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WORKS OF 
 PROFESSOR J. A. MANDEL 
 
 PUBLISHED BY 
 
 JOHN WILEY & SONS, INC. 
 
 TRANSLATIONS 
 A Text-book of Physiological Chemistry. 
 
 By Olof Hammarsten, Emeritus Professor of Medi- 
 cal and Physiological Chemistry in the University 
 of Upsala, with the Collaboration of S. C. Hedin, 
 Professor of Medical and Physiological Chemistry 
 in the University of Upsala. Authorized translation 
 from the Author's enlarged and revised 8th German 
 edition, by John A. Mandel, Sc.D., Professor of 
 Chemistry in the New York University and Bellevue 
 Hospital Medical College. viii+1026 pages. 6 by 
 9. Cloth, $4.00 net. 
 
 A Compendium of Chemistry, Including General, 
 Inorganic, and Organic Chemistry. 
 
 By Dr. Carl Arnold, Professor of Chemistry in the 
 Royal Veterinary School of Hannover. Authorized 
 translation from the eleventh enlarged and revised 
 German edition, by John A. Mandel, Sc.D. xii + 
 627 pages. 51 by 8. Cloth, $3.00 net. 
 
A TEXT-BOOK 
 
 OF 
 
 PHYSIOLOGICAL CHEMISTS! 
 
 BY 
 
 OLOF HAMMARSTEN 
 
 EMERITUS PROFESSOR OP MEDICAL AND PHYSIOLOGICAL CHEMISTRY~IN THE UNIVERSITY OP UPSALA 
 
 WITH THE COLLABORATION OF 
 
 S. G. HEDIN 
 
 PROPE890R OP MEDICAL AND PHYSIOLOGICAL CHEMISTRY IN THE UNIVERSITY OP UPSALA 
 
 JVutljoriseb translation 
 
 FROM THE A UTHOR'S ENLARGED AND REVISED 
 EIGHTH GERMAN EDITION 
 
 BY 
 
 JOHN A. MANDEL, ScD. 
 
 PBOPE8SOR OP CHEMISTRY IN THE NEW YORK UNIVERSITY AND BELLEVUE HOSPITAL MEDICAL COLLSGX 
 
 SEVENTH EDITION 
 TOTAL ISSUE, THIRTEEN" THOUSAND 
 
 NEW YORK 
 
 JOHN WILEY & SONS, Inc. 
 
 London: CHAPMAN & HALL, Limited 
 

 Copyright. 1893. 1898, 1900, 1904, 1908, 1911, 1914, 
 
 BY 
 
 JOHN A. MANDEL 
 
 ¥¥ 
 
 
 \3\A- 
 
 PRISB or 
 
 BRAUNWORTH l CO. 
 
 BOOK MANUFACTURER* 
 
 BROOKLYN, N. V. 
 
PREFACE TO THE EIGHTH GERMAN EDITION 
 
 The revision of this edition has been accomplished with the collabora- 
 tion of Professor S. G. Hedin, and the work has been divided so that 
 Hedin has revised Chapters I, III, VIII, XII and XVI, besides the Index 
 of Authors, while Hammarsten has revised Chapters II, IV, V, VI, VII, 
 IX, X, XI, XIII, XIV, XV, and XVII, besides the General Index. 
 The numerous recent developments in physiological chemistry have made a 
 thorough revision and reconstruction necessary in all the chapters, and in 
 order to prevent a noticeable increase in the size of the work, it was also 
 necessary to change the arrangement of the foot-notes more economically 
 The number of chapters in this edition is XVII instead of XVIII as in the 
 seventh edition, because for several reasons it was found advisable to 
 combine the first two chapters of the seventh edition into one chapter, and 
 at the same time certain parts of the first chapter have been incorporated 
 into other chapters, thus for example the oxidation processes have been 
 introduced into Chapter XVI (on respiration and oxidation). In general 
 the plan of the work remains unchanged. 
 
 Olof Hammarsten. 
 
 Upsala, September, 1913. 
 
 iii 
 
TRANSLATOR'S PREFACE TO THE SEVENTH 
 AMERICAN EDITION 
 
 Workers in Biochemistry are to be congratulated on the appearance 
 of a new edition of Hammarsten's " Physiologischen Chemie." At this 
 time, when so many new and important biochemical facts are being 
 published and when so many older theories and deductions are found 
 more or less erroneous, due to recent investigations using, new methods, 
 it is very fortunate that we have this complete and critical compilation 
 from the master hand of Professor Hammarsten, now in his 73d year. 
 We all owe him a great debt of gratitude for his painstaking work for so 
 many years. 
 
 I take great pleasure in expressing my indebtedness to my assistant, 
 Dr. A. O. Gettler, for the help he has given me in revising the proof and for 
 making the Indexes. 
 
 John A. Mandel. 
 New York, June, 1914. 
 
 v 
 
CONTENTS 
 
 CHAPTER I 
 
 PAGE 
 
 General and Physico-chemical 1 
 
 CHAPTER II 
 The Proteins 77 
 
 CHAPTER III 
 The Carbohydrates 196 
 
 CHAPTER IV 
 Animal Fats and Phosphatides 232 
 
 CHAPTER V 
 The Blood 250 
 
 CHAPTER VI 
 Chyle, Lymph, Transudates and Exudates 345 
 
 CHAPTER VII 
 The Liver 381 
 
 CHAPTER VIII 
 
 PlGESTION , 451 
 
 vii 
 
Tin CONTENTS. 
 
 CHAPTER IX 
 
 PAGE 
 
 Tissues of the Connective Substance 514 
 
 CHAPTER X 
 The Muscles 565 
 
 CHAPTER XI 
 Brain and Nerves 604 
 
 CHAPTER XII 
 Organs of Generation 620 
 
 CHAPTER XIII 
 The Milk 643 
 
 CHAPTER XIV 
 The Urine 672 
 
 CHAPTER XV 
 The Skin and its Secretions 837 
 
 CHAPTER XVI 
 Respiration and Oxidation 850 
 
 CHAPTER XVII 
 Metabolism 878 
 
 Index to Authors 945 
 
 General Index 983 
 
PHYSIOLOGICAL CHEMISTRY. 
 
 CHAPTER I. 
 GENERAL AND PHYSICO-CHEMICAL. 
 
 I. OSMOTIC PRESSURE. 
 
 When certain substances are placed in contact with water they 
 dissolve therein and finally a liquid is obtained which contains an equal 
 quantity of the dissolved substance in each unit volume. There exists 
 between the water and the soluble body a certain attractive force. Upon 
 this force depends also the so-called diffusion, which manifests itself when 
 two different solutions of the same or different substances are brought 
 into immediate contact with each other. The dissolved molecules and 
 the water intermingle with each other so that finally the dissolved 
 bodies are equally divided in the entire quantity of water. Imagine 
 a cane-sugar solution in contact with pure water; the equilibrium or the 
 homogeneity of the system can then be brought about in two ways; 
 namely, the sugar molecule can migrate in part into the water, and sec- 
 ondly, the water can pass into the solution. If the two fluids at the 
 beginning are in immediate contact with each other then the two proc- 
 esses take place simultaneously. 
 
 The conditions change when the two liquids are separated from 
 each other by a membrane, which allows of the passage of water but 
 not of the dissolved substance (in this case cane-sugar) . In the presence 
 of such a so-called semipermeable membrane the equilibrium can only 
 be established by the water passing into the cane-sugar solution. Semi- 
 permeable membranes have been artificially prepared, and they also 
 occur in nature, or conditions exist which give results like those of the 
 membranes. To the first group belong Tratjbe's so-called precipitation 
 membranes. 1 Such a membrane, for example can be produced by care- 
 fully dropping a concentrated solution of copper sulphate into a dilute 
 
 1 Arch. f. (Anat. u.) Physiol., 1867, pages 87 and 129. 
 
2 GENERAL AND PHYSICO-CHEMICAL. 
 
 solution of potassium ferrocyanide. Thereby the drop of copper sulphate 
 is surrounded by a membrane of copper ferrocyanide, which is imper- 
 vious to copper sulphate as well as to potassium ferrocyanide, but allows 
 water to pass. The drops retain their blue color in the yellow solution 
 but increase in volume, due to the taking up of water, until the tension of 
 the membrane prevents the further increase in size. If the difference in 
 concentration of the two solutions is great enough, the membrane is 
 ruptured by the pressure. 
 
 In order to give the copper-ferrocyanide membrane a greater rigidity, 
 Pfeffer has suggested forming the precipitate on a porous, rigid wall. 1 
 For this purpose he makes use of a small, porous earthenware cell which, 
 after careful cleaning, is treated with copper sulphate and potassium 
 ferrocyanide so that the membrane is precipitated on the inner wall 
 of the cell. The membrane thus obtained is impervious to the cane- 
 sugar. If the cell is filled with a cane-sugar solution and then placed 
 in pure water, no sugar leaves the cell, while water passes into the cell, 
 and this continues until the opposite pressure produced prevents the 
 further passage of water. If the cell is completely closed and in. con- 
 nection with a manometer, then on the establishment of an equilibrium 
 the manometer indicates the force with which the inclosed solution 
 attracts water. 
 
 As the sugar is attracted with the same force by the water as the water 
 is by the sugar and also as the sugar cannot pass through the membrane 
 therefore the sugar exerts a pressure upon the membrane equal to the 
 pressure indicated by the manometer. This pressure is called the 
 osmotic pressure of the enclosed solution. For dilute cane-sugar solutions 
 Pfeffer's determinations show that the osmotic pressure is approx- 
 imately proportional to the concentration and slowly rises with the 
 temperature. 
 
 Experiments with other semipermeable membranes have also been 
 carried out by de Vries, and these will be discussed on page 5. De 
 Vries' experiments have led to the following result: Solutions of analo- 
 gously constructed bodies having the same molecular concentration give the 
 same osmotic pressure. 
 
 Van't Hoff first called attention to the analogy which exists between 
 the laws of osmotic pressure of a dissolved substance and of gases, 2 
 namely, that the osmotic pressure is proportional (or inversely propor- 
 tional to the volume of the solution) to the concentration, and corre- 
 sponds completely with Boyle-Mariotte's law on the relation between 
 the volume and pressure of gases. Also, that equimolecular solutions 
 
 1 Osmotische Untersuchungen, Leipzig, 1877. 
 
 2 Zeitschr. f. physik. Chem., 1, 481 (1887). 
 
OSMOTIC PRESSURE. 3 
 
 have the same osmotic pressure, corresponds to Avogadro's law, that 
 equal volumes of different gases under the same pressure contain the 
 same number of molecules. 
 
 From Pfeffer's results of the osmotic pressure of cane-sugar solu- 
 tions van't Hoff has calculated that it is the same as the pressure exerted 
 by any gas of the same molecular concentration and temperature. In 
 general the following is true: 
 
 Dissolved bodies exert in solution the same osmotic pressure they would 
 exert if they were gases at the same temperature and in equal volume. 
 
 Recently Morse, Frazer and collaborators have brilliantly substan- 
 tiated the theory of van't Hoff for solutions of cane-sugar and glucose, 
 by making use of Pfeffer's method but using a very refined technique. 1 
 
 From what has been given, the osmotic pressure of a solution, sepa- 
 rated from the surrounding pure solvent by a semipermeable membrane, 
 exerts its effects in two ways. First the pure solvent tries to enter 
 the solution and secondly the dissolved substance presses upon the 
 membrane with a force equal to the gas pressure. According to whether 
 we consider either one or the other of these ways, the osmotic pressure 
 of a solution can be considered as its ability to attract the solvent, or as 
 a pressure directed toward the outside. This last conception seems prob- 
 ably for the present to be the most acceptable, nevertheless, the fact that 
 the pure solvent enters through the unmovable semipermeable membrane 
 (as in Pfeffer's experiments) is difficult of reconciliation with this mode 
 of explanation. Obviously, and for physiological purposes, it seems best 
 to make use of the former explanation, in which the osmotic pressure 
 is considered as a measure of the force with which a solution attracts 
 the solvent. 
 
 Pfeffer's above-described method of directly determining the 
 pressure can only be used in exceptional cases, first because the prepara- 
 tion of the semipermeable membrane is connected with difficulties, and 
 second, because there are only a few crystalline bodies for which imper- 
 meable membranes have been found. There are other quicker and easier 
 ways of determining the osmotic pressure. 
 
 Solutions of non-volatile substances boil at a higher temperature 
 than the pure solvent. This is due to the fact that the dissolved sub- 
 stance, because of the osmotic pressure, holds on to the solvent with 
 a certain force. As in boiling a part of the solvent is separated from 
 the dissolved body, and as the osmotic pressure can be considered as a 
 measure of the attractive power between the solvent and the dissolved 
 substance, then it is clear that solutions which are prepared with the 
 same solvent and have the same osmotic pressure (isosmotic solutions) 
 
 1 Amer. Chem. Journ., 37, 425, 558 (1907); 41, 1, 257 (1909). 
 
4 GENERAL AND PHYSICO-CHEMICAL. 
 
 must also boil at the same temperature. The rise in the boiling-point 
 of a solution above the boiling-point of the solvent (elevation of the 
 boiling-point) is also, like the osmotic pressure, for dilute solutions pro- 
 portional to the concentration. 
 
 Solutions have a lower freezing-point than the pure solvent, and as 
 in dilute solutions the solvent can be frozen out from the dissolved body, 
 then isosmotic solutions have the same freezing-point. The depres- 
 sion of the freezing-point is also proportional to the concentration. 
 
 The determination of the elevation of the boiling-point for the esti- 
 mation of the osmotic pressure of animal fluids is applicable only in 
 exceptional cases, because on heating, precipitates often form. The 
 determination of the depression of the freezing-point has been found of 
 much greater use. This can be accomplished in an easy manner by aid of 
 the apparatus suggested by Beckmann. In regard to the use of this 
 method we must refer to more complete works. 1 
 
 The above rule that equimolecular solutions of different bodies have 
 the same osmotic pressure is only applicable to non-electrolytes. The 
 electrolytes (bases, acids, salts) show in aqueous solution a much greater 
 pressure (i.e., a much lower depression of the freezing-point) than equi- 
 molecular solutions of non-electrolytes. As is known, Arrhenius has 
 explained this lack of correspondence by the assumption that the mole- 
 cule of the electrolyte is divided or dissociated into so-called ions hav- 
 ing an opposed electric charge. An ion exerts upon the osmotic pressure 
 the same influence as the non-dissociated molecule. The larger the 
 number of dissociated molecules the more does the osmotic pressure 
 of the solution rise above the pressure of an equimolecular solution of a 
 non-dissociated body. The osmotic action of a dissociated body is equal to 
 that of a non-dissociated body which in a given volume contains as many 
 molecules as the dissociated body contains ions plus non-dissociated mole- 
 cules. If we assume that a is the degree of dissociation, i.e., the number of 
 the molecules that are dissociated, then 1— a is the number that is not 
 dissociated. If in the dissociation of a molecule n ions are formed 
 then the relation of the molecules present before the dissociation to the 
 ions + molecules present after the dissociation is 1:(1— a-\-na) or 
 = l:(l-r-[n — l]a). The expression (l+[n — l]a) is generally denoted by 
 the letter i, and can be directly determined by estimating the freezing- 
 point of a solution of known molecular concentration. 
 
 A gram-molecule aqueous solution (one that contains as many grams per 
 liter as the molecular weight of the substance) of any non-electrolyte freezes 
 at about - 1.86°, or, the depression of the freezing-point A is =1.86°. For example, 
 
 1 Ostwald-Luther, Hand- und Hilfsbuch zur Ausfuhrung physik.-chemischer 
 Messung, 3 Aufl., 1910. 
 
OSMOTIC PRESSURE. 5 
 
 if we find that A for a gram molecular solution of NaCl is 3.40° then we have 
 according to the above 1 : (l+[n — l]o) =1.86 : 3.40. In the dissociation of 
 NaCl two ions are formed, therefore n = 2, and from the above equation the degree 
 of dissociation can be calculated, a =0.83. The degree of dissociation can also 
 be calculated from the electrical conductivity. Only the ions take part in the con- 
 duction of electricity, and the molecular conductivity ( = — i i — — — : : — } 
 
 * V molecular concent nit ion/ 
 
 is proportional to the degree of dissociation. The dissociation increases with the 
 dilution and at infinite dilution all molecules are dissociated (a = l). If we desig- 
 nate with n*> the limit value which the molecular conductivity approaches in 
 infinite dilution and with y.v the molecular conductivity at some definite dilution 
 
 v, then the degree of dissociation at this dilution is a = — . 
 
 The positively charged ions are called cations, and the negatively 
 charged ones anions. Common for all acids are the positively charged 
 H-ions while the negatively charged OH-ions are common for all bases. 
 
 Osmotic Experiments with Plant Cells. We often meet the 
 word osmosis in literature without understanding exactly what is meant 
 thereby. As a rule diffusion streams are meant, w-hich are modified 
 by means of the permeability conditions of an inclosing membrane. 
 We now know that the driving force, namely, the streaming, is brought 
 about by the differences in concentration, i.e., by difference in the osmotic 
 pressure on the two sides of the membrane. 
 
 After Nageli found that certain plant cells, when they were treated 
 with a sufficiently concentrated solution of certain substances, changed 
 their appearance so that the protoplasm retracted, 1 de Vries studied 
 this phenomenon further. 2 He called it plasmolysis. The most important 
 substances for bringing about plasmolysis are the salts' of the alkalies and 
 alkaline earths, varieties of sugars, polyatomic alcohols, and neutral amino- 
 acids. An indispensable condition for bringing about plasmolysis is that 
 the solution must not have any destructive action upon the cells. Xageli 
 gave the correct interpretation of plasmolysis, which is that those bodies 
 which plasmolyze plant cells pass through the cell membrane of the cell, but 
 not through the protoplasmic layer which follows. Instead of this the sub- 
 stance attracts water from the inner parts of the cell. The cell contents 
 surrounded by protoplasm therefore diminish in volume and the protoplasm 
 recedes more or less from the cell membrane. From this it follows that 
 only those solutions whose power of attracting water is greater than that 
 of the cell contents can bring about plasmolysis. As the ability to attract 
 water (or the osmotic pressure) increases with concentration, there must 
 be a limit solution for every substance above which all higher concentra- 
 tions plasmolyze. The limit solution is called isotonic with the cells; 
 
 1 Pflanzenphysiol. Untersuch., 1855. 
 
 2 Eine Analyse der Turgorkraft, Jahresber. f. Wissensch. Botanik, 14, 427 (1884). 
 
6 GENEKAL AND PHYSICO-CHEMICAL. 
 
 weaker solutions are called hypotonic, and stronger hypertonic. De 
 Vries, with the aid of equal cells (cells of the epidermis of the lower 
 side of the leaf of the Tradescantia discolor) has, for various substances, 
 determined the concentration of this limit solution. It was found that 
 the limit solution of analogously constructed salts had the same molec- 
 ular concentration. Thus the alkali salts of the type NaCl (haloid 
 salts, nitrate, acetate) plasm olyzed at one molecular concentration and 
 the salts of the type Na2SC>4 (sulphate, oxalate, diphosphate, tartrate) 
 at another concentration. If the plasmolyzing power of a molecule 
 of the first group is equal to 3, then the molecule of the second group 
 equals 4. The concentration of the limit solution varied in de Vries' 
 experiments between the limits corresponding to a NaCl solution of 
 0.6-1.3 per cent. 
 
 As above mentioned, only those substances bring about plasmolysis 
 which cannot themselves pass through the protoplasm envelope of the 
 cell content, and these substances only in the case that the concentration 
 is sufficient. If a body is taken up by the protoplasm it produces no 
 plasmolysis, because its tendency to attract water has been satisfied 
 by its own passage into the cell. These substances do not produce 
 plasmolysis in any concentration. If a body slowly passes in, then 
 at first it causes plasmolysis, but this then ceases later. The plasmolytic 
 methods have been used by de Vries, and especially by Overton. 1 
 
 Experiments with Blood Corpuscles. Over a hundred years ago 
 Hewson observed that the blood corpuscles were destroyed in water, 
 and that salts in certain concentrations prevented destruction. 2 Ham- 
 burger 3 has carefully and systematically investigated the action of 
 salts of the alkalies and alkaline earths, and concludes that when blood 
 is mixed with certain volumes of solutions of different concentrations 
 of the same salt, all solutions whose concentration lie below a certain 
 limit cause the exudation of haemoglobin. On comparing the molec- 
 ular concentration of the limit solution of different salts it was found 
 that they bore the same relation to each other as the relative figures 
 found by de Vries for the molecular concentration of the plasmolytic 
 salt solutions. From this it probably follows that the protective action 
 of the salts upon the blood corpuscles depends upon the same reason 
 as the plasmolysis. This conclusion is also supported by the fact that 
 those substances which, according to de Vries, in proper concentration 
 cause plasmolysis in living plant cells, can also under similar conditions 
 prevent the exudation of haemoglobin. Those bodies, on the contrary, 
 
 " Vierteljahwchr. d. Naturf. Gesellsch. zu Zurich, 40, 1 (1895); 41, 383 (1896). 
 
 'Phil. Trans., 1773, p. 303. 
 
 1 Arch. f. (Anat. u.) Physiol., 1888, p. 31; Zeitschr. f. Biol., 26, 414, (1889). 
 
OSMOTIC PRESSURE. 7 
 
 which do not cause plasmolysis, act in aqueous solution in the same 
 manner upon the blood corpuscles as pure water. This has been espe- 
 cially shown by the investigations of Gryns. 1 
 
 Different investigators have attempted to perform plasmoh tic 
 experiments with animal cells, but without any special result. With 
 the microscope one can often observe that the red blood corpuscles 
 shrink under the influence of a strong salt solution, but the limit solu- 
 tion when the shrinking just begins cannot be exactly determined because 
 the changes in volume are so very small. If we summate the changes 
 in volume of many corpuscles, which can be done by centrifuging the 
 blood mixture in a graduated tube, then very small changes can be detected. 
 Such determinations have been made by Hedin, 2 Koeppe 3 and others. 
 It was found that the blood corpuscles swell in a weak salt solution, 
 shrink in a stronger solution, and there is a certain concentration which 
 does not change the volume. By determining the freezkig-point Hedin 
 found that this concentration for NaCl was nearly isosmotic with the 
 serum of the blood corpuscles used. The depression of the freezing- 
 point was about 0.56° and the concentration of the NaCl solution is 
 0.9 per cent, or about 0.15 normal. 
 
 The question as to the permeability of the blood corpuscles has been 
 investigated by Hedin, using a method depending upon the following: 4 
 
 The depression of the freezing-point of a solution is proportional to its con- 
 centration. A certain amount of the substance to be tested is dissolved in blood. 
 The serum of this treated blood freezes at a lower temperature than before the 
 salt was added. The depression of the freezing-point can be designated as a. 
 Now the same amount of substance is dissolved in serum using the same volume 
 of serum as blood was previously used. The depression of the freezing-point 
 of this serum can be designated as b. From this it is evident that a =6 if 
 the blood corpuscles take up just as much dissolved substance from the blood 
 as an equal volume of serum. If the blood corpuscles take up less than the serum 
 
 then a> b or-r > 1 , and when they take up more than the serum then a <b or-? < 1. 
 
 The result r, in the calculation of which the change taking place in the volume 
 
 of the blood corpuscles on the addition of the substance must be considered, 
 gives immediately an approximate idea of the quantity of substance which has 
 passed into the blood corpuscles. 
 
 The results were as follows: 
 
 The salts of the fixed alkalies and alkaline earths, neutral amino- 
 acids, varieties of sugars as well as hexatomic and pentatomic alcohols 
 pass into the blood corpuscles only to a slight degree. Erythrite (tetra- 
 
 1 Pfluger's Arch., 63, 86 (1896). 
 
 2 Skand. Arch. f. Physiol., 5, 207, 238. 377 (1895). 
 
 3 Arch. f. (Anat. u.) Physiol., 1895, 154. 
 
 4 Pfluger's Arch., 68, 229 (1897); 70, 525 (1898). 
 
8 GENERAL AND PHYSICO-CHEMICAL. 
 
 tomic alcohol), passes slowly, and glycerin (triatomic) also passes slowly, 
 but faster than erythrite. Ethylene glycol (diatomic alcohol) passes rather 
 rapidly, and the monatomic alcohols immediately divide themselves 
 equally in the serum and blood corpuscles. Ether, esters, aldehyde, 
 and acetone divide themselves so that the blood corpuscles contain more 
 than does an equal volume of serum. These bodies are equally absorbed 
 by the blood corpuscles. Ammonium salts with univalent anions pass 
 in quickly while with divalent or polyvalent anions the greater part 
 remains in the serum; still they pass in to a greater extent than do the 
 corresponding salts of the fixed alkalies. 
 
 Overton had previously arrived at the same results, using plant 
 cells and chiefly by making use of the plasmolytic method. Urea is prob- 
 ably more quickly taken up by the blood corpuscles than by plant cells, 
 and ammonium salts also seem to pass more easily into the blood cor- 
 puscles than into the plant cells. 
 
 In regard to other salts Hedin's results have been substantiated 
 by Oker-Blom, 1 by estimating the electrical conductivity of the blood. 
 
 It must also be stated that according to Hedin, only those bodies 
 which do not pass, or pass slowly into the cells, can essentially alter 
 the volume of the cells. A close correspondence exists in this regard 
 between the plant and animal cells. 
 
 Gurber found that when blood corpuscles are repeatedly washed 
 with salt solution until the wash solution does not show any alkaline 
 reaction, and are then suspended in NaCl solution and treated with 
 CO2, the alkaline reaction increased while the blood corpuscles became 
 richer in chlorine. No exchange of K or Na took place. 2 Gurber 
 explains the experiment as follows: the carbonic acid set a small amount 
 of HO free from the salt, and this HC1 was taken up by the blood corpuscles. 
 The Na2CC>3 formed at the same time gave the alkaline reaction to the 
 solution. Koeppe 3 as well as Hamburger and v. Lier 4 claim, on the 
 contrary, that an exchange of HC03-ions and Cl-ions takes place between 
 the blood corpuscles and the solution, and Hamburger and v. Lier 
 claim to have shown that the blood corpuscles are permeable only for 
 anions, while the cations do not pass in. 
 
 Hamburger 5 and his collaborators have also found about the same 
 osmotic phenomena with other free mobile cells such as leucocytes, 
 spermatozoa as with the red blood corpuscles. The osmotic relations 
 have also been tried with intact parts of organs, therefore with cells 
 
 1 Pfliiger's Arch., 81, 167 (1900). 
 
 2 Sitzungsber. d. med. phys. Gesellsch. zu Wurzburg, 1895. 
 » Pfliiger's Arch., 67, 189 (1897). 
 
 <Arch. f. (Anat. u.) Physiol., 1902, 492. 
 
 'Osmotischer Druck und Ionenlehre, Wiesbaden, 1902, 1, 401. 
 
OSMOTIC PRESSURE. 9 
 
 in connection with other tissue constituents. By investigations on the 
 changes in the weight (instead of the volume changes in the above-mentioned 
 experiments with plant cells and blood corpuscles) which frog muscles 
 undergo in solutions, various experimenters, Nasse, 1 Loeb, 2 and Over- 
 ton, 3 have tried to prove the ability of muscle to take up various substances. 
 Overton found that as long as the irritability of the muscle was retained 
 the muscle took up the same bodies as the plant cells. The sarcolemma 
 is not responsible for the permeability, but the outer layers of the muscle 
 protoplasm are. 
 
 The skin of amphibians seems according to Overton to behave like the muscles 4 
 in regard to permeability. 
 
 Theories of Admissibility. On what does the permeability or non- 
 permeability of membranes and of cells for certain bodies depend? The 
 discoverer of precipitation membranes, M. Traube, considered the mem- 
 brane as a sort of molecular sieve. The relation of the size of the particles 
 passing and the width of the pores of the membrane is important. 5 This 
 view cannot be contested. The copper ferrocyanide membrane may be 
 considered to act in this way and the non-permeability of most mem- 
 branes for colloid substances depends upon the fact that the pores are 
 too narrow for the particles. 
 
 The question as to the occurrence of a special outer limiting layer 
 of the cells is of interest for the understanding of the metabolism of the 
 cells as well as for the knowledge as to the manner in which the cells take 
 up and give out substances. In this connection it must be recalled that 
 in the protoplasm of certain cells we find an outer dense layer or a true 
 membrane which seems to consist of protein substances. Still, even in cells 
 in which no special outer limiting layer can be seen, the presence of such a 
 limiting layer must be admitted because of the permeability condi- 
 tions of these cells. 
 
 Nernst 6 has shown, by special experiments, that the permeability 
 of a membrane for a certain substance is essentially dependent upon 
 the solvent power of the membrane for this substance. This question 
 which is very important for the study of the osmotic phenomenon in 
 living cells has been especially studied by Overton. 7 From the behavior 
 
 1 Pfluger's Arch., 2, 114 (1869). 
 
 i Ibid., 69, 1; 71, 457 (1898). 
 
 J Ibid., 92, 115 (1902); 105, 176 (1904). 
 
 * Verhandl. d. phys. med. Gesellsch. zu Wurzburg (N. F.), 36, 277 (1904). 
 
 5 Arch. f. Anat. Physiol, u. Med., 1867, 87. 
 
 6 Zeitschr. f. physikal. Chem., 6, 37 (1890). 
 
 7 Vierteljahrsschr. d. Naturf. Gesellsch. in Zurich, 44 (1899) and Overton, Studien 
 uber die Narkose, Jena, 1901. 
 
10 GENERAL AND PHYSICO-CHEMICAL. 
 
 of living cells to dye-stuffs, as well as the special ease in which certain 
 substances, which are not soluble in water or only slightly so, but are readily 
 soluble in fats or fat-like bodies, pass into animal and plant protoplasms 
 has led Overton to the conclusion that the protoplasmic limiting layer 
 behaves like a substance layer having the solvent properties similar to the 
 fatty oils. According to Overton the protoplasmic layer is probably 
 impregnated with lipoids, i.e., bodies more or less similar to the fats 
 in regard to their solubilities and their solvent power upon certain sub- 
 stances. The lipoids do not form a chemically definable class of bodies. 
 Certain of them are still of an unknown constitution while others are 
 known, especially the lecithins (the phosphatides as a group) and the 
 cholesterin are to be especially mentioned on account of their great 
 importance. 
 
 The assumption that an accumulation of lipoids occurs, as a special 
 limiting layer, in the cells is not sufficiently founded and not generally 
 true at least for the animal cells. Still this assumption is not absolutely 
 necessary for a comprehension of the action of lipoids in the above 
 sense. Objections have been raised by a few investigators against 
 Overton's theory, which has found general acceptance. 1 Thus it fails 
 to explain all cases, although this was suggested by Overton himself, 
 for instance according to Cohnheim, it does not explain the absorption 
 processes in the intestinal canal, and according to Moore and Roaf it 
 cannot explain certain properties of the cells, namely the varied composi- 
 tion of the electrolytes within and outside of the cells, and the selective 
 taking up of certain soluble substances such as food products, drugs, 
 toxins and antitoxins by the cells. The investigations of the last men- 
 tioned experimenters are based essentially upon investigations of the 
 behavior of mineral substances, and they show that the above theory 
 offers certain difficulties in explaining the exceedingly important exchange 
 of mineral substances between the cells and the external fluid. Also the 
 fact that the cells are readily permeable for water is explained with diffi- 
 culty by Overton's theory. 
 
 J. Traube 2 especially has put forth objections to Overton's theory. 
 According to him, the passage of a substance from a watery solution 
 into the cells, is in the first place due to its so-called solution tenacity in the 
 watery solution. This solution tenacity is according to Traube the attrac- 
 tion bf 'tween the solvent and the solute; and is not identical with the 
 osmotic pressure, but is measured by the surface tension of the solution. 
 
 1 See O. Cohnheim, Die Physiologie der Verdauunp; u. Ernahrung (1908). J. Loeb 
 in Oppenheimer'8 Handbuch der. Biochem. Bd. 2, 105. T. B. Robertson Journ. of 
 biol. Chem., I L908). B. Moore and H. Roaf, Biochem. Journ., 3 (1908). 
 
 M'fluger's Arehiv., 105, 541 (1904); 123, 419 (1908); 132, 511 (1910); 140, 109 
 (1911). 
 
OSMOTIC PRESSURE. 11 
 
 It has been shown that those substances which are not taken up by the 
 cells at all or only slightly, do not lower the surface tension of the water 
 when dissolved therein. On the contrary, those substances which lower 
 the surface tension, pass into the cells. According to Gibbs those sub- 
 stances, which when dissolved in water lower the surface tension, occur 
 in greater concentration on the surface as compared with the interior. 
 Thus according to Traube the solution tenacity is less the lower the 
 surface tension of the watery solution. Otherwise the direction of 
 movement of a substance in the boundary between two phases (watery 
 solution and cells) is determined by the relationship between the solution 
 tenacity of the substances in the two phases. However, the solution tenac- 
 ity of a substance can only be directly measured in the watery solution. 
 Traube supports his theory. upon different experiments in which mem- 
 bers of the same homologous series were dissolved in water in such con- 
 centration # as to have the same surface tension and also showed the 
 same ability to pass into the cells. The disagreement in other cases can 
 be explained by the unknown solution tenacity in the cell phase. As we will 
 show below Traube's proposition calls to mind the accepted views as to 
 the origin of the adsorption phenomena or the taking up of dissolved sub- 
 stances by solid bodies. Lowe l has also found, in studying the taking 
 up of different dissolved substances by lipoids, that the process does not 
 take place as called for by Overton's theory according to Henry's law 
 of absorption but rather an adsorption. 
 
 Certain substances which are of the very greatest importance for life 
 processes and which probably are burned to a great extent within the cells, 
 have according to the above experiments only a limited ability to enter 
 the cells. These bodies are the sugars and the amino-acids. Also the 
 presence of salts within the cells is not easily understood in view of the 
 above experiments. In consideration of this it must be remarked that 
 the above described experiments on the permeability of animal cells have 
 been carried out with cells that were removed from their attachment to 
 the living animal. Although these cells are not considered as physio- 
 logically dead cells still it is very probable that certain life functions 
 have been arrested. It is readily conceivable that the oxidation processes, 
 whereby the organic substances taken up within the cells are trans- 
 formed into simpler products, are at least partly brought to a standstill 
 (see Chapter XVI). That, nevertheless, at least salts and sugar also 
 attract water in the living organism and therefore only pass into the 
 cells in small quantities follows from the experiments of Heidenhain, 
 according to whom these substances are designated as lymph forming 
 agents of the second order (Chapter VI). This action is also explained 
 
 1 Bioch. Zeitschr., 42; 150, 190, 205, 207 (1912). 
 
12 GENERAL AND PHYSICO-CHEMICAL. 
 
 by Heidenhain as being dependent upon their power of abstracting 
 water from the tissues. 
 
 If we admit that the cells normally contain only small amounts of 
 sugar and amino-acids at any one time, then, if these substances are being 
 continuously burned within the cells, new quantities must constantly 
 be taken up and in this way gradually large quantities of the mentioned 
 substance would be taken up and burned. If the combustion is arrested 
 no new quantities are taken up. The fact that certain substances 
 are only taken up in small quantities at a time does not prove that they 
 are not burned within the cells. 
 
 According to Moore and Roaf 1 the salts exist in the blood cor- 
 puscles in the form of " adsorpates;" these are adsorbed by the solid 
 constituents of the blood corpuscles. As we will see further on (page 
 27) an adsorbing substance can only take up a limited amount of another 
 substance. If, after the saturation limit is reached, more of the adsorbed 
 substance is added then practically no more is taken up. In this way 
 we can explain why the blood corpuscles only take up very little of the 
 salts added. The slight ability of the sugars and amino-acids to be 
 taken up can perhaps be explained in a similar manner. 
 
 Osmotic Pressure of Animal Fluids. As is apparent from the 
 above, a substance exerts upon living cells an entirely different influence, 
 depending upon whether the substance is able to pass into the cell or 
 not, and whether the substance which does not pass in has the ability 
 of attracting water or not. Therefore that part of the osmotic pres- 
 sure of body fluids which is caused by bodies not passing in is called 
 the effective osmotic pressure. In this manner therefore the salts of the 
 alkalies and alkaline earths and the sugars act. As sugar, as well as the 
 bodies which according to the just mentioned experiments are readily 
 taken up by the cells, occurs under ordinary conditions only in very 
 small amounts in the blood, and also as the proteins are practically with- 
 out influence upon the osmotic pressure, the normal osmotic pressure of 
 the blood is chiefly due to the salts. As the depression of the freezing- 
 point is almost the only method used for animal fluids, therefore ordinarily 
 the freezing-point depression (A) is given as a measure of the osmotic 
 pressure. For mammalian blood A is constant with the exception of slight 
 variations flue to the food and perhaps also to other circumstances. It 
 is 0.56 , 2 which corresponds to a 0.90 per cent NaCl solution and to an 
 osmotic pressure of about 6| atmospheres. In lower animals A may 
 be slightly lower, for example, in the frog A = 0.46°. In invertebrate 
 sea animals the body fluid is equal to the osmotic pressure of the sur- 
 
 1 Bioch. Journ., 3, 55 (1908). 
 
 J Hamburger, Osmotischer Durck u. Ionenlehre, 1, 456. 
 
colloids. ia 
 
 rounding sea water (A = 2.3°) and varies with the quantity of salt in the 
 water (Bottazzi). Irf lower fishes (Selachii) the osmotic pressure 
 of the blood is equal to the surrounding medium, and in higher fishes 
 (Teleostomi) lower (A = 1.0°) (Bottazzi). In Selachii the osmotic 
 pressure of the blood is chiefly due to urea (Schroeder). 1 
 
 In sea fishes as well as fresh-water fishes, for example, the eel, a lower 
 osmotic pressure (A = 0.41°) is found when kept in fresh water than 
 when kept in sea water (A = 0.55°) 2 . In lower sea animals the osmotic 
 pressure is equal to the surrounding medium, while higher animals are 
 independent of the surroundings. Hober calls attention to this condi- 
 tion and points out the analogy with the body heat of the various 
 animals. 3 
 
 If we pass to other body fluids we must mention that the lymph 
 shows a somewhat higher osmotic pressure than the blood, and this is 
 due to the lymph taking up from the tissues metabolic products hav- 
 ing a low molecular weight. 4 Milk and bile have the same osmotic 
 pressure as the blood, 5 while saliva has a lower pressure. 6 The urine 
 of man and mammalia generally has a much higher osmotic pressure 
 than the corresponding blood. 7 For human urine A varies between 1.3 
 and 2.3°. After abundant drinking as well as under pathological con- 
 ditions (diabetes insipidus) the osmotic pressure cf the urine can be lower 
 than the blood. In regard to the osmetic pressure of animal fluids under 
 normal and pathological conditions we refer to the work of Koranyi 
 and Richter. 8 
 
 n. COLLOIDS. 
 
 The word colloid originated with Graham, who included in this name 
 different substances which did not have the property of diffusing through 
 an animal membrane. In opposition to this Graham called those 
 bodies which passed through a membrane, crystalloids, because they 
 were as a rule crystalline, a property which with few exceptions does 
 not belong to the colloids. 9 Graham included soluble silicic acid among 
 
 1 Bottazzi, Archives ital. de biol. 28, 61 (1897). Schroeder, Zeitschr. f. physiol. 
 Chem. 14, 576 (1890). 
 
 2 Dekhuisen, Arch, neerland, 10, 121 (1905); Quinton, Compt. rend. boc. biol., 
 57, 470, 513 (1904). 
 
 3 Physik. Chem. d. Zelle u. Gewebe, 3. Aufl. 353, (1911). 
 4 Leathes, Journ. of Physiol., 19, 1 (1895). 
 
 6 Dresser, Arch. f. exp. Path. u. Pharm., 29, 303 (1892). 
 . 6 Nolf, Traveaux du lab. de phys. de Liege, 6, 225 (1901). 
 
 7 Kordnyi, Zeitschr. f. klin. Med., 33, 1 (1897), 34, 1 (1898). 
 
 8 Physikalische Chemie und Medizin. Leipzig (1907). 
 
 s Ann. d. Chem. u. Pharm., 121, 1 (1862) as well as Ann. de chim. et de Phys. (4)„. 
 3, 127 (1864). 
 
14 GENERAL AND PHYSICO-CHEMICAL. 
 
 the colloids and also analogous forms of stannic acid, titanic acid, 
 molybdic acid and tungstic acid, aluminium hydroxide and analogous 
 metallic oxides, when they exist in the soluble form, and also starch, dex- 
 trins, the gums, caramel, tannin, albumin and gelatin. 
 
 Some colloids are characterized by the fact that under certain con- 
 ditions they solidify into a gelatinous form containing considerable water. 
 In the case where water is the solvent then Graham called the soluble 
 form hydrosol and the gelatinous form hydrogel. 
 
 By diffusion through a membrane (called dialysis by Graham) colloid sub- 
 stances can be separated from crystalloids. Colloidal silicic acid as well as 
 corresponding forms of certain other bodies are obtained by treating the soluble 
 alkali salt with hydrochloric acid, then removing the excess of hydrochloric acid 
 as well as of chlorides, by means of dialysis. Colloidal alumina was obtained by 
 Graham by dissolving aluminium hydroxide in aluminium chloride. This last salt 
 was removed by dialysis and the hydroxide remained with more or less HC1 com- 
 bined in solution. 
 
 Various metallic sulphides can be obtained in colloidal solution. Such solu- 
 tions of As 2 S 3 and Sb 2 S 3 can be obtained by passing H 2 S into dilute solutions 
 of the respective metallic oxide, 1 and colloidal CuS can be prepared by washing 
 the precipitated compound with water, by which treatment thej,CuS finally becomes 
 soluble in water. 2 
 
 The metals can be obtained as hydrosols, and indeed in two ways: 
 
 1. By treating a salt with various reducing agents (for example formaldehyde, 
 hydrosulphurous acid, hydrazine, Irydroxylamine) the various metals are obtained 
 in colloidal solution. 3 As the solutions thus obtained are often very unstable, 
 it has been found advisable to help their stability by the addition of organic 
 colloids (gelatin). We will discuss the mode of action of these so-called pro- 
 tective colloids on page 23. 
 
 2. Bredig 4 has discovered a method which makes possible the production 
 of pure metallic sols by the cathode spraying of metallic wires under water. 
 Svedbeug 5 prevents the heating of the fluid in this spraying by using the 
 induction current. This makes the spraying also possible under organic fluids 
 and sols of the light metals have also been prepared. Practically sols of all 
 metals and metalloids can be prepared in this way. 
 
 Among those bodies which can be obtained in the colloidal state 
 we have acids as veil as bases, and the chemical elements are also known 
 as colloids, as well as bodies of more complex molecular structure like 
 the proteins and starches. The colloid bodies, therefore, have from a 
 chemical standpoint nothing in common. More likely the colloidal con- 
 dition is due to physical properties, and this follows from the researches 
 of Graham. The crystalloids and the colloids are therefore not to 
 be considered as chemically different classes of bodies, but rather only 
 as different physical conditions of matter and the boundary between 
 
 1 II. Schulze, Journ. prakt. Chem. (N.F.), 25, 431 (1882), and 27, 320 (1883). 
 •Spring, Ber. d. d. chem. Gesellsch., 16, 1142 (1883). 
 ' Miillr-r, Allg. Chemie d. KoHoide. Leipzig (1907), 6. 
 
 * A&arganiflcfae l ermente. Leipzig (1901), 24. 
 
 * Ber. d. d. chem. GeBellach., 38, 3616 (1905); 39, 1705 (1906). 
 
COLLOIDS. 15- 
 
 these two conditions is often very indefinite. Certain chemically definable 
 classes of substances, such as proteins, occur only or chiefly in the col- 
 loidal condition while others, such as the inorganic salts, occur as crys- 
 talloids. Finally we find others that can occur in both forms, namely 
 the soaps (page 17). In short the difference between the crystalloid and 
 colloidal condition may be considered in that the crystalloids occur in 
 solution as molecules of medium size while the colloids are either very large 
 molecules, molecular aggregations or at least particles of a larger spacial 
 volume than the crystalloids. According to such a conception many 
 properties of the colloids can be explained. 
 
 In order to give a better review we will give a classification of the 
 colloids which seems, for the present, to be rather universally accepted. 
 This was first suggested by Perrin 1 and later accepted by Hober, 2 
 A. Muller, 3 and Wo. Ostwald, 4 although different authors use different 
 names for the two classes. The classifications of Hardy 5 and Zsig- 
 mondy 6 have also much in common with the classification given below. 
 
 One of the two groups of colloids is called hydrophile colloids (emul- 
 sion colloids, emulsoides) because in the aqueous solution a certain rela- 
 tion still exists between the dissolved substance and the solvent which 
 is evident especially by a certain viscosity of the solution. The hydro- 
 phile colloids often gelatinize on cooling, the gel is again soluble in 
 water (reversible), and in general the hydrophile colloids are separated 
 from their solution by electrolytes wnth greater difficulty than the col- 
 loids of the second group. Bodies of the greatest importance for phys- 
 iological chemistry like the proteins, starch, glycogen, and soaps in 
 watery solution belong to the hydrophile colloids. 
 
 Contrary to the hydrophile colloids, the colloids of the colloidal metal 
 type are called suspension colloids (suspensoids) as they must be con- 
 sidered as suspended solid particles in a solvent and have no close 
 relation to the solvent. The viscosity of the solution does not differ 
 much from that of the pure solvent; besides this, the suspension col- 
 loids do not gelatinize, do not swell up, and are readily precipitated 
 by electrolytes. To this group belong the metallic sols, the colloidal 
 metallic sulphides, and certain typical suspensions obtained by dissolving 
 water-insoluble substances in another liquid (alcohol, acetone) and then 
 pouring this solution into a large volume of water. In this way the 
 substance is precipitated in a finely divided condition. Such suspensions 
 
 1 Journ. de Chimie phy., 3, 84 (1905). 
 
 2 Physik. Chem. d. Zelle u. Gewebe, 2 Aufl. (1906), 208. 
 s Allg. Chemie d. Kolloide (1907), 187. 
 
 * Zeitschr. f. Chem. u. Ind. d. Koll., 1, 331 (1907). 
 
 5 Proc. Roy. Soc, 66, 95 (1899). 
 
 6 Zur Erkenntnis d. Koll. (1905), 16. 
 
16 GENERAL AND PHYSICO-CHEMICAL. 
 
 behave in many respects like suspension colloids. Suspensions of mastic, 1 
 colophony, 2 and cholesterin 3 belong to this class. 
 
 The hydrophile colloids stand closer to the crystalloids than do the 
 suspension colloids, and the transition between the crystalloids and the 
 hydrophile colloids is only gradual. At the boundary we find the pep- 
 tones and proteoses which belong to the proteins, but at the same time 
 dialyze rather well. On the other hand, we also have colloids which to a 
 certain extent form intermediary steps between the hydrophile colloids 
 and suspension colloids. Finally, there are also numerous intermediary 
 members between the suspension colloids and the finely divided substances 
 suspended in water (kaolin). 
 
 Osmotic Pressure. As above stated, the osmotic pressure of solu- 
 tions of crystalloids can be determined only in exceptional cases by 
 means of the semipermeable membrane, because it is very difficult to 
 prepare membranes which are impermeable for crystalloids. As pre- 
 viously stated, most membranes are impermeable for colloids, and the 
 osmotic pressure of the colloids can be best directly determined by the 
 aid of a membrane in a so-called osmometer. As shown bjr Moore and 
 Roaf, in such an apparatus changes in pressure can be determined which 
 are not detectable by the determination of the freezing-point. 4 
 
 Equimolecular solutions of various non-electrolytes give the same 
 osmotic pressure. From this it follows that when different non-elec- 
 trolytes exist in solutions with the same percentage concentration, the 
 osmotic tension of these solutions must be in inverse proportion to their 
 molecular weights. Certain colloids which will be discussed in another 
 connection (proteins, glycogen, etc.) must have a very large molecule. 
 From this it follows that these bodies must exert a very low osmotic 
 pressure. The proteins always contain a small amount of salts which 
 «xist either in a sort of combination with the colloids or are to be con- 
 aidered as contaminations which are difficult to remove. For this reason 
 it has been repeatedly stated that these salts are responsible for the 
 small differences in the osmotic pressure. By carefully washing crys- 
 stalline proteins from serum and egg-white, Reid was able to prepare 
 bodies which gave finally no osmotic pressure in the osmometer. 5 In 
 opposition to this, Mooue and Roaf as well as Lillie call attention to 
 the fact that the osmotic pressure of protein solutions is influenced by 
 the tn-atmcnt which the protein received before the determination. 
 
 1 Zeitsc.hr. f . physik. Chem., 57, 47 (1906). 
 
 2 Ibid., 38, 385 (1901). 
 
 ■ Bioch. Zeitschr., 7, 152 (1908). 
 
 * Bioch. Journ., 2, 34 (1906). 
 
 • Journ. of Physiol., 31, 438 (1904). 
 
COLLOIDS. 17 
 
 Starling, 1 Moore and Parker, 2 Moore and Roaf 3 and Lillie, 4 using 
 protein preparations which had not been exposed to any strong treat- 
 ment before use (serum proteins, ovalbumin), as well as Reid 5 (with 
 haemoglobin), have been able to detect a low osmotic pressure and 
 indeed by the aid of osmometric methods. According to Starling, 
 the proteins of the serum correspond to a pressure of 30-40 mm. Hg. 
 and Reid 6 found a pressure of 3-4 mm. Hg. for a 1 per cent haemoglobin 
 solution. 
 
 Th© influence of added bodies upon the osmotic pressure has been tested by 
 Lillie by adding the substance to be tested in the same percentage concentration 
 to the inner and outer fluids. It was found that non-electrolytes were without 
 action while acid and alkalies increased the osmotic pressure of gelatin solutions, 
 while salts lowered the pressure of gelatin as well as ovalbumin solutions. Adam- 
 son and Roaf 7 arrived at similar results in regard to alkalies and acids. Besides 
 this, Lillie found that the osmotic pressure was dependent upon the past history 
 of the colloid. Warming as well as shaking the solutions seems to change the 
 aggregate condition, which returns very slowly or not at all. The changes 
 in the osmotic pressure produced by salts, Lillie explains by a change in the 
 aggregate condition of the colloid, by the addition of salts it is brought closer 
 to its precipitation point and is probably united in large aggregations. In this 
 way the number of particles is diminished and, as this number must be important 
 for the osmotic pressure, this pressure is lowered. In agreement with this 
 the above mentioned influence of acids and alkalies upon the osmotic pressure of 
 gelatin can be explained by an increase in the particles. 8 
 
 As we have seen above the determination of the elevation of the boil 
 ing-point or the depression of the freezing-point is the simplest way 
 for estimating the osmotic pressure of a crystalloid substance in solution. 
 If such determinations are made with a colloidal solution then unmeas- 
 urable results are found for the elevation of the boiling-point or the depres- 
 sion of the freezing-point. This indicates, as above stated, that the 
 molecules or the particles must be very large. F. Kraft 9 found no 
 elevation of the boiling-point for soaps in watery solution but obtained 
 values which correspond to the calculated molecular weights when the 
 soaps were dissolved in alcohol. Therefore the soaps are colloidal in 
 watery solution and crystalloidal bodies in alcoholic solution. 
 
 Filterability. Large particles suspended in a liquid can be removed 
 from the fluid by filtering. The finer the suspended particles are the 
 
 1 Journ. of Physiol., 19, 322 (1896). 
 
 2 Amer. Journ. of Physiol., 7, 261 (1902). 
 
 3 Bioch. Journ., 2, 34 (1906). 
 
 * Amer. Journ. of Physiol., 20, 127 (1907). 
 
 6 Journ. of Physiol., 33, 12 (1905). 
 
 8 Bioch. Journ., 3, 422 (1908). / 
 
 'Ibid. 
 
 "Pauli, Koll. Zeitschr., 7, 241 (1900). 
 
 •Ber. d. d. chem. Gesellsch., 29, 1328 (1896); 32, 1584 (1899). ' 
 
18 GENERAL AND PHYSICO-CHEMICAL. 
 
 closer must the filter be Extensive experiments on the filtering of 
 colloids have been carried out by Bechhold. 1 He used paper filters 
 which were impregnated with collodion dissolved in glacial acetic acid. 
 According to the concentration of the collodion solution filters of dif- 
 ferent porosity were obtained. The colloid solutions were pressed 
 through the filter by a pressure up to five atmospheres. It was shown 
 that all colloid solutions contained particles of various sizes. Never- 
 theless for every solution a filter could be prepared whose pores were 
 small enough to retain all the particles. In this manner Bechhold was 
 able to classify the colloids in a series according to the size of the smallest 
 particles. He found that in general the inorganic colloids (Prussian 
 blue, platinum, iron oxide, gold, silver) form larger particles than the 
 organic colloids (gelatin, haemoglobin, seralbumin, proteoses, dextrin). 
 Still it must be remarked that according to Zsigmondy 2 the size of the 
 particles of the same colloid are larger in one preparation than in another 
 and that the size can change on keeping. 
 
 On filtering proteose solutions through filters of unequal thickness 
 Bechhold was able to show that the larger the particles of the proteoses, 
 the easier are they precipitable by ammonium sulphate. 
 
 Diffusion. We have already seen that the osmotic pressure of a 
 colloid solution is very small and also that the osmotic pressure of a solu- 
 tion is the cause for the diffusion of the particles, therefore it is evident 
 that the diffusion ability of colloids can only be very slight. This is 
 not only true for the free diffusion but also for the diffusion through a 
 membrane. Both of these was first studied by Graham. The first was 
 found very slight but measurable in several cases while the fact that 
 the colloids did not diffuse through membranes (non-dialysable) was 
 given as the most constant difference between colloids and crystalloids. 
 Nevertheless, there does not exist any sharp boundary and dialysis depends 
 principally upon the size of the particles as well as upon the character of 
 the membrane. 
 
 Internal Friction. By the internal friction of a fluid we mean the 
 force whiph resists the displacement of the particles of the fluid among 
 one another. The internal friction is therefore an expression for the 
 great thickness or viscosity of the fluid. 
 
 For physiological purposes the internal friction is determined by 
 measuring the time which a given volume of the fluid requires to flow 
 through a capillary tube under a pressure of its own weight. 
 
 It is generally accepted that the internal friction of suspension col- 
 
 ' ZeilM hr. f. physik. Chem., 60, 257 (1907). 
 
 2 Zur Erkenntnis d. Koll., (1905), 104 as well aB Zeitschr. f. Elektrochem., 12, 
 681 Ifl 
 
COLLOIDS. 19 
 
 loids is equal to that of the pure solvent or differs from it only slightly. 
 On the contrary hydrophile colloids are, in proper concentration, very 
 viscous which is probably the reason that they gelatinize under certain 
 circumstances. Pauli as well as Pauli and Handovsky 1 have inves- 
 tigated strongly dialyzed serum in regard to its internal friction. The 
 addition of a little salt (to 0.05 normal) causes a lowering of the internal 
 friction below that of a pure albumin solution, while acids and alkalies 
 in small amounts cause a powerful rise in the viscosity. 
 
 Optical Properties. Colloidal solutions are opalescent by reflected 
 light, which depends upon the fact that the ligljt is reflected by the sus- 
 pended particles. The reflected light is partly polarized. This phenom- 
 enon, called Tyndall's phenomenon, depends upon the presence of 
 small particles in the liquid, and is considered as a test for colloid solu- 
 tions. Still there are colloid solutions (certain gold solutions, Zsig- 
 mondy), which do not give Tyndall's phenomenon, and on the other hand 
 we also have solutions of certain high molecular crystalloids (cane 
 sugar, rafnnose), which produce this phenomenon. 2 
 
 With the aid of the ultramicroscope of Siedentopf and Zsigmondy, 
 it has been made possible to see the colloidal particles directly. 3 In 
 this apparatus the colloidal particles are strongly illuminated by direct 
 light, so that no ray of light falls directly into the eye of the observer. 
 The particles are hereby made visible on account of the formation of 
 diffraction disks which are visible through the miscroscope. In colloidal 
 solutions where the particles are close together, a more or less intense, 
 homogeneous, polarized sphere of light is seen in the microscope where 
 the individual particles cannot be distinguished from each other. This 
 is possible on diluting the solution. Those particles which are only 
 made visible by dilution are called submicrons, while those that gradually 
 disappear on dilution are called amicrons. 
 
 The investigations of Zsigmondy and others upon the growth of colloidal 
 metallic particles are also interesting. Thus the reduction of gold chloride by 
 formaldehyde, whereby colloidal gold is formed, is accelerated by the addition of 
 colloidal gold, and the added particles indeed grow at the cost of the newly 
 reduced gold. 4 In a similar manner the reduction of silver nitrate with ammonia 
 and formaldehyde is helped by the addition of colloidal gold when the reduced 
 silver precipitates upon the gold particles. 5 In such processes the amicrons can 
 enlarge so that they can be observed by the ultramicroscope (submicrons). 
 
 'Pauli, Koll. Zeitschr., 3, 5 (1908); Pauli and Handovsky, Biochem. Zeitschr., 
 18, 340 (1909); 24, 239 (1910). 
 
 2 Lobry de Bruyn and Wolff, Rec. trav. chim. des Pays-Bas., 23, 155 (1904). 
 
 3 Zsigmondy, Colloids and the Ultramicroscope, translated by Alexander, New York, 
 1909. 
 
 * Zsigmondy, Zeitschr. f. physik. Chem., 56, 65 (1906). 
 5 Zsigmondy and Lottermoser, ibid., 56, 77 (1906). 
 
20 GENERAL AND PHYSICO-CHEMICAL. 
 
 According to the manner of preparation the colloids may have particles of different 
 sizes. (See page 00.) 
 
 Submicrons have also been detected in solutions of organic colloids. The 
 work of Gatix-Gruzewska and Biltz, * who used a specially pure glycogen, 
 must be especially mentioned. They found that the aqueous solution of glycogen 
 contained amicrons as well as easily recognizable submicrons, whose presence 
 was only evident by a homogeneous sphere of light, but on the addition of alcohol, 
 conglomerate into detectable submicrons. 
 
 Molecular Movement. R. Brown 2 first found that small particles 
 
 suspended in water showed a quivering motion, and this phenomenon 
 
 has been called, from its discoverer, Brownian molecular motion, although 
 
 the particles in no manner are to be considered as molecules. This 
 
 phenomenon has been observed since then by many investigators in 
 
 fluids having suspended solid particles as well as in substances dissolved 
 
 in colloidal condition. 
 
 The Brownian movement is considered by some as a manifestation of a general 
 molecular movement of matter. According to this view it is comparable with 
 the supposed motion of gas molecules according to the kinetic theory of gases. 
 Perrix as well as Svedberg 3 claim that the law of gases also holds for very 
 dilute colloidal solutions. 
 
 Electrical Transportation of Suspended Particles. A not too weak 
 electric current has the power of causing motion in small quantities 
 of fluid enclosed in a capillary tube or in a porous diaphragm. The 
 particles suspended in a fluid also wander under the influence of the 
 electric current, and indeed to the anode or cathode, according to the 
 nature of the fluid and the particles. This phenomenon is called cata- 
 phoresis. Such movements have also been found in colloidal solutions. 
 According to Biltz, 4 in dialyzed aqueous solution, the colloidal metallic 
 hydroxides wander to the cathode, and the other colloids (metals, 
 metallic sulphides, acids) wander to the anode. The colloidal particles 
 in water are therefore probably electrically charged, hence the nega- 
 tively charged wander to the anode and the positively charged to the 
 cathode. Dialyzed protein solutions show according to older investiga- 
 tions no cataphoresis. The addition of acid or alkali gives to the pro- 
 tein a positive or negative charge respectively, hence an alkaline solu- 
 tion wanders to the anode and an acid solution to the cathode (Hardy, 5 
 Pal li 6 ). According to Michaelis 7 the proteins in perfectly neutral 
 
 1 Pfliiger's Arch., 105, 115 (1904). 
 
 »Edinb. Phil. Journ., 5, 358 (1828); 8, 41 (1830). 
 
