can be detected by this method is about an M/5000.
The limit with phosphotungstic acid is about an M/3000, although a very
faint and transient blue color is developed in M/4000.

Experiment 16. Reduction of phosphotungstic acid.—Glucose and
other reducing sugars have the power also of reducing phosphotungstic
acid with the production of a deep blue color of wolframic oxide.

To 1 cc. of M/5 glucose in a test tube add 3 drops of 10 per cent.
NaOH and heat to boiling or until it becomes yellow. Then add 1 drop
of 10 per cent. phosphotungstic acid (made as described on page 1063).
A deep blue color supposed to be due to the formation of W,O, is pro-
duced. This reaction is very feeble if the phosphotungstic acid is added
to the sugar solution before heating, for if the phosphotungstie acid is
heated first with sodium hydrate the reaction is not given, hence the
PRACTICAL WORK AND METHODS 875

necessity of heating the glucose solution first. The limit of sensitivity
of this reaction is about M/8000 glucose solution.

Experiment 17. Reduction of permanganate.—Glucose and many
other organic substances reduce potassium permanganate in an acid or
alkaline solution.

To 1 cc. of M/5 glucose or other sugar solution add 2 drops of 10
per cent. H,SO, or 16 per cent. NaOH and then drop by drop N/20
KMn0O,. The prink color of the KMnO, is discharged, due to the reduc-
tion of the manganese in KMnO, where it has a positive valence of 7,
to MnSO,, where it has a valence of 2.

Reaction:

2KMn0, -+ 3H,SO, + glucose —_- K,SO, + 2MnSO, + glucose(O,)

Experiment 18. The reduction of an acid cupric salt solution.
Barfoed’s solution.—In the preceding experiments the reduction has
always been carried out in an alkaline medium. In Barfoed’s solution
it is carried out in an acid medium. Barfoed’s reagent is prepared by
dissolving 13.3 grams of crystallized neutral cupric acetate in 200 cc.
of distilled water. This is filtered if necessary and to each 200 ce. of
filtrate are added 5 c.c. of a 38 per cent. acetic acid solution.

Determine the speed in which the following solutions reduce 5 ce.
of the reagent: To 5 ce. of M/100 solutions of arabinose, glucose,
levulose and galactose, also M/100 and M/25 solutions of maltose, lac-
tose and saccharose add 5 e.c. of the reagent. Immerse in boiling water
and continue heating therein for 25 minutes. Observe the time at which
perceptible reduction occurs in the different tubes. (Read page 40.)

It will be observed that in this case the monosaccharides reduce far
faster than the disaccharides and Barfoed’s solution may be used as
a rough indication of whether monosaccharides or disaccharides are
present. The reduction in the monosaccharides is, however, far slower
than in an alkaline solution. This is owing to the fact that the sugar
is not fragmented to the same degree here as in an alkaline solution.
The cupric ions are present in larger numbers, but the oxygen or
hydrate ions are present in very much smaller amount. The total speed
is probably a function of the product of the number of active sugar
particles, the concentration of the cupric ions and of the hydrate ions.
The product is lower in acid than in alkali. The sugar in an acid
solution is oxidized almost quantitatively to gluconic acid C,H,,0,.

Reactions :

tt = + ~—
2 Cu+4 0H +C,H,,0, —-C,H,,0,+H,042Cu+2 0H

6 12 6 6°12 7

+ —
2 Cu+2 OH —- Cu,0 + H,0
red
ppt.
876 PHYSIOLOGICAL CHEMISTRY

—Barfoed’s test is interfered with by any but small amounts of sodium
chloride so that it cannot be used to detect glucose in the urine.

Experiment 19. Reduction of bismuth salts to metallic bismuth.
(Béttger’s, Nylander’s and Almen’s tests.)—An alkaline solution of
reducing carbohydrates reduces bismuth salts to black metallic oxide.

(Béttger’s test.) To a pinch of bismuth subnitrate, BiONO,.H,O,
add 3 drops of 10 per cent. NaOH and 2 e.c. M/5 glucose. Heat to
boiling. The solution turns first yellow and ultimately a black precipi-
tate appears of metallic bismuth. The appearance of this black precipi-
tate is the reaction sought. If but little sugar is present, the black
bismuth will mix with unchanged bismuth subnitrate to make a gray
powder.

Nore: If sulphides or protein are present a black precipitate of bismuth
sulphide will be obtained. The test can only be used in their absence.

Experiment 20. Nylander’s modification of this test is as follows:
To 2 ec. of M/S glucose, or other sugar solution, add 2 drops of
Nylander’s reagent and heat five minutes in a boiling water bath. The
solution darkens and on standing a black precipitate of bismuth appears.

(Nylander’s reagent: 2 grams bismuth subnitrate, and 4 grams of
Rochelle salts are heated in 100 cc. 10 per cent. KOH. Cool and
filter.)

The advantage of this and Bottger’s reaction is that bismuth sub-
nitrate is not reduced by uric acid, creatinine and some other reducing
agents.

Experiment 21. Reduction of mercury salts to metallic mercury.
(Knapp’s and Sachsse’s reactions.)—To 1 ¢.c. of potassium mercuric
iodide solution add 3 drops 10 per cent. NaOH and 1 ¢.c. M/5 glucose
solution. Heat to boiling. Black precipitate.

Experiment 22. Reduction of silver salts to metallic silver—
To 1 ce. of AgNO, solution add 1 ce. dilute NH,OH and 1 ec. M/5
glucose solution. Warm gently and set aside. Note the mirror formed
on standing. This is due to the precipitation of metallic silver on the
glass.

Experiment 23. Reduction of methylene blue or safranine.—To
1 ee. M/5 glucose add 2 drops of 0.1 per cent. methylene blue or
sapranine and 3 drops of 10 per cent. NaOH and heat. The dye will
be decolorized.

For information on the behavior of carbohydrates in alkaline solu-
tion and on the use of Barfoed’s solution, study the following: H.
McGuigan: Am. Jour. Physiol., Vol. 19 (1907), p. 175; J. Nef: Annalen
der Chemie, Vol. 357 (1907), p. 214; A. P. Mathews and H. McGuigan:
Am. Jour. Physiol., Vol. 19 (1907), p. 199; A. P. Mathews: Jour. Biol.
PRACTICAL WORK AND METHODS 877

Chem., Vol. 6, p. 3, 1909; Nef: Annalen der Chemie, Vol. 403 (1913),
p. 204; H. H. Bunzel: Jour. Biol. Chem., 7, p. 157 (1910).

Exercise VIII. IDENTIFICATION OF PARTICULAR CARBOHYDRATES

In this exercise we will take up the reactions and characteristics
which may be used for the identification of particular carbo-
hydrates. ,

The reactions which are included in this exercise all depend on the
fact that all carbohydrates without exception when treated with strong
solutions of mineral acids lose water and are converted into furfural, or
derivatives of methyl furfural. This conversion is almost quantitative
in the case of the pentoses, but in the case of rhamnose, which is a
methyl pentose, we have methyl furfural produced, whereas with the
hexoses the principal product is not furfural but 4-methyl-2 hydroxy-
furfural. (See page 37.) The ketose hexoses produce this most readily.
The methyl-hydroxy-furfural when acted on further by acids is con-
verted in large part into levulinic and formic acids. The furfurals thus
formed interact with numerous aromatic substances to produce pig-
ments. Among the substances found to be most delicate and satisfactory
in coupling to make pigments are aniline acetate, alpha naphthol,
resorein, phloroglucinol, orcinol, diphenylamine and naphthoresorcinol.

It is probable that some intermediate products other than furfural
will also react with the aromatic compounds coupling up to produce
pigments of a different tint than that due to furfural itself. This may
account for the different tints produced by heating the carbohydrates
with aromatic substances in the presence of strong acids.

Experiment 24. Alpha naphthol reaction. (Molisch reaction for
carbohydrates.)—This is the most general test for any carbohydrate
whether it is free or combined.

To the carbohydrate solution in a test tube add 2 drops of a 5 per
cent. solution of alpha naphthol in alcohol. Mix and then allow 5 ce.
concentrated H,SO, to flow down the side of the test tube so as to keep
the solution and acid in separate layers. In some cases it may be
necessary to agitate a little so as to have a slight mixing at the junction
of the two liquids. Let stand at room temperature. If there is a
carbohydrate present, there will be formed a purple or pinkish ring at
the junction of the acid and the carbohydrate solution.

A negative result by this reaction is very good evidence of the
absence of carbohydrates, but a positive one is simply an indication of
the probable presence of carbohydrates. The test is probably of more
value if used as modified by E. Pinoff (Berichte 38, p. 3308), who found
that by heating in test tubes definite quantities of carbohydrates, alcohol,
878 PHYSIOLOGICAL CHEMISTRY

sulphuric acid and 5 per cent. alcoholic alpha naphthol solution for
definite lengths of time one obtains a colored solution, which, when
examined for its absorption spectrum, gives characteristic bands with
different classes of carbohydrates. Among the substances which give this
test are various furfurals, glycuronic acid, glycolaldehyde, glycerose,
erythrose, oxygluconic acid and glycerol aldehyde.

Reaction :
cH O 3H.0 —_~ H.C — C.OH

6 12-6 * 2
on, cao
\ 7
Oo
4-methyl-2-hydroxy furfural.

To show that wood is a carbohydrate and gives this test, take a
small sliver of wood, a small sliver of a match will do. Place in the
bottom of a test tube, add 1 cc. of water, 2 drops of a-naphthol and
then run down the side of the tube, the latter being held slantwise, so
that the acid comes to lie beneath the water, about 1 c.c. of concentrated
‘H.SO,. Agitate a little and gently so that the end of the splinter comes
into the strong acid and is attacked by it. Then agitate gently again so
that a partial mixture of the top layer of the acid and the bottom layer
of the water occurs. If the experiment is successful a pink ring will
form at the zone of contact, showing the presence of a carbohydrate in
the wood.

Try the same experiment with a small pellet, about as big as a small
pea, of absorbent cotton. This is also a carbohydrate. Repeat also with
a small pellet of filter paper. On gently agitating a red ring develops
at the junction of the acid and the water.

Use milk also. Milk contains a sugar called lactose.

Repeat with starch and a pinch of flour. Saliva also contains a
carbohydrate group in the mucin it contains. To a little saliva add
three drops of alpha naphthol and then as before concentrated sulphuric
acid. Agitate very slightly and let stand. <A pink ring will develop at
the junction.

Repeat with a little agar agar. Agar agar is derived from a Japanese
seaweed and is used in bacteriology to make a jelly and in medicine
as a soft mechanical laxative. As an example of a gum, test a small
fragment of gum arabic in the same way. If furfural can be obtained,
as in experiment 32, show that this also gives a pink color.

All of these things give a Molisch reaction. That shows they contain
a carbohydrate. This is, therefore, a universal test for all manner of
carbohydrates. All are positive.

The foregoing reaction will serve to detect a carbohydrate of some
PRACTICAL WORK AND METHODS 879

kind. But it is desired to be specific. How can one tell whether the
substance is a pentose or a hexose?

Experiment 25. Pentose reactions. Phloroglucinol reaction.
(Tollen’s.)—This reaction consists in the development of a cherry red
pigment, soluble in amyl alcohol and showing a strong absorption band
in the green between the D and E line. All substances which contain
pentoses, such as most gums, cherry, gum arabic, etc., give this reaction.
The color is probably due to the coupling of the phloroglucine with
some aldehyde decomposition product other than furfural of the pentose
with the production of a pigment.

Gum arabic is a gum which is excreted as an adhesive juice from
the bark or roots of various African aceacias. On hydrolysis it yields,
among other products, d-glucose and the pentose sugar, arabinose. It
may be used here as the substance which will yield a pentose and give
this reaction.

Prepare 27 cc. of Tollen’s reagent by adding 3 e.c. of 2 per cent.
phloroglucinol solution in alcohol to 15 ¢.c. of concentrated HCl and
dilute to 27 cc. with distilled water.

To 3 ec. of the reagent in a test tube add three drops of a dilute
(5 per cent.) gum arabic solution and heat to boiling. The solution
becomes a deep cherry red after a few moments of heating. This is the
test. When examined with the spectroscope it is seen to have a strong
absorption band in the green. If desired the solution may be shaken
with about 2 «ec. of amyl aleohol. The color will go entirely into the
alcohol and the spectrum may be seen there also.

This reaction needs to be carefully controlled, since other sugars,
notably hexose ketoses, will also give a red coloration, but the color is
slightly different and has no absorption band in the green. Test in the
same manner the reaction with M/5 solutions of levulose, glucose, maltose
and galactose. Also with starch. Take of the reagent 2 c.c. in a test
tube and add two drops of M/5 solutions of the different sugars, and
boil. It will be observed that levulose changes at once to a red which
is very easily confused with the pentose reaction, but the color gives no
absorption band in the green. None of these sugars give this reaction,
showing that they are not pentoses.

Wood also contains a pentose, xylose. So take a splinter of a match
and boil it in 2 cc. of this reagent. It will be observed that the splinter
changes color to a deep pink.

This reaction is not due to furfural itself formed from the sugar by
the acid, as might be supposed, for if furfural is boiled with the reagent
a deep bluish green pigment is produced soluble in amyl aleohol but
without definite absorption bands. If furfural can be obtained try
this.
380 PHYSIOLOGICAL CHEMISTRY

Experiment 26. Orcinol test for pentoses.—Orcinol is methyl
resorcinol. It is dioxytoluene.

Orcinol:
OH

CH,

wen

Pentoses, when warmed with orcinol and strong hydrochloric acid,
give a violet followed by a blue, red, and finally a green color and a
bluish green precipitate. This precipitate redissolved in amyl alcohol
gives an absorption band in the red between lines D and C.

The reagent is made by mixing 1 volume of half-saturated orcinol
solution, 5 volumes concentrated HCl and diluting to 9 volumes with
water.

Take into a test tube about 2 c.c. of the oreinol reagent and add to
it two drops of 5 per cent. gum arabic solution and heat to boiling.
Observe the change of color characteristic for a pentose.

Repeat using in each case 2 ¢.c. of the reagent and adding three
drops of M/5 solutions of the sugars to be tested. Among them test
arabinose if this is obtainable. Test the same substances as were tested
in the phloroglucinol experiment and see which contain pentoses.

CH,C,H, (OH),

3.6 3

 

Nore.—In both of the Tollen’s reactions above it is very important to observe
the concentrations of acid and orcinol or phloroglucinol, otherwise the velocity of
the reaction and the colors obtained are very different from those described in the
literature. Various modifications of these tests have been made, the most important
being the shaking out of the colored solution with amyl alcohol and the examina-
tion of the absorption spectrum of the amyl alcohol solution after proper dilution.
When thus employed one can more readily and accurately distinguish pentoses
from hexoses; these reactions, however, do not distinguish pentoses from glycuronic
acid.

By the use of naphthoresorcin and HCl (Tollens: Berichte 41, pp. 1783-87 and
1788-90, 1908) glycuronic acid may be detected more accurately in that the colored
substances formed by the interaction of glycuronic acid with naphthoresorcin in
presence of 18 per cent. HCl are readily soluble in ether, while the pentoses, hexoses
and rhamnose yield colored substances difficultly soluble in ether. The ether solu-
tion of the former shows a characteristic absorption spectrum. (See experiment
185.)

The following articles deal with the above reactions: V. Udransky: Zeit.
Physiol. Chem., 12, pp. 355-95; Roos: Ibid., 15, pp. 518-38; Treupel: /bid., 16, pp.
47-67; Neuberg: Ibid., 31, p. 564, 1901; Tollens: Annalen, 286, p. 301; Ber. 29,
p. 1202, 1896; Fleig: Ann. Chim. Analyt. 13, p. 427, 1908.

Experiment 27. The detection of galactose. Mucic acid reaction.
PRACTICAL WORK AND METHODS _. 881

—We may now pass on to consider special reactions for the detec-
tion of particular carbohydrates.

Galactose may be detected whether it is present either in a glucoside
or free state by its yielding on oxidation with nitric acid, a crystalline
acid, mucie acid, COOH.(CHOH),COOH, which is isomeric with sac-
charie acid, but unlike the latter is almost insoluble in cold water and
alcohol.

To 10 c.c. M/5 lactose solution in a 100 ¢c.c. beaker, add 5 c.c. distilled
water and 15 e.c. concentrated HNO;. Heat on the water bath until
concentrated to about 1/5th of the original volume. Now add 15 ee.
distilled water, mix well and set aside in the desk until the next day.
Recrystallize in the following way: Filter off the crystals of mucic acid,
suspend in 10 ¢.c. water and add 1 c.c. strong NH,OH (No. 5). To
the solution add'1 ec. strong HNO, (sp. gr. 1.42), stir again and set
aside for crystallization. Examine the crystals under the microscope.
Filter off the crystals, wash in cold water, dry over CaCl, in desiccator.
Glucose, and polysaccharides containing it, give, when similarly treated,
an isomeric acid, saccharic acid, which is soluble and hence does not
crystallize out. The reaction is as follows:

6 HNO, + C,H,,0, —~ C,H, ,0, + 6NO, + 41,0

12 1 3
Mucic acid

Experiment 28. Detection of starch by iodine.—Iodine reacts with
starch to form a deep blue colored substance, starch iodide. The color
is discharged on heating and reappears on cooling, although in dimin-
ished intensity. The exact nature of the reaction is unknown. Bromine
and chlorine also unite with starch, but oxidize it, the elements being
changed to their colorless condition. It is possible that the intermediate
compound persists longer with iodine, which is a weaker oxidizing agent,
and that the blue color is due to the particular condition of the iodine
atom. Iodine has a violet color in many solvents, usually those which
contain no hydroxyl groups. Other substances than starch will give a
blue color with iodine. An amyloid so reacting may be prepared from
cellulose, and choleic acid also yields a blue compound. Starch is prac-
tically the only common. carbohydrate which gives this reaction. The
color of starch iodide is discharged by NaOH, sodium thiosulphate and
alcohol. ;

To 5 ec. 0.1 per cent. starch solution add a few drops of I,KI solu-
tion. Starch gives a blue color. Heat and observe the disappearance
of the color on heating and its return on cooling. Erythrodextrin gives
a red reaction with iodine, and glycogen a brown red.

Experiment 29. Seliwanoff’s reaction for ketoses.—This reaction
consists in the development of a deep red color followed by the appear-
882 PHYSIOLOGICAL CHEMISTRY

ance of a brownish precipitate when a ketose, such as levulose, is heated
with strong HCl and resorcinol.

The reagent is made by dissolving 0.5 gram of resorcinol in 100 e.c.
concentrated HCl and diluting to 300 c.c. by addition of water.

5 «ec. of the reagent is placed in a test tube, 1 ¢.c. of the sugar
solution to be tested is added and immersed in boiling water. If a
ketose is present the solution will quickly turn red and a red precipitate
will appear.

To test the reaction of glucose, levulose and galactose with this
reagent measure 5 c.c. portions of the reagent into each of 4 test tubes,
add respectively to the different tubes 1 ¢.c. of M/5 solutions of glucose,
levulose and galactose, and 1 ¢.c. of M/100 levulose, immerse all the
tubes at the same time in boiling water and heat for several minutes.
Observe how quickly the two levulose tubes react as compared with the
aldehyde sugars.

This test is often described as a specific test for levulose. It is
better to call it an indicative test for ketose, since other keto-hexoses,
like sorbose, as well as keto-trioses, keto-tetroses, d-oxygluconic acid and
formose give the test readily. All hexoses give the test very faintly and
very slowly if proper precautions are observed as to concentrations of
the acids and carbohydrates. The color developed here is due to the
formation of 4-methyl-2-hydroxy-furfural and the reactions of this with
the resorcin in presence of a 12 per cent. solution of hydrochloric acid.
The same reactions are involved in the Boas test for a certain hydrogen
ion concentration in gastric juice. Boas’ reagent is a solution of cane
sugar and resorein in 95 per cent. alcohol; the presence of a certain
hydrogen ion concentration is indicated by the developing of a red color.
(See experiment 132.)

The Seliwanoff reaction has been modified in various ways, using
lower concentrations of hydrochloric acid, alcoholic solutions of sulphurie
acid, or a slightly ionized acid like acetic acid instead of the 12
per cent, hydrochloric acid solution employed above. In any ease,
due precautions as to length of time of heating and concentration
of carbohydrate must be taken to make the reaction of diagnostic
value. The test is carried out more satisfactorily when the precipi-
tate, formed after heating, is filtered off, dissolved in 95 per cent.
aleohol and this alcoholic solution then examined for its absorption
spectrum.*

Experiment 30. Identification of carbohydrates by means of their
osazones.—Any carbohydrate which is a good reducing agent and con-
sequently easily oxidized will combine with phenyl hydrazine to form
osazones. These are insoluble substances which form long yellow crystals

*For detailed information read: (1) Seliwanoff: Berichte 20 (1887), p. 181;
(2) Ekenstein and Blanksma: Ber. 48, p. 2355, 1910; (3) Pinoff: Ber. 38, p. 3308,

 

ee
PRACTICAL WORK AND METHODS 883

and these crystals have definite melting points. The formation of
these crystals constitutes one of the best ways of detecting and
identifying carbohydrates. All aldoses and ketoses form these osazones
and many other ketones and aldehydes will react to form hydrazones
and if they contain in addition an alcohol group will form also
osazones.

Weigh out 1.0 gram of phenyl hydrazine hydrochloride and 1.5
grams of sodium acetate on the torsion balance. Transfer to a clean
mortar and grind together until the mixture is uniform in appearance.
Now weigh a 0.5 gram portion into each of four dry test tubes and to
the tubes add respectively and accurately by a pipette 2 cc. of M/5
solutions of glucose, mannose, levulose and lactose. Label carefully,
and immerse all the tubes at the same time in boiling water. At intervals
of one-half minute remove all from the bath at the same time, shake
for an instant and return to the bath at once. Repeat this three times.
Observe if any white crystalline precipitate, hydrazone, appears in the
mannose tube. If it does remove a little with a pipette, observe under
the microscope and draw some of the crystals. What is a hydrazone?
(See page 43.) Continue boiling for 25 minutes and note the time when
precipitation occurs in the different tubes during that period. Now
remove all the tubes from the water bath and, while cooling, note the
time when a precipitate appears in those tubes which were clear. The
osazones appear in about the order of the reactivity of the different
sugars, levulose first. Saccharose forms no osazone. Examine a little
of the precipitate from each tube on a slide under the microscope and
draw the crystals of osazones formed. They are yellow in color. Man-
nose is at first precipitated as a colorless hydrazone, but later changes
to the yellow osazone. The other hydrazones are scluble. Mannose,
glucose and levulose form the same osazone. What does this show as
regards the constitution of the last four carbon atoms of these sugars?
The object of adding the sodium acetate is to reduce the acidity, thus
increasing the concentration of the free base, phenyl hydrazine, which
is more reactive than the salt. ;

Experiment 31. The solubility of cellulose in ZnCl, and other so-
lutions.—Cellulose is a polysaccharide. It is readily soluble in ZnCl,
solution in concentrated HCl and in an ammoniacal CuCO, solution
(Schweizer’s reagent).

The Zine Chloride solution is made by dissolving ZnCl, in twice its
weight of concentrated HCl. Filter paper and other cellulose will
dissolve readily in this solution and may be precipitated out on diluting
with water.

In place of ZnCl, solution, Schweizer’s reagent may be used. This
is made as follows:
884 PHYSIOLOGICAL CHEMISTRY

Dissolve 3 grams of moist cupric hydroxide, formed by addition
of KOH solution to a solution of cupric sulphate containing some
ammonium chloride and filtering and washing, in a liter of 20 per cent.
hydroxide.

EXxErcIsE IX. DECOMPOSITION OF CARBOHYDRATES BY ACIDS AND
ENZYMES.

Acids hydrolyze di-, tri- and polysaccharides when in aqueous solu-
tion into simpler sugars and if they act long enough decompose the
monosaccharides with the formation of furfural from pentoses and
methyl-hydoxy furfural, levulinie acid and formic acid and humus sub-
stances from hexoses. These aldehydes are the causes of the color reac-
tions given in the various color tests already studied in the last exercise,
showing formation of furfural from pentoses.

The following experiment is to be done in groups of three, one of
the three distilling bran, another glucose and another levulose. The
object of this experiment is to show that pentoses yield large quantities
of furfural, C,H,O,, when distilled with strong HCl, whereas hexose
sugars (glucose and levulose) do not yield furfural except in very small
traces but produce humic acid and levulinic acid instead.

Experiment 32. Decomposition of monosaccharides by acids.—
Connect three 250 c.c. flasks, A, B and C, with condensers and receiving
flasks ready for distillation, as shown in Figure 68:

|

 

 

 

 

 

Fig. 68.—Apparatus arranged for the distillation of furfural.

Place in one flask (A) 5 grams wheat bran; into (B) 5 grams glucose;
(C) 5 grams levulose. Add to each flask 70 e.c. HCl (concentrated HCl
diluted with equal amount of water). Heat each to boiling and boil

 
PRACTICAL WORK AND METHODS 88y

vigorously until about 15 ¢.c. distillate have come over. While the
distillation is going on observe and note the red color developed in the
glucose and levulose solutions on heating, the appearance of black humus
substances in all flasks, and by holding a piece of filter paper moistened
with aniline acetate over the end of the condenser observe that the paper
touched to the bran condenser gives a bright pink color, but not when
touched to the others. This is a test for furfural.

When the 3 distillates are collected, smell and note nutty odor of
furfural, and the sharp smell of formic acid in the distillates from the
hexoses.

Examine each distillate for furfural by aniline acetate and by the
orcinol test such as was carried on in experiment 26. The bran distillate
gives the furfural tests, ie., aniline acetate and the orcinol reaction
(blue color) but the distillates of the hexoses do not.

In the hexose flasks there has been formed in place of furfural the
methyl hydroxy furfural and this has broken down with the formation
of levulinie and formic acids according to the following reaction:

HC — COH H.C — CH
“ | 2
cut b_ CHO +2H,0 —- CH,COH C=0 + HCOOH
\ 7 NZ
O 0
Methyl-hydroxy- Levulinic acid Formic
furfural

The levulinic acid undergoes rearrangement to CH,-CO-CH,-CH,
COOH.

The formation of levulinic acid in an acid hydrolysis is an indication
that a hexose has been present. Pentoses yield furfural.

Aniline acetate paper: To prepare this place a drop of 50 per cent.
aniline acetate solution on a small piece of filter paper and allow to
become partially dry. Furfural turns it pink.

After removing the greater part of the furfurals, by distillation, and
the humie acids by filtration, extract the levulinic acid from the filtrate
by ether. The ether solution on evaporation on the steam bath yields
erude levulinic acid, which even in the cold readily forms iodoform
when made alkaline with NaOH and a few drops of KI, solution (No.
45) added. What other common substances readily yield iodoform by
similar treatment? Are these substances likely to be present here? To
further identify levulinic acid, it is usually separated as the zine salt
and then identified as the silver salt. (Annalen, 206, pp. 207 and
226.)

Experiment 33. The decomposition of sugars by enzymes,—In the
886 PHYSIOLOGICAL CHEMISTRY

body the sugars are not decomposed by acids and alkalies but by other
reagents of an unknown nature supposed to be enzymes. Such an
enzyme, called zymase, is found in yeast. By its action dextrose and
similar hexoses are converted into alcohol and carbon dioxide. This is
the fermentation of dextrose by yeast which is at the foundation of bread
making, brewing, wine and alcohol manufacture. To test the decom-
position of various sugars proceed as follows:

Make a suspension of half a cake of compressed yeast in 10 c.c.
of water by triturating very gently in a small mortar. To 15 cc.
portions of the carbohydrate solution (DO NOT USE THE TOLUENE
PRESERVED SOLUTIONS HERE), add 1 ec. of the well-mixed suspension,
mix well and at once transfer to the fermentation tube. For carbo-
hydrate solutions use M/25 solutions of arabinose, levulose, glucose,
mannose, galactose, maltose, saccharose, lactose and 0.70 per cent. starch
paste. Prepare the starch paste by the method outlined. Set aside the
tubes and note the amount of gas liberated after 6-8, 10-12 and 18-24
hours. Note which of the sugars is fermented. What do the fermented
sugars have in common which determines fermentability ?

Products of fermentation. CO,. Into a tube in which artive
fermentation has taken place introduce 1 ec. strong NaOH solu-
tion (No. 86) into the closed end of the tube by means of a bent pipette.
Next add enough water completely to fill the shorter end of the tube,
‘place the thumb over the opening without introducing air and invert
the tube two or three times without collecting any of the gas under the
thumb in the shorter arm of the tube. Set the tube upright and note
the suction on the thumb. When the thumb is removed the fluid rises
in the closed arm, due to the absorption of the CO, from the fermenta-
tion gas. Different kinds of bacteria and yeasts form gas with more or
less H, intermixed with the COQ,.

Products of fermentation. Alcohol. Filter a portion of the solu-
tion in the fermentation tube and add 5-20 drops of I,KI solution
(No. 45) to 5 ee. thereof. Sufficient I,KI should be added to render the
entire mixture light yellow for about one-half minute. Warm slightly,
note the odor of iodoform, and set aside. Examine the separated yel-
low, iodoform erystals with a microscope. Rosettes. Acetone and some
other substances will give the iodoform reaction with iodine.

Does yeast contain invertin? Does it contain lactase?

Experiment 34. Inversion of cane sugar by acids.—Acids
hydrolyze disaccharides into monosaccharides.

To 10 ec. of a M/5 solution of saccharose add an equal quantity
in a test-tube of N/2H,SO, and immerse in boiling water for 45 minutes.
While this is heating take in another test-tube 10 ¢.c. of M/5 saccharose,
dilute it with an equal quantity of water and determine its specific rota-

ee
PRACTICAL WORK AND METHODS 887

tory power at 20° C. by means of the polariscope (see page 25). At
the end of the 45 minutes of heating the original solution, cool it to
the temperature of the solution you have just examined, about 20° C.,
and determine the specific rotatory power of this and compare it with
the other. If the solution is still dextro-rotatory, return it to the water
bath for longer heating. Explain the change in the rotatory power pro-
duced by the acid. Write the reaction. This change in rotatory power
from dextro to levo is called inversion.

After recording the polariscope reading of the solution which has
been heated with acid, neutralize the acid exactly by adding carefully
from a burette N/2 NaOH or cold, saturated barium hydrate, using
litmus as an indicator. Do not add an excess of alkali. Filter from
the barium sulphate, if Ba(OH), has been used, and test the reducing
power of the solution with Benedict’s and Barfoed’s reagent. The
reducing power will be found greatly increased by the acid treatment
_ due to the formation of glucose and levulose. Test some of the filtrate
for the presence of levulose. (Seliwanoff’s reaction.) Compare thus the
reducing reaction of the saccharose before and after acid has acted.

Experiment 35. Hydrolysis of polysaccharides by acids.—Take
2 cc. 2 per cent. starch solution in test-tube. Add 1 cc. concentrated
HCl, heat to boiling and about 30 seconds longer. Notice solution clears
up. Then take out 1 «c., add 3 drops CuSO, and NaOH to alkaline
reaction shown by development of deep blue color. Heat and note deep
red precipitate due to reduction by sugar set free.

To the clear starch solution remaining and cooled add a couple of
drops of I,KT solution. If the boiling has been long enough the solution
will turn a deep red, due to the erythro-dextrin formed. The starch
has disappeared, as shown by loss of blue reaction with iodine and
erythro-dextrin and a reducing sugar (glucose) have taken its place.

Experiment 36. Alkalies do not hydrolyse starch, other poly-
saccharides and cane sugar.—Indeed these are all remarkably resistant
to the action of alkalies although so easily split by acids.

To 1 «ce. of 1 per cent. solutions of starch and M/5 cane sugar in
separate test-tubes add 3 drops concentrated NaOH and heat to boiling.
Observe that no brown color develops on heating as would be the case if
glucose, levulose or other monosaccharide had been set free. After
boiling gently 2 or 3 minutes add a couple of drops of 2 per cent. CuSO,
solution, and again heat to boiling. There is no reduction, showing that
no monosaccharide has been formed.

EXERCISE X. QUANTITATIVE DETERMINATION OF REDUCING SUGARS.

The commonest sugar to be determined is glucose (dextrose) and a
very good method is that of Benedict. This method is used to measure
888 PHYSIOLOGICAL CHEMISTRY

the sugar in the urine and is described on page 1111. One of the most
accurate methods is that of Munson and Walker. Two or three of these
other standard methods are given here. Others will be found in the
section on the blood, page 1021, 1028.

Experiment 37. Quantitative determination of reducing sugars in
urine and other fluids. Bertrand method (Bertrand, Bull. de la Soc.
Chimique de Paris, XXXV, p. 1285, 1906).—In the absence of other
reducing substances in the solution the amount of glucose, or other
reducing sugar, can be best determined by the Bertrand method or by
a combination of the Munson and Walker and Bertrand methods which
follow (38).

Principle. The sugar contaniing liquid is boiled with a definite
amount of Fehling’s solution for a specific time. There must always be
a large excess of Fehling’s solution used, so that the blue color is little
changed. The cuprous oxide formed is filtered off on an asbestos filter,
and the amount determined by redissolving it in a solution of ferric .
sulphate containing sulphuric acid and titrating the amount of ferrous
sulphate formed by standard permanganate of potassium solution. From
the amount of copper thus determined one finds by means of tables the
amount of glucose or other sugar present in the quantity of original
solution used.

Process. Place in a 150 ¢.c. Erlenmeyer flask 20 c.c. of the neutral
sugar solution (urine) to be determined. These 20 c.c. must not contain
more than 100 mgs. of glucose or other sugar: in other words, it must
not be more than 0.5 per cent. It is preferable to have between 10 and
90 mgs. Dilute the solution if more concentrated than this, but always
take 20 cc. Add 20 cc. of the cupric-sulphate solution (1) and 20
e.c. of the alkaline liquid (2) and heat to boiling. Boil gently, so as
not to concentrate the solution too greatly, for exactly 3 minutes. With-
draw the flask from the fire, and after waiting a moment for the cuprous
oxide to settle, filter while still hot under suction through a previously
prepared Gooch crucible with an asbestos mat. The way to make this
filter is described in the following modification of this method, p. 996.
Bring as little as possible of the cuprous oxide on the filter, but wash
the cuprous oxide by decantation with distilled water, decanting through
the asbestos filter each time. After several washings by decantation
and suction, when the asbestos filter is free from blue color and any
tartrate, remove and rinse carefully the filter flask so that it is quite
clean and then replace the filter over it. Now dissolve the Cu,O in the
Erlenmeyer flask by adding successive portions little by little, about 5 ¢.c.
at a time, of the ferric-sulphate solution (8). About 10-20 e.c. will be
necessary. The precipitate changes to a blue black and dissolves to a

 
PRACTICAL WORK AND METHODS 889

green solution. When this is dissolved pour this solution carefully, using
mild suction, through the asbestos filter so as to dissolve any cuprous
oxide which may have been caught in the filter. It may be necessary
to add a little fresh ferric-sulphate solution to bring this all in solution.
Rinse the Erlenmeyer carefully with distilled water and pour the wash-
ings through the filter. Now titrate the filtrate in the suction flask by
adding from the burette the potassium permanganate solution (4) until
a single drop gives a permanent pink color. The end point is very
sharp.

Calculation. Multiply the number of c.c. of permanganate used by
the figure expressing the value of each c.c. of permanganate in copper,
and look up in the table the amount of reducing sugar corresponding
to this amount of Cu. For the determination of the value of the per-
manganate in copper see the following paragraph.

Solutions needed:

1. Pure crystalline CuSO, ..........- eee eee eee eee 40 g.
Distilled water to ...... 0. cece eee e cece ee eee eee 11

2. Pure Rochelle salts ..........eseeeeeee eect eee enes 200 g.
NaOH in sticks ........ce cece ee see e cece cece cece 150 g.
WAbOE 607 secede asavecis sco @ taal hae wvelere sie easeevarrattouselene Ge ids

8. Ferric sulphate .......... cc cece cece eee eee eee 50 g.
Con. HjSO, .. see e cece cece eee ee eee aera phere, beg wiahshas 200 ee.
Water to sranesc sageay cases. vestige ines Sve ate 11.

4. Potassium permanganate ............2.0.eeeeeeee 5 g.
Water 10) <i.ios ise ee BORE S Rey Sa eRe ees 11

Determine the permanganate as follows: 0.250 g. ammonium oxalate
is warmed with 100 c.c. water and 2 ¢.c. con. H,SO, in a beaker to 60-80".
Run in permanganate from a burette until a rose color remains (about
22 ¢.c. will be needed). Multiply the amount of ammonium oxalate by
63.62

———- (=0.8951). This gives the amount of Cu which the amount of
142.1

permanganate used in the titration represents. In round figures 1 liter
of permanganate solution equals 10 gr. of copper. Reactions:
1, Cu,0 + Fe,(SO,), + H,SO, = 2CuSO, + H,0 + 2FeSO,
2. FeSO, + 2KMn0, + 8H,SO, —5Fe,(SO,), + K,SO, + 2MnSO, + 8H,0
3. 50,H,0, + 2KMn0, + 3H,SO, —10CO, + 2MnSO, + K SO, + 8H,0
1 molecule (NH 4) 2,9 i is equivalent to 2Fe.
TABLE FOR THE DETERMINATION oF REDUCING SuGAaRS BY THE BreRtRaAND MerHop.

(Note.—This table can only be used when the directions of Bertrand as to the con-
centration of the Fehling’s solution and the time of heating have been exactly
followed. Different values are obtained if the heating is for another time and
if the relative concentrations of sugar, copper sulphate and alkali are different
from those specified.)
 

890 PHYSIOLOGICAL CHEMISTRY

Cu. IN Mes. FoR THE FoLLowine SuaaRs:
Sugarin Glucose Invert sugar Galactose Maltose Lactose Mannose Xylose Arabinose
mngs, Cu. Cu. Cu. Cu. Cu. Cu. Cu. Cu.
10 20.4 20.6 19.3 11.2 14.4 20.7 20.1 21.2
11 22.4 22.6 21.2 12.3 15.8
12 24.3 24.6 23.0 13.4 17.2
13 26.3 26.5 24.9 14.5 18.6
14 28.3 28.5 26.7 15.6 20.0
15 30.2 30.5 28.6 16.7 21.4
16 32.2 32.5 30.5 17.8 22.8
17 34.2 34.5 32.3 18.9 24.2
18 36.2 36.4 34.2 20.0 25.6
19 38.1 38.4 36.0 21.1 27.0
20 40.1 40.4 37.9 22.2 28.4 40.5 39.6 41.9
21 42.0 42.3 39.7 23.3 29.8
22 43.9 44.2 41.6 24.4 31.1
23 45.8 46.1 43.4 25.5 32.5
24 47.7 48.0 45.2 26.6 33.9
25 49.6 49.8 47.0 27.7 35.2
26 61.5 51.7 48.9 28.9 36.6
27 53.4 53.6 50.7 30.0 38.0
28 55.3 55.5 52.5 31.1 39.4
29 57.2 57.4 54.4 32.2 40.7
30 59.1 59.3 56.2 33.3 42.1 59.5 58.7 62.0
31 60.9 61.1 58.0 34.4 43.4
32 62.8 63.0 59.7 35.5 44.8
33 64.6 64.8 61.5 36.5 46.1
34 66.5 66.7 63.3 37.6 47.4
35 68.3 68.5 65.0 38.7 48.7
36 70.1 70.3 66.8 39.8 50.1
37 72.0 72.2 68.6 40.9 51.4
38 73.8 74.0 70.4 41.9 52.7
39 75.5 75.9 72.1 43.0 54.1
40 77.6 17.7 73.9 44.1 55.4 78.0 77.3 81.5
41 79.3 79.5 75.6 45.2 56.7
42 81.1 81.2 174 46.3 58.0
43 82.9 83.0 79.1 47.4 59.3
44 84.7 84.8 80.8 48.5 60.6
45 86.4 86.5 82.5 49.5 61.9
46 88.2 88.3 84.3 50.6 63.3
47 90.0 90.1 86.0 51.7 63.3
48 91.8 91.9 87.7 52.8 65.9
49 93.6 93.6 89.5 53.9 67.2
50 95.4 95.4 91.2 55.0 68.5 95.9 95.4 100.6
51 97.1 97.1 92.9 56.1 69.8
52 98.9 98.8 94.6 57.1 71.1
53 100.6 100.6 96.3 58.2 72.4
54 102.3 102.3 98.0 59.3 73.7
55 104.1 104.0 99.7 60.3 74.9
56 105.8 105.7 101.5 61.4 76.2
57 107.6 107.4 103.2 62.5 77.5
58 109.3 109.2 104.9 63.5 78.8
59 111.1 110.9 106.6 64.6 80.1
60 112.8 112.6 108.3 65.7 81.4 113.3 113.2 119.3
61 114.5 114.3 110.0 66.8 82.7
62 116.2 115.9 111.6 67.9 83.9
63. 117.9 117.6 113.3 68.9 85.2
64, 119.6 119.2. 115.0 70.0 86.5
65 121.3 120.9 (116.6 71.1 87.7
66 123.0 122.6 118.3 72:2 89.0
PRACTICAL WORK AND METHODS 891

Cu. rn Mes. ror THe FoLLowine Sucars:

Sugarin Glucose Invert sugar Galactose Maltose Lactose Mannose <Xylose Arubinose
Migs. Cu. Cu. Cu. Cu. ib. Cu. Cu. Cu.
67 124.7 124.2 120.0 73.3 90.3
68 126.4 125.9 121.7 74.3 91.6

69 128.1 127.5 123.3 75.4 92.8
70 129.8 129.2 125.0 76.5 94.1 130.2 130.6 137.5
71 131.4 130.8 126.6 77.6 95.4
72 133.1 132.4 128.3 78.6 96.6
73 134.7 134.0 130.0 79.7 97.9

74 136.3 135.6 131.5 80.8 99.1
75 137.9 137.2 133.1 81.8 100.4
76 139.6 138.9 134.8 82.9 101.7
17 141.2 140.5 136.4 84.0 102.9
78 142.8 142.1 138.0 85.1 104.2
79 144.5 143.7 139.7 86.1 105.4
80 146.1 145.3 141.3 87.2 106.7
81 147.7 146.9 142.9 88.3 107.9
82 149.3 148.5 144.6 89.4 109.2
83 150.9 150.0 146.2 90.4 110.4
84 152.5 151.6 147.8 91.5 111.7
85 154.0 153.2 149.4 92.6 112.9
86 155.6 154.8 151.1 93.7 114.1
87 157.2 156.4 152.7 94.8 115.4
88 158.8 157.9 154.3 95.3 116.6
89 160.4 159.5 156.0 96.9 117.9
90 162.0 161.1 157.6 98.0 119.1 163.3 164.2 172.7
91 163.6 162.6 159.2 99.0 120.3
92 165.2 164.2 160.8 100.1 121.6
93 166.7 165.7 162.4 101.1 122.8
94 168.3 167.3 164.0 102.2 124.0
95 169.9 168.8 165.6 103.2 125.2
96 171.5 170.3 167.2 104.2 126.5
97 173.1 171.9 168.8 105.3 127.7
98 174.6 173.4 170.4 106.3 128.9
99 176.2 © 175.0 172.0 107.4 = 130.2
100 177.8 176.5 173.6 108.4 131.4 179.4 180.5 189.8

TABLE FoR GLUCOSE BY BERTRAND FOR SMALLER AMOUNTS oF CU. (Moeckel and

Frank).
Cu Glucose Cu Glucose
Mg. NM. Mg. Ng.
Lid esccdis doe aba 24 huaneiee & axatheave-n nisea 0.5 WO aii og cated Bia ocean rhe de ete eee 5.5
DED) valet ch saccie dicar doanteresteecauanane tanletiocdceiiera ss 1.0 VD} os soca eitase tenn aeeee gaGay daraaiaaseee 6.0
DD sip abistin 3 4 Ng eaied es vis fencored ncete anaes ocala 1.5 TBO) ia eeeaalagiea eons fad a acish ee Ne weet eas 6.5
GW) wonid's jaGiatus a ios Gene DE Raaaerdee 2.0 VAD a ties ake eevee hee as a Si avs 7.0
DO. Aveta yee Se he ee N eees eee 2.5 UD soo ci darth tt Settee d aelp vichvocd aabviove 7.5
Get Tirta Seay aaeccerahettetasnsnal @atGoreaent a eae 3.0 1G air ak apd areieraeddgee ts oe a ckeacahcces oid 8.0
hap uancascss Sranaercenen a tyevsausee.t Cine scene aueie 3.5 DGD. ue ea bud Bevel eek a aWieg eds cccura vores 8.5
IED. © sg tiasliecery. 4 labsaetes gataldatledaahebaeh 48. 4.0 WB IES eid Sore ashentie daveticcsapeat atte Maton 9.0
DED scsriamer wie: bcc cadet aes, aeenee sn evar meoatgaisests 4.5 19.5: ascoceriewi sate with, meena ans ba 9.5
NOS) Sv wade deadeaas areki os Ree Ree 5.0 20:5 cas eons eal age dacs te acke ates 10.0

experiment 38. Quantitative determination of reducing sugars in
urine and other fluids by a combination of the Munson and Walker
and Bertrand methods (Bertrand, loc. cit. Munson and Walker: U. 8.
Department of Agriculture, Bulletin 107, 1912, p. 240; see also Cir.
cular 82).—In the experience of this laboratory this method is the most
892 PHYSIOLOGICAL CHEMISTRY

accurate and convenient of all the methods of sugar analysis. It differs
from the Munson and Walker method in that the cuprous oxide is not
weighed but titrated by permanganate as in the Bertrand method. It
differs from the Bertrand method in that the composition of the Fehling
solution is different and the time of heating is more precisely defined.

Principle. This is the same as the Bertrand method.

Process. Transfer 25 ¢.c. each of the copper (1) and alkaline tar-
trate solutions (2) to a 400 c.c. Pyrex beaker and add 50 e.c. of the
neutral sugar solution, or 10 cc. if urine be used and add water to
make the final volume 100 e.c. Cover the beaker with a watch crystal
and heat on an asbestos gauze over a Bunsen burner so regulated that
boiling begins in exactly 4 minutes, and continue the boiling for exactly
2 minutes. Filter the cuprous oxide at once through an asbestos felt in
a porcelain Gooch crucible, using suction. (A device which has been
found useful in this laboratory to control the time of heating is to use
a CaCl, solution, boiling at 112° C. This is brought to boiling and
while boiling the beaker with the sugar and Fehling’s solution is im-
mersed in it for exactly 6 minutes. It takes 4 minutes to come to the
boil and boiling lasts 2 minutes. Another device has been suggested by
Cole: namely, to connect a water manometer with the gas supply and,
having once found what pressure of gas with a given burner will boil the
beaker in 4 minutes, this gas pressure is obtained each time by regulat-
ing a thumbscrew.) Wash the cuprous oxide and the filter thoroughly
with water at a temperature of about 60°, using suction. (At this point
in the Munson and Walker method the precipitate is washed with alcohol
and ether, dried at 100° and weighed as Cu,O.) In the Bertrand modifi-
cation proceed as follows: Transfer the asbestos and precipitate as
soon as clean to the beaker in which the precipitation was conducted,
suspend in water and then add 10 e.c. of the Bertrand ferric-sulphate-
sulphuric-acid solution, or 10 cc. of M/4 ferric-ammonium-sulphate-
sulphuric-acid solution (4). Stir until all of the Cu,O is dissolved,
taking care to dissolve all the Cu,O on the stirring rod or adherent to
the inside of the crucible or the sides and lip of the beaker. A green
solution is obtained. Titrate at once with continual stirring until the
pink due to the permanganate persists for about 10-15 seconds. 1 ee.
N/20 KMn0, is equivalent to 0.00315 gram of Cu. Calculate the amount
of Cu reduced and from this weight find the amount of reducing sugar
to which it is equivalent in the appended tables (Bulletin 107, Dept. of
Agriculture, pp. 243-251. For lactose see the correction in Jour. Am.
Chem. Soc., XXXIV, p. 202, or Circular 82). In the tables appended
here the lactose correction has not been made.

Solutions and preparation of the asbestos filter. 1. Copper-sulphate
solution (Soxhlet’s modification of Fehling’s) :
PRACTICAL WORK AND METHODS 893

CuSO oH,O (Baker’s best quality) ......+..... 34.639 grams.
Water to) x. cavers oS cai sawn co Tailed stems Re eee 500 ec.

2. Alkaline tartrate solution:

Rochelle ‘salts. cs vases ve wsesie es dae ee dade sane ss 173 grams.
NaOH (best quality) ......... cece cece ee ee eee ee 50
Water? LOS ies caaats-aveishua cera e keene a 5 neces oa Mcanargin eases 500 c.c.

8. Potassium permanganate solution: Instead of the solution rec-
ommended by Bertrand, a N/20 solution is generally used in this labo-
ratory, as it is weaker:

WOMNO caste test aie een dee ale a aloes eeeen te oie eevee 3.16 grams.
Waiter” sss an sawed ye eecthy Sst paw Peas Vie 2 liters

Dissolve the KMnO, in 2,000 «c. distilled water, allow to stand in the
dark for 2-3 days, then filter through asbestos or glass wool or decant
the supernatant clear fluid into a clean, dry bottle. Keep in a bottle
covered with black paint or black paper to protect from light. Standard-
ize against sodium oxalate. Dry overnight about 0.66 gram of sodium
oxalate (pure) in a weighing tube in a steam oven and carefully weigh
off three samples of 0.10-0.15 gram each of this dried sodium oxalate.
Dissolve each sample in 100 cc. of water, add 5 «e. of H,SO, (1:1 by
volume), warm to 70° C. and titrate the KMnO, against this. 1 ce.
N/20 KMn0O, is equivalent to 0.0035 gram sodium oxalate, or 0.00315
gram copper.

4. Ferric-ammonium alum and sulphuric acid: In place of the
ferric sulphate recommended by Bertrand the ferric-ammonium alum
may be used. The solution used is M/2.

(NH, Fe(SO,),).24H,0 savas ialiseer eter ale G soneen'e eateeere's © 240.9 grams.
Sulphuric acid (Con.) 2.2... ce cece cece eee eee 200 c.c.
Water to)... isaewsscuens s sarce evs tents ev eeees sate 1 liter

5. Asbestos filter: Digest the amphibole variety of asbestos, cut in
pieces about 1 inch long, for 3 days with 1:3 HCl. Wash free from
acid and digest for a similar period with soda solution, after which
treat for a few hours with hot alkaline copper tartrate solution of the
strength employed in sugar determinations. Wash the asbestos free
from aikali, digest with nitric acid for several hours and after washing
free from acid break into shreds by shaking for some time in water.
Folin suggests suspending it in water and forcing a brisk air current
through it. Load the Gooch crucible with a layer of the washed asbestos
about % inch thick, wash it repeatedly with water to remove fine asbestos
and then with alcohol. Dry at 100° if the gravimetric Munson and
894 PHYSIOLOGICAL CHEMISTRY

Walker method is to be used. With the Bertrand method it is not neces-
sary to weigh or dry. The filters improve with use. In the gravimetric
method the cuprous oxide may be washed out with nitric acid after
each determination and the felts used over and over. In the Bertrand
method the asbestos is filtered off after each titration, washed thor-
oughly and used for the next filtration.

The lactose figures in the following tables are slightly erroneous and cor-
rections have been published in Circular 82, and in the Journal Amer. Chem. Soc.,
34, p. 204, 1912. The correction is generally less than 1 per cent.
895

PRACTICAL WORK AND METHODS

[For correction of lactose figures see Cir. 82.]

[Expressed in milligrams.]

sucrose (0.4 gram and 2 grams total sugar), lactose (three forms), and maltose
(anhydrous and crystallized).

Table for calculating dextrose, invert sugar alone, invert sugar in the presence of

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

*(O80D) prro snomdng

 

here ANE WOhOR = . rt 2
(o'N9) pyro dnoidng | SASRS BSHV& RAARA RRRRA SSRBS SSSSS VTVRT BVVSRS SaSRS BESVS Sssss
aks : grea Sa a See poy Bee eee) secrerer an. nara cverse pea 120. Se eas Pe eset agen
g OH +HOs HD SANSS SES Sodsdd Addds SNSSS SddaS SSssae Seadd asides eeesq
3
&

3 AMON HBOWND WOHNS BWOWVWND ODOM PMOMHRW LOMARQ KHIYNSDW OFfFNOW OFNOSCH OVOHOr
. poy piace oan. ees OS: RNS see Se earns tere or ete
Nowy | SSNs sori Gugss ndtsy dddds RSHAS SHHS SSHSS SAsse aasss Feses
Orta WNWWN BOMOChR WHEW DNONRWOH MOrMS Mito NOMAD COMOOM CRM DMHwonon
. Seas DIY: OOO UN ia Qa a eens Se a See ve en ee ret Goin eae eae S ames
OH +NOe HED V¥HSS NHS Sides Moser HsIig ANNRA SARSEN RAASS SSSSS SASES SSSRS
2 OWOMAO MOONS OMOnNDM OCO%MONM BOW M MMH OM HOMO WHO wMet COdeoow e100 1 e100 IN NOON OV
Bo || Se BOOS. DOE: BOSS Nt Serene. Tate ee. eee Ape es wet oes
B fOr E+NoRytg | SFOSS MOMSS SAANS SAGES SARAR RANAR AARRA NRARS RSASH ABBSKS BRIBE
a
a
00 1 1.00 = weet WHONMO OMOCOM BPONMSH NHN WOO Met COM et wt Ornmowo IMONAD wINGwS
sopccales ce |) SOMREMO NE TE SEARS Storie oeen vee BO Seen Sucre pegias ton oan ota are ee etre ae gcuers ce Seaham eet
UpkyVIg | SASS Kevddc Srridse Gaggs SUSKS SEANN KSSKS KARRA SSSSS SRA88 RSSSR
as “re3ng

3S | 1830} sures Z
23

£a
eu -redns

a | (8102 ured 50

in OS HOO CD RNR Otamrm MQwoona TOON © tin oF st DOOD NO PINOMD OCRed wonawt DWHrmAD
taiagicvas || ae e Go Ga Bese BONO SCOR Race Se Oo ee een Oe eas Aes
weSns weary | VSSss Srrdd Sass FANN ANS SESE RARAR RRARN RRARKA ARRRR RAARA
CHIAMr~ NOON| MrADD NQMon OnmOSG CONOR OWONr root MrNOS U9 2 OD COCR oCrwWownt
Dias CPCS: «NOCH C2) ASAD Si oer see eeace a cae Banos caneaer ce cacrercaen ae
*(esoon[Z-p) osorqxag. | WSS Sore Beds Sona SSVAy TERS BAAR AARRR AANKAR RRAAR RRR
Admins MAnHOD Ormiotos AOMO owowseon mONMD WtONe OOorow WOANS arrows MA OCO
nn) addon | SAGAN Sxvisod 5 i 5x as no S MANS HSrao SSH MISS 55 T+] Rete) 5
(np) addon | “FSHAN SHSSS HYSSR ARAAA RARRS BHRSS SSSRS SSsSSS FSSSR GIS Boas
Ouanva=s wWoOrda et oo 69 (Oh 0O D> aow wD 1 al
Saat Hene> SaRes REARR SSSBS SSSSS SAAGS VSSSS BaSVS BRERS SSSes

 

 
 

*(OfND) Pyxo snozdng

 

 

 

), lactose (three forms), and maltose
[For correction of lactose figures see

 

 

 

invert sugar alone, invert sugar im the presence of

[Expressed in milligrams.]

 

 

3 “OTH FOR AND
3
‘a
a “HQ AED

“OH +NOR AND
3 [On E4+NOSHMND
A

“MORAY

i
ao “1830s
26 18103 surei3 zg
S38
82 *1e3ns
HA | 18307 Wes FO
as

 

PHYSIOLOGICAL CHEMISTRY

*183N8 JOA]

 

*(asoon|8-p) o6013x0q.

 

*(nQ) seddop

 

 

sucrose (0.4 gram and 2 grams total sugar
(anhydrous and crystallized )—Continued.

Cir. 82.]

*(omg) pro snordng

 

Table for calculating dewtrose,

896

 

 
PRACTICAL WORK AND METHODS 897

Table for calculating dewtrose, invert sugar alone, invert sugar in the’ presence of
sucrose (0.4 gram and 2 grams total sugar), lactose (three forms), and maltose
(anhydrous and crystallized)—Continued. [For correction of lactose figures see

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Cir. 82.] [Expressed in milligrams.]
: . | Invert sugar. :
e $ Broyles Lactose. Maltose., a
a 3 ;
g 3 aie ei 6 aS
3 a i) : ° is] a cl
% s & a [ee es “ Hi a | y
3 5 Ey a oa 3 + + : + 3
ei, |2]2/88| 2) é)é]é)]é]é]s
° g ts & >? 5° 3 R e a z 2
a | & | & 3 i [| me lm | &
5 e 4 ~ s © By s 3 5
5 ° Aa = So a 5 ° 5 5 ° o
120} 106.6 52.3 54.3 52.2 46.0 75.8 77.8 79.8 93.1 98.0 | 120
121) 107.5 52.7 54.7 52.7 46.5 ‘76.5 78.5 80.5 93.9 9) 1b
108. 53.2 55.2 53.1 46.9 W1 79,2 81.2 94.7 99.7 | 122
123 | 109.3 53.6 55.7 53.6 47.4 . 9 8t.9 95.5 | 100.5 | 123
1247 110.1 $4.1 56.1 4.1 47.9 . 5 82.6 96.3] 101.4] 124
125 | 111.0 54.5 56.6 54.5 48.3 79.1 81.2 83.3 97.1 | 102.2 | 125
126 | 111.9 55.0 57.0 55.0 48.8 79.8 81.9 84.0 97.9| 103.0 | 126
127} 112.8 55.4 57. 55.5 49.3 80.4 82.5 84.7 98.7] 103.9 | 127
128 | 113.7 55.9 . 55.9 49.8 81.1 83,2 85.4 99.4) 104,7°|, 128
129] 114.6 56.3 58.4 56.4 50.2 81.7 83.9 86.0 | 100.2; 105.5 | 129
130) 115:5 58.8 58.9 56.9 50.7 82,4 84.6 86.7 | 101.0] 106.4] 1
131 | 116.4 57.2 59.4 57.4 61.2 83. 85.2 |. 87.4] 101.8] 107.2] -131
132 | 117.3 57.7 59.8 57.8 51. 83.7 85.9 88.1) 102.6] 108.0} 132
133} 118.1 58.1 3 58.3 52.1 84.4 86.6 .88.8} 103.4] 108.9] 133
134] 119.0 58.6 60.8 58.8 52. 85.0 87.3 89.5) 104.2] 109.7] 134
135 | 119.9 59.0 61.2 59.3 53.1 85.7 87.9 90.2} 105.0) 110.5] 135
136 | 120. 59.5 61.7 59.7 53.6 86.3 88.6 90.9 | 105.8) 111.4] 136
137 | 121.7] °60.0 62.2 60.2 64.0 | .87.0 89.3 91.6 112.2] 137
138 | 122.6 60.4 62.6 60.7 54.5 87.7 90.0 92.3 113.0 | 138
139 | 123.5 .9 63.1 61.2 55.0]- 88.3 90.6 93.0 / 108.2} 113.9} 139
140 | 124.4 61.3 63.6 61.6 55.5 89.0 91.3 93.6 | 109.0] 114.7] 140
141) 125.2 61.8 64.0 62.1 55. 89.6 92.0 94.3) 109.8| 115.5| 141
142) 126.1 62.2 64.5 62.6 4 90.3 92.6 95.01 110.5] 116.4| 142
143} 127.0 62.7 65.0 63.1 56.9 90.9 93.3 | 95.7} 111.3] 117.2) 143
144| 127.9 63.1 § 63.5 57.4 91.6 94.0 96.4 | 112.1] 118.0) 144
145 | 128.8 68.6 65.9 64.0 57.8 92.2 94.7 97.1) 112.9] 118.9] 145
146 7 0 66.4 64.5 58.3 92.9 95.3 97.8] 113.7] 119.7| 146
147 | 130.6 64.5 66.9 65.0 58.8 93.5 96.0 98.4) 114.5] 120. 147
148} 131.5 65.0] 67.3 65.4 59.3 94.2 96.7 99.1] 115.3] 121.4] 148
149 | 132.4 65.4 67.8 65.9 59.7 94.8 97.3 99.8 . 122. 149
150 | 133.2 65.9 68.3 66. 4 60.2 95.5 98.0] 100.5] 116.9] 123.0} 150
151 | 134.1) ¢ 66.3 68.7 66.9 60.7 96. 2 98.7 | 101.2 | 117.7 | 123.9] 151
152 | 135.0 66.8 2 67.3 61.2 96.8 99.3] 101.9} 118.5] 124.7] 152
153 | 135.9] .67.2 69.7 67.8 61.7 97.6) 100.0] 102.6 | 119.3] 125.5] 153
154.) 136.8 67.7 7.14 -68.3 62.1 98.1 | 100.7} 103.3] 120.0] 126.4] 154
155°] 137.7 68,2 70.6 68.8 62.6 98.8] 101.4] 104.0] 120.8] 127.2] 155
138.6 68.6 TWL1 69. 63.1 99.4 | 102.0). 104.7 | 121.6] 1280), 1
157 | 139.5 69.1 7L.6 69.7 63.6 | 100.1] 102.7 3} 122.4] 128.9} 167
140.3 69.5 72.0 . 64.1] 100.7} 103.4] 106.0] 123.2] 129.7] 1!
159 | 141.2 70.0 72.5 0. 64.5 | 101.4} 1041) 106. 124.0 | 180.5) 159
160 | 142.1 70.4 73.0 71.2 65.0 | 102.0} 1047] 107.4] 124.8] 131.4] 1
161 | 143.0 70.9 7334 71.6 65.5 | 102.7} 105.4] 108.1 | 125.6) 132.2] 161
162 9 71.4 73.9 1 66.0 | 103.4] 106.1 |} 108.8] 126.4 1
163 | 1448 71.8 744 72.6 66.5 | 104.0] 106.7 | 109.5 | 127.2) 133.9] 163
145.7 . 74.9 73.1 66.9} 1 107.4 | 110.2 134.7 | 164
165 | 146.6 72.8 75.3 73.6 67.4] 105.3; 1081] 110.9} 128.8] 135.5] 165
166 | 147.5 73.2 75.8 74.0 67.9 | 106.0] 1088] 111.5 . £ 166
167 | 148.3 73.7 76.3 74.5 68,4 | 106.6) 109.4] 112.2 . 137.2 | 167
168 | 149.2 74.1 75.0 68.9 | 107.3] 110.1] 112.9] 131.1] 138.0] 168
169 | 150.1 74.6 77.2 76.5 69.3 9} 110.8 3.6] 131.9 | 138.9] 169
170 | 151.0 76.1 77.7 76.0 69.8) 108.6) 111.4) 114.3} 132.7 | 139.7] 170
171 | 151.9 75.6 78.2 76. 4 70.3 | 109.2) 112.1} 115.0) 183.5) 140.5] 171
172 | 152.8 76.0 78.7 76.9 70.8 | 109.9) 112.8] 115.7] 1843; 141.4] 1
173 | 153.7 76.4 . 1 B 74.3] 110.5] 113.5} 116.4] 135.1 | 142.2] 173
174| 1646 76.9 79.6 | 77.9 71.7) W112 | 141] 117.1 | 135.9] 143.0] 174

 
898 PHYSIOLOGICAL CHEMISTRY

Table for calculating dextrose, invert sugar alone, invert sugar in the presence of
sucrose (0.4 gram and 2 grams total sugar), lactose (three forms), and maltose
(anhydrous and crystallized)—Continued. [For correction of lactose figures see

 

 

 

 

Cir. 82.] [Expressed in milligrams.]

t . Invert suger 0
§ 3 aid ernie, Lactose. Maltose. é
s : | &
2 5 2 13 ei 6¢ |=

. ic) . to a a

i438 S |e ee = | & a
c S 3 y as & S t Ba a + ©
s ) 3 8 | 2 | ah | gP] 6 | &é}] 6] 6] 6é!/ 8
£ & $ 5 m? | 22) & 8 8 g a °
a) eee eo Le |e |e |) ele hes
5 8 A a 3 a 3° ° Oo o 3 }
175 | 155.5 77.4 80.1 78.47 72,2} 111.9] 114.8] 117. 136.7) 143.9} 175
176| 156.3 77.8 60.6 78.8 72.7} 112.5 | 115.5 | 118.4] 187.5] 1447] 176
177 | 157.2 78.3 81.0 79.3 73.2 | 118.2} 116.1 | 119.1} 188.3) 145.5] 177
178 | 158.1 78.8 81.5, 79.8 73.7 | 118.8} 116.8] 119.8] 139.1] 1444] 178
179} 159.0 79.2 82.0 8.3 74.2) 114.5] 117.5] 120.5} 139.8; 147.2] 179
180 | 159.9 19.7 82.5 80.8 74.6) 115.1] 1182] 121.2] 140.6) 1480] 1
181 | 160.8 80.1 $2.9 81.3 75.1) 115.8] 1188 | 121.9] 141.4] 1489] 181
182 | 161.7 80.6 83.4 E 7 76.6) 116.5] 119.5 | 122.6] 142.2) 149.7] 1
182 | 162.6 81.1 83.9 2 76.1) 127.1} 120.2 | 123.3] 143.0] 150.5] 183
184 | 1634 81.5 844 82.7 76.6 | 117.8} 120.9| 123.9 { 143.8] 151.4] 184
18} 164.3 82.0 84.9 83.2) .77.1 | 118.4] 121.5] 1346] 1446] 152.2] 185
186 | 165.2 82.5 85.3 83.7 77.6) 119.1) 122.2) 126.3) 145.4] 1530] 186
187 | 166.1] .82.9 85.8 [ 84.2 78.0 | 119.7] 122.9] 126.0] 146.2) 153.9
188 | 167.0 83.4 ge. 3 84.6 78.5 | 120.4] 123.5 | 126.7] 147.0 | 154.7] 188
189 | 167.9 83,9 . 8 85.1 79.0) 121.0] 1242] 127.4] 147.8) 155.5
1 168.8 84.3 87.2 85.6} 79.5 | 121.7] 124.9 | 1281] 148.6] 1564] 190
191 | 169.7 84.8 87.7 86.1 80.0 | 122.3) 125.5] 128.8{ 149.3] 157.2] 101
I 170.5 85.3 88.2 86.6 80.5} 128.0) 126.2) 129.5 158.0 | 192
193 | 171.4 85.7 88.7 87.1 81.0] 123.6} 126.9] 130.1 | 180.9] 168.9 | 193
194 | 172.3 86.2 89. 2 87.6 81.4] 124.3} 127.6] 180.8) 151.7 | 159.7] 194
195 | 173.2 86.7 89.6 88.0 81.9] 125.0] 128.2] 131.5] 1525) 160.5] 195

174.1 87.1 90.1 885 82.4) 125.6} 128.9] 132.2] 153.3) 161.4) 196
197 | 175.0 87.6 90.6 89.0 82.9 | 126.3) 129.6] 133.9} 154.1] 162.2] 1
198 | 175.9 88.1 91.1 89.5 838.4 | 126.9) 130.3] 183.6] 154.9 | 163.0) 198
199 | 176.8 88.5 91.6 90.0 83.9 | 127.6) 190.9} 1343] 155.7 | 1639} 199
200 | 177.7 89.0 92.0 90.5 84.4] 1282] 131.6] 135.0] 156.5] 1647] 200
201) 178.5 89.5 92.5 91.0 848] 128.9} 132.3] 18.7] 157.3) 1655] 20
202 | 1379.4 89.9 93.0 91.4 85.3 | 129.5] 132.9] 186.3) 1581] 166.4] 22
203 | 180.3 90. 4 93.5 91.9 85.8 | 130.2 | 183.6] 137.0] 1588) 167,2) 20
204) 181.2 90.9 94,0 92. 4 86.3 | 130.8} 134.3] 137.7] 159.6] 168.0 | 204
205 | 182.1 91. 4 94.5 92.9 86.8 | 131.5] 185.0] 138.4) 160.4] 168.9] 205
206 | 183.0 91.8 94.9 93.4 87.3 | 132.1] 135.6] 139.1] 161.2] 169.7] 206
207 | 183.9 92.3 95.4 93.9 87.8 | 132.8) 136.3] 139.8] 162.0] 170.5] 207
208 | 184.8 92.8 95.9 94.4 88.3! 133.4] 137.0] 140.5] 162.8} 171.4] 208
209 | 185.6 93. 2 96. 4 94.9 88.8} 134.1) 187.6] 141.2] 163.6] 172.2] 209
210 | 186.5 93.7 96.9 95.4 89.2} 134.8 | 138.3] 141.9] 1644] 173.0} 210
211) 187.4 94.2 97.4| 95.8 89.7 | 1 139.0 5] 165.2] 1738] 211
212 | 188.3 94.6 97.8 96.3 90.2 | 136.1 6] 1432] 166.0] 1747] 212
213 | 189.2 95.1 98.3 96.8 90.7 | 1386.7] 140.3] 143.9] 166.8] 1755] 213
214) 190.1 95.6 98.8 97.3 91.2] 187.4] 141.0] 144.6] 167.5] 176.4] 214
215 | 191.0 96.1, 99.3 07.8 91.7 | 188.0] 141.7] 145.3] 1683] 177.2] 216
216 | 191.9 96.5 99.8 98.3 92.2 | 188.7] 142.3] 146.0{ 169.1] 1780] 216
217 | 192.8 97.0 | 100.3 98.8 92.7 | 139.3 | 143.0] 146.7] 169.9} 1789] 217
218 | 193.6 97.5 | 100.8 99.3 93.2) 140.0] 143.7) 147.3] 170.7] 179.7] 218
219 | 194.5 98.0] 101.2 99.8 93.7 | 140.6] 1443] 148.0] 171.5 5] 219
220 | 195.4 98.4 | 101.7] 100.3 94.2} 141.3] 145.0] 1487] 172.3] 181.4
221 | 196.3 98.9 | 102.2] 100.8 94.7 | 141.9] 1457] 149.4] 1731) 182.2 | 221
222 | 197.2 99.4 | 102.7] 101.2 95.1 146.3} 150.1 | 173.9] 1830] 222
223} 198.1 99.9] 103.2 | 101.7 95.6 | 143.2| 147.0] 150.8] 174.7 9] 223
224; 199.0] 100.3] 103.7); 102.2 96.1 | 143.9 | 147.7] 151.5] 175.5] 1847] 224
225 | 199.9] 100.8 { 1042] 102.7 96.6 | 1446; 148.4] 152.2] 176.2] 185.5] 225
226 | 200.7] 101.3) 1046] 103.2 97.1} 145.2 | 149.0] 152.9] 177.0) 186.4 | 226
907 | 201.6] 101.8 | 105.1) 103.7 97.6 | 1439] 149.7 58.6 | 177.8 | 187.2 | 227
228 | 202.5 ee 2] 1056] 1042 98.1 180. 4 54.2 | 1786) 189.0] 228
229 | 203.4 02.7 | 106.1 | 104.7 98.6 | 147.2] 151.1) 1849 79,4 8). 2

 

 

 

 

 

 

 

 

 

 

 

 

 

 
PRACTICAL WORK AND METHODS 899

Table for calculating dewtrose, invert sugar alone, invert sugar in the presence of
sucrose (0.4 gram and 2 grams total sugar), lactose (three forms), and maltose
(anhydrous and erystallized)—Continued. [For correction of lactose figures see

 

 

Cir. 82.] (Expressed in milligrams.]

: ‘ Invert sugar is
eS s and sucrose. Lactose. Maltose. é
g g re 8
= 5 s s 9 o é g
SI x x 8 3 3 iq 2 2, =
ig a 3 a : s ian i] Si g
Se ge ego a eee be ee
3 2 3 g 5 = = = = a
| 8] | 2 ]8/a2/%/2)/%)a)a]eé
eB) 8 | B | E eo) ee po ee
S 2° o q ~ a a a A a
° 5 A a s a Oo Oo Oo S S F

 

234 . 105.1] 108.6 | 107.2], 101.1] 150.5] 154.4] 158.4] 183.4| 193.0| 234
235 | 208.7] 105.6) 100.1] 107.7] 101.6) 151.1] 155.1] 159.1] 184.2] 193.8] 2385
236] 209.6) 106.0] 109.5] 1082] 102.1) 151.8] 155.8] 159.7] 184.9 236
237 | 210.5) 106.5) 110.0] 1087] 102.6) 152.4 | 156.4] 160.4) 185.7] 195.5 | 237

241) 214.1) 108.4] 112.0] 110.6} 104.5) 155.0] 159.1] 163.2) 188.9 | 1988] 241
242) 215.0] 108.9] 112.5) 111.1] 105.0] 155.7 163.9 | 189.7 | 199.7 | 242
243 | 215.8 113.0 | 111.6] 105.5 56.3} 160.5) 164.6] 190.5 5| 243

251 113.2 | 116.9} 115.6 09.5 | 161.6) 165.8] 170.1 | 196.8] 207.2} 251
252 | 223.8) 113.7] 117.4) 116.1} 110.0] 162.2) 166.5 | 170.8] 197.6 0 2
253] 224.7) 114.2] 117.9 16,6 | 110.5} 162.9) 167.2] 171.5 | 1984] 2088] 253
254) 225.6] 114.7] 118.4] 117.1] 111.0] 163.5} 167.9} 172.2 254

266 | 236.3] 120.5] 124.4] 123.1] 117.0] 171.4] 175.9] 180.4] 208.7 |, 219.7] 266
267 | 237.2 | 121.0] 124.9] 123.6] 117.5] 172.0] 176.6] 181.1] 209.5] 220.5] 267
268 | 238.1) 121.5] 125.4] 124.1] 118.0] 172.7] 177.2] 181.8} 210.3] 221.3) 268
269 | 258.9] 122.0] 125.9] 1246) 1185) 173.3] 177.9) 1825) 211.0) 222.1) 269
270 | 239.8] 122.5] 126.4] 125.1] 119.0] 174.0] 178,6| 183.2) 211.8} 223.0} 270
271 | 240.7] 122.9] 126.9] 125.6] 119.5) 1746] 179.2] 183.8] 212.6 | 223.8) 271
272 | 241.6] 123.4) 127.4] 126.2] 120.0) 175.3} 179.9] 184.5] 213.4} 224.6) 272
9731 242.5 | 1293.9] 127.9] 126.7] 120.6] 176.0} 180.6 | 185.2] 214.2} -225.5| 273
974| 243.4) 124.4] 128.4] 127.2] 121.1] 176.6/ 181.3] 185.9] 215.0) 226.3) 274
275 | 244.3! 124.9] 128.9] 127.7| 121.6] 177.3] 181.9] 186.6] 215.8; 227.1] 275

6 | 245.2] 125.4] 129.4] 128:2) 122.1] 177.9| 1826) 187.3) 216.6) 228.0) 276
277 | 246.1] 125.9] 129.9] 128.7] 122.6] 1786] 183.3] 188.0] 217.4) 228.8) 277
978 | 246.9| 126.4| 130.4] 129.2] 123.1] 179.2] 184.0] 188.7] 2182) 220.6] 278
279 | 247.8] 126.9] 130,9] 129.7] 123.6] 2179.9) 184.6] 189.4] 2189] 230.6] 270
280 | 248.7] 127.3] 131.4] 130.2] 1241] 180.6] 185.3} 190.1] 219.7] 231.3) 280
281 | 249.6] 1 131.9 | 130.7 | 124. 181.2 86.0] 190.7] 220.5) 232.1) 281
282 | 250.5 | 128.3 131.2 | 125.1] 181.9) 186.6} 101.4] 221.3] 233.0) 282
283 | 251.4] 128.8) 132.9 125.6 | 182.5 | 187.3 222.1) 233.8 | 283
284] 252.3) 129.3] 133.4] 132, 126.1] 183.2] 188.0] 192.8] 222.9) 2346) 284

 

 

 

 

 

 

 

 

 

 

 

 

 

 
900 PHYSIOLOGICAL CHEMISTRY

Table for calculating dextrose, invert sugar alone, invert sugar in the presence of
sucrose (0.4 gram and 2 grams total sugar), lactose (three forms), and maltose
(anhydrous und ecrystallized)—Continued. [For correction of lactose figures see

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Cir, 82.] [Expressed in milligrams.]
. i Invert sugar “3
8 $ ‘and sucrose. Lactose. Maltose. 8
3 =
S 3 3 3 2 : ; oS
Silay peie | se l8 a | g fis
a a & a ee ee re se) q 2
2 2S o = aa | 23 rs t + i ft 2
- $ a 5 g = = zs a = 3
ela |e)e/e|#?/313e12e)e)a)3
ee By Ee: ee ee ee
5 5 a a oS a 5 5 5 5 5 °
285 | 253.2] 129.8] 133.9] 132.7] 126.6} 183.8] 188.7] 193.5 | 223.7] 235.5] 285
286 | 254.0) 180.3) 134.4] 133.2] 127.1] 184.5] 189.3] 194.2] 224.5] 236.3) 286
287 | 254.9] 130.8] 134.9 33.7 | 127.6 | 185.1] 190.0] 194.9] 225.3) 237.1 | 287
288 | 255.8 | 181.3] 135.4] 134.3] 128.1} 185.8) 190.7] 195.5 | 226.1] 238.0) 288
289 | 256.7] 131.8] 135.9 8] 128.6} 186.4) 191.3] 196.2] 226.9] 238.8] 289
290} 257.6) 132.3] 136.4] 135.3] 129.2] 187.1] 1920] 196.9] 227.6] 239.6] 290
201 | 258.5] 182.7] 136.9] 135.8] 129.7] 187.7} 192.7] 197.6] 228.4| 240.5| 291
202 | 259.4) 183.2] 137.4} 136.3} 180.2] 188.4] 193.3] 198.3; 229.2] 241.3] 292
293 | 260.3) 183.7] 137. 136.8} 130.7 | 189.0| 194.0] 199.0] 230.0; 242.1 293
294 | 261.2] 134.2] 1384] 137.3] 131.2] 189.7] 194.7] 199.7| 230.8] 242.9] 294
295 | 262.0) 184.7] 138.9 | 137.8} 131.7| 190.3] 195.4] 200.4] 231.6] 243.8] 295
296 | 262.9] 135.2] 139.4 2] 191.0} 196.0] 201.0} 232.4] 244.6; 296
297 | 263.8] 135.7] 140.0] 1388 | 132.7] 191.7] 196.7] 201.7{ 233.2] 245.4] 297
298 | 264.7] 136.2] 140.5] .139.4| 133.2] 193.3] 197.4] 202.4| 234.0] 246.3] 208
299 | 265.6) 136.7] 141.0] 1 133.7 | 193.0) 198.0] 203.1); 234.8) 247.1] 299
300 | 266.5 | 187.2] 141.5} 140.4] 134.2] 193.6] 198.7] 203.8] 235.5] 247.9] 300
301 | 267.4] 187.7 . 140.9 | 134.8] 194.3] 199.4) 204.5] 236.3] 248.8] 301
302 | 268.3] 188.2] 1425] 141.4] 185.3] 194.9] 200.0) 205.2] 237.1] 249.6] 302
303 | 269.1] 138.7] 143.0] 141.9] 135.8] 195.6] 200.7] 205.9] 237.9] 250.4} 303
304) 270.0 | 189.2] 143.5] 142.4} 186.3) 196.2] 201.4) 206.5) 238.7] 251.3] 304
305 | 270.9] 139.7] 144.0] 142.9] 136.8] 196.9 | 202.1] 207.2] 239.5) 252.1] 305
306 | 271.8] 140.2] 1445] 143.4. 3 5} 202.7 | 207.9) 240.3] 252.9] 306
307 | 272.7] 140.7] 145.0 | 1440] 137.8] 198.2] 203.4| 2086] 241.1 | 253.8] 307
308 |} 273, 141.2} 145.5 | 1445) 188.3] 198.8] 204.1] 209.3] 241.9] 254.6] 308
309 | 274.5] 141.7] 146.1] 145.0] 138.8 99.5} 204.7 { 210.0} 242.7] 255.4] 309
310 | 275.4 | 142.2] 146.6] 145.5] 139.4] 200.1 | 205.4] 210.7] 243.5) 256.3] 310
311 | 276.3 | 142.7] 147.1 | 146.0 139.9} 200.8] 206.1 | 211.4] 2442] 257.1] 311
312 | 277.1] 143.2] 147.6 | 146.5] 140.4] 201.4 | 206.7) 212.1) 245.0; 257.9] 312
313 | 278.0] 143.7] 148.1] 147.0] 140.9 | 202.1] 207.4) 212.7] 245.8] 258.8] 313
314 | 278.9] 1442] 1486] 147.6] 141.4] 202.8] 2081] 213.4] 2466] 2596/ 314
315 | 279.8] 1447] 1492] 1481] 141.9] 2034 208.8} 2141! 247.4] 2604] 315
316 145.2 | 149.6] 148.6) 142.4] 2041) 200.4] 2148] 248.2] 261.2] 316
317 | 281.6} 145.7] 150. 149.1} 1420] 204.7) 210.1 | 215.5] 249.0] 262.1) 317
318 | 282. 146.2 | 150.7 9. 143.5 | 205.4] 210.8) 216.2] 249.8 9} 318
319 | 2834] 146.7] 151.2] 150.1) 1440] 206.0) 211.5| 216.9] 250.6] 2637) 319
320 | 284.2] 147.2] 151.7 | 150.7] 1445 ao 7 | 212.1 | 217.6) 251.3) 2646] 320
321 | 285.1 | 147.7] 152.2°} 151.2] 145.0 3 8 18.3] 252.1 | 265.4] 321
286.0} 148.2] 152.7] 151.7] 145.5} 2080] 213.5] 218.9] 252.9] 266.2
323 | 286.9] 148.7] 1532 146.0} 208.6] 214.1) 219.6) 253.7 | 267.1 |) 323
324 | 287.8} 149.2] 153.7 7| 146.6] 200.3] 214.8] 220.3] 2545] 267.9] 324
288.7} 149.7] 1543] 153.2] 147.1] 210.0; 215.5 | 221.0] 255.3 | 268.7] 325
326 | 289.6} 150.2] 164.8} 153.8] 147.6] 210.6] 216.2! 221.7] 256.1 / 269.6] 326
290.5) 150.7 | 155.3] 1543] 148.1,) 211.8) 216.8 | 222.4} 256.9] 270.4 | 327
291.4 | 151.2] 155.8] 1548 48. 211.9 | 217.5 | 223.1) 257.7] 271.2 | 328
829 | 292.2] 151.7] 156.3] 155.3 | 149.1 12.6:| 218.2) 223.8] 2585) 272.1} 329
‘330 | 293.1 |) 152.2] 156.8] 155.8] 149.7] 213.2] 2188] 224.4] 259.3] 272.9] 330
331 | 294.0] 152.7] 157.3} 156.4] 150.2] 213.9} 219.5] 225.1] 260.0) 273.7] 331
332 | 204.9] 153.2] 1679] 156.9] 180.7} 214.5] 220.2] 225.8] 260.8] 2746] 332
333 | 205.8) 153.7{ 1584] 157.4] 151.2°| 215.2] 220.8] 226.5 | 261.6 | 2754] 333
334 | 206.7) 1542] 1589] 157.9 in 215.8 | 221.5] 227.2 | 262.4] 276.2) 334
335 | 207.6} 154.7] 159.4] 1584] 152.8] 216.5) 222.2] 227.9] 263.2] 277.0! 335
336 | 298.5 . . 159.0 | 152.8) 217.1 | 222.9 | 2286) 264.0] 277.9 | 336
337 | 299.3 5| 159.5] 153.3] 217.8 | 223.5] 229.2) 2648) 278.7) 337
338 | 300.2] 156.3] 161.0] 160.0] 153.8] 2184] 2242] 220.9] 265.6] 279.5] 338
339 | 301.1 5 161.5 | 160.5] 1543] 2191) 2249] 2306] 266.4] 280.4) 339

 

 
PRACTICAL WORK AND METHODS 901

Table for calculating dextrose, invert sugar alone, invert sugar in the presence of
sucrose (0.4 gram and 2 grams total sugar), lactose (three forms), and maltose
(anhydrous and erystallized)—Continued. [For correction of lactose figures sec

 

 

 

 

 

 

 

Cir. 82.] [Expressed in milligrams]
: 5 Invert sugar Fi
a 3 and euierone! Lactose. Maltose. S
a g = 3
g a z 2 : : =
3 , % u 3 s is] Q 2 5
% S s a we |) Phe ra >] >) a
>a en ea Pe eee ee eo
g a 8 = | 2 | eo] 6 6 6 6 3
€ 2 5 § m* | B82 a 8 8 a a 8
es; ei eB |e ls B/S); 8)3) 8) &
a) 3 A als n 5 | 3 3 5 51 8
340 | 302.0] 157.3] 162.0] 161.0 | 154.8] 219.8} 225.5 | 231.3] 267.1] 281.2| 340
341 | 302.9; 157.8] 162.5] 161.6 | 155.4] 220.4] 226.2 | 232.0] 267.9] 282.0] 341
342 | 303.8] 158.3] 163.1] 162.1 | 155.9! 221.1 | 226.9] 232.7] 268.7) 282.9] 342
343 | 304.7 | 1588] 163.6) 162.6 | 156.4] 221.7] 227.5 | 233.4] 269.5] 283.7) 343
344] 305.6] 159.3] 1641.) 163.1] 156.9 | 222.4] 228.2) 234.1] 270.3 | 284.6] 344
345 | 306.5 | 159.8] 1646) 163.7] 157.5] 223.0) 228.9) 2347] 271.1 | 2854] 345
346 | 307.3 | 160.3 | 165.1 | 164.2 | 158.0} 223.7 | 229.6) 235.4] 271.9| 286.2] 346
347 | 308.2] 160.8] 165.7 | 164.7] 168.5] 224.3 | 280.2 | 2361] 272.7] 287.0; 347
B48 | 309.1 | 161.4] 166.2} 165.2 | 159.0 | 226.0] 230.9} 236.8) 273.5} 287.9] 348
349 | 310.0] 161.9) 166.7 { 165.7 | 169.5) 225.6 | 231.6 | 237.5] 274.3 | 288.7] 349
310.9 | 162.4) 167.2] 166.3] 160.1 | 226.3] 232.2) 2382 275.0| 289.5] 350
351 | 311.8 | 162.9] 167.7} 166.8] 160.6] 226.9] 232.9] 2389] 275.8] 290.4] 351
352 | 312.7 | 163.4 3) 167,38] 161.1] 227.6 | 233.6] 239.6) 276.6] 291.2] 352
353 | 313.6 | 163.9] 168.8} 167.8] 161.6) 228.2] 234.2) 240.2] 277.4] 292.0] 353
354) 314.4] 164.4) 169.3 | 168.4 | 162.2 | 2289] 234.9) 240.9) 2782] 292.8) 354
355] 315.3 | 1649] 169.8) 168.9] 162.7] 229.5| 235.6} 241.6] 279.0| 293.7] 355
316.2 | 165.4] 170.4] 169.4) 163.2) 230.2 | 236.3} 242.31 279.8) 204.5] 356
357 1 0| 170.9] 170.0 | 163.7'| 230.8 | 246.9 | 243.0) 280.6 | 205.3] 357
318.0 | 166.5 | 171.4] 170.5 | 16437) 231.5] 237.6 | 243.7] 281.4| 296.2) 358
359 | 318.9 | 267.0) 171.9] 171.0) 1648] 232.1 | 24833] 2444] 282.2) 297.0] 359
360] 319.8 | 167.5] 172.5 | 171.5] 165.3] 232.8] 238.9] 245.1 | 282.9] 207.8] 360
361 | 320.7) 168.0 | 1730] 172.1) 165.8] 233.5] 239.6] 245.8 oo 7| 298.7 | 361 |
362 | 321.6 | 168.5) 173.5 | 172.6 | 166.4] 234.1] 240.3] 246.4 4.5] 299.5 | 362
169.0 | 174.0] 173,1 | 166,9| 234.8] 241.0] 247.1 | 285.3] 300.3] 363
364) 323.3 | 169.6) 1746) 173.7 | 167.4) 285.4 | 441.6 | 247.8 | 286.1] 301.2] 364 |
365 | 324.2] 170.1] 175.1] 1742 | 167.9] 236.1) 242.3} 248.5) 286.9 | 302.0] 365 |
366 | 325.1) 170.6] 175.6 | 174.7] 168.5] 236.7) 243.0) 249.2] 287.7] 302.8] 366
367 | 326.0) 171.1] 176.1 | 175.2] 169.0) 237.4) 243.6 | 249.9] 288.5] 303.6] 367. .
368 | 326.2 176.7 | 175.8 | 169.5] 2381] 2443] 250. 289.3 | 304.5] 368 |
369 | 327.8) 172.2 | 177.2 | 1763] 170.0) 238.7} 245.0] 251.3) 290.0} 305.3] 369
370} 328.7 | 172.7 | 177.7] 1768 | 170.6 | 239.4 {| 246.7] 252.0] 290.8] 306.1] 370 |
371 | 329.5] 173.2) 1783] 177.4] 171.1 | 240.01 246.3] 252.7] 201.6| 307.0] 371 °
372 | 330.4} 173,71 178,8| 177.9 | 171.6] 240.7] 247.0] 253.3| 292.4) 307.8] 372
373 | 331.3} 174.2] 179.3 | 1784] 172.2] 241.3) 247.7 | 254.0) 293.2] 3086) 373 ;
374 2) 174.7 | 179.81] 179.0 | 172.7] 242.0) 248.4] 2547 | 2940] 309.5] 374 ;
375 | 333.1 | 175.3 | 180.4] 179.6] 173.2) 242.6) 249.0] 255.4] 204.8) 310.3] 375
376 | 334.0] 175.8} 180.9] 180.0] 173.7 | 243.3 | 249.7] 256.1] 295.6] 311.1] 376 '
377 | 334.9] 176.3] 181.4] 180.6] 1743 250.4 | 256.8] 296.4 | 312.0] 377 .
378 | 335.8] 176.8] 182.0] 181.1] 1748 6| 251.0} 257.5 | 297.2] 312.8] 378 ©
379 | 336.7] 177.3 | 1825} 181.6] 175.3] 245.2] 251.7} 258.2) 297.9] 313.6] 379 i
380 | 337.5] 177.9] 183.0] 182.1] 175.9} 245.9] 252.4} 258.8) 298.7) 3145] 380 |
381 | 338.4] 178.4] 183.6) 182.7] 176.4] 246.6 | 253.0] 259.5) 200.5) 315.3) 381 °
382 | 339.3] 1789] 1841] 183.2] 1769] 247.2| 253.7] 260.2] 300.3] 316.1] 382 -
383 179.4 | 184.6] 183.8] 177.5 | 247.9] 254.4) 260.9] 301.1] 316.9] 383 :
384] 341.1] 180.0] 185.2] 1843] 178.0) 2485] 2551) 261.6] 301.9] 317.8] 384
3. 342.0 | 180.5] 185.7] 1848| 1785] 240.2] 255.71) 262.3] 302.7] 3186] 885
386 | 342.9] 181.0] 186.2] 185 179.1 | 249.8] 256.4! 263.0) 303.5] 319.4] 386
387 181.5} 186:8| 185.9} 179.6] 250 257.1 | 263.6] 3042] 320.3) 387
388 | 3446] 182.0] 187.3] 186.4] 180.1} 251.1] 257.7| 2643) 305.0] 321.1) 388
389 | 345.5 | 182.6] 187.8] 187.0] 180.6 258.4 | 265.0] 305.8 | 321.9) 389
390 183.1 | 188.4] 187.6] 181.2] 252.4] 259.1 | 265.7} 306.6] 322.8] 390
391; 347.3] 183.6] 188.9] 188.0 81, 253,1 | 259.7) 266.4! 307.4] 323.6] 391
392 | 348.2) 1841] 189.4] 1886 253.7 | 260;4| 267.1.| 308.2.) 3244] 392
393 | 349.1] 1847 | 190.0] 1891] 182.8) 2544 267.8 | 309,01] 325.2] 303
394 | 350.0] 185.2] 190.5 183,3 | 255.0 | 261.8| 268.5] 3802.8] 326.1} 93094
1

 

 

 

 

 

 

 

 

 

 
7
i

 

Comp) prxo snoring | BARRE SSSSS VSSSS GIST GSHIS SSNS FSHSI Hess BSHSS IIIs

 

 

 

{For correction of lactose figures see

 

@ROTN TAROT ACANG CACAR BNSM NUNN WrNA Owen ~THEN maaDe

g | ‘outnomH"o| SSNS2 RESUS SERRE GSsad LE¥S¢ ELS8a FekRe SSREE Sease BeSEs
3 STI BRTAn BASES TACwE A|rane query Aowoe Horan waroe woes
NoPH"o | SASS SHSas SH88S SHSSS SHSSA SHES8 SSSR FBSS= LAES¥ SESEE

ARONA ONSSD OHTA DADA AOMCO MONT DANTE AMON OVOhS ITM

‘ofu+tomnto | SSSER NESSES SENESS ERREN RKRKK RRLLA KRAAR SHSKR RRSSS SSzSS

g SHOT OTH TOGA GAMA DHAMH AMG NADA SMARS AROS OAmORM
3 ‘OfH E+" OF HD HERES BEERS 28ShN SESE ESESS SERRX SARRS AARRA RAKAE RARZ

 

worH"O | ESRRS SGSS8 SEA22 SEER SESE ANGER ASSHE SQSEE SSKRR ARERR

 

total sugar), lactose (three forms), and maltose

invert sugar alone, invert sugar in the presence o

[Expressed in milligrams.]

ror Stig ,| Gaddd duudd dees dddud sedge ssgdd dedcs sages geuq gages

 

Invert sugar
and sucrose,

 

 

PHYSIOLOGICAL CHEMISTRY

swing | 35923 Sagae geece geese es iddds aaaca
mio mus yo] $8558 S8SS8 Besse SISSS RRRGR RARSE SRRRN RENEE NARA SHEER
oO

SeesS eos st ae ew K %
‘vans woant | SSS FSSES SHSTS SSSR RENE RASRR RRASR RAKE SRRSA SAARS

 

RA
*(esoon|s-p) esor]x0q BS

 

HOWMOD MVMOAM CORON WHAHO

“too) too! Bag

 

 

(anhydrous and crystallized )—Continued.

sucrose (0.4 gram and 2 grams
Cir, 82.]

355.
356.
357.
358.
368,
359.
360.
361.
362.
363.
364.2
365.1
366.0
366. 9
414) 367.7

Satan WoeNe weaan cHnes qaans enoce
SESE8 EESES ESSSH 88882 SRSSE S2SzR
3 3

Baaaa SHSSS SS5SF FESR FGRRS IIRY

 

(omg) prro moidng | BBBSE SSSSS GSSSS FHI:

 

Table for calculating dextrose,

902

 
903

PRACTICAL WORK AND METHODS

t sugar in the presence of

inver
sucrose (0.4 gram and 2 grams total sugar), lactose (three forms), and maltose

(anhydrous and crystallized )—Continued.

Yable for calculating dewtrose, invert sugar alone,
Cir. 82.]

[For correction of lactose figures see

[Expressed in milligrams.]

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

“(o™Mp) pixo snoring | SERRE BSEBS SESSS SSSSS SESHS SSSSSE SERFS BBHSS S

g | O'H+NOFHMO | SESSS SESSS SHR8S BESES SESE8 BASE BASES SSSSS 8

a} nu, | S3282 ausee Zegse Geceg Suda suis Seles gay

onn"o | $2885 S88Ss S888 $8588 SSEES SSSES ESSE HSSZS 8

ACSCMOM BHOSMe WDiOHANHWOG COMO mer MOON ONG Ohm <T

‘onm+omH" | $5882 SiS Skass HSss8 SASAS SAMSH SSASS ERRSS 8

g | BRERS ONSEN GERSR MENMe HRehY Suyeh wonen Renee
§ fout+torno| £8858 $8288 S88S8 SSSGh SSH SSHSS SSSAN ASSESS

womn"o| S888 SkRkR RARSS SESS SSSSH BSSES EHSSS SHSSE H

beg] seme. | SSIS BOSGy SS8ae SounS ARate emnae Magee quawe o

BE | min sums c| RASSH SSESN SSRN SANNA NNWEN RAGAN REREN NEAEN &

We ein NOLS SENny HMA CARMA TOBE awYae Team agoon &

BE | rior mes yo| SSNSN RASS ASSNS SSSSS SNRAX RRAKR RRARE KSREN Q

segs aroaur | ARANN QASSS SNNSS SSSHR SASS RASRN RASRS SERKE X

: AWMHMAANW OHHON MMOH BWOOK& NOW OCSeired eaea “ON OA = i

“(esconj#-p) sonmeg | BASSE SESHR NANNA ASASS RENAN ASKER SHARE SARS §

(no) weddoo | ZESSE SESE SSEGT GEIS SSTSS SASH SHHSs sagss g

-(otng) pixo snoring | SSHSS BSHSS SSTSS BSSRS SESS SEGSS SARE BSSRR S

 

 
CHAPTER XXIV.

LIPINS. NEUTRAL FATS AND OILS. PHOSPHOLIPINS.
STEROLS.

Neutral fats mixed with other lipins may be obtained from plant
and animal tissues both by physical and chemical means. The physical
means are by warming and pressing. In this way oils and fats are ob-
tained from nuts, seeds and from animal tissues containing a large
amount of neutral fat, as in fatty tissue. The chemical means are by
extraction with lipin solvents such as ether, carbon tetrachloride, alcohol,
gasoline, benzene, etc. Whichever method is used, the resulting fatty
matter is a mixture and generally contains, in addition to neutral fats
and fatty acids, phospholipins, sterols and lipases. The neutral fats
are separated from the phospholipins most readily by precipitation of
the ether solution by acetone. Soaps and phospholipins are insoluble
in acetone; fats and sterols are soluble in acetone. The separation of
cholesterol from neutral fat cannot be made without saponification of
the latter and the isolation of cholesterol from the soap by ether.

EXERCISE XI. THE PHYSICAL PROPERTIES OF OILS AND FATS AND THE
ACIDITY OF ORDINARY FATTY OILS.