 * Perrin, Colloid-chem. Beihefte, 1, 221 (1910). Svedberg, KoU. Zeitschr., 7, 1 
 (1910). 
 
 * Ber d. d. chem. Gesellsch., 37, 1095 (1904). 
 
 * Joum. of Physiol., 24, 288 (1899). 
 
 « Hofrneister's Beitrage, 7, 531 (1906). 
 
 'Biochem. Zeitschr., 16, 81 (1909); 19, 181 (1909); 24, 79; 27, (38; 28, 193; 
 29, 439 (1010); 33, 456 (1911); 41, 373 (1912). 
 
COLLOIDS. 21 
 
 solution wander to the anode in the case when the experiment is so 
 carried out that the formation of acid or alkali is prevented at the anode 
 or cathode respectively. If the neutral protein solution is treated with 
 a trace of acetic acid then the particles wander to the cathode. With 
 a certain very slight degree of acidity the direction of the wandering of 
 the particles is reversed. With this reaction no wandering or a double- 
 sided wandering of the protein bodies can be detected. This so-called 
 isoelectric point has been determined by Michaelis, Rona and their 
 collaborators for different protein substances. 1 Michaelis and Rona 
 claim to have found in the isoelectric point the most favorable reaction 
 for the heat coagulation of the protein substances, while Sorensen and 
 Jurgensen consider the reaction which the pure protein substance 
 gives to pure water as the optimal precipitation reaction. 2 
 
 According to Gatin-Grtjzewska 3 pure glycogen wanders distinctly 
 to the anode. 
 
 Precipitation of the Colloids. 
 
 The colloids can be separated from their solutions in various ways. 
 Many colloidal solutions are so unstable that they flock out after a 
 time without the addition of anything (silicic acid, metallic hydroxides). 
 Certain colloids appear as flocculent precipitates on heating their solu- 
 tions (certain proteins, see Chapter II). Others solidify on cooling 
 from hot concentrated solutions, as semisolid forms, so-called jellies 
 or hydrogels, containing considerable water (glue, starch, agar). 
 
 On evaporating the hydrosols at ordinary temperature we obtain 
 a residue which Zsigmondy divides into reversible and irreversible col- 
 loids, according whether they are again soluble in water or not. 4 Accord- 
 ing to this definition starch, dextrin, agar, gum, and proteins belong to the 
 reversible colloids while colloidal silicic acid, stannic acid, colloidal metallic 
 hydroxides and sulphides, and the pure colloidal metals belong to the 
 irreversible colloids. The former are relatively non-sensitive toward 
 the addition of electrolytes, while the latter flock out on the addition 
 of the smallest quantity of electrolyte, and indeed again in an irreversible 
 form. This classification stands in accord with what was given above 
 (page 15), as the reversible colloids coincide in a measure with the 
 hydrophile colloids and the irreversible with the suspension colloids. 
 
 Electrolyte Precipitation of Suspension Colloids. It must be 
 remarked that for every precipitating electrolyte a certain minimal con- 
 
 1 See page 74. 
 
 * See Ergebnisse d. Physiologie, 12, 506 which also gives the literature. 
 
 J Pfliiger's Arch., 403, 287 (1904). 
 
 4 Zur Erkenntnis d. Koll., page 21. 
 
22 GENERAL AND PHYSICO-CHEMICAL. 
 
 cent rat ion is necessary to bring about flocking. In comparing the 
 precipitation ability of various electrolytes the concentration of that 
 solution which is just sufficient to cause a visible cloudiness is given in 
 millimolls ( = roVir gram-molecule) per liter. 
 
 Hardy 1 has also found that colloids which wander to the anode 
 are chiefly flocked out by the cations of the precipitating electrolyte, 
 and colloids wandering to the cathode are chiefly flocked out by the anions. 
 H. Schultze 2 has proven that the precipitating ability is influenced 
 greatly by the valence of the precipitating ions, as the divalent ions act 
 much stronger than the monovalent and the trivalent are still more 
 active than the divalent. This rule has been substantiated by Hardy. 3 
 
 This valence rule becomes clear by the following experiment of Freundlich. 4 
 The figures give the lowest precipitation concentration expressed in millimolls 
 per liter. The hydrosol was As 2 S 3 (negative) and the valence of the cations is 
 applicable chiefly for the precipitating action. 
 
 KgSOj __ _ MgCl 2 0.717 
 
 ~2~ b5b MgSO-4 0.810 
 
 KC1 49.5 CaCl 2 0.649 
 
 KNO, 50.0 SrCl 2 0.635 
 
 NaCl 51.0 BaCl 2 .0.691 
 
 LiCl 58.4 Ba(N0 3 ) 2 0.687 
 
 H*SOi ^"Cl 2 0.685 
 
 — 2~ 30.1 U0 2 (N0 3 ) 2 0.642 
 
 HC1 30.8 
 
 AICI3 0.0932 
 
 A!(NO s ) 3 0.0982 
 
 The precipitating action of anions upon a positive hydrosol (Fe[OH] 3 ) is shown 
 in the following experiment of Freundlich : 
 
 KCJ 9.03 K2SO4 0.204 
 
 KNO, 11.90 T1 2 S0 4 0.219 
 
 NaCl 9.25 MgS0 4 0.217 
 
 BaGk 
 
 .9.64 K 2 Cr 2 7 0.194 
 
 Freundlich has extended the valence rule by the fact that with a negative 
 sol, II ions, the ions of the heavy metals, as well as organic cations in weaker con- 
 centration, have a greater precipitating action than other cations; OH ions as well 
 SB organic anion- act against the precipitating action of the cations. The reverse 
 own with a positive sol; OH ions and organic anions of smaller precipitation 
 concentration than corresponds to their valence; H ions and organic cations 
 gainst the precipitating properties of the anions. 
 
 Certain above-mentioned suspensions (mastic), as well as other particles 
 suspended in water, act the same as suspension colloids. Schulze 5 has found 
 thai cloudiness due to 'lav particles on the addition of clarifying bodies (alum, 
 lime) give a voluminous deposition. Schloessing • found that clay suspensions 
 
 1 Zeitschr. f. physik. Chem., 33, 385 (1900). 
 
 'Journ. prakt. Chem. (2),. 25, 431 (1882). 
 
 ■Proc. Roy. 80c., 66, 110 (1899). 
 
 4 Zeitschr. f. Chem. u. Ind. d. Koll., 1, 323 (1907). 
 
 •Ann. Phys. (2), 12'.), 366 (1866). , 
 
 •Compt. rend., 70, 1345 (1870). 
 
COLLOIDS. 23 
 
 which do not settle after months are precipitated in 24-48 hours by a minimum 
 quantity of lime or magnesia. Be also calls attention to the essential role which 
 the salts of sea water must play in the sedimentation of the cloudy fresh water 
 flowing into the sea (delta formation). 
 
 In consideration of the conditions just mentioned, under which the 
 suspension colloids are precipitated by electrolytes, the mutual precipita- 
 tion ability of suspension colloids is of considerable interest. Accord- 
 ing to what lias been stated previously, the colloids are considered as carriers 
 of electricity, and it has been proved that the oppositely charged col- 
 loids can act precipitatingly upon each other. This rule was first pro- 
 posed by Linder and Picton, 1 and has subsequently been substantiated 
 by many investigators. Biltz 2 has made especially systematic investiga- 
 tions on this subject and finds that colloids carrying 'the same kind of 
 charge do not precipitate each other. For the mutual complete precipita- 
 tion of opposed electrically charged colloids, a certain quantitative rela- 
 tion is necessary. On the action of two colloids with opposite charges in 
 variable quantities an optimum of the precipitation action is noticed; while 
 on overstepping the desirable precipitation conditions in both directions 
 no precipitation occurs at all. 
 
 In analogy with the mutual precipitation ability of the colloids, Biltz believes 
 that the especial great ability of most salts of the heavy metals to precipitate 
 colloids lies in the hydrolytically split and colloid-dissolving metallic hydroxides. 
 
 Protective Colloids. Certain hydrophile colloids, which are precip- 
 itated with difficulty by electrolytes, have the power of protecting 
 suspension colloids against the precipitating action of electrolytes. Meyer 
 and Lottermosser 3 have found with silver hydrosol that the presence 
 of protein prevented the flocking out by electrolytes. Zsigmondy 4 
 has investigated the relative action of the protective colloids and has 
 found considerable differences. The figure in milligrams of colloid which 
 is just insufficient to protect 10 cc. of gold solution (0.0053-0.0058 
 per cent) against the action of 1 cc. 10 per cent NaCl solution is called the 
 gold equivalent for the respective colloid. Gelatin offers the best pro- 
 tection, then comes isinglass, casein, ovalbumin, gum arabic, Irish moss, 
 dextrin, starch. The colloidal sulphides (As 2 ^3. Sb 2 S3, CdS) are also 
 protected in the same manner against the influence of electrolytes (A. 
 Miller and Artmaxx 5 ). Inorganic colloids may also act as protective 
 
 1 Journ. chem. Soc, 71, 572 (1897). 
 
 2 Ber. d. d. chem. Gesellsch., 37, 1095 (1904). 
 
 3 Journ. prakt. Chem. (2), 56, 241 (1897). 
 
 4 Zeitschr. analyt. Chem., 40, 697 (1901). 
 5 Oester. Chem. Ztg., 7, 149 (1904). 
 
24 GENERAL AND PHYSICO-CHEMICAL. 
 
 colloids. Thus according to Biltz l zirconium hydroxide protects gold 
 better than does gelatin. 
 
 By the addition of organic protective colloids, the inorganic colloids 
 which on evaporation otherwise become irreversible, are made reversible, 
 in that the dry residue is soluble in water again. On this depends the 
 use of the protective action in the preparation of permanent inorganic 
 hydrosols, and this is of importance in many cases. 
 
 According to Bechhold 2 the filterability of suspension colloids through 
 collodion filters is increased by the addition of organic colloids. It is also 
 well known that certain finely divided substances (carbon) pass more 
 easily through a filter in the presence of protein than without protein. 
 
 The action of the protective colloids is ordinarily explained accord- 
 ing to the theor3 r 'of Quincke 3 on the mutual surface tension of the 
 active bodies, and the process belongs accordingly to the adsorption 
 phenomenon which will be discussed later. According to this theory 
 the protective colloid under certain conditions spreads like an envelope 
 around the particles. In this wise the entire mass takes the properties 
 of the protective colloid and is therefore not precipitated by the elec- 
 trolyte any more than the protective colloid itself. In filtration the pro- 
 tective colloid acts to a certain extent like a lubricant. This theory of 
 colloid envelope has recently received support by experiments of Michaelis 
 and Pincussohn. 4 They found that when suspensions of indophenol 
 and mastic were mixed together the number of particles visible in the 
 ultramicroscope diminished; after mixing, the physical properties of 
 the indophenol (pseudofluorescence, positive cataphoresis) were not 
 evident. 
 
 Electrolyte Precipitation of Hydrophile Colloids. The salts of the 
 alkalies precipitate the suspension colloids even in low concentrations. 
 The alkali salts behave differently toward the hydrophile colloids. This 
 may in part be due to the fact that hydrophile colloids have much less 
 of a certain electric charge than the suspension colloids. For this reason 
 the hydrophile colloids are often precipitated from their solution by alkali 
 salts. For this purpose, firstly, certain concentrations are necessary; 
 secondly, the precipitates of the hydrophile colloids are again solul le 
 in water (reversible) in opposition to those of the suspension colloids. 
 In regard to the ability of different alkali salts to act precipitatingly 
 certain laws have been formulated, but they cannot be arranged in a 
 general rule. 
 
 1 Ber. d. d. chem. Gesellsch., 35, 4431 (1902). 
 
 Zeitechr. f. physik. Chem., 60, 301 (1907). 
 ' Ann. I'liys. (3), 3. r >. 580 (1888). 
 1 Bioch. Zeitechr., 2, 251 (1907). 
 
COLLOIDS. 25 
 
 On comparing the concentration of various salts just sufficient for precipita- 
 tion, where at one time the same anion with different cations was tested and 
 another time the same cation with different anions, Pauli has arranged the cations 
 and anions in the following order in increasing precipitation ability: 
 
 CNS <I <Br <XO, <C1 <OCO.CH 3 <HPO< <S0 4 
 XH 4 <K<Xa<Li. 
 
 The protein used in these experiments was white of egg. According to Pauli 
 certain ions have a precipitating action and others a solvent action. The action 
 of a salt corresponds to the algebraic sum of the action of the ions. 1 Pauli has 
 attempted to associate the precipitation ability of the salts in relation to their 
 action upon the coagulation temperature, but without any positive results. 2 
 
 Nevertheless Spiro ! has shown that the kind of protein as well as its con- 
 centration are of importance for the precipitation action, and Hober * has recently 
 shown that the series I<Br<Cl<S0 4 and Li<Xa<K <Rb <Cs is valid in 
 alkaline reaction, but that the series is reversed in acid reaction. In nearly 
 neutral reaction irregularities in the ion series occur which can be considered as 
 a transition series between the two just-mentioned series. That the reaction must 
 be of great importance in the precipitation of proteins seems very probable in 
 consideration of the fact that the proteins take a decided electric charge on the 
 addition of acid or alkali. In regard to the precipitation by salts of the heavy 
 metals, the hydrophile colloids do not seem to differ essentially from the suspension 
 colloids. 5 
 
 On boiling a protein solution the protein suffers an irreversible change 
 and under certain circumstances flocks out. Boiled but not flocked 
 egg-white behaves with precipitating substances, like a suspension colloid. 6 
 In regard to the precipitation of proteins see Chapter II. 
 
 Theories of Precipitation Phenomena. 
 
 At least for the suspension colloids there is no question that they 
 are flocked out by ions which carry an electric charge opposite to the 
 colloid particles, and also by other colloids having an opposite charge. 
 This fact follows from Hardy's theory, according to which the flocking 
 out is a neutralization process in which the charge of the colloid is 
 just neutralized and the colloid therefore precipitates. 7 The mixture 
 formed on precipitation has been showm to be electrically neutral (iso- 
 electric) as the precipitated particles show no cataphoresis. In this 
 manner it is easily understood that polyvalent ions have a stronger 
 precipitating action than monovalent, as the electrical charge in, for 
 
 1 Hofmeister's Beitrage, 3, 225 (1902). 
 
 2 Pfluger's Arch., 78, 315 (1899). 
 
 3 Spiro, Hofmeister's Beitrage, 4, 300 (1903). 
 * Ibid., 11,35 (1908). 
 
 5 Pauli, Ibid., 6, 233 (1905). 
 
 6 Hardy, Proc. Roy. Soc, 66, 110 (1900). 
 
 7 Zeitschr. f. physik. Chem., 33, 385 (1900). 
 
26 GENERAL AND PHYSICO-CHEMICAL. 
 
 example, a trivalent ion is three times greater than in a monovalent ion. 
 Otherwise greater precipitation ability of polyvalent ions can also be 
 explained by a greater hydrolytic cleavage of the salts (page 23). 
 
 The mechanism of the precipitation of the isoelectric solution accepted 
 in Hardy's theory is explained by Bredig x as follows: At the boundary 
 between suspended particles and solvent a certain surface tension exists 
 which tries to diminish the total contact surface between the two media, 
 which can happen by the small particles uniting to form larger ones, 
 when nocking is brought about. The electrical charge of the particles 
 acts against the surface tension so that equally charged particles repel 
 each other. If the electrical charge is discharged, as takes place in the 
 isoelectric point, then the surface tension reaches its highest value and 
 the precipitation may occur. 
 
 The correctness of Hardy's claim that precipitation occurs just in 
 the isoelectric fluid is disputed on special grounds by Billitzer. He 
 believes that the ions have a much greater charge than the colloid par- 
 ticles. An ion collects the oppositely charged colloid particles around 
 itself, and during these neutralization processes it may occur, that the 
 entire complex may become so large as to become visual and on 
 account of the gravity it precipitates out. 
 
 In general it can be stated that the stability of a colloid is greater the smaller? 
 cet. par., the particles are; as the probability that the number of particles sufficient 
 for the precipitation is then less. With equal size of particles the stability of a 
 colloid is dependent upon the size of the charge which the particles carry. Too 
 weak and very strongly charged colloids are relatively more stable; the first 
 because of the large number which must collect around an ion when flocking takes 
 place and the second because the number of particles required for the neutrali- 
 zation is perhaps too small, so that the necessary size of the complex for precipita- 
 tion is not attained. 2 
 
 The findings of Linder and Picton 3 that when colloidal AS2S3 is 
 precipitated with BaCk> the solution becomes acid, and a small quantity 
 of barium remains in the precipitate, corresponds to Billitzer's theory. 
 This quantity of barium cannot be removed by water, but can be replaced 
 by the corresponding cation by washing with a solution of another salt. 
 According to Billitzer in the mutual precipitation of colloids a quan- 
 tity relation exists which is dependent upon the electrical charges 4 (see 
 also page 22). 
 
 The fact that the precipitation of colloids is a manifestation of 
 processes which occur in a homogeneous medium, makes the understand- 
 ing of these especially difficult. If, as is generally accepted, we consider 
 
 1 Anorganische Fermente (1901), 15. 
 
 Zeitechr. f. physik. Chem. Soc, 45, 327 (1904); 51, 129 (1905). 
 Mourn. Chem. Soc, 67, 63 (1895). 
 •Zeitechr. f. physik. Chem., 51, 141 (1905). 
 
COLLOIDS. 27 
 
 the colloid BOlution as a homogeneous fluid of suspended solid or fluid 
 particles, then in the " solution " there occur at least two special con- 
 stituents] separated from each other — the colloid particles and the sol- 
 vent. This is expressed as follows: the system contains two phases. 
 The solvent is often more correctly called the dispersion means and the 
 colloid particles called the disperse phase. If to such a system a new 
 sul stance is added, then the reaction which follows, depends essentially 
 upon the division of the new substance between the two phases. In 
 regard to the possible division two cases will be presented: 
 
 1. The process can be similar to the division of a soluble substance 
 between two solvents. If a substance is brought in contact with two 
 solvents at the same time, then it divides itself so that the relation 
 between the concentration in the two solvents remains the same but 
 independent of the total quantity of the dissolved substance. If the 
 quantity of substance in each 100 cc. of the two solutions 1 and 2 is 
 
 Cl 
 
 designated bv ci and c-2, then it follows that — =k where k is a constant. 1 
 
 C-2 
 
 The first example where this law was shown to be correct was the divi- 
 sion of succinic acid between water and ether (Berthelot and Jung- 
 fleisch -'.). This law was also shown to be true for the division of 
 a gas between a gaseous and a fluid phase, i.e., for the absorption of a 
 gas in a fluid (Henry's law of absorption). The conditions for the cor- 
 rectness of this law are that the temperature remains the same in experi- 
 ments with different quantities of substance as well as that the substance 
 has the same molecular size in the two phases. 
 
 2. In those cases where finely divided solids take up dissolved sub- 
 stances or gases the division is generally not independent of the total 
 quantity of the dissolved substance or of the gas. This is often called 
 adsorption. 3 For example, if we are dealing with the adsorption of a 
 dissolved substance by a finely divided solid occurring in a solution, 
 then a greater percentage is taken up from a dilute solution than from a 
 concentrated one. On increasing concentration the adsorbed fraction 
 becomes continuously less so that the absolute quantity taken up reaches 
 a maximum which corresponds to the greatest adsorption ability of the 
 solid body. 
 
 This is expressed by the formula — = k, where Ci and c 2 indicate the concentra- 
 tion of the solid body and in the solution; n and k are constants and indeed, n is 
 
 'Nenut, Zeitschr. f. physik. Chem., 8, 110 (1891). 
 
 2 Ann. Chim. phys. (4>. 26. 396 (1872). 
 
 3 It must be remarked that in the older literature oftentimes no difference was 
 made between adsorption, and absorption, in which case both processes were included 
 under the name absorption. 
 
28 GENERAL AND PHYSICO-CHEMICAL. 
 
 always > 1. (If n = \ then the formula would be —=k and we would be dealing 
 with a so-called solid solution.) 
 
 Appleyard and Walker l have studied the adsorption of organic 
 acids from aqueous and alcoholic solutions by means of silk; the divi- 
 sion was found to correspond to the above formula for adsorption. 
 Freundlich 2 has also carefully tested the adsorption of crystalloids 
 by carbon. From these experiments it was shown that the equilibrium 
 could be quickly attained from both sides, i.e., that the process was readily 
 reversible. The above-given formula was found sufficiently accurate 
 for the case w r here only the total quantity of the dissolved (to adsorb) 
 substance varied. The series in which the organic acids were adsorbed 
 by silk, as found by Appleyard and Walker, were pratically the same 
 as with carbon. The influence of temperature was slight. 
 
 According to Kuster, 3 the combination between starch and iodine 
 is to be considered as an adsorption compound, and Biltz 4 finds for the 
 division of AS2O3 between iron hydroxide (1) and water (2) the for- 
 mula — = 0.631. 
 
 The theoretical foundations for the adsorption phenomenon are 
 not especially clear. Generally the adsorption is considered as con- 
 nected with segregation and surface tension phenomenon. At the con- 
 tact surface between a solid body and solution a surface tension exists 
 which is considered as positive, i.e., this attempts to diminish the 
 contact surface. The surface energy used thereby tends to be a min- 
 imum potential energy. As the product from size of surface and surface 
 tension are the same, and as the first cannot change, the surface energy 
 can only be diminished by a reduction of the tension. If, therefore, 
 the tension is diminished by increasing the concentration of a sub- 
 stance dissolved in a fluid, then this substance tries to collect itself 
 at the surface in greater concentration than in other parts of the fluid 
 (Ostwald, 5 Freundlich 6 ). In regard to the surface tension of solid- 
 fluid we only know that it is positive, but can otherwise show great 
 differences (Ostwald, 7 Hulett 8 ). According to this theory the facts 
 are that certain solid substances possess the ability of adsorbing dis- 
 
 1 Journ. Chem. Soc, 69, 1334 (1896). 
 
 2 Debar die Adsorption in Losungen, Leipzig (1906). 
 » Ann. d. Chem. u. Pharm., 283, 360 (1894). 
 
 * Ber. d. d. chem. Gesellsch., 37, 3138 (1904). 
 
 •Lehrh. d. allg. Chem., 2. Aufl., 2. Bd., 3. Teil, 237 (1906). 
 
 8 Ueber Adsorption in Losungen, 50-51. 
 
 7 Zeitschr. f. physik. Chem., 34, 495, 1900. 
 
 •Ibid., 37, 385 (1901). 
 
COLLOIDS. 29 
 
 solved bodies, and for this reason the ads >rbed substance lowers the 
 surface tension of the solid-fluid, and indeed, the more the greater con- 
 centration in which it occurs. That especially carbon and colloid sub- 
 stances are adsorption bodies lies in the fact that they have an especially 
 large surface due to their finely divided state or porosity, which there- 
 fore, cet. par., must give them a great surface energy. 
 
 That proteins, on precipitation, carry down other bodies with avidity 
 is well known; inorganic hydrogels also take up dissolved substances 
 with energy. The curves obtained for the latter process by van Bem- 
 melen x show a close analogy with the characteristic curves for the 
 adsorption compounds. It often occurs that the body taken up homo- 
 
 geneouslv saturates the hydrogel, in which case — = k, and a sort of 
 
 solid solution is the result. In certain cases, undoubtedly, chemical 
 combinations with quite positive conditions are formed. 
 
 The precipitation of colloids by electrolytes has also been discussed 
 by Freundlich 2 from the standpoint of the adsorption hypothesis. 
 Thus, for the precipitation ability of an electrolyte, the electric charge 
 of the precipitating ion comes first into consideration and secondly, the 
 ability of the precipitating colloid to adsorb the same. According to 
 Moore and Roaf 3 the salts of the red corpuscles are retained as adsorp- 
 tion compounds (adsorpates) by the proteins. 
 
 Thus far only the adsorption of crystalloids has been considered. 
 Colloids are also taken up by solid substances or by other colloids. Still in 
 these cases the conditions are more complicated than in the above- 
 mentioned adsorption phenomena, as the combinations formed are in special 
 cases irreversible or gradually become irreversible. It is well known that 
 carbon takes up colloidal colored substances, and we have numerous exam- 
 ples of the combination of dissolved colloids with solid colloids in technology. 
 Biltz 4 has been able to show that many dyeing processes are to be 
 considered as adsorption phenomena, and later Freundlich and Losev 5 
 have measured the adsorption of basic and acid pigments by carbon 
 and also by fibers (wool, silk, cotton), and have shown the correspondence 
 of the two processes. With the basic pigments, which were used as 
 salts, a splitting occurred into a pigment base, which was taken up by 
 the fibers as well as by carbon, and an acid which quantitatively remained 
 behind. This is similar to the cleavage which precipitating electrolytes 
 undergo in the precipitation of the suspension colloids (see page 26). 
 
 1 Zeitschr. anorg. Chem., 23, 111, 321 (1900). 
 
 2 Zeitschr. f. Chem. u. Ind. d. Koll., 1, 321 (1907). 
 
 3 Bioch. Journ., 3, 55 (1908). 
 
 4 Ber. d. d. chem. Gesellsch., 37, 1766 (1904); 3S, 2963, 2973, 4143 (1905). 
 5 Zeitschr. f. physik. Chem., 59, 284 (1907). 
 
30 GENERAL AND PHYSICO-CHEMICAL. 
 
 Tanning is also brought about by adsorption processes, as the prepared skins 
 adsorb the tanning substance. 1 
 
 The precipitation of portein by adding finely divided solids (carbon* 
 kaolin 2 ) or by suspended solids (mastic 3 ) precipitated in the liquid, 
 as well as the action of protective colloids as already mentioned are also 
 due to adsorption processes. The precipitation of protein, which occurs 
 on shaking the protein solution with liquids, in which the protein is 
 not soluble, is also to be considered as a surface tension action (Ramsden 4 ). 
 
 Bechhold, 3 in his above-mentioned experiments on the filtration of 
 colloids, has observed conditions which he considers as adsorption phe- 
 nomena. Under certain circumstances a colloid can prevent the filtra- 
 tion of another colloid. A filter which was permeable for colloidal AS2S3, 
 but retained colloidal Prussian blue, did not allow a clear mixture of 
 the two to pass through. The particles of AS2S3, were adsorbed by the 
 particles of Prussian blue, and could therefore not pass through the 
 filter. 
 
 Gels. We have often mentioned gels or jellies (page 14). Only 
 certain colloids can occur in the form of gels. Certain gels are spon- 
 taneously formed in sufficiently concentrated solutions (silicic acid, 
 certain metallic hydroxides) and these do not redissolve in water. Other 
 gels, like gelatin and agar, are formed on cooling of the hot, concentrated 
 solutions, and are again soluble in water. 
 
 According to Hardy 6 the gel formation of gelatin is to be considered 
 as a segregation process whereby a separation into two fluids occurs, 
 one of which solidifies. The two phases are only differentiated by the 
 microscope, and the chemical testing of the theory fails because of the cir- 
 cumstances that the two phases cannot be analyzed separately. In opposi- 
 tion to this Pauli claims that the gel passes through all of the intermediary 
 steps into the corresponding sol and is therefore homogenous in the same 
 sense as these. 7 
 
 When gels are freed from water by evaporation or in other ways, 
 they show a special ability to take up water, which is brought about 
 by different processes which are included in the ordinary term imbibition. 
 The views on this imbibition are indefinite. Surface phenomena play 
 a role here. According to van Bemmelen 8 the water is not chemi- 
 
 1 See Zeitschr. f. Chem. u. Ind/d. Koll., 2, 257 (1908). 
 
 2 Bioch. Zeitschr., 5, 365, 1907. 
 
 • Ibid., 2, 219 (1906); 3, 109 (1906). 
 
 • Zeitschr. f. physik. Chem., 47, 343 (1904). 
 1 Ibid., 60, 299 (1907). 
 
 • Ibid., 33, 326 (1900). 
 
 ■> Bioch. Zeitschr., 18, 367 (1909). 
 
 'Zeitschr. anorg. Chem., 13, 233 (1896); 20, 185 (1899). 
 
COLLOIDS. 31 
 
 cally combined in definite proportions, hut the quantity continually 
 changes with the temperature and the vapor pressure. On the other 
 hand, the imbibition stands in close relation to the osmotic pressure 
 which is evident, if we define the osmotic pressure of a substance as 
 its ability to attract water. The relation between imbibition and 
 osmotic pressure is still closer in those cases when the Substance finally 
 is dissolved in water. 
 
 If a hydrogel is placed in a salt solution instead of in pure water, 
 the imbibition phenomena essentially change. This was first studied 
 by Hofmeister, 1 using gelatin plates. The process is rather com- 
 plicated, as salt is taken up by one side of the gelatin plate and water 
 by the other, and the taking up of water is influenced by the quantity 
 of salt taken up. It has also been found that when gelatin plates are 
 treated with solutions of increasing concentration of the same salt, 
 the taking up of salt increases at first with the salt concentration, then 
 becomes slower, and attempts to reach a maximum and then remains 
 almost stationary. As long as the taking up of salt increases, the quan- 
 tity of water passing into the gelatin also increases; when the salt fails 
 to pass then the water also ceases to pass. It has also been found that 
 the maximum of salt absorption for sulphate, tartrate and citrate can 
 be attained with much lower molecular concentrations than with chloride, 
 nitrate and bromide. From this it follows that the sulphate, tartrate 
 and citrate have a retarding action upon imbibition within certain limits 
 of concentration, while the chloride, nitrate and bromide have an 
 accelerating action. 
 
 Pauli 2 has investigated the influence of salt solutions upon the solid- 
 ification and melting-point of gelatin. If the salts are arranged in the 
 order of their ability to lower the solidification point of gelatin we 
 come to the series sulphate, citrate, tartrate, acetate (water), chloride, 
 chlorate, nitrate, bromide, iodide. This series corresponds well with 
 that of Hofmeister. 
 
 Acids and alkalies exert a special influence upon gelatin, as they 
 both, in very dilute solutions, strongly accelerate imbibition (Spirq, 3 
 Wo. Ostwald 4 ). From the previously mentioned investigations of 
 Lillie, on the osmotic tension of gelatin solutions, it was found that the 
 addition of acids and alkalies increased it (page 17). 
 
 Since Graham's fundamental experiments it was believed that col- 
 loidal sols could not diffuse into gels while crystalloids could pass just 
 
 1 Arch. f. exp. Pathol, u. Pharm., 28, 210 (1891). 
 
 2 Pfluger's Arch., 71, 333 (1898). 
 
 3 Hofmeister's Beitriige, 5, 276 (1904). 
 
 4 Pfluger's Arch., 108, 563 (1905). 
 
32 GENERAL AND PHYSICO-CHEMICAL. 
 
 as quickly into gels as into pure water. Nevertheless, Spiro * has observed 
 that dissolved ovalbumin as well as haemoglobin could pass into gelatin 
 plates. On the other hand K. Meyer 2 as well as Bechhold and Zeigler 3 
 have found that the distance passed by a crystalloid in gelatin may be 
 much shorter than in pure water. In such experiments no doubt adsorp- 
 tion processes must be considered. 
 
 m. CATALYSIS. 
 
 When two bodies which can act chemically upon each other are 
 brought together the reaction generally takes place so fast that it can- 
 not be measured. In other cases, by special means, we can observe 
 how the reaction gradually proceeds. When cane-sugar is inverted 
 by weak acid, the decrease in the rotation of the solution can be fol- 
 lowed with the polariscope; and when an ester is decomposed by alkali 
 the quantity of still free alkali can be determined by titration. The 
 quantity of substance measured in gram-molecule per liter (mole) 
 which is decomposed in the unit of time, is called the reaction velocity 
 of the system. The so-called law of mass action, as proposed by Gtjld- 
 berg and Waage, states that the reaction velocity is every moment 
 proportional to the molecular concentration of the reacting bodies. A 
 mixture cf alcohol and acetic acid is transformed into acetic ether and 
 water, especially in the presence of some mineral acid. If the molec- 
 ular concentration of the alcohol and acid be designated by C A and C s , 
 then according to the law of mass action the reaction velocity is 
 vi=ki.C A -C s , where &i indicates a constant which is independent of 
 the quantity of reacting substances and the time limit is so short that 
 the concentration can be considered as constant. This reaction, like 
 many others, is reversible, #i.e., two reactions occur simultaneously: 
 one between the alcohol and acetic acid, producing acetic ether and 
 water, and second, between acetic ether and water, re-forming alcohol 
 and acetic acid. This is expressed as follows: 
 
 C 2 H 5 .()H+HO.CO.CH3^C2H 5 .O.CO.CH3+H 2 0. 
 
 The velocity of reaction when it passes from left to right is called 
 V\. If the velocity in the reverse reaction is called v<z and the molecular 
 concentration of the acetic ether and water is called C B and C w , 
 then we obtain V2 = ko- Ce-C w . At the beginning when C E as well as 
 
 1 Hofmeister's Beitriige, 5, 294 (1904). 
 
 2 Ibid., 7, 393 (1905). 
 
 1 Zeitschr. f. physik. Chem., 56, 105 (1906). 
 
CATALYSIS. 33 
 
 C w = 0, the velocity of the ester formation is expressed by the formula 
 Vi = ki-C A -C s \ afterward it is expressed by the difference v\ — V2 or 
 ki'C A 'C s —k2-C E -C w . Of the two reaction velocities V\ and V2 at the 
 begining vi always diminishes while V2 increases. When k\-C A -C s = 
 A*2"Ce-CV is attained, then the velocity of both reactions is the same; 
 no measurable decomposition occurs and the system is in equilibrium. 
 The equilibrium condition is the same irrespective of whether we start 
 from alcohol +acetic acid or from the corresponding quantity of acetic 
 ether + water. On equilibrium it is 
 
 h.C A .C s = h.C E .C w or ^^-J^ = K. 
 t Le-^w m 
 
 K is called the equilibrium constant; as is apparent it can be determined 
 in two ways — either from the concentration of the reacting bodies when 
 equilibrium is present or from the velocity coefficient k\ and fo as deter- 
 mined in a manner given below. 
 
 In the above-mentioned transformation of alcohol and acetic acid 
 these two bodies are simultaneously used up. The reaction is therefore 
 called bimolecular, and a reaction is called mono-, bi-, tri-, etc., molecular 
 according to the number of the kinds of molecules which diminish their 
 concentration thereby. 1 
 
 Berzelius 2 found that certain bodies by their mere presence, and 
 not by their affinity, have the power of awakening the dormant affinity 
 at a certain temperature, i.e., the power of starting a reaction. These 
 phenomena were called catalytic by Berzelius. 
 
 According to Ostwald 3 catalysis is the acceleration (or retardation) 
 of a slow-proceeding chemical change by the presence of a foreign body. 
 That body which influences a reaction in this manner is called a catalyst. 
 It does not itself undergo any appreciable change by the reaction. 
 
 Catalytic reactions have been studied, especially by Wilhelmy. 4 
 van't Hoff, 5 Ostwald, 6 Arrhenius 7 and Bredig. 8 Of all other sub- 
 stances the acids and alkalies seem to act most catalytic. A well-known 
 
 1 It is assumed here that of every kind of molecule one molecule of each takea 
 part in the reaction. 
 
 o 
 
 2 Berzelius, Arsberattelse om framstegen i Fysik och Kemi., 13, p. 245 (1836). 
 
 3 Lehrb. d. aUg. chem. 2. Aufl. II., 1, 515. 
 
 4 Poggendorff's Ann., 81, 413 (1850). 
 
 5 Etudes de dynam. chim. (1884). 
 
 ' 6 Lehrb. d. aUg. Chem., 2. Auf. II, 2, 199. 
 7 Zeitschr. f. physik. Chem., 4, 226 (1889). 
 8 Anorganische Fermente (1901); Bioch. Zeitschr., 6, 283 (1907). 
 
34 GENERAL AND PHYSICO-CHEMICAL. 
 
 example is the inversion of cane-sugar by means of acid. This reac- 
 tion is monomolecular because only the cane sugar is consumed. If 
 the concentration of the cane-sugar at the beginning is C moles, and if 
 x moles are transformed in t time, then at that time there are (C — x) 
 moles remaining. If dx indicates the quantity which is transformed 
 
 dx 
 in dt time, then the reaction velocity is — . According to the law 
 
 dt 
 
 of mass action this is at every moment proportional to the concentration 
 
 of the decomposing substance, or 
 
 ~=k.(C-x) '. . . (1) 
 
 dt 
 
 For practical use this equation is integrated into the following: 
 
 A,- = -nat. log. -— (2) 
 
 t C — x 
 
 If the theoretical considerations upon which this formula is based 
 
 are correct, then the x values determined by the polariscope after various 
 
 times must give the same figure for k. This is indeed the case. 1 k is 
 
 called the velocity coefficient (also velocity constant or specific reaction 
 
 velocity). If in the equation (1) C — x or the concentration of the still 
 
 dx 
 undecomposed cane-sugar =1, then the equation becomes ~r = k, from 
 
 which it follows that k indicates the reaction velocity if the concentra- 
 tion of the substrate could be kept the entire time at =1. 
 
 In these experiments k retains the same value. If in different experi- 
 ments the quantity of catalyst (acid) varies, then the obtained value 
 for k is proportional to the concentration of the H ions. This is so 
 prominent that the catalytic action of acids is due to the H ions (Ar- 
 rhenius 2 ) . Still irregularities occur as the anions of acids as well as 
 of salts present can under certain circumstances influence the action of 
 H ions (see page 70). 
 
 Frankel 3 has recently studied the decomposition of diazoacetic ether under 
 the influence of different acids. The reaction is as follows: 
 
 N a : HC.CO.O.C 2 H & +H 2 0=HO.CH 2 .CO.O.C 2 H5+i\ 2 . 
 
 1 See Poggend. Ann., 81, 413 and 499 (1850). 
 2 Zeitschr. f. physik. Chem., 4, 226 (1889). 
 3 Ibid., 60, 202 (1907). 
 
CATALYSIS. 
 
 35 
 
 The progress of the reaction can be determined by measuring the nitrogen 
 
 set free. The following figures explain the results: 
 
 Acid. 
 
 Cone, of the 
 
 Acid in Mol. 
 
 per Liter. 
 
 'On- 
 
 Cone, of the 
 
 H ions liy 
 
 Electric 
 
 Conductivity. 
 
 K 
 
 Velocity 
 Coefficient. 
 
 K 
 
 Nitric acid 
 
 0.001820 
 
 . 000909 
 I) 000909 
 . 000364 
 . 009900 
 0.003640 
 0.009090 
 0.018200 
 
 001820 
 0.000909 
 0.000909 
 
 0.000364 
 0.001680 
 0.001460 
 0.C00724 
 0.000563 
 
 . 0703 
 0.0346 
 
 0.0356 
 0.0140 
 . 0632 
 0.0571 
 
 . 0285 
 0.0218 
 
 38.7 
 
 Picric acid 
 
 w-Nitrobenzoic acid 
 
 Fumaxic acid 
 
 38.0 
 39.2 
 38.3 
 37.7 
 39.1 
 
 Succinic acid 
 
 38.5 
 
 Acetic acid 
 
 38.7 
 
 7.' 
 
 As 77-for the different acids and different quantities of acid is the same, then 
 the velocity coefficient is here also proportional to the concentration of the H ions. 
 
 As the catalytic action of acids is caused by the H ions, so are the 
 catalytic properties of bases due to the OH ions. The first determined 
 case of this kind was the transformation of byoscyamine into the stable 
 atropine. 1 
 
 Koelichen 2 has studied a specially pretty case of the catalytic action of 
 OH ions in the decomposition of diacetonalcohol into acetone : 
 
 CH 3 .CO.CH,.C(CH 3 ) 2 .OH = 2CH3.CO.CH3. 
 
 The reaction is reversible, and from the following table it is seen that the 
 velocity constant for various concentrations of the same catalyst remains the same 
 as well as by using different bases. 
 
 Catalyst. Cone, of the Velocity 
 
 Catalyst. Constant. 
 
 Piperidine 0.1090 0.038 
 
 Triethylamine . 4900 . 036 
 
 Ammonia 0.5500 0.038 
 
 Tetraethvlammonium / 0.0760 0.037 
 
 hydroxide \ 0.0076 0.037 
 
 « j. , , • , / 0.0725 0.036 
 
 Sodium hydroxide | Q m2 Q Q35 
 
 By this a rule which van't Hoff and Ostwald 3 proved by thermo- 
 dynamic means, is substantiated, namely, that the equilibrium at con- 
 stant temperature does not change with the quantity and kind of catalyst 
 when the catalyst is not changed by the reaction. 
 
 Among other kinds of ions which act as catalysts we must mention (1) iodine 
 ions, which decompose H 2 2 in proportion to their concentration, 4 and (2) cyan- 
 
 1 Ber. d. d. chem. Gesellsch., 21, 2777 (1888). 
 2 Zeitschr. f. physik. Chem., 33, 129 (1900). 
 
 3 Van't Hoff, Vorlesungen, 1, 211. 
 
 4 Walton, Zeitschr. f. physik. Chem., 47, 185, 1904. 
 
36 GENERAL AND PHYSICO-CHEMICAL. 
 
 ions, which transform benzaldehyde into benzoin according to the following 
 equation: 
 
 2C 6 H 5 .COH =C 6 H 6 .CO.CH(OH).C 6 H5. 1 
 
 If those bodies which accelerate a reaction are to be considered as catalysts, 
 then certainly the solvents must belong to the catalytes. Attention must be 
 called to the enormous influence which the solvent can exert upon the velocity 
 of a reaction under otherwise equal conditions. Thus Menschutkin 2 found for 
 the reaction 
 
 (C 2 H 5 ) 3 .N+C 2 H 5 .I = (CJBi)4.N.I. f 
 
 the following velocity in different solvents: 
 
 Hexane 0.00018 
 
 Heptane .000235 
 
 Xylene 0.00287 
 
 Benzene 0.00584 
 
 Ethyl alcohol 0.03660 
 
 Benzyl alcohol 0. 13300 
 
 Recently Bredig and Fajans 3 have been able to show that an optically active 
 solvent can help in the decomposition of optical antipodes to a varying extent. 
 Of the optical antipodes of campho-carboxylic acid, the d-form is 17 per cent 
 more quickly decomposed than the Z-form, when they are dissolved in nicotine 
 or when nicotine is present, dissolved with the catalyte, while in an optically 
 indifferent solvent and without any nicotine the catalyte decomposes both forms 
 with equal rapidity. The reaction proceeds differently with or without catalyst, 
 and hence the catalyst brings about changes in reaction other than those of velocity. 
 It is apparent that this does not conform with Ostwald's definition of a catalyst 
 (page 33). It must be mentioned that Bredig and Fiske have been able to 
 perform the asymmetric synthesis of benzaldehyde and hydrocyanic acid by means 
 of quinine and quinidine as catalysts (page 60). 
 
 Catalysis in Heterogeneous Systems. The above-treated catalytic 
 processes all occur in homogenous systems, i.e., the systems which by 
 mechanical means cannot be separated into different constituents. In 
 heterogeneous systems with phases which can be separated from each 
 other by mechanical means, catalytic reactions can also occur, and 
 indeed, in such cases the substances taking part [in the reaction and 
 the catalyst occur in different phases. Such a reaction is the union of 
 detonating gas, the synthesis of SO3 (from SO2 and O), and the decom- 
 position of H2O2 by platinum. In case the system is two-phased, and 
 the reaction takes place only at the boundary between both phases, or 
 in the one we can differentiate two simple limits: 
 
 1. The accumulation of the bodies which are necessary for the 
 reaction at the proper place takes such a short time that in comparison 
 
 1 Stern, ibid., 50, 513 (1905). 
 
 2 Ibid., 6, 41 (1890). 
 
 8 Ber. d. d. chem. Gesellach., 41, 752 (1908). 
 
BNZYME8. 37 
 
 with the real chemical reaction it can be neglected. In these cases 
 the reaction velocity behaves similarly to a homogeneous system. 1 
 
 2. The chemical reaction occurs at a rate which in comparison with 
 the time necessary for the accumulation can be neglected. In this 
 case the time necessary can be generally compared with a diffusion 
 process. 2 
 
 The catalytic processes in heterogeneous systems have excited 
 interest since Bredig 3 showed that the colloidal metals prepared by 
 him showed catalj'tic properties. The best-studied process is the decom- 
 position of H2O2 by colloidal platinum, gold, and other metals or oxides 
 (Mn02, Pb02). Attention must be called to the small quantity of 
 catalyst sufficient to decompose H2O2. The action of 1 gram atom 
 platinum in 70 million liters of reaction mixture has been detected. The 
 decomposition of H0O2 by platinum catalyst in nearly neutral or faintly 
 acid solution has been shown to be a monomolecular reaction. 
 
 Still certain differences occur from the conditions formed in the 
 homogeneous catalysis. At one time in certain experiments the value 
 for A; rises considerably during the catalysis, and secondly, k is not 
 proportional to the ferment concentration, but rises more quickly than 
 this. 
 
 In connection with these experiments Bredig has expressed the 
 view that an analogy exists between the catalytic processes of the inor- 
 ganic world and the enzyme action of the organic. 
 
 The following important facts give support to Bredig's view: 
 
 1. In both cases we are dealing with catalytic processes; the metallic sol and 
 the enzyme are active in very small quantities and during the reaction they do 
 not undergo any appreciable change. 
 
 2. In the decomposition of H 2 2 by platinum sols or by the enzyme haemase, 
 the reaction is monomolecular. 
 
 3. The action of metallic sols as well as enzymes is paralvzed by certain 
 poisons (HCN, H 2 S). 
 
 4. Both classes of bodies are colloid substances and possess an enormous 
 surface upon which their catalytic action depends. 
 
 According to Neilson, 4 ethyl butyrate, salicin and amygdalin are decom- 
 posed by platinum black as well as by enzymes. 
 
 IV. ENZYMES. 
 
 Chemical Processes in Plants and Animals. It follows from the law 
 of the conservation of matter and of energy that living beings, plants 
 and animals, can produce neither new matter nor new energy. They 
 
 1 Goldschmidt. Zeitschr. f. physik. Chem., 31, 235 (1899). 
 
 2 Nernst and Brunner, ibid., 47, 52 and 56 (1904). 
 
 3 Anorganische Fermente, Leipzig, 42 (1901). 
 
 4 Amer. Journ. of Physiol., 10, 191 (1904); 15, 148 (1906). 
 
38 GENERAL AND PHYSICO-CHEMICAL. 
 
 are only called upon to appropriate and assimilate material already exist- 
 ing and to transform it into new forms of energy. 
 
 Out of a few relatively simple combinations, especially carbon 
 dioxide and water, together with ammonium compounds or nitrates, 
 and a few mineral substances, which serve as its food, the plant builds 
 up the extremely complicated constituents of its organism — proteins, 
 carbohydrates, fats, resins, organic acids, etc. The chemical work 
 which is performed in the plant must, therefore, in the majority of cases, 
 consist in syntheses; but besides these, processes of reduction take 
 place to a great extent. The radiant energy of the sunlight induces 
 the green parts of the plant to split off oxygen from the carbon dioxide 
 and water and this reduction is generally considered as the starting- 
 point in the syntheses that follow. According to a hypothesis suggested 
 by A. Baeyer, 1 formaldehyde is first produced, C02+H20 = CH20+02, 
 which by condensation is transformed into sugar. From the sugar other 
 bodies can then be built up. 
 
 With the aid of the silent electric discharge W. Loeb 2 has succeeded 
 in obtaining from carbon dioxide and water, formaldehyde, and as a 
 product of polymerization, also glycolaldehyde, CH2OH.CHO, from 
 which sugar can be readily produced. Still the conditions under which 
 these bodies were formed cannot be applied to the conditions in the 
 plants. The investigations of Usher and Pristley 3 are of greater 
 interest in that they show the formation of formaldehyde in the photo- 
 lytic decomposition of moist carbonic acid in the presence of chloro- 
 phyll. These investigations also do not seem to be entirely free from 
 exception. The conception as to the formation of sugar from formalde- 
 hyde is also often different from that explained by v. Baeyer's idea, 
 and his view as to the assimilation of carbonic acid constitutes a hypoth- 
 esis which requires further proof. The essentials of this hypothesis, 
 namely, a formation of formaldehyde with a subsequent sugar formation 
 by condensation of the aldehyde groups, is still very generally accepted 
 as probably correct. Independent of the ways and means of how the 
 assimilation processes in the plants originate, it is obvious that the free, 
 radiant energy of the sun is hereby bound and stored in a new form, as 
 chemical energy, in the combinations formed by the syntheses. 
 
 In animal life the conditions are not the same. Animals are depend- 
 ent either directly, as the herbivora, or indirectly, as the carnivora, 
 upon plant-life, from which they derive the three chief groups of organic 
 nutritive matter — proteins, carbohydrates, and fats. These bodies, 
 of which the protein substances and fats form the chief mass of the 
 
 1 Ber. d. d. chem. Gesellseh., 3. 
 *Zeitschr. f. Electrochem., 12. 
 
 1 Proc. Roy. Soe. London, 78, Series B. 
 
ENZYMES. 39 
 
 animal body, undergo within the animal organism a cleavage and oxi- 
 dation, and yield as final products exactly the above-mentioned chief 
 components in the nutrition of plants, namely, carbon dioxide, water, 
 and ammonia derivatives, which are rich in oxygen and have little energy. 
 The chemical energy, which is partly represented by the free oxygen 
 and partly stored up in the above-mentioned more complex chemical 
 compounds, is transformed into other forms of energy, principally heat 
 and mechanical work. While in the plant we find chiefly reduction 
 processes and syntheses, which by the introduction of energy from 
 without produce complex compounds having a greater content of energy, 
 we find in the animal body the reverse of this, namely, cleavage and oxi- 
 dation processes, which, as we used to state, convert chemical tension 
 into living force. 
 
 This difference between animals and plants must not be overrated, 
 nor must we consider that there exists a sharp boundary line between 
 the two. This is not the case. There are not only lower plants, free 
 from chlorophyll, which in regard to chemical processes represent inter- 
 mediate steps between higher plants and animals, but the difference 
 existing between the higher plants and animals is more of a quantitative 
 than of a qualitative kind. Plants require oxygen as peremptorily as 
 do animals. Like the animal, the plant also, in the dark and by means 
 of those parts which are free from chlorophyll, takes up oxygen and 
 eliminates carbon dioxide, while in the light the oxidation processes going 
 on in the green parts are overshadowed or hidden beneath the more intense 
 reduction processes. As in the animal, Ave also find a heat production 
 in fermentation produced by plant organisms; and even in a few of the 
 higher plants — as the aroidcce when bearing fruit — a considerable develop- 
 ment of heat has been observed. On the other hand, in the animal 
 organism, besides oxidation and splitting, reduction processes and syn- 
 theses also take place. The contrast which seemingly exists between 
 animals and plants consists merely in that in the animal organism the 
 processes of oxidation and splitting are predominant, while in the plant 
 chiefly those of reduction and synthesis have thus far been studied. 
 
 Wohler l in 1824 was the first to observe an example of the syn- 
 thetical processes within the animal organism. He showed that 
 when benzoic acid is introduced into the stomach, it reappears as hippuric 
 acid in the urine after combining with glycocoll (aminoacetic acid). 
 Since the discovery of this synthesis, which may be expressed by the 
 following equation : 
 
 CoH5.COOH+XH 2 .CH 2 .COOH = NH(C 6 H 5 .CO).CH 2 .COOH+H 2 0, 
 
 Benzoic acid Glycocoll Hippuric acid 
 
 1 Berzelius, Lehrb. d. Chemie, iibersetzt von Wohler, 4, p. 356, Abt. 1, Dresden 
 (1831). 
 
40 GENERAL AND PHYSICO-CHEMICAL. 
 
 and which is ordinarily considered as a type of an entire series of syn- 
 theses occurring in the body, where water is eliminated, the number of 
 known syntheses in the animal kingdom has increased considerably. 
 Many of these syntheses have also been artificially produced outside 
 of the organism, and numerous examples of animal syntheses of which 
 the course is absolutely clear will be found in the following pages. Besides 
 these well-studied syntheses, there also occur in the animal body similar 
 processes unquestionably of the greatest importance to animal life, but 
 of which we know nothing with positiveness. We enumerate as examples 
 of this kind of synthesis the re-formation of the red-blood pigment (the 
 haemoglobin), the formation of the different proteins from simpler sub- 
 stances, and the production of fat from carbohydrates. This last- 
 mentioned process, the formation of fat from carbohydrates, is also an 
 example of reduction processes which occur to a considerable extent in 
 the animal body. 
 
 Certain reactions, which are either not reproduceable with dead ma- 
 terial or are only possible under conditions which destroy the cells, belong 
 to the chemical decompositions going on within the living organism. 
 Thus the synthesis of glycogen or of protein has not been accomplished 
 outside of the organism or without the aid of agents prepared by the 
 cells. On the other hand proteins and starches can be split into simpler 
 products without these agents, but for this purpose the action of acids or 
 alkalies of a concentration which would kill the cells is necessary. In 
 certain cases it is possible to bring about such reactions outside of the 
 organism without any injurious effect upon the cells. This is accomplished 
 by the aid of substances which are formed within the cells but have the 
 power of being active after they have left the cells. These substances 
 have been called enzymes or ferments. 
 
 Enzymotic Processes. We must now mention a group of reactions 
 which are more or less related to enzyme action. 
 
 In the first place the so-called hydrolytic cleavage processes in which 
 complex substances are divided into simpler substances with the simul- 
 taneous decomposition of water and the taking up of its constituents. 
 These processes are of the greatest importance in the digestion of the 
 food-stuffs and for making them of value but they are also important for 
 the metabolic processes in general. As examples of such cleavages 
 we will mention the division of proteins into simpler products, the trans- 
 formation of starch into sugar and the cleavage of neutral fats into the 
 corresponding fatty acid and glycerin: 
 
 (•,,n5^i.sH,,50 2 ),+3H20 = C3F5rOH)34-3C 1 8H 3 o02 
 
 Tristearin Glycerin Stearic Acid. 
 
 The importance of the hydrolytic cleavage processes for digestion 
 will be discussed in detail in Chapter VIII. 
 
ENZYMES. 41 
 
 Other cleavage processes are certain so-called fermentation processes, 
 
 which are connected with the presence of living organisms, fungi and 
 bacteria of various kinds. Among these we include chiefly the alcoholic 
 fermentation and butyric acid fermentation of carbohydrates. Accord- 
 ing to the view based upon Pasteur's investigations it has been gen- 
 erally considered that these processes are phases of the life of these 
 organisms and the name organized ferments or ferments have been given 
 to such organisms, especially to the ordinary yeast fungus. 
 
 A ferment, according to this view, is a living organism. By the name 
 enzyme, as introduced by Kuhne, we mean a product of the chemical 
 processes in the cells, which is active without the life of the cell and which 
 can be separated from the cell. The decomposition of invert sugar into 
 carbon dioxide and alcohol in fermentation is considered as a fermentative 
 process closely connected with the life of the yeast fungus. The inver- 
 sion of cane-sugar previous to fermentation is on the contrary, an enzymotic 
 process which is brought about by a body or mixture of bodies which 
 are formed in the fungus and which can be removed from the fungus 
 and are still active after the death of the fungus. Consequently fer- 
 ments and enzymes are capable of manifesting a different behavior toward 
 certain chemical reagents. Thus there exist a number of substances, 
 among which we may mention arsenious acid, phenol, toluene, salicylic 
 acid, boracic acid, sodium fluoride, chloroform, ether, and protoplasmic 
 poisons, which in certain concentration kill ferments, or at least retard 
 their action, but which do not noticeably impair the action of the enzymes. 
 
 The above view as to the difference between ferments and enzymes 
 has lately been essentially shaken by the researches of E. Buchner : 
 and his pupils. He has been able to obtain from beer-yeast, by grind- 
 ing and strong pressure, a cell-fluid rich in protein, and which when intro- 
 duced into a solution of a fermentable sugar caused a violent fermenta- 
 tion. The objections raised from several sides that the fluid expressed 
 still contained dissolved living cell substance has been so successfully 
 answered by Buchner and his collaborators that there is at present 
 no question that alcoholic fermentation is caused by a special enzyme or 
 mixture of enzymes called zymase, which is formed in the yeast-cell. 
 
 As from the yeast-cells so also from ether lower organisms, indeed 
 
 l E. Buchner, Ber. d. deutsch. chem. Gesellsch., 30 and 31; E. Buchner and Rapp, 
 ibid., 31, 32, 34; H. Buchner, Stizungsber. d. Gesellsch. f. Morphol. u. Physiol, in 
 Munehen, 13 (1897), part 1, which also contains the discussion on this topic. See also 
 E. and H. Buchner and M. Hahn, Die Zymasegarung, Munehen (1903); Stavenhagen, 
 Ber. d. deutsch. chem. Gesellsch., 30; Albert and Buchner, ibid., 33; Buchner. ibid. 
 33; Albert, ibid., 33; Albert, Buchner, and Rapp, ibid., 35; in regard to the opposed 
 views see Macfadyen, Morris, and Rowland, ibid., 33; Wroblewski, Centralbl. f. Physiol., 
 13, and Journ. f. prakt. Chem. (N. F.), 64. 
 
42 GENERAL AND PHYSICO-CHEMICAL. 
 
 from the lactic-acid bacilli and beer vinegar bacteria, it is possible to 
 separate the specific fermentative principle of these organisms from the 
 living organism and to bring about changes with the dead organism 
 (E. Buchner, and Meisenheimer and Gaunt, Herzog x ) . The ques- 
 tion whether there exist ferment processes which, in Pasteur's sense, 
 are the result of the biological phenomena connected with the metab- 
 olism of the micro-organism and which we can directly identify with the 
 life processes, is very difficult to answer; hence for the present we have 
 no foundation for a sharp differentiation between the organized ferments 
 and enzymes. The metabolic processes of the living organisms which 
 we recognize as fermentation phenomena must as a rule be ascribed to 
 enzymes acting within the cell. If such processes are closely connected 
 with the life of the cell, then this is explained in part by the fact that 
 this special enzyme is produced only by living cells and in part by the 
 fact that it cannot be separated from the living cells or that it is readily 
 destroyed on the death of the cell. The names enzyme and ferment are 
 now generally used in the same sense. 
 
 Formerly the view was generally accepted that animal oxidation 
 takes place in the fluids, while to-day we are of the opinion, derived 
 from the investigations of Pfluger and his pupils, 2 that it is connected 
 with the form-elements and the tissues. The question as to how this 
 oxidation in the form-elements is induced and how it proceeds cannot 
 be answered with certainty. On the other hand, it is accepted that the 
 living protoplasm in some manner or other takes part, in which case the ' 
 oxidation processes must cease with the life of the cells while ion the 
 other hand it has been found that certain oxidative processes can be 
 brought about by means of catalyts in the ordinary sense as well as by 
 enzymotic substances. If in the latter case the oxygen of the air is 
 directly transported to the oxidizable substance then we call this a direct 
 oxidation. Ordinarily the oxidative processes take place in the follow- 
 ing way. First a peroxide is formed by taking up oxygen, like in 
 the formation of hydrogen peroxide, H-O-O-H, which then transfers 
 oxygen to the oxidizable substance by aid of the mentioned substances. 
 In these cases the oxidation is indirect. All these oxidative processes 
 will be treated in detail in Chapter XVI. At the same time also other 
 enzymes will be discussed which decompose hydrogen peroxide, with 
 the setting free of oxygen, without oxidizing at the same time. These 
 
 1 E. Buchner and J. Meisenheimer, Ber. d. d. chem. Gesellsch., 36; 634 (1903); 
 and Annal. d. Chem. u. Pharm., 349; with Gaunt, ibid., 349; Herzog, Zeitschr. f. 
 plivMol. f.'hern., 37. 
 
 2 Pfliiger, Pfiuger's 'Archiv, 6 and 10; Finkler, ibid., 10 and 14; Oertmann, ibid., 
 14 and 15; Hoppe-Seyler, ibid., 7. 
 
ENZYMES. 43 
 
 have been called catalases. Also reduction processes will be mentioned 
 which seem to be brought about by enzymes. 
 
 Besides these processes just mentioned the following processes, 
 namely, autolysis and putrefaction, are to be considered as due to enzyme 
 action entirely or in part. 
 
 If an animal organ is kept in water at 37° C. under conditions so 
 that no micro-organisms are active then the organ gradually dissolves 
 in great part under the influence of the contained enzymes. This 
 process is called autodigestion or aidohjsis. The action of micro- 
 organisms can be prevented either by removing the organ under strictly 
 aseptic conditions or by allowing the digestion to take place in the 
 presence of antiseptic substances (toluene, chloroform, etc.). As the 
 animal organs consist chiefly of protein substances the autolysis con- 
 sists chiefly in the action of enzymes which dissolve proteins. Autol- 
 ysis was first observed and studied by Salkowski and his pupils 
 with liver, muscle and supra-renal capsule. 1 Jacoby then showed 
 that the enzymes active in autolysis do not orginate in the digest- 
 ive tract and are not identical with trypsin or pepsin. 2 Biondi found 
 that hydrochloric acid had a favorable influence upon the autolysis 
 of the liver while Hedin and Rowland 3 observed that the organic 
 acids accelerate the autolysis of nearly all organs. This has been sub- 
 stantiated by several authors (Wiener, Arinkin 4 ). The findings of 
 Lane-Claypton and Schryver 5 that the autolysis of the liver and 
 kidneys begins only after a latent period of from two to four hours 
 when the post mortem formation of acid is at its height, substantiates 
 the influence of the acid reaction. 
 
 The autolysis is retarded to a great extent by an alkaline reaction. 
 This is shown by the experiments of Schwienig with the liver as well 
 as those of Hedin and Rowland with several other organs. Hedin 
 has also shown by experiments with various organs that a preliminary 
 treatment with acetic acid markedly helps the autolysis in alkaline reac- 
 tion, which for the spleen at least is explained by a destruction through 
 the treatment of acetic acid of a substance which has an inhibiting action 
 in. alkaline solution. Such an inhibiting substance destroved by acetic 
 
 ^eitschr. f. klin. Med., 1890, Suppl., Schwiening, Yirchow's Arch., 136, 444 (1894), 
 Biondi, ibid, 144, 373 (1896). 
 
 2 A complete summary of the literature of intracellular enzymes and autolysis may 
 be found in Jacoby, Ueber die Bedeutung der intrazellularen Fermente, etc., Ergeb- 
 nisse der Physiologie, Jahrg. 1, Abt. 1, 1902. 
 
 3 Zeitschr. f. physiol. chem., 32, 341, 531 (1901). 
 
 4 Wiener, Centralbl. f. Physiol., 19, 349 (1905); Arinkin, Zeitschr. f. physiol. Chem., 
 53, 192 (1907). 
 
 6 Journ. of Physiol., 31, 169 (1904). 
 
44 GENERAL AND PHYSICO-CHEMICAL. 
 
 acid is also found in serum. 1 The serum also inhibits the autolysis of 
 the liver (Baer, Longcope and others) 2 and also the thymus under 
 certain circumstances (Rhodin 3 ). 
 
 Experience has shown that the post-mortem autolytic process may 
 also be influenced by many other bodies and indeed in various ways. 
 For example, according to Hess and Saxl, arsenious acid exerts a 
 retarding action on the first stages of autolysis, while phosphorus accel- 
 erates it. Izar as well as Laqueur and Ettinger 4 obtained with small 
 quantities of different arsenic preparations an acceleration of the autol- 
 ysis and with larger amounts a retardation. Laqueur 5 obtained a 
 retardation with oxygen and an acceleration with carbon dioxide. 
 Ascoli and Izar 6 have thoroughly investigated the action of inorganic 
 colloids upon autolysis. Radium rays as well as radium emanations 
 accelerate post-mortem autolysis of normal as well as carcinoma tissues 
 (Wohlgemuth, Neuberg, Lowenthal and Edelstein 7 ). 
 
 The products of the activity of the different enzymes dissolving pro- 
 teins in autolysis have been studied by Hedin and his collaborators, by 
 studying the action of organ press juices upon protein added, or upon 
 the protein contained in the juice. The same cleavage products were 
 found as in the deep-seated cleavage of proteins in the digestive canal. 8 
 Similar investigations have also been carried on by Levene and Jones 9 
 who chiefly considered the decomposition of the nuclein substances. 
 The combined action of various enzymes in autolysis also explains to us 
 why, as especially shown by Levene and by Jones, the products obtained 
 by the hydrolytic cleavage of an organ by means of an acid are some- 
 what different from those products produced on autolysis. In autol- 
 ysis we are not only dealing with the cleavage of the proteins, but other 
 enzymotic processes also occur such as the splitting of fats and carbo- 
 hydrates, the splitting off of NH2 groups from amino-acids, oxidations, 
 reductions and perhaps also syntheses. 
 
 1 Hammarsten's Festschr., 1906. 
 
 2 Baer, Arch. f. expt. Path. u. Pharm., 56, 68 (1906); Longcope, Journ. nied. 
 Research, 13, 45 (1908). 
 
 3 Zeitschr. f. physiol. Chem., 75, 197 (1911). 
 
 4 Hess and .Saxl, Zeitschr. f. expt. Path. u. Therapie, 5 (1908); Izar, Bioch. Zeitschr., 
 21, 46 (1909); Laqueur and Ettinger, Zeitschr. f. physiol. Chem., 79, 1 (1912). 
 
 5 Zeitschr. f. physiol. Chem., 79, 82 (1912). 
 8 Bioch. Zeitschr., 17, 361 (1909). 
 
 7 Wohlgemuth, Berl. klin. Wochenschr., 26, 704; Neuberg, Zeitschr. f. Krebsfor- 
 schung, 2, 171 (1904); Lowenthal and Edelstein, Bioch. Zeitschr., 14, 484 (1908). 
 
 8 Leathes, Journ. of Physiol., 28, 360 (1902); Dakin, ibid., 30, 84 (1904); Hedin,. 
 ibid., 30, 155 (1904); Cathcart, ibid., 32, 299 (1905). 
 
 8 Levene, Amer. Jour, of Physiol., 10, 11, 12 (1904); Jones, Zeitschr. f. physiol.. 
 Chem., 42, 35 (1904). 
 
ENZYMES. 45 
 
 It is at present impossible to state what part autolytic processes 
 take in life under physiological conditions, and we can have only con- 
 jectures on this subject. In the autolysis of a removed organ or of one 
 through which the blood is not flowing, the conditions in many ways are 
 quite different from the conditions in life. The products which appear 
 after weeks or months of autolysis, sometimes in very small quantities, 
 do not give any clue to the nature of the vital processes, and conclusions 
 must be drawn very carefully from these results. 
 
 For the present it is impossible to judge of the importance of the 
 enzymes active in autolysis for physiological conditions, but this does 
 not exclude the possibility that in normal cell life the enzymes play a 
 very important role. Numerous observations show this to be true, and 
 we tend more and more toward the view that the chemical transforma- 
 tions in the living cells are brought about by enzymes, and that these 
 latter are to be considered as the chemical tools of the cells (Hofmeister 
 and others. 1 ). 
 
 From this standpoint the enzymes are of especial interest because 
 to-day it is the general belief that nearly all chemical processes of great 
 importance do not occur in the animal fluids, but on the contrary in the 
 cells, which are the real chemical workshops of the organism. It is also 
 chiefly the cells, which by their more or less active efficiency regulate 
 the extent of the chemical processes and thereby also the intensity of 
 the general metabolism. The folloAving will be given as special examples 
 of the action of such enzymes under pathological conditions. The 
 changes of the liver and blood in acute phosphorus intoxication and 
 in acute yellow atrophy of the liver, where we find, in the urine, the 
 enzymotic decomposition products of the proteins. 2 Another example 
 is the solution of pneumonic infiltrations by the enzymes of the migrated 
 and inclosed leucocytes as studied by Fr. Muller, 3 and this is at the same 
 time an example of heterolysis, i.e., of a solution or a destruction in an 
 organ by enzymes not belonging therein but introduced from without. 
 An autolysis, although not very marked, occurs in those organs or parts 
 of organs which have not been normally nourished because of a dis- 
 turbance in the circulation, and they are gradually consumed by this 
 action. The part injured undergoes solution, while the healthy part 
 remains unattacked. By this solvent action as well as by the forma- 
 tion of bactericidal bodies, as observed by Conradi, 4 and of antitoxins 
 
 1 F. Hofmeister, Die chemische Organisation der Zelle, Braunschweig, 1901. 
 
 2 Jacoby, Zeitschr. f. physiol. Chera., 30, 174 (1900). 
 
 3 Verhandl. d. naturforsch. Gesellsch. zu Basel, 1901. See also O. Simon, Deutsch 
 Arch. f. klin. Med., 1901. 
 
 4 Hofmeister's Beitrage, 1. 
 
46 GENERAL AND PHYSICO-CHEMICAL. 
 
 (Blum ! ) by means of autolysis, we can consider this autolysis as a 
 remedy and perhaps also as a protective agent for the animal body. 
 In this connection the investigations of Billard 2 must be mentioned 
 where the autolytic fluid from the pig liver was strongly antitoxic 
 toward viper poison, cobra poison, tetanus toxin and also toward 
 cocaine, curare and strychnin. 
 
 As above stated, the chemical processes in animals and plants do 
 not stand in opposition to each other; they offer differences indeed, 
 but still they are of the same kind from a qualitative standpoint. Pflu- 
 ger believes that there exists a blood-relation between all living cells 
 of the animal and vegetable kingdoms, and that they originate from the 
 same root. The animal body is a complexity of cells, hence study of the 
 chemical processes must not only be made upon higher plants, but also 
 upon unicellular organisms in order that we get a proper explanation 
 of the chemical processes in the animal organism. Although a bio- 
 chemical study of the micro-organisms is very important, we must bear 
 in mind also the important role played by such organisms in animal 
 life, chiefly as exciters of disease; hence the study of the conditions of 
 life of these micro-organisms and the chemical investigation of the prod- 
 ucts produced by them must be of infinite importance. 
 
 If in the autolysis of animal tissues micro-organisms are added and 
 if no antiseptic is present which prevents their development, then they 
 increase abundantly because of the favorable conditions for development. 
 At the same time the enzymes are also formed to a great extent and by 
 whose aid the exchange of matter takes place in the bacteria. It follows 
 that many chemical processes occur depending upon the kind of bacteria 
 present and which are foreign to bacteria-free autolysis. The entire 
 process has been called putrefaction. Among the products formed we 
 will mention the sulphureted hydrogen, indol and skatol which chiefly 
 give the odor to putrefying proteins. In regard to other putrefactive prod- 
 ucts we refer to Chapter VIII. Under ordinary circumstances compounds 
 of a basic nature may also be produced by putrefaction. To this class 
 belong the cadaver alkaloids called ptomaines, first found by Selmi in 
 human cadavers and then specially studied by Brieger and Gautier. 3 
 Certain of these are poisonous, designated as toxines, while others are 
 non-poisonous. They all belong to the aliphatic compounds and gen- 
 erally do not contain oxygen. As an example of these basic substances 
 
 ■Hofmeister's Beitrage, 5, p. 142. 
 
 2 Cornpt. rend. 8oc. Biol., 70, 623 (1911). 
 
 : ' Selmi, Sulle ptomaine od alcaloidi cadaverici e loro importanza in tossicologia, 
 Bologna, 1878; Ber. d. d. chem. Gesellsch., 11, Correspond, by H. Schiff; Brieger, 
 Ueber Ptomaine, Parte 1, 2, and 3, Berlin, 1885-1886; A. Gautier, Traits de chimie 
 ppliquee a la physiologic, 2, 1873, and Compt. rend., 94. 
 
ENZYMES. 47 
 
 Ave must mention the two diamines, cadaverine or pentametbylenediamine, 
 C5H14X2, and putrescine or tetramethylenediamine, C4H12N2, which 
 have awakened speeial interest because they occur in the contents of the 
 intestine and in the urine in certain pathological conditions, especially 
 in cholera and cystinuria. 1 The putrefaction bases marcitine, CsHigNa, 
 putrine, C11H26X2O3, and riridinine, C8H12N2O3, isolated by AcKER- 
 mann, also belong to this group. Of special interest is the bacterial 
 poison isolated by Faust, 2 called sepsine, C8H14N2O2, which is the sub- 
 stance producing the characteristic toxic action of putrefactive masses. 
 Sepsine was prepared by Faust as a crystalline sulphate which, on repeated 
 evaporation of its solution, was readily converted into cadaverine sulphate. 
 
 Of especially great interest are the toxines which are found in the 
 higher plants and animals, like the jequirity-bean and castor-seed, in 
 the poison of snakes and spiders, in blood-serum, etc., and particularly 
 those produced by pathogenic micro-organisms have an unmistakable 
 relation to the enzymes. A closer study of these various bodies, lysines, 
 agglutinines, toxines, etc., as well as of the antitoxines and the theory 
 of immunity, does not lie within the scope of this work, but on account 
 of the great importance of the subject it will be briefly discussed on 
 page 66. 
 
 Classification of the Enzymes. If we exclude those processes which 
 are the result of several enzymotic reactions (i.e. autolysis, putrefaction) 
 then the most important enzymotic processes studied so far are the fol- 
 lowing : 
 
 1. Hydrolytic cleavage processes. 
 
 2. Cleavages of another variety (fermentation). 
 
 3. Oxidations. 
 
 We have no general chemical reaction in the ordinary sense which 
 is common to all enzymes or ferments and each enzyme is characterized 
 by its action and by the conditions under which this action is developed. 
 As the action of an enzyme upon a substance, or related substances, or 
 groups is limited therefore these substances or groups are called the 
 substrate of the enzyme. 
 
 In regard to the terminology it must be remarked that an enzyme 
 is often named after the substrate (amylase, protease, lipase); in other 
 cases the kind of action determines the name (oxidase, reductase) and 
 finally one of the products produced by its action forms the basis for 
 the name (alcoholase). 
 
 ^ee Brieger, Berlin, klin. Wochenschr., 1887; Baumann and Udransky, Zeitschr. 
 f. physiol. Chem., 13 and 15; Brieger and Stadthagen, Berlin, klin. Wochenschr.. 1889. 
 
 2 Faust, Arch. f. exp. Path. u. Pharm., 51; Ackermann, Zeitschr. f. phvsiol. Chem., 
 54 and 57. 
 
48 GENERAL AND PHYSICO-CHEMICAL. 
 
 Of the above mentioned enzymotic reactions the hydrolytic cleavage 
 processes have been best studied, and the general properties of the 
 enzymes which will be given apply chiefly to the hydrolytically splitting 
 enzymes. Among these the following are to be mentioned especially: 
 
 1. Enzymes which split fats and other esters with the formation of 
 the corresponding alcohol and acid. They are called lipases or esterases. 
 
 2. Enzymes which split complex carbohydrates with the formation 
 of simpler ones. To these belong: 
 
 a. Disaccharide splitting enzymes for instance saccharase (invertase, 
 invertin), maltase, lactase which act upon the corresponding disaccharide 
 saccharose (cane-sugar) maltose and lactose (milk sugar); 
 
 b. Polysaccharide splitting enzymes such as amylase, ptyalin. The 
 name diastase is often used to designate all the enzymes of this group. 
 In close relation to these enzymes stand the glucoside splitting enzymes 
 which occur especially in higher plants and the best known of which is 
 amygdalase (emulsin) occurring in the almond. 
 
 3. Enzymes which act upon the proteins or their related cleavage 
 products. Of these we have : 
 
 a. Peptidases and erepsin which split polypeptides or peptones; 
 
 b. Proteases which act upon proteins as substrate (pepsin, trypsin, 
 autolytic enzymes). 
 
 Among the hydrolytic enzymes of the animal kingdom we also 
 include the arginase, which splits arginine into urea and ornithin and the 
 histozym, which splits hippuric acid. The two following groups also 
 belong here, namely, the nucleases which split nucleic acids and which 
 will be discussed in Chapter II, and the coagulating enzymes, rennin 
 and thrombin, which are probably active as proteases. The deamidizing 
 enzymes which split off the NH2 group from amino combinations are, 
 at least in certain cases, to be classed as hydrolytic enzymes. This is 
 for example the case with the adenase and guanase which splits off ammonia 
 from the two bodies adenine and guanine converting them into hypoxan- 
 thine and xanthine respectively. The urease which splits urea also belongs 
 to this group. 
 
 General Properties of the Enzymes. When possible we make use 
 of watery solutions of enzymes in experimentation. In case they are 
 insoluble in water (certain lipases) we use them in the form of more or 
 less purified powders or together with the tissue where they are formed. 
 We have no general method for preparing enzyme solutions. In certain 
 cases they are contained in secretions (gastric and pancreatic enzymes); 
 in others they are prepared from the cells by crushing and pressing out 
 the cell juice (zymase, organ enzymes), and finally, most enzymes can 
 be extracted from the cells with water or glycerin, and as this last gives 
 permanent solutions it has found great use as an extraction medium. 
 
ENZYMES. 49 
 
 The aqueous solutions can be kept at low temperatures for a long time 
 after the addition of toluene or chloroform. 
 
 In all these cases the enzymes are obtained strongly contaminated 
 with other bodies, especially by proteins. Only in exceptional cases is 
 it possible to free the enzyme solution from protein so that the solution 
 does not give the ordinary protein reactions. This is true for the solu- 
 tion of saccharase obtained from yeast by treatment with water; if 
 this is shaken with kaolin the protein is adsorbed by the kaolin while the 
 solution contains the enzymes. 1 
 
 No enzyme has thus far been obtained in a perfectly pure form, 
 and the chemical constitution as well as structure is therefore unknown. 
 The enzymes probably belong to the colloids; if they themselves are not 
 colloids, they occur at least with colloids, from which they may be sepa- 
 rated only with difficulty, if at all. The enzymes are characterized by 
 the fact that they are readily taken up by finely divided substances 
 (inorganic precipitates, carbon, kaolin, infusorial earth and other col- 
 loids such as alumina, iron hydroxide, proteins 2 ) . This process may 
 act selectively, as from a solution certain enzymes can be taken up and 
 others not at all, or only to a slight extent (Hedin, 3 Michaelis and 
 Ehrenreich 4 ). The adsorption process is more or less irreversible 
 and differs in this from the adsorption of crystalloid substances. Still 
 the trypsin and rennin adsorbed by charcoal can be to a slight extent 
 expelled from the charcoal by means of other adsorbable substances such 
 as casein and albumin (Hedin). 5 Rennin taken up by charcoal can to 
 a very slight extent be set free by the addition of glucose (Hedin) and 
 saccharase adsorbed by charcoal can be set free by cane-sugar (Eriks- 
 son). 6 As we will learn below, the adsorbed enzyme is inactive. The 
 so-called shaking inactivation of enzymes or the loss in activity of enzymes, 
 which occurs on shaking their solution seems to be due to an adsorp- 
 tion of the enzyme when it is either taken up by the precipitate 
 formed on shaking (Abderhalden and Guggenheim) or is concentrated 
 at the surface between the solution and the froth (S. and S. Schmidt- 
 Nielsen). 7 These latter found the inactivation of rennin by shaking 
 was regained if the froth was allowed to subside. 
 
 All enzymes lose their specific action on sufficiently heating their 
 
 1 Michaelis, Bioch. Zeitschr., 7, 488 (1907). 
 
 2 Dauwe, Hofmeister's Beitrage, 6, 426 (1905). 
 
 3 Bioch. Journ., 2, 112 (1907). 
 
 « Bioch. Zeitschr., 10, 283 (1908). 
 
 6 Bioch. Journ., 2, 81 (1906); Zeitschr. f. physiol. Chem., 63, 143 (1909). 
 
 6 Hedin, ibid., 63, 143 (1909); Ericksson, ibid., 72, 313 (1911). 
 
 7 Abderhalden and Guggenheim, Zeitschr. f. physiol. Chem., 54, 352 (1907); S. and 
 S. Schmidt-Nielsen, ibid., 68, 317 (1910) which also contains the literature. 
 
50 GENERAL AND PHYSICO-CHEMICAL. 
 
 aqueous solutions, and even at ordinary temperature the enzymes are 
 gradually decomposed. In general the enzymes lose their activity by 
 heating for a short time to 70° C. Madsen and Walbum have followed 
 this process at different temperatures and found that the decomposition 
 of trypsin, pepsin and rennin at given temperatures proceeds mono- 
 molecularly, i.e., that the velocity of reaction at every moment is pro- 
 portional to the concentration of the enzyme (page 34). l The readiness 
 with which an enzyme is decomposed is nevertheless to a great extent 
 dependent upon the presence of other bodies (page 54). 
 
 Certain enzymes are also sensitive to light. According to Schmidt- 
 Nielsen 2 rennin is injured by light and in particular, by the ultra-violet 
 rays. The experiments of Jodlbauer and Tappeiner 3 with invertin 
 have led to the same results; the visible rays can also in some cases 
 (peroxidase, haemase) in the presence of oxygen or certain fluorescent 
 substances exert an injurious action. 4 
 
 According to Schmidt-Nielsen 5 the weakening in the rennin under 
 the influence of light proceeds like a monomolecular reaction. 
 
 Experiments on the cataphoresis of enzymes have been made by 
 Bierry, Henry and Schaeffer 6 as well as by Michaelis. . These 
 investigators found that saccharase migrates to the anode. Michaelis 7 
 found that the migration direction of other enzymes was dependent upon 
 the reaction, namely in faintly acid reaction they moved to the cathode 
 and in faintly alkaline reaction to the anode. Recently Pekelharing 
 and \Y. E. Ringer 8 have observed that the migration direction of pig 
 pepsin was very materially influenced by the addition of small amounts 
 of proteoses. From what was previously stated (page 20) the saccharase 
 must therefore have a negative charge. As Michaelis, 9 has on the other 
 hand, found that the saccharase can be adsorbed by the positively 
 charged aluminium hydrate and not by the negatively charged kaolin, 
 he concludes that the formation of adsorption compounds, at least in 
 certain cases, depends upon an opposed electric charge of the two com- 
 ponents. 
 
 1 Arrhenius, Immunochemie, Leipzig, 1907, 58. 
 
 2 Hofmeister's Beitrage, 5, 355(1904); 8,481(1906); Zeitschr. f. physiol. Chem., 58, 
 233 (1908). 
 
 Arch. f. klin. Med., 87, 373 (1906). 
 
 * Bioch. Zeitschr., 8, 61 and 84 (1908). See also Agulhon, Compt. rend., 153, 979 
 (1911;. 
 
 5 Zeitschr. f. physiol. Chem., 58, 232 (1908). 
 
 • Compt. rend. boo. biol., 68, 226 (1907). 
 
 7 Bioch. Zeitschr., 16, SI, 486; 17, 231 (1909). 
 8Zeitsfhr. f. physiol. Chem., 75, 282 (1911). . 
 9 Bioch. Zeitchr., 10, 299 (1908). 
 
ENZYMES. 51 
 
 Like the colloids the enzymes only diffuse very slowly and the dif- 
 fusion through membranes does not occur in most cases; only certain 
 membranes such as collodion tubes allow certain enzymes to pass 
 through. The collodion tubes can be impregnated in such a way with 
 lecithin or cholesterin that the diffusion is very slight. The same 
 applies to the filtration through collodion membranes (Bierry and 
 S( iiaeffer). 1 It must not be forgotten in such experiments that the 
 membrane can adsorb a considerable part of the enzyme (Bechhold). 2 
 
 Just as it is difficult to prepare an enzyme free from non-enzymotic 
 contaminations, so also is it difficult to exclude the possibility that a 
 so-called enzyme is not a mixture of several related enzymes. In fact 
 the several enzymotic processes proceed step by step, and it is possible 
 that the various steps are caused by different enzymes. Thus the 
 decomposition of protein into amino-acids, with proteoses, peptones, 
 and polypeptides as intermediary products, may be the result of the 
 activity of several enzymes which are active one after another or are 
 parallel with one another in activity. Erepsin does not attack genuine 
 proteins, but completes the decomposition which has been begun by 
 other enzymes (pepsin, trypsin). 
 
 The enzymes are formed within the living cells. In certain cases 
 the cells do not secrete the complete enzyme, but substances which are 
 transformed first outside of the cells into active enzymes. These pre- 
 liminary steps or mother substances of the enzymes have been called 
 proenzymes or zymogens. These under certain conditions are changed 
 into enzymes and in certain cases this is brought about by the inter- 
 action of special but not well known substances which have been called 
 kinases (see Chapters V and VIII). In other cases the transformation 
 of the zymogen into the active enzyme is brought about by well defined 
 chemical substances. Thus the proenzymes of pepsin and of rennin are 
 activated by acids (see below on the retardation of enzyme action and 
 also Chapter VIII). 
 
 In certain other cases the presence of bodies which resist temperature 
 and are dialyzable and therefore not enzymes, are necessary or helpful 
 besides the real organic enzyme. Thus the presence of an acid is neces- 
 sary for the action of pepsin and hydrocyanic acid, according to Mendel 
 and Blood, 3 favors to a high degree the action of papain (a plant pro- 
 tease). R. Magnus 4 has been able to separate by dialysis, from a solu- 
 tion of liver-lipase, a body which is necessary for the action upon amyl 
 
 ^ompt. rend. soc. biol., 62, 723 (1907). 
 
 2 Zeitschr. f. physik. Chem., 60, 257 (1907). 
 
 3 Journ. of biol. Chem., 8, 177 (1910). 
 
 4 Zeitschr. f. physiol. Chem., 42, 149 (1904). 
 
52 GENERAL AND PHYSICO-CHEMICAL. 
 
 salicylate. Enzymes made inactive by dialysis can be activated again 
 by the addition of boiled enzyme or the concentrated dialysate. Harden 
 and Young l on filtering yeast-press juice through earthenware filters 
 impregnated with gelatin, have found different constituents of the zymase 
 on the filter and in the filtrate. The true enzyme remains on the filter. 
 This alone is inactive, but becomes active when the other part which 
 has passed through the filter, and which is dialyzable and resistant to 
 temperature, is added. This part is consumed during fermentation 
 and therefore the enzyme becomes inactive. After the addition of, best, 
 boiled press-juice to this the fermentation begins again (see also Chapter 
 III). Certain of the just-mentioned substances which are resistant 
 to heat, whose presence are necessary for the action of certain enzymes, 
 are ordinarily called co-enzymes. As they are not to be classified with 
 the enzymes, they are more correctly called activators, as suggested by 
 Etjler. 2 Their action is probably different in different cases, and 
 differs also from the activating action of the kinases. 
 
 Many enzymes are secreted by the cells as such or as proenzymes. 
 They act outside of the cells in which they were formed, or they act after 
 having been transformed into the enzyme, and hence are called secre- 
 tion enzymes or extracellular enzymes. Besides these extracellular 
 enzymes we also have another group which acts within the cells, hence 
 are intracellular and therefore are called intracellular enzymes or endo- 
 enzymes. To this group belongs, beside the yeast zymase, numerous 
 enzymes such as oxidases and hydrolytic enzymes. 
 
 Formation and Secretion of Enzymes. The investigations of Pawlow 3 
 and his pupils upon the formation and secretion of the enzymes active 
 in the alimentary tract are very important. According to these investiga- 
 tions the amount of secretion of the glands and the behavior of the enzymes 
 contained in the secretion are dependent upon the amount and com- 
 position of the food taken and in such a manner that the kinds and 
 amounts of enzymes are appropriate for the digestion of the food- 
 stuffs (see Chapter VIII). Similar results were also obtained by Wein- 
 land 4 who found that the pancreas does not normally contain any 
 lactase but did contain this enzyme after feeding the animal with 
 milk or milk sugar. This has been substantiated by Bainbridge. 5 
 Analogous experiments have been made with salivary ptyalin by Neil- 
 
 ^roc. Physiol. Soc, 32 (1904); Proc. Chem. Soc, 21, 189 (1905); Proc. Roy. 
 Soc, 77 (ser. B), 405 (1906); ibid., 78, 369 (1906). . 
 
 2 Zeitschr. f. physiol. Chem., 57, 92 (1908). 
 
 3 Arbeit der Verdauungsdriisen, Wiesbaden, 1898, s. 51. 
 ♦Zeitschr. f. Biol., 38, 607 (1899); 40, 386 (1900). 
 
 6 Journ. of Physiol., 31, 98 (1904). 
 
ENZYMES. 53 
 
 son and Lewis 1 with the same results. On the other hand the cor- 
 rectness of these observations is disputed by Bierry, 2 Plim.mer, 3 Wohl- 
 gemuth 4 and Popielski 5 as they could not find any accommodation. 
 Mendel 6 and his co-workers by careful investigations on certain enzymes 
 obtained from embryonal intestine and other embryonal tissues could 
 not find any marked difference between these enzymes and the enzymes 
 of the full grown animal. These results speak against the accepted influence 
 of the food and of the processes depending upon the taking up of food, 
 upon the formation of enzymes. Recently the investigations of Lon- 
 don 7 and his collaborators upon the influence of the food upon the 
 digestion juices have shown that the amount of juice secreted is dependent 
 upon the constitution of the food but not the ferment content of the 
 same. The observations of Cohnheim 8 also speak against the view 
 that the kind and quantity of enzymes secreted in the intestinal tract 
 accommodate themselves to the digestion, as he found that the organism 
 secretes as much fluid (gastric juice, pancreatic juice and bile) when 
 already digested food is introduced into the stomach as when undigested 
 food is introduced. Arrhenius 9 has calculated from London's figures, 
 that the total amount of digestive juice secreted was proportional to the 
 quantity of food-stuffs. From experiments which Euler and his 
 collaborators have made upon the formation of inverting enzymes he 
 concludes that we have inverting enzymes whose formation is specific 
 by getting accustomed to the substrate, while the formation of others 
 is in no wise thus influenced. 10 
 
 In this connection we will call attention to the appearance of enzy- 
 motic substances in the blood after the subcutaneous or intravenous 
 (parenteral) introduction of certain food-stuffs. Weinland first showed 
 that the parenteral introduction of cane-sugar caused the appearance 
 in the serum of a cane-sugar splitting enzyme. 11 Abderhalden and 
 Kapfberger 12 have substantiated and developed these observations. 
 Bodies having a similar action also appear after the injection of milk 
 
 I Journ. of Biol. Chem., 4, 501 (1908). 
 2 Compt. rend. soc. biol., 58, 701 (1905). 
 
 3 Journ. of Physiol., 34, 93 (1906). 
 
 4 Bioch. Zeitschr., 9, 1 (1908). 
 
 5 Pfliiger's Arch., 127, 443 (1909). 
 
 6 Amer. Journ. of Physiol., 20, 81, 97 (1907); 21, 64, 69, 85, 95 (1908). 
 
 7 Zeitschr. f. physiol. Chem., 68, 366 (1910). 
 8 Ibid., 84, 419 (1913). 
 
 9 tbid., 63, 323 (1909), see also London, ibid., 65, 189 (1910). 
 
 10 Ibid., 70, 279; 76, 388; 78, 246; 79, 274; 80, 241 (1912). 
 
 II Zeitschr. f. Biol., 47, 279 (1905). 
 
 12 Zeitschr. f. physiol. Chem., 69, 23 (1910). 
 
54 GENERAL AND PHYSICO-CHEMICAL. 
 
 sugar and of starch. Abderhalden and his co-workers have shown 
 that the parenteral introduction of protein or peptone gives the blood 
 serum of the animal the power of splitting proteins, which power is 
 destroyed on heating to 60-65° C. 1 The introduction of very large quan- 
 tities of sugar or proteins per os (over feeding) has the same effect as 
 the parenteral introduction. Abderhalden considers the active sub- 
 stances thus obtained as enzymes. The question is still undecided whether 
 the substances introduced bring about a formation of the enzymes or 
 whether they only transport the already formed enzymes to the blood. 
 
 Heat Production. The question whether in the hydrolytic processes 
 with the aid of enzymes heat is given off or taken up has been attacked 
 in two different ways. Grafe 2 could not find either any setting free or 
 taking up of heat in the digestion of protein in a Rubner calorimeter. 
 On the other hand Hari 3 by determining the calorific values of albumin 
 before and after digestion came to about the same results. If we exclude 
 the work developed in the process then it follows that the energy supply 
 of the organism is not perceptibly changed by the hydrolytic cleavages 
 of the protein. The chief source of energy is to be sought in the oxida- 
 tion processes that follow the cleavages. 
 
 Modes of Action of the Enzymes. The enzj r mes do not suffer any 
 appreciable change during the reaction they perform, and insignificant 
 amounts of the enzyme are able to decompose relatively enormous amounts 
 of the substrate. For example, 1 part of saccharase can invert 100,000 
 parts of cane-sugar (O'Sullivan and Thompson) 4 and 1 part of rennin 
 can decompose more than 400,000 parts of casein (Hammarsten) 5 . 
 For these reasons the enzymes have for a long time been considered as 
 catalytic substances. Nevertheless the enzyme reactions always take 
 place in heterogeneous media where on one hand the enzyme exists as 
 colloid and on the other the substrate in many cases is a colloid (starch, 
 proteins). As above mentioned, the enzymotic decompositions are 
 often complicated by their taking place over several intermediary steps 
 to the final product. As indicated by several conditions, the enzymes 
 also, before they act upon the substrate, combine therewith in some way 
 or another. The fact that the action of an enzyme is dependent upon the 
 stereometric construction (page 61) of the substrate speaks essentially 
 for this view. The substrate also protects certain enzymes against destruc- 
 
 » Ibid., 61, 200; 62, 120, 243 (1909); 64, 100, 423, 426, 427; 66, 88; 69, 23 (1910); 
 71, 110, 307, 385 (1911). See also 77, 250 (1912). 
 2 Arch. f. Hygiene, 62, 216 (1907). 
 •ranger's Arch, 115, 11 (1906); 121, 459 (1908). 
 * Journ. ohem. Soc, 57, 926 (1890). 
 6 See Maly's Jahresber, 7. 
 
ENZYMES. 55 
 
 tive influences (heat, alkalies) 1 ; According to this only that part of the 
 added enzyme which is combined with the substrate is active. In 
 judging of the rapidity of enzyme reactions the following must he con- 
 sidered: 
 
 1. The velocity with which the enzyme combines with the substrate. 
 
 2. The result of the division, i.e., how much of the added enzyme 
 is combined with the substrate. 
 
 3. The velocity of the chemical processes produced by the enzyme. 
 
 The velocity of the combination of the enzyme with the substrate (1) can at 
 least in many cases be ignored in consideration of the time necessary for the 
 chemical reaction (see page 37). This applies to those cases where the chemical 
 transformation in the presence of an excess of substrate at the beginning of the 
 processes remains the same in each successive time interval. The quantity of 
 enzyme combined with the substrate, does not, in these cases, increase with the 
 time, which would be the case if the time necessary for the combination is not in 
 comparison with that for the chemical reaction. Equal decomposition for equal 
 time at the beginning of the processes have been found for the following enzymes — 
 invertase, 2 diastase, 3 trypsin with casein, 4 as substrate. 
 
 The second question, as to the division of the enzyme between different phases 
 we will discuss after we have spoken of the velocity of the real chemical reaction 
 (page 58) as well as the retardation of enzyme action (page 62). 
 
 In regard to the chemical reaction they proceed probably in a different 
 manner according to the kind of combination between the substrate and 
 the enzyme. In one case we can consider that the combination of the 
 enzyme with the substrate is of such a kind that both form a homogeneous 
 phase and that one serves as solvent for the other (page 27). In this 
 case the chemical reaction produced by the enzyme takes place in a 
 homogeneous medium. Secondly, we can consider the combination of 
 the substrate and enzyme as an adsorption combination (see page 27) 
 in which case the combination does not form a homogeneous phase and 
 the reaction differs more or less from one taking place in a homogeneous 
 system. Bearing this in mind it would be interesting to investigate 
 whether the facts found for enzymotic reactions correspond with 
 catalytic reactions in homogeneous media. 
 
 For these latter the following laws (see page 33) have been found: 
 
 1. When the quantity of catalyst remains constant, the reaction 
 
 1 O'Sullivan and Thompson, Journ. Chem. Soc, 57, 926 (1890); Bayliss and Starling, 
 Journ. of Physiol., 30, 71 (1903); Hedin, ibid., 30, 173 (1903); 32, 474 (1905); Taylor, 
 Journ. of biol. Chem., 2, 90 (1906). 
 
 2 O'Sullivan and Thompson, Journ. Chem. Soc, 57, 926 (1S90); Ducleau, Traite. 
 de microbiologie II, 137; Brown, Trans. Chem. Soc, 81, 373 (1902); Armstrong, 
 Proc. Roy. Soc, 73, 500 (1904); Hudson, Journ. Amer. Chem. Soc, 30, 1160, 1564 
 (1908). 
 
 3 Brown and Gliddinning, Proc Chem. Soc, 18, 43 (1902). 
 • Hedin. Journ. of Physiol., 32, 471 (1905). 
 
56 GENERAL AND PHYSICO-CHEMICAL. 
 
 velocity for every moment is proportional to the concentration of the 
 body decomposed, which is shown by the velocity coefficient in the same 
 experiment being constant at different times. 
 
 2. The velocity coefficient or the reaction velocity with constant 
 concentration of substrate is proportional to the quantity of catalyst. 
 
 The first law has been shown for certain enzymes in case an excess 
 of enzyme is present, namely for saccharase, 1 lactase 2 and trypsin. 3 It 
 was found that the decomposition in a certain time was proportional to 
 the substrate. In other cases the determination of the correctness of 
 the law was accomplished with difficulty. A part of the enzyme may 
 during an experiment be either destroyed or in other ways (combining 
 with the product) be put out of action; then reverse reactions may take 
 place (page 11) and finally in many cases our analytical methods are 
 incapable of obtaining comparative results for different decompositions, 
 as the reaction in many cases takes place step by step, or several reac- 
 tions occur at the same time. 4 Only in a few cases with especially 
 simple reactions have constant values been found for the velocity 
 coefficient at the beginning, as long as the quantity of reaction product 
 was small and the active quantity of enzyme remained unchanged 
 according to the formula (see page ll). 5 
 
 t X i C 
 k= — log 
 
 t b C-x 
 
 Recently Hudson 6 has found constant values for k for the entire 
 process of inversion of cane-sugar by saccharase in a faintly acid reac- 
 tion. The reason for the different results of earlier investigators 7 is 
 due, in part, according to Hudson, to the fact that the multirotation 
 of the glucose formed was not considered by these experimenters before 
 the extent of inversion was determined polariscopically. In the cleavage 
 of salicin by emulsin Hudson and Paine 8 obtained constant values 
 for k in the entire process. 
 
 1 Brown, Proc. Chem. Soc, 18, 14 (1902). 
 
 2 Armstrong, Proc. Roy. Soc, 73, 500 (1904). 
 
 * Hedin, Journ. of Physiol., 32, 475 (1905). 
 
 * Hedin, Zeitschr. f. physiol. Chem., 57, 468 (1908). 
 
 6 Senter, Zeitschr. f. physik. Chem., 44, 257 (1903); Issajew, ibid., 42, 102; 44, 
 546; Eider, Ilofrneister's Beitrage, 7, 1 (1906); Dietz, Zeitschr. f. physiol. Chem., 
 52, 301 (1907); Taylor, Journ. of biol. Chem., 2, 93 (1906); Nicloux, Compt. rend, 
 soc. biol., 56, 840 (1904); Rona, Bioch. Zeitschr., 33, 413 (1911); 39, 21 (1912); Euler, 
 Zeitschr. f. physiol. Chem., 51, 213 (1907). 
 
 6 Journ. Amer. Chem. Soc, 30, 1160, 1564 (1908). 
 
 7 Bee Henri, Zeitschr. f. physik. Chem., 39, 194 (1901) also A. J. Brown, Trans. 
 Chem. Soc, 81, 373 (1902). 
 
 8 Journ. Amer. Chem. Soc, 31, 1242 (1909). 
 
ENZYMES. 57 
 
 The second law for catalytic reactions which we have formulated, 
 that with constant quantities <>f substrate the reaction velocity is pro- 
 portional to the quantity of enzyme, has been shown in certain cases 
 where the substrate was in excess (practically constant quantity) namely 
 with kephir lactase, 1 trypsin with casein as substrate. 2 In the just- 
 mentioned inonomolecular enzyme reactions the velocity coefficient in 
 a few cases was found proportional to the quantity of enzyme (catalase 
 from blood, 3 erepsin with glycyl-glyeine as substrate, 4 pancreatic lipase 5 ) 
 and in others not (catalase from Boletus scaber, lipase from pig fat). 6 
 It has been shown for several enzymotic reactions that with the same 
 substrate the same decomposition can be obtained if the time of action 
 varies in inverse proportion to the added quantity of enzyme. If p is 
 the quantity of enzyme and t the time of action, then the decomposition 
 is the same in all tests where p.t is the same figure. This rule has been 
 found true for the following enzymes: saccharase (O'Sullivan and 
 Thompson as well as Hudson 7 ), pepsin (Sjoqvist 8 ), rennin (especially 
 Fuld 9 ), peptone-splitting enzyme (Vernon 10 ), fibrin ferment of snake 
 poison (Martin 11 ), trypsin (Hedin 12 ), pepsin, rennin, trypsin, pyocy- 
 aneus protease (Madsen 13 ). On the action of trypsin upon casein this 
 law has been shown correct for different stages in the reaction. 14 This 
 indicates that the progress of the entire reaction remains the same with 
 different quantities of enzyme, only that the time for the same decom- 
 position is inversely as the quantity of enzyme. As clearly shown by 
 Hedin, this indicates that the velocity coefficient is proportional to the 
 quantity of enzyme which is called for by the second law. If we start 
 with the above-mentioned assumption that only that enzyme is active 
 which is combined, then it follows from the proportionality between the 
 velocity coefficient and the quantity of enzyme, that always the same 
 fraction of the enzyme is combined with the substrate, or that the divi- 
 sion of the enzyme remains independent of the quantity. 
 
 Armstrong, Proc. Roy. Soc, 73, 500 (1904). 
 
 2 Hedin, Journ. of Physiol., 32, 471 (1905). 
 
 3 Senter, Zeitschr. f. physik. Chem., 44, 257 (1903). 
 
 4 Euler, Zeitschr. f. physiol. Chem., 61, 213 (1907). 
 
 5 Kastle and Loevenhart, Amer. Chem. Journ., 24, 491 (1900). 
 
 6 Euler, Hofmeister's Beitriige, 7, 1 (1906). 
 
 7 Trans. Chem. Soc, 57, 926, 1890; Journ. Amer. Chem. Soc, 30, 1160, 1564 (1908). 
 
 8 Skand. Arch. f. Physiol., 5, 358 (1895). 
 
 9 Hofmesietr's Beitriige, 2, 169 (1902). 
 
 10 Journ. of Physiol., 30, 334 (1903). 
 
 11 Ibid., 32, 207 (1905). 
 
 12 Ibid., 32, 468 (1905); 34, 370 (1906). 
 
 13 Arrhenius, Immunochemie, Leipzig, 1907, 46. 
 
 14 Zeitschr. f. physiol. Chem., 57, 478 (1908). 
 
58 GENERAL AND PHYSICO-CHEMICAL. 
 
 In determining the quantity of enzyme the so-called Schutz's rule plays 
 an important part. In its newest form this is, that the decomposition is pro- 
 portional to the square root of the quantity of enzyme and the time, or decom- 
 position = kV pt where A; is a constant, p the quantity of enzyme and t the time 
 of the action. This was first _shown by Schutz * for pepsin and also, 
 in this form, decomposition =kvp as the time (t) was constant. The form 
 decomposition =fcv pt was given by Schutz, and Huppert. 2 According to 
 Pawlow this rule also applies to trypsin digestion. 3 Schutz's rule is good for 
 a certain stage of digestion only and it indicates that the extent of the validity 
 must be very dependent upon the method used for the determination of the 
 decomposition as the different digestion products are determined by different 
 methods. It must also be remarked that within the entire domain where 
 Schutz's rule is applicable the same value for pt must correspond to the same 
 decomposition, and necessarily the above-discussed enzyme-time rule must also 
 be valid. Schutz's rule has also been proved for the action of gastric and pan- 
 creatic lipase. 4 According to Arrhenius 5 the validity of the rule can be explained 
 by the assumption that the enzyme combines with the reaction products so that 
 the active mass of enzyme changes in inverse proportion to the auantity of 
 reaction products. 
 
 Reversibility of Enzyme Action and Enzymotic Syntheses. Many 
 catalytic processes have been shown to be reversible, i.e., the same 
 catalyst can influence the reaction in different directions according to 
 the concentration of the substances present. Thus far we have only 
 spoken of enzymotic cleavages; according to the above it is to be expected 
 that synthetical processes can also be produced by enzymes. 
 
 The first example of such a reaction was given by Croft-Hill. 6 
 He treated a 40 per cent glucose solution with maltase at 30° C. for a 
 very long time and concluded from the change in rotation and reducing 
 power that some maltose was formed from the glucose. Emmerling 7 
 showed afterward that a synthesis of maltose did not occur, but rather 
 an isomeric carbohydrate, isomaltose was formed, which is not split by 
 maltase. According to Armstrong 8 emulsin splits isomaltose, but 
 not maltose, and therefore it can synthesize maltose from glucose. A 
 similar reaction had previously been shown by E. Fischer and Arm- 
 strong, 9 that kefir-lactase produced isolactose and not lactose from 
 galactose and glucose. According to Cremer l0 yeast-press juice has 
 the power of forming glycogen from glucose or fructose. 
 
 1 Zeitschr. f. physiol. Chem., 9, 577 (1885). 
 2 Pfliiger's Arch., 80, 470 (1900). 
 3 Arbeit der Verdauungsdriisen, Wiesbaden, 1898, 33. 
 
 • Stade, Hofrneister's Beitrage, 3, 318 (1903); Engel, ibid., 7, 77 (1906), see Fromme, 
 ibid., 7, 77, (1906). 
 
 5 Immunochemie, 1907, 43. 
 
 6 Journ. of chem. Soc, 73, 634 (1S98). 
 
 > Ber. d. d. chem. Gesellsch., 34, 600 and 2207 (1901). 
 8 Proc. Roy. Soc. (ser. B), 76, 592, (1905) 
 ' Her. d. d. chem. Gesellsch., 35, 3151, (1902). 
 '"Ibid., 32, 2062 (1899). 
 
ENZYMES. 59 
 
 A. Danilewski first made the observation that concentrated solutions 
 of peptic cleavage products of protein substances separates an insol- 
 uble substance under the influence of rennin. This phenomenon has 
 since been observed by various investigators and the precipitate has 
 been called plastein by Sawjalow ' and coagalose by Lawrow. 2 The same 
 phenomenon is also obtained by other proteolytic enzymes. 3 The plas- 
 tein* are considered by various investigators as synthetically formed 
 protein. The best proof for this view has been given by Henriques 
 and Gjalback. They show with the formol titration (see Chapter II) 
 that the nitrogen titratable by formol diminishes in the reaction and 
 they also found that the nitrogen precipitatable by tannic acid was 
 increased in the plastein formation. In a later work these authors 
 find that peptic cleavage products from proteins show a plastein for- 
 mation when under the influence of pepsin-hydrochloric acid in con- 
 centrated solution while in dilute solution the cleavage goes further and 
 they conclude from this that the process is reversible. Even protein 
 which has been partly split by acid or alkali shows a plastein formation 
 with pepsin-hydrochloric acid. 4 
 
 The behavior of amygdalin and its cleavage products with enzymes 
 requires special mention. The cleavage takes place step by step as 
 follows : 
 
 C:oH 2 -NOi 1 +H 2 = Cr,H5.CH(OCr ) H 11 05).CN+C G H 12 OG. . (1) 
 
 Amygdalin Mandelic acid nitrileglucoside Glucose 
 
 C6H5.CH(OC6H„0 5 ).CX + H 2 = Cr,H5.CH™+C6H 12 6 . . (2) 
 
 Uri 
 
 Mandelic acid nitrileglucoside Mandelic acid nitrile Glucose 
 
 CoH 5 CH^ = CY,H5C<(°+KCX (3) 
 
 Mandelic acid nitrile Bcnzaldehyde Hydrocyanic acid 
 
 The entire process with the formation of the end products sugar, 
 benzaldehyde and hydrocyanic acid takes place under the influence of 
 emulsin from almonds. The first part of the process can be especially 
 brought about by the influence of yeast (Fischer) 5 and the second and 
 third parts under the influence of prunase from the leaves of Pruneae. 6 Of 
 
 1 Zeitschr. f. physiol. Chem., 54, ,119 (1907). 
 
 2 Ibid., .51, 1: 53, 1 (1907); 56, 343 (1908); 60, 520 (1909). 
 
 3 Kurajeff, Hofmeister's Beitriige, 4, 476 (1904); Numbers, ibil., 4, 543 (1904). 
 
 4 Zeitschr. f. physiol. Chem., 71, 485 (1911); 81, 439 (1912). 
 
 5 Ber. d. d. chem. Gesellsch., 28, 1508 (1896). 
 
 6 H. E. Armstrong, E. F. Armstrong and Horton, Proc. Roy. Soc, 85, 359, 363, 
 370 (1912). 
 
60 GENERAL AND PHYSICO-CHEMICAL. 
 
 the three above given reactions 1 and 3 can be reversed by enzymes and 
 indeed 1 even by using yeast (Emmerling) 1 and 3 with emulsin (Rosen- 
 thaler 2 ) . In the last instance the reaction is asymmetric in that 
 the d-form of the mandelic acid nitrile is formed. The asymmetric C 
 atom is marked in the above formula. Subsequently Rosenthaler was 
 able to divide the emulsin into a splitting component (5-emulsin) and 
 a synthetical form (or-emulsin) 3 . In connection with the views on the 
 structure and mode of action of enzymes it is of special interest that 
 recently Bredig and Fiske 4 have been able to prepare the two optical 
 antipodes of mandelic acid nitrile from benzaldehyde and hydrocyanic 
 acid by means of optically active catalysts. By using quinine as 
 catalyst the dextro-rotatory nitrile was formed and by quinidine (iso- 
 meric with quinine but opposed in regard to rotation power) the laevo- 
 rotatory nitrile was formed. This indicates that possibly the enzymes 
 also have an asymmetric structure. The synthetic formation of gluco- 
 sides by the aid of emulsin has also been performed by van't Hoff. 5 
 
 An undoubted synthesis of fat and other ester-like combinations 
 of fatty acids is also known. Kastle and Loevenhart 6 have shown 
 the formation of ethyl butyrate from ethyl alcohol and butyric acid 
 under the influence of a pancreas enzyme. In an analogous manner 
 Hanriot 7 obtained monobutyrin from butyric acid and glycerin with 
 blood serum. Pottevin 8 by means of a pancreas enzyme transformed 
 oleic acid and glycerin into mono- and triolein as well as oleic acid esters 
 with monatomic alcohols. The synthetical action of the pancreas has 
 been closely studied bj r Dietz. 9 
 
 The enzyme used by Dietz was insoluble in water, and its action was tested 
 with z'-amyl alcohol and n-butyric acid or the corresponding ester. It was shown 
 that the reaction took place in the insoluble phase (enzyme). From the formula 
 alcohol -f acid tester + water, it follows that when the molecular concentrations 
 of alcohol, aeid, ester and water are designated Ca, Cs. Ce. Cw, the reaction velocity 
 
 dx 
 of the ester formation for a homogeneous system is -j7=k 1 .CA-Cs—ki.CE.Cw (see 
 
 dx 
 page 32), which equation can be simplified to -rr = ki.Cs—k 2 .CE as the alcohol 
 
 and water were in excess and their concentration considered as constant and 
 included in the constants h and k 2 . At equilibrium we have kiCs=k 2 Cs or 
 
 1 Ber. d. d. chem. Gesellsch., 34, 3810 (1901). 
 
 * Bioch. Zeitschr., 14, 238 (1908). 
 » Ibid., 17, 257 (1909). 
 
 ♦Bioch. Zeitchr, 46, 7 (1912). 
 
 8 Sitzungsber. preuss. Akad. Wiss., 1909, s. 1065; 1910 s. 963. 
 
 8 Arner. Chem. Journ., 24, 491 (1900). 
 
 7 Compt. rend., 132, 212 (1901). 
 
 »nnd., 136, 1152 (1903), 138, 378 (1903); Ann. Inst. Past., 20, 901 (1906). 
 
 • Zeitschr. f. physiol., Chem., 52, 279 (1907). 
 
ENZYMES. 61 
 
 j-=7T =K (page 32). It follows that the same equilibrium is attained irrespective 
 
 of whether we start with alcohol+acid, or ester+H 2 0. The equilibrium is also 
 independent of the antecedents as well as the quantity of enzyme. 
 
 On comparing the equilibrium constants (A') which are obtained with dif- 
 ferent quantities of ester or acid, it is shown that in the above equation V(7s 
 must be introduced instead of Ce in order to obtain constant values for A'. In 
 the saponification of the ester the reaction velocity is proportional to \^Cs, 
 and not Ce. According to Dietz this is due to the fact that the system is a 
 heterogeneous one, and that only that part of the ester which is absorbed by 
 the solid phase (enzyme) takes part in the reaction. The velocity constant of 
 the ester formation is shown to be proportional to the quantity of enzyme. 
 
 According to what was stated above (page 35), the equilibrium in a reversible 
 reaction must be independent of the nature of the catalyst. This was not the 
 case in Dietz's experiments. With picric acid as the catalyst a different equilib- 
 rium was obtained than with the pancreas enzyme. With the acid as catalyst 
 the equilibrium was moved toward the direction of the ester. While this action 
 is not understood it may perhaps be explained by the fact that the system in one 
 case was homogeneous and in the other case heterogeneous. 
 
 Similar observations that the enzymotic end-condition can be different from 
 the stabile end-condition of the same system have previously been made by 
 Tammann, 1 but in these cases generally so-called false equilibrium existed, which 
 for example, by the addition of more enzyme changed, so that the cleavage pro- 
 ceeds further. These false equilibria are generally caused by the enzyme being 
 destroyed or put out of action in other ways. 
 
 Among the enzymotic-ester syntheses we must also include! he or- 
 mation of carbohydrate phosphoric acid ester in fermenting sugar solu- 
 tions in the presence of soluble phosphates, as first observed by Harden 
 and Young. 2 These will be discussed in detail in Chapter III. 
 
 It is seen that enzymotic syntheses are known. From this it fol- 
 lows that the questionable enzyme reactions are to be considered as 
 reversible. In certain cases another substance which cannot be split 
 by the enzyme is formed while in other cases the opposite direction of the 
 reaction can be detected by means of various constituents of the same 
 enzyme solution. 
 
 Specificity of Enzyme Action. It has been known for a long time 
 that a great difference exists in regard to the action of enzymes in the 
 sense that different enzymes act only upon certain classes of bodies (pro- 
 teins, carbohydrates, fats). Then there also exist differences in the 
 manner in which different enzymes of the same group influence different 
 members of the same class (maltase, lactase, saccharase). Finally, it is 
 possible for one enzyme to attack one of two optical antipodes and the 
 other not at all, or only to a slight degree. That optical antipodes 
 are burned with unequal facility in the organism was shown by E. Fischer, 
 and that of the numerous aldohexoses only three, rf-glucose, d-mannose 
 
 1 Zeitschr. f. physiol. Chem., 16, 271 (1892). 
 
 2 Proc. Roy. Soc. B, 1908 p. 209. 
 
62 GENERAL AND PHYSICO-CHEMICAL. 
 
 and d-galactose, and of the ketohexoses only one, d-fructose are fer- 
 mentable; and then that the synthetically prepared stereoisomeric 
 glucosides behave differently with the enzymes. Thus of two isomeric 
 glucosides, one methyl-d-glucoside, the (a) was attacked by yeast and 
 the other (j3) only by emulsin, while the corresponding methyW-glucosides 
 were not split by either of these enzymes. The corresponding glucoside 
 obtained from galactose behaves in a similar manner. 1 On the behavior 
 of amygdalin to various enzymes see page 59. In connection with 
 these observations Fischer presents the theory that for the action of 
 an enzyme a certain correspondence in stereometric structure of the 
 enzyme and substrate must exist; the enzyme must fit the substrate 
 somewhat like a key fitting a lock. 
 
 Then followed similar observations of Darin, 2 who found that racemic 
 mandelic acid ester, on incomplete hydrolysis by liver press-juice, yielded 
 a strongly dextrorotatory acid, while the ester remaining was levorotatory. 
 The dextrorotatory ester was more quickly hydrolyzed than the levoro- 
 tatory ester. Finally, we must mention the investigations of Fischer 
 and Abderhalden 3 on the cleavage of polypeptides by pancreas 
 press-juice. From abundant material they concluded that those polypep- 
 tides which consist entirely of the optical forms of amino-acids occurring 
 in nature are hydrolyzed and the others not. If in a racemic form besides 
 a polypeptide consisting of natural amino-acids, another occurs also, 
 then only the first is hydrolyzed. Besides this, other factors are also 
 of importance. Thus Z-leucyl-glycine is not hydrolyzed, although both 
 constituents occur in nature. The size of the molecule seems also to be 
 of importance, as mono-, di- and triglycyl-glycine are not split, while 
 tetraglycyl-glycine is. See also Chapter VIII. 
 
 Retardation of Enzyme Action. There are several reasons for the 
 assumption that the hydrolytic enzymes are only active after they have 
 combined with the substrate. From this it follows that those substances 
 which prevent the formation of such combination may cause the retarda- 
 tion of enzyme action. For this reason the enzyme action is retarded 
 by such substances which adsorb the enzyme (page 49). Hedin 4 has 
 made experiments on the retarding action of charcoal upon the action 
 of trypsin upon casein, and the action of rennin upon milk and it was 
 shown that the retardation was more pronounced if the powder and 
 enzyme were allowed to act upon each other before the substrate was 
 added than if this was present from the beginning. This fact indicates 
 
 1 Zeitschr. f. physiol. Chem., 26, 60 (1898) (collection of Fischer's works). 
 Mourn, of Physiol.', 30, 253 (1903); 32, 199 (1905). 
 •Zritechr. f. physiol. Chem.,' 46, 52 (1905); 61, 264 (1907). 
 
 •Bioch. Jpurn., 1, 484; 2, 81 (1906); Zeitschr. f. physiol. Chem., 50, 497 (1907); 
 60, 143 (1909). See also Jahnson-Blohm, ibid., 82, 178 (1912). 
 
ENZYMES. 63 
 
 that the adsorption process is only reversible with great difficulty or 
 that the enzyme to a certain extent is fastened to the charcoal. That 
 the substrate influences the formation of adsorption combination is 
 shown by the fact that the substrate is also adsorbed by the charcoal. 
 A small part of the adsorbed enzyme can indeed be subsequently 
 displaced on the charcoal by other adsorbable. substances and in this way 
 become active again. As various substrates are unequally adsorbed 
 by charcoal the retardation is, therefore, also different in degree. The 
 retardation of the saccharase action by charcoal is the same as for the 
 retardation of the trypsin or rennin action (Eriksson j ). 
 
 The action of several enzymes is retarded by normal serum. This 
 was first observed by Hammarsten and Roden 2 for the action of rennin. 
 Besides this certain constituents of the serum as well as other protein 
 containing fluids have a retarding action and in many such cases the 
 order of the addition of the bodies is important. The retardation by 
 charcoal corresponds to this retardation in several ways and this has 
 led Hedin 3 to the assumption that the retardation in both cases is brought 
 about by a colloidal reaction (adsorption) between the enzyme and a 
 solid or colloid phase. The facts correspond to this assumption namely 
 that during the action of the retarding substance upon the enzyme 
 the amount of water present is without importance for the final result 
 of retardation. Such a retardation by normal serum or fluids con- 
 taining protein has been observed in the following cases: retardation of 
 trypsin digestion of casein by native seralbumin, 4 retardation of the action 
 of rennin by neutral serum and by white of egg, 5 and the action of sac- 
 charase by serum. 6 Besides this Hedin 7 found a similar retardation by 
 seralbumin upon the digestion of casein by means of the a-protease of the 
 spleen. The retardation by normal serum or seralbumin has been shown 
 in the cases investigated not to be a specific kind, i.e., a given enzyme 
 is retarded about to the same extent regardless from what species of 
 animal it was prepared. 
 