Experiment 39. Solubility—To one drop of olive oil in a test-tube
add about 5 c.c. of the solvent. Allow to act in the cold. If solution is
not evident from the usual physical changes, filter off the clear solvent
and place a few drops on a piece of paper. In case some of the fat has
dissolved, a translucent spot (grease spot) is left on evaporation of the
solvent. Test in this way the solubility in hot and eold 95 per cent.
and 70 per cent. ethyl alcohol; and in cold ether, chloroform, gasoline
or naphtha, carbon tetrachloride, ethyl-acetate, water and acetone.

‘Test the solubility of castor oil in cold alcohol. How does castor oil
differ in its composition from olive oil? What effect would this differ-
ence probably have on its solubility in alcohol, and why?

Experiment 40.—Crystallization of neutral fats.—Dissolve a little
beef tallow, or tristearine if it can be obtained, in about 5 ¢.c. warm
benzene, and allow to cool slowly. Examine under the microscope. Note
the long feathery and needle-shaped crystals.

Experiment 41. Surface tension of neutral oils—Count the num-

ber of drops which will be formed from 1 ce. of pure distilled water
: 904
PRACTICAL WORK AND METHODS 905

when issuing from a perfectly clean 1 c.c. pipette. Now dry the same
pipette by alcohol and ether, fill it accurately to the mark with olive
oil at room temperature and count the number of -drops of olive oil
delivered from the same pipette. For the relation between the number
of drops and surface tension, see page 207. As performed in this way
this method is: only approximately accurate. The lower the surface
tension of the liquid the larger the number of drops. Olive oil has a
lower surface tension than water.

Experiment 42. The surface tension of water is lowered by a
small amount of oil or soap.—After the pipette used in experiment 34
has drained off its oil a very thin layer of oil still remains in it. Without
cleaning it now fill it again to the mark with clean water and again
count the number of drops issuing from the pipette. The number
should be greater than before, for when the surface tension of the water
is reduced, the film of water on the surface which supports the drop is
no longer able to sustain as great a weight as before, and the drop falls
when its weight is less and it is smaller. Repeat with slightly soapy
water. Soap lowers the surface tension even more than oil.

Experiment 43. The surface tension of water is lowered by a very
small amount of soap or oil so that movements due to surface tension
may be checked.—Place in a perfectly clean beaker, that is a beaker
which has been cleaned with soap and water, then by alcohol and ether,
and then by cleaning fluid and finally thoroughly washed with distilled
water, clean distilled water until half full. Sprinkle a few small pieces
of camphor on the surface. If the water is perfectly clean the camphor
will dart here and there on the surface. While it is moving, rub a
glass rod on the side of. the nostril and then touch it to the surface of
the water. The fat of the skin is sufficient, usually, to add in this way
enough oil to the surface of the water to make the camphor at once come
to rest. If the camphor has already come to rest it will be observed
usually that when the rod touches the surface of the water the camphor
fragments move away from the rod in all directions. This is owing to
the stretching of the surface due to the lowering of its tension at the
point where the oil touches it. To get a clean surface of water in a
laboratory is very difficult.

_ Experiment 44. Free acids in oils—Most commercial oils and fats
undergo in the light and moisture a partial decomposition (rancidity)
so that they have an acid reaction. Moreover, lipases are present in
many oils which in the presence of moisture decompose the oil, setting
free some fatty acid. To get a neutral oil precautions must be taken
to separate the free acid thus formed. The acid is determined as follows:

To about 25 ¢.c. 95 per cent. aleohol in a 100-150 c.c. flask add about
% «ec. phenolphthalein solution. Heat to boiling on a steam bath and
906 PHYSIOLOGICAL CHEMISTRY

gradually add from a burette N/10 NaOH until a faint pink coloration
remains. Now add 5 cc. olive oil by means of a pipette. Again heat
to boiling, stopper the flask and shake gently; the alkaline alcohol be-
comes colorless. Continue the addition of N/10 NaOH and shaking,
until after a vigorous shaking the pink color in the alcohol layer just
remains. Record the c.c. of NaOH required. Allow the two layers to
separate in a large test-tube. Draw off, by means of a pipette, the oil
layer for use as neutral olive oil in a subsequent experiment, and dis-
card the alcoholic layer. Calculate the per cent. of acid in the fat,
assuming the acid measured to be oleic acid and the specific gravity of
the oil to be 0.90 at room temperature. All fats, even when freshly
prepared, contain titratable amounts of acid. The amount of titratable
acid increases slightly with development of rancidity. It is very doubt-
ful, however, whether the acidities in the fresh and rancid fats are due
to the same acids.

Exercise XII. THE SAPONIFICATION OF FAT.

To show the composition of the fat the glycerol and fatty acid are
separated by saponification with alkali and both are isolated. Saponifica-
tion in animals and plants is not produced by alkali but by substances
of an unknown nature called lipases. The action of the steapsin, a
typical lipase, is shown in experiment 138.

Experiment 45. Saponification of fat—Weigh off 10 grams mutton
tallow, render it by heating gently and pouring off the melted fat and
transfer to a 500 c.c. flask, add 8 e.c. of 40 per cent. NaOH solution and
50 ec. aleohol. Fit with a reflux air condenser (a glass tube about 8 mm.
diameter and about 2 feet long), boil gently for an hour on the water
bath and then evaporate to about 50 ¢.c. in a porcelain dish. Add
100 c.c. hot water and again evaporate to about 50 ec. to get rid of
alcohol. Next dissolve completely in about 500 c.c. water. The saponifi-
cation has now taken place. To separate the soap and glycerol formed
proceed with the following experiment.

Experiment 46. Separation of the soap by salting out.—Take 5 c.c.
of the above solution, add 50 cc. of water and saturate with NaCl.
Filter off the precipitated soap. Dissolve in about 100 c.c. of water
and test a part of the solution with a few drops of CaCl,, MgSO, and
Pb acetate. The calcium, magnesium and lead soaps, being insoluble,
are precipitated. Substances which lower the surface tension of water, ,
such as soaps, lipins, ether, gases, etc., may be salted out by the addition
of some neutral salt, since the latter increases the surface tension and
drives the former into the surface film.

Experiment 47. Separation of the fatty acid—To the remaining
PRACTICAL WORK AND METHODS 907

495 c.c. solution above, add concentrated HCl until the water solution
is distinctly acid to litmus paper and a sharp separation of fatty acid
as an oil occurs on heating on the steam bath. After heating set aside
to cool, or draw off the lower layer by means of a separatory funnel,
saving both it and the upper layer of fatty acid. Wash the fatty acids
twice with 100 c.c. hot water. Then remove the fatty acid layer. Dis-
solve a little in hot alcohol and show that it will neutralize NaOH and
is acid to phenolphthalein. See experiment 44.

Experiment 48. Separation of glycerol.—Filter the aqueous layer
from 47 above and neutralize the filtrate with 10 per cent. NaOH solu-
tion. Evaporate on the steam bath. Grind the residue, salt and glycerol,
to a fine powder; moisten with alcohol, and again evaporate to dryness.
This is done to remove water as completely as possible. Next add_
25-50 ¢.e. alcohol, heat to boiling on the steam bath, filter and treat the
undissolved residue once more with 25-50 c.c. alcohol in the same way.
Combine the filtrates and evaporate to a syrup in a small dish on the
water bath. The residue should be syrupy glycerol. It should have
a sweet taste and give the acrolein test, as in experiment 49.

Experiment 49. Acrolein test for glycerol—To about 2 drops
glycerol in a dry test-tube add an equal quantity of solid KHSO, or
P,O;. Heat carefully and gradually, without charring, over a burner
and note the penetrating, sharp odor of the vapor given off. This char-
acteristic odor is due to acrolein which is formed by the dehydrating
action of P,O, and heat on the glycerol. Perform the same test on the
original fat and on. the fatty acids obtained in 47.

Exercise XIII. MeEtruHops OF IDENTIFICATION OF FATS.

One of the best ways of determining the identity of an oil or fat is
to determine its power of absorbing iodine. This is an indication of its
degree of saturation. See page 70.

Experiment 50. Absorption of iodine by unsaturated fatty acids.—
Dissolve a small amount (a couple of drops of oily acids) of the
fatty acids in chloroform, then add thereto 2-3 drops of Hubl’s iodine
solution and shake. The iodine will be absorbed by the fatty acid and
the solution decolorized if unsaturated acids are present. Try this
test also on the original fat as well as the fatty acid and make
a control, shaking the chloroform and iodine alone. The iodine
unites with the unsaturated carbons of the fatty acids to make the
iodized fatty acids. For the composition of V. Hubl’s solution see next
experiment.

Experiment 51. Determination of the iodine number.—The iodine
number is the per cent. of iodine absorbed by the fat. That is, it is the
908 PHYSIOLOGICAL CHEMISTRY

number of centigrams of iodine absorbed by 1 gram of fat. Neutral fats
of saturated fatty acids absorb no iodine; but neutral fats, which contain
some unsaturated acids, will absorb iodine at the unsaturated bond, two
atoms of iodine being absorbed to each double bond.

The solutions needed in this determination are made up and titrated
by the instructors and the titration values of the thiosulphate in mgs.
of iodine and of the Wijs solution (iodine dissolved in iodine trichloride,
ICl,) are marked on the bottles. In accurate work the oil or fat must
be weighed, but in the class work it may be measured by a pipette and
the weight calculated from the specific gravity. (See page 66.)

Weigh a dry 100 cc. flask. Add to it about 0.28 ¢e.c. of olive oil
and weigh again carefully so as to obtain the amount of oil used. Dis-
solve the oil in 10 ec. of carbon tetrachloride and add after solution
by a volumetric pipette 25 e¢.c. of Wijs’ iodine solution, shake to mix,
stopper the flask and allow it to stand in the dark for from one to two
hours. Then pour the contents of the flask without loss into a 750 e.e.
Erlenmeyer flask, adding to the small flask 10 cc. of KI solution to
wash out the iodine left in it and wash the potassium iodide quantita-
tively into the 750 c.c. flask. The total volume of fluid should be about
300 ec. Now titrate the iodine solution with the standard thiosulphate
solution, using starch as indicator. Do not add the starch until the
amount of iodine remaining is sufficient to give only a light-brown color.
The calculation should be recorded as follows:

Amount of iodine in 25 c.c. Wijs solution .......... mgs.

Thiosulphate used in the titration .......... CC a3 aes mgs. I not absorbed.
1 cc. thiosulphate equals .......... mgs. iodine.

Mgs. of iodine absorbed by the fat—.......... Seas cutencuelers cgs.

Weight of fat taken—.......... grams.

Iodine number of fat=.......... egs. of iodine absorbed ~ wt. of fat in grams.

' Standard thiosulphate solution for the iodine value of fats. Dis-
.solve 48 grams of sodium thiosulphate in 2 liters of water. Allow to
stand about 24 hours and then standardize it according to Vollhard as
follows: Dissolve 3.8657 grams accurately weighed of potassium bichro-
mate in water and make up to 1 liter. 10 ec. of 10 per cent. KI solu-
tion, 5 ec. strong HCl and exactly 20 ce. of the bichromate solution
are mixed in a 750 c.c. Erlenmeyer flask and then diluted with about
300 ¢.c of water. This amount of bichromate solution when acidified
with HCl liberates exactly 0.2 gram of iodine from the KI. Now deter-
mine by titration how many e.c. of the thiosulphate solution it takes
to reduce exactly this amount of iodine. To do this run in from a
burette carefully some of the thiosulphate until the iodide solution is
only faintly yellow from the iodine, and then add a little starch solu-
tion.’ Now carefully add the thicsulphate until the blue color of the
PRACTICAL WORK AND METHODS -, 909

starch is just lost. The number of ¢.c. of thiosulphate equivalent to
0.2 gram iodine is thus determined. From this, by division, the amount
of iodine equivalent to 1 ¢.c. thiosulphate may be calculated.

Iodine solution of Wijs. This is an iodine monochloride solution
made as follows: Weigh into a 300 cc. flask 9.4 grams of iodine tri-
chloride and then pour in about 200 c.c. of glacial acetic acid. Fit the
flask with a cork carrying a CaCl, tube and heat on the water bath
till all is dissolved. Rub 7.2 grams iodine to a fine powder in a mortar
and wash it with glacial acetic acid into a similar flask and heat this in
the same manner to solution. Pour the contents of both flasks into a
stoppered graduated liter flask, add glacial acetic acid in portions to the
iodine and reheat until all the iodine has been dissolved and carried to
the liter flask. Stopper, allow to cool, make up exactly to 1 liter with
acetic acid and titrate on the following day by the standard thiosul-
phate solution. To do this measure exactly with a pipette 10 or 20 cc. of
the solution into a large Erlenmeyer flask, add 10 ¢c.c. 10 per cent. KI
solution and dilute with 200 or 400 ¢.c. water. Then run in from a
burette the thiosulphate until pale yellow, then add some starch, and
add thiosulphate carefully to the discharge of the blue color. The
strength of the iodine chloride solution may alter a little in the first
week, but thereafter, Leathes says, it remains very nearly constant for
some weeks. Restandardize from time to time. The glacial acetic acid
should be twice recrystallized and the mother liquor poured off from
the crystals before use. Care must be taken to prevent the absorption
of water.

Lewkowitsch modifies Wijs solution by adding the iodine and iodine
trichloride in molecular proportions : 7.9 grams ICI, and 8.7 grams I,.

Von Hubl’s iodine solution. Dissolve 25 grams of iodine in 500 «.c.
95 per cent. aleohol mixed the day before being used with an equal
‘quantity of a solution of 30 grams mercuric chloride in 500 e.c. of 96
per cent. alcohol. Waller recommends adding 50 cc. strong HCl to
1 liter of the mixed iodine and mercuric chloride solution. This keeps
its titer better as it prevents the reaction of the ICl with water (IC]+
H,O=IOH+HCl). von Hubl’s solution gives lower values for the
iodine number in linseed oil and cholesterol than Wijs.

Experiment 52. Determination of the saponification number.—
Obtain a sample of oil, the saponification number of which is known to the
instructor, from the storeroom. Read under general directions as to the
use of pipettes. Accurately measure out 5 ¢.c. of the oil with a clean,
dry 5 ce. graduated pipette and transfer to a clean, dry 250 ¢.c. Erlen-
meyer flask. (If facilities for weighing exist it is better to weigh about
5 grams ‘of oil instead of measuring it. This can be done by placing a
910 PHYSIOLOGICAL CHEMISTRY

small amount of oil with a medicine dropper in a small flask, weighing
all accurately, then transferring about 5 c.c. of oil to the Erlenmeyer
flask and reweighing the medicine dropper and flask. The difference in
the weighings gives the weight of oil taken.) Now add 50 cc. of the
alcoholic N/2 KOH solution (N/2 KOH in 90 per cent. alcohol), this
also to be measured accurately with a dry 25 c.c. pipette. Into another
flask of the same kind also measure accurately 50 c.c. of the alcoholic
KOH solution with the same 25 c.c. pipette. Fit each flask with a reflux
air condenser and boil gently over a wire gauze for 30 minutes; next
cool, add about 1 ¢.c. phenolphthalein solution and titrate still warm
with N/2 H,SO,. Titrate the blank flask in the same way.

By saponification number is meant the number of mgs. of KOH
necessary to neutralize the fatty acids in 1 gram of fat, phenolphthalein
being the indicator. Record your measurements and calculations in some
such way as the following: (Specific gr. of oil may be obtained from
instructor.)

Oil taken 5 cc. sp. gr. =... eee eee Saeed Grams oil.

Burettte readings in titrating the blank ..........

Burette readings in titrating the saponified oil ..........

e.c. N/2 H,SO, equivalent to the KOH used in the saponification ..........
c.c.N/2 KOH required to saponify .......... grams of oi] ..........
1 ce. N/2 KOH contains .......... grams of KOH

Saponification number of the oil=..........

Experiment 53. The emulsification of fats by soaps. Cleansing
action of soaps.—In this experiment, use the oil layer, described above,
experiment 44; before using, however, heat on the water bath until it
has become clear. For the rancid oil use the olive oil on the side shelf.
Prepare tubes as follows:

 

Tube Oil added Other addition State of emulsion after 30 minutes

 

3 drops rancid |5 c.c. H,O

oe neutral cE de

“rancid [5 cc. H,O + 0.3 cc. N/10 NaOH
ct neutral ec 6 “ ae 17 “ “

“« rancid |5 ¢.c. 0.05% Soap Sol.

“ce neutral cE ty ris “

moAndan#n
wwww w

 

 

Shake all tubes equally thoroughly. Note the appearance immediately
after shaking and 30 minutes later. The fat is distributed through the
liquid in tubes containing soap.

Write a brief explanation of the manner in which soaps emulsify
fats. See page 222.
PRACTICAL WORK AND METHODS 911

EXERCISE XIV. PREPARATION AND PROPERTIES OF PHOSPHOLIPINS.
QLYCOLIPINS AND STEROLS.

Experiment 54. Preparation of a glycolipin, cerebrin. (Phrenosin
and kerasin mixture.)—Obtain 200 grams finely chopped calves’, pigs’
or sheep brains. Transfer to a 10-12-inch porcelain dish and gradually
add, while stirring well, 200 c.c. 95 per cent. aleohol. Next transfer the
mixture to a 750-1,000 e.c. flask; add 200 e.c. 95 per cent. alcohol, mix,
fit flask with a reflux condenser, heat on the steam bath for %4 hour,
agitating the flask from time to time. Filter hot, through a dry, creased
filter paper, into a dry flask, removing as much of the liquid as possible
from the undissolved tissue. Again add 250 ¢.c. 95 per cent. alcohol to
the residue which has been returned to the flask, boil on the steam bath
20 minutes and filter into the same filtrate as before. Return the residue
to the flask and extract again with 250 c.c. fresh alcohol. Combine the
three filtrate and set aside in a covered beaker in a cool place until the
next day to permit of crystallization. Save the residue, return it to
the flask and add 250 c.c. ether, allow to extract at room temperature
for several hours, filter off the ether and extract once more with the
same amount of ether. Save the residue for experiment 75, p. 933.
Unite and save ether filtrates. The hot alcohol takes out cholesterol,
lecithin, and the cerebrosides, besides other substances. The ether will
take out an additional amount of cephalin.

Separation of cerebrin. The next day filter off the cold alcohol from
the white crystalline precipitate (protagon) which has separated out,
using the Buchner suction funnel. The filter paper in the funnel is cut
just large enough to cover the bottom and must be first moistened with
water, suction applied and then the water removed from the paper
with acetone or alcohol. The filtrate contains phospholipin and may be
united with the ether extract. The white precipitate consists of some
phospholipin, cerebrin and some cholesterol and other substances. It
is sometimes called impure protagon. Transfer the white material to
a small beaker and dissolve in 100 ¢.c. hot methyl or ethyl alcohol. Filter
hot. Allow the hot, clear solution to cool slowly and thoroughly. Im-
pure cerebrin and cholesterol crystallize out. Filter off, adding the
filtrate to the ether extract above. Wash the crystals in ether. Choles-
terol dissolves in ether and will go into solution, while cerebrin is insolu-
ble. Add the ether washings to the ether extract. Recrystallize the
cerebrin crystals once more from hot methyl alcohol. The result is an
impure phrenosin and kerasin (cerebroside). To remove the last traces
of phospholipin it would be necessary to recrystallize in hot glacial acetic
acid. The following experiments can be tried with the cerebrin thus
obtained.
912 PHYSIOLOGICAL CHEMISTRY

Experiment 55. Hydrolysis of cerebrosides (glycolipin).—The
object of this experiment is to show that the glycolipin from brain is
hydrolized by acid and yields galactose. For composition of phrenosin
see page 579.

To about 0.5 gram cerebrin in a 100-150 cc. flask add about 50 cc.
distilled water and 0.5 ¢.c. con. H,SO, (measured by a 5 c.c. graduated
cylinder). Then proceed with the hydrolysis as in the hydrolysis of a
polysaccharide, experiment 35. Do fatty acids form on the surface?
Remove the sulphuric acid in the same way. Test a portion of the clear
water solution by Fehling’s solution in the usual way. A reduction
should be obtained. ‘Test another small portion for phosphoric acid.
This should be negative. Evaporate the remainder to about 5 c.c. and.
proceed with the mucie-acid test for galactose as given in experiment 27.

Experiment 56. Preparation of phospholipins (phosphatides).—
Concentrate the united ether extracts and cold alhoholic filtrates from
experiment 54 in a porcelain dish on the water bath. Evaporate to the
consistence of a salve or paste. Avoid overheating or baking. Add
25 «ec. 95 per cent. alcohol, moistening all the solid substance in the
dish therewith. Try to redissolve the material as much as possible so
as to cover less space in the dish. Again evaporate as above to a thick
syrup. Again take up in 95 per cent. alechol and repeat the evapora-
tion. This is to remove the water. Allow to cool and immediately on
cooling extract the residue in the dish three times, using 20 e.c. ether
each time. Decant the ether off each time into a large test-tube. Be
very careful to avoid getting water into the material in the dish and
be sure to break up the lumps as much as possible while treating with
ether. The milky ether extracts, combined in a large test-tube, stoppered,
are set aside in a cool place to settle. After a day or two the clear super-
natant liquid is poured into a 300 c.c. beaker and then, while stirring
well, three volumes of anhydrous acetone added. Be sure to use acetone
which has not been allowed to absorb moisture by standing exposed to
the air or by being measured with vessels which contain water or alcohol.
The precipitated phospholipin (lecithin, cephalin and various myelins)
is allowed to settle out and the supernatant acetone-ether solution poured.
into a 10-12-inch porcelain dish and allowed to evaporate spontaneously.
forthe cholesterol preparation. (Experiment 58). The phospholipin.
precipitate is now. kneaded with a horn spoon or a glass rod until all the
particles. are collected.into one mass. Knead this mass well with two
15:.c.c. portions of acetone, adding the acetone washings to the acetoné-
ether solution, evaporating for cholesterol. The phosphatide prepara-
tion may be: kept on a watch glass in a desiccator. Examine a portion
of:it; ta determine. its composition as follows:

Experiment 57. Tests for glycerol, fatty acids and organic phos:
PRACTICAL WORK AND METHODS 913

-phoric acid in phospholipin.—Glycerol. Take a‘portion of phosphatide
about equal in bulk to a sphere with a diameter of one-eighth inch, place
in dry test-tube and cover with a little P,O, and heat gently in the
flame, smelling from time to time as soon as white fumes come off from
the fused phosphatide. Note the irritating, penetrating odor of acrolein,
showing the presence of glycerol. Fatty acid. Take another portion
of phospholipin of the same amount, dissolve this in a test-tube in about
6 c.c. 95 per cent. alcohol. Heat to boiling in a steam bath and add an
equal quantity of concentrated HCl and heat on the steam bath for 20
minutes. Transfer to a 100 cc. beaker and evaporate until the alcohol
has been boiled off. Then shake the residue in 20 c.c. ‘ether in a separa-
tory funnel, separate the ether layer from the other and evaporate both
separately on the steam bath until the ether and HCl have been removed.

Note the character of the material, fatty acid, left from the ether.
Dissolve this in the least quantity of dilute NaOH. Acidify again and
heat to boiling. Fatty acids separate out.

Phosphoric acid. The residue from the evaporation of the aqueous
solution above should be tested for phosphoric acid. It is dissolved in
a little water, transferred to a test-tube, most of the water evaporated
and 10 drops of concentrated H,SO, added to destroy organic matter.
Heat in the flame until charred, then add one drop of concentrated
HNO, and again heat until charred. Be very careful to hold the mouth
of the test-tube directed away from you when you add the nitric acid; in
applying this test to fatty substances or to substances containing
glycerol, alcohol or ether one must be very cautious, as a violent reaction
as likely to take place at the beginning of digestion. Repeat the addition
of one drop of nitric acid and the heating until the material no longer
chars on heating sufficiently to give off white sulphuric-acid fumes.
Allow to cool, then carefully add about 5 e.c. distilled water and strong
NH,OH (No. 5) until faintly alkaline. Now again add just enough
concentrated NHO, (one drop at a time) to render acid. To this solu-
tion warmed to about 70° C. add a few c.c. ammonium molybdate solu-
tion (No. 6). Agitate with a stirring rod. If phosphorus is present in
the original substance it will be precipitated here as the yellow, am-
monium phosphomolybdate.

Experiment 58. Preparation of cholesterol—To the residue left
on evaporating the acetone-ether solution decanted above, add 25 cc.
95 per cent. alcohol and 5 ¢.c. strong NaOH solution (No. 86). Mix well.
transfer to a 150 «ec. Erlenmeyer flask, using 10 ¢.c. more 95 per cent.
alcohol to rinse the dish with. Fit the flask with a reflux air condenser
and boil on the water bath for 30 minutes. Then evaporate in a 10 cm.
dish to remove most of the alcohol; neutralize to litmus by gradually
adding, while stirring, concentrated HCl. Cool and extract with three
914 PHYSIOLOGICAL CHEMISTRY

20 «ec. portions of ether. Allow the combined clear ether extracts to
evaporate spontaneously to a small volume in a 100 cc. beaker. The
erystals of cholesterol may be recrystallized from 95 per cent. alcohol.
They should be colorless plates.

Experiment 59. Cholesterol reactions.—(a) I0DINE-SULPHURIC-
ACID TEST. Prepare a mixture of 5 c.c. concentrated H,SO, + 1 ee.
water. Cool and add a few small white crystals of cholesterol. Allow
to act at room temperature until the edges of the crystals are colored
pink. Then add 3 drops of a dilute iodine solution (1 vol. No. 45 + 10
vol. water), agitate and note the colors developing. Gradually add 5 to
10 drops more of the same iodine solution, agitating after each addition.
Observe under microscope. The crystals are colored on the edges.

(b) SALKOWSKI’S REACTION. Dissolve a small crystal of cholesterol
in about 1 ¢.. chloroform in a dry test-tube. To this solution at room
temperature add an equal volume concentrated H,SO,. Do not agitate at
once, but note the red color of the upper chloroform layer and the
fluorescence of the sulphuric-acid layer after standing a minute or so.
Agitate to mix the two layers and set aside. Note the intense cherry-red
coloration of the upper layer on separating into two layers.

(e) LIEBERMANN-BURCHARD REACTION. In a dry test-tube dissolve a
few small crystals of cholesterol in about 2 ¢.c. chloroform. Next add
15 drops acetic anhydride, mix and then add concentrated H,SO,, drop
by drop, 2-5 drops. Shake the contents of the tube. Allow to act
further without heating and note the changes in color. A deep blue
color develops.
CHAPTER XXV.
THE PROTEINS.

EXercisE XVI. ELEMENTARY COMPOSITION AND TESTS FOR DETECTION.

Experiment 60. Elementary composition of the proteins.—Care-
fully heat a small amount of dried casein or other dried protein in a
dry test-tube until the characteristic odor of burning hair is developed.
Note the drops of water on the sides of the tube. The presence of this
water, if the protein has been thoroughly dried at 100°, means that
proteins contain hydrogen. The charred black mass shows the presence
of carbon. To show the presence of nitrogen now add to another por-
tion of casein about 1 «ec. of a mixture of equal parts of powdered
iron or iron filings and dry sodium hydrate. Heat carefully in a
crucible until the material has well charred and fused. Now allow to
cool and then extract with 10 cc. distilled water. Filter through a
small filter from the iron and carbon. By heating with iron and by dry
distillation of proteins, hydrocyanic acid is formed. Ferrous salts unite
with the sodium cyanide to make ferrocyanide. Now add to the solution
concentrated HCl until just acid. If the dark-blue ferric-ferrocyanide
is not formed at once, add a drop of ferric chloride when it will appear.
The presence of nitrogen in proteins is shown by the formation of the
ferrocyanide. Many proteins contain also sulphur in an unoxidized
form, which may be detected by the formation of lead sulphide as in

experiment 61.
Experiment 61. Methods of detecting proteins in solutions or

solids. Color reactions of the proteins—For these experiments take
portions of blood serum which has been diluted 10 times with water; or
egg white, diluted with five volumes of water; or a 2 per cent. solution
of commercial peptone.

*A. Biuret (Piotrowskw’s) reaction. To 2 ¢.c. portions of the solu-
tions of blood albumin, egg white and commercial peptone add 2 e.c.
10 per cent. NaOH and then one drop of a 0.5 per cent. CuSO, solution.
Mix well and add more CuSO,, drop by drop, until a distinct pink or
violet color is developed or until a precipitate of Cu(OH), is formed.
If the rose or violet color which constitutes the biuret reaction does not

come at once, allow to stand 15-20 minutes.
915
916 PHYSIOLOGICAL CHEMISTRY

This reaction is interfered with if much ammonium salt is present.
In its presence it is necessary to add a large amount of strong NaOH
to get rid of the influence of the NH,OH. If NaOH is added to a solu-
tion containing an ammonium salt, such as (NH,),S0,, sodium sulphate
will be formed and ammonium hydrate. The ammonium hydrate is a
very weak base, particularly in the presence of ammonium salts; it
combines with the cupric ions, removing thém from the solution, so
that it is necessary to add NaOH until nearly the whole of the am-
monium sulphate or other ammonium salt has been decomposed. To
show this add to the peptone test above, drop by drop, a saturated solu-
tion of ammonium sulphate until the blue color of the ammonium cupric
salt replaces the pink color, due to the biuret reaction. Now add 40 per
cent. NaOH (No. 86) until the pink color returns.

Read on page 144.

Repeat this test with biuret. Make biuret from urea as follows:

* A. 1. Formation of biuret and cyanuric acid from urea. Care-
fully heat over a low flame a layer of urea about one-eighth inch deep
in a dry test-tube. The urea melts, then the liquid boils and finally
if the heating is continued a white solid remains on the sides and bottom
of the tube. During the heating note the odor of the vapor (ammonia)
escaping from the tube. After cooling add about 5 cc. of water, warm
slightly and decant from the undissolved residue into another tube and
then make the biuret test in the aqueous solution. A pink color should
be obtained. Biuret is formed according to the following reaction:

NH,—CO—NH, + NH,—CO—NH 2. N H,—CO—NH—CO—NH, +NH,
Urea. Urea. Biuret.

*B. Xanthoproteic reaction (see page 147). This reaction, the
yellow protein reaction, is give both by solid and dissolved protein, but
is most delicate when applied to solid protein. To a small amount of
the solid protein or its. solution add about 1 ¢@e. concentrated nitric
acid and boil until dissolved. Cool. Note the lemon-yellow color which
develops in case protein is present containing a benzene nucleus. Cool
the solution and add strong NaOH solution until the solution is slightly
alkaline. The color should change to a deeper orange if the test is posi-
tive. See if solid gelatin, casein, solutions of the proteins, a few drops
of 2 per cent. phenol solution and a little salicylic acid give the same
reaction. This reaction is not given by proteins which lack the aromatic
nucleus. Explain the reaction (see page 148). Tyrosine; phenylalanine
and tryptophane in the protein molecule give this reaction.

*C. Millon’s reaction. For the composition of Millon’s reagent
see page 147. To apply this test to solutions add a few drops of the
PRACTICAL WORK AND METHODS 917°

reagent to four or five c.c. of the solution and then heat to boiling.
Generally there is first formed a white precipitate which turns pink or
red slowly on heating. In case a pink or red color does not develop it
is best to add a few more drops of the reagent and again heat to boiling
and allow to stand. The amount of reagent to be added varies con-
siderably, depending on the concentration and nature of the solution.
Try this reaction on 2 c.c. of the 2 per cent. gelatine and casein solutions,
and on solid gelatin and casein and on the albumin solutions. Test
also 2 drops of 2 per cent. phenol solution added to 3 ¢.c. of water and
test also a few crystals of resorcine, phloroglucine, thymol, vanilline
and tyrosine. For an explanation of the reaction see page 147.
Tyrosine is the only amino-acid in protein which gives this reaction.

a. The reaction is interfered with by the presence of sodium
chloride. Repeat the phenol or any of the other reactions which have
been positive, but add to the solution some solid NaCl first. It will be
found necessary to add. much more Millon’s solution before a red color
is developed if NaCl is present.

*D. Tryptophane reaction. (Adamkiewicz reaction.) To 5 ee.
glacial acetic acid add 3 to 10 drops of the 2 per cent. protein solution,
mix well and pour concentrated H,SO, down the side of the tube so
that it forms a layer underneath the acetic acid. A violet ring should
develop at the plane of contact if the test is positive. This generally
develops after standing a few minutes. After it appears, or if the test
is negative, agitate the tube so as to mix the sulphuric acid and acetic.
The whole solution may become violet.

Try this test on gelatin and casein in particular. Gelatin is negative.
If the test is negative, it is well to repeat it by dissolving some of the
dry protein in glacial acetic acid by warming, then cooling and adding
the H,SO,. A violet ring or a violet solution should be obtained on
mixing if the test is positive. If tryptophane and indole can be pro-
cured, repeat this test on small quantities of these substances or their
solutions. For explanation of the reaction see page 148. What proteins
will not give this reaction?

*E. Glyoxylic acid reaction (Hopkins-Cole reaction). To about 2
e.c. of glyoxylic acid reagent (No. 38) add 2 «.c. of the 2 per cent. pro-
tein solutions, or of the protein solutions to be tested, mix and pour
concentrated H,SO, down the side of the tube. A purple-violet ring at
the plane of contact is a positive reaction. Repeat, mixing the contents
of the tube. This reaction does not always come immediately, so if nega-
tive allow it to stand 15 minutes, If still negative, repeat, using a little
of the solid protein matter dissolved in glyoxylic acid and sulphuric.

The glyoxylic-acid reagent is made in the following way:

In a 500 cc. flask place 10 grams powdered magnesium, cover with
918 PHYSIOLOGICAL CHEMISTRY

distilled water and slowly add 250 cc. of saturated oxalic-acid solution,
cooling from time to time under the tap. Filter off the magnesium
oxalate, acidify slightly with acetic acid, dilute to one liter and keep
in a stoppered bottle, with a little chloroform added. The solution
contains oxalic acid and glyoxylic acid, CHO.COOH.

This reaction, like the preceding, is for tryptophane. See page 148.

Cole states that this reaction is interfered with by the presence of
chlorides in excess, and in the presence of chlorates, nitrates and nitrites.
It is also important to use pure sulphuric acid.

*F, Acree-Rosenheim formaldehyde reaction. To 2 ¢.c. of the 2
per cent. solution add 3 drops of formaldehyde solution 1:5,000 (No.
33). Mix and pour concentrated sulphuric acid down the side of the
tube, as in the preceding. Note the purple ring which forms after
standing about 5 minutes if the test is positive. This is also a trypto-
phane reaction. For explanation see page 149. A trace of iron is
necessary.

*@. Liebermann’s reaction. This test can be made in two forms..
Boil the solid protein with 5 c.c. concentrated HCl for about a minute
with a few drops of dilute saccharose solution. A violet color develops
if the test is positive. Test in this way dry egg albumin, casein and
gelatin.

In the other way, treat the same proteins by boiling with alcohol and
ether in the water bath first, pour off the alcohol and ether before boil-
ing with concentrated HCl and do not add the saccharose. The solid
protein often takes a beautiful blue coloration if it does not dissolve,
and the solution becomes violet if it does dissolve. For an explanation
of these reactions read page 149. In the first case the aldehyde is sup-
plied by the decomposition of the saccharose; in the latter case it is
present already in the alcohol or ether or both, so that the addition of
glyoxylic acid or formaldehyde is unnecessary. Sometimes carbohydrate
is already present in the protein molecule and it is only necessary to
boil with hydrochloric acid to develop the violet color.

*H. Ehrlich’s p-dimethyl-ammo-benzaldehyde reaction. In place
of formaldehyde, or glyoxylic acid or the aldehyde developed from the
carbohydrates by the action of strong acids, we may use also aromatic
aldehydes for the detection of tryptophane. Among these the p-dimethyl-

amino-benzaldehyde is often used. This reagent has the formula:
C—CHO

un
HO CH

nin

\ 7
Cc

kicu,),
PRACTICAL WORK AND METHODS 919

To 1 c.c. of the protein solution add an equal volume of concentrated
HCl and boil for one-half minute; note the color developed, if any
(Liebermann reaction). Then add two drops of the 5 per cent. solution
of p-dimethyl-amino-benzaldehyde in 10 per cent. H,SO, (No. 59), mix
and again note the color. A red to violet color develops in case trypto-
phane or other indole derivatives are present. A few drops of 0.5 per
cent. NaNO, (No. 87) solution added now changes the color to a blue.
Another method of making this test is described on page 988. This
reaction is also used for the detection of indole substances in the urine.

I. -Ehrlich’s diazo reaction. Diazo-benzene-sulphonic acid. This
is a histidine and tyrosine reaction. The formula of diazo-benzene-
sulphonic acid is C,H,N,SO,H. Take about 1 ec. of 0.5 per cent.
sulphanilic-acid solution in 2 per cent. HCl (No. 91), add an equal
volume of 0.5 per cent. NaNO, (No. 87) solution. Mix well and after
about one-half minute add 1 ec. of the 1 per cent. protein solution.
Again mix and then add enough NH,OH or Na,CO, to make the mixture
distinctly alkaline. Histidine gives a red to orange color and tryosine
gives an orange color, but less intense. Tyrosine when converted into the
benzoyl derivative no longer gives the test, but benzoyl histidine does (K.
Inouye, Zetts. f. physiol. Chem., 83, 1913, p. 79). Carry out this test
with gelatin and casein solutions, with distilled water and with tyrosine
and histidine, if they can be provided by the instructor.

* J. Molisch reaction. See the directions in experiment 24. Try
this test on solutions of casein, egg albumin, blood proteins, gelatin and
Witte’s peptone. Note which are positive. A positive reaction is an
indication of the presence of carbohydrate in the protein molecule or
solution.

*K. Reduced sulphur reaction. To the protein solution, and for
this test it is better to take a fairly concentrated solution of egg white,
add four volumes of 10 per cent. NaOH and boil for a few moments,
1-2 minutes. Then add a few dops, 3-10, of lead-acetate solution. A
brown color or a black precipitate formed on the addition of Pb acetate
shows the presence of unoxidized sulphur in the molecule. It is split
off as the sulphide and precipitated as sulphide of lead. Test also
gelatin, casein and Witte’s peptone solution by this test. Has casein
sulphur in it? If the instructor can supply some cystine, repeat this
test with a little cystine dissolved in sodium hydrate containing some
lead acetate.

* Ninhydrin (triketohydrindenehydrate) reaction (Ruhemann, Trans.
Chem. Soc., 97, p. 2025, 1910; Abderhalden and Schmidt, Zeit. f. phys.
Chem., 72, p. 837, 1911). Ninhydrin is triketohydrindenehydrate. This
reaction is one of the most sensitive of the protein reactions. It is
positive with proteins, peptones and amino-acids, with the exception of
920. PHYSIOLOGICAL CHEMISTRY

those which have an imino instead of an amino group. One carboxyl
and one @-amino group must be free for protein or peptide to give a
positive test. The reaction is made as follows: 0.1 gr. of the reagent
is dissolved in 30-40 ¢.c. of water. One or two drops of this solution
is added to 1 ¢.c. of the solution to be tested and heated a short time to
boiling. On cooling a more or less intense blue color develops, if the
test is positive. It is necessary that the fluid should be neutral in reac-
tion. If acid, the color is more red-violet and the reaction is retarded
by alkali. See page 150. The solution of ninhydrin does not keep well,
so it is well to make it up in small amounts. It comes in the trade in
0.1 gram vials. In using this test for the presence of amino-acids in
dialyzate Abderhalden recommends that it be made as follows: To 10
e.c. of the dialyzate 0.2 ¢.c. of the 1 per cent. solution of ninhydrin is
added. A boiling stick is then added and the solution boiled for exactly
one minute from the appearance of the first air bubble on the side of
the test-tube. A control should be run at the same time, the control
having no substance in it giving the test. A blue color develops on
cooling if the test is positive (Abderhalden, Abwehrfermente des
tierischen Organismus, Berlin, 1912).

“Exercise XVIII. Meruops OF PRECIPITATING PROTEINS.
HEAT COAGULATION.

.,.The protemms may also be detected in solution by precipitation by
heat, or combination with other substances. Insoluble precipitates are
thus formed. It is often desirable to remove proteins from solution in
‘order that the non-protein constituents may be detected and deter-
mined, .as for example in the examination of blood plasma for sugar,
amino-acids and so on.

The principal precipitants of the proteins are various acids such as
picric, tannic, tungstic, bichromic, etc.; various metals, such as mercury,
silver, platinum, copper and lead; and various colloids such as colloidal
iron, cream of alumina, ete.

‘Experiment 62. Acid precipitants of proteins.—In all of these
tests the reaction of the medium must be acid. To 2 «ec. of a solution
of any protein in a test-tube add a drop of a saturated, aqueous solution
of picric acid. A yellow precipitate of the picrate of protein is thrown
down. This precipitation may be accomplished by adding sodium
picrate solution and then acidifying by acetic acid.

In place of sodium picrate and picric acid we may use sodium
tungstate, sodium bichromate, sodium ferrocyanide, or a phosphomolyb-
date, or phosphotungstate, trichloracetate or metaphosphate. To any
protein solution add a few drops of any of these salts and acidify
PRACTICAL WORK AND METHODS 921

with acetic acid. The protamines alone are precipitated without acidifi-
cation. The free acids precipitate all proteins. The precipitates all
dissolve if the solution is made alkaline but the protein is often changed
by the acid.

Reaction : Na picrate + protein + acetic acid___Na acetate + albumin picrate.

Experiment 63. To precipitate by the metals take any protein so-
lution which is neutral, such as white of egg, peptone, or blood serum
or plasma diluted, add to a few c.c. of the solution a few drops of lead ,
acetate, mercuric chloride, copper sulphate, or other heavy metal salt.
A partial precipitation will often occur if the solutions are very faintly
alkaline, as white of egg normally is, but the precipitation is much more
complete if a distinct alkalinity is had by adding sodium hydrate. Try.
the various metals in this way. The lighter metals such as calcium,
magnesium, ete. do not precipitate.

Reaction: Pb acetate + protein + NaOH Pb proteinate + Na acetate.

 

Experiment 64. Colloidal precipitation of the proteins——To any
protein solution which contains electro-negative proteins, i.e., faintly
alkaline solutions, add a few drops of dialysed colloidal iron, or other
colloidal metal or cream of alumina. A heavy precipitate results
formed of the union of the colloidal iron (or other metal) with the
colloidal protein.

To make colloidal iron, precipitate ferric hydrate in a little alkali,
filter, suspend the precipitate in ferric chloride solution and dialyse
after shaking well. Colloidal ferric hydrate remains in the tube.

In place of electro-positive colloids we may precipitate in an acid
solution, in which the proteins become electro-positive colloids them-
selves, by adding an electro-negative colloid such as colloidal arsenious
sulphide, colloidal silicic acid, fullers’ earth, or other electro-negative
colloid. The precipitate is composed of the united negative and positive
colloids. Protein can be completely precipitated by these means.

Experiment 65. Heat coagulation of the proteins —For the study
of the conditions governing heat coagulation, either egg white diluted
with a double volume of distilled water or blood serum similarly diluted
may be used. To show that not all proteins are coagulated by heat, a
2 per cent. albumose or gelatin or casein solution should be heated under.
similar conditions. Many. of the proteins are denatured by heat. That
is, they become insoluble metaproteins. There are two distinct changes
involved in coagulation: a chemical and a physical. The chemical
change will ‘occur in the absence of salt or electrolytes, but the physical
change, the agglomeration into a coagulum, will only happer if -elec-
trolytes are present. The chemical change occurs best if the solution
922 PHYSIOLOGICAL CHEMISTRY

is very faintly acid, or neutral. The nature of the chemical change
involved is unknown. If the solution of a protein is dialyzed or diluted
with distilled water so that it contains no electrolyte, or very little, then
if it is heated the solution generally becomes slighly opalescent, but
there is no precipitation. If, however, salt: is added to the previously
heated but cooled solution, the protein now precipitates, although it
would not precipitate before heating. The coagulation and precipita-
tion are most complete if both salt and very little acid are present. If
the reaction is very alkaline, no precipitation occurs, even though the
salt be present, but the protein is converted by heating into a soluble
metaprotein. If it is more than very faintly acid, also, the same result
will be obtained. Both of these metaproteins, acid and alkali, will be
precipitated if the solution is made exactly neutral. The dependence of
the coagulation on salts, acids and alkalies is shown in the following
experiments:

*1. Heat to boiling 5 cc. of the undiluted egg albumin and blood
serum, after assuring yourself that they are faintly alkaline to litmus.
They will be found to coagulate, although faintly alkaline.

*2. Repeat, using the diluted serum and egg albumin. It will be
found that the solution coagulates very imperfectly. Now add to the
opalescent tubes some powdered, solid, sodium chloride, about the
amount on a knife point. On standing the proteins are precipitated.
This shows that heating has changed the proteins and made them more
easily precipitated by salt, although it has not coagulated them.

*3. Now take four tubes containing each 5 c.c. of the diluted serum,
or egg white, add to (1) a couple of crystals, about a centigram, of
solid sodium chloride; to (2) add two drops of 10 per cent. acetic acid;
to (3) add two drops of 10 per cent. NaOH; to (4) add a eg. of NaCl
and two drops of 10 per cent. acetic acid; mix contents of each tube;
immerse all four in boiling water and note which coagulate best, and
how the coagulation compares with that in (*2). Number 4 will gen-
erally coagulate most firmly and completely.

After heating and cooling neutralize (3) exactly with acetic acid.
A precipitate of metaprotein, alkali albumin, is now obtained at the
neutral point.

*4. Is water involved in heat coagulation? Place a small amount of
finely divided dry egg albumin into each of two dry test-tubes. To one
add 5 cc. of a 1 per cent. NaCl solution. Immerse both tubes in a bath
of boiling water and heat thus for 10 minutes, agitating frequently.
One has been heated while dry; the other while moist. Allow to cool
and then add 5 cc. of a 1 per cent. NaCl to the tube containing the
dry egg albumin and allow to stand for 10 minutes at room temperature,
with frequent agitation. Next decant the clear liquid from each tube
PRACTICAL WORK AND METHODS 923

or filter if necessary and by the biuret test tried in exactly the same
way on equal volumes of each solution ascertain whether the albumin
heated dry is now equally as insoluble as that heated moist. To another
portion of each solution add 1 drop 10 per cent. acetic acid and heat to
boiling. Do not conclude that prolonged heating of the dry protein will
be equally inactive, as the solubilities of heat-coagulable proteins are
distinctly altered by heating the dry proteins at 100° C. for periods of
one hour or longer.

EXeERcIsE XVIII. MeEtTnHops oF ISOLATING AND PREPARING DIFFERENT
KINDS OF PROTEINS.

Experiment 66. Preparation of simple albumins and globulins.—
For this work we shall use blood serum which contains both these pro-
teins. To obtain blood serum ox blood is collected in pans. It clots and
then the clot shrinks, squeezing out a clear, yellowish-colored liquid
called the serum. This serum has in it two proteins, or groups of pro-
teins, called serum globulin and serum albumin.

Obtain 100 ce. of the serum. Test its reaction to litmus paper and
to phenolphthalein. It is usually alkaline to litmus and acid to phenol-
phthalein. Test its specific gravity by means of a specific gravity bulb.
For this, place the serum in a measuring cylinder and immerse a clean
sp. gravity bulb in the scrum. Read the specific gravity from the neck
of the sp. gravity bulb. Record the results in your notebook for future
reference.

(a). Precipitation of the globulins by dialysis—The serum con-
tains salts (test a little for chlorides and phosphates) and these salts
hold the globulm in solution. A portion of the globulin is precipitated
if these salts are removed. Place 100 cc. of the serum in a parchment
dialyzing tube, examining the tube first to see that it does not leak,
and dialyze the serum for 24 hours against running tap water. At the
end of that time remove the tube, notice if there is in it a deposit of
protein and pour the contents into a clean beaker. A portion of the
protein should now be in suspension in the liquid. This portion is called
euglobulin. Remove this by filtration, through a small folded filter.
Save both the filtrate and the precipitate. After filtration take some
of the precipitate, suspend it in distilled water. It will not dissolve.
Now add to the suspension a crystal of sodium chloride. Both salt and
euglobulin dissolve. This illustrates the fact that globulins are insoluble
in distilled water, but soluble in neutral salts. Test the solution by the
biuret test to make sure that the precipitate was protein in nature,
and boil some after adding 1 drop of acetic acid to see that it is
coagulated.
924 . PHYSIOLOGICAL CHEMISTRY

(b). The precipitation of globulins by salts.—Globulins are not
only insoluble in water; they are more or less completely precipitated by
half saturating their solution with ammonium sulphate, and some are
precipitated by saturation with sodium chloride. Fibrinogen is one of
these, but serum globulin is not thus precipitated by NaCl. The filtrate
from (a), that is the dialyzed serum, still contains some globulin, indeed
the. greater. part, because there are substances in the serum (phospho-
lipins) which hold the globulin in solution in the absence of salt. The
fact that this globulin is there can be shown by salting it out. To show
this, dilute the filtrate with an equal amount of distilled water, and
then add to the diluted serum an equal volume of saturated ammonium
sulphate solution. A precipitate of globulin appears and it is complete
after standing for some hours. The solution may, if necessary, be left
covered with a watch glass until the next morning, but not longer, since
concentration of the sulphate by evaporation will precipitate some of
the albumin. Filter off the precipitate of serum globulin, and wash the
‘precipitate twice with half-saturated (NH,).SO,, saving both precipitate
and filtrate. The filtrate contains serum albumin and is used in (d).
Test the precipitate as follows:

(c). Dissolve the precipitate on the filter with some distilled water.
There is generally enough salt in the precipitate to bring it into solu-
tion. Make a biuret test in a little of the solution. Boil about 3 ce.
‘of the remainder. It should coagulate. Keep the remainder of the solu-
-tion for comparison with the albumin solution.

(d). Preparation of serum albumin.—The filtrate from the half-
saturated, diluted blood serum in (b) contains still some protein, serum
albumin. To separate this, saturate the solution by the addition of
powdered ammonium sulphate. Saturating with ammonium sulphate
in a faintly acid solution removes all proteins from solution, except the
peptones. To the saturated solution add enough 10 per cent. acetic
acid to have the resulting solution contain 1 per cent. acetic acid. In
saturating a solution with a solid substance this substance must be
added in a finely powdered form in small quantities at a time with very
frequent (best continual) stirring or shaking until considerable of the
solid remains undissolved. Each 100 cc. of half-saturated (NH,).S0,
solution will dissolve about 35 grams (NH,),.SO,. Allow to stand for
2 to 24 hours, then filter through a ereased filter and allow to drain
as well as possible. Transfer the precipitate to 50 c.c. distilled water,
neutralize toward litmus by the addition of 10 per cent. Na,CO, solu-
‘tion and then dialyze until free from sulphates. Observe the same
precautions in making the test here as above in the preparation of serum
‘globulin. Remove the solution from the tube to a clean beaker. Test
3 «ce. of the solution by the biuret test and another 8 c.c. heat to boiling.
PRACTICAL WORK AND METHODS 925

If it does not coagulate, take another 8 ¢.c., add to it a little ammonium-
sulphate solution, or a crystal of the solid, and repeat.

(e). Having made the above tests, secure solid serum globulin and
albumin in a coagulated form by precipitating the solutions with alco-
hol. To do this pour each solution into an equal volume of 95 per cent.
ethyl alcohol. If the solutions are very poor in salts, the alcohol does
not readily precipitate the proteins, but a milky solution may result. If
this happens, add to the alcohol 1 c.c. of 10 per cent. acetic acid to 100
c.c of alcohol used. Allow to settle, filter the white precipitate through
a creased filter, or through a small suction filter, drain well and either
dry with suction, using alcohol and ether, or spread the moist precipi-
tate in a thin layer on a watch glass and dry in a desiccator. Test the
solubility of the dry powders in water and dilute salt. They will not
dissolve. They have been coagulated, changed to metaproteins by the
action of the alcohol. Test the two preparations for carbohydrate,
tryptophane, oxyphenyl and unoxidized sulphur groups.

Experiment 67. Preparation of edestin—Grind 25 grams hemp
seed to a fairly fine powder, transfer to a 250-500 c.c. flask and add
200 cc. of a 5 per.cent. NaCl solution, previously heated to 60° C. Do

 

 

 

Fie. 69.—Crystals of edestin. (Osborne.)

not heat above 65° or the edestin will be coagulated. Keep immersed in
a bath at 60-65° C. for 4% to 1 hour with very frequent stirring. Prepare
a creased filter, and just before beginning the filtration moisten the
paper with 5 per cent. NaCl solution heated to 70° C. Filter the ex-
traction until about 100-150 c.c. have been collected. Warm the filtrate
to 60-65° C. and a milky solution should remain. Now cool rapidly by
immersing and agitating in cold tap water or in ice water. Allow the
crystals to settle out and decant the supernatant liquid. Filter off the
926 ' PHYSIOLOGICAL CHEMISTRY

white or grayish solid, redissolve in the smallest volume of 5 per cent.
NaCl solution kept at 60-65° C. and allow to cool very gradually by
keeping the container immersed in a large bath kept at 60-65° C. and
then allow bath and all to cool gradually. The next day, or when
cooled to room temperature, examine the crystals with a microscope (see
figure 69), filter off the main bulk of the crystals and wash with 95 per
cent. aleohol. Spread out on a watch glass and dry in a desiccator.
Test portions of the filtrate by boiling, by adding an equal volume of
saturated (NH,),SO, solution and by adding a few drops 1.0 per cent.
acetic acid. Test a part of your preparation for ‘‘ reduced ”’ sulphur,
for histidine and for tryptophane.

Experiment 68. Preparation of excelsin—Excelsin, a globulin
from Brazil nuts, is very easily obtained crystalline (see Osborne, The

 

 

 

 

Fic. 70.—Crystals of excelsin. (Osborne.)

Plant Proteins, 1909). The ground-up nuts are freed from fat by extrac-
tion with gasoline, or petroleum ether, or benzene. The dried powdered
residues are then extracted with 10 per cent. NaCl. The excelsin is
dissolved. Filter. Dialyze the filtrate against water. The excelsin will
often crystallize out in the dialyzer. Crystals are small, hexagonal plates.
Redissolve the precipitate from the dialysis tube in dilute ammonium
sulphate and precipitate with an equal volume of saturated ammonium
sulphate. Filter, redissolve in dilute ammonium sulphate and dialyze.
The excelsin crystallizes out in the dialyzer. Figure 70.

Experiment 69. Preparation and properties of a prolamine.
Gliadin—Preparation of gliadin. To 100 grams wheat flour gradually
add enough water to make a thick dough. Then knead in the hand in a
stream of cold water until all the starch is washed out. Now cut the
protein into small pieces with a knife or scissors and extract twice (14
PRACTICAL WORK AND METITODS 927

hour each time) with 200 ¢.c. portions of boiling 70 per cent. alcohol,
by boiling in a flask on the steam bath. The gliadin dissolves in the
alcohol. Filter hot each time and. evaporate the alcoholic filtrates to
about %4 of the original volume, then allow to cool and add while
stirring 10 ¢.c. 10 per cent. NaCl solution. Allow to settle out, filter
off and dehydrate with cold 95 per cent. alcohol in the usual way.

Carry out the xanthoproteic, biuret, Millon and glyoxylic-acid tests
on your preparation.

Experiment 70. Preparation and properties of the nucleoproteins.
Nucleic acid.—Nucleic acid is, as its name implies, the acid of the
nucleus. It constitutes the part of the chromatin of the nuclei which
has an affinity for basic stains. It is, hence, in that part of the nucleus
which appears'as chromosomes in cell division, and it is this substance
which the histologist by his basic dyes follows in his study of inheritance
and mitosis. Nucleic acid has been described on page 163. It yields,
it will be remembered, phosphoric acid, purine and pyrimidine bases
and a carbohydrate group. Its structural formula is that indicated on
page 172. The nucleic acid of yeast has a pentose in it, d-ribose. That
of the thymus gland has a hexose sugar. Both are polynucleotides.

Preparation of nucleic acid from compressed yeast.1 To 150 grams
finely divided compressed yeast in a liter beaker add 450 cc. of a 1.5
per cent. NaOH solution. Stir well while heating on a boiling water
bath and digest thus for 30 minutes. Stir frequently. While still hot
filter through a creased filter in a 6-inch funnel. To the cooled filtrate
obtained after 1 hour’s filtration add glacial acetic acid, drop by drop,
until faintly acid to litmus. Stir well and again at once filter through
a creased filter. Now make this filtrate slightly alkaline by the addition
of a 10 per cent. NaOH solution. Concentrate on the water bath in a
porcelain dish to about one-half of the original volume. Cool this solu-
tion and again add glacial acetic acid until just acid to litmus. Filter
rapidly again if necessary and then pour the solution, while stirring,

into two volumes cold 95 per cent. alcohol containing 1 ¢.c. concentrated
~ HCl per 100 ce. aleohol, Transfer the milky solution at once to 1-inch
test-tubes and allow to settle out therein. Decant the supernatant alco-
holic layer from the separated nucleic acid as soon as possible and trans-
fer all of this solid material to one of the tubes, using in all 50 ce.
95 per cent. alcohol in so doing. Mix this well and again allow to
settle out; decant the supernatant alcohol. Again add 50 c.c. 95 per
cent. aleohol to the precipitate, mix well and allow to settle out. Repeat

1In this preparation yeast must be taken which is free from starch. Most
compressed yeast now contains much starch. If yeast contains starch, the starch
must be removed before the yeast is extracted with alkali, as the starch will swell
and make a non-filterable mixture with the alkali.

\
928 PHYSIOLOGICAL CHEMISTRY

this treatment with 95 per cent. alcohol twice. Finally gradually add
50 c.c. ether. to the precipitate in the tube, mix well, allow to settle out
and decant as before. Again add 25 cc. ether, mix well and transfer
rapidly on to a suction filter of hardened filter paper moistened with
aleohol. Wash twice on the filter with 25 ¢.c. portions of ether. Suck
dry by suction. When dry remove from the paper and expose on a
watch glass until all ether is removed. Note the solubility of
your product in water, dilute acids and bases. Note that it does not
coagulate on heating. Carry out the biuret test on your prepara-
tion. Heat a small amount of your product with 10 per cent. H,SO,
for a minute or two, then cool and apply the a-naphthol test. Test
a small amount also for organic phosphorus, as given under phospho-
lipin, experiment 57. Observe the precautions in the. digestion with
nitric acid.

Experiment 71. Preparation and properties of the secondary-
derived proteins.—In this group are the products of hydrolytic cleavage
known as albumoses, or proteoses and peptones. These substances, it
will be recalled, are soluble in water, not coagulated by heat, and all
but the peptones are separated by saturating their solutions with am-
monium sulphate. They may be prepared either by the hydrolytic action
of sulphuric or other acids, or by digesting proteins with pepsin-hydro-
chloric acid. For the experiments which follow obtain the materials
from the storeroom. Witte’s peptone is a commercial product which
consists chiefly of proto- and deutero-proteose. There is relatively little
peptone in it. Armour’s peptone consists chiefly of peptone, with little
of the albumose.

* A. Albumoses. Obtain 5 grams of Witte’s peptone and dissolve
it in 100 ec. distilled water by heating, slightly acidify with acetic acid
and filter.

1. Take about 1 ¢.c. for each test and add about twice the volume
of water and test the solutions for tryptophane, tyrosine, unoxidized
sulphur and by the biuret and xanthoproteic and Molisch test. Record
your observations.

2. To separate the various albumoses mix the filtrate in a beaker
with an equal volume of saturated (NH,),SO, (No. 7). A precipitate
forms which sticks to the rod on stirring. Remove this from the solu-
tion. It is primary albumose and consists of a mixture of proto- and
hetero-albumose. Save the filtrate which contains the secondary
albumoses.

3. Proceed with the primary albumoses. Dissolve them in a little
water.

a. Heat to boiling a portion of the solution. It does not coagulate.

b. Acidity slightly with acetic acid and add a drop of ferrocyanide
PRACTICAL WORK AND METHODS 929

of potassium. A precipitate is formed. Distinction from secondary
albumoses.

ce. Add a drop of copper-sulphate solution. <A precipitate forms.

d. Add a few drops of concentrated nitric acid. <A precipitate
forms which dissolves on heating.

e. Add some tannic acid. A precipitate forms.

f. Add some lead acetate. A precipitate forms.

g. Add a few drops of sodium or potassium bichromate. A pre-
cipitate forms if the solution is acid, but not if it is neutral.

h. Make a test for unoxidized sulphur.

*4. Secondary albumoses. Deutero-albumoses. Thio-albumose,.
synalbumose, etc. To the filtrate from the separation of the primary
albumoses add an equal volume of saturated ammonium sulphate in a
beaker and stir. Thio-albumose (and possibly other albumoses) are pre-
cipitated. Remove the precipitate; save the filtrate.

Precipitate. Thio-albwmose. Dissolve the precipitate in a little
water and make the test for unoxidized sulphur. It should be particu-
larly strong. Hence this is called thio-albumose. Repeat the tests a, b,
c, d of the preceding experiment with this albumose.

Snyalbumose, etc. The filtrate or the solution after the separation
of the thio-albumose is saturated while warm and stirring constantly by
the addition of powdered ammonium sulphate. The remainder of the
deutero-albumoses separate out and generally stick to the rod or the
sides of the beaker. Pour off and keep the solution. Dissolve the pre-
cipitate of deutero-albumose in distilled water, and test small portions
as follows:

a. Heat to boiling. No coagulation.

b. Biuret test. Pink and positive.

ce. Acidify slightly with acetic acid and add a drop or two of potas-
sium ferrocyanide. No precipitate. Difference from protoproteose.

d. Add a few drops of copper sulphate. No precipitate.

e. Add a few drops of nitric acid. No precipitate forms.

f. Add a few drops of tannic acid. A precipitate forms.

g. Add a few drops of lead acetate or mercuric chloride. <A pre-
cipitate forms.

h. Acidify and add a few drops of potassium-bichromate solution.
A precipitate forms. :

i. Make a test for unoxidized sulphur, tryptophane and iis
sine. How do aey HOnIPETE with the - similar tests of the ober
albumoses ?

*B. Peptone. There is still left in the solution: which has ‘been
saturated with ammonium sulphate some substances which give a biuret
test. Test some of the solution for these substances: Use 40 per cent.
930 PHYSIOLOGICAL CHEMISTRY

NaOH after a drop or two of CuSO,, since in the presence of so much
ammonium sulphate a large amount of NaOH must be used. A pink
biuret test shows the probable presence of peptone. To isolate the pep-
tone is a matter of difficulty.

Peptone. Its properties. To study the properties of peptone take
two grams of Armour’s peptone from the storeroom and dissolve in 50
c.c. distilled water.

a. Dilute a small portion and make in it the usual protein
reactions.

b. Saturate 5 cc. with powdered (NH,),SO,. There will be little
or no precipitate showing the absence of all albumoses.

e. Add a few drops of picric acid. There will be little precipitate
showing the absence of albumoses.

d. Add a few drops of ferrocyanide to the solution slightly acidified
with acetic acid. It is negative. ,

e. Add to the solution three volumes of alcohol. <A precipitate
shows that the peptones are not soluble in strong alcohol.

f. Add a few drops of lead acetate. A white precipitate. From
these reactions it appears that the peptones differ from the albumoses
in their solubility in saturated ammonium sulphate. They are still
precipitated by lead acetate and tannic acid, and these reagents are
among the best methods of separating the proteins completely from a
solution.

Experiment 72. Albuminoids.—A. Gelatin. Gelatin is in reality
a derived protein, being formed by the partial hydrolysis of the sub-
stance, collagen, found in white connective tissue and in the ground
substance of cartilage and bone. A very pure gelatin may be obtained,
also, from the scales of fishes after the removal of the other albuminoid,
icthylepidin. Take some of the 2 per cent. solution of gelatin and make
with it the following tests:

a. The various protein reactions. Observe whether the sulphur,
tyrosine and tryptophane reactions are positive or negative. ,

b. Obtain some pieces of gelatin from the storeroom. Test the solu-
bility of some small pieces in cold water. They swell but do not dissolve.
Heat. They go into solution. Cool the tube under the tap. If the solu-
tion is concentrated enough, it will set or gel. This is the best test for
gelatin. The connective tissues of the invertebrates do not yield on
boiling with water any substance which will set or gel.

b. If the solution is very dilute, say 1 per cent., the solution will
not gel. Make such a solution or obtain some of the stock 2 per cent.
solution and make with it the following tests:

ce. The various protein reactions. Observe whether the sulphur,
tyrosine and tryptophane reactions are positive or negative.
PRACTICAL WORK AND METHODS 93)

d. Is gelatin precipitated by half saturating the solution with
ammonium sulphate? By lead acetate? By tannic acid? By ferro-
cyanic acid? By picric acid?

e. If boiled with acid, gelatin loses its power of gelatinizing. Make
some of the solution distinctly acid by adding to 5 ¢.c. of a 10 per cent.
solution 1 ¢.¢c. of 10 per cent. HCl, heat to boiling for 2 minutes and
then cool under the tap. If it does not set, neutralize with 10 per cent.
NaOH and test its setting powers. If it sets, reacidify and heat again
for 2 minutes.

Exercise XIX. QUANTITATIVE DETERMINATION OF NITROGEN AND.
PHOSPHORUS IN PROTEINS.

Experiment 73. Determination of the amount of nitrogen in pro-
tein bodies by the Kjeldahl method.—The Arnold-Gunning modification
of the Kjeldahl method. Determine the nitrogen in a sample of protein
material given you by the instructor. When you have made the de-
termination, compare your results with the correct values to be obtained
from the instructor. The sample given you is probably not a pure simple
protein, but a mixture; possibly a ground-up, alcohol-extracted, dried
tissue.

Procure a weighing tube of the unknown substance from the store-
room. Transfer the contents to a clean, dry 75-100 e.c. beaker and dry
in an oven at 100-105° ©. for 1 hour; then remove from the
oven, break up any caked masses which may have formed, transfer
the finely divided material to the weighing tube and again dry
for 1 hour at 100-105° C. In the meantime, if your desiccator is not
already in good condition, clean and dry same, then transfer thereto
fresh granular CaCl,; also apply a very small amount of vaseline to the
desiccator cover to make it tight. After drying in an oven for 1 hour,
cool the loosely stoppered tube in the desiccator. Next weigh the tube
and contents carefully on an analytical balance, then transfer, without
loss, about 0.5 gram of powder to a clean, dry, 500 to 800 ce. Kjeldahl
flask, stopper the tube and weigh again carefully. Keep all these
records in your notebook in ink. With a fine jet of pure water wash
down the powder adhering to the neck of the flask (use as little water as
possible). Add 5 grams potassium sulphate, or 10 grams crystalline
Na,SO, 20 ¢c. pure concentrated H,SO, (Sp. G. 1.84) and 10 ce. of
the HgSO,+CuS0, solution (containing 1 gram HgSO, and 1 gram
CuSO, per 10 e.e.).

Heat on the digestion shelf until the water has been removed, then
add 15 grams more of potassium sulphate or 30 of Na,SO,.10H,O and
continue the digestion for 214-3 hours. To have the digestion complete,
932 PHYSIOLOGICAL CHEMISTRY

the mixture must, however, have been boiling for at least 2’2 hours and
the hot solution left must not be colored yellow, but simply green,
due to the CuSO,. After the digestion is complete, remove from the
digestion shelf, cool, and just as the contents of the flask begin to
erystallize gradually add, while agitating 250 ¢.c. distilled water;
stopper loosely and set aside until it is to be treated as indicated
below.

While your digestion is under way, clean out the distilling apparatus
as follows: To a clean 800 e.c. Kjeldahl flask transfer 300 c.c. distilled
water, add a knife point of granular zinc, connect with the proper dis-
tilling bulb and distill about 150 ¢.c. into a clean receiver. Throw
this away and then proceed with the distillation of the ammonia as
follows:

Having cleaned and prepared the distilling apparatus, measure off
exactly 25 ¢.c. N/2H,SO, into a 250-300 ¢.c. Erlenmeyer flask by means
of a burette. Place the flask so that the glass adapter end of the distilling
apparatus is just sealed by the acid. Dry the Kjeldahl flask on the
outside and carefully wipe the first inch of the inside of the neck of the
flask with a piece of filter paper (this is done to insure a good tight
joint with the rubber stopper later on). Add a small knife point of
granular zine to the contents of the flask to insure quiet ebullition and
just before adding the alkali and steadily (not so rapidly as to mix
the liquids) pour down the side of the neck of the flask (avoid moisten-
ing the first inch of the neck) 100 ce. of the special 40 per cent.
NaOH-+K,S solution (see page 860 for directions for making) in such
a way that the two solutions form distinct layers. Now rapidly connect
the flask with the distilling apparatus. In making the connection do
not try to twist the stopper into the flask, but hold the stopper firmly
and twist the flask on to the stopper. Now start the burner with a
medium-sized flame and at once rotate the flask to insure thorough
mizing of the contents. After the first 5-10 minutes’ heating the liquid
can be boiled rather vigorously until about 150 ¢.c. have distilled over.
30 minutes’ rapid distillation should suffice. Then lift the end of the
adapter out of the distillate by lowering the receiving conical flask and
continue the distilling for about 5 minutes longer. Wash off the tip of
the adapter with a jet of distilled water, the washings going into the dis-
tillate. Then remove the conical flask and turn off the gas last of all.
Titrate the distillate with n/2 NaOH, using congo red as indicator.
Make a correction of about 0.5 ¢c.c. N/2 solution for the ammonia in @
blank test. Caleulate the per cent. of nitrogen in the dry substance.
This has been determined to be about the correction necessary for an
ordinary distillation carried out in this way. In accurate work it is
necessary to make a blank distillation with the reagents used (H,SO,,
PRACTICAL WORK AND METHODS 933

ete.) to determine the amount of ammonia in them. 1 ee. N/2H,SO,
=1ee. N/2 NH,OH. 1 c.c. N/2 NH,OH contains .007 gram N.

Experiment 75. Determination of phosphorus quantitatively in
nucleoproteins.—All cells contain nucleoproteins and also phospho-
proteins, so that, if the residue after exhausting the tissue with alcohol
and ether is examined, it will be found to contain organically bound
phosphoric acid. Determine what per cent. of phosphorus there is in
the brain residue after complete extraction by alcohol and ether. Obtain
the sample of the tissue from the storeroom. This is the residue from
completely extracted sheep’s brains or other tissues and the phosphorus
content has been determined by the instructor. The Pemberton-
Neumann method is sufficiently accurate if all conditions are carefully
observed. It is more rapid than the magnesium-phosphate method, but
not so reliable. In practice it must be frequently controlled by gravi-
metric determinations. The point of difficulty seems to be in getting the
molybdate-phosphoric-acid precipitate to have always the same com-
position.

Weigh off into a 300 ec. Kjeldahl flask 0.4 to 0.5 gram of the sub-
stanee previously dried as directed in the Kjeldahl method. To this
then add 5 e.e. concentrated H,SO, and heat under a hood over a free
flame until well charred. From a separatory funnel, furnished for the
purpose, now gradually add, drop by drop, concentrated HNO, until
the char has disappeared. CAUTION! IN HEATING AFTER ADD-
ING THE NITRIC ACID, OR WHILE ADDING THE ACID, HOLD
THE MOUTH OF THE FLASK DIRECTED AWAY FROM YOUR
FACE! Heat again carefully until the nitric oxide fumes are boiled off.
The mass will char again. Repeat in the same way the gradual addi-
tion of the nitric acid and the heating until, after the last addition and
heating, white fumes of sulphuric acid appear and there is no further
eharring of the liquid contents of the flask. Heat on the digestion shelf
for about 10 minutes longer. Then allow to cool.

Next transfer, without loss, the contents of the flask to a 250 ce.
beaker and also rinse the flask 5 times with 15 e.c. portions distilled
water, adding each washing to the beaker. Gradually add, while stir-
ring, concentrated NH,OH until the solution is very slightly alkaline to
litmus, then add sufficient HNO, to make the solution slightly acid. To
this solution now add 15 grams NH,NO, and heat to 65° C.

To 30 ee. ammonium molybdate solution (see below) add 1.5 ee.
concentrated HNO, and filter, then add this to the above solution while
stirring and keep in the bath at 65° for 15 minutes.

Filter this with suction through a prepared asbestos filter in a Gooch
crucible (p. 893) and wash beaker and precipitate three times with 10
per cent. HNO,. Follow this by 5 washings with 2 per cent. NH,NO,
934 PHYSIOLOGICAL CHEMISTRY

solution. All of this manipulation must be done neatly and without
loss. See that your filtrates are clear.

Transfer the percipitate and asbestos by means of a glass rod from
the crucible to a beaker in which the precipitation was conducted. Add
exactly 20 ¢.c. N/2 NaOH measured from a burette or pipette to the
mass in the beaker, stir with a glass rod and remove the adhering pre-
cipitate from the funnel by means of a drop of the mixture in the
beaker. Finally rinse the funnel with a jet of distilled water, allowing
the washings to flow into the beaker. Stir until all is dissolved and
add enough water to make about 100 ¢.c. volume. Add phenolphthalein
solution (1 ¢e.) and titrate with N/2 H,SO,. Calculate the per cent.
of phosphorus in the dry substance. The precipitate obtained above,
if the instructions as to time, temperature and other conditions of pre-
cipitation are followed, is of the formula (NH,),P0,.12Mo0O,. Each
cubic centimeter of the N/2 NaOH is equivalent to 0.674 milligram phos-
phorus.

a. The ammonium molybdate solution used in the above method is
made as follows: Dissolve 100 grams of molybdiec acid in 144 ce. of
ammonium hydrate, sp. g. 0.90, and 271 ¢.c. water; slowly and with
constant stirring, pour the solution thus obtained into the mixture of
489 ¢.c. of nitric acid (sp. gr. 1.42) and 1,149 c.c. of water contained in
a large porcelain dish. Keep the mixture in a warm place for several
days, or until a portion heated to 40° deposits no yellow sediment, and
preserve in glass-stoppered bottles. Before using a portion of this solu-
tion for the precipitation of PO,, add 5 ec. con. nitric (sp. gr. 1.42)
to each 100 c.c., mix well and filter.

The solution may also be made by mixing 489 e.c. HNO, (sp. er.
1.42) and 1,149 cc. H,O in a large porcelain dish (18-inch) and then
pouring into this while stirring a solution made as follows: powder 121
grams ammonium molybdate, dissolve in 355 c.c. water, stirring fre-
quently to hasten solution and then add 60 ¢c.c. strong ammonium hydrate
(sp. gr. 0.90). This solution is then poured into the nitric acid as
indicated above.

EXErcisE XX. Wan SLYKE AMINO NITROGEN DETERMINATION.

Experiment 76. Determination of amino nitrogen by the Van
Slyke method.—(Van Slyke, Jour. Biol. Chem., 12, 1912, p. 275; 16,
1913, p. 121; 23, 1915, p. 408.) Principle. Nitrous acid acting on
amino-nitrogen sets free nitrogen gas which is collected over water and
its volume determined.

RNH, + HNO, — ROH +N, + H,0.
PRACTICAL WORK AND METHODS 935

    

To Hempet
Pipette

 

 

 

Fie. 71. Fig. 72.

Fig. 71.—Van Slyke animo-nitrogen apparatus.
Fig. 72.—Detail of Van Slyke apparatus (Van Slyke).
936 PHYSIOLOGICAL CHEMISTRY

There are two forms of apparatus, a larger and smaller, differing
only in capacity.

Process. The following description of the method is taken from Van
Slyke, Jour. Biol. Chemistry, 12, 1912, pp. 277-284:

“‘ The structure of the apparatus and the manner in which it ‘is
set up are apparent from the accompanying cut and photograph.

D is of 40-45 c.c. capacity, A of about 35 ¢.c. and the burette B of 10 cc. The
wire from which the deaminizing bulb D is suspended should be fairly stiff, and
rigidly fastened in position from above so that the loop about the capillary acts as
a fixed center. A is then so placed that its center of gravity comes near this center
and the shaking of D is accomplished with a minimum motion in A, and, con-
sequently, without putting a dangerous strain on the tube which connects A with
D. This tube is strong-walled and of 3 mm. inner diameter. It is essential that the
bore of cock @ should also be 3mm. The reason for this is that during the analysis
gas containing some nitrogen collects in the tube. Unless a is of as wide bore as
the tube the liquid from A may flow around the bubble instead of forcing it into
BD at the end of the reaction. The cock d is also of large bore in order to facilitate
emptying D. The neck connecting D and B must be of at least 8 mm. inner diameter
in order to allow free circulation of the solution in D up to the cock B. The small
bulb at the top of D keeps the reacting solution from splashing into the capillary.

In order to insure tightness of the cocks and to prevent their becoming loosened
by vigorous shaking it is well to lubricate them with a paste made by dissolving
together over a flame one part of rubber, one part of paraffin and two parts of
vaseline.

The structure of the modified Hempel pipette is entirely apparent from the
photograph. This form would undoubtedly facilitate absorption in all gas analyses
where shaking is necessary.

The driving wheel, as can be seen from the photograph, is so arranged that it
can be used alternately to shake the deaminizing bulb or the Hempel pipette. The
driving rod is shown in position for shaking the deaminizing bulb. By lifting the
rod from the shoulder of D and placing the other hook, at the end of the rod, over
the horizontal lower tube of the pipette, the power is transferred to the latter.
Rubber tubes drawn over the hooks at the end of the driving rod and those from
which the Hempel pipette is suspended make the apparatus almost noiseless. For
power one can use a good water motor. Still more convenient is a small electric
motor, particularly when connected with a rheostat enabling one to regulate the
speed. The gearing should be so arranged that the driving wheel, to which the
driving rod is eccentrically attached, makes 300 to 500 revolutions per minute. The
driving rod is attached about 1.5 em., in no case over 2 cm., from the center of
the wheel.

The manipulation is in principle the same as described in this
Journal, ix, pp 189-91. As there are slight variations due to the differ-
ent form of the present apparatus, however, we describe the present
technique, dividing the determination into three stages as in the original
description.

1. Displacement of air by nitric oxide. Water from F fills the
capillary leading to the Hempel pipette and also the other capillary
PRACTICAL WORK AND METHODS 937

as far as c. Into A one pours a volume of glacial acetic acid sufficient
to fill one-fifth of D. For convenience, A is etched with a mark to
measure this amount. The acid is run into D, cock c being turned so as
to let the air escape from D. Through A one now pours sodium nitrite
solution (30 gms. NaNO, to 100 e.c. H,O) until D is full of solution
and enough excess is present to rise a little above the cock into A.
It is convenient to mark A for measuring off this amount also. The gas
exit from D is now closed at c, and, a being open, D is shaken for a few
seconds. The nitric oxide, which instantly collects, is let out at c, and
the shaking repeated. The second crop of nitric oxide, which washes
out the last portions of air, is also let out at c. D is now connected
with the motor and shaken till all but 20 c.c. of the solution have been
displaced by nitric oxide and driven back into A. A mark on D indi-
cates the 20 ¢.c. point. One then closes a and turns c and f so that D
and F are connected. The above manipulations require between one and
two minutes.

2. Decomposition of the amino substance. Of the amino solution
to be analyzed 10 c.c. or less, as the case may be, are measured off in B.
Any excess added above the mark can be run off through the outflow
tube. The desired amount is then run into D, which is already con-
nected with the motor, as shown in the photograph. It is shaken, when
a-amino-acids are being analyzed, for a period of three to five minutes.
With a-amino-acids, proteins or partially or completely hydrolyzed pro-
teins, we find that at the most five minutes’ vigorous shaking completes
the reaction.1 Only in the cases of some native proteins which, when
deaminized, form unwieldy coagula that mechanically interfere with the
thorough agitation of the mixture, a longer time may be required. In
case a viscous solution is being analyzed and the liquid threatens to foam
over into f’, B is rinsed out and a little caprylic alcohol is added through
it. For amino substances, such as amino-purines, requiring a longer
time than five minutes to react (cf. p. 191, former article), one merely
mixes the reacting solutions and lets them stand the required length of
time, then shakes about two minutes to drive the nitrogen completely
out of solution.

When it is known that the solution to be analyzed is likely to
foam violently, it is advisable to add caprylic alcohol or kerosene through
B before the amino solution. B is then rinsed with alcohol and dried
with ether or a roll of filter paper before it receives the amino solution.

3. Absorption of nitric oxide and measurement of nitrogen. The

*Only 95 per cent. of the lysine nitrogen reacts in five minutes, but the

remaining one-twentieth of the lysine nitrogen is a practically negligible propor-
tion of the total nitrogen of a complete protein.
938 PHYSIOLOGICAL CHEMISTRY

reaction being completed, all the gas in D is displaced into F by liquid
from A and the mixture of nitrogen and nitric oxide is driven from F
into the absorption pipette. Solution in absorbing pipette is KMnO, 50
grams -+ 25 KOH (or 18 grams NaOH) in one liter. The driving rod is
then connected with the pipette by lifting the hook from the shoulder of
d and placing the othcr hook, on the opposite side of the driving rod,
over the horizontal lower tube of the pipette. The latter is then shaken
slowly by the motor for a minute, which, with any but almost com-
pletely exhausted permanganate solutions, completes the absorption of
nitric oxide. The pure nitrogen is then measured in F. During the
above operations a is left open, to permit displacement of liquid from D
as nitric oxide forms in D.

Testing completeness of reaction. Particularly when the mechanical
shaker is used, there is little danger of failing to obtain a complete
evolution of nitrogen. The point may be tested, however, as follows.
The nitrogen from F is driven out at c; a is closed and D connected with
F. The gas which has formed in the nitrous-acid solution in D during
the absorption of the nitric oxide and measurement of nitrogen is shaken
out and driven over into F and then into the Hempel pipette as before.
After absorption of the nitric oxide, the gas left should not measure more
than that obtained in blank tests, usually less than 0.1 cc. After the
gas has all been forced from D over into F at the end of the reaction,
the nitrous solution is run out from D, by opening d, through a tube
leading to a drain. B is rinsed and dried with a roll of filter paper or
with alcohol and ether and the apparatus is immediately ready for use
again.

Blank determinations, performed as above except that 10 cc. of
distilled water replaces the solution of amino substance, must be per-
formed on every fresh lot of nitrite used. The amount of gas obtained
on a five-minute blank is usually 0.3 to 0.4 ¢.c., with very little increase
for longer tests. Nitrite giving a much larger correction should be re-
jected.

The following determinations, performed with an 3% solution of
leucine, indicate the speed of the reaction. The correction applied for
reagents was 0.40 cc. Ten cubic centimeters of 74 leucine solution,
containing 14.01 mgs. of nitrogen, were used for each determination.

 

 

 

 

 

 

 

eee N Temperature} Pressure N obtained avond. evalkins caotaes
: of solution
Minutes C.c. Degrees Mm. Mgs. Ce. gs.
2 24.38 23 762 13.71 0.45 13.97
3 24.65 22 762 13.93 0.20 14.03
4 24.80 22 762 14.01. 0.00 ‘ 14.1
10 | 25.07 24 762 14.03 0.00 if 14.03

 
PRACTICAL WORK AND METHODS 939

The driving wheel was making 300 revolutions per minute. At
speeds of 400 or 500 revolutions the reaction can be driven to completion
in three, or, with higher room temperature, in two minutes.

The rate of reaction of ammonia is shown in the following table.
Ten c.c. portions of 4; ammonium sulphate solution, containing 28.02
mgs. of nitrogen each, were used.

 

 

* Per cent. of
Time of ‘ A
sanction N Temperature Pressure Weight of N a tGnen
Minutes Cc, Degrees C. Mm. Mys.
3 12.1 24 752 6.86 21.6
5 18.4 24 752 10.16 36.3
10 31.5 24 752 17.38 62.1

 

 

 

 

As pointed out before, ammonia reacts slowly compared with the
amino-acids. For accurate determination of NH, nitrogen in digesting
solutions, etc., it is advisable to first remove the ammonia; although good
comparative results can be obtained, in the presence of the relatively
small proportion of ammonia usually present, if reaction conditions of
time, temperature and concentration of solutions are kept constant, so
that the proportion of the ammonia decomposed is the same in each de-
termination. The ammonia can be conveniently removed and deter-
mined by distillation with Ca(OH), under diminished pressure, as de-
scribed on page 21, Vol. X, of this Journal. After the distillation the
excess Ca (OH), is dissolved with acetic acid. It is essential that all
the ethyl alcohol should be distilled off, as it decomposes nitrous acid
with formation of large volumes of gases which can be removed with
permanganate only with difficulty and by the use of perfectly fresh
permanganate solution. The point at which the alcohol has all been
boiled off is usually indicated when the solution begins to foam in the
distilling flask.

The following results were obtained with lysine picrate. Lysine, as
previously stated, reacts more slowly than the other amino-acids be-
cause it contains not only an @-amino group, but also an @-amino group.
In the fifteen- and thirty-minute determinations the solution was shaken
only during the last five minutes.

 

 

Weight of Ti f NH,-N NH,-N
waine ‘pieraté feaetion N Temperature) Pressure found calculated
Gram Minutes C.c. Degrees C. Mm. Per cent. Per cent.
0.200 5 25.4 24 764 7.13 7.47
0.200 15 ' 26.7 24 764 7.49 TAT
0.200 30 26.7 24 764 7.49 TAT

 

 

 

Solutions to be analyzed should be free of ethyl alcohol and acetone.
These substances when mixed with nitrous acid give off gases or vapors
which are with difficulty absorbed by the permanganate,
940 PHYSIOLOGICAL CHEMISTRY

Amyl alcohol, which in the original description of the amino method
was recommended to prevent the foaming of viscous solution, must be
replaced for this purpose by caprylic alcohol [Kahlbaum’s ‘‘ octyl-
alkohol (sekundir)I’’]. Amy! alcohol, boiling at 131°, has the dis-
advantage of a very noticeable vapor tension. Permanganate solution
apparently possesses the power to absorb slight amounts of amyl alcohol
vapor. Particularly on hot days, however, and when relatively much of
the alcohol is used, it is nevessary to change the permanganate with
every analysis or else reduce the volume of gas observed by multiplica-
tion with an empirically determined factor.

For convenience in calculating results the following table is ap-
pended. The figures are calculated by dividing by 2 those for moist
nitrogen given by Gattermann in the Praxis des organischen Chemikers,
ninth edition. They represent the weights of amino-nitrogen in milli-
grams which correspond to 1 c.c. of nitrogen gas, obtained by the action
of nitrous acid and measured over water, at the temperatures and at-
mospheric pressures indicated.’’

Van Slyke micro-apparatus for amino-nitrogen.—The smaller
apparatus has the following dimensions: gas burette, 10 c.c., the upper
part measuring 0.02 ¢.c. The Ceaminizing bulb is 11-12 ¢.c. and the
10 cc. burette is replaced by one of 2 cc., only 10 cc. of nitrite
solution and 2.5 ¢.c. acetic acid are required and the correction for the
reagents is 0.06-0.12 c.c., according to the quality of the nitrite used.
‘* With the micro-apparatus the error need not be more than 0.005 mg.
of N when 2 c.c. or less of N gas are measured. One can analyze 1/5th
of the amount of substance needed for the larger apparatus. 0.5 mg. of
amino N can be measured to an accuracy of 1 per cent. The only
change of procedure from that already described is in the speed at which
the deaminizing bulb is shaken and the Hempel pipette. The deaminiz-
ing bulb should be shaken at a very high rate of speed; about as fast as
the eye can follow, or an unnecessary amount of time is lost in freeing
the apparatus from air. This stage is accelerated by warming the nitrite
solution to 30° before it is used. During the absorption of the nitric
oxide the Hempel pipette should be shaken not faster than twice per
second. Becanse of the small amount of nitrogen to be measured it is
especially necessary that the removal of air in the first stage be com-
plete. This is assured by shaking the solution in the deaminizing bulb
back each time in this stage until the bulb is two-thirds filled with
nitric oxide. The hook or wire loop from which the deaminizing bulb is
suspended should be perfectly rigid and hold the capillary outlet tube
tightly. Binding the tube to the holder with a strip of rubber band is a
satisfactory method of insuring a firmly held apparatus.’’ ‘‘ Clean whole
appatatus with bichromate-sulphuric-acid mixture. For especially ac-
PRACTICAL WORK AND METHODS 941
curate results measure the amino-acid solution with an Ostwald 1 or
2 @c. pipette graduated to deliver between 0.001 and 0.002 c¢.c. into the
2 ¢.¢. burette and wash the burette twice with 6 or 7 drops of water dis-
tributed about the entire inner walls of the burette for each washing.’’

MILLIGRAMS oF AMINO NITROGEN CORRESPONDING TO 1 c.c. OF NITROGEN GAS AT
11°-30° C; 728-772 mM. PRESSURE.*

 

 

 

 

 

 

¢| 728 | 730 | 7382 | 784 | 736 | 7388 | 740 | 742 | 744 | 746 | 748 | 750
11° | 0.5680 | 0.5695 | 0.5710 | 0.5725 | 0.5745 | 0.5760 | 0.5775 | 0.5740 | 0.5805 | 0.5820 | 0.5840 | 0.5855
12° | 0.5655 | 0.5670 | 0.5685 | 0,5700 | 0.5720 | 0.5735 | 0.5750 | 0.5765 | 0.5780 | 0.5795 | 0 5x15 | 0.5830
18° | 0.5630 | 0.5645 | 0.5660 | 0.5675 | 0.5695 | 0.5710 | 0.5725 | 0.5740 | 05755 | 0.5770 | 0.5745 | 0 5805
14° | 0.5605 | 0.5620 | 0.5635 | 0.5650 | 0.5665 | 0.5680 | 0.5700 |] 0.5715 | 0.5730 | 0.5745 | u.576U | 0 577
15° | 0.5580 | 0.5595 | 0.5610 | 0.5625 | 05640 | 05655 | 0.5670 | 0.5685 | 0.5705 | 0.5720 | 0.5735 | 0 5750
16° | 0.5555 0.5645 | 0.5660 | 0.5675 | 0.5690 | 0 5710 | 0.57.5
17° | 0.5525 0.5620 | 0.5635 | 0.5650 | 0.5665 | 0.5680 | 0 5695
18° | 0.5300 0.5595 | 0.5610 | 0.5625 | 0.5640 | 0.5655 | 0.5670
19° | 0.5475 0.5565 | 0.5580 | 0.5595 | 0.5610 | 0.5680 | 0.5645
20° | 0.5445 0.5540 | 0.5555 | 0.5570 | 0.5585 | 0.5600 | 0 5615
21° | 0.5420 0.5510 | 0.5525 | 0.5540 | 0.5555 | 0.5575 | 9.5590
22° | 0.5395 0.5485 | 0.5500 | 0.5515 | 0.5530 | 0 5545 | 0.5560
23° | 0.5365 0.5455 | 0.5470 | 0.5485 | 0.5500 | 0.5515 | 0.5530
24° | 0.5335 0.5430 | 0.5445 | 0.5460 | 0.5475 | 0.5490 | 0.5505
25° | 0.5310 0 5400 | 0.5415 | 0.5480 | 0.5445 | 05460 | 05475
26° | 0.5260 0.5370 | 05365 | 0.5400 | 0.5415 | 0.5480 | 0.5445
27° | 0.5250 0.5840 | 0.5355 | 0.5370 | 0.5385 | 0.5400 | 0.5415
28° f 0.5220 0.5310 | 0.5825 | 0.5340 | 0.5855 | 0.5370 | 0.5385
29° | 0.5195 0.5280 | 0.5295 | 0.5810 | 0.5825 | 05340 | 0 5355
30° | 0.5160 0.5250 | 0.5265 | 0.5280 | 0.5295 | 0.5310 | 0.5325

e| 728 740 742 | 744 | 746 748 750

 

 

 

 

 

«| 752 | 754 | 756 | 758 | 760 | 762 | 764 | 766 | 768.) 770 | 7172 |

11° | 0.5870 | 0.5885 | 0.5900 | 0.5915 | 0.5935 | 0.5950 | 0.5965 | 0.5980 | 0.5995 | 0.6010 | 0.6030 |

 

15° | 0.5765 | 0.5765 | 0.5795 | 05810 | 0.5830 | 0.5845 | 0.5860
16° | 0.5740 | 0.5755 | 0.5770 | 0.5785 | 0.5800 | 0.5815 | 0.5830

 

 

aoe | 05630 | 0.5645 | 0.5660 | 0.5675 | 0.5690 | 0.5705 | 0.5725 | 05740 | 0.5755 | 0.5770 | 0.5785

21° | 0.5605 | 0.5620 | 0.5635 | 05650 | 9.5665 | 0.5680 | 0.5695 | 0.5710 | 0.5725 | 0.5740 | 0.5755
22° | 0.5575 | 0.5590 | 0.5605 | 0.5620 | 0.5635 | 0.5650 | 0.5665 | 0.5680 | 0.5695 | 05715 | 0.5730

 

 

95° | 0.5490 | 0.5503 | 0.5520 | 0.5585 | 0.5550 | 0.5565 | 0.5590 | 0.5595 | 0.5610 | 0.5625 | 0.5640 |:

26° | 0.5460 | 0.5475 | 0.5490 | 0.5505 | 0.5520] 0.5535 | 0.5550 | 0.5565 | 05580 | 0.5595 | 0.5610] .. ....
27° | 0.5430 | 0.5445 | 0.5460 | 0.5475 | 0.5490 | 0.5505 | 0.5520 | 0.5535 | 0.5550 | 0.5565 | 0.5580 |.... ..

28° | 0.5400 | 0.5415 } 0.5430 | 0.5445 | 0.5460 | 0.5475 | 0.5490 | 05505 | 0.5520 | 05535 | 0.5550 |.......,
29° | 0.5370 | 05885 | 05400 | 0.5415 | 0.5430 | 0.5415 | 0.5460 | 0.5475 | 0.5490 | 0.5505 | 0.5520 |........
30° | 05340 | 05355 | 0.5370 | 0.5885 | 0.5400] 05415 | 0.5430 | 05445 | 05460 | 0.5475 | 0.5490 |..... ..

e| 752 | 754 | 756 | 758 | 760 | 762 | 764 | 766 | 768 | 770 | 772

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

* Journal of Biological Chemistry, xii, No. 2. Van Slyke: The Quantitative
Determination of Amino Groups. II.

Exercise XXJ. PREPARATION oF TRYPTOPHANE,
Experiment 77. Tryptophane from casein. (Hopkins and Cole:

Jour. Physiol.; Dakin: Biochem. Jour., 12, 1918.) Dissolve 200 grams
easein in 2 liters of..3 per cent. Na,CO,. Adjust the alkalinity so that
942 PHYSIOLOGICAL CHEMISTRY

the most favorable H ion concentration for trypsin exists, i.e., about
P,, =8.1. Add an active pancreatic powder (pancreatine) about 2 grams
and digest for 4 days at 37° C. in a stoppered flask to which toluene has
been added to prevent putrefaction. At the end of 4 days add another
two grams of pancreatine and put back for another four days. Acid
hydrolysis cannot be used if tryptophane is desired, since this amino-
acid is destroyed by acids. The mixture is then heated to 80° C. and
filtered. To the clear filtrate add concentrated H,SO, about 15 to 18 c.c.
Allow to stand and if a precipitate appears filter it off (CaSO,). Pre-
cipitate the tryptophane as follows: To the filtrate add with brisk
stirring about 200 c.c. of the mercuric sulphate reagent made as de-
scribed below. This should be added until precipitation begins and
then an additional quantity of about 50 ¢.c. This precipitates trypto-
phane, cystin and some other substances in small amounts. Allow to
stand in a cool place for 12 hours or more for the precipitata of a yellow
lemon color to settle. Filter, using suction. Wash the precipitate with
5 per cent. H,SO, until the washings are free from Millon reaction or
at best give but a brownish yellow color. Suspend the precipitate in
about 500 ¢.c. water and pass H,S until decomposition is complete. Do
not heat on water bath. Filter from the mercuric sulphide, and re-
saturate the filtrate with H,S to make sure the decomposition is complete
and all mercury removed. Suspend the precipitate of HgS in water
and pass H,S until saturated. This is done to make sure that all the
tryptophane is removed from the ppt. Filter and add the filtrate to
the original filtrate which has been resaturated. Remove H,S from the
solution by aeration with a lively current of air at room temperature.
Remove the H,SO, by the addition of exactly enough Ba(OH), solution,
filter from the BaSO, (avoid any excess of Ba(OH),), concentrate on
the water bath to about 30 c.c. under reduced pressure.

Extraction of the tryptophane. From here on proceed by the Dakin
method. Place the solution in a small extraction flask of a Steudel-
Kutscher extraction apparatus arranged as in figure 73. The solution
is placed in flask A and in flask B there is placed 200 cc. of butyl
alcohol. Heat the butyl alcohol flask by a low flame and start the
extraction. The extraction should be continued for about 12 hours.
Tryptophane begins to crystallize out in flask B in about 1 hour. The
yield should be about 1.75 per cent. of the weight of the casein taken.
When the extraction is complete, cool the flask and allow to stand.
Pour off the residual butyl alcohol from the crystals, filter off the
erystals, using a small suction filter, and wash the crystals with ether.

Purification. The tryptophane thus obtained is almost pure and may
be purified by a single crystallization from dilute alcohol. Dissolve the
erystals in a minimal quantity of hot distilled water, add an equal bulk
PRACTICAL WORK AND METHODS 943

of 90 per cent. alcohol, a little animal charcoal, heat to boiling a moment
and filter boiling hot with suction. Transfer to a crystallizing dish or
beaker and concentrate on the water bath, adding from time to time a
little 90 per cent. alcohol until crystallization begins. Remove and allow

   

 

A848 AAD =
Fn ee ee eee he et el

X I actg =

i

 

 

 

Fig. 73.—Kutscher-Steudel extractor. Extractor is A; flask to right is B.

to cool and crystallize. Filter on small suction filter plate. Fine, white,
pearly plates.

Mercuric sulphate reagent for precipitation of tryptophane. (Hop-
kins and Cole.) 250 ¢.c. con. H,SO, added to 4750 e.c. H,O. 500 grams
HgS0O, are weighed out. 50 grams at a time are ground up in a mortar
with the dilute acid and poured into a five liter flask containing the
greater part of the acid. This is repeated until all of the salt is trans-
944 PHYSIOLOGICAL CHEMISTRY

ferred to the flask with the acid. Stir briskly for a brief time and allow
to settle. Pour off the reagent and filter from the precipitate.

ExercisE XXII. IsoLaTIoN OF AMINO-ACIDS BY THE DAKIN METHOD.
(Dakin: Biochem. Jour., xii, 1918, pp. 290-317.)

Experiment 78. Separation of amino-acids by percolation with
butyl alcohol.—Principle. The completely hydrolyzed protein is ex-
tracted in a continuous extraction apparatus by butyl alcohol. The
proline and practically the, whole of the monoamino acids are extracted by
the alcohol, leaving the diamino and the dicarboxylic acids unextracted.
The further separation of the amino-acids is accomplished by special
methods to be described. By this means the troublesome and destructive
distillation in the Fischer method is avoided and much better yields
obtained.

Apparatus. Some form of continuous extraction apparatus must be
used. That described by Steudel and Kutscher and shown in figure 73
is very convenient.

Procedure. By the method which follows the amino-acids are to be
separated into five groups:

1. Monoamino acids, both aromatie and aliphatic, insoluble in butyl
alcohol but extracted by it.

2. Proline, soluble in butyl alcohol and extracted by it.

38. Peptide anhydrides: (diketopiperazines) extracted by butyl
alcohol but separated from (2) by sparing solubility in water and
alcohol.

4. Dicarboxylic acids, not extracted by butyl alcohol.

5. Diamino acids, not extracted by butyl alcohol, but separable
from (4) by phosphotungstic acid and other means.

While the amino-acids are insoluble in butyl alcohol they are soluble
in butyl alcohol containing some water. Since salts greatly reduce the
miscibility and mutual solubility of butyl alcohol and water, the pres-
ence of salt, and particularly of calcium chloride, is to be avoided during
the extraction.

Hydrolysis. Heat to boiling in a flask 200 grams casein suspended
in 5 to 6 times its weight of sulphuric acid prepared by adding con-
centrated acid: to 3 volumes of water, for 12 to 16 hours. Dilute by
adding 5 times the volume of distilled water, mix well, and remove the
excess of sulphuric acid by adding little by little saturated Ba(OH),
solution until the reaction is acid to litmus but no longer acid to congo
red. Concentrate the filtrate somewhat and allow some tyrosine to
crystallize out. Filter and again concentrate. Make now approximately
neutral to litmus by addition of dilute Ba(OH),. Filter from any
PRACTICAL WORK AND METHODS 945

precipitate. Concentrate the amino-acid solution on the water bath
until leucine begins to crystallize out, and then transfer the mixture of
liquid and erystals while still warm to flask A of the extraction
apparatus.

Extraction. The Kutscher-Steudel apparatus is arranged as shown.
Figure 73. A 350 ¢.c. extraction flask is large enough for the amino-
acids from 100 grams of casein. The aqueous layer should occupy
3/4ths or 5/6ths of the available space and the volume of supernatant
butyl alcohol is relatively small. Rubber connections can be employed,
but they are attacked by the butyl alcohol and their surface exposed
should be as small as possible. Heat the extraction flask on a sand
bath, with rather high sides so as to avoid condensation in the flask,
and the flame is so adjusted that a reasonably rapid flow of alcohol
returns into the flask. If a 300 or 400 c.c. apparatus is used the aqueous
solution soon acquires a temperature of 60-80° and no further heating
is required, but when larger quantities of fluid are being extracted it
is well to heat the aqueous flash A to 75° or 80° by means of a water
bath. |

The separation of the amino-acids as a cream-colored granular pre-
cipitate soon begins in the extraction flask B. Allow extraction to con-
tinue for the day. Turn off at night. Next morning filter off the
amino-acids which have accumulated by a Buchner funnel, washing them
with butyl alcohol and ether, and then continue the extraction with the
combined butyl alcohol filtrates, which are light brown. This reduces
risk of breakage due to accumulation of amino-acids in the flask. Con-
tinue the extraction until no additional amounts of amino-acids separate
out in the extraction flask. The extraction will take about 36 hours.
There is thus obtained in a erystaline form the monoamino acids. The
aqueous solution is kept for future separations of glutaminie and
diamino acids. The butyl alcohol contains the proline in solution.

Proline separation. The filtered butyl alcohol extract is evaporated
to dryness under reduced pressure. Boil the sticky, brownish residue
with about 10 parts of absolute alcohol. With casein the whole of the
residue (proline) dissolves. Allow solution to stand over-night. A small
quantity of peptide anhydrides crystallize out. (About 2 per cent. of
casein taken) this is group 3. Filter with suction. Filtrate has alcohol
evaporated under reduced pressure. The residue is dissolved in a small
amount of hot water, decolorized by boiling for a minute or so with a
little animal charcoal, then filtered with suction, the filtrate concentrated
on the water bath in an evaporating dish, to a syrup. On allowing to
stand a further crystallization of peptides takes place. Filter from these,
using a small suction filter. All told 2 to 3 per cent. of peptides are
obtained from casein. The clear aqueous solution now contains the
946 PHYSIOLOGICAL CHEMISTRY

whole of the proline. The proline can be separated for purposes of
identification most easily and conveniently as the hydantoine, C,H,NO,,
or it may be separated by making the copper salt. (See below under
preparation of proline.)

Preparation of prolylhydantoine. The crude proline from 200 grams
of casein should weigh about 16 grams. Take an amount equivalent to
about 5 grams. Add 5 grams potassium cyanate and 50 ¢.c. H,O and
evaporate to dryness on the water bath. This makes the uramido com-
pound. Dissolve in water, about 50 c.c., acidify to congo red with
sulphuric acid and extract with ether in the extractor for 7 hours. This
removes the uramido compounds of leucine, valine, etc., present as
small amounts of impurities. To the extracted aqueous solution then
add 1/5th volume of 50 per cent. H,SO, and heat on water bath for
1 to 2 hours, and again extract with ether as before for 12 hours. By
this heating the prolylhydantoine is formed and the ether now takes this
out. On evaporation of the ether about 4.5 grams of prolylhydantoine
should be obtained. It cones out as thick, transparent prismatic needles.

Preparation of proline. If proline is desired it is prepared by taking
the aqueous solution above, adding to it while boiling freshly precipi-
tated and washed Cu(OH), as long as it dissolves. Evaporate the dark
blue solution to dryness. Extract the dry residue with absolute alcohol
as long as any redissolves. The active, l-proline copper salt is soluble
in absolute alcohol whereas the racemic salt is insoluble. Evaporate off
the alcohol in the water bath and take up the residue in a little water,
carefully concentrate and allow to crystallize. The copper salt erys-
tallizes out. If the free proline is desired the copper salt may be freed
from copper by passing H,S, filtering and evaporating on the water
bath to a small volume and allowed to crystallize. The crystals are flat
needles. Taste sweet. [a]}—= —17.4.

EXxercisE XXIII. SEPARATION OF GLUTAMINIC ACID.

Experiment 79. Glutaminic acid—Glutaminic acid separation.
The aqueous solution left after the butyl alcohol has extracted the mono-
amino acids from it is concentrated under reduced pressure on the
water bath to remove the butyl alcohol. The final volume should be
about 150 ec. Transfer to a 250 c.c. Erlenmeyer flask and pass dry
hydrochloric acid gas through the mixture until it is saturated. Stopper
the flask and set in the ice box for crystallization. After a day or so,
when the glutaminic acid hydrochloride has erystallized out, add an
equal volume of ice-cold alcohol, and filter, using suction and a Buchner
funnel. Keep the filtrate for separation of basic amino-acids. Wash
the erystals with small amounts of concentrated hydrochloric acid previ-
PRACTICAL WORK AND METHODS 947

ously cooled nearly to zero. The filtrate may be resaturated with HCl
and a new crop of giutaminic acid obtained, but this yield will not be
large. :

To recrystallize the glutaminic acid hydrochloride disssolve the
erystals in about 100 c.c. water, decolorize by boiling with a little char-
coal, filter, cool, saturate the filtrate as before with dry HCl gas and
allow to stand in the ice box until crystallization is complete (about 24
hours). Then add an equal volume of ice-cold alcohol and filter with
suction as before. Drain as dry as possible.

To free the glutaminic acid from HCl dissolve it in a minimum
amount of water. Add sufficient NaOH to neutralize the hydrochloric
acid (about 5.44 «ec. of N NaOH for every gram of the dry salt will be
necessary). Add the NaOH until the reaction is no longer acid to
congo red but is strongly acid to litmus. Evaporate in vacuum until
the bulk is about 75 ¢.c. Transfer to a beaker and allow to stand and
erystallize in the ice box. Glutaminic acid should crystallize out
promptly. Filter from the crystals, using suction, wash quickly under
suction with a little water, and dry the crystals with filter paper.
Crystals are rhombic tetrahedra.

ExeErcisE XXIV. PREPARATION OF TYROSINE, CYSTINE AND CYSTEINE.

Experiment 80. Tyrosine and cystine.—These are readily pre-
pared from ox horns. Allow horns to dry so that the bony core can be
easily slipped out. Cut horn into small pieces with saw and hatchet.
1 kilo dried horn is placed in a 8 liter flask and extracted 2 or more
days at room temperature with 10 per cent. HCl. Pour off the acid,
add 2 liters half-concentrated HCl and heat on steam bath with reflux
condenser for 7 days at temperature of 90°. Then filter from undis-
solved. Partially decolorize once with charcoal. Refilter. Evaporate
deep red liquid in vacuo in water bath until a fairly thick syrup of
about 700 cc. remains. Pour syrup into a large beaker and add strong
NaOH carefully, little by little, with constant stirring to the solution
until reaction is faintly acid or neutral to litmus. Cool. Add ethyl
alcohol until solution contains about 50 to 70 per cent. by volume and
allow to stand at a low temperature for two days. Filter the voluminous
brownish ppt., which may have already begun to appear before the
addition of the alcohol, on a suction filter. Bring the ppt. after washing
with water into a large evaporating dish and extract four or five times
with a liter of boiling water each time to extract the tyrosine and
leucine, etc. The cystine remains undissolved. Keep the filtrates for
tyrosine. Extract until the residue gives only a faint reaction with
Millons solution.
943 PHYSIOLOGICAL CHEMISTRY

Cystine. The portion which is insoluble is mainly impure cystine.
Suspend in about 500 c.c. boiling water, add enough concentrated HCl
to dissolve the ppt. Decolorize with charcoal. Neutralize the clear
filtrate with pure 10 per cent. NaOH. From the clear solution the
cystine begins to crystallize out in large hexagonal plates separating as
a sandy ppt. before the neutral point is reached. When the crystalliza-
tion has begun examine a little under the microscope. As long as the
crystals of cystine alone are appearing continue to add little by little
the NaOH, but just as soon as the first tyrosine needles appear filter
quickly, using suction through a Buchner funnel. The tyrosine con-
tinues to come out in the filtrate. Neutralize still further while this is
coming out and prepare a new filter. As soon as cystine begins to
appear again filter quickly. This will give principally tyrosine with a
little cystine. The solution when neutral is allowed to stand in the cold.
It will settle out nearly all of the remainder of the tyrosine and cystine.
- Filter and keep this mixture for further separation. To purify the first
cystine redissolve in a little water to which strong HCl is added just
enough to dissolve it, neutralize with ammonia. Cystine crystallizes out,
leaving the tyrosine in the solution. The purity of the cystine is shown
by the fact that it gives no Millon reaction, but only a white ppt. with
the Millon reagent. From 1 kilo of horn about 40 grams of cystine can
be obtained.

Tyrosine. Tyrosine is separated from the 5 liters of water which
has been used to dissolve it from the cystine, and from the united filtrates
from the cystine precipitations, by concentrating and cooling. Recrys-
tallize by dissolving in hot water and cooling. Observe under the micro-
scope. Tyrosine crsytallizes in needles and balls of needle-shaped
crystals. By concentrating these extracts further erystallizations are
obtained. By dissolving in HCl and neutralizing and filtering while
the character of the crystals is being watched in the microscope, the
tyrosine may be separated from a remnant of cystine.

Cysteine. Cysteine is very easily made by dissolving the cystine in
HCl, adding some tin, which reduces the cystine to cysteine. Freeing
from tin by saturating with H,S, filtering from the sulphide of tin,
and evaporating the filtrate on the water bath to crystallization. Trans-
parent prismatic needles and prisms. Dissolved give a blue color wit!
FeCl, and a brilliant cherry with sodium nitroprusside.
CHAPTER XXVI.
COMPOSITION OF THE FOODS.
EXeERcIsE XXV. MILK.

Experiment 81. Reaction.—Test the reaction of some fresh milk
to litmus. Perfectly fresh milk is amphoteric, but market milk is usually
slightly acid due to lactic acid. ;

Experiment 82. Microscopic appearance.—Place a drop of milk
on a Slide, cover with a coverslip and observe under low and high power
of the microscope. Prick your finger, obtain a little blood, touch a pin
to the blood and then to the milk so as to obtain a few corpuscles and
compare the size of the corpuscles and the milk globules. The corpuscles
are 7 mu in diameter.

Experiment 83. Salting out casein and fat.—Casein, the principal
protein of milk, or caseinogen as it is also called, may be separated from
the other constituents by salting out with NaCl.

To 10 c.c. milk in a test-tube add 3.6 grams of powdered NaCl.
This is the amount necessary to saturate the milk. Close the mouth of
the tube with the thumb and shake. After the NaCl has dissolved, the
casein and fat will separate as a curd and the milk becomes so thick
that it flows very slowly. Take a small funnel, arrange a simply folded
10 em. filter paper, leaving the filter paper dry, and pour on the milk.
Collect the filtrate in a test-tube. It should be perfectly clear but a
slight yellow in color. In the filtrate is lactalbumin, lactose, extractives,
lactic acid, citric acid, etc. Save the filtrate for the following experi-
ments.

Experiment 83. Properties of casein.—After the filtration is
ended, remove the filter paper with the casein and fat from the funnel,
open it out so that it is flat and transfer a little of the casein on the
point of a knife to a test-tube, add about 3 ¢.c. of water and shake. The
easein redissolves and the fat is again distributed through the solution
with the casein. There appears to be some sort of a union, physical or
chemical, between the casein and the fat. Heat the solution to boiling.
Observe that it does not coagulate. Casein does not coagulate on heating.
Cool the heated solution and add to it a drop of dilute acetic acid. The
easein flocks out of the solution. After observing this redissolve by a
drop of Na,CO, solution. Add a drop of 10 per cent. NaOH and a

couple of drops of 2 per cent. CuSO,. Observe the biuret reaction.
949
950 PHYSIOLOGICAL CHEMISTRY

After this has appeared, heat the solution to boiling. The copper will
be reduced by a small amount of lactose which has remained in the curd. .

To another small pinch of the precipitate in a test-tube add glacial
acetic acid, or glyoxylic acid 2 to 3 ¢.c., shake, then add an equal amount
of concentrated H,SO,. Observe the plain tryptophane reaction.

The main bulk of the casein precipitate should be brought into a
small beaker or a large test-tube, about 15 c.c. of acetone added and
heated by immersion in hot water. This dissolves out the fat. Decant
the acetone through a dry filter paper, and re-extract the casein with a
new lot of acetone. The filtrate, containing the fat, should be evaporated
on the water bath. Observe the butter fat, with its typical odor, remain-
ing after evaporation of the acetone.

The casein may be dried by allowing the acetone to evaporate from
it. It is a clean white powder, soluble in water, precipitated by dilute
acid but redissolved in excess. If desired it may be dissolved in water,
a little rennin added and the solution coagulated by its action. Para-
casein is formed.

A solution of casein in lime water does not precipitate calcium
phosphate if a dilute phosphoric acid solution is added to neutrality.
Liquid becomes turbid.

Experiment 84. Lactalbumin.—The lactalbumin is in the filtrate
from the saturated salt milk of experiment 83. It may be separated by
coagulating it by heating. For this purpose suspend the test-tube by
means of a clamp on the lamp stand in a beaker of water, so arranged
that the bottom of the test-tube is a little above the bottom of the
beaker. A thermometer is placed’ in the test-tube so as to observe the
temperature of coagulation. Now heat the water in the beaker and
observe the temperature at which the albumin coagulates. It is between
74 and 80° C. Heat until the temperature in the tube is about 95°
so as to be sure that the coagulation is complete. Observe how copious
the precipitate is but that it is much less than the casein. Filter through
a small filter paper and collect the filtrate for the lactose. Take a little
of the lactalbumin from the filter paper and make a biuret test with it,
and any other protein tests desired.

Experiment 85. Lactose.—The filtrate from the lactalbumin con-
tains the lactose and also lactic acid. To test for lactic acid take 1 c¢.c.
of the filtrate, add a couple of ¢.c. of water and then with a small pipette
add to it 4 or 5 drops of FeCl, solution. Observe the lemon yellow
color which develops. Make a control tube containing only 3 ¢.c. water
with the same amount of FeCl, added for comparison. Lactic acid
gives this lemon yellow color with ferric chloride. Test for lactose in
the filtrate by taking 1 ¢c.c., adding 4 ¢.c. of a mixture of equal parts of
Fehling’s solutions A and B and heating. A copious reduction occurs.
.

PRACTICAL WORK AND METHODS 951

Experiment 86. Preparation of lactose.—Dilute 500 ¢.c. of milk
with 1 liter of water, add gradually enough dilute acetic acid to pre-
cipitate the casein. When this flocks readily, filter. Place the filtrate
in an evaporating dish, heat to boiling to coagulate the lactalbumin.
Filter. Evaporate the filtrate on the water bath to about 75 ¢c.c. Filter
trom calcium phosphate. Evaporate the filtrate to a syrup and set aside
for lactose to crystallize. Purify if necessary by recrystallizing, using
charcoal.

Experiment 87. Determination of the amount of fat by Meigs’
method.—The advantage of this method over the Soxhlet is its greater
ease. It gives results usually about 1 per cent. lower than the latter.

Process. The glass stopper cylinder shown in Figure 74 is pre-

 

 

 

 

Set
J
! I

Fig. 74.—Pipette arranged to remove the upper layer in Meigs’ method of fat
extraction. The end of the tube, A, is placed at the surface of the division between the
two layers of the mixture of milk treated with the water, alcohol, and ether mixture
and the upper layer is then forced out through it by forcing air into the space above the
mixture through tube B.

pared. This cylinder is a 100 cc. cylinder. The arrangement of the
tube bent up at the end is sufficiently obvious. It makes possible a very
clean separation of the ether fat layer. To 10 cc. of milk measured
from a pipette into the 100 cc. glass stoppered cylinder add 20 ec. of
distilled water and 20 cc. ethyl ether and shake for 5 minutes. Add
then 20 e.c. 95 per cent. alcohol and shake again for 5 minutes. Allow
952 PHYSIOLOGICAL CHEMISTRY

to stand until contents separate into two distinct layers. Remove the
upper layer by the pipette, as shown in Figure 74, into a weighed
evaporating dish. Add 5 «cc. ether to the cylinder so as to wash down
the sides of the cylinder; remove by the pipette to the evaporating dish;
repeat this process of washing 5 times, using 5 e.c. each time and
removing each time to the evaporating dish. Evaporate the washings
and upper layer to dryness on the water bath, cool over sulphuric
acid in a desiccator and dry at room temperature to constant weight.
The weight of the residue may be taken as very nearly that of the total
lipin. It contains a slight amount of lactose, and the extraction of lipin
material is not quite so complete as the Soxhlet method.

Experiment 88. Determination of fat by Babcock method.—
Place 10 cc. of milk into the Babcock bottle (Figure 75) which has

 

Fia. 75.—Babcock’s cream test bottle.

a long, slender, graduated neck. Add 2 e.c. of a mixture of amyl alcohol
37 parts, methyl alcohol 13 parts and hydrochloric acid 50 parts; fill to
the neck with concentrated sulphuric acid, mixing as it is gradually
added. Centrifugalize rapidly for 3 minutes. This separates the fat.
Then add warm water sufficient to force the fat into the neck of the
tube and the percentage of fat can be read from the scale.

Experiment 89. Determination of lactose.—Transfer 2 c.c. of milk
by a volumetric pipette to a 100 ec. volumetric flask. Add saturated
NaCl solution to the mark. Shake. This separates casein and fat.
Filter through a dry filter paper. Take 50 ¢.c. of the filtrate (corre-
sponding to 1 cc. of milk) and determine the lactose by the Munson
and Walker-Bertrand method described in experiment 38, page 891.
The result gives the mgs. lactose in 1 ¢.c. milk, or grams in a liter.

Experiment 90. Determination of lactose in milk—(Folin and
Denis, J. Biol. Chem., 33, p. 521 (1918).)

Principle Lactose reduces the copper solution of Folin and McEIll-
roy and proteins do not interfere.
PRACTICAL WORK AND METHODS 953

\ Method. A 5 ee. burette is provided with a finely drawn out tip
attached to the ordinary tip by a piece of rubber tubing. The top
is stoppered and a glass tube is passed through it with a rubber
tube attached in order to fill the burette by suction (Figure 77). The

 

 

 

=)

 

Fie. 77.—Special sugar pipette used in Folin’s method (after Cole).

burette is filled to the zero mark by milk diluted 1:5 with water (10 c.c.
milk to 50 ¢.c. by water). Make the copper solution by placing in a
large test-tube 5 grams of the phosphate, thiocyanate mixture and 5 e.c.
accurately measured of 5.9 per cent. CuSO, solution and 1 ce. sat.
Na,CO, solution. Heat in micro burner until a clear blue solution is
obtained. Run in now from the burette 4 c.c. cow’s milk (about 3 cc.
954 PHYSIOLOGICAL CHEMISTRY

woman’s milk) and boil gently for 4 minutes. If the color nearly dis-
appears and the tube is filled with a white precipitate add a drop more
of milk at a time, boiling each time for 1 minute until the end point
is reached. The end point is shown by turning from a green to a yellow.
If too much sugar is added the first time so that the tube is completely
reduced by the first addition, discard the tube and begin again with
milk diluted somewhat more. This amount of copper solution, i.e.,
5 «c., is exactly reduced by 40.4 mgs. of anhydrous lactose under the
conditions of the experiment. This is about the amount of lactose
present in 1 cc. of milk. This amount of lactose is present in the
amount of diluted milk added. 4.04 x 5 (milk diluted 5 times) divided
by c.c. diluted milk used gives grams lactose in 100 ¢.c. milk.

1. Phosphate-thiocyanate mixture. 200 grams cryst. HNa,PO,.
12H,O powdered in a mortar are covered with 50 grams sodium
thiocyanate (60 grams potassium salt). Mix in mortar with spatula for
about 10 minutes until uniform liquid paste. Then add 120 grams
Na,CO,.H,O (or 100 to 110 of anhydrous salt) and mix with spatula
until a rather light granular powder is obtained. Cover with paper
and let stand over-night. Mix once more. Transfer to stoppered bottles.
It keeps indefinitely.

2. Saturated sodium carbonate solution. (14 to 20 per cent.
Na,CQ,.)

3. Copper sulphate solution. 59 grams CuSO,.5H,O and 2 ee.
concentrated H,SO, per liter.

Experiment 91. Determination of freezing point of milk—The
freezing point of milk is that of the blood and varies from —0.55° to
—0.60°. Any addition of water to milk is easily determined by a change
in the freezing point toward that of water. The apparatus most con-
venient for this purpose is that of Hortvet (Figure 76). The description
of the method is-that published to accompany the instrument.

Procedure. Insert a small caliber thistle-tube or funnel-tube into the
vertical portion of the T-tube at one side of the apparatus and pour in
about 400 ¢c.e. of ether previously cooled to 15° C. or lower. Close the
vertical tube by means of a small cork and connect the pressure bulb
or pump to the air inlet tube of the air-drying attachment on the op-
posite side of the apparatus.

Measure into the inner test-tube 30 to 35 ec. of boiled distilled
water, previously cooled to 10° C. or lower. Enough water should be
measured in to fairly submerge the thermometer bulb. Insert the ther-
mometer, which together with the stirring device is mounted in a sound
stopper, and lower the test-tube into the larger tube, which is tightly
fitted into the apparatus. A small quantity of alcohol, sufficient to fill
the space between the two test-tubes, will serve to complete the con-
PRACTICAL WORK AND METHODS 956

 

s

Fig. 76.—Hortvet cryoscope for determining freezing point of milk.

ducting medium between the interior of the apparatus and the liquid to
be tested. . A sufficiently tight connection between the inner and outer
tubes is afforded by means of a narrow section of thin walled tubing.
The thermometer and stirring device should fit accurately in the stopper
and the entire arrangement should be in a vertical position.

By means of the pressure pump maintain a steady current of air
through the apparatus, thereby vaporizing the ether at a fairly rapid
rate. Arrangement may be made so as to conduct the ether vapors away
from the operator and the APPARATUS SHOULD NOT BE USED
IN THE VICINITY OF A FLAME. Keep the stirring device in
steady up-and-down motion at a rate of approximately one stroke each
two or three seconds, or even at a slower rate providing the cooling
proceeds satisfactorily. Maintain passage of air through the apparatus
until the temperature of the ether-cooling bath approaches 3° below
zero, as indicated on the control thermometer, or until the top of the
mercury thread in the special freezing-point thermometer recedes to a.
point in the neighborhood of the probable freezing point of water. Con-
tinue the manipulation of the stirring device until a supercooling of sam-
$56 PHYSIOLOGICAL CHEMISTRY

ple from 1.2° to 1.3° is observed. Also note the temperature recorded on
the control thermometer in order to guard against excessive supercooling
and a consequent too low convergence temperature. As a rule, by this
time the liquid will begin to freeze, as may be noted by, the rapid rise
of the mercury thread. Manipulate the stirring device slowly and care-
fully two or three times while the mereury column approaches its highest
point. By means of a suitable light-weight mallet tap the upper end of
the thermometer cautiously several times in order to insure a permanent
position of the top of the mercury column. Observe the exact reading
on the thermometer scale and estimate to 0.001° C. After a few minutes’
time the mercury may begin to recede owing to the cooling effect of the
ether in the interior of the apparatus. When the above observation
has been satisfactorily completed make a couple of duplicate determina-
tions, then remove the thermometer and stirring device and empty the
water from the inner tube.

Rinse out the test-tube with about 20 ¢.c. of the sample of milk to
“be tested, measure into the tube about 35 ec. of the milk, or enough to
fairly submerge the thermometer bulb, and insert the tube into the ap-
paratus. In the meantime by lowering a narrow tube into the ether
bath, then closing the top end by means of the forefinger and raising
to a suitable height, a: judgment may be obtained as to whether an
additional supply of ether is necessary for the next determination.
Usually an additional 50 to 75 ¢.c. of cold ether should be, poured in
at this stage. .When the apparatus,has.once been cooled down to low
temperature an additional 50 c.c. of ether is sufficient on an average for
every four or five succeeding determinations.

Make the determination on the sample of milk, following the same
procedure as that employed in determining the freezing-point of water.
As a rule, however, it is necessary to start the freezing action in the
sample of milk by dropping in a small fragment of ice (approximately
0.05 gram) at the time when the mercury column has receded to a place
from 1.2° to 1.3° below, the probable freezing-point. A rapid rise of the
mercury column results almost immediately. Manipulate the stirring
device slowly and carefully two or three times while the mercury column
approaches its highest point. Complete the adjustment of the mercury
column in the same manner as in the preceding determination, then
observe the exact reading on the thermometer scale and estimate to
0.001°. THE ALGEBRAIC DIFFERENCE BETWEEN THE
READING OBTAINED ON THE SAMPLE OF WATER AND THE
READING OBTAINED ON THE SAMPLE OF MILK REPRE-
SENTS THE FREEZING-POINT OF MILK.

For deducing the proportion of. added water from the determined
freezing-point use Winter’s Table .ag published in extended form in
PRACTICAL WORK AND METHODS 957

The Chemical News, Vol. 110, 1714, pages 283-284, or use the porcelain
scale accompanying the cryoscope. The percentage of added water (W)

may also be calculated as follows:
100(T—T*)

w= F

in which T represents the average freezing-point of normal milk
(—0.550) and T* the observed freezing-point on a given sample.

EXERCISE XXVI. Mzat

To obtain fresh muscle kill a rabbit by a blow on the head, insert
a cannula in the carotid artery, and wash out the blood vessels after
opening the right auricle by injecting 0.9 per cent. NaCl solution. Skin.
Remove the intestines. (Glycogen can be recovered from the liver.)
Remove leg and back muscles quickly into twice their weight of ice-cold
5 per cent. MgSO, solution; when chilled put through a meat chopper
or mill. Put through at least twice, grinding them up with the cold
sulphate solution. This extracts from the muscle some of the proteins
in an unchanged form. Allow to stand in the sulphate in the ice box
for 12 to 24 hours. Filter cold through muslin. 10 cc. of filtrate is
given each student.

Experiment 92. Reaction—Test reaction of extract to litmus.
Usually amphoteric. An H ion determination can be made with the indi-
cator method.

Experiment 93. Clotting.—This extract,- like the blood, has the
power of clotting. (Myosinogen converted to myosin.) Dilute 2 e.c.
with 8 ¢.c. water. Take 5 ¢.c. in each of two test-tubes. Place one in
the incubator at 37°. Keep the other at room temperature. Both clot
and the myosin separates from the serum. The one at 37° clots first.
Take the reaction of the serum after clotting. It is acid. Add to it
3 or 4 small drops FeCl,. Observe lemon yellow color of lactic acid.
Evaporate another portion of serum or water bath. Lactic acid in
residue by Hopkin’s method.

Experiment 94. Myosin.—The clot consists of myosin. Separate it.
Observe that it is soluble in 10 per cent. NaCl.

Experiment 95. Temperature of coagulation—The remainder of
the undiluted plasma extract (8 ¢.c.) place in test-tube with thermometer,
suspend in a clamp and immerse in beaker of boiling water with the
bottom of the tube above the bottom of the beaker. Heat the water
gradually. Observe the temperatures of coagulation. 47° paramyo-
sinogen (Filter). 57° myosinogen.

The extractives of muscle——The principal substances in the ex-
tractives are creatine (the name of which signifies that it comes from
958 PHYSIOLOGICAL CHEMISTRY

muscle), hypoxanthine, inosite, and numerous other substances. Cre-
atine and hypoxanthine may be prepared in the following manner:
Experiment 96. Preparation of creatine.—Take 10 grams of Lie-
big’s beef extract, dilute to 200 c.c. with water. Add Ba(OH), solution
until plainly alkaline. Filter off the precipitate of phosphates, car-
bonates, etc. Free the filtrate from excess barium by saturating the solu-
tion with a stream of CO,. Filter from BaCO,. Evaporate filtrate in
evaporating dish on the water bath to a syrup, filtering from any further
precipitate of BaCO, which may come out on heating. Set aside the
syrup for a few days. Crystals of creatine appear in it. These are
rhombic crystals. See Figure 78. When crystallization appears to be

 

Fic. 78.

Fic. 78.—Crystals of creatine.

complete filter off through a small suction filter, wash the crystals with
alcohol, adding the wash water to the original filtrate. Save the filtrate
for hypoxanthine. The crystals may be recrystallized by dissolving
them in hot water, filtering and decolorizing if necessary with a small
pinch of charcoal, and evaporate the filtrate again on the water bath to
erystallization.

‘Experiment 97. Conversion of creatine to creatinine—Dissolve
the creatine obtained in 20 c.c. hot water. Make in 5 ce. of this
amount the tests for creatinine (Jaffe’s, Weyl’s). Creatine does not
give these tests. The other 15 c.c. convert into creatinine by heating
on the water bath in a small flask provided with a reflux condenser
after the addition of 15 c.c. N HCl for three or four hours. This makes
creatinine. At the end of that time neutralize the acid, and repeat
the creatinine tests with this solution.
PRACTICAL WORK AND METHODS 959

Experiment 98. Hypoxanthine.—The filtrate from experiment 96,
after crystallizing the creatine, contains a small amount of the purine
bases, xanthine and hypoxanthine. The latter used to be called sarcine
to indicate its origin in flesh. To separate hypoxanthine evaporate the
filtrate on the water bath to get rid of the alcohol, dilute with 20 c.c.
water and add to the solution ammoniacal silver nitrate solution as long
as a precipitate forms. This precipitates a compound of xanthine and
hypoxanthine and silver oxide, and silver compounds of any other purine
bases which may be present. Filter off the precipitate and wash with
water; transfer with the filter paper to an evaporating dish, add a few
c.c. 15 per cent. HNO,, heat to boiling for a moment to dissolve the pre-
cipitate and filter hot through a small folded filter paper. The hypo-
xanthine silver nitrate is insoluble in the cold and crystallizes out on
standing. Observe the crystalline form under the microscope. Long
slender prisms. Filter off the crystals through a small filter and save
the filtrate for xanthine. To prepare hypoxanthine, wash the crystals
with water, suspend in a little water, warm to dissolve same and pass
H,S to remove silver. Filter off silver sulphide, evaporate on water
bath until free from H,S, make slightly alkaline with ammonia, con-
centrate on water bath until odor of ammonia is gone, filter if necessary,
concentrate to a small bulk and put aside to erystallize. It comes out
in crusts or flakes made of small, colorless crystals. It is soluble in
300 parts of cold water.

Experiment 99. Xanthine.—The filtrate from the hypoxanthine
silver nitrate containing the xanthine silver nitrate is made slightly alka-
line with ammonia. The silver oxide xanthine is precipitated. Filter.
Wash with water. Suspend the precipitate in a little water and pass
H.S to free from silver. Have the solution warm. Filter from silver
sulphide and evaporate the solution to a small bulk on the water bath.
Put aside for crystallization. If xanthine does not crystallize out
evaporate further on the water bath until the xanthine begins to
come out in white deposits on the side of the dish. It is very
insoluble in water. With the xanthine make the murexide test, and
compare the reaction with that of uric acid. Also try the following
reaction, which is an oxidation to dialurie acid and alloxan with the
formation of ammonium purpurate. (Weidel’s reaction.) Oxidize the
xanthine to alloxan by dissolving it in a little bromine water and
evaporating on the water bath to dryness; now blow a little ammonia
fumes across the residue. They turn red, due to the formation of
ammonium purpurate (page 725).
960 PHYSIOLOGICAL CHEMISTRY

EXERCISE XXVII. Bone.

Bone is composed of an organic matrix which consists of protein
materials, collagen, elastin, chondrogen, mucoid, ete., and deposited in
this are crystalline inorganic salts of phosphate and carbonate of lime.

Experiment 100.—Place a small piece of bone (20 to 50 grams), rib
or femur of the rabbit killed for muscle, in 100 ¢.c. 10 per cent. HCl
and allow to stand at room temperature. Note the effervescence of the
carbonates. The acid gradually dissolves out the salts. After several
days when the salts are fairly well removed take out the organic matrix
of the bone and filter. Save filtrate for 102.

Experiment 1o1.—Organic matrix. Gelatine.—Observe that the
bone is still of its former shape and size but is now flexible and trans-
lucent. It is the organic matrix which gives toughness to the bone,
while the salts give rigidity. Wash off the acid, place the bone in 50 ce.
water in beaker and boil. Most of the bone will go into solution. The
solution on cooling gels. This is due to the formation of gelatine from
collagen. :

Experiment 102. The salts——The acid solution filtered in experi-
ment 100 contains abundant phosphates and calcium. There is not
enough phosphoric acid to form calcium phosphate with all the calcium
present so that some of the calcium must have been present as the car-
bonate. Determine the amount of phosphates in 10 ec.c. and also the
amount of Ca in the same amount. Determine the phosphates by the
method given on page 1109, experiment 304. The calcium is estimated
as given on page 1108, experiment 302. Calculate the amount of Ca
necessary to form Ca,(PO,), with the phosphoric acid present. The
excess has been present as carbonate. A little magnesium is present also.

EXeERcIsE XXVIII. Potato

Experiment 103. Reaction—Apply azolitmin paper or litmus to
the freshly cut surface of the potato. An acid reaction is usual in most
vegetables. Test for lactic acid by the thiophene test or Uffelman in
the filtered extract of experiment 104.

Experiment 104. Starch.—The principal constituent of the potato
is starch. Scrape into a fine pulp some of a potato and collect the
scrapings in a small beaker with a little water. The starch settles
quickly to the bottom. Take a drop out and observe under the micro-
scope. To the drop on the slide add a drop of KI, solution. Observe
the deep blue color of the grains.

Experiment 105. Sugar.—The potato also contains a good deal of
sugar, dextrose. Filter off a little of the potato juice and extract from
PRACTICAL WORK AND METHODS 961

the beaker, add a couple of drops of copper sulphate and make alkaline
with NaOH two drops, and heat. Reduction occurs.

Experiment 106. Protein——Transfer some of the decanted starch
and potato suspension to a test-tube, add a couple of ce. of glyoxylic
acid and pour underneath some concentrated sulphuric acid for the
tryptophane test. It is positive. Try also the Millon (strong) and
biuret.

Experiment 107. Oxidase—The fresh potato contains oxidase.
This is shown by its power of bluing tincture of guaiac without the
addition of peroxide or turpentine: To 3 ce. of the filtered extract of
the shredded potato add a couple of drops of guaiac tincture and shake.
Observe the deep blue color which develops. This is the sign of an
oxidase.

Experiment 108 Catalase.—Potato contains also catalase. To 3 cc.
of the filtered extract add a little of hydrogen peroxide. Observe the
effervescence of gas due to the catalase present. The gas is oxygen.
Catalase is found in all living tissues.

Experiment 109. Bread.—Test some small pieces of bread for re-
ducing sugar, starch and protein. See experiment 69 for isolation of
gliadin from fiour.
CHAPTER XXVII.
SALIVARY DIGESTION.
EXERCISE XXIX. SaALiva

Collect some saliva by chewing paraffine and expectorating into a
beaker. Collect about 25 c.c. of filtered saliva.

With this saliva carry out the following experiments:

Experiment 110. Reaction.—Test its reaction to litmus, phenol-
phthalein and congo red. Determine the concentration of hydrogen
ions in saliva by adding 1 ¢.c. phenolsulphonephthalein to 2 ec. of the
filtered saliva and comparing with the standard. See page 322.

Experiment 111. Mucin.— Precipitate 20 c.c. filtered saliva by add-
ing 4 volumes of 95 per cent. alcohol. Allow to settle, then filter
through a 7 em. filter and identify the mucin in the precipitate by the
following tests:

1. Dissolve the precipitate as fully as possible in a little water. To
a portion of the solution add, drop by drop, dilute acetic acid. A stringy
precipitate insoluble in acetic acid but soluble in 0.1 per cent. HCl
indicates mucin. In a little dilute sodium-carbonate solution the pre-
cipitate from the acetic acid redissolves to a slimy solution.

2. Make the biuret, Molisch and Millon tests with the solution.
The positive Molisch test shows the presence of carbohydrates in the
molecule. Mucin is a glycoprotein.

Experiment 112. Digestive action.—Saliva has the property of
dissolving starch and converting it to maltose, a reducing sugar. The
active principle, or enzyme, which causes the transformation of the
starch is called ‘‘ ptyalin.’’ For a fuller account of this substance and
the conditions of its activity see page 331. To illlustrate this action and
to study the conditions of activity of ptyalin, prepare about 100 cc. of
starch paste by the method given on page 963.

Experiment 113. Raw starch is not digested by ptyalin, except
very slowly.—Place a little raw starch, as much as can be held on the
point of a knife, in 5 c.c. of water in a test-tube and add 1 ec. of the
filtered saliva. Mix and allow the two to act together for 30 minutes or
longer. At the end of that time test the reducing action of a portion of
the solution by Fehling’s solution as indicated in experiment 11. Com-

pare the result with the action on boiled starch. The failure of the
962
PRACTICAL WORK AND METHODS 963

enzyme to digest the uncooked starch is due to the fact that the outside
of the grain of starch is either cellulose or some other carbohydrate not
easily penetrated by the ptyalin. If this is broken by chewing, etc.,
then the starch digests. To show this take some of the same kind of
starch used in the preceding experiment, chew it thoroughly for a few
moments, expectorate it into a test-tube, dilute with a little water and
test for a reducing sugar. The test should be positive.

Experiment 114. Starch paste is digested with great speed by
ptyalin.—Take 5 c.c. of the 1 per cent. starch paste in a test-tube, add
about 1 ec. of the filtered saliva, mix thoroughly by inverting the tube
with the thumb held over the end. Then at once heat the solution to
boiling in the Bunsen burner in order to stop the action of the ptyalin.
Notice the very rapid clearing of the starch solution when the saliva
is added, if the saliva is active. Now take about 3 c.c. of the solution,
add 3 ec. of the Fehling mixture and boil. A copious reduction to the
red cuprous oxide is obtained if the saliva is normally active. This
shows that saliva forms a reducing sugar from the starch at the very
start of its action. Try to see how short the time of contact of the
saliva and starch can be and still result in the formation of a reducing
sugar, maltose. The action of the saliva can be stopped most easily
and instantaneously by making the mixture fairly alkaline with NaOH.
The great speed of the action on cooked starch contrasts strongly with
the slowness of digestion of uncooked.

Experiment 115.—Formation of dextrins from starch.—Place on a
white porcelain plate several drops of KI, solution. Now place in a test-
tube 10 c.c. of starch paste and 2 ¢.c. of saliva, mix well and place in
the water bath at 38-40° C. From time to time remove a drop on a glass
rod and touch it to a drop of KI, on the plate. At first the color will
be pure blue, that of starch iodide itself. Gradually the color will
change to a violet, then to a reddish or reddish brown and finally the
red color will be given more and more faintly and ultimately it will dis-
appear. The time when this happens should be noted. It is the achromic
point. The time taken to reach this point under similar conditions of
experimentation may be taken as a measure of the digestive strength of
the saliva. The red color is due to erythrodextrin, or red dextrin, the
colorless solution still contains a colloidal dextrin, achroodextrin.

Experiment 116. Determination of the H ion concentration most
favorable to the digestion of starch by saliva—Saliva digests best in
a very feebly acid medium, that is a medium acid to litmus but alkaline
to congo red.

Take of the buffer solutions into separate test-tubes 5 c.c. each of the
buffers containing the following H ion concentrations (P,), ie., 8.0, 7.4,
6.8, 6.0, 5.3, 5.0. To each tube add 1 ec. of 1 per cent. starch paste and
1 ce. of NaCl 0.9 per cent. solution. Then add to each tube, as nearly
964 PHYSIOLOGICAL CHEMISTRY

simultaneously as possible, 1 ¢.c. of fresh saliva diluted to 5 volumes with
water. Immerse all in a beaker of water at 37° and from time to time
remove a drop of the mixture of each tube by means of a glass rod-and
mix it with a very small drop of KI, solution on a porcelain plate.
Observe which tube gives a pink and then a colorless solution in the
shortest time. When one of the tubes has apparently reached, or nearly
reached, the achromic point, remove all from the water bath (this will
be usually after about 10-15 minutes) and add to each tube two drops
of KI,, adding with a 1 «ec. pipette so that the drops are not large.
Now compare the colors of the different tubes after mixing. One tube,
i.e., that of the most favorable H ion concentration, will be nearly color-
less, the others will be the deeper blue the slower they have digested.
Record which tube digests best. Michaelis states that 6.7 is the most
favorable concentration. Do you agree with this? This experiment is
of importance since digestion takes place by the saliva in the stomach
under conditions of slight acidity.

Directions for making the buffer solutions are xivcen on pages 544
and 1122.

Experiment 117. The action of the saliva is lost by boiling.—Heat
some saliva to boiling and then mix it with starch paste under the same
conditions as in experiment 115. From time to time test the iodine
reaction and see if any reducing substance (maltose) appears in the
mixture. The fact that the property of the saliva is lost by heating
shows that the substance which is active is unstable, heat labile and, since
a little of it converts a great deal of starch, it is called an enzyme.

Experiment 118. Salts are necessary for the action of ptyalin.—
Dialyse some saliva until it is free from chlorides. Prepare two tubes
of 5 ec. each of 1 per cent. starch solution. To one (A) add 1 ec.
N NaCl solution; to the other add 1 e.c. distilled water. Now add to
each tube 1 ¢.c. of the dialysed saliva and immerse in water heated to
37°. Observe the course of the digestion in each by taking a drop from
each tube from time to time and touching the drop to a drop of KI, solu-
tion on a porcelain plate. The digestion goes most rapidly in the tube to
which salt has been added and the color changes from a blue to a red and
finally the achromic point is reached. The dialysed saliva loses its activ-
ity but this is restored by the addition of NaCl, among other salts.

Experiment 119. Excretion of salts in saliva—The saliva contains
many substances which are adventitious, excretory substance. Iodides
pass readily into the saliva. By swallowing sodium iodide (3 grains) in
capsules and noting the time at which the iodine reappears in the saliva,
an idea may be had of the motor activity of the stomach, since the absorp-
tion is largely from the intestine. The iodides must be passed through
the stomach before they are reabsorbed. To test for iodine in saliva add
to it a little starch paste and at once thereafter a drop of FeCl, solution
or a drop of HNO, solution and acidify.
CHAPTER XXVIII.
GASTRIC DIGESTION.

Exercise XXX. PEPSIN, PEPSINOGEN, RENNIN AND HCl.

On account of the difficulty of obtaining sufficient quantities of
gastric juice, the experiments which follow will be made with artificial
gastric juice. In making this juice it is best, if the mucous membranes
of the hog’s stomach can be obtained, to make the juice by extracting
the dried or undried mucous membrane with 0.4 per cent. HCl. A large
amount of the mucous membrane can be dried on glass plates, then
extracted with gasoline and ground and kept on hand. If mucous mem-
branes cannot be obtained, a similarly acting juice may be made from
commercial pepsin preparations, which are generally made from hog’s
stomach mucosa by allowing it to digest with acid, dialyzing out much
of the protein digestive products and drying the pepsin solution on
glass plates at low temperatures. This makes scale pepsin.

Experiment 120. Reaction of the mucous membrane.—Test the
reaction of the fresh mucous membrane of the hog’s stomach to litmus.
It will be found to be either very faintly acid or slightly alkaline. There
is no storage of hydrochloric acid in the membrane, although it secretes
a juice strongly acid with HCl.

_ Experiment 121. The active digestive principle of gastric juice
is stored in the mucosa and digests proteins.—That the mucous mem-
brane contains pepsin which digests proteins in an acid solution may be
shown as follows:

Take 50 grams of hashed pig’s stomach mucosa, add to it 250 cc.
of 0.4 per cent. HCl, place it in a flask and allow it to digest itself for
24 hours at 37-40° C. .At the end of that time filter the solution from
the undigested residue and test the solution for the metaproteins: acid
albumin (precipitation on exact neutralization to limus), protalbumose,
deutero-albumose and peptone. The peptone and peptides can be re-
moved from the solution without injuring the pepsin by dialysis. The
presence of these digested proteins, when the digestion has occurred in
4 per cent. HCl, shows the enzyme to be pepsin.

Experiment 122. Existence of pepsinogen.—Principle and object.
This experiment constitutes the chief evidence that pepsin exists in

the mucous membrane of the stomach in an inactive, more resistant
965
966 PHYSIOLOGICAL CHEMISTRY

form, which has been called pepsinogen. The evidence consists in the
fact, shown by this experiment, that a neutral infusion of the stomach
mucosa can be made alkaline by sodium carbonate without permanently
losing its activity. That is, digestive action returns when it is reacidi-
fied; whereas an acid infusion, or a neutral infusion which has been
made acid by HCl and consequently contains active pepsin, cannot
be made alkaline without losing the greater part of its activity. The
pepsin is evidently very sensitive to alkalies; whereas the substance in
the neutral infusion is not nearly so sensitive. This inactive precursor,
or form, of pepsin is called pepsinogen, the theory being that it goes
into pepsin by the action of acid.

To make the neutral infusion. To 0.5 gram of dried, fat-free, pow-
dered hog stomach mucosa add 50 e.ec. distilled water. Mix well and
allow to stand for 20 minutes. Then strain through cheese cloth.

The experiment. Measure four 5 c.c. portions of this infusion into
four test-tubes and label them (a), (b), (c) and (d).

(a) To (a) add 4 ¢c. water and 1 ec. N Na,CO,. Allow to stand
for 15 minutes at room temperature, then make it acid by the addition
of 3 «ec. NHCI and dilute to 20 cc. This tube now contains N/10 HCl.

(b) To (b) add 2 ce. N HCl and dilute to 20 cc. This tube also
contains N/10 HCl, approximately 0.36 per cent., but the infusion has
not been alkaline at any time.

(ec) To (ce) add 0.6 «ce. N HCl and 0.4 cc. H,O, so that the total
volume is 6 ce. After standing 15 minutes at room temperature, add
1.6 «ce. N Na,CO, and dilute to 10 cc. Allow to stand for 15 minutes.
This tube now contains the same concentration of N Na,CO, that (a)
did, but the solution has been exposed to acid for a time, so as to form
pepsin. Now add to this tube, after standing 15 minutes, 3 ec. N HCl
and dilute to 20 ec. with water. This tube also now contains
N/10 HCL

(d) To (d) add 1 ee. N NaCl and dilute to 20 ce.

Transfer 10 ¢.c. samples of these solutions to four dry test-tubes,
label each accurately, add a piece of fibrin about the size of a pea to
each and digest at 35-40° C. at the same time and for the same length
of time. Observe the digestion in each tube. (b) should be the fastest,
but compare especially the rate of digestion in (a) and (b).

Experiment 123. To show the optimum concentration of acid and
hydrogen ions for peptic activity and the action of different kinds of
the same concentration. —Principle. This experiment illustrates a very
interesting fact which is apparent everywhere in nature: namely, that
all kinds of living phenomena take place at their optimum under the
conditions which prevail in nature. This is called ‘‘ adaptation.’’ \t
may be observed in this experiment that the digestion goes best at the
PRACTICAL WORK AND METHODS 967

concentration of HCl normally present in the stomach; and that the
most favorable acid is HCl, the acid normally present.

Prepare the following mixtures in large test-tubes. For the pepsin
solution take some of the artificial juice prepared in expt. 121 and add
thereto Na,CO, solution until the solution is no longer acid to congo red,
but is still acid to htmus. After the contents of each tube have been
well mixed, add to each an amount of fibrin equal in volume to a small
pea. Shake each tube from time to time and digest at 35-40° C. for
10-20 minutes.

 

 

ae wjtiee Firet addition Second addition Calculated HCl waishivecuetuits
BD. “Sehacereie 5 5 H,0 5 H,0

DD ena 5 98 “ 0.2 N HCl

Crd arsvseaueves 5 95 “ 05“

Oxo seee 5 93 “ O7“«

Cl ~ S46 wdtiete 5 90 * qeOre 86

Peed Saks 5 8251-7 La #

BD ieiac g 8.0 20 «

Be aaierd seers 5 6.0 “ 4:0) 6

 

 

Determine the hydrogen ion concentration in that tube which has
digested best by filtering off 5 ¢.c., adding to it 2 drops of 0.01 per cent.
solution of gentian violet and matching the color of the tube with
a series of tubes each containing 5 c.c. and 2 drops of 0.01 per cent.
gentian violet but containing the following concentrations of HCl:
0.01N; 0.03; 0.05; 0.07; 0.10; 0.15N. What is the most favorable H
ion concentration for peptic digestion?

The action of other acids in peptic digestion. Prepare a set of large
tubes as above, one with the optimum amount of N HC] added and the
others with the same volumes of N HNO,, N H,SO,, N oxalic, N lactic
and N acetic respectively. Then add fibrin and digest as above. Do
all the acids act equally well?

Exercise XXXI.

Experiment 124. Pepsin quantitative determination.—The quanti-
tative determination of pepsin is made by one of the following methods.
Most of these are adapted for clinical examination of the gastric juice.
The activity of the juice is compared with the activity of a standard
solution of pepsin.

a. Jacoby’s method. 0.5 gram of ricin are dissolved in 50 cc. of
a 5 per cent. NaCl solution and filtered. To the opalescent liquid
enough N/10 HCl is added to make it slightly cloudy, and 5 cc. is
placed in each of 10 test-tubes in the water bath at 38° C. The stomach
juice, or the pepsin solution, is added in decreasing amounts to each
‘of the tubes and the tubes are made up to the same volume by the addi-
968 PHYSIOLOGICAL CHEMISTRY

tion of distilled water or cooked juice. The greatest dilution which
after three hours in the thermostat clarifies the ricin shows the lowest
concentration of juice which can fully digest this amount of protein.
If 1 ce. of juice diluted 100 is able to do this, it contains 100 pepsin
units. If the total acidity of the juice is normal and equal to 40-60 c.c.
of N/10 NaOH, the juice, if normal, should contain 100-200 peptic
units. In hypo acidity the pepsin is generally reduced; in hyper acidity
it remains about the same. In the absence of HCl there may still remain
a very weak digestive action.

*b. Mett’s method. Procure two glass tubes (2-8 mm. internal
diameter and 7 em. long) from the storeroom, draw up fresh egg white
in the same until filled; then place horizontally in water which is boiling
hot, remove the flame and allow the tubes to remain in the water for
10 minutes. Remove and allow to cool. When the solutions, etc., below
are all ready for use, cut the capillary tubes into lengths of approxi-
mately 1 em. each. The coagulated egg albumen must be free
from air bubbles and the broken surfaces must be even with the glass
ends.

Prepare two 34-inch flat-bottom vials as follows:

(a) 0.5 ee. 0.3 per cent. solution of 1:3,000 U.S.P. pepsin in 0.2
per cent. HCl plus 4.5 ¢.c. 0.2 per cent. HCl and 2 Mett’s tubes.

(b) 1.0 ¢.c. unknown solution in 0.2 per cent. HCl plus 4.0 ce. 0.2
per cent. HCl and 2 Mett’s tubes.

Rotate gently, stopper lightly with cork and set aside at 35-40° C.
for 24 hours. Gas bubbles may cling to the ends of the tubes, be sure
to brush these off with a fine wire before setting aside to digest. The
Mett’s tubes must lie flat on the bottom of the tube. Remove all the
tubes at the.same time and place on a millimeter scale, then by means
of a lens measure the lengths of albumen dissolved at both ends of each
tube. This gives four readings for each solution tested. Take the aver-
age of the four readings.

The above conditions are such that the Schiitz law holds. What is
this law and what are the conditions under which it holds? Calculate
the per cent. concentration of the unknown solution in 1:3,000 U.S.P.
pepsin. The U.S.P. standard of 1:3,000 means that 1 part of pepsin
dissolves 3,000 parts of coagulated egg white in 24% hours when-tested
by the U.S.P. assay method.

ce. Edestin method (Fuld). The most accurate, quick and con-
venient method for estimating the amount of pepsin present in gastric
contents or any other liquid is by means of edestin. Since the early
stages of digestion are most accelerated by pepsin the method chosen
should show this change. This is the case in the edestin method. The
following method is practically that of Fuld.
PRACTICAL WORK AND METHODS 969

Dissolve 0.1 gram of edestin in 100 ¢.c. 0.1 N HCl. Heat if necessary
to dissolve. If 2 ec. of this solution are superimposed in a test-tube
above 2 ¢.c. of saturated NaCl solution, a white ring of edestin hydro-
chloride appears at the zone of contact, and if the solutions are mixed
a white cloudiness is produced. When edestin is digested this ring or
cloudiness no longer appears and the method consists in comparing the
time necessary for the unknown to digest the edestin to a point where
it no longer forms the ring and comparing this time with the time taken
by a solution of pepsin of known strength. The amount of pepsin in
the two solutions is inversely as the time.

To make the test, first prepare a standard solution of pepsin by
dissolving 0.01 gram of a good commercial pepsin (strength of 1:3,060.
See final paragraph of the Mett method) in 10 «ec. of N/10th HCl. Do
not. heat this. Transfer of the edestin solution 25 ec. to each of two
90 ec. flasks. To one of these add 1 ¢.c. of the standard pepsin solution
and 4 cc. of H,O, and to the other 5 c.c. of the unknown solution
(filtered stomach contents, etc.) note the time of addition, mix, and let
stand at 35° C. At 15 minute intervals remove 2 ¢.c. from each flask
and superimpose it upon 2 ¢.c. saturated NaCl in a test-tube and note
the time at which the edestin is so digested that it no longer gives a
white ring. This will take for the standard about 25 minutes. If the
unknown takes twice as long, the concentration of pepsin in it is 1/10th
that in the standard, since the amount taken was 5 c.c. as compared
with 1 ¢.c. of the standard.

The following experiment (Miss Kronacher) shows the relationship
between the concentration of pepsin and the time of digestion of edestin:
(Temp. 24° C.)

 

 

Concentration of pepsin a Time in a to " Time x
sappearance 0 estin pptn.
(Armour’s scale) 01% edestin in M/10 HCl concentration
.008% 25 0.200
004% 50 0.200
.002% 100 0.200
.001% 205 0.205

 

 

Experiment 125. The coagulation of milk by rennin. Rennin is
activated by HCl, like pepsin.—Principle. This experiment is de-
signed to show that mucous membranes of calves’ stomachs contain an
enzyme or active principle which coagulates milk in a neutral solution,
and that it exists in the mucosa in an inactive form, being activated
by HCL.

(a) To 0.5 gram dried, fat-free, powdered calves’ rennets add
x @c. of water. The value of x will be given you by the instructor. It
970 PHYSIOLOGICAL CHEMISTRY

has to be determined by a preliminary experiment, as the rennet activity
is variable, and the condition of the milk varies. It should be so chosen
that the time of coagulation in the following most favorable tube is
between 5 and 10 minutes. After standing 15 minutes strain through
cheese cloth. Take three 5 ¢.c. samples and treat as follows, making all
measurements very carefully:

(b) To 5 ce. add exactly 45 cc. distilled water and mix well.

(ec) To5 ce. add 1 ec. N/10 HCl, and after 5 minutes gradually
add while stirring 1 c.c. N/10 Na,CO,;. Next add 48 c.e. distilléd water
and mix well.

(d) To5 cc. add 1 ee. N/10 NaCl and after 5 minutes add 44 ce.
distilled water and mix well.

Now determine how long it takes for each (b), (ce) and (d) to curdle
milk when the following conditions are observed. Measure into three
clean, dry, large (l-inch diameter) test-tubes (large tubes are used so
as to permit of instantaneous mixing) 10 ¢.c. samples of sweet, well-
mixed milk. Heat each to 40° C. When all the tubes are ready
add 1.0 ec. of (b), (ce) and (d) to the respective tubes; mix
immediately after the addition and accurately note the time to within
5 seconds.

_ Place all in the bath at 40° C. Now do not shake the mixture, but
remove the tubes one by one from the bath from time to time (every
30 seconds), incline the tubes gently and note the time in each tube
when the curd breaks away smoothly from the side of the tube. Write
your own explanation of why the milk is solidified at different rates in
these tubes.

Experiment 126. Relation of the time of coagulation to the
amount of rennin. Time law.—Take 5 ¢.c. samples of (c) above and
prepare dilutions as follows:

(e) 5 «ae. plus 2% c.c water and mix well.

(f) 5 «ec. plus 5 ¢.c. water and mix well.

(g) Now determine how long it takes for 1.0 e.c. of (ce), (e) and (f)
to eurdle 10 cc. of milk at 40° C. Is the time of coagulation inversely
proportional to the amount of rennin?

Experiment 127. Calcium salts are necessary for the coagulation
of milk, but not for the action of the rennin on casein.—Prepare large
test-tubes as follows:

 

 

Tube Milk Other addition
Bs cee Gab eaeinedten nin cone 10 cc. [2 ce. 2% (NH,),C,0, Sol.
Big notes doasstarns ors warner “«“ l4 ec, H,0
Gog Ane at ae had eres Mes neh “ © Wee, 5% CaCl, Sol. + 2 ¢.c.2% (NH,),C,0, Sol.

 
PRACTICAL WORK AND METHODS 971

‘Warm to 40° C. and keep in a bath at 40° C. To each tube now add
sufficient of solution (c), experiment 125 above, to cause the contents
of the tube (b) to curdle in 7-12 minutes. As soon as (a) has
‘stood with the enzyme solution the length of time it required (b) to
curdle, at once add 2 ec. 2 per cent. CaCl, solution to (a),
mix well and note whether coagulation now occurs. This tube should
coagulate at once on the addition of CaCl, if the experiment is accurately
performed.

Experiment 128. The calf’s stomach extract has a weak peptic
--action, but a strong rennin action; the conditions are reversed in the
pig’s stomach.—Prepare 50 ¢.c. 1 per cent. water extract of dried calves’
rennets and 50 «.c. 1 per cent. extract of dried hogs’ stomach mucosa.
Allow each to digest. for 15 minutes, then filter and measure off accu-
rately (without contaminating one solution with the other) into dry
vessels two 10 ¢.c. portions of each. To each 10 c.c. portion now add
2ec. N HCl. After standing 1 to 5 minutes, add carefully 2 cc.
N Na,CO, to one tube from each set and, after mixing each well, com-
' pare these as to curdling action on milk.

After 5 to 30 minutes add to the other 10 ¢e. portions of acid’
solutions 10 ¢.c. distilled water, mix each well, warm to 40° C. and
add the same amount.of well-washed fibrin to each. Digest at 40° C.
and note from, time to time the relative rates at which the fibrin is
dissolved.

Exercise XXXII. ExXaMINATION OF GASTRIC CONTENTS.

Test meal. The supper should have been light and nothing should
have been eaten for 12 hours before the test meal. In the morning a
roll and a piece of bacon are thoroughly chewed and swallowed and a
cup of weak tea or coffee or water taken. Forty-five or sixty minutes
later the stomach tube is swallowed, the Rehfuss tube (Figure 79), and
the contents obtained by suction by a syringe. The amount, clarity, odor,
appearance of the contents should be noted. Any blood or hematin
should be noted. Occult blood may be tested for by the benzidine
reaction. The principal information desired is as to the hydrochloric
acid. The following determinations should be made:

1. Total acidity.

2. Free hydrochloric acid.

3. H ion concentration.

4. Presence of lactic, butyric or other acids showing fermentation.

5. Pepsin may be tested for in the manner described but usually
the pepsin content goes parallel with the acid, so that if the HCl is
normal the pepsin is also.
972 PHYSIOLOGICAL CHEMISTRY
6. A microscopic examination should be made for parasites, sar-

cine, molds, ete.
7. Combined HCl may also be determined.

nA A

 

 

 

 

 

 

 

 

A B
Fic. 79.—Stomach tube. A, Rehfuss. B, Lyon’s modification of Rehfuss.

Practice may be obtained in making these tests by examining a
sample of artificial gastric juice made up as described in experi-
ment 123.

Method of obtaining stomach contents. Stomach contents for
analyses are readily obtained without serious discomfort to the patient
by the use of the Rehfuss tube or the Lyon modification of the samé.
This tube is a very small soft rubber tube having on the end a metal
attachment provided with holes or slits through which the stomach
contents enter the tube. To obtain stomach contents proceed as follows:
The contents of the stomach should be withdrawn about 45 minutes,
usually after the meal, but of course it may be desirable in special cases
to allow other times to elapse. The patient is seated in a chair with
PRACTICAL WORK AND METHODS 973
a towel to protect the clothing in case of vomiting and with a receptacle
of some sort to receive the vomit in case of vomiting. The tube is
cleaned and sterilized, preferably while it is wrapped in a towel or a
piece of muslin. It is moistened with glycerine to make swallowing
easy and the patient is instructed to swallow promptly when told to
do so. The metal bob at the end of the tube is then carefully intro-
duced into the mouth, but is not permitted to touch the fauces until just
at the moment of swallowing. It is then laid on the back of the tongue
and, at the moment of laying, the patient is told to swallow and as he
does so the tube is passed quickly back through the fauces. At this
moment there will be some sensation of gagging, but as soon as the
metal part is in the esophagus that sensation passes. From time to time
after the tube is passed the patient is told to swallow and also to take a
deep breath. Usually little or no discomfort is experienced after the
first moment and the tube passes freely down to the mark placed upon
it. A Luer or other syringe is then introduced into the end of the
tube, which falls to the patient’s lap, and the flow of gastric contents is
started by withdrawing the plunger of the syringe. The contents may
be pretty thick and come through the tube with some little difficulty.
About 20 to 50 e.c. is ample for all the analyses. Having obtained the
contents the tube is withdrawn, usually producing gagging again when
it passes the fauces. The tube should be at once cleaned by a rapid
stream of hot water being forced through it. The Lyon modification
of the Rehfuss tube is easier to clean and does not so easily choke as
the original Rehfuss.

For the analyses of the contents, filter the same and use the ‘filtrate.
Determine in it the total acidity, the acidity by Tépfer reagent (dimethyl
amino azobenzene), free hydrochloric acid by Giinzberg, the hydrogen
ion concentration, and make tests for lactie acid. This is sufficient for
ordinary clinical purposes. The presence of a mineral acid, probably
hydrochloric, can be almost immediately determined by touching a drop
to congo red paper and if this turns blue free hydrochloric acid is
present.

Experiment 129. Total acidity—(Normally this should be 60-120
ec. for 100 ec. juice.) Titrate 10 ec. of the filtered gastric contents
with N/10 NaOH, using phenolphthalein as an indicator. The total
acidity is often expressed as grams of HCl in 100 ec. of juice or as c.c.
of N/10 NaOH necessary to neutralize 100 cc. of juice. To get the
former number multiply the number of ¢.c. of NaOH required to neu-
tralize the juice with the factor 0.0365, since each ec. of N/10 HCl
would contain this number of grams of the acid. The acidity is some-
times expressed as c.c. of N/10 NaOH required for neutralizing 100 e.c.
of juice. Why is phenolphthalein chosen as the indicator for total
974 PHYSIOLOGICAL CHEMISTRY

acidity? It is usually advisable to filter the juice, as the particles in
suspension in the juice may make a considerable part of its volume,
although on filtering a slight loss occurs in the acid attached to the sub-
stances in suspension.

Experiment 130. Free HCl.—The estimation and detection of free
HCl is made by means of the Giinzberg test as follows (see page 368
for the composition of the reagent): One drop of the reagent is placed
on a white porcelain plate or evaporating dish and dried very cautiously
over a free flame or better on a water bath. It must not be heated toc
high. Then add one drop of the filtered juice to the clear yellow spot
left by the evaporation of the indicator and warm again carefully. If
hydrochloric acid is present in the free state, a purplish red color
develops on heating. If the spot is heated too much, it will be brown.
On diluting the gastric juice and repeating the test, a limit will be
found when the reaction is just perceptibly positive. At this dilution
the juice contains about N/2,500 HCl.

a. Confirm the accuracy of this test for free HCl by repeating it,
using N/300 and N/2,000 HCl; with a mixture of N/30 acetic acid in
which a little NaCl has been dissolved; and with N/10 lactie acid.
Which is positive?

b. To show that the presence of proteins reduces the amount of
free hydrochloric acid, add to 10 c.c. N/30 HCl 10 e.c. of a 1 per cent.
solution of Witte’s peptone. Mix. Determine by the Giinzberg reagent
what the amount of free HCl is now in the solution.

ec. _ Titrate 5 ce. of the mixture from b with N/10 NaOH, using
phenolphthalein as an indicator. Has the total acidity been reduced by
the addition of the peptone?

Experiment 131.—Titrate another 10 ¢.c. with N/10 NaOH, using
di-methyl-amino azobenzene as an indicator (Tépfer’s test). How do
these figures compare with the total acidity and with the acidity as
determined by Ginzberg? Normal juice has an acidity of from 50-90
c.c. N/10 HCl by this reagent, depending on period of digestion. It is
higher at the end of digestion.

In place of determining the free HCl by the dilution of the gastric
juice, it can also be determined by titrating by the addition of N/10
NaOH and determining the end point at which the free HC! is neu-
tralized by testing from time to time 1 drop of the liquid by the
Giinzberg method. This method is perhaps more tedious than the dilu-
tion method.

For clinical purposes the Giinzberg, the total acidity, and the Tépfer
titration are all that are required. By consulting the tables on page 368
it will be seen that the Topfer titration, ie., the dimethyl-amino azo-
benzene, shows an amount of acid between the free and the total acidity.

aside -
% PRACTICAL WORK AND METHODS 975
It gives an imperfect notion of the amount of free HCl. It is not
affected by organic acids, except when they are present in high concen-
tration. The concentration of hydrogen ions at which it changes color
may be seen in table, page 371, to be N/10#°—N/10*. It requires a
little higher concentration than congo red.

Experiment 132. Free HCl by Tropxolin OO.—The determina-
tion of the free HCl by tropxolin gives the same results as Giinzberg,
but the latter is sharper. Some drops of saturated solution of tropeolin
OO in 94 per cent. alcohol are placed on a porcelain plate and dried
at 40° C. To the dried spot a drop of the liquid to be tested is added
and again dried at 40° C. In the presence of as much as 0.006 per cent.
HCl a violet spot is left when the liquid evaporates.

a. Control this test with dilute HCl, lactic acid, ete.

Boas reagent for free HCl is made by dissolving 5 grams resorcinol
and 3 grams sucrose in 100 e.c. 50 per cent. alcohol. Used like Giinzberg.
Free HCl gives a rose red.

The student should read the action of indicators in one of the following:
Stieglitz’s Qualitative Analysis, Part I; Jones’ Elements of Physical Chemistry ;
Smith’s Introduction to Inorganic Chemistry.

Experiment 133. Hayem and Winter method for the determina-
tion of hydrochloric acid—This method requires the facilities of a
chemical laboratory. It is based on the determination of chlorine.
Three portions of 5 ¢.c. each of stomach contents are placed in three
crucibles, a, b and «. To (a) is added an excess of sodium carbonate
and all three are dried on the water bath and then in an oven at 100°
until completely dry. The crucible (a) is then ashed over a free flame,
very lightly, and the ash extracted with water. The chlorine is then
determined in the water by silver nitrate titration. This gives the total
chlorine, both acid, free and combined, and sodium chloride. The
second crucible (b) as soon as completely dry has Na,CO, added and
ashed and the chlorine is determined as in (a). By heating on the
water bath and at 100° the free hydrochloric acid is driven off. The
difference between (a) and (b) gives, then, the free hydrochloric acid.
The third crucible (c) is ashed directly without the addition of the
carbonate. By this only the chlorine of the sodium chloride is left.
The difference between (b) and (c) gives, then, the combined HCl.
This method, however, is only approximate, since in the ashing some
acid phosphate is formed which will expel some of the chlorine from
the sodium chloride. Moreover, in the drying some of the combined
hydrochloric acid is set free and goes off.

Experiment 134. Lactic-acid detection—By fermentation of the
carbohydrates in the stomach, when the hydrochloric acid is low, a
considerable degree of acidity may be formed, due to lactic or other
976 PHYSIOLOGICAL CHEMISTRY

organic acids. It is sometimes desirable to examine the contents for
lactic acid. The lactic acid may be detected either by the decolorization
of phenol-ferrie-chloride solution (Uffelmann’s reaction) or by Hopkins’
method. The latter is the better.

a. By Uffelmann’s reaction. The reagent is made by taking 10 c.c.
of 4 per cent. solution of carbolic acid, 20 c.c. of distilled water and 1
drop of a 2 per cent. ferric-chloride solution. This solution has a deep
violet color, due probably to the partial reduction of the iron. The
addition to this solution of a solution of lactic acid, even very dilute
(1 part in 10,000), causes the color to change to a yellow. To 3 cc. por-
tions in test-tubes add respectively 1 drop of N/10 and N/100 lactic
acid and N/10 and N/100 HCl. Note yellow color with lactic. The re-
action is not specific for lactic acid, but it is not given by dilute HCl.
Now see if the filtered gastric contents discharge the color from Uffel-
mann. It is better to shake out the contents with ether, in which the
lactic acid is soluble, evaporate the ether on the steam bath in a beaker,
dissolve the syrup, if any, which is left in a little water and repeat the
test with this. Keep a portion of the ether extract for testing by
Hopkins’ method, which is as follows:

*b. Hopkins’ method of detecting lactic acid. This depends on
the cherry-red color developed by thiophene.

Into a clean, dry test-tube place 3 drops of a 1 per cent. solution
of lactic acid in alcohol, add 5 e.c. of concentrated sulphuric acid and
3 drops of a saturated solution of CuSO,. Mix and place in a beaker
of boiling water and heat for 5 minutes. Cool under the tap and add
2 drops of a 2 per cent. alcoholic solution of thiophene and shake. Re-
place the tube in boiling water. A cherry-red color develops.

ce. Repeat by using in place of the lactic acid that recovered from
the gastric contents by ether. It is not probable that this method either
is specific for lactic acid, but it is the only substance in gastric contents
which gives the reaction.

Experiment 135. H ion concentration.—This is most easily made
by placing 5 ec. of the filtered contents in a test-tube and adding 2
drops of 0.10 per cent. gentian violet solution. This tube is then
matched by comparing it with a series of tubes containing each 5 c.c.
of HCl of the following concentrations: 0.01N; 0.02N; 0.04N; 0.06N;
0.1N; 0.15N. If the color is a blue or green it shows that the concentra-
tion of the H ions is more than 0.05N; normal stomach contents have
a concentration of H ions, usually between 0.03 and 0.05 or even 0.1 at
the end of digestion. If the color of the gential tube is purple it means
that the acidity is lower than normal.

/

!
CHAPTER XXIX.
INTESTINAL DIGESTION. BILE. PANCREAS.
EXERCISE XXXII. PANCREAS AND DUODENAL SECRETION.

Pancreas.—The three juices bile, pancreatic and duodenal are mixed
with the chyme, and digestion is the result of their combined actions.
The: different juices will here be studied separately. Owing to the
impossibility of obtaining pancreatic juice for experimental work, it is
necessary to use extracts of the pancreas and the duodenal mucosa.
These act, on the whole, like the secretions, since the active principles
or enzymes are stored in the glands and may be extracted. In some
particulars, however, the actions may not be strictly comparable, since
by extraction substances may be obtained which are not normally
secreted. Pancreatic juice digests starches and dextrins, proteins and
fats.

Experiment 136. Amylolytic activity of pancreatic extracts.—
Qualitative detection. Prepare some 1 per cent. starch paste as in
experiment 114. Take about 10 grams of fresh pig’s pancreas which
has been freed as far as possible from fat and been finely hashed in a
meat chopper. Add 40 c.c. of 35 per cent. ethyl alcohol and grind and
mix well in a mortar or mill. Both the lipolytic and the amylolytic
enzymes are readily soluble in dilute alcohol. Filter off the extract.
Test its reducing action on Fehling’s solution. It should be negative.

To 5 cc. starch paste in a test-tube add 1 cc. of the extract and
mix. Observe the very rapid clearing of the starch due to the formation
of soluble starch. Place in beaker of water heated to 40° C. At the end
of 1 minute of action remove a drop to a drop of dilute I,KI solution
and test for starch. Remove also 2 cc. and see if this will reduce
Fehling’s solution. After 10 minutes repeat both tests. The pancreatic
extract contains an active principle, amylopsin, probably a mixture of
enzymes, which converts starch to dextrins and maltose.

Experiment 137. Proteolytic activity of the pancreas.—The pro-
teolysis caused by extract of the pancreas differs from that produced by
pepsin in that there are set free amino-acids and tri- and di-peptides.
The amount of albumoses formed is relatively very little. One of the
amino-acids thus set free is tryptophane, so that the pancreatic digest

forms free tryptophane, and by this it is easily distinguished from the
977
978 PHYSIOLOGICAL CHEMISTRY

digest of the stomach. To show this add to 20 grams of hashed, fresh
pig’s pancreas 100 cc. of 1 per cent. NaHCO, solution and 10 c.c. of
a fresh 10 per cent. aqueous extract of duodenal mucosa, add toluene,
stopper the flask with a good-fitting cork, shake well and allow to digest —
24 to 48 hours at 40° C. At the end of that time filter off a little of
the extract; make in a portion of it a biuret test; in another portion
test for free tryptophane in the following way: Acidify about 5 c.c. of
the filtered extract with acetic acid and add to it, drop by drop, a
solution of bromine in water. A violet or pink color develops in the
presence of free tryptophane. When the maximum color is developed
add 2-3 e.c. amyl aleohol and shake. The color dissolves in the amyl
alcohol.

Permit the rest of the digestion mixture to digest and test it from
time to time by the biuret test. After a few days (4-5) this will be
almost gone. When this is faint remove from the thermostat. Observe
any white deposits (tyrosine or leucine) and examine under the micro-
scope to see if they are crystalline. Test some of the white matter by
the tyrosine test. Heat the remainder of the digestion mixture to
boiling, filter while hot and evaporate on the water bath to a small bulk,
20 c.c. <A erystalline crust and deposit will form if the digestion has
been sufficiently prolonged. Identify tyrosine and leucine in the crust.
Tyrosine crystallizes in balls of needle-shaped crystals; leucine in solid
conglomerates having a radiate structure.

Experiment 138. Lipolytic activity—Take 2 c.c. of olive oil, 3 ce.
of water in a test-tube. Shake thoroughly. Then add 1 drop of litmus
solution. The reaction will probably be slightly acid. If it is, add now
from a pipette or burette enough N/10 NaOH just to make the reaction
of the litmus blue. Now add to this tube 1 c.c. of the dilute alcoholic
extract of the pancreas just prepared, shake well again and place in
the beaker of water at 40° C. Shake from time to time. Observe if
the litmus changes to an acid reaction, due to the splitting of the fats
by the lipase, and also observe if on shaking the emulsion becomes more
permanent, the fat not separating as at first. If the litmus becomes
red, make it blue again by the addition of a little NaOH and place
in the bath again. Write the reaction for the splitting of the neutral
fat by lipase.

Experiment 139. The activity of both amylase and lipase depends
on the presence of salts.—To show this take the remnant of the alcoholic
extract of the pancreas from experiment 186, place it in a small dialyz-
ing, parchment tube and dialyze against running water for 24 hours.
Then remove the contents of the tube to a beaker and test the activity
on starch paste and on neutral fat suspended in distilled water. Make
the tests just as in experiments 136 and 138, but neutralize the olive oil
PRACTICAL WORK AND METHODS 979

separately by shaking it with a little dilute sodium hydrate and remov-
ing 2 e.c. of the neutral oil to a tube of distilled water. Repeat the
experiment, having added a drop of CaCl, solution to each tube. The
activity should have disappeared on dialysis and return on the addition
of a little salt, such as CaCl, (No. 18).

Exercise XXXIV. Pancreas (Cont’d).

Experiment 140. The Roberts method for amylase determina-
tion.—Prepare 100 cc. of a 1 per cent. starch paste as previously
directed. Take 10 cc. of this paste and add 90 ce. boiling hot water.
Cool to 40° C. and set aside in the water bath kept at 40° C. Now at
a noted time add 1 ¢.c. of the enzyme solution and mix well by stirring.
After acting for 3 minutes remove 1 ¢.c. of the mixture and add thereto
1 ce. of the following iodine solution (dilute 0.5 c.c. of the N/10 iodine
solution on the side shelf with 100 c.c. distilled water). Note the time
when, on mixing the solutions as indicated, a colorless or yellowish
solution remains. Repeat the digestion tests on 100 c.c. portions of the
diluted starch paste with varying quantities of the amylase solution
until the achromic point is reached in 4 to 6 minutes after addition of
the enzyme solution. The activity is then calculated by the formula
D=10/V X5/n where V=volume of enzyme solution and n=time in
minutes required to reach the achromic point. D then is the number
of c.c. 1 per cent. starch paste hydrolyzed to the end point here taken,
in 5 minutes, by 1 cc. of the enzyme solution.

Determine the relative activities of two substances or solutions by the
above method.

Experiment 141. Most favorable hydrogen ion concentration for
pancreatic amylase.—Like ptyalin the pancreatic amylopsin digests
most rapidly in a faintly acid medium. Digestion in the duodenum
takes place in such a reaction and the digestive enzymes are all adapted
to work at their optimum near the neutral point. The starch and
carbohydrate enzymes all act best in a feebly acid medium, having a
hydrogen ion concentration of about N/100,000, or P,=5. This is just
about the concentration at which dimethyl amino azobenzene changes
from a clear yellow to a slight orange. Prepare an extract of the
panereas by grinding 2.5 to 5 grams of fresh hog pancreas with a small
amount of sand and gradually add thereto 100 e.c. distilled water.
Strain through cheese cloth. (It is often more convenient to make a
glycerine extract of the pancreas and keep this on hand. The enzymes
retain their activity for a long period in such a medium and they are
well extracted by glycerine). Note the reaction of this extract to litmus;
methyl orange and dimethylamidoazobenzene. Now take of the extract
980 PHYSIOLOGICAL CHEMISTRY

10 c.c. portions in 4 test-tubes. To A and B add 1 drop of dimethyl-
amino-azobenzene; to C 1 drop of good litmus solution; to D 1 drop
of phenolphthalein. Add to A and B from a burette N/10th HCl until
A is a plain pink and B is just orange; C make neutral (violet) to
litmus; to D add N/10 NaOH until it is just pink to phenolphthalein.
The following is now the concentration of H ions in these tubes: P,,,
A=; B=5; C=7; D=8.3. Add to each tube 3 c.c. 1 per cent. starch
paste and allow to stand after mixing at room temperature. Determine
by removing a drop to a porcelain plate and touching a small drop of
KI, at what time the achromic point is reached in the different tubes.
The tube reaching this point in the shortest time is the most
favorable H ion concentration of those tested. B will probably be the
best.

Experiment 142. Enterokinase and trypsinogen.—Principle. The
object of this experiment is to show that a mixture of the extract of
the intestinal mucosa and the extract of the pancreas digests proteins
very much faster than either alone. This fact is interpreted to mean
that there is in the intestinal secretion a heat-sensitive substance, which
has been named enterokinase, which has the function of activating the
inactive proferment of the pancreas so that it will digest proteins. The
inactive proferment is called trypsinogen.

Experiment. Prepare 50 cc. of a 1 per cent. water extract of dried
fat-free hog pancreas and also 25 c.c. of a 1 per cent. water extract of
dried, fat-free hog duodenal mucosa.

Instead of the above, one may prepare 10 per cent. water extracts of
the fresh tissues as directed under experiment 141. This is preferable.

Now prepare the following mixtures in large test-tubes.

 

 

 

Tube fener ae ne Water Treatment
a 10 cc. 9 5 ee. Xeep at 40°C for 20 min.
b 10 ¢.e. 5 C.c. 0 os “ “ ce ivy
e 0 5 c.c. 10 ae, “oe “ce “ ee “c “
d 10 c.e. 5 ae. 0 (Heat in boiling water for
5 min., cool to 40°C and
5 ce, keep at 40° for 20 min.,
Oo cadens sss 10c.c. | (boiled) 0 Keep at 40°C for 20 min.

 

 

 

 

To each tube now add 5 cc. 10 per cent. Na,CO, solution and mix
well immediately after the addition. Now add to each tube the same
amount (about the size of a hazel-nut) of well-washed fibrin. Agitate
well and digest at 40° C. Note the changes in 2 to 40 minutes. Tube b
should digest very much faster than the others.

Experiment 143. Determination of the optimum alkalinity for
tryptic digestion Principle. The object of this experiment is to de-
PRACTICAL WORK AND METHODS 981

termine whether trypsin digests better in an acid or alkaline medium,
and what the optimum concentration of alkali or acid is. (H ion econ-
centration. )

Experiment.

a. The optimum Na,CO, concehtration for trypsin action. Prepare
100 ¢.c. of an activated trypsin solution. For the preparation of this
extract see experiment 144. Take 5 c.c. samples and add thereto 14.5;
14.0; 18.5; 18.0; 12.5; 12.0 and 11.5 ce. water, and 0.5; 1.0; 1.5; 2.0;
2.5; 3.0 and 3.5 ec. N Na,CO, solution respectively. Add the water
first. Shake well immediately after each addition and add to each a
piece of well-washed fibrin about the volume of a pea. Digest at 35-40° C.
Calculate the concentration in Na,CO, in each case above and note the
optimum concentration for trypsin action. Also determine by the indi-
eator method (phenolsufonephthalein) the concentration of H_ ions.
The most favorable reaction is said to be P,—8.

b. Effect of hydrogen ions on trypsin action. In the same way as
above determine the effect of adding to 15 c.c. of the activated trypsin
solution 14.9; 14.8; 14.7; 14.6 and 14.5 ec. water, and 0.1; 0.2; 0.3;
0.4 and 0.5 N/10 HCl respectively. Determine the H ion by indicator
(sodium alizarin sulphonate).

Experiment 144. Preparation of trypsin solutions free from
lipase and amyopsin.—By the action of acid the trypsinogen is activated
and the trypsin is preserved while the lipase and amylopsin are de-
stroyed by an acid solution of P, 5.5. (Method of Mellanby and
Wooley.) Hash pigs pancreas freed as far as possible from fat. Weigh.
To each gram of hash add 3 ¢.c. 0.5 per cent. HCl. (Concentrated HCl
(sp. gr. 1.16) is approximately loN, or 31.5 per cent. by weight. 36.6
grams per liter.) Dilute 1 volume of the concentrated acid to 63
volumes with water, to make a 0.5 per cent. solution. After mixing stir
frequently and allow to stand for 30 minutes. Add 3.2 «ce. of 10 per
" cent Na,CO, for every 100 c.c. of HCl which was added. This reduces
the H ion to about P,, 4.7. Filter. The filtrate should be clear. Reduce
the acidity to P,—5.5 by the addition of 10 per cent. Na,CO,. The
solution gives at this acidity a faint red to methyl red. Add toluene
and shake. Keep in ice box in stoppered flask. The lipase and amy-
lopsin are destroyed by the acid and 5.5 is the optimum for the stability
of trypsin. It keeps well at this acidity.

Experiment 145. Steapsin is destroyed by heat and its activity is
greater in the presence of bile——Prepare 100 c.c. of a 10 per cent. water
extract of fresh hog pancreas by grinding in a mortar with sand and
gradually adding the water. Saturate with toluene and set aside for 24
hours. After standing for 24 hours, strain through cheese cloth and
make the following tests, adding one drop toluene to each tube and
982 PHYSIOLOGICAL CHEMISTRY

digesting at 37-40° for 24 hours. Use large test-tubes all of the same
diameter and stopper them. After the digestion cool, add a drop of
phenolphthalein and titrate with N/20 NaOH until a permanent pink
remains. Shake well while titrating.

 

 

Tnhe Olive oil Water Other addition Enzyme rolution
AS. Fok Date aicosnsiicanitanns 0.5 4.5 0 5 ¢.c.

DE enero ees 0.5 4.5 0 “boiled
Co. Gheegad xGiha inet adie 0 4.5 0 5 cc.

Co serdineiens eo geans 0 4.5 0 “boiled
@: ssc hes eee eeee 0.5 9.5 0 0

ff iainch enn 9 ates 0.5 8.5 1 «ce. bile 0

@ bos watgcacsrenae 0.5 3.5 LS 5 cc.

Th ean cmion 0 8.5 Leone AS 0

iid baste uanevatede sehse 0 3.5 ste SS 5 c.c.

 

 

 

 

Record for each tube the amount NaOH necessary to neutralize the
fatty acid set free by the action of lipase. g should be most active.

Experiment 145(a). Following the course of a protein digestion by
trypsin by formol titration (Sérensen method).—Object: This experi-
ment illustrates a very important method, that of formol titration of
amino-acids (see page 363), and at the same time affords a proof that in
the course of a proteolytic hydrolysis there is a steady increase in the
number of free amino groups.

Experiment. The Sodrensen method depends on the amino-acids
having their basic character destroyed by formaldehyde and thus bring-
ing about a greater ionization into H+ and RCHN (:CH,) COO-.
Consequently the greater the concentration of the amino-acids produced
by the hydrolysis of the protein the greater the acidity, and thus from
a measure of the increased acidity we have a measure of the degree of
hydrolysis.

Prepare 40 c.c. of a 10 per cent. extract of fresh hog pancreas. Also
prepare 10 c.c. of a 10 per cent. extract of duodenal mucosa and mix
the two. ;

Take 10 c.c. of the above and add to 100 cc. of a 4 per cent. casein
solution in 0.5 per cent. Na,CO, solution. Immediately after mixing
remove 25 ¢.c. by means of a pipette, then add 10 ¢«.c. neutralized forma-
lin (a 40 per cent. formaldehyde solution rendered neutral toward
phenolphthalein by adding N NaOH), and set aside the remaining solu-
tion at 35-40° C. Titrate the 25 cc. sample by N/10 NaOH or HCl,
as the ease may be, using phenolphthalein as indicator. In this titration
a definite pink color should be chosen as end point. The titration
should be carried to match this control each time. A buffer solution
of P,=8.3 makes a suitable control. At the end of %, 1% hours and
24 hours remove 25 c.c. samples and titrate these in the same way. As
PRACTICAL WORK AND METHODS 983

the digestion proceeds and more NH, groups are set free from union
with COOH groups the acidity increases.

Experiment 146. Invertin in intestinal mucosa.—Besides the pro-
teolytic activator, enterokinase, the mucosa contains also invertin and
the proteolytic enzyme erepsin. These may be demonstrated as follows:
Prepare 100 cc. of a 10 per cent. water extract of fresh intestinal
mucosa in the same way as directed for pancreatic amylase above.
Detect invertin in this solution by adding 2 per cent. of cane sugar to
10 c.c. of this solution and one drop of 10 per cent. acetic acid, and after
standing % hour at 37° C remove 1 cc. to Fehling’s solution and test.
by reduction for glucose.

Experiment 147. Erepsin in duodenal mucosa.—To 10 c.c. of the
above extract add in a flask 50 ¢.c. of a 2 per cent. peptone solution, add
1 c.c. toluene, stopper and shake. Prepare another mixture in the same
way, but boil the intestinal extract first. This will be the control. At
once remove exactly 10 c.c. from each flask, add phenolphthalein and
titrate with N/10 H,SO, or N/10 NaOH to the neutral point. Now add
formalin as in experiment 145 and titrate the acidity which develops.
Keep a record of your results. Place both flasks in the incubator at 37°
and remove 10 ¢.c. portions and titrate this same way after a period of
digestion of 3 hours, 24 hours and 48 hours. If erepsin is present, there
should be a digestion of the peptone with the setting free of amino-acids
and dipeptides, and these will be detected by the acidity which develops
when formalin is added.

Experiment 148. Lactase.—To 10 c.c. of the intestinal extract pre-
pared in 146 add 4 per cent. by weight of lactose and allow to digest
for 1 hour at 37° C. At the end of that time transfer to a fermentation
tube, add a little yeast and put back in the incubator. Lactose is not
fermented by yeast. The lactase of the intestine will convert the lactose
to galactose and glucose and the latter will be fermented and gas pro-
duced.

EXeERrcisE XXXV. THE BILE.

The bile has very little digestive action, its main function being to
act in co-operation with the pancreatic and duodenal juices and to assist
in absorption, particularly of the fats. It is also an excretory substance.
The constituents which are important in digestion and absorption are
the bile salts, sodium glycocholate and taurocholate, and the alkali,
sodium carbonate. Its pigments make it very striking. So far as known
these are excretions and have no function. The bile contains also some
phosphatide and cholesterol, the function of which is unknown.

Experiment 149. The physical properties of bile—Obtain 25 c.c.
ox bile. (Note—This is best kept and transported in the ligated gall
984 PHYSIOLOGICAL CHEMISTRY

bladders.) Note the specific gravity, the viscidity, slimy character, the
taste, smell and color. Test its reaction to litmus paper and to
phenolphthalein.

Experiment 150. Powers of solution.—To 5 c.c. of bile in a test-
tube add 0.5 c.c. of oleic acid and shake. The oleic acid partially dis-
solves and is held in solution in the bile.

Experiment 151. Reactions by which its pigments can be de-
tected.—The bile pigments have the property when oxidized of passing
through a series of brightly colored compounds, finally becoming
yellow. The final yellow color is called choletelin. Bilicyanin, a blue
color, and bilifuscin are intermediate oxidation products. The two
principal pigments of the bile are biliverdin, C,,H,,N,O,(?), and
bilirubin, C,,H,,N,O,(?). Bilirubin by oxidation is converted into
biliverdin.

Experiment 152. Gmelin’s test for bile pigments.—This test may
be tried in various ways. It depends on the oxidation of the pigments
by concentrated fuming nitric acid: i.e, nitric acid containing some
nitrous acid.

a. Place a thin layer of bile on a porcelain dish and place on it a
drop of fuming nitric acid. The drop becomes surrounded by a series
of various colors, blue, pink, orange, etc.

b. Place 3 e.c. of fuming nitric acid in a test-tube and carefully

place above it a layer of bile. On agitating very gently a series of colors
develops at the zone of contact. Repeat this test, diluting the bile many
times with water.
' Experiment 153. Huppert-Cole test for bilirubin.—To about 50 c.c.
of diluted bile add an excess of baryta water or milk of lime. The
bilirubin is precipitated as an insoluble barium or calcium compound.
It coheres. Allow to settle. Remove most of the supernatant liquid with
a pipette, transfer the remainder to a small filter. Remove the precipi-
tate from the filter; place it in a test-tube; add about 5 cc. of 95 per
cent. alcohol, 2 drops of strong sulphuric acid and 2 drops of a 5 per
cent. solution of potassium chlorate and boil for a minute. After the
settling of the precipitate of calcium or barium sulphate, the super-
natant liquid will be an emerald or blue-green.

Experiment 154. Hammarsten’s reaction for bilirubin.—Take one
volume of the acid mixture (see below) and add to it 4 volumes alcohol.
If a drop of a solution containing bilirubin is added to a few e.c. of
this mixture, a beautiful, permanent green color at once develops. By
the addition of more of the acid mixture to the green solution the other
colors of Gmelin’s test to choletelin can be obtained. The acid mixture
should be prepared beforehand. It is made by mixing 1 volume of nitric
acid and 19 volumes of HCl, each acid being about 25 per cent. in
PRACTICAL WORK AND METHODS 985.

strength. It is used when it has become yellow by standing. It will keep
at least a year.

THE BILE SALTS. SODIUM SALTS OF GLYCOCHOLIC AND TAUROCHOLIC ACIDS.

Experiment 155. Preparation of glycocholic acid—From some
biles rich in glycocholic acid the acid will crystallize. Place 100 cc. of
ox bile in a stoppered cylinder, add 5 c.c. of con. HCl and 12 «.c. of ether
and shake vigorously. Put aside in a cool place. In some cases the
glycocholic acid will crystallize out in a few minutes or hours. If this
does not happen, make the solution faintly alkaline with sodium-hydrate
solution and precipitate the glycocholic acid by lead acetate or ferric
chloride. Filter. Decompose the precipitate with a small amount of
soda; filter; and treat the solution with HCl so that it is plainly acid.
Glycocholie ciad should crystallize out. It may be recrystallized from
hot water.

Experiment 156. Preparation of bile salts. Platner’s bile—Mix
40 ec. bile with enough powdered charcoal to make a thin paste, about
10 grams, and evaporate to dryness on the water bath. Grind the residue
in a mortar, transfer it to a flask and extract it with about 75 c.c. of
absolute alcohol by boiling it on the water bath for a few moments. Cool
and filter through a dry filter into a dry beaker. Add ether to the alcohol
until a permanent cloudiness results and put in a cool place, covering
the beaker with a plate. The pile salts should erystallize out as long
needles, or a thick hard mass. At times their crystallization is delayed.
Filter off and dry in the desiccator.

Dissolve in water a little of these salts for the tests for bile salts
which follow:

Experiment 157. Pettenkofer’s test for bile salts—To 5 ¢.c. of the
solution of bile salts, or diluted bile, in a test-tube add a small crystal
of cane sugar and shake until dissolved. Then holding the tube inclined
allow concentrated sulphuric acid, about 5 ¢.c., to run down beneath the
bile salt solution. In the presence of the salts there develops at the place
of junction a purple color. This reaction is the same as the Molisch reac-
tion, only in this case we use the bile salts, the cholic part of the
molecule, as the chromogenic substance in place of a-naphthol. The
reaction is due to the production of methyl hydroxy furfural from the
sugar by the sulphuric acid and the union of this with the bile acid to
give the color.

Note—This reaction is also given as Raspail’s reaction by some fatty acids:
namely, by those of sphingosine and lecithin,
986 PHYSIOLOGICAL CHEMISTRY

Experiment 158. Gallstones.—Gallstones can usually be obtained
from the dissecting room. Htman gallstones are usually composed prin-
cipally of cholesterol, but phosphate and pigment stones are also found.
The cholesterol stones are sometimes white. They may rarely be found
as large as acorns and translucent when the cholesterol is unusually
pure. More commonly they consist of cholesterol with pigment. Very
frequently at the center will.be found microscopic, highly colored casts
of the bile capillaries. On section, the cholesterol stones generally show
a erystalline or radiate structure. Often there are rings of pigment
shown in cross-section, as if the stone had grown in definite periods.
Take a small stone, section it with a sharp knife. Powder a portion,
extract it with ether. Filter. Evaporate the ether, dissolve in chloro-
form and make the cholesterol tests, page 914. If the stone is a phos-
phate stone it will not disappear when it is burned. To find out whether
the stone is chiefly organic or inorganic heat a fragment on a cover of
a porcelain crucible. If it is organic it will char and burn, but if it is
a phosphate stone it will leave a large residue. In that case dissolve
the residue in a little nitric acid and add to the solution some ammonium
molydate solution and warm. A yellow precipitate will form if phos-
phate is present.

Experiment 159. Hay’s test for bile salts—Bile salts like soaps
have the property of greatly lowering the surface tension of water.
This may be used as a very delicate test for their presence, although it
is of course not specific. Prepare two perfectly clean beakers and fill
them about half full of perfectly clean, not greasy or soapy, water. To
one of them add a little of the bile salt solution. Now sprinkle on the
top of each some flowers of sulphur. If the water is clean, the sulphur
will float on the top of one, but will sink in the one to which the bile
has been added, owing to the lowering of its surface tension.

Experiment 160. Bile salts precipitate proteins—The bile salts
have the property, common to many acids, of forming insoluble precipi-
tates with the proteins. Slightly acidify a filtered solution of Witte’s
peptone, about 1 per cent. solution, and add to it a drop or two of bile
salt solution or of diluted bile. A white precipitate should form. The
bile probably thus precipitates and unites with the proteins in the
chyme when it is discharged from the pylorus.

Experiment 161. Cholesterol in bile—Hvaporate 10 c.c. bile to
dryness on the water bath. Extract twice with small quantities of ether
and evaporate the ether extracts to dryness in another dish. DO NOT
WORK NEAR A FLAME WHEN USING ETHER. CARRY ON
ALL SUCH WORK ON THE STEAM BATH. Dissolve the residue
left from the ether solution in about 2 ec. chloroform and apply the
Salkowski test.
PRACTICAL WORK AND METHODS 987

Exercise XXXVI. THE FECES.