 A specific retardation due to kind have been observed in the following 
 cases : 
 
 1. The antienzyme obtained by immunization (see page 66) retards 
 in those cases tested, only or chiefly the enzyme used in the immuniza- 
 
 l Ibid., 72, 313 (1911). 
 
 2 Upsala lakarefor. forh., 22, 546 (1887). 
 
 3 Bioch. Journ., 1, 484 (1906); Zeitschr. f. physiol. Chem., 60, 364 (1909); Ergebn. 
 d. Physiol., 9, 433 (1910). 
 
 * Journ. of Physiol.. 32, 390 (1905); Bioch. Journ., 1, 474 (1906). 
 
 5 Zeitschr. f. physiol., 60, 85, 364; 63, 143 (1909). 
 
 6 Ibid., 72, 313 (1911). 
 
 7 Hammarsten's Festschr., 1906. 
 
64 GENEEAL AND PHYSICO-CHEMICAL. 
 
 tion. Hildebrandt l first produced an anti-enzyme toward emulsin; 
 and Morgenroth 2 obtained in a similar manner an anti-rennin in goats' 
 serum; Bordet and Gengou 3 immunized against fibrin ferment, Sachs' 4 
 against pepsin, Schutze as well as Bertarelli 5 against various 
 plant lipases, Schutze 6 against lactase, Preti as well as Schutze and 
 Braun 7 against diastase, £. Meyer 8 against the proteases of bacillus 
 prodigiosus and bacillus pyocyaneus. 
 
 2. The retarding body of the rennin enzyme which was obtained 
 by treating a neutral infusion of the mucous membrane with dilute 
 ammonia and neutralizing, has been recently shown by Hedin 9 to 
 chiefly retard the enzyme of the same species (see Chapter VIII). In 
 these cases the importance of the order of treatment was also evident. 
 
 Most of the retarding substances contained in the serum lose their 
 retarding power on sufficiently heating them. This also occurs in cer- 
 tain cases by treatment with acid. Thus normal horse serum as 
 well as egg-white lose their ability to retard rennin by treatment 
 with very dilute hydrochloric acid and for this reason rennin which 
 has been inactivated by serum or egg-white can be set free again 
 by the use of hydrochloric acid (Hedin) 10 . Native seralbumin loses 
 its power of attaching itself to trypsin by treatment with dilute 
 acetic acid. 
 
 Certain proteins which are digested with difficulty retard the diges- 
 tion of more readily digestible ones without the order-phenomenon being 
 observed. In such cases the total digestion is probably diminished 
 because the more difficultly digested protein as substrate attracts a 
 part of the enzyme. As the order-phenomenon does not exist, the enzyme 
 is taken up in a complete and readily reversible manner (enzyme devia- 
 tion Hedin). 11 It is easily understood that the retardation must be 
 less effective than in those cases where the enzyme is attached to the 
 retarding substance. The tryptic digestion of casein in the presence 
 of seralbumin, treated with acid, is diminished by enzyme deviation as 
 well as the digestion of readily split proteins is retarded by egg-white 
 
 1 Virchow's Arch., 131, 33 (1893). 
 
 2 Centralbl. f. Bakt., 26, 349 (1899); 27, 357 (1900). 
 
 'Ann. inst. Past. 15, 129 (1901). 
 
 * Fortschr. d. Med., 20, 593 (1901). 
 
 6 Deutsch. med. Wochenschr., 1904; Centralbl. f. Bakt., 40, 231 (1905). 
 
 'Zeitschr. f. Hyg., 48, 457 (1904). 
 
 7 Bioch. Zeitschr., 4, 6 (1907); Zeitschr. exp. Pathol, u. Therap., 6, 307 (1909). 
 
 »Bioch. Zeitschr., 32, 280 (1911). 
 
 •Zeitschr. f. physiol. Chem., 72, 187; 74, 242; 76, 355 (1911). 
 
 "Zeitschr. f. physiol. Chem., 60, 85, 364 (1909). 
 
 » Ibid., 52, 412 (1907). 
 
ENZYMES. 65 
 
 which is difficult to digest (Delezenne and Pozerski, 1 Vernon, 2 
 Qompel and Henri, 3 Hedin 4 ). 
 
 At this time we must also mention the retarding action which the 
 proteolytic primary cleavage products (proteoses, peptones) exert upon 
 digestion. These products are further split; a part of the enzyme is 
 combined with the products and in this way prevented from dissolv- 
 ing new protein (Hedin). 5 The retarding power of proteoses and pep- 
 tones upon rennin action is probably similar to the above. 6 
 
 Finally, the end products of enzymotic activity i.e., bodies which 
 cannot be further split by the enzyme, have also a retarding action on 
 the enzyme action. That the inversion of cane-sugar is retarded by 
 invert sugar has been claimed by many (Henri, 7 A. J. Brown, 8 Baren- 
 drecht, 9 Armstrong 10 ), and indeed Barendrecht claims that glucose 
 as well as fructose has a retarding action, and that galactose has an 
 even stronger retarding action than the direct cleavage products of cane- 
 sugar. H. E. and E. F. Armstonng 11 found that saccharase, maltase and 
 lactase are retarded by just those varieties of sugar which are produced 
 by their activity. The accumulation of the amylolytic cleavage prod- 
 ucts have according to Sh. Lea, 12 a retarding action upon saliva. 
 
 The retarding action of amino-acids upon the decomposition of glycyl- 
 Z-tyrosine by yeast-press juice has recently been studied by Abderhalden 
 and Gigon. 13 They found that cleavage of peptides is retarded by 
 those optically active amino-acids which occur in the proteins. This 
 result is remarkable in consideration of the observations of Fischer 
 and Abderhalden that only those polypeptides were split by pancreatic 
 juice which are composed of natural optically active amino-acids (page 62). 
 
 The retardation of the action of papain by egg protein and by serum, which 
 is prevented by heating or action of hydrochloric acid, as shown by the investiga- 
 tions of Delezenne, Mouton and Pozerski as well as by Jonescu and Sachs u 
 is a peculiar behavior. 
 
 1 Compt. rend. soc. biol., 55, 935 (1603). 
 
 2 Journ. of Physiol., 31, 495 (1904). 
 
 3 Compt. rend. soc. biol., 58, 457 (1906). 
 
 4 Zeitschr. f. physiol. Chem., 52, 422 (1907). 
 
 5 Zeitschr. f. physiol. Chem., 52, 422 (1907). 
 
 6 Ibid., 46, 307. 
 
 7 Zeitschr. f. physik. Chem., 39, 194 (1901). 
 
 8 Journ. Chem. Soc, 81, 382 (1902). 
 
 9 Zeitschr. f. physik. Chem., 49, 456 (1904). 
 
 10 Proc. Roy. Soc. (ser. B), 73, 516 (1904). 
 
 11 Ibid., 79,360 (1907). 
 
 12 Journ. of Physiol., 1911. 
 
 13 Zeitschr. f. physiol. Chem., 53, 251 (1907). 
 
 14 Delezenne, Mouton and Pozerski, Compt. rend., 142; Jonescu, Bioch. Zeitschr., 
 2; Sachs, Zeitschr. f. physiol. Chem., 51, 488 (1907). 
 
66 GENERAL AND PHYSICO-CHEMICAL. 
 
 In consideration of what has been said (page 58) about enzymotic 
 syntheses it seems very possible in the retardation of enzymotic cleavages 
 by means of cleavage products that we are dealing with synthetic 
 processes where the cleavage products supply the material. This is espe- 
 cially shown by the above-mentioned investigations of Rosenthaler 
 on emulsin that the retarding action of benzaldehyde or of hydrocyanic 
 acid upon emulsin action, as shown by Tammann, 1 is explainable by 
 syntheses. Lichwitz 2 considers the interaction of the products as a 
 reversible paralyzation of the enzyme. 
 
 Appendix : Antigens and Anti-bodies. In connection with the retar- 
 dation of enzyme action we can also call attention to other similar proc- 
 esses. Under the name antigen we include those substances which, 
 when injected into animals, cause the formation of bodies in the organ- 
 ism with which they can in some way or another react. The process 
 is called immunization and the bodies formed are called anti-bodies or 
 in certain cases immune bodies. General)}'' these anti-bodies are specific 
 in the sense that they only react with the corresponding antigen. The 
 chemical constitution of the antigen as well as of the anti-body is not 
 known; they belong perhaps to the colloids, or at least they occur asso- 
 ciated with colloids. 
 
 The antigens are either substances soluble in water or occur as 
 constituents of the cells. We will first discuss the antigens soluble in 
 water. 
 
 To this group belong, in the first place, certain poisonous substances 
 of animal or plant origin (toxins), for example, snake poisons, bacterial 
 poisons, ricin (from the seeds of Ricinus communis), also enzymes as well 
 as certain proteins without special action. The reaction with the anti- 
 bodies (which are obtained in the blood serum of animals) manifests 
 itself with the poisons by the suppression of the poisonous action, with 
 the enzymes by retardation of the enzyme action, and with certain pro- 
 teins by formation of a precipitate which contains the antigen as well 
 as the anti-body. Anti-bodies of this last type are called precipitins. 
 
 The longest known (due to the epoch-making investigations of v. 
 Behring 3 ) and best studied are those anti-bodies which are produced 
 by toxins and which neutralize the action of the toxins upon the animal 
 organism (antitoxins). According to the older view this takes place 
 by some sort of an action of the anti-body upon the cells sensitive to the 
 toxins. After it was shown that the toxins could also be neutralized 
 in vitro by the anti-bodies, it is now generally accepted that the neu- 
 
 l IbirL, 16, 271 (1892). 
 - Ibid., 78, 128 (1912). 
 * Deutsch. ix.ed. Wochenschr., 1892; Zeitschr. f. Hygiene, 12 (1892). 
 
ENZYMES. 67 
 
 tralization is brought about by some sort of a combination between the 
 
 toxin and the anti-body. The views are very contradictory in regard 
 to the nature of this combination and the manner in which it is formed. 
 
 The oldest theory, which has contributed much to our knowledge 
 of these conditions, is that of P. Ehrlich, whom we must thank for the 
 method of measuring the quantity of toxin by injection into an ani- 
 mal. The quantity of toxin which is just sufficient to kill a guinea-pig 
 of given weight in a certain time is selected as the unit. According to 
 so-called side-chain theory of Ehrlich l the toxins firstly have a so- 
 called haptophore group, by means of which the toxin can attach itself 
 to a certain cell, and secondly, a so-called toxophore group, by which 
 the toxin exerts its poisonous action. The formation of anti-body 
 after the injection of the toxins Ehrlich explains by the fact that those 
 cells which are attacked by the toxins are supplied with so-called recep- 
 tors, which just fit the haptophore group- of the toxins; the toxins are 
 thus anchored on the questionable cells and can then begin their action 
 by aid of the toxophore group. By the attachment of the receptors, the 
 cells are induced to produce new receptors, and indeed, so many recep- 
 tors are produced that they are thrown off and appear free in the blood 
 plasma. The receptors circulating in the blood are the anti-bodies. 
 As these are able to combine with the toxins they can protect against 
 the toxin those cells which are supplied with the same receptor under 
 whose influence they were found. The toxophore group of the toxins 
 can gradually be destroyed on keeping. A toxin so changed can be 
 continuously anchored to cell-receptors and in this way form anti-bodies, 
 but cannot produce any poisonous action. A toxin without toxophore 
 groups is called a toxoid by Ehrlich. It follows that the toxoids can 
 combine with the anti-bodies. 
 
 According to Ehrlich, on the neutralization of a toxin a chemical 
 combination takes place between the toxin and the anti-body, and so 
 much of this combination is formed that either the toxin or the anti- 
 body is completely consumed. Now the bacterial poisons are not 
 simple bodies, but mixtures of several poisons of different toxicity and 
 different avidity toward the anti-bodies. Generally the most poison- 
 ous is first neutralized, but it also occurs that a less poisonous or indeed 
 a non-poisonous body is first combined with the anti-body (proto-toxoids) 
 or that non-poisonous bodies are combined parallel with the true toxins 
 (syntoxoids). The less poisonous or non-toxic bodies first combined 
 after the binding of the true toxins are called toxons (also epitoxoids). 
 According to the relative quantity and the avidity of the different con- 
 stituents of the toxic solution, the addition of a certain quantity of anti- 
 body can produce entirely different results. 
 
 1 See Michaelis, Die Bindungsgesetze von Toxin und Antitoxin, Berlin, 1905. 
 
68 GENERAL AND PHYSICO-CHEMICAL. 
 
 Arrhenius opposes Ehrlich's theory that the combination between 
 toxin and anti-body is of a chemical nature, but claims, that their for- 
 mation does not proceed until one of the components has been used up. 
 An equilibrium is established between the free toxin and the free anti- 
 body on one side and the combination of the two on the other, which 
 the law of mass action requires according to the formula: 
 
 C . C = K . C N (page 32). 
 
 toxin anti-body toxin +anti-body 
 
 For tetanolysin (a substance obtained from tetanus cultures, which dissolves 
 red-blood corpuscles) and its anti-body, as well as for diphtheria toxin and the 
 corresponding anti-body, n=2 was found, i.e., in the combination of a molecule 
 of toxin with a molecule anti-body two molecules toxin-antitoxin combination 
 was formed. 
 
 The toxic action which a mixture of toxin and anti-body exerts 
 depends upon the quantity of toxin which, according to the above 
 formula, must always remain free. 1 According to this theory the toxin 
 is a unit poison, as Arrhenius 2 now admits with Ehrlich, that the 
 poison is gradually transformed into a non-toxic or only slightly toxic 
 substance which has the same ability to combine with antitoxin as the 
 toxin itself. 
 
 Ehrlich's theory, as well as that of Arrhenius admits of a chem- 
 ical combination between the antigen and the anti-body. According 
 to Ehrlich besides this the substrate (or the cells sensitive to the anti- 
 gen) combines with the antigen, which is not conformable with the theory 
 of Arrhenius. 
 
 The combination toxin-anti-body is first gradually produced, and 
 then it is taken up from all sides so that the toxin is fastened to the 
 anti-body by a secondary process (exception, cobra poison). The com- 
 bination toxin-antitoxin is not reversible in the ordinary sense. This 
 is most easily shown by the fact that to a certain limit more toxin is 
 neutralized according to the time allowed to elapse before the quantity 
 of toxin remaining free is determined by injection into an animal or in 
 other ways. 3 In certain cases it is possible to obtain the toxin again in 
 an active form from the toxin-antitoxin combination, and indeed by 
 treatment with very dilute hydrochloric acid (Morgenroth 4 ). See 
 also page 64 on the setting free of rennin from its combination with 
 normal scrum and with egg-white. Hedin 5 has also been able to obtain 
 
 « Zeitschr. f. physik. Chem., 44, 7 (1903). 
 3 Immunochemie, Leipzig, 1907, 132. 
 
 3 Martin and Cherry, Proc. Roy. Soc, 1898, 420'. 
 
 4 Berl. klin. Wochenschr., 1905, No. 5; Festschr. z. Eroffnung d. pathol. Instit. 
 Berlin, 1906; Virchow's Arch., 190, 371 (1907). 
 
 * Zeitschr. f. physiol. Chem., 77, 229 (1912). 
 
ENZYMES. 69 
 
 the rennin again in an active form, from the combination of the rennin 
 with anti-rennin obtained by immunization by treatment with hydro- 
 chloric acid and then neutralizing. 
 
 Recently a third manner of considering the toxin-antitoxin reaction 
 has been presented which is based on the fact that the reaction takes 
 place in a heterogeneous system. According to this the reaction is con- 
 sidered as an adsorption process, and in support of this assumption, sev- 
 eral examples can be given where finely divided solids or colloid sub- 
 stances take up toxins or enzymes, in an irreversible manner (Nernst, 1 
 Biltz, 2 Landsteiner 3 ). 
 
 In reference to the formed antigens we must call attention to the fol- 
 lowing : 
 
 If certain cells, for example, bacteria, blood-corpuscles, and sperma- 
 tozoa are injected into animals, then anti-bodies are formed which have 
 been called immune bodies (also amboceptors or sensibilizators) . By 
 themselves the immune bodies are inactive, but form with complements, 
 substances occurring in normal serum, so-called cytotoxins, which destroy 
 the kind of cells active in their formation. These cytotoxins are called 
 bacteriolysins, hemolysins, etc., according to the kind of cells used. 
 The immune bodies are specific in that they together with the com- 
 plement only attack those cells from which they are formed and 
 they are also stable against heat; the complements can act together 
 with different immune bodies and are very unstable, as they are gen- 
 erally destroyed by heating to 56° C. for one-half hour. Other anti- 
 bodies, produced under the influence of injected cells, show their action 
 by flocking together and agglutinating the cells set free in their forma- 
 tion. These anti-bodies are called agglutinins. 
 
 In regard to the immune bodies, Ehrlich believes that they com- 
 bine with those cells under whose influence they have been formed 
 and also with the complements. They serve to fasten (amboceptors) 
 the complement, which produces the real poisonous action, to the cells. 
 The immune bodies correspond therefore to the haptophore groups of 
 the toxins and the complements of the toxophores. According to 
 Bordet the immune bodies act upon the cells in the way that the latter 
 are sensitive toward the complements (sensibilizators). 
 
 If a certain immune serum is heated to 56° then, according to what 
 has been given, the complement is destroyed and the serum now con- 
 tains only the amboceptor of the original cyto-toxin and this amboceptor 
 can be made active again by the addition of normal serum (complement). 
 
 » Zeitschr. f. Elektrochem., 10, 379 (1904). 
 
 2 Ber. d. d. Chem. Gesellsch., 37, 3147 (1904); Beitr. z. exp. Therapie, 1, 30 (1905). 
 
 3 Zeitschr. f. Chem. u. Ind. d. Koll., 3, 221 (1907); Bioch. Zeitschr., 15, 33 (1908). 
 
70 GENERAL AND PHYSICO-CHEMICAL. 
 
 If. therefore, an antigen of the corresponding immune serum be headed 
 to 56° (amboceptor) and mixed with sufficient amount of normal serum 
 (complement), then the complement is bound up so that when subsequently 
 serum-free red-blood corpuscles and a certain quantity of immune serum, 
 obtained by immunization with these and after losing its complement 
 by heating to 56°, are added, no solution of the red-blood corpuscles 
 (haemolysis) takes place. If in the first mixture either the antigen or the 
 corresponding amboceptors are absent then the complement is not 
 combined and a haemolysis occurs because the complement cannot unite 
 with the haemolytic amboceptors added. In this manner it has been 
 attempted to determine the presence of an antigen or of amboceptors 
 which fit with the antigen (method of complement deviation). 
 
 The protective substances formed by immunization can protect the 
 organism against many fatal doses of the antigen and this protective 
 power can be brought about by the parenteral introduction of the immune 
 serum of another organism. The immunity is called active when the 
 organism obtains the antigen and itself produces the corresponding 
 protective substance. On the contrary the immunity is called passive 
 if the organism receives the anti-body formed in another living being 
 by active immunization. 
 
 During immunization under certain circumstances it is observed 
 that a condition of super-sensitiveness toward the antigen exists. This 
 super-sensitiveness occurs only toward the antigen used and is therefore 
 specific. The same has been observed in using the soluble as well as the 
 formed antigens. This mysterious phenomenon has been called anaphyl- 
 axis. 
 
 V. IONS AND SALT ACTION. 
 
 We have previously mentioned various processes which depend upon 
 the influence of ions. To these belong the precipitation of suspension 
 colloids by electrolytes as well as different catalytic processes. That 
 in the last case we are dealing with the action of ions is proven by the 
 fact that the velocity coefficient is proportional to the concentration 
 of a certain kind of ion. Nevertheless it has been shown, that the 
 velocity coefficient in the inversion of cane-sugar, by acid, is only propor- 
 tional to the H ions when dilute acids are used. With greater concen- 
 tration disturbances occur which can be ascribed to the action of the 
 negative ions of the acids. The catalytic processes can be influenced 
 by salts in a similar manner (salt action). 
 
 The enzyme action has shown itself proportional to the quantity of enzyme 
 in certain cases. Euler ' has attempted to show a correspondence between ion- 
 
 1 Zeitsehr. f. physik. Chem., 36, G41 (1901). 
 
IONS AND SALT ACTION. 71 
 
 action and enzyme action by the assumption that the enzymes cause an increase 
 in those ions, which could cause the reaction without the presence of the enzyme. 
 On the other hand J. Loeb 1 believes that the enzymes can also be electrolytically 
 dissociated and that their action depends on the amount of ions. Tims pepsin 
 is a weak base which forms a salt with the hydrochloric acid added and thai this 
 salt is more strongly dissociated than the base; for this reason the action of pepsin 
 is increased by acid. 
 
 Many enzymotic processes are influenced by the presence of salts 
 of the alkalies or alkaline earths. According to Bierry, Giaja and Henri 
 as well as Preti 2 pancreatic juice dialyzed for a long time has no action 
 upon starch, but becomes active again on adding NaCl or other salts. 
 According to Wohlgemuth 3 the diastatic power of saliva is increased 
 ten-fold by the addition of NaCl. The anions are the active part in both 
 cases (see page 52 on co-enzymes). The strong retarding action which 
 NaFl exerts upon the enzymotic cleavage of esters is also to be men- 
 tioned (Loevenhart and Pierce, Amberg and Loevenhart 4 ). 
 
 Other actions of salts are also ascribed to ion-action. To these 
 belong the experiments of Dresser 5 according to which mercury salts, 
 which are relatively strongly dissociated, have a poisonous action upon 
 organic formations (yeast, frog heart), while potassium-mercury hypo- 
 sulphite was nearly non-toxic. As the last-mentioned salt contains 
 very few free Hg ions the posionous action of the first salt is ascribed to 
 the ions. Paul and Kronig 6 have arrived at similar results by inves- 
 tigating the poisonous action of mercury salts upon spores. They found 
 that KoCy4Hg, which hardly contains any Hg ions, is much less poison- 
 ous than an equivalent solution of HgCy2- The same conditions were 
 observed by Maillard 7 for copper salts. 
 
 This leads us to the question as to the importance of water and the 
 mineral bodies, which are of just as great moment for the life of the 
 cells and their metabolism as the organic constituents. In regard to 
 the water this follows from the fact that the animal body consists of 
 about two-thirds water. If we also recall that water is of the 
 greatest importance for the normal physical condition of the tissues, 
 that the solution of numerous bodies and the dissociation of chemical 
 compounds, that all flow of juices, all exchange of material, all supply 
 of food, all growth or destruction and all removal of destructive prod- 
 
 1 Bioch. Zeitschr., 19, 534 (1909). 
 
 2 Compt. rend. soc. biol., 60, 479 (1906); 62, 432, (1907); Bioch. Zeitschr., 4, 1 
 (1907); 40, 357 (1912). 
 
 3 Bioch. Zeitschr., 9, 1 (1908). 
 
 4 Journ. of Biol. Chem., 2, 397 (1907); 4, 149 (1908). 
 6 Arch. exp. Pathol, u. Pharm., 32, 456 (1893). 
 
 6 Zeitschr. f. physik. Chem., 31, 411 (1896). 
 7 Compt. rend. soc. biol., 50, 1210 (1898). 
 
72 GENERAL AND PHYSICO-CHEMICAL. 
 
 ucts, are connected with the presence of water, and that besides this 
 the water by its evaporation is an important regulator of temperature, 
 it is evident that water must be a necessity of life. 
 
 The mineral substances found habitually in the cells of higher plants 
 and of animals are potassium, sodium, calcium, magnesium, iron, phos- 
 phoric acid, sulphuric acid, chlorine, and perhaps also iodine (Justus). 1 
 Besides, in certain cells or organs we also find manganese, lithium, barium, 
 silicium, fluorine, bromine, and arsenic. 
 
 We are chiefly indebted to Liebig for showing that the mineral 
 bodies are as important for the normal constitution of the organs and 
 tissues, as well as for the normal performance of the processes of life, 
 as the organic constituents of the body. The importance of the mineral 
 constituents is evident from the fact that we know no animal tissue 
 and no animal fluid which is free from mineral bodies, and also from 
 the fact that certain tissues or tissue elements contain chiefly certain 
 mineral bodies and not others. In regard to the alkali compounds this 
 division is, in general, as follows: The sodium compounds occur chiefly 
 in the fluids, while the potassium compounds occur especially in the 
 form-elements. Corresponding to this, the cells contain chiefly potas- 
 sium as phosphate, while they are less rich in sodium and chlorine com- 
 pounds. The fundamental experiments of Forster 2 have shown us that 
 inorganic salts, as constituents of the food are necessary for the animal 
 organism. 
 
 We have already called attention to the importance for every organ- 
 ism of the salts for the production of a rather constant osmotic pressure. 
 That the importance of the salts is not limited to the maintenance 
 of the osmotic pressure follows from the fact that different salt solutions 
 of the same osmotic pressure are not of the same value for the main- 
 tenance of the functional powers on extirpated organs. Since S. 
 Ringer 3 showed that various organic structures retained their best 
 functional activity in a solution which contained NaCl, CaCl? and 
 KC1 at the same time, various investigators have given the most suit- 
 able composition of such solutions. For the transfusion fluid for the 
 mammalian heart Locke 4 suggests the following composition; NaCl 
 
 1 Justus, Virchow's Arch., 170, 176 and 190. In regard to arsenic see the works 
 of Gautier, Compt. rend., 129, 130, 131, 139; Bertrand. ibid., 134; Segale, Zeitschr. 
 f. physiol. Chem., 42; Kunkel, ibid., 44. In regard to the barium see Schultze and 
 Thierf elder, Sitzungsber. d. Gesellsch. naturforsch. P'reunde, 1905, No. 1, and in 
 regard to lithium see Hermann, Pfluger's Arch., 109; and in regard to manganese see 
 Bradley, Journ. of Biol. Chem., 3. 
 
 2 Zeitschr. f. Biol., 9, 297 (1873); 12, 464 (1877). 
 
 'Journ. of Physiol., 6, 154, 361 (1885); 7,118(1886); 16, 1, 17, 23 (1895); 18, 
 425 (1896). 
 
 * Centralbl. f. Physiol., 14, 672 (1900). 
 
IONS AND SALT ACTION. 73 
 
 0.9-1 per cent, CaCl 2 0.02-0.024 per cent, KC1 0.02-0.042 per cent, 
 XaHCO:j 0.01-0.03 per cent. Each of the salts NaCl, CaCl 2 and KC1 
 individually has a poisonous action upon the organ but this action is 
 counteracted by the presence of the two other salts (antagonistic salt 
 action). 
 
 This neutralizing action of salts has been studied during recent 
 years especially by J. Loeb and his collaborators. As general results 
 it has been found that the most favorable quantity relations of the 
 three salts NaCl, KC1 and CaCk for the maintenance of life is the same 
 as exists in blood. Especially interesting are the experiments with the 
 Fundulus heteroclitus, a genus of killifish. This fish, it is remarkable, can 
 also live in distilled water and is therefore within wide limits, not depend- 
 ent upon the osmotic pressure of the surrounding medium. For this 
 reason it is specially suited for the study of the poisonous action of salts 
 or mixture of salts. KC1 in concentrations in which it exists in sea water 
 acts as a poison upon these fishes, if it is alone in solution. The same 
 is true for NaCl. On the contrary these fishes live for an indefinite 
 time in a pure CaCl2 solution in a concentration similar to sea water. 
 One mol. KC1 can be very nearly de-toxicated by 17 mol. NaCl or by 8^ mol. 
 Na2S04. \ mol. K2SO4 is just as poisonous as 1 mol. KC1. The toxicity 
 of the potassium salts is therefore dependent upon the K ions and the 
 de-toxicating substance on the Na ion. CaCl2 de-toxicates a KC1 solu- 
 tion even when ^ mol. CaCb to 1 mol. KC1 is present. SrCl2 shows 
 almost as great a de-toxicating action as CaCk- NaCl in concentra- 
 tions, in which it occurs in sea-water can only be incompletely de-toxi- 
 cated by KC1; only by the addition of CaClo can the complete de- 
 toxication be brought about. The poisonous action of acids upon 
 Fundulus can be arrested by neutral salts. 1 Fundulus can accommodate 
 themselves to a rise in temperature; a rise in temperature can be more 
 easily endured when the concentration of the surrounding medium is raised 
 at the same time (Loeb and Wasteneys). Can fishes also accommodate 
 themselves to an abnormal concentration of the surroundings as long 
 as the rise in concentration takes place gradually? In both cases the 
 accommodation, according to Loeb, 2 depends upon a slow proceeding 
 process, possibly a tanning of the surface of the animal. 
 
 The fertilized eggs of the Fundulus develop, according to Loeb, just 
 as well in water free from salt as in sea-water. If the fertilized eggs are 
 placed in a NaCl solution of the same osmotic pressure as the sea-water 
 
 ^ioch. Zeichr., 31, 450; 32, 155, 308; 33, 480- 489 (1911); 39, 167; 43, 181 
 (1912). 
 
 2 Loeb and Wasteneys, Journ. of exp. Zool., 12, 543 (1913) and Loeb, Bioch. 
 Zeitschr., 53, 391 (1913). 
 
74 GENERAL AND PHYSICO-CHEMICAL. 
 
 they die; the toxicity of the NaCl solution can be arrested by small 
 quantities of almost any salt with polyvalent cations. Not only the salts 
 of the alkaline earths, but also those of the heavy metals (for instance 
 zinc sulphate or lead acetate) can neutralize the toxicity of the NaCl in 
 proper concentration. 1 The eggs can develop in solutions which kill the 
 completed fish. 
 
 The antagonistic action of salts upon organic structures depends, 
 according to Loeb, upon the fact that the salts mixed in proper propor- 
 tions causes a " tanning " of the protoplasmic surface of the cells whereby 
 the cells become impermeable for certain destructive substances to which 
 the salts also belong. The fertilized eggs of Fundulus can. be tanned 
 by NaCl + a heavy metal but not the completed fish. 2 Many observa- 
 tions indicate that the egg is more permeable after fertilization than 
 before. 3 
 
 Appendix 21 Determination of the Reaction of a Solution. The 
 reaction of the solution, in which a chemical reaction takes place, plays 
 an important role in many cases. As the acid or alkaline reaction of a 
 solution depends upon the amount of H or OH ions it is often of import- 
 ance to be able to determine the concentration of these ions in solution. 
 These cannot be determined by titration with alkali or acid in the pres- 
 ence of organic salts. In this titration the existing equilibrium in the 
 solution is disturbed and therefore also other decompositions occur 
 besides the neutralization of H or OH ions. The quantity of alkali or 
 acid used does not therefore correspond to the original concentration 
 of H or OH ions. 
 
 According to the law of mass action there exists, between the H and 
 OH ions formed by the dissociation of the water on the one hand and 
 the concentration of the non-dissociated molecules on the other, the 
 following equation 
 
 Ch-Coh = Ki-Ch>o 
 
 where C H , Coh represents the concentration of the H and OH ions, 
 < ii o the non-dissociated water molecules and Ki a constant. As Cr 2 o 
 can only be considered as constant in certain dilute solutions we have 
 Ch-Coh = K) where K is called the dissociation constant of the water. 
 As K is a constant it follows that the figures for Cr- and Coh can be 
 calculated, if the other is known. As it is more convenient to determine 
 Ch than C'oh> therefore Cr is also ordinarily determined for solutions 
 
 1 PfliiKer's Arch., 88, 68 (1901). 
 ^Science, 84, 653 (1911). 
 
 3 Lillie, Amer. Journ. of Physiol., 27, 289 (1911); McClendon, ibid., 27, 240; 
 Science, 32, 122, 317; Lyon and Shackell, ibid., 32, 249 (1910). 
 
IONS AND SALT ACTION. 75 
 
 with alkaline reaction. Complete investigations on this subject have 
 been carried out by Sorensen. 1 He found the value 10 ~ 14,14 for K 
 at 18° C. Ch is determined in either of two ways. The best method, 
 the electromotive, is based upon the electromotive force of gas chains, 
 as developed by Nernst; 2 namely, if platinum foil covered with 
 platinum black is introduced into a watery solution and this saturated 
 with hydrogen, then a difference of electrical potential is produced between 
 the platinum and the solution and this potential is theoretically propor- 
 tional to the concentration of the hydrogen ions in the solution. We 
 cannot give any further detail as to this theory or to the performance 
 of the measurement of the difference in potential. 3 If the concentration 
 of the hydrogen ions Ch is expressed in gram ions per liter by the figure 
 10~ p , then according to the suggestion of Sorenson the name hydrogen 
 ion exponent and the symbol Pr- is used f° r the numerical value of the 
 exponents of this potence. The relationship between p H and the electro- 
 motive force -k at the contact between the platinum and the solution 
 can be expressed graphically by a straight line; hence it follows that if 
 r is known then / H can be very easily found (the exponential line). 
 
 The other method used by Sorensen 4 for the determination of C H 
 is a colorimetric method and depends on the use of indicators. After 
 much investigation 20 indicators are recommended, of which certain ones 
 require strictly fixed methods of use. As soon as more than a qual- 
 itative approximation is required then the shade of color produced by 
 the indicator must be compared with a shade of color produced by the 
 same indicator in a solution of known concentration of H ions. Such 
 standard solutions which allow of a variation in the concentration of the 
 H ions at one's pleasure have been given by Sorensen, and the original 
 article gives a table of curves from the corresponding value for p H which 
 can be read off, when the composition of a standard solution is known. The 
 figure j H for the standard solutions is determined by aid of the electro- 
 motive method. Standard solutions are selected so that they serve as 
 natural protectors against too sudden changes in pn (so called buffer) 5 . 
 
 As above stated the dissociation constant according to Sorensen 
 for water is 10 -1414 at 18° C. or C H - C o h = 10~ 1414 . In neutral reac- 
 tion C H = C H and therefore C H = 10~ 7-07 or pn = 7.07. Smaller values 
 for ;>h correspond to acid and greater values to alkaline reaction. 
 
 Hasselbach 6 has suggested a modification of Sorensen's method 
 
 1 Bioch. Zeitschr., 21, 131 (1909) also Ergebn. d. Physiol. Vol. 11. 
 
 2 Zeitschr. f. physik. Chem., 4, 129 (1889). 
 
 3 In regard to the determination see the work of Sorensen cited on page 74. 
 
 4 Sorensen, Enzymstudien, Bioch. Zeitschr., 21, 253. 
 
 5 Ibid., 167. 
 
 6 Bioch. Zeitschr., 30, 317 (1910). 
 
76 GENERAL AND PHYSICO-CHEMICAL. 
 
 for the electrometric determination of reaction in fluids containing 
 carbon dioxide. By the aid of this method Hasselbach and Lunds- 
 gaard l have made determinations of the reaction of the blood. From 
 these it follows that at a temperature of 38.5° C. where the value for 
 p H = 6.78 corresponds to the neutral reaction the figure obtained for pn 
 with defibrinated ox-blood was 7.36 showing therefore a slight alkaline 
 reaction. The influence of the respiratory variation in the CO2 tension 
 upon the H ion concentration of the blood is of a measurable size. 
 The total blood has a greater H ion concentration than the serum at 
 equal CO2 tension but less than the blood corpuscles. For human blood 
 saturated with CO2 under 40 mm. tension at 38° C. Lundsgaaed 2 
 found p H = 7.19. Michaelis and Davidoff 3 found the average values 
 of normal venous blood for p H = 7.35 at 37.5° C. 
 
 1 Biochem. Zeitschr., 38, 77 (1911). 
 
 2 Ibid., 41, 2641(1912). 
 
 3 Ibid., 46, 131 (1912).. 
 
CHAPTER II. 
 THE PROTEIN SUBSTANCES. 
 
 The chief mass of the organic constituents of animal tissues consists 
 of amorphous nitrogenized, very complex bodies of high molecular weight. 
 These bodies, which are either proteins in a special sense or bodies nearly 
 related thereto, take first rank among the organic constituents of the 
 animal body on account of their great abundance. For this reason they 
 are classed together in a special group which has received the name 
 protein group (from irpayrtvw, I am the first, or take the first place). 
 The bodies belonging to these several groups are called protein sub- 
 stances, although in a few cases the protein bodies in a special sense are 
 designated by the same name. 
 
 The several protein substa?ices x contain carbon, hydrogen, nitrogen, and 
 oxygen. The majority contain also sulphur, a few phosphorus, and a 
 few also iron. Copper, chlorine, iodine, and bromine have been found 
 in some few cases. On heating the protein substances they gradually 
 decompose, producing a strong odor of burned horn or wool. At the same 
 time they produce inflammable gases, water, carbon dioxide, ammonia, 
 and nitrogenized bases, besides many other substances, and leave a large 
 quantity of carbon. On deep hydrolytic cleavage they yield abundance 
 of a-monamino-acids of various kinds as decomposition products. 
 
 The nitrogen occurs in the protein bodies in various forms, and this 
 is also revealed in the division of the nitrogen among the cleavage prod- 
 ucts. On boiling with dilute mineral acids we obtain (1) so-called amide 
 nitrogen, which is readily split off as ammonia; (2) a guanidine residue 
 which is combined with diaminovaleric acid as arginine, and which 
 has also been called the urea-forming group; (3) basic nitrogen or diamino- 
 acid nitrogen, or hexone bases nitrogen, which is precipitated by phos- 
 photungstic acid as basic products (to which also the guanidine residue 
 of arginine belongs); (4) monamino-acid nitrogen; and (5) the nitrogen 
 
 1 See " Eiweisskorper," Ladenburg's Handworterbuch der Chemie, 3, 534-589, 
 which gives a complete summary of the literature of protein substances up to 1885. 
 The more recent literature may be found in O. Cohnheim, Chemie der Eiweisskorper, 
 Braunschweig, 1911. See also Oppenheimer's Handbuch der Biochem. der Menschen. 
 und der Tiere, 1908. 
 
 77 
 
78 THE PROTEIN SUBSTANCES. 
 
 in variable amounts which appears as humus-like melanoidins, which 
 seem to be of only secondary formation as products of elaboration. 
 
 The quantitative division of the total nitrogen between the above 
 five groups is different in the various protein substances, and more- 
 over cannot be given with certainty, because of the above-mentioned 
 melanoidin formation and the errors in the methods used. 1 The follow- 
 ing gives at least an approximate idea of this division. 2 The loosely 
 combined so-called amide nitrogen seems to be entirely absent in the 
 protamines. In the gelatins we find 1-2 per cent, and 5-10 per cent 
 in other animal protein substances, 3 in certain plant proteins, the 
 prolamines (see page 106), 13-25 per cent of the total nitrogen is amide 
 nitrogen. The guanidine nitrogen may amount in the protamines to 
 22-44 per cent of the total nitrogen, in the histones to 12-13 per cent, 
 in the gelatins about 8 per cent, and in the other protein bodies about 
 2-5 per cent. As basic nitrogen precipitable by phosphotungstic acid 
 (including the guanidine residue) we find 35-88 per cent in the protamines, 
 35-42.5 per cent in the histones, 15-30 per cent in the other animal pro- 
 tein substances. In the prolamines 3-6 per cent of the total nitrogen is 
 found as products precipitable by phosphotungstic acid but in plant 
 globulin (globulin of the wheat) indeed 37 per cent. The chief quantity 
 of the nitrogen, 55-76 per cent, occurs, with the exception of the pro- 
 tamines, as the monamino-acid groups. The results for the melanoidin 
 nitrogen vary so considerably that they will not be mentioned. 
 
 Recently D. v. Slyke 4 has perfected a method which is based upon "the 
 deamidation of the amino-acids by HN0 2 (see below) and which allows of a still 
 more detailed differentiation of the nitrogen partition. In this method the 
 nitrogen of the ammonia, the melanines, the cystine, arginine, histidine, proline and 
 oxyproline besides one-half of the tryphtophane nitrogen as well as the nitrogen 
 of the remaining amino-acids can be specially determined. 
 
 From recent as well as older observations it follows as chief result 
 that the nitrogen in the proteins occurs in such combinations so that 
 on hydrolysis with acids, its chief amount splits off in the form of amino- 
 acids. 
 
 1 See the work of Hausmann, Zeitsehr. f. physiol. Chem., 27 and 29; Henderson, 
 ibid., 27; Kossel and Kutscher, ibid., 30; Kutscher, ibid., 31, 38; Hart, ibid., 33 
 Giimbel, Hofmeister's Beitriige, 5; Rothera, ibid. 
 
 2 See the works given in footnote 1 and Blum, Zeitsehr. f. physiol. Chem., 30 
 Kossel, Her. d. d. chem. Gesellsch., 34, 3214; Hofmeister, Ergebnisse der Physiol. 
 Jahrg. I, Abt. 1, 759, which also contains the literature; Osborne and Harris, Journ 
 Amer. Chem. Soc, 25; and Giimbel, I.e. 
 
 3 Skraup and v. Hardt-Stremayr, Monatsh. f. Chem., 29, found lower results than 
 other investigators arid they found also that about two-thirds of the amide nitrogen 
 ■was readily split off and one-third slowly. 
 
 4 Ber. d. d. chem. Gesellsch, 43 and 44 and Journ. of biol. Chem., 9, 10 and 12. 
 
NITROGEN DISTRIBUTION. 79 
 
 By the action of nitrous acid upon proteins at least a partial deamidation 
 takes place and so-called desamino proteins arc obtained. The nitrogen expelled 
 originated from the XII,. groups according to the formula RNHi+HNOj=»ROH+ 
 
 Xj+IIjO. The amount of such nitrogen is generally only small, 1-2 per cent, 
 and for this reason it has been accepted thai such groups only occur in small 
 amounts in the proteins. This is probably true for a large number of proteins 
 but not for all and as example of these we will recall that Kossel and CAMERON ' 
 have shown that those protamines which contain no other hexone base besides 
 arginine although they have NH a groups at the ends in the guanidine residue 
 HN.CNH.NHj of the numerous arginine groups, do not yield any nitrogen on 
 Using v. Sltke's method while those protamines containing lysine do. We must 
 be very careful in drawing certain conclusions from the results obtained by the 
 action of nitrous acid upon proteins. 
 
 The nitrous acid can develop nitrogen from the XTH 2 groups of the acid amides 
 as well as from the XH 2 groups of the amino-acids. On the contrary no nitrogen 
 is evolved in v. Slyke's method from the guanidin groups and from the peptic! 
 combinations containing imid groups (see below). This is also the reason, as 
 remarked above, why those protamines containing only arginine do not jdeld 
 any nitrogen while those protamines which also contain lysine where there exist 
 free XH 2 groups do give off nitrogen. On hydrolyzing these deamidized pro- 
 tamines and also other deamidized proteins we therefore do not obtain any lysin 
 as shown by Skraup and collaborators and by Levites for certain proteins. 
 The quantity of monamino-acid nitrogen is therefore in such cases found to be 
 increased. 
 
 According to Osborne, Leavenworth and Brautlecht, 2 who worked 
 with plant proteins, the splitting off of XH 3 on the acid hydrolysis of the proteins 
 was very similar to the splitting off of X T H 3 from the acid amide asparagine, so 
 that the binding of XH 2 groups on the carboxyl groups seems very probable. 
 The quantity of NH 3 split off in the hydrolysis ran parallel With the amount 
 of asparagine and glutamic acid present and the quantity of XH 3 , split off by 
 hydrolysis with alkali corresponded nearly to the sum of the ammonia that was 
 split off by acid hydrolysis and one-half of the arginine nitrogen. According to 
 these investigators the XH 2 groups occur chiefly as acid-amide combinations. 
 
 A part of the nitrogen in the proteins occurs from the above, undoubt- 
 edly as NH 2 groups; the extent of this part, which is different in different 
 proteins cannot be positively given. The chief mass of the nitrogen 
 in the proteins, although other forms of binding occur, exists as imide- 
 like combinations of amino-acids united together and this will be com- 
 pletely developed in the following pages. 
 
 The sulphur occurs in the different proteins in very different amounts. 
 Certain of them, such as the protamines and apparently also certain 
 
 1 In regard to the action of nitrous acid upon proteins, their deamidation and cleav- 
 age products see C. Paal, Ber. d. d. Chem. Gesellsch., 29; H. Schiff, ibid., 1354; 0. 
 Loew, Chemiker Ztg., 1896 and O. Nasse, Pfliiger's Arch., 6; Treves and Salomone, 
 Bioch. Zeitschr, 7; Skraup, Monatsh. f. Chem., 27 and 28, with Hoernes, ibid., 27, 
 with Kaas, Annal. d. Chem. u. Pharm., 351; Lampel, Monatsh. f. Chem., 28; Traxl, 
 ibid., 2ft; Levites, Zeitschr. f. physiol. Chem., 43, and Bioch. Zeitschr., 20; D. v. 
 Slyke, foot-note 4, page 78; Kossel and Cameron, Zeitschr. f. physiol. Chem., 76; Kossel 
 and F. Weiss, ibid., 78. 
 
 2 Amer. Journ. of Physiol., 23. 
 
80 THE PROTEIN SUBSTANCES. 
 
 bacterial proteids, 1 are free from sulphur; some, such as gelatin and 
 elastin, are very poor in sulphur, while others, especially horn sub- 
 stances, are relatively rich in sulphur. On hydrolytic cleavage with 
 mineral acids, the sulphur of the protein substances is regularly, at 
 least in part, split off as cystine (K. Morner) or, with bodies poorer in 
 sulphur, as cysteine (Embden) , but this, according to Morner and Patten, 
 is a secondary formation. From certain protein substances a-thiolactic 
 acid (Suter, Friedmann, Frankel), which Morner claims is also pro- 
 duced secondarily, mercaptans and sulphureted hydrogen (Sieber and 
 Schoubenko, Rubner), and a body having the odor of ethyl sulphide 
 (Drechsel) have been obtained. 2 
 
 A part of the sulphur separates as potassium or sodium sulphide on 
 boiling with caustic potash or soda, and may be detected by lead acetate 
 and quantitatively determined (Fleitmann, Danilewsky, Kruger, 
 Fr. Schulz, Osborne, K. Morner 3 ). What remains can be detected 
 only after fusing with potassium nitrate and sodium carbonate and 
 testing for sulphates. The ratio between the sulphur split off by alkali 
 and that not split off is different in various proteins. No conclusions 
 can be drawn from this • in regard to the number of forms of combination 
 which the sulphur has in the protein molecule. As shown by K. Morner, 
 only about three-fourths of the sulphur in cystine can be split off by 
 alkali, and the same is true for the cystine-yielding complex of the pro- 
 tein substances. If the quantity of lead-blackening sulphur in a pro- 
 tein body be multiplied by f, we obtain the quantity corresponding to 
 the cystine sulphur in the body. By such calculation Morner found 
 in certain bodies, such as horn substance, seralbumin and serglobulin, 
 that the quantity of cystine sulphur and total sulphur were identical, 
 and therefore we have no reason for considering the sulphur in these 
 bodies as existing in more than one form of combination. In other 
 proteins, such as fibrinogen and ovalbumin, on the contrary, only one- 
 half or one-third of the sulphur appeared as cystine sulphur. 
 
 Just as in the products of acid hydrolysis of proteins we know of 
 two forms of oxygen bondage, the hydroxyl form OH and the carbonyl 
 
 1 See Nencki and Schaffer, Journ. f. prakt. Chem. (N. F.), 20, and M. Nencki, Ber. 
 d. d. chem. Gesellsch., 17. 
 
 2 K. Morner, Zeitschr. f. physiol. Chem., 28, 34, and 42; Patten, ibid., 39; Embden, 
 Urid., 32; Suter, ibid., 20; Friedmann, Hofmeister's Beitriige, 3; Sieber and Schou- 
 benko, Archiv d. sciences biol. de St. Pdtersbourg, 1; Rubner, Arch. f. Hygiene, 19; 
 Drechsel, Centralbl. f. Physiol., 10, 529; Frankel, Sitzungsber. d. Wien. Akad., 112, 
 II b, 1903. 
 
 * Fleitmann, Annal. dor Chem. und Pharm., 6fi; Danilewsky, Zeitschr. f. physiol. 
 •Chem., 7; Kruger's, Pflliger's Archiv, 43; F. Schulz, Zeitschr. f. physiol. Chem., 25; 
 Osborne, Connecticut Agric. Expt. Station Report 1900; Morner, 1. c. 
 
HYDROLYSES OF PROTEINS. 81 
 
 form in CONH; so according to Treat B. Johnson l two analogous forms 
 of sulphur bondage exist in the proteins, namely the mercaptan form 
 SH as in cystine and the form NH.CH.CS.NH corresponding to the oxygen 
 binding in the polypeptids (see page 86). He has in fact also prepared 
 thio-polypeptides from glycocoll and these were analogous to the corre- 
 sponding glycin polypeptids (see page 88) and like certain proteins 
 gave HoS on acid hydrolysis. 
 
 The constitution of the protein bodies is still unknown, although 
 the great advances made in the last few years have brought us very much 
 closer to the elucidation of the question. In studying the constitution 
 of the protein bodies they have been broken up in various ways into 
 simpler portions, and the methods used for this purpose have been of 
 different kinds. In such decompositions, for which only purified proteins 
 are to be used, first large atomic complexes — proteoses and peptones — 
 are obtained which still have protein characteristics, and these then 
 suffer further decomposition until finally we obtain simpler, generally 
 crystalline, or at least well-characterized, end products. 
 
 As to the products obtained by hydrolytic cleavage with mineral acids, 
 important investigations have been carried out by numerous older and 
 more recent experimenters. 2 Besides certain acids, which will be men- 
 tioned later and which occur in few cases only, we obtain the following: 
 monamino-acids such as glycocoll, alanine, aminovaleric acid, leu- 
 cine, isoleucine, serine, aspartic and glutamic acids, cysteine and its disul- 
 phide cystine, phenylalanine, tyrosine, pyrollidine — and oxypyrollidine 
 carboxylic acid, tryptophane and also the three hexone bases, histidine, 
 arginine and lysine, the two latter being diamino-acids. Besides these 
 also ammonia, sulphureted hydrogen, ethyl sulphide and melanoidins, 
 which latter seem to be secondary products, have been obtained. 
 
 On the hydrolysis with alkalies we obtain, after a preliminary forma- 
 tion of intermediary steps which will be discussed later, chiefly the same 
 cleavage products as in acid hydrolysis but with the exception that in 
 the alkali hydrolysis a considerable part of the amino-acids become 
 racemerized and therefore appear in optically inactive form while in the 
 acid hydrolysis chiefly optically active acids are obtained. Because 
 of the action of the alkali a part may suffer further decomposition which 
 loads to the formation of simpler cleavage products and ammonia. 
 
 On fusing proteins with caustic alkali, ammonia, methyl mercaptan and other 
 volatile products are evolved and other products are produced such as leucine, 
 
 1 Journ. of biol. Chem., 9. 
 
 2 In regard to the literature see O. Cohnheim, Chemie der Eiweisskorper, Braun- 
 schweig, 1911, and F. Hofmeister, Ergebnisse der Physiologie, Jahrg. I, Abt. 1, 759, 
 1902; E. Fischer, Untersuchungen uber Aminosauren, Polypeptide und Proteine (1899- 
 1906), Berlin, 1906. See also special references. 
 
82 THE PKOTEIN SUBSTANCES. 
 
 from which then volatile fatty acids such as acetic acid, valeric acid and also 
 butyric acid are formed, also tyrosine from which latter phenol is formed and 
 indol and skatol. 
 
 Most proteins are split by proteolytic enzymes in the same manner 
 as on hydrolysis with acids or alkalies, but more or less completely depend- 
 ent upon the kind of enzymes. In the first place proteoses and peptones 
 (see below) are formed, then also polypeptids and amino-acids of 
 various kinds, in certain cases also oxyphenylethylamine, diamines, and 
 a little ammonia and other bodies. 
 
 A great many substances are produced in the putrefaction of pro- 
 teins. First the same bodies as are formed in the decomposition by 
 means of proteolytic enzymes are produced, and then a further decom- 
 position occurs with the formation besides ammonia, carbon dioxide 
 and hydrogen, of a large number of bodies belonging in part to the aliphatic 
 and in part to the aromatic and heterocyclic series. 
 
 'To the aliphatic series belong volatile fatty acids and as shown 
 by Neuberg l and collaborators not only fatty acids of the normal chain 
 but also with branched chains, also optically active acids, also succinic 
 acid, methane, methyl mercaptan and others. To this series belongs 
 also the two putrefaction bases cadaverine and putrescine, produced from 
 the diamino acids, and also the so-called ptomaines or cadaver alkaloids 
 which may originate, at least in part, from other tissue constituents 
 and not from proteins. 
 
 The putrefactive products of the aromatic and heterocyclic series 
 originate from the corresponding amino-acids. From tyrosine the 
 aromatic oxy-acids such as p-oxyphenyl-propionic acid, the p-cresol, 
 phenol and oxyphenylethylamine are formed. The phenylalanine is the 
 mother substance of the phenylpropionic acid, the phenylacetic acid and 
 the phcnylethylamine. Indolpropionic acid, indolacetic acid, skatol 
 and indol originate from the tryptophane (indolaminopropionic acid) ; the 
 imidazolpropionic acid and imidazolethylamine originate from the histi- 
 dine. 2 
 
 By the moderate action of chlorine, bromine, or iodine upon proteins, 
 these halogens enter into more or less firm combination with the proteins 
 and according to the method of procedure we can prepare derivatives 
 having different but constant amounts of halogens. The proteins are 
 so changed that they do not split off sulphur on treatment with alkali, 
 nor do they respond to Millon's or Adamkiewicz-Hopkins reaction. 
 Side processes, oxidations and cleavages may also take place here. The 
 most striking fart seems to be a substitution of hydrogen by iodine in the 
 
 1 Bioch. Zeitsohr., 37, where the earlier works of Neuberg are cited. 
 : A< kermann, Zeitschr. f. physiol. Chem., (55. 
 
OXIDATION OF PROTEINS. 83 
 
 aromatic nucleus of tyrosine and also perhaps in the indol nucleus of 
 tryptophane and the imidazol nucleus of histidine. 1 Halogen proteins 
 occur, as will be shown later, in the animal kingdom, especially in the 
 albuminoid group and indeed iodized tyrosine (3-5 di-iodotyro-incj 
 has been isolated. 
 
 By the oxidation of protein by means of potassium permanganate, Maly 
 obtained an acid, ojcyprotosulphonic acid, C 51.21, H 6.89, X 14.59 8 1.77, O 23.21 
 per cent, which is not a cleavage product, but an oxidation product in which 
 the group SH is changed into SO;. OH. This acid does not give the proper color 
 reaction with Millon's reagent, yields no tyrosine or indol, but gives benzene 
 on fusing with alkali. On continued oxidation Maly obtained another acid, 
 peroxyproteic acid, which gives the biuret reaction, but is not precipitated by 
 most protein precipitants. The oxyprotein obtained by Schulz on the oxida- 
 tion of protein by hydrogen peroxide is closely related to oxyprotosulphonic 
 acid in composition and general characteristics, but contains lead-blackening 
 sulphur and gives Millon's reaction. The oxyprotein is claimed to be a pure 
 oxidation product, while in the production of oxyprotosulphonic acid Schulz 
 claims that a cleavage takes place. According to Buraczewski and Krauze 
 the oxyprotosulphonic acid is a mixture of several substances. According to 
 the investigations of v. Furth - there exist at least three different peroxyproteic 
 acids (from casein) which differ from each other by a different division of the 
 nitrogen in the molecule. On treatment with baryta-water we find that they 
 split off basic complexes and oxalic-acid groups, and new bodies, the dcsamino- 
 proteic acids, which give the biuret reaction, are produced. These later acids, which 
 on hydrolysis give benzoic acid but no diamino-acids, may be further oxidized, 
 which is not true of the peroxyproteic acids, and yield a new group of acids, the 
 kyroproteic acids, which give the biuret reaction, hold about one-half of their 
 nitrogen (11.08 per cent total nitrogen) in acid-aniide-like combination, but 
 yield neither basic products nor benzoic acid. 
 
 On the oxidation of gelatin or protein with permanganate we also obtain 
 oxaminic acid, oxamide, oxalic acid, oxaluric-acid amide, succinic acid, several 
 volatile fatty acids, and guanidine, which was first shown by Lossen as an oxida- 
 tion product. 3 
 
 On the oxidation of gelatin by ferrous sulphate and hydrogen peroxide 
 Blumenthal and Xeuberg have obtained acetone as a product, and Orgler 
 the same from ovalbumin. The action of ozone upon casein has been studied 
 
 1 In regard to the action of halogens upon proteins see Loew, Journ. f. prakt. 
 Chem. (N. F.), 31; Blum, Munch, med. Wochenschr., 1896; Blum and Vaubel, Journ. 
 f. prakt. Chem. (X. F.), 57; Liebrecht, Ber. d. deutsch. chem. Gesellsch., 30; Hop- 
 kins and Brook, Journ. of Physiol., 22; Hopkins and Pinkus, Ber. d. deutsch. chem. 
 Gesellsch., 31; Hofmeister, Zeitschr. f. physiol. Chem., 24; Kurajeff, ibid., 26; Oswald, 
 Hofmeister's Beitriige, 3; C. H. L. Schmidt, Zeitschr. f. physiol. Chem., 35, 36, 3"; 
 Xeuberg, Biochem. Zeitschr., 6: Pauly and Gundermann, Ber. d. d. chem. Gesellsch., 
 41, 43; Krzemecki, Chem. Centralbl., 1912; Pauly, Zeitschr. f. physiol. Chem., "6. 
 
 ' Maly, Sitzungsber, d. k. Akad. d. Wissenseh., Wien, 91 and 97. Also Monatshefte 
 f. Chem., 6 and 9. See also Bondzynski and Zoja, Zeitschr. f. physiol. Chem., 19; 
 Bernert, ibid., 26; Schulz, ibid,, 29; Buraczewski and Krauze, ibid, 76; v. Furth, Hof- 
 meister's Beitriige, 6. 
 
 3 Lessen, Annal. d. Chem. u. Pharm., 201; Kutscher, Zeitschr. f. physiol. Chem., 
 32; Zicksraf, ibid., 41; Seemann, ibid., 44; Kutscher and Schenck, Ber. d. d. chem. 
 Gesellsch.. 37 and 38. 
 
84 THE PROTEIN SUBSTANCES. 
 
 by Harries and Langheld j and the action of chlorine by Habermann and 
 Ehrenfeld and Panzer. 2 
 
 Nitric acid gives various yellow products, which turn reddish-brown in 
 alkaline solution. Of these we must especially mention the so-called xantho- 
 protein, besides nitrated proteoses and peptones. The xanthoprotein does not 
 yield any tyrosine on acid hydrolysis and it does not give the Millon or the lead- 
 blackening reactions. Among the cleavage products v. Furth 2 has obtained a 
 melanoidin substance, xanthomelanoidin. 
 
 On the nitration of the protamines (see below) Kossel 4 and co-workers have 
 obtained nitroprotamines which give nitroarginine on hydrolysis which shows that 
 the nitro groups have entered the guanidine groups of the arginine. 
 
 By the dry distillation of proteins we obtain a large number of decomposition 
 products having a disagreeable burned odor, and a porous glistening mass of carbon 
 containing nitrogen is left as a residue. The products of distillation are partly 
 an alkaline liquid which contains ammonium carbonate and acetate, ammonium 
 sulphide, ammonium cyanide, an inflammable oil, and other bodies, and a brown 
 oil which contains hydrocarbons, nitrogenized bases belonging to the aniline and 
 pyridine series, and a number of unknown substances. 
 