Experiment 162. Microscopic examination.—Collect a small
amount of feces on toilet paper. Transfer a little with a glass rod to
a small drop of water on a slide and rub up with the water, place over
it a cover glass and observe with a good microscope. Observe the
enormous number of bacteria of various kinds and sizes, yeasts, ete.
There are also present undigested remnants of foods, principally of
vegetable fibers, sieve tubes, ete. After examining carefully, touch a
drop of KI, to one edge of the cover slip so that it flows under. Observe
as it goes under that some of the organisms take a deep blue stain, as
if they contained starch, and that other fragments stain a brownish
red.

Experiment 163. Intestinal putrefaction products.—As may be
seen by the examination of the feces in experiment 162 the large intcs-
tine contains myriads of bacteria which act upon the food products and
intestinal -secretions which have escaped absorption and produce by
putrefaction and fermentation various products, some of which are of-
fensive and harmful. Among these products which give to feces their
characteristic odor are indole and scatole. The method of separating
these substances from the feces is by distillation. This method and some
of the properties of these bodies are illustrated in the following experi-
ments, but instead of using feces,’ a putrefying protein solution, blood,
fibrin or casein, is used instead. This is inoculated by Bacillus coli
communis, the principal inhabitant of the colon. This bacillus makes
indole from tryptophane. The protein chosen to be putrefied should be
one containing tryptophane, and casein is chosen for this reason.

Obtain 100 c.c. of the putrefied protein from the storeroom. Distill
off about 50 ¢.c., using a water-cooled condenser. It may be necessary
to coagulate and filter before conducting the distillation.

Acidify the distillate with hydrochloric acid and extract three times
in a separatory funnel with 20 ¢.c. ether each time. Allow the ether
extract to evaporate spontaneously at room temperature. Save also the
acid aqueous layer for the work below.

' Dissolve the residue from the ether extract in about 10 ce. water,
filter if necessary and test for phenol and cresol and for indole and
scatole. For the two former apply the Millon test. To another portion
add saturated bromine water, a crystalline precipitate indicates tribrom-
phenol and tribrom-cresol formation.

Indole gives a red coloration by glyoxylic acid + concentratedH,SO,,
and a violet blue with formaldehyde +H,SO,. Scatole requires the
addition of a drop of very weak. FeCl, solution (or other oxydizing
agent) to give the test with formaldehyde +H.SO,. Apply these tests
988 PHYSIOLOGICAL CHEMISTRY

on 1 ¢.c. portions of your solution above (Dakin, Jour. Biol. Chem., Vol.
II., 1907, p. 289). ;

With para-dimethyl-amido-benzaldehyde (Ehrlich’s reagent. Para-
dimethylamidobenzaldehyde 4 parts; alcohol 95-98 per cent. 380 parts;
con. HCl 80 parts) indole gives a red color which becomes darker red
on the addition of sodium nitrite. Scatole yields a blue-violet color
before adding the nitrite and a deep blue after addition thereof. This
blue color may be extracted by chloroform (Steensma, Zetts. f. Phycvol.
Chem., Vol. XLVII, 1906, p. 25).

The original acid-water solution above may contain sulphides and
ammonia. Test directly for sulphides therein. Concentrate the re-
mainder of the solution to about 5 to 10 cc. and test the same for
ammonia.

The graphic formulas of indole and seatole are given on page 441.

Experiment 164. Examination of feces for occult blood.—The
presence of blood or its decomposition products in the feces is of
diagnostic importance in various pathological states. One-of the im-
portant methods of detecting such blood is by means of the benzidine
reaction. The reaction depends on the development of a blue color in
benzidine by hematin or iron salts. The test may be applied as follows
or by using the benzidine tablets supplied by various drug houses.

Place in a dry test-tube about 1 eg. of benzidine (a pinch), add
3 ce. glacial acetic acid and mix for about one minute. Add an equal
volume of hydrogen peroxide. Mix and pour one-half of the solution
into a clean dry test-tube, keeping the other half for a control, to see
that it does not turn blue without any addition. Now add to one of
the tubes about 1 c.c. of a suspension of feces in distilled water. If
blood is present or its decomposition products, the tube becomes greenish
blue. Make the test first with a very dilute blood solution.

Experiment 165. Occult blood in stools. (Method of Lyle and
Curtman: J. B. C., 33, 1918, p. 1.)

Principle. A part of the stool is boiled with acetic acid, extracted
in ether and the presence of hemin in the ether detected by means of a
modified guaiaconic acid-peroxide of hydrogen method. This method is
more certain than the guaiac, but not quite so sensitive as, and more
reliable than, the benzidine test.

Method. Stools should be obtained from patients when on a meat
free and soup free diet, since meat extract contains substances which
will give this test. The test is used for the purpose of detecting blood
in the stool for the diagnosis of ulcer and cancer. About 10 grams of the
stool are placed in a 50 or 100 cc. beaker, 25 c.c. distilled water are
added and the whole stirred to uniform consistence and then boiled for
several minutes over a low flame. Cool and transfer to a small stoppered
PRACTICAL WORK AND METHODS 989

bottle of about 100 ¢.c. capacity, or less, add 5 cc. of good grade glacial
acetic acid and 25 ¢.c. ether and shake thoroughly and allow to stand
until the ether has separated. Draw off about 2 c.c. of the ether extract
into a test-tube, add 0.5 c.c. of the guaiaconic acid reagent (1:60) and
finally 1 to 5 drops of 30 per cent. perhydrol are added from a pipette,
shaking after each addition. A decided green, light blue or purple
color indicates the presence of blood of sufficient quantity to be of
clinical significance.

Guaiaconic acid. This may be purchased or prepared from gum
guaiac. In the latter case proceed as follows: Grind 50 grams crude
gum guaiac and extract it in a beaker at room temperature with 20
grams KOH dissolved in 200 c.c. water. Stir thoroughly during the
extraction. After thorough stirring filter with suction on Buchner
through thin layer of cotton. Wash residue with water and filter also,
repeating until the combined filtrates are about 114 liters. Run into
the filtrate dropwise from a burette 21 ¢c.c. glacial acetic acid, stirring
constantly. Allow to settle, decant the supernatant liquid, saving the
ppt., wash with water by decantation. Then filter through Buchner
by suction. The precipitate cannot be dried in this way. To separate
the water remove from the filter and heat ppt. gently in small portions
in evaporating dish. The water separates and may be removed by filter
paper. Spread in thin sheets on glass and dry in air. Grind the dried
masses to powder and.treat with 300 cc. 95 per cent. alcohol, stirring
constantly. Filter from a dark brown flocculent ppt., and distill off
the alcohol from the filtrate. Redissolve residue with 20 grams of KOH
dissolved in 200 e.c. water and reprecipitate with glacial acetic acid.
Filter and dry as before. Yield should be 30 grams. Grind it and
keep in stoppered bottle or in a desiccator.

The reagent is made by dissolving 1 gram of this substance in 60 e.c.
95 per cent. alcohol. Kept in stoppered, ordinary glass bottle no diminu-
tion of reactivity was noted in several weeks.
CHAPTER XXX.
THE BLOOD.

EXERCISE XXXVII.

Vertebrate blood consists of a liquid called the plasma, which holds
in suspension a vast number of small bodies: the red corpuscles, the
white corpuscles and the blood platelets. There are in 1 ¢.mm. of normal
human blood about 5,500,000 red corpuscles and 8,000 whites. The
plasma consists of water holding in solution about 8 per cent. of coagula-
ble proteins, serum globulin, fibrinogen and serum albumin,—salts, a
little glucose (about 0.1 per cent.) and a great number of other sub-
stances, amino-acids, urea, ete., which are present in very small quanti-
ties. The red corpuscles owe their color to hemoglobin, a protein having
the power of forming a loose union with oxygen and which carries
oxygen to the tissues. The number of white and red corpuscles varies
under different conditions of health and their number aften furnishes
a valuable means of diagnosing disease and of showing the state of
health or disease of the blood. For this reason simple and expeditious
methods have been devised for the determination of the number of
white and red corpuscles in the blood and of the amount of hemoglobin,
and also of the relative amount of corpuscles and plasma. The study
of the blood may be begun with the determination of these factors.

Experiment 166. Determination of the number of red corpuscles
in the blood. Method of using the hemacytometer of Thoma-Zeiss.—
The apparatus consists of two pipettes for dilution and a slide ruled in
squares. The pipette for the determination of the red corpuscles dilutes
the blood 100 times; that for the white, 10 times. Prick the finger and
when a good drop of blood has collected draw it up into the pipette for
the reds to the mark 1. Wipe the blood off the exterior of the pipette
and then draw up quickly some Hayem’s solution to the mark 101.
Mix the contents thoroughly at once in the bulb by shaking the pipette
back and forth so that a uniform suspension of corpuscles is obtained.
Allow some of the mixture to flow out of the pipette and then transfer
a small drop to the ruled platform of the cell. But before dsing this
see that the cell is perfectly clean and that there is no dust either on
the slide or the cover slip. The drop should be so large that when the
cover slip is put on it does not overflow into the moat, but completely

covers the ruled platform. Allow the slide to stand for a few moments
990
PRACTICAL WORK AND METHODS 991

for the corpuscles to settle and then count 16-20 of the small squares,
beginning at one corner and going straight across the cell and then the
next tier and so on. The average number of corpuscles per square is
determined. The squares are ruled so that each square is one-four-
hundredth of a square mm. and the depth of the space above the ruled
platform is one-tenth of a mm., so that each square represents the
number of corpuscles in one-four-thousandth of a emm. The average
number of corpuscles per square multiplied by 4,000 and by 100, since
the sample of blood was diluted 100 times, gives the number of red
corpuscles in 1 cmm. of blood. Hayem’s solution in the following:
Na,SO,, 5 grams HgCl,, 0.5 gram; NaCl, 1 gram; distilled water, 200 c.c.

The determination of the white corpuscles is made in a similar way,
except that the other pipette is used which dilutes only 10 times. It is
filled with blood to the mark 1 and then the point is wiped off and at
once the solution of 0.5 per cent. acetic acid is drawn up to the mark 11,
and the blood mixed with the solution by shaking and rotating the
pipette. It is necessary to work rapidly to prevent the clotting and
sticking together of the corpuscles. It is difficult to get a uniform dis-
tribution of the corpuscles. After thoroughly mixing place on the slide
as before and count the corpuscles. The red corpuscles are dissolved by
the acid and the whites are fixed. The number per square multiplied
by 40,000 gives the number of corpuscles per c.mm. It is well to count
the whole of a large square. This determination is a difficult one to
make accurately.

Experiment 167. Oliver’s hemacytometer.—The Thoma-Zeiss
method of directly counting the corpuscles is trying on the eyes and
consumes much time. A simple, accurate clinical method of determining
the number of red corpuscles is that of Oliver. The principle of this
method consists in diluting a given amount of blood with a solution
until the flame of a candle makes just the image of a line through it.
The apparatus is simple. It consists of a measuring, capillary pipette,
a pipette like a medicine dropper and a test-tube graduated and having
a rectangular cross-section. The method of using this apparatus is
described by Cabot as follows:

Clean and dry the capillary pipette by drawing through it a needle carrying
thread or darning cotton saturated with water and then with alcohol and ether.
The medicine dropper is then filled with Hayem’s solution. The capillary pipette is
then filled with blood in the usual way, any superfluous blood on the outside being
quickly removed, the pipette connected at the blunt end with the rubber on the end
of the medicine dropper and the blood washed out by means of the Hayem’s solution
into the graduated test-tube. If the previous hemoglobin estimation has shown
nearly the normal amount of hemoglobin, the blood may now be diluted to about 80
with Hayem’s solution. Hold it.in the hand in the manner indicated, with the
thumb and first finger extending up the sides of the tube, and holding the tube close
992 PHYSIOLOGICAL CHEMISTRY

to the eye in a dark room and about 9-10 feet from the small wax (Christmas)
candle, see if the image of the candle suddenly appears as a bright line across the
tube. Dilute by adding a few drops of Hayem’s solution, hold the finger over
the mouth of the tube and invert once or twice, wiping each time the thumb on the
mouth of the tube so that the liquid sticking to it goes back in the tube, and continue
diluting and testing until the bright line suddenly appears. This dilution is such
that the solution contains approximately 52 corpuscles to 36 squares of the Thoma-
Zeiss counter. If enough fluid has been added to make the volume in the tube 100,
the blood contains 5,000,000 corpuscles per c.mm. If it has been necessary to dilute
it to 80, it will contain 80 per cent. of 5,000,000 or 4,000,000; if only diluted to
50, the number is 2,500,000. The values obtained by this instrument with human
blood after a little practice are usually correct within 1 per cent., unless a very
great number, 100,000 per c.mm., or more, leucocytes are present when somewhat
higher figures are obtained. The tube held in the hand in the manner indicated
shuts off with the hand the light of the candle, except that coming through the tube.

Experiment 168. Hematokrit method.—In this method the relative
volume of plasma and corpuscles is determined by the separation of the
corpuscles by rapid centrifugalization in a Daland hematokrit. The
finger or ear is pricked and a sample of blood is sucked up into the
capillary, graduated tube of the hematokrit. The tube must be exactly
filled. To do this grease the first finger with a little vaseline and, as
soon as the tube is removed from the blood-drop, slip the greased finger
tightly over the end. Holding it there, detach the rubber tube from
the other end and slip the tube into the hematokrit, with the beveled
end toward the axis of rotation. An empty tube has previously been put
into the other side of the centrifuge for balancing purposes. Now rotate
the centrifuge for two minutes at the rate of 70 revolutions of the handle
per minute. Ifthe work has been rapidly done, the blood is centrifuged
before it has clotted, the corpuscles are packed as a solid plug at the
outer end of the tube. They make, as a rule, in normal blood about 40
per cent. of the length of the tube. This corresponds to about 5,000,000
corpuscles per cmm. Since many factors influence the volume of the
corpuscles, the method is not accurate for determining the number
of corpuscles. The centrifuge used for the purpose is noisy, crude and
generally unsatisfactory.

Determination of hemoglobin.—The most convenient clinical method
for this purpose is that of Tallqvist. The most elaborate is that of
von Fleischl, but as the latter method is inaccurate, cumbersome and
expensive, it possesses no superiority over and should be replaced by the
Tallqvist and Dare methods. Normal human blood contains 14-15 per
cent. of Hb.

Experiment 169. Tallqvist method.—The advantage of this method
is its cheapness, the Tallqvist scale costing but $1.25, and its extreme
simplicity. A determination of the hemoglobin within at least 10 per
PRACTICAL WORK AND METHODS 993

cent. can be made at the bedside and in a few moments. A drop of
undiluted blood is soaked into a piece of filter paper and after a moment
or so, and before it has dried, it is matched with the color of the scale
against a white background and in ordinary daylight. The scale has
been carefully prepared by matching the tint of blood of various degrees
of anemia and known hemoglobin content when soaked into filter paper.
The colors were lithographed and the lithographed scale bound up with
50 pieces of filter paper. It can easily be slipped in the pocket. The
errors are not greater than with the von Fleisch] apparatus in the usual
hands.

Experiment 170. Oliver’s hemoglobinometer.—This has, in gen-
eral, the same principle as the foregoing, but uses diluted blood. The
capillary pipette is filled with blood from a pricked ear or finger and
washed as in the case of the hematocytometer into the cell by some water.
The cell is then filled with water, the cover put on so that only a very
small bubble remains under the cover slip, to show that it is not overfull,
and compared with the scale. The color of the cell is matched with the
colors of the scale. Two riders are supplied which, when placed above
the scale, make the tjnt somewhat deeper and so increase the number of
subdivisions. For example, if the sample has a tint between 80 and 90,
a rider placed on 80 makes it 85. If the test comes between 85 and 90,
it could be called 87.5. 100 per cent. is taken as the normal. This cor-
responds to 14 per cent. of hemoglobin in the blood. Reflected candle-
light is used. By this a reading may be had to 2 per cent. A set of
disks can be had adjusted to daylight readings.

Experiment 171. Dare’s hemoglobinometer.—This instrument. is
extremely convenient. It uses undiluted blood so that it avoids the errors
due to dilution. It is compact, but it costs $20. It is more accurate
than Tallqvist’s method, but not so convenient. It is probably the best
of the methods here given.

The film of blood is drawn between two plates of glass by capillarity,
and the tint compared with a revolving scale of a circular disk of glass.
The two colors are matched and the per cent. of Hb in terms of the
normal read off. The colors are compared by candlelight and the
observation made through a telescopic attachment so that light is cut off
other than that transmitted. A dark room is not necessary. The instru-
ment is simply pointed at some dark corner. The candle is attached to
the instrument. A more accurate method, using the Duboseq colorimeter,
is described by Newcomer. .

Experiment 172. Hemoglobin by Newcomer’s method. (New-
comer: Jour. Biol. Chem., 37, p. 465, 1918.)

Principle. The color of hematin hydrochloride is matched in a
‘Duboseq or Benedict and Bock colorimeter against a special colored
994 PHYSIOLOGICAL CHEMISTRY

glass (1 mm. thickness), furnished by A. H. Thomas Co., Philadelphia,
or by Bausch and Lomb.

Method. On top of one of the plungers of the Duboscq place the
special glass and partly fill the cup on that side with distilled water.
Into the cup of the other side place 5 ¢.c. of 0.1IN HCl (1 per cent. C.P.
HCl) and add 20 emm. of blood obtained by puncture measured by a
capillary pipette. Stir. Balance the color of the two sides of the
colorimeter. The reading darkens slowly and it is better to make the
readings after 40 minutes, but a correction can be made for times less
than this.

Calculation. Grams of Hb in 100 e«.c. blood are given by the formula:
one where t is the thickness of the hematin solution, that is, the
reading of the colorimeter on the hematin side, and d is the dilution.
This is the formula if the reading is made at least 40 minutes after the
dilution of the blood. As d is the dilution, in this case 251, the formula
95.38

i

 

with this dilution would be

formula would bea aS) where x is the time in minutes after the

addition of the blood and t is as before the reading of the colorimeter.
The oxygen capacity of the blood can be calculated, since 1 gram

of Hb yields 1.34 ¢.c. of O, measured under standard conditions, by the

0.38d 0.51d
r xX 134= eo

When corrected for the time, the

 

 

formula: The capacity of normal blood is about

18.75 c.e.

Since correction must be made for the exact thickness of the plate
of glass, which is seldom exactly 1 mm. and for the time, the following
tables will be found useful. The thickness of the glass is along the top
of the table and the time along the ordinate. These tables are for the
dilution of 251. The Duboseq colorimeter will read without difficulty
the dilution even when the blood is 50 per cent. normal content of Hb.
The Benedict-Bock may be used even for 20 per cent. Hb content. For
more abnormal bloods the dilution must be reduced. When using the
table look along the top to the column having the right thickness of the
glass, then down this to the figure opposite the right time. Divide the
number thus found by the reading of the colorimeter. The result will
be the grams of Hb in 100 cc. blood. The other table gives oxygen
capacity of the blood.
PRACTICAL WORK AND METHODS 995

TABLE 1.

To obtain gm. of hemoglobin per 100 c.c. of whole blood, divide the colorimeter
reading into the figure of the appropriate square, the choice of the square depending
upon the thickness of the glass used and the length of time since dilution of the
blood was made.

 

Thickness of colored glass in mm.
Time
since

dilution {| 0.95 | 0.96 | 0.97 | 0.98 | 0.09 | 1.00 | 1.01 | 1.02 | 1.03 | 1.04 | 1.05

 

 

10 94.4 | 95.4 | 96.4 | 97.4 | 98.4 | 99.4 | 100.4} 101.4
15 93.1 | 94.1 | 95.0 | 96.0 | 97.0 | 98.0 | 99.0] 100.0
20 92.5 | 93.5 | 94.5 | 95.4 | 96.4 | 97.4 | 98.4] 99.4
30 91.8 | 92.8 | 93.8 | 94.8 | 95.7 | 96.7.| 97.7] 98.6
40 91.6 | 92.5 | 93.5 | 94.5 | 95.4 | 96.4 | 97.4] 98.3
Final ...] 90.6 | 91.6 | 92.5 | 93.5 | 94.4 | 95.4 | 96.4] 97.3

 

 

 

 

 

 

 

 

 

 

 

 

 

TABLE 2.

To obtain the per cent. hemoglobin, Haldane scale, divide the. jorimeter reading
into the figure of the appropriate square, the choice of the sqys#? depending upon
the thickness of the glass used and the length of time since /#lution of the blood
was made. ,

 

Thickness of colored rian mm.
Time whe
since f J
dilution 0.95 | 0.96 0.97 | 0.98 | 0.99 1.00,4 4.01 1.02 1.03 1.04 1.05
POEL.

 

 

 

 

726 | 733 | 740 | 748 | 755
716 | 723 | 730 | 737 | 744
711 | 718 | 725 | 732 | 739
706 | 718 | 720 | 727 | 734
704 | 711] 718 | 725 | 732
697 | 704 | 711 | 717 | 724

10 683 | 690 | 697 | 704
15 673 | 680 | 687 | 694
20 669 | 676 | 683 | 690
30 664 | 671 | 678 | 685
40 662 | 669 | 676 | 683
Final ...| 655 | 662 | 669 | 676

 

 

 

 

 

 

 

 

 

 

 

 

 

; i
ExercisE XJ¥XVIII. HeEmoGLOBIN.

This, the red coloring mftter of the blood, is confined in the verte-
brate blood to the red blog4 corpuscles. If small quantities are present
in the plasma, the amoyfit is so small that at present we cannot detect
it. The function of #4is red conjugated protein is to convey oxygen
from the lungs to e tissues. It has the power of formirg a loose,
dissociable union with oxygen, the compound being known as oxy-
hemoglobin. It hés also the power of combining with other substances.
The absorption spectra due to the absorption of light of these various
compounds di er somewhat and the compounds may be detected and

Zz

4
996 PHYSIOLOGICAL CHEMISTRY

distinguished by these spectra. We will consider, first, influences which
cause the hemoglobin to leave the corpuscles and become free in the
plasma. This process is called laking the blood; second, we will consider
methods of crystallizing hemoglobin; third, the spectra of various com-
pounds of hemoglobin with oxygen, carbon monoxide, of hemoglobin
itself and methemoglobin and some other derivatives; and fourth, some
of the decomposition products of hemoglobin.

Influences which cause the hemoglobin to leave the corpuscles.
Laking the blood.—The separation of hemoglobin from corpuscles is
a matter of very great interest because, owing to the color cf the hemo-
Sobin, the process can be very easily followed and the causes and nature
of the processes involved can be studied. It is not probable that the
P&oe out of hemoglobin is at all peculiar, but that all cells in a similar
MANN Jose some of their constituents when subjected to these same
Process, We cannot, however, sc easily detect them, owing to the lack
of color. tm studying the laking of the blood, therefore, it is probable
that the "stter is of greater interest as a type of cellular reaction than
it Is for it, immediate significance for the physiology of the blood.
Laking may bx ‘produced by the following agents: by warming, or freez-
ing and thawing blood; by the action of anesthetics of ali kinds; by the
pumping of the wases out of the blood, particularly by taking out the
oxygen; by the aCBaa of very dilute alkalies; by the action of bile salts
BOSD by the ‘ction of specific hemolytic agents such as the

emolysins; by dilutin, the blood with water so that the blood is hypv-
tonic; by condenser discharges through the blood; by various toxic sub-
stances, such as certain s ke venoms, and saponins.

; It will be observed thatmost of these agents cause either stimula-
tion or depression of Protop'asmie processes. Since the discharge of
hemoglobin from the corpuscle \-eans that a change in the distribution
of the constituents of the proto} sm has occurred under the influence
of the reagent, it has been sugges\ that the nature of the process of
stimulation consists in the physica ange in distribution of the con-
stituents of the protoplasm caused by Awe laking agent or the stimulus.
The hemoglobin is generally supposed t\ e held in the corpuscle by the
limiting membrane, which is of such a na e that it cannot go through.
Many authors accordingly speak of the hem \vtic agents as affecting the
permeability of the sheaths of the corpuscles\ As pointed out on page
498, the phenomena may also, and perhaps mre correctly, be inter-
preted on the hypothesis that the hemoglobin is \& loose aatod with the
constituents of the stroma of the protoplasm and these various reagents
alter the stability of that union.

Experiment 173. Influence of hypotonicity. Laking by dilution
with water.—To illustrate this take 7 test-tubes and place in each 10 ec.

 
PRACTICAL WORK AND METHODS 997

respectively of the following solutions: a, distilled water; b, 0.2 per
cent. NaCl solution; ¢, 0.4 per cent. NaCl; d, 0.5 per cent. NaCl; e, 0.7
per cent. NaCl; f, 1.0 per cent. NaCl; g, 1.2 per cent. NaCl. Most easily
prepared by running the proper amounts of 2 per cent. NaCl solution
and water from two burettes. To each tube add from a pipette two
drops of defibrinated blood. Mix well and allow to stand for a few
minutes. Record the results. The laked blood is a darker color and
more transparent. Which tubes lake the blood?

Experiment 174. Laking by anesthetics.—Anesthetics unfortu-
nately have the property of laking blood. Take two tubes containing

ar
#

anesthetic and shake. Then add to each three drops of defibrinate fe

10 ¢.c. 0.9 per cent. NaCl solution. To one add a drop or two of wf

blood and mix. Allow both to stand. Observe the laking in the ana
thetic tube. Test in this way ether, chloroform, naphtha, tolu#”
acetone and alcohol. a

Experiment 175. Laking by warming.—To 5 cc. of 0.9 pe cent.
NaCl add 8 drops of defibrinated blood and warm carefully to aout 50°,
not higher, holding the blood for a few minutes at that teperature.
Laking will occur. Laking will also occur by freezing the blood and
thawing. The laking in this case is possibly accelerated by the fact that
on melting the ice crystals local differences of a occur. But
this it not the only explanation. £

Experiment 176. Laking by bile salts—To 1/ ¢.¢. of a 0.3 per
cent solution of bile salts in 0.9 per cent. NaCl add 3 drops of defibri-
nated blood. Observe the laking. Repeat, using 2 little soap solution
in place of bile salts. v

Experiment 177. Laking by saponins,—J0 10 «c. of a 0.9 per cent.
solution of NaCl containing 0.1 per cent, of saponin add 3 drops of
defibrinated blood. A

Experiment 178. Laking by diz falkalies.—Make a precipitate
of sodium magnesium phosphate by’@#ing to a solution of MgCl, some
solution of Na,HPO,, and if nee¢ #y a little NaOH. Filter off and
wash with water. Now suspend some of the precipitate in 10 c.c. of
0.8 per cent. NaCl, add 3 drops of defibrinated blood, mix well by invert-
ing the tube twice and allow fo stand. Have a control tube with the
blood and sodium chloride, alone. The hemolysis may be slow, so
observe for some time. i

Experiment 179. Crystallizing hemoglobin.—Various bloods crys-
tallize with different ease. Guinea-pig blood crystallizes with the
greatest ease and crystals can be obtained by mixing a drop of the
defibrinated blood with a drop of water on the microscopic slide and
putting on a cover glass. The erystals form very quickly. Rat and
mouse blood erystalize readily, and also the blood of the amphibian,

 
    
298 PHYSIOLOGICAL CHEMISTRY

Necturus. Dog’s blood oxyhemoglobin crystals may be obtained by
adding a few drops of ether, or better toluene, to 10 c.c. of defibrinated
dog’s blood in a test-tube and mixing until laked. Then add a little
powdered ammonium oxalate, shake once or twice and place in the ice-
box or a cool place for a few hours. Crystals of hemoglobin will gen-
erally be found. Examine them microscopically and draw some of them.
The other proteins and ghosts of corpuscles may be removed from laked
blood, leaving Hb alone, by addition of Al(OH), freshly pptd. and
filtering.

The forms of the crystals are, as shown by Reichert, characteristic

_for different animals. See Figure 50.
\. Experiment 180. Spectra of various compounds of hemoglobin.—
emoglobin, both solid and when in a solution, has a purplish-red color.
Te colors of the various compounds of hemoglobin differ somewhat
from, each other and from hemoglobin. Oxyhemoglobin is a bright
scarlet: earbonyl-hemoglobin is more of a cherry red; methemoglobin
has a biownish-red color; and so on. The difference in color is due to
the fact that the different compounds absorb light of different wave
lengths so ‘that the light which comes through the solution, or the
crystals, has Yost vibrations of certain wave lengths and so no longer
appears white but colored. Most of the red is obviously transmitted,
but some of the green or blue must be absorbed. By passing the light
which has traversed a hemoglobin solution through a prism which
spreads it into a spectrum it is easy to see which of the rays have been
absorbed. An instrument for thus determining the absorption or emis-
sion spectra is called a spectroscope. These are of two forms, but the
one of a very convenient form for such work is a direct vision spectro-
scope provided with a wave-length scale. The direct vision spectroscope
of Zeiss is a convenient form. \

The direct vision pee The instrument consists of a train
of crown and flint glass prism arranged that the course of the rays,
though scattered to form a spectrum, is not bent, but passes directly to
the eye in line with the object looked at. At one end is the eyepiece.
At the other end is a small slit or opening which can be narrowed by
the use of a screw. At the side of the main tube is a small secondary
tube which contains the wave length scale which by means of a mirror
is sent into the eyepiece so that the wave-length scale and the spectrum
appear to lie side by side. In using the instrument, point it at the
source of sunlight, with the small tube at the lett. Have a very narrow
slit so that the spectrum is barely visible. Now draw out the tube
gently until the spectrum appears to be traversed in a vertical direction
with very fine, dark lines (Frauenhofer lines) ; adjust the slit and the
focus of the tubes until these are very sharp. One very plain one will
PRACTICAL WORK AND METHODS $99

be noticed in the orange just to the left of the yellow. This is the D line
of the spectrum and corresponds to the main line of sodium. The wave
length of the light at this point of the spectrum is 0.59 w or A 589. By
means of the screw adjust the wave-length scale so that the line of the
scale 0.59, or A 589, comes exactly above the D line of the spectrum.
The wave length of any of the other dark lines can now be determined
very readily by reading off the scale.

The sample of blood, hemoglobin or other substance of which it is
desired to determine the absorption spectrum, is placed in a test-tube or
beaker, or better in a small flat-walled glass, and this is placed just in
front of the slit and between it and the source of light. A bright day-
light is good, but any other bright white light may be used. If the
solution is too concentrated, too little light will come through, most of
the field appearing dark. The more dilute the solution, the narrower
will the absorption bands become. With a proper dilution of the
oxyhemoglobin a broad absorption band will be seen in the violet
and two narrow absorption bands in the green between the D and E
lines of the spectrum. These bands are blackest in their centers
and shade off somewhat toward the edges. In recording your
observations, record the wave length read from the scale correspond-
ing to the center of the band, and the wave lengths at the sides of each
band.

The width of the absorption bands will vary with the concentration
of the solution looked through. The absorption spectra are very com-
monly plotted, therefore, in the manner indicated in Figure 51, p. 505,
the absorption being indicated by the shaded portions, the wave lengths
being marked ‘along the abscissa and the concentration of the solution
along the ordinate. The solution is examined in a layer 1 centimeter
thick. The point of maximum absorption is usually given by the tip
of the plot.

In making the experiments which follow, it is not necessary to pre-
pare the hemoglobin in a pure form first; for experiment has shown
that the absorption spectra of the isolated hemoglobin and the hemo-
globin of laked blood are the same. Make some laked blood by adding
a little blood to distilled water in a test-tube, filtering the solution and
then examining the absorption spectra of various dilutions. It willbe
observed that if the solution is strong enough the various absorption
bands fuse together, giving for example one broad absorption band for
oxyhemoglobin.

Experiment 181. Spectrum of oxyhemoglobin.—Shake the laked
blood with some air in a test-tube. Examine the spectrum. Make a
scale in your note-books and plot on it the absorption bands. The bands
of oxyhemoglobin can be distinguished in a solution which contains one
1000 PHYSIOLOGICAL CHEMISTRY

part of oxyhemoglobin to 10,000 of water. The spectroscope could be
used for the estimation of thc amount of oxyhemoglobin in a
solution. A 0.3 per cent. solution of oxyhemoglobin gives a good
spectrum. The Hb in blood is 14 per cent. From this complete the
dilution necessary to give 0.3 per cent. Map the absorption bands.
The @ band (the one toward the red end) has its center at A 578,
that of the & band is about y 540. By holding the hand in the sunlight
and examining the light reflected upward from the palm of the hand
the two oxyhemoglobin bands may be seen. The one toward the red is
strongest.

Experiment 182. Reduced hemoglobin.—Take 5 c.c. of a solution
of oxyhemoglobin so dilute that the two absorption bands are fairly far
apart and not fused in one. Add to this solution two drops of
ammonium-sulphide solution and warm very gently, not over 55°. Ob-
serve if there is any darkening of the tint of the solution. Now without
shaking the tube examine its spectrum. It should now show the single
broad absorption band of reduced hemoglobin. The center is about

565. Instead of ammonium sulphide as a reducing agent, Stokes’
reagen may be used. It is a freshly prepared solution of ferrous
sulphate o which some tartaric acid has been added and which is made
slightly alkaline with ammonium hydrate just before use. Map the
Spectrum of kemoglobin. Sodium thiosulphate solution will also reduce
blood. It is the most convenient to use.

Stokes’ Solution is the following: 3 grams ferrous sulphate dis-
solved in cold waxer; add a cold aqueous solution of tartaric acid,
2 grams, and mak) up to 100 c.c. with water. Just before using
add enough strong amonium hydrate just to redissolve the precipitate
which is first formea, ‘1 rapidly oxidizes itself, so must be freshly
prepared. Its advantage is that it reduces the oxyhemoglobin without
warming.

Experiment 183. Carbonylhemoglobin—This is the union of
hemoglobin with carbon monoxide in place of the oxygen. Its spectrum
is like that of oxyhemoglobin, but the two bands are shifted slightly
toward the violet end of the spectrum, Prepare a solution of carbonyl-
hemoglobin by passing a little illuminating gas through a solution of
laked blood under the hood. Notice the change in tint of the blood. It
has a bluish tint. Dilute if necessary and examine the spectrum. The
middle of a is at 572 and of B at 535.

i Experiment 184.—Carbonylhemoglobin is uot reduced by ammonium
sulphide. To show this repeat experiment 182, using the carbonyl-
hemoglobin in place of the solution of oxyhemoglobin. See that the
spectrum remains unchanged after warming with (NH,).S8.

Experiment 185.— Another very convenient way of distinguishing

 
PRACTICAL WORK AND METHODS 1001

carbonyl- and oxyhemoglobin is to take two solutions of oxyhemoglobin
and carbonylhemoglobin of about the same tint and dilute equally with
water. It will be seen that the solution of oxyhemoglobin becomes
yellowish, while that of carbonylhemoglobin acquires a carmine tint.
Try this.

Experiment 186.—Another test for carbonylhemoglobin in the blood
is Katyama’s. Add 5 drops of blood to 10 cc. of water. Then add 5
drops of orange-colored ammonium sulphide. Mix and make faintly
acid with strong acetic acid. Blood containing CO develops a rose-red
color; normal blood, a dirty greenish-gray. The difference is perceptible
with one part of CO blood to 5 of normal blood.

Experiment 187. Methemoglobin.—This is a union of oxygen and
hemoglobin, but the union is firmer than oxyhemoglobin. The gas cannot
be pumped out. Oxyhemoglobin passes with great ease into methe-
moglobin if made slightly acid, or alkaline, or in the presence of various
oxidizing agents such as ferricyanide, chlorates, etc. It is formed in the
blood in nitrite and chlorate poisoning and by a great variety of other
poisons such as acetanilide.

The exact difference in composition between oxy- and methemoglobin
is unknown, but probably oxyhemoglobin is a union of a molecular
nature, the union involving the residual valences of the oxygen. The
difference may be schematically represented as follows:

79 0
Hb = 0O—O Hb=O0=0 3 Hb | : Hb
Oxyhemoglobin if Oxyhemoglobin if \O \o
oxygen is univalent. oxygen is bivalent. Oxyhemoglobin. Methemoglobin.

Probably none of these representations is correct, for the oxidation
involves water, and probably the first union is a union between water,
oxygen and hemoglobin.

Experiment 188.—To a few c.c. of water add three or four drops of
blood. Mix. This lakes the blood. Now add two drops of a saturated
solution of potassium ferricyanide. The blood color changes to a choco-
late brown. Examine the solution spectroscopically. Methemoglobin
has an absorption band in the red, 4 630. There is marked absorption
of the blue end.

_Experiment 189.—It is possible by reducing agents to restore the
oxyhemoglobin and then to produce reduced hemoglobin. To show this
take 5 c.c. of the methemoglobin solution just prepared and add to it
a few drops of ammonium sulphide. It will be seen that the color
changes to a red. Examine by the spectroscope and see that the red
absorption band of methemoglobin disappears and the bands of oxy:
hemoglobin appear. On warming gently the bands of oxyhemoglobin
1002 PHYSIOLOGICAL CHEMISTRY

disappear and reduced hemoglobin appears. Now shake strongly with
some air. The bands of oxyhemoglobin reappear. It is probable that
small amounts of methemoglobin appear at times in normal blood. This
experiment is particularly instructive, as it shows that in the act of
oxidation of substances several intermediate stages occur. Always a
molecular union is made first.

Another very singular fact is that the addition of potassium ferricy-
anide to blood causes the liberation of an amount of oxygen equal to the
oxygen combined as oxyhemoglobin in the blood. This fact is the basis
of the method generally used at present for the estimation of the oxygen
in blood. No air-pump is needed.

Haldane has given the following provisional equation to represent
what happens, but it is not satisfactory:

HbO, + 4Na,FeCy, + 4NaHCO, — HbO, + 4Na,FeCy, + 4CO, + 2H,0 +0,

Perhaps a molecular union of HbO, and ferricyanide occurs first and
then this is rearranged, oxyhemoglobin being changed to methemoglobin,
the ferricyanide reduced and and oxygen set free.

Experiment 190.—Take 3 c.c. of defibrinated blood, lake by diluting
with 3 cc. water and warming gently. Then add 6 ec.c. of saturated
potassium ferricyanide solution, mix by inverting and observe the libera-
tion of bubbles of gas. O,.

Decomposition Products of Hemoglobin.

The relation of the various decomposition products of hemoglobin
may be seen in the following scheme of Halliburton and Rosenheim:

 

Oxyhemoglobin —___, -Hematin .) —_~_ __~ -Hematoporphyrin
| (acid)
pi : 2 By strong
By reducing agents }By acids } By reducing agents aids
Hemoglobin ——— | Reduced hematin Hematoporphyrin
(hemochromogen ) (acid)

Experiment 191. Hemin crystals (Teichman’s crystals).—Prick
the finger. Collect a drop of blood on a microscope slide. Dry. Put
on a drop of glacial acetic acid, cover with a cover glass and boil. Ex-
amine for crystals of hemin. Dark-brown plates and prisms. See
figure 80. In examining old blood stains by this method it is necessary
to add a minute-crystal of salt. There is salt enough in fresh blood.
These crystals are acetylated hematin chloride. The hydroxy] of the
PRACTICAL WORK AND METHODS 1003

hematin is replaced by chlorine and one acetyl group enters from the
acetic acid. Hematin is C,,H,,N,0,Fe.

 

Fie. 80.
Fig. 80.—Hemin crystals.

Experiment 192. Reduced hematin (hemochromogen).—A_ solu-
tion of oxyhemoglobin made alkaline with NaOH forms alkaline hematin
(band from D to A 630). Reduce this by adding a few drops of
(NH,).S. This forms the red hemochromogen. Bands in the green.
a at 7 558; @ at 520.

Experiment 193. Acid hematin—Hematin split off from oxy-
hemoglobin by the action of acids is soluble in ether. Heat a few c.c. of
defibrinated blood with a drop of strong HCl and a few e.c. of acetic
acid. Cool and extract with 5 c.c. of ether. Examine the spectrum of
acid hematin in ether. Red, 4 638. On dilution other bands appear.

Experiment 194. Hematoporphyrin.—This is iron-free hematin. It
is prepared by the action of strong acids or alkalies on hemoglobin.

Acid hematoporphyrin.—The strong acid at first splits off hematin
from the globin and then this hematin loses its iron and is converted to
hematoporphyrin. As reduced hemoglobin is far less stable than oxy-
hemoglobin, the iron is split off easiest from the reduced hemoglobin.
Add a couple of drops of putrefying blood (the putrefaction reduces
the hemoglobin) to a few c.c. of concentrated sulphuric acid and mix
by gently shaking. The solution has a purple color. Examine spectro-
1004 PHYSIOLOGICAL CHEMISTRY

scopically. Center of absorption bands: a at A 600 relatively fainter,
and # at A 554.

Experiment 195. Alkaline hematoporphyrin.—Make a somewhat
stronger solution of acid hematoporphyrin than the preceding, but in
the same way, and pour it into 50 c.c. of water in a beaker. Stir well.
Collect the precipitate which rises to the surface on a rod, transfer to a
test-tube, add a few c.c. of alcohol and boil. Make alkaline with a few
c.c. of NaOH. Examine the spectrum of alkaline hematoporphyrin thus
obtained. 4 absorption bands: A 622, y 576, A 539, and A 504.

EXERCISE XXXIX. COAGULATION OF THE BLOOD.

Clotting of the blood depends on the interaction of four elements:
Fibrinogen, calcium salts, prothrombin and tissue fibrinogen or thrombo-
kinase. The fibrinogen, calcium and prothrombin are in the blood; the
other element, tissue fibrinogen, is in the tissues.

For these experiments one can use ox or horse’s blood rendered non-
coagulable by receiving it into a saturated solution of sodium or potas-
sium fluoride or mixing it as it is shed with an amount of powdered
sodium oxalate equal to .01 to .02 gram for each 10 c.c. of blood.

Experiment 196. Fibrinogen.—Centrifugalize 20-30 c.c. of oxalate
or fluoride plasma. Note the relative volume, approximately, of plasma
and corpuscles. The plasma should be a light-yellow color and clear.
Pipette it off from the corpuscles very carefully without getting an
admixture of corpuscles. This is best done by connecting a test-tube
with the suction water pump by a tube (a) which reaches just through
the cork. Another.tube (b) goes through the cork, but ends nearer the
bottom. The other end of the tube (b) is attached to a flexible piece of
rubber tubing. The pump is started going and one end of this rubber
tubing is introduced into the plasma just below the surface of the
plasma. The plasma is sucked over into the the second tube. As it is
drawn over follow it down with the end of the rubber until no more
can be taken off without mixing with corpuscles.

Experiment 197.—Take 5 c.c. of the plasma in a test-tube and add
an equal volume of saturated NaCl solution. Fibrinogen will be pre-
cipitated.

Experiment 198.—Take another 3 ¢.c. in a test-tube and immerse in
a beaker partly filled with water. Heat slowly with a thermometer in
the fibrinogen solution. The tube must not touch the bottom of the
beaker. Note the temperature of coagulation. About 56°-58°; and a
more copious at 75°-80°. Fibrinogen coagulates at 56°.

Experiment 199.—To another 5 cc. of the plasma add sufficient
CaCl, to precipitate all the oxalate and leave a very small excess of
PRACTICAL WORK AND METHODS 1005

calcium salt in the solution. The solution will usually clot on standing.
If not, add to it some serum or a few c.c. of 0.9 per cent. NaCl solution
which has been rubbed in a mortar with the fresh fibrin of defibrinated
blood. Allow to stand and observe clotting. By extracting the fibrin a
solution of thrombin is obtained.

The power of fibrinogen solutions to clot on the addition of thrombin
as the real test for this substance.

Experiment 200.—Clotting is also prevented by receiving the blood
at once in strong solutions of magnesium sulphate. A saturated solution
of magnesium sulphate is placed in a bottle, or jar, and blood run in
directly from the veins, mixing well as it flows, until the combined
volume of blood and sulphate solution is about five times that of the
sulphate solution alone. The blood is then centrifugalized to remove the
corpuscles and the plasma drawn off and kept in a cool place. It is
salt plasma. It may be used in place of the oxalate or fluoride plasma.

Experiment 201.—Dilute some of the salt plasma by adding about 4
times its volume of distilled water. Divide into two portions in two
test-tubes. To A add nothing; to B add some of the fibrin ferment solu-
tion. Place both in the bath at 38° C. Observe that both clot, but that
B clots the sooner.

Experiment 202. The tissue element in clotting.—In order that
the blood may clot as soon as shed on rupture of the blood vessels, but
will not clot while in the blood vessels, one element of the clotting has
been put in the tissues and the others in the blood. The tissue element
was named “‘ tissue fibrinogen ’’ by its discoverer, Wooldridge, and that
is the name adopted here. It has also been called coagulin, thrombo-
plastic substance, thrombokinase, cytozyme, and by others it has been
supposed to be cephalin, a phospholipin. The name ‘‘ tissue fibrinogen ”’
is best chosen, in the author’s opinion, for the reasons that it resembles
blood fibrinogen in all its main characteristics except that it will not
clot. Its coagulation temperature is the same, i.e., 56°; it is precipi-
tated by half saturation with NaCl, or by weak acid, or CO,, like blood
fibrinogen; like blood fibrinogen also it is composed of a union of a
phospholipin and protein. It is present in the greatest amounts in those
tissues and organs in which hemorrhage is most to be feared, i.e., lungs,
brain, kidneys. Muscles have much less. Probably injured blood vessel
wall will be found to have much of it. The addition of this substance
to blood, instantly clots it either within or without the body. It is the
most powerful of styptics. To show its presence grind a small amount
of lungs or brain of any mammal with 1 per cent. NaCl. Strain through
cheese cloth, or filter through coarse paper. Take two test-tubes, each
containing 3 c.c. of oxalate plasma. Add to each sufficient CaCl, solu-
tion to precipitate the oxalate, about 1-3 drops 1 per cent. CaCl, per e.e.
1006 PHYSIOLOGICAL CHEMISTRY

of blood; to one tube add in addition 1 drop of the tissue fibrinogen
solution just prepared. This tube will clot promptly; the other after
some delay.

Exercisk XL. THE QUANTITATIVE CHEMICAL ANALYSIS OF THE BLOOD.

The determination of the simpler substances, the extractives and
sugar of the blood plasma, has recently become of great value in giving
a measure of the condition of the kidneys and the condition of the
carbohydrate metabolism. Methods have now been worked out so simple,
exact and expeditious and requiring so little blood that the blood ex-
amination is becoming more and more a routine diagnostic procedure.
It is not possible in any other way to secure accurate information as to
the state of the kidney. Especially in cases of nephritis the determina-
tion of the total non-protein nitrogen gives exact information as to the
condition of the patient as regards the approach of uremia. Similarly
unexpected shortcomings in the carbohydrate metabolism are. often re-
vealed by a determination of the blood sugar, since if the kidney is not
functioning normally there may be an accumulation of sugar in the
blood without any indication of sugar in the urine. The determination
of the non-protein nitrogen in the blood is often of value also in dif-
ferentiating between different comas. A coma of unknown origin may
be of traumatic origin, of a diabetic or uremic or some other toxic type,
or due to some vascular change in the brain, such as clot, pressure, etc.
The examination of the blood extractives will often give aid in dif-
ferentiating between various comas. The analysis is now easily made
and requires but 5 «ec. or so of blood and only a few hours for a
determination of all the more important constituents. The method is
also applicable to other fluids such as spinal liquid, exudates, etc., and
its application has thrown much light on the physiology of the blood.
There are three main systems of blood analysis in general use, that of
Greenwald, based on the Lewis-Benedict method, the Lewis-Benedict
and the Folin-Wu. In all these systems the first procedure is to obtain
a filtrate which is completely free from protein. Lewis-Benedict does
this by the addition of picrie acid to the blood; Greenwald uses trichlor
acetic acid and Folin uses tungstic acid. Hither of these procedures
is good. They each have special points of advantage. The Folin-Wu is
perhaps the more reliable and simpler and takes less blood. It relies,
however, altogether on colorimetric methods, and the Nessler reagent in
inexperienced hands is very troublesome.

Of the various constituents uric acid is earliest affected by an im-
pairment of kidney function and may be abnormally high while the other
eonstituents are normal; urea is the next substance affected and cre-
PRACTICAL WORK AND METHODS 1007

atinine is the last of the substances to accumulate. However, since both
urea and uric acid are greatly dependent on the diet, whereas creatinine
is not affected by diet but is of endogenous origin, it is often found that
a small urea and low total non-protein nitrogen are associated with high
creatinine. For this reason the creatinine offers a particularly valuable
index of the extent of kidney impairment of function since it will not
accumulate until the kidney has been impaired for a long time. Ac-
cording to Myers and Killian an amount of creatinine more than 5 mgs.
in 100 c.c. blood represents a very serious impairment of kidney function
with the probability of a fatal termination within a few months. The
prognosis becomes worse the higher the creatinine is above this figure.
In general high creatinine and failure of excretion of sulphonephthalein
are corroborative of each other.

Experiment 203. System of blood analysis (Folin and Wu: Jour.
Biol.. Chem., 38, p. 90, 1919; ibid., 41, p. 367, 1920).

 

Normal human blood Abnormal blood
mgs. in 100 c.c.

 

Total non-protein N .......... 26-43 50-275
Wea NG si.5 sca wieticak blerata a atneianess'6 10-22 30-235
Uric acid. si saiacs saat payed 1.0-4.2 5-14.3
Creatinine 6 s.4 ss ox dawcasaeeees 1.2-2.5 3-13

Creatine and creatinine ....... 53.-6.7 7-27.2
Dextrose... ce4-53.4 ease eens 2 77-119 157-400

 

 

(The foregoing figures for abnormal bloods have been taken from cases reported
by different investigators. The amounts may no doubt be even larger than those
here reported.)

For total non-protein N; urea; creatinine; creatine; uric acid; and
sugar 5 ¢.c. of blood required.

Method of obtaining a sample of blood. A dry test-tube or small
flask capable of holding 20 c.c. and provided with a tightly fitting cork
is prepared to receive the blood. Into this test-tube is placed 20 mgs.
of finely powdered potassium or sodium oxalate. This is to precipitate
the calcium and prevent the blood from clotting. This is enough oxalate
for 10 c.c. of blood. The blood is drawn into a sterile syringe through
a needle inserted into the vein at the elbow. About 7 to 10 cc. should
be drawn. It is usually best to moisten the inside of the barrel of the
syringe with two or three drops of a 20 per cent. potassium oxalate
solution. The blood is drawn and discharged as quickly as possible into
the test-tube containing the oxalate and the oxalate is mixed with the
blood: by inverting the tube once or twice. Coagulation is thus pre-
vented. - -
1008 PHYSIOLOGICAL CHEMISTRY

The protein free filtrate. To get rid of the corpuscles and the pro-
teins proceed as follows: Measure accurately with a 5 e.c. volumetric
Ostwald pipette, 5 c.c. of the oxalated blood into a 100 ¢.c. flask. Empty
the pipette by blowing it gently into the flask. Measure with a burette
or another pipette into the flask 35 c.c. of distilled water and mix well

 

 

 

 

 

 

 

 

 

 

 

Fig. 81.—Needle arranged for drawing blood. (Van Slyke and Cullen.)
For determination of alkali reserve see p. 1043.
with the blood. This lakes the blood and causes the discharge from
the corpuscles of amino-acids, sugars, salts, urea and other constituents.
With another 5 c.c. volumetric pipette measure accurately into the flask
5 «ec. of a 10 per cent. solution of sodium tungstate (Na,W0O,2H,0)
and mix. With another pipette add 5 c.c. of two-thirds normal sulfuric
acid, shaking the flask gently while adding the acid. Close the mouth
of.the flask with a rubber stopper and give a few vigorous shakes.
Proper coagulation of the blood by. the tungstic acid is shown by no
air bubbles, or only one or two, forming on shaking, and also by a
gradual change in the color of the precipitate from a red to a brown.
If the latter change does not occur, sufficient acid has not been added,
and in that case add while shaking vigorously, and drop by drop (at
the most not more than 2 drops should be required), 2NH,SO, solution,
allowing to stand a few minutes between-each addition of a drop and
before adding more, until the coagulation is complete and the color is
brown. The amount of acid added is just sufficient to set free the
PRACTICAL WORK AND METHODS ‘1009

tungstic acid from the sodium but an excess must be avoided. The
filtrate should be not more than neutral or barely acid to congo red.
The blood has now been diluted from 5 ec. to 50 «c., that is, 10 times.
If a larger amount of filtrate is desired, more blood may be taken than
5 @e., and all the other solutions increased in proportion, so that the
final dilution is to 10 times that of the blood taken. 5 e.c. of blood
should give enough filtrate for all the determinations.

Prepare a funnel with a dry, folded filter paper large enough to
take the whole volume of the 50 c.c. at once, a paper 12 ems. in diameter

  

Fie. 82.

Fig. 82.—_-Duboseq colorimeter.

is large enough. Do not wet the filter paper with water, but moisten
it with a few drops of the clearer supernatant liquid in the. flask,
allowing it to wet the whole paper by capillarity before pouring the
rest of the liquid on. In this way the first drops will be clear. If the
first filtrate is not clear, pour it back on the filter until a clear filtrate
is obtained. Collect the clear filtrate in a small, clean, dry flask. This
gives the protein free filtrate for the determinations which follow.
Testing the acidity of the filtrate. It is necessary in the determina-
tion which follows that the filtrate shall be nearly neutral. Test a drop
1010 PHYSIOLOGICAL CHEMISTRY

of the filtrate with congo red paper. It should be neutral or barely acid.
10 c.c. of the filtrate should require about 0.2 ¢.c. of 0.1N NaOH to neu-
tralize, using phenolphthalein as indicator. If the filtrate is to be kept
any time before using, a couple of drops of toluene or xylene should be
added and the flask stoppered, to preserve it from putrefaction.

Non-protein nitrogen. The total non-protein nitrogen is determined
by setting free the nitrogen as ammonia by the Kjeldahl process,
Nesslerizing the ammonia thus set free and comparing the color with
that of a standard ammonium sulphate solution.

Solutions required:

1. Standard ammonium sulphate solution. Recrystallize twice a
good grade of (NH,),SO,. Dry the crystals at 100°. Weigh off 0.4715
gr., dissolve in distilled water and make to 1 liter. This solution con-
tains 1 mg. of N in 10 «ec. 3 @e. are required for each determination.
(See also page 1084.)

2. Sodium tungstate (Na,WO,, 2H 20) solution 10 per cent. 5 ec.
at each determination.

8. 2/3 N H,SO,. (5 «ce. each determination.)

4. Nor 2N H,SO0,.

5. Sulphuric-phosphoric acid mixture. For hydrolyzing and oxi-
dizing in the Kjeldahl method. Mix 300 c.c. 85 per cent. H,PO, (sp. gr.
1.71) and 100 ec. concentrated H,SO,. Let stand to sediment any
CaSO,. To 100 c.c. of clear upper layer add 10 c.c. 6 per cent. CuSO,
and 100 c.c. water.

6. Nessler solution,

(a) Mercurie potassium iodide.

Make a solution of NaOH completely saturated at room temperature.
This can be done by dissolving about 120 grams NaOH (good grade)
in 100 c.c. water. It is better to make about 10 or 20 times this amount
and keep as a stock solution. Dissolve 2,400 grams NaOH in a beaker
in 2 liters of water. Transfer all to a large bottle provided with a
rubber stopper with 2 holes. Shake and allow to stand. Through one
hole goes a glass tube which reaches to near the bottom, bends at a
right angle above the cork, and then after about 1 foot bends again at
right angles, the downward part being about 2 inches long. This is the
delivery tube. The other hole has in it a glass tube reaching just
through the stopper and bent at right angles. This tube connects by
rubber tubing with the compressed air, but between the bottle and the
air is inserted a T tube. One arm of the T has attached a piece of
rubber tubing ending in a soda lime tube. By holding the thumb over
the end of the soda lime tube and turning on the air, NaOH is driven
out of the bottle. (A bicycle pump will do if compressed air is not
available.) The solution is allowed to stand at room temperature. The
PRACTICAL WORK AND METHODS 1011

excess of NaOH and any Na,CO, crystallizes out. The upper layers
become clear in a day or two. Use this layer. This solution, when
properly made, is at 15 degree approximately 50 per cent. NaOH and
has a sp. gr. of 1.53. It is very important that this solution be in reality
a saturated solution of sodium hydrate free from carbonate. Take 800
grams of the clear supernatant liquid (delivered by closing the one
tube while turning on the compressed air), add it to 3,200 ec. of dis-
tilled water. This will give approximately a 10 per cent. solution. It
is best to take the sp. gr. of this solution (1.116) or to titrate it to make
sure that it is 10 per cent. Take of this solution 3,500 ec. in a large
bottle. Add to it slowly while stirring 750 c.. of the mercuric
potassium iodide solution described below and 750 «.c. distilled water,

 

Fig. 83.
Fig. 83.—Bock-Benedict colorimeter.

making in all 5 liters. This Nessler solution contains enough alkali
in 15 ec. to neutralize 1 ¢.c. of the sulphuric-phosphorie acid mixture
and to give a suitable alkalinity for color by ammonia at a volume of
50 cc. To make sure that this is 30, measure out accurately with a
burette 2 ¢.c. of the acid mixture, add 3 «ce. of the standard ammonia
solution, add 55 ¢.c. water and then 30 e.c. of the Nessler, diluting then
to 100 ¢¢., and see if a clear deep yellow turning to yellowish-brown
1012 PHYSIOLOGICAL CHEMISTRY

solution is obtained. If it is not obtained it means that the solution has
not sufficient alkali or has carbonate in it. It is important that these
directions be accurately followed and that alkali free from carbonate
be used, as otherwise a deep brown-red turbid solution is obtained,
which is unfit for Nesslerization. In other Nesslerizations, where there
is no acid to be neutralized, 10 c.c. of the Nessler reagent per 100 c.c. of
Nesslerized ammonia is correct.

Mercuric potassium iodide. Into a 500 ¢.c. Florence fiask measure
150 gms. KI and 110 gms. I,, add 100 cc. water and 150 gms. of
metallic Hg and shake vigorously for about 10 minutes until the dis-
solved I, has nearly disappeared. The solution heats. When the red
solution begins to pale, but is still red, cool in running water and con-
tinue shaking until the red has disappeared and been replaced by the
greenish color of the double iodide. The whole time is about 15 minutes.
Decant from the mercury into a 2 liter volumetric flask; wash the mer-
cury repeatedly with distilled water and add the washings to the flask
and dilute finally with ammonia-free distilled water to the 2 liter mark.
If the cooling has been begun in time the resulting solution is clear
enough for immediate dilution with the 10 per cent. alkali, as described
above, and can be used at once for Nesslerization.

Ammonia-free water. Acidify distilled water with H,SO, and
redistill.

Special apparatus required.

1. Duboseq or other colorimeter.

2. One 100 c.c. volumetric flask. One 50 ¢.c. volumetric flask.

3. Large Pyrex glass test-tube, 200 mm. x 22 mm., holding 75 e.c.

4. Quartz pebbles.

5. Ostwald pipettes, 3 c.c. and three 5 ec.

Method. With a5 cc. Ostwald pipette measure accurately 5 e.c. of
the protein free blood filtrate into a 75 c.c. hard glass dry test-tube
(Pyrex glass, 200 mm. x 22 mm.) which has been graduated by marking
it at 35 c.c. and at 50 c.c. To prevent bumping this tube and the pebbles
must be washed with HNO, and carefully dried. Add 1 eae. of the
phosphoric sulphuric acid mixture described above, then 2 or 3 small dry
quartz pebbles to prevent bumping, support the tube by clamping to a
lamp stand in a vertical position, and boil vigorously over a micro-
burner until dense white fumes begin to fill the tube, 3 or 7 minutes;
then cut down the flame, so that the contents just boil, and continue
boiling gently for 2 minutes from the time that the fumes appear. As
soon as the fumes appear cover the mouth of the tube with a clean dry
watch glass. The tube contents may become colorless in a few seconds
but the heating must be continued for two minutes, nevertheless. If
the solution is not nearly colorless at the end of 2 minutes it must be
PRACTICAL WORK AND METHODS 4013

heated until it is. Allow to cool for at least 70 to 90 seconds (longer is
better) and then add 15 to 25 «.c. of distilled water free from ammonia.
(The water should be distilled from over sulphuric acid.) Cool again
to room temperature and then fill to the 35 ¢c.c. mark with water. Now
add 15 c.c. of the Nessler solution described above, using a pipette, close
the tube with a clean rubber stopper and invert the tube to mix. The
solution becomes a deep yellowish color. If it is turbid, centrifuge a
portion of it before placing it in the colorimeter for comparison with
the standard.

Preparation of the standard. The preparation of the standard solu-
tion of ammonium sulphate is described on page 1010. This standard
contains 1 mg. of N in 10 cc. With a 3 ce. pipette measure 3 c.c. of
the ammonium sulphate solution into a 100 ec. volumetric flask. This
contains 0.3 mg. of nitrogen as (NH,),SO,, which is about the amount
needed for normal bloods. Add to it 2 ¢.c. of the sulphuric-phosphoric
atid mixture, about 60 c.c. of water and 30 ¢.c. of the special Nessler
solution and make up to the mark of 100 ec. with ammonia-free dis-
tilled water. Mix. This standard and the unknown should have the
Nessler solution added to them at about the same time and the dilutions
completed at as nearly the same time as possible.

Comparison in the colorimeter. Test the colorimeter by introducing
into the clean and dry cylinders of the Duboseq colorimeter some of the
standard solution and match the two sides. The readings of the two
sides should be identical. If not, correct the illumination until they
are, or repair the colorimeter. Sometimes one of the prisms may loosen
and drop down a little unperceived. Leave the standard in one side,
and rinse out the other cylinder with water and then twice with the
unknown, then fill with the unknown. Set the standard at 20 mm. and
move the unknown up or down until the two sides have the same color
in the eyepiece. Then read the unknown.

Computation. If the standard is set at 20 mm. for the color com-
parison, 20 divided by the reading and multiplied by 30 gives the non-
protein N in mgs. per 100 e.c. blood.

20 X 30

 

Non-protein N, mgs. per 100 cc. blood = where y is the

reading of the unknown. This is derived as follows:

The 5 cc. of filtrate taken carried the non-protein N of 0.5 ec. blood.
This was diluted to 50 c.c., while the standard was diluted to 100 e.c.
Suppose the unknown had read 20, that is, just the same as the standard.
Then it would contain 0.3 mg. N in 100 c«.c., but it has been diluted only
to 50 c.c., that is, one-half, so it has 0.3 mg./2. That is, it has in 50 ce.
only % the nitrogen in the standard. The 5 ec. of filtrate contained
then 0.15 mgs. N, but this wes in 0.5 «ec. of blood. 100 cc. of blood
1014 PHYSIOLOGICAL CHEMISTRY

would then contain 200 x 0.15 mgs., or 30 mgs. If instead of 20 the
unknown had read 30, the standard being 20, then it would contain
_ 20/30 of 30 mgs., or 20 mgs.

Urea in blood. Normal blood contains 10-22 mgs. urea N.

Principle. Urea in the blood filtrate is hydrolyzed to ammonia by
the addition of urease, the ammonia is removed by distillation or aera-
tion into acid, and the ammonia is determined by Nesslerization and
colorimetric comparison with a standard ammonia solution.

Special reagents required in addition to those used in the total non-
protein nitrogen determination.

1. Urease solution. To get rid of any ammonia in the urease, per-
mutit powder is employed. Place in a 200 c.c. flask 3 grams of permutit
powder, wash by decantation once with 2 per cent. acetic acid and then
twice with water. Add 100 «ec. of 30 per cent. aleohol (35 ¢.c. of 95
per cent. alcohol mixed with 70 ¢.c. water), and 5 grams of jack bean
meal and shake for 10 minutes. Filter and collect filtrate into 3 or 4
different small, 25 to 50 ¢.c., bottles. Set one aside for immediate use;
the others should be stoppered and placed on ice. They will remain
good for at least three or four weeks. The one which is currently used
can be kept serviceable for at least a week at room temperature if pro-
tected from direct sunlight. The urease solution is sufficiently strong
_.8o that 1 ¢.c. added to 300 mgs. of urea N in 200 c.c. solution will yield
37 to 22 mgs. urea N in one hour at 20 degrees. Complete in 18 hours.

2. Buffer solution. (a) Pyrophosphate solution. 140 grams sodium,
pyrophosphate and 20 g. glacial phosphoric acid per liter.

(b) Phosphate solution. A solution containing 1/3 og a gram mole-
cule of NaH,PO, and 2/3 of a gram molecule of Na,HPO, per liter.
(46.03 grams NaH,PO,, H,O and 238.8 grams Na,HPO,, 12H,O per
liter.) The pyrophosphate is preferable.

3. Saturated borax solution.

Method. Clean a 75 c.c. (200 mm. X 23 mm.) Pyrex, or other hard
glass test-tube by rinsing it out with nitric acid and then thoroughly in
water and drying. The rinsing in nitric acid is necessary to remove any
copper, mercury or other metal of a poisonous kind which may have
been in solutions previously in the tube and which stick so closely to
the glass that the metal cannot be removed except by washing in nitric
acid. If the metal is not removed it may poison the urease and give
false results. Measure accurately into the dry tube 5 ¢.c. of the tungstic
acid blood filtrate obtained, as stated on page 1008, using an Ostwald
pipette for the transfer; then add as buffer two drops of the pyro-
phosphate or ordinary phosphate solution described above. This addi-
tion is made to give the most favorable hydrogen ion concentration for
the urease action. The reaction is kept nearly neutral by them. Now
add from 0.5 to 1 ¢.c. of the urease solution, from a pipette (the prepara-
PRACTICAL WORK AND METHODS 1015

tion of the urease is given above). Immerse the tube in a water bath
at 40° to 50° C. for 10 minutes. This is sufficient to convert the urea to
ammonia under these conditions. Then add to the solution one, or several,
small dry quartz pebbles, to prevent bumping, 2 c.c. of the saturated
borax solution to set free the ammonia, and 1 drop of paraffine oil,
connect up the test-tube by means of a rubber stopper and a glass tube,
as shown in Figure 84. The receiving or second tube should be smaller

 

Fie. 84.

Fie. 85.

Fig. 84.—Test-tubes arranged for distillation of urea nitrogen. A, First position.
B, Second position. (After Folin.)

and lighter than the first, should have a capacity of about 40 cc. and
the cork should have two holes in it or a channel cut at one side so as
to permit escape of steam. Place in the second tube exactly 2 ce. of
0.05 normal HCl, measured from a small burette or accurate pipette.
This second tube should have a mark on its side showing where 25 c.e.
would come to. The delivery tube from the first tube dips below the
surface of the acid in the second tube. In this distillation care must
be taken to prevent sucking back, owing to drafts blowing the flame of
the micro burner. Use a wind shield. Boil over a micro burner rather
vigorously for 4 minutes to distill over the ammonia. The flame should
be so high that the first steam comes from the second tube before the
end of 3 minutes. Continue the distillation for 1 minute more and then
slip the receiving tube into the position shown by figure 85. Distill for
1016 PHYSIOLOGICAL CHEMISTRY

one minute more in this position to wash out the inside of the tube.
Then with the wash bottle and a fine stream wash off the end of the
receiving tube, collecting the small amount of washings into the second
test-tube. Put out the flame. Cool the distillate under the tap, dilute to
20 c.e. with distilled ammonia free water, prepare the standard and when
it is ready add 2.5 ¢.c. of the Nessler reagent, fill to the 25 ¢.c. mark
with water, insert a clean rubber stopper, and invert tube to mix con-
tents. This tube is now Nesslerized. Meanwhile, during the distillation,
proceed to prepare the standard for comparison.

Preparation of standard. With a 8 ¢.c. pipette measure accurately
3 c.c. of the standard (1 mg. N to 10 cc.) ammonium sulphate solution
into a clean 100 ec. volumetric flask, and when the distillate just
obtained is ready, add as nearly as possible at the same time as the
Nessler is added to the distillate 10 ¢.c. of Nessler solution and fill to
the mark with ammonia free water. Stopper the flask, and mix.

Comparison. Compare the two solutions in the Duboseq colorimeter
in the manner described on page 1013, always testing the instrument first
by reading the standard against itself.

Computation. The computation is made as in the total non-protein
nitrogen except that inasmuch as the standard is diluted to 100 ce,
while the unknown, which corresponds to 0.5 c.c. of blood is diluted
only to 25 e«.c., the ratio of the standard to the unknown in mm. is
multiplied by 15 instead of 30.

Reading of standard

Mgs. of urea nitrogen in 100 c.c. blood = 15

Reading of unknown

The reading of the standard should be about 20. Usually the standard
is set at 20 mm. and the unknown matched with it.

Modtfication. Instead of distilling off the ammonia into the second
tube it can be carried over by aeration with a current of air. In this
case, after the urease has acted, set up the apparatus as indicated on
page 1082. When all is ready and the acid in the receiving tube,
which is prepared as before, add to the first tube 1 or 2 «ec. of NaOH,
10 per cent., to set free the ammonia and aspirate at a lively rate into
the test-tube graduated at 25 e.c. The rubber tubing should be washed
thoroughly before beginning the aspiration to guard against any con-
tamination with ammonia. The glass tubes should in all cases come
tight together within the rubber tubing used for connections. Con-
tinue the aeration at room temperature for 10 minutes. Then discon-
nect and Nesslerize, as in the preceding.

Instead of using urease the urea may be hydrolyzed by autoclaving.
In this case to 5 c.c. of the blood filtrate add 1 ¢.. normal HCl, cover
the mouth of the test-tube with tinfoil and heat in autoclave at 150° C.
PPACTICAL WORK AND METHODS 1017

for 10 minutes. Allow autoclave to cool below 100 degrees before open-
ing. Distill as in the first process, except that 2 cc. of Na,CO,, 10 per
cent. solution, is added in place of the borax. This method is no better
than the other.

Creatinine determination in blood.-Amount in normal human
blood: 1 to 2 mgs. per 100 e.c. blood. Over 5 mgs. represents a very
serious impairment of the kidney with a prospect of fatal termination
within a few months. The prognosis is worse the greater the excess
above 5 mgs. (See Meyers and Killian.)

Solutions needed:

1. Standard creatinine solution. 6 ¢.c. of the standard creatinine
solution for urine (page 1095), containing 1 mg. per c.c. of creatinine,
are measured into a volumetric liter flask. 10 c.c. of N HCl are added
and 4 to 5 drops of toluene and made to the mark with water. This
solution will keep indefinitely. 5 ¢.c. of this solution contain 0.03 mgs.
of creatinine. This is the amount needed for the standard for normal
human blood.

2. Saturated, purified picric acid solution, kept protected from
light. The picric acid develops, when exposed to light, substances which
give a color like picramic acid. The picric acid of commerce is often
impure. It should be carefully recrystallized from hot water and its
purity tested by the method of Folin and Doisy (J. B. Chem., 28,
1916-17, p. 349).

Apparatus needed:

1. Large test-tubes holding 50 e.c. and graduated at 25«¢. 4.

2. Colorimeter: Duboscq, Benedict-Bock or Hellige.

Method. 50 c.c. of the saturated, purified, aqueous solution of picric
acid are placed in a small clean flask, and 10 c.c. of 10 per cent. NaOH
are added and mixed. This solution should be made fresh each time.
Take 4 large test-tubes graduated at 25 c.c. Into one place 10 cc. of
the blood filtrate from the tungstic acid precipitation (page 1008) ; into
the others introduce respectively 5 ce, 10 «ce. and 15 ec. of the
standard creatinine solution containing 0.03 mgs: creatinine in 5 c.c.
Then add to these three tubes respectiveiy 15 «c., 10 cc. and 5 ce. of
water and mix. The four tubes are now ready for the alkaline picrate
solution. Add to each of the 4 tubes as nearly simultaneously as possi-
ble of the alkaline picrate solution 5 cc. to the blood tube; and 10.c.c.
to each of the three standard tubes. Mix by stoppering and inverting
the tubes once or twice. Allow to stand at room temperature for 8 to 10
minutes. Then select the standard which is nearest the blood tube in
depth of tint and compare the two in the colorimeter, having the
standard set at 20 for the Duboscq colorimeter. Always read the
standard against itself before making the reading with the blood. This
1018 PHYSIOLOGICAL CHEMISTRY

is to check the accuracy of the instrument. The two sides should agree
with an error of not more than 0.1mm. The reading must be completed
within 15 minutes from the time the alkaline picrate is added, as the
color fades, and also for the reason that other reducing substances may,
on standing, produce color. The creatinine coloration comes promptly
at room temperature but is not permanent.

Of these three standards the first will correspond to the content of
1 to 2 mgs. creatinine in 100 ¢.c. blood; which is the normal content;
the second covers the range from 2 to 4 mgs.; and the third from 4 to 6
mgs. In eases of long-continued nephritis the creatinine content may
rise, however, to 10 mgs. or more. It is well, if such a condition is sus-
pected, to have a fourth standard tube which contains 20 c.c. of the
standard creatinine solution to which no water is added. This would
give a range of 6 to 10 mgs.

In case a higher creatinine content than this is encountered, when
no standard sufficiently high is available for comparison, the determina-
tion can be saved by diluting the alkaline picrate solution with 2 volumes
of water and then diluting the blood tube with an equal, or some other
known volume, of this solution.

Computation. The standard tubes contain in 30 e.c. 0.03, 0.06, 0.09
and 0.12 mgs. creatinine respectively. In 10 cc. they contain, then,
0.01, 0.02, 0.03 and 0.04 mgs. of creatinine. The blood tube contains
15 cc. Hence 1.5 times these figures represents the amount in the
blood tube if it exactly matches the standards. If it does not match,
the figures just obtained must be multiplied by the ratio of the reading
of the standard to the reading of the unknown. Hence

Megs. creatinine in 100 c.c. blood = 1.5, 3, 4.5, or 6 (depending on
which standard is used) X 20 (the reading of standard) ~ the reading
of the unknown.

Creatine and creatinine determination. (Total creatinine.)—There
is much more creatine than creatinine in blood. To determine the cre-
atine it is converted over into creatinine and the total measured as
creatinine. In normal human blood there are about 6 mgs. total cre-
atinine in 100 cc. blood. In uremia the amount may be more than
three times this figure.

Solutions required. In addition to those required for creatinine a
N HCl solution.

Additional apparatus required. -An autoclave capable of heating to
155° C.

Method. To a large, 50 to 75 cc. capacity, hard glass test-tube
(Pyrex glass) introduce by a pipette exactly 5 ¢.c. of the tungstie acid
blood filtrate. The test-tube should be graduated at 25 c.c. In ease of
uremic bloods with much creatine and creatinine take 1, 2, or 3 cc. of
PRACTICAL WORK AND METHODS 1019

the filtrate and dilute to 5 ¢.c. with water. Add 1 ¢.c. N HCl, cover the
mouth of the tube with tinfoil; autoclave it at 130° C. for 20 minutes; or
at 155° C. for 10 minutes. Let the autoclave cool below 100° before
opening it. Remove and cool the tube. Prepare the standard by placing
20 c.c. of the standard creatinine solution in a volumetric flask (50 e.c.) ;
add 2 ec. N HCl. Now add to the standard 10 cc. of the alkaline
picrate solution (page 1017) and to the unknown 5 e.c. of the picrate solu-
tion. Let stand for 8 to 10 minutes, then dilute the standard to 50 c.c.
and the unknown to 25 e.c. and compare in the colorimeter as for the
creatinine, first comparing the standard against itself.
Computation. If 5 e.c. of blood filtrate taken, then:

by Reading of standard
Mgs. total creatinine per 100 ¢.c. blood =———-—______
Reading of unknown

If 1 ¢.c. was taken it will be 5 times this amount.

Determination of uric acid.

Principle. Uric acid is precipitated from the tungstic acid filtrate
by silver lactate and then estimated colorimetrically by the production
of a blue color with phosphotungstic acid and compared with color pro-
duced by a standard uric acid solution.

Apparatus needed:

1. Duboseq or other colorimeter.

2. Centrifuge and centrifuge tubes.

Solutions:

1. Standard uric acid sulfite solution. Make 1 to 3 liters of 20
per cent. solution of sodium sulfite. Let stand over night and filter.
Dissolve 1 gram uric acid, accurately weighed, in 125 to 150 cc. of 0.4
per cent. Li,CO, solution and dilute to 500 c.c. 50 cc. of this solution
containing 100 mgs. uric acid are accurately measured into a series of
volumetric 1 liter flasks; 200 to 300 ¢.c. of H,O added to each, and then
500 ec. of filtered 20 per cent. sodium sulfite solution. Make up to
volume, with water and mix well. Fill with this solution a series of
200 ¢.c. bottles and stopper very tightly with rubber stoppers. This
is done to preserve the uric acid from oxidation by air. In these bottles
the uric acid will keep for a long period. One of these bottles is kept
for daily use. When opened and in use it should keep 3 to 4 months.
This gives a stable standard uric acid solution. 10 c.¢c. contain 1 mg.
urie acid.

2. 5 per cent. lactate in 5 per cent. lactic acid. (4 or 5 ce. for a
determination.) Make up 1 liter.

3. 10 per cent. sodium sulphite solution. Made by diluting the 20
per cent. solution already described and transferring to a similar series
1020 PHYSIOLOGICAL CHEMISTRY

of small tightly stoppered bottles to keep from oxidation by air. (2 ee.
for each determination. )

4. 5 per cent. NaCN solution. To be added from a burette. 2.5 to
5 ee. for each determination.) Poisonous. Do not measure from pipette.

5. 10 per cent. NaCl solution in 0.1N HCl. (10 to 20 cc. for each
determination.)

6. Urie acid reagent of Folin and Denis. 100 grams sodium
tungstate, 80 ¢.c. 85 per cent. phosphoric acid (sp. gr. 1.71) and 700 e.c.
H,0O. Boiled for at least 2 hours. Dilute to 1 liter. (2.5 cc. for each
determination.) ‘

7. 20 per cent. solution of Na,CO,. (15 ¢.c. for each determination.)

Method. Measure 10 «ec. by a volumetric pipette into each of 2
centrifuge tubes. To each add 2 cc. of the 5- per cent. silver lactate
lactie acid solution and stir with a fine glass rod. This precipitates the
uric acid. Wash the rod with a drop or two of water, the washing being
added to the tubes. Centrifuge until the precipitate is well separated
and packed. Add a drop of the lactate solution to the supernatant
liquid. If it remains clear, enough of the lactate has been added. If
it becomes cloudy, add another e.c. of the lactate solution and recentrif-
ugalize after stirring once more. Decant the clear supernatant liquid,
taking care to lose none of the precipitate which contains the urie acid.
To separate the uric acid from the precipitate add to each tube 1 «ec.
of the 10 per cent. NaCl solution in HCl and stir thoroughly with the
glass rod. Then add 5 to 6 c.c. of water and stir again. By this means
the uric acid is brought into solution and the silver left as silver
chloride. Centrifuge again. Transfer the supernatant liquids contain-
ing the uric acid to a 25 c.c. volumetric flask, or a test-tube graduated
to 25 ec. Add 1 ce. of the 10 per cent. sodium sulphite; 0.5 cc. of
5 per cent. NaCN; and 3 e.c. 20 per cent. Na,CO,. The sodium cyanide
is added to make the blue color which develops later more permanent;
the carbonate prevents the sulphite from itself reducing the reagent and
making a blue color. Now prepare simultaneously 2 standard uric
solutions as follows: Two 50 c.c. volumetric flasks are taken; into one
is introduced 1 c.c. (0.1 mg. uric acid) and into the other 2 ¢c.c. (0.2 mg.
uric acid), measured by pipettes, of the standard uric acid sulphite
solution. To the first flask add also 1 c.c. of 10 per cent. sodium sul-
phite. Add to each flask 4 cc. of the acidified NaCl solution, 1 c.c. of
the NaCN solution, and 6 e.c. of the Na,CO, solution. Dilute each with
water to about 45 ¢.c. To the two standards and the unknown now add
the uric acid reagent. 0.5 ¢.c. of this is added to the unknown and 1 cc.
each to the two standards, and content of each is mixed. It is important
always not to add the uric acid reagent until the sodium carbonate is
added, since otherwise the sulphite may itself reduce the reagent and
PRACTICAL WORK AND METHODS 1021

produce a blue color. Let stand for 10 minutes, dilute each to the mark
with water, mix and compare the unknown in the colorimeter with that
standard which is nearest it in depth of tint.

Computation. The blood filtrate taken corresponds to 2 ec. of
blood. The standard is diluted to twice the volume of the unknown and
contains either 0.1 or 0.2 mg. of uric acid. If the unknown reads the
same as the weaker standard it will contain 14 of 0.1 mg. of uric acid
or 0.05 mg. This is in the filtrate from 2 ¢.c. blood. 1 ¢.c. would contain,,
then, 0.025 mg. And 100 e.c. blood would contain 2.5 mg. One cor-
responding to the 2d standard would contain 5 mgs. We have then de-
pending on the standard used

Reading of standard

Mgs. uric acid in 100 e.c. blood = < 2.5 (or 5)

Reading of unknown

Determination of sugar in blood. (Folin and Wu: Jour. Biol.
Chem., 41, p. 367, 1920.)

Principle. The tungstic acid blood filtrate is boiled with feebly
alkaline copper tartrate solution at the same time as a standard dextrose
solution similarly treated. Phosphomolybdic-phosphorie acid is added
to dissolve the cuprous oxide. This is oxidized and the molybdic reagent
reduced with the production of a blue color which is compared with the
standard.

This is undoubtedly one of the best methods of determining sugar
in blood.

Apparatus required:

1. Duboseq colorimeter, or some other colorimeter, such as the
Benedict-Bock.

2. Special sugar tubes. (See Figure 86.) 3 or 4.

Solutions required:

1. Standard sugar solution. Dissolve 1 gram pure anhydrous dex-
trose in water and dilute to a volume of 100 cc. Keep in stoppered
bottle in cool place, having added 3 to 4 drops of toluene. If pure
dextrose is not available the standard can be made from cane sugar.
1 gram cane sugar and 20 ¢.c. N HCl are put into a 100 e.c. volumetric
flask and allowed to stand over night at room temperature, or flask and
contents may be rotated at 70° in a water bath for 10 minutes. This
hydrolyzes the sugar. Add 1.68 gms. NaHCO, and about 0.2 grams
sodium acetate to neutralize the HCl. Shake a few minutes to remove
most of the CO,, then fill to 100 ¢.c. mark with water. Then add 5 c.c.
more of water (1 gr. of cane sugar gives 1.05 of invert sugar) and mix.
Place in bottle with a few drops of toluene, shake and stopper well.
This solution keeps indefinitely. To make the first standard take of this
stock solution 5 ¢.c. and dilute it to 500 c.c. To make the second take
1022 PHYSIOLOGICAL CHEMISTRY

5 c.c. and dilute to 250 c.c. Preserve all by toluene or xylene. These
are the standards. No. 1 contains 1 mg. of dextrose or invert sugar in
10 ce. (For each determination 2 ¢.c. required.) No. 2 has 2 mg. in
10 c.c.

 

 

 

 

Fig. 86.
Fig. 86.—Special tube for micro blood sugar method. (After Folin.)

2. Alkaline copper tartrate solution. Dissolve 40 gms. anhydrous
Na,CO, in about 400 ¢.c. of water and transfer to liter flask. Add
7.5 gms. tartaric acid and when this has dissolved add 4.5 gms. of
erystallized CuSO,.5H,O, mix and make up to 1 liter with water. De-
cant from any sediment (impure carbonate) which may form. Keep in
a bottle.

3. Molybdate-phosphate mixture. (This is a modification of the
Folin-Denis phenol reagent. Mix in a liter beaker 35 grams molybdic
acid, 5 grams sodium tungstate, and 200 cc. 10 per cent. sodium
hydrate and 200 c.c. water. Boil vigorously 20 to 40 minutes so as to
remove ammonia. Cool, dilute to about 350 cc. and add 125 c.c. con-
eentrated (85 per cent.) H,PO, (sp. gr. 1.71). Dilute to 500 cc. Keep
in a stoppered bottle.

Method. Take 3 sugar test-tubes (Figure 86), each graduated at
PRACTICAL WORK AND METHODS 1023

25 c.c. (In case these are not to be had ordinary test-tubes capable of
holding 25 cc. and not more than 17 mm. in diameter may be used
with very little error from reoxidation.) To one add 2 c.c. blood filtrate ;
to one of the others 2 ¢.c. of standard glucose containing 1 mg. in 10 c.c.;
and to the other 2 cc. of the standard twice as strong as this. To each
tube add 2 cc. of the alkaline copper tartrate. Each tube must now
be filled so that the surface of the liquid lies in the constricted neck. If
bulb of tube is a little too large, a little, but not more than 0.5 e.c. of
alkaline tartrate diluted 1:1 may be added, to bring the solution into
the neck. If it fills above the neck discard the tube. Immerse all three
tubes simultaneously in a beaker already boiling of water and continue
boiling for 6 minutes. Transfer all to cold water bath and let cool
without shaking for 2 to 3 minutes. Add to each test-tube 2 c.c. of
molybdate-phosphate mixture. The Cu,O dissolves rather slowly but the
whole up to the amount given by 0.8 mgs. dextrose will dissolve within
2 minutes. When Cu,O is dissolved dilute resulting blue solution to
25 e.c. with water. Stopper with rubber stopper and invert to mix
thoroughly. This must be done thoroughly, as most of blue color is in
bulb. Compare tubes in colorimeter after full color develops, i.e., in
from 5 minutes to 1 hour. These two standards cover the range from
70 to nearly 400 mgs. dextrose per 100 c.c. blood.

Computation. If the tube having 0.2 mg. dextrose is chosen as the
nearest for comparison, the standard being set at 20

20
mgs. of dextrose per 100 c.c. blood = — x 100
y

y being the reading in mms. of the unknown. If the 0.4 mg. standard
is taken this must of course be multiplied by 2. The 2 ee. of blood
filtrate taken correspond to 0.2 ¢.c. of blood.

Experiment 204. System of blood analysis by the methods of
Greenwald, Lewis and Benedict and Meyers and Bailey.—Non-protein
nitrogen is determined by the method of Greenwald; creatinine and
sugar by the Meyers and Bailey modification of the Lewis-Benedict
method; urea by the van Slyke modification of the Marshall method;
uric acid by Benedict’s modification of Folin’s method. (Greenwald:
Jour. Biol. Chem., 21, p. 61, 1915.)

Non-protein nitrogen. (Greenwald, Jour. Biol. Chem., 21, p. 61,
1915.)

Principle. The proteins and corpuscles are precipitated by tri-
chloracetic acid. The filtrate is digested with sulphuric acid by Kjeldahl
method, the ammonia distilled off and the ammonia determined by
titration.
1024 PHYSIOLOGICAL CHEMISTRY

Solutions needed:

1. Standard acid and alkali. 0.01N NaOH and 0.01N HCl. (15 ec.
of each for a determination.)

2. Indicators. 0.1 per cent. solution of methyl red; 0.1 per cent.
methylene blue.

3. Concentrated H,SO,, 5 per cent. CuSO, and solid K,SO,, or the
phosphoric sulphuric acid mixture of Folin and Wu (page 1010).

4, 2.5 per cent. solution of trichloracetic acid.

Apparatus:

1. Condenser.

2. 200 c.c. Kjeldahl flask; a 50 ¢.c. volumetric flask.

Method. 5 cc. of oxalated blood (see page 1007) are accurately
measured by an Ostwald pipette into a 50 c.c. volumetric flask and filled
to the mark with 2.5 per cent. trichloracetic acid. After standing 30
minutes it is centrifuged and filtered through a dry filter paper. If
greater accuracy is desired the filtrate may be shaken with a small
quantity of kaolin (4 mgs. per 100 ¢.c.) and again filtered. This removes
a small amount of a colloidal nitrogenous substance. An error of not
more than 2 mgs. of N in 100 ce. blood will be caused by omitting the
kaolin. If the ammonia is to be distilled and titrated, measure accurately
20 ¢.c. of the blood filtrate by a pipette into a small (200 cc.) Kjeldahl
flask, add 2 cc. concentrated H,SO, and 4 drops of 5 per cent. CuSO,
and heat under the hood, using a micro burner until the water is gone
and white fumes begin to fill the flask, then add 0.3 gram of powdered
K,SO,, or an equivalent amount of Na,SO, which has been found to
be free from ammonia, and heat for 5 minutes, with the flame turned so
low that the boiling is moderate, after the solution has become colorless.
Allow to cool and while cooling arrange the condenser for distillation, as
shown in figure 87. The flask which is to receive the distilled ammonia
has measured into it from a burette exactly 15 cc. of 0.01N acid. A
100 c.c. Erlenmeyer flask should be taken. When the still is ready, add
to the Kjeldahl flask a clean, dry, quartz pebble to prevent bumping
and then 10 ec. of saturated NaOH solution. Connect immediately
with the condenser and heat cautiously at first and then rapidly with a
small burner. 10 minutes vigorous distillation will be sufficient to distill
over the ammonia. While the distillation is going on or before it has
begun add to the distillate 3 drops of methyl red (1 per cent. solution).
After 10 minutes remove the end of the distilling tube from the acid
so that it now no longer dips below the surface and distill for two
minutes more so that the steam and water wash out the inside of the
tube. Turn out the flame. With a few c.c. from a wash bottle wash
out the tube and the. outside of it, receiving the washings into the flask.
Add 1 drop of methylene blue and titrate with 0.01IN NaOH. At the
PRACTICAL WORK AND METHODS 1025

end point the color changes sharply from a purple to a green. In case
of uremic blood distill in to 0.05N HCl in place of 0.01N and use 0.05N
NaOH for titration.

Computation. The 20 cc. of filtrate taken correspond to 2 ¢.c. of
blood. 1 cc. of 0.01N acid neutralized by the ammonia is equivalent

)

 

 

 

Fig. 87.

Fic. 87.—Apparatus arranged for Kjeldahl ammonia distillation.

to 0.14 mgs. N. If 15 e.c. of the acid were taken and 10 ¢.c. 0.01N NaCH
were required to titrate the excess, then 5 ¢.c. would be neutralized by
the N from 2 ce. blood. This would be 0.7 mg. And 100 ec. blood
would have 50 times this or 35 mgs. N. A table prepared by F. Hulton-
Frankel and very convenient enables a reading to be made without
ealculation. For example, if 10.4 e.c. of 0.01LN NaOH were used in the
titration, by inspection of the table it appears at once that the blood
must contain 32.2 mgs. non-protein N in 100 c.e.

Tables I and II. (Taken from Hulton-Frankel: Jour. of Laboratory
and Clinical Medicine, III, p. 3, 1918.)
1026 PHYSIOLOGICAL CHEMISTRY

TABLE I. NON-PROTEIN NITROGEN.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Take 20 c.c. filtrate for micro Kjeldahl, digest and distill into 15 c.c. N/100 acid.
Figures give mg. N per 100 c.c. blood.
Alkali used for titration

¢.c. 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

O° sah Ger gues 105.0] 104.3 |103.6 [102.9 |102.2 ]101.5 |100.8 |100.1 | 99.4 | 98.7
dt nee dslacinstnaie 98.0 | 97.3 | 96.6 | 95.9 | 95.2 | 94.5 | 93.8 | 93.1 | 92.4 | 91.7
DZ; iosensesenrocs a 91.0 | 90.3 | 89.6 | 88.9 | 88.2 | 87.5 | 86.8 | 86.1 | 85.4 | 84.7
B- sacerommyeee 84.0 | 83.3 | 82.6 | 81.9 | 81.2 | 80.5 | 79.8 | 79.1 | 78.4 | 77.7
Oh seiisatant easyer 77.0 | 76.3 | 75.6 | 74.9 | 74.2 | 73.5 | 72.8 | 72.1 | 71.4 | 70.7
Disa su eevajseaneiGie 70.0 | 69.3 | 68.6 | 67.9 | 67.2 | 66.5 | 65.8 | 65.1 | 64.4 | 63.7
Gi eeieatoeundenes 63.0 | 62.3 | 61.6 | 60.9 | 60.2 | 59.5 | 58.8 | 58.1 | 57.4 | 56.7
SE fayaeeeneaasetemesees 56.0 | 55.3 | 54.6 | 53.9 | 53.2 | 52.5 | 51.8 | 51.1 | 50.4 | 49.7
BS) ses siectiwnes 49.0 | 48.3 | 47.6 |] 46.9 | 46.2 | 45.5 | 44.8 | 44.1 | 43.4 | 42.7
Ol ke.amhene cies 42.0 | 41.3 | 40.6 | 39.9 | 39.2 | 38.5 | 37.8 | 37.1 | 36.4 | 35.7
10) ga demuveacuc 35.0 | 34.3 | 33.6 | 32.9 | 32.2 | 31.5 | 30.8 | 30.1 | 29.4 | 28.7
Ll) asamesitet se 28.0 | 27.3 | 26.6 | 25.9 | 25.2 | 24.5 | 23.8 | 23.1 | 22.4 | 21.7
1D) vascesessceeesee 21.0 | 20.3 | 19.6 | 18.9 | 18.2 | 17.5 | 16.8 | 16.1 | 15.4 | 14.7

TABLE II. NON-PROTEIN NITROGEN.
Take 20 c.c. filtrate for micro Kjeldahl, digest and distill into 10 cc. N/20 acid.
Figures give mg. N per 100 c.c. blood.
Alkali used for titration
c.c. 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

O ese stsvoersies 350.0 | 346.5 | 343.0 | 339.5 | 336.0 | 332.5 | 329.0 | 325.5 | 322.0 | 318.5
Ty seen atravaty 315.0 | 311.5 | 308.0 | 304.5 | 301.0 | 297.5 | 294.0 | 290.5 | 287.0 | 283.5
D caybecoces sccitatecs 280.0 | 276.5 | 273.0 | 269.5 | 266.0 | 262.5 | 259.0 | 255.5 | 251.0 | 248.5
B eetrdstlsimacvac 245.0 | 241.5 | 238.0 |} 234.5 | 231.0 | 227.5 | 224.0 | 220.5 | 217.0 | 213.5
Be Se acnnemdatsy 210.0 | 206.5 | 203.0} 199.5 | 196.0 | 192.5 | 189.0} 185.5 | 182.0 | 177.5
Ds cvauacauaumioheda 175.0 | 171.5 | 168.0 | 164.5 | 161.0} 157.5 | 154.0] 150.5 | 147.0 | 143.5
Gi ietesics, 0 pearstoncye 140.0 | 136.5 | 133.0] 129.5 | 126.0 | 122.5] 119.0 | 115.5 | 112.0] 108.5

 

 

 

 

 

 

 

 

Determination of creatinine and sugar. Lewis-Benedict method
modified by Myers and Killian.

Chem., 1915, 20, p. 61. Meyers and Killian: Amer. J. Med. Sci., 157,

1919, p. 674.)

(Lewis and Benedict: Jour. Biol.

Principle. The proteins and corpuscles are precipitated by saturat-
ing with picric acid. The filtrate is made alkaline in the cold for
creatinine, the comparison being made with a standard in the colorimeter.
The alkalinized filtrate is warmed to give the color with sugar. In this
case the creatinine color fades and the reduction to picramic acid due to
PRACTICAL WORK AND METHODS 1027

the dextrose comes to the fore. This is estimated in the colorimeter by
comparison with a standard solution.

Solutions needed:

1. Standard creatinine solution. See page 1017. The stock solution
contains 1 mg. creatinine to 1 ¢c.c. of 0.1IN HCl. Appropriate amounts
may be taken from this stock solution and made up to 100 ce. by
addition of saturated picric acid solution to make the standards for
comparison.

2. Picrie acid which has been purified and kept protected from the
light. See page 1017.

Apparatus:

1. Centrifuge and tubes.

2. Duboseq or other colorimeter.

Method. To 20 cc. distilled water in a 50 cc. centrifuge tube add
5 ¢.¢. oxalate blood. (See page 1007.) Stir with a small glass rod until
the blood is completely laked, then add about 1 gram dry, purified
(Note: picrice acid is an explosive. Do not pulverize in a mortar) picric
acid. The mixture is thoroughly stirred for several minutes until the
proteins are coagulated and the liquid uniformly yellow and saturated
with picrie acid. It is then stirred at intervals for from 20 to 30 minutes
longer to make sure that coagulation and saturation is complete.
It is very important that the liquid be saturated. Centrifuge until
well separated. Decant supernatant liquid through a dry filter
paper and finally bring all of the liquid and the precipitate on to
the filter paper. The filtrate may be used for both creatinine and
sugar.

To determine the creatinine. Measure 10 cc. of the filtrate by an
Ostwald pipette into a small flask or large test-tube. Then make three
(or 5) standard solutions of creatinine in saturated picric acid. 10 e.c.
of each of the standards are taken and similarly placed in tubes or small
flasks. These standards contain, respectively, 0.2, 0.4, 0.6, 2.0 and 4.0
mgs. creatinine in 100 cc. saturated picric acid solution. They are
easily prepared from the stock solution which contains 1 mg. per c¢.c. by
adding the appropriate amount of creatinine to 100 ec. volumetric
flasks and diluting to 100 «cc. by saturated picric acid. When the
standards are ready add to each test-tube 0.5 ¢.c. of 10 per cent. NaOH
solution. Mix by inverting the tube once. The standard is selected
which comes nearest in color to the unknown and these two are com-
pared in the colorimeter. If the standard is set at 20, the mgs. of
creatinine in 100 mgs. blood can be read directly from the accompanying
table taken from Hulton-Frankel. The reading must be made about
8 minutes after the addition of the alkali and never later than 15
minutes. The unknown should never read more than 3 mm. below or
1028 VYAHYSIOLOGICAL CHEMISTRY

about the standard. That is, if the standard is 10 it should not read
below 7 or above 13 mm.

 

 

TABLE III. CREATINE.

i i ‘ J m .O mg. fl ,

Standard set at 20) Igy Pe | OF poe Der | OF Oe Per | Soe PT | Ao Goe oer
Unknown reading Standard Standard Standard Standard Standard

LOO} cred os cassie’ 2.00 4.00 6.00 20.0 40.0
VEO) aed sa ares 1.82 3.64 5.45 18.2 36.4
12.0) ead netineces 1.67 3.34 5.00 167 33.3
DO) Se as orake saute 1.54 3.08 4.60 15.4 30.8
DAO asia eaten oes 1.43 2.86 4.29 143 28.5
TADS ectees aaueniae 1.38 2.76 4.14 13.8 27.6
V5.0) iciecmeonviereceis 1.33 2.66 4.00 13.3 26.6
VOID). sasscveatds-b seve 1.29 2.58 3.87 12.9 25.8
16.08 siding os aaart 1.25 2.50 3.75 125 25.0
W636: chests sence 1.21 2.42 3.64 12.1 24.2
VO! xe eee ea. cin bes 1.17 2.34 3.53 ice 23.5
DUB te sete 3s detiens 1.14 2.28 3.43 11.4 22.8
VSO) ede cecotais poenecs 1.11 2.22 3.33 11.1 22.2
TSB ie Scucracrovadteed 1.08 2.16 3.24 10.8 21.6
PDO) seuss. shee. habe bets 1.05 2.10 3.16 10.5 21.0
V9.5) hstiiersvt-ew avgutiore 1.02 2.04 3.08 10.2 20.4
200 printed ase 1.00 2.00 3.00 10.0 20.0
205 5 ssiacss exe ssataroe 0.97 194 2.92 9.7 19.4
2V.O8 cn cain tic awies 0.95 1.90 2.85 9.5 19.0
PEO sec nats ral eo acon 0.91 1.82 2.73 9.1 18.2
23.0) a4 Soe aeae 0.87 1.74 2.60 8.7 17.4
EOE ces Scorn i acatea ss 0.83 1.66 2.50 8.3 16.6
ZOO cia a5 Rua Apu 0.80 1.60 2.40 8.0 16.0
ZO O% 65.8 connie tinue voosuels 0.77 1.54 2.30 7.7 15.4
ZT crahassien Soh eacsero 074 1.48 2.22 74 14.8
280) airintaeesaietins 071 1.42 2.14 7.1 14.2
29,08 ceeiceceiie cones 0.69 1.38 2.07 6.9 13.8
30,0 setiney Secs ahons 0.67 1.34 2.00 6.7 13.4

 

 

 

 

 

 

Determination of sugar.
24, p. 147, 1916.)

Solutions needed:

1. Standard dextrose solution. 0.2 gram pure dextrose dissolved
in saturated picric acid and made up to 1,000 cc. Each ee. of this
solution will contain 0.2 mgs. of dextrose. (3 ¢.c. are needed for each
determination.)

2. 20 per cent. Na,CO, solution.

Apparatus:

1. Colorimeter.

2. Narrow test-tube graduated at 3, 4, 10, 15 and 20 ce.

Method. Take 3 c.c. of the picrie acid blood filtrate measured by an
Ostwald pipette into a tall, narrow test-tube graduated at 3, 4, 10, 15
and 20 ¢.c. Into a similar tube measure 8 ¢.c. standard glucose solution.

(Myers and Bailey: Jour. of Biol. Chem.,
PRACTICAL WORK AND MET'HODS 1029

The standard tube then contains 0.6 mgs. dextrose. Now add to the
standard and the blood filtrate 1 ¢c.c. of 20 per cent. Na,CO, solution
and immerse in a beaker of boiling water for 15 minutes. This is
sufficient to develop the full strength of the color, but longer heating
does no harm. Cool and with water dilute the standard to 10 ce. and
the blood tube to 4, 10, 15 or 20, depending on the depth of color. Now
compare in the colorimeter, setting the standard at 20 if a Duboscq is
used. If the dilution has been. made to 10, the mgs. of dextrose in
100 ¢.c. blood can be read directly from the accompanying table IV

TABLE IV. SUGAR IN BLOOD.

 

Strength of Standard 200 mg. per 1,000 c.c.

 

 

Colorimeter Reading
Percentage of Sugar in Blood
(Standard set at 20)

7.0 0.286
7.5 0.267
8.0 0.250
8.5 0.235
9.0 0.222
10.0 0.202
11.0 0.182
12.0 0.167
13.0 0.154 ‘
14.0 . 0.143
15.0 0.133
16.0 0.125
17.0 0.117
18.0 0.111
19.0 0.105
20.0 0.100
21.0 0.095
22.0 0.091
23.0 0.087
24.0 0.083
25.0 0.080
26.0 0.077
27.0 0.074
28.0 0.071
29.0 0.069
30.0 0.067
31.0 0.064

 

 

(Hulton-Frankel). When the standard is set at 10 mm. a blood con-
taining 0.1 per cent. of dextrose manipulated as above with dilution
to 10 ¢.c. will:mateh at 10 mm., since the 3 cc. of filtrate employed are
the equivalent of 0.6 ¢.c. of blood and the standard contained 0.6 mgs.
dextrose.

If the Hellige colorimeter is used with a wedge of standard picramic
acid, the latter is prepared by dissolving 0.10 gram of picramiec acid
1030 PHYSIOLOGICAL CHEMISTRY

and 0.2 gram of anhydrous Na,CO, in 30 ¢.c. warm distilled water and
final dilution to 1 liter. The scale is read most accurately between 40
and 65. This would require with normal blood a dilution to 10 cc. If
the standard Hellige instrument is used with the zero at the top of the
scale, the value of the colorimeter reading in per cent. of blood sugar
may be obtained by subtracting the colorimetric reading from 100 and
multiplying by the factor 0.002 if the dilution was to 10 ec.; by 0.003
if the dilution was to 15 cc. and so on.
When the Duboseq is used the following formula may be used:

 

: Dx0.1
Grams of dextrose in 100 c.c. blood = R

where D represents the dilution and R the colorimeter reading, the
standard being at 10 mm.

Determination of uric acid in blood. Benedict’s modification of
Folin-Denis method. (Benedict: Jour. Biol. Chem., 1915, 20, p. 629.
Meyers and Fine: Arch. Int. Med., 17, p. 573, 1916.)

Principle. 10 c.c. or 20 ¢.c. of blood are coagulated by adding to
boiling acetic acid. The filtrate is concentrated and the uric acid deter-
mined by development of color in the Folin reagent of phosphotunstate.

Solutions needed:

1. Alumina cream. (15 e.c. needed for determination.) See fur-
ther on. oe

2. Ammoniacal silver solution. (For preparation, see further on.)

8. KCN 5 per cent.

4. Na,CO, (22 per cent.). Saturated solution.

5. Urie acid reagent. Preparation is given at the end.

6. Standard uric acid. 5 ¢.c. contain 1 mg. uric acid. Preparation
described at end of this method.

Apparatus:

1, Colorimeter.

2. Centrifuge.

Method. 10 e.c. (or 20 cc.) oxalated blood are brought into a cas-
serole of 375 c.c. capacity and 50 cc. (or 100 cc.) .0.01N acetic acid
added. Heat on water bath and bring to boil over free flame, stirring
continuously. Add 4 cc. of fairly thick alumina cream (made by pre-
vipitating 8 per cent. solution of aluminum acetate in acetic acid with
sodium bicarbonate, washing by decantation with large volume of water
repeatedly). Stir continuously and from time to time wash down sides
of casserole with water. Filter through hardened filter paper. Coagu-
lum is returned to casserole with about 150 c.c. hot water, heated and-
filtered through same paper. Combined filtrates are evaporated to 1 or
2 cc. (should be protein free) (final evaporation in qmall flask or
beaker) and transferred to 15 c.c. conical centrifuge tube, washing with 2
PRACTICAL WORK AND METHODS 1031

or 3 portions of hot water but keeping volume at or below 10 cc. Add
about 15 drops ammoniacal-silver magnesium mixture. (This is made
by mixing 70 c.c. 3 per cent. AgNO,, 30 c.c. magnesia mixture, and 100
¢.c. concentrated ammonia. Filter if necessary. Magnesium mixture is
prepared by dissolving 35 grams MgSO, and 70 gms. NH,ClI in 280 e.c.
H,0 and adding 140 c.c. con. NH,OH.) Stir, place in refrigerator for 15
minutes to allow precipitation of purines. Centrifuge. Decant super-
natant liquid and allow to drain in inverted position about 5 minutes.
Tip of the tube is wiped with filter paper and allow NH, to evaporate.

For development of color prepare a 100 c.c. graduated cylinder for
the standard and a 50 «.c. cylinder for the unknown. 5 ec. of the uric
acid standard (5 c.c.—1 mg. uric acid) are pipetted into the 100 ee.
cylinder. To the standard are added 2 drops of 5 per cent. KCN, 2 «.c.
of uric acid reagent, 20 c.c. of saturated Na,CO, (22 per cent.) and in
about 1 minute water to the 100 mark. The precipitate in the centrifuge
tube is stirred with 1 or 2 drops of KCN, 2 c.c: of the uric acid reagent
added, stirred, and 15 or 20 c.c. of saturated Na,CO,, depending on the
amount of ppt. and whether the color is subsequently diluted to 50 or
100 ec. After 40 to 60 seconds transfer to 50 c.c. or 100 c.c. cylinder,
dilute with water until the intensity of color is similar to the standard
and then match in the Duboscq colorimeter. It is necessary to note to
what point the unknown has been diluted.

Computation, The amount of uric acid in 10 c.c. of blood, if .the
standard is 100 c.c. and the blood has been diluted to 50 c.c. and the
reading of the unknown is the same as the standard, would be 1 mg.
X 50/100 or 0.5 mg. 100 ec. blood would contain 5 mgs. If it were

diluted to 100 ¢.c. it would contain 1 mg. in 10 c.c. or 10 mgs. in 100.

uM 100 blood Reading of standard __ 50 or 100 10
eae ee ee Reading of unknown X00 *

 

If standard is made to 50 e.c., 10 or 20 c.c. blood taken and final dilution
to 25 or 50 c.c. the mgs. uric acid in 100 ¢.c. blood may be read directly
from table VII. (Hulton-Frankel.)

Standard uric acid. 9 gms. erystalline HNa,PO, and 1 gm. H,NaPO,
are dissolved in 250 to 300 c.c. hot water. Filter and make up to about
500 e.c. with water. Pour this warm, clear solution on 200 mgs. uric
acid suspended in a little water in a liter volumetric flask. Agitate
until completely dissolved. Add at once exactly 1.4 cc. glacial acetic
acid. Make up to 1 liter with water and -add 5 ¢.e. chloroform: 5 e.c.
of this solution contain 1 mg. uric acid. Solution should be freshly
prepared every 2 months.

Uric acid reagent (Benedict’s modification of Folin and Denis).
Boil 100 gms. sodium tungstate, 20-¢.c. concentrated HCl and 30 ce.
85 per cent. phosphoric acid in 750 c.c. distilled water for two hours,
preferably under reflux condenser. Make up to 1 liter with water.
1032 PHYSIOLOGICAL CHEMISTRY

TABLE VII. URIC ACID.
Standard set at 20.

 

 

Dilution of unknown 25 50 50 100 25
Volume of blood 10 20 10 20 20
Colorimeter reading
10.0 10.00 20.00 5.00
10.5 9.50 19.00 4.70
11.0 9.10 18.20 4.50
11.5 8.70 17.40 4.30
12.0 8.30 16.70 4.10
12.5 8.00 16.00 4.00
13.0 7.70 15.40 3.80
13.5 7.40 14.80 3.70
14.0 7.10 14.30 3.50
14.5 6.90 13.80 3.40
15.0 6.70 13.30 3.50
15.5 6.40 12.90 3.20
16.0 6.20 12.50 3.10
16.5 6.00 12.10 3.00
17.0 5.87 11.75 2.90
17.5 5.70 11.40 2.80
18.0 5.55 11.10 2.75
18.5 5.40 10.80 2.70
19.0 5.20 10.50 2.60
19.5 5.12 10.25 2.55
20.0 5.00 10.00 2.50
21.0 4.70 9.50 2.30
22.0 4.50 9.10 220
23.0 4.30 8.70 2.10
24.0 4.10 8.30 2.05
25.0 4.00 8.00 2.00
26.0 3.80 7.70 1.90
27.0 3.70 7.40 1.85
28.0 3.60 7.10 1.80
29.0 3.40 -690 1.70
30.0 3.30 6.70 1.60

 

 

 

 

Determination of urea in blood. Van Slyke modification of the
Marshall method. (Van Slyke and Cullen: Jour. Biol. Chem., 1914, 19,
p. 211.)

Principle. Urea in blood is converted into ammonia by urease, the
ammonia aerated into acid and the ammonia determined by titration.

Solutions needed:

1. Jack bean powder, urease, or urease solution prepared according
to Folin and Wu.

2. Methyl red, 0.1 per cent.; methylene blue, 0.1 per cent.

3. Standard acid and alkali 0.01N HCl and NaOH; 0.05N HCl and
NaOH.

4. 10 per cent. Na,CO, solution.

Apparatus:

1. Suction or blast for aeration.

2. Test-tubes with tops for aeration, as in figure, page 1082.

Method. Measure with Ostwald pipette 2 cc. of oxalate blood (see
PRACTICAL WORK AND METHODS 1033

page 1007) into a 75 ce. hard glass (Pyrex) test-tube which has been
previously thoroughly washed with nitric acid to remove any mercury,
or other toxic metal, and then rinsed in water. Dilute with water to
10 ce., add 1 gram Jack bean powder, or 25 mgs. pure urease (or 1 ¢.¢.
of urease solution of Folin and Wu, page 1014). Add 2 drops buffer
(page 1014). Place in water bath at 40° to 50° C. for 10 minutes to con-
vert urea to ammonia. Connect up the tube as shown in figure 69B for
aeration and aerate the ammonia into 10 ¢.c. of 0.01N HCl or H,SO,. In
case of uremic bloods use 10 c.c. of 0.05N acid. 2 drops of methyl red are
added to the acid. To prevent foaming add to the blood 1 ¢.c. of tur-
pentine containing 20 per cent. turpentine resin, or capryllic alcohol or
kerosene. Aerate with a rapid current of air for 20 minutes or more.
Towards the end of the aeration add to the blood enough 10 per cent.
Na,CO, solution to make the blood alkaline. This will free the last trace

TABLE V. UREA.

 

 

 

10 c.c. Acid N/100 2 ¢c.c. Blood Mg. Urea per 100 ¢.c. of Blood
00IN NaoH | 9% | 01 | 02 | 08 | o4 | 05 | 06 | 07 | 08 | 09
OP Aan tents 150.0 | 148.5 | 147.0 | 145.5] 144.0} 142.5] 141.0] 139.5 | 138.0} 136.5
acer ts ctonnen se 135.0} 133.5 | 132.0] 150.5 | 129.0} 127.5] 126.0] 124.5 | 123.0) 121.5
DF cd aeatien a46 120.0] 118.5] 117.0] 115.5] 114.0} 112.5] 111.0] 109.5 | 108.0 | 106.5
DO sig encei a leneats 105.0 | 103.5 | 102.0] 100.5] 99.0] 97.5} 96.0] 94.5; 93.0] 91.5
Boas an ae 90.0| 88.5] 87.0| 85.5] 84.0} 82.5] 81.0] 79.5] 78.0] 76.5
Oe ware a nui eee: 75.0) 73.5| 72.0| 70.5] 69.0] 67.5! 66.0] 64.5] 63.0] 61.5
Ge os oat ee Reanecdng 60.0| 58.5] 57.0] 55.5] 54.0] 52.5{ 51.0] 49.5] 48.0] 46.5
ats BAN Ecoais 45.0| 43.5| 42.0] 40.5] 39.0] 37.5] 36.0} 34.5] 33.0] 31.5
Bret each 30.0] 28.5) 27.0] 25.5) 24.0] 22.5] 21.0} 19.5) 18.0] 16.5
DS sea itatecniyeneiny 15.0} 13.5] 12.0] 10.5 9.0 75 6.0 4.5 3.0 1.5

 

 

 

 

 

 

 

of ammonia. At the end of the aeration disconnect, add to the acid a
drop of methylene blue solution and titrate with 0.01N or 0.05N NaOH,
depending on which strength of acid had been placed in the receiving
flask. The end point is shown by change from purple to green.

Computation. Read from Table V or Table VI the mgs. of urea N
per 100 ec. blood corresponding to the ¢.c. of 0.01N or 0.05N NaOH
used to titrate the excess of acid. The computation is made in the same
manner as in total non-protein N on page 1025.

Nessler reagent. Modified according to Myers and Fine for urea.
(Arch, Int. Med., 17, p. 574, 1916.)

Place 100 gms. mercuric iodide and 50 gms. KI, both “inely pow-
dered, in a 1 liter flask (volumetric) and add about 400 ¢.c. water.
Dissolve 200 gms. KOH in about 500 ¢.c. water, cool thoroughly and
add with constant shaking to the mixture in the flask and then make up
1034 PHYSIOLOGICAL CHEMISTRY

to the mark with water. This usually becomes perfectly clear. Keep
at body temperature over night or until the yellowish-white precipitate
which may settle out is thoroughly dissolved and only a small amount
of dark brownish red precipitate remains. The solution is now ready
to be siphoned off and used. In using this take 10 c.c. of this solution,
dilute about five times with water and add 20 to 25 ec. of the diluted

TABLE VI. UREA.

 

 

 

 

100 ¢.c. N/20 Acid 2 cc. Blood s Mg. Urea per 100 c.c. of Blood
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 | 0.8 0.9
O} eens c eee 750.0 | 742.5 | 735.0] 727.5 | 720.0 | 712.5 | 705.0 | 697.5 | 690.0 | 682.5
dL = sera tessbssiaindeoes 675.0 | 667.5 | 660.0 | 652.5 | 645.0 | 637.5 | 630.0 | 622.5 | 617.0 | 609.5
Deane teahtuanite 600.0 | 592.5 | 585.0 | 577.5 570.0 562.5 | 555.0 | 547.5 | 540.0 | 532.5
BP desea Sod: wrtnleboeey 525.0 | 517.5 | 510.0 | 502.5 | 495.0 | 487.5 | 480.0] 472.5 | 465.0 | 457.5
BY sehen tvneiata ne 450.0 | 442.5 | 435.0] 427.5 | 420.0 | 412.5 | 405.0} 397.5 | 390.0 | 382.5
DY teks Ss evar eave 375.0 | 367.5 | 360.0 | 352.5 | 345.0 | 337.5 | 330.0 | 322.5 | 315.0] 307.5
Go sg ina wares 300.0 | 292.5 | 285.0 | 277.5 | 270.0 | 262.5 | 255.0] 247.5 | 240.0 | 242.5
i eaislaneaieadt 225.0 | 217.5 | 210.0 | 202.5 | 195.0 | 187.5 | 180.0 | 172.5 | 165.0 | 155.5
8: cng ese wee 150.0 | 142.5 | 135.0] 127.5 | 120.0 | 112.5] 105.0] 97.5 90.0] 82.5
OF sae tana 75.0] 67.5| 60.0] 52.5] 45.0] 37.5] 30.0) 22.5} 15.0 75

 

 

 

 

 

 

 

solution to the standard ammonium sulphate, the whole being diluted’
to 100 c.c. later with water. This is for use in the urea determination
in blood by the Marshall and Van Slyke method where the ammonia is
Nesslerized instead of being titrated.

Experiment 205. Chlorides in blood plasma. Microchemical
method of McLean and Van Slyke. Jour. Biol. Chem., 21, 1915, p. 361,
corrected and modified by Van Slyke and Donleavy: Jour. Biol. Chem.,
37, 1919, p. 551.)

Principle. The chlorides of oxalate plasma are precipitated by
standard silver nitrate and excess of silver nitrate is titrated by iodide
and starch.

Reagents and apparatus:

1. Standard silver nitrate. Solution contains per liter 5.812 grams
pure, fused AgNO, ; 250 ¢c.c. HNO, (sp. gr. 1.42) ; and 7.5 grams of pure
picric acid. The silver nitrate must be accurately weighed. This pre-
cipitates both proteins and chlorides.

2. Starch-citrate-nitrite solution. Dissolve 2.5 grams of soluble
starch in about 500 cc. boiling water, add 446 grams of crystalline
sodium citrate (Na,C,H,0,.542H,O) and 20 grams of sodium nitrite
after the starch solution has been boiled for several minutes. When all
is dissolved cool the flask, transfer to a volumetric, 1 liter flask, and
make up to the mark by the addition of distilled water. 4 cc. of this
PRACTICAL WORK AND METHODS 1035

solution contain enough citrate to react with the acid of 1 cc. of HNO,
of a sp. gr. 1.42, so that the resulting solution will have the optimum
acidity for the production of the blue color with starch and iodine. If
ordinary starch is used instead of the soluble variety it should be boiled
for one hour before adding the other constituents.

3. KI solution. This is an M/73.1 solution of KI. 1 ee. of it is -
equivalent to 0.8 mgs. of NaCl. It is standardized so that 2.5 cc. is
equivalent to 1 ¢.c. of the AgNO, solution. The solution must contain
2.27 grams KI per liter. To make it, weigh out about 2.4 grams of KI
and add nearly a liter of water. Standardize it against the AgNO,
solution and dilute it so that 2.5 cc. are just equivalent to 1 cc. of the
AgNO,. To make this standardization take 5 e.c. of the AgNO, solution
measured with a 5 c.c. standardized pipette (the pipette should have an
error not greater than 0.01 c.c. That is, it delivers 4.97 grams of pure
water at 20 degrees C.) into a small Erlenmeyer flask (50 c.c.). Add
5 «ec. of the starch citrate solution and 5 cc. of distilled water. Now
run in the KI solution from a burette until a permanent faint but un-
doubted blue end point is reached. The amount required when the
solution is accurately made up and diluted should be 12.65 e.c., 12.50 ce.
being required to precipitate the standard silver nitrate and 0.15 c.c. to
give the blue end point with the starch.

Method. 5 .c.-10 c.c. blood is drawn, as described on page 1007, and
mixed with powdered potassium oxalate to prevent clotting. Centri-
fuged until a clear plasma is obtained. 2 c.c. oxalate or citrate plasma
are drawn into a dry Ostwald pipette calibrated to contain 2 ¢c.c. + .005.
(The pipette must be calibrated before using. It must weigh 1.994 +
.005 gm. more when filled with water at 20 C. than when dry and
empty.) Transfer the 2 ec. plasma to a 50 ec. volumetric flask
(calibrated) half full of distilled water, and the pipette is rinsed by
drawing the water up into the pipette and re-expelling it once or twice.
10 ce. of standard silver nitrate-picric acid mixture are accurately
measured with a volumetric calibrated pipette into the flask and the
mixture is diluted to the 50 ¢c.c. mark and shaken at intervals for several
minutes until the coagulation of the precipitate is complete and it settles
clear. The addition of a drop or two of capryillic alcohol prevents
foaming and facilitates coagulation. Filter solution through a dry,
chloride-free filter paper. Pour back the first filtrate until it is per-
fectly clear. 20 ¢.c. duplicate portions of the filtrate are measured with
a calibrated pipette into 100 ec. Erlenmeyer flasks, 4 c.c. of the starch
citrate indicator are added to each and the standard KI is run in from
a burette until a permanent blue end point is reached. The titration
goes best if performed over a sheet of light yellow paper lying on the
table under the burette. If the KI is run in fast a false end point
1036 PHYSIOLOGICAL CHEMISTRY

will be obtained, the blue color fading after a time. The true end point,
when reached, does not fade but deepens slightly. If the KI is run in
slowly the color disappears after each drop until the end point is
reached. If it is desirable to use less than 2 ¢.c. of plasma, 1 c.c. meas-
ured within + 0.002 c.c. may be precipitated with 5 c.c. of standard
- silver solution and diluted to 25 ¢.. instead of 50 cc. This will yield
one 20 c.c. portion instead of duplicates.

Computation. 1 ¢.c. of the KI solution is equivalent to 0.8 mgs. of
NaCl. Since 2 ¢.c. plasma were taken, diluted to 50 ¢.c. and 20 cc. of
filtrate used for analysis, the filtrate taken corresponded to 0.8 c.c. of
plasma. 10 cc. of AgNO, solution were taken. This corresponds to
25 ¢c.c. of the KI solution. 2/5 were taken for titration, corresponding
to 10 ec. KI if none had been precipitated. Consequently, subtract
from 10.15 the number of c.c. taken of the KI to give the blue color.
This gives the mgs. NaCl in 1 «ec. of plasma or grams NaCl in 1 liter
of plasma.

Example. Suppose 4.35 c.c. KI were needed to give the end point,
.. 10.15 — 4.35 = 5.80 mgs. NaCl per c.c. plasma. Normal human
plasma contains between 5.50-6.0 grams NaCl per liter.

The normal chloride ‘‘ threshold level ’’ of McLean is 5.62 grams
of NaCl per liter and the significant figure is the difference between this
and the results obtained. Hence the necessity of the extreme accuracy
and the use of calibrated apparatus. (McLean: Jour. Expt. Med., 1915,
22, pp. 212 and 366.) Errors of more than 0.10 gram are undesirable.

Experiment 206. Determination of chlorides in whole blood.
(Austin and Van Slyke: Jour. Biol. Chem., 41, 1920, p. 345.)

Principle. Same as in determination of chlorides in plasma, see
experiment 205, except that whole blood is taken and laked.

Solutions needed:

1. Standard silver nitrate: Same as in plasma method, except that
picric acid is eliminated.

2. Standard KI solution same as in plasma method.

8. Citrate-starch-nitrite mixture: Same as in plasma method.

4. Saturated solution of picric acid.

Method. Measure accurately 3 c.c. of oxalated blood (see page 1007)
into a 60 cc. volumetric flask. If a 60 ¢.c. flask is not procurable use
a 100 c.c. Florence flask and add from a burette exactly 27 cc. water
to lake the blood, and then 30 e.c. of saturated picrie acid solution.
Mix contents and after 10 minutes filter through a dry filter paper.
The picric acid precipitates the proteins. To 40 cc. of the filtrate add
10 c.c. of the standard silver nitrate solution and 2 dops of ecapryllic
alcohol or other anti-foaming solution. Mix the solutions thoroughly
and allow them to stand in the dark over night for the AgCl to separate
PRACTICAL WORK AND METHODS 1037

and give a clear solution. Decant the clear supernatant liquid through
a dry, chloride free filter paper. 20 c.c. of the filtrate are titrated, as in
the preceding method, with the standard KI to permanent blue iodide
color.

Computation. Since the 20 c.c. of the filtrate represent, as before,
0.8 c.c. of blood the calculation is the same as before, namely,

10.15 —c.c. of KI used = mgs. NaCl in 1 ¢.c. of whole blood.

Ox blood contains 4.53 mgs. in 1 gram of blood.

Experiment 207. Determination of Mg in blood. (Method of
Denis: Jour. Biol. Chem., 41, p. 363, 1920.)

Principle. This method is used in conjunction with Lyman’s method
for the determination of Ca in the blood. See page 1039. The organic
material in the filtrate from the calcium precipitation is removed, the
magnesium is precipitated as magnesium ammonium phosphate, and
the phosphate in this compound is determined nephelometrically by the
reagent of Pouget and Chouchak, Bull. Soc. Chem., 1909, Vol. 5, p. 104.
Normal blood contains 1.6-3.5 mgs. Mg in 100 c.c.

Method. 5 cc. of the citrated plasma or whole blood, as the case
may be (for preparing citrated blood, see page 520), are measured
accurately with an Ostwald pipette into 15 cc. of 6.5 per cent. tri-
chloracetic acid solution in a 25 or 50 e.c. Florence flask. The flask
is stoppered and shaken so as to mix thoroughly and hasten flocculation
and is then allowed to stand for at least, 30 minutes and is then filtered
through a dry filter which is free from salts. 10 c.c. of the filtrate,
which is equivalent to 2.5 ¢.c. of the blood or plasma, are taken for the
determination of the calcium by the method of Lyman. The calcium
is precipitated by the method of Lyman—experiment 208. The crystals
of calcium oxalate are collected by centrifuging the blood in a small
centrifuge tube. The supernatant liquid is decanted or removed by a
small siphon into a flat-bottomed platinum dish, and the washings of
the precipitate made by adding 5 ee. ammonium oxalate solution, stir-
wing the crystals and recentrifuging, this baing repeated twice, are
added to the dish. 3 ac. of 10 per cent. H,SO, is then added and the
whole evaporated to dryness on the water bath; the residue is ignited
over a free flame for 2-3 minutes until it is white. When cool the white
residue is dissolved in about 5 ¢.c. of distilled water and 10 per cent.
HCl is added drop by drop until the solution of the ash is acid to methyl
orange. The solution is then transferred without loss to a 100 ee.
beaker and the platinum dish carefully washed with distilled water,
collecting the washings in the beaker. The solution in the beaker is
then evaporated on the water bath to a volume of 2 to 3 «c.; concen-
1088 PHYSIOLOGICAL CHEMISTRY

trated NH,OH is then added drop by drop until the solution is alkaline
and finally 0.5 ¢.c. of 10 per cent. ammonium phosphate solution, con-
taining 50 c.c. concentrated ammonia per liter is added. The beaker is
then covered with a watch glass and allowed to stand over night. The
next day the liquid and precipitate is poured into a conical centrifuge
tube and the last traces of the precipitate are carried into the tube by
washing the beaker with 20 per cent. alcohol containing 50 c.c. NH,OH
(concentrated) per liter. After centrifuging until the precipitate is
thoroughly packed the supernatant liquid is removed by a small siphon,
and the beaker and tube again washed with about 10 c.c. alcohol am-
monia mixture, the precipitate being stirred up with a-very fine (2 mm.)
stirring rod. The washing should be repeated with centrifuging be-
tween three times. (This part of the determination is identical with
the method of Marriott and Howland: J. Biol. Chem., 32, 1917, p. 233.)
This is a most important part of procedure.

Determination of the phosphate in the- precipitate. After removal
of the last washings, the tube and beaker are allowed to stand until the
ammonia has evaporated, preferably on the water bath. The NH,MgPO,
is then dissolved in 10 cc. of 0.1 N HCl and transferred without loss
to a 100 c.c. volumetric flask, the tube and beaker being washed carefully
with distilled water, the washings being added to the flask. The flask is
filled to the mark with distilled water, stoppered, mixed by inverting
several times, and the phosphate determined by adding the strychnine-
molybdate reagent of Bloor (Bloor: Jour. Biol. Chem., 1918, 36, p. 34).

Nephelometry. For normal plasma 25 c.c. of foregoing solution is
usually the most convenient. Transfer then 25 c.c. by means of an
accurate pipette from the 100 c.c. flask to another 100 c.c. flask. Add
25 e.c. of distilled water, and then 25 ec.c. of the strychnine molybdate
solution, stopper the flask and mix. Meanwhile there should have been
prepared the comparison standard flask. Into another 100 ce. volu-
metric flask measure accurately by a pipette 5 cc. containing 0.01 mg.
Mg in the form of ammonium magnesium phosphate dissolved in hydro-
chloric acid, from the diluted standard solution mentioned below, add
‘45 e.c. distilled water and then 25 c.c. of strychnine molybdate reagent.
Mix. This solution should be made and the molybdate added to it at
the same time as the unknown. Allow both solutions to stand for 5
minutes after mixing and then read the unknown in the nephelometer
against the standard.

Calculation of results. The magnesium has been precipitated from
2.5 ec. of blood. 1/4th of this amount was taken, corresponding to
0.625 ¢.c. of blood. If this reads the same as the standard which has
0.01 mg. Mg, then the blood would contain in 100 e@c. 1.6 mgs. Mg.
The amount in normal blood varies from 1.6-3.5.
PRACTICAL WORK AND METHODS 1039

Reading of standard

mgs. Mg in 100 c.c. blood =2_W_—+__
8 8 7 Reading of unknown

Standard Mg solution. Dissolve 1.02 gram pure air dried NH,Mg
PO,.6H,O in 100 e.c. N HCl and dilute to 1 liter with distilled water.
1 c.c. of this solution is equivalent to 0.10 mg. of.magnesium. To make
the dilute standard take 2 c.c. of this stock solution and dilute to 100 c.e.
with 0.1 N HCl. 5 ¢.c. of the resulting solution contains 0.01 mg. of
magnesium.

Sirychnine-molybdate reagent. (Bloor: J. Biol. Chem., 1918, 36,
p. 34.)

Experiment 208. Determination of calcium in blood. (Method for
urine, see page 1108.) (Lyman: Jour. Biol. Chem., 29, p. 169, 1917.)

Principle. Calcium is precipitated as calcium oxalate, redissolved in
dilute acid, and reprecipitated as calcium ricinate and determined by
comparison with the cloud formed by a standard calcium solution in
a nephelometer.

Solutions needed. ,

1. Ammonium stearate reagent. Dissolve 4 gms. stearic acid and
0.5 oleic acid in 400 c.c. of hot aleohol. Add 20 gms. ammonium car-
bonate dissolved in 100 c.c. hot water and allow mixture to boil a few
moments. Cool. Add 400 cc. alcohol, 100 ¢.c. water and 2 c.c. NH,OH
(sp. gr. 0.9). Filter. Should be perfectly clear and colorless. Well
stoppered keeps indefinitely. Before using, test as follows: Into 2 flasks
pipette respectively 10 and 5 c.c. of calcium oxalate standard and to the
5 ec. add 5 cc. of HNO,, 0.05N. Treat both with 25 c.c. of ammonium
stearate reagent and compare in nephelometer. If they do not read
exactly 2 to 1 there is impurity in some of the chemicals. Alcohol used
should be redistilled over calcium carbonate. Stearate acid recrystallized
from boiling alcohol.

2. Prichloracetic acid, 6.5 per cent.

3. Nitric acid, 0.1N and 0.05N; NH,OH, 2N (138.5 ec. con. made
to 100.)

4. Methyl orange, 0.1 per cent. (0.1 gm. dissolved in 10 e.c. alcohol
made to 100 with water).

5. 4 per cent. oxalic acid (1 cc. for a determination). i

6. 20 per cent. sodium acetate (1 ee. for a determination). . (20
grams crystallized sodium acetate to 100 c.c. of water . 4 a. tea.

7. .5 per cent, ammonium oxalate (20 ¢.c. for a determination). ae

8. Standard calcium oxalate in HNO,. (0.0730. gram of calcium
oxalate (CaC,0,.1H,0) in 25 c.c. 2N HNO,, made to 1 liter. with, water:
Contains 0.2 mg. Ca in 10 c.c. (10 e.c. for a. determination). . :

Apparatus: year
_. 1. Nephelometer. (Kober; J. Biol. Chem., 1917, 29, p. 155.). eae

 

 

 
1040 PHYSIOLOGICAL CHEMISTRY

2. Conical centrifuge tubes.

Method. 10 ec. of blood filtrate obtained in experiment 207 are
pipetted into a 50 ¢.c. Erlemeyer flask. Add 1 drop 0.1 per cent. methyl
orange and then 2N NH,OH drop by drop until just yellow. Add
0.05 N HNO, until just pink and then Ic.c. more. Add I c.c. 4 per cent.
oxalic acid. Add 1 c.c. sodium acetate, 20 per cent., drop wise. Cool
under tap until faint cloud appears. Shake 10 minutes, or let stand
over night as convenient. Rinse stopper with few drops of ammonium
oxalate, 0.5 per cent., pour into a centrifuge tube. Centrifuge. Pipette
off supernatant liquid. Rinse flask with 5 c.c. ammonium oxalate, 0.5
per cent., pour into centrifuge tube, stir, rinse rod with the oxalate
solution, and again centrifuge. Pipette off supernatant liquid. Dis-
solve precipitate in 5 c.c. of 0.1N nitric acid by stirring, and pour into
original flask. Agitate to dissolve any precipitate adhering to the walls.
Rinse rod and centrifuge tube with 5 c.c. of water and pour into flask.

Into another flask of about 100 c.c. capacity, pipette 20 c.c. of the
standard oxalate solution. Pipette 50 c.c. and 25 c.c. respectively of the’
ammonium stearate reagent into two clean dry beakers. Pour the
standard solution into the 50 ¢.c. of the reagent, and the unknown into
the 25 e«.c., and pour back twice. Stopper and let stand 10 minutes.
Set left side of nephelometer at 32 mm. Fill both tubes with standard
and compare to make sure both are the same. Replace standard on left
with unknown and read. Remove any bubbles adhering to walls of
tubes.

Computation. If the unknown is set at 32 mm. and the standard is
read, the reading divided by 4 will equal the number of mgs. Ca in
100 «.c. blood.

Experiment 209. Determination of sodium and potassium in
blood.—(Kramer: J. Biol. Chem., 41, 1920, p. 263.)

Both sodium and potassium may be determined in 3 e¢.c. of serum
with an error of + 5 per cent. K content of normal human serum
varies between 16 and 22 mgs. per 100 c.c. serum. Na content has been
found with normal children and adults to vary between 280-310 mgs.
per 100 c.c. serum. Blood is collected, as in experiment 210, over am-
monium oxalate from median basilic vein.

Method: Potassium. 1 «.c. of blood, 3 to 5 e.c. clear plasma, or equal
amount of serum are dried in a platinum dish on the steam bath, then
at 110° for one-half hour. The dish or crucible is then placed in a flat
bottom quartz dish 10 em. diameter and 6 cm. deep in which have been
placed some pieces of porcelain. Heat outer dish with low flame of
Meeker burner until fumes begin to come off, and continue gentle heat
until no more fumes, then hot until charred material is immobile. Cover
the large dish with a quartz plate and continue until ashed. If coal
remains, dissolve in HCl, concentrated, evaporate on water bath, dry at
PRACTICAL WORK AND METHODS 1041

105° and heat until white ash. Remove, cool, dissolve ash in 0.5 cc.
H,0 with aid of 1-2 drops of glacial acetic acid; add 0.5 to 1.0 ce.
sodium cobalti-nitrite reagent drop by drop, stirring and allow to
stand at least 10 minutes. Filter through Gooch crucible having in
bottom a piece of hardened filter paper and a good mat of well washed
asbestos 2 mm. thick. Wash with water and transfer precipitate to
Gooch, wash. The precipitate and pad are transferred to 50 c.c. beaker,
the paper removed from the solution by forceps. 25 c.c. 0.01N potassium
permanganate and 5 c.c. 25 per cent. H,SO, are added, heated for just
3 minutes on steam bath, and sufficient 0.01N oxalic acid added from
burette to decolorize solution. Titrate back by 0.01N potassium per-
manganate. Total number of c.c. of KMnO, added less the number of
e.c. of 0.01N oxalic acid added, multiplied by 0.071, gives the number of
mgs. of K in the sample analyzed. Caution: Remove all ammonia.

Reagent:

Solution A. 50 gms. cobalt nitrate crystals are dissolved in 100 e.c.
water and to this solution 25 ¢.c. of glacial acetic acid are added.

Solution B. 50 gms. sodium nitrite (potassium free) are dissolved
in 100 c.c. water. Mix 6 volumes of A and 10 volumes of B. Aerate
solution to remove all nitric oxide gas. Let stand in ice box for 24 hours
at least. Filter before using. It will keep for at least a month in the
ice box and often longer. For quantities between 1 to 3 mgs. of
potassium, use 1 c.c. reagent. For smaller quantities use 0.5 c.c.

2. 25 per cent. H,SO,.

3. 0.01N KMn0O, and 0.01N oxalic acid.

Sodium. The ash of 1 or 2 ce. of blood, serum or plasma, obtained
as described in the potassium method, is dissolved in water in a platinum
dish using 0.5 ¢.c. of water for each c.c. of serum, plasma or blood. If
necessary add a drop or two of N HCl. Make slightly alkaline with
freshly prepared KOH solution, 10 per cent. 15 ce. of the potassium
pyroantimonate reagent and one-fifth of the entire volume of absolute
aleohol are then added. (Care not to add more than this of alcohol,
as some of reagent will be precipitated.) Precipitation occurs at once.
Stir. Let stand two hours at least, filter through wet asbestos mat in a
Gooch crucible previously weighed. Wash 4 or 5 times with 3 ¢.c. por-
tions of 30 per cent. alcohol, dry at 110° C., cool in dessicator and weigh.
1 mg. of sodium yields 11.08 mgs. of the sodium pyroantimonate
(Na,H,Sb,0,,6H,O).

Caution. Ammonia precipitates the reagent. All chemicals must be
tested for ammonia and sodium, particularly the KOH. Run a blank.

Reagents:

1. Potassium pyroanimonate. (J. T. Baker, analyzed C.P. re-
agent.) 2 gms. of the powder are added to 100 cc. boiling water in a
350 ¢.c. pyrex flask, and heating continued until no more dissolves.
1042 PHYSIOLOGICAL CHEMISTRY

Cool rapidly under tap, and 3 ¢.c. 10 per cent. KOH are added, stir and
filter. The clear filtrate is the reagent. The potassium pyroantimonate
need be weighed only roughly. It is better to prepare fresh frequently.

Experiment 210. Determination of CO, in blood. The alkali re-
serve. Macro method. (Van Slyke and Cullen: J. B. Chem., 30, pp.
289, 347, 1917.)

Principle. A very convenient method for the determination of car-
bonate and CO, in the blood is that of van Slyke and Cullen. It will
be recalled (see page 539) that the principal alkali which is used in the
neutralization of acid in the body is bicarbonate of sodium. In all cases
in which there is a production of non-volatile acid, that is, acid which
cannot be excreted from the lungs like carbon dioxide, the sodium is
drawn away from the sodium bicarbonate and the acid,—lactic, sul-
phuric, acetoacetic or other acid,—is excreted with the sodium through
the kidneys. In this way there is a depletion of the alkali reserve of
the body and such a depletion is a sure sign that there has been an °
acidosis. Normal human blood plasma contains enough bicarbonate to

RANGE OF RESULTS OBTAINED WITH NORMAL AND PATHOLOGICAL
PLASMAS. Van Slyke, et al.

 

Plasma CO, capacity Corresponding
values of acid
index of urine

 

 

Condition of subject Reading of {CO, reduced to 0°, 7S
apparatus. 760 mm., bound wVe
Gas from 1 cc. |as bicarbonate by Deviations
plasma 100 cc. plasma jof +10 must be
allowed for.
c.c. c.c.
Normal resting adult,*  ex-
treme limits .............. 0.90-0.65 77-53 3-27
Mild acidosis. No visible
SyMptoms ........ cece e eee 0.65-0.52 53-40 27-40
Moderate acidosis. Symptoms
may be apparent .......... 0.52-0.41 40-30 40-50
Severe acidosis. Symptoms of
acid intoxication .......... Below 0.41 Below 30 Over 50
Lowest. CO, observed with re-
COVETY® esses Guordns gant ea eas 0.26 16 64

 

 

 

 

*Schloss (Am. J. Dis. Child., 1917, 13, p. 218) finds the carbon dioxide bound
by the plasma of normal infants to be 46 to 63 cc. per 100 ce. of plasma, about
10 cc. lower than in adults. Acid index of urine, see Fitz, Jour. Biol. Chem.. 30,
p. 389, 1917. Plasma. CO, capacity = 80 /%s- D is rate of excretion of N/10
NH, plus acid per 24 hours time unit: W is body weight; C the N/10 NH, plus
acid per liter. Figures from the acid and ammonia of urine agree closely with
direct determinations.
PRACTICAL WORK AND METHODS 1043

yield 65-90 ¢.c. of carbon dioxide gas, when the latter is measured at
room temperature and at atmospheric pressure, per 100 c.c. of plasma.
Practically all of this bicarbonate is in the plasma. The method of its
determination is to receive the blood on a little powdered oxalate of
sodium or potassium, about 20 mgs. for 10 ¢.c. of blood. The blood is
centrifuged to remove the plasma. The oxalate plasma thus obtained
is then drawn into a separatory funnel or other receiver which is filled
with alveolar air by expiring through it so as to displace its air with
expired air. The plasma is then rolled around the sides of the vessel
in order that it may saturate itself as far as possible with bicarbonate
when exposed to this amount of carbon dioxide. The plasma is then
measured carefully (1 ¢.c.) into the apparatus pictured, acid is added to
liberate the CO, it contains; the CO, is pumped out and its volume is
measured. The solution is shaken at room temperature in the evacuated
space, where volume relations between liquid and free space are known,
a definite proportion of the gas escapes into the free space and the
residue remaining in solution is calculated from its solubility coefficient.

Solutions:

1. Caprylie alcohol.

2. Ammonia free from carbonate. This igs prepared by adding a
small quantity of saturated Ba(OH), to ordinary 1 per cent. ammonia
water. BaCO, is filtered off and an excess of Ba is removed by pre-
eipitating with a little ammonium sulphate.

Apparatus. Van Slyke CO, apparatus. See figure 90.

Method. Draw 10 e.e. of blood into, a centrifuge tube containing
oxalate and paraffine oil, as shown in Figure 1, p. 1008, and centrifuge
after mixing with the oxalate until a clear plasma is obtained. Transfer

 

Fia. 89.
Fig. 89.—Separatory funnel arranged for alveolar air. (After Van Slyke.)

about 5 c.c. of the clear plasma into a clean and dry separatory funnel
of a capacity about 100 times that of the volume of plasma taken, in this
case a 500 c.c. separatory funnel. (See Figure 89.) Turn the sep-
aratory funnel on its side, insert in the path of the alveolar air a bottle
filled with dry glass beads to collect moisture, and after inhaling a deep
breath exhale through the funnel. Repeat two or three times until the
1044 PHYSIOLOGICAL CHEMISTRY

air has been replaced by alveolar air. Then close the cocks of the funnel
and rotate the funnel. The object of this is that the blood may saturate
itself with CO, at a pressure of the latter equal to that of the alveolar
air of the lungs. Then set the funnel upright.

Preparation of the mercury pump. Shown in Figure 90.

Position 2
5

 

 

 

  
 

 

Position 3
ts 60cm below
pasition 2

 

Fie. 90.
Fig. 90.—Van Slyke macro carbon dioxide apparatus. (After Van Slyke.)

The mercury pump has the cocks held in place by rubber bands. Lift
bulb to position 1 and completely fill the pump and the capillaries
about e with mercury. Test the pump now to see if free from air. With
e turned at right angles and f as shown lower bulb to position 3. Tor-
ricellian vacuum is produced, the mercury falling to the middle of d.
Now slowly raise the bulb to 1. If all the air is out the mercury will
PRACTICAL WORK AND METHODS 1045

come up against the cock e with a sharp click. If it does this the pump
is ready; if it does not, discharge a little air and mercury through a,
eatching in a bottle any mecrury. Turn e so that it cuts off the air
from entering and lower bulb again. It is difficult to get all air out.
It adheres to glass. Sometimes cocks leak. If so, lower mercury below
f and remove and wipe and regrease e and f and reinsert. Having
freed the pump from all air, a little mercury being in the capillaries
a and b, wash out 6 by letting a few drops of ammonia free from CO,
flow down the side of the glass and wipe out with a little absorbent
cotton. ,

Introduction of plasma. In an Ostwald 1 ce. pipette draw up
quickly 1 ¢.c. of plasma from the separatory funnel and transfer to b.
Put the end of the pipette close to the mercury so that as the solution
flows out it covers the end of the pipette and thus diminishes its ex-
posure to the air and prevents loss of CO,. To discharge last drop
from the pipette, put thumb on end and warm bulb with left hand.
Quickly lay down the pipette, lower bulb to 2, and by cautiously opening
e allow the plasma to flow into the pipette; take in all but a little in
the capillary. No air must go in. Wash down the sides of b with 0.5
e.c. of water and draw it in; repeat with another 0.5 c.c. and draw this
in. Add one drop of capryllic aleohol to prevent foaming. It must
lie in the capillary. Then introduce about 1 c.c. of 0.5 per cent. H,SO,.
This carries the alcohol in ahead of it. Allow the acid to enter until the
liquid in the pipette is exactly 2.5 cc. Close e and put a drop of
mercury in b to seal. Now lower bulb to position 3, until the level of
the mercury is exactly at 50 cc. Close f. Replace bulb at 2. Remove
pipette from the clamp, put thumb over b and turn pipette upside down
15 times to secure thorough equilibrium between the gas in solution and
in the space above it. Replace pipette in clamp. Lower bulb to posi-
tion 8. Turn cock f to position shown in the cut. Allow all the liquid
but none of the gas to pass into d. Raise bulb to 2. Turn f so that it
connects c with the pipette and bring mercury back by raising bulb in
left hand until the level of the mercury in the bulb is exactly the same
as that in the pipette. There will be about 10 mm. of liquid above the
mercury. Read the level of the liquid to get the cc. of gas. Note
barometric pressure and temperature of the gas.

A. For calculation of CO, combining power of plasma use Table 1.
Example. At 746 mm. and 25°, the observed gas reading was 0.60.

746

6.60 Xiz59= 0.58.

In Table I, the corresponding figure for 0.58 at 25° is 46.6, which is
therefore volume per cent. of CO, found bound as bicarbonate in plasma.
1046 PHYSIOLOGICAL CHEMISTRY

TABLE I.
Tapuy FOR CALCULATION OF CARBON DioxmpE COMBINING PoWER OF PLASMA.

_

 

 

 

 

 

 

 

 

 

 

C.c. of CO,, reduced to 0°, C.e. of CO,, reduced to 0°,
pesca 760 mm., bound as bicarbo- pag 760 mm., bound as bicarbo-
vere Baer nate by 100 cc. of plasma sa ee nate by 100 c.c. of plasma
com Cy a0"

15° 20° 25° | 30° 15° 20° 25° | 30°

0.20 9.1 9.9 | 10.7 | 11.8 0.60 47.7 | 48.1 | 48.5 | 48.6
1 10.1 | 10.9 | 11.7 | 12.6 ] 48.7 | 49.0 | 49.4 | 49.5
2 11.0 | 11.8 | 12.6 | 13.5 2 49.7 | 50.0 | 50.4 | 50.4
3 12.0 | 12.8 | 13.6 | 14.3 3 50.7 | 51.0 | 51.3 | 51.4
4 13.0 | 13.7 | 14.5 | 15.2 4 51.6 | 51.9 | 52.2 | 52.3
5 13.9 | 14.7 ] 15.5 | 16.1 5 52.6 | 52.8 | 53.2 | 53.2
6 14.9 | 15.7 | 16.4 | 17.0 6 53.6 | 538 | 54.1 | 54.1
7 15.9 | 16.6 | 17.4 | 18.0 7 54.5 | 54.8 | 55.1 | 55.1
8 16.8 | 17.6 | 18.3 | 18.9 8 55.5 | 55.7 | 56.0 | 56.0
9 17.8 | 18.5 | 19.2 | 19.8 9 56.5 | 56.7 | 57.0 | 56.9
0.30 18.8 | 19.5 | 20.2 | 20.8 0.70 57.4 | 57.6 | 57.9 | 57.9
1 19.7 | 20.4 | 21.1 | 21.7 1 58.4 | 58.6 | 58.9 | 58.8
2 20.7 | 21.4 | 22.1 | 22.6 2 59.4 | 59.5 | 59.8 | 59.7
3 21.7 | 22.3 | 23.0 | 23.5 3 60.3 | 60.5 | 60.7 | 60.6
4 22.6 | 23.3 | 24.0 | 24.5 4 61.3 | 61.4 | 61.7 | 61.6
5 23.6 | 24.2 | 24.9 | 25.4 5 62.3 | 62.4 | 62.6 | 62.5
6 24.6 | 25.2 | 25.8 | 26.3 6 63.2 | 63.3 | 63.6 | 63.4
7 25.5 | 26.2 | 26.8 | 27.3 7 64.2 | 64.3 | 64.5 | 64.3
8 26.5 | 27.1 | 27.7 | 28.2 8 65.2 | 65.3] 65.5 | 65.3
9 27.5 | 28.1 | 28.7 | 29.1 9 66.1 | 66.2 | 66.4 | 66.2
0.40 28.4 | 29.0 | 29.6 | 30.0 0.80 67.1 | 67.2 | 67.3 | 67.1
1 29.4 | 30.0 | 30.5 | 31.0 1 68.1 | 68.1 | 68.3 | 68.0
2 30.3 | 30.9 | 31.5 | 31.9 2 69.0 | 69.1 | 69.2 | 69.0
3 31.3 | 31.9 | 32.4 | 32.8 3 70.0 | 70.0 | 70.2 | 69.9
4 32.3 | 32.8 | 33.4 | 33.8 4 71.0 | 71.0 | 71.1 | 70.8
5 33.2 | 33.8 | 34.3 | 34.7 5 71.9 | 72.0 | 72.1 | 71.8
6 34.2 | 34.7 | 35.38 | 35.6 6 72.9 | 72.9 | 73.0 | 72.7
7 35.2 | 35.7 | 36.2 | 36.5 7 73.9 | 73.9 | 74.0 | 73.6
8 36.1 | 36.6 | 37.2 | 37.4 8 74.8 | 74.8 | 74.9 | 74.5
9 37.1 | 37.6 | 38.1 | 38.4 9 75.8 | 75.8 | 75.8 | 75.4
0.50 38.1 | 38.5 | 39.0 | 39.3 0.90 76.8 | 76.7 | 76.8 | 76.4
1 39.1 | 39.5 | 40.0 | 40.3 1 717.8 | 77.7 | 77.7 | 77.3
2 40.0 | 40.4 | 40.9 | 41.2 2 78.7 | 78.6 | 78.7 | 78.2
3 41.0 | 41.4 | 41.9 | 42.1 3 79.7 | 79.6 | 79.6 | 79.2
4 42.0 | 42.4 | 42.8 | 43.0 4 80.7 | 80.5 | 80.6 | 80.1
5 42.9 | 43.3 | 43.8 | 43.9 5 81.6 | 81.5 | 81.5 | 81.0
6 43.9 | 44.3 | 44.7 | 44.9 6 82.6 | 82.5 | 82.4 | 82.0
7 44.9 | 45.3 | 45.7 | 45.8 7 83.6 | 83.4 | 83.4 | 82.9 --
8 45.8 | 46.2 | 46.6 | 46.7 8 84.5 | 84.4 | 84.3 | 83.8
9 46.8 | 47.1 | 47.5 | 47.6 9 85.5 | 85.3 | 85.2 | 84.8°
0.60 47.7 | 48.1 | 48.5 | 48.6 1.00 86.5 ] 86.2 | 86.2 | 85.7

 

 

 

 

 

 

 

 

 

 
PRACTICAL WORK AND METHODS 1047

B. For calculating the amount of CO, present in the solution, use
Table II.

 

 

 

TABLE II.
Carson DioxipE INDICATED BY READING oF V ©.0. OF GAS AFTER 4 SINGLE
EXTRACTION.

Air dissolved in 2.5 ¢c.c. H,0

Subtract this from V and

Temperature multiply result by A to

of analysis calculate mg. CO,; by C s ¢
to calculate c.c. CO, re-
duced to 0°, 760 mm.
°C. c.c. i mg. ‘ ec.

15 0.051 Te 1.935 a X 0.985
16 0.050 = 1.924 ef 0.980
17 0.049 re 1.912 se 0.974
18 0.048 of, 1.900 a 0.968
19 0.048 “« -1.889 . 0.962
20 0.047 ae, 1.877 - 0.956
21 0.046 * 1.866 & 0.950
22 0.045 fe 1.854 « 0.944
23 0.045 66 1.842 0.938
24 0.044 6 1.831 as 0.932
25 0.043 . 1.819 ne 0.927
26 0.042 . 1.808 se 0.921
27 0.041 1.796 < 0.915
28 0.040 « 1.784 “ 0.909
29 0.040 ee 1.773 se 0.903
30 0.039 se 1.761 r 0.897

 

 

 

 

(a) If mg. CO, is desired, subtract amount of air dissolved in 2.5
c.c. H,O (2nd column of Table II) from observed volume and multiply
by A.

Example. At 750 mm. and 22°, 0.69 c.c. of gas is measured, then

750

(0.690 — 0.045) X7e0% 1.854 = 1.180 mg. co,.

(b) If e.c. CO, reduced to 0°, at 760 mm. is desired, subtract amount
of air dissolved in 2.5 e.c. H,O (2nd column of Table II) from the
observed volume and multiply by C.

Example

(0.690 — 0.045) xen X 0.944 = 0.601 c.c.

Experiment 211. Micro determination of CO, and CO, in solu-

tion by Van Slyke’s method. (Jour. Biol. Chem., 30, p. 362, 1917.)
Principle. The apparatus given in Fig. 91 is used, taking 0.2 c.c.
*B is the barometic reading.
1048 PHYSIOLOGICAL CHEMISTRY

of the fiuid to be analyzed. The only difference in procedure is that
extracted gas, instead of water, is removed to another chamber.

      

i |_2Setution abaut'
to be extracted

 

 

_ Fig. 91.—Van Slyke micro carbon dioxide apparatus. (After Van Slyke.)

Process. Fill the apparatus with Hg. 0.2 c.c. of solution or plasma
is measured into cup, the tip of the pipette being kept in contact with
the liquid during the delivery. The solution is washed from a into b
with 2 portions of about 0.1 ¢.c. of H,O each, the H,O being distributed
about the lower part of the wall of a with a fine-pointed medicine
dropper. Enough 5 per cent. H,SO, is admitted to fill b down to 0.5
c.c. mark. The admission of successive portions of liquid from @ into b
is best controlled by leaving open the connection between a and } and
governing the inflow with cock e. Cock d is now turned to connect @
with c and a little Hg is forced up into a to make good Hg seal. Turn
cock d to the position shown in the figure, which represents the apparatus
at this stage of the determination. Chamber b is then evacuated till Hg
PRACTICAL WORK AND METHODS 1049

has fallen to the 10 ¢c.c. mark. Close cock c, loosen clamp about c and
shake it by removing it to the horizontal position and back a dozen
times. Return the apparatus to the upright position, and c, which has
hitherto been full of Hg, is now evacuated. Then leveling bulb being
raised to about the level of ¢, d is turned to connect b and ¢ and Hg is
at once admitted through e into b. When the solution in 6 has risen
to its narrowed upper position of the chamber, rate of flow is retarded
and is cautiously allowed to progress until its meniscus of H,O just or
almost reaches cock d. Cock e is then closed and d is turned to connect
a and 6. Hg from a flows into evacuated bore of d and seals the cock
with completeness. Turn cock e to admit Hg into c, and the volume of
gas trapped in the calibrated capillary at the top of ¢ is read off at
atmospheric pressure. Read barometric pressure and temperature.

Barometric pressure must be corrected for the effect of capillary
attraction on Hg in the calibrated capillary. For this, connect c with
outer atmosphere through d, hold the levelling bulb near to the eali-
brated capillary and measure the difference between the level of Hg
surface of the bulb and capillary. Subtract this difference from
barometric pressure.

Calculation. The different parts of the micro apparatus are in the
same relative proportions as the corresponding parts of the macro
apparatus, being 1/5th as large. Consequently if the solution is intro-
duced to exactly 0.5 ¢.c. mark, the same tables given under macro
method can be used for calculating results, each 0.002 c.c. on the smaller
corresponding to 0.01 c.c. on the larger.

In using this apparatus, it must be calibrated. Weigh Hg or water
from it. (See original article.)

Experiment 212(a). Hydrogen ion concentration of the blood.
(Levy, Rowntree and Marriott, Archives Int. Med., 16, pp. 389-405,
1915; Marriott, ibid., 17, p. 840, 1916.)

Principle. The alkali reserve may be considerably reduced without
any marked change of the hydrogen ion concentration of the blood.
Nevertheless, so far as the life of the tissues are concerned, the actual
hydrogen ion content of the blood and liquids is very important. Par-
ticularly in the case of the spinal liquid the determination of this factor
may be of considerable importance in distinguishing between epidemic
and tuberculous meningitis in the early stages. The principle of. the
present method is to determine the hydrogen ion concentration of the
blood by means of indicators. For this purpose it is necessary to work
with the plasma of the blood or to free the blood of its pigment. This
is done by dialyzing the blood through a collodion tube and measuring
the hydrogen ion in the dialysate by means of matching the tint -pro-
duced in solutions of known hydrogen ion concentration which contain
1050 PHYSIOLOGICAL CHEMISTRY

a certain indicator. The indicator chosen for this work is preferably
sodium phenolsulfonephthalein, since this indicator has a marked
change of color for a very small change of hydrogen ions.

Apparatus needed:

1. Collodion sacks. These can either be purchased from Hynson,
Westcott and Dunning, Baltimore, or they can be made with some
trouble and a little experience. They are made as follows: Select a
small test-tube of 9 mm. diameter and 120 mm. long. Pour into this
a strong, 30 per cent. solution, of collodion in alcohol and ether, equal
parts; see that the whole of the inside of the tube is wet by the collodion
and then pour out all that will come out. Now slowly rotate the tube,
holding it horizontal or slightly inverted so that any excess of the col-
lodion comes out and what remains is evenly distributed over the walls.
A small current of air may be blown into the tube to hasten the drying.
Do not, however, allow the collodion to become dry. When the collodion
is so dry that it will no longer run out, but before it is quite dry (about
10 minutes) run a little distilled water into the tube. This is to fill
the pores of the collodion with water. Pour out most of the water.
Now detach the collodion sack by means of a pair of forceps or a knife
from the neck of the tube at one side and gently force down between
‘the wall of the tube and the collodion sack a fine stream of water from
a wash bottle. This will gradually separate the sack from the tube
and ultimately the sack will come out whole. Fill it with water to
‘see that it does not leak and then immerse it in a beaker containing a
neutral solution of .9 per cent. NaCl and allow it to remain until needed.
‘The making of these small sacks is a difficult operation until one becomes
skilled at it.

2. Procure by purchase a set of standard hydrogen ion tubes with
phenolsulphonephthalein in them. These standards can be made by
making mixtures of Na,HPO, and NaH,PO,, as directed on page 544.
It is easier to purchase them. They may be obtained from Hynson,
Westcott and Dunning, Baltimore. (Price $5.)

Method. <A few c.c. of oxalated blood (drawn under paraffine) or
other liquid of which it is desired to test the hydrogen ion concentra-
tion are obtained. Introduce into one of the sacks, prepared as de-
scribed, 3 c.c. of the liquid and place it in a small test-tube, 100 x 10
mm., which contains 3 ¢.c. of 0.9 NaCl solution. Allow to dialyze for
5-10 minutes. Then remove the collodion- tube. Now add to the
dialysate 0.2 ¢.c. of phenolsulphonephthalein (0.1 per cent.) and com-
pare in: the: comparator with the standards. The hydrogen. ion
concentration is shown as P,. given by the tube which matches most
closely. The normal is about 7.3-7.4 for normal blood or 7.6-7.7 for
normal serum. as
PRACTICAL WORK AND METHODS 1051

Experiment 212(b). Hydrogen ion determination by the gas
shain method. vee

Principle. This, the direct method of determining the hydrogen ion
concentration, depends on the fact that a platinum electrode, when
saturated with hydrogen gas, acts as if it were a hydrogen electrode.
By measuring the difference of potential such an electrode shows when
partly immersed in an unknown solution and in H, gas, we may dis-
cover what the concentration of hydrogen ions is in the solution, for the
difference of potential depends on this concentration, as is shown on
page 940.

Apparatus needed:

a. Hildebrand method. As this is in many ways the simplest and
easiest of the various devices suggested it is placed first. (See Figure
93.)

   

   
 

 
  

Hi
Ty

  

tii
Fie. 93.
Fic. 93.—Hydrogen electrode. (After Hildebrand.)

1. A dry cell or other form of battery or a lead storage cell.

2. A voltmeter graduated to hundredths of a volt and permitting
thousandths to be estimated.

3. A rheostat.

4. <A calomel electrode.

5. A hydrogen electrode.

6. A capillary electrometer or some form of galvanometer of
D’Arsonval type.

7. A microscope to observe capillary electrometer.

8. Three keys, with wire for connections.
1052 PHYSIOLOGICAL CHEMISTRY

9. A standard cell is not necessary but is useful in checking volt-
meter.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

a AWWW b
cS
V
E
Cc
K
Fie. 94.

Fic. 94.—Diagram of connections for Hildebrand method of determining bydrogen
ions. (After Hildebrand.)

10. A beaker or other glass vessel for concentrated KCl solution.

11. A eylinder of pure H, gas; or an electrode cell for generating it.

b. If the Hildebrand method is not used then the following ap-
paratus is needed:

1. A storage cell. (Lead accumulator.)

2. A Weston standard cell. Voltage = 1.019.

3. Electrometer, calomel electrode and hydrogen electrode, as in the
Hildebrand method.

4. Three resistance boxes in place of the rheostat. These should
have at least 110 ohms each.

5. Keys, binding posts and wire, as in other method.

Procedure:

a. Platinizging the electrodes. (Figure 95.) Clean electrodes by
chromic acid. The electrode which is to be used in the hydrogen cell is
D. Immerse both platinum electrodes in a small vial, B, containing a
solution of 3 gms. PtCl, and 0,.02-0.03 gm. lead acetate to 100 ¢.c. H,O.
Pass current from battery of 2 or 3 dry cells or lead accumulator, D
PRACTICAL WORK AND METHODS 1053

being the cathode, for 10 to 15 minutes until the electrode is well cov-
ered with a black velvety coat of spongy platinum. The gas evolved
should escape freely. To free from traces of chlorides, etc., dip in the

 

 

 

H2S80,' BAT TERY

 

 

 

 

 

 

 

 

 

 

A B

Fig. 95.

Fic. 95.—Diagram of connections for platinizing electrodes.

other vial containing 10 per cent. H,SO, and continue to pass the current
for several minutes. Wash the electrodes with CO,-free distilled water
until all soluble matter has been removed. The electrode is then ready.

b. Preparation of the calomel electrode. Cover the wire at the
bottom of the electrode with mercury. Make a paste by rubbing to-
gether mercury, mercurous chloride (calomel) and a little 0.1N KCl (or
saturated KCl, depending on whether the electrode is to be 0.1N or
saturated KCl electrode) and place a layer of about % inch deep above
the mercury (in the second bulb of the electrode), fill the rest of the
electrode with 0.1N or saturated KCl, as the case may be. It is perhaps
better, on the whole, to use saturated KCl in this electrode, and the
figures in Table I, p. 1058, are given for this concentration. Be sure that
the electrode is filled with the solution so that there is no break in the
column of liquid clear to the end of the side tube. Have the cock of
the electrode closed but not greased. Having done this, and after cork-
ing the top, clamp electrode in place so that the end of the side tube
dips into the beaker of saturated KCl solution or in the Hildebrand
method directly into the solution to be measured.

e. The capillary electrometer or galvanometer is set up and con-
nected as shown in Figures 94 or 96. A microscope will be necessary to
read the capillary electrometer.

d. The hydrogen electrode (if blood or animal liquids containing
1054 PHYSIOLOGICAL CHEMISTRY

CO, and O, are to be examined) is set up by filling the electrode with
the liquid to be determined so full that the liquid just touches the
platinum or wets it only about a mm. in depth. The space above the
liquid is filled with pure hydrogen. (A very good form of electrode is

 

 

 

 

 

 

 

 

 

 

 

 

 

 

KEY

 

 

 

 

 

+ —
STANDARD
CELL

Fie. 96.

Fig. 96.—Diagram of connections for determining hydrogen ions using resistance
boxes. R,. R,. R,, resistance boxes. Hg, calomel electrode. H, hydrogen electrode. KCL,
saturated KCl solution. The capillary electrometer is shown as shortJcircuited in key 3.
B, lead accumulator.

that of Clark, shown in Figure 96. It is not necessary to keep
hydrogen bubbling through after the gas has been brought in. It is
well to fill the electrode full of the liquid and to displace it to the
proper level by allowing hydrogen to come in. It is necessary for a
few minutes to elapse after the hydrogen has been brought in before
the electrode comes to equilibrium with the hydrogen. The electrode
should be frequently replatinized. For using Clark electrode, see J. Biol.
Chem., 1915, 28, p. 478.

e. Connect the hydrogen electrode in the manner shown in Figures
94 and 96 so that the current from the gas chain goes through the
capillary electrometer in the direction indicated. The key on the capil-
lary electrometer must always be kept closed so that the electrometer is
short cireuited except at the moment of testing. The current is then
sent through the electrometer only for an instant while the mercury
meniscus is being observed.
PRACTICAL WORK AND METHODS 1055

f. Now connect up the rheostat or resistance boxes and the dry cell
or accumulator and the Weston standard cell in the manner indicated
in the diagrams. If the resistance boxes are used a large amount of
resistance, 1,000 to 5,000 ohms, is taken out of box 3 and kept out. R.,
1,000 ohms out. R, has at first no resistance out. The plugs are in.
Be careful that the accumulator is connected as shown so that the
current goes through the capillary electrometer in the opposite directior

Pres
SL iets bel

 

Fig. 97.

Fic. 97.—Potentiometer, for use in hydrogen ion determination, made by Leeds and
Northrup. Bridge, resistance boxes and keys are included in the potentiometer.

to that coming from the gas chain, since we are to balance these two
currents against each other. By having no resistance out of R, and
much out of R, and R,, practically no current will be flowing through
the galvanometer or electrometer. See that the standard cell is con-
nected up in the manner shown with the correct polarity. Key 2 must
be so arranged that the current from either the gas chain or the standard
cell can be sent through the galvanometer or electrometer as desired:
Key 3, Figure 96, is so made that breaking the short circuiting key
automatically connects the negative poles of the gas chain and the
standard cell.

_ The following solution may be used for testing the gas chain ap-
paratus: 50 c.c. N NaOH; 100 ce. N acetie acid; 350 ec. H,O. This
gives H+ = 2.35 X 10-° or P,, = 4.63 at 18°. It should give at 18°
604.5 mv. with the N/10 calomel electrode, and 517 mv. with the sat-
urated KCI calomel electrode.

g. Having set up the apparatus as indicated, begin by measuring
the hydrogen ion concentration of some solution of which the hydrogen
1056 PHYSIOLOGICAL CHEMISTRY

ion is known. For this purpose make up the solutions just described
or a buffer as shown in Table II. Fill the nydrogen electrode in the
manner indicated and see that it is filled with pure aydrogen gas. (This
gas can now be purchased sufficiently pure, or nearly so, in cylinders.
It has been prepared by electrolysis.) If not on hand, make it by
electrolysis of an NaOH solution, the gas being collected from a porus
cup about the platinum electrode. Purify the gas by running it through
NaOH, over a heated, reduced copper spiral to remove the last traces
of oxygen, and through water and then into the electrode. It must be
at room temperature. when it goes into the electrode. The apparatus
having stood after admission of the hydrogen for a couple of minutes,
momentarily open the key of the electrometer so that the current can
flow through the electrometer. Observe that the meniscus of the mercury
at once moves. Close key. Now remove a plug from R, and insert it
into R, in a corresponding hole, so that the total resistance of R, and R,
remains unchanged. Begin with a small plug. This causes some current
to flow from the accumulator in the reverse direction through the elec-
trometer. Momentarily open the key while observing the electrometer.
Close instantly if the mercury moves. (During all this time Key 2 is
so set that the standard cell is cut out. Key 1 is kept closed.) Remove
another plug from R, to its corresponding hole in R, and repeat the
electrometer observation. And proceed in this fashion by constantly
increasing the resistance taken out of R, and reducing that out of R.
until the eurrent from the accumulator exactly balances the current
from the gas chain and the mercury in the manometer, or the galvanom-
eter mirror, 1f that has been used, shows no deflection.

‘We have now balanced the gas chain against the accumulator. We
record the resistance out of each of the boxes, and now proceed to test
the voltage of the accumulator by balancing it against a known source
of electromotive force, the standard Weston cell. This has a voltage of
1.019. To do this replace in R, all the plugs transferred to R., turn
the switch key 2 so that the gas chain is no longer in the circuit but
the standard cell is in its place, and begin now and balance the standard
cell against the accumulator. Be very careful not to switch too high
a voltage through the cadmium eell, for it will spoil it. In just the same
way remove plugs from R, into R, until the current from the Weston
is just balanced. Record the resistance in each box, R,, R, and R,.

h. Computation. The voltage of the standard cell is known. It is
1.019. To balance this we have had to take, let us say, all the resistance
from R, and place it in R,. The totai resistance in the circuit of the
accumulator, the sum R,, R, and R, is, let us say, 6,000 ohms. We have
in R, 1,110 ohms resistance out. Then the fall of potential being uni-
form, we have the equation
PRACTICAL WORK AND METHODS 1057

1,100
1.019 — voltage of standard cell — aauee the voltage of the accumulator.

This gives the voltage of the accumulator. We now take this value and
substitute it in the equation:

Voltage of the gas chain =} of the voltage of the accumulator

where x is the resistance of R, and y is the sum of R,, R, and R, when
the gas chain was balanced. This gives the E.M.F. in volts.

To find the H ion concentration we have to consult Table I, where
the temperature correction is made. We have the equation

E is the E.M.F. just measured; F is the voltage of the calomel saturated
KCl electrode against N H+, and d is a numerical factor which includes
the gas constant, the Faraday constant, the number of valences, and
also the temperature. Its value is 0.0001983T. When F is measured
in millivolts, as in the table, we have 0.1983T; in that case E is also in
millivolts (thousandths of a volt). d is equal to the fraction x where
n

R is the gas constant, F, the Faraday constant, T the absolute tempera-
ture, and n, the valence, in this case 1. The T in the foregoing is the
absolute temperature or 273 plus degrees Centigrade. At 20°, T is 293.
The values of d are given for millivolts in column 5, Table‘I.

In case the Hildebrand method is used, much of this calculation is
avoided, but the method is the same, in its essentials. A dry cell may
be used in place of the lead accumulator. The apparatus is set up as
‘shown in Figure 94. The voltage of the dry cell is measured directly.
Key 1 is only closed for a moment of making the observations, as these.
cells easily polarize. In case liquids like the urine or sea water are ex-
amined the simple electrode of Hildebrand, figured as h, may be used. In
that case the liquid is placed in the beaker and the calomel electrode.
dips directly into it. H gas is passed continuously, escaping through the
notches cut in the bell at the lower end of the electrode. In. this case it
is only necessary to slide the contact S along the rheostat a-b until there
is no longer -any deflection in the capillary electrometer and read off
the voltage by the voltmeter V. This gives the voltage directly. The
standard cell is not necessary.

In all eases, and particularly when blood or other fluids containing
1058 PHYSIOLOGICAL CHEMISTRY

TABLE I.

Table for calculation of PR from measured E.M.F. (F) with different electrodes.
+ E—F

Py=lg H =

 

d

F — difference of potential between calomel electrode and normal H ions.
It involves gas constant, Faraday constant, number
Voltage Weston cell at 20° = 1.0191.

also a function of temperature.

of valences, and factor changing log , to log, ,.

d is

 

 

 

F in millivolts
Temp. ;
af 8G N/10 KCl N/1 KCl Saturated d
calomel calomel KCl calomel
electrode electrode electrode

15 252.5 57.1
16 251.7 57.3
17 250.9 57.5
18 336. 277. 250.3 57.7
19 249.5 57.9
20 248.8 58.1
21 248.2 58.3
22 247.5 58.5
23 246.8 58.7
24 246.3 58.9
25 245.8 59.1
26 244.7 59.3
27 244.4 59.5
28 243.3 59.7
29 242.6 59.9
30 241.9 60.07
31 241.2 60.27
32 240.5 60.47
33 239.8 60.66
34 239.2 60.86

- 35 238.5 61.06
36 237.8 61.25
37 235.5 61.45
38 235.0 61.64
39 61.85
40 62.08

 

 

 

 

 

oxygen are used, it is desirable to replatinize frequently, or at any rate
to immerse the electrode as the cathode in dilute acid. This recharges

it with ‘hydrogen.

‘If the lead accumulator is used in the Hildebrand method, put in
series with thé rheostat sufficient resistance so that the fall in potential
between the ends. of the rheostat is about 1 volt;. with a dry cell or
other battery of a little over a volt, this. will not be necessary. The
capillary electrode must be sensitive to 1-2 millivolts. Its capacity is sO
little that, the stop cock (not. greased) of the calomel electrode may
remain closed during the ‘observation, ‘thus preventing diffusion of the

KCL
PRACTICAL WORK AND METHODS 1059

TABLE II. (Clark and Lubs, J. B. C., 25, 1915, p. 503.)

Composition of mixtures giving PR values at 20° C. at intervals of 0.2 P

 

 

 

 

 

 

‘ Phthdlate-HC] mixtures
2.2 50 c.c. 0.2 m KH Phthalate 46.70 cc. 0.2 N HCl Diluted to 20U c.c.
2.4 50 oc 0.2 ee ee cc 39. 60 ce 0. Dp 6 “e ct cs 4 ce
26 50 “ 0.2 cc “cc “ 32. 95 “ 0. 2 19 ce cc ce ce cc
2.8 50 “ 0.2 “cc ce oe 26 42 “cc 02 “ cc (13 r13 [17 “
3.0 50 “ce 0.2 ce “cr oe x 20.32 ce 02 oe {3 o ce ce ee
3.2 50 ec 0.2 cc “ ce 14.70 “ 0.2 oe [t7 cc “cr 17 ce
3.4 50 “ce 0.2 ce ce ce 9.90 it3 0 2 ee ce “ “ “ “
3.6 50 ee 0.2 ce ce cc 5.97 “ 02 “cc ee “ oe ce ft
3.8 50 cc 0.2 “cc rT7 “ec 2.63 iT7 0.2 cc ce “cc cc “ “ce

a Phthalate-NaOH mixtures
4.0 50 cc. 0.2 m KH Phthalate 0.40 c.c. 0.2 N NaOH|Diluted to 200 ce.
4.2 50 ce 0.2 “ ec “ec 3.70 “ce 0.2 “ce iT? “
4.4 50 “e 0.2 “cr “ ce 7.50 be 0.2 ce 77 ce cc ch “a
4.6 50 “cr 0.2 cc 119 cc 12.15 oc 0.2 cc ce cc ce “ce cc
4.8 50 ce 0.2 se TF rt7 17.70 ee 0.2 “ ce cc [13 “ ce
5.0 50) 82 0:2) st * 23.85 “ 0.2 “ se s Ca
5.2 50 “ 0.2 oe “ ce 29.95 ee 0.2 “ “ cc “ ce “
5.4 50 cc 0.2 “ (17 “ 35.45 “ce 0.2 ce cc cc [19 13 ce
5.6 50 iy 0.2 ce ee cc 39.85 ce 0.2 ce “ce “cc 17 ““c “
5.8 bor Ot s 43.00 “ 02% ce a a
6.0 50 ce 0.2 rt? “ cc 45.45 ce 0.2 “ ci4 ce 6c “ ec
6.2 50 “ee 0.2 cc cc oe 47.00 “ 0.2 19 “ce cc [<7 ee “

rs KH,PO,-NaOH mixtures

50 ce. 0.2 m KH,PO 3.72 cc. 0.2 N “Or Diluted to 200 c.c.
50 ce 0 2 “ fa s 5.70 “ 0. 2 “ “ “ce “cc 6c

: 50 « 02 “ “ 8.60 “ 02 “ “ “ «“ «6
6.4 50 oc 0.2 ee “cr 12.60 “ 0.2 ce oc it? “c cc “
6.6 50 “ 02 7 “ 17.80 “ 92 7 “ “ “ “ ‘“c
6 8 50 cc 0 2 “cc “ 23.65 “ce 0.2 “ce “ (13 rt ri7 oh
7.0 50 “e 02 ce (13 29.63 ce 0.2 oc “ “ ce ce ce
7 2 50 cc 0.2 “ ce 35.00 ec 0.2 cc “ [73 “ “ce “
74 50 © 02 « ey 39.50 “ 0.2 “ 7 6c co 6
76 50 “ 02 “c 6c 42.80 “ 0.2 “ “ “ ow 6 “
78 50 “ 02 6“ “ 45.20 “ 0.2 “ “ec 7 “6
8.0 50 “« 92 « “ 46.80 “© 0.2 “ “ “ cc “ “

 

 

‘

 

_

P Borie acid, KCI-NaOH mixtures
H :

7.8 | 50 cc. 0.2 m H, BO, 0.2 m KCl] 2.61 ec. 0.2 N NaOH|Diluted to 200 ce.
8.0 50 “ 0.2 “ O22 8 3.97 © 0.2 “ = & «6
8.2 50 “ 0.2 “ my on “ 5.90 “ 0.2 “ ee ns i
8.4 50 “ 0.2 “ “ 02% « 8.50 “ 0.2 “ “ cE
8.6 50 “ 0.2 “ $ 0:23 = 12.00 “* 02° >> ae 3E Se
8.8 50 “ 0.2 “ se O22 16.30 “ 0.2 “ «6 es co
9.0 | 50 “ 0.2 “ as 02 21.30 “ 0.2 “ ss ve a
9.2 50 ce 0.2 “ “ce 0.2 ce 6c 26.70 “ 0.2 74 “ “ “ “ o
94 |)50" 02% & 0.2 32.00 ©“ 02% “. “ “« 6 6
9.6 50 “ 0.2 “ «f o2 36.85 “ 0.2 “ *6 ss . :
98 50 rt 3 0.2 “ce cif 0.2 “ce cc 40.80 “ 0.2 cc “ “ “ec ‘=
0.0

 

 

 

nO 0.2 “cc « 0.2 “ “ 43.90 “ 02 * “ce “cc be “

 
CHAPTER XXXII.
THE URINE.

EXERCISE XLII. PREPARATION AND PROPERTIES OF THE PRINCIPAL
URINARY CONSTITUENTS. UREA AND URIC ACID.

The urine is a most important excretion. In it the nitrogenous
wastes and most of the mineral and some of the carbon wastes are
excreted. The determination of the character and amount of these is
of great importance in throwing light on the physiology and pathology
of the body. Among the nitrogenous wastes urea, uric acid, creatinine,
ammonia and hippuric acid are the more abundant, although a very
large number of nitrogen-containing substances occur in the urine in
small amounts. Of the inorganic wastes, chlorides, sulphates and phos-
phates may be mentioned, and of the substances containing carbon,
various aromatic compounds, such as phenyl-acetic acid, and aliphatic
compounds such as acetone, hydroxybutyric acid, lactic acid, acetoacetic
acid. There are, however, a large number of other substances present
in very small amounts.

Experiment 213. Collect a sample of urine and note its color, odor,
transparency, reaction to litmus and to phenolphthalein and its specific
gravity.

Experiment 214. Make qualitative tests for the presence of chlo-
rides, sulphates and phosphates. For the chlorides acidify with nitric
acid and add a few drops of silver nitrate. A white precipitate indi-
eates the presence of chlorides. For the sulphates, acidify with
hydrochloric acid and add a few drops of barium chloride. A white
precipitate shows the presence of inorganic sulphuric acid. For the
phosphates add to the clear urine made alkaline by ammonia some
ammonia-magnesia mixture used for the determination of phosphates.
A white precipitate indicates phosphates. Or add to the urine some
drops of nitric acid and then some ammonium-molybdate solution. A
yellow precipitate of phosphomolybdate occurs in the presence of
phosphates.

Experiment 215. Preparation of urea and uric acid from urine.—
Evaporate 500 cc. of fresh urine in evaporating dish to dryness on
steam bath. Extract residue with three successive portions of 95 per

cent. alcohol, or acetone, using 25 ¢.c. each time and heating to boiling
1060
PRACTICAL WORK AND METHODS 1061

on the water bath to facilitate solution of the urea. Decant the hot
alcohol or acetone from the insoluble residue each time, pouring the
hot solution containing the urea into a small evaporating dish. The
uric acid is left in the residuc in the lurge evaporating dish, while the
urea, separated from most of the salts, is in the alcohol or acetone.
Save the residue with the urie acid and proceed with the preparation
of the urea.

Preparation of urea. Evaporate the alcoholic or acetone urea solu-
tion to a syrup on the water bath, add 10 ¢.c. of water, and after thor-
ough mixing add, little by little, while stirring and keeping the
evaporating dish cold by floating it in cold water, half concentrated,
white, not fuming, nitric acid as long as crystals of urea nitrate con-
tinue to separate out. When no more crystals form, filter through a
small suction filter, using suction flask and the little filter plate, and
suck the nitrate of urea dry. Most of the color remains in the filtrate.
To the filtrate add a little strong nitric acid to make sure that all urea
has been precipitated. There is dungcr of oxidizing the urea, so do not
use fuming nitric acid; be sure to cool the urea solution between each
addition of nitric acid. Now transfer crystals of urea nitrate from the
filter paper to small evaporating dish. Add to the erystals in the
evaporating dish 15 ¢.c. distilled water and then, little by little, powdered
barium carbonate, stirring between each addition, as long as efferves-
cence lasts, and then add more to make sure that all the urea nitrate is
decomposed and that there is an excess of barium carbonate present.
The solution will then be very faintly acid, due to the carbonic acid.
By this one forms barium nitrate, carbon dioxide and free urea. The
carbonate must be really in excess, or the urea will be oxidized on
warming. Now add about half a level teaspoonful of good powdered
bone black and heat on water bath for about fifteen minutes. Mean-
while prepare the small filter plate for suction filtering, placing a test-
tube in the filter flask to catch the filtrate, and at the end of fifteen
minutes filter the hot solution and transfer the filtrate, which should
be clear as water, to a small evaporating dish, and evaporate nearly to
dryness on the water bath. Crystals of barium nitrate separate out.
Extract the residue without separating the crystals, with two 10 cc.
portions of 95 per cent. ethyl alcohol, heating on the water bath each
time and transferring the hot alcohol containing the urea by decanta-
tion to another small evaporating dish. Barium nitrate remains undis-
solved. Evaporate the alcoholic solution of urea on the water bath
until it begins to crystallize and then remove it from the bath and allow
it to erystallize at room temperature. Urea crystallizes in long prismatic
erystals. The preparation should be white. If it is yellow it will not
erystallize so well. If this should be the case dissolve in a little alcohol
1062 PHYSIOLOGICAL CHEMISTRY

and decolorize again with powdered animal charcoal. The yield should
be 6-10 grams.

Uric acid. To the residue in the evaporating dish from which the
urea was extracted, add 50 c.c. distilled water distinctly acid with hydro-
ehloric acid. All.the inorganic salts dissolve and leave the uric acid as
a small, reddish, granular, crystalline precipitate. Decant and discard
the liquid but keep the crystals. Examine the crystals microscopically.
Wash them once or twice with fresh water by decantation. Finally add
about 30 ¢.c. of water and then, drop. by drop, saturated Na,CO, solution,
stirring after each addition. Seven or eight drops are usually enough,
and warm for a minute or so on the water bath to hasten solution. Add
a pinch of animal charcoal. Filter through a small suction filter,
transfer without loss to a small beaker and acidify with hydrochloric
acid. The uric acid will crystallize out promptly as a sandy precipitate,
perhaps not entirely white. After standing for half an hour or over
night, filter through a small suction filter. Allow the crystals to dry
on the filter, then remove them to a small vial. Make the following
various tests for uric acid with them. Yield about 0.2 gram.

Reactions for the identification of uric acid.

Experiment 216. Murexide test—See page 720 for the reaction in-
volved. Place a few crystals of uric acid in a porcelain dish, moisten
them with a drop or two of concentrated nitric acid and dry on the
water bath until the nitric acid is completely gone or heat very carefully
over a flame until HNO, is nearly gone and it begins to turn red on
the edges. A reddish residue remains. Now cool this and moisten it
with a very small drop of dilute ammonia solution or blow ammonia
vapor over it. The residue becomes a violet or purple red, due to
formation of ammonium purpurate. Add a little 10 per cent. NaOH,
the residue becomes a bluish violet. On heating the color disappears.
Of the other purine bases adenine and hypoxanthine do not give the
murexide test. Guanine and xanthine give the reaction forming the
yellow nitroxanthine first, which turns violet or purple when moistened
with sodium hydrate but remains yellow on addition of ammonia. The
color does not disappear on heating.

Experiment 217. Reducing reactions. Reduction of Fehling’s so-
lution.—Urie acid is easily oxidized and it is accordingly a reducing
substance. Many tests have been devised for its detection based upon
this property. None of these are specific, since they are given by other
reducing reagents. Uric acid reduces Fehling’s solution. Take a few
crystals, dissolve in a little sodium hydrate and assure yourself that
they reduce Fehling’s solution. To another portion of the sodium-
PRACTICAL WORK AND METHODS 1063

hydrate solution add some powdered bismuth subnitrate and heat. This
is not reduced.

Experiment 218. Reduction of silver nitrate. Schiff’s reaction. —
Dissolve a few crystals of uric acid in a few c.c. sodium-carbonate solu-
tion (sodium hydrate cannot be used because it precipitates the silver
as brown silver hydrate), the solution being distinctly alkaline. Pour
a drop of the solution on a filter paper moistened with silver nitrate
solution. A black spot will be formed in the presence of uric acid.
This reaction depends on the power of the uric acid to reduce alkaline
silver solutions.

Experiment 219. Reduction of phosphotungstic acid. Folin re-
action.—This reaction depends on the power of uric acid to reduce
sodium phosphotungstate solution. To a very small amount of uric
acid in a beaker is added 20 c.c. saturated sodium-carbonate solu-
tion. It may be warmed to hasten solution. As soon as the uric
acid is dissolved add 1 c.c. of sodium phosphotungstate reagent
(Folin’s). A blue color is obtained, due to the formation of oxide of
tungsten W,O,.

Folin’s sodium phosphotungstate reagent is the following: 100 grams
pure sodium tungstate, 80 c.c. 85 per cent. orthophosphoriec acid and 750
c.c. distilled water are boiled gently or in a flask with a reflux con-
denser for 112-2 hours. Cool and dilute to 1 liter. This solution is
reduced by other compounds, for example by polyphenols. It is used
in the microchemical estimation of uric acid.

Experiment 220. Benedict’s solution not reduced by uric acid.—
The reduction by uric acid is most rapid in an alkaline solution. Bene-
dict’s solution, which is reduced by carbohydrates, is not so alkaline as
Fehling’s and is hence reduced by uric acid at a very much slower rate.
Test the reducing action of a sodium-carbonate solution of uric acid on
Benedict’s solution. Experiment 12.

Experiment 221. Precipitation reactions of uric acid.—The in-
solubility of the free acid has already been noted. The ammonium salt
is also very insoluble, particularly in the presence of other soluble am-
monium salts. This method is the basis of the quantitative method for
the estimation of uric acid of Hopkins.

Make a saturated solution of uric acid by heating some crystals
with 10 cc. of 2 per cent. Na,CO,. After adding two drops of ammonia,
saturate the solution with ammonium chloride. Note the white, amor-
phous precipitate of ammonium urate which forms. If desired,. the
precipitate may be identified as uric acid.

Experiment 222. Precipitation by ammoniacal silver solution.—
Add an excess of ammonia to the sodium-carbonate solution of urie aeid,
and then a few drops of silver nitrate. A white precipitate is formed.
1064 PHYSIOLOGICAL CHEMISTRY

This is the silver compound of the uric acid. Other purines are precipi-
tated by this method (see Salkowski’s method).

Experiment 223. Detection of uric acid in urine and other fluids.—
To show the presence of uric acid in small quantities of urine, Folin’s
method may be used. Place 1-2 c.c. of urine in an evaporating dish, add
1 drop of saturated oxalic-acid solution and evaporate to complete dry-
ness on the water bath. Cool, add 10 cc. 95 per cent. alcohol and
allow to stand for 5 minutes to extract phenols which will also, if present,
give the reduction reaction with phosphotungstate. Pour off the alcohol.
Add to the residue 10 cc. of water and a drop of saturated sodium-
carbonate solution. Stir to complete the solution of the uric acid;
transfer to a small beaker; add 1 ¢.c. of Folin’s reagent (sodium phos-
photungstate) and 20 e.c. of saturated sodium carbonate. A blue color
indicates the presence of uric acid.

Experiment 224. Another method is the following (Cole): Take
50 ¢.c. of urine, add 2 drops of ammonia and then saturate with pow-
dered ammonium chloride. Allow the excess of ammonium chloride to
settle for 15 seconds and pour off into another beaker. Allow to stand.
Note the gelatinous precipitate of ammonium urate. Filter: scrape the
precipitate from the filter and transfer it to an evaporating dish. Add
3 or 4 drops of strong nitric acid and evaporate to dryness, then with
ammonia make the murexide test. If urates are present in the precipi-
tate, they will give a positive reaction.

EXERCISE XLVI. CREATININE, HIPPURIC ACID AND INDICAN.

Creatinine.
NH — C=O
7
NH —C
N(CH,) —CH,

Experiment 225. Preparation of creatinine. Zinc-chloride method.
—Make 500 ¢.c. of urine alkaline with milk of lime and add CaCl, solu-
tion to completely precipitate the phosphates. Filter, acidify the filtrate
with acetic acid and evaporate to a syrup. Extract the creatinine from
the syrup by treating it with warm 95-99 per cent. alcohol, 100 ¢.c. in
two 50 ¢.c. portions. Allow alcohol extract to stand 8-24 hours in a cool
place. Filter. A little sodium acetate is added to the alcoholic filtrate
to reduce the acidity and about 1 c.c. of strong, acid-free zinc-chloride
solution, sp. gr. 1.2. Stir and allow to stand 2 to 3 days in a cool place.
Creatinine zine chlortde crystallizes out as a sandy, yellowish powder
PRACTICAL WORK AND METHODS 1065

composed of fine needles in rosettes or balls. Collect by filtering on a
small suction filter, wash with alcohol, suspend the crystals in a little
(100 c.c.) warm water and add some freshly precipitated lead hydrate,
or red lead oxide, warm to about 75° for a minute or so while stirring
to decompose the zine chloride-creatinine, and filter, using suction. The
precipitate is lead chloride and zine oxychloride. Decolorize the filtrate
with a pinch of animal charcoal, warming while stirring, filter (using
suction), evaporate the solution to dryness, extract with strong alcohol
(creatinine dissolving, creatine remaining insoluble) and evaporate the
alcoholic extract to beginning crystallization and then allow to stand
and crystallize. Make some of the following tests with the crystals.
The crystals when pure are colorless, monoclinic prisms.

Experiment 226. Preparation of creatinine from the urine by the
picrate method.—(See experiment 285, page 1095.)

Experiment 227. Reactions for the detection of creatinine.
Weyl’s reaction. Nitroprusside reaction.—Like some other reducing
substances, creatinine gives a red color with sodium nitroprusside. See
the test for acetone (Legal’s). Cysteine gives a similar reaction. To
5 e.e. of urine in a test-tube add a few drops of a dilute, fresh solution
of sodium nitroprusside and make alkaline with sodium hydrate. A
ruby-red color which fades to yellow is the result. If to this solution,
cold, is added.an equal quantity of glacial acetic acid, a precipitate of
the nitroso compound (C,H,N,O,) results. If acidified with acetic acid
and heated, the solution becomes green, then blue and: Prussian blue
separates. Dissolve one crystal of the creatinine in water and repeat
the test with it.

Experiment 228. Picramic-acid reaction. Jaffé.—Creatinine com-
bines with picric acid. If made alkaline, it very rapidly reduces the picric
acid at room temperature forming the red-colored picramic acid. See
page 41. To 5 ec. of urine in a test-tube add a few drops of picric-acid
solution and make alkaline with sodium hydrate. <A red color is pro-
duced. This reaction is the basis of Folin’s creatinine quantitative
method. It is not specific for creatinine, but depends on the reducing
action of creatinine in alkaline solution.

a

Hippuric acid.

Experiment 229. Preparation of hippuric acid from urine. Roaf.—
To 500 e.c. of the urine of a horse, or cow, add 125 grams of ammonium
sulphate and 7.5 concentrated sulphuric acid. Hippuric acid crystallizes
out. Filter. Wash the crystals with a little cold water, redissolve in
a small amount of hot water, boil with animal charcoal to decolorize,
filter and allow to cool. The hippuric acid crystallizes in standing. If
1066 PHYSIOLOGICAL CHEMISTRY

some of the crystals are evaporated to dryness on the water bath with
1-2 ¢.c. concentrated nitric acid and then the residue heated by a flame,
an odor of nitrobenzene may be perceived.

Experiment 230. Detection of indican.—Indican is the potassium
salt of indoxyl-sulphurie acid.

CH CH CH
HO” No—c-0-s0,k af Net_co ood S cH
nh § Qn ne Ybid l de

Ni Na \a@ Xa Xa Ge
Indican. Indigo blue.

{ndican is formed from the indoxyl which has been formed from indole
by oxidation. Indole is derived from the tryptophane of proteins by
processes of putrefaction. The presence of indican in larger than usual
quantities in the urine generally means excessive putrefaction in the
alimentary canal. In constipation the indican is usually increased.
Indican is detected by converting it to indigo blue by oxidation. Various
oxidizing agents may be used, but, ferric chloride or hypochlorite is
usually employed. The oxidation takes place in strong acid solution,
the acid splits off sulphuric acid, leaving indixyl which is oxidized to
indigo blue. :

Experiment 231. Jaffé’s indican test—Take 5 cc. of urine in a
test-tube and add 5 e.c. of concentrated HCl and then 1 drop of a 3 per
cent. KC1O, solution. By the action of the acid chlorate is formed.
Add a little chloroform, 2-8 ¢.c., and shake. If the chloroform settles
out colorless, add another drop of hypochlorite and shake again. If
indican is present, the chloroform should be colored blue. If thymol
has been added to the urine for preserving it, the chloroform will be a
reddish or violet color if indican is present. Keep on adding drop by
drop of the chlorate and shaking until a maximum blue is obtained.
The oxidizing agent must be added cautiously, since the addition of too
much carries the oxidation to indigo white which is colorless. Calcium
hypochlorite or bleaching powder may be used in this test in place of
the chlorate. The result is the same.

Experiment 232. Detection of indican by ferric chloride. Ober-
mayer’s test.—This may be performed either directly on the urine or
after partial purification by precipitation with lead acetate. The test
in the latter case is performed as follows: Add to 50 cc. acid urine 5 c.c.
basic lead acetate. Filter. Take 10 c.c. of the filtrate in a test-tube,
add an equal volume of concentrated HCl containing 2-4 grams FeCl,
per liter, and 2-3 ¢.c. chloroform and shake thoroughly. The chloro-
form which settles out should be more or less blue if indican is present.
PRACTICAL WORK AND METHODS 1067

There is not so much danger in this test of overoxidation as there is in
the hypochlorite test.

Exercises XLIV-XLVII. DeEtTEecTION OF PATHOLOGICAL CONSTITUENTS
OF URINE.

Detection of proteins.—Normal urine does not contain protein. In
abnormal conditions various proteins may occur. Coagulable proteins
derived from the blood plasma may be present in inflammation of the
kidney, or nephritis; the nephritis being either chronic, due to a chronic
infection of the kidney, or acute. Acute nephritis may accompany
scarlet fever and other diseases. A deutero-albumose, generally called
peptone, may be present particularly during the absorption of partially
digested pus from abscesses ‘or in the absorption of pneumonia exudate.
An especial protein called the Bence-Jones protein appears in the urine
in osteomalacia or multiple myeloma. This is more nearly related to
a hetero-albumose. The nitrogen in the protein in the latter case may
amount to as much as one-third of the total nitrogen of the urine.