 The occurrence of protein substances which contain a carbohydrate 
 group has been known for a long time. The nature of this carbohydrate, 
 which can be split off by acid and which may amount to as much as 35 
 per cent, has been explained chiefly by the investigations of Friedrich 
 MfJLLER 5 and his students. They have shown that it is always an 
 amino-sugar, and generally glucosamine and perhaps galactosamine 
 as |an exception. That so-called true proteins also yield a carbohydrate 
 on hydrolytic cleavage was first shown by Pavy, using ovalbumin. The 
 continued investigations of Fr. Muller, and others have demonstrated 
 that in these cases the carbohydrate is also glucosamine. A carbohy- 
 drate complex, although sometimes only to a very slight amount, has 
 been detected in other proteins, ovoglobulin, serglobulin, seralbumin, 
 peaglobulin, albumin of the gramineae, yolk-proteid, and fibrin. In 
 other proteins, on the contrary, such, as edestin (of the hemp-seed) and 
 casein, myosin, pure fibrinogen, and ovovitellin, carbohydrates have 
 been sought for with negative results. All proteins hence do not contain 
 a carbohydrate group, and future investigators must therefore decide 
 whether the carbohydrate groups belong positively to the protein com- 
 
 1 Blumenthal and Neuberg, Deutsch. med. Wochenschr., 1901; Orgler, Hofmeister's 
 Beitrage, 1; Harries and Langheld, Zeitschr. f. physiol. Chem., 51. 
 
 2 Habermann and Ehrenfeld, Zeitschr. f. physiol. Chem., 32; Panzer, ibid., 33 
 and 34. 
 
 3 See Maly's Jahresber, 30, p. 24. 
 
 4 Kossel and Kennaway, Zeitschr. f. physiol. Chem., 72, with E. Wechsler, ibid., 
 78 and with F. Weiss, ibid., 78. 
 
 6 In regard to the literature on this subject see the work of Fr. Muller, Zeitschr. 
 f. Biologie, 42, and Langstein, Ergebnisse der Physiologie, Jahrg. I, Abt. 1, 63, Zeitschr. 
 f. physiol. Chem., 41, and Hofmeister's Beitrage, 6. See also Abderhalden, Bergell, 
 and DOrpinghaus, Zeitschr. f. physiol. Chem., 41. 
 
CARBON NUCLEI. 85 
 
 plex or whether they are united with the protein only as impurities. Sev- 
 eral observations 1 show that in working with crystalline proteins a con- 
 tamination with other protein substances is unfortunately not excluded, 
 and this must not be lost sight of, especially as the quantity of carbohy- 
 drates obtained is often very small. In this connection we must call 
 attention to the findings of Osborne and collal (orators that on recrystalliz- 
 ing ovalbumin six times they found that the glucosamine content was 
 reduced to 1.23 per cent while other investigators give 7-8-15 per cent. 
 Under these circumstances we are not warranted in considering the 
 carbohydrate groups as belonging to the carbon nucleus produced on 
 the destruction of the real protein complex. 
 
 The previously mentioned methods used in studying the structure 
 of the protein substances are not of the same value, but they in part 
 substantiate each other. Of these we must mention the hydrolysis 
 by means of boiling dilute mineral acids, or by proteolytic enzymes, 
 as the best methods for obtaining the carbon nuclei in the protein mole- 
 cule. The most important of the carbon nuclei obtained are as follows: 
 
 I. The Nuclei belonging to the Aliphatic Series. 
 
 A. Sulphur free, but containing nitrogen: 1. A guanidine residue (combined 
 with ornithine as arginine). 2. Monobasic monami no-acids: Glycocoll, alanine, 
 valine (amino valeric acid), leucine, and isoleucine. 3. Bibasic monamino- 
 acids: Aspartic acid and glutamic acid. 4. Oxymonamino-acids: serine oxy- 
 aminosuccinic acid and oxyaminosuberic acid. 5. Monobasic diamino-acids: 
 Diaminoacetic acid, ornithine (from arginine) and lysine. 6. Oxydiamino-acids: 
 Oxydiaminosuberic acid, oxydiaminosebacic acid, diaminotrioxydodecanoic acid, 
 caseanic and caseinic acids. 
 
 B. Sulphurized: Cysteine and its sulphide cystine, thiolactic acid (mercaptans, 
 and ethyl sulphide). 
 
 II. The Nuclei belonging to the Carbocyclic Series. 
 Phenylalanine and tyrosine. 
 
 m. The Nuclei belonging to the Heterocyclic Series. 
 Proline, oxyproline, tryptophane and histidine. 
 
 In regard to these carbon nuclei it must be remarked that they are 
 not all found in every protein body thus far investigated, and also that- 
 one and the same cleavage product, such, for example, as glycocoll, 
 leucine, tryosine, etc., is obtained in very variable amounts from differ- 
 ent protein substances. 
 
 It is very difficult to say to what extent all the above-mentioned 
 carbon nuclei exist in the protein molecule. It is not inconceivable 
 
 1 Osborne, D. B. Jones and Leavenworth, Amer. Journ. of Physiol., 24. 
 
86 THE PROTEIN SUBSTANCES, 
 
 that in the hydrolysis certain carbon nuclei may be secondarily formed 
 from others. Even if we admit the above, still it is undoubtedly true 
 that the chief cleavage products of the protein substances are amino- 
 acids. Emil Fischer has shown that the amino-acids have the property 
 of readily grouping together when water is split off and the amide group 
 of one amino-acid unites with the carboxyl group of the other. In 
 accord with this behavior we can, as Hofmeister 1 and others have 
 explained, but which was first proved by the epoch-making investiga- 
 tions of Emil Fischer, consider the proteins as chiefly formed by the 
 condensation of aminoTacids, where the amino-acids are united to each 
 other by means of imino-groups according to the following scheme: 
 
 — NH.CH.CO— NH.CH.CO NH.CH.CO— NH.CH.CO— 
 
 C4H9 CH 2 .C 6 H 4 (OH) CH2.COOH C3He.CH2.NH2 
 
 (Leucine) (Tyrosine) (Aspartic acid) (Lysine) 
 
 Such chaining of animo-acids is for the synthesis of protein-like bodies 
 of the very greatest importance. The older statements of Grimaux, 
 Schutzenberger and Pickering on the artificial preparation of pro- 
 tein-like substances where these investigators were able to prepare sub- 
 stances, which in many properties are similar to the proteins, from various 
 amino-acids either alone or mixed with other bodies such as biuret, alloxan, 
 xanthine, or ammonia. Of special interest are the investigations of 
 Curtius and his collaborators, in which they were able to prepare syn- 
 thetically the so-called biuret base (triglycyl-glycine ethyl ester) and sub- 
 sequently many other bodies which were related to the proteins. The 
 most important work on the chaining of amino-acids has been per- 
 formed by E. Fischer 2 and his pupils but especially by Abderhalden. 
 They have prepared a large number of complex bodies called polypeptides 
 by Fischer, which according to whether they contain two or more 
 amino-acid groups united together, are called di-, tri-, tetrapeptides, 
 etc. As examples of polypeptides we will mention — dipeptides: glycyl- 
 tvrosine, alanylglycine, leucylglycine, leucylcystine, prolylphenylalanine, 
 
 1 " Ueber den Bau des Eiweissmolekiils." Gesellsch. deutsch. Naturforscher und 
 Aertze, Verhandl. 1902, and Ergebnisse der Physiologie, Jahrg. I, Abt. 1, 759. 
 
 See Pickering, King's College, London, Physiol. Lab. Collect. Papers, 1897, which 
 also cites Grimaux'e work; also Journ. of Physiol., 18, and Proceed. Roy. Soc, 60, 
 1897; Schutzenberger, Compt. rend., 100 and 112; Curtius, Journ. f. prakt. Chem. 
 (N. li. 26 and 70, and Per. d. d. chem. Gesellsch., 37; Fischer and collaboral >rs, 
 Untersuchungen iiber Aminosauren, Polypeptide und Proteine (1S99-1906) Berlin 190G 
 and 1'ir. d. d. chern. Gesellsch., 39, 40, 41, 42, und Annal. d. Chem. u. Pharm., 354,. 
 :{.")7. :{<;:{. :{C>r>. :{*;*.>, 37."); see also Abderhalden, Ber. d. d. chem., Gesellsch, 40 to 43 and 
 Zeitschr. f. physiol. Chem., 72, 75, 77. 
 
POLYPEPTIDES. 87 
 
 leueylhistidine: tripeptides: di-glycylglycine, alanylglycyltyrosine, leu- 
 cyltryptophyl.ulutaniic acid; tetra peptides: glycyfglutamyldiglycine, dileu- 
 cylglycylglycine; pentapeptidee: tetraglycylglycine and leucyltriglycylgly- 
 cine; hexar and heptapeptides: leucyltetraglycylglycine and leucylpentagly- 
 cylglycine. The most complex polypeptide thus far prepared is an 
 octadecapeptide with 15 glycocoll and 3 leucine residues namely: Meucyl- 
 triglycyl-/-leucyltriglycyl-/-leucyl-octaglycylglycine = 
 
 XHoCH(C 4 H 9 )( X ).[XHCH L »C , ( )] 3 .XHCH(C 4 H 9 )CO. 
 [NHCH 2 CO]3.NHCH(C4H9)CO.[NHCH2CO]8.NHCH 2 COOH. 
 
 with the supposition that the amino-acids are here also combined together 
 in the imide binding. 
 
 The large number of amino-acids isolated from the proteins make 
 a large number of bindings possible. The number of possible combina- 
 tions is still further increased by the fact that all the amino-acids with 
 the exception of glycocoll contain at least one asymmetric carbon atom, 
 and this leads to the possible formation of stereochemically different 
 peptides. Thus in order to give a simple example, from two optically 
 active amino-acids, four different isomeric forms of clipeptides may occur, 
 namely (if we designate the optical antipodes by d- and /-) dd, 11, dl and Id. 
 Of these forms two can form a racemic dipeptide, thus c/-alanyl-c?-leucine + 
 /-alanyl-Meucine and rf-alanyl-Meucine + /-alanyl-f/-leucine. As the pro- 
 teins are optically active and on hydrolysis yield chiefly optically active 
 amino-acids, those polypeptides which can be built up from the natural 
 amino-acids of the proteins are of special importance in the study of the 
 constitution of the proteins. 
 
 Most of the artificial polypeptides are constructed from monamino- 
 mono-carboxylic acids, but polypeptides have also been prepared which 
 contain diamino-acids or aniino-dicarboxylic acids, and in this way the 
 number of possible polypeptides becomes still greater. With an aminodi- 
 carboxylic acid such as aspartic acid, other amino-acids can be bound 
 with one carboxyl group or with both, but also, if we start with aspara- 
 gine, they can be anchored with the amide group. If we start from the 
 acid amides we can also obtain a peptide which still contains the CONH2 
 group and on total hydrolysis yields NH3, like most proteins. A poly- 
 peptide of this kind is the tripeptide, glycl-/-asparaginyl-Meucine prepared 
 by E. Fischer and Koenigs. 
 
 NH 2 CH 2 CO.NHCHCO.NHCH(C4H 9 )COOH 
 
 CH2CONH2 
 
 In consideration of the form of binding of the sulphur in the proteins 
 it is interesting to consider the preparation of thiopolypeptids as performed 
 
88 THE PROTEIN SUBSTANCES. 
 
 by Treat B. Johnson. 1 He has prepared the following: thioglycylglycin- 
 thioamide NH2CH2CS.NHCH2CSNH2 which is analogous to glycylgly- 
 cinamide, NH2CH2CO.NHCH2CONH2 and also dithiopiperazine 
 
 .CH2.C0V 
 
 hn/ ">nh. 
 
 X CS.CH 2 
 
 Polypeptides of higher amino-fatty acids such as a-aminolauryla- 
 lanine, a-aminolaurylleucine and others have been prepared by Hop- 
 wood and Weizmann. 2 These peptids are different from the so-called 
 lipopeptids prepared by Bondi and his collaborators 3 which are not 
 chains of only amino-acids but combinations between a high fatty acid, 
 such as lauric- or palmitic acid and an amino-acid (glycocoll or alanine) 
 or a dipeptide (lauryl-alanylglycine) . 
 
 Methylated polypeptides such as methyl- and dimethylleucylglycine (E.. 
 Fischer and Gluud) and betaindiglycylglycine 
 
 (CH 3 ) 2 N. CH2CO.NHCH2CO.NHCH2CO 
 
 (Abderhalden and Kautzsch) are also known. 4 Amides of amino-acids and 
 dipeptides have been prepared by Bergell 5 and his co-workers. 
 
 The methods used by E. Fischer in the synthetical preparation of 
 polypeptides are chiefly as follows: 
 
 The first dipeptide prepared by him, glycylglycine, he obtained from glycocoll 
 ethyl ester which in water is transformed into a diketopiperazine, glycine anhy- 
 dride, according to the following equation: 
 
 /CH 2 .CO x 
 2(XH 2 CH 2 CO.O.C 2 H 6 ) = 2C 2 H 6 OH+ NH< >NH. 
 
 NCO.CH/ 
 
 By the action of dilute alkali upon this anhydride with the taking up of water the 
 glycylglycine NH 2 CH 2 CO.NHCH 2 COOH is formed, and according to this prin- 
 ciple other dipeptides can also be prepared. 
 
 Another method which has much greater application consists in the anchoring 
 of an amino-acid to a halogen of an acid radical, for example, by the action of 
 brompropionyl bromide or chloride upon glycocoll according to the following 
 equation : 
 
 CH 3 CHBrCOCl+NH 2 CH 2 COOH=HCl+CH 3 CHBrCO.NHCH 2 COOH 
 
 1 Journ. of biol. Chem., 9. 
 
 2 See Chem. Centralbl., 1911. 
 
 3 Biooh. Zeitschr., 17 and 23; see also Abderhalden and C. Funk, Zeitschr. f. physiol. 
 Chem., 66. 
 
 4 Fischer and Gluud, Annal. d. Chem. u. Pharm., 369; Abderhalden and Kautzsch, 
 Zeitschr. f. physiol. Chem., 72 and 75. 
 
 6 Ibid., 64, 65 and 67. 
 
POLYPEPTIDES. 89 
 
 (brompropionyl glycine). On subsequent treatment with ammonia the halogen 
 (Br) is replaced by XH 2 and the dipeptide alanylglycine 
 
 CH 3 CHXH 2 CO.XHCH 2 COOH+XHJ3r 
 
 is obtained. By the second action of brompropionylchloride and then treatment 
 with NH 3 we introduce a new alanyl group and the tripeptide alanyl-alanyl 
 glycine is prepared. By the action of a halogen derivative of an acid radical 
 another amino-acid residue can be introduced, and the chain of amino groups 
 can be thus extended. 
 
 The prolongation of the chain on the other side, namely, at the carboxyl, 
 Fischer has accomplished by chlorination of the amino-acids by special treatment 
 with phosphorus pentachloride. The carboxyl is thus transformed into COO, 
 while the acid at the same time fixes a molecule of HC1, for example CH3CHXH2HCI 
 
 COC1 
 Just as in the case of the carboxyl group of an amino-acid, so also can a poly- 
 peptide or its halogen acyl combination be chlorinated and then combined with 
 a new amino-acid, or a new peptide. As an example, Fischer, from a-brom- 
 isocapronyldiglycyl glycine, first prepared a-bromisocapronyldiglycylgh'cyl chlo- 
 ride, and then with diglycylglycine he obtained the heptapeptide leucyl- 
 pentaglycylglycine, 
 
 C4H 9 CH(XH 2 )CO.(NHCH,CO) 5 .XHCH 2 COOH. 
 
 For the various combinations of the optically active amino-acids to poly- 
 peptides it was important to possess methods of preparation of these amino-acids, 
 and for this purpose Fischer in many cases used the so-called Walden's reversion. 
 This consists in that one optically active amino-acid, for example the /-form, is 
 transformed into the corresponding halogen fatty acid by the action of nitrosyl 
 bromide, yielding the optical antipode the d-form. By the action of ammonia 
 the c/-amino-acid is now obtained which in the above-mentioned manner can be 
 retransformed into the /-form. Thus from (/-leucine we first obtain /-bromiso- 
 caproic acid and then by the action of ammonia /-leucine and in the preparation 
 of the polypeptides the same occurs. Thus, for example, if by reversion (/-leucine 
 is changed first into /-bromisoeapronyl chloride, if this last is combined with 
 /-leucine, then we obtain the dipeptide /-lcucyl-/-leucine. On combination with 
 diglycylglycine the tetrapeptide /-leucyl-diglycyl glycine is produced. Walden's 
 reversion does not take place with all amino-acids; other methods can also be 
 used to obtain the optical antipodes, such as the preparation of the alkaloidal 
 salts of the benzoyl or formyl combinations of the racemic amino-acids. 
 
 The /3-naphthalinsulpho combination of the polypeptides and peptones may 
 serve, as Fischer, Abderhalden and Funk l have shown, in explaining the 
 structure of these bodies. By the action of ^-naphthaline sulphochloride the 
 NHg groups existing at the beginning of the chain in the amino-acids react there- 
 with and on subsequent total hydrolysis this naphthaline-sulpho combination 
 remains unsplit. Thus for instance we can differentiate between glycylalanine 
 and alanylglycine because after hydrolysis in the first case we obtain naphthalin- 
 sulphoglycine and alanine and in the second naphthalin-sulphoalanine and 
 glycocoll (glycine). Tyrosine may, depending upon whether the XH 2 as well 
 as the OH groups are free or not or if only one is available, yield di- or mononaph- 
 thalinsulpho-derivatives and in this way we can also draw conclusions as to the 
 structure of tyrosine containing peptides. 
 
 The previously mentioned deamidation method of van Slyke (page 781 where 
 oxyacids are formed by the action of HX0 2 upon the XH, groups can also give 
 
 1 E. Fischer and Abderhalden, Ber. d. d. Chem. Gesellsch., 40; Abderhalden and 
 C. Funk, Zeitschr. f. physiol. Chem., 64. 
 
90 THE PROTEIN SUBSTANCES. 
 
 certain conclusions as to the structure of the peptides by comparing the hydro- 
 lytic products before and after deamidation. 
 
 A comparison of the artificially prepared polypeptides with the pro- 
 teins, and especially with the cleavage products of these last, the so-called 
 preoteoses and peptones, is of great interest in several respects, especially 
 in connection with certain reactions. For instance there are several 
 polypeptides which give the biuret reaction which is characteristic of 
 the proteins in general, and also several (polypeptides containing tyrosine), 
 which give Millon's reaction (see further on). The above-mentioned 
 octadecapeptide is precipitated by phosphotungstic acid, tannin and 
 ammonium sulphate ; we also know tri- and pentapeptides containing 
 tyrosine, which are very similar in properties to the proteoses. 
 
 The behavior of the polypeptides with proteolytic enzymes is of 
 great interest. As this interesting question will be thoroughly treated 
 in other chapters (I and VIII) it is sufficient here to recall that the 
 possibility that polypeptides as well as proteins are hydrolyzed by the 
 same enzymes, yielding amino-acids, is a weighty proof of the prob- 
 ability that in the proteins the amino-acid chains are of the same kind 
 as in the polypeptides. 
 
 A very important support for such a view is found in the occurrence 
 of polypeptides among the cleavage products of proteins, a find which 
 to a certain extent forms the reverse of the above-mentioned syntheses. 
 Such polypeptides are chiefly di- but also tri- and tetrapeptides. They 
 have been obtained in the hydrolytic products of silk waste, silk fibroin 
 and elastin (Fischer, Abderhalden), gelatin (Levene, Wallace and 
 Beatty) and of gliadin (Osborne and Clapp) 1 . Of special interest in 
 this connection are those polypeptides which like glycyl-d-alanine, 
 d-alanyl-glycine, glycyl-Z-tyrosine, Z-prolyl-Z-phenylalanine and d-alanyl- 
 glyeyl-Z-tyrosine, are identical with the corresponding synthetically pre- 
 pared polypeptides or at least very closely related. 
 
 We have therefore conclusive reasons for the assumption that in the 
 proteins, peptide bindings chiefly occur, i.e., a combination of the a- 
 amino-acids by means of the imide binding. It is also possible that 
 other linking may occur, and Fischer has also given expression to such 
 a possibility. Besides the above-mentioned imide binding another kind 
 must also without doubt exist in the proteins, namely, the anchoring 
 of the urea-forming group (the guanidine residue) with the ornithin 
 (diamino-valeric acid) by the imide binding. This imide linking is 
 
 1 Fischer and Abderhalden, Sitz. Ber. d. d. Berl. Akad. d. Wissensch, 30, and Ber. 
 d. d. Chem. Gesellsch., 89, 40; Abderhalden, Zeitschr. f. physiol. Chern., 02, 03 and 72; 
 Levene and Wallace, ibid., 47, with Beatty, Ber. d. d. Chem. Gesellsch., 39, and Bioch. 
 Zcitsrhr., 4; Osborne and Clapp, Amer. Journ. of Physiol., 18. 
 
CLASSIFICATION. 91 
 
 not, like the «-amino-acids, broken by trypsin, but rather by an enzyme 
 arginase, discovered by K08SEL and Dakin. 1 
 
 If the proteins are considered as consisting chiefly of peptide-like 
 complexes consisting of amino-acids united and containing also several 
 NH2 groups at the ends, it is readily understood that the proteins are 
 amphoteric electrolytes, like the amino-acids, which form salts with bases 
 as well as with acids and undergo hydrolytic dissociation. As we also 
 accept the theory that the protein molecule contains a large number of 
 ( ( M >II as well as XH L » groups, it follows that the proteins may be poly- 
 basic acids as well as polvacidic bases. The different proteins act in 
 this regard somewhat differently, thus the protamines are strongly basic 
 while casein behaves strikingly acid, and others take a certain mean 
 position. It is unfortunately impossible to base a classification of the 
 proteins upon this behavior, as well as upon chemical constitution. 
 The general properties, such as solubility and precipitation properties, 
 are too uncertain to aid us, and especially as in the investigations of 
 proteins we, as a rule, cannot decide whether we are dealing with a pure 
 or with a contaminated substance, namely, with mixtures. Experience 
 has shown that the solubility and precipitation properties of the proteins 
 are strongly influenced by the presence of other bodies, and under such 
 circumstances a proper classification, as demanded by science, is impos- 
 sible. On the other hand, a classification is important, and as the 
 ones used up to the present time" were based upon the solubility and 
 precipitation properties, we give the following schematic summary of the 
 chief groups of protein bodies: 
 
 I. Simple Proteins. 
 A. True Albuminous Bodies or Proteids. 
 
 Albumins, 
 Globulins 
 
 Seralbumin, 
 
 Lactalbumin, and others. 
 Fibrinogen, 
 Serglobulins, and others. 
 
 Phosphoproteins (Nucleoalbu- I Ovovitellin, 
 
 mins) [ Casein, and others. 
 
 (Coagulated proteins.) 
 
 Histones. 
 
 (Protamines?) 
 
 1 Zeitschr. f. physiol. Chem., 41. 
 
92 THE PROTEIN SUBSTANCES. 
 
 B. Albuminoids or Albumoids. 
 Keratins. 
 Elastin. 
 
 Collagen and glutin. 
 Reticulin. 
 
 (Fibroin, Sericin, Coilin, Cornein, Spongin, Byssus, and others.) 
 
 C. Cleavage Products of True Albuminous Bodies. 
 
 Alkali and Acid Albuminates. 
 Proteoses, Peptones, Polypeptides. 
 (Amino-acids.) 
 
 II. Compound Proteins. 
 
 . \Mucin substances, 
 
 [ Ichthulin, and others. 
 
 Nucleoproteins. 
 
 Chromoproteins 
 
 J Hcemoglobin, 
 | Hcemocyanin. 
 
 As there are two classifications recognized by English-speaking scien- 
 tists we will give the classifications adopted by the American Physio- 
 logical Society and the American Society of Biological Chemists and 
 also the British Medical Association. 
 
 Classification adopted by the American Physiological Society and 
 the American Society of Biological Chemists: 
 
 I. Simple Proteins. 
 
 A. Albumins. 
 
 B. Globulins. 
 
 C. Glutelins. 
 
 D. Prolamins (Alcohol-soluble proteins). 
 
 E. Albuminoids. 
 
 F. Histones. 
 
 G. Protamines. 
 
 II. Conjugated Proteins. 
 
 A. Nucleoproteins. 
 
 B. Glycoproteins. 
 
 C. Phosphoproteins. 
 
 D. Haemoglobins. 
 
 E. Lecithoproteins. 
 
CLASSIFICATION. 93 
 
 HI. Derived Proteins. 
 
 1. Primary Protein Derivatives. 
 
 A. Proteans. 
 
 B. Metaproteins. 
 
 C. Coagulated proteins. 
 
 2. Secondary Protein Derivatives. 
 
 A. Proteoses. 
 
 B. Peptones. 
 
 C. Peptides. 
 
 Classification of proteins adopted by the British Medical Association: 
 
 I. Simple Proteins. 
 
 1. Protamines. 
 
 2. Histones. 
 
 3. Albumins. 
 
 4. Globulins. 
 
 5. Glutelins. 
 
 6. Alcohol-soluble proteins. 
 
 7. Scleroproteins. 
 
 8. Phosphoproteins. 
 
 n. Conjugated Proteins. 
 
 1. Glucoproteins. 
 
 2. Nucleoproteins. 
 
 3. Chromoproteins. 
 
 HI. Products of Protein Hydrolysis. 
 
 1. Infraproteins. 
 
 2. Proteoses. 
 
 3. Peptones. 
 
 4. Polypeptides. 
 
 To this summary must be added that we often find in the investiga- 
 tions of animal fluids and tissues protein substances which do not fall 
 in with the above schemes, or are classified only with difficulty. At the 
 same time it must be remarked that bodies will be found which seem 
 to rank between the different groups, hence it is very difficult to sharply 
 divide these groups. 
 
94 THE PROTEIN SUBSTANCES. 
 
 I. Simple Proteins. 
 
 A. True Albuminous Bodies. 
 
 The albuminous bodies are never-failing constituents of the animal 
 and vegetable organisms. They are especially found in the animal 
 body, where they form the solid constituents of the muscles and of the 
 blood-serum, and they are so generally distributed that there are only 
 a few animal secretions and excretions, such as the tears, the perspira- 
 tion, and perhaps the urine, in which they are entirely absent or occur 
 only in traces. 
 
 All albuminous bodies contain carbon, hydrogen, nitrogen, oxygen 
 and sulphur; 1 a few contain also phosphorus. Iron is generally found 
 in traces in their ash. The elementary composition of the different 
 albuminous bodies varies a little, but the variations are within relatively 
 close limits. For the better-studied animal albuminous bodies the 
 following composition of the ash-free substance has been found: 
 
 C 50.5 —54.6 percent 
 
 H 6.5 — 7.3 " 
 
 N 15.0 —17.6 " 
 
 S 0.5 — 2,2 " 
 
 P 0.42— 0.85 " 
 
 21.50—23.50 " 
 
 The animal proteids are odorless, tasteless, and ordinarily amorphous. 
 The crystalloid spherules (D otter plattchen) occurring in the eggs of certain 
 fishes and amphibians, do not consist of pure proteids, but of albuminous 
 bodies containing large amounts of lecithin, which seem to be combined 
 with mineral substances. Crystalline proteids 2 have been prepared 
 from the seeds of various plants, and crystallized animal proteids (see 
 seralbumin and ovalbumin, Chapters V and XII) can be readily pre- 
 pared. In the dry condition the proteids appear as white powders, 
 or when in thin layers as yellowish, hard, transparent plates. A few 
 are soluble in water, others only soluble in salt or faintly alkaline or acid 
 solutions, while others are insoluble in these solvents. Solutions of 
 proteids are optically active and turn the plane of polarized light to the 
 left. All proteids when burned leave an ash, and it is therefore ques- 
 
 1 See foot-note 1, p. 80. 
 
 2 See Maschke, Journ. f. prakt. Chem., 74; Drechsel, ibid. (N. F.), 19; Griibler, 
 ibid. (N. F.), 23; Ritthausen, ibid. (N. F.), 25; Schmiedeberg, Zeitschr. f. physiol. 
 Chem., 1; Weyl., 1; ibid., 1. 
 
PROPERTIES. 95 
 
 tionable whether there exists any proteid body which is soluble in water 
 without the aid of mineral substances. Nevertheless it has not been 
 thus far successfully proved that a native proteid body can be prepared 
 perfectly free from mineral substances without changing its constitution 
 or its properties. 1 
 
 As previously stated, the albuminous bodies are amphoteric elec- 
 trolytes, and are polyacidie liases as well as polybasic acids. The base- 
 and acid-combining powers of various proteids have been the subject of 
 numerous investigations which cannot be given in short. In regard to 
 various methods used in such investigations as well as to the dissociation 
 of protein salts we refer especially to the work of T. B. Robertson. 2 
 
 The proteids can be salted out from their neutral solutions by neutral 
 salt? (NaCl, Na 2 S0 4 , MgS0 4 , [NH 4 ] 2 S0 4 , and many others) in sufficient 
 concentrations. By this salting out the properties remain unchanged 
 and the process is reversible, as on diminishing the concentration of the 
 salt the precipitate redissolves. The various proteids act in an entirely 
 different manner toward the same salt, and also for one and the same 
 proteid the behavior toward different neutral salts is different, as some 
 cause a precipitate, while others on the contrary do not precipitate. 
 
 The behavior of various proteids with one and the same salt, such 
 as MgSCU or (NEU^SO-i, is often made use of in the isolation of the 
 proteid, and special methods of separation are based' upon fractional 
 precipitation. It has been shown that these methods may lead to great 
 errors, and give good results only under special conditions. 3 
 
 The conditions are different from those of salting out, when the pro- 
 teid solution is precipitated by salts of the heavy metals. Here the 
 precipitates (often called metallic albuminates) are not true combina- 
 tions in constant proportions, but are rather to be considered as loose 
 adsorption compounds of the proteid with the salt. 4 These reactions 
 are irreversible in so far that dilution with water or removal of the salt 
 by means of dialysis does not restore the unchanged proteid. On the 
 other hand the precipitate, at least in certain cases may be redissolved 
 in an excess of the salt solution or of the proteid solution, and in this 
 sense the process is a reversible one. 
 
 1 See E. Harnack, Ber. d. d. chem. Gesellsch., 22, 23, 25, and 31; Werigo, Pfliiger's 
 Archiv, 48; Biilow, ibid., 58; Schulz, Die Grosse des Eiweissmolekuls, Jena, 1903. 
 
 2 Ergeb. d. Physiol. 10; Journ. of physical Chem., 14, 15, and Journ. of biol. Chem., 
 9. 
 
 3 See Cohnheim, Chemie der Eiweisskorper, 3. Aufl., 101 1 ; Pinkus, Journ. of Physiol., 
 27; Pauli, Hofmeister's Beitrage, 3, p. 225; Halsam, Journ. of Physiol., 32. 
 
 4 See Galeotti, Zeitschr. f. physiol. Chem., 40, 42, 44, and 48 and Bonamartini and 
 Lombardi, ibid., 58. See also the opposed views of Linpirh. ibid, 74. 
 
96 THE PROTEIN SUBSTANCES. 
 
 The precipitation of proteids and also other soluble proteins by salts 
 stands in close relation to their colloidal nature, and in this connec- 
 tion we refer to what has been said in Chapter I. The proteids do not 
 as a rule diffuse through animal membranes, or only to a very slight 
 extent, and hence have in most cases a pronounced colloidal nature in 
 Graham's sense. They belong to the hydrophile colloids; their solu- 
 tions show properties in common with those of typical colloids and also 
 true solutions. Certain of them, especially the peptones and a few 
 proteoses, which will be discussed later, seem to occupy an intermediate 
 position, as their solutions are characterized by a lesser viscosity and 
 greater diffusibility and filtration ability, are not readily precipitable 
 by alcohol or coagulable by heat, and are only partially precipitable 
 by salts. 
 
 The solutions (or suspensions) of proteids in water, the proteid hydro- 
 sols, are converted by various means into proteid hydrogels. Of these 
 means we must specially mention the following: nocking out with salts, 
 precipitation with alcohol, gelatinization of a gelatin solution on cool- 
 ing, and coagulation by the action of enzymes or heat. 
 
 Those proteids which occur, according to the common views, pre- 
 formed in the animal fluids and tissues, and which have been isolated 
 from these by indifferent chemical means without losing their original 
 properties, are called native proteids. New modifications having other 
 properties can be obtained from the native proteids by heating, by the 
 action of various chemical reagents such as acids, alkalies, alcohol, and 
 others, as well as by proteolytic enzymes. These new proteids are called 
 modified (" denaturierte ") proteids, to differentiate them from the native 
 proteids. 
 
 The precipitation with alcohol is a reversible reaction, as the pre- 
 cipitate redissolves on subsequent dilution with water. The proteids 
 are changed by the action of alcohol, some readily and quickly, others 
 with difficulty and very slowly; the proteid then becomes insoluble in 
 water and is modified. 
 
 On heating a solution of a native proteid it is modified at a different 
 temperature for each different proteid. With proper reaction and other 
 favorable conditions, for instance in the presence of neutral salts, most 
 proteids can in this way be precipitated in a solid form as coagulated 
 proteid. The hydrosol is converted into hydrogel, but as' a modification 
 takes place, this process is irreversible. The temperature at which 
 coagulation occurs is a variable one for the same protein under different 
 conditions of the experiment. The various temperatures at which 
 coagulation of different proteids occurs in neutral solutions containing 
 salt have in many cases given us good means for detecting and separating 
 proteids. The views in regard to the use of these means are somewhat 
 
PROPERTIES. 97 
 
 4 
 
 divided ' and the same applies to the question as to manner of heat 
 coagulation and the conditions under which it takes place. 
 
 The heat coagulation of a protein solution is dependent upon the hydrogen 
 ion concentration of the solution. According to Michaelis and co-workers 
 the optimum of the hydrogen ion concentration falls in the coagulation of a 
 protein solution (precipitation of the modified protein) with the isoelectric point 
 of the solution and the optimum of the flocking is not changed in regard to the 
 hydrogen ion concentration by changes in the protein concentration. According 
 to Sorensen and Jurgexsen, 2 on the contrary the optimal hydrogen ion con- 
 centration is the same as contained in a solution of the pure protein in pure 
 water caused by the electrolytic dissociation of this protein and is therefore 
 independent of the protein concentration. This hydrogen ion concentration, 
 according to these workers diminishes during the heat coagulation which they 
 consider as a proof of the diminution in the protein concentration of the solution. 
 
 A modification can be brought about also by the action of acids, 
 alkalies, or salts of the heavy metals, in certain cases by water alone, 
 and also by the action of alcohol, chloroform (Salkowski), and ether, 
 by violent shaking (Ramsden 3 ), etc. 
 
 An adsorption of proteids by a suspension colloid such as silicic acid, 
 colloidal ferric hydroxide and kaolin, can easily take place, and indeed the 
 proteid of a solution can be removed by the use of colloidal ferric hydrox- 
 ide or shaking with kaolin (Rona and Michaelis 4 ) . That the proteids 
 can serve as preventives in the precipitation of suspension colloids has 
 been mentioned in Chapter I. In the same manner a mastic suspension 
 is protected from the precipitating action of an electrolyte by an excess of 
 a proteid solution, while the reverse may be brought about, namely, a 
 proteid solution can be precipitated by a large quantity of mastic emulsion 
 in the presence of a proportionately small amount of electrolyte. The 
 method for the removal of proteid from solutions, as suggested by 
 Michaelis and Rona, 5 is based upon this behavior. 
 
 We have already discussed in Chapter I the electric charge of the 
 proteins under various conditions and the migration of these in electric 
 fields of currents. 
 
 1 See Halliburton, Journ. of Physiol., 5 and 11; Corin and Berard, Bull, de 1'Acad- 
 roy. de Belg., 15; Haycraft and Duggan, Brit. Med. Journ., 1890, and Proc. Roy. 
 Soc. Edin., 1889; Corin and Ansiaux, Bull, de l'Acad. roy. de Belg., 21; L. Fredericq, 
 Centralbl. f. Physiol., 3; Haycraft, ibid., 4; Hewlett, Journ. of Physiol., 13; Duclaux, 
 Annal. Institut Pasteur, 7. In regard to the relationship of the neutral salts to the 
 heat coagulation of albumins see also Starke, Sitzungsber. d. Gesellsch. f. Morph. u. 
 Physiol, in Munchen, 1897; Pauli, Pfliiger's Arch., 78. 
 
 2 See Ergeb. d. Physiol., 12 which contains the pertinent literature. 
 
 3 See Salkowski, Zeitschr. f. physiol. Chem., 31; Fr. Kriiger, Zeitschr. f. Biologie, 
 41; Loew and Aso, Bull. Coll. Agric, Tokio, 4; Ramsden, Zeitschr. f. physik. Chem., 
 47 and Arch. f. (anat. u.) Physiol., 1894. 
 
 4 Biochem. Zeitschr., 5. 
 
 6 Biochem. Zeitschr., 2, 3 and 4. 
 
98 THE PROTEIN SUBSTANCES. 
 
 The determination of the molecular weight of the proteids has been 
 attempted by various methods which are more or less uncertain. 1 There 
 is no doubt that the molecular weight of the proteids is very high, but 
 the statements about the size vary considerably. For the true proteids 
 thus far investigated, values ranging from 4000 — 6000 — 10,000 have 
 been found. 
 
 The general reactions for the proteids are very numerous, but only 
 the most important will be given here. To facilitate the study of these, 
 they have been divided into the two following groups. It must be 
 remarked that the precipitation reactions are not only applicable for 
 the soluble true proteids but also, more or less, for other soluble proteins 
 in general. The color reactions are applicable to all soluble or insoluble 
 proteins with few exceptions, which will be mentioned later. 
 
 Precipitation Reactions of the Proteid Bodies. 
 
 1. Coagulation Test. An alkaline proteid solution does not coagulate 
 on boiling, and a neutral solution only partly and incompletely; the reac- 
 tion must therefore be acid for coagulation. The neutral liquid is first 
 boiled and then the proper amount of acid added carefully. A fiocculent 
 precipitate is formed, and with proper technique the filtrate should be 
 water-clear. If dilute acetic acid be used for this test, the liquid must 
 first be boiled and then 1, 2, or 3 drops of acid added to each 10-15 cc, 
 depending on the amount of proteid present, and boiled before the addi- 
 tion of each drop. If dilute nitric acid (25 per cent) be used, then to 
 10-15 cc. of the previously boiled liquid }5-20 drops of the acid must 
 be added. If too little nitric acid be addei, a soluble combination of the 
 acid and proteid is formed, which is precipitated by more acid. A pro- 
 teid solution containing a small amount of salts must first be treated with 
 about 1 per cent NaCl, since the heating test may fail, especially on using 
 acetic acid, in the presence of only a slight amount of proteid. 
 
 2. Precipitation by Alcohol. The solution must not be alkaline, 
 but must be either neutral or faintly acid. It must, at the same time, 
 contain sufficient quantity of neutral salts. 
 
 3. Neutral Salts, such as Na2S(>4 or NaCl, when added to saturation 
 precipitate certain proteids but not others. Ammonium sulphate when 
 dissolved to saturation in the liquid is considered as the general pre- 
 cipitant for proteids. In the presence of free acetic or hydrochloric 
 acid the above-mentioned salts, NaCl or Na2S04, in sufficient con- 
 centration, are also general precipitants for the proteids. 
 
 4. Precipitation by Metallic Salts such as copper sulphate, ferric 
 chloride, neutral and basic lead acetate (in small amounts), mercuric 
 
 1 See especially F. N. Schulz, Die Grosse des Eivveissmoleciile, Jena, 1903. 
 
COLOR REACTIONS. 99 
 
 chloride and others. On this is based the use of proteids as antidotes 
 in poisoning with metallic salts. 
 
 5. Precipitation by Mineral Acids at Ordinary Temperatures. The 
 proteids are precipitated by the three ordinary mineral acids in proper 
 amounts, hut not by orthophosphoric acid. If nitric acid be placed in a 
 test-tube and the proteid solution be allowed to flow gently thereon, a 
 white opaque ring of precipitated proteid will form where the two liquids 
 meet (Heller's albumin test). 
 
 6. Precipitation by the so-called Alkaloid Reagents. To these belong 
 the precipitation by metaphosphoric acid and by hydroferrocyanic acid, 
 which is carried out by the aid of potassium ferrocyanide in a liquid 
 containing acetic acid; precipitation by phosphotungstic acid or phos- 
 phomolybdic acid in the presence of free mineral acids; precipitation 
 by potassium-mercuric iodide or potassium-bismuth iodide in solutions 
 acidified with hydrochloric acid; precipitation by tannic acid in acetic 
 acid solutions. The absence of neutral salts or the presence of free 
 mineral acids may prevent the appearance of the precipitate, but after 
 the addition of a sufficient quantity of sodium acetate the precipitate 
 will in both cases appear; precipitation by picric acid in solutions acid- 
 ified by organic acids. Proteids are also precipitated by trichloracetic 
 acid in 2-5 per cent solutions, by phenol, salicyl sulphonic acid, nucleic 
 acid, taurocholic acid and by chondroitin sulphuric acid in acid solutions. 
 
 Color Reactions for Proteid Bodies. 
 
 1. MUlon's Reaction. 1 A solution of mercury in nitric acid contain- 
 ing some nitrous acid gives a precipitate with proteid solutions which 
 at the ordinary temperature is slowly, but at the boiling-point more 
 quickly, colored red; and the solution may also be colored a feeble 
 or bright red. Solid albuminous bodies, when treated by this reagent, 
 give the same coloration. This reaction is due to the tyrosine and is 
 also given by other monohydroxyl benzene derivatives. According to 
 O. Nasse 2 it is best to use a solution of mercuric acetate which is treated 
 with a few drops of a 1 per cent solution of potassium or sodium nitrite; 
 previous to use a few drops of acetic acid are added. 
 
 2. Xanthoproteic Reaction. With strong nitric acid the albuminous 
 bodies give, on heating to boiling, yellow flakes or a yellow solution. 
 
 1 The reagent is prepared in the following way: 1 pt. mercury is dissolved in 2 pts. 
 nitric acid (of sp.gr. 1.42), first cold and then warmed. After complete solution of 
 the mercury add 1 volume of the solution to 2 volumes of water. Allow this to stand 
 a few hours and decant the supernatant liquid. 
 
 2 See O. Nasse, Sitzungsb. d. Naturforsch. Gesellsch. zu Halle, 1879, and Pfluger's 
 Arch., 83; see also Vaubel and Blum, Journ. f. prakt. Chem. (N. F.), 57. 
 
100 THE PROTEIN SUBSTANCES. 
 
 After making alkaline with ammonia or alkalies the color becomes orange- 
 yellow, due to the nitroderivatives of the benzene and indol groups. 
 
 3. Adomkieivicz's Reaction. If a little proteid is added to a mixture 
 of 1 vol. concentrated sulphuric acid and 2 vols, glacial acetic acid a 
 reddish-violet color is obtained slowly at ordinary temperatures, but 
 more quickly on heating. According to Hopkins and Cole x this reaction 
 takes place only on using glacial acetic acid containing glyoxylic acid. 
 According to them it is better to use a solution of glyoxylic acid, which 
 can be readily prepared by adding sodium amalgam to a concentrated 
 solution of oxalic acid and filtering after the discharge of the gas. A 
 dilute aqueous solution of the acid or some of the solid acid is added to the 
 proteid solution and sulphuric acid allowed to flow down the side of the 
 test-tube, when the reddish-violet color will appear at the point of con- 
 tact of the two liquids or on shaking the mixture. This color reaction, 
 which is generally called the Adamkiewicz-Hopkins reaction depends 
 upon the tryptophane and therefore gelatin (which does not contain 
 •any tryptophane) does not give this reaction. 
 
 As further color reactions we will mention: 4. Biuret Test. If a 
 proteid solution be first treated with caustic potash or soda and if then 
 a dilute copper-sulphate solution be added drop by drop, first a reddish 
 then a reddish-violet, and lastly a violet-blue, color is obtained. 5. Pro- 
 teids are soluble on heating with concentrated hydrochloric acid, produc- 
 ing a violet color, and when they are previously boiled with alcohol and 
 then washed with ether (Liebermann 2 ) they give a beautiful blue 
 solution. This blue color is due, according to Cole, 3 to a contamination 
 of the ether with glyoxylic acid, which reacts with the tryptophane 
 groups split off by the hydrochloric acid. The violet color obtained 
 with proteins not purified with ether is also considered as a tryptophane 
 reaction with the furfurol (oxymethylfurfurol) formed from the hexose 
 containing protein by the action of the concentrated hydrochloric 
 acid. Reaction 6 with concentrated sulphuric acid and sugar (in small 
 quantities) is explained in the same way. The beautiful red coloration 
 is connected with the formation of furfurol from the sugar. 7. With 
 7>-dimethylaminobenzaldehyde and concentrated sulphuric acid thepro- 
 teids give a beautiful reddish-violet or deep-violet coloration (0. Neu- 
 bai"er and E. Rohde 4 ). Other aldehydes also give color reactions 
 by virtue of the tryptophane group in proteins. Other reactions are 8; 
 Arnold's reaction 5 is a purple-violet coloration which the proteins give 
 
 1 Proceed. Roy. Soc, 08. 
 
 2 Centralbl. f. d. ined. Wissensch., 1887. 
 
 3 Journ. of Physiol., 30. 
 
 4 Zeitsche. f. physiol. Chem., 44. 
 
 Arnold, Zeitschr. f. physiol. Chem., 70. 
 
PROTEIN REACTIONS. 101 
 
 with sodium nitroprusside and ammonia. This reaction is not given by 
 all proteins and is due to the cystine groups. 9, Abderhalden and 
 Schmidt's reaction with triketohydrindenhydrate which gives a blue 
 coloration on boiling. The triketohydrindenhydrate (also called "Nin- 
 hydrin ") reacts with all compounds which have an amino group in 
 the a-position to the carboxyl, is according to Abderhalden and 
 Hchmidt l an excellent reagent for the detection of dialyzable amino- 
 acids and non-biuret giving amino-acid derivatives. They have been 
 able to detect by this reagent such non-biuret giving substances in the 
 dialysate on the dialysis of different animal fluids. They have also 
 determined the delicacy of this reagent with different amino-acids. 
 
 The biuret reaction is not only given by protein substances, but also by many 
 other bodies. According to H. Schiff 2 this reaction occurs with those bodies 
 containing amino groups, COXH 2 , CSNH«, C(NH)XH 2 or also CHaNHj, united 
 either directly by their carbon atoms or by means of a third carbon or nitrogen 
 atom. As examples of such bodies we can mention several diamines or amino- 
 amides, such as oximide, biuret, glycinamide, a- and /3-aminobutyramide, aspartic- 
 acid amide, etc., although we are not certain as to the conditions necessary for the 
 bringing about of this reaction. The biuret reaction alone is therefore no proof 
 as to the protein nature of a substance — for example, urobilin gives a very similar 
 color reaction — and a protein substance can still retain its protein nature, as by 
 the action of nitrous acid or by a splitting off of ammonia, although it does not 
 give the biuret reaction. 
 
 The delicacy of the various reagents differs for the different proteids, 
 and on this account it is impossible to give the degree of delicacy for 
 each reaction for all proteids. Of the precipitation reactions. Heller's 
 test (if we eliminate the peptones and certain proteoses) is recommended 
 in the first place for its delicacy, though it is not the most delicate reac- 
 tion, and because it can be performed so easily. Among the precipita- 
 tion reactions, that with basic lead acetate (when carefully and exactly 
 executed) and with alcohol and the reactions given under 6, are the most 
 delicate. The color reactions 1 to 4 show great delicacy in the order in 
 which they are given. 3 
 
 No proteid reaction is in itself characteristic, and, therefore, in testing 
 for proteids one reaction is not sufficient, but a number of precipitation 
 and color reactions must be employed. 
 
 For the quantitative estimation of coagulable proteids the determina- 
 tion by boiling with acetic acid can be performed with advantage, for 
 by operating carefully, it gives exact results. Treat the proteid solution 
 with a 1-2 per cent common-salt solution, or if the solution contains 
 large amounts of proteid dilute with the proper quantity of the above 
 salt solution, and then carefully neutralize with acetic acid. Now deter- 
 
 1 Zeitschr. f. physiol. Chem., 72 and 85. 
 
 2 Ber. d. d. chem. Gesellsch., 29 and 30. 
 
 3 In regard to the precipitation and color reactions of proteids with aniline dyes 
 zee Heidenhain, Pfluger's Arch., 90, 96. 
 
102 THE PROTEIN SUBSTANCES. 
 
 mine the quantity of acetic acid necessary- to completely precipitate 
 the proteids in small measured portions of the neutralized liquid which 
 have previously been heated on the water-bath, so that the nitrate does 
 nut respond to Heller't test. Now warm a larger weighed or meas- 
 ured quantity of the liquid on the water-bath, and add gradually the 
 required quantity of acetic acid, with constant stirring, and continue 
 heating for some time. Filter, wash with water, extract with alcohol 
 and then with ether, dry, weigh, incinerate, and weigh again. With 
 proper work the filtrate should not give Heller's test. This method 
 serves in most cases, and especially so in cases where other bodies are 
 to be quantitatively estimated in the filtrate. 
 
 In many cases good results may be obtained by precipitating all the 
 proteid with tannic acid and determining the nitrogen in the washed 
 precipitate by means of Kjeldahl's method. On multiplying the quan- 
 tity of nitrogen found by 6.25 we obtain the quantity of proteid. Many 
 other methods for the quantitative estimation of proteins have been 
 suggested. 
 
 The removal of proteids from a solution may in most cases be per- 
 formed by boiling with acetic acid. Small amounts of proteid which 
 remain in the filtrates may be separated by boiling with freshly pre- 
 cipitated lead carbonate or with ferric acetate, as described by Hof- 
 meister. 1 . If the liquid cannot be boiled, the proteid may be precipi- 
 tated by the very careful addition of lead acetate, or by the addition 
 of alcohol. If the liquid contains substances which are precipitated 
 by alcohol, such as glycogen, then the proteid may be removed by tri- 
 chloracetic acid as suggested by Obermayer and Frankel. 2 Recently 
 Michaelis and Rona have suggested a method for the removal of proteids 
 by using kaolin, colloidal ferric hydrate or a mastic emulsion. The 
 principle of these methods has already been given on page 97 and in 
 regard to the practical execution of the method we refer to the works 
 there cited. 
 
 In the precipitation of proteid as well as the quantitative estimation by means 
 of heat, it must be borne in mind, as shown by Spiro, 3 that several nitrogenous 
 substances, such as piperidine, pyridine, urea, etc., disturb the coagulation of the 
 proteids. 
 
 Synopsis of the Most Important Properties of the Different Groups of 
 
 Albuminous Bodies. 
 
 As it is not possible to base the classification of the different proteid 
 groups according to their constitution, we are obliged to make use of 
 their different solubilities and precipitation properties in their general 
 characterization. As there exist no sharp differences between the various 
 groups in this regard it is impossible to draw a sharp line between them. 
 
 Albumins. These bodies are soluble in water in neutral reaction and 
 are not precipitated by the addition of a little acid or alkali. They are 
 
 1 Zeitschr. f. physiol. Chem., 2 and 4. 
 
 2 Oberrnayer, Wien. med. Jahrb., 1888; Frankel, Pfluger's Arch., 52 and 55. 
 1 Zeitschr. f . physiol. Chem., 30. 
 
GLOBULINS. 103 
 
 precipitated by the addition of large quantities of mineral acids or metallic 
 -alts. Their solution in water coagulates on boiling in the presence of 
 
 neutral salts, but a weak saline solution does not. If NaCl or MgS< '4 
 is added to saturation to a neutral solution in water at the normal tem- 
 perature or at 30° C. no precipitate is formed; hut if acetic acid i> added 
 to this saturated solution the albumins readily separate. When ammonium 
 sulphate is added to one-half saturation the albumin solutions are not 
 precipitated at ordinary temperatures. Of all the native proteids the 
 albumins are the richest in sulphur, containing from 1.6 per cent to 2.2 
 per cent. So far as they have been investigated they do not yield any 
 glycocoll on acid hydrolysis. 
 
 Globulins. These substances are, as a rule, insoluble in water, but 
 dissolve in dilute neutral salt solutions. The globulins are precipitated 
 unchanged from these solutions by sufficient dilution with water, and 
 on heating they coagulate. The globulins dissolve in Avater on the addi- 
 tion of very little acid or alkali, and on neutralizing the solvent they 
 precipitate again. The solution in a minimum amount of alkali is pre- 
 cipitated by carbon dioxide, but the precipitate may in certain cases be 
 redissolved by an excess of the precipitant. The neutral solutions of the 
 globulins containing salts are partly or completely precipitated on satura- 
 tion with NaCl or MgSC>4 in substance at normal temperatures, depending 
 upon the kind of globulin. The globulins are completely precipitated 
 by half-saturating with ammonium sulphate. The globulins contain an 
 average amount of sulphur generally not below 1 per cent. As a differ- 
 ence between the albumins and globulins the latter yield glycocoll among 
 the hydrolytic cleavage products, and according to Obermayer and 
 "Willheim l they contain fewer NH2 groups at the end of the chain, as 
 determined by formol titration, as compared to the total number of N- 
 atoms. 
 
 A sharp line cannot be drawn between the albumins and globulins 
 from their properties and this is shown from the researches of Moll, 2 
 which show r that by the action of dilute alkalies and warmth upon 
 seralbumin it attains the properties of serglobulin. It is evident that 
 we are here dealing with a change of the external properties of the 
 albumins to a greater similarity to those of the globulins, and not with 
 a true transformation of the albumin, which is free from glycocoll, into 
 globulin which contains glycocoll. The same follows from the observa- 
 tions of others. 3 This is an instructive example of the subordinate impor- 
 tance the solubility and precipitation properties have in the differentia- 
 tion of various groups of proteids. 
 
 1 Bioch. Zeitschr., 38. 
 
 2 Moll, Hofmeister's Beitrage, 4 and 7; also Breinl, Arch. f. exp. Path. u. Pharm., 65. 
 •Obermayer and Willheim, 1. c; R. Gibson, Journ. of biol. Chem., 12. 
 
104 THE PROTEIN SUBSTANCES. 
 
 It is just as difficult to draw a sharp line between the globulins and 
 albuminates as it is between the globulins and albumins. Several globu- 
 lins are very readily changed by the action of very little acid, as also by 
 standing under water when in a precipitated condition, into albuminates, 
 and then become insoluble in neutral salt solutions. Osborne, 1 who has 
 closely studied this property in connection with edestin (from hemp-seed) , 
 considers the globulin, " globan," which has been made insoluble in salt 
 solution, as an intermediate step in the formation of the albuminate which is 
 produced by the hydrolytic action of the H ions of water or of the acid. 
 
 Phosphoproteins are a group of phosphorized proteids which occur 
 extensively in the animal and plant kingdoms and which include the 
 nucleoalbumins and the little-studied lecithalbumins. 
 
 Nucleoalburnins. These proteids behave like rather strong acids, 
 are nearly insoluble in water, but dissolve easily with the aid of a little 
 alkali and, in the* entire absence of phosphatides, contain also 
 phosphorus. Certain of the nucleoalbumins, resemble the globulins by 
 their solubility and precipitation properties. Others resemble the 
 albuminates, but differ from both of these groups by containing phos- 
 phorus. The> T stand close to the nucleoproteins by their content of 
 phosphorus, but differ from these in not yielding any purine bases on 
 cleavage. It has not yet been found possible to obtain from the neucleo- 
 albumins any proteid-free pseudonucleic acids corresponding to the 
 nucleic acids, but only acids rich in phosphorus, which always give the 
 proteid reactions. 2 For this reason the nucleoalbumins cannot be classed 
 as compound proteins. In peptic digestion a proteid rich in phosphorus 
 can be split off from most nucleoalbumins, and this has been called 
 para- or pseudonuclein. The claim made that the pseudonuclein is a 
 combination of proteid with metaphosphoric acid has been shown to be 
 incorrect by the investigations of Giertz. 3 
 
 ' The separation of pseudonuclein in peptic digestion is no doubt characteristic 
 of the nucleoalbumin group, but the non-appearance of the pseudonuclein pre- 
 cipitate does not entirely exclude the presence of a nucleoalbumin. The extent 
 of such a formation is dependent upon the intensity of the pepsin digestion, the 
 degree of acidity, and the relation between the nucleoalbumins and the digestive 
 fluids. The separation of a pseudonuclein may, as shown by Salkowski, 
 not occur even in the digestion of ordinary casein, and Wr6blewski and others 4 
 did not obtain any pseudonuclein at all in the digestion of the casein from human 
 milk. The most essential characteristic of this group of proteids is that they con- 
 tain phosphorus, and that the purine bases are absent in their cleavage products. 
 
 1 Zeitschr. f. physiol. Chem., 33. 
 
 2 Levene and Alsberg, ibid., 31; Salkowski, ibid., 32; Levene, ibid., 32; A. Reh, 
 Hofmeister'e Beitrage, 11; Dietrich, Bioch. Zeitschr., 22. 
 
 3 Giertz, Zeitschr. f. physiol. Chem., 28. 
 
 4 Salkowski, Pfliiper's Arch., 63; Wr6blewski, Beitrage zur Kenntnis des Frauen- 
 kasei'ns, Inaug.-Diss., Bern, 1894. 
 
LECITHALBUMINS. 105 
 
 The nucleoallmmins arc often confounded with nucleoproteina and 
 also with phosphorized glucoproteins. From the first class, they differ 
 by not yielding any purine bases when boiled with acids, and from the 
 second group by not yielding any reducing substance on the same treat- 
 ment. The best studied member of this group is the casein of milk, 
 which will be discussed, in detail in chapter XIII. 
 
 Neuberg and Pollak ' have artificially prepared phosphoproteins by the 
 action of phosphorus oxychloride upon an alkaline solution of lactalbumin or blood 
 globulin. The product obtained from lactalbumin was rather close to casein 
 in regard to composition and other properties. 
 
 Lecithalbumins. In the preparation of certain protein substances, 
 products are often obtained containing lecithin, and this lecithin (see 
 the phosphatides, Chapter IV) can be removed only with difficulty or in- 
 completely by a mixture of alcohol and ether. Ovovitellin (Chapter XII) 
 is such a protein body containing considerable lecithin, and Hoppe-Seyler 
 considers it a combination of proteid and lecithin. Similar substances 
 occur in fish-eggs. These last lecithalbumins often have the solubilities 
 of the globulins and are readily soluble in dilute salt solutions. The 
 behavior of the nucleoalbumin of the eggs of the perch shows how easily 
 this solubility may be changed. This nucleoalbumin, which contains con- 
 siderable amounts of lecithin, is readily soluble in dilute NaCl solution, 
 but at ordinary temperatures it is changed by 0.1 per cent HC1 almost 
 instantaneously and without splitting off lecithin, so that it becomes in- 
 soluble in dilute salt solutions (Hammarsten) . Liebermann 2 has obtained 
 ];roteids containing lecithin as an insoluble residue on the peptic digestion 
 of the mucous membrane of the stomach, liver, kidneys, lungs, and spleen. 
 He considers them as combinations of proteid and lecithin and calls them 
 lecithalbumins. Further investigation of these bodies is desirable. 
 
 Mayer and Terroine ' have shown that from lecithin emulsified in water and 
 a dialyzed solution of ovalbumin or dialyzed blood serum a precipitate can be 
 obtained which has some similarity to the lecithalbumins, but which in other 
 respects is so strikingly different that we are not justified in calling this pre- 
 cipitate lecithalbumin. 
 
 Nothing characteristic has thus far been found which differentiates 
 this group from others in the quantity of amino-acids split off on hydrol- 
 ysis. The members of this group differ essentially among themselves, 
 e.g., vitellin yields glycocoll while casein does not. 
 
 In order to give a review of the three above-mentioned groups of pro- 
 teids we give (page 106) a tabulation of the amounts of the amino-acids 
 
 1 Ber. d. d. chem. Gesellsch., 43 and Bioch. Zeitschr., 26. 
 
 2 Hoppe-Seyler, Med. chem. Untersuch., 1868; also Zeitschr. f. physiol. Chem., 13, 
 479; Hammarsten, Skand. Arch. f. Physiol., 17; Liebermann, Pfluger's Archiv, 50 
 and 54. 
 