Experiment 233. Coagulable protein.—Very little of this may
appear in the urine, even though pretty extensive chronic nephritis
prevails. The presence of any in the urine should be regarded with
suspicion. If the urine is clear and acid, place about 7 c¢.c. in a test-
tube and, while holding the tube inclined, heat the upper parts of the
urine to boiling. If coagulable protein is present, this part of the urine
should appear cloudy as compared with the unboiled part. If it be-
comes turbid, add a drop of dilute acetic acid. The turbidity might be
due to phosphates, but these dissolve in the presence of dilute acid. The
protein coagulum does not dissolve. If the urine is alkaline, make it
before boiling very faintly acid with dilute acetic acid.

Experiment 234. Heller’s test.—Place in the bottom of a test-tube
about 4 ¢.c. concentrated nitric acid and holding the tube inclined pour
carefully down the side of the tube some urine. The urine should float
on top of the nitric acid. In the presence of albumin a white ring
appears at the junction of the liquids. This is a very delicate test. It
may happen that there is a ring of urea-nitrate or uric acid in concen-
trated urines. If the ring is due to these substances, it will disappear
on diluting the urine and repeating the test. Urines preserved with
thymol may give a ring of nitrothymol. The thymol may be separated
from the urine by extracting it with a little naphtha. Resinous sub-
stances which appear in the urine after a person has been treated with
balsams also may produce a white ring. It is well, therefore, to further
identify the protein, if the test is positive. The colored ring which
develops due to the oxidation of various chromogens may be disregarded.
1068 PHYSIOLOGICAL CHEMISTRY

Experiment 235.—Various modifications of these tests have heen
proposed. Various salts such as sodium chloride are added to the urine,
or to the nitric acid to increase the delicacy of the test. The Heller test
is, however, already sufficiently delicate for all ordinary purposes. The
identification of the protein can be confirmed by trying the precipitation
test with potassium ferrocyanide and acetic acid. Make the urine faintly
acid with acetic acid and add a drop of potassium ferrocyanide solution.
In the presence of albumins or protalbumose a white precipitate appears.

A case came under the author’s observation in which the urine con-
tained so little protein that it was with great difficulty that its presence
could be certainly established. At times the urine was free from pro-
tein. The person died not long afterwards of an acute infection, and
on autopsy the kidneys were found to have been the seat of a long-
standing and extensive nephritis.

Experiment 236. Quantitative estimation of albumin present.
Esbach’s method.—Principle. The method consists in precipitating the
protein with picric acid and estimating the volume of the precipitate. A
graduated tube known as Esbach’s albumimometer is used.

AT

ul

      

 

 

 

 

Fia. 98.

Fig. 98.—Esbach albuminometer.

Process. Fill the tube with urine to the mark U and then add the
picric-acid reagent to the.mark R. Cork, mix by inverting a couple of
times and then allow to stand upright for 24 hours. At the end of
PRACTICAL WORK AND METHODS 1069

that time read off on the scale etched on the tube the height of the
column of precipitate. The figures give the number of grams of dried
protein in a liter of urine. If thé amount is less than 0.05 per cent.,
it cannot be accurately determined in this way. If the precipitate comes
above 5, it is better to dilute the urine once and repeat.

Picric-acid reagent. Dissolve 10 grams picric acid and 20 grams
citric acid in 800 c.c. boiling water, transfer to a 1,000 ¢c.c. volumetric ©
flask, cool and make up to the mark.

Experiment 237.—The coagulable protein can also be directly deter-
mined by boiling 50 ¢.c. of urine, slightly acidified with acetic acid, till
the separation is complete, filtering through a dried weighed filter paper,
washing thoroughly with hot water and alcohol, drying and reweighing.

Experiment 238. Bence-Jones protein.—This protein is detected by
its peculiarity of becoming insoluble on heating to 60° and the precipitate
redissolving on heating to boiling.

If the urine is acid, it may be heated directly; if alkaline, make it
faintly acid with acetic acid. Heat slowly. The urine becomes turbid
at about 45° and a precipitate appears at 60° which redissolves on
further heating to boiling. On cooling the reverse phenomena appear.

Experiment 239. Albumose.—This can be detected if in sufficient
quantity by the rose-colored biuret test in urine which will not coagulate
on heating. It may also be detected as follows: Remove the coagulable
protein by heating to boiling the faintly acid urine and filtering. In
the filtrate a biuret reaction or a ring test with Spiegler’s reagent indi-
cates the presence of albumose or peptone. To detect any albumin by
Spiegler’s reagent be sure that the urine is acidified first. Alkaline
urine will give a precipitate even when free from albumin and albumose.

Spiegler’s reagent. HgCl,, 40 grams; tartaric acid, 20 grams; NaCl,
50 grams; glycerine, 100 grams; H,O, 1,000 ec. This reagent is a very
delicate test for proteins, as both albumoses and peptones are precipi-
tated by it. It is used in the same way as the nitric acid in Heller’s
test. 1 part protein in 250,000 can be detected by it. Control it by
normal urine.

Experiment 240. Detection of glucose in urine.—Glucose appears
in the urine under the circumstances already described on page 757. If
the urine is light colored, of large volume, three liters or more per day,
and with a nomal specific gravity, the presence of glucose is indicated.
To detect glucose three or four methods may be used: i.e., reduction test
with Fehling’s or Benedict’s solution, reduction of bismuth subnitrate,
polarimeter examination, the fermentation by yeast, formation of phenyl-
glueosazone. The best method is Benedict’s. For the quantitative esti-
mation use the Benedict method of experiment 305.

Experiment 241.—To 4 c.c. of urine add an equal quantity of Feh-
1070 PHYSIOLOGICAL CHEMISTRY

ling’s mixture, page 873, and heat to boiling. Boil for a few moments.
In the presence of glucose, lactose and some other substances a
yellow or red precipitate of cuprous oxide is formed. Normal urine
has some powers of reduction, but not sufficient to form a precipitate
of ecuprous oxide under these conditions. Caution: If chloroform has
been added to the urine as a preservative, it will itself reduce Fehling’s
solution.

Experiment 242. Benedict’s qualitative reaction—To 5 cc. of
Benedict’s reagent (see experiment 12) heated to boiling, add 8 drops
of the urine and boil vigorously for two minutes. The fluid will contain,
after spontaneous cooling, a dense but very finely divided red, yellow
or green precipitate if glucose to the extent of 0.2 per cent., or more is
present. The green precipitate appears with the lower concentrations
and the red with the higher. It is best ordinarily not to use more than
8 drops of the urine, although if the urine is very dilute as to uric acid,
creatinine and phosphates, one may use as much as 16 drops of the urine
to 5 «ec. reagent. Lactose also reduces Benedict’s solution.

Experiment 243.—Many substances besides glucose reduce Fehling’s
solution. The reduction might be due to lactose, pentoses, aromatic
bodies, urates, creatinine or glycuronic acid, for example. If glucose is
present, the urine will be dextro-rotatory and will reduce bismuth sub-
nitrate and ferment with yeast. Nylander’s reaction. Bismuth sub-
nitrate reaction. To 5 c.c. of urine in a test-tube add a little powdered
bismuth subnitrate, make the urine alkaline with sodium hydrate and
heat to bciling for a few minutes. If glucose is present, the subnitrate
will be reduced to the black bismuth. Uric acid and creatinine do not
give this reduction.

Experiment 244. Fermentation test.—Fill a fermentation tube with
urine which has not been preserved by the addition of formol or other
preservative, and in which 4 cake of yeast has been suspended. Put in
a warm place (40°). If the reducing body is glucose and more than 0.5
per cent., the yeast will ferment and a gas will collect in the course of
1 to 2 hours in the closed arm of the tube. Lactose, pentoses and
glycuronic acid or aromatic substances will not ferment.

Experiment 245. Form the osazone in the method described in ex-
periment 30. If the osazone, the reduction and the fermentation tests
are positive, it may be concluded that the substance is glucose or levulose.
The rotation will distinguish between these two. The melting point of.
the osazone can also be determined.

Experiment 246. The identification of small amounts of glucose
and lactose in urine (Cole).—This is probably the best method of dis-
tinguishing between small amounts of glucose and lactose in urine in the
shortest time of high grade charcoal can be had. Experiment 247 gives
a better method.
PRACTICAL WORK AND METHODS 1071

Principle. The use of Fehling’s solution for the detection of glucose
is open to the following disadvantages: 1. Other substances than glucose
reduce this solution; namely, pentoses, glycuronic acid, lactose, urates,
creatinine and some oramatic bodies. Moreover, creatinine holds the
cuprous oxide in solution so that it does not precipitate. 2. The strong
alkali used will destroy small amounts of glucose. To many samples of
urine as much as 0.5 per cent. of glucose can be added without produc-
ing more than a greenish cloud with Fehling. Normal urine contains
between 0.03 and 0.08 per cent. of glucose. Benedict’s solution removes
many of these difficulties because by the use of carbonate in place of
sodium hydrate the reducing action of uric acid and creatinine is elimi-
nated. Cole proposes the following improvement on Benedict (Cole,
Lancet, September 20, 1913). The principal improvement introduced
is that glucose is not adsorbed by good blood charcoal in the presence
of acetic acid, whereas other reducing substances, and particularly lac-
tose, are. In the absence of acetic acid small amounts of glucose are
adsorbed. In the actual reduction also sodium carbonate is used in place
of sodium hydrate.

Procedure. ‘‘ In a dry boiling tube or large test-tube place about 1
gram of good absorbent blood charcoal (Merck’s or some other as good.
The charcoal should be tested first to see that it will absorb small
amounts of lactose). (A spatula about three-eighths of an inch broad,
well piled up with the charcoal for just over an inch, carries about 1%
gram.) Add 10 «ec. of the urine and shake from side to side to mix
thoroughly. Heat to boiling point, shaking the whole time. Cool thor-
oughly under the tap and shake at intervals for about five minutes.
Filter through a small paper (9 to 11 em. in diameter) into a rather
wide test-tube containing about half a gram of anhydrous sodium car-
bonate. When the fluid has filtered through, add 6 drops of pure
glycerine (the glycerine used must not itself reduce the copper), shake
and heat to boiling. Note the time when boiiing commences. Maintain
active boiling for 50 seconds, shaking from side to side to prevent spurt-
ing. Immediately add 4 drops of a 5 per cent. solution of crystallized
copper sulphate. Shake for a moment to mix the solutions, and allow
the tube to stand without further heating for one minute. With normal
urine the fluid remains blue, with a variable amount of a grayish precipi-
tate of the earthy phosphates. If glucose is present to the extent of
0.02 per cent. or more, above the average normal amount, the blue color
is discharged, and a yellowish precipitate of cuprous hydroxide forms.
The rapidity with which the precipitate forms is a rough measure of
the amount of glucose present. With 0.05 per cent. it appears in a few
seconds. With 0.02 per cent. it may not appear till 50 seconds. A
yellowish precipitate or coloration appearing after 60 seconds must, not
1072 PHYSIOLOGICAL CHEMISTRY

be taken as evidence of abnormal glycosuria. It may be due to the
normal amount of sugar in the urine.

Remarks. The method is more sensitive than Benedict’s and Nylan-
der’s. CHCl, does not give a positive result even when present in con-
siderable excess. Urines of patients treated with chloral hydrate and
containing glycuronates are:also negative. There is no necessity to
remove albumin, but it is advisable to do so by boiling and filtering
before treatment with charcoal. If specific gr. of urine is more than
1,025, dilute with equal volume of water and take 10 c.c. of the diluted
urine. With a modification of this method Cole detects one part of
glucose in a million parts of water, when glucose is present alone. But
this cannot be done in the urine.

Experiment 247. Method of distinguishing lactose and glucose in
urine and estimating dextrose in the presence of lactose. (Mathews.)
—In the case of urine from a pregnant or nursing woman which con-
tains a reducing substance proceed as follows:

Estimate total reducing power as glucose by Benedict’s method,
experiment 305. Then take 20 c.c. of urine in a fair-sized test-tube,
mix it with half a cake of compressed yeast by placing yeast in urine,
stopping tube with the thumb and shaking until the yeast is uniformly
mixed and no more lumps. Place test-tube at angle of about 45° in a
vessel of water (beaker), heated to 42° C. If more than 1 per cent.
dextrose is present there will begin in a couple of minutes an evolution
of gas, the bubbles rising along the inclined upper surface of the tube.
With less than 1 per cent. this will hardly be visible, and if no dextrose
is present, after about 2 minutes when some air occluded during the
shaking is given off, there will be no evolution of gas. With 3 per cent.
or more of dextrose the fermentation is stormy. Lactose is not fermented.
Leave tube 50 minutes at 42° C. All dextrose up to a content of 6 per
cent. will then be destroyed. Take out tube twice during this period, put
thumb over end and invert to remix the yeast, and replace in bath.
All dextrose is now destroyed. Filter through dry folded filter. Fil-
trate will probably be opalescent but this does not matter. Determine
reducing power in filtrate by Benedict’s method. The difference be-
tween the original and this determination gives amount of dextrose.
The remainder may be lactose. To identify it make the osazone in
filtrate after fermentation by method in experiment 30.

Experiment 248. Identification of lactose in urine (Cole).—Princi-
ple. Adsorption of lactose by charcoal and the formation of lactosazone
in the presence of acetic acid. The surest indication that a reducing
sugar is not glucose and may be lactose is its failure to ferment and
produce CO, gas with yeast. But this method gives a positive proof of
lactose.

Procedure. To 1 gm. of good charcoal add 25 c.c. of suspected urine,
PRACTICAL WORK AND METHODS 1073

mix by shaking, boil for a few seconds, cool thoroughly and shake at inter-
_ vals for 10 minutes. Filter through a small paper or use a filter pump.
’ When the charcoal has completely drained, transfer it to a porcelain dish
containing 10 c.c. of water and 1 «ee. glacial acetic acid. This is best
done by opening the paper, holding it by the clean half and moving it
about in the liquid. The greater part of the charcoal is thus removed
from the paper. Stir the charcoal with a glass rod and transfer the
mixture to a boiling tube. Heat to boiling for about 10 seconds and
filter the hot solution through a small paper into a test-tube containing
as much solid phenyl-hydrazine-hydrochloride as will lie on a quarter,
and twice this amount of solid sodium acetate. Mix thoroughly and
filter from any insoluble oily residue. Place the tube in a boiling water
bath and leave it there for 45 minutes. Remove the tube and allow it
to stand at room temperature for at least one hour. It is advisable to
allow it to stand longer if possible. Pipette off a little of the deposit,
if any, and examine it on a slide under the high power of the microscope.
Lactosazone crystallizes in characteristic clumps with projecting spines
(“* hedge-hog ”’ crystals). If desired filter off crystals, redissolve in hot
water and allow to reerystallize. Melting point of crystals 200° C.

_ Experiment 249. Detection of acetone in urine. Detection in the
undistilled urine. Legal’s nitroprusside reaction—To 5 cc. of urine
in a test-tube add a few drops of a fresh solution of sodium nitroprusside
and then make the urine slightly alkaline with sodium hydrate. A ruby-
red color results both in normal and abnormal urine, due to the cre-
atinine of the urine. If the urine is now made acid with acetic acid, if
creatinine alone is responsible for the color, the solution will become
yellow, whereas if acetone is present in sufficient amount the red color
is intensified by the addition of acetic acid. Control m8 test with
normal urine and with an acetone solution.

Experiment 250. Rothera’s nitroprusside reaction—This test is
better than Legal’s. To 5 cc. of urine add a little solid ammonium
sulphate and 2-3 drops of a fresh 5 per cent. solution of sodium nitro-
prusside and 1-2 cc. of concentrated ammonium hydrate. If acetone
is present, a permanganate color develops.

Acetone may be more accurately detected in the distillate from urine.

Distill about 20 c.c. of liquid from 200 e.c. of urine acidified by the
addition of several drops of concentarted HCl. Use a good water con-
denser, as acetone is volatile. The distillate may be subjected to the
following examination.

Experment 251. Iodoform test.—Gunning’s. To 5 ¢.c. of the dis-
‘tillate (undistilled urine may be used) add a few drops of KI, solution
(Lugol’s = 4 grams iodine; 6 grams KI; 100 c.c. water) and enough
NH,OH (5-10 drops) to make a black precipitate of nitrogen iodide. On
standing this is changed to iodoform (CHI,). Detect this by the odor
1074 PHYSIOLOGICAL CHEMISTRY

and by examining the crystals. They are yellow in color and form
hexagonal plates and rosettes. Neither alcohol nor aldehyde form iodo-
form under these conditions. Acetoacetic acid is decomposed by heating -
with acid, so that the distillate contains acetone from this as well as
preformed acetone.

Experiment 252. Detection of acetoacetic acid—CH,.CO.CH.,.
COOH. Diacetic acid is readily decomposed, forming acetone if the
acid urine is boiled. It gives a much more sensitive reaction with
sodium nitroprusside than does acetone in the Le Nobel test. The test
may be made in the following way (Harding and Ruttan, Biochem.
Journal, VI, p. 445, 1912). Acidify the urine with acetic acid, add
0.5 ec. N/10 sodium nitroprusside (1 c.c. = 0.0298 grs. Na,Fe(CN),
NO.2H,0) and then overlie the solution with concentrated aqueous
NH,OH. A violet ring is produced. This is a modification of Taylor’s
method of carrying out the reaction. Acetoacetic acid gives this
reaction far better than acetone. A solution of acetone 0.057 per cent.
gives a very faint reddish-violet ring after about 20 minutes. Aceto-
acetic acid gives it at once. Acetoacetic acid 1 pt. in 30,000 will give
a positive reaction. It is far more delicate than the Gerhardt ferric-
chloride test.

Experiment 253. Gerhardt’s reaction for acetoacetic acid.—To 5
¢c.c. of urine in a test-tube add ferric-chloride solution, drop by drop,
as long as a precipitate forms. An addition of more FeCl, to the
filtrate produces a Bordeaux red color if acetoacetic acid is present. 1 pt.
in 7,000 is the limit of the reaction. Many other substances give this
reaction with FeCl,, such as salicylic acid, and substances excreted after
the ingestion of antipyrin, phenacetin and thallin. If the color is due to
acetoacetic acid it will disappear on boiling. It is an enol reaction.
Acetoacetic acid may be extracted from the urine by acidifying and
shaking with ether and the test made in the ether extract. Acetone does
not give this reaction.

Experiment 254. Salicylaldehyde reaction for acetone.—To 5 e.c.
urine (or better distillate) add 1 ¢.c. of a 10 per cent. alcoholic solution
of salicylaldehyde, shake well and introduce a piece of solid NaOH about
the size of a large pea. Set aside. Prepare a control tube with dis-
tilled water or normal urine in the same way. In ease acetone is present
to the extent of 0.01 per cent. or more a deep orange-red ring is formed
at the junction of the two layers. Normal urines and urines containing
aldehydes give a yellowish to brown ring. The red-colored substance
formed with acetone is 0.0-dioxydibenzolacetone.

Experiment 255. Black’s reaction for beta-hydroxybutyric acid.—
Concentrate 15 ¢.c. urine to 5 cc. on the stearn bath; this is done to
remove any acetoacetic acid which may be present. Now add two drops
concentrated HCl and the least quantity of plaster of Paris necessary
PRACTICAL WORK AND METHODS 1075

to form a solid‘ mass. Pulverize this mass and extract twice with ether.
Evaporate the ether extract without loss, add 5 cc. water and a small
amount of BaCO,. To the almost neutral water layer now add 2 or 3
drops hydrogen-peroxide solution, shake and add a few drops of 5 per
cent. FeCl,. Set aside for a few minutes. If beta-hydroxybutyric acid
was present, it has been oxidized to acetoacetic acid and gives the usual
red color with ferric salts. Black claims this method will detect the
acid in concentrations of 1: 10,000.

Experiment 256. Detection of glycuronic acid.—CHO.(CHOH),,.
COOH. See page 759. This acid gives many of the reactions of the
pentoses. It may be differentiated as follows (Tollen’s reaction): Add
to equal quantities of urine and concentrated hydrochloric acid 0.5-1 ¢.c.
of a 1 per cent. naphthoresorcin solution in alcohol. The solution is
heated slowly to boiling and boiled one minute, shaking the tube con-
stantly. Allow to stand and cool for four minutes, then cool under the

_ tap and shake out with ether. The red or violet color goes into the ether
if a glycuronate was present, but is insoluble in ether if the color is due
to a pentose or hexose. The ether solution examined spectroscopically
shows two bands, one on the D line and one between D and E.

Glycuronates reduce Fehling’s solution, but they do not ferment
with yeast. On heating with strong acid the glycuronic acid is set free.
In the free state it is dextro-rotatory. In the paired condition it, is
levo-rotatory. It occurs in the urine normally in very small amounts; in
larger quantities after certain drugs are taken.

Experiment 257. Detection of glycuronic acid in urine (Tollens-
Neuberg and Schwket, Biochem. Zeitschrift, 44, 1912, 502).—In a small
separatory funnel place 10 ¢.c. fresh urine with 2 «ec. dilute H,SOQ,.
Add at once 10 ¢.c. ordinary alcohol and 20 ¢.c. ether. After several
strong shakings, hasten the separation by the addition of a few c.c. of
water or NaCl solution. As soon as the ether layer is separated, draw
off the alcohol-water layer and shake the ether with 2-3 cc. H,O or
NaCl solution. Separate the ether carefully and filter through a small,
dry filter into a porcelain dish. Add 5 cc. water and evaporate the
ether on the water bath. Divide the fluid which remains, and which
may have some oil drops or be slightly cloudy, into two parts and test
one with orcin and the other with naphthoresorein. By using 10 c.e.
of normal urine both tests are positive. By shaking out with ether the
glycuronie acid esters are separated from the pentoses which may be
present and which give similar reactions. If the amount of glycuronic
acid is small, the amyl-alcohol extract of the orcin test only gives a plain
absorption band after standing.

Experiment 258. Detection of pentoses.—These occur in the urine
in disease of the pancreas and in some other conditions, the amount
depending on the amount eaten. There is still some doubt about the
1076 PHYSIOLOGICAL CHEMISTRY

character of the pentose found in the urine in endogenous pentosuria.
The presence of pentoses may be inferred if the urine reduces Fehling’s
solution, does not ferment with yeast, gives a positive reaction by Tollen’s
naphthoresorcin reaction, the color being insoluble in ether; and forms
furfural when the urine is made strongly acid with phosphoric or hydro-
chloric acid and distilled. Furfural comes off both from pentoses and
glycuronates. The other pentose reactions may also be tried: ie., with
phloroglucin and orcinol. To make the phloroglucin test, take 1 c.ce.
of 2 per cent. phloroglucin solution, 5 c.c. of concentrated HCl and 6 e.c.
of urine, mix well and immerse in boiling water. <A red color indicates
pentose or glycuronic acid. In using orcinol proceed in the same way,
but use half-saturated orcinol solution, 1 ¢.c., in place of the phloroglucin.
A blue color and precipitate indicates pentoses (see page 880). Extract
the color with amyl alcohol and examine spectroscopically.

Experiment 259. Detection of bile salts. ‘Reduction of surface
tension of urine. Hay’s test.—Bile lowers the surface tension of water.
This lowering may be detected by sprinkling some flowers of sulphur
on the surface of the urine. In the presence of bile the sulphur falls
at once to the bottom of the beaker. In normal urine it floats on the
surface. Other greases act in the same way as bile. The reaction is not
specific.

_Experiment 260. Pettenkofer’s test for bile salts—This has al-
ready been described in experiment 157, page 985. A red ring is
formed at the junction of the sulphuric acid and the urine after the
addition of a crystal of saccharose. Keep the temperature below 70°
in making this test. A little furfural may be added in place of the
saccharose. The test remains essentially the same.

Experiment 261. Detection of bile pigments in urine——Gmelin’s
test already described can be applied to the urine. Pour 5 ce. of urine
over some concentrated nitric acid in a test-tube, holding the tube
inclined while pouring so that the liquids do not mix. At the zone of
contact a series of violet, green-blue and red colored rings develop in
the presence of bile. The blue-green ring is characteristic. Indoxyl
and scatoxyl also produce colors in this test. Urobilin does not.

Experiment 262.—The bile pigments can also be detected by the
Huppert-Cole reaction described in experiment 153.

Experiment 263. Examination for other pigments——(a) Uro-
bilin. To 5 ¢.c. urine add an equal*volume of a 10 per cent. solution of
zine acetate in absolute alcohol and filter. The filtrate should possess a
reddish-green fluorescence. In case this gives a negative result shake
10 ¢.¢c. urine with 5 ¢.c. warm amyl alcohol after acidifying with hydro-
ehlorie acid. Remove the amyl-alcohol layer, filter if necessary, render
it alkaline with NH,OH and add 1% volume of the 10 per cent. alcoholic
PRACTICAL WORK AND METHODS 1077

zine-acetate solution. Shake. Green fluorescence if urobilin present.

Experiment 264. Bile pigments (Gmelin-Rosenbach reaction).—
Saturate a piece of filter paper with urine by filtering much urine
through it. Allow it to dry partially and drop thereon a few
drops of concentrated nitric acid. Around each drop of nitric acid
soon appears a series of concentric rings—yellow-red, red, violet, blue
and green.

Experiment 265. Huppert-Nakayama reaction for bile pigments.—
To 5 c.c. urine in a centrifuge tube add 5 cc. 5 per cent. BaCl, solution,
mix and centrifuge. Pour off the supernatant clear solution, add to
the precipitate 2 ¢.c. of the special ferric-chloride reagent (4.0 grams
FeCl, dissolved in 990 c.c. 95 per cent. alcohol + 10 ¢e. concentrated
HCl) and heat to boiling. The solution becomes green to bluish green
and after adding a drop of concentrated nitrie acid (yellow) the solu-
tion becomes violet and red. This reaction is stated to detect 1 part
bilirubin in 1,000,000 parts urine.

Blood in Urine.

Experiment 266. Benzidine reaction—To 1 eg. (a pinch) of
benzidine in a dry test-tube add about 3 ec. of glacial acetic acid and
mix. After a moment add an equal volume of hydrogen peroxide.
Divide into two portions and to one add 1 ¢.c. of urine. The other serves
as a control. If there is any blood in the urine, or the decomposition
products of hemoglobin the benzidine tube will be colored greenish
blue.

Experiment 267. Guaiac reaction.—To 5 c.c. slightly acid urine
add about 0.5 c.c. of a freshly prepared tincture of guaiac, mix and then
add about 1 «ec. old turpentine. Shake thoroughly and set aside for
afew minutes. If a green to blue color has not developed, add a few c.c.
of alcohol and again shake gently. In case the result is still negative
one can conclude that hemoglobin is absent from the urine. A blank
test, using water in place of the urine, should be made also. The
turpentine can be prepared by exposing it in a flat dish for some
hours to direct sunlight. Or in making the test add a drop of
hydrogen peroxide. It is necessary that the terpentine be partially
oxidized.

Experiment 268. Teichmann hemin reaction.—In case 267 above
is positive it is best to confirm it by this test. To 10 to 15 cc. urine add
10 per cent. NaOH to make it strongly alkaline, heat to boiling and set
aside to cool. Filter off the precipitate of phosphates, ete. Wash with
distilled water and finally transfer the precipitate to a microscopic slide.
add a very small crystal of NaCl and very carefuliy evaporate to dry-
1078 PHYSIOLOGICAL CHEMISTRY

ness over a low flame. Cover the dry material with a cover glass and
allow to flow under it a drop or two of glacial acetic acid. Now again
warm gently over a free flame until the acetic acid begins to boil gently.
Allow to cool and examine under the microscope for the reddish-brown
hemin crystals. Page 1003.

V.Adoman.

Fie. 99.—Uric acid crystals deposited from urine.

 

Experiment 269. Spectroscopic examination—Examine by a
direct-vision spectroscope for oxyhemoglobin and methemoglobin. Page
493.

Exercis—E XLVIII. QUANTITATIVE EXAMINATION OF THE URINE.

For the quantitative experiments all the urine for 24 hours must be
carefully collected. Two kinds of urine are to be examined: (a) while
the student is on a low protein diet and (b) while he is on a high protein
diet. ‘

In this part of the work students may work in pairs, provided
they alternate in making the various determinations. The urine must,
PRACTICAL WORK AND METHODS 1079

however, be collected in both periods from the same individual and
during 24 consecutive hours each time. The same amount and kind of
exercise must be taken during both diet periods. Bottles provided for
the urine are to be properly cleaned and rinsed with a saturated alco-
holic solution of thymol before taking from the laboratory. During the
first period the subject is to take a low protein diet for two days. On
the second day of this period the urine voided before breakfast is to be
discarded, but all passed during the next 24 hours is to be collected.
Other hours may be more convenient, but consecutive 24 hours must be
taken each tvme. Also have the urine as fresh as possible for analysis.
Uric acid is to be determined first, as it settles out on standing; then
creatinine and then ammonia. The thymolized urine must be kept in the
refrigerator. The uric acid should be determined within the first 24
hours after collection. In the same way collect a second sample of urine
two weeks later on the second day of a two-days’ diet on high protein.
If possible weigh the food taken and from the record obtained calculate
the caloric value in both diets. Submit your proposed diet to an in-
structor before beginning thereon.
The following determinations are to be made in each of the two
samples of urine.
1. Volume, specific gravity, odor, color, transparency and any sedi-
* ments.
Uric acid. By experiment 288.
Creatinine. By experiment 283.
Ammonia. By experiment 282.
Total nitrogen. By experiment 273 or 275,
Creatine. By experiment 286.
Urea. By experiments 277 and 280.
Total acidity. By experiment 293.
9. Sulphates and total sulphur. By experiments 299 and 300.
10. Chlorides. By experiment 301.
11. Phosphates. By experiment 304.
Experiment 270. Volume.—Determine the volume of the well-
mixed 24-hour sample by means of a 1,000 or 2,000 ¢c.c. cylinder.
Experiment 271. Specific gravity—Determine the specific gravity
by means of a urinometer. Note the temperature and for every 3° C.
over the temperature recorded on the urinometer add 0.001 to the
observed specific gravity. For every 3° below subtract 0.001 from the
observed specific gravity. It is better, however, to cool the urine to
the temperature at which the urinometer is to be employed and then
determine the specific gravity.
Experiment 272. Odor, color and transparency.—Note these care-
fully and interpret your observations.

VY OF: G8. INS
1080 PHYSIOLOGICAL CHEMISTRY

TOTAL NITROGEN.

Experiment 273. Total nitrogen by the Gunning-Arnold modifica-
tion of the Kjeldahl process.—Carefully measure into a long-necked
800-1,000 ¢.c. Kjeldahl flask 5 ¢.c. of urine by means of a 5 c.c. pipette.
With a fine jet of water wash any urine in the neck of the flask down
into it. Use as little water as possible. Add 5 grams potassium sulphate,
or 10 grams sodium sulphate, 20 c.c. pure concentrated H,SO, (Sp.
G. 1.84) and 10 c.c. of the HgSO,+CuS0O, solution (containing 1 gram
HgSO, and 1 gram CuSO, per 10 c.c.).

 

 

 

 

 

Fic. 100.
Fie. 100.—Arrangement for rapid distillation of ammonia. (After Folin.)

Heat on the digestion shelf until the water has been removed, then
add 175 grams more of potassium sulphate or 30 grams Na,SO,10H,0,
and continue the digestion for 24% to 3 hours. To have the digestion
complete, the mixture must, however, have been boiling for at least 2%
hours and the hot solution left must not be colored yellow, but simply
green, due to the CuSO,. After the digestion is complete, remove from
the digestion shelf and, just as the contents of the flask begin to crystal-
lize, gradually add, while agitating, 250 ¢.c. distilled water; stopper
loosely and set aside until it is to be distilled as described in experi-
ment 73.

Experiment 274. Simplified macro Kjeldahl method for urine.
(Folin and Wright, J. B. C., 38, 1919, PP. 461- noe Takes only 20 to
25 minutes for determination,
PRACTICAL WORK AND METHODS 1081

Method. 5 ¢.c. undiluted urine are measured by volumetric pipette
into a 300 cc. Pyrex Kjeldahl flask, 5 ¢.c. of phosphoric sulphuric
acid mixture’ are added and 2 ec. of 10 per cent. FeCl, solution
and 4 to 6 small pebbles to prevent bumping. Boil in hood over a
microburner. Top of burner not more than 1 em. from bottom of flask.
Boil vigorously. After 3 to 4 minutes flask becomes filled with dense
white fumes. When this happens cover the mouth of the flask with a
small watch glass and continue vigorous heating for 2 minutes. Then
turn flame very low and boil gently 2 minutes more, making total of
boiling 4 minutes from appearance of fumes. Remove flame and cool

Air.

 

 

 

 

 

 

 

 

 

 

 

t
a 7
1
Lt
wy, .
Fre. 101. Fie. 102.

Fig. 101.—Adapter to be placed in the top of the Kjeldahl digestion flask or large test-
tube to be connected with the suction for the removal of SO, or other harmful fumes

(Folin).
Fia. 102.—Apparatus set up for the aération of the ammonia into the sulphuric acid.

The test-tuve contains the Kjeldahl digestion made alkaline. The air is blown through
this and into the 10U ¢e.c. volumetric flask containing the sulphburic-acid solution. A per-
forated cork disk is placed part way down the tube to prevent foam going over (Folin).

for 4 to 5 minutes. After 4 but not more than 5 minutes add first 50 ¢.c.
of water, then 15 c.c. sat. NaOH solution and connect promptly (Figure
100) the flask by rubber stopper and ordinary glass tubing with receiver
containing 35 to 75 ce. of 0.1IN H,SO, together with water enough to
make total volume 150 c.c. and a drop or two of alizarin red. 300 ee.
Florence Pyrex flasks are best for receivers. As soon as connection is

made with the end of the tube below the surface of liquid apply flame

*To 50 c.c. 5 per cent. CuSO, add 300 cc. 85 per cent. H,PO, and 100 cc. con.
H,S0..
4

2
1082 PHYSIOLOGICAL CHEMISTRY

with full force but not directly under the center until acid and alkali
have had time to mix. After 4 to 5 minutes boiling remove stopper from
flask and then turn off the flame. Leave the lower part of the tube in
the flask. Cool flask under the tap, then titrate the excess acid with
0.JN NaOH.

This method not applicable to proteins, milk, ete., in which longer
heating is necessary. Sugar urines require about 6 minutes of heating.

Experiment 275. Determination of total nitrogen in urine. Micro-
chemical. (Folin and Farmer, Jour. Biol. Chem., XI, 1912, p. 493.)—

AIR

FROM ~~ &
WASHBOTTLE —— sucTION

[

 

 

 

= =

 

 

 

 

 

 

 

UO) UJ
Fig. 103.

ia. 108.—The same as Wig. 69, except arranged for suction in place of a blast
(Folin).

5 «.c. of urine are measured into a 50 ¢.c. measuring flask, if the specific
gravity of the urine is over 1.018, or into a 25 c.c. flask if the specific
‘gravity is less than 1.018 (the urine should be so diluted that 1 ee.
contains from 0.75-1.5 mgs. of N). Fill the flask to the mark with water
and invert a few times to secure a thorough mixing. 1 cc. of this
diluted urine is measured into a large test-tube of Jena glass (size 20
to 25 mm. by 200 mm.). To the urine in the test-tube add 1 cc. of
concentrated sulphuric acid, 1 gram of potassium sulphate, 1 drop of
5 per cent. CuSO, solution and a small, clean quartz pebble to prevent
bumping. Boil over a micro-burner for about 6 minutes: i.e., about 2
minutes after the mixture has become colorless. As soon as the water
is boiled off and white fumes begin to fill the tube cover the mouth

t
PRACTICAL WORK AND METHODS 1083

with a water crystal, turn the micro-burner down until the solution
just boils and boil at this rate until the end. Allow to cool about 3
minutes until the digestion mixture is beginning to become viscuous,
but it must not be allowed to solidify. Add about 6 c.c. of water at
first, a few drops at a time, then more rapidly so as io prevent the
mixture from solidifying. To the acid solution is then added an excess
of sodium hydrate (3 ¢.c. of saturated solution) and the NH, is aspirated
by means of a rapid air current (see Figure 102) into a 100 cc. measur-
ing flask containing about 20 c.c. of water and 2 cc. of N/10 H.S0,.
The air current used for driving off the ammonia should be rather

 

LAE | oe

Fre. 104.—Apparatus as set up in Folin’s laboratory, showing the small Kjeldahl
digestion flasks or test-tube at the right with the adapter above and micro-burner beneath.
Other solutions givimg off odors or irritating fumes are shown over the other burners.
The large bottle contains an alkaline solution, and the whole is connected with the water

pump at the left (Folin). i
moderate for the first 2 minutes, but thereafter for 8 minutes it should
be as rapid as the apparatus can stand. Compressed air is best used,
but in the absence of this an air current may be drawn through by
a pump. In that case the NH, is not received directly into the measur-
ing flask, since the mouth of the latter is too narrow to take a double-
bored stopper, but is received in a second test-tube and the contents of
this afterwards washed into a 100 ¢.c. measuring flask. (Figure 103.)
Nesslerizing. After 10 minutes disconnect; dilute the contents of
the flask to about 60 ¢.c. and dilute similarly the standard ammonium-.

 
1084 PHYSIOLOGICAL CHEMISTRY

sulphate solution, of which enough has been taken to contain 1 mg.
of N, to about the same volume in a second 100 c.c. volumetric flask.
Nesslerize both solutions as nearly as possible at the same time with
5 «ce. of Nessler’s reagent diluted immediately beforehand with about
25 «ec. of water (5 c.c. of Nessler’s reagent gives the maximum color
with 1 to 2 mgs. of ammonia and when diluted, as indicated, turbidity
is avoided). The color produced does not reach its maximum for about
half an hour, but the increase is small and is immaterial when both
are Nesslerized at the same time. Fill the two flasks at once to the
mark with distilled water and, after mixing each, determine the relative
intensities of color by a Dubose colorimeter. Set the standard at 20
and match the unknown with it by moving the prism up or down. The
calculation is easy. If the unknown is at 14 when the two have the
same tint in the colorimeter, the amount of nitrogen is 20/14X1 mg.
in the amount of urine taken. It is important in reading the colorimeter
to bring the two sides to equality of color, first from below and then
-from above, or vice versa, and to take the mean of several readings.
In the case cited 1 ¢.c. contains 1.43 mgs. N. If the urine was diluted
10, then each c.c. of the original urine contained 14.3 mgs. 100 ec.
of urine would contain 1.43 grams of N.

Preparation of the standard solution of ammonium sulphate. Am-
monium salts generally contain pyridine bases. To prepare pure
ammonium sulphate decompose a high-grade ammonium sulphate with
caustic soda and pass the ammonia by means of an air current into
pure sulphuric acid. Precipitate the salt so obtained by the addition
of alcohol, redissolve in water and again precipitate with alcohol and
finally dry in a desiccator over sulphuric acid.

9.4285 grams of ammonium sulphate are dissolved in water and the
volume made up to 1 liter (stock solution).

100 c.e. of the stock solution are diluted to form 1 liter. This is the
standard solution. 5 ¢.c. contain 1 mg. of nitrogen.

Nessler reagent. Dissolve 62.5 gm. KI in about 250 c.c. distilled
water. Set aside a few c.c. and add gradually to the larger part a cold
saturated soluton of mercuric chloride until a faint permanent precipi-
tate is produced (about 500 e.c. required). Add the reserve portion of
KI and then more HgCl, solution gradually until a slight permanent
precipitate is again formed. Dissolve 150 grams of solid KOH in 150 e.c.
distilled water, allow to cool and add gradually to the above solution
and make the volume finally to 1 liter with water. After settling, decant
the clear liquid into another bottle and keep in the dark. Reagent im-
proves on keeping.
PRACTICAL WORK AND METHODS 1085
UREA.

Quantitative determination of urea—The best method for the
quantitative determination of urea is probably the urease method. The
other methods which are given here are, however, good, particularly
when applied to human urine which has little or no allantoine. The
Benedict method determines not only the urea, but about 50-70 per cent.
of the allantoine. Allantoine is present in the urine of the lower animals
in larger amounts than in human urine, so that the urease method is to
be preferred in examining all urines. In human urines there is so little
allantome that the Benedict method may be used without appreciable
error.

Experiment 276. Estimation of urea by the Benedict method.—
Principle. The urea is hydrolyzed to ammonium carbonate by the acid

 

 

| aot! |
I My |

Fig. 105..—Copper baths designed by Koch for use in the Benedict method. They are
of copper, and Merauker is partially filled with paraffin. ‘The test-tubes, with the urine,
ete., go into the copper tubes, which they closely fill, but they do not come in contact
with the oil, Two such baths are in the laboratory; one kept at 140-150° and the other
at 165-170° C.

developed from KHSO, and ZnSO, by heating for a certain time at

definite temperatures. The solution is then made alkaline and distilled

as usual into a measured amount of sulphuric acid as in the Kjeldahl
process.

Method. Weigh off 3 grams KHSO, and 2 grams ZnSO, and transfer

to a large (1 inch) test-tube. Add 5 c.c. urine, a pinch of paraffin and

of pumice. Boil almost to dryness in the oil bath kept at 130-150° C.
1086 PHYSIOLOGICAL CHEMISTRY

Now transfer to another bath kept at 165-170° C. and heat thus for 1
hour. Allow to cool partly, then transfer the contents of the tube to
an 800 ¢.c. Kjeldahl flask, using hot distlled water and diluting to about
400 ¢.c. with cold distilled water. Add a small amount of granular zine
and 5 ce. of the 40: 100 NaOH solution. Distill into 50 ¢.c. n/10 H,SO,

 

 

 

 

 

 

 

 

 

 

 

 

Fig. 106.—Koch’s aération tube for use in Folin’s microchemical or macrochemical
method of aérating ammonia from a solution to acid. D is a section of a wooden base
containing, in the Chicago laboratory, 12 aération apparatus, as indicated. Only half
of the wooden base is shown in the cut. The other half is like this and faces it, and
the air inlet is a metal pipe provided with cocks running between the two halves. A is
the aération tube containing the alkaline solution to be aérated and filled to the mark
shown and covered with a little paraffin oil. C is a cylinder containing the acid, the
amount of which is known; B is a Folin aération tube. An upright metal rod carries
metal clips shown, but not lettered, which support the tubes.

as usual. Use congo red as indicator. Correct for the ammonia in the
urine and also make a blank test on your reagents and process. Caleu-
late as urea and urea N both in grams per 100 ce. urine, and grams
per 24 hours. 1 cc. n/10 H,SO, is equivalent to 0.0014 grams of N.
If x ec, of the n/10 H,SO, are neutralized by the ammonia from the
PRACTICAL WORK AND METHODS 1087

urea and ammonia combined, then 0.0014 + — ammonia N will be the

urea N in 5 «ec. of urine. The urea is Saths of the urea N.

Experiment 277. Quantitative determination of urea by the
urease method (Marshall; Van Slyke and Cullen).—Principle. The
urea is decomposed to ammonium carbonate by the action of the specific
enzyme, urease, found in the castor or jack bean. The dried urease is
prepared and put on the market but finely ground jack bean does as
well. See page 1014. This is in many ways the ideal method, for it
requires no heating, no addition of salt and the action of the enzyme
is specific, not affecting or hydrolyzing the other constituents of the
urine. The ammonia nitrogen must be separately determined, as in the
other methods, and the amount subtracted from the amount given by
this method. The ammonia set free by the urease is carried over by an
air stream into standard acid and determined either by titration, if
large amounts have been taken, or better by Nesslerizing if small
amounts of urine are used.

Experiment: Determination of urea in urine. Into each of two
clean absorption cylinders (Figure 106) (or large test-tubes) measure off
carefully by a burette 20 ec. N/10 H,SO,, then add enough distilled
water just to cover the bell of the absorption tube, B, when the same
is attached to the Koch ammonia aération tube, A (Figure 106). Hav-
ing these prepared, now very carefully measure off into the aération
tube, A, (or into a large test-tube previously washed with HNO, to
remove any toxic metals absorbed by the glass (Figure 102), 1 ¢.c. urine,
10 ce. water and 5 cc. of a 5 per cent. urease solution, or 1 gram jack
bean powder, mix well, add 5 «cc. paraffin oil and at once connect with
the absorption tube. Prepare another aération tube in the same way,
using 1 ¢.c. water instead of 1 cc. urine. After both tubes are con-
nected, at once aérate at a fair rate for 30 minutes. Next rapidly add
to each aération tube 5 grams dry Na,CO,, again connect and again
aérate vigorously (avoid foaming) for 1 hour. Titrate the acid solution
in the absorption cylinder or test-tube with N/10 NaOH, using congo red
as indicator and leaving the absorption tube in the cylinder. Allow for
the blank titration and calculate to urea nitrogen after correcting for the
ammonia nitrogen as estimated below. Calculate the per cent. of the
total nitrogen found as urea nitrogen and also the grams of urea
nitrogen and urea as such in the 24-hour sample. For calculation, see
experiment 276.

Experiment 278. Determination of urea by urease.—The following
deseription of the method has been taken from the circular sent out
with the commercial preparation ‘‘ arleo-urease ’’ and may be followed
in the absence of the aération device just described:
1088 PHYSIOLOGICAL CHEMISTRY

“ Standardization of the enzyme. One-tenth gram of arlco-urease
dissolved in 1 ¢.c. of water and added to 5 c.c. of 1 per cent. solution of
pure urea will hydrolyze .0168 gram of urea, yielding .00953 gram of
ammonia, which is equivalent to 28 ¢.c. of fiftieth normal acid, in 15
minutes at 25° C., or in correspondingly less time as the temperature
approaches 50° ©. as a maximum. Therefore, from these standardiza-
tion data one can calculate the amount of arlco-urease required to
decompose a given quantity of urea in a solution where the analysis
is desired.

“ Determination of urea (in urine). Dilute the urine ten times.
Take 5 cc. of the diluted urine for analysis. Add to this, in a 100 cc.
test-tube a few drops of caprylic aleohol or 1 ¢.c. of amyl alcohol or
kerosene to prevent subsequent foaming; then add 1 ec. of a 15 per
cent. solution of arleo-urease. Close with stopper, and allow to stand
until the reaction is complete. Allow 15 minutes at 20° C. (68° F.) or
10 minutes at 25° C. (79° F.); 3 minutes suffice at 50° C. (122° F.).
The tube is connected through its stopper with a second tube containing
25 c.c. of fiftieth normal hydrochloric acid. Aérate the digestion mix-
ture for half a minute after the reaction is complete and before the
tube is opened, in order to prevent loss of any possible ammonia fumes
in upper part of tube. Open tube, add dry postassium carbonate (4 to 5
grams), close quickly and aérate for about 15 minutes, as in Folin’s
micro-method for ammonia determination, or until all ammonia gas has
been carried over into the standard acid. Then to complete the analysis,
titrate the acid solution to neutrality with fiftieth normal sodium hydrox-
ide, using as indicator 1 drop of a 1 per cent. sodium alizarin sulphonate
solution. The number of c.c. of fiftieth normal acid neutralized by the
ammonia from the urea is multiplied by .12 to give the per cent. urea
in the urine, or by .056 to give the per cent. urea nitrogen. Under ordi-
nary conditions, the total time for a complete analysis need not exceed
30 minutes, and it can be reduced considerably below this limit.

‘“ (In blood). Urease is particularly valuable in permitting a sim-
ple and accurate determination of urea in the blood. Five e.c. of freshly-
drawn blood are mixed in a 100 c.c. test-tube with 1 @c. of 5 per cent.
potassium-citrate solution to prevent clotting, a few drops of caprylic
alcohol to prevent foaming during subsequent aération, and 1 c.c. of a
10 per cent. solution of arlco-urease. The remainder of the procedure
is the same as described for urine, except that only 10 ec. of fiftieth
normal acid need ordinarily be used. In this case the number of c.c.
of fiftieth normal acid neutralized is multiplied by .012 to give the per
cent. of urea, or by .0056 to give the per cent. of urea nitrogen.”’

Experiment 279. Quantitative determination of urea by Folin’s
microchemical method (Jour. Biol. Chem., XI, 1912, p. 513).—Princi-
PRACTICAL WORK AND METHODS _ 1089

ple. The urea is decomposed, forming ammonia, by heating with solid
potassium acetate and a little acetic acid at 153-160° C. The ammonia
is then liberated from the ammonium acetate by the addition of sodium
hydrate and carried over by a strong air current into a measured amount
of standard acid in a volumetric flask. The contents of the flask are
filled up to the mark and the amount of ammonia determined by
Nesslerizing as in the total nitrogen method.

Procedure. The urine is diluted so that 1 ¢.c. contains 0.75 to 1.5
mgs. of urea nitrogen. Dilutions of 1 in 20, 1 in 10 or rarely 1 in 5
are usually adequate for this purpose. One cubic centimeter of the
diluted urine is then transferred by means of an accurate pipette
(Ostwald) to a large, dry Jena-glass test-tube (200 mm. by 20 mm.)
previously charged with 7 grams of dry potassium acetate (free from
lumps), 1 cc. of 50 per cent. acetic acid, a small sand pebble or better
a little powdered zine (not zine dust) to prevent bumping during the
boiling, and a temperature indicator. Close test-tube by means of a
rubber stopper carrying an empty, narrow calcium-chloride tube with-
out a bulb (size 25 em. by 1.5 em.) to act as a condenser. Suspend the
tube by means of a burette clamp so that it can be raised or lowered
and heat over a micro-burner. As soon as the acetate dissolves and the
mixture begins to boil, usually in about 2 minutes, the indicator begins
to melt, showing that the desired temperature (153-160°) has been
reached. Continue gentle boiling for 10 minutes, when decomposition
of the urea is complete. Remove apparatus from the flame and dilute
the contents of the tube with 5 e.c. of water added by a pipette partly
through the CaCl, tube so as to wash off the bottom of the rubber stopper
and the sides of the tube. Use not more than 5 «ce. for this purpose.
Add 2 ce. of saturated NaOH or K,CO, solution and drive the liberated
NH, over into a 100 c.c. volumetric flask containing about 35 e.c. water
and 2 ¢.c. N/10 sulphuric acid, by means of an air current as in the total
nitrogen determination. (See Figure 102.) 10 minutes is sufficient if
a rapid air current is used. Determine the N in the flask after making
up to 100 ee. by the Nessler and colorimetric method as described under
experiment 275.

This method determines both urea and ammonia nitrogen. ‘This
method may be used when urease is not available. Neither creatinine
nor hippuric acid gives a trace of ammonia. Allantoine may give off
about % its N in the presence of zinc, but in its absence it behaves like
urea, provided its quantity does not exceed 0.5 mgs. of allantoine N.
Urie acid may give a little N, but it is quantitatively imperceptible,
in the presence of the urea.

It is important to have potassium acetate which is dry and free from
ammonia. This is made by J. T. Baker Chemical Company, Phillips-
1090 PHYSIOLOGICAL CHEMISTRY

burg, N. J. The best German product is generally dry enough, but it
should be tested for ammonia. The American acetate, except that men-
tioned, is apt to have too much water in it. It may be dried by placing
about a pound of it in a large porcelain dish and letting it stand on a
warm plate (at about 115°) for about 24.hours. The plate must not be
too hot or acetic acid will be driven off.

Temperature indicator. This consists of a small amount of HgICl
sealed into a small glass bulb not over 1 mm. in diameter. They may
be obtained from Eimer and Amend in New York. This salt is bright
red at ordinary temperatures. At 118° it turns lemon yellow and at
155° melts to a clear dark red liquid. It solidifies on cooling at 148° C.
and slowly takes again its red color in about 24 hours. The acetate
begins to cake and solidify at 160° C. So this temperature must be
avoided. The indicator is made by heating in a dry state intimately

 

 

 

 

 

 

 

 

   

Fic. 107.—Arrangement for the determination of urea by the hypobromite method.

mixed molecular proportions of mercuric chloride and iodide at 150-
160° for 6 to 8 hours. It is then cooled, powdered and kept dry until
sealed up in bulbs for use. The advantage of this method is that a ther-
mometer need not be introduced.

It is important in making the determniation that no bumping or
spattering occur. The test-tube must be dry. If too much heat is
applied the acetate cakes at the bottom, if too little it cakes at the top.
PRACTICAL WORK AND METHODS 1091

The bottom of the test-tube should be some distance above the micro-
flame, which should be about 0.5 em. long. It should boil gently with-
out caking. Bottomless beakers make excellent wind shields for these
micro-flames.

Computation. If the colorimeter tube A, the standard, has been set
at 20 and the other tube B reads 15 when both colors match, then the
amount of nitrogen in the 1 ¢.c. of diluted urine taken would be 20/15
mg. If the urine was diluted 10 times, the amount of urea N in 1 ec.
of undiluted urine would be 200/15 mgs. And in 100 cc. of urine it
would be 100 times this amount.

  

weap

  

   
  

    

reid

  

ul

 
 

Oo eee [TT

Fre. 108.—Doremus ureometer.

Experiment 280. Estimation of urea by the hypobromite method
(Krogh, Zeit. f. physiol. Chem., LXXXIV, 1913).—Principle. The
urea is oxidized by bromine, or more properly by sodium hypobromite
with the formation of carbon dioxide and nitrogen gas. The carbon
dioxide is absorbed by strong alkali and the nitrogen collected and
measured volumetrically. The reaction is as follows:

CO(NH,), + 3NaOBr = 3NaBr + CO, + 2H,0 +N,

Even with pure urea and with temperature and barometric pressure cor-
rection this method gives inaccurate results, but in the urine as ordi-
1092 PHYSIOLOGICAL CHEMISTRY

narily tried it gives very inaccurate results. Not only urea but also
ammonia salts and other nitrogen constituents of the urine give off their
N so that the amount of N found is generally nearer the total N than
it is the urea N. It gives, however, a rapid, not too difficult, approxi-
mate determination of the total N of the urine which can be used
clinically. Some CO and NO gases are formed also.

Process. Use the apparatus figured in cut 107. Various forms of
ureometers have also been proposed, of which one is figured in Figure
108. They are very inaccurate, but convenient. A 50 ¢.c. burette (a) is
supported in a clamp attached to a lampstand and immersed in a tall
measuring cylinder filled with water. The upper end of the burette
carries a one-hole rubber stopper through which passes a T tube, the
free end of the tube being closed by a rubber tubing clamped by a screw
clamp (e). The other arm of the T is connected by a piece of rubber
tubing to a glass tube which goes through the one-hole rubber stopper
in the neck of the wide-mouth bottle C. The bottle C is supported in a
erystallizing dish resting on a ring of the lampstand. Water is put in
the crystallizing dish so as nearly to cover the bottle. A short glass vial
(d) of about 10 cc. capacity is placed in the bottle.

Place 25 ce. of hypobromite solution in the bottle C. Into the vial
place 5 cc. of the urine. With forceps carefully place the vial upright
in the hypobromite bottle so that the urine does not come in contact
with the solution. While the screw clamp (e) is open place the stopper
in the bottle and the bottle in the jar of water and leave for a few
moments to acquire the temperature of the water. Adjust the burette
so that the level of the water in the burette is a little below the begin-
ning of the scale on the burette, screw the screw clamp tight shut and
read the burette. The level of the water should be the same within and
without the burette when the reading is made. Now raise the bottle
and capsize the vial and mix the hypobromite and urine thoroughly.
Gas is given off. Place the bottle back in the water and after three
minutes again adjust the burette by raising it so that the level of the
water within and without the burette is again the same. Read the
burette carefully and subtract from this the former reading. The result
is the c.c. of N, gas which have been evolved.

Calculation. The reading is corrected for temperature and pressure
and the pressure of aqueous vapor as follows: If v is the actual reading,
if t is the temperature at which the reading was made, p’ the pressure
due to aqueous vapor and p the barometric pressure, then at 0° and 760
mm. barometric pressure the real volume of N gas evolved measured at
0° and 760 mm. is v’.

, VX 273 x (p—p’)
~~ (273 +t) x 760
PRACTICAL WORK AND METHODS 1093

' 1 gram of urea evolves 357 c.c. of N, measured under standard condi-
tions. v'/357 represents, then, the amount of urea in 5 c.c. of urine.
And 20 v’/357, the grams of urea in 100 ¢.c. of urine.

Hypobromite solution. Add slowly 25 ¢.c. of bromine to the cooled
solution of 100 grams NaOH in 250 c.c. of water. Cool under the tap
while adding the bromine. The solution must be made shortly before
use, since on standing a part is converted to bromate. The reaction is
as follows:

2Na0H + Br = 2NaOBr
3NaOBr — 2NaBr + NaBrO iE

AMMONIA.

Experiment 281. Estimation of ammonia nitrogen by the Folin
macrochemical method.—Principle. The ammonia of the urine is freed
by making the urine alkaline by the addition of Na,CO,. A strong
current of air is then blown or drawn through the apparatus and carries
the ammonia over into a second tube which contains a measured amount
of N/10 H,SO,.

Procedure. Set up the apparatus as shown in Figure 103 or 106.
Introduce 20 e.c. N/10 H,SO, into the absorption cylinder and add
enough distilled water to just cover the bell of the absorption tube when
the same is attached to the ammonia aération tube. Carefully measure
off by means of a pipette 25 ¢.c. urine into the aération tube, also 5 e.c.
paraffin oil and 5 grams dry Na,CO,;. At once connect with the absorp-
tion tube and aérate vigorously for 142 hours. Titrate in the absorption
cylinder as indicated above, using congo red as indicator. Calculate the
total grams of nitrogen found as ammonia, also calculate what per cent. of
the total nitrogen this represents, as well as the total grams of ammonia
found in the 24-hour sample. For calculation see experiment 276.

Experiment 282. Microchemical method for the estimation of
ammonia in urine (Folin and Macullum, Jour. Biol. Chem., XI, 1912,
p. 523).—Into a large 75 c¢.c. test-tube measure by means of an accurate
Pipette 1 to 5 ec. of urine. The volume taken should give 0.75 to 1.5
mgs. ammonia nitrogen. With normal urines 2 e.c. will most often give
the amount. With very dilute urines 5 ¢.c. will be necessary, and with
diabetic urine with much ammonia 1 ¢.c. may be too much and the
urine must be diluted. Add to the urine in the tube enough water to
make the total about 5 ¢.c., then a few drops of a solution containing
10 per cent. of K,CO, and 15 per cent. potassium oxalate, and a few
drops of keroesne or heavy, crude machine oil to prevent foaming.
Introduce a tube through a cork as in the determination of the total
nitrogen (Figure 103) and pass a strong current of air through the
1094 PHYSIOLOGICAL CHEMISTRY

mixture for 10 minutes, or as long as it is necessary to drive off all the
NH,, and collect the NH, in a 100 ¢.c. measuring flask containing about
20 «ec. of water and 2 ec. N/10 H,SO,. Nesslerize as in the total N
method (page 1083) and compare the color with 1 mg. of nitrogen ob-
tained from a standard ammonium-sulphate solution and similarly
Nesslerized. This method is very convenient and rapid. The computa-
tion is made as in the other colorimetric determinations. (Experiment
275.)

CREATININE AND CREATINE.

Experiment 283. Estimation of creatinine by the Folin colori-
metric method.—The significance and chemistry of creatinine is dis-
cussed on page 709.

Principle. The principle of the method consists in the reduction of
picrie acid, C,H,(NO,),0H, to picramic acid, C,H,(NO,),(NH,) OH.
Picramic acid in alkaline solution has a reddish-brown color and its
amount is determined colorimetrically by comparing it in a Duboseq
colorimeter with a standard of N/2 potassium bichromate solution, or
with a standard formed by reducing picrice acid by a known amount of
creatinine.

Process. Before making the estimation, learn to use the Duboseq
colorimeter and compare two samples of N/2 potassium-bichromate solu-
tion with each other, setting the one at a depth of 8 mm. and adjusting
the other until the two fields appear the same in intensity. Then read
the seale; the variation should not be more than 0.2 mm. Continue this
drill until you have accustomed your eyes to the instrument.

Into a 500 c.c. volumetric flask introduce 10 ¢.c. urine, measured by
a pipette. Now add by means of a pipette 15 ¢.c. saturated (aqueous)
picric-acid solution, mix well and then add 5 c.c. of a clear 10 per cent.
NaOH solution. At once mix well, note the time and allow to stand at
room temperature for exactly 5 minutes. In the meantime prepare the
Duboseq colorimeter with a standard tube of N/2 potassium-bichromate
solution and set the scale at 8 mm. After standing the 5 minutes, add
enough water to make up to 500 ¢.c., mix well and compare the solution
with the bichromate solution, taking at least three readings, but keeping
the standard set at 8 mm. Match the colors both when approaching the
correct point from above as well as from below. Slightly different values
will be obtained. The three readings should not vary more than 0.2
to 0.3 mm. from each other and the color of the creatinine solution should
be of such intensity that the reading on the colorimeter scale will be
somewhere between 6 and 12 mm. If the reading is not between these
extremes, it is necessary to repeat the test with more or less urine,
PRACTICAL WORK AND METHODS 1095

depending on whether the color is too weak or too intense. Be sure to
avoid having air bubbles under the surface of the movable glass prisms.

It has been determined by experiment that 10 mg. of creatinine
treated as above and diluted to 500 c.c. gives a colored solution of such
intensity that it requires a layer 8.1 mm. deep to give the same intensity
as the 8 mm. layer of N/2 potassium bichromate. A layer of 8.1 mm.
is then equivalent to 10 mg. creatinine in the orignial solution under the
conditions given here, or the constant 8.110=—81 holds for all dilu-
tions of creatinine within the limits stated. If the reading X is known,
then 81/X=megs. of creatinine in the original urine taken. This method
is very good for normal urines, but in certain abnormal urines where
acetoacetic acid is present this may give low results. Dextrose up to 5
per cent. is said not to interfere (Greenwald, Jour. Biol. Chem., XIV,
1913, p. 87). Calculate the grams of creatinine found in the total 24-
hours’ sample, also the grams of nitrogen corresponding thereto and
the per cent. of this in terms of total nitrogen.

Note. The color involves the dissociation of the salt of picramic
acid and of the bichromate. These do not change their color in the
same way with the temperature. The standard bichromate solution has
been made to compare with the picric acid-creatinine mixture at a tem-
perature of about 20° C. The reading should be made at this tempera-
ture. At higher or lower temperatures the bichromate solution no
longer corresponds to the 10 mg. of creatinine ‘when set at 8.1. Each
colorimeter, also, should be standardized to make sure that when set at
8.1 it really matches the bichromate solution and a standard creatinine-
picric acid solution of the strength indicated. To avoid these difficulties
Folin has recently suggested that for comparison a standard solution of
creatinine should be treated as is the urine and compared in the
colorimeter with it. Variations in temperature will not then be im-
portant. Probably picramic acid could itself be used to advantage.
Bichromate has the great advantage of convenience and stability. (See
next experiment. )

Bichromate solution. 24.55 grams of pure potassium bichromate dis-
solved in water and made up to 1,000 e.c.

Experiment 284. Microchemical colorimetric method for cre-
atinine using creatinine as a standard (Folin).—Principle. If ere-
atinine is available to make a standard it is much better to use the
following method. In case creatinine is not available the bichromate
method described in 283 may, however, be used.

Solutions needed. 1. Standard creatinine solution. Dissolve 1.61
gram of creatinine zine chloride, or 1 gram pure creatinine, in 1 liter
of N/10 HCl. This solution keeps well. It contains 1 mg. creatinine
per c.c.
1096 PHYSIOLOGICAL CHEMISTRY

Method. Measure by a 1 c.c. Ostwald pipette 1 c.c. or 2 ¢.c. of urine
(for most urines the former) into a 100 c.c. volumetric flask. To an-
other 100 c.c. flask measure 1 c.c. of the standard creatinine solution for
a standard. To each flask add 20 ¢.c. saturated picric acid solution, and
then 1.5 cc. clear 10 per cent. NaOH by a burette to each. Mix and let
stand 8 minutes. Dilute to the mark with water and mix. Compare
the standard against itself in the colorimeter, then with the standard
set at 20 mm. compare with it the unknown. 20 divided by the reading
of the unknown gives the quantity of creatinine in mgs. in the amount
of urine taken. The unknown must not deviate from the standard more
than 5 mm. above or below. If it does repeat the determination using
more or less urine as required.

Experiment 285. Preparation of pure creatinine for standard
creatinine solution. (Benedict, Jour. Biol. Chem., XVIII, 1914, p. 183.)
—Add to 10 liters fresh urine 180 gr. picric acid dissolved in 450 e.e.
boiling aleohol. Stir. Allow to stand over night. Decant, filter with
suction and wash precipitate with cold, saturated picric acid and suck
dry. To each 100 g. of creatinine picrate add 60 ¢.c. con. HCl; stir to
thin paste in mortar for from 3 to 5 minutes, filter with suction through
hardened filter paper and wash twice with suction with enough H,O to
cover precipitate. Filtrate contains creatinine. Transfer to large flask,
neutralize with excess of MgO (solid) added little by little while cooling
flask under tap. Filter with suction and wash residue twice with water.
Add a few cc. glacial acetic acid to filtrate to strong acid reaction,
dilute with 4 vols. 95 per cent. alcohol and after 15 minutes filter with
suction. Treat final filtrate with 30 to 40 cc. of 30 per cent. ZnCl,
solution in alcohol, stir and allow to stand over night for creatinine
zine chloride to crystallize out in a cool place. Preferably in
refrigerator. Collect the creatinine ZnCl, on a Buchner filter, wash
once with H,O, then thoroughly with 50 per cent. alcohol,
finally with 95 per cent. aleohol and dry. Yield is 90-95 per cent.
of the total creatinine. To obtain creatinine from this compound
recrystallize by treating 10 grams with 100 cc. water and 60 ec. N
H,SO, and heat to boiling until clear solution obtained. Add about
4 gms. purified animal charcoal, boil one minute longer and filter with
suction through small Buchner, pouring back until colorless. Wash
charcoal with hot water and treat filtrate while hot with 3 ¢.c. con.
ZnCl, solution and 7 gms. potassium acetate dissolved in a little water.
After 10 minutes dilute to twice its volume with 95 per cent. alcohol and
allow to stand in ice box or cool place for several hours to crystallize.
Decant supernatant liquid. Wash crystals of creatinine ZnCl, free
from potassium acetate by stirring with twice their weight of water,
filter, and wash with cold water and then 95 per cent. alcohol. Yield
should be 8 to 9 gms, snow white, crystalline ZnCl,-creatinine.
PRACTICAL WORK AND METHODS 1097

To obtain creatinine: Powder the white crystals just obtained, trans-
fer to a clean dry flask and treat with 7 times its weight by volume of
con. NH,OH. Warm the mixture and shake gently until a clear solution
is obtained, taking care to drive off as little NH, as possible. Stopper
flask, allow to cool and place in ice box for 1 to 2 hours. Pure ereatinine
erystallizes out; yield, 60-80 per cent. Decant through a dry Buchner
filter of small size, filter quickly and wash once with distilled water
cooled to low temperature. If product is yellow, it may be recrystallized
by dissolving in 5 vols. warm, concentrated NH,OH and recooling. It
may finally be recrystallized from a small quantity of boiling alcohol.
The product is pure and may be used for a standard in the various
determinations of creatinine.

Experiment 286. Estimation of creatine by the Folin-Benedict
method.—Principle. The preformed creatinine is separately deter-
mined in the manner just mentioned. Then the urine is heated with
acid in an autoclave which converts all the creatine to creatinine. The
creatinine is now redetermined. This figure gives the combined creatine
and creatinine. The difference between this and the preformed cre-
atinine is considered to represent the creatine present.

Process. Introduce 20 c.c. of urine, or less if the urine is concen-
trated and contains much creatinine, into a 50 e.c. volumetric flask, add
an equal volume of N HCl and heat in an autoclave at 117-120° C. for
¥% hour. Dilute the cool mixture to 50 c.c., mix well and introduce by
means of a pipette 25 c.c. of the mixture into a 500 ¢.c. volumetric flask
and proceed with the estimation of the creatinine as described in experi-
ment 283, or better by 284. Calculate the amount of creatine present.

In place of the HCl picric acid may itself be used to convert the
creatine to creatinine. Folin recommends the following quick procedure:
Measure 1 ec. urine into a 100 ¢.c. volumetric flask, add 20 c.c. sat-
urated picriec acid solution, cover the flask mouth with tinfoil and heat
in autoclave to 115°-120° C. for 20 minutes. Cool autoclave before open-
ing. Remove flask, cool to room temperature, prepare a standard cre-
atinine flask as in experiment 284 and add 1.5 c.c. 10 per cent. NaOH at
the same time to each and read in colorimeter after standing 10 minutes.

Caution. By this method the creatine is determined indirectly. The
difference between the two readings is supposed to represent creatine.
It has been found that when acetoacetic acid is present in the urine the
first, or creatinine, determination is low. By heating with acid the
acetoacetic acid is destroyed so that the second reading of creatinine is
now higher. The difference would be counted as creatine, whereas it is
due to another substance. Other oxidizing substances which are de-
stroyed by acid may behave like aceoacetic acid. Reducing substances
will increase the reduction of picric acid and make the creatinine appear
1098 PHYSIOLOGICAL CLEMISTRY

high. The interpretation of results must be made with these circum-
stances in mind.

URIC ACID.

Experiment 287. Quantitative estimation of uric acid by the
Folin-Shaffer method.—Principle. The uric acid is precipitated from
the urine as ammonium urate and the amount in the precipitate deter-
mined by titration with a standard potassium-permanganate solution in
the presence of acid. Phosphates and some organic matter are first
precipitated by uranium acetate and (NH,),SO,.

Process. Into a 350 ¢.c. beaker introduce 100 cc. urine (more if
the specific gravity is less than 1.020) and while stirring add by means
of a pipette 25 ¢.c. Folin-Schaffer reagent (500 grams ‘ammonium sul-
phate and 5 grams uranium acetate in 710 cc. of an 0.84 per cent.
acetic-acid solution). Allow to stand until the precipitate has separated
fairly well. Then filter through a dry filter paper into a dry beaker
or flask. Now measure off 100 c.c. of the filtrate (or more if the urine is
dilute) by means of a pipette into a 300 cc. beaker. To this solution
then add while stirring 5 ¢.c. of concentrated ammonium hydroxide and
set aside for 24 hours. Filter through a well-washed, properly prepared
asbestos filter in a Hirsch funnel or Gooch crucible and wash rapidly
with a 10 per cent. ammonium-sulphate solution until the filtrate is
free from chlorides. Now by means of 100 c.c. hot water, transfer the
asbestos and precipitate back into the 350 ¢.c. beaker. After cooling
add gradually while stirring 15 ¢.c. concentrated H,SO, and titrate at
once with n/20 KMnO, solution from a glass stopcock burette.

Titrate to the point where, on.vigorously stirring, the entire solution
remains faintly pink for an instant after adding two drops of the
standard permanganate solution. The end point is somewhat difficult
to determine.

Each c.e. of the n/20 KMn0O, is equivalent to 0.00375 gram uric acid.
Caleulate the grams of uric acid in the original urine sample taken,
adding 0.003 gram to correct for the solubility of ammonium urate in
100 cc. Calculate the total grams of uric acid, total grams of uric
acid nitrogen and the per cent. of the total nitrogen found as uric acid
nitrogen in the 24-hours’ sample.

N/20 Permanganate solution. 1.581 grams of pure potassium per-
manganate are dissolved in pure water and made up to 1,000 cc. Keep
protected from light. Standardization page 893.

Experiment 288. Quantitative microchemical determination of
uric acid. (Folin and Wu, Jour. Biol. Chem., 38, 1919.) This is the
PRACTICAL WORK AND METHODS 1099

most convenient and rapid of the methods of determination of uric acid.
It is a modification of the methods of Folin and Macallum: Jour. Biol.
Chem., 18, 1913, p. 863; and Folin and Denis: Jour. Biol. Chem., 14,
1913, p. 97. Benedict modification.

Principle. The uric acid is precipitated from the urine by silver
lactate-lactic acid mixture, and the uric acid determined colorimetrically
by the blue color it develops in phosphotungstie acid. A standard uric
acid solution is compared with the original.

Solutions needed. The same as in uric acid in blood, page 1019.

Apparatus:

1. Duboseq or other colorimeter.

2. Centrifuge.

Method. Measure with an Ostwald pipette 1 to 3 ¢c.c. of urine, de-
pending on the concentration, into a centrifuge tube. Add water to a
volume of about 6 e.c. Add 5 e.c. of the silver lactate-lactic acid solu-
tion (page 1019) and stir with a fine glass rod. Wash the rod with a few
drops of water into the tube. By this means the uric acid is completely
precipitated as the silver salt and freed from other substances which
might give a blue color with phosphotungstic acid. Put in a counter-
balancing tube and centrifuge for 2 to 3 minutes. To the clear super-
natant liquid add a drop of the silver lactate solution. If enough has
been added it will remain clear. If a turbidity appears on the addition
of the lactate add 2 c.c. more of the silver lactate solution and centrifuge
again. When no further precipitate forms, decant the supernatant
liquid, as clearly as possible. The uric acid is in the precipitate. Now
dissolve the precipitate in the tube by adding from a burette 4 cc. of
a5 per cent. NaCN solution. (Poison. Caution: do not draw it up in
a pipette.) Stir until a perfectly clear solution is had. Wash the
stirring rod, collecting the washings into a 100 c.c. volumetric flask ; pour
into the flask the solution from the tube without loss, and wash by
rinsing the tube at least 3 times into the same flask with 5 ¢.c. water
each time. Add 5 e.c. of the sodium sulphite solution to balance that in
the standard and dilute to about 40 ¢.c. In another 100 e.c. volumetric
flask prepare the standard by measuring 5 c.c. of the standard uric acid
sulphite mixture (page 1019) containing 0.5 mgs. uric acid; add 4 c.c. of
cyanide solution and 35 cc. of water. Add 20 cc. of 20 per cent.
Na,CO, solution to each flask and finally add while shaking 2 c.c. of the
urie acid reagent (phosphotungstie acid, page 1020). A blue color de-
velops. After 3 to 5 minutes dilute to the mark and mix.

Colorimetric comparison. Set the standard at 20 mm. and add more
of the standard solution to the other cylinder, matching it against the
standard to check the instrument. Now replace the standard in one tube
by the unknown and read against the standard at 20.
1100 PHYSIOLOGICAL CHEMISTRY

Computation:

1 Standard reading

Mgs. of uric acid in vol, of urine taken = Unknown reading

HIPPURIC ACID.

Experiment 289. Quantitative determination of hippuric acid in
urine (Folin and Flanders, Jour. Biol. Chem., 11, 1912, p. 257).—
Principle. The hippuric acid is hydrolyzed to benzoic acid first by
treatment of the urine with NaOH and then by boiling with strong nitric
acid and a little Cu(NO,),. The benzoic acid is shaken out of the mix-
ture with chloroform, the chloroform washed and then the benzoic acid
it contains determined by titrating with N/10 Na alcoholate, using
phenolphthalein as indicator.

Process. Measure 100 ¢.c. of urine into a porcelain evaporating dish
by means of a pipette. Add 10 @e. of 5 per cent. NaOH and evaporate
to dryness on the steam bath. Transfer the residue to a 500 cc. Kjeldahl
flask by means of 25 «ec. of water and 25 cc. of concentrated nitric
acid. Add 0.2 gram Cu(NO,),, a couple of pebbles to prevent bumping
and boil very gently four and one-half hours over a micro-burner. The
necks of the flasks are fitted with Hopkins condensers, that is, large
test-tubes of Jena glass carrying a double-holed rubber stopper, through
which one tube goes nearly to the bottom, the other to the top of the
tube and a stream of cold water is kept circulating through the tubes.

After cooling rinse the condensers with 25 ¢.c. water and transfer
contents of flask to a 500 ¢.c. separatory funnel by the use of 25 ¢.c. more
water. The total volume of the solution is now 100 ec. Just saturate
the solution with powdered (NIH,).SO, (about 55 grams). Extract 4
times by shaking with freely washed chloroform, using 50, 35, 25 and -
25 e.c. portions. The first two portions may be used to rinse out further
the Kjeldahl flask before they are added to the separatory funnel. Col-
lect the successive portions of CHCl, in another separatory funnel and
wash the chloroform by adding to the combined extracts 100 c.e. of sat-
urated solution of pure NaCl, to each liter of which has been added 0.5
e.c. concentrated HCl. Shake well and draw the chloroform, which
contains the benzoic acid, into a dry 500 ¢.c. Erlenmeyer flask and titrate
with N/10 sodium alecoholate, using 4-5 drops of phenolphthalein as
indicator. The first distinct end point is taken, although the color may
fade on standing a short time.

The sodium ethylate is made by dissolving 2.8 grams of cleaned
metallic sodium in 1 liter of absolute alcohol. It is advisable that it be
slightly weaker rather than stronger than N/10. It may be standardized
against purified benzoic acid in washed CHCl,. It may also be standard-
PRACTICAL WORK AND METHODS _1101

.ized against N/20 HCl provided the ethylate is free from carbonic acid.

As a rule, some carbonate will be present and then the titrations in
aqueous media will show the ethylate somewhat stronger than in the
CHCl,. The carbonate does not titrate in the latter case.

ALLANTOINE,

Experiment 290. Determination of allantoine in the urine.
Urease method.—Allantoine is most easily determined by determining
the urea, allantoine and preformed ammonia by Benedict’s urea method
and then the urea and preformed ammonia by the urease method.
Benedict’s method detects not only the urea nitrogen, but about 70 per
cent. of the allantoine nitrogen as well. The urease detects only the
urea nitrogen.

Method. In one sample of urine the preformed ammonia is de-
termined by Folin’s method, experiment 281. In another sample the
urea and allantoine are determined by Benedict’s method, experiment
276. In a third sample the urea and preformed ammonia are deter-
mined by hydrolyzing the urea by the soy bean and drawing air through
to remove the ammonia formed, as in Folin’s method. To determine the
urea and preformed ammonia by the soy bean urease introduce 5 c.c. of
urine into a Folin ammonia cylinder, add 50-60 e.c. of water, and 1
gram of finely ground soy bean. Keep in the water bath at 35-40° with
an air current going through for one hour. (Experiment 277.) The air
current must be freed from ammonia as usual. Then disconnect, add
1 gram anhydrous Na,CO, to the cylinder, some liquid petroleum and
determine the ammonia as described in experiment 276. Receive the
ammonia in 25-50 cc. n/10 H,SO,, of which the volume has been accu-
rately measured. Titrate with n/10 NaOH, using alizarin or congo red
as an indicator. In dog’s urine in 5 cc. about 1.3-1.9 ec. of N/10 acid
is needed to neutralize the ammonia from the allantoine. The difference
between the combined ammonia, urea and allantoine obtained in Bene-
dict’s method and the urease urea and ammonia gives the allantoine
ammonia. Since this detects only about 70 per cent. of the allantoine
the result must be multiplied by 100/70 to give the amount of ammonia
from the allantoine in 5 c.c. of urine.

Experiment 291. Allantoine determination by the Wiechowski
method.—This is an accurate but rather long method. It gives the
allantoine directly and is hence superior in accuracy to the method de-
scribed in 290. (Hofmeister’s Beiirdge, 11, 1907-08, 109).

Principle. A dilute solution of mercuric acetate in the presence of a
large amount of sodium acetate precipitates the allantoine as a white
precipitate. The urine is first purified from basic substances, chlorine
1102 PHYSIOLOGICAL CHEMISTRY

and ammonia, by phosphotungstic acid, lead and silver acetate; then
sodium acetate is added and the allantoine precipitated by 0.5 per cent. .
mercuric acetate.

Method for dog urine. (Hofmeister’s Beitrage, 11, 129, 1907-08.)
Allantoine is most easily prepared from dog urine in which it occurs in
considerable amount. In human urine only very small quantities are
present.

‘Dog and rabbit urine are to begin with diluted. In general I
have diluted the 24-hour urine of a rabbit to 150 c.c., that of a dog to
300 ¢.c., utilizing also the cage washings.—For the allantoine determina-
tion 100 ec. are taken, 10 cc. of 8 per cent. H,SO, added, transferred
to a volumetric flask of appropriate size, precipitated with the exactly
necessary amount of phosphotungstic acid 10 per cent. (a preliminary
test showing how much it is necessary to add) and the flask filled to the
mark with water. After standing at least an hour it is filtered through
a thick, folded filter into an evaporating dish, and the clear, generally
dark-colored filtrate treated with lead carbonate and rubbed as long as
CO, is evolved and until the liquid reacts weakly acid or neutral. Filter
under sharp suction. As large a rounded amount as possible of the fil-
trate, which is often weakly blue but always neutral, is measured off
into a volumetric flask of sufficient size, and precipitated, while avoiding
an excess, by basic lead acetate. The amount necessary to add is de-
termined by a preliminary test. The volume is made up to the mark
with water. The filtrate from the lead precipitate is treated with H,S
and the filtrate from the lead sulphide freed from H.S by a current of
air. If chlorine is present an aliquot rounded portion of the acid filtrate
is taken and precipitated in a volumetric flask with silver acetate solu-
tion and made up to the mark with water. The filtrate from the silver
chloride is handled, in the same manner as that from the lead precipita-
tion, with H,S and the filtrate freed from H,S with air. This filtrate
is now tested in small portions to see that the precipitation with phos-
photungstic acid; basic lead acetate and silver nitrate has been complete,
and now the reaction is absolutely negative. If this is the case two
rounded aliquot portions of the filtrate are taken after preliminary
neutralization with chlorine-free NaOH (prepared from Na) and the
allantoine precipitated by mercuric acetate and sodium acetate.

‘* This reagent is best made as follows: commercial mercuric acetate
(Merck) is dissolved in water to 1 per cent. and pure sodium acetate
added to saturation and diluted to such a degree with water that the
content of mercuric acetate is 0.5 per cent. The completeness of the
allantoine precipitation is determined by the addition of some of the
reagent or allantoine to a small filtered test portion. After at least one
hour standing the precipitate is brought on a filter and washed with
PRACTICAL WORK AND METHODS 1103

water until the disappearance of a precipitate or of a yellow color on
the addition of Na,S. One of the precipitates is now taken and its N
determined by Kjeldahl. The precipitate from the second portion is
washed by a stream of water into a beaker, and while heated to boiling
completely decomposed with H,S. The mixture is then evaporated to
dryness on the water bath, the residue digested with water, quantita-
tively transferred to a small measuring cylinder or volumetric flask and
the volume increased with water to 25 ¢.c. generally, or 50 ¢.c. if much
allantoine is present. The final filtration is made through very thick
filter paper and repeated until the filtrate is quite clear. A rounded
volume of the same is evaporated in a weighed glass dish, dried at 100°
and reweighed. The remainder of the filtrate serves for testing the
identity and purity of the allantoine. If the allantoine crystals are
colored they may be decolored with H,O, in the method indicated.
Hither at once or after one recrystallization the melting point of the
erystals is determined (230-232° with decomposition) and a small por-
tion burned on platinum foil (no residue). Finally the value of allan-
toine and allantoine nitrogen obtained is multiplied with the total
volume numbers (volume of urine, volume after addition of phospho-
tungstic acid, volume after basic lead acetate, after addition of silver
acetate and final volume after decomposition of the allantoine precipi-
tate) and divided by the value of the aliquot parts taken, so as to get
the value for the total urine. The determination takes not more than
6-12 hours.’’ The large number of dilutions makes it necessary to be
extremely careful with the measuring. Carefully tested and calibrated
pipettes and flasks must be used. The precipitation of the allantoine
is quantitative. The result is exact only to about 0.01 gram allantoine
per day. For human urine the method must be changed as described
_in Biochem. Zischr., 19, 1909, 378.

PURINES.

Experiment 292. Quantitative estimation of total purines and of
purines other than uric acid. Salkowski-Arnstein—Principle. The
total purines are precipitated by ammoniacal silver nitrate, the precipi-
tate washed and treated with magnesia to free from ammonia and the
nitrogen determined in it by the Kjeldahl method. The uric-acid
nitrogen is separately determined by Folin’s method and subtracted
from the total. The difference is computed as xanthine.

Process. Measure 200 ¢.c. of protein-free urine into a 300 ec. volu-
metric flask, add 50 ¢.c. of magnesia mixture, make up to the mark with
water, mix well and allow to stand for a little. Filter through a dry
filter paper into a dry 500 c.c. flask. To 200 cc. of the filtrate add 10-15
1104 PHYSIOLOGICAL CHEMISTRY

ec. of a 3 per cent. silver nitrate solution. This precipitates all the
purines, including uric acid. Filter and wash the precipitate thor-
oughly with water to free it from ammonia, finally place the funnel in
the neck of a Kjeldahl flask, puncture the end of the filter and with a
wash bottle wash the whole of the precipitate into the flask. Wash
down the neck of the flask. Add to the flask a little magnesia and heat
to boiling. Boil for 20 minutes to free from ammonia. Proceed to
determine the nitrogen remaining by the Kjeldahl method, described in
experiment 274. From the nitrogen thus determined calculate the total
purine nitrogen in 100 cc. of urine. Subtract from this the uric-acid
nitrogen in 100 ¢.c. of urine, as determined by the Folin method (experi-
ment 288). Compute the remaining nitrogen as xanthine, or leave it as
purine base nitrogen.

Magnesia mixture. 100 grams of crystallized magnesium chloride
are dissolved in water, ammonia added until liquid smells strongly of it,
and then ammonium chloride sufficient to dissolve the precipitate.
Make up to a liter.

AMINO ACIDS.

Experiment 294. Determination of amino-acids by the formol
titration of Sérensen.—Immediately after the titration of acid, Experi-
ment 316, add to the sample you have just titrated 50 c.c. of a fresh
formalin solution, made by taking 15 c.c. of formalin, 30 c.c. of water and
enough N/10 NaOH to make it just very faintly alkaline to phenolph-
thalein. Mix and now titrate again, noting the reading of the burette
when you start, by adding N/10 NaOH until a faint permanent pink re-
mains. Compare the color with a standard tube prepared beforehand, as
the end point is not sharp. This standard is best made by taking one of
the definite buffer mixtures on page 1059 which is just faintly pink with
phenolphthalein. (P,—8.3-8.4.) Each ce. of the N/10 NaOH is equiva-
lent to 1/10,000 of a gram equivalent to amino-acid. How much
nitrogen does this represent calculated for a mono-amino-monocarboxylic
acid? This titration (the last one) gives us a measure of the NH, and
amino-acids together, according to the following equations:

4NH,Cl + 6HCHO + 4NaOH ——~ N ,(CH,) 6 + 10H,O + 4NaCl
Hexamethylenetetramine.
R R

| |
CHNH, + CH, +Na0H —~ CH—N — OH, + 2H,v

woe as II |
.». ,COOH O COONa
PRACTICAL WORK AND METHODS 1105

How do you account for the increased acidity developed after adding
the formaldehyde? Calculate the grams of nitrogen in the form of
amino-acid nitrogen in the total 24-hour sample, also calculate the per-
cent. of the total nitrogen found as amino-acid nitrogen. (See p. 121.)

Experiment 295. Amino-acid nitrogen in urine. Quantitative
determination. Formol titration after removal of ammonia (Benedict
and Murlin, Jour. Biol. Chem., 16, p. 386, 1914). Principle. Precipita-
tion of ammonia and basic substances in the diluted urine by phos-
photungstic acid; removal of excess of phosphotungstic acid with
Ba(OH), and titration of the amino-acid by formol and N/10 NaOH.

Process. Measure into a 500 ¢.c. Erlenmeyer flask 200 c.c. of a 24-
hour human urine which has been previously diluted to 2,000 ce. Add
an equal quantity of 10 per cent. phosphotungstic acid (Merck) (note,
Kahlbaum’s preparation is a very different substance) in 2 per cent.
HCl. Let stand at least 3 hours; better overnight.

Pour off 250 ¢.c. of the clear fluid; add 1 cc. of a 0.5 per cent. solu-
tion of phenolphthalein and barium hydrate in substance until the whole
fluid turns decidedly pink. The Ba(OH), should be added a very little
at atime. Let stand one hour. Filter off two 100 ¢.c. samples (=50 c.e.
urine). Neutralize to litmus (Squibb’s papers answer for all practical
purposes) with N/5 HCl. Add 10-20 cc. neutral formalin and titrate
cautiously with N/10 NaOH, to a deep red color, i.e., until a drop pro-
duces no additional color.

Correct by deducting the amount of N/10 NaOH necessary to pro-
duce the same depth of color in an equal quantity of CO,-free water
with the same quantity of neutral formalin added.

SULPHUR.

Experiment 296. Quantitative estimation of total sulphates in
urine (Folin, Jour. Biol. Chem., 1, 1905, 6, 153). Gently boil in a 200-
250 c.c. Erlenmeyer flash for 20-30 minutes 25 ¢.c of urine and 20 ee.
of dilute HCl (1 part HCl, sp. gr. 1.20 to 4 pts. water) ; or 50 cc. urine
and 4 «ec. con. HCl. Keep covered with a small watch glass during
boiling. Cool for 2-3 minutes under the tap and dilute with cold water
to 150 cc. To this cold solution add 10 ec. of 5 per cent. BaCl, slowly
drop by drop, without shaking or stirring the solution during the addi-
tion, and allow to stand without shaking for one hour. After one hour,
or later as convenient, shake the mixture and filter through a Gooch
weighed crucible with an achestos mat, wash with about 250 ¢.c. eold
water, dry and ignite, cool and weigh. Ten minutes’ ignition is sut-
ficient unless organic matter is present.

Preparation of the asbestos mat. Cut a good grade of fibrous asbestos
1106 PHYSIOLOGICAL CHEMISTRY

with scissors into lengths of 50-70 mm. Place a few grams at a time in
a cylinder with 300 c.c. of 5 per cent. HCl and pass a strong air current
through. This separates the fibers. Keep ready for use in dilute HCl.
From 50-100 mgs. of asbestos is used for each mat. By a good vacuum
pump the asbestos mat is packed firmly in a thin but compact layer. It
is washed finally with water with only enough vacuum to make the
water go through slowly. Dry, ignite and weigh. The same mat may
be used repeatedly until about 1 gram BaSO, has collected in it. It is
somewhat difficult to prepare such mats, which show no loss on filtering
and ignition. On igniting do not apply the flame directly to the bottom
of the crucible, or losses will occur. To ignite, place the lid of a platinum
crucible on a triangle and let the crucible stand upright on the lid.
Another platinum lid should cover the crucible. The flame is applied to
the lower platinum lid. Ten minutes’ ignition is sufficient.

Experiment 297. Estimation of inorganic sulphates of urine
(Folin, Jour. Biol. Chem., 1, 1905, 151).—Measure 25 c.c. of urine, 100
e.c. of water and 10 c.c. of HCl (1 vol. concentrated HCl to 4 vols. of
water) into a 250 ¢c.c. Erlenmeyer flask. If the urine is dilute 50 ce.
may be taken instead of 25 and correspondingly less water. Add
drop by drop, preferably by means of an automatic dropper, 10 c.c.
of 5 per cent. BaCl, solution. The urine solution is not to be shaken
or stirred during the addition of the BaCl, nor for an hour afterward.
At the end of the hour proceed with the determination of the precipi-
tated sulphates in the manner already described for the estimation of
the total sulphates.

Experiment 298. Estimation of the conjugated sulphates.—The
conjugated sulphates are estimated by taking the difference between the
total sulphates as determined in experiment 296 and the inorganie sul-
phates in experiment 297.

Experiment 299. Estimation of total sulphur by the Benedict-
Denis method.—

Reagent: 25 grams Cu(NO,),

25 “ NaCl
10 =“ NH,NO,
100 c.e. H,0

Process. In a 4-inch porcelain dish evaporate 10 to 25 ec. urine
with 5 ¢.c. of the above reagent. When dry, heat carefully over a free
flame, gradually raising the temperature until heated to a dull red heat.
Keep at this temperature for 10-15 minutes. Cool and dissolve in 10-20
ec. 10 per cent. HCl solution. Warm and dilute to about 150 ee.
Filter if necessary and determine the SO, as described in 297.

Experiment 300. Ethereal and inorganic sulphates. Volumetric
PRACTICAL WORK AND METHODS 1107

method (Rosenheim and Drummond, Bioch. Journal, viii, 148, 1914) —
Principle. Precipitation of the sulphates with benzidene hydrochloride
and titration of the acid in the benzidene sulphate with KOH.

Process. Make the benzidene (p-diamino-diphenyl, NH,.C,H,.C,H,.
NH,) solution by rubbing 4 grams of Kahlbaum’s benzidene to a fine
paste with about 10 ¢c.c. of water and transfer to a 2-liter flask with about
500 cc. of H,O; 5 ec. con. HCl (sp. gr. 1.19) are added and the solu-
tion made to 2 liters with water. 150 ¢.c. of this solution, which keeps
indefinitely, are sufficient to precipitate 0.1 gr. H,SO,.

Inorganic sulphates.—Measure 25 c.c. of urine with a pipette into a
250 c.c. Erlenmeyer flask, add dilute (1:4) HCl until the reaction is
distinctly acid to congo paper. 1-2 c.c. of acid is usually enough. 100
c.c. of benzidene solution is then run in and the preciptiate which forms
in a few seconds is allowed to settle 10 minutes. Filter the precipitate
under suction, wash with 10-20 ec. H,O saturated with benzidene sul-
phate; transfer the precipitate and filter paper to the original flask
with about 50 ¢.c. of water and titrate hot with 0.1 N KOH after adding
a few drops of a saturated alcoholic solution of phenolphthalein. 1 c.c.
N/10 KOH corresponds to 4.9 mgs. H,SQ,.

Total sulphates.—Measure 25 ¢.c. urine into a 250 c.c. flask, add 20
cc. dilute (1:4) HCl and boil for 15-20 minutes to hydrolyze the
ethereal sulphates. The authors state that a smaller amount of acid,
namely 2-5 c.c., will do as well. If the larger-amount of acid is used
carefully neutralize the acid after boiling with KOH and then again
add HCl until the reaction is acid to congo red. Cool solution and
precipitate at once with benzidene solution. Proceed from this on as in
the inorganic sulphate determination. Ethereal sulphates are given by
difference between the total and inorganic. Method is an accurate as
the gravimetric and much easier.

CHLORIDES.

Experiment 301. Quantitative determination of the chlorides of
urine. Vollhard’s method.—Principle. All the chlorides in the urine
are precipitated as silver chloride by the addition of a known amount
of silver nitrate to the urine after the addition of nitric acid. The
excess of silver remaining in solution is then titrated with a standard
solution of potassium sulphocyanate, using a ferric salt as an indicator.

Process. Introduce into a 100 ¢e.c. volumetric flask 10 cc. of urine
with a pipette, add 4 ec. concentrated nitric acid and 20 cc. of a
standard solution of silver nitrate. Fill the flask to the mark with dis-
tilled water, mix well, and filter through a dry filter paper into a dry
beaker or flask. Take accurately 50 c.c. of the filtrate, add to it 5 ee.
1108 PHYSIOLOGICAL CHEMISTRY

of iron alum solution and titrate with the standard sulphocyanate solu-
tion until a permanent red color is obtained. Record the reading cf the
burette containing the sulphocyanate at the beginning and end of the
titration.

Calculation. On standardization of the KCNS it was found that
1 ee=ex ee. AgNO, solution. In the above titration y ec. KCNS
solution have been required to titrate the excess AgNO, in 50 ec. of
the filtrate. 100 c.c. or the whole filtrate would require 2y ¢.c. KCNS.
This is equivalent to 2zy ¢c.c. AgNO, solution. The whole amount of
AgNO, solution added was 20 ce., hence 20—2zy e.c. is the amount
which has been used to precipitate the chlorides. 1 ¢.c. of the standard
AgNO, is equivalent to .01 gram NaCl or .00606 gram Cl. The total Cl
in 10 «ec. of urine is hence (20-2xy) X.00606 grams. In 100 cc. it is
10 times this amount.

Standard solutions. Silver nitrate. Dissolve 29.063 grams fused
AgNO, in distilled water and make up to 1 liter. 1 ¢.c.=.00606 gr. Cl.

Potassium sulphocyanate. 8 grams of the salt dissolved in water and
made to 1 liter, Standardize by titrating against AgNO, solution.

Iron alum. Concentrated solution. Pure HNO, free from chlorine.

Standardization of sulphocyanate solution. Measure accurately with
a burette or pipette 10 c.c. of the standard AgNO, solution into a clean
300 c.c. beaker or flask, add 5 ¢.c. of pure nitric acid, 5 ¢.c. of iron alum
solution and 80 ¢c.c. of water. From a burette run in the solution of
sulphocyanate carefully until a permanent faint red tinge is obtained.
Note the reading of the burette before and after the addition of the
sulphocyanate to the silver nitrate. Two determinations should be made
and the mean taken.

x ec. KONS = 10 ec. AgNO,.*, 1 ¢.c, KCNS = 10/x cc. AgNO...

CALCIUM.

Experiment 302. Estimation of calcium. Method of McCrudden.
(Jour. Biol. Chem., X, p. 187, 1911.)—(a) Solutions required. 2.5 per
cent. oxalic acid, 20 per cent. sodium acetate, 0.5 per cent. (NH,),C,0,
sol,

(b) Process. Before measuring off a sample of urine, determine the
reaction toward litmus paper. If neutral or alkaline, make slightly acid
by the addition of cone. HCl. Mix well and filter a portion. Measure
off 200 ¢.c. of the filtered urine; if only faintly acid to litmus paper
add 10 drops cone. HCl; if the filtered urine is strongly acid make
slighty alkaline with NH,OH and then acidify as above indicated, finally
adding 10 drops conc. HCl (sp. gr. 1.19) or 10 ec. N/2 HCl,
PRACTICAL WORK AND METHODS 1109

Now add 10 c.c. of the 2.5 per cent. oxalic acid drop by drop to the
cold solution; then in the same way add 8 c.c. of the 20 per cent. NaC,
H,0, solution. Now allow to stand overnight or shake vigorously for
10 minutes.

Filter off on ashless paper, wash free from chlorides with 0.5 per
eent. (NH,),C,0, solution. Ignite in a porcelain crucible and weigh
as CaO. Ignite strongly until constant in weight. Or filter through
asbestos, transfer precipitate, washed with dilute ammonium oxalate and
3 times with cold water, and asbestos to a beaker with a little water,
acidify with sulphuric acid and titrate with N/20 permanganate.