 3 Compt. rend. soc. biol., 62. 
 
106 
 
 THE PROTEIN SUBSTANCES. 
 
 obtained on cleavage, but we must bear in mind that the figures, because 
 of the difficulty in the quantitative estimation, are not quite exact, but 
 must be considered as minimum values. As a representative of the glob- 
 ulin group we give fibrin, which is a coagulated globulin; and as repre- 
 sentative of the phosphoprotein group, besides casein also ovovitellin, 
 although not quite pure. The results are based on 100 parts of the 
 substances. 
 
 The proteins occurring in the plant kingdom either in the seeds or 
 tuberes belong chiefly to the globulins, which correspond essentially in 
 properties to the animal globulins. Besides these many other less 
 abundant proteins occur, which like the albumins are soluble in water, 
 while toward certain salts they behave like globulins. It is not clear 
 whether the phosphorized plant proteids contain their phosphorus as 
 impurities or whether they are the same as the animal phosphoproteins. 
 In seeds there occur also proteins, which are not represented in the animal 
 kingdom and of these we must especially mention the prolamines. They 
 are soluble in alcohol and besides this they are characterized by not 
 yielding any lysine on hydrolysis. 
 
 
 Lact- 
 albumin. 1 
 
 Ser- 
 albumin. 2 
 
 Ov- 
 albumin. 
 
 Ser- 
 globulins. 2 
 
 Fibrin. 5 
 
 Casein. 7 
 
 Vitelline* 
 
 Glycocoll 
 
 0.0 
 
 2.5 
 
 0.9 
 
 19.4 
 
 1.0 
 10.1 
 
 2.4 
 
 0.85 
 
 4.0 
 
 3.07 11 
 
 0.0 
 
 2.7 
 
 20.0 
 
 0.6 
 3.1 
 
 7.7 
 
 2.5 3 
 
 3.1 
 
 2.1 
 
 1.04 
 
 0.0 
 
 2.2 
 
 2.5 
 
 10.7 
 
 2.2 
 
 9.1 
 
 0.3 3 
 
 5.17 
 
 1.77 
 
 3.56 
 
 1.71 
 4.91 
 3.76 
 1.34 
 
 3.5 
 
 2.2 
 
 18.7 
 
 2.5 
 
 8.5 
 
 1.51 3 
 
 3.8 
 
 2.5 
 
 2.8 
 
 3.0 
 
 3.6 
 
 1.0 
 
 15.0 
 
 0.8 
 2.0 
 10.4 
 1.17 3 
 2.5 
 3.5 
 3.6 
 
 3.0 6 
 4.0 6 
 
 0.00 
 1.50 
 7.20 
 9.35 
 1.43 10 
 0.50 
 1.39 
 15.55 
 0.07 
 3.20 
 4.50 
 6.70 
 0.23 
 1.50 
 2.50 
 3.81 
 5.95 
 1.60 
 
 1.1« 
 
 Alanine 
 
 0.75 9 
 
 Valine 
 
 2.40 8 
 
 Leucine 
 
 11. 8 
 
 Isoleucine 
 
 Serine 
 
 Aspartic acid 
 
 Glutamic acid 
 
 Cystine 
 
 2.13 9 
 12. 95 9 
 
 Phenylalanine 
 
 Tyrosine 
 
 2.S 8 
 3.37 9 
 
 Proline 
 
 4.18 9 
 
 Oxvproline 
 
 Tryptophane 
 
 Histidine 
 
 1.90 9 
 
 
 7.46 9 
 
 
 4.81 9 
 
 Ammonia 
 
 1.25 9 
 
 
 
 1 Abderhalden and H. Pribram, Zeitschr. f . physiol. Chem., 21. 
 
 2 Abderhalden, Lehrb. d. physiol. Chem., 1909. 
 8 K. Morner, Zeitschr. f. physiol. Chem., 34. 
 
 4 Osborne and co-workers, Amer. Journ. of Physiol, 24. 
 
 6 Abderhalden and Voitinovici, Zeitschr. f. physiol. Chem., 52. 
 
 * Kutscher, Endprodukte der Trypsin Verdauung, Habit. Schrift., Marburg, 1899. 
 
 7 Osborne and Guest, Journ. of biol. Chem., 9. 
 
 8 Abderhalden and Hunter, Zeitschr. f. physiol. Chem., 48. 
 'Osborne and D. B. Jones, Amer. Journ. of Physiol., 24. 
 
 10 Levene and D. v. Slyke, Journ. of biol. Chem., 6. 
 
 11 Colorimetric determinations by Fasal, Bioch., Zeitschr, 44. 
 
COAGULATED PROTEINS. 
 
 107 
 
 In the tabulation of the hydrolytic products of plant proteins we 
 give edestin, of the hemp-seed, and legumin of the pea as examples of 
 globulins. The other three, hordein of barley, gliadin of wheat and zein 
 from corn belong to the prolamine group. 
 
 
 Edestin. 1 
 
 Legumin. 4 
 
 Hordein. 8 
 
 Gliadin. 9 
 
 Zein. 10 
 
 Glycocoll 
 
 3.8 
 
 3.6 
 
 5.6 2 
 20.9 
 
 0.33 
 
 4.5 
 18. 74 3 
 
 0.25 
 
 2.4 
 
 2.1 
 
 1.7 
 
 2.0 
 
 0.38 12 
 
 1.1 
 11.7 
 
 1.0 
 
 0.38 
 2.08 
 1.0 8 
 8.0 
 0.53 
 5.3 
 13.8 
 
 3.75 
 1.55 
 3.22 
 
 2.42 
 
 10.12 
 
 4.29 
 
 1.49 
 
 0.0 
 0.43 
 0.13 
 5.67 
 
 43. 19 7 
 
 5.03 
 
 1.67 
 
 13.73 
 
 1.28 
 2.16 
 0.00 
 
 4.87 
 
 0.68 9 
 
 2.0 
 
 3.34 
 
 6.62 
 
 0.13 
 
 0.58 
 
 43.66 
 0.45 
 2.35 
 1.20 
 
 13.22 
 
 1.00 
 0.61 
 3.16 
 0.00 
 • 5.22 
 
 0.0 
 
 Alanine 
 
 9.79 
 
 Valine 
 
 1.88 
 
 Leucine 
 
 19.55 
 
 Serine 
 
 1.02 
 
 Aspartic acid 
 
 Glutamic acid 
 
 Cystine 
 
 1.71 
 
 26.17 
 
 Phenylalanine 
 
 Tyrosine 
 
 Proline 
 
 6.55 
 
 3.55-10. i 11 
 
 9.04 
 
 Oxyproline 
 
 Histidine 
 
 0.82 
 
 Arginine. . . ; 
 
 1 55 
 
 Lysine 
 
 00 
 
 Ammonia 
 
 3 61 
 
 
 
 Coagulated Proteins. Proteins may be converted into the coagu- 
 lated condition by different means: by heating, by the action of alcohol, 
 especially in the presence of neutral salts, by chloroform, ether, and 
 metallic salts, and by the prolonged shaking of their solutions and in 
 certain cases, as in the conversion of fibrinogen into fibrin (Chapter V), 
 by the action of an enzyme. The nature of the processes which take 
 place during coagulation is unknown. The coagulated albuminous 
 bodies are insoluble in water, in neutral salt solutions, and dilute acids 
 or alkalies, at normal temperature. They are dissolved and converted 
 into albuminates by the action of dilute acids or alkalies, especially 
 on heating. 
 
 Coagulated proteins also seem to occur in animal tissues. We find, 
 at least in many organs such as the liver and other glands, proteins 
 
 1 Abderhalden, Zeitschr. f. physiol. Chem., 37 and 40. 
 
 2 Levene and D. v. Slyke, Journ. of biol. Chem., 6. 
 
 3 Osborne and Biddle Amer. Journ. of Physiol, 26. 
 
 4 Osborne and Clapp, Journ. of biol. Chem. 3. 
 8 Abderhalden and Babkin, ibid., 47. 
 
 6 Osborne and Clapp, Amer. Journ. of Physiol., 19. 
 
 7 Osborne and Jones, ibid., 26. 
 
 8 Osborne and Guest, Journ. of biol. Chem., 9. 
 
 9 Abderhalden and Samuely, Zeitschr. f. physiol. Chem., 44. 
 
 10 Osborne, Jones and Clapp, Amer. Journ. of Physiol., 26. 
 
 11 Kutscher, Zeitschr. f. physiol. Chem., 38. 
 
 12 Fasal, Bioch. Zeitschr., 44. 
 
10S THE PROTEIN SUBSTANCES. 
 
 which are not soluble in water, dilute salt solutions, or very dilute alkalies, 
 and only dissolve after being modified by strong alkalies. 
 
 Histones are basic proteins which stand to a certain extent between 
 the strongly basic protamines (see below) and the true proteins. Their 
 content of nitrogen varies between 16.5 and 19.8 per cent, and in certain 
 instances is not higher than in other proteins, especially vegetable pro- 
 teins. According to Kossel and Kutscher and Lawrow they are, 
 on the contrary, richer in basic nitrogen, and especially yield more arginine 
 than other proteins. Kossel first isolated a peculiar protein substance 
 from the red corpuscles of goose blood which was precipitated by ammonia, 
 and because of its similarity in certain regards to the peptones (in the 
 old sense) he called it histone. At the present time a number of very 
 different bodies are described as histones, such as those obtained from 
 nucleohistone (Lilienfeld), from haemoglobin (globin according to 
 Schulz), from mackerel spermatozoa (scombron according to Bang), 
 from the codfish (gadushistone according to Kossel and Kutscher), 
 from the burbot (lotahistone, Ehrstrom), and from the sea-urchin 
 (arbacin, Mathews), although probably not all are true histones, 
 especially the above mentioned globin. 1 
 
 Sulphur has been found in those histones in which it has been tested 
 for, but they do not, at least not all, give the lead-blackening test with 
 alkali and lead acetate. They give the biuret test, but as a rule only 
 a faint Millon's reaction. The goose-blood histone first studied by 
 Kossel gives the three following reactions: First, the neutral salt- 
 free solution does not coagulate on boiling; second, gives a precipitate 
 with ammonia which is insoluble in an excess of the precipitant; third, 
 gives a precipitate with nitric acid which disappears on heating and 
 reappears on cooling. 
 
 The different histones behave differently with these three reactions, 
 and hence they are not specific. On the other hand, all histones seem 
 to be precipitated from neutral solution by alkaloid reagents, and they 
 also produce precipitates in protein solutions. These two reactions are 
 likewise not specific for the histones, as the protamines have a similar 
 behavior. The histones differ from the protamines by having a much 
 lower content of basic nitrogen, and also probably by always containing 
 sulphur. True proteins, as Osborne's 2 edestan, also give these two 
 reactions; therefore it is impossible by qualitative tests alone to identify 
 
 1 Kossel Zeitschr. f. physiol. Chem., 8, and Sitzungbers. der Gesellsch. zur Beford. 
 d. ges Naturwiss. zu Marburg, 1897; Kossel and Kutscher, ibid., 1900, and Zeitschr. 
 f physiol. Chem., 31; Lawrow, ibid., 28, and Ber. d. d. chem. Gesellsch., 34; Lilienfeld, 
 Zeitschr. f. physiol. Chem., 18; Schulz, ibid., 24; Bang, ibid., 27; Ehrstrom, ibid.,,. 
 32; Mathews, ibid, 23. 
 
 2 Zeitschr. f. physiol. Chem., 33. 
 
HISTONES AND PROTAMINES. 109 
 
 a substance as a histone with positivencss. The large content of basic 
 nitrogen and of arginine is not a sure point of difference between hist ones 
 and other bodies. Histone yields little more than 40 per cent basic 
 nitrogen, while a heteroproteose yields about the same, namely, 39 
 per cent. Histone yields 14-15.5 per cent arginine (gadushistone), 
 and the lotahistone only 12 per cent. The vegetable proteid excelsin 
 is just as rich in arginine, namely, 14.14 per cent (Osborne and Clapp 1 ). 
 The characteristics of the histones, according to Kossel, are the above- 
 given reactions and the high amount of hexone bases, especially arginine. 
 The arginine nitrogen amounts to about 25 per cent of the total nitrogen, 
 the lysine N = 7-8.5 per cent and the histidine N = 1.8-4.5 per cent. No 
 proteids, with the exception of certain protamines, are known for the. 
 present, which contain as much arginine and lysine as the histones. On 
 hydrolytic cleavage the histones, like other proteins, but unlike the pro- 
 tamines, yield a large number of monamino-acids. Abderhalden and 
 Rona 2 obtained from thymus histone the following: leucine 11.8, 
 alanine 3.46, glycocoll 0.50, proline 1.46, phenylalanine 2.20, tyrosine 
 5.20, and glutamic acid 0.53 per cent. 
 
 On pepsin digestion the histones, according to Kossel and Pringle 3 
 yield so-called histone-peptone, which also contains 25 per cent of the 
 total nitrogen as arginine nitrogen. This histone-peptone differs from 
 the protamines in not giving a precipitate with proteid in neutral or 
 ammoniacal solution, but is precipitated in neutral reactions by sodium 
 picrate. This property is used in its isolation. 
 
 According to Kossel the histones are probably intermediate bodies 
 between the protamines and protein bodies on the demolition of the 
 latter, and if this be true, then it is not to be expected that a sharp dif- 
 ferentiation exists between histone and proteid, and for this reason it 
 is hardly possible for the present to give a precise definition for the 
 histones. 
 
 Protamines. In close relation to the proteins stands a group of 
 substances, the protamines, discovered by Miescher, which are desig- 
 nated by Kossel as the simplest proteins or as the nucleus of the pro- 
 tein bodies. Thus far they have been found only in combination with 
 nucleic acids in fish spermatozoa, 4 and the investigations of Kossel and 
 Weiss 5 have shown that the material from which the protamines are 
 
 1 Amer. Journ. of Physiol, 19 and 23. 
 
 2 Zeitschr. f. physiol. Chem., 41. 
 
 3 Ibid., 49. 
 
 4 Nelson, Arch. f. exp. Path. u. Pharm., 59, has recently shown that the body 
 called by him thymamine and prepared from the thymus glands, is a protamine, still 
 he has not given sufficient evidence of the protamine nature of the substance. 
 
 6 Zeitschr. f. physiol. Chem., 52. 
 
110 THE PROTEIN SUBSTANCES. 
 
 formed, at least in the salmon, is the muscle proteid. The question has 
 been raised whether the protamines are true proteids or not, and whether 
 it would not be more correct to consider them as cleavage products of 
 proteid, or as fractions thereof. According to the generally accepted 
 view we will treat them as true proteids. 
 
 Protamine was discovered by Miescher * in salmon spermatozoa. 
 Later Kossel and his pupils isolated and studied similar bases from the 
 spermatozoa of herring, sturgeon, mackerel, and other fishes. As all 
 these bases are not identical, Kossel uses the name protamines to designate 
 the group, and calls the individual protamines according to their origin 
 salmine, clwpeine, scombrine, sturine, cyprinine, cyclopterine, crenilabrine 
 etc. 
 
 They differ materially from the proteins by the fact that they yield 
 chiefly diamino-acids (always abundant arginine) as cleavage products, 
 and only a small amount of monamino-acids. They are strongly basic 
 substances rich in nitrogen (about 30 per cent or more) and have high 
 molecular weight. 
 
 The percentage composition of these bodies has not been satisfactorily 
 determined. As probable formulae we have for salmine C32H54N18O4 
 (Miescher, Schmideberg, Nelson), or C30H57H17CV (Kossel and Goto), 
 for clupeine C30H62N14C9, and for sturine C36H69H19O7 (Kossel) or 
 C34H71N17O9 (Goto), or according to Malenuck C27H55H13O7 for 
 sturine from Accipenser Guldenstadtii. On boiling with dilute mineral 
 acids as also by tryptic digestion, the protamines first yield peptone- 
 like substances called protones, from which simple products (amino-acids) 
 are derived on further cleavage. All protamines yield arginine, the four 
 protamines salmine, clupeine, cyclopterine, and sturine, yielding 87.4, 
 82.2, 62.5, and 58.2 per cent respectively. In the three protamines sal- 
 mine, clupeine and scombrine the arginine nitrogen, according to Kossel 
 and Pringle 2 , amounts to about 89 per cent of the total nitrogen. 
 Sturine yields besides this the two hexone bases lysine, 12 per cent, and 
 histidine, 12.9 per cent. Histidine has not been found in any other pro- 
 
 1 In regard to protamines, see Miescher, Histochemische und Physiologische 
 Arbeiten, Leipzig, 1897; Piccard, Ber. d. deutsch. chem. Gesellsch., 7; Schmiedeberg, 
 Arch. f. exp. Path. u. Pharm., 37; Kossel, Zeitschr. f. physiol. Chem., 22 (Ueber die 
 basischen Stoffe des Zellkerns), 25, 165 and 190, 26, 40, 44, and 69; and Sitzungsber. 
 der Gesellsch. zur Beford. der ges. Naturwiss. zu Marburg, 1S97; Berl. klin. Woch- 
 enschr., 1904; Kossel and Mathews, Zeitschr. f. physiol. Chem., 23 and 25; Kossel 
 and Kutscher, ibid., 31; Goto, ibid., 37; Kurajeff, ibid., 32; Morkowin, ibid., 28; 
 Kossel and Dakin, ibid., 40, 41, and 44; Malenuck, ibid., 57; Pringle, ibid., 49; Ken- 
 naway, ibid., 72; Cameron, ibid., 76; F. Weiss, 59, 60 and 78; Nelson, Arch. f. exp. 
 Path. u. Pharm., 59. 
 
 2 Zeitschr. f. physiol. Chem., 53. 
 
PROTAMINES. 
 
 Ill 
 
 tamine. The carp protamine, cyprinine, occurs in two different modi- 
 fications, namely, a- and jS-cyprinine. The a-cyprinine yields only little 
 arginine, 4.9 per cent, but the lysine content is pronounced, 28.8 per cent. 
 Of the total nitrogen 30.3 per cent exists as Lysine. Kossel and Dakin 
 have obtained from salmine the following cleavage products, namely, 
 arginine 87.4, serine 7.8, aminovaleric acid 4.3, and a-pyrrolidine-car- 
 boxylic (proline) acid 11 per cent, and according to them the salmine 
 contains about 10 mol. arginine, 2 mol. serine, 1 mol. aminovaleric acid, 
 and 2 mol. proline. Scombrine contains only arginine, alanine, and pro- 
 line. According to Kossel, every protamine contains only 2 or 3 mon- 
 amino-acids (clupeine contains 4) and for every 2 molecules of arginine 
 cnly 1 molecule of monamino-acid occurs. The above-mentioned pro- 
 tones (of the salmine group) are symmetrically constituted diarginides 
 with a monamino-acid — for example diarginylserine, diarginylproline etc., 
 — and these diarginides are united together forming the protamine. 
 Thus according to Kossel in clupeine we can accept the presence of 
 diarginylalanine, diarginylserine, diarginylproline, and diarginylvaline 
 (Kossel and Pringle). 
 
 The following summary according to Kossel gives a view of the 
 cleavage products of the protamines thus far investigated: 
 
 Alanine 
 
 Serine 
 
 Valine 
 
 Leucine 
 
 Arainine 
 
 Lysine 
 
 Histidine. . . . 
 
 Proline 
 
 Tyrosine . . . . 
 Tryptophane 
 
 Scom- 
 brine. 
 
 Salmine. 
 
 + 
 
 
 
 
 
 4- 
 
 
 
 + 
 
 
 
 
 
 
 
 + 
 4- 
 
 
 
 + 
 
 
 
 
 + 
 
 
 
 
 Clupeine. 
 
 Sturine. 
 
 Cyelop- 
 terine. 
 
 a-Cyp- 
 
 rinine. 
 
 0-Cyp- 
 rimne. 
 
 + 
 
 + 
 
 ? 
 
 ? 
 
 ? 
 
 + 
 
 
 
 ? 
 
 ? 
 
 ? 
 
 + 
 
 
 
 ? 
 
 + 
 
 4- 
 
 
 
 + 
 
 ? 
 
 9 
 
 ? 
 
 + 
 
 + 
 
 + 
 
 + 
 
 + 
 
 
 
 4- 
 
 
 
 + 
 
 4- 
 
 
 
 + 
 
 
 
 
 
 
 
 4- 
 
 
 
 ? 
 
 ? 
 
 ? 
 
 
 
 
 
 4- 
 
 6 
 
 6 
 
 
 
 
 
 + 
 
 
 
 
 
 Creni- 
 labrine. 
 
 ? 
 ? 
 ? 
 ? 
 
 + 
 
 
 ? 
 
 4- 
 
 
 
 Solutions of these bases in water are alkaline and have the property 
 of giving precipitates with ammoniacal solutions of proteins or primary 
 proteoses, but the researches of Hunter l show that these precipitates 
 are not histones, as generally considered. The salts with mineral acids 
 are soluble in water, but insoluble in alcohol and ether. They are more 
 or less readily precipitated by neutral salts (XaCl). Among the salts 
 of the protamines, the sulphate, picrate, and the double-platinum chloride 
 are the most important, and are used in the preparation of the protamines. 
 The protamines are, like the proteins, levogyrate; but by the action of 
 alkali the rotation is reduced or made to disappear, which according 
 
 1 Zeitschr. f. physiol. Chem. 53. 
 
112 THE PROTEIN SUBSTANCES. 
 
 to Kossel and Weiss l depends at least in part, to a racemisation of the 
 hexone bases, especially arginine within the protamine molecule. They 
 give the biuret test beautifully, but with the exception of cyclopterine, 
 /3-cyprinine and crenilabrine do not give Millon's reaction. The pro- 
 tamine salts are precipitated in neutral or even faintly alkaline solutions 
 by phosphotungstic acid, picric acid, chromic acid, and alkali ferrocy- 
 anides. 
 
 The protamines are prepared, according to Kossel, by extracting 
 the heads of the spermatozoa, which have previously been extracted 
 with alcohol and ether, with dilute sulphuric acid (1-2 per cent), filtering, 
 and precipitating with 4 vols, of alcohol. The sulphate may be purified 
 by repeated solution in water and precipitation with alcohol, and if 
 necessary, conversion into the picrate. For more details see the works 
 of Kossel and Malenuck. The double-platinum salt is best suited 
 for analysis and can be obtained, according to Goto, by precipitating 
 the methyl-alcohol solution of the protamine hydrochloride with plat- 
 inum chloride. Miescher also precipitates the base as a double-plat- 
 inum salt. 
 
 B. Albuminoids or Albumoids. 
 
 Under this name we collect into a special group all those protein 
 bodies which cannot be placed in either of the other groups. Most and 
 best studied of the bodies belonging to this group are important con- 
 stituents of the animal skeleton or the cutaneous structure. Some are 
 hardened secretions, and all occur as a rule in an insoluble state in the 
 organism, and they are distinguished in most cases by a pronounced 
 resistance to reagents which dissolve proteins, or to chemical reagents 
 in general, and it is due to these external properties that they are put 
 in a special group. From a purely chemical standpoint there is no 
 reason why they should be separated from the true proteids in a special 
 group. Most of the bodies belonging to the albuminoids have been 
 given on page 92. 
 
 The Keratins. Keratin is the chief constituent of the horny struc- 
 ture of the epidermis, of hair, wool, of the nails, hoofs, horns, feathers, 
 of tortoise shell, etc., etc. Keratin is also found as neurokeratin (Kuhne) 
 in the brain and nerves. The shell membrane of the hen's egg seems 
 also to consist of keratin, and according to Neumeister 2 the organic 
 matrix of the eggshells of various vertebrate animals belongs in most 
 cases to the keratin group. 
 
 1 Zeitschr. f. physiol. Chem., 59, 60, and 78. 
 
 2 Kuhne and Ewald, Verh. d. naturhistor.-med. Vereins zu Heidelberg (N. F.) ; 1; 
 also Kuhne and Chittenden, Zeitschr. f. Biologie, 26; Neumeister, ibid., 31. 
 
KERATINS. 113 
 
 It seems that there exist a number of keratins, and these form a special 
 group of bodies. This fact, together with the difficulty in isolating the 
 keratin from the tissues in a pure condition without a partial decom- 
 position, is sufficient explanation for the variation in the elementary 
 composition given below. As examples the analyses of a few tissues 
 rich in keratin and of keratins are given: 1 
 
 c h n s o 
 
 Human hair 43.72 6.34 15 06 4.95 29.93 (Rutherford 
 
 and Hawk) 
 
 Nail 51.00 6.94 17.51 2.80 21.75 (Mulder) 
 
 Neurokeratin.. . . 56.11-58.45 7.26-8.02 11.46-14.32 1.63-2.24 (Kuhne) 
 
 Neurokeratin 56.61 7.45 14.17 2.27 (Argiris) 
 
 Horn (average) . . 50.86 6.94 3.20 (Horbaczewski 
 
 Tortoise shell 54.89 6.56 16.77 2.22 19.56 (Mulder) 
 
 Shell membrane. . 49.78 6.64 16.43 4.25 22.50 (Lindvall) 
 
 Egg membrane . . 53.92 7.33 15.08 1.44 (Pregl) 
 
 (Scy Ilium) 
 
 Mohr 2 has determined the quantity of sulphur in various keratin 
 substances. Sulphur is in great part in loose combination, and it is 
 removed principally by the action of alkalies (as sulphides), or indeed in 
 part by boiling with water. Combs of lead after long usage become black, 
 and this is due to the action of the sulphur of the hair. On heating keratin 
 with water in sealed tubes to a temperature of 150° C. or higher, it dis- 
 solves with the elimination of sulphureted hydrogen or mercaptan 
 (Batjer), and the solution contains proteose-like substances (Kruken- 
 berg) called atmidkeratin and atmidkeratose by Bauer. 3 Keratin is 
 dissolved by alkalies, especially on warming, producing besides alkali 
 sulphides also proteose substances. 
 
 Besides the well-known cleavage products such as leucine, tyrosine, 
 aspartie acid, glutamic acid, arginine, and lysine, Fischer and Dorping- 
 haus 4 have found glycocoll, alanine, valine, proline, serine, phenyla- 
 lanine, and pyrrolidone-carboxylic acid (secondary from glutamic acid) 
 among the cleavage products of horn substances. Emmerling claims 
 to have found cystine as a sulphurized cleavage product, but K. Morner 
 
 1 Rutherford and Hawk, Journ. of biol. Chem., 3; Mulder, Versuch einer allgem. 
 physiol. Chem., Braunschweig, 1844-51; Kiihne, Zeitschr. f. Biologie, 26; Horbaczew- 
 ski, see Drechsel in Ladenburg's Handworterbuch. d. Chem., 3; Lindvall, Mary's 
 Jahresbericht, 1881; Argiris, Zeitschr. f. physiol. Chem., 54; Pregl., ibid., 56. 
 
 2 Zeitschr. f. physiol. Chem., 20. 
 
 3 Krukenberg, Untersuch. iiber d. chem. Bau d. Eiweisskorper, Sitzungsber. d. 
 Janaischen Gesellsch. f. Med. u. Naturwissensch., 1886; Bauer, Zeitschr. f. physiol. 
 Chem., 35. 
 
 4 Zeitschr. f. physiol. Chem., 36, which contains also the older literature. 
 
114 THE PROTEIN SUBSTANCES. 
 
 was the first to prove positively the abundant occurrence of cystine in 
 the cleavage products. Morner obtained from ox horn, human hair, 
 and the shell-membrane of the hen's egg 6.8, 13.92, and 7.62 per cent 
 cystine calculated on the basis of the dry substance. Buchtala l obtained 
 the following amounts of cystine from the respective keratin forma- 
 tions, namely, 12.98-14.53 per cent from human hair, 5.15 per cent from 
 nails, 7.98 per cent from horsehair, 3.20 per cent from horse hoofs, 7.27 
 per cent from ox hair, 5.37 per cent from ox hoofs, 7.22 from pig bristles, 
 2.17 per cent from pig hoofs, 6.30 per cent from goose feathers, 2.14 
 per cent from chicken spurs, 1.88 per cent from the epidermis scales of 
 chicken feet and 4.7 per cent from elephant epidermis. From the amount 
 of sulphur split off by alkali, Morner concludes that, at least in ox 
 horn and human hair, all the sulphur exists as cystine. Galimard 2 
 was able to get only a qualitative test for cystine in the keratin of the 
 adder eggs. Suter, Morner, and Friedmann 3 have obtained ct-thio- 
 lactic acid as a hydrolytic cleavage product of the keratin substances. 
 The last-mentioned investigator was also able to detect thioglycolic acid 
 in the cleavage products of wool. 
 
 The shell membrane of the hen's egg, and the eggshells of amphibians 
 and certain fishes are, as above mentioned, ordinarily classified as kera- 
 tins. These bodies among themselves, as well as on comparison with 
 other keratins, show a marked difference in properties, this being very 
 evident from the tabulation on page 115. 
 
 The large quantity of cystine in the keratins is considered as espe- 
 cially characteristic, and they differ in this regard from^he other proteins. 
 The shell membrane of the hen's egg behaves like a keratin in regard to the 
 large amount of cystine contained, but differs essentially by the absence 
 of tyrosine. It is remarkable that the egg membrane of the Selachii, 
 which biologically is analogous with ovokeratin, differs from the typical 
 keratins by the absence of cystine, while it contains, on the contrary, 
 large amounts of tyrosine. The typical keratins differ among them- 
 selves in regard to composition, thus the keratin from the sheep hoofs 
 contains 2 per cent phenylalanine, while this ammo-acid is absent in the 
 keratin of hair and feathers. It is difficult to say whether or not this 
 is due to a difference in the purity of the bodies or not. The keratins 
 investigated chemically, thus far, do not form a sufficient characteristic 
 group. 
 
 1 Morner, ibid., 34 and 42; Emmerlinp:, Ref. in Chemiker Zeitung, 1894; Buchtala, 
 Zeitschr. f. physiol. Chem., 52, fii*, and 78. 
 
 *Chem. Centralbl, II, 1905. 
 
 1 Suter, Zeitschr. f. physiol. Chem., 20; Morner. ibid., 42; Friedmann, Hofmeister's- 
 Beitrage, 2. 
 
KERATINS. 
 
 115 
 
 
 from 
 
 Ho ' 
 
 hair. 1 
 
 Keratin 
 from 
 Sheep 
 
 Keratin 
 from 
 
 Goose 
 Feathers 1 
 
 Keratin 
 from 
 Sheep 
 H< rn ' 
 
 Shell 
 
 MlMII- 
 
 brane 
 of the 
 Ben - 
 egg. 6 
 
 Mem- 
 brane 
 
 lin m 
 alt liar • .• 
 
 Tortoise 
 
 • 
 
 ( 'ht lone 
 
 Glycocoll 
 
 4.7 
 
 1.5 
 
 0.9 
 
 7 1 
 
 0.6 
 
 0.3 
 
 3.7 
 
 : 98 s 
 
 0.0 
 
 3.2 
 
 3.4 
 
 O.til 3 
 
 4.45* 
 
 1.12 3 
 
 0.58 
 
 1 10 
 
 2.80 
 11.5 
 
 0.1 
 
 2.3 
 12.9 
 
 7.3 
 
 2 . 9 
 
 4.4 
 
 2.6 
 1.8 
 
 ii g 
 8.0 
 0.4 
 1.1 
 2.3 
 
 0.0 
 3.6 
 
 3.5 
 
 0.45 
 1 .6 
 4.5 
 
 15.3 
 1.1 
 2.5 
 
 17.2 
 7.5 
 1.9 
 3.6 
 3.7 
 
 2.7 
 
 0.2 
 
 3.9 
 
 3 5 
 
 1.1 
 
 7 1 
 
 1.1 
 8.1 
 7.62' 
 
 0.0 
 
 4 
 
 2.6 
 3.2 
 
 5.8 
 
 2.3 
 
 7.2 
 
 ■; 
 
 3.3 
 
 10.6 
 4.4 
 1 7 
 3.2 
 3.7 
 
 l.i 36 
 
 .Alanine 
 
 
 
 5 ■•:; 
 
 Leucine 
 
 :; 26 
 
 Serine. 
 
 
 
 
 
 
 Glutamic acid 10 
 
 
 
 ( \>t ine 
 
 5.19 
 
 Phenylalanine 
 
 Tyrosine 
 
 1 (is 
 
 13 59 
 
 Proline 
 
 
 Histidine 
 
 
 
 Argininc 
 
 Lysine 
 
 — 
 
 
 
 Bodies occur in the animal kingdom which form to a certain extent 
 intermediate substances between coagulated protein and keratin. C. 
 Th. Morner u has detected such a body (albumoid) in the tracheal car- 
 tilage which forms a net-like trabecular tissue. This substance appears 
 to be related to the keratins on account of its solubilities and the quan- 
 tity of the sulphur (lead-blackening) it contains, while according to its 
 solubility in gastric juice it must stand close to the proteins. Another 
 substance, nearly like keratin, is the horny layer in the gizzard of birds. 
 According to J. Hedenius this substance is insoluble in gastric or pan- 
 creatic juice, and acts quite like keratin. According to K. B. Hofmann - 
 and Pregl, 12 who call this substance koilin, it does not yield any cystine 
 on hydrolysis, or at least not a determinable quantity. According to 
 others the quantity of cystine is very small. Buchtala 13 obtained only 
 
 1 Abderhalden and Wells, Zeitschr. f . physiol. Chem., 46. 
 
 2 Buchtala, ibid., 52. 
 * Argiris, ibid., 54. 
 
 4 Abderhalden and Voitinoviei, ibid., 52. 
 
 5 Abderhalden and Le Count, ibid., 46. 
 
 6 Abderhalden and Ebstein, ibid , 48. 
 
 7 Korner, ibid., 34 and 42. 
 
 8 Pregl, ibid., 56. 
 
 8 Buchtala, ibid., 74. 
 
 10 Abderhalden and Fuchs, Zeitschr. f. physiol. Chem., 57, have shown that the 
 same variety of keratin, on ageing of the horn structure, becomes somewhat poorer 
 in glutamic acid. 
 
 11 See Mary's Jahresber., 18. 
 
 12 Hedenius, Skand. Arch. f. Physiol., 3; Hofmann and Pregl, Zeitschr. f. physiol 
 Chem., 52. 
 
 13 Zeitschr. f. physiol. Chem., 69. 
 
11C THE PROTEIN SUBSTANCES. 
 
 a little more than 0.5 per cent pure crystalline cystine and on account 
 of the low cystine content as well as for other reasons the koilin differs 
 from the keratins. 
 
 Keratin is amorphous or takes the form of the tissues from which 
 it was prepared. It is insoluble in water, alcohol, or ether. On heating 
 with water to 150-200° C. it dissolves. It also dissolves gradually in 
 caustic alkalies, especially on heating. It is not dissolved by artificial 
 gastric juice or by trypsin solutions. Keratin gives the xanthoproteic 
 reaction, as well as the reaction with Millon's reagent, although the 
 latter is not always typical. 
 
 In the preparation of keratin a finely divided horny structure is 
 treated first with boiling water, then consecutively with diluted acid, 
 pepsin-hydrochloric acid, and alkaline trypsin solution, and, lastly, with 
 water, alcohol, and ether. 
 
 Elastin occurs in the connective tissue of higher animals, sometimes 
 in such large quantities that it forms a special tissue. It occurs most 
 abundantly in the cervical ligament (ligamentum nuchae). 
 
 Elastin used to be generally considered as a sulphur-free substance. 
 According to the investigations of Chittenden and Hart, it is a question 
 whether or not elastin contains sulphur, as it may have been removed by 
 the action of the alkali in its preparation. H. Schwarz has been able 
 by another method, to prepare an elastin containing sulphur, from the 
 aorta, and this sulphur can be removed by the action of alkalies, without 
 changing the properties of the elastin; and Zoja, Hedin, Bergh, 
 and Richards and Gies } have found that elastin contains sulphur. The 
 most trustworthy analyses of elastin from the cervical ligament (Nos. 
 1 and 2) and from the aorta (No. 3) have given the following results, 
 which compare well with each other: 
 
 s o 
 
 .... 21.94 (Horbaczewski 2 ) 
 
 .... 21.79 (Chittenden and Hart) 
 
 0.38 (H. Schwarz) 
 
 Zoja found 0.276 per cent sulphur and 16.96 per cent nitrogen in 
 elastin. Hedin and Bergh found different quantities of nitrogen in 
 aorta-elastin, depending upon whether Horbaczewski's or Schwarz's 
 method was used in its preparation. In the first case they found 15.44 
 per cent nitrogen and 0.55 per cent sulphur, and in the other 14.67 per 
 
 1 Chittenden and Hart, Zeitechr. f. Biologie,' 25; Schwarz, Zeitschr. f. physiol. 
 Chem., 18; Zoja, ibid., 2'.l; Bergh, ibid., 25; Hedin, ibid.; Richards and Gies, Amer. 
 Journ. of Physiol., 7. 
 
 2 Zeitschr. f. physiol. Chem., 6. 
 
 
 C 
 
 H 
 
 N 
 
 1. 
 
 54.32 
 
 6.99 
 
 16.75 
 
 2. 
 
 54.24 
 
 7.27 
 
 16.70 
 
 3. 
 
 53.95 
 
 7.03 
 
 16.67 
 
ELASTIN. 117 
 
 cent nitrogen and 0.66 per cent sulphur. Richards and Gies found 
 0.14 per cent sulphur and 16.87 per cent nitrogen in (last in. The ques- 
 tion whether elastin is a unit body still remains open. 
 
 The quantity of hydroh tic cleavage products are given in the table 
 on page 125. It is sufficient to here call attention to the fact that no 
 aspartic acid and only very little glutamic acid have been found. The 
 hexone bases have been obtained, but only in very small amounts, so 
 that the basic nitrogen represents only 3.34 per cent of the total nitro- 
 gen (Richards and Gies). From an elastin proteose, Wechsler l 
 obtained 1.86 per cent arginine, 0.5 per cent, histidine and 2.48 per cent 
 lysine. 
 
 Indol and skatol have not been found on the putrefaction of elastin, 2 
 but Schwarz, on the contrary, obtained indol, skatol, benzene, and 
 phenols on fusing aorta-elastin with caustic potash. On heating with 
 water in closed vessels, on boiling with dilute acids, or by the action of 
 proteolytic enzymes, the elastin dissolves and splits into two chief prod- 
 ucts, called by Horbaczewski hemielastin and elastinpeptone. Accord- 
 ing to Chittenden and Hart, these products correspond to two proteoses 
 designated by them protoelastose and deuteroelastose. The first is soluble 
 in cold water and separates out on heating, and its solution is precipi- 
 tated by mineral acid as well as by acetic acid and potassium ferrocyanide. 
 The aqueous solution of the other does not become cloudy on heating, 
 and is not precipitated by the above-mentioned reagents. 
 
 Pure elastin when dry is a yellowish-white powder; in the moist 
 state it appears like yellowish-white threads or membranes. It is insol- 
 uble in water, alcohol, or ether, and shows a resistance toward the action 
 of chemical reagents. It is not dissolved by strong caustic alkalies at 
 the ordinary temperature and only slowly at the boiling temperature. 
 It is very slowly attacked by cold concentrated sulphuric acid, but it 
 is relatively easily dissolved on warming with strong nitric acid. Elastins 
 of different origin act differently with cold concentrated hydrochloric 
 acid; for instance, elastin from the aorta dissolves readily therein, while 
 elastin from the ligamentum nucha?, at least from old animals, dissolves 
 with difficulty. Elastin is more readily dissolved by warm concen- 
 trated hydrochloric acid. It responds to the xanthoproteic reaction, 
 and to that with Millon's reagent, but not to the Adamkiewicz- 
 Hopkins reaction. 
 
 On account of its great resistance to chemical reagents, elastin may 
 be prepared (best from the ligamentum nuchae) in the fallowing way: 
 First boil with water, then with 1 per cent caustic potash, then again 
 
 1 Zeitschr. f. physiol. Chem., 67. 
 
 2 See Walchli, Journ. f. prSkt. Chem. (N. F.), 17. 
 
118 THE PROTEIN SUBSTANCES. 
 
 with water, and lastly with acetic acid. The residue is treated with cold 
 5 per cent hydrochloric acid for twenty-four hours, carefully washed 
 with water, boiled again with water, and then treated with alcohol and 
 ether. 
 
 In regard to the methods used by Scrwarz and by Richards and Gies, which 
 are somewhat different, we refer to the original publications. 
 
 Collagen, or gelatin-forming substance, occurs very extensively in 
 vertebrates. The flesh of cephalopods is also said to contain collagen. 1 
 Collagen is the chief constituent of the fibrils of the connective tissue and 
 (as ossein) of the organic substances of the bony structure. It also occurs 
 in the cartilaginous tissues as chief constituent; but it is here mixed 
 with other substances, producing what was formerly called chondrigen. 
 Collagen from different tissues has not quite the same composition, and 
 probably there are several varieties of collagen. 
 
 By continued boiling with w r ater (more easily in the presence of a 
 little acid) collagen is converted into gelatin. Hofmeister 2 found that 
 gelatin on being heated to 130° C. is again transformed into collagen; 
 and this last may be considered as the anhydride of gelatin. Collagen 
 and gelatin have about the same composition. 3 
 
 Collagen -50 . 75 
 
 Gelatin (commercial) .... 49.38 
 
 Gelatin from tendons.. . . 50.11 
 
 Gelatin from ligaments. . . 50.49 
 
 Fish glue (isinglass) 48.69 
 
 Gelatins of different origin show a somewhat variable composition, 
 which seems to indicate the occurrence of different collagens. It is diffi- 
 cult to say whether the variable content of sulphur is due to a contami- 
 nation with a substance rich in sulphur or to a splitting off of loosely 
 combined sulphur during the purification. C. Morner 4 has prepared 
 a typical gelatin containing only 0.2 per cent of sulphur by a method 
 which eliminated any possible changes due to reagents. 
 
 Sadikoff 5 has prepared gelatins by various methods from tendons and 
 from cartilage. Those from tendons, some of which were prepared after pre- 
 vious tryptic digestion, some after treatment with 0.25 per cent caustic potash, 
 and some after treatment with sodium hydroxide and then carbonate, showed 
 
 6.47 
 
 17.86 
 
 24.92 (Hofmeister) 
 
 6.80 
 
 17.97 
 
 0.70 25.13 (Chittenden) 
 
 6.56 
 
 17.81 
 
 0.26 25.26 (van Name) 
 
 6.71 
 
 17.90 
 
 . 57 24 . 33 (Richards and Gies) 
 
 6.76 
 
 17.68 
 
 — — (Faust) 
 
 1 Hoppe-Seyler, Physiol. Chem., p. 97. 
 
 2 Zeitschr. f. physiol. Cheni., 2. 
 
 a Hofmeister, 1. c; Chittenden and Solley, Journ. of Physiol., 12; van Name, 
 Journ. of Exper. Med., 2; Richards and Gies, Amer. Journ. of Physiol., 8; Faust, 
 Arch. f. exp. Path. u. Pharm., 41. 
 
 4 Zdtechr. f. physiol. Chem., 28. 
 
 f Tbid., 89 and 41. 
 
COLLAGENS. 119 
 
 somewhat differenl physical properties among each other, hut had about the same 
 elementary composition, with 0.34 <!.;">:! per cent sulphur. Sadikoff stems to 
 
 think that the gelatins prepared up to this time were perhaps not unit bodies 
 but were possibly mixtures. The bodies prepared by Sadikoff from cartilage 
 he calls glutting, because they were essentially different from the other gelatins 
 or glutins. They were poorer in carbon and nitrogen, 17.17 to 17. s7 per cent, 
 but somewhat richer in sulphur, 0.53-0.718 per cent, than the tendon glutin. 
 The gluteins differ also from the glutins in thai on boiling with a mineral acid 
 they have a faint reducing action, and also in that they give a color reaction 
 with phloroglucin-hydrochloric acid which is probably due to contamination. The 
 glutins differ from the gluteins by a different behavior with certain salts. 
 
 The decomposition products of the collagens are the sum" as those of 
 the gelatins ami will be found in the table on page 1?5. ( >f special 
 mention is the fact that gelatin contains no tyrosine and tryptophane 
 but does yield considerable glycocoll. This latter substance has, because 
 of its sweet taste, been called gelatin sugar. Skraup l has obtained on 
 the hydrolytic cleavage of gelatin a crystalline acid having the formula 
 C12H25N5O10, which he calls glutinic acid. Gelatin yields considerable 
 basic nitrogen, according to Hausmann, 2 35.83 per cent of the total 
 nitrogen. It also yields considerable arginine (9.3 per cent), lysine 5-G 
 per cent, but only little histidine (0.4 per cent). The aromatic group in 
 g latin is therefore, as directly shown by Fischer and also by Spiro, 3 
 represented by phenylalanine. 
 
 Collagen is insoluble in w r ater, salt solutions, and dilute acids and 
 alkalies, but it swells up in dilute acids. By continued boiling with 
 water it is converted into gelatin. Various collagens are converted into 
 gelatin with varying readiness; the formation of gelatin occurs also 
 from difficultly soluble collagens by continuous boiling with water. 
 Collagen is dissolved by the gastric juice and also by the pancreatic 
 juice ( trypsin solution) when it has previously been treated with acid 
 or heated with water above 70° C. 4 By the action of ferrous sulphate 
 corrosive sublimate, or tannic acid, collagen shrinks greatly. Collagen 
 treated by these bodies does not putrefy, and tannic acid is therefore of 
 great importance in the preparation of leather. 
 
 Gelatin or glutin is colorless, amorphous, and transparent in thin 
 layers. It swells in cold water without dissolving. It dissolves in warm 
 water, forming a sticky liquid, which solidifies on cooling when sufficiently 
 concentrated. As Pauli and Rona 5 have shown, various bodies may 
 have a different influence upon the gelatinization-point of a gelatin 
 
 1 Monatshefte f. Chem., 26. 
 
 2 Zeitsehr. f. physiol. Chem., 27. 
 
 3 Fischer, Levene and Aders, Zeitschr. f. physiol. Chem., 35; Spiro, Hofmeister's 
 Beitrafre, 1. 
 
 4 Kiihne and Ewald, Verh. d. Xaturhist. Med. Vereins in Heidelberg, 1877, 1. 
 
 5 Hofmeister's Beitrage, 2. 
 
120 THE PROTEIN SUBSTANCES. 
 
 solution; thus certain substances such as sulphates, citrates, acetates, 
 and glycerin may accelerate, while the chlorides, chlorates, bromides, 
 alcohol, and urea retard, this power. 
 
 Gelatin solutions are not precipitated on boiling, or by mineral 
 acids, acetic acid, alum, basic lead acetate, or metallic salts in general. A 
 gelatin solution acidified wjth acetic acid may be precipitated by potas- 
 sium ferrocyanide on carefully adding the reagent. Gelatin solutions 
 are precipitated by tannic acid in the presence of salt, and according to 
 Trunkel l completely if the gelatin and tannic acid are in» the propor- 
 tion 1 : 0.7. According to him the precipitation is not due to a chemical 
 combination but to an adsorption phenomenon. Solutions of gelatin 
 in water are also precipitated by acetic acid and common salt in sub- 
 stance; mercuric chloride in the presence of HC1 and NaCl; by meta- 
 phosphoric acid and phosphomolybdic acid in the presence of acid; 
 and lastly also by alcohol, especially when neutral salts are present. 
 Gelatin solutions do not diffuse. Gelatin gives the biuret reaction, 
 but not Adamkiewicz-Hopkins reaction. It gives Millon's reaction 
 and the xanthoproteic reaction so faintly that they probably occur from 
 impurities consisting of proteids. According to C. Morner, pure gelatin 
 gives a beautiful Millon's reaction, if not too much reagent is added. 
 In the other case no reaction or only a faint one is obtained. 
 
 By continued boiling with water gelatin is converted into a non- 
 gelatinizing modification called /3-glutin by Nasse. According to Nasse 
 and Kruger the specific rotatory power is hereby reduced from — 167.5° 
 to about— 136°. 2 According to Trunkel, who has especially studied 
 the rotation behavior of gelatin, the rotation of /3-glutin is less than the 
 ordinary a-glutin. On prolonged boiling with water, especially in the 
 presence of dilute acids, also in the gastric or tryptic digestion, the gelatin 
 is transformed into gelatin proteoses, so-called gelatoses and gelatin 
 peptones, which diffuse more or less readily. 
 
 According to Hofmeister two new substances, semiglutin and hemicollin, 
 are formed. The former is insoluble in alcohol of 70-80 per cent and is precipitated 
 by platinum chloride. The latter, which is not precipitated by platinum chloride, 
 is soluble in alcohol. Chittenden and Solley 3 have obtained in the peptic 
 and tryptic digestion a proto- and a deutero-gelatose, besides a true peptone. The 
 elementary composition of these gelatoses does not essentially differ from that of 
 the gelatin. 
 
 Paal * has prepared gelatin-peptone hydrochlorides from gelatin by the 
 action of dilute hydrochloric acid. These salts are partly soluble in ethyl and 
 
 1 Bioch. Zeitschr., 26. 
 
 2 Nasse and Krtiger, Mary's Jahresber., 19, p. 29. In regard to the rotation of 
 /3-glutin. see Framm, Printer's Arch., 68; Trunkel, 1. c. 
 
 3 Hofmeister, 1. c; Chittenden and Solley, 1. c. 
 1 Her. d. deutsch. chern. Gesellsch., 25. 
 
RETICUUN. 121 
 
 Diethyl alcohol, and partly insoluble 1 herein. The peptones obtained from 
 these salts contain less carbon and more hydrogen than the gelatin from which 
 they originated, showing that hydration has taken place. The molecular weight 
 of the gelatin peptone as determined by PaAL, by RaOULT's cryoseopic method, 
 was 200 to 352, while that for gelatin was 878 to 960. The gelatin peptones 
 isolated by Siegfried and his pupils which will be discussed below, are of great 
 interest. 
 
 Collagen (contaminated with mucoid) may be obtained from bones by 
 extracting them with hydrochloric acid (which dissolves the earthy 
 phosphates) and then carefully washing the acid out with water. It 
 may be obtained from tendons by extracting with lime-water or dilute 
 alkali (which dissolve the proteids and mucin), and then thoroughly 
 washing with water. Gelatin is obtained by boiling collagen with water. 
 The finest commercial gelatin always contains a little proteid, which 
 may be removed by allowing the finely divided gelatin to swell up in 
 water and thoroughly extracting with large quantities of fresh water. 
 Then dissolve in warm water and precipitate with alcohol. 
 
 Collagen may also be purified from proteids, as suggested by van 
 Name, by digesting with an alkaline trypsin solution or by extracting 
 the gelatin for many days with 1-5 p. m. caustic potash, as suggested 
 by C. Morner. The typical properties of gelatin are not changed by 
 this. 
 
 Chondrin or cartilage gelatin is only a mixture of gelatin with the specific 
 constituents of the cartilage and their transformation products. 
 
 Reticulin. The reticular tissues of the lymphatic glands contain a 
 variety of fibers which have also been found, by Mall in the spleen, intes- 
 tinal mucosa, liver, kidneys, and lungs. These fibers consist of a special 
 substance, reticulin, investigated by Siegfried. 1 
 
 Reticulin has the following composition: C 52.88; H 6.97; N 15.63; 
 S 1.88; P 0.34; ash 2.27 per cent. The phosphorus occurs in organic 
 combination. It yields no tyrosine on cleavage with hydrochloric acid. 
 It yields, on the contrary, sulphureted hydrogen, ammonia, lysine, 
 arginine, and valine. On continued boiling with water, or more readily 
 with dilute alkalies, reticulin is converted into a body which is precipitated 
 by acetic acid, and at the same time phosphorus is split off. 
 
 Reticulin is insoluble in water, alcohol, ether, lime-water, sodium 
 carbonate, and dilute mineral acids. It is dissolved, after several weeks, 
 on standing with caustic soda at the ordinary temperature. Pepsin- 
 hydrochloric acid or trypsin does not dissolve it. Reticulin responds 
 to the biuret, xanthoproteic, and Adamkiewicz-Hopkins reactions, 
 but not to Millon's reagent. 
 
 1 Mall, Abhandl. d. math.-phys. Klasse d. Kgl. sachs. Gesellsch. d. Wiss., 1891; 
 Siegfried, Ueber die chem. Eigensch. der retikulirten Gewebe, Habil.-Schrift, Leipzig, 
 1892. 
 
122 
 
 THE PBOTEIN SUBSTANCES. 
 
 According to Tebb reticulin is only a somewhat changed, impure collagen 
 but this is disputed by Siegfried. » 
 
 It may be prepared as follows, according to Siegfried: Digest intes- 
 tinal mucosa with trypsin and alkali. Wash the residue, extract with 
 ether, and digest again with trypsin and then treat with alcohol and ether. 
 On careful boiling with water the collagen present either as contamina- 
 tion or as a combination, with recticulin is removed. The thoroughly 
 boiled residue consists of reticulin. 
 
 Ichthylepidin is an organic compound, so-called by C. Morner, 2 which occurs 
 with collagen in fish-scales and forms about one-fifth of their organic substance. 
 This compound, with 15.9 per cent nitrogen and 1.1 per cent sulphur, stands on 
 account of its properties rather close to elastin. . It is insoluble in cold and hot 
 water, as well as in dilute acids and alkalies at the ordinary temperature. On 
 boiling with these it dissolves. Pepsin-hydrochloric acid, as well as an alkaline 
 trypsin solution, also dissolves it. It responds beautifully to Millon's reagent, 
 the xanthoproteic reaction, and the biuret test. At least a part of the sulphur 
 is split off «by the action of alkali. Ichthylepidin stands very close to elastin 
 in regard to its solubilities; but it differs essentially in composition as it is markedly 
 poorer in glycocoll, but much richer in proline and glutamic acid (Abderhalden 
 and Voitinovici 3 ). 
 
 As skeletins, Krukenberg 4 has designated a number of nitrogenized 
 substances which form the skeletal tissue of various classes of inverte- 
 brates. These substances are chitin, spongin, conchiolin, byssus, cornein, 
 and crude silk {fibroin and sericin). Of these, chitin does not belong to 
 the protein substances, and silk is hardly to be classed as a skeletin. 
 Only those so-called skeletins will be discussed that actually belong to 
 the protein group, and chitin will be discussed in another chapter. 
 
 The elementary composition of certain of the bodies belonging to 
 this group is as follows : 5 
 
 C H N 
 
 Conchiolin (from the shells of pinna) .. . 52.70 G.54 1G.G0 
 
 Spongin 46.50 G.30 16.20 
 
 48.75 6.35 16.40 
 
 Cornein 48.96 5.90 16.81 
 
 Fibroin 48.23 6.27 18.31 
 
 " 48.30 6.50 19.20 
 
 Sericin 44.32 6.18 18.30 
 
 44.50 0.32 17.14 
 
 s 
 
 85 
 50 
 
 (Wetzel) 
 
 (Crookewitt) 
 
 (Posselt) 
 
 (Krukenberg) 
 
 (Cramer) 
 
 (Vignon) 
 
 (Cramer) 
 
 (Bondi) 
 
 1 Tebb, Journ. of Physiol., 27; Siegfried, ibid., 28. 
 
 2 Zeitschr. f. physiol. Chem., 24 and 37. See also Green and Tower, ibid., 35. 
 ' Zeitschr. f. physiol. Chem., 52, p. 368. 
 
 4 Grundziige einer vergl. Physiol, d. thier. Geriistsubst. Heidelberg, 1885. 
 
 ■• Krukenberg, Her. d. d. chem. Gesellsch., 17 and IS, and Zeitschr. f. Biologie, 22; 
 Croockewitt, Annal. d. Chem. u. Pharrn., 48; Posselt, ibid., 45; Cramer, Journ. f. 
 prakt. Chem., 96; Vignon, Compt. rend., 115; Wetzel, Zeitschr. f. physiol. Chem., 29 
 and CentralU. f. Physiol., 13, 113; Bondi, Zeitschr. f. physiol. Chem., 34. 
 
CORNEIN. 123 
 
 Spongin forms the chief muss of the ordinary sponge. It dissolves with 
 difficulty in concentrated mineral acids but dissolves with readiness in caustic 
 alkalies. It does not give the MlLLON reaction or An aukiewicz's. It gives 
 no gelatin. On hydrolysis spongin yields considerable glycocoll 13.9 per cent, 
 glutamic acid l.S.l per cent, leucine 7.f> per cent, proline 6.3 per cent, lysine 
 3-4 per cent, and arginine 5 6 per cent. 1 Tyrosine and phenylalanine could 
 
 not be detected. After Hundeshagen bad shown the occurrence of iodine 
 
 and bromine in organic combination in different sponges and designated the albu- 
 moid containing iodine, iodospongin, IIaknack - later isolated from the ordinary 
 sponge, by cleavage with mineral acids, an iodospongin which contained about 
 II per cent iodine and 4.5 per cent sulphur. Strauss 3 has obtained sponginoses 
 of various kinds from spongin by dilute acids. The heterosponginose contained 
 the greater part of the iodine and sulphur, while the deuterosponginose contained 
 the carbohydrate groups. Iodospongin is considered as a derivative of the 
 heterosponginose. Conchiolin is found in the shells of mussels and snails and 
 also in the eggshells of these animals. It yields, according to Wetzel, 4 glycocoll, 
 leucine, and abundance of tyrosine. The quantity of diamino-nitrogen amounts 
 to 8.7 per cent and the amide nitrogen 3.47 per cent (from the shell of pinna). 
 The Byssus contains a substance, closely related to conchiolin, which is soluble 
 with difficulty. According to Abderhalden 5 it yields considerable glycocoll 
 and tyrosine and also alanine, aspartic acid and very large amounts of proline. 
 
 Cornein is the name given to the substance of the axial system of 
 certain Anthozoa. The substance occurring in the groups of Gorgonia 
 and Antipathes has been called gorgonin by C. Morner 6 and differs from 
 the pen not id in of the Pennatulidea? by the latter being readily soluble 
 in pepsin-hydrochloric acid. The cleavage products have not been care- 
 fully studied; one of the crystalline products, called cornicrijstalline by 
 Krikenberg, is nothing but iodine crystals, as shown by Morner. 
 After Drechsel 7 found nearly 8 per cent iodine in the dry substance of 
 the axial system of the Gorgonia Cavolini, C. Morner showed that in 
 the Anthozoa in general the organic skeletal substance contains halogens 
 in organic combination. Iodine was found in all varieties, and indeed 
 in amounts from traces up to 7 per cent. Eromine w r as found, with the 
 exception of two Antipathes, in amounts of 0.25 to 4 per cent, while 
 chlorine, which was never absent, occurred as a few tenths per cent. 
 The halogens occur in the organic skeletal substance as gorgonin and 
 pcnnatulin. 
 
 Drechsel obtained leucine, tyrosine, lysine, ammonia and an iodized 
 amino-acid, iodogorgonic acid, as cleavage products of gorgonin. This last 
 
 'Abderhalden and Strauss, Zeitschr. f. physiol. Chem., 48; Kossel and Kutscher, 
 end., :$!, 20.-). 
 
 2 Zeitschr. f. physiol. Chem., 24; Hundeshagen, Maly's Jahresber., 25, 394; see 
 also L. Scott, Biochem. Zeitschr., 1. 
 
 ' Biochem. Centralbl., 3. 
 
 4 Zeitschr. f. physiol. Chem. 29, and Centralbl. f. Physiol., 13, 113. 
 
 5 Zeitschr. f. physiol. Chem., 55. 
 
 6 Zeitschr. f. physiol. Chem., 51 and 55. 
 
 7 Zeitschr. f . Biol., 33. 
 
124 THE PROTEIN SUBSTANCES. 
 
 is identical with 3-5 di-iodo-tyrosine, HOI 2 C6H2.CH 2 .CHNH 2 COOH, 
 synthetically prepared by Wheeler and Jamieson. 1 On acid cleavage 
 of gorgonin, Henze 2 obtained the three hexone bases, abundant tyrosine 
 and 'very little leucine. On cleavage with barium hydroxide he obtained 
 only lysine, besides tyrosine and glycocoll in larger amounts. 
 