Experiment 303. Estimation of calcium in urine by Lyman’s
method (Lyman, Jour. of Biol. Chem., 21, p. 551, 1915).—Principle.
This is the same in all particulars as given for blood filtrate on page
1039.

Solutions needed:

1. Potassium ricinate. Dissolve 15 grams KOH in a mixture of
25 ec. of water and 100 ec. 95. per cent. alcohol. Warm. Add 100 e.e.
castor oil. Shake well. Heat with reflux condenser on steam bath until
a sample dissolves in water with no free oil showing. Takes usually 7
hours. This is the stock solution. Pipette 35 cc. of stock solution into
a flask, add 965 ¢.c. of NaOH solution containing 9 grams of NaOH.
Reagent should be perfectly colorless and perfectly clear. Must be made
fresh from stock. Will not keep more than a week.

2. 2 per cent. oxalic acid (1 ¢.c. for a determination).

8. 10 per cent. sodium acetate (1 cc. for a determination). 10
grams crystallized sodium acetate to 100 c.c. of water.

4, 0.5 per cent. ammonium oxalate (20 c. ¢. for a determination).

5. Standard calcium oxalate in 2.5 per cent. HCl. [0.5475 gram
ealcium oxalate (CaC,0,.H,O) in a liter of 2.5 per cent. HCl]. Contains
1.5 mgs. of Ca per 10 e.c.

6. 5 per cent. HCL

Method. If urine is alkaline make it slightly acid. Filter to make
Ca oxalate centrifuge out better. Make just alkaline with concentrated
NH,OH added drop by drop. Make just acid with concentrated HCl
(sp. gr. 1.20). Cool. For every 100 ¢.c. urine add now 5 more drops of
acid. If cloud of phosphates produced by alkalinity is easily seen use
it as indicator. If not use litmus paper. If cloud is heavy take 5 c.c.
of urine; if light take 8 ¢.c., 10 e.c. or more. Usually with normal adults
8 «ec. is best. Pipette 8 ce. into small Erlenmeyer flask. Add 1 cc.
2 per cent. oxalic solution. Add 1 ¢.c. 10 per cent. sodium acetate solu-
tion. Stopper. Shake well 10 minutes by the watch. Rinse stopper
with a little 0.5 per cent. ammonium oxalate solution. Pour into centri-
fuge tube. Rinse flask with 2 c.c. of the ammonium oxalate. Centrifuge
1110 PHYSIOLOGICAL CHEMISTRY

until supernatant liquid is perfectly clear. (2 to 3 minutes). Pour
off, being careful not to disturb the precipitate. Wash the precipitate
with 10 ¢.c. of 0.5 per cent. ammonium oxalate solution. Centrifuge and
decant. By stirring with rod dissolve precipitate in 5 c.c. of 5 per cent.
HCl. If precipitate dissolves with difficulty, place tube in hot water for
a minute or two until dissolved and then recool. Pour into original
flask. Rinse tube and stirring rod with 5 c.c. distilled water. Agitate
liquid in flask to dissolve any precipitate clinging to walls. In another
flask take 10 c.c. of a standard calcium oxalate solution. From a pipette
from which the tip has been broken run into each flask 20 ec. of
potassium ricinate reagent, agitating meanwhile. Mix. Allow to stand
for two minutes and read the clouds in Duboscq colorimeter with the
shade of the latter in place. Read standard against itself first. If 10
¢.c. urine are taken and unknown is set at 15 mm. the reading of the
standard will equal the number of mgs. of calcium in 100 cc. urine. In
any case where unknown is set at given height and the standard read
against it, the following formula serves: Mgs. of calcium in 1 ee.
urine = Reading of standard 1.5 divided by Reading of unknown X
no. of ¢.c. of urine taken.

If urine contains less than .75 mg. Ca or more than 2.5 mgs. the
readings are not quantative and another determination is necessary,
with a volume of urine containing an amount between. these.

PHOSPHATES.

Experiment 304. Estimation of total phosphates by the uranium
acetate titration—Measure off by a pipette 50 ¢.c. urine into a 150 to
300 c.c. beaker, add 5 c.c. sodium-acetate solution (1 liter contains 100
grams sodium acetate and 30 grams acetic acid) and heat to boiling.
Now while boiling gently and agitating, add drop by drop of the
standard uranium-acetate solution (35.461 grams made up to 1 liter)
until one is no longer able to see the formation of a distinct precipitate
as the solution is added. Now remove a drop on to a titration plate
containing drops of 10 per cent. K,FeCy, solution. The end point is
reached when the mixture of the drops at once yields a brownish-red
precipitate.

It may be necessary to repeat the titration to arrive at a more
accurate end point. Calculate the total grams of phosphorus in the
24-hours’ sample. Each c.c. of the uranium-acetate solution is equiva-
lent to 0.002183 gram phosphorus. This must be determined by
standardizing the uranium solution against a standard KH,PO, solution
(4.39 grams in 500 ¢.e. solution) containing 2 mgs, P in 1 ee.
PRACTICAL WORK AND METHODS 4111

This method is not so accurate as the gravimetric, but is sufficiently
accurate for all ordinary purposes and much easier than the gravimetric.

DEXTROSE.

Experiment 305. Glucose by Benedict’s method (Journal Amer.
Med. Ass., LVII, 1193, 1911).—Principle. Complete reduction of a
standard cupric sulphate to a colorless cuprous state by urine run in
from a burette.

Process. 25 ¢.c. of the reagent are measured by a pipette accurately
into a 25-30 em. evaporating dish. Add 10-20 grams of crystalline
Na,CO,, or half the quantity of the anhydrous salt, some powdered
pumice or taleum, and heat to boiling over a free flame until the car-
bonate has completely dissolved. Dilute 10 cc. of urine to 100 ee.
unless the amount of sugar in it is small, when it can be used sitio
dilution, fill a burette with the urine, or diluted urine, to the zero mark
and run into the boiling copper solution rather rapidly at first, then
more slowly as the color grows less, then add but a few drops at a time
until the solution is colorless. A white precipitate forms. If the mix-
ture becomes too concentrated during the process, add water to replace
that evaporated.

Calculation: The reduction of the 25 ¢.c. of the copper reagent is
accomplished under these conditions by exactly 50 mgs. of glucose. The
amount of urine run in from the burette contained this amount of glu-
cose, provided of course there was no other reducing substance in it. If
the urine was diluted 10 times, then the per cent. of glucose in the
original urine was 0.0501,000/x, where x was the number of c.c. used
from the burette.

Standard solution. CuSO, (erystalline), 18.0 grams; Na,CO, (erys-
talline), 200 grams; sodium or potassium citrate, 200 grams; KCNS, 125
grams; potassium ferrocyanide, 5 per cent. solution, 5.0 ¢.c.; distilled
water to make a total volume of 1 liter. Dissolve the carbonate, citrate
and thiocyanate in about 700 c.c. water and filter if necessary. Dis-
solve the copper sulphate in 100 ¢.c. water and add slowly little by little,
stirring constantly, to the 700 c.c. Add the ferrocyanide and make up
exactly to 1 liter. The copper salt must be weighed exactly; the other
constituents need not be so exactly measured.

The advantage of this method over the original titration by Fehling’s
solution in a similar way is that the solution is less alkaline so that the
sugar is less profoundly decomposed by the alkali and there is less
danger of oxidation by the oxygen of the air. The end point in this
method is sharp, whereas in the titration by Fehling’s solution it is
often very indefinite. The method is probably as accurate as any
1112 PHYSIOLOGICAL CHEMISTRY

volumetric method. The drawback to the method lies in the changing
concentration of the copper as the titration proceeds, so that relatively
less copper is used to oxidize each molecule of glucose and the danger
of oxidation by the air increases. It is, however, the most convenient
and exact of the volumetric methods, except the method of Bang, which
has some points of superiority, but is not so simple.

Other methods have been suggested, but they are inferior to those
given here and are accordingly omitted. This second method is superior
to Benedict’s earlier method.

Experiment 306. Determination of glucose in urine. (Folin and
Peck: J. Biol. Chem., 1919, 38, p. 287.)

The solutions required are the same as described in experiment 90,
pages 952-954.

A special sugar burette reading to 0.02 cc. furnished by Emil
Greiner Company, New York, is recommended. See Figure 77, page 953.

The undiluted urine is run in from the burette, 0.4 to 1 c.c., to clear,
warm copper-salt solution in a large, previously dried, Pyrex glass tube.
This solution is made by transferring to the test-tube 5 c.c. of the 5.9
per cent. copper sulphate solution, adding approximately 1 c¢.c. saturated
sodium carbonate, shaking for a moment, then adding 4 to 5 gms. of
the phosphate-carbonate-thiocyanate mixture, warming gently and shak-
ing until all is dissolved. Add 0.4 to 1 e.c. of the urine, heat fairly
rapidly to boiling and then boil gently. When the mixture begins to
bump add a dry pebble. If the boiling contents of the test-tube do not
become suddenly filled with cuprous thiocyanate precipitate within
the first 15 seconds of boiling, then less than half the required sugar
has been added and more urine should be added without delay. If an
excess is added at the start so that the solution becomes colorless before
4 minutes of boiling discard and begin again, adding less of the urine.
The volume of solution in tube should not become less than 6 to 7 e.c.

Experiment 307. Quantitative determination of glucose by Bang’s
method. (Bioch. Ztschr. 2 and 11; and 49, 1913.)\—Principle. A
standard solution of Cu(CNS),, cupric thiocyanate, made alkaline by
K,CO, and KHCO, is reduced by glucose to colorless CuCNS, euprous
thiocyanate. An excess of the reagent is used and the excess of the
cupric salt is titrated to the colorless cuprous salt by a standard solution
of hydroxylamine sulphate. From the amount of hydroxylamine re-
quired to reduce the excess of cupric salt the amount of cupric salt
reduced by the sugar can be found and from this by reference to a table
which has been prepared by determining the reducing power of known
amounts of dextrose, the amount of dextrose in the solution is deter-
mined.

Process. Measure from a pipette 10 cc. of urine, which must not
PRACTICAL WORK AND METHODS 1113

contain more than 0.6 per cent. of glucose, into a flat-bottomed 200 «.c.
flask. If the urine contains more than this amount of sugar take a
proportionately smaller amount of it, 2-5 ¢.c., and dilute with water so
that the bulk of fluid added to the flask is:10 cc. Add 50 «ec. of the
copper solution and heat to boiling on a wire gauze and boil for exactly
three minutes. Remove and cool at once under the tap to stop the re-
action. Now run in slowly from a burette, the reading of which has
been taken, the standard hydroxylamine solution, shaking the flask with
‘each addition, until the solution loses its blue color and becomes color-
less or has a slight yellow tint. The end point is not very sharp. An
error of 0.5 c.e. of hydroxylamine is possible for beginners. From the
amount of hydroxylamine solution required the amount of dextrose can
be found by consulting the accompanying table:

 

 

 

 

 

 

 

 

 

 

 

 

 

droxylami ylami: i i
MreTation | Suxar [verorsiemine| sugar [[Bydrozzlamine| sugar [[Hyaroxylemine] sugar
Ce. Ms. C.c. Mgs. Cc. gs. Ue. . Mys.
43.85 5 29.60 19 17.75 33 | 7.65 | 47
42.75 6 28.65 20 16.95 34 |] 7.05 48
41.65 7 27.75 21 16.15 Ju 6.50 49
40.60 8 26.85 22 15.35 36 5.90 50
39.50 9 26.00 23 14.60 37 5.35 61
38.40 10 25.10 - 24 13.80 38 4.75 52
37.40 Il 24.20 25 13.05 39 4.20 53
36.40 12 23.40 26 12.30 40 3.60 54
35.40 13 22.60 27 11.60 41 3.05 55
34.40 14 21.75 28 10.90 42 2.60 56
33.40 15 . 21.00 29 10.20 43 2.15 57
32.45 16 20.15 30 9.50 44 1.65 58
31.50 17 19.35 31 8.80 45 1.20 59
30.55 18 18.55 32 8.20 46 0.75 60
(Bang.)

Standard solutions: Copper solution. Dissolve 12.5 grams of copper
sulphate (CuSO,+5H,O) in 60 c.c. water, warming if necessary, and
after cooling to room temperature dilute to 75 ¢c. Place in a large
evaporating dish or beaker 200 grams of powdered potassium thiocya-
nate, 250 grams of potassium carbonate and 50 grams of potassium bicar-
bonate, and about 600 cc. of water. Stir and if necessary aid. the
solution by gently warming on the water bath, but not over 50°. Cool
the solution under the tap to the usual temperature, add the copper-
sulphate solution very slowly, a little at a time while stirring. Transfer
to a 1 liter volumetric flask, washing the evaporating dish or beaker out
carefully into the volumetric flask and make up to 1 liter. The solution
slowly changes on standing and in three months is useless. With a
little care the solution may be made directly in the flask. The process
outlined above must be followed exactly or a false titer will be found.

Hydroxylamine solution. Dissolve 200 grams potassium thiocyanate
in 1,500 c.c. of water in a 2-liter volumetric flask. Add to it a solution
1114 PHYSIOLOGICAL CHEMISTRY

of 6.55 grams hydroxylamine sulphate in a little water, carefully wash-
ing all the solution into the flask, and make up to 2 liters.

Standardization. These two solutions should exactly correspond.
Titrate 50 c.c. of the copper solution, to which 10 c.c. water is added,
with the hydroxylamine-sulphate solution, to make sure that they do
correspond.

The advantages of this method are that the solution is less alkaline
so that the sugar is not broken up readily by the alkali and no precipi-
tate forms so that the end point is sharper. The reducing action of the
uric acid and creatinine is small in this faintly alkaline solution, so that
they introduce less error.

Experiment 308. Quantitative determination of glucose by Bang’s
micro-method (Bang, Biochemische Zeitschrift, XLIX, p. 1, 1913; LVII,
301, 1913).—Principle. The macrochemical method is expensive on
account of the cost of hydroxylamine and thiocyanate and the copper
solution is not stable. By this method very minute amounts of glucose,
even 0.1 mg., may be accurately determined. The copper solution is
modified from the former method in that less carbonate is taken and
potassium chloride is substituted for potassium thiocyanate. The copper
is reduced in the absence of air and the reduced cuprous chloride, which
is kept in solution by the large amount of KCl present, is titrated directly
with N/100 or N/25 or N/10 I solution, soluble starch being used as the
indicator. The iodine solution under these conditions oxidizes the
cuprous chloride back to the cupric condition.

Process. Place in a 100 cc. Jena-glass, flat-bottomed flask having
a neck without a turned-over edge 0.1-2 c.c. of the sugar solution to
be examined. The solution must not contain more than 10 mgs. of
glucose, and it is better to have not more than 5 mgs. If the sugar
solution is more dilute, more than 2 ¢e.c. of the solution may be added.
Then add 55 c.c. of the copper solution, measured with a 55 c.c. pipette,
and draw over the neck of the flask a closely-fitting, good rubber tube,
leaving about 2 ems. projecting above the neck of the flask. A good
Mohr pinch cock is laid ready to put on the rubber tubing so as to close
the flask air-tight at the end of boiling, or the special apparatus figured
in Figure 109, which enables one to lift the flask from the flame and close
the mouth at the same instant, is adjusted on the flask. Heat over a
flame so high that it takes 314 to 3% minutes to come to the boil and
then boil vigorously for exactly 3 minutes. At the end of 3 minutes
exactly, clamp air-tight the rubber tube on the neck of the flask, lift the
flask from the flame and cool at once under the tap. The object of the
clamp is to prevent any oxidation by air while cooling. After cooling
to room temperature, open the rubber tube and add 14 to 1 ae. of a
1 per cent. solution of soluble starch in a saturated KCl solution to the
PRACTICAL WORK AND METHODS 1115

contents of the flask and titrate with N/100 or N/25 iodine solution
run in from a glass-stoppered, small burette. At first the decolorization
of the starch takes place at once and the iodine may be run in freely.
When the blue color of the starch iodide appears, shake the flask gently
once and allow it to stand. Near the end point the color persists 2-3
seconds and then disappears. Add iodine solution until the color per-
sists 5-10 seconds. Particularly in titrating urine a slow decolorization
of the starch due to the absorption of the oidine goes on. This is to be
disregarded and the end point taken as that at which the color persists
for 5-10 seconds.

Computation. There is a direct proportionality between the amount
of sugar oxidized under these circumstances and the amount of iodine

 

Fic. 109.—WMask for use in Bang’s micro-method.

solution used, at least up to 9.5 mgs. of sugar. It is only necessary,
therefore, in order to find the mgs. of glucose in the amount of solution
taken, to divide the number of ¢.c. of N/100 I used in the titration by
the factor 2.7. If the titration is by N/25 I divide by the factor 0.7, and
if N/10 I by the factor 0.285. Since in making the copper solution 16.5
c.c. of stock solution are diluted by the addition of 38.5 ¢.c. of KCl solu-
tion, it is possible to replace the 38.5 ¢.c. of KCl by 38.5 «.c. of the sugar
solution to which has been added 11-11.5 grams of solid KCl. In this
way 0.1 mg. of glucose may be accurately determined when present in
a concentration of 1: 300,000.

Copper solution. Dissolve first 160 grams KHCO,, 100 grams K,CO,
and 66 grams KCl with about 700 c.c. water in a 1-liter flask. Since the
bicarbonate is not readily soluble, pulverize and dissolve it first at a
temperature of about 30° C. Then dissolve the KCl and finally, under
cooling, the carbonate. Now add 100 cc. of a 4.4 per cent. solution of
CuSO,+5H,0 and fill to the mark when the slight evolution of CO, is
at an end. Shake the solution only gently so as not to absorb much
air. Use the solution on this account only after 24 hours’ standing.
1116 PHYSIOLOGICAL CHEMISTRY

This is the stock copper solution. To use, dilute 300 cc. to 1,000 e.c.
by the addition of saturated KCI solution. Shake this mixture also very
little, and use only after standing some hours.

Iodine solution. This is a N/100 I solution. This may be made by
diluting a N/10 I solution with boiled water. Such a solution in a
dark-colored bottle keeps its strength unchanged for 3 months. One can
make it also from iodide and iodate. Pour 1 «ce. of a 2 per cent. KIO,
solution in a 100 ¢.c. volumetric flask, add 2-2.5 gr. KI and exactly 10
e.c. N/10 HCl. The acid sets free an equivalent amount of I. The
reaction is instantaneous and the flask is filled to the mark with water.

Starch solution. 1 per cent. soluble starch in saturated KCl solution.

This keeps perfectly.
Note. 55 c¢.c. of the copper solution is reduced completely by 10 mgs.
glucose.

Experiment 309. Microdetermination of glucose in blood (Bang,
Bioch. Zeits., XLIX, 1913, p. 23; LVII, 1913, p. 301.—Principle. Two
or three drops of blood are received on a previously weighed, small piece
of blotting paper and rapidly weighed. The blotting paper is then
heated with slightly acid salt solution to coagulate the proteins, which
remain in the blotting paper while the glucose goes into the salt solution.
The glucose is then determined by the method given in 308, except that
smaller amounts of copper solution and a weaker iodine solution, N/200,
are employed. Microchemical methods are also given for the determina-
tion of water and chlorides in blood.

Process. Small pieces of a good, thick blotting paper are prepared
about 16 x 28 mm. in area and weighing about 100 mgs. The paper must
be extracted thoroughly first with hot water acidified with acetic acid to
remove any iodine-binding substances, chlorides and reducing matters.
(Papers all prepared, weighed and having attached to them a small wire
for attaching them to the balance may be had from Warmbrunn and
Quilitz, Berlin, who also have the other apparatus recommended.) The
‘ blotting paper is weighed beforehand. Two or three drops of blood
weighing about 100-130 mgs. are drawn into the paper and immediately
weighed. The weighing is very conveniently done on a small torsion
balance of a special type recommended in the original article. Weighing
to 1 mg. is sufficient. The blotting paper with the blood is now placed
in a test-tube and 6.5 cc. of boiling hot salt solution containing HCl
is added and, after standing ¥ hour, the clear liquid is poured carefully
without loss into a 50 cc. Jena glass flask with a neck without a rim
and provided with a piece of rubber tubing like that described in the
previous method—308. The salt solution with HCl consists of 1,360
c.c. of saturated KCl + 640 ec. H,O + 1.5 ee. 25 per cent. HCl in a
2-liter flask. The salt solution may be measured into a test-tube with a
PRACTICAL WORK AND METHODS 1117

mark at 6.5 ¢.c. in which it ig boiled. The blotting paper is now washed
out again with another 6.5 c.c. of hot salt solution and this is added to
the 50 c.c. flask. The flask is cooled and after cooling add 1 ¢.c. of the
copper stock solution described in 308. This is sufficient for at least
0.8 mg. of glucose. The flask provided with the rubber tubing and with
the holder ready to clamp the tubing (Figure 109) is now heated to boil-
ing so that it takes 1 min. 30 sec. to come to the boil (a variation of
5 seconds more or less permitted) and then boiled gently for exactly 2
minutes. At the end of 2 minutes exactly the rubber tube on the neck
of the flask is clamped air-tight, the flask lifted from the flame and
cooled under the tap. The reduced cuprous chloride is now titrated
with N/200 iodine solution after the addition of 1-2 drops of starch
solution and while the air in the flask is being driven out by a gentle
stream of CO, which is carried to the bottom of the flask by a small tube.
It is very necessary to prevent the entrance of air during the titration,
as this oxidizes some of the cuprous chloride. The N/200 I is run in
from a small, sharp-pointed, glass-cock burette of about 2 ¢.c. capacity,
graduated to one-fiftieth of a c.c. One adds the iodine solution until
the slight blue color persists for from 30-60 seconds. At first the color
will disappear in 2-3 seconds as one draws near the end point.

Computation. Since, for some reason, the salt solutions and copper
solutions have the power of binding about 0.12 cc. of N/200 I, this
amount is first subtracted from the amount of N/200 I consumed in
the titration and the result divided by 4 gives the number of mgs. of
glucose in the amount of blood or liquid taken. 0.01 mg. of glucose is
equivalent to 0.04 c.c. of N/200 I solution. For example, if 0.72 e.c.
of N/200 I has been used, the amount of glucose will be 0.72—0.12—-4, or
0.15 mg. of glucose. The results thus obtained with blood are a little
higher than results given by Bertrand or the macro-method. This is due
to the presence of some I-binding material in blood. Experience shows
that this is about equivalent to 0.01-0.015 per cent. of glucose, so that
this amount should be subtracted from the results obtained to give the
true value of the glucose content.

Solutions. The N/200 I is made fresh since it is not stable. Into
a 100 cc. volumetric flask bring 1-2 ¢.c. 2 per cent. KI,, about 2 grs.
KI, exactly 5 cc. of N/10 HCl and fill to the mark with water. The
copper solution has already been described in 308.

ACETONE; DIACETIC ACID; HYDROXYBUTYRIC.

Experiment 310. Determination of acetone. Micromethod.
(Folin.)—-Principle. The acetone is absorbed in bisulphite solution and
then determined by nephelometric comparison between this solution and
1118 PHYSIOLOGICAL CHEMISTRY

a standard acetone solution using Scott-Wilson reagent for acetone.

Solutions needed:

1. Scott-Wilson reagent. Add a cold solution of 180 grams NaOH
in 600 c.c. water to 10 grams mercuric cyanide dissolved in 600 ¢.c. H,0.
Add slowly to the solution thus obtained and while vigorously stirring
a solution of 2.9 grams AgNO, dissolved in 400 ¢.c. water. This is best
done in a strong jar not easily broken. The silver should dissolve com-
pletely to a clear solution. If it is turbid allow to stand until turbidity
is settled and take the clear supernatant part. The reagent gradually
deteriorates and is not usable for quantitative work after a few months.
(45 e.c. needed for each determination. )

2. 2 per cent. sodium bisulphite solution. This must be made fresh.
(30 ¢.c. needed for each determination. )

3. Standard acetone solution. 2.5 grams acetone sulphite are dis-
solved in 50 ¢.c. water, transferred to a 1,000 c.c. volumetric flask and
filled to the mark with 1:5 HCl. Standardize this solution by titrating
25 «ec. of it. Add to the 25 cc. in a flask 20 cc. of N/10 I solution.
After 5 minutes titrate the unreduced iodine by N/10 thiosulphate solu-
tion. This will give the SO,.

To determine the acetone take another 25 ¢.c., add to it 50 cc. N/10 I,
allow to stand 5 minutes, then add 10 ec.c. 40 per cent. NaOH and after
5 minutes add 18 ¢.c. concentrated HCl. Titrate surplus I with thio-
sulphate. From the 50 ¢e.c. N/10 I taken subtract the c.c. of thiosul-
phate used in the last titration and the c.c. of iodine used in the SO,
determination above. The result gives the iodine taken up by the
acetone. Prepare from this stock solutions by dilution a solution which
contains 1 mg. acetone in 10 c.c.

Apparatus. 1. Nephelometer.

Method. To get an idea how much urine to take so that it will
contain about 0.5 mg. of free acetone make a preliminary test as follows:
Place 5 cc. of the urine in a large test-tube, add a few drops of dilute
HCl, connect up the tube for aération. In a second tube place 5 ee.
H,O and 5 ce. Scott-Wilson reagent. Connect up this tube as in
Figure 103 with the other. Aérate briskly from the first to the second
tube, warming the first tube to about 35°, but keeping the second one
cool. If the urine contains much acetone the second tube will become
turbid at once; if only very little it will take 5 to 10 minutes. Having
thus found the approximate richness in acetone of the urine take some-
thing between 0.5 and 5 c.c., the more, the longer it has taken to develop
turbidity; add this to 1 cc. 10 per cent. H,SO, in a large test-tube
arranged for aération. Connect the test-tube, as in Figure 103, with a
second receiving tube which contains 10 c.c. of the freshly made 2 per
cent. sodium sulphite solution. Warm the first tube to 35°, keeping
PRACTICAL WORK AND METHODS 1119

the second cool, and aspirate air through it at a moderate rate for 10
minutes. Disconnect and pour without loss the acetone sulphite mixture
into a 100 ec. volumetric flask, dilute with H,O to 50 cc. To make
the standard for comparison: into two 100 ¢.c. volumetric flasks measure
an amount of the stock solution to contain 0.5 mg. of acetone, add to
each 10 e.c. of 2 per cent. sodium bisulphite and dilute each to 50 e.c.
with water. Now to each flask add 15 «ec. of a clear Scott-Wilson
reagent and at once, before turbidity, dilute to the mark with water.
Mix. Take one of the standards and put it into the two tubes of the
nephelometer and adjust the light so that the two cylinders match when
each is set at 20. Empty out the cups and into one put the other
standard and into the other the unknown from the urine, set the
standard at 20 and match the unknown against it.
Computation:

: : 20 x 0.5
Mgs. acetone in number of c.c. uf urine taken = —_____“~_____,
Reading of unknown

Experiment 311. Acetone. Quantitative determination. Folin
method (Jour. Biol. Chem., ITI, 1907, 177).—Principle. The acetone
is carried over by a strong air current from the urine and is absorbed
and changed to iodoform by an alkaline hypoiodite solution of known
strength. The amount of iodine remaining is then titrated with a
standard thiosulphate solution with starch as an indicator. The method
is accurate, rapid and simple, and requires only 20-25 ¢c.c. urine.

Process. Into an aérometer cylinder like that described in experi-
ment 277, or a large test-tube, measure accurately 20-25 ¢.c. of urine
with a pipette, add 0.2-0.3 gram of oxalic acid or a few drops of 10 per
cent. phosphoric acid, 8-10 grams of solid NaCl and a little petroleum.
The salt renders the acetone less soluble. Connect with the absorbing
bottle or test-tube which is like that described for the ammonia ap-
paratus, experiment 275. In the absorbing bottle place about 150 e.c.
water, 10 ce. 40 per cent. KOH solution and an excess of standardized
solution of N/10 I, the amount being carefully measured from a burette
or pipette. An excess of 10-15 c.c. of the standardized iodine solution
should be added. Hawk recommends that to get an idea of the amount
of iodine solution necessary to add, take in a test-tube 10 e.c. of urine
and add 1 ee. of ferric-chloride solution (100 grams ferric chloride
in 100 c.c. water). Compare the color with 10 ¢.c. of the original ferric-
chloride solution in a similar test-tube. If the colors about match, then
take 20 c.c. of the iodine solution. If the urine is darker so that it has
to be diluted once with water, then take 35 ¢.c.; if still darker take more.
It is important to connect the apparatus and begin the air current at
once after adding the strong alkali to'the iodine, since the hypoiodite
1120 PHYSIOLOGICAL CHEMISTRY

goes over very rapidly into iodate. Run the air through briskly, but
not so fast as for ammonia, for 20-25 minutes. Then disconnect, acidify
the contents of the hypoiodite tube by the addition of 10 ¢.c. concen-
trated HCl for each 10 ¢.c. of strong alkali added at the start, and titrate
the excess of iodine by means of the N/10 thiosulphate solution with
starch as indicator. The sodium thiosulphate solution must have been
titrated against the iodine beforehand. It must be kept in the dark.
For standardization of the thiosulphate see experiment 51.

Experiment 312. Determination of acetone and diacetic acid
(Folin-Hart).—Principle. The acetone is first determined as in experi-
ment 310 or 311, then the urine is acidified and heated while air carries
over the acetone which is set free from the diacetic acid in these circum-
stances. The two determinations may be made as one, the total acetone
being received in a single solution of hypoiodite, or the preformed acetone
may be separately determined first.

Method. The arrangement of the apparatus is as in experiment 311,
except that in place of the cylinder for holding the urine, the latter is
placed in a large Pyrex test-tube which is supported in such a way that
it can be heated. The air inlet tube dips to the bottom of the Pyrex glass
test-tube. The air is passed through a hypoiodite solution first to remove
any substance which may reduce iodine. Place in the large test-tube,
which carries the stopper and tubes for aérating the urine, 20 c.c. of
urine, and the phosphoric acid, oxalic acid, salt and petroleum as de-
scribed in 311 and without heating the tube air is passed through for
25 minutes. The air with the acetone passes into a measured amount
of fresh hypoiodite as described. Then disconnect the hypoiodite, place
a fresh cylinder of a known amount of hypoiodite solution to receive
the acetone and heat the tube just to boiling for 25 minutes while the
air is going through. By this heating the acetoacetic acid is decom-
posed, acetone is liberated and absorbed by the second hypoiodite tube.
The first hypoiodite tube being titrated in the manner indicated in 311
gives the preformed acetone; the second hypoiodite tube gives the
acetone from the diacetic acid. The sum of the two gives the total
acetone in 20 c.c. urine.

Experiment 313. Organic acids in urine by titration. (Van
Slyke and Palmer, Jour. Biol. Chem., 41, 1920, p. 567.) —Method. 100
c.c. of urine are thoroughly mixed with 2 gms. finely powdered calcium
hydroxide, and filtered after standing with occasional stirring for 15
minutes. Carbonates and phosphates removed. 25 c.c. of the filtrate
accurately measured are taken in a 125 or 150 c.c. test-tube, 0.5 cc. of
1 per cent. phenolphthalein added, and .2 N HCl run in from a burette
until the pink color is just gone. (Amount of acid need not be meas-
ured.) This makes P,, about & 5 ec. of 0.02 per cent. troprolin OO
PRACTICAL WORK AND METHODS 1121

are added little by little with stirring so as to prevent precipitation, and
now the solution is titrated by running in 0.2 N HCl until the red color
equals a standard solution made of 0.6 c.c. of 0.2 N HCl, 5 ec. tropxolin
0O solution and water to a total volume of 60 c.c. When the end point
is approached sufficient water is added to the tube to make the volume
60 c.c. equal to the standard. The two tubes may be placed for com-
parison in.titration in a comparator.

Calculation. From the number of c.c. of 0.2 N HCl needed to titrate
from the end point of phenolphthalein to that of tropzolin, the amount,
usualiy 0.7 ¢.c., is subtracted, which is necessary to titrate a control tube
of water between these limits. The volume of 0.2 N HCl remaining
represents approximately the amount of organic acids plus the creatine
and creatinine and a negligible amount of amino acids. The figure may
be expressed in c.c. of 0.1N organic acid per liter by multiplying the
number of e.c. of 0.2N HCl used in titrating the organic acids by 80
(1,000/25 multiplied by 2).

The result obtained indicates very closely the amount of acetone
bodies (acetoacetic acid and hydroxybutyric acid) in the urine and is an
easy substitute for clinical purposes for the more elaborate and difficult
methods of determining acetone and these acetone bodies. In normal
individuals the amount obtained is about 412 to 748 c.c. per 24 hours
(0.1N HCl) or an average of. 8.2 (from 6.1 to 9.7) ce. per kg. Ina
case of diabetic acidosis the figure rose from 26 cc. 0.1N HCl per kilo
per 24 hours to 180 ¢.c. per kilo per 24 hours in the coma, some 3 weeks
later.

Experiment 314. Quantitative determination of acetone and
acetoacetic acid and hydroxybutyrc acid in the same sample of urine.
Method of Shaffer and Marriott (Jour. Biol. Chem., XVI, p. 276,
1914).—Principle. The urine is distilled with acid after precipitation
of sugar and other substances by basic lead acetate and ammonia. This
separates the acetone, both that preformed and that arising from the
acetoacetic acid. The acetone is titrated with standard iodine and thio-
sulphate. The urine filtrate, from which the acetone has been distilled,
is oxidized by the cautious addition of bichromate to the boiling urine
containing the oxybutyrie acid. This oxidizes the oxybutyric acid to
acetone, the acetone is distilled off and titrated in the same way as the
performed acetone. The yield is about 90-95 per cent. of the theo-
retical.

Process. From 25-100 ¢c.e. of urine (usually 50 cc.) are measured
with a pipette into a 500 ¢.c. volumetric flask containing 200-300 e.c.
of water. Add basic lead acetate solution (U.S.P.) in amounts equal to
the urine taken and mix the liquid well. If the urine contains but little
sugar, only half the amount, or less, of the lead acetate should be used.
1122 PHYSIOLOGICAL CHEMISTRY

Next pour into the flask strong ammonia water equal to about half
the volume of lead-acetate solution taken, dilute to the mark with water,
shake and after a few moments’ standing filter through a folded filter.
Measure 200 ec.c. of the filtrate into a round-bottom flask (800 cc. or
liter Kjeldahl), dilute with water to about 600 c.c., add 15 c.c. concen-
trated sulphuric acid and tale or boiling stone and distill the mixture
until about 200 ¢.c. of the distillate has been collected. (Distillate A.
This contains the preformed acetone and that derived from the aceto-
acetic acid.) The distilling flask must be fitted with a dropping tube
and water run in from time to time to prevent the volume in the flask
from becoming less than 400-500 ¢c.c. The tube at the end of the con-
denser should dip below the surface water in the receiving flask, a second
Kjeldahl, to prevent loss of acetone.

Distillate A is redistilled for about 20 minutes after addition of
10 c.c. of 10 per cent. sodium hydroxide. If a high degree of accuracy
is not required, distillate A may be titrated directly with standard
iodine solution, N/10, and thiosulphate. The results are a little higher
than after redistillation from an alkaline solution. The distillate from
A, which may be called A,, is titrated with iodine and standard
thiosulphate.

After A has been distilled off, a new receiving flask is adjusted, the
end of the tube dipping below the water in the receiving flask and
bichromate solution as indicated below is slowly added to the distilling
flask while the distillation is continued to give B.

The residue of the urine plus sulphuric acid from which distillate
A was obtained is again distilled, adding water or bichromate solution
when necessary to keep the volume between 400 and 600 cc. From
0.5-1 gram of bichromate will usually be sufficient and not more than
1 gram should be added, unless the liquid turns green, indicating a great
reduction to chromium sulphate: very rarely 2 or 3 grams of bichromate
may be necessary, especially if the sugar has not been completely
removed. To make the distillation proceed as follows:

A 10 per cent. solution of potassium bichromate is kept on hand and
10 cc. of this, diluted to 100 ¢.c., are measured out for each determina-
tion. 20 ec. of the dilute solution (0.2 gram K,Cr,O,) are first added
slowly through the dropping tube and then 10 c.c. portions every 15 or
20 minutes until the whole has been added. Should the liquid become
markedly green the bichromate must be added at correspondingly
shorter intervals and in amount sufficient to maintain a slight red-
yellow color of the chromic acid, which may be detected even in the
presence of the green. The distillation is continued with moderate boil-
ing for from 2-3 hours. The distillate B, which should be collected in
a liter flask to avoid transference, is again distilled for about 20 minutes
PRACTICAL WORK AND METHODS 1123

after adding 10 ¢.c. of 10 per cent. sodium hydroxide and 25 «ac. of
3 per cent. H,O,. The flask must be heated cautiously until the peroxide
has been decomposed. This distillate, B,, is titrated with the standard
iodine and thiosulphate.

Computation. 1 ec. of N/10 iodine=0.968 mg. acetone=1.736 mg.
oxybutyric acid, or

1 ee. of 1.035/10 iodine (=13.13 mg. I,)=1 mg. acetone=1.793
Ing. oxybutyrie acid.

Note——This method gives results usually a little lower than the
extraction and polarization method of Black, but is more accurate for
small amounts of oxybutyric acid and somewhat more convenient.

In making this titration an excess of N/10 iodine solution is added
and then the solution made alkaline by the addition of 10 e.c. 60 per
cent. NaOH, the flask stoppered, shaken and allowed to stand 5-10
minutes, then acidified by the addition of 15 ¢.c. concentrated HCl and
the liberated iodine titrated with standard thiosulphate in the usual way.

For the determination of acetone in blood, or when small quantities
are present, see Marriott, Jour. Biol. Chem., 16, p. 284 and p. 289, 1913.
For determination of oxybutyric acid in blood and tissues, see Marriott,
ibid., page 293.

Experiment 315. Quantitative determination of saccharose in
urine (Jolles, Biochem. Ztschr. 43, p. 56, 1912).

Determine polarimetrically after treating the urine with 0.1 per cent.
NaOH 24 hours in the thermostat at 37°. All other mono- and di-sac-
charides become inactive under these circumstances. Saccharose is not
affected.

ACIDITY.

Experiment 316. Total acidity of urine by Folin’s method.—By
means of a 25 c.c. pipette measure off 25 ¢.c. urine into a 250 c.e. conical
flask, add 25 c.c. distilled water, 5 drops phenolphthalein solution and
15 grams finely powdered potassium oxalate. Rotate the contents of the
flask for 2 minutes and titrate at once with n/10 NaOH required to
neutralize the entire 24-hour sample of urine.

The end point is not sharp and it will vary with different. observers.
It is a good plan to have a standard color and go to this point for the
different urines examined. The oxalate is added to precipitate the lime
salts which. otherwise precipitate the phosphates when the hydrate is
added.

Experiment 317. Quantitative determination of the hydrogen ion
concentration of urine. Sdrensen indicator method. (Henderson and
Palmer, Jour. Biol. Chem., 13, 398, 1918).—Principle. Matching the
1124 PHYSIOLOGICAL CHEMISTRY

color of the urine, diluted if necessary and containing a known amount
of some indicator, with the color developed by a similar concentration of
the indicator in a series of solutions containing known concentrations of
hydrogen ions.

Solutions needed:

 

1. M/10 KH,PO, (13.6 gram per liter).
2. M/10 Na,HPO,.12H,O (35.8 grams per liter).
3. N/5 acetic acid. (By titration.)
4. N/5 Na acetate (27.3 grams per liter).
5. 2 per cent. aqueous solution sodium alizarin sulphonate.
6. 2 per cent. aqueous solution neutral red.
7. 1 per cent. alcoholic solution phenolphthalein.
8. Toluene.
H ron SoLutions. For Comparison.
Solution H,O to
No. M/10 KH, POs M/10 Na, HPOs make Pu Indicator
: sina at Ree ayes no ec. + oa ee; 500 c.c. 8.7 t Phenolphthalein
eee 5 + a sos 8.0
De marksmen 5.0 “ 4 25 « i a 7.4 t ho
he eeenieeaas 5.0 “ + 11.5“ caries 7.0 )
1
N/5 CH, COONa N/5 CH? COOH |
By dushiseeceney: 225 cc. + 230 ce. tS 6.7 :
BY cs oevearon 5.75 ob a ie i ke 6.3 ee,
(ae 11.50 “© 4 cw i 6.0 + cutehanete
ener 23.00 “ + sei 8 tes 5.7 P
DO ccaearenas 57.50 “ + ees tt * See 5.3
10, eden: 115.00 “ + 200 “ to ae 5.0 | | Methyl red
Ll. snanvacse 230.00 “ 4+ 230 “ Ne eae 4.7 J

Process. In a series of exactly similar 250 c.c. flasks place 10 c.c.
of each of the standard solutions indicated above, make up the solutions
to 250 ¢.c. with distilled water and add enough of an aqueous solution of
alizarine sulphonate of sodium so that the concentration of the latter is
about 0.0003 per cent. and is exactly equal in all cases. 10 cc. of
urine are now introduced into another 250 cc. flask and the same
amount of distilled water and indicator added. The color of the diluted
urine with its indicator is now matched with one of the standard
series. If the reaction as thus measured is more acid than P,,=—5.3
(H ion=NX50X10-") further tests are made with methyl red; for the
range 5.3—6.7 with p-nitrophenol and with neutral red; for more alkaline
urines phenolphthalein is used.

(a) H iron concentrations greater than 50X10—*. ‘‘ 10 ec. portions
of standard solutions are introduced into carefully selected colorless
test-tubes (15.51.7 cm.) and 10 ec. urine into another tube. To
match the color of the urine add to the standard solutions enough of
p-nitrophenol, methyl orange or bismarck brown. In case the pigment
is itself an indicator within this range of acidity the necessary amount
PRACTICAL WORK AND METHODS 1125

will vary with the reaction of the standard solution. Now add to the
standard solutions and to the urine 0.15 e.c. of a saturated solution
in 50 per cent. alcohol of methyl red and match the color with the
standards.

(b) H ion between 5010-7 and 2X10~-*. Carry out the estima-
tion in flasks as with the alizarine sulphonate, but use in place of the
latter p-nitrophenol 0.08 per cent.

(ec) Hion between 2xX10—7 and 0.8X10~-7. The estimation is made
as with alizarine sulphonate, but employ neutral red, 0.0006 per cent.

(d) H ion less than 0.3X10—-7. Match undiluted urine in test-
tubes against undiluted standard solutions, using phenolphthalein as
indicator, without previous coloration of the standard solutions.

Make all estimations in duplicate.

Note.—Albuminous urine presents certain difficulties with alizarin
sulphonate. Methyl red and p-nitrophenol are little affected by the
presence of albumin. Alizarine sulphonate changes too gradually be-
tween 0.3 and 2X10—". ‘Neutral red is used for this interval. Methyl
red is superior to p-nitrophenol for the greater acidities.

ADRENALINE.

Experiment 318. Quantitative determination of adrenaline (Folin,
Cannon and Denis, Jour. Biol. Chem., 13, 1913, p. 479).—Principle.
The method depends on the development of a blue color with phospho-
tungstic acid in alkaline solution. The adrenaline is estimated
colorimetrically by comparison with the color of a standard uric-acid
solution similarly treated with phosphotungstic acid. The reaction is
not specific. Other substances give the blue color also.

Process. Determination of the amount in the supra-renals. Thor-
oughly rub the weighed gland in a mortar with sand and N/10 HCl,
using about 15 e.c. of the acid for each 2 grams of gland, and about
45 e.c. of water. Transfer to a beaker and heat to boiling, to dissolve
the adrenaline. Add to the boiling mixture 5 ¢.c. of 10 per cent. sodium-
acetate solution for each 15 e.c. of HC] taken and again heat to boiling.
Transfer the whole mixture, except the sand, to a volumetric flask of a
capacity of 100 c.c. for each 2 grams of gland taken and dilute to the
mark with water. Filter or centrifuge as much of the solution as is
needed.

Colorimetric determination. Pipette 5 e.c. of the clear extract just
made into a 100 ce. volumetric flask, and into another similar flask
pipette 1 ec. of a fresh uric-acid solution containing 1 mg. of uric acid.
Add to each flask 2 ¢.c. of the uric-acid phosphotungstic reagent (ex-
perim nt 219) and 20 c.c. of saturated Na,CO, solution. After standing
2 or 3 minutes dilute each to the 100 c.c. mark with water, shake and
at once transfer to the Duboscq colorimeter and compare after setting
1126 PHYSIOLOGICAL CHEMISTRY

the standard at 20 mm. Calculate the adrenaline as if it were uric
acid and then divide the result by 3, since the adrenaline makes a color
6 times as intense as an equal weight of uric acid.

Tf the reading of the unknown tube in the colorimeter were 25
then the mgs. of adrenaline in the weight of gland taken would be
20/25X1/3 mgs.X20 (since 5 «ec. of the extract had been diluted to

100).
Abderhalden, diagnosis of
pregnancy, 550

Abomasum, 339

Abeseprion of foods, 451-
45

in cephalopods, 453

in intestine, 451

in stomach, 451

ee parts of tract,
5s

not due to osmotic pres-
sure, 452

of carbohydrates, 453

of fats, 452

of proteins, 453

of undigested protein and
anaphylaxis, 454

of water, 456

polyfistula dog, 455

role of leucocytes in, 455

ge as and literature,

Absorption of water by cells,
212-215

action of acid on, 213
ssbserpeion of water by gels,

Acetic acid, melting and boil-
ing points,
conductivity and inverting
power, 195
in chitin, mucin and mu-
coid, 325
Acetoacetic acid, from pro-
tein, 813
from phenylalanine, 815
from fatty acids, 75
Gerhardt’s reaction, 1074
Harding and Ruttan meth-
od, 1074
in urine, 759, 1074
salicylic aldehyde reaction,
1074

Acetone in diabetes, 781
in urine, detection, 1073
in urine, 759
iodoform test for, 1074

quantitative determina-
tion, 1116
Acetyl glucosamine ; see
in mucin, 325

Acetylation, process of, 824
Acetyl number, 74
Achroodextrin, 58, 327
Acid albumin, 113, 360
‘Acid fuchsin ‘as indicator of
acidity, 246
Acid, rigor of muscle, 628
Acidity, of blood, 539
of fasting stomach, 367
of Sastre contents, 346
364,
of sani “contents to any
ous indicators, 368
of muscle, 246
of stomach in various con-

ditions,

of urine, 688, 689

of urine gente
(Folin), 112

 

INDEX.

Acidity, réle in_protoplasmic
- motions, 628-630
Acidosis, and ammonia, 746
in diabetes, 760
Acids, action on water ab-
sorption, 213
effect on cell metabolism,

247
ionization of, 195
fatty, formulas and melt-
ing points,
neon of in cells,

Acree-Rosenheim tryptophane
reaction, 918
Acrolein, 66, 907
Acromegaly, 645
Acrose,
Adamkiewicz reaction
proteins, 148, 881
Adelomorphic cells, 353
Adenase, 166, 731
distribution’ of, in tissues,

for

Adenine, amount in nucleic
aci

chemistry and properties,
165

decomposition by acids,
166

decomposition by adenase,
166

derivation of word, 165
transformation to hypoxan-
thine, 731
Adenosine, 172

Adenosine desamidase, 732
Adrenaline, action on vari-
ous tissues, 674
amount in n glands, 671
formula, 6
cancion of oe 672
yperglycemia by,
liver necrosis by, 672

properties and prepara-
tion,

quantitative estimation,
671, 1124

quantitative estimation,
(Folin, Cannon = and
Denis), 4,

relation to sugar metabo-
lism,

ny to sympathetic,

secretion during anger,
675

Adsorption, 220, 241-242
Aerobic respiration 257
Aerotonometer, 479
@sthesin, 579

anaes of Wooldridge,

influence _on creatine
execution, 711
Agamatine, 441
Alanyl-leucine, formula, 131

Age,

Albumins, definition, 112
in urine, detection, 1067,
1069 :
Albumins, in urine, quanti-

1127

 

tative determination,
1060
Alanine, formula, 116
amount in various pro-
teins, 128, 129
Alanine hydrochloride, 120
Albuminoids, 112, 635
Alanyl- -alanine, 126
hydrochloride, 133
Albumose, Alberhalden meth-
od of preparation, 551
of whey, 376
Albumoses, formation in pep-
tic digestion, 349, 351
formation in tryptie diges-
tion,
occurrence in urine, 1069
physiological action, 452
preparation, 928
properties, 928
relation to blood coagula’
tion, 517
Albumosease of stomach, 381
aleaptons see Homogentistic

dAleaptonutia. T52
slecnol burned by muscle,

not a food, 301
Alcohols from waxes,
Alcoholic fermentation,
Alcoholysis, 99
Aldose, definition, 19

Separation from ketose, 44

ees to ketose,

Alexis St. Martin, 339 F
Alizari as indicator, 871
Alkali albumin, 113

Alkali reserve of blood, 1042
Alkalinity of blood, 539
Alkalinity of protoplasm,

247
Alkaline tide in the body,

Allantoine, chemistry, 740
determiniation, quantita-
tive, 110
from _urie acid, 734, 735,
740

in plants, 741
in urines, 721, 740

80
886

in various mammals’
ee 6 ;

synthesis from 0! lic
acid, 740 e70ry

Allanturie acid, formula, 740

Alloxan, formula, from uric
acid, 724

Alloxantin, formula, 725 _.

aor bases; see Purine -

base
Allozyproteic acid: in ae as
746

Ally! mustard oil,: 53 :

Allyl sulphide in mustard
ofl, 820

Almen’s test for

 

zlueose,
1m a 4

aimond oil, fodine number.
1128
Amann composition, 110,
mol. weight, 139
a o nitrogen, definition,
amount in various pro-
teins, 144

Amido; see Amino
Amines produced by
boxylase, 249
Amino acetic acid, 116
Amino-acids, absorption of,

453

car-

amount of, in various pro-
tamines, 128

amount excreted per day, |
746

amount in various pro-
teins, 129

isolation of by Dakin’s
method, 944

carbamino reaction of, 121

condensation with alde-
hydes, 121

definition, 116°

dissociation, 119

sormias of, graphic, 116-

deamidization

by
tion,
formation from ammonia
and glucose, 186
formation from
acids,
formation of lactams from,
124

oxida-

in metabolism, specific ac-
tion, 82: :

in urine, 746

necessary for growth, 822

nitrogen in urine, quanti-

tative, 1104
number of, in protein
molecule, 14
oxidation of, 123
pre-existence in protein

molecule, 115
precipitation by mercuric
aceatate, 12
properties, 118-126

optical properties, 125

racemization by alkali,
125, 126

resyothesis in intestinal
mucosa, 454

solubility, 118
taste of, 124
union with acids,
and salts, 119
Amino-benzoic acid, 764
Amino-caproic acid, 117
Amine ety! alcohol, 100,
oe
Amino-glutaric acid, 117
Amino groups, free, in pro-
tein molecule, 137
Amino groups determination
by Van Slyke method,
137,
Amino-guanidine
acid,
eng noole propionic acid,

bases

valerianic

Amino-lipins, 62

amine Hpptliniee see Amino-
pins

Amino-malonic acid, 184

Amino-malonic nitrile, 184

Amino-myelin and Petten-
kofer’s reaction, 577

Amino-myelin from _ brain,

/ 70, 577
Amino-myelin cadmium chlo- |:

.. Vide, 577

ketonic |

 

INDEX

Amino-nitrogen in proteins,

5;

Amino-nitrogen, _determina-
tion by Van Slyke
method, 935

Amino-succinic acid, 117

Amino-propionic acid, 116

Amino-valerianic acid, 116

Ammonia, amount excreted
per day, 691...
angst fo various tissues,

amount in various bloods,
702

by

excretion
746

in blood, 375

in intestinal mucosa, 454
in mesenteric vein blood,

autolysis of cells,

in urine, 689,

454
in mucosa, 375
in portal and hepatic vein
blood, 701 ’
oe after Eck fistula,
in urine after liver cor-
rosion, 699, 700
in _urine in liver disease,
699
quantitative determina-
tion, 1093
origin of, in liver, 701
variation with acidity, 689
Ammonium chloride in HCl
secretion, 374
Ammonium cyanate, 692
Ammonium sulphate, separa-
Gee of albumoses, 361

8
standard solution, 1084,
1010

Ameeba, surface _ tension,
movements of, 211
Amygdalin, 54
Amylase, action not acceler-
ated by bile, 412
determination of activity,

in blood, 550
of hemp seed, composition,
330

of malt, composition, 330
of pancrease; see Amylop-

sin
of pancreas, discovery, 329
of saliva; see Ptyalin
quantitative determination
(Robert’s method), 979
protection by  carbohy-
drates, 979
Amyloid from cellulose, 59
Amylolytic enzymes, distri-
bution in tissues, 329
aoe 398
iffers from ptyalin, 399

favorable reaction for,
979

properties and  prepara-
tion,

Ansrobie respiration, 41,

Anaphylaxis, by  artifictal
peptide, 135

none by racemized pro-

tein,
Anesthesia, increased excre-
tion of neutral sulphur

: n,
Anesthetics, effect on respi-

ration of brain, 5938
ane. with hemoglobin,

 

}

Animal proteins,
tion, table, 110
Animal heat, 269-297
a combustion, 270
history of discovery of na-
ture, 269
aud derivation of word,

compost-

Anthracene glucosides, 53
Antiaris toxicaria, protein

in, 143
Antithrombin, 534
Antiglyoxalase, 785
Antimony sulphide solution,

Autipyrine, excretion of, 762
Antoxyproteic acid in urine,

14
Appetite secretion of gastric
juice, 34
Aporrhegmas, 442
methylated, 442

Apricot kernel oil, iodine
‘number, 70 7
Araban gum in_ diatase,
330
Arabinose, 19, 59
formula,

Arabinulose, 19
Arachidic acid,
in butter, 64

formula, 64

_Arbacin, 179

Arbutin, 53
Arginase, 708
Arginine, amount
tamines, 178
amount in various blood
proteins, 553
amount in various
teins, 128, 129
formula, 118
seeempoat ion by bacteria,
4

in pro-

pro-

decomposition to ornithine
and urea,
aromee acids, fate in body,

Aromatic oxy acids of urine,
T47

Arsenic in glucose, 303
Artificial muscle, 629
Asbestos alter preparation

of,
Ascoli and Izar’s work on
uric acid,
Asparagine, 145
reaction with ninhydrin,

150

Aspartic acid, amount in
various proteins, 129

formula, 117

Asses’ milk, 314

Association of water, 191

Assurin, 570, 578

Asters in cells, dependence
on oxygen, 181

Asymmetrical carbon, action
on light, 22-25

Asymmetrical carbon com-

pounds, 22
Sota of linseed oll,

Autolysis of cells, action of
acid on, 248

Autolytic enzymes, 107

Auto-oxidation of cephalin,

575
of bilirubin. 416
of linseed oi], 68
of phospholinins, 97, 98
Azelaic acid. 67

Bacteria, to feces, 488, 445
putrefaction by, 488
Bacterial decomposition of
foods, 437
Bang’s method for elucure
(hydroxylamine), 1111
microchemical, 1113, 1115
Barfoed’s solution, 875
composition, 875
theory of action, 40
Basedow's disease, 659
Basic nitrogen, in various
proteins, 144
Sane goes, in chromatin,

Basic substances in urine,
746

Beaumont and Alexis St.
Martin, 339

Beech nut oil, iodine num-
er, 70

Beet, allows iodine number,

Bees vee composition, 80,
iodine number, 71
Behenic acid,
Bence-Jones protein in urine,
157, 9
Benedict and Denis’ method
for total sulphur in
urine, 1106
Benedict and Murlin’s amino-
acid method, 1104
Benedict’s copper’ solution
for carbohydrates, 873
method for glucose in
urine, 1109
method for urea, 1085
quantitative determination
of glucose, 1109
Benzene, fate in metabolism,

A
Benzoic acid, ingestion, ef-
fect on hippuric acid ex-
cretion, 743
ingestion, effect on urea
excretion, 70
source of, in body, 744
Beri-beri, 339
Bernard's work on source
oes HCl of stomach,

work on sugar metabolism,
768

Beta-albumosease in gastric
juice, 381
Beta-alanine from carnosine,

Beta-hydroxybutyric acid
(also called _beta-oxy-
butyric acid), 75

Betaine, an aporrhegma, 442

formula, 92

leta-keto palmitic acid, 76

Beta-oxy-a-amino butyric
acid in cephalin, 573

Beta oxybutyrie acid;
Oxybutyric acid

Beta - parahydroxyphepy! -
amino propionic acide

see

1
Beta phenyl-a-amino
pionic acid, 117
Bile. 406-437
acids: see Bile salts
ae on fat digestion,
amount increased by bile
ingestion,
cout secreted per day,
4

0
bladder bile, 408, 433
cholesterol in, amount,

origin of, 432

pro-

32, 986
cholesterol,

 

INDEX

Bile, circulation of, 412
composition, 407

dissolves cholesterol, 414,

dissolves fatty acids, 413
ae acids in, amount of,

fistula bile, 408
freezing point, 407

function in absorption,
411, 412

functions of, 411

general properties, 407

Gmelin’s test for,

Hammarsten reaction, 984

Huppert-Cole test for, 984

hydrogen ion  concentra-
tion, 407

icterus, 407

a aa putrefaction,

laxative action, 414
lecithin in, 41

method of obtaining,
of new born, 427
phospholipins in, 435
practical work on, 983
reaction of, 407

ge a into stomach,

437
secretion of,
secretion of, increased by
blood poisons, 419
sulphur in, in various ani-
mals, 427
summary of functions, 414
taste, 407
Bile pigments, 411-423
amount depends on blood
decomposition, 414
bilipurpurin, £17
bilirubin, compesition, 414
biliverdin,
cholohematin, 418
hemibilirubin, 417
hydrobilirubin, 417
literature references, 448
origin in blood, 416
origin in hemoglobin, 419
oxidation products, 415
relation to chlorophyll, 423
stercobilin, 417
urobilin, 417
relation to hemoglobin, 423
Bile salts, 422-432
amount of various acids in
bile, 428
choleie acid,
431

407

phosphoprotein a

properties,

eholic acid, properties, 428
functions, 423
glycocholic acid, decompo-

sition, 425

glycocholeic acid, proper-
ties, 430

glycocholic acid, prepara-
tion, formula, proper-
ties, 425, 985

glycocholic acid, separa-
tion from taurocholic,
425

Hay’s test for, 984

laking of blood by, 497

place of formation. 424

Pettenkofer’s reaction,
424, 985

Platner's bile, 424, 985

preparations and _ proper-
ties, 424, 985

precipitation by ammonium
sulphate, 425

Telation to fat digestion
and absorption, 412

 

1129

Bile salts, taurocholeic acid,
properties 431
taurocholic acid, increased
by ingestion of cholic,
428
taurocholic acid, proper-
ties, 426
time of appearance in
ontogeny, 424
variation in different ani-
mals,
Biliary fistula, 408
Billpurpurin, 418
Bilirubin, formula and prop-
erties, 416
Huppert-Cole reaction, 984
Billverdin, formula, proper-
ties and preparation,
416-417
in sea-anemone, 417
Biogene, 261, 631, 852
Bioses, 19
Biot, rotatory action of dex-
trins on light, 327
Birds and reptiles, nitrogen
excretion, 700
Birotation ; see Mutarotation
Bismuth subnitrate test for
glucose, 876
Biuret base, 133
Biuret, formula and forma-
tion from urea, 144, 692
reaction of proteins, 144,

Black’ S reaction for oxybuty-
ric acid, 1074
Blaze current, 245
Blood, 458-562
alkali reserve, 1042
alkalinity of, 539
amino-acids in, 472
amount in body, 466
amount of plasma, 468
amylase in, 55'
analysis, quantitative, sys-
tem, 1007
arterial and venous, differ-
ence, 473
as a food carrier, 469
as living protoplasm, 459
carbon dioxide in, 476,
1042

changes by fasting, 836
cholesterase, 551
cholesterol in, 468
circnlation of, 458
composition of,
461-463, 468
conductivity, 549
creatine and creatinine in,

709
detection of, 988

general,

dextrose in, 470, 1021,
1028

dissociation of oxyhemo-
globin,

enzymes in, 549

fat in, 470

freezing point, 547-549

freezing point changed by
secretion of gastric:
juice, 346

functions of. 459

gases in, 47°

glycolysis, 550

hemoglobin, decomposition
products, 508

hydrogen ion content. 540,

hydrogen ions, effect of
CO, on, 545

invertin in. 550

laking of, 497

Hpase, 550
1130

Blood, metabolic co-ordina-
tion by,
microscopical _ test
1003
mrp nology of, 463-468
occult, in feces, 988
benzidene reaction, 988
osmotic pressure, 547
oxygen carrier, 473, 496
physico-chemical factors of
circulation, 512
practical work on, 990-

for,

protease, 550

reaction of, 539

respiration. of, 490

respiratory exchange, 482

respiratory function, lit-
erature, 563

ema of oxygen carry-
ng,

urea content before and
aaa kidney extirpation,

urea in, 472

urea in, before and after
perfusion, 696

viscosity, 512-514

vividiffusion, 469-470

waste substances in, 509

waste substances in, in
various diseases, 509

Blood clotting, 514-538

a crystallization, 523

antithrombin, 535

author’s suggestion of na-
ture of, 5388

ealcium salts in, 534

cepa action on, 518

contact with foreign sub-
stances, 518

fibrin, 522

fibrinogen, 522

Fuld and Spiro and Mora-
witz view, 534

hemophylia, 519

hemorrhage, effect on, 518

hirudin,

Howell's view, 534

influence of contact with
- tissue, 515, 519

intravascular and blood
plates, 467, 531

literature, 558

Nolf’s view, 534

not due to thrombin, 533

optical phenomena, 523

physiological variation in
tendency to clot, 519

practical experiments,
1004, 1006

prevention by albumose,
517

prevention by leech ex-
tract, 518
prevention by

oxalates,
fluorides and
520

citrates,

prevention by salt solu-
tions, 520
prothrombin, 532
relation to laking, 525
a of structural elements
n,
summary of, 536
syneresis of clot, 514
thrombin, 5 4
thrombosis of veins, 517
tissue extracts, action of,
518-520, 1005
Wooldridge’s view, 535
Blood corpuscles, determina-
tion of number, 990

 

INDEX

Blood corpuscles, destruction
lait ea bilit
ng an permea) y,
497, 996

Pe action on alkalinity,

red, amount of surface,
466
red, as source of blood
proteins, 557
red, composition, 461, 499
red, length of life, 496
red, lipins in, 499
red, motility, 465
red, number of, 474
red, volume in arterial and
venous, 476
white, composition, 463
white, motility, 464
Blood plasma, a dilute pro-
toplasma, 554, 536
amino acids in, 472
composition, 461, 462
enzymes in, 551
fluoride, 520
origin and function of pro-
teins of,
oxalate, 530
peptone, 518
proteins in, 551
salt, 520
specific gravity, 461
Blood platelets, 466, 529
composition of, 531
liquid crystals, 530
literature, 558,, 562
preparation of, 468
relation to blood proteins,

557
relation to clotting, 521
Blood serum, composition,

468
formation, 514
Boas reagent as indicator,

370, 97

Body, resembles a magnet,
266

considered as a machine,

Boiling point and osmotic
pressure, 204

Bombicesterol, 87

HOME somporiion of, 636-

inorganic constituents, 640
Bone growth, 640
Bone marrow, relation to
fibrinogen, 554, 557
Bone oil, fodine number, 71
Borget breakfast, 369
BOE: nee: influence on hair,

influence on oiliness of
skin,
Béttger’s test for glucose,

Brain, 565-598
esthesin, 579
amino-lipins or

lipotides, 580
amino-myelin, 577
assurin, 578
autolysis of, 589
bregenin, 578, 580
“buttery substance,” 569
cephalin ; see Kephalin
cerebronic acid, 76
cerebrosides or

sides, 578
cerebrospinal fluid, 585
cerebro-suluphatides, 580
chemical difference from

other tissues, 568
cholesterol, amount, 581

amino-

glacto-

 

Brain, copper in, 574
corpus callosum, composi-
tion, 583
oe amount of, 582,

d ot in o-diphosphatides,
oa
diem inio-menuphoaphatides,

energy involved in, 594
extractives, 570, 581
formic acid, 582

aly teneine (caprine),

glycolipins, 578
erey matter, composition,

gray and white matter,
difference, 583

human, composition, 584

hypoxanthine, 581

inorganic salts, 583

inosite, 582

kephalin, preparation and
properties, 573

kerasin, 578

krinosin, 580

lactic acid, 582

lecithin cadmium chloride,
properties, 571

lecithin, preparation and
properties, 570-572

lignocerie acid, 578, 579

medullary sheaths, compo-
sition and function of,
586

myelin, 576

neuroplastin, 583

no neutral fat in, 568

nucleie acid of, 585

“oily substance,” 569-570

ore of psychic qualities,

ee consumption by,
paramyelin, 576
phospholipin, method of

separation, 568
piseynenus, importance of,

phrenosin, 578
phrenosterol, amount, 581

physiological interpreta-
tion of chemical compo-
sition, 586

protagon, 580

proteins of, 584

protoplasm, physical con-
sistence of, 585

psychosin, 579

reserve carbohydrate lack-
ing, 588

respiration of, 590-593

specific gravity, 584

sterols, 581

sphingol, 577

sphingomyelin, 577

spingomyelinie acid, 577

spingosin, 577

structure of nerve cells
andfibers, 566

succinic acid, 582

sulpholipins, 480

sulphur of, 580

summary, 595

taurine in, 580

water in, 568

weight of human, 584

whe matter, composition,

““white substance,” 569
Brazil nut ofl, fodine num-

ber, 70
Bregenin, 62, 578, 580
BEoHunes oxidizing power,
Brom phenrhmercaptarie acid,

Bromphopionyl chloride, 134
Brownian movement, 243
Brunner’s glands; see Duo-

denum
Buffer solutions, 544, 1059,
1122

for urease, 1014 c
Burchard-Liebermann_choles-
terol reaction, 82
Butter, coun matter, 64
composition, 64
Butter fat, 64
iodine number, 71
Reichert-Meissl number, 74
relation to growth, 844-

848
saponification number, 74
Buttery substance from
- brain, 569-570
n-Butyl mercaptan, 820
Butyric acid, formula and
‘properties, 74
oxidation of, 75
amount in butter, 64
Butyro-betaine, 442

Cadaverine ; see Pentamethy-
lenediamine
Cadmium sulphide solution,

2
Caffeine, fate in body, 765
Calcium, quantitative deter-

mination, blood, 1039;
urine, 1108
in urine, 756
Caleium salts, relation to
bone growth, 6
relation to clotting of
blood, 520

Calorimeter of Atwater, Rosa
and Benedict, 283-286
ice, of Lavoisier, 272
of Rubner, 281
water, of Dulong, 278
Camphor, excretion of, 759,

formula, 79
movement on water, 209
Can gastrie acidity in,

Cane sugar; see Saccharose

Capillary method of surface
tension, 207

Capillarity ; see Surface ten-
10n

s
Caprine, formula, 117, 582
Caproic acid, formula, 64

in butter, 64
Capryliec acid, formula, 64
Carbamic acid in urine, 764
Carbamie acid and urea, 691

Carbamino compounds in
blood, 477
Carbamino -_ reaction of

amino-acids, 121
Carbodiimide, 692
Carbohydrates, 16-60

absorption of, 453
ae in blood plasma,

amount in  mucoids and
mucin, 32:

bacterial decomposition,
440

classification, 19
decomposition by acids, 35-
decomposition by alkali,

30-34
definition, 16-18

 

INDEX

Carbohydrates, dissociation
constants of, 18
excretion in intestine, 453
fermentation by yeast, 886
influence on intestinal pu-
trefaction, 445
blood, determination,
1021, 1028
in nucleic acid, 169
in urine, 758, 1109
metabolism, 771-797
metals reduced by, 38
Molisch reaction for, 151
occurrence, 16
osazones, 43
oximes, 42
pee! experiments on,

in

properties, 17

reaction with hydrocyanic
acid, 42

reduction of, 41-42

nou of muscle energy,
union with acid, 18
union with alkali, 17
Carbon, dioxide, acceleration
foe digestion by,
action on blood clotting,
526
amount excreted per day,
293
in blood, 475,
amount produced by vari-
ous tissues, 490
combined in blood, 476,
1041

hemoglobin, 495
pee in alveolar air,

amount
1041

pressure in blood, 477

pressure in tissues, 477

production by nerves, 592

relation to uric acid syn-
thesis, 739

Téle in dissociation of oxy- |:

hemoglobin, 476
secretion of by lungs, 480
meen with amino-acids,

Carbon income and outgo,
292

Carbon monoxide hemochro-
mogen, 508 i
Carbon monoxide, method of
determination of satura.
tion of blood, 480
toxicity, 495
Carbon monoxide
globin, 140
see Carbonyl hemoglobin
Carbonyl hemoglobin, prop-
erties, 494
recognition in blood, 1000
spectrum, 493, 1000
Carboxyl groups, number in
protein molecule, 137

hemo-

Carboxylase decomposition of

amino-acids, 441
Cardiod condenser, 216
Carnauba wax, 8

iodine number, 71
Carnaubon, 99, 100
Carnaubyl alcohol, 81
Carnie acid, 310, 534, 613
Carniferrin from milk, 310
Carnine; see Inosine
Carnitine, in muscle, 610
Carnosine, 610
Carnutine,- 92
Carotin in butter, 64
Carotin in milk, 311

 

1131
Cartilage acid; see Chon-
droitic acid
ene chondromucoid,
come on 324, 325,
637
relation to chitin and
mucin,
structure, 637
oe autolysis in,

chemical changes during,
182

sap,

sensitivity to erpers 182

Caseaniec acid, 30:
i in clotting,

relation to nuclear
181

composition, 110, 308
ease of digestion, 356
ee weight, 139, 142

of different animals, 308

preparation, 94

properties, 308

structure of clot, 282
Caseinic acid, 308
Caseinogen ; see Casein
Castor oil, 65, 66

iodine number, 70
Catabolism, 9

of amino-acids, 810

of glycocoll, 810

of proteins, 807
Catabolism of tyrosine, 812
Catalase, 256

in saliva, 334
Catalysis, definition, 10

explanation, 250-256
Cataphoresis of colloids, ap-

paratus, 218

Cation, derivation of word,

Cell, colloidal
12

organization of, 11, 12

phospholipins in, 101
Cellulose, 19, 58, 59

in animals, 59
Cephalin; see also Kephalin
cm amount in milk,

constitution,

amount of glycerol in, 99
bases in, 100
relation to clotting, 516
Cerebric acids, 570
Cerebrin, 570, 578
preparation of, 911
Cerebron, 578
Cerebronic acid, 579
Cerebrosides, 570, 579
hydrolysis of, 912
see Glycolipins
Cerebrospinal fluid, composi-
tion, fe
Ceryl alcohol, 81
Cetyl alcohol, 81
Chemical resistance, 253
Chemotaxis of leucocytes,

Chenocholie acid, 432
Chevreul,
Chicken muscle,
tion, 605
Chitin, composition, 325
relation to mucin, 324
Chitosan, 326

composi-

Chloral hydrate, excretion
of, 759, 763
Chlorides, determination,

in blood, 1034, 1036
Vollhard method. 1107
In urine, 756, 1107
1132

Chlorine, amount in various
tissues, 372
in gastric juice, 346-
relation of jamount_ se-
_creted in gastric juice
to all in body, 347
in cells, distribution,
microchemical, 373
Chloroform poisoning, disap-
pearance of fibrinogen,

Chlorophyll, chemistry, 48
eo to bile pigments,

relation to synthesis of

dextrose,
Cholalic acid; see Cholic
acid
Choleic acid, 431
Choleprasin, 416
Cholesterase in blood, 551
Cholesterin; se¢ Cholesterol
Cholesterinic acid,
cholic, 429
Cholesterinie acids 85
Cholesterol, 81-86
absent in hippopotamus
Dile, 433
amount in different brains,

from

amount in nervous tissue,
84
amount in various tissues

chemistry of, 85-86
ae reactions, 82, 83,

discovery of, 81

Cholesterol, in atheromatous
arteries, 8

in bile, 432, 986

in blood serum, 85

in brain, 570, 581

in butter, 64

in cerebrospinal fluid, 87

in egg ofl, 84

in food and excretions, 435

in natural oils, 64

physjological action of, 86

preparation from _ brains,

properties, 81

quantitative determina-
tion,

quantitative determina-
tion, colorimetric, 84

Cholesterol esters, 82

in brain, 84

in egg oil, 84

in commercial lecithin.

85
Coole acid, amount in bile.
color reaction with HCl,

430
formula and _ properties,

ingestion, effect on _ bile,
preparation, 428
pele to cholesterol,

separation from choleic,

etc.,
Choline, amount in various
tissues, 95
chemistry of, 92. 93
decomposition, 94
from lecithin, 91
in sinalbin, 53
in sphingomyelin, 577
microchemical tests

for,
95
Choline pertodide, 94

 

INDEX

Cuelte physiological action,

quantitative determina-
tion, 94
Choline PtCl, contamina-

tion by KCl, 572
Cholohematin, 418
Chondroitic acid, chemistry,

324, 325

graphic formula, 325
in eggs,
in mucin and mucoid, 324

Chondroitin, 325
Chondromucoid, 637, 639
Chondrosin, 325
Cbondroproteins, 637
Chromafiine tissue, 668
Chromatin, basic constitu-
ents, 176
composition, 162-179
in living nucleus, consist
ence, 158
of sperm, 178
nucleic acid in, 164
Chromoproteins, 113, 155

Chromosomes, sudden ap
pearance, 159
Chyle, 452

Chyme, reaction of, 365
Chymosin; see Rennin
Cirrhosis of liver, and urea,

Citrates, influence on blood
clotting, 520
Citric acid in milk, 310
Citronellol, 80
Classification of
hydrates, 19
Classification of fats, 61
of ee 61-62
Classification of oils, 62
of proteins, 112, 113, 114
Clotting of blood, practical
work,
see Blood clotting
Ce of milk, in stomach,
349, 376-380

earbo-

by proteases, 377
Clupanodonie acid, 65
fodine number of, 71
Clupein, amino-acids in, 128
composition, 11
elementary analysis, 177
Coagulated proteins, 113
Coagulins, 516
Cocceryl alcohol, 80
Coccoon, amino-acids in, 129
Cocoa butter, iodine number,

a
specific gravity, 66
Cocoanut oil, 64, 71
Reichert-Meiss] number, 74
saponification number, 74
Rone yer oil, iodine number,

valuable in growth, 846
Coenzyme of pancreas, 397
Cole’s method of distinguish-

ing lactose and glucose
in urine, 1070
Collagen, composition, 110,
636
precipi-

Collodial albumin,
ated by salts, 224
Colloidal metals, size of par-

ticles, 215
Colloidal nature of enzymes,

Collotds, 218-244
Brownian movement, 243
cataphoresis of, 218
erystalloid gels, 230

degree of dispersion, 215

emai and suspensoids,
Colloids, gel structure, 229-
230

inua ace of valence in pre-
cipitation, 224

importance in cell life, 12

laws of precipitation, 225

meaning of word, 214

origin of electric charges,
219-222

osmotic presure of, 2438

particles _ electrically
charged, 217

preepation by salts, 224,

properties of, 214
size of particles, 215
syneresis of gels, 233,
235
Tyndall phenomenon, 217
Colorimetric wethod of esti-
é mating saturation of
hemoglobin, 480
Color reactions of proteins,
144-152
Colostrum, composition, 307
Combustion and_ electrical
phenomena, 258
Compound proteins; see Con-
jugated proteins
Conalbumin in egg white, 315
Condensation of amino-acids
to proteins, 133
Conductivities, equivalent of
various acids, 195
Conductivity of blood, 549
Conglutin, composition, 110
Congo red as indicator, 371
Congo red in stomach titra-

tion,
Coniferin, 53
Conifery] alcobol, 53

Conjugated proteins,
fication, 113
Connective tissues, composi-

tion, 634-642
white, 634
Conservation of energy in
the body, 276, 281, 290
Contractile tissues, 600
Contraction, mechanism

627
Convicin, 168
Cooking, importance in
gestion, 319
Copper in brain, 574
Coprosterol, 87, 434, 435
comroues amino-acids

8
Cornaro, Louis, 801
Corn oll; see Maize oil
Corpora lutea, action of ex-
tracts of, 678
ree to milk secretion,

classi-

of,

di-

in.

relation to pregnancy, 679
Corpus callosum, 567, 583
Corpuscles, birds, method of

separating nuclei, 162
blood ; see Blood corpuscles
of pees determination of,

Corylin, composition, 110
Cotton-seed oil, 67
jodine number, 70
Creatine, acetoacetic acid af-
fects determination, 712
amount in blood, 609, 718
amount in muscle, 609
sa in various tissues,

dostruction in tissues, 717

 

determination of, 712
Creatine, execretion after in-
Paro 2 creatine, 711,
713,

eeureton. during menstrua-
tion,

excretion in children, 711
712

excretion in fasting, 714

excretion, influence of thy-
roid,

excretion under
conditions, 711

in blood before and after
kidney excision, 717

in brain, 582

influence of carbohydrate
food ou excretion, 715

in formative metabcilsm
of muscle, 619

in liver autolysis, 717

in men's urine, 706

in muscle extract, 609

in urine, chemistry of,
705-721

in various muscles, 610

origin, 609, 71

preparation from meat,
958

various

properties, 707

sexual difference in excre-
tion of, 711

significance of, 709

transformation to crea-
tinine, 717
variation with stimula-
tion, 609

Creatinine, amount a blood,
amount in various tissues,
709
chemistry and properties,
708-709

coefficient, 711

excretion, after
ingestion, 716

execretion after guanidine
acetic acid ingestion, 716

arginine

excretion, after ingestion
of lecithin, 717

excretion after methyl
guanidine ingestion,
T17

excretion, effect of crea-
tine ingestion, 711,
714

excretion, effect of, crea-

tinine ingestion, 711.
excretion, cifect of fasting

on, 714

excretion, effect of thyroid
on

excretion, independent of
protein intake, 706,
710

excretion, variation with
weight, 706

in blood, 1017, 1027
in mascle, 660
in urine, amount per day,

691,

Jaffé’s reaction, 1065
literature, 769 .
picramic acid reaction,

preparation of, from urine,
1095

quantitative determination

(Folin), 1093, 1017,
1026

relation to muscle metabo-
lism, 719

summary, 720

Weyl's nitroprusside reac-
tion, 1065

 

INDEX

Creatinine zine chloride,
1064

Crenilabrin, amino-acids in,
128

Cresol in urine, 749

Cretinism, 659

Croton oil, 65

Cryptorrhetic tissues, 643-
684

scheme of interrelation,

no

Crystallization of fibrin,
523

Crystalloids, distinction from
colloids, 214

Cucurbitol, 87

Cuorin, preparation, proper-
lies, 95, 96” /

Current of Ae 245-248

Cyanamide, 692,
Cyanaminoe as idleniae, 246
microchemical indicator of
acid, 374
Cyanides, relation to respira-
tion, 592
Cyanurie acid, formula, from
urea, 692
Creliteria, amino-acids in,

Cymene, fate in body, 763
Cyprinin, amino-acids in, 128
Cysteine, conversion to tau-
rine, 426
formula, 117
nitroprusside reaction, 135,
151 es
Cystine, amount in various
proteins, 129
bacterial decomposition of,
442

catabolism of, 816
crystals of, 117
formula, 117
ingestion increases tauro-
cholic acid, 428
mercapitans from, 442
Cystinuria, 818
Cytoplasm, nucleic acid in,
174
phospholipin in, 101
phosphoric acid in, 174
proteins in, 155
Bag to nuclear sap,

Cytosine, amount in nucleic
acid, 169
color reaction (Murexide),
168
formula and _ properties,
168
origin in nucleic acid, 164
Cumiec acid,
Cuminurie acid, 763

Dakin, racemization method,
amino-acid method, 944
Deen aten, by bacteria,

by oxidation, 122
during absorption of pro-
teins, 454
Defibrination
554
Dehydrocholic acid, 429
Demethylation in body, 764
Deoxycholeic acid, 430
Derived proteins, classifica-
tion, 113
Desoxycholic acid; see Cho-
leie acid
Deutero-albumose, 361, 929
Deutero-protose; see Deu-
_teros albumose

of animals,

 

1133
Dextrins, 19
achroo-, 58, 327
Dextrins, by pancreatic di-
gestion, 399

by salivary eerien 327
erythro-, 58, 327
from starch, 327
practical work on, 963
Dextrose, acids from, by ox-
idation, 33
amount in blood, 470
changed to levulose, 31-32
condensation with am-
pute to amino-acids,

condition in blood, 471
cons:mption by heart, 783
Heceu eaten by alkalies,

decomposition
790, TOA

dissociation constant,

Hap mentaeen

in muscle,

18
in muscle,
formed from amino-acids,
TT

glycolysis in muscle, 624
mutarotation of, 50
osazone, 43

oxidation by bromide, ete.,

and I ehling's solution,
38-39
oxidation by ‘body, rela-

tion to pancreas, 781
oxime, 43
properties, 50
reduction of, 41
relation to anwrobic respi-

ration, 42
structural formula, 28
synthesis in plants, 44
union with hydrocyanic

acid, 42

Dextrose metabolism ;
Carbohydrate
lism

Dextrose to nitrogen ratio,
in urine, 776

Diabetes _ insipidus,
peepee

651,

gee
metabo-

after

extirpation,
Diabetes Te itn TH
cause of, 787
excretion of acetone bodies
in, 781
relation to pancreas, 781
Diabetes, phlorizin, 776
Diacctic acid; see Acetoace-
tie acid
quantitative determination,
1116
Diacid amides give biuret
test, 145
Dialuric acid, from uric acid,
720

Dialysis, definition, 217

Dialysis experiment, 923

Diamino-diphosphatides,

Diamino-monocarboxylic
acids, 118

Diamino-monophosphatides,
577

578

Dia min o-trioxydodecanoic -
acid, 308

Diastase, definition, 329

see Amylase

Diazo-henzene sulphonic acid,

Diner. ey: of indoie,

Dielectric constants. of vari-
ous liquids, 192

Diet, carbohydrate and
amount of ptyalin, 334
1134

Diet, effect of, on bile secre-
tion, 410
effect of,
cosity, 5 .
effect of, on intestinal pu-
trefaction, 445
importance of lipins, 309
low protein, effect on uri-
nary nitrogen, 710
relation to longevity, 801
sterilized milk, 313 :
Diethyl methyl sulphonium
hydrate, 820
Digestion, bacterial decompo-
sition,
party transformation in,

on blood vis-

formation of feces, 438

general discussion, 319

of fats, in stomach, 350

of potene in stomach,
34

peptic, increase of acid
combining power during,

peptic, increase of free
amino groups, 363

peptic, of proteins, 360

protein, by pancreas, 400

rate of, as shown by car-
bamino reaction, 122

salivary, 319-337

salivary, inhibited
products of
333

by
digestion,

starch, by saliva, 327
stomach, 338-386 ‘
summary of digestive
changes in food, 446
Dihydroxystearic acid, 65
Diketopiperazine from am-
ino-acids, 124
Dimethy! amino-benzaldehyde
reaction of mucin, 326
Dimethyl amino-azo-benzene
as indicator, 371
Dimethyl ethyl pyrrol, for-
mula, 415
Die guanidine in urine,

Dioxyacetone, 19, 66

Digxy eauucsubcre acid,
8

Dioxyphenyl alanine, 676
Direct vision spectroscope,

998
Disaccharides, classification,
19, 54

composition of various, 19
Disaccharides, from mono-
saccharides by alkalies,
ahcompeateen by alkalies,
properties of various, 54-
relation of Barfoed’s solu-

tion, 40
Hydrolysis by acids, 35-37,

Dissociation constants of
carbohydrates, 18
Dissociation constants of

various acids, 257
D:N ratio in diabetes, 776,

793
Delpnin oll, iodine number,

Doremus ureometer, 1091

Drechsel’s theory of urea
formation, 704

Drying oils, composition, 67

Ductus choledochus, 407

Dulong and Depretz, experi-

 

INDEX

ments on animal heat,
247
Dulong calorimeter, 278
Duodenal secretion, 387
enterokinase in, 387
invertin in, 388
lactase in, 388
H ion content, 387
maltase in, 388
quantity, 387
Duodenum, excretion of
monosaccharides in, 453
excretory function, 389
extirpation of,
other functions than di-
gestion, 388
paslalne, ae ;
ysplasia adiposo genitalis
646, 647 ‘

2

Eck fistula, 697

metabolic changes by,
698

Edestan, 113

Edestin, composition, 110,

dichloride, 137
molecular weight, 139
monochloride, 137
preparation, 925

Egg oil, composition, 317
iodine number, 71

Eggs as food, 314
Bee ig white,
Sone tten, yolk, 314,

conalbumin in, 315
hematogen in, 317
literature, 318

mineral substances in
yolk, 317

ovalbumin, 315
ovoglobulin, ovomucoid,

ovomucin, 315
proteins in white, 315
proteins in yolk, ovovitel-

nD,
Ehrlich, diazo reaction, 919
Ehrlich’s dimethyl-amino-
pean nyae reaction,
reaction of chitin, 326
reaction of mucin, 326
Elasmobranchs, function of
urea in, 5
Elasmobranch muscle, urea

in, 6
Elastin, composition, 110,
129, 636

preparation, 636

Elastin, use in detecting
pepsin, 357
Electrical basis of oxida-

tions, 256-257
charges on colloids, 218
Mouele layer on colloids,

phopenena blaze current,
phenomena, of protoplasm,
244 ‘ ‘

Eleomargaric acid, 65
Emulsification of fats; by
pancreas, 395
of fats by soaps, 222, 910
Emulsin, 54
Emulsion, definition, 230
structure of protoplasm,

Emulsoids, 217

Enamel, composition, 641

Energy changes of proteoly-
sis, 365 ;

 

Energy, conservation of, In
animals, 8, 276-288
of living matter, 8
Endothelium of blood ves-
sels, function, 420, 558
Ethol of acetoacetic acid, 75
of peptides, 126
of xanthine, 167

reaction; see Gerhardt’s
reaction
Enterokinase, 387
action on trypsinogen,
401, 980
experiment on, 980

Enzymes, active principle a

protein, 330, 400

combine with substrate,
331, 357

definition, 10, 256

determination of activity
of amylase, 3.

endocellular, 256

glycolytic, 550

in blood, 549

in nucleus, 182

kinds of, in cells, 256

a velocity of amylase,

nuclein cuore _ 131
pepsin an pepsinogen,
353-354
pepsin, law of velocity,
358
protective action of sub-
strate, 331
ptyalin, conditions of ac-
oe 3
resemble substrate, 330
reversible action of, 255
Episaccharic acid, 169
Hauiierinne of reactions,

Erepsin, in intestine, 403
in pancreas, 402
of pancreas, not activated
by enterokinase, 405
in saliva, 334
in stomach, 381
intestinal mucosa, 983
Erucic acid,
iodine number of, 71
Erythrodextrin, hexose
groups in, 58
Erythrose, 19
Erythrulose, 19
Esbach's method for albumin
in urine, 1068
Esocin, amino-acids in, 128
Essential oils, 62, 78-80
Esterases, 256
Eaten Seeton of amino-acids,

Ethal, 81
Ethereal sulphate, excretion
amount, per day, 748,
821
in bile, 427
in urine, 744

in urine quantitative de-
termination, 1106
Ethyl ,2cetate, equilibrium,

5
Ethyl amine from alanine,
Ethyl

Ethyl-methyl-amino propionic
acid, 117
Ruglobulin, 552
Ewald test breakfast, 342
Execlsin, 197
composition, 110, 129, 189
molecular weight, 139
preparation, 926

mercaptan in feces,
Exeretions, carbon dioxide
per day, 6
nitrogen per day in skin,

of the body, 685

urine; see Urine

water in urine, feces, ete.,
686

Exocellular enzymes, 256
Ege es: of brain, 570
of connective tissue, 636
of muscle, 608-615

Fasting, chart of fasting ex-
periment, facing, 836
effects of, 835
effect on creatinine and

creatine, 714
effect on uricolysis, 738
from water and mineral
substances, 837
loss of weight of different
organs during, 835
metabolism in, 833-836
rejuvenescence by, 835

Fats, absorption of, 452
acetyl number, 74
absent from brain, 568
as muscle foods, 291
crystallization of, 65
definition, 61
digestion and absorption

influence by bile, 411
digestion of, by pancreas,

digestion of, in stomach,
35

emulsification of, by soap,
222

heat by metabolism, 295
history of discovery of na-
ture, 62

identification of, 69-71

in surface films, 65

iodine number, 72

iodine numbers of, 72

location in See 78

melting points,

oxidation of, 75

physiological value, 74
Reichert-Meiss] number, 74

relation to leucocytes, 454

resynthesis of, 452

saponification of, 72

saponification number, 73

see also Lipins

staining reactions of, 78

syuthesis of, 76-78

volatile, in butter, 64
FE acids, from waxes,

identification of, 69-71

in bile, amount, 432

iodine numbers of, Ti

melting points and for-
mulas, 64, 65

oxidation of, 75

synthesis of, 76-77

Fatty oils, 61

classification, 62

influence on surface ten-
sion, 209

see Oils

Feces, amount per day, 292

bacteria in, number of,
439

basterial nitrogen, 439

composition, in absence of
bile, “411

composition of, 294

decomposition of
acids in, 441-443

energy in, 292

amino-

=)

 

INDEX

Feces, formation of, 438
nitrogen in, per ‘day, 4389
“water in, 292
weight per day, 439

Fehling’s solution, composi-
tion of, 38
explanation of action on
carbohydrates, 38-40

practical, 872
Fermentation, alcoholic, 886
ot glucose, 794
of various by
yeast, 24

sugars

Ferments; see Enzymes
Ferric chloride,

indican test,

1
Ferric hydrate as_ colloid,
221

Ficoceryl alcohol, 81

Fibrin, amount in arterial
and venous blood, 555
composition, 110, 139, 144
distribution of nitrogen in,

molecular weight, 139
origin of, 524, 554
origin of, in blood plates,
530
phospbolipin in, 525
relation to fibrinogen, 524
reformation of, 555
variation in amount in
leucocytosis, 552
ferment ; see Thrombin
Fibrinogen, amount in blood
plasma, 552
composition, 139
Fibrinogén, A-, of Wool-
dridge, 467
molecular weight, 139
properties, 526, 552
relation to blood clotting,

relation to blood viscosity,

51
relation to fibrin, 523
Fibrinoglobulin; see Serum
fibrinogen
Fisher’s hydrolysis of pro-
teins, 127

Fish muscle, amino-acids in,
608

Fish oil, iodine number, 70
Fish scales, guanine in, 165

Fletcher, and diet, 8

Fluorides and blood clotting,
5

Folin, ammonia, aération

method, 1081

and Benedict, creatine de-
termination, 1096

and Denis, uric acid in
blood, determination,
1030

and Farmer, microchemi-
eal nitrogen method,

108

Folin and Hart, determina-
tion of acetone and
diacetic acid in urine,
1118

and Macallum, uric acid,
microchemical determi-
nation. 1098
and Shaffer, uric acid de-
termination, 1097
Folin-Wu ‘system of blood
analysis, 1007
creatinine determination,
1093, 1017
determination inorganic
sulphates urine, 1105
determination of acetone
in urine, 1115

 

1135

Folin-Wu, determination of
ammonia in urine. ma-
crochemical, 1093

determination of ammonia
in urine, microchemical,
1093

determination of total
acidity, 1121

determination of total sul-
phates of urine, 1105

phosphotungstate reagent
for urie acid, 1063

reaction for uric acid, 1063

temperature indicator,
1089

urea, microchemical deter-
mination, 108

urea, quantitative method,
1088

Foods, amount required per

day,

artificial, 303

composition of various,
304, 305

definition of, 300

diversity of, importance,
302 :

heat equivalent of, in

body, 290-292
in growth, 297

Food poisonings, 443
Formaldehyde,

color  reac-

tions with tryptophane,
47, 918

condensation to
hydrates, 47

detection in milk, 149

from COz and H20
light, 45

in plants, 45-46

réle in eeren in
body,

Sérensen
amino-acids
982

earbo-

by

titration of
by, 363,

union with amino groups,

Formic ‘acid, 33
from adenine by hydro-
lysis,
from nucleic acid, 164
in brain, 581,
Formamide, molecular
pene of proteins in,

molecular weight of starch
in, 14
Formol titration of protein
hydrolysis, 363
Freezing point, and osmotic
pressure, table, 201
anomalous of salt  solu-
tions, 197
apparatus, Bartley, 201
apparatus, Beckmann, 196
of! blood, increased by- gas-
tric secretion, 346
of CaCl solutions, 196
of urine, 690
of various bloods, 547, 549
of various solutions of
glucose, 196
Fructose; see Levulose
ae composition of vari-

306
Furfural formed from pen-
toses, 35, 885
Fundus mucosa of stomach,
chlorine in, 372

Galactase, 313

Galactogogic action of pitu-
itrine, 651

Galactonie acid. 41
1136

Galactosamine,
mucoid, 324 7
Galactosazone, melting point,

Galactose, 19, 51
detection, 880
in brain, 578
in glycolipins, 100
in milk as brain food, 30
in mucoid, 324
method of detection, 880
mucic acid from, 41, 880
properties, 51
structural formula, 28
Galactosides in brain, 578
Gall atone, cholesterol in,
86, 6
iveibentse of bilirubin
from, 416
origin of, 433
Gas-chain method for hydro-
gen ions, 540, 1051
Gas seeprede for blood, 542,

Gases, in blood, 473
analy composition of,

in tendon

Gastric contents, acid in, de-
termination, 366
determination of acid in,
practical, 973
hydrogen ions in, 368
ee of obtaining, 366,

reaction to various indi-
cators, 366
Sastre digestion, of milk,

optimum concentration of
HCI for, 965-977 ea

practical work on,
977
Gastric juice, acidity of, 346
action on carbohydrates,
350
action on fats, 350
action on wilk,
action on proteins, 349,
351
amount per day, 347
antiseptic action, 350
appetite secretion, 343
freezing point of, 345
human, St, Martin's, 340
hydrochlorie acid in, meth-

od of determination,
366, 974
hydrogen ion  concentra-

tion of human, 346, 358
manner of obtaining, 342
pepsin and pepsinogen, 353
proteolysis by, 361
pure, of dog, 345
nelatlon to amount of food,

secretion of ere 372-373
sham feeding, 3 3
test meals, 342
cara in acidity after
meals, 369
variation with diet, 348
Gastrin, 348
Gelatin, amino-acids in, 129
composition, 110, 637

Gelatin, distribution of
nitrogen in, 144
nutritive value of, 803,

823.
Gels, casein, 232
definition of, 229
fibrin, 231
nuclele acid, 163
of emulsofd colloids, 217
of gelatin, 231

INDEX

Gels, protoplasmic, 214, 233
: reversible and . irreversible,
reversible,

of
myelin, 75
' structure of, 229, 232
swelling of, 235-238
Ceraniol,
Gerhardt’s reaction for
acetoacetic acid, 1074

amino-

Clee and hypophysis,
Giladin, composition, 110,
112, 129, 189, 144

molecular weight, 139
preparation of, 926
definition, 114
Globin, composition, 139, 505
from hemoglobin, amino-
acids in, 129
molecular weight, 139
Globulin, cotton seed, com-
position, 110
from squash seed, compo-
sition, 129
precipitation by dialysis,

by

925

precipita tion

definition, 112
dissolution by salts, 227
from blood plasma, 552
from tissues, 105, 519
wheat, 110
Glucal, in nucleic acid, 169
Gluconic acid, formula, 33
peeo gave, in cartilage,

in chitin, 325
osazone from, 43
in ovalbumin, 315
ee ees melting point,

salts,

Glucose. commercial. 50, 303
deterinination, Ban
metbod, 1110, 1112

determination, Bang'‘s

microchemical method,

4

determination
method, 1109

determination, Bertrand
and Munson and Walker
method, 891

determination,
method, 888

determination in blood,
Bang's microchemical
method, 1114

d-glucose ; see also Dextrose

in urine, Benedict's meth-
od for, 1109

in urine, detection, 1069

in urine, fermentation
test, 1070

in urine, identification of
small amounts, Cole’s
method, 1070

in urine, osazone forma-
tion, 1070

nitrile.