 Fibroin and sericin are the two chief constituents of raw silk. By 
 the action of boiling water the sericin (silk gelatin) dissolves and can be 
 obtained by a method suggested by Bondi, 3 while the more difficultly 
 soluble fibroin remains undissolved in the shape of the original fiber. 
 The sericin, whose sufficiently concentrated hot solution gelatinizes on 
 cooling, is precipitated by mineral acids, several metallic salts, and by 
 acetic acid and potassium ferrocyanide. The spider silk investigated 
 by Fischer 4 yielded fibroin but not sericin. 
 
 Abderhalden and his collaborators 5 have investigated a great 
 number of varieties of silk and found sericin in varying amounts (15 to 
 28 per cent). The composition of the various kinds of silk is char- 
 acterized, especially, by a varying amount of glycocoll and in this regard 
 we can differentiate between two chief groups. The one group is, like 
 the Italian silk, very rich in glycocoll while the other group, like the 
 Tussah silk, contains a much smaller quantity of glycocoll. 
 
 Sericin, whose proper concentrated warm solution gelatinizes on 
 cooling, is precipitated by mineral acids and several metallic salts and 
 by acetic acid and potassium ferrocyanide. In regard to the products 
 of hydrolysis it differs very essentially from fibroin by being much poorer 
 in glycocoll, alanine and tyrosine. 
 
 Fibroin is soluble in concentrated acids and alkalies and reprecipitable 
 (in a modified form) on neutralization. It gives the biuret test and 
 Millon's and Adamkiewicz-Hopkiih's reactions, the last but faintly. 
 Fibroin has an especially great interest because of the hydrolyses per- 
 formed by Fischer and his co-workers, and especially by the finding of 
 the previously mentioned polypeptides by these workers. Of the cleavage 
 products which characterize fibroin we must mention the large amount 
 of glycocoll, alanine and tyrosine, and the very small amounts of hexone 
 bases, besides the almost complete absence of monamino-dicarboxylic 
 acids. The quantity of the hydrolytic cleavage products of the three 
 silk substances, in so far as they have been investigated, are given in the 
 following table, which also includes the results for elastin, gelatin, and 
 
 1 Wheeler and Jamieson, Amer. Chem. Journ., £3; Wheeler, ibid., 38. 
 
 2 Henze, Zeitechr. f. physio). Chem., 38 and 51. 
 ' Zeitschr. f. physiol Chem., 34. 
 
 * Ibid., 58. 
 
 8 See Zeitschr. f. physiol. Chem., 59, 61, 62, 64, 71, 74, 80. 
 
ALBUMINATES 
 
 125 
 
 koilin. The fibroin A came from ordinary silk; fibroin B and the sericin 
 
 originated from Indian Tussah .silk. 
 
 Glycocoll 
 
 Alanine 
 
 Valine 
 
 Leucine 
 
 Serine 
 
 Aspartic acid . 
 Glutamic acid. 
 
 Cystine 
 
 Phenylalanine 
 
 Tyrosine 
 
 Proline 
 
 Oxyproline. . . 
 
 Histidine 
 
 Arginine 
 
 Lysine 
 
 Elastin. 1 
 
 Gelatin." 
 
 Koilin.' 
 
 Fibroin A 1 
 
 Fibroin B* 
 
 Sericin. 7 
 
 25.75 
 
 19.25 
 
 1.2 
 
 36.0 
 
 9 5 
 
 1 5 
 
 6.6 
 1.0 
 
 21.1 
 
 3.0 
 
 5.8 
 
 21.0 
 
 24 o 
 
 9 . S 
 
 9.2 3 
 
 13.2 
 
 15 
 
 1.5 
 
 4.8 
 
 — 
 
 0.4 
 
 — 
 
 1.6 
 
 2 
 
 5.4 
 
 — 
 
 1.2 3 
 
 2.3 
 
 — 
 
 
 •_' s 
 
 0.8 
 
 16. 8 3 
 
 5.2 
 
 — 
 
 10 
 
 1.8 
 
 — 
 
 — 
 
 0.7 10 
 
 — 
 
 — 
 
 — 
 
 3.9 
 
 1.0 3 
 
 2 3 
 
 1.5 
 
 0.6 
 
 0.3 
 
 0.34 
 
 — 
 
 5.4 
 
 10.5 
 
 9.2 
 
 10 
 
 1.7 
 
 7.7 
 6.4 
 0.4 
 
 5.5 
 
 — 
 
 1.0 
 
 3.0 
 
 
 
 0.03 5 
 
 
 
 
 
 — . 
 
 0.3 
 
 9.3 
 
 3.60 s 
 
 1.0 
 
 — 
 
 — 
 
 — 
 
 5.6 
 
 1.64 5 
 
 ■ — ■ 
 
 — 
 
 — 
 
 35.13 
 
 23 . 4 
 
 1.76 
 11 70 
 
 S 20 
 
 3.68 
 
 5.24 9 
 
 C. Cleavage Products of Simple Proteins. 
 
 On the hydrolysis of proteins by the aid of acids, alkalies or by 
 enzymes, cleavage products are obtained which represent various inter- 
 mediary steps between the native proteins on one side and the simple 
 cleavage products, the amino-acids, on the other side. Among these 
 products we have for a long time known two chief groups which still 
 retain, to a high degree, their protein character, namely, the albuminates 
 and the proteoses (and peptones). 
 
 1. Albuminates. 
 
 Alkali and Acid Albuminates. The native proteins are modified 
 by the action of sufficiently strong acids or alkalies. By the action of 
 alkalies all native albuminous bodies are converted, with the elimina- 
 tion of nitrogen, or by the action of stronger alkali, with the extraction 
 of sulphur also, into a new modification, called alkali albuminate. If 
 caustic alkali in substance or in strong solution be allowed to act on a 
 
 1 Cited from Abderhalden's Lehrbuch d. physiol. Chem., 1909. 
 
 2 Cohnheim, Chemie d. Eiweisskorper 3 d. Aufl. 
 z Skraup and Biehler, Monatsh. f. Chem., 30. 
 
 4 K. B. Hoffmann and Pergl. Zeitschr. f. physiol. Chem., 52. 
 6 v. Knaffl-Lenz, ibid., 52. 
 
 6 Abderhalden and Spack, ibid., 62. 
 
 7 Strauch, ibid., 71. 
 8 E. Fischer, ibid., 53. 
 
 8 Calculated as arginine. 
 
 10 This figure is somewhat uncertain. 
 
126 THE PROTEIN SUBSTANCES. 
 
 concentrated protekl solution, such as blood-serum or egg-albumin, the 
 alkali albuminate may be obtained as a solid jelly which dissolves in 
 water on heating, and which is called " Lieberkuhn's solid alkali 
 albuminate." By the action of dilute caustic alkali solutions on dilute 
 proteid solutions we have alkali albuminates formed slowly at the ordinary 
 temperature, but more rapidly on heating. These solutions may vary 
 with the nature of the proteid acted upon, and also with the intensity 
 of the action of the alkali, but still they have certain reactions in common. 
 
 If proteid is dissolved in an excess of concentrated hydrochloric acid, 
 or if we digest a proteid solution acidified with 1-2 p. m. hydrochloric 
 acid in the thermostat, or digest the proteid for a short time with 
 pepsin-hydrochloric acid, we obtain new modifications of proteid which 
 may show somewhat varying properties, but have certain reactions in 
 common. These modifications, which may be obtained in a solid gelat- 
 inous condition on sufficient concentration, are called acid albuminates 
 or acid albumins, and sometimes syntonin, though we perfer to apply 
 the term syntonin to the acid albuminate, which is obtained by extract- 
 ing muscles with hydrochloric acid of 1 p. m. 
 
 The alkali and acid albuminates have the following reactions in 
 common: They are almost insoluble in water and dilute common-salt 
 solution (see page 104), but they dissolve readily in water on the addi- 
 tion of a very small quantity of acid or alkali. Such a solution as nearly 
 neutral as possible does not coagulate on boiling but is precipitated at 
 the normal temperature on neutralizing the solvent by an alkali or an 
 acid. A solution of an alkali or acid albuminate in acid is easily pre- 
 cipitated on saturating with NaCl, but a solution in alkali is precipitated 
 with difficulty or not at all, according to the amount of alkali it contains. 
 Mineral acids in excess precipitate solutions of acid as well as alkali 
 albuminates. The nearly neutral solutions of these bodies are also pre- 
 cipitated by many metallic salts. 
 
 Notwithstanding this agreement in the reactions, the acid and alkali 
 albuminates are essentially different, for by dissolving an alkali albumi- 
 nate in some acid no acid, albuminate solution is obtained, nor is an alkali 
 albuminate formed on dissolving an acid albuminate in water by the 
 aid of a little alkali. In the first case we obtain a combination of the 
 alkali albuminate and the acid, soluble in water, and in the other case a 
 soluble combination of the acid albuminate with the alkali added. The 
 chemical process in the modification of proteids with an acid is essentially 
 different from the modification with an alkali, hence the products are 
 of a different kind. The alkali albuminates are relatively strong acids. 
 They may be dissolved in water with the aid of CaC03, with the elimina- 
 tion of ( '<>_>. which floes not occur with typical acid albuminates, and 
 they show in opposition to the acid albuminates also other variations 
 
PROTEOSES AND PEPTONES. 127 
 
 which stand in connection with their strongly marked acid nature. Dilute 
 solutions of alkalies act more energetically on proteida than do acids 
 of corresponding concentration. In the first case a part of the nitro- 
 gen and often also the sulphur, is split off, and from this property we may 
 obtain an alkali albuminate by the action of an alkali upon an acid albu- 
 minate; but we cannot obtain an acid albuminate by the reverse reac- 
 tion (K. Mokner l ). This does not exclude the possibility that, by 
 a more severe acid treatment, products can be obtained which perhaps 
 correspond to those products obtained by a more mild alkali treatment. 
 
 The preparation of the albuminates has been given above. The 
 corresponding albuminate obtained by the action cf alkalies or acids 
 upon a proteid solution may be precipitated by neutralizing with acid 
 or alkali. The washed precipitate is dissolved in water by the aid of a 
 little alkali or acid, and again precipitated by neutralizing the solvent. 
 If this precipitate, which has been washed in water, is treated with 
 alcohol and ether, the albuminate will be obtained in a pure form. 
 
 In the preparation of acid as well as of alkali albuminates, proteoses and the 
 closely related albuminates are formed. The " alkali albumose " obtained by 
 Maajb 2 belongs to this class. The lysalbinic acid and protalbinic acid obtained 
 by Paal ' from ovalbumin are likewise alkali albuminates. These have been 
 can tally studied by Skraup and his co-workers. 4 
 
 Desaminocdbtiminic acid is an alkali albuminate which Schmiedeberg 5 obtained 
 by the action of such weak alkali that a part of the nitrogen was evolved but the 
 quantity of sulphur remained the same. The proteid combination obtained by 
 Blum' by the action of formol on proteid and called by him protogen, has similarities 
 with the alkali albuminates in regard to solubilities and precipitation, but is not 
 identical therewith. 
 
 2. Proteoses and Peptones. 
 
 Peptones were formerly designated as the final products of the decom- 
 position of protein bodies by means of proteolytic enzymes in so far as 
 these final products are still true proteins, while the intermediate prod- 
 ucts produced in the peptonization of proteins, in so far as they are 
 not substances similar to albuminates, were designated as proteoses 
 (albumoses, or propeptones) . Proteoses and peptones may also be 
 produced by the hydrolytic decomposition of the proteins with acids 
 
 1 Pfluger's Arch., 17. 
 
 2 Zeitschr. f. physiol. Chem., 30. 
 
 3 Ber. d. d. chem. Gesellsch., 35. 
 
 4 Hummelberger, Lampel and Woeber, Monatsh. f. Chem., 30. 
 6 Arch. f. exp. Path. u. Pharm., 39. 
 
 6 Blum, Zeitschr. f. physiol. Chem., 22. The older investigations of Loew may 
 be found iu Maly's Jahresber., 18S8. On the action of formaldehyde see also Benedi- 
 centi, Arch. f. (Anat. u.) Physiol., 1897; S. Schwarz, Zeitschr, f. physiol. Chem., 30; 
 Bliss and Xovy, Journ. of Exper. Med., 4. 
 
128 THE PROTEIN SUBSTANCES. 
 
 or alkalies, and by the putrefaction of the same. They may also be 
 formed in very small quantities, as by-products, in the investigations 
 of animal fluids and tissues, and the question as to the extent to which 
 these exist preformed under physiological conditions requires very 
 careful investigation. 
 
 Between the peptone, which represents the final cleavage product, 
 and the proteose, which stands closest to the original protein, we have 
 undoubtedly a series of intermediate products. Under such circum- 
 stances it is a difficult problem to try to draw a sharp line between the 
 peptone and the proteose group, and it is just as difficult to define our 
 conception of peptones and proteoses in an exact and satisfactory manner. 
 
 In the past we used to consider the peptones as the end products 
 in the hydrolysis, they still being true proteins, but we must call atten- 
 tion to the fact that since that time we have learned of polypeptide- 
 like cleavage products of the proteins, and also that polypeptides have 
 been prepared synthetically. With this in mind it is not possible to 
 say what we understand by the conception true proteid, and also that 
 possibly there exists a large number of intermediary steps between the 
 original modified proteid and the simplest cleavage products. There 
 is no doubt that those bodies which have been called proteoses and 
 peptones are chiefly mixtures; and the question has been proposed by 
 Abderhalden l whether it is not best to drop the conception of pro- 
 teoses and to call all products precipitable by ammonium sulphate, etc., 
 and previously described as proteoses, peptones. 
 
 Although there is much in favor cf such a proposition, still on account 
 of the great importance which the conception of the proteoses has gen- 
 erally received, it is probably too early to drop the question of proteoses 
 entirely from a text-book, and we will therefore, as in the past editions, 
 discuss the historical development of the proteoses and peptones in 
 the ordinary sense. 
 
 The proteoses (or albumoses) used to be considered as those protein 
 bodies whose neutral or faintly acid solutions do not coagulate on boil- 
 ing and which, to distinguish them from peptones, were characterized 
 chiefly by the following properties: The watery solutions are precipitated 
 at the ordinary temperature by nitric acid, as well as by acetic acid and 
 potassium ferrocyanide, and this precipitate has the peculiarity of dis- 
 appearing on heating and reappearing on cooling. If a proteose solu- 
 tion is saturated with NaCl in substance, the proteose is partly pre- 
 cipitated in neutral solutions, but on the addition of acid saturated 
 with salt it is more completely precipitated. This precipitate, which 
 dissolves on warming, is a combination of the proteose with the acid. 
 
 1 Oppenheirner's Handb. der Biochern., Bd. 1, 1908. 
 
PROTEOSES AND PEPTONES. 129 
 
 We formerly designated as peptones those protein bodies which are 
 readily soluble in water and which are not coagulated by heat, whose 
 solutions are precipitated neither by nitric acid, nor by acetic acid and 
 potassium ferrocyanide, nor by NaCl and acid. 
 
 The reactions and properties which the proteoses and peptones have 
 in common were formerly considered as the following: They all 
 give the color reactions of .the proteins, but with the biuret test they give 
 a more beautiful red color than the ordinary proteins. They are pre- 
 cipitated by ammoniacal lead acetate, by mercuric chloride, tannic, phos- 
 photungstic, and phosphomolybdic acids, by potassium-mercuric iodide 
 and hydrochloric acid, and also by picric acid. They are precipitated 
 but not coagulated by alcohol, that is, the precipitate obtained is soluble 
 in water even after being in contact with alcohol for a long time. The 
 proteoses and peptones also have a greater diffusive power than native 
 proteins, and the diffusive power is greater the nearer the questionable 
 substance stands to the final product, the now so-called true peptone. 
 
 These old views have gradually undergone an essential change. After 
 Heynsius' * observation that ammonium sulphate was a general pre- 
 cipitant for proteins, and for peptones in the old sense, Kuhne and his 
 pupils 2 proposed this salt as a means of separating proteoses and pep- 
 tones. Those products of digestion which separate on saturating their 
 solution with ammonium sulphate, or can indeed be salted out at all, 
 are considered, by Kuhne and also by most of the modern investigators, 
 as proteoses, while those which remain in solution are called peptones 
 or true peptones. These true peptones are formed in relatively large 
 amounts in pancreatic digestion, while in pepsin digestion they are formed 
 only in small quantities or after prolonged digestion. 
 
 According to Schutzenberger and Kuhne 3 the proteins yielded 
 two chief groups of new protein bodies wdien decomposed by dilute 
 mineral acids or w r ith proteolytic enzymes; of these the anti group shows 
 a greater resistance to further action of the acid and enzyme than the 
 other namely, the hemi group. These two groups are, according to 
 KtiHNE, united in the different proteoses, even though in various relative 
 amounts, and each proteose contains the anti as well as the hemi group. 
 The same is true for the peptone obtained in pepsin digestion, hence he 
 calls it amphopeptone. In tryptic digestion a cleavage of the ampho- 
 
 1 Pfluger's Archiv, 34. 
 
 2 See Kuhne, Yerhandl. d. naturhistor. Vereins zu Heidelberg (N. F.), 3; J. Wenz, 
 Zeitschr. f. Biologie, 22; Kuhne and Chittenden, Zeitschr. f. Biologie, 22; R. Neu- 
 meister, ibid., 23; Kuhne, ibid., 29. 
 
 3 Schutzenberger, Bull, de la Soc. chimique de Paris, 23; Kuhne, Yerhandl. d. 
 naturhist. Vereins zu Heidelberg (N. F.), 1, and Kuhne and Chittenden, Zeitschr. f. 
 Biologie, 19. See also Paal, Ber. d. deutsch. chem. Gesellsch., 27. 
 
130 THE PROTEIN SUBSTANCES. 
 
 peptone takes place into aniipeptone and hemipcptone. Of these two 
 peptones the hemipeptone is further split into amino-acids and other 
 bodies while the antipeptone is not attacked. By the sufficiently 
 energetic action of trypsin only one peptone remains to the last — the so- 
 called antipeptone. 
 
 Kuhne and his pupils, who have conducted extensive investiga- 
 tions on the proteoses and peptones, classify the various proteoses accord- 
 ing to their different solubilities and precipitation properties. In the 
 pepsin digestion of fibrin l they obtained the following proteoses : (a) 
 Heteroproteose, insoluble in water but soluble in dilute salt solution; 
 (6) Protoproleose, soluble in salt solution and water. These two pro- 
 teoses are precipitated by NaCl in neutral solutions, but not completely. 
 Heteroproteose may, by being in contact with water for a long time 
 or by drying, be converted into a modification, called (c) Dysproteose, 
 which is insoluble in dilute salt solutions, (d) Deuteroproteose is a pro- 
 teose which is soluble in water and dilute salt solution and which is 
 incompletely precipitated from acid solution by saturating with NaCl, 
 and is not precipitated from neutral solutions. 
 
 The proteoses obtained from different protein bodies do not seem to be 
 identical, but differ in their behavior to precipitants. Special names have been 
 given to these various proteoses according to the mother-protein, namely, albu- 
 moses, globidoses, vitelloses, caseoses, myosinoses, elastoses, etc. These various 
 proteoses are further distinguished, as proto-, hetero-, and deuterocaseose, for 
 example. Chittenden 2 has suggested the common name proteoses for the prod- 
 ucts formed intermediary between the proteins and peptones in the digestion of 
 animal and vegetable proteins. We have made use of it in this sense in pref- 
 erence to the word albumose (which is used in the German and by some other 
 writers), but which will be used in this book as indicating the intermediary 
 products in the hydrolysis of albumins and not as a general term. Certain 
 proteoses have also been obtained in a crystalline state (Schrotter) . 
 
 Neumeister 3 designates as atmidalbumose that body which is obtained by 
 the action of superheated steam on fibrin. At the same time he also obtained a 
 substance called atmidalbumin, which stands between the albuminates and the 
 proteoses. 
 
 Of the soluble proteoses Neumeister designates the protoproteose 
 and heteroproteose as primary proteoses, while the deuteroproteoses, 
 which are closely allied to the peptones, he calls secondary proteoses. As 
 essential differences between the primary and secondary proteoses he 
 
 'See Kuhne and Chittenden, Zeitschr. f. Biologie, 20. 
 
 * Kuhne ami Chittenden, Zeitschr. f. Biologie, 22 and 26; Neumeister, ibid., 23; 
 Chittenden and Hartwell, Journ. of Physiol., 11 and 12; Chittenden and Painter, 
 Studies from the Laboratory, etc., Yale University, 2, New Haven, 1887; (l.ittenden, 
 ibid., .'5: Sebelien, Chem. Centralblatt, 1890; Chittenden and Goodwin, Journ. of 
 Physiol.. 12. 
 
 3 Zeitschr. f. Biologie, 26. See also Chittenden and Meara, Journ. of Physiol., 
 15, and Salkowski, Zeitschr. f. Biologie, 34 and 37. 
 
PROTEOSES .WD PEPTONES. 131 
 
 BUggeste the following. 1 The primary proteoses are precipitated by 
 nitric arid in salt-free solutions, while the secondary proteoses arc pre- 
 cipitated only in salt solutions, and certain deuteroproteoses, such as 
 deuterovitellose and deuteramyosinose, are precipitated by nitric acid 
 only in solutions saturated with Na( '1. The primary proteoses arc pre- 
 cipitated from neutral solutions by copper-sulphate solution (2:100), 
 and by NaCl in substance, while the secondary proteoses are not. The 
 primary proteoses are completely precipitated from a solution saturated 
 with NaCl by the addition of acetic acid saturated with salt, while the 
 secondary proteoses arc only partly precipitated. The primary proteoses 
 are readily precipitated by acetic acid and potassium ferrocyanide, while 
 the secondary are only incompletely precipitated after some time. The 
 primary proteoses are also, according to Pick, 2 completely precipitated 
 by ammonium sulphate (added to one-half saturation), while the second- 
 ary proteoses remain in solution. 
 
 The true peptones, as they were formerly considered to be, are exceed- 
 ingly hygroscopic, and if perfectly dry, sizzle like phosphoric anhydride 
 when treated with a little water. They are exceedingly soluble in water, 
 diffuse more readily than the proteoses, and are not precipitated by 
 ammonium sulphate. In contradistinction to the proteoses, the true 
 peptones are not precipitated by nitric acid (even in solutions saturated 
 with salt), by sodium chloride and acetic acid saturated with salt, 
 potassium ferrocyanide and acetic acid, picric acid, trichloracetic 
 acid, potassium-mercuric iodide, and hydrochloric acid. They are 
 precipitated by phosphotungstic acid, phosphomclybdic acid, corrosive 
 sublimate (in the absence cf neutral salts), absolute alcohol, and tannic 
 acid, but the precipitate may redissolve on the additicn of an excess of 
 the precipitant. As an important difference between amphopeptone and 
 antipeptone we must also mention that the former gives Millon's 
 reaction, while the antipeptone does not. 
 
 In regard to the precipitation by alcohol we must call attention to the observa- 
 tions of Frank el that not only are the acid combinations of peptone (Paal) 
 soluble in alcohol, but also the free peptone, and Frankel has even suggested a 
 method of preparation based on this behavior. Schrotter 3 has also prepared 
 crystalline proteoses which were soluble in hot alcohol, especially methyl alcohol. 
 
 The views on the hydrolytic cleavage products of peptic and tryptic 
 digestion which were accepted until a few years ago have recently been 
 considerably modified in several points. 
 
 1 Xeumeister, Zeitschr. f. Biologic, 24 and 26. 
 
 2 Zeitschr. f. physiol. Chem., 24. 
 
 3 Frankel, Zur Kenntnis der Zerfallsprodukte des Eiweisses bei peptischer und 
 tryptiecher Verdauung, Wien, 1896; Schrotter, Monatshefte f. Chem., 14 and 16. 
 
132 THE PROTEIN SUBSTANCES. 
 
 The older view that in peptic digestion only proteoses and peptones, 
 but no simpler cleavage products, are formed, has been shown not to be 
 true. The works of Zunz, Pfaundler, Salaskin, Lawrow, Lang- 
 stein, 1 and others have shown that by very lengthy digestion amino- 
 acids may in part be formed and also other products such as oxyphenyl- 
 ethylamine, tetra- and pentamethylenediamine. The biuret reaction 
 does not disappear and the above mentioned products seem to be formed 
 only under special conditions. In ordinary, not too lengthy pepsin, 
 digestion, it is generally admitted that no amino acids are formed but 
 only proteoses and peptones. 
 
 In connection with the above-mentioned experimental results it must be 
 remarked that not all the products found, for example, the oxyphenylethylamine 
 and the diamines, are produced by the action of pepsin, but rather by the action of 
 other enzymes. In certain cases, undoubtedly, impure pepsin was used, or indeed 
 autodigestion of the stomach was carried on, and the action of other enzymes 
 was not excluded. In other cases the digestion with pepsin and considerable 
 acid (even 1 per cent H 2 S0 4 ) was continued for a very long time, indeed for an 
 entire year, without controlling the influence of the acid alone upon the proteoses. 
 
 Kuhne's view that in tryptic digestion (pancreatic digestion) a 
 peptone, so-called antipeptone, always remains which cannot be further 
 split is not strictly true. By sufficiently long autodigestion of the pan- 
 creas, Kutscher 2 was able to obtain, as final products, a mixture of 
 digestion products which failed to respond to the biuret test, and the same 
 results have been obtained by others. An antipeptone in the old sense, 
 i.e., a digestion product which is resistant to tryptic digestion but which 
 still gives the biuret test, is without question not always obtained as end 
 product in trypsin digestion. On the contrary as Fischer and Abder- 
 halden 3 have shown, polypeptide-like bodies are produced in tryp- 
 tic digestion (and the same is probably true also for peptic digestion) 
 which do not give the biuret test, i.e., " abiuret " products, and which 
 are resistant to further tryptic digestion but yield amino-acids on 
 hydrolysis with acids. This behavior stands in close relation to the 
 observation that in tryptic digestion certain amino-acids, such for example, 
 as tyrosine, tryptophane and leucine are split off earlier and more readily 
 than the others of the protein molecule. 
 
 Antipeptone, which is only attacked with great difficulty by trypsin 
 has in fact been isolated by Siegfried (see below) and although the 
 
 ' Zunz, Zeitschr. f. physiol. Chem., 28, and Hofmeister's Beitrage, 2; Pfaundler, 
 Zeitschr. f. physiol. Chem., 30; Salaskin, ibid., 32; Salaskin and Kowalewsky, ibid., 
 38; Lawrow, ibid., 33; Langstein, Hofmeister's Beitrage, 1 and 2. 
 
 2 Zeitschr. f. physiol. Chem., 25, 26, 28, and Die Endprodukte der Trypsinver- 
 dauung, Habilitationsscbxift Strassburg, 1899. 
 
 3 Zeitschr. f. physiol. Chem., 39. 
 
PROTEOSES AND PEPTONES. 133 
 
 views of Kuhne are not in all points correct still the fact remains that 
 under certain circumstances I he protein can be split into fractions, of which 
 the hemi group is further easily decomposed by enzyme action while the 
 other, the anti group, is very much more resistant to such action. It 
 also seems as if the first group is characterized by a larger content of 
 tyrosine, tryptophane and the latter by its content of glycocoll, phenyl- 
 alanine and proline. 
 
 By the use of the methods specially worked out by the Hofmeister 
 school, of fractionally salting out with ammonium sulphate or zinc sul- 
 phate or also by Siegfried's iron-alum method, numerous attempts to 
 separate the various proteoses and peptones have been made. 1 Not 
 only have we learned by these methods of a larger number of proteoses, 
 but our older conception of the products formed primarily has been 
 materially modified. Immediately at the commencement of diges- 
 tion, even in peptic digestion, a splitting of the protein molecule into 
 several complexes takes place. In opposition to the view of Huppert, 2 that 
 the proteoses, in pepsin digestion, are always derived from the primarily 
 formed acid albuminate, Pick and Zunz have shown that several pro- 
 teoses, as well as acid albuminate, appear as primary products at the 
 commencement of the digestion. According to Goldschmidt 3 a splitting 
 off of proteoses and the formation of acid albuminate takes place simul- 
 taneously by the action of dilute acids alone. Besides the proteoses 
 we also have, according to Zunz and Pfaundler, even at the beginning, 
 other primary bodies, which cannot be salted out and which do not 
 give the biuret reaction, but are in part precipitated by phosphotungstic 
 acid. These little-known products seem to be intermediate between 
 the peptones and the amino-acids, and they correspond probably to 
 the polypeptide bodies obtained by Fischer and Abderhalden in tryptic 
 digestion. 
 
 By fractional precipitation of Witte's peptone with ammonium sulphate 
 Pick has obtained various chief fractions of proteoses. The first contains the 
 proto- and heteroproteoses whose precipitation limit lies at 24-42 per cent satu- 
 ration with ammonium sulphate solution, i.e., the presence of 24-42 cc. of the 
 saturated ammonium sulphate solution in 100 cc. of the liquid. Then follows 
 a fraction A at 54-62 per cent saturation, then a third fraction B. with 70-95 
 per cent saturation, and finally fraction C, which precipitates from the saturated 
 solution on acidification with sulphuric acid saturated with the salt. 
 
 1 Umber, Zeitschr. f. physiol. Chem., 25; Alexander, ibid., 25; Pfaundler, ibid., 
 30; Zunz, ibid., 28, and Hofmeister's Beitriige, 2; Pick, ibid., 2, and Zeitschr. f. physiol. 
 Chem., 24 and 28; Siegfried, see footnote 3, p. 136. 
 
 2 Schiitz and Huppert, Pfliiger's Arch., 80. 
 
 * F. Goldschmidt, Ueber die Einwirkung von Sauren auf Eiweisstoffe, Inaug.- 
 Diss. Strassburg, 1898. 
 
134 THE PROTEIN SUBSTANCES. 
 
 The hetero- and protoproteoses *are not, according to our present 
 views, the only primary proteoses. In the proteose fraction obtained 
 on saturating with ammonium sulphate in neutral liquids, which should 
 contain secondary proteoses only, primary proteoses such as the gluco- 
 proteose (Pick), which contains a carbohydrate group and the so-called 
 synproteose (Hofmeister l ) occur. It is no longer sufficient to consider 
 an unequal ability to be salted out, as an essential difference between 
 the primary and secondary proteoses. 
 
 There is no doubt that there exists a large number of so-called pro- 
 teoses having various precipitation properties, and different other prop- 
 erties and new differences appear, while working with them according to 
 different methods. For example Rona and Michaelis 2 find that cer- 
 tain proteoses are precipitated by mastic emulsion while others are not. 
 Those that are precipitatable by mastic, can all be salted out, while all 
 those that can be salted out are not all precipitated by mastic. The 
 hetero- and protoproteoses act, according to Zunz 3 like strong protec- 
 tion colloids toward colloidal gold, which is not the case with the others, 
 and also, according to this worker, the so-called proteoses are more readily 
 precipitated by chondroitin-sulphuric acid and acetic acid than the so- 
 called secondary proteoses. According to Hunter 4 only the primary 
 proteoses are precipitated by protamines while the secondary are not. 
 It is also possible that numerous intermediary members exist between 
 those proteoses which stand close to the original protein and those that 
 are further removed. The difficulties in isolation and purification of 
 these different members are so very great that the proteoses thus far 
 isolated must not be considered as chemical individuals. Under these 
 circumstances the above-mentioned differentiation and classification 
 of the various proteoses is of little value and a more detailed discus- 
 sion of the properties of the various proteoses thus far isolated is with- 
 out interest. 
 
 It would be of great interest if certain differences in the chemical 
 structure of the different proteoses could be determined with certainty. 
 Such differences are claimed to have been found in certain cases. Thus 
 Hart has found that the heteroproteose (from muscle syntonin) was 
 considerably richer in arginine and poorer in histidine than the proto- 
 proteose, and Pick has also found marked differences between the hetero- 
 and protoproteose from fibrin. The hetero-proteose yields very little 
 
 1 Ueber Bau unci Gruppirung der Eiweisskorper, Ergebnisse der Physiol., Jahrg. I, 
 Abt. 1 , 783. 
 
 "- Biochem. Zeitsohr., 3. 
 
 3 Arch, internat. d. Physiol., 1 and 5, and Bull. Soc. Scienc. med. et natur. Brux- 
 elles, (54. 
 
 Mourn, of Physiol., 37. 
 
PROTEOSES AND PEPTONES. 135 
 
 tyrosine and indoi but abundant leucine and glycocoll, and about 39 
 per cent of the total nitrogen in a basic form. The protoproteose, 
 according to Pick, on the contrary yields considerable tyrosine and indol, 
 only little leucine but no glycocoll, and contains only about 25 per cent 
 basic nitrogen. Friedmann, Hart, and Levene have obtained very 
 similar results in regard to the quantity of basic nitrogen in the two-pro- 
 teoses, although Leyene as well as Adler l did not find the same results 
 as Pick in regard to the amounts of monamino-acids in the two proteoses. 
 The work of Levene, v. Slyke and Birchard 2 show, in many important 
 points, a decided contradiction to the statements of Pick and these 
 divergent results may possibly be explained by the fact that they were 
 not working with pure substances, but rather with mixtures. 
 
 According to Pick the heteroproteose is also more resistant toward 
 tryptic digestion than the protoproteose, a behavior which coincides 
 with Kuhne's view of a resistant atomic complex, an anti group, in the 
 protein bodies. Kuhne and Chittenden 3 regularly obtained on the 
 tryptic digestion of heteroproteose a separation of so-called antialbumid, 
 a body which is attacked with great difficulty in tryptic digestion, but 
 which separates as a jelly-like mass and which is richer in carbon (57.5- 
 58.09 per cent), but poorer in nitrogen (12.61-13.94 per cent), than the 
 original protein. The occurrence of such resistant complexes in diges- 
 tion has also been repeatedly observed. 
 
 This antialbumid later attracted increased interest, because as 
 first found by Danilewsky and later other investigators have shown, 
 that solutions of rennin, gastric juice, pancreatic juice, and papain cause 
 a similar coagulum in not too dilute proteose solutions. These coagula, 
 called plasteines (coagulum by rennin) by Sawjalow, and coaguloses 
 (coagulum by papain) by Kurajeff, 4 are similar in many respects to 
 antialbumid, having a higher content of carbon (57-60 per cent) and 
 nitrogen (13-14.6 per cent). In other cases the quantity of carbon as 
 well as nitrogen is lower (Lawrow). 
 
 We cannot for the present make any positive statement as to the 
 importance and mode of formation of the coaguloses or plasteins. It 
 
 1 Hart, Zeitschr. f. physiol. Chem., 33; Pick, ibid., 28; Friedmann, ibid., 29; Levene, 
 Joum. of Biol. Chem., 1; R. Adler, Die Heteroalbumose und Protalbumose des Fibrins 
 Dissert. Leipzig, 1907. 
 
 2 Joum. of Biol. Chem., 8 and 10. 
 
 3 Zeitschr. f. Biol., 19 and 20. 
 
 4 The works of Danilewsky and Okunew are cited and reviewed in the following 
 Sa\Vjalow, Pfliiger's Arch., 85, and Centralbl. f. Physiol., 16; and Zeitschr. f. physiol. 
 Chem., 54; Lawrow and Salaskin, Zeitschr. f. physiol. Chem., 36; Lawrow, ibid., 51, 
 53, 56 and 60; Kurajeff, Hofmeister's Beitrage, 1 and 2: see also Sacharow, Biochem. 
 Centralbl., 1, 233; Levene and v. Slyke, Biochem. Zeitschr., 13. 
 
136 THE PROTEIN SUBSTANCES. 
 
 is rather generally admitted that they are formed by a synthesis, a view 
 which has received support by the investigations of V. Henriques and 
 Gjaldbak. 1 According to Sawjalow a plastein is not formed from a 
 proteose alone, but always from a mixture of proteoses. Lawrow claims 
 that they may be produced from proteoses as well as from polypeptide 
 substances, and correspondingly we must differentiate between the coag- 
 uloses or coagulosogens from the proteose group coaproteoses, and from 
 the polypeptide group or coapeptides. The latter yield on hydrolysis 
 chiefly monamino-acids, while the first yield also basic nitrogenous prod- 
 ucts. Perhaps the plasteinogen investigated by Bayer 2 which differs 
 essentially from the true proteid in its elementary composition as well 
 as from other coaguloses, belongs to the coapeptides. 
 
 The different behavior on saturating their solution with ammonium 
 sulphate has been generally used, as above remarked, for years to dif- 
 ferentiate between the proteoses and peptones. Those precipitable 
 by this salt were called proteoses, and those not were called peptones. 
 This method of division, which never had sufficient support and which 
 was perfectly arbitrary, cannot be considered at the present time. We 
 know now, thanks to the works of Emil Fischer and his co-workers, 
 that there are polypeptides either prepared artificially or found among 
 the cleavage products of the proteins, which are precipitated by ammo- 
 nium sulphate. At the present it is generally conceded that the peptones 
 in the ordinary sense are only a mixture of different bodies. The chief 
 step in these investigations must be the isolation from this mixture 
 of unit bodies with definite chemical characteristics. Of such bodies, 
 besides the polypeptides previously mentioned and studied by Fischer 
 and others, we must mention the products isolated by Siegfried and 
 his pupils. 3 
 
 These so-called peptones are in part peptic-peptones and partly 
 tryptic-peptones, and some are prepared from proteid (fibrin) and others 
 from gelatin. The tryptic fibrin-peptones are antipeptones in Kuhne's 
 sense because they are very resistant to the further action of trypsin. 
 They are according to Neumann simultaneously bibasic acids and mono- 
 acidic bases. They give the biuret reaction, but not Millon's reaction; 
 they contain no tyrosine and yield on hydrolysis, arginine, lysine, glutamic 
 acid, and it seems also aspartic acid. A pepsin-glutin peptone isolated 
 by Siegfried and Schmitz yielded arginine, lysine, glutamic acid, gly- 
 
 1 Zeitschr. f. physiol. Chem., 71 and 81. 
 
 2 Hofmeister's Beitriige, 4; see also L. Rosenfeld, ibid., 9; J. Lukomnik, ibid., 9 
 and F. Micheli, Biochem. Centralbl., 6, p. 562. 
 
 ' The works of Siegfried and his pupils, Fr. Muller, Borkel, Miihle, Kriiger, Scheer- 
 rnesser, Neumann, H. Schmitz, may be found in Arch. f. (Anat. u.) Physiol., 1894 and 
 Zeitschr. f. physiol. Chem., 21, 41, 43, 45, 48, 59, and 65 and Pfliiger's Arch. 136. 
 
PROTEOSES AM) PEPTONES. 137 
 
 cocoll and besides these also leucine and proline although not in quan- 
 tities that could be determined. Of the total nitrogen they found 
 i'9-7 per cent arginine, 9.1 per eent lysine, 19.2 per cent glycocoll, 9.3 
 percenl glutamic acid and 12.7 per cent proline and leucine together. 
 Siegfried has given proof in several ways as to the purity and unity 
 of the peptones isolated by him. 
 
 In another manner, namely by fractional precipitation with metallic salts, 
 especially with mercuric-potassium iodide and the preparation of phenyliso- 
 cyanate compounds, Hofmeister and his pupils Stookey, Raper and Rogo- 
 zinski i have isolated peptones or polypeptide-like bodies from blood proteid. 
 One of these, called arginine-histidine peptone, yielded arginine and histidine as 
 basic hydrolytic products while another yielded chiefly lysine^as basic product 
 and hence was called ly sine-peptone. 
 
 From glutin-pcptone, Siegfried, on warming with hydrochloric 
 acid, obtained a base, C21H39N9O8, which can also be directly obtained 
 from gelatin. This he calls a kyrin, because it is to be considered as a 
 basic protein nucleus, and he calls this special one glutokyrin. The 
 glutokyrin gives the biuret reaction and is considered as a basic peptone. 
 On complete hydrolytic cleavage it yields arginine, lysine, glutamic 
 acid, and glycocoll. Of the total nitrogen two-thirds belong to the 
 bases and one-third to the amino-acids. Recently he with 0. Pilz 
 on further hydrolysis has prepared a /3-glutokyrin, which only yielded 
 arginine, lysine and glutamic acid. Similar basic nuclei, protokyrins, 
 have recently been obtained by Siegfried 2 from fibrin and casein, using 
 the same method. Caseinokyrin gives a non-crystalline sulphate, but 
 a crystalline phosphotungstate. The free caseinokyrin has an alkaline 
 reaction, gives the biuret test, and its composition corresponds to the 
 formula C23H47N9O8. It yields arginine, lysine, and glutamic acid on 
 cleavage. The basic nitrogen amounts to about 85 per cent of the total 
 nitrogen, and caseinokyrin, behaves in this respect like a protamine. 
 
 Among the known cleavage products of proteins, arginine is the only 
 one which, up to the present, is never absent, and for this reason Ave 
 designate as proteins only those atomic complexes which contain, besides 
 chained monamino-acids, also arginine, or, more simply, show the prev- 
 iously mentioned imide bindings. Hence caseinokyrine, which yields 
 only arginine, lysine and glutamic acid, and scombrin, which yields 
 only arginine, proline, and alanine, are the simplest known proteins. 
 
 Scombrin belongs to the previously mentioned group of protamines 
 which, according to Kossel, 3 are formed by a successive cleavage of the 
 
 1 Hofmeister's Beitrage, 7, 9, and 11. 
 
 2 Kgl. Sachs. Ges. d. Wiss., Math.-Phys. Klasse, 1903, and Zeitschr. f. phvsiol 
 Chem., 43, with Pilz., ibid., 58. 
 
 3 Zeitschr. f. phvsiol. Chem., 44. 
 
138 THE PROTEIN SUBSTANCES. 
 
 typical protein. The occurrence of basic protokyrins in the hydrolytic 
 cleavage of genuine proteins like gelatin has given valuable support to 
 Kossel's theory as to a basic nucleus in the protein bodies. 
 
 On account of the cleavage taking place in digestion, the digestive 
 products should have a lower molecular weight than the original protein. 
 This is really the case as shown by molecular weight determinations. 
 As these determinations have been made upon impure substances or 
 mixtures, the results 1 obtained are only of little value. The same is 
 true for the elementary analysis of the proteoses and peptones. 2 
 
 ■v — 
 
 In the preparation and separation of various proteoses and peptones 
 all precipitable protein is always removed first by neutralization and 
 then by boiling. The proteoses may then be separated from the pep- 
 tones by means of ammonium sulphate according to Ivuhne's method, 
 and divided into different fractions according to the method of Pick 
 and the Hofmeister school. The separation and preparation of pure 
 hetero- and protoproteoses can be best performed by the method sug- 
 gested by Pick, but this method, as well as that with ammonium sulphate, 
 gives good results only when the precautions suggested by Haslam 3 
 are carefully followed. We can here only refer to the cited works of 
 Kuhne and co-workers, of E. Zunz and especially those of the Hof- 
 meister and the Siegfried schools. In regard to the literature on the 
 detection of proteoses and peptones in animal fluids we refer to Chapters 
 V and XIV. 
 
 If we wish to detect the presence of so-called true peptone, by means 
 of the biuret reaction in a solution saturated with ammonium sulphate, 
 we add a slight excess of a concentrated solution of caustic soda and 
 cool, and then add a two per cent solution of copper sulphate drop by 
 drop, after the sodium sulphate has separated out. 
 
 In the quantitative estimation of proteoses and peptones we make 
 use of the nitrogen estimation, the biuret test (colorimetric), and the 
 polarization method. These methods do not give exact results. 
 
 The polypeptides have had their most important properties dis- 
 cussed on pages 85-91, and of the cleavage products of the proteins only 
 the amino-acids remain to be discussed. 
 
 1 Sabanejew, Ber. d. d. chem. Gesellsch., 26, 385; Paal, ibid., 27, 1827; Sjoqvist, 
 Skand. Arch. f. Physiol., 5. 
 
 - Klernentary analyses of proteoses and peptones will be found in the works of 
 Kuhne and Chittenden and their pupils, cited in footnote 2, p. 130; also by Herth, 
 Zeitschr. f. physiol. Chem., 1, and Monatshefte f. Chem., 5; Maly, Pfluger's Arch. 
 9, 20; Henninger, Compt. rend., 86; Schrotter, 1. c, Paal, 1. c. 
 
 3 Journ. of Physiol., 32 and 36. 
 
GLYCOCOLL. 139 
 
 3. The Amino-acids. 1 
 
 CH 2 (NH 2 ) , „ , , 
 
 Glycocoll (ami no-acetic acid), Cl>H5N ( >2 = /, __ , also called gly- 
 
 COOH 
 
 cine or gelatin sugar, is found in the muscles of the invertebrates, 
 
 but has chief interest as a hydrolytic decomposition product of protein 
 bodies, especially fibroin, spider-silk elastin, gelatin, and spongin, as 
 well as of hippuric acid and glycocholic acid. 
 
 Glycocoll forms colorless, often large, hard rhombic crystals or four- 
 sided prisms. The crystals have a sweet taste and dissolve readily in 
 cold water (4.3 parts). Glycocoll is insoluble in alcohol and ether and 
 dissolves with difficulty in warm alcohol. Like the amino-acids in gen- 
 eral it combines with acids and alkalies. With the latter compounds 
 we must mention those with copper and silver. Glycocoll dissolves 
 cudHc hydroxide in alkaline liquids, but does not reduce at boiling heat. 
 A boiling-hot solution of glycocoll dissolves freshly precipitated cupric 
 hydroxide, forming a blue solution, which in proper concentration deposits 
 blue needles of copper-glyeocoll on cooling. The compound with hydro- 
 chloric acid is readily soluble in water but less soluble in alcohol. 
 
 Sorensen 2 finds that phosphotungstic acid does not precipitate 
 glycocoll from dilute solutions but only from concentrated ones. By the 
 action of gaseous HC1 upon glycocoll in absolute alcohol, beautiful 
 crystals are obtained of the hydrochloride of glycocoll-ethyl ester, which 
 melts at 144° C. and from which the glycocoll-ethyl ester can be obtained 
 by the method suggested by E. Fischer 3 for the separation of glycocoll 
 from the other amino-acids. On shaking with benzoyl chloride and 
 caustic soda, hippuric acid is formed, and this is also made use of in 
 different ways in detecting and isolating glycocoll (Ch. Fischer, Gox- 
 nermaxn, Spiro 4 ). The /3-naphthalene-sulpho-glycine with a melt- 
 ing-point of 159°, the 4-nitro-tolulene-2-sulpho-glycine, melting at 180°, 
 and the a-naphthylisocyanate compound melting at 190.5-191.5° are 
 also of importance. On putrefaction methane is probably produced 
 from glycocoll. 
 
 Glycocoll can be best prepared from hippuric acid by boiling it with 
 4 parts dilute sulphuric acid (1:6) for ten to twelve hours. After cooling 
 
 1 In regard to the division of the amino-acids among the three chief groups of 
 ■organic compounds we refer to pages 85-86. 
 
 2 Meddelelser, fraa Carlsberg-laboratoriet, 6, 1905. 
 
 3 Ber, d. d. chem. Gesellsch., 34. 
 
 4 Ch. Fischer, Zeitschr. f. physiol. Chem., 19; Spiro, ibid., 28; Gonnermann, 
 Pfliiger's Arch., 59. 
 
140 THE PROTEIN SUBSTANCES. 
 
 the benzoic acid is removed, the filtrate concentrated, the remaining 
 benzoic acid removed by extracting with ether, the sulphuric acid pre- 
 cipitated by BaCOs, and the filtrate evaporated to the point of crys- 
 tallization. (In regard to its preparation from protein substances see 
 below.) 
 
 CH 3 
 d-Alanine(a-aminopropionic acid), C3H 7 N02 = CH(NH 2 ). The d-alanine 
 
 COOH 
 is obtained in relatively small amounts from the true proteids, but in 
 larger quantities from the albuminoids, especially from fibroin, spider- 
 silk and elastin. 
 
 d-alanine has been prepared from Z-serine by E. Fischer and K. 
 Raske, 1 and Fischer has also obtained it from racemic alanine by split- 
 ting the benzoyl combination, or from Z-alanine by splitting with yeast 
 by Walden's reversion. 
 
 Alanine generally crystallizes in needles or oblique rhombic columns. 
 It is very readily soluble in water, having a sweetish taste, and dissolves 
 cupric hydroxide on boiling, producing a deep blue solution of a crystalliza- 
 ble copper salt. Alanine is insoluble in absolute alcohol. The rota- 
 tion of alanine at 20° C. in aqueous solution is (a) D =+2.7° and for a 
 solution in hydrochloric acid (9-10 per cent solution) is (ct) D = +10.3°. 
 
 The /3-naphthalene-sulpho-d-alanine melts, when dry, at about 123° 
 and sinters at 117° C. The phenylisocyanate melts at 168° and the 
 a-naphthylisocyanate alanine melts at 198°. On putrefaction alanine 
 yields propionic acid. 
 
 CH3CH3 
 
 V 
 
 ClT 
 d- Valine (a-amino-valeric acid), C5HnN02 = ^ TT , ATTT N , u 
 
 n CH(NH 2 ), has been 
 
 COOH 
 detected several times among the cleavage products of protein sub- 
 stances, although only in small quantities. Kossel and Dakin obtained 
 4.3 per cent valine from salmine, and E. Fischer and Dorpinghaus 2 
 5.7 per cent from horn substance. The largest quantity has been obtained 
 from casein and edestin, namely, 7.20 and 5.6 per cent respectively. 
 Because of the difficulty in separating valine from the two leucines 3 
 the figures given are somewhat uncertain. The acid isolated by H. 
 and E. Salkowski 4 from putrefying proteid or gelatin seems to have 
 been 5-amino-n-valeric acid. 
 
 1 Ber. d. d. chem. Gesellsch., 40. 
 
 2 Kossel and Dakin, Zeitschr. f. physiol. Chem., 41; Fischer and Dorpinghaus, 
 i\nd., .'{<>. 
 
 -<•<• Levene and v. Slyke, Journ. of Biol. Chem., 6. 
 4 Ber. d. d. chem. Gesellsch., 16 and 31. 
 
LEUCINE. HI 
 
 d-valine can be obtained aa microscopic crystalline leaves. It iq 
 rather readily soluble in water and the solution lias a faint sweetish taste 
 and at the same time somewhat bitter. The solution has a rotation 
 of (a) D = +6.42°. The hydrochloric acid solution (20 per cent) shows, 
 according to Fischer, a rotation of (r;) D =+28.8°. The copper salt, 
 which forms leaves which are rather soluble in water, is very easily soluble 
 in methyl alcohol (Schulz and Winterstein 1 ). 
 
 The phenylisocvanate melts at 147°, and on boiling with 20 per cent 
 hydrochloric acid for a short time, it is changed into rf-phenylisopropyl 
 hydantoin, which melts at 131-133° C. 
 
 On putrefaction valine yields isobutylamine and isovaleric acid. 
 /-Leucine (aminocaproic acid, or, more correctlv, a-aminoisobutylacetic 
 CH3CH3 
 
 \y 
 
 CH 
 
 acid), CeHi3N02= CH2 , is produced from protein substances in 
 
 CH(NH 2 ) 
 
 COOH 
 their hydrclytic cleavage by proteolytic enzymes, by boiling with dilute 
 acids or alkalies or by fusing with alkali hydroxides, and by putre!: ttion. 
 There are also observations that indicate that in the hydrolysis besides 
 the ordinary leucine perhaps also normal leucine may be formed (Heckel 
 and Samec 2 ). 
 
 Because of the ease with which leucine (and tyrosine) are formed 
 in the decomposition of protein substances, it is difficult to decide pos- 
 itively w T hether these bodies when found in the tissues are constituents 
 of the living body or are to be considered only as decomposition products 
 formed after death. Leucine, it seems, has been found as a normal 
 constituent of the pancreas and its secretion, in the spleen, thymus, and 
 lymph glands, in the thyroid gland, in the salivary glands, in the kidneys 
 and in the liver. It also occurs in the wool of sheep, in dirt from the 
 skin (inactive epidermis), and between the toes, and its decomposition 
 products have the disagreeable odor of the perspiration of the feet. 
 It is found pathologically in atheromatous cysts, ichthyosis scales, pus, 
 blood, liver, and urine (in diseases of the liver and in phosphorus poison- 
 ing). Leucine often occurs in invertebrates and also in the plant king- 
 dom. On hydrolytic cleavage various protein substances yield different 
 amounts of leucine, as shown in the tables given on pages 106, 107, 115 and 
 125. From the figures, there given, we call attention to the following: 
 Erlenmeyer and Schoffer obtained 36-45 per cent leucine from the 
 cervical ligament. E. Fis^ her and Abderhalden 20 per cent from haemo- 
 
 1 Zeitschr. f. physiol. Chem., 35. 
 
 2 Heckel, Monatsh. f. Chem., 29; Samec, ibid., 29. 
 
142 THE PROTEIN SUBSTANCES. 
 
 globin, and Fischer and Dorpinghaus 18.3 -per cent from horn sub- 
 stance. 1 . 
 
 The leucine obtained by cleavage of protein substances is generally 
 Z-leucine, which is levorotatory in water solution and dextrorotatory 
 in acid solution. The leucine prepared synthetically by Hufner 2 
 from isovaleraldehyde, ammonia, and hydrocyanic acid is optically 
 inactive. Inactive leucine may also be prepared, by the cleavage of pro- 
 teins with baryta at 160-180° C, because of a ready racemation. The 
 e/-/-leucine may be split into the two components by various means, espe- 
 cially by the preparation of the formal combination. 3 
 
 On oxidation the leucines yield the corresponding oxyacids (leucinic 
 acids). Leucine is decomposed on heating, evolving carbon dioxide, 
 ammonia, and amylamine. On heating with alkalies, as also in putre- 
 faction, it yields valeric acid and ammonia. On putrefaction it yields 
 isoamylamine and isocaproic acid. 
 
 Leucine crystallizes when pure in shining, white, very thin plates, 
 usually forming round knobs or balls, either appearing like hyaline, or 
 with alternating light and dark concentric layers which consist of radial 
 groups of crystals. By slow heating, leucine melts and sublimes into 
 white woolly flakes, which are similar to sublimed zinc oxide. At the 
 same time an odor of amylamine is developed. Quickly heated in a 
 closed capillary tube, it melts with decomposition at 293-295°. 
 
 Leucine, as obtained from animal fluids and tissues is always impure, 
 and is very easily soluble in water and rather easily in alcohol. Pure 
 leucine is soluble with difficulty. Pure /- and d-leucine dissolve in 4C- 
 46 parts water, more readily in hot alcohol, but with difficulty in cold 
 alcohol. The (/-/-leucine is much less soluble. According to Haber- 
 mann and Ehrexfeld 4 100 parts of boiling glacial acetic acid dissolve 
 29.23 parts of leucine. The specific rotation of /-leucine, dissolved in 
 hydrochloric acid (20 per cent solution) is (a) D = +15.6° according to 
 Fischer and Warburg. In aqueous solution it is (a) D = —10.40°, 
 according to F. Ehrlich and Wendel. 5 
 
 The solution of leucine in water is not, as a rule, precipitated by 
 metallic salts. The boiling-hot solution may, however, be precipitated 
 by a boiling-hot solution of copper acetate, and this fact is made use of 
 in separating leucine from other substances. If the solution of leucine 
 
 1 Erlenmeyer and Schoffer, cited from Maly, Chem. d. Verdauungssiifte, in Her- 
 mann's Handb. d. Physiol., 5, Theil 2, p. 209; Fischer and his collaborators, ibid., 36. 
 
 -Jouni. f. prakt. Chem. (N. F.), 1. 
 
 ■ Fischer and Warburg, Ber. d. d. chem. Gesellsch., 38. 
 
 *Zeitschr. f. physiol. Chem., 37. 
 
 R Fischer and Warburg, Ber. d. d. chem. Gesellsch., 38; E. Ehrlich and Wendel, 
 Biochem. Zeitschr., 8. 
 
I80LEUCINE. 143 
 
 is boiled with sugar of lead and then ammonia be elded to the cooled 
 solution, shining crystalline leaves of leucine-lead oxide separate. Leucine 
 dissolves cupric hydroxide, but dors not reduce on boiling. 
 
 Leucine is readily soluble in alkalies and acids. It gives crystalline 
 compounds with mineral acids. If Leucine hydrochloride is boiled with 
 alcohol containing 3-4 ]><•!■ cenl BC1, long narrow crystalline prisms of 
 leucine-ethyl-ester hydrochloride, melting at 134° ('.. an- formed. The 
 picrate of the leucine ester melts at 128°. The phenylisocyanate of 
 '/-/-leucine melts at 165° and its anhydride at 12o° G. The o-naphthyl- 
 isocyanate leucine melts at 163.5°, the naphthalene-sulpho-Meucine 
 at 68° C. 
 
 Leucine is recognized under the microscope by the appearance of bail- 
 or knobs, by its action when heated (sublimation test), and by its 
 compounds, especially the hydrochloride and picrate of the ethyl ester 
 and the phenylisocyanate compound of the racemic leucine obtained 
 on heating with baryta water, the a-naphthylisocyanate compound and 
 the p-naphthalene-sulpho-leucine. According to the method suggested 
 by Lippich l the leucine can be transformed into isobutylhydantoin, 
 having a melting-point of 205°, by boiling with an excess of urea and 
 baryta water. For the preparation and separation of leucine from the 
 other amino-acids of the leucine fraction special methods have been 
 suggested by F. Ehrlich and Wendel, Levene and v. Slyke. 2 
 
 Leucinimide, CuH^N^ = " 9 ' •, „ ' TTT '% u ~ „ , was first obtained by Ritt- 
 
 HAU8EN in the hydrolytic cleavage products on boiling proteins with acids, and 
 subsequently by R. Cohx. Salaskix 3 obtained it in the peptic and tryptic 
 digestion oi haemoglobin. As an anhydride of leucine (2.5-diacipiperazine) it 
 is probably formed by a secondary change, from leucine. 
 
 It crystallizes in long needles and sublimes readily. The melting-point has 
 not been found constant in the different cases. The leucinimide (3.6-diisobutyl- 
 2.5-diacipiperazine) prepared synthetically by E. Fischer 4 from leucine-ethyl 
 ester melted at 271° C. 
 
 i-Isoleucine (/3-methyl-ethyl-a:-amino-propionic acid), 
 
 CH3C2H5 
 
 v 
 
 C6Hl3N ° 2 = CHNH 2 
 COOH 
 
 1 Ber. d. d. chem. Gesellsch., 39. 
 
 - F. Ehrlich and Wendel, 1. o.; Levene and v. Slyke, Journ. of Biol. Chem.. 6. 
 
 3 Ritfhausen, Die Eiweisskorper der Getreidearten, etc., Bonn, 1872; R. Cohn, 
 Zeit?chr. f. physiol. Chem., 22 and 29; Salaskin, ibid., 32. 
 
 4 Ber. d. d. chem. Gepellsch., 34. 
 
144 THE PROTEiN SUBSTANCES. 
 
 is an isomer of leucine discovered by F. Ehrlich, 1 who first isolated 
 it from the mother-liquor after removing the sugar from beet-sugar 
 molasses. He also found it in the hydrolysis of several proteins, and 
 recently it has been found by others among the products of hydrolysis 
 of the proteins. The largest amount thus far found was 2.6 per cent 
 by Levene, v. Slyke and Birchard 2 in a heteroproteose. It seems 
 to be associated regularly with ordinary leucine, forming mixed crystals, 
 which give an impression of a chemical combination and which are dif- 
 ficult to separate. On this account the earlier claims as to the quantity 
 of leucine are somewhat uncertain, as they always refer to leucine 
 containing isoleucine. 
 