Glicendes,

Benedict's

Bertrand’s

42
classification of,

hydrolysed by emulsin, 54

hydrolysed by invertin, 54

hydrolysed by maltase, 54

of pyr enone 168
Glucosone,

Glucothlonic Sold, 312, 638
Glutamic acid, ‘amount. in
various prote’ ns, 129
conversion to pfoline, 124

formula, 117

 

lactam form, 124

Glutamic acid, reaction with
ninhydrin, 150

Glutelins definition, 112

Glutenin, composition, 110

Glycerie acid, 66

Glycerides, 63 —

Glycerin; see Glycerol

Glycerol, absorption of, 452
fern test for, 66,

in cephalin, 574

oxidation of, 66
Glycerose, 19, 66
Glyceryl aldehyde, 66
Glycine; see Glycocoll
Glycinin, composition, 110
Glycocholeic acid,

Glycocholic acid, formula,
properties, preparation,
425

preparation of, 985
re-excretion after inges-
tion, 428

Glycocoll, amount formed,
744

amount in various pro-

teins, 129
conjugated in urine, 763
cme action of,

ethyl ester, formula, 127

formula, 116

from adenine by hydro-
lysis, 166

in bile salts, 424

in muscle, 612

in hippuric acid, 743

origin from NHs3
glyoxal, 704

origin in body, 744

pairing with acromatic oxy

and

acids, 743
pairing with phenyl acetic
acid, 743
relation to urea, 704
Glycocyamine, relation to

creatine, 706
Glycogen, 19,
affected by adrenaline, 623

amount in heart wuscle,
602, 620
amount in muscle, after

phlorhbizin, 623
amount in skeletal muscle,
.
amount in various mus-
cles, 621
and diabetes, 772
chemistry of, 774
conversion to glucose, 717
Meee psarince on death,

discovery of, 172-774

from amino-acids, 777

from carbohydrate, 775

from fat, 776

from other substances, 776

from protein, 774

importance in anzrobic
respiration, 42

in various tissues, 774

localization in muscle, 620

of fasting muscle, 620, 623

origin of, 774

relation to anesthesia, 778

relation to emotions, 778

Glycogen, relation to gly-

oxalase, 785

relation to pancreas, TS85

relation to —_ splanchnic
stimulation, 778

rele ton to supra-renals,

 

resistant to alkali, 35
Glycol aldehyde, 19, 31
Glycoleucine, 117, 582
Glycol, from choline, 94
GiyeoR pie: definition,

in brain, 570, 578
preparation of, 911
sugar group in, 100
Glycolysis in blood, 550
relation to pancreas, 784
Glyco-phospholipins, 100
a definition,

62,

mucin, composition, 323
Glycosuria, ailmentary, 758
emotional, 758
sugar puncture, 772
Glycosurias, kinds of, 758
Glycuronates, in urine, 762
Glycuronie acid, distinguish-
ed from pentoses, 759
from mucin and cartilage,

325
in urine, 758, 762
origin, 759
Glyoxalase, 785
Glyoxal carbonic acid, 185
formula, 31
Glyoxylic acid, in  trypto-
phane test, 148, 917
Gmelin-Rosenbach test for
bile in urine, 1077
Gmelin’s test for bile pig-
ments, 984
erates relation to iodine,

Gold, oe ae size of par-|

ticles
Goose fat, iodine number, 71
Grape seed oil; iodine num-
ber, 70
Grape sugar; see Dextrose
Gray matter of brain, com-
position, 583

see Brain
Grigant’s colorimetric de-
termination of choles-

terol, 84

Growth, 296
importance of lipins for,
844

importance of vitamines
in, 838, 844
of bones, 640
specific amino-acids neces-
sary for, 82
Guaiac teaeren for blood,
Guaiaconic acid, ear for oc-
eult Beet a
Guanase, 165,
distribution Me 732
Guanidine, formula, 186
propionic acid, 185
Guanine, amount in nucleic

acid, 169
compounds of, 165
formula and _ properties.
165
gout, 165
occurrence in tissues, 165
a in nucleic acid.
4

products of oxidation. 165
transformation to  xan-
thine, 731
Guanosine, 171
desamidase. 732
tuanyvlie acid. 170
gel property of, 173
location in cell, 173
Guldhers and Waage

2
«29.

law.

Gulose

 

INDEX

Gum, animal, from chitin,
326
Gum arabic, 59
colloidal solution of, 221
Gum, in animal ‘amylase,

in invertin, 330
in malt diastase, 330
Gums, 19, 58-59
Gun cotton, 59
Gundang wax, 81
Gunning’s iodoform
1073
Giinzberg
HCl,

test,

reagent for free
367, 371, 974
Gynesin, in urine, 747

Hair, dog, composition, 144
Hammarsten’s bilirubin reac-

tion, 984

Harding and Ruttan, aceto-
acetic acid detection,
1074

Harvey and Bensley's work

on LC) secretion, 373

Hayem and Winter method
for LIC], 975

Ilayem’s solution for blood
corpuscles, 991

Hay’s test for bile salts in
urine, 1076

Heart muscle, glycogen in,

Ileat coagulation of amino-
myelin, 577
Heat, animal, 269-279
animal, balance of, 282
animal, history of discov-
ery of cause, 269
animal origin of, 275
animal, production
various diets, 282
animal, where produced in
body, 274
equivalents
foods, 295
liberated in swelling proc-

on

of various

esses, 241

Fleller’s protein test, 1067

Hemacytometer of Oliver,

of

Hemacytometer of Thoma-
Zeiss,

Hea acid, formula, 415,
4

Ifematin, formula and prop-
erties, 506

spectrum. 493
Tlematocrit method, 992
IIematogen, 317
Wematoporphyrin, acid, 1003

alkaline, 1004

formula and__ properties,

415, 596, 507. 1003

physiological orton, 422

spectra, 193, 50
Hemibilirubin, 417,
Hemicellulose. 51, 59
Hemin, 506, 507

erystals, 1003
Tlemochromogen, 508

practical work on. 1003

spectrum, 493

Bpecinen and
Hemochromoproteins, 155
Hemocyanino, 113

formation,

composition, 144, 500
Hemoglobin, carbonyl, 140
chemistry of, 499
Hemoglobin, composition of
ox, 110, 139
crystalline form of vari-
ous, 500

 

1137

ce crystallization

definition, ‘118

derivatives, spectra, prac-
tical work, 01

determination of amount,
922, 99

determination, Tallqvist
method, 992

dissociation of, influence

of salts, 484

evolution of, 491, 499

in muscle and lower ani-
mals, 492

molecular weight, 140

nature of union with oxy-
gen, 483

relation to development of
nervous system, 500

spectra of various com-
pounds of, 493, 505

transformation to bile pig-
ments, 419

union with
95

union with stroma of cor-
puscles, 490

Hemoglobinometer of Oliver,

993

anesthetics,

Hemolysis ;
blood

Memophilia, 517

{lemopyrrol, 415

Ilemp seed, amylase, 330

Ilemp-seed oil, iodine num-
ber, 70

Ilenderson and Palmer, hy-
drogen ion method, 1122

Mepatothrombin, 583

Ileptose, formation
hexose, 42

in urine, 30

Ilerring oil,

77

see Laking of

from

iodine number,
sperm heads composition,
178
sperm, protamine in, 128
Herzig and Meyer method
for methyl groups, 99
Tletero-cyclic amino-acids in
proteins, 118
IIleteroproteose, 361
liexahydromethylene ;
Inosite
TIexone bases, 131
Hexoses, decomposition by
acids, 35
differentiation from pen-
tdses, 35
in nucleic acid, 169
isomerism, 19-29
osazones. 43
oximes 42
pyrimidine glucosides, 168
structural formulas, 28, 29
varietics of, 19
Hippocoprosterol. 87. 435
Hippuric acid, 741-746
amount in urine and ori-
gin, 743
formula and properties, 742
literature, 769
method of isolation from
urine, 745, 1065
quantitative determina-
tion, 745, 1099
synthesis in kidney, 743
THirudin, 518
Histamine, formula, 394
see Imidazylethyamine

see

Histidine, amount in various
blood proteins, 553
amount in various pro-

teins, 128
1138

Histidine, dichloride,
talline form, 119
formula, 118
in carnosine, 610
Hlpeane of birds’ corpuscles,

crys-

thymus, amino-acids in,
1

Histones, definition, 112
Homogentisic acid, 752, 813
reaction with iron, 753
Hopkins-Cole tryptophane re-

action, 148, 917
Hopkins’ lactic acid reac-
tion, 976
work on growth, 822, 843
Horbaczewski's work on uric
acid, 730
Hordein, composition, 110,
112, 129, 139
Hordenine, 442
Hormone, derivation of word,

in stomach, 348
Horses’ foot oil, iodine num-

ber,

v. Hubl’s iodine solution,
909
Bully of
ene acid, from hexoses,

milk globules,

Humors, cardinal of Hippoc-
rates, 406

Humus formed from amino-
acids, 127

Hunger, cause of, 342

Hunter and Given’s work on
purine nitrogen distri-
bution, 732

Huppert-Cole reaction for
bilirubin, 984
Nakayama reaction for

bile pigments in urine,
1077

Hydantoic acid, 741
Hydantoine, relation to al-
lantoine, 741
Hydrazones, 43
Hydrobilirubin, 417
Hydrochinon acetic acid, 752
in urine, 749
Hydrogen ions, and various
indicators, 371
Hydrocholesterol, 85
Hydrochloric acid, amount in
gastric juice, 357
determination, methods in
gastric juice, 366
free, determined by Gtinz-
berg reagent, 367
in castes juice, practical,

in gastric juice, variation
in disease, 366, 370
in stomach, 351

in stomach, theory of for-
mation from NH,Cl, 374

orig in stomach, 371-
374

Hydrocyanic acid, relation to
urines, 165, 184
a to pyrimidines,
strength and conductivity,
do
from xanthine, 167
union with hemoglobin,

495

Hydrogel, definition, 214

Hydrogenation of  unsatu-
rated fats, 71

Hydrogen fons, concentration
of, in plasma, 469

 

INDEX

Hydrogen ions, concentration

of, in bile, 40

concentration of, in blood,
540, 545 7

concentration of, in pan-
creatic juice, 390; in
intestinal juice, 387

cencentration of, in pro-
toplasm, 4

concentration of, in saliva,
322

concentration, of various
standard solutions, 543-
544, 1059, 1122
determination by Giinz-
berg, reagent, 367, 368
determination, gas chain
method, 540, 1051
determination, indicator
method, 542, 1122
favorable concentration
ne salivary digestion,

2 2
in stomach contents, 357,

of pure gastric juice, hu-
man, 34
optimum for pepsin, 356,
358
optimum for steapsin,
397
Hydrogen number of fats
and oils, 71
Hydrogen peroxide,
in oxidations,
formed in starch synthe-
sis, 47-4
in vital oxidations, 762
Hydrogen, solubility
serum,
Hydrolysis, by pepsin, 364
definition, 9
Fisher’s method, 127
of epee Rtlaee by trypsin,

formed
‘0

in

of polypeptides by various
proteases, 40.
of proteins, 116
Hydrone, 191
Hydrophil colloids, 229
Hydrosol, definition, 214
Hydroxy-amino propionic
acid, 11
Hydroxybutyric acid, in dia-
betes, 7
in urine, 759, 760, 1116
see also Oxybutyric acid
Hydroxyl ions, in blood
serum, 247
in pancreatic juice, 390
in water, 7é
Hydroxypentacosanic
578

Hyocholic acid, 430
Hyoglycocholic acid, 432
Hypaphorine, 442
Ply peresycemiae: 779
Hypertonic solutions, 206
Hypobromite determination
of urea, 1090
Hypoglycemia after  phlor-
hizin, 795
Hypophysis, 643-653
acromegaly, 64/
and dwarfs, 645
and growth, 646
and sexual organs, 646
effects of feeding, 649
extirpation and persistence
of thymus, 648
extirpation, effect on genl-
tals, 649-650
extirpation, effect on ni-
trogen metabolism, 647

acid,

 

Ilypophysis, extirpation of,
results, 647
phylogeny, history and
morphology, 644-645
a a composition,

posterior lobe, action of,
651

relation to adrenaline, 648

structure of, 643
Hypotonicity and laking, 996
Hyptonic solutions, 206
a azo compound,

eolor reaction, 167

does not give Weidel re

action, 167
formula and _ properties,

67,
from adenine by adenase,
166

from brain, 581

from inosinic acid, 172

in muscle,

in urine, 721

preperation from meat,

Hypoxanthine picrate, 167

Hy perantnne silver nitrate,
transformation to

thine, 731

Icterus, 407

Icthulin, 113

Idose, 29

Imbibition; see Swelling

Imidazole synthesis from
ammonia and carbohy-
drates, 185

Imidazolyl carbonic
185

xan-

acid,

Imidazolylacetic acid, 747
Imidazolylacrylic acid, 747
Imidazolyl-ethyl amine; see
Imidazyl-ethy! amine
Imidazyl-ethyl amine, 441
and secretin, 394
Physiological action, 442
Imino group, definition, 137
os molecule, 137,

Immunity and protective en-
zymes, 550

Indican in urine, detection,
1066

Jaffés test for, 1066
Indican, of urine, 749
of urine, amount per day,

75!
Indicator method for H ions,
542

Indicators, H ion concentra-
tion of, color change,
371, 546

table of, Salm’s, 546
Indigo blue, amount in hu-
man urine, 751
formula, 750
Indole acetic acid. 440
et color reaction, 150,

formation
phane, 44
formula, 441
reparation, 987
Tndole-ethyl amine, 442
Indole propionic acid, from
tryptophane, 440
Indoxyl, 750
Inductance, 69
Tatar item and hypophysis,

65
Inogen, 625, 631

arom trypto-
Inosine, 172, 612
hydrolase, 732
Inosinic acid, 171, 612
Inosite, fate in body, 613
formula in brain, 582
in muscle, 612
Inosit¢, in urine, 612
Insect wax, iodine number,

71
Intermediary metabolism,
method of study, 819
aera) pressure of liquids,

pressure of salt solutions,
198

Internal secretion, of mus-

cle,
off pancreas, 782
pancreatic and
Langerhans,
see Cryptorrhetic tissues
Interstitial hepatitis and
urea, 699
Intestinal digestion, 387
bacterial decomposition in,
7

isles of

character of contents at
end of small intestine,

438
erepsin in, 403
gases in, 440
in stomach, 381
invertin in, 388
pepsin in, 365

practical work, 983
waa e" passage of food,

Intestinal gases, 440
Intestinal mucosa, ammonia
in
content of chlorine, 372
a to lack of On:,

Intestinal putrefaction, and
arterial sclerosis,
bacteria, 438
detection by indican of

urine. 750
influence of bile, 413
influence on skin, 444
methods of reducing, 445
aug ueniity to disease,

of tryptophane, 441
practical work, 987
sulphur compounds. 816
Intestine, excretion of mono-
saccharides, 453
small, reaction ofi contents,

438

Inulin, 19, 49

Inversion coefficient of vari-
ous acids,

Inversion of cane sugar, 55,
886

Invertin, 54, 55
in blood, 550
in duodenal secretion, 388
Ta intestine! mucosa, 388,

in pancreas, 400
Invert sugar, 55-56
Todine in thyroid, 664
Iodine number, definition, 70
determination of, 907
of cuorin, 96
Iodine numbers, of various
fats and waxes, 70-71
Iodine reaction of cholic

acid, 429
Iodine trichloride solution,

lodine solution of v. Hubl,
909

 

INDEX

Todine solution of Wijs, 908
Iodine-starch reaction, 881
Iodoform test for acetone,

1073
Iodoform test, Gunning’s,
1073

7
Todothyrin, 667
Tonic theory, 195
Ionization, of carbohydrates,

219
of colloids, 220
of glass, 219
of proteins, 153
Ions, definition, 193
Iron in nucleus, 176

Iron reaction, of pyrocate-
chol, 749

Irreversible gels, 230

Irritability,

due to oxygen
compound, 2
Islets of Langerhans, 785
Isoamyl amine, 442
Heeb cee sects acid,
Isocholesterol, 81

in brain, 581

in wool wax, 85
Isocreatinine, 609
Isocyclic amino acids in pro-

teins, 117

teen? value of foods,

Isolectric point of oxyhemo-
globin, 503

Iso-leucine, formula, 117

Iso-linolenic acid,

Isomaltose, 55

Isomers, definition, 19

Teepe pyrauunodcerts acid,

Isopropyl iodide, 575
Tsosmotic solutions, 206
Isovaleric acid, 64

Jacobi’s method,
quantitative, 967
J aE es creatine reaction,

pepsin,

5
Japan wax, iodine number,
71

Jaundice; see Icterus
Jecorin, 100

Ketose, definition, 19
from an aldose, 31-32, 44
reaction, 881

Kephalin, amino ethyl alco-

hol in, 575 .
amino-oxy-butyric acid in,
”

auto-oxidation of, 575
bases in, 574
ebloride, 574
neurine in, 574
hydrolysis of, 574 ;
preparation from _ brain,
properties, 574
see also Cephalin
serine in, 575

Kephalinie acid, 573, 575

penal phosphoric acid,
574

Kerasin, 100, 578

se composition, 112,

Ketostearic acid, 72
ee creatine, amount in,

sxtippation, result of, 694
morphology, 686

perfusion of, 695
secretion; see Urine

 

1139

Kidneys, synthesis of hip-
puric acid by, 743
work done per day, 689
Kjeldahl - Gunning - Arnold
method for nitrogen, 931
Kjeldabl-Gunning, method,
composition of NaOH,
Na,8, solution, 860
Knapp and Sachsse reaction
for glucose, 876
Koch aération apparatus,

1086
Kossel-Neumann method of
preparing nucleic acid,

Kossel's theory of proteins,

Koprosterol, 87
Krinosin, 570, 580
Kupfer cells of liver, 420
Kyrins, 136

Laboratory equipment, 858
Laccase, 613
Bae
Lactam and lactim form of
urie acid, 722
Lactase, 56
in duodenal secretion, 388
in gpa secretion,

Lacteals, 451
Lactic acid, 19
in brain, 582
in muscle, 625
in stomach, detection of,

Lactose, 19
action of alkali on, 34
adsorption by charcoal,
1071

composition,

amount in cow and human

_ milk, 306

distinction from glucose in
urine, 1071 °

absorption of, 453

identification in urine,
1072

in blood, 471
in urine, 758
Munson and Walker tables
of Cu equivalent, 895
mutarotation, 56
osazone melting point, 43
preparation from milk, 951
properties of, 56
structural formula, 57
Lactosuria, 758
Laking of blood, 497
a type of cell stimulation,

498

by anesthetics, 997

practical work on, 996
Lanolin, 80, 85
Lanolin alcohol, 81
Lard, 63

iodine number, 71
Lard oil, 63
Laurel oil, iodine number, 71
Laurie acid, in butter, 64
Lavoisier and animal heat,

4, 270
Law of peptic activity, 358-
Law of Schiitz and Boris-
sow,
Lecithin, amount in milk.
309
becithen CdClz, properties,
Lecithin chloride, 574

Lecithin, colloidal, 218
derivation of word, 90
1140

Lecithin, discovery, formula
and Gee 90-91
gives PeLienROrene reac-
tion, 572
hydrolysis, 91
in bile, 436
in brain, 570, 571-573
in pepsin, 356
poseyly a polyphosphatide,
5
preparation of, 571
properties, 97, 571-572
separation of oleic acid
from, 572
Lecitho-proteins, 113
Leech extract, action on
blood, 518
Legal’s acetone reaction,
1073

Legumelin, composition, 110
Legumin, composition, 110,
129, 139

Leucemia (Leucocythemia),
Leucinamide, biuret test,
145

Leucine, amount of, in vari-
ous proteins, 128, 129
baer decomposition of,

formula, 117
Leucinimide, formula, 124
Leucocytes, intravascular in-

jection, effect on clot-
ting, 528

motility of, 464
‘pumber in blood, 464, 562

relation to blood clotting,

see also Blood, corpuscles,
white
Leucocythemia, effect on uric
acid secretion, 730
Leucocytosis, digestive, 454
Leucothrombin, 535
Levulinic acid, formula, 35
from carbohydrates, 35, 885
from nucleic acid, 164
Levulose, 19, 49
decomposition by alkali,
30-34

dissociation of, 30
properties, 49
reduction of, 41
Seliwanoff's reaction, 881
Lewis-Renedict method for
dextrose, 1023
Liebermann-Burchard choles-
terol reaction, 82, 914
Liebermann, protein reac-
tion, 149, 918
Life. definition, 3
origin of,
Meamentam nuche, compo-
sition, 635
Light. sensitivity to, 442
Ldguger le acid, 64, 578,
Limonene, 79
Linkires in protein molecule,

135
Linolenie acid, iodine num-

ber,

Linvlenic acid, 65

iso, 65

auto-oxidation, 67

from kephalin, 71, 575
Linolic acid, 65

idoine number, 71

iso, 65
Linolinic acid; see .Linolenic

ac
Linseed of], composition, 67
iodine number, 70

 

INDEX

Linseed oil, melting point, 66
respiration of,

Lipase, in blood, 4550
accelerated by bile, 411
gastric, 350
pancreatic, 3895
reversible reaction of, 255,

452.
Lipins, 61-102
acetyl number, 74
amino, 62
amount in muscle, 602
amount in tissues, 62
classification, 61
distribution in cells, 78
glyco, 62
in bile, 436
iodine numbers, 70-71
literature, 102
of brain; see Brain
of milk, importance in
diet, 309
phospho, 62, 95, 96, 97
Drupertice and’ detinition,

saponification, 72
saponification numbers, 73
specific gravities,
Lipoids; see Lipias
Lipo-proteins, 113, 469
amount in blood, 461
see also Serum globulin
Litmus as indicator, 371
Liver, amount of Ammonia
in, 702
amount of creatinine in,
706

arginase, 703

corrosion of, effect on ni-
trogen excretion, 699

destruction of red corpus-
cles in, 419

detoxicating function, 699,

disease of, effect on urine,

698

Eck fistula, 697

extirpation in birds, effect
on uric acid,

eet neon of, in birds
41

forms urea, 696
glyogenie function, 773
meeraels of, by adrenaline,

necrosis in absence of
sugar, 790

origin of bile pigments, 418

perfusion and urea forma-
tion, 696

relation to fibrinogen for-
mation, 555

relation to uric acid de-
struction, 734

removes ammonia from
blood, 701

réle in sugar metabolism,
788

transforms creatine to
creatinine, 717

Living matter, chemical re-
actions In, 8-11

collodial constitution of,

combustions in, 8

composition, 12-14

contains ions, 197

difference from lifeless, 6

distribution of phospho-
lfpins in, 100

energy of, 6, 7, 276

enzymes in,

fats in, 78

foam structure of, 213

 

Living matter, gcneral prop-
erties, 3-1

organic matter in, 15
organization of, 11, 213
origin of, 15
osmotie pressure, 199, 205
phosphoric acid in, 160
pipes chemistry of, 190-

possible chemical nature,
2D
psychical phenomena of, 7
reaction of, and its con-
trol, 246-250
result of destruction of
organization of, 213
réle of oucteus, 159
réle of water in, 190-192
salts in, 15, 193-197
water, amount in, 14
Liquid crystals of phospho-
lipin, 97
Tana: Seer tOLy action of,

i

watcr secreted per day, 685

Lupeose, 19, 52

Lymph, 459

Lymphocytes, influence on
clotting, 527

Lysine, amount in_ various
proteins, 128, 129

formula 118
Lyxose, formula, 30

Macallum’s method for chlo-
rides 373
Mace butter, 64, 71
Magnesium, in blood, 1037
Magnesium in urine 757
Magnet resemblance to or-
ganism, 266
Maize oil, 65
iodine number, 70
Malic acid, 338
Malonamide relation to
biuret test, 145
Malonic acid, 33
Malt amylase, 229
Maltase, in duodenal juice,
glucosides split by, 54
in pancreas, 400
in saliva, 334
reversible action of, 255
Malt diastase; see Amylase
Malt extract. digestion of
starch by, 327
Maltose, 19, 30
absorption, 453
action of alkali on, 34-

35
from starch, 327
in blood, 471
properties of, 57
Mannitol, 4
Mannoheptose, 19
Mannose, 19
bydrazone, 43
structural formula, 28
Mass action, law of, 252
Mastication, importance in
digestion, 335
McCollum and -Davis work
on growth, 844
Medullary sheaths of nerves,
composition and fune-
tion, 567, 5
Meigs’ method for milk
analysis, 951
Melanin nitrogen in various
proteins, 144
Melibiose, 19, 14, 56
Melissyl alcohol, 81
Melitose. 19, 58
Melizitose, 19)
Mewarys an auto-catalysis,

in linseed oil, 68
physical basis of, 69, 590
Mendel and Osborne’s work
on growth, 823, 844
Menhaden oil, iodine num-
ber, 70
Menthol, 79
_excrecion of, 762
Mercaptans, in feces, 442,
820 .
Mercuric acetate precipita-
tion of amino-acids, 121
Mesitylene, fate in body, 763
Mesitylenic acid, 763
Mesityluric acid, 763
Meso-tartarie acid, 22
Mesoxalic acid, formula, 66
Mesoxalylurea; see Alloxan
Metabolic co-ordination by
blood, 510
Metabolism, balance of en-
ergy and matter, 293
carbohydrate, glyoxalase,
785 .
carbohydrate, historical,
71
carbohydrate,
diabetes, 781
carbohydrate,
d abetes, 778
carbohydrate, sugar punc-
ture, 772
experiment in calorimeter,
291

pancreatic

phlorhizin

in fasting 833,

of body as whole, 771-851

of brain, 591, 594

of carbohydrates, 771-798

of milk glands, 312

of muscle, 615, 623

of nerve fibres, 588

of nerve fibers and rela-
tion to rate of trans-
mission, 588

of proteins, 799-832

of substances not foods,

761
sulphur, 816
under various conditions,
833-857
vitamines, 837
Metaphosphoric acid in nu-
eleic acid, 1
Metbemoglobin, 503
practical work, 1001
Methoxyfurfural, 32, 37
Methylation, in animals, 718-

719 .
ofi Pe nOlee tellurium, ete.,

Methylene alinine, 121
Methylene blue, reduction by
glucose, 876
Meuiyh stir! maleic imide,
Methyl furfural reaction for

cholesterol, 83

Methyl groups, by Herzig
and Meyer method, 99
Methyl guanidine acetic

acid: see Creatine
Methyl guanidine, and para-
thyroidectomy, 656
ingestion no_ effect
creatine, 717
in muscle, 609
in parathyroidectomy, 656
in urine, 656, 746
‘Methyl! hydantoic, acid, from
creatine, 707
Methyl-n-nonyl ketone, 79

on

 

INDEX

Mistay) Onmnee: as indicator,

Methyl-phenyl hydrazine, 44
Methyl pyridine chloride in
urine, 747
Me violet, as indicator,
Methoxyfurfural from  hex-
oses, 885
Mett’s quantitative determi-
nation of pepsin, 968
Mett’s tubes, for measuring
amylolytic activity, 331
Mett’s tubes for pepsin ac-
tivity, 359
Microchemical determination,
ammonia, 109
elgcose in blood, 1021,
1028, 1112, 1114
urea, Folin, 1088
uric acid, 1098
uric acid in blood, 1030
Microchemical method,
chlorides, 373

for

for protoplasmic acidity,
247 :

urea, by urease, 1014, 1086,
1087

nitrogen, 1010, 1081
Microchemical, Prussian blue
reaction for acid, 373
Miescher, discovery of nu-

clein, 160
Migraine and food poison-

ing, 4 :
Milk, as a food for young,
306

ash compared with body
ash,

asses’, 314

bacteria_in, 310

casein,

citric acid in, 310

clotting in stomach, 349,
376-380

colostrum, 307

coagulation by rennin,
practical work, 949-954

coloring matter of, 310

composition of, 306

enzymes in, 313

fat by Meigs’ method,

goats’, 314

hulls of globules, 310
human and cows’, 306
inorganic substances,
lactalbumin, 309

lipins of, 309

orotic acid in, 310
proteins of, 308

purine nitrogen in, 310
souring of, 313
unknown organic constitu-

310

ents,
various ‘kinds ‘of, com-
pared, 313
Mk, cere: composition,

metabolism, 312
relation to copora lutae,
312
‘relation to growth, 844
ei reaction, 146, 147,

Millon’s reagent, directions
for making, 147
Mingin, in urine, 747
Mitchondria contain
pholipins, 102, 585
Molecular ae importance

phos-

of, 20-
Molecular weight of proteins,
138-142

 

1141

Molecular weight, by
motic pressure, 140
from sulphur content, 139
Molisch carbohydrate reac-

tion, 151, 877
Molybdate-phosphate mixture
of Folin and Wu, 1022

os-

Mono-amino dicarboxylic
acids in proteins, 117
diphospha tides,

Mono-amino
96

Mono-amino monocarboxylic
acids in proteins, 116

Mono-amino _ monophospha-
tides, 573, 576

Mononucleotides, 164, 171

Monosaccharides, acids
formed by oxidation, 33

Monosaccharides, _classifica-
tion, 19

aero upeRt sen by acids, 35-

decomposition by alkalies,
30-34, 874
dissociation, 30
isomerism, 19-30
oxidation of, 38-40
oximes, 42-43
structural formulas,

28,
synthesis in plants, 44-46
union with HCN, 42
reduction of Barfoed'’s, 40
reduction of Fehbling's so-
lution, 38, 872
synthesis by ultra-violet
light, 45
Moore’s carbohydrate reac-
tion,
Moore's test for sugars, 30-34
Movements by surface ten-
sion, 209
Mucic acid, from galactose,

7
Mucilages, 19
Mucin, composition and prep-
aration, 326
chemistry of, 323
in bile, 437
preparation from saliva,
962

with ‘dimetbyl-
326

322,

reaction
aminobenzaldebyde,

relation to chitin, 325

SUE Tae ' 110,

5
tendon, 110, 322, 325
Mucoid, composition, 110, 324
in connective tissue, 636
preparation from tendon.

relation to eartilage, 324
sulphur in, 325
Muconic acid, from benzene,

815
Mucosa of stomach, ammonia
in,
chlorine in, 372
Murexide reaction, 724
for urie acid. 724. 1062
given by cytosine, 168
Muscarine, formula, 92
Muscle, acidity of, 626
amino-acid content of vari-
ous muscles, 608
amount of lipins in, 603
autolytic enzymes, 618
earbohydrates vs. fat as
source of energy, 291
carnie acid. 613
carnitine, 610
earnosine, 610

changes on activity, 620-
622
 

1142

Muscle, chemistry of, 600-633
eblorine in, 372
clotting of, 605
creatine, 609, 708
double refraction, 607
energy metabolism of, 620
extractives, 608-613
formative metabolism, 616
general composition, 603
glycocoll, 612
glycogen in, 620-622
glycolysis, 622
nypoxantbine and muscle

work,

ignotine, 610
dey aie constituents of,

inosine, 612

inosinie acid, 612

inosite, 612

internal secretion of, 615

lactic acid, rdle of, 625

lipins of, 614

literature, 632

maintenance of neutrality,
625

mechanism of contraction,
627

metabolism, creatine and
creatinine, in,

methylation in, 718

methyl guanidine, 610

myogen, 605

myogen fibrin, 605

myoproteid, 606

myosin, 604

myostromin, 607

mytilite, 613

novain, 611

nucleoproteins
60!

6
oblitin, 611
of scallop, amino-acids in,
129

oxygen consumption, 489

phosphocarnic acid, 613

phospholipins, 606

physical consistence, 602

proteins extracted by
-NHiCl, 604

proteins of, 603-607

purines, 610

purine‘nitrogen, 611

se of contraction,

rigor of, 628
smooth, inorganic constitu-
ents, 615
stroma proteins, 606
structure,
succinic acid in, 614
taurine in, 611
urea in, 612
xylose in, 613
Muscle plasma, 604
Muscle stroma, 606
Mustard: active principle of,

in stroma,

oil, 53
Muta rotation, 49, 50
of levulose, 49
Musto. tallow, iodine num-

Myelin,” brain, preparation
and properties, 76
brain, 571
forms, 97, 574
sheaths; see Medullary
sheaths

Myogen fibrin, soluble, 605

Myogen, preparation and
properties,

Myosan. 113

Myosin fibrin, 606

 

INDEX

Myosin fibrin, preparation
and properties,

Myostromin,
Myrcene, 79
Myricy! alcohol,
Myristic acid,
Myronic acid, 53
Myrosin, 53
Mytilite, 613
Myxeedema, 659

Naphthalene, excretion, 761
methylated in body, 717
Naphthol-benzylamine, 44
Naphthoresorcin, Tollen’s re-
action for glycuronic
acid,
Naphthoresorcin, Tollen’s re-
action for pentoses, 880
Narcosis, effect on _ respira-
tion, 594
Nerve cells, consistence of
protoplasm of, 585
mitochondria in, 585
Nerve fibers, carbon dioxide
production, 593
metabolism, 586, 593
oxygen consumption, 594
Nervous system ; see Brain
Neat's-foot oil, iodine num-

ber, 71
Nesslerizing, 1013, 1083
NESS Ere PeaBERS: 1010, 1033,

81

an

Neuberg and Rauschwerger'’s
cholesterol reaction, 83
Neumann method for pre-
paring nucleic acid, 163
Neurine, from cephalin, 574
from lecithin, 91, 572
from sphingomyelin, 578
Neuroplastin, 58
Newiet eee crystallization
of,
Neutral red, as indicator of
protoplasmic reaction,

Nickel in hydrogenized fats,
Nicotinie acid
vitamine, 843
Ninhydrin protein reaction,
150, 919

Nissl substance, 585
Nitric gnide hemochromogen,

in beri-beri

bemoglobin, 494
Nitrile reaction for thyroid,

663
Nitriles, fate in body of,
764

Nitrogen, distribution of, in
protein molecule, 1

urine, microchemical
quantitative method,
1081

in

method. of detecting in or-
ganic matter, 109

non-protein, in blood, 1010,
1023

quantitative determina-
tion of, by Kjeldahl-
Gunning, 931
equilibrium, 802
Nitrogen excretion,
creased in
work, 620
by skin, 686
by urine, 691
distribution, in
urine, 691
Nitrogen gas,
blood,
table, 941

not In-
muscular

normal

amount in

Nucleases,
Nucleases,

 

Nitrogen income and outgo
of body, 292
Nitronsusaite reaction,
acetone, 1073
for creatinine, 1065
of cysteine, 185, 151
Nitrous oxide hemoglobin,

for

494
Nonylic acid, 67
Novain, 610

Nuclear division; see Caryo-

kinesis
Nuclear membrane,
ence, 158
results of rupture, 180
Nuclear sap, 180
changes produced in cyto-
plasm by, 180
res to caryokinesis,

71
distribution in
various organs, 732

consist-

Nuclease, in pancreatic juice,
405

Nucleic acid, adenine in, 166

amount in thymus gland,
163-

eee ediate. radicle of,
composition, 162-167
deep upopltton by enzymes,

decomposition, diagram of,
733

decomposition in body, 731

discovery by Altmann, 161

distinction from phospho-
proteins, 174

empirical formula, 164

ease of hydrolysis. of
purines, 163

formation and destruction
of, 182

from brain, 585

function of, 175

from human placenta, 164

from pancreas, 171

guys acid, 170, 171,

identity in different nu-
clei, 174-176

in chromatin, 162

inosinie acid, 171

levulinie acid, from, 165

location in cell, 173

Method of preparation,
163

physical and chemical

properties, 163
Pert of forming gels, 158,

preparation of, from yeast,
893

products of decomposition
of, 164, 170

purine bases in, 165-166

pyrimidine bases in, 168

ae decomposition,

hates proper of, 176
erent formula, 170-

ayntneat of, in nuclei,

thymus nucleic acid, 170,
172

triticonucleic acid. 169
xanthine and _hypoxan-
thine from, 166
yeast nuclele acid, 169
Nuclein, 118
catabolism,
732

enzymes in,
Nuclein, composition of,"
diet, effect on purine ex-
cretion, 7
digestion by pancreas, 405
discovery of, 160
distinction from
nuclein, 174
from milk: glands, 312
from salivary glands, 336
of muscle stroma, 606
metabolism, see Purine
metabolism
separation of, method, 161
Bin eleOnr pee ney definition,
1

para-

Nucleoprotein; see Nuclein

Nucleo- ee composition,
Nicleo - protamine, Herring,

composition, 11
Nucleosides, 170
Nucleotides, 164
Nucleus, absence of potas-

sium in, 176 .

anaerobic respiration of,

182

basic constituents of, 176
ee composition,

composition of, 157-186

eee no pbospholipins,

enzymes in, 180-182

from birds blood
puscles, 179

from spermatozoa, 162

function of, 159

histone in, 179

iron in, 176

method of obtaining for
chemical analysis, 160

morphology of, 157

nuclein in, 161

of starfish egg, 180

physical structure, 158

ag muscle contraction,

cor-

role in synthesis, 160
Nutrition, balance of energy
and matter in, 292
Nylander’s test for glucose,
876, 1070

Obermayer’s indican reaction,
437

Oblitin, 611
Octodecyl alcohol, 81
Qidema and swelling, 239
Oil films, thickness of, 209
Oil of bay, 79

of cardamom, 78

of cedar, 78

of cloves, 62

of peppermint, 78

of turpentine, excretion,
758

of wintergreen, 78

Oils, acetyl number, 74
drying, 61, 67
essential, 62, 78-80
fatty, 61, 62-66
formed from

drates, 76-78
influence on surface ten-

carbohy-

sion, 209
iodine numbers of vari-
ous, 70-71
‘ location in cells, 78
methods of identification,
69-71 i

ozonides of, 72
physical properties of, 65
rancidity of,

 

INDEX

Oils, Reichert-Meiss] number
f, 74

oO
saponification number of,

saponification of, 72
semi-drying, 61, 67
specific gravities of, 66
synthesis of, 76-78
temperature of decompo-

sition, 66

Oily eee from brain,

Oleic acid, formula, 63, 67
iodine number, 71
ozonide of, 71
properties of, 65

Olcin, formula, 63

melting point, 70
ozonide,
Olcomargarine, 64

nutritive value, 64
saponification number, 74
Oleo oil, 64
Olive oil, iodine number, 70
specific gravity, 66

saa hemoglobinometer,
hemacyiometer method,
O91
Omasum, 338

Orcin reaction for pentoses,
880

Organism, like magnet, 266
Ornithurie acid, 742

Orotic acid, in milk, 310
Orthonitro toluene, excretion

ol,
Osazones, 43-44

oa directions for,
Osmometer of Hiifner and
Gansser,
Osmotic pressure, and gas
pressure, 200, 203
Bartley’s freezing point

apparatus, 201
calculation from freezing
point, 201
cause of, 203
definition, 199
hematocrit method, 206
method for determining
molecular weight, 140-

141 :

method of determining, of
cells, 205

of blood, 547

of colloids, 243

of colloids, method,
243

of
14

140,

Pemuelonn solution,

of mammalian corpuscles,

of salt solutions, 199-206

of sea water, 201

of urine, 690

of various sugar solutions,
200

Pfeffer’s osmometer, 198

relation to concentration,
200

re to freezing point,

relation to number of par-
ticles, 244

relation to temperature,

relation to vapor pres-
sure, 203

réle in vital phenomena,

semipermeable membranes,
199

 

1143

Osmotic pressure, table of,
and freezing point, 201
Osones, 38
Osseo-albuminoid, 639
Osseo-mucoid, 63
Ovalbumin, amino-acids in,
129
amount in eggs, 315
composition, 139
Ovaries; see Sexual glands
Ovoglobulin, 315
Ovomucin, 315
Ovomucoid, 315, 316
Ovovitellin, composition, 139
properties and amount in
eggs, 316
Oxalates, influence on blood
clotting, 520
Oxalic acid, from carbohy-
drates, 33
strength,
etc., 195
Oxamide, formula, 860
Ox bile, 425

conductivity,

Oxidase, in saliva, 335
Oxidase reaction, with p-
phenylendiamine, 3385
Oxidases, various kinds in

cells, 68
Oxidation, in body, 762, 850

Oxidation of cells, reduced
by acid, 248

Oxidation, physical chemis-
try of, 256-261

Oximes, 42

Oxonium salts, 36
Ox me composition, 603

Oxyacetone, formula, 31
Oxyamino butyric acid, in
phospholipins, 100

Oxybutyric acid, 75
detection in urine, 1074
Oxycholesterol, 83
in vernix caseosa, 85
reaction, Lifschiitz, 83
Oxycrotonie acid, 75
Oxydiamine pyrimidine, 184
Oxyethyl amine in cephalin,

Oxygen, amount in blood,
473
consumption, by brain,
594 4 1a
consumption, by nerve
fibers, 594
consumption, by various

tissues, 489,
consumption of men at
rest, digesting, ete., 273
292
consumption of muscle,
336
consumption,
glands, 336
electrical phenomena of,
257-258
extra valences on. 191
meaning of word, 270
oxidizing power of, 258
solubility in serum, 475
Oxyhemoglobin, absorption
bands, 503
composition, 139, 144, 504
erystalline form of, 501

of salivary

erystals of, properties,
502

dissociation, influence of
salts, acid, alkalies,

COz on, 484-487

dissociation of, biological
importance, 489
dissociation of, influence

of various factors, 488
1144

Sap nemcelene. isoelectric
point, 503

method of crystallizing,

Rr

10
molecular weight, 129, 140,
485

nature of, 483
nitrogen in, by Van Slyke,
144
properties of, 503
quantitative determina-
tion, 503
spectrum, 4938, 505, 999
temperature coefficient of
reduction, 483
Oho ced 10 acid, 53
Oxylic acid,
Oxymandelle acid,

of

in urine,

Oxyene éells stomach,

on acetic acid, 749,
Oxypheny!- ethyl amine, for-
mula, 6 749

Oxyphenyl glycolic acid, 752
Oxypheny! propionic acid, in
a ot hs

xyproteic acid,
746

in urine,

On naines: excretion of,
Oxystearic acid, 72
Oxytrimethyl - fie nabetuine:

Ozonides of oleic acid and
olive oil, 71, 72

Painting, chemistry of, 67
resemblance to metabolism,
67-68
Palmitin, 63
melting point, 70
Palmitic acid, 62, 64
Palm kernel oil, Reichert-
Meiss] number, 74
saponitication number, 71

Palm oil, iodine number, 71
saponification number, 74
Pancreas, joo of am-

monia in, 702
anitiglyoxalase in, 785
control of secretion of, 391
digestive function, discov-

ery of, 394
internal secretion of, 782
literature on, 449
morphology of, 390
relation to carbohydrate

metabolism, 781

Pancreatic juice, 389-406
acon on carbohydrates,

action on nucleins, 406
action on proteins, 400
amylopsin, 398, 979
composition of human,

8!
eS action on fats,
freezing polnt of, 389

general properties, 389-

hydroxyl ions in, 390
OO of ‘bile on, 411,

loss of power on dialysis,

method of obtaining for
Geamibantion (Boldyreff),

nuclease in, 405

practical work on, 977-
983

 

INDEX

Pancreatic juice, relation to
intestinal juice, 401
secretion of,

summary of actions, 406
DParacasein, -376
VParacresol, from _ tyrosine,
41
Paramyelin, 576
Paramyelin cadmium chio-
ride, 576

Paramyelin from brain, 570
Pata esyphenys acetic acid,

Parathyroidectémy, 655
peo Downe excretion in,
Parathyroids, chemistry, 654
relation to teeth, 656
Parathyroid tetany, 655
influence of alkalies,
cium, ete., on, 655
influence of bone injury

eal-

on, 655
methyl-guanidine in, 656
Parietal cells of stomach,

alkaline, 373
secretion, 354

Pasteur, discovery of molec:
ular form,

Pathological constitutents of
urine, 757

Paunch; see Jtumen

Peach-kernel oil, iodine num-
er,

Pellagra, 844

Tentacosanic acid, from

sphingomyelin, 578
Pentamethylenediamine,
in urine, 747
Pentose, from yeast. 169
Pentose reaction, practical
directions for, f
Pentoses, from nucleic acid,
164, 169
in urine, 759
isomerism, 29
kinds of, 19
occurrence, 30
structural formulas, 30
Pentosides, 171, 172
Pentosuria, 158
Pepper, 53
Pepsin, artificial polypep-
eae not digested by,

442

character of linkings at-
tacked by, 362

conditions of action. 357

determination of, Jacoby's
method, 967

determination of, Mett’s
method, 968

discovery and name, 349

ate of, in intestine,

hydrolysis of proteins by,
in embryonic development,
in urine, 365

law of action, 358

method of obtaining, 355
nature of, 354, 355

optimum H ion, 357, 367 |

relation to rennin, 377
salmin not digested by,

62
sensitivity to alkalies, 352,
sensitivity to CO. 352
unites with substrate, 357
eee in different animals,

Pepsinogen, 351
destroyed by CO:, 352

Pepsinogen, difference from
pepsin, 352-353
practical work on, 965
Pepa. eestor of proteins,

energy of transformation,

Peptides, definition, 114
formula of, 2
in urine, 746
Peptone blood, 517
Peptone, definition, 114
in gastric digestion, 361
T’eptone plasma, 517
VPeptone, properties, 897
Witte’s, digestion by pep-
sin, 364
Percin, amino-acids in, 128
I’erfusion method, 695"
Permeability of cells, 101

I’crspiration, amount per
day, 686
nitrogen in, per day, 294
nrea in, 4
bile

TPettenkofer’s reaction,
salts, 424, 985
by amido myelin, 577
by cephalin, 574
by lecithin, 572
by myelin, 576
Phaseolin, composition, 110
Phenol, in urine, 748
origin from tyrosine, 749
Phenolphthalein, as _ indi-
eator, 371
Pheny! acetaldehyde, fate in
ody, 763, 814
Phenyl-a-lactie acid, fate in
body. 4
Phenyl amino acetic acid,
fate in body, 814
Phenylalanine, amount
various proteins, 129
formula, 117
Phenyl! f-alanine, fate
body, 814
PHeovieaenle acid, fate in
body,
Phenyl ee alcohol,
tn body, 763, 814
Phenyl ethyl! amine, 442
Phenyl glucosazone, proper-
ties, 43
Phenythydrazine, condensa-
tion with sugars, 882
Pheny! hydroxypropionic
ale fate in body, 763

in

in

fate

Phenyl osazones, properties,

Phenyl ras acid, fate in
ody,

Phenyl perie, fate in body,

763,
Phloretin, 53
Polo composition of,
>
derivation of word, 795

glucosuria, 758, 776, 795
method of administering,

 

Phloroglucin reaction, Tol-
len's,_ 87

Photosynthesis, 44

: Phosphates, total in urine,

etermination of, 1109

| sisi Sa see Phospho-
ns
Phosphocarnic acid, 618
Phosabolpins : see also
Lecithin, Cephalin,
Mien. Pharamyelein,

omyelin
Phosoheliy pins, 62, 88

 
enoe) holipins, absent in nu-
eic of sperm, 162

feos in, 100
classification of, 62,
definition, 88
functions ee 98, 99
hemolysis b
bile 436 33-94, 99

in blood platelets, 467

in butter,

in fibrin and fibrinogen,
525

89

in milk, amount and kind,
309

in muscles, 614
in natural oils, 64

method of extraction, 89

physical and chemical
properties, 97

preparation of, from
brain, 912

separation from brain

method, 568, 912
solubilities, 88°
staining reactions, 101
Phosphonuclease, 731
ea veD Eee definition,

of phosphoric

hydrolysis
by alkali,

acid from,

174
in bile, 437
leeegoe in cell of, 156,

17
Phosphoric acid, control of
cell activity,
determination Pemberton-
Neumann, 933
in enzymes, 330
in gums, 59
in living matter, 160, 183
in nucleic acid,. 164
in starch, 58
inversion coefficient,
195
organic, test for, 913
me in HCl formation,

75
Phosphorus excretion, after
parathyroidectomy, 755
dependence on diet, 756

of,

ee with disease,
“T55

Phosphorus, importance in
brain, 595

in feces, 755
in urine, amount, 758
poisoning, secretion of
amino-acids in, 742, 746
Phrenosin, 570,
Phrenosinic acid ; see Cere-
bronic acid
Phrenosterol, 570
Phycocyan, 48, 113, 155
Piyepecreeln, 48, 113, 155,

Phyllocyan, 423
Phylloerythrin, 418
Phylloporphyrin, 423
Phyllopyrrol, formula, 415
Physetoleic acid,
Physical chemistry of pro-
toplasm, 190-262
of oxidation, 256
Phytin, formula, 613
Phytosterol, sone 87
from soil, 8
Picramic Rokt formuia, 41
Picrate method of prepar-
creatinine from
urine, 1095
Pierie acid, formula, 41
reagent, Esbach’s 1069

 

INDEX

Pigments, of bile: see, Bile
igments
urinary, 765
Pike pereh putaming, amino-

Peat Sind influence on
growth, cena develop-
ment, ete.,

Pinene,.

Piperazine, formula, 124

nuclei, in proteins, 124

Pisan alcohol, 81

hone: composition of,

Plant. proteins, composition,
table,

Tlasmolysis, method of de-
err osmotic. pres-

Platelets of blood ;
Blood platelets

Plattner’s bile, 424, 985

Poison, definition of, 301

po description of,

see

Pelmnpeleouees, definition,
Polysaccharides, action of
acids on, -
classification of, 19
colloidal, 19, 58
digestion of, 326, 440, 398
formation of, 58
wee by alkali,

7
properties of, 58
Populin, 53
Potassium, absent
nucleus, 176
distribution of, in various
cells, 14
distribution by

from

surface

tension in Acineta, 242

in blood, 1040
in lecithin, 572
in myelin, 574
Potatoes, amylase in, 329
composition of,
Pneumonia, influence
chloride excretion, 756
hole dog, of London,

on

Polyneuritis, of birds, 841
Bolypergae’ formula, graphic

of,
Polypeptides, artificial, not
Cereted by pepsin, 136,
definition, 114 5
digested by pancreatic
juice, 40:
Posterior. lobe of hypophy-
sis, 65
Practical work and methods,
858-1124
eee ee reactions of
proteins, 152
Precipitation, of colloids by
salts, 223-228
Pregnancy, diagnosis of, by

enzymes, 550
Primary. protein derivatives,

Protamines, definition of, 112
Proline, amount in various
proteins, 129
formula, 118
no reaction with
hydrin, 150

nin-

origin from glutamic acid,:
124

Prosthetic 2 Broun, definition

of, 11
of mucoid, 324

 

1145
Protagon. 569, 530
Protalbamase ; see Pro-
toproteose
Protamine ucleate, from

herrfng sperm, 178

Protamine, reversible syn-
thesis of, by trypsin,
255

Protamines, amino-acids i
various, 128 -

composition and _ proper-

ties, 177
definition, 112
origin of name, 177
method of extraction from
sperm, 177

anipeyede structure of,
Proteans, 113
Protease, 361
in blood, 538, £50
in blood plates, 538,
Protein, amount needed per
ay,
Bence-Jones, 757
catabolism of, 807
Protein ingestion, increased
nent production by,

Protein metabolism, 799-831

amount of protein necded
per day, 800

catabolism, 807

catabolism of
812

heat production by, 806

homogentisic acid, 812

is minimum protein desir-
able?

kind ofi protein important,
803

tyrosine,

literature of, 825
low protein, Fletcher, 800
nitrogen minimum, 804

course of oxidation of
amino-acids, 809
oxidation of benzene

nucleus, 815
protein sparing by carbo-
Bpanates and fat, 802

sulphur metabolism, 816
al of amino-acids,

Protein sparing action of
carbohydrates and fats,

02, 83
Proteins, 104-189
absorption of, 453-455
acid combining powers of,

Adamkiewicz reaction of,
sinine acids found in, 114-

amounts of amino-acids in
various, 128, 12

basic, in nucleus, 162

biuret reaction of, 145

chromo, 113

classification,

112
classification, English, 114
colloidal nature of, 110
colar reacHoue of, 144-151,

American,

color reactions with vari-
ous aldehydes, 150°

composition, 109-110, 144
composition, determina-
tion of, pratical, 915

conjugated,

crystalline, 106, 143°
1146

Proteins, decomposition prod-
ucts of, 114-118

definition,

derived, 113 :

distribution of nitrogen
in, 143

electrical charge on mole-
cules_ of,

formaldehyde reaction

(Acree), 149
heat coagulation of, 921
Bopene Sole reaction of,

in protoplasm, 105

in seeds, 107

in urine, 757

lecitho, 113
Hees reaction of,

lipo, 113

living and dead, 104
meaning of word, 104
meee ofi extraction, 105-

08
Millon’s reaction of, 146
molecular weight of, 138-

Molisch reaction of, 151

ninhydrin reaction, 150

nucleo, 113, 161, 166-183

number of amino-acids in
molecule, 142

number of free amino and
carboxyl groups in, 137

of blood, 469, 551, 561

of brain, 584

of brazil nut, 107

of cytoplasm and nucleus,

of milk, 308

of muscle, 603

of smooth muscle, 608
origin of, 6

oxygen compounds of, 492
phospho, 113
pes reaction of,

properties of, 110-111

putrefaction of, 440

racemization by alkali,
125-126

Separation by fractional
precipitation, 10

simple, 112

solubilities, 112, 113

storage of, by body, 805

storage of, in cells, 106-

107
structure of, 130-132
sulphur, amount of in, 139

SUlEne detection of in,

synthesis of, 133, 183

triketohydrindene reac-
tion, 150

united with lipins, 469
mene proteic reaction,

Proteoses,
tion,
Proteolytic enzymes; see
Proteases. Erepsing,
Trypsin, Pepsin
_Proteoses, definition of, 113
deutero, 361, 929
hetero, 361, 928
primary, 361, 928
Secondary, 361, 929
thio, 929
Prothrombin, 527, 532, 533
| Protones, 136
' Protoplasm ; sec

by peptic diges-
361

Living mat-
ter
a gel, 214, 233

Ptyalin,

 

INDEX

Protop iam, and blood plasm,
36

an emulsion, 234
conception of
structure, 25)
control ofi reaction of, 246
enucleate, powers of, 159
osmotic pressure of, 205
pee chemistry of, 190-

158, 234
surface films in, 210
chemistry of, 157, 255-256
Protoproteose, 361
Prussian blue and secretion
of acid, 373
Pseudoglobulin, 552
Psychic qualities of brain,
basis of, 597
Psychism of molecules, 267
Psychosin, 579
Psylla alcohol, 87
Psylla wax, 81

chemical

structure of,

Psyllostearyl alcohol, 81

Ptomaines,
in urine, 747
amount
with diet, 334
composition, 329
conditions of activity, 331,

variable

964
difference from amylopsin,
399

derivation of word, 328

favorable hydrogen ion
for, 331, 964

inhibited by products of
digestion, 333

Tor velocity of action,

practical work on, 963

time of appearance in
ontogeny,
various kinds of, 334
Purine bases, adenine, 166

amount excreted in vari-
ous animals, 736

chemistry of, 165-168

from nucleic acid, 164

guanine, chemistry, 165

hypoxanthine, 167

of muscle, 610

possible __ destruction
body, 727

pacducke of oxidation of,

in

oo
quanitative determina-
tion, 1103
reaction with cupric salts
and bisulphite, 167
relative amounts in differ-
ent tissues, 174
BvD thea of, in cells, 182,

xanthine, 167
Purine catabolism, 731
Purine coefficient, 736
Purine metabolism of body,
721-741

Purine nitrogen, in urine, 736

in various tissues, 736
Purine nuclease, 732
Purine, structural

of, 165

Purpurie acid, possible for-
mula, 725 ’

ce intestinal, 437,

intestinal, bacteria in fn-
testine, 438
intestinal influence of bile

formula

j eeinal f tryptopl
otestinal. o tryptophane,
750 oP

,

 

Putrescine ; see Tetramethyl-
enediamine
in urine, 746
Pylorus eee secretion of

stomach, amount of chlo-
rine in,
Pyridine, methylation of, in
_body, 71
Pyrimidine bases, cytosine,
168

in nucleic acid, 164, 168
origin of, in cells, 183
thymine, 168
uracil, 168
Pyrimidine glucosides, 168
Pyrocatechol, in urine for-
mula, 749
iron reaction of, 749
Ey carboxylic acid,

Pyrrol nuclei in hematopor-
phyrin, 508

Pyruvic acid, by oxidation
of carbohydrates, 33

Pyruvie acid, rdle in pro-
tein synthesis, 186

Pyruvic aldehyde from car-
bohydrates, 186

Quantitative determination.
acetone and_ diacetic
acid, Folin-Hart, 1118

acetone, diacetic acid and
hydroxybutyric acid.
1115

acetone, Folin, 1115

acidity of urine, 1122

adrenaline, 1124

allantoine, urease method,
1100

Wiechowski,

amino-acids, after removal
1104

Snamtaine

amino-acids, formal meth-

od, 1104

amino nitrogen, Van Slyke
method, 935

amino nitrogen, micro-
chemical, Van _ Slyke,
940

ammonia, Folin macro-
chemical, 1092

ammonia microchemical,
1093

ealcium, 1039, 1108

chlorides, Vollhard, 1107

conjugate sulphates, 1106

creatinine, Folin, 1093,
1017, 1026

creatine, Foiln-Benedict,
1017

ethereal

urine,
glucose,
glucose,
glucose,

sulphates of
Folin, 1096
Benedict. 1109
Bertrand, 888
Bertrand and
Munson and = Walker,
891

glucose by Bang, 1110

glucose, microchemical,
Bang, 1112

glucose, Bang microchemi-
cal in blood, 1114 .

hippuric acid, 1099

hydrogen ions in urine,

1122
inorganic and_ total sul-
hates. volumetric

method, 1106

milk fat, Meigs’, 951

of nitrogen Kjeldahl-Guan-
ning, 931
Quantitative determination,
of nitrogen, microchemi-
eal, 1010, 1023, 1081
urea, Benedict's method,
1084
of urea, by ureometer,
1090 .
of urea, hypobromite
method, 1090 :
of urea, microchemical,
Folin, 1088
of urea, urease method,
1086, 1087
urea, urease, microchemi-
cal, 1088, 1032, 1014
phosphates _ b: uranium
acetate, 110
phosphoric acid, Neumann-
Pemberton, 933
saccharose in urine, 1121
purines in urine, 1103
total sulphates of urine,
1105

uric acid, microchemical,
1098, 1019, 1030
Quantitative methods, blood,
1007-1059

urine, 1078-1124
Quince-oil acid, 65

Racemic acid, 20, 22
Racemized casein not digest-
7 ed, 403
Raffinose, action of invertin
and emulsin on, 732
see Melitose
Rape oil, 65
iodine number, 70
saponification number, 73
Rapie acid, 65
ee fat, iodine num-
er,
Reaction of protoplasm, 245-
250

Reductions in body, 764

in living matter, 9
Reductonovain, 747
eter ees double, of myosin,

Regnault and Reiset, 279
Rehfuss tube, 972
Reichert-Meiss] number, 74
Rennin, law of action, 379
in stomach, 349, 376-
382
practical work on, 969-
971
Rennin time law, 970
Resorcin, reaction for ke-
toses, 881
Respiration, a combustion,
274
anerobic, relation to car-
bohydrates, 41
apparatus of
and Reiset,
blood gases, 473
Respiration calorimeter of
Rubner, 281
mes - Atwater - Benedict,

Regnault

83
Respiration, consumption of
oxygen by various tis-
sues, 489
exchange in the tissues,
489

mechanism of in
478

of blood, 491
of brain, 585

lungs,

 

INDEX

Respiration, of brain, effects
of anesthetics on, 594
of ceuh relation to nucleus,
1

of nerve fibers, 593

of tissues, 850

physical chemistry of oxi-
dation, 256-261

possible réle of oxidases

in respiratory exchange,
483

reduced .by acid, 247
Respiratory quotient, 278
variation with the diet,

280
Reticulum (Stomach), 338
Reversible action of en-
zymes, 255

Reversible gels, 230

Rhamnose, formula, 17
inglucosides, 53

Rhizocholie acid, from cholic,

480
Ribonie acid, formula, 33
Ribose, 19!
in muscle, 612
in nucleic acid, 169, 171
structural formula, 30
Rice, relation to _ beri-beri,

Ricinoleic acid, 65, 72

Rigor mortis of muscle, 675

Robert’s amylopsin quantita-
tive method,

Rosenheim and Drummond’s

volumetric sulphate
method, 1106
Rosolie acid as _ indicator,

371
noe ioe of polarized light,
Rothera’s acetone reaction,
1073

Rubner, conservation of en-
ergy law, 281

Rubner calorimeter, 281

Rumen, 338

Saccharic acid, from nucieic
acid,
formula, 33
Sacchrose, 19, 55
absorption of, 452
in urine, quantitative,
1121
inversion of in .intestine,
388
oe of in stomach,
properties and formula, 55
quantitative determina-
tion in urine, 1121
synthesis from glucose by
alkali, 34
Salicin, 53
Salicylic aldehyde reaction
for acetone, 10
Saligenin, 53
Saliva, 320-336
ash in, 322
chemistry of mucin, 323-
325

composition of mixed hu-
man,
digestion of starch by, 326
digestive action of, 326-
336
enzymes in, other than
ptyalin, 334 .
excretory substances in,
functions of, 323
ion concentration in,
322

 

1147

Saliva, importance of, 337
physiology of secretion of,
320-322 ;
practical work on, 962-964
ptyalin in, 327-333
reaction of, 322
sulphocyanate in, 335
Salivary digestion, 326-336
in stomach, 382
Salivary glands, composition
and metabolism, 335
Salkowski, cholesterol reac-
tion, 82, 914
Salmin, amino-acids in, 128,
129

elementary analysis, 177
formula, 178
tripeptide structure, 136

Salts, action on  dissocia-
tion of oxyhemoglobin,
488

action on salivary diges-
tion,

influence of solution ten-
sion on __ precipitating

power of, 228
influence of valence of, on
colloids, 224
in living matter, 13
inorganic, of brain, 583

precipitation of colloids
by, 224-228
of bile; see Bile salts

relation to blood clotting,
520

Salt solutions, electrical con-
ductivity of, 193
nature of, 193
supmplouy freezing points,

internal pressure of, 198
ionization of, 194
Salvelin, amino-acids in, 128
Santalol, 80
Sapogenin, 54

Sepeniien Ton: of fats, 72,
Saponification number, de-

termination of, 909

fats and oils, 72-73
Saponin, laking by, 997
Sarcode, 4
Sarco-lactic acid, 626
Sarcosine,

relation to creatine, 707
Sardine oil, iodine number,

Scallop muscle, composition,
608

Scatole, isolation and color
reaction, 987
from tryptophane, 441
Seatoxyl sulphate, 751
Schiff cholesterol reaction,

83,
Schiff’s reaction
acid, 1063
Sclero proteins,

for uric

definition,

Schiitz and Borissow, law of
enzyme action, 334,
338

Scombrin, amino-acids in,
128

Scurvy, 848

Scymnol, 427

Secretin, 391

composition and prepara-
tion,

Secretion a syneresis, 234

Selective adsorption, by gela-
tin, 229

Seliwanoff’s reaction, 881

Semi-drying oils, 67
1148

Semipermeapie membranes,
Senility and intestinal pu-
trefaction, 443
Seralbumin; see Serum al-
bumin
Serglobulin ;
globulin
Serine, amount in various
proteins, 128, 129
formula, 117
in phospholipins, 100
Serum albumin, amount in
plasma, 551
composition, 110, 139, 553
origin of, 553
preparation of, “24
Serum fibrinogen, 469
Serum globulin, amount in
plasma, 551
preparation, 923
properties, 552
aa oil, iodine number,

see Serum

Sexual difference in metabo-
lism, 712

Sexual glands, internal secre-
tion,

Schaffer and Marriott meth-
od, hydroxybutyrie acid
and acetone in urine,

1119 -
Sham feeding, 343
Shark-liver oil, iodine num-

ber, 7
Siegfried carbamino reaction,
Silk, India tussa, amino-acids

n,
Ttallan, amino-acids, in,

aa amino-acids in,
Simple proteins, classifica- ||
tion of, 112
Sinalbin, 53

Sinapinie acid, 53

Sinapin i 53
Sinigrin, 5

Sitosterol, 87

Se ae secreted by,

water secreted by, 686
Skunk, n-butyl mercaptan in,

Smooth muscle, proteins in,
Soap, cleansing action of,
222

colloidal nature, 222
Soap films, thickness, 215
Soaps, 72

emulsifying action on fats,

in bile, 432
influence on surface ten-
sion, 209
Sodium fn blood, 1040
Bele, bean, fodine number,

Soluble starch, 328

Solution tension, influence
on precipitation, 228

Serbite, 41

Sorbose, 19, 29

Sérensen method, amino-
acids in urine, 1104

Strensen titration, of pep-
tie hydrolysis, 363

practical, 982

Soy bean, urease, 1014

Spallanzani, 349

Specific heat of water and
{mportance fn iife, 193

 

INDEX

Specific rotatory power, 25

Spectra of blood and other
pigments, 493

Spectra of hemoglobin deriv-
atives, 493, 10038

Spectrophotometer, 503

Spectrum of oxyhemoglobin,

Speigler's reagent for al-
bumose in urine, 1069
Spermatozoa, composition of
heads, 161, 162, 177
free from nuclei, 160
method of analysis, 161
protamines from, 128, 177
Spermaceti, 62, 71, 80, 81

SPO heads, composition,
free from  phospholipin,
100

method of separating from
tails, 161

Sperm oil, 62, 80
iodine number, 71

Sphingol, 577

Sphingomyelin, 570, 577
lignoceric acid from, 578

Sphingomyelinic acid, 577

Sphingosin, 577, 579

Sphingostearic acid, 577

Spongosterol, 87

Stachydrine, 48, 442, 708

Stachyose, 19, 52

Stains, _ basic

acid, 176

Stalagmometer of

207,

Starch, 19
chemistry of, 58
digestion by pancreas, 398
ae by saliva, 326-

digestion in stomach, 335
soluble, 58, 328
Steapsin, action of bile on,

and nucleic

Traube,

adsorption of by filter.
397 :

coenzyme, 397
conditions of action of,

395
effect of bile on, 981
manner of action, 398
optimum hydrogen ion con-
centration, 327
Stearic acid, 62, 63, 64
Stearin, 63, 64, 66
Stercobilin, 417
Stercorin, 87, 4384, 4385
influence of diet on, 435
Stereoisomers, 22
Sterols, 62,
table of, melting points, 87
Stigmasterol,
Soe acidity of contents,

appetite secretion, 343

bile in, 382

contents, theory of_ titra-
tion of acidity, 370

control of secretion,

in,
digestion in, 838-386

digestion of carbohydrates
In, 350
fats

digestion of
350

digestion of proteins in,
351

gas-

in,

aoe practical work,
fasting, acldity of, 367
free HCl tn, 367

human, general physiology

 

of, Alexis St. Martin,
339
Stomach, in coustipation, 341
influence of ardent spirits
on, 341
tae ek digestion in,
lactic acid in, 975
morphology of, 338
mucosa, chlorine in, 372
nerves of, 345
optimum acidity, 367
Tawlow pouch, 343, 344
phenomena of secretion in,
relation of amount of food
aud amount of juice,

salivary digestion in, 382

secretion after mechanical
stimulation, 341

secretion of HCl in, 371-
374

see also Gastric juice
aueneny of digestion in,

time of digestion, 340
Sturine, amino-acids in, 128
sia a analysis of,

Styreolene glucosides, 53

Submaxillary gland, oxygen
consumption of, 48

aves acid, from brain,

in muscle, 614
See ues, and biuret test,

Sucrose ; see Saccharose

Sugar, in blood, amount, 471

Sugar metabolism, 771
literature, 797

Sugar puncture, 772

Sugar tolerance, after Eck

fistula, 789
Sugars, eperetion in intes-

By
Sulphates, ethereal, in urine,

in urine, 754
of urine, determination,
1105-1106
Sulphatides ; see Sulpholipins
Sulphocyanate in saliva. 335
Sulphocyanpate solution,
Standardization of, 908
Sulpholipins, 62, 570, 580
Sulphur, amount in various
piles, 427
amount in various
teins, 139
amount of,
324

pro-
in mucoids,

condition of, in oxyhemo-
globin,
detection of, in proteins,
, 151
excretion under various
conditions, 754
income and outgo of body,

753
in urine, 753
neutral, of urine, 754
compounds in _ proteins,

135
Sulphuretted hydrogen, ac-
tion on ptyalin diges-
tion, 332
unton with hemoglobin,

Sulpburte acid, conductivity
and inverting power,

195
in mucin and mucolds, 325
Sunflower oil, iodine num-
ry
Suppuration, action of, on
a content of blood,
je)

Supra-renal capsules, adrena-
ne in,
amount of lipin in, 62
ee and histology,

chromaffine tissue, 668
embryology,

functions of, 669

oxygen consumption of,

4
relation to blood pressure,
670

relation to emotions, 675
relation to glycogen and
sugar metabolism, 778
results of extirpating, 669
Surface film, accumulation
of substance in, 209, 210
of fat, 65
Surface films, contractile ac-
tion of, 233
in gels, 232
Surface tension, 206-213
adsorption, 241
eapillary method of de-
termining, 207
drop method of determin-
Dg, .
influence of fats and soaps
on,
in muscle contraction, 630
of water and salt solu-
tions, 208
relation to gels, 232
relation to precipitation of
colloids, 226-227
role in determining distri-

pution of cell  sub-
stances, 242
Suspensoids, definition, 217

Srveer oly specific gravity of,

Swelling, and muscle con-
traction, 62
by capillarity, 236
By molecular imbibition,

by osmosis, 236
heat set free by, 236, 241
influence of salt on, 238
laws of, 236
nature of process, 239
of cellulose, 241
of fibrin in acid, 235
processes in protoplasm,
rate of, 237
various kinds of, 236
Synalbumose, 929
Syneresis, and secretion by
protoplasm, 23!
of gels, 233 , .
Synthesis, dehydration, in
living matter, 9, 10, 256
in fats, 76-78
Synthesis, relation to nu-
7 cleus, 160
Syntonin, 360

Tagatose, fromula, 28

Tallow, 63,

Tallqvist method for hemo-
globin, 992

Talose, structural formula,
2

Taraxasterol, 87

Taririce acid, 65
stereoisomerism in, 21, 22

Tartaric acid, 22

 

INDEX

Tartronic acid, 33

from glyerol, 66

from uric acid, 724
Taurine, 426

in brain, 580

in metabolism, 818

in muscle, 611
Taurocarbamiec acid, 821
Taurocholeic acid, 431

determination of, 426

properties and  prepara-
tions, 426
Tautomeric rearrangements

of xanthine, 167
Teeth, composition of, 644
Teichman reaction for hemin,

1003
Teichman’s crystals, 1003
Tellurium, methylation of,
in body,
Temperature, coefficient, ex-
planation of, 253
of reduction of oxyhemo-
globin, 483 :
of vital, reactions, 253
Tendo Achillis, composition
of, 635
Tendomucoid, 6389
decomposition
324
Terpenes, 78, 79, 80 .
excretion of, as glycuro-

products,

nates, 758

Terpin, 79

Testes of fish, method of
separating spermatozoa
from, 161

Testis, amount of creatine

in, 9
Test meal of Ewald, 342
Tetramethylenediamine, 442

in urine, 747

Tetra nucleotide, 172
Tetra nucleotidase, 171, 406
Tetrasaccharides, 19
Tetramethyl putrescine, 442
Tetroses, 19
Thioalbumose, 929
Tule gane propionic acid,

117
Thioethyl amine, 442
Thiopyruvic acid, 819
Thiosulphate solution, stand-
ard, making of, 908
none Bets hemacytometer

9
Thoracic duct, 452
Thrombin, does not cause
gn LO EBCUIAT clotting,

(fibrin ferment), origin in
plood plates, 527, 532
not a ferment, 527
preparation of, 532
Thrombogen; see Prothrom-
in
Meron PopieeHe substances,
Thrombokinase, 534
Thymic acid, 164
Thymine, amount in nucleic
acid, 169
formula, composition and
properties,
origin in nucleic acid, 164
Thymol, excretion, 762
Thymus gland, physiology
of, 675
Thymus nucleic acid, decom-
position of,
structure of. 172, 173
Thynnin, amino-acids in, 164
Thyreoglobulin, 664
Thyroids, 657-666

 

1149

Thyroids, active principle of,
= 666 , p

Basedow’s disease, 659
colloid, 665

exophthalmic goitre, 659
extracts, action of, on
metabolism

6
extracts, action on blood
pressure, 661
function, cretinism, 658
659
influence of ingestion on
creatine excretion, 716
iodine in, 664
iodothyrin, 667
morphology, embryology
and histology, 657, 658
myxedema, 659
nitrile reaction for, 663
results of extirpation of,

660
thyreglobulin, 664, 665
Thudichum, on the brain, 568
Tiglic acid, 65
Tissue extracts, influence on
_ ,blood clotting, 518
Tépfer reagent for free HCl,

Tollens’ glycuronic acid re-
action, 1075
Tollens-Neuberg _glycuronic
acid reaction, 1075
Tollens’ . phlorogiucin reac-
tion, 879
Toluic acid, in urine, 763
Tolurie acid, 762
Toluylendiamine, action on
bile secretion, 419
Toxicity and solution ten-
sion,

Trehalose, composition, 19, 54
Trichloracetic acid, inver-
sion coefficient, 195
Triketohydrindene hydrate,
formula of, and protein

reaction, 150, 919

Trimethyl amine, from
lecithin, 93
in blood, 93

in cerebro-spinal fluid, 586
in urine, 93, 747
Trimethyl-amino acetic acid,

92
butyric acid, 442
Tele oxybutyro betaine,

tryptophane betaine, 442
Trinucleoside, 175
Trioses, 19
Tripeptide, 134

in protamine, 136
Triticonucleic acid, 169
Triolein; see Olein
Trioxypyrimidine, 185
Brlenechander classification,

Tristearin; see Stearin
Tropexolin as indicator, 371
Tripalmitin; see Palmitin,
Trypsin, 401
action of bile on, 402
digestion of artificial poly-
peptides, 403
Jaw of rate of action, 405
cope alkalinity for,

products of tryptic diges-
tion, 402 en oe
Preparation of, free from
lipase and amylopsin,

Trypsinogen, 401

seg vetion by Ca salts,
1150

Tripsinogen, activation b:
enterokinase, 401 :
practical work on, 977-980

Tryptic digestion, bromine

reaction of, 149
of ji peace polypeptides,

optimum alkalinity for,
980

Tryptopbane, amount in
various proteins, 128
a necessary constituent of
food, 82:

color reactions of, 148,
color reaction with bro-
mine, 1

eolor reaction with p-

dimethyl-amino-benzalde+
hyde, 149

decomposition by bacteria,
441 :

derivation of word, 400
formula, 1
in tryptic digestion, 400
putrefaction of, 441, 750
Tung oil, 65
Turpentine oil of, 6, 79
oil of, autooxidation, 68
Tyndall phenomenon of col-
loidal solutions, 217
Tyrosine, amount in various
proteins, 128, 129
crystals, 119
decomposition by bacteria,

formula, 117
orieeton and fate in body,

Uteiaanns lactic acid reac-

on,

Ulcer of stomach, acidity in,
370

Ultra microscope, 216
Ultra microscopic picture of
gels, 231,
Ultra violet light, physio-
logical action of, 46
synthetic action of, 46
Uracil, chemistry and for-
~ mula, 168
origin of, 164
Uramil, 184
Uraminobenzoie acid, 764
Uranium acetate method for
_ phosphates, 1109
Ores arer liver corrosion,

after liver disease, 699
ammonia in portal and
hepatic blood, 701
amount in blood after kid-
ney extirpation, 694
Amount secreted per day,

chemistry of, 691

commensurate with uric
acid in birds, 700

conversion to biuret and
cyanuric acid, 702

determination by  hypo-
bromite, 1090

determination, microchem-

. ileal, Folin, 1088

determination, urease
method, 1086

excretion after ingestion
of ammonia and amino-
acids, 703

excretion in perspiration,

694
excretion in saliva, 694

 

INDEX

Urea, excretion reduced after
benzoic acid, 70
oo varies with diet,

formation from ammonium
carbamate, 704

from cyanamide, 704

in blood before and after
perfusion, 696

in blood after adding am-
monium carbonate, 696

in muscle, 613

in urine, 691

in Eine after Eck fistula,

69:

literature, 766

nitrate, 692, 1061

nitrogen in blood, 509

origin from arginine, 703

origin of, in mammals, 693

origin, summary, 703

other sources of, 701

oxidation of, 692, 703

physiological action of,
704

percursors of, 701

preparation from _ urine,
1060

quantitative determination
of, 1085

quantitative determina-
tion, Benedict’s method,
1085

quantitative determina-
tion, Folin method, 1088
union with salts, 693
Urease, in soy bean, 693
method of determining
urea, 1084
Ureometer, Doremus, 1091
Urethane, formula, 122
Urie acid, allantoine from,

a small part of ingested
purine, 727

amount in urine, 691, 721,
738

chemistry of, 722

Cole’s method of detec-
tion, 1064

crystalline form, 723, 1078

destroyed by alkali, 723

destruction of, by various
organs, 724-725

detection of, in liquids,
1064

diagram of catabolism of
nucleic acid,

endogenous and exogen.
ous, 726, 729
enzymes concerned in

formation, 732
excretion, birds, after liver

extirpation, 700
excretion by birds, after

ingestion of amino-acids,

excretion on various diets,

formation from _ spleen

pulp,
formation from xanthine,
732

formation in birds and
reptiles, 70

lood, microchemical
determination. 1019, 1080
ne and lactim forms,

literature, 766
methyl glyoxalidine salt,

in

murexide reaction, 724
murexide test for, 1062

 

Uric acid, not destroyed by

human tissues (7?) 734

origin in nucleins, 724

oxidation of, 734

piperazine salt of, 723

possible origin from non-
purine bodies, 737

precipitation reaction, 1063

pres from urine,
060

quantitative determina-
tion, by microchemical
method, 1019, 1030,
1097

quantitative determina-
tion, Folin-Shaffer meth-
od, 1097

relation to  leucocytosis.
7

reducing actions, 1062

Schiff’s reaction, 1062

standard solution, sulphite
reagent, Folin's, 1019

standard solution of, Fo-
lin, 1019

synthesis from _ dialuric
acid in liver, 740

synthesis from uramil and

urea, 183

synthesis in birds and rep-
tiles, 737

synthesis in dog’s and

bird’s livers, 738-739
time of -excretion in rela-
tion to eating, 728
uricolysis, 734
variation with age, 730
variation with disease, 72%
Uricase, 734
effect of fasting on, 738
Uricolysis, 734
Uricolytic index, 736
Urine, b
acetoacetic acid,
nation, 6
acetoacetic and hydroxy-
butyric acid, determina-
tion of, 1119
acetone, determination.
1116

acetone in, 759. 1076, 1116
acidity of, 688, 689
acidity, variation in dis-
ease, 689
albumin, in, detection, 1067
allantoine in, 740. 1100
amino-acids in, determina-
tion, 1104
ammonia in, 689, 746
amount per day, 292, 687
amount secreted and rela-
tion to urea, 705
aromatic oxy acids in.
746

746

determi-

basic substances in,

cadaverine in,

calcium and magnesium
in, T57

carbon in, per day, 292

chlorides. 756

composition in high and

low protein, 754
creatine and _ creatinine.
705-721

detection of glucose in,
1069

determination of H ions,
1123

differentiation of glucose
and lactose in, 1072

distribution of purine ni
trogen in different mam-
malia, 736

energy in, 292
Urine, energy expended in

secretion of, 690

ethyl sulphide in, 816

excretion of various drugs
in,

freezing point, 690

general composition, 691

glucose in, quantitative
determination by vari-
ous methods, 1109-1113

glycuronic acid in, 759,
1075

gynesin in, 747

hippuric acid in, 741-746
hydrogen ions in, 689
imidazol-acetic acid

T47
indican in, 749
indoxyl in, 748
inosite in, 611
Methylated excretory
products in, 718 |
methyl guanidine in, 747
methyl pyridine chloride
in, 747
mingin in, 747
nitrogen in, per day, 292

in,

nitrogenous constituents
in,

nitrogenous substances
present in small

amounts in, 746
novain in, 747
ornithine conjugates, 763
osmotic pressure of, 690
oxybutyric acid in, 1119
oxymandelic acid in, 747
pathological constituents

of, 757, 1067
paraoxyphenyl

acid in, 748
pentoses

propionic

in, detection,
pepsin in, 365
phenol in, 748
phenyl acetic acid in, 748
phosphorus in, 755

Urine pigments in, 765
practical work on, 1060-

1123
protein in, 757, 1067
ptomaines in, 747
ptreszine in, 747

purine bodies in, 721-
738, 1103

qualitative examination
of, 1067

reaction, 688, 1122

reductonovain in, 747
result of Eck fistula on,

saccharose in, determina-
tion, 1121

sactoxyl in, 748

specific gravity, 688

substances paired with gly-
cine, 763

sugars in, 758, 1069, 1109

sulphuric acid conjugates,
763, 1106

sulphur in, 753-754, 1105

trimethyl amine in, 747

uramido conjugates, 764

urea, chemistry of, 691

urie acid, 721

urobilin in, 417

vitiatin in, 747

Urobilin, 417, 765
absorption spectrum, 418
detection, 1076

 

INDEX

Urobilin, gives Ehrlich’s re-
action, 418

Urobilinogen, 417

Urocanic acid, 747

Urochrome, 765

Uroferric acid, 746

Urorosein, 751

Urotoxie coefficient, 747

Uroxanic acid, 735

Valeric acid, iso, 64
Valine, amount in various
proteins, 128,
formula, 116
Van Slyke method for amino
nitrogen, 137, 935
Van Slyke method for carbon
dioxide, 1042-1048
Velocity, of chemical reac-
tions, 251
of reaction of first order,

of salivary digestion, rela-

tion to concentration of
ptyalin, 333

Mencue vives: gases of, 489,

Vertebrate skeleton, relation
of composition of, to in-
vertebrate, 324-325

Vicilin, composition, 110

Vicin,

Vidin, 100

Villi, 451

Vignin, composition, 110
Vital .phenomena, rdle of

phosphoric acid in, 160
Vitamines, 838
literature, 853
Vitellin, 113, 129
Vitiatin, in urine, 747
Vitreous humor, composition,
637
Viscosimeter

of
pitz, 511
Viscosity of blood, 510
effect of diet on, 513
effect of gases on, 513
on of temperature on,

Burton-

5
plasma, 512 _
relation to fibrinogen, 513
Vivi-diffusion of blood, lit-
erature, 561
method, 469
Volemose, 30
Vollhard’s chloride method,
1107

Von Mering, discovery of
pancreatic diabetes, 781

walaue oil, iodine number,

7

SYa ECE) SO PSG EDEN by gels,

amount excreted‘in urine,
686

amount in various tissues,

aay ae excreted in feces,

amount needed per day,
301

as a food, 301

cohesive pressure of, 198

composition of, 190, 191

dielectric constant, 192

excretion in perspiration
oat respiration, 293,

 

1151

Water, in brain,
with age, 568
ionizing power, 193

mee uae association of,

variation

power of accelerating re-
actions,

powers of solution, 192

properties of, 190, 192

role in vital reactions, 190

specific heat, 192

oP inductive capacity,

Waxes, 62. 80
iodine numbers, 71

Weidel's reaction ;
Murexide reaction

Weyl’s nitroprusside reac-
tion for creatinine,
1065

Whale oil, iodine number, 71

Whey albumose, 376

White connective tissue, 634

White matter of brain, com-
position, 583

White matter of brain, spe-
cific gravity, 584

eRe from brain,

de-

see

Wiechowski, allantoine
termination, 1101
Wijs iodine solution, 909
Wooldridge, biography of, 535
Wooldridge’s view of clot-
ting, 535
of difference between fi-
brin and fibrinogen, 525
Wool wax, S80, 81
iodine number, 71

Xanthine bases; see Purine
bases

Xanthine decomposition on
dry heating, 167

Xanthine, discovery,

prop-
erties and
167

chemistry,

enol form, 167
ibm euanine by guanase,

from nucleic acid, 166

in urine,

origin in nuclein, 161
Xanthine hydrolase, 732
Xanthine oxidase, 73

distribution in tissues, 730
Xanthophyl] in butter, 64
Xantho-proteic reaction of

proteins, 147. 916 .

Xiphin, amino-acids in, 128
Xylene, fate in body, 763
Xylose, 19, 29

in muscle, 613

Yeast nucleic acid, 175
Yellow atrophy of liver, ef-
fect on nitrogen excre-

tion, 699
Yellow connective tissue,
composition, 635
composition,

Yolk of eggs,
316

Zaltty, eompOstHOe, 112, 129,

Zymase, 51, 625
Zymogen, conversion to en-
zyme by acid, 3382, 352
in stomach, 354
nature of, in amylase, 331
INDEX. PRACTICAL WORK AND METHODS.

determination

Adrenaline,
Cannon and

of, Folin,
Denis, 124

Alkali reserve of blood, 1042

Amino-acid nitrogen, Van
Slyke microchemical
method, 940

Amino-nitrogen
y Van Slyke method,
9

Bile, 983-986
cholesterol in bile, 987

Hammarsten’s_ test for
bilirubin, 984

Gmelin’s test for bile pig-
ments, 984

Hay’s test for bile salts,
986

Huppert-Cole test for bili-
rubin,

Pettenkofer’s test for bile
salts, 985

physical properties, 985

powers of solution, 984

preparation of bile salts,
Fler uer? pe aoe. *

preparation of glycocholic
acid, 985

reactions to detect pig-
ments, 984

ae precipitate proteins,

Blood, 990-1059
acid hematin, 1003
alkali reserve, 1042
analysis of, 1007
039

calcium in,

chlorides in, 1036

clotting, fibrinogen, salt
plasma, etc., 1004

orystalileing hemoglobin,

determination of hemo-
globin by Tallqvist
method, 992

glucose in, determination,
microchemical of Bang,

1114
glucose In, 1021, 1029
hematokrit method for cor-
puscles, 992
hematoporphyrin, acid and
alkaline, 1003
hemin crystals, _Teich-
man’s crystals, 1003
hemochromogen, 1003
hemoglobin by Dare’s
hemoglobinometer, 998
hemoglobin by Newcomer’s
method, 993
hemoglobin, by Oliver's
hemoglobinometer, 993
laking of, 996
laking by hypotonicity, an-

esthetics, bile salts,
saponins and dilute al-
kalles, 996-997

magnesium In, 1038
methemoglobin, 1001

determined,

 

Blood,
in,
Oliver’s
9!

potassium in, 1040

sodium in, 1040

spectra of various deriva-
tives of hemoglobin and
compounds, 998

i a a direct vision,

Ten prota nitrogen
1007, 1023
hemacytometer,

spectrum carbonyl hemo-
globin, 505, 1000

spectrum of oxy and re-
duced hemogolbin, 505,

1

Stoke's solution, composi-
tion, 1000

Thoma-Zeiss determina-
oD of blood corpuscles,

uric acid in, microenemical
determination, 1019,
1030

Bone, practical work on, 960

Carbohydrates, action of
acids on pentoses, 884
action of strong acid on
disaccharides, 886
action of strong acids on
monosaccharides, 884
increase of reducing power
by peat treatment, 871,

Benedict’s solution for
quantitative determina-
tion of, 1109

cane sugar, starch, and
gum not hydrolysed by
alkall, 887

caramel and humus form-
ed by alkali, 871

decomposition of. by alka-
lies, 871

detection of galactose,
mucie acid, 881

detection of pentoses by

phloroglucin, Tollens,
879-880

detection of starch by
lodine, 881
disaccharides. action of

acid on, 886
experiments on, 870-903
Fehling’s solution, S72
fermentation by yeast, 886
formation of alcohol and

CO. from, by yeast, 886
methoxyfurfural. forma-

tion from hexoses, 885
formation of methoxyfur-

fural, levulinie acid,

and humic acid from

hexoses, 885
formation of methoxyfur-

fural from __levulose,

88h
furfural reactions for de-
tecting pentoses, hexoses

1152

 

and glycuronates, 879,

Carbohydrates, hexoses, ac-
tion of acids on, 884
ketose reaction, Seliwan-
off’s, 881

lactose, preparation from
cow's milk,

methods of identifying and
detecting, 877

Molisch reaction, 877

Moore’s test for, 871

paphthoresorcin reaction
to distinguish pentoses

and glycuronates, 880,
1075

orein reaction for pen-
toses, 880 -

osazones, formation from
hydrazine, 882

pentoses, action of acid
on,

pentoses, formation of fur-
fural,

polysaccharides, action of
strong acid on, 887

saccharose, inversion of,
886

starch lodine reaction, 881

reaction with resorein, 882

reducing actions of, 870

reduction of Barfoed’s
solution, 875

reduction of bismuth salts
by, Bottger’s Nylander’s
and Allman’s reactions,

reduction of cupric salts,
reduction of mercury salts,
876

reduction of
blue, 876

reduction of picrie and
phosphotungstic acid,
873-874

near of silver salts

methylene

Seliwanoff’s reaction, 881
literature, 882
starch and gum _ arabic,
hydrolysis by acid, 887
Tollen's reaction, 879
Coagulation of blood. 1004

Creatine, from meat, 958

Dakin separation of amino
acids, §$
Direction for work, 861

Equipment, student and gen-
eral, of laboratory, 858

Fats; see Lipins
Feces, practical
87-989

occult blood in, 988

work on,

Gastric contents, method of
obtaining, 972
Gastric digestion, 965-976
any of gastric juice,

active principle stored in
mucosa, 965

coagulation of milk by
rennin, 969

comparison of action of
pig's and calve’s stom-
ach mucosas, 971

determination of total
acidity, free HCl,
amount of chlorine, 971:
975

existence of
965
lactic acid, detection by
Hopkins’ method, 976
lactic acid in, detection by
Uffelmann’s method, 976
optimum acidity for pep-
tic action, 966 .
preparation of artificial
gastric juice, 967
quantitative determination
of pepsin, 967
pepsin, quantity determin-
Saat Jacoby’s method,

pepsinogen,

pepsin, quantity deter-
inet by Mett’s method,

reaction of mucous mem-
brane, 965

rennin activated by HCl,
969

time law of peptic diges-

tion, 9

time law of rennin action,
970

Glucose, determination by

Bang’s pearoxzlaniine
method, 11

determine Oe Bertrand
method, 8

determination by Bertrand
and Munson and Walker
method, 891

determination by  micro-
ebemical method, Bang,

1112

determination in blood
microchemical method,
Bang, 1112

microchemical, Folin-Wu,
1021

ee by Bene-

dict, 1109
by Lewis Benedict, 1023
Glutaminic .acid, prepara-
tion, 946

Hydrogen ions, method of
determining, 1049

Intestinal digestion, 977-988

Intestine, enterokinase, 980
erepsin in mucosa. Séren-

sen titration, 982

intestinal putrefaction
products, 987

invertin in mucosa, 983

tests for indole and sca-
tole, 987

Lipins, 904-914
absorptions of fodine by
unsaturated fatty acids,
907
acrolein test for glycerol,
907 :
preparation of,

914

chlesterol,

cholesterol reactions,

 

INDEX

Lipins, cleansing action of
soaps, 910
determination of saponifi-
cation numper, 909

emulsification of fal vy
soaps, 905

free acid in oils, 905

see in neutral fat,

hydrolysis of glycolipin,
912

iodine number, determina-
tion of, 907
Liebermann-Burchard cho-
lesterol reaction, 914
neutral fats, crystalliza-
tion of, 904
Lipins, neutral oils, surface

tension of, iD

phospholipins, preparation
of, §

preparation of glycolipin,
eerebrin from brain,
911

Salkowski cholesterol re-
action,

saponification of fat, 906

separation of by
salting out, 906

showing how to identify a
neutral fat, 90

ee obuity, of fats and oils,

surface rengien vf water
lowered by, 9

tests for Piyecrol, fatty

phosphoric

acids and

acid in pbospholipin,
912

thiosulphate solution,
Standardization of, 908

Meat,
95

Milk, fat, determination by
Meis’s method, 953
fat, by Babcock, 952
freezing point of, 955
MUR zapetede in by Folin,

practical work on,
7-96

practical work, 949-954

by Van

935,

ammonia, methods, 1093

Kjeldahl - Gunning- Arnold
method, 931

Nessler method, 1083,

method,

1084-1092

Pancreas, amylolytic activ-
ity, 977-979
amylolytic and lipolytic
hydrolysis depend on the
popence of salts,

amino,

Nitrogen,
method,

Slyke ©
940

1007
microchemical

urea, methods,

determination of optimum
alkalinity for tryptic di-
gestion, 980
effect of acids and alkalies
on amylase, 979
lipolytic activity, 978
preparation of _ tryptic
solution free from li-
pase, 981
977,

Bretee lene

Robert’s method for amy-
lase determination. 979

steapsin is destroyed by

action,

 

1153

heat influence of
bile

Potato,

960,-

Proteins, 915-948
albuminoids, preparation
and properties, 930

biuret reaction, 915

and

, 98.
practical work on,

color reactions of, 915-
920

detection of organic phos-
phorus,

determination of nitrogen
in, by Kjeldahl-Arnold-
Gunning, 931

determination of  pbhos-
phorus in nucleic acid.
by Neumann-Pemberton
method, 933

deutero-proteoses, 929

dimethyl - amino - benzalde-
hyde reaction, 918

Ebrlich's reaction, diazo,
919

elementary composition of,

formaldehyde reaction
Acree, 918

formation of biuret from
urea, 916

gelatin, 930

globulin, Pre euen by

dialysis,
soph ac of recipitation by
salts, 9
heat coagulation of. 921
hydrolysis. by Dakin’'s
method, 4
reaction,

Liebermann’s
918

methods. of detecting in
solution, 5
Millon reaction, 916
Molisch reaction, 919
ninhydrin reaction, 919
peptone preparation, 929
precipitation by alkaloid
reagents, 921
precipitation by
metals, 92
preparation and properties
of albumoses, 928
preparation of casein, 949
preparation of edestin, 925
preparation of excelsin,

gliadin,

of nucleo-pro-
nucleic acid,

heavy

preparation of
926

preparation
tein and
927

preparation of secondary-
derived proteins, 928

preparation of serum al-
uate and — globulin,

preparation of
phane, 941

Hopkins-Cole reaction, 917

neduced sulphur reaction,

trypto-

tryptophane reactions, 917
ea eoproterc reaction,

Reagents, desk and
Shelf, 858,-859

Saliva, 962-964
excretion of salts in, 964
digestive action, 962
formation of dextrins from
starch,
mucin in, 962

side
1154

Saliva, ptyalin, conditions of
activity,
ptyalin killed by heat, 963
reaction,
Seliwanoff's reaction, 881

Urine, 1060-1123
acetoacetic acid, detection
py Gerhardt’s _ ferric
chloride test, 1074
acetoacetic acid, detection
ene and Ruttan,

acetoacetic acid, detection
by salicylaldehyde re-
action, 1074

acetone, acetoacetic acid
and hydroxybutyric acid,
determination by Shaffer
and Marriott, 1119

acetone and diacetic acid,
Folin-Hart method, 1118

acetone determination by
Folin method, 1115, 1117

acetone, detection, Legal’s
nitroprusside reaction
1073

acetone, detection, by Ro-
thera’s nitroprusside re-
action, 1073

acetone detent test,

method,

Gunning’s, 1073
acidity, Feltas
1122
in, detection of,

albumin
1067
albumin, quantitative de-
termination of, Esbach’s

method, 1068
Urine, eo ferrocyanide

test,

albumoses, detection of,

1069

allantoine determination,

Wiechowski method,

1101

allantoine, determination
method,

by urease
1100

amino-acids in, determina-
tion by formal titration,
- 1104
amino-acids in, formol
titration after removal
of ammonia, 1104
ammonia, Folin method,
macrochemical, 1092-3
ammonia, microchemical
determination, Folin
and Macallum, 1093
blood in, detection of, by
guaiac reaction, 1077
blood in, detection of; by
Teichmann hemin reac.

tion,
Bence-Jones protein, detec-
tion of, 9
pile pigments, detection by
ita ert-Cole method,
ae pigments, detection
MEpere Rama emH
: esmad, 7

bile salts, dewestion of, by
Hay’s method, 1076

bile ‘salts, detection by
oe method,
calcium, determination,
1108

 

INDEX

Urine, chlorides, determina-
tion by Vollhard, 1107
creatine, determination by
Folin-Benedict method,
1096
creatinine determination,
Folin colorimetric
method, 1094
creatinine, nitroprusside
reaction, Weyl, 1065
creatinine, picramic acid
reaction, Jaffé, 1065
creatinine, preparation of,
from urine by _ ainc-
chloride, 1064, 1095
creatinine, preparation by
Ppicrate method, 1096
excretion by a high and
low protein diet, 1078
Esbach’s picric acid re-
agent, 1069
Folin aération apparatus,
1081-1082

glucose by Benedict’s
method, 1070, 1110

glucose, by Folin and
Peck, 1110

glucose. detection of,
Benedict’s, Fehling’s

fermentation and _ osa-
zone tests, 1070

glucose, quantitative de-
termination, Folin meth-
od, 1110

glycuronic acid, detection
by Tollens-Neuberg
method, 1075

glycuronic acid, detection
hs Tollens”” method,

Heller’s albumin test, 1067

hippuric acid, determina-
tion, Folin and Flan-
ders, 1099

hippuric acid, preparation
from cow’s urine, 1065

indican, detection of, Jaf-
fé’s test, 1066

indican, Obermayer ferric-
chloride Method, 1066

hydrogen ion determina-
von indicator method,

identification of small
amounts of glucose and
lactose, 1071-1073

Koch and Folin aération
apparatus, 1086

lactose in, identification,
1072

method of distinguishing
lactose and glucose in
urine, 10

Nassler reagent, 1018, 1033,
1084

Nessterizing method, 1083

nitrogen, total by Kjel-
dahl - Gunning - Arnold
method, 1079

nitrogen, total, Folin and
Farmer, microchemical
method, 1081

oxybutyric acid, detection
by Black’s method, 1074

pathological constituents,
detection of, 1067

meee detection of,
0
phosphates, uranium ace-

tate method, 1109

 

Urine, purines, determination
a Salkowski-Arnstein,
qualitative examination,
1067

quantitative determination
of its constituents,
1078-1124

reaction and specific grav-
ity, 1079

saccharose in,
tion,

specific gravity, determina-
tion of, 1079

sulphates, conjugated sul-
phates, 1106

sulphates, ethereal and in-
organic, by volumetric
method, 1106

sulphates, inorganic, 1105

sulphur, total by Bene-
dict-Denis method, 1106

sulphates, total determina-

determina-

tion, Folin method,
1105

tests, qualitative, for chlo-
rides, phosphates and

sulphates, 1060

urea, Benedict’s method,
copper baths for, 1085

urea, determination by
urease method, 1085

urea, determination by
Benedict method, 1085

urea, Folin, temperature
indicator, 1089

urea, Folin microchemical
method, 1088

urea, hypobromite method,
1090

urea, preparation of, by
nitrate, 1060

urea, quantitative, by
Folin method, 1088

urea, urease microchemical
method, 1087

uric acid, detection of,

in urine and _ other
fluids, Cole’s method,
1064

uric acid, determination by
ca method,

uric acid, determination,
microchemical, Folin-
Wu method, 1098

urie acid, Folin's phos-
photungstate reagent,
1020

uric acid, Folin, reaction,
1063.”

uric acid, precipitation by
ammoniacal silver solu-
tion, 1063

urle acid, precipitation re-
actions, 1063

me ae preparation of,

uric acid, quantitative de

termination, micro-
chemical, Folin-Macal-
lum, 1098

uric acid, reactions for
identifying, murexide
test, 1062

uric acid, reducing reac-

tions,
pn acid, Schift's reaction,

urobilin, detection, 1076
 

 

R te EN, Prk
Eg pts peters saa eee ae

ey ee er
ana i Se bi: ake?
br bvareantensns et
na pamatoe ined bihsee Se Le
pk door oe ae em

eisai re ft
mas as Hanne
"a ii if Se ith sacasbe I eae

Hage spel nats erie Mia
ie sadn ct Vevic ape

ER af Psd ‘Vea menecea eh
ce An