 The constitution of isoleucine has been explained by Ehrlich through 
 its relation to d-amyl alcohol. Just as according to F. Ehrlich valine 
 3'ields the isobutyl alcohol in alcoholic fermentation so isoleucine yields 
 "d-amyl alcohol in the fermentation of sugar with yeast. On the other 
 hand, it can also be obtained, in a manner analogous to the synthesis 
 of leucine, from d-amyl alcohol (as a mixture of isoleucine and alloiso- 
 leucine, the latter is levogyrate and has a different stereometric configura- 
 tion from the isoleucine). The synthesis of isoleucine has been accom- 
 plished in other ways by several investigators. 3 On putrefaction d-ca- 
 proic acid and d-valeric acid have been obtained from isoleucine. 4 
 
 Isoleucine crystallizes in leaves or rods and plates of the rhombic 
 form. It is more soluble in water than leucine (1:25.8). Its solutions 
 have a bitter taste and are astringent. It is dextro-rotatory in aqueous 
 as well as in acid solution. In aqueous solution it has a specific rotation 
 f ( a j D =+9.74° and in 20 per cent hydrochloric acid (a) D = +36.8°. 
 Like valine its copper salt is readily soluble in methyl alcohol. The 
 benzoyl combination melts at 116-117°, the benzene sulphoisoleucine 
 at 149-150°, the phenylisocyanate combination at 119-120°, and the 
 naphthylisocyanate combination at 178° C. 
 
 In the leucine fraction, from the amino-acids contained in nerve 
 substance, Abderhalden and Weil 5 have obtained a new amino-acid, 
 C6H13NO2 which is isomeric with leucine and which seems to be d-a- 
 amino-n-caproic acid and called d-caprine by them. When crystallized 
 from water it forms six-sided plates which unite to tufts having a faint 
 sweet taste. At 280° (uncorrected) it softens and at 285° (uncorrected) 
 
 1 Felix Ehrlich, Her. d. d. chem. Gesellsch., 37. 
 
 2 Journ. of Biol. Chem., 8. 
 
 1 Ehrlich, Ber. d. d. chem. Gesellsch., 40 and 41; Brasch and Friedmann, Hof- 
 meister'e Beitrage, 11; Bouveault and Locquin, Compt. rend., 141, and Bull. soc. 
 fhim. (3), :{•">: Locquin, Bull. boc. chim. (4), 1. 
 
 ; C. Neuberg, Bioch. Zeitachr., 37. 
 
 *Zeitschr. f. physiol. Chem., 81 and 84. 
 
SERINE. 145 
 
 it sublimes. Its solubility in water is 1.5:100; at 20° in aqueous so- 
 lution (a) D +6.53° and in 20 per cent hydrochloric acid-f-14.1°. It gives 
 a copper salt crystallizing in needles. 
 
 CH 2 (OH) 
 /-Serine (a-amino-/3-oxypropionic acid), C3H7N03 = CH(NH 2 ), was 
 
 COOH 
 obtained by Fischer and his collaborators as a cleavage product of 
 several proteins, generally only in small quantities. The largest quan- 
 tity, 6.6 per cent, was obtained by Fischer and Skita from sericine; 
 Kossel and Dakin l obtained a still larger amount from salmine, namely 
 7.8 per cent. The racemic serine is the one generally obtained. From 
 fibroin Fischer 2 obtained a mixture of active and inactive serine anhy- 
 dride from which he finally prepared /-serine by hydrolysis. Serine has 
 also been found by G. Embden and Tachau 3 in fresh perspiration. 
 Synthetically c/-/-serine has been prepared by Fischer and Leuchs 
 from ammonia, hydrocyanic acid and glycol aldehyde, and also in other 
 ways by others. 4 Fischer and Jacobs 5 have prepared /-serine from 
 (/-/-serine by the preparation of the alkaloid salt of the p-nitro-benzoyl 
 combination. On reduction serine is transformed into alanine, and on 
 oxidation with nitrous acid it yields glyceric acid. The relation of serine 
 to alanine, lactic acid and glyceric acid is evident from the following for- 
 mulae: 
 
 CH 2 (OH) 
 
 CH 3 
 
 CH 3 
 
 CH 2 (OH) 
 
 CH(NH 2 ) 
 
 CH(NH 2 ) 
 
 CH(OH) 
 
 CH(OH) 
 
 COOH 
 
 COOH 
 
 COOH 
 
 COOH 
 
 Serine 
 
 Alanine 
 
 Lactic acid 
 
 Glyceric acid 
 
 The /-serine crystallizes in thin leaves or crusts. It is rather readily 
 soluble in water; the (/-/-serine is soluble in 23 parts water at 20° C. 
 The solution of /-serine has a sweet taste with an insipid after taste. 
 The specific rotation in aqueous solution at 20° C. is (a) D =— 6.83° 
 and the hydrochloric acid solution at 25° C. is (a) D = +14.45°. The 
 /3-napthalene-sulpho-serine melts at 220° C. when anhydrous. The 
 /-serine anhydride, which is identical with that obtained from fibroin, 
 forms thin, colorless needles which melt at 247° with decomposition. 
 Its specific rotation in aqueous solution at 25° C. (a) D = —67.46°. 
 
 1 Fischer and Skita, Zeitschr. f. physiol. Chem., 35; Kossel and Dakin, ibid., 41. 
 
 2 Ber. d. d. chem. Gesellsch., 40. 
 
 3 Bioch. Zeitschr., 28. 
 
 4 Fischer and Leuchs, Ber. d. d. chem. Gesellsch, 35; Erlenmeyer and Stoop, ibid., 
 35; Leuchs and Geiger, ibid., 39. 
 
 6 Ber. d. d. chem. Gesellsch., 39. 
 
146 THE PROTEIN SUBSTANCES. 
 
 Isoserine (£-amino-a-oxypropionic acid) has been prepared by Ellinger 
 from diamino-propionic hydrobromide and silver nitrite, and by Neuberg and 
 Silbermann from the hydrochloric acid combination of diamino-propionic acid. 
 Other syntheses have been made by Neuberg and Mayer and by Neuberg 
 and Ascher. 1 
 
 COOH 
 Z-Aspartic acid (aminosuccinic acid), C4H7N04=att , has been 
 
 CH.2 
 
 COOH 
 
 obtained on the cleavage of protein substances by proteolytic enzymes as 
 well as by boiling them with dilute mineral acids in comparatively small 
 quantities. This acid also occurs in secretions of sea-snails (Henze 2 ) 
 and is very widely diffused in the vegetable kingdom as the amide 
 Asparagine (aminosuccinic-acid amide), which seems to be of the 
 greatest importance in the development and formation of the proteins 
 in plants. d-Z-Aspartic acid has been prepared synthetically from fumaric 
 acid and alcoholic ammonia. On putrefaction of aspartic acid, propionic 
 acid and succinic acid are formed. 
 
 /-Aspartic acid dissolves in 256 parts water at 10° C. and in 18.6 parts 
 boiling water, and on cooling crystallizes as rhombic prisms, and its 
 4 per cent solution acidified with HC1 has the rotation («)d = +25.7°; 
 in alkaline solution the acid is levo-rotatory. It forms with copper 
 oxide a crystalline compound which is soluble in boiling-hot water and 
 nearly insoluble in cold water, and which may be used in the prepara- 
 tion of the pure acid from a mixture with other bodies. 
 
 The benzoyl-Z-aspartic acid melts at 184-185°. For identification 
 we make use of the analysis of the free acid and the copper salt, as well 
 as of the specific rotation. 
 
 COOH, 
 CH(NH 2 ) 
 ^/-Glutamic acid (a-aminoglutaric acid), CsH9N04 = CH2 , is 
 
 CH 2 
 COOH 
 obtained from the protein substances under the same conditions as the 
 other monamino-acids (see tables on pages 106, 107, 115 and 125) and 
 from the peptones (Siegfried). It is absent in the protamines and in the 
 varieties of silk, it occurs only in small amounts with the exception of spi- 
 der's web. Hlasiwetz and Habermann obtained 29 per cent from casein 
 by cleavage with hydrochloric acid, while Kutscher could obtain only 
 1.8 per cent glutamic acid by cleavage with sulphuric acid. Other 
 
 'Ellinger, Ber. d. d. chem. Gesellsch., 37; Neuberg and Silbermann, ibid., 37;, 
 Neuberg and Mayer, Biochem., Zeitschr 3; Neuberg and Ascher, ibid., 6. 
 2 Ber. d. d. chem. Gesellsch., 34. 
 
GLUTAMIC ACID. 147 
 
 investigators such as Abderhalden and Ft nk and Skraup and r l i'KK 
 have shown that the same quantities of glutamic- acid can be obtained 
 by the use of the two mineral acids. Skraup and Turk obtained on the 
 hydrolysis of casein 20.3-22.3 per cent glutamic acid hydrochloride 
 
 corresponding to about 17 per cent glutamic acid. Abderhalden and 
 Sasaki 1 obtained 13.6 per cent glutamic acid from meat syntonin. It 
 occurs most abundantly In the plant proteins where the quantity may 
 lie more than 40 per cent. Levene and Mandel 2 have obtains i a strik- 
 ingly large quantity of glutamic acid, namely 25 per cent, from a nucleo- 
 protein of the spleen. 
 
 On heating glutamic acid to 180-190° it is converted into pyrrolidon- 
 carboxylie acid, which latter can be retransforn ed into glutamic acid 
 by HO gas; therefore, a formation of pyrrolidon-cnrbow lie acid at the 
 same time, or in place of glutamic acid, in the hydrolases, is not excluded. 
 
 On putrefaction glutamic acid gives 7-aminobutyric acid, n-butyric 
 acid and succinic acid. 
 
 tf-Glutamic acid crystallizes in rhombic tetrahedra or octahedra or 
 in small leaves. It dissolves in 100 parts water at 16° C, and the solu- 
 tion has an acid taste with a peculiar after-taste. It is insoluble in 
 alcohol and in ether. 
 
 In water it has a rotation of (a) D = +12.04°. Strong acids increase 
 the rotation, and a 5 per cent solution of glutamic acid containing 9 per 
 cent HO has a rotation (a) D = +31.7°, while that obtained by heating 
 with barium hydroxide is optically inactive. (/-Glutamic acid forms 
 a beautifully crystalline combination with hydrochloric acid, which is 
 almost insoluble in concentrated hydrochloric acid. This compound 
 is used in the isolation of glutamic acid. On boiling with cupric hydroxide 
 a beautiful crystalline copper salt, which is soluble with difficulty, is 
 obtained. 3 The benzoyl-d-glutamic acid melts at 130-132° C. The 
 hydrochloride, the a-naphthylisocyanate of glutamic acid, which melts 
 at 23(3-237° C, the analysis of the free acid, and the specific rotation 
 are used in its detection. 
 
 As previously stated monamino-oxydicarboxylic acids have also 
 been found among the cleavage products of the proteins. To these belong 
 the following: 
 
 That oxyaminosuccinic acid, C u H-XOi occurs among the hydrolytic cleavage 
 products of proteids has been shown to be probable by Skraup. This acid has 
 
 1 Hlasiwctz and Habermann, Annal. d. chem. u. Pharm., 159; Kutscher. Zeitschr., 
 f. physiol. Chem., 28; Abderhalden and Funk, ibid., 53; with Sasaki, ibid., 51; Skraup 
 and Turk, Monatseh. f. Chem., 30. 
 
 2 Bioch. Zeitschr, 5. 
 
 3 Several salts of £lutamic acid have been prepared and studied by Abderhalden 
 and Kautzsch, Zeitschr. f. physiol. Chem., 64, 68, and 78. 
 
.148 THE PROTEIN SUBSTANCES. 
 
 been prepared synthetically by Neuberg and Silbermann from diaminosuccinic 
 acid and barium nitrite in sulphuric acid solution. Oxyaminosuberic acid, 
 C 8 HisN0 6 , has been detected by Wohlgemuth l in the cleavage products of a 
 liver nucleoprotein. 
 
 /-Cystine, C6H12N2S2O4 (a-cliamino-/3-dithiolactolic acid), the disulphide 
 
 CH 2 — S— S— CH 2 
 of cysteine (a-amino-|3-thiolactic acid), CH(NH 2 ) CH(NH 2 ), was first 
 
 COOH COOH 
 
 obtained, with certainty, as a cleavage product of protein substances by 
 K. Morner, and then also by Embden. Kulz 2 obtained it once as a 
 product of tryptic digestion of fibrin. The quantities found by Morner 
 and Buchtala in the various proteins are given in the tables on pages 
 106, 107, 115 and 125. 
 
 According to Neuberg and Mayer 3 two kinds of cystine occur in nature, 
 namely, stone-cystine, designated /3-cystine, and protein-cystine, called a-cystine 
 
 CH 2 NH 2 CH 2 NH» 
 Stone-cystine is the disulphide of /3-amino-a-thiolactic acid, CH — S — S — CH 
 c COOH COOH 
 
 The protein-cj'stine has been chiefly obtained from the protein substance, 
 but also from calculi, while the stone-cystine has been obtained from urinary 
 calculi only. 
 
 Many objections have been raised from many sides as to the correctness of 
 this assumption. Rothera could not find any difference between the stone- 
 cystine and the cystine prepared from hair, and Fischer and Suzuki, and recently 
 also Abderhalden, 4 arrived at similar results, which seems to place the exist- 
 ence of stone-cystine in doubt. The occurrence of two structurally isomeric 
 cystines is not improbable, from certain observations of Morner, but Friedmann 
 and Baer 5 have shown that these observations do not lead to this assumption 
 and at the present time we cannot admit of the occurrence of two different cystines; 
 
 Cystine probably occurs normally as traces in the urine. In rare 
 cases, in cystinuria, it occurs in larger quantities in the urine, the sediment 
 or in calculi. Traces have also been found in the ox-kidney, in the liver 
 of the horse and dolphin, and in the liver of a drunkard. Abder- 
 halden 6 has found cystine in the urine and also abundantly in the 
 organs (spleen) in a case of parental cystine diathesis. 
 
 The constitution of cystine has been explained by Friedmann, 7 and 
 
 1 Skraup, Zeitschr. f. physiol. Chem., 42; Neuberg and Silbermann, ibid., 44; 
 Wohlgemuth, ibid., 44. 
 
 2 K. Morner, ibid., 28, 34, and 42; Embden, ibid., 32; Kulz, Zeitschr. f. Biologie, 
 27. 
 
 3 Zeitschr. f. physiol. Chem., 44. 
 
 4 Rothera, Journ. of Physiol., 32; Fischer and Suzuki, Zeitschr. f. physiol. Chem., 
 45; Abderhalden, ibid., 51. 
 
 Friedmajon, Hofmeister's Beitrage, 3. With Baer, ibid., 8. 
 • Zeitschr. f. physiol. Chem., 38. 
 7 Hofmeister's Beitrage, 3. 
 
CYSTINE. 149 
 
 he has also established the relation between cystine and taurine. Cys- 
 tine is the disulphide of cysteine, which is a-amino-/3-thiola<t ic acid. 
 From cysteine by oxidation Friedmann obtained cysteinic acid, 
 
 CH2SO2OH 
 C3H7NS0 6 =CH(NH 2 ), from which taurine CH 2 (S0 2 OH) is produced'by 
 
 COOH CH 2 (NH 2 ) 
 
 splitting off C0 2 . 
 
 Cystine has also been prepared synthetically in several ways. For 
 example, Fischer and Raske * have prepared cystine from a-amino- 
 /S-chlorpropionic acid (obtained from Z-serine) by the action of barium 
 hydrosulphide and a subsequent oxidation in the air. 
 
 /-Cystine crystallizes in thin, colorless, hexagonal plates. It is not 
 soluble in water, alcohol, ether, or acetic acid, but dissolves in mineral 
 acids and oxalic acid. It is also soluble in alkalies and ammonia, but 
 not in ammonium carbonate. Cystine is optically active, being levorota- 
 tory. Morner found it to be (a) D = —224.3°. On heating with hydro- 
 chloric acid it can, according to Morner, be changed into a modifica- 
 tion crystallizing in needles and with a weaker levorotatory power, or 
 indeed dextrorotatory, composed of a mixture of the two optically active 
 cystines. On heating with HC1 to 165° for 12-15 hours Neuberg and 
 Mayer obtained inactive cystine. By fungus fermentation with Asper- 
 gillus niger they obtained dextrorotatory cystine. Cystine has no 
 melting-point but slowly decomposes at 258-261°. On boiling cystine 
 with caustic alkali it decomposes and yields alkali sulphide, which can 
 be detected by lead acetate or sodium nitroprusside. According to Mor- 
 ner 2 75 per cent of the total sulphur is separated. Cystine treated 
 with tin and hydrochloric acid develops only a little sulphureted 
 hydrogen, and is converted into cj^steine. Cystine yields sulphureted 
 hydrogen and methyl mercaptan on putrefaction. 
 
 On heating upon platinum-foil cystine ignites and burns with a bluish- 
 green flame, with the generation of a peculiar sharp odor. When warmed 
 with nitric acid it dissolves with decomposition, and leaves on evapora- 
 tion a reddish-brown residue, which does not give the murexid test. 
 
 Cystine is gradually precipitated from its sulphuric acid solution by 
 phosphotungstic acid. Cystine forms crystalline salts with mineral 
 acids and with bases. For isolating and separating cystine the precipita- 
 tion with mercuric acetate is especially suited. The benzoyl cystine 
 (Baumann and Goldmann 3 ) melts at 180-181°; the phenylisocyanate 
 compound at 160°. On boiling with 25 per cent hydrochloric acid this 
 
 1 See Erlenmeyer and Stoop, Ber. d. d. chem. Gesellsch., 36; Gabriel, ibid., 38; Fischer 
 and Raske, ibid., 41. 
 
 2 Zeitschr. f. physiol. Chem., 34. 
 
 3 Ibid., 12. 
 
150 THE PROTEIN SUBSTANCES. 
 
 compound passes to the anhydride, which is a hydantoin melting at 
 119° C. By the action of potassium cyanide Mauthner l obtained 
 a-amino-jS-suphocyanpropionic acid, CH 2 (SCN).CH(NH 2 )COOH. 
 
 Stone-cystine, according to Neuberg and Mayer, differs in many respects 
 from the ordinary cystine, among which the following may be mentioned: The 
 optically active stone-cystine crystallizes in needles, the specific rotation is 
 (a)D=-206°; it melts' at 190-192° with marked swelling up. The benzoyl 
 compound melts at 157-159°; the phenylcyanate compound melts at 170-172°, 
 and it is not changed on boiling with hydrochloric acid, 
 
 In the detection and identification of cystine we make use of the 
 crystalline form, the behavior on heating on platinum-foil, and the sul- 
 phur reaction after boiling with alkali. As to its preparation from protein 
 substances see K. Morner and Folin 2 . In regard to the detection 
 of cystine in the urine see Chapter XIV. 
 
 CH 2 .SH 
 
 Cysteine (a-amino-/3-thiolactic acid), C 3 H 7 NS02=CH(NH 2 ), is formed from 
 
 COOH 
 cystine by reduction with tin and hydrochloric acid. It is also produced in the 
 cleavage of protein substances not as Embden believes as a primary formation 
 but according to Morner and Patten 3 as a secondary formation. Cysteine 
 can be easily converted into cystine by oxidation. 
 
 According to V. Arnold 4 cysteine occurs as a constituent of the press-juice 
 or extracts of various animal organs. He has found it especially in the hair and 
 he considers it as a primary cell constituent. 
 
 Toward alkalies and lead acetate it acts like cystine. With sodium nitro- 
 prusside and alkali it gives a deep purple-red coloration; with ferric chloride 
 the solution gives an indigo-blue coloration which quickly disappears. 
 
 CH 3 
 
 Thiolactic acid (a-thiolactic acid), C 3 K 6 S0 2 =CH(SH), has been found once 
 
 COOH 
 as a cleavage product of ox-horn by Baumann and Sitter. Morner, Fried- 
 mann and Baer obtained it from cystine. It has been shown by Friedmann 
 that this acid is a regular cleavage product of keratin substances, and that it 
 can also be obtained from the proteins. Frankel 5 obtained the acid from 
 haemoglobin. The pyroracemic acid obtained by Morner as a decomposition 
 product from several protein substances originates, according to Morner, only 
 in part from the cystine. 
 
 Taurine (aminoethylsulphonic acid), C2H7NS03 = Act - (SO Vym ' ^ as 
 not been obtained as a cleavage product of protein substances; still its 
 
 1 Zeitschr. f. physiol. Chem., 78. 
 
 2 Morner, Zeitschr. f. physiol. Chem., 34; Folin, Journ. of Biol. Chem., 8. 
 
 3 See foot-note 2, rage 80. 
 ♦Zeitschr. f. physiol. Chem., 70. 
 
 5 Morner, Zeitschr. f. physiol. Chem., 42; Suter, Zeitschr. f. physiol. Chem., 20; 
 Friedmann, Hofmeister's Beitrage, 3; with Baer, ibid., 8; Frankel, Sitzungsber. d. 
 Wien. Akad., 112, II, b, 1903. 
 
TAURINK. 151 
 
 origin from proteins has been shown by Friedmann by the close rela- 
 tion that taurine bears to cysteine; and this is the reason why it is 
 treated here in connection with the amino-acids. 
 
 Taurine is especially known as a cleavage product of taurocholic 
 acid, and may occur to a slight extent in the intestinal contents. Taurine 
 has also been found in the lungs and kidneys of oxen and in the blood 
 and muscles of cold-blooded animals. 
 
 Taurine crystallizes in colorless, often in large, shining, 4- or 6-sided 
 prisms. It dissolves in 15-16 parts of water at ordinary temperatures, 
 but rather more easily in warm water. It is insoluble in absolute alcohol 
 and ether; in cold alcohol it dissolves slightly, but better when warm. 
 Taurine yields acetic and sulphurous acids, but no alkali sulphides, 
 on boiling with strong caustic alkali. The content of sulphur can be 
 determined as sulphuric acid after fusing with saltpeter and soda. 
 Taurine combines with metallic oxides. The combination with mercuric 
 oxide is white, insoluble, and is formed when a solution of taurine is boiled 
 with freshly precipitated mercuric oxide (J. Lang 1 ). This compound 
 may be used in detecting the presence of taurine. Taurine is not pre- 
 cipitated b} r metallic salts. 
 
 The preparation of taurine from ox-bile is very simple. The bile 
 is boiled a few hours with hydrochloric acid. The filtrate from the 
 dyslysin and choloidic acid is concentrated well on the water-bath, and 
 filtered hot so as to remove the common salt and other substances which 
 have separated. The solution is evaporated to dryness and the residue 
 dissolved in 5 per cent hydrochloric acid, and precipitated with 10 vols. 
 95 per cent alcohol. The crystals are readily purified by recrystalliza- 
 tion from water. 
 
 The acid alcoholic solution can be used for the preparation of glycocoll. After 
 the evaporation of the alcohol, the residue is dissolved in water, treated with a 
 solution of lead hydroxide, filtered, the lead removed by H 2 S, and the filtrate 
 strongly concentrated. The crystals which separate are dissolved and decolor- 
 ized by animal charcoal and the solution then evaporated to crystallization. 
 
 Though taurine shows no positive reactions, it is chiefly identified 
 by its crystalline form, by its solubility in water and insolubility in 
 alcohol, by its combination with mercuric oxide, by its non-precipitability 
 by metallic salts, and above all by its sulphur content. 
 
 /-Phenylalanine (phenyl-a-aminopropionic acid), 
 
 C 6 H 5 
 CH 2 . 
 C 9 HnN02 = CH(NH 2 )i 
 
 COOH 
 
 1 See Maly's Jahresber, 6. 
 
152 THE PROTEIN SUBSTANCES. 
 
 was first found by E. Schulze and Barbieri l in etiolated lupin sprouts. 
 It is produced in the acid cleavage of protein substances in quantities 
 rarely above 5-6 per cent. It has been prepared synthetically in several 
 ways by Erlenmeyer, Jr., Sorensen and E. Fischer, Wheeler and 
 Hoffman. 2 
 
 The /-phenylalanine crystallizes in small, shining leaves or fine needles 
 which are rather difficultly soluble in cold water but readily soluble in 
 hot water. The solution has a faint bitter taste. A 5-per cent solution 
 acidified with hydrochloric acid or sulphuric acid is precipitated by 
 phosphotungstic acid, while a more dilute solution is not precipitated. 
 On putrefaction, phenylalanine yields phenylacetic acid. On heat- 
 ing with potassium dichromate and sulphuric acid (25 per cent) an odor 
 of phenylacetaldehyde is produced and benzoic acid is formed. In 
 aqueous solution it has a rotation of (a) D = — 35.1°. The phenyliso- 
 cyanate-1-phenylalanine melts at about 182° C. 
 
 /-Tyrosine (p-oxyphenyl-a-aminopropionic acid), 
 
 C 6 H 4 (OH) 
 
 C 9 HiiN0 3 = 
 
 CH 2 
 
 CH(NH 2 )' 
 COOH 
 
 is produced from most protein substances under the same conditions 
 as leucine, which it habitually accompanies. The largest quantity of 
 tyrosine obtained from animal proteins was about 10-13 per cent (see 
 tables, pages 106, 107, 115 and 125). In gelatin and a few keratins 
 tyrosine is absent. It is especially found with leucine, in large quantities, 
 in old cheese (Tupos), from which it derives its name. Tyrosine has not 
 been found with certainty in perfectly fresh organs. It occurs in the 
 intestine during the digestion of protein substances, and it has about 
 the same physiological and pathological importance as leucine. 
 
 Tyrosine was prepared by Erlenmeyer and Lipp from p-amino- 
 phenylalanine by the action of nitrous acid, and according to another 
 method by Erlenmeyer and Halsey. 3 On fusing with caustic alkali 
 it yields p-oxybenzoic acid, acetic acid, and ammonia. On putrefaction 
 it may yield oxyphenylethylamine, oxyphenylpropionic acid, oxyphenyl- 
 acetic acid, p-cresol and phenol. 
 
 1 Ber. d. d. chem. Gesellsch., 14, and Zeitschr. f. physiol. Chem., 12. 
 
 2 Erlenmeyer, Annal. d. Chem. u. Pharm., 275; Sorensen, Zeitschr. f. physiol. 
 Chem., 44; E. Fischer, Ber. d. d. chem. Gesellsch., 37; Wheeler and Hoffman, Amer. 
 Chem. Journ., 45. 
 
 3 Erlenmeyer and Lipp, Ber. d. d. chem. Gesellsch., 15; Erlenmeyer and Halsey, 
 ibid., 80. 
 
TYROSINE. 153 
 
 Naturally occurring tyrosine and that obtained by the cleavage of 
 protein substances by acids or enzymes, is generally /-tyrosine, while 
 that obtained by decomposition with baryta-water or prepared syn- 
 thetically is inactive, v. Lippmann l has obtained d-tyrosine from 
 beet-sprouts. The statements as to specific rotation of tyrosine are 
 somewhat variable. For tyrosine from proteins E. Fischer has found 
 a rotation of (a) D =— 12.56 to 13.2° for the hydrochloric acid solution, 
 while Schulze and Winterstein 2 obtained higher results using tyrosine 
 from plants, namely, (a) D =— 16.2°. 
 
 Tyrosine in a very impure state occurs in the form of balls similar 
 to leucine. The purified tyrosine, on the contrary, appears as colorless, 
 silky, fine needles which are often grouped into tufts or balls. It is diffi- 
 cultly soluble in water, being dissolved by 2454 parts of water at 20° C, 
 and 154 parts boiling water, separating, however, as tufts of needles on 
 cooling. It dissolves more easily in the presence of alkalies, ammonia, 
 or a mineral acid. It is difficultly soluble in acetic acid. Crystals of 
 tyrosine separate from an ammoniacal solution on the spontaneous 
 evaporation of the ammonia. One hundred parts glacial acetic acid 
 dissolve on boiling only 0.18 part tyrosine, and by this means, especially 
 on adding an equal volume of alcohol before boiling, the leucine can be 
 quantitatively separated from the tyrosine (Habermann and Ehren- 
 feld 3 ) . The Z-tyrosine-ethyl-ester crystallizes in colorless prisms which 
 melt at 108-109° C. The naphthylisocyanate-Z-tyrosine melts at 205- 
 206°. Tyrosine can be oxidized with the formation of dark-colored 
 products by various plant as well as animal oxidases, so-called tyro- 
 sinases (see Chapters XV and XVI). In alcoholic fermentation of sugar 
 the tyrosine present at the same time is transformed according to F. 
 Ehrlich 4 into tyrosol (p-oxyphenylethyl alcohol), C8H10O2. Tyrosin is 
 identified by its crystalline form and by the following reactions: 
 
 Piria's Test. Tyrosine is dissolved in concentrated sulphuric acid 
 by the aid of heat, by which tyrosine-sulphuric acid is formed; it is 
 allowed to cool, diluted with water, neutralized by BaCC>3, and filtered. 
 On the addition of a solution of ferric chloride the filtrate gives a beautiful 
 violet color. This reaction is disturbed by the presence of free mineral 
 acids and by the addition of too much ferric chloride. 
 
 Hofmann's Test. If some water is poured on a small quantity of 
 tyrosine in a test-tube and a few drops of Millon's reagent added and 
 
 1 Ber. d. d. chem. Gesellsch., 17. 
 
 2 See Hoppe-Seyler-Thierfelder,-Handb. d. physiol. u. pathol. chem. Analyse, 8. 
 Aufl., 1909. Also E. Fischer, Ber. d. d. chem. Gesellsch., 32; Schulze aud Winter- 
 stein, Zeitschr. f. physiol. Chem., 45. 
 
 3 Zeitschr. f. physiol. Chem., 37. 
 
 4 Ber. d. d. chem. Gesellsch., 44. 
 
154 THE PROTEIN SUBSTANCES. 
 
 then the mixture boiled for some time, the liquid becomes a beautiful 
 red and then yields a red precipitate. 
 
 Deniges' Test, modified by C. Morner, is performed as follows: 
 To a few cubic centimeters of a solution consisting of 1 vol. formaline, 
 45 vols, water, and 55 vols, concentrated sulphuric acid add a little 
 tyrosine in substance or in solution and heat to boiling. A beautiful 
 permanent green coloration is obtained. 
 
 Folin and Denis's test. The reagent consists of a solution containing 
 10 per cent sodium tungstate, 2 per cent phosphomolybdic acid and 
 10 per cent phosphoric acid. In performing the test mix 1-2 cc. of the 
 reagent with an equal volume of the tyrosine solution and then add 3-10 
 cc. saturated sodium carbonate solution when a beautiful blue color 
 results. Its delicacy is 1:1000000. The reagent can also be used for the 
 colorimetric quantitative estimation of tyrosine in proteins. According 
 to Abderhalden and Fuchs and to Abderhalden 1 the reagent suggested 
 by Folin and Denis for tyrosine also gives a blue coloration with trypto- 
 phane, oxytryptophane and Z-oxyproline and the value of this reagent for 
 quantitative tyrosine determinations requires further testing. 
 
 H2C — CH2 
 
 I I 
 /-Proline (a-pyrolidine carboxylic acid), C 5 H 9 N02 = H 2 C CH.COOH, 
 
 NH 
 
 was first obtained by E. Fischer and then by Fischer and collabora- 
 tors from several proteins as a primary cleavage product (Abder- 
 halden and Kautzsch 2 ). The proline here obtained was generally 
 the laevo-rotatory modification. The largest quantity of proline was 
 secured from the vegetable proteins hordein and gliadin, namely, 13.7 
 per cent and 13.2 per cent, and also from gelatin, 7.7 per cent (see table 
 pages 106, 107, 115 and 125). Kossel and Darin 3 obtained 11 per cent 
 from salmine. Proline also occurs in scombrine and clupeine, but not in 
 sturine, which, according to Kossel, seems to contradict the view as 
 to the common origin of ornithine and proline. 
 
 Sorensen, 4 by means of a general method of preparing a-amino- 
 acids synthetically, has prepared a-amino-5-cxy valeric acid from phthali- 
 midemalonic ester and has obtained proline from this by evaporating with 
 
 1 Deniges, Compt. rend., 130; C. Th. Morner, Zeitschr. f. physiol. Chem., 37; Folin 
 and Denis, Journ. of Biol. Chem., 12; Abderhalden and Fuchs, Zeitschr. f. physiol. 
 Chem., 83 and Abderhalden, ibid., 85. 
 
 2 E. Fischer, Zeitschr. f. physiol. (.'hem., 33 and 35. See also footnote 2, p. 86, 
 and Abderhalden and Kautzsch, Zeitschr. f. physiol. Chem., 78. 
 
 1 Zeitschr. f. physiol. Chem., 41. 
 
 4 Zeitschr. f. physiol. Chem., 44; with A. C. Anderson, ibid., 56. 
 
TRYPTOPHANE. 155 
 
 hydrochloric acid, at the same time splitting off water. Recently he 
 has suggested another method which yields good rasults. Other syn- 
 theses of proline have also been performed by E. Fischer and Will- 
 statter. 1 By the reduction of the ethyl ester of pyrrolidon carboxylic 
 acid (see glutamic acid) E. Fischer and Boehner 2 have obtained racemic 
 a-proline. On putrefaction proline yields 5-amino-valeric acid and n- 
 valeric acid (Neuberg and Ackermann 3 ). 
 
 /-Proline crystallizes in flat needles. It is readily soluble in water 
 and alcohol. The solution has a sweet taste; the specific rotation at 
 20° C. is (a) D = —77.40°. The solution acidified with sulphuric acid is 
 precipitated by phosphotungstic acid. In the detection of this acid 
 we make use of the copper salt, the anhydride of the phenylisocyanate 
 compound (melting-point 14-i°), and the picrate. The inactive acid and 
 its compounds show somewhat different properties. 
 
 Oxyproline (oxy-a-pyrolidine carboxylic acid), C5H9NO3. This acid, 
 whose constitution is not understood was first obtained by E. Fischer 
 on the hydrolysis of casein and of gelatin. It dissolves readily in water; 
 has a specific rotation of (a) D = —81.04°, and the solution has a sweet 
 taste. Oxyproline crystallizes in beautiful colorless plates and gives 
 a readily soluble copper salt. The constitution of natural oxyproline 
 has recently been explained by Leuchs and Brewster. 4 They find 
 that the natural oxyproline is a 7-oxy-derivative of pyrrolidine-a-carbox- 
 ylic acid. Leuchs found the specific rotation of ^-oxyproline to be 
 ( a ) D =_76° at 20° C. 
 
 /-Tryptophane (indol-a-aminopropionic acid), 
 
 C.CH 2 .CH(NH 2 )COOH 
 CnHi 2 N202 = C 6 H4<^CH 
 NH 
 
 is one of the cleavage products of the proteins formed in tryptic diges- 
 tion and other deep decompositions of the proteins, such as putrefaction, 
 cleavage with baryta-water or sulphuric acid. It gives a reddish-violet 
 product with chlorine or bromine which is called yroteinochrome. Nencki 5 
 considered tryptophane, which name is generally given to this acid, 
 as the mother-substance of various animal pigments. 
 
 1 Ber. d. d. chem.. Gesellsch., S3. 
 
 2 Ber. d. d. Chem., Gesellsch., 44. 
 
 ' Neuberg, Bioch. Zeitschr, 37; Ackermann, Zeitschr. of Biol., 57. 
 
 4 Fischer, Ber. d. d. chem. Gesellsch., 35 and 36; Leuchs and Brewster, Ber. d. d. 
 Chem., Gesellsch.. 46. 
 
 5 In regard to tryptophane, see Stadelmann, Zeitschr. f. Biologic, 26; Neumeister, 
 ibid., 26; Nencki, Ber. d. d. chem. Gesellsch.,' 28; Beitler, ibid., 31; Kurajeff, Zeitschr. 
 f. physiol. Chem.. 26; King, Pfliiger's Arch., 86. 
 
156 THE PROTEIN SUBSTANCES. 
 
 Tryptophane was first prepared in a pure form by Hopkins and 
 Cole, 1 and they considered it as skatolaminoacetic acid. After Ellin- 
 ger showed that skatolcarbonic acid (Salkowski) and skatolacetic 
 acid (Nencki) were indolacetic acid and indolpropionic acid respectively, 
 and after the synthesis of d-Z-tryptophane by Ellinger and Flam and, 2 
 the nature of this substance as indolaminopropionic acid was established. 
 
 By condensation of /3-indolaldehyde with hippuric acid Ellinger and Flamand 
 prepared the azlactone (lactimide) : 
 
 N 
 CsHeNCHO+^Qg 6 6 =C 8 H 6 N.CH ; C C .C 6 H 5 +2H 2 0. 
 
 CO— O 
 
 On boiling with dilute caustic soda, with the taking up of water, the sodium 
 salt of indoxyl-a-benzoylaminoacrylic acid, 
 
 C 8 H 6 N.CH : C.NH.COCeH, 
 COONa 
 
 is obtained, from which by reduction and splitting off of the benzoyl group by 
 the action of sodium alcoholate the tryptophane is obtained : 
 
 C 8 H 6 N.CH : C.NH.COC 6 H 5 
 
 | +H 2 +H 2 =C 8 H 6 N.CH 2 .CH.NH 2 +C 6 H5COOH. 
 
 COOH COOH 
 
 The trytophane formed in digestion is /-tryptophane, which is lsevoro- 
 tatory in aqueous solution (Hopkins and Cole). Racemic ^-trypto- 
 phane has also been obtained by digestion in certain cases by Allers 
 and Neuberg, this is probably formed from the /-tryptophane (Abder- 
 halden and L. Baumann 3 ), which very readily undergoes racemization. 
 
 Tryptophane crystallizes in silky rhombic or six-sided leaves. It 
 does not have a sharp melting-point, and according to the rapidity of heat- 
 ing melts at 252°, 273° and 289°, according to various authorities. 
 Tryptophane is readily soluble in hot water, difficultly soluble in cold 
 water, and* only slightly soluble in alcohol. The solution of d-Z-trypto- 
 phane has a faintly sweetish taste, and /-tryptophane a faintly bitter taste. 
 The statements as to the optical behavior of tryptophane differ some- 
 what, which, according to Abderhalden, is probably due to the readiness 
 with which it undergoes racemization. According to Abderhalden 
 and L. Baumann, 4 at 20° C. the aqueous solution has a rotation of 
 
 1 Journ. of Physiol., 27. 
 
 2 Ellinger, Ber. d. d. Chem. Gesellsch., 37 and 38. With Flamand, ibid., 40, and 
 Zeitschr. f. physiol. Chem., 55. 
 
 3 R. Allers, Biochem. Zeitschr., 6; C. Neuberg, ibid., 6; Abderhalden and Baumann, 
 Zeitschr. f. physiol. Chem., 55. (Literature on the specific rotation.) 
 
 * See Abderhalden and Baumann, Zeitschr. f. physiol. Chem., 55 (literature). 
 
INDOL AND SKATOL. 157 
 
 («)d= — 30.33°. Hopkins and Cole give ( a ) D =-33° for the watery 
 
 N N N 
 
 solution. It is dextrorotatory in — or — NaOH as well as in — HC1. 
 
 \ z 1 
 
 Tryptophane yields indol and skatol when sufficiently heated. It 
 gives the Adamkiewicz-Hopkins * reaction and a rose-red color on the 
 addition of chlorine or bromine water (tryptophane reaction). The 
 brom-tryptophane is readily soluble in amyl alcohol or acetic ether 
 and on shaking with these solvents the reaction is more delicate. 2 If 
 a pine stick previously moistened with hydrochloric acid and washed 
 with water is introduced into a concentrated tryptophane solution, 
 it becomes purple (pyrrole reaction) on drying. The melting-points 
 of the benzoylsulphotryptophane, the /3-naphthalenesulphotryptophane 
 and the naphthylisocyanatetryptophane are according to Ellinger 
 and Flamand, 3 185°, 180° and 158° C. respectively. Several compounds 
 of tryptophane have been prepared by Abderhalden and Kempe. 4 
 Among these we will mention the tryptophane chloride hydrochloride, 
 because it is used as the starting material for the synthesis of trypto- 
 phane polypeptides. In the alcoholic fermentation of sugar, as found 
 by F. Ehrlich 5 the tryptophane present is transformed into tryptophol 
 (/3-indoxylethyl alcohol). 
 
 In regard to the rather complicated method for preparing trypto- 
 phane we must refer to the original work of Hopkins and Cole, of 
 Neuberg, and of Abderhalden and Kempe. Fasal 6 has suggested a 
 quantitative colorimetric method for estimating tryptophane based 
 upon the Adamkiewicz-Hopkins reaction. 
 
 As shown by Hopkins and Cole, 7 tryptophane on anaerobic putre- 
 faction yields indolpropionic acid and indolacetic acid, and indol and 
 skatol on aerobic putrefaction. Among these putrefactive products the 
 indol and skatol will be specially discussed. 
 
 CH 
 
 Indol, C8H 7 N = C 6 H 4 <^\CH, and Skatol, or /3-methylindol, 
 NH 
 
 1 In regard to this reaction see also Dakin, Journ. of Biol. Chem. 2, and O. Rosen- 
 heim, Biochem. Journ., 1. 
 
 2 Neuberg, Bioch. Zeitschr., 24. 
 »1. c. 
 
 4 Zeitschr. f. physiol. Chem., 52, and Ber. d. d. chem. Gesellsch., 40. 
 
 5 Ber. d. d. chem., Gesellsch., 45. 
 
 6 Hopkins and Cole. Journ. of Physiol., 27 and 29; Neuberg and Popowsky, Biochem. 
 Zeitschr., 2; Abderhalden and Kempe, Zeitschr. f. physiol. Chem., 52; Fasal, Bioch. 
 Zeitschr., 44. 
 
 7 Journ. of Physiol., 29. 
 
158 THE PROTEIN SUBSTANCES. 
 
 C.CH 3 
 C9HgX = C6H4<^ /CH, are formed in variable quantities from pro- 
 
 NH 
 
 tein compounds under different conditions. Hence they occur habitually 
 in the human intestinal canal, and, after oxidation into in doxy 1 and 
 skatoxyl respectively, pass, at least partly, into the urine as the cor- 
 responding ethereal sulphuric acids, and also as glucuronic acids. 
 
 Indol and skatol crystallize in shining leaves, and their melting- 
 points are 52° and 95° C. respectively. Indol has a peculiar excremen- 
 titious odor, while skatol has an intense fetid odor. Both bodies are easily 
 volatilized by steam, skatol more easily than indol. They may both be 
 removed from the watery distillate by ether. Skatol is the more insoluble 
 of the two in boiling water. Both are easily soluble in alcohol and give 
 with picric acid a compound crystallizing in red needles. If a mixture 
 of the two picrates be distilled with ammonia, they both pass over with- 
 out decomposition; while if they are distilled with caustic soda, the indol 
 but not the skatol is decomposed. The watery solution of indol gives 
 with fuming nitric acid a red liquid and then a red precipitate of nitroso- 
 indol nitrate (Nencki 1 ). It is better first to add two or three drops 
 of nitric acid and then a 2-per cent solution of potassium nitrite, drop 
 by drop (Salkowski 2 ). Skatol does not give this reaction. An alcoholic 
 solution of indol treated with hydrochloric acid colors a pine chip cherry- 
 red. Skatol does not give this reaction. Indol gives a deep reddish-violet 
 color with sodium nitroprusside and alkali (Legal's reaction). On 
 acidifying with hydrochloric acid or acetic acid the color becomes pure 
 blue. Skatol does not act the same. The alkaline solution is yellow 
 and becomes violet on acidifying with acetic acid and boiling. With a 
 few drops of a 4-per cent formaline solution and concentrated sulphuric 
 acid indol gives a beautiful violet color while skatol gives a yellow or 
 brown color (Kondo 3 ). On warming skatol with sulphuric acid a 
 beautiful purple-red coloration is obtained (Ciamician and Magnanini 4 ). 
 According to Sasaki skatol, in methyl alcohol free from aldehyde, 
 gives with concentrated sulphuric acid containing ferric salt a violet- 
 red ring at the juncture of the two liquids. Indol and tryptophane 
 do not give this reaction. Deniges has carefully studied the behavior 
 of these two bodies with Ehrlich's reagent, dimethylaminobenzaldehyde, 
 or with cinnamic aldehyde and vanillin. Comparative investigations on 
 
 1 Ber. <1. d. deutsch. chem. Gesellsch., 8, 727, and ibid., 722 and 1517. 
 
 2 Zeit.^flir. f. phyBiol. Chem., 8, 447. In regard to newer reactions for indol and 
 skatol, see Bteensma, ibid., 47, and Deniges, Compt. rend. soc. biol., 64. 
 
 3 Zeitschr. f. physiol. C\\em., 48. 
 
 * Ber. (1. d. chem. Gesellsch., 21, 1928. 
 
HISTIDINE. 159 
 
 the behavior of indol and skatol with the aromatic aldehydes have been 
 carried out by Blumenthal. 1 
 
 For the detection of indol and skatol in, and their preparation from, 
 faeces and putrefying mixture-, the main points of the usual method are 
 as follows: The mixture is distilled after acidifying with acetic acid; 
 the distillate is then treated with alkali (to combine with any phenols 
 which may lie present) and again distilled. From this second distillate 
 the two bodies, after the addition of hydrochloric acid, are precipitated 
 by picric acid. The precipitated picrate is then distilled with ammonia. 
 The two bodies are obtained from the distillate by repeated shaking 
 with ether and evaporation of the several ethereal extracts. The residue, 
 containing indol and skatol, is dissolved in a very small quantity of 
 absolute alcohol and treated with 8-10 vols, of water. Skatol is precip- 
 itated, but not the indol. The further treatment necessary for their 
 separation and purification will be found in other works. 2 
 
 Skatosine, GoHieNoOo, is a base first obtained by Baum in the pancreas auto- 
 digestion and later studied by Swain. It develops an indol- or skatol-like odor 
 on fusing with potassium hydroxide. Langstein 3 obtained a substance which is 
 perhaps identical with skatosine, in the very lengthy peptic digestion of blood 
 proteins. ' 
 
 /-Histidine, C6H9X3O2, is j3-imidazol-a-aminopropionic 4 
 
 CH-NHX 
 
 C N^ CH 
 
 acid, =CHo 
 
 CH(NH 2 ) 
 
 COOH 
 Histidine was first discovered by Kossel in the cleavage products 
 of sturine. It was found at the same time by Hedin in the cleavage 
 products of proteins by acid hydrolysis, and by Kutscher among the 
 products of tryptic digestion, and finally also as a cleavage product of 
 many different animal and plant protein substances. It does not occur 
 in the protamines, with the exception of sturine. Of the protein bodies 
 globin (from horse-haemoglobin) seems to be richest in histidine, as 
 
 'Sasaki, Bioch. Zeitschr. 23, 29; Deniges, Compt. rend. soc. biol., 64; Blumenthal, 
 Bioch. Zeitschr., 19. 
 
 2 For quantitative, colorimetric determinations of indol in feces see Einhorn and 
 Hiibner, Salkowski's Festschrift, Berlin, 1904; C. A. Herter and Foster, Journ. of 
 biol. Chem., 2. 
 
 3 Baum, Hofmesister's Beitrage, 3; Swain, ibid.; Langstein, see Hofmeister, Ueber 
 Bau und Gruppierung der Eiweisskorper, in Ergebnisse der Physiologie, I, Abt. 1, 
 1902. 
 
 1 Sec Pauly, Zeitschr. f. physiol. Chem., 42; Knoop and Windaus, Hofmeister's 
 Beitrage, 7 and 8; Knoop, ibid., 10; Ackermann, Zeitschr. f. physiol. Chem., 65. 
 
16a THE PROTEIN SUBSTANCES. 
 
 Abderhalden found 10.96 per cent. It also occurs in germinating plants 
 (E. Schulze x ). 
 
 Histidine has been prepared synthetically by Pyman. 2 4 (5) chlormethyl 
 glyoxalin yields with sodium chlormalonic ester the glyoxalinmethylchlormalonic 
 ester. 
 
 CH.NHx 
 
 II > 
 
 C N^ 
 
 CH 2 .CC1(C0 2 .C 2 H 6 ) 2 
 
 >CH , which on 
 
 hydrolysis gives cW-a-chlor-/3-glyoxalin-4 (5) propionic acid, 
 
 CH— NHs 
 
 > 
 
 >CH 
 
 C N 
 
 CH 2 CHCI.COOH 
 
 This latter treated with NH 3 yields d-Z-histidine, which is changed into the active 
 forms by means of tartaric acid. 
 
 In the anaerobic putrefaction of histidine, /3-imidazolylethylamine 
 and imidazolylpropionic acid are formed (Ackermann 3 ). 
 
 Histidine crystallizes in colorless needles and plates and is readily 
 soluble in water, but less soluble in alcohol, and has an alkaline reaction. 
 It is precipitated by phosphotungstic acid, but this precipitate is soluble 
 in an excess of the precipitant (Frankel). With silver nitrate alone 
 the aqueous solution is not precipitated; on the careful addition of 
 ammonia or baryta-water an amorphous precipitate, which is readily 
 soluble in an excess of ammonia, is obtained. Histidine can be pre- 
 cipitated by mercuric chloride, or, still better, by the sulphate acidified 
 with sulphuric acid, and can in this way be separated from the other 
 diamino-acids (Kossel and Patten). The hydrochloride crystallizes in 
 beautiful plates (Bauer), dissolves rather readily in water, but is insolu- 
 ble in alcohol and ether. With hydrochloric acid and methyl alcohol 
 it gives the dihydrochloride of histidine methyl ester, which melts at 
 196°. Histidine is lsevorotatory, (a) D = —39.74°, while its solution in 
 hydrochloric acid is dextrorotatory. On warming it gives the biuret test 
 (Herzog), and it also gives Weld el's reaction if performed as sug- 
 gested by Fischer (see Xanthine, Chapter V) (Frankel 4 ). On adding 
 
 1 Kossel, Zeitsflir. f. physiol. Chem., 22; Hedin, ibid., Kutscher, ibid., 25; Wetzel, 
 ibid., 26; Lawrow, ibid., 28, and Ber. d. d. chem. Gesellsch., 34; Kossel and Kutscher, 
 Zeitschr. f. physiol. Chem., 31; Hart, ibid., 33; Abderhalden, ibid., 37; Schulze, ibid. r 
 24 and 28. 
 
 - Cited from Chem. Centralbl., 1911, 2, p. 760. 
 
 3 Zeitsr-lir. f. physiol. Chem., 65. 
 
 * Kossel and Patten, Zeitschr. f. physiol. Chem., 38; Bauer, ibid., 22; Herzog, ibid. y 
 37; Frankel, Sitz.-Ber. d. Wiea. Akad., 112, II. B., 1903, and Hofmeister's Beitrage, 8. 
 
ARGININE. 161 
 
 sufficient bromine water and warming, a reddish coloration ensues 
 which turns deep wine-red, later becoming cloudy, due to the forma- 
 tion of dark amorphous particles (F. Knoop ] ). It gives a very beautiful 
 diazo-reaction with diazobenzenesulphonic acid, in solutions made alkaline 
 with sodium carbonate, which according to Pauly is deep cherry-red 
 in dilutions of 1:20000 and still markedly red in 1:100000 (tyrosine 
 gives a similar reaction). 
 
 Several salts of histidine are known; H. Pauly 2 has especially 
 studied the iodized derivatives of histidine and imidazole. 
 
 On feeding ^-/-histidine to rabbits Abderhalden and Weil 3 obtained 
 from the urine tf-histidine which was crystalline, was as sweet as sugar 
 and showed a specific rotation (a) D = +40.15° at 20° C. 
 
 Histidine is sometimes classified in a group, with the two diamino- 
 acids, arginine and lysine which Kossel has called the hexone bases. 
 
 (/-Arginine ( 5-guanido-a-aminovaleric acid), 
 
 ( HN ) C <NH 2 CH 2 
 C 6 H 14 N40 2 = (CH 2 ) 2 , 
 
 CH(NH 2 ) 
 COOH 
 
 first discovered by Schulze and Steiger in etiolated lupin- and pumpkin- 
 sprouts, has later been found in other germinating plants, in tubers and 
 roots. Gulewitsch has found arginine in the ox-spleen, and Totani 
 and Katsuyama have found it in ox-testicles. It was first found 
 by Hedin as a cleavage product of horn substance, gelatin, and several 
 proteins, and then by Kossel and his pupils as a general cleavage prod- 
 uct of protein substances as a class. The greatest quantity was obtained 
 from the protamines; but the histones and certain plant proteins, 
 edestin and the protein from pine seeds and especially excelsin (14.14 
 per cent), also yield abundant arginine. Arginine also occurs among 
 the products of tryptic digestion (Kossel and Kutscher 4 ). 
 
 On boiling with baryta-water, as well as by the action of an enzyme, 
 arginase, discovered bv Kossel and Dakin, 5 arginine yields urea and 
 ornithine. 
 
 1 Hofmeister's Beitrage, 1 1 . 
 
 2 Ber. d. d. chem. Gesellsch., 43. 
 1 Zeitschr. f. physiol. Chem., 77. 
 
 4 Schulze and Steiger, Zeitschr. f. physiol. Chem., 11; Schulze and Castoro, ibid., 41; 
 Gulewitsch, ibid., 30; Totani and Katsuyama, ibid., 64; Hedin, ibid., 20 and 21; Kossel 
 and Kutscher, ibid., 22, 25, 26. 
 
 6 Zeitschr. f. physiol. Chem., 41, and Dakin, Journ. of biol. Chem., 3. 
 
162 THE PROTEIN SUBSTANCES. 
 
 Arginine has been prepared synthetically from ornithine (a-5-diamino- 
 valeric acid) and cyanamide by Schulze and Winterstein. Recently 
 Sorensen and Hoyrup x have prepared oM-arginine from ornithuric 
 acid. The a-monobenzoyl ornithine obtained by splitting ornithuric 
 
 N 
 acid with — barium hydrate yields a-benzoylamino-5-guanido-valeric 
 
 5 
 acid with cyanamide and this on boiling with hydrochloric acid gave 
 S-guanido-a-aminovaleric acid (eW-arginine) . 
 
 Arginine crystallizes in rosette-like tufts, plates, or thin prisms, is readily 
 soluble in water with alkaline reaction and almost insoluble in alcohol. 
 With several acids and metallic salts it forms crystalline salts and double 
 salts respectively. Its acidified watery solution is precipitated by phos- 
 photungstic acid. The most important salts are the copper-nitrate 
 (C 6 Hi4N 4 02)2.Cu(N03)2+3H20 and the silver salts 
 
 C 6 Hi4N 4 02.HN03+AgN03 
 
 (the more readily soluble) and CeHuN^.AgNOa+^O (the more 
 difficultly soluble), and its compound with picrolonic acid (Steudel 2 ). 
 
 Arginine is dextrorotatory. For arginine-chloride in watery solu- 
 tion with excess of hydrochloric acid, Gulewitsch 3 found (a) D = +21.25° 
 at 20° C. The arginine obtained by Kutscher in the tryptic digestion 
 of fibrin was racemic arginine. As found by Kossel and Weiss (see 
 page 112) arginine or more properly the ornithine is very easily 
 racemerized within the protein molecule by the action of alkali. The 
 racemic arginine can, as Riesser 4 has shown, during cleavage by means 
 of arginase, yield Z-arginine, which is an asymmetric change. In^putre- 
 faction arginine yields ornithine, guanidine, putrescine and 5-amino- 
 
 valeric acid. 
 
 /NH 2 
 Agmatine (guanidobutylamine), C 6 H 14 N4 = HN.C<; 
 
 \NH.CH 2 (CH 2 ) 2 .CH 2 NH 2 , 
 
 is a base obtained by Kossel in the hydrolysis of herring sperm, and later by 
 Kutscher and Engeland 5 from ergot. Kossel has also obtained it synthetically 
 from cyanamide and tetramethylendiamine, and in this manner proved its con- 
 stitution. It is produced from arginine by splitting off C0 2 and bears the! same 
 relation to arginine that putrescine does to ornithine and cadeverine does to 
 lysine (see below). Agmatine gives several crystalline salts as described by 
 Kossel. It is precipitated by phosphotungstic acid. 
 
 Schulze and Winterstein, Ber. d. d. chem. Gesellseh., 32 and Zeitschr. f. physiol- 
 Chem., 34; Sorensen and Hoyrup, Ber. d. d. chem. Gesellseh, 43 and Zeitschr. f. physiol- 
 Chem., 76. 
 
 2 Zeitschr. f. physiol. Chem., 37 and 44. 
 
 3 Ibid., 27. 
 
 4 Kutscher, Zeitschr. f. physiol. Chem., 28 and 32; Riesser, ibid., 49. 
 
 6 Kossel, ibid., 66 and 68; Engeland and Kutscher, Centralbl. f. Physiol., 24, 479. 
 
ORNITHINE AND LYSINE. 163 
 
 CH 2 .(NH 2 ) 
 
 (('IT ^ 
 
 (/-Ornithine (a-8-diaminovaleric acid), CsHtsNjOa' /...V.,. kl is not a primary 
 
 Crl(.\ rl^) 
 
 COOH 
 cleavage product of proteins, but is formed from arginine on boiling with baryta- 
 water. Jaffe, 1 who first discovered this body, obtained it as a cleavage product 
 
 from ornithinic acid, which is found in the urine of hens fed with benzoic acid. 
 The ornithine which E. Fischer and later Sorensen, 2 have prepared syn- 
 thetically yields, as shown by Ellinger, putrescine (tetramethylene diamine), 
 (\H,iX1Ij)j, on putrefaction. A. Loewy and Neuberg 3 have shown that 
 ornithine is split into putrescine and CO> in the organism of cystinuria patients. 
 
 Ornithine is a non-crystalline substance which dissolves in water, giving an 
 alkaline reaction, and yields several crystalline salts. It is precipitated by 
 phosphotungstic acid and several metallic salts, but not by silver nitrate and 
 baryta-water (differing from arginine). Ornithine hydrochloride is dextrorotatory; 
 the synthetically prepared one is inactive. On shaking ornithine with benzoyl 
 chloride and caustic soda it is converted into dibenzoylornithine (ornithuric 
 acid). On splitting artificially prepared racemic ornithuric acid Sorensen has 
 shown that the naturally occurring ornithuric acid is identical with the dextro- 
 rotatory a-5-dibenzoyldiaminovaleric acid. Salts and derivatives of ornithine 
 have been described by Kossel and his collaborators 4 and they have given a 
 method for its isolation from mixtures. 
 
 Diaminoacetic acid, C , jH 6 X..Oj=CH(XHj)oCOOH was obtained by Drechsel 5 
 as a cleavage product of casein by boiling with tin and hydrochloric acid. It 
 crystallizes in prisms and gives a monobenzoyl compound which is not very soluble 
 in cold water and is almost insoluble in alcohol, and can be usi'd in the isolation 
 of the acid. 
 
 CH 2 (NH 2 ) 
 
 d-Lysine (a-c-diaminocaproicacid), C6Hi4N20o= ; ltJ ^L \, was first 
 
 C rl(J\ri2/ 
 
 COOH 
 
 obtained by Drechsel as a cleavage product of casein. Later he and 
 
 his pupils, as well as Kossel and others, found it among the cleavage 
 
 products of various proteins. It has not been detected in some vegetable 
 
 proteins such as the prolamines (page 1C6). E. Schulze found lysine 
 
 in germinating plants of the Lupinus luteus, and Winterstein found 
 
 it in ripe cheese. It has been obtained in largest amounts (28.8 per cent) 
 
 by Kossel and Dakin from the protamine a-cyprinine. From a gliadin 
 
 which was not contaminated and which they considered as a unit substance 
 
 although obtained from different fractions having different solubilities 
 
 in alcohol, Osborne and Leavenworth 6 found a small amount of lysine 
 
 1 Ber. d. d. chem. Gesellsch., 10 and 11. 
 
 2 Fischer, Ber. d. d. chem. Gesellsch, 34; Sorensen, Zeitschr. f. physiol. Chem., 44. 
 
 3 Ellinger, Zeitschr. f. physiol. Chem., 29; Loewy and Neuberg, ibid., 43. 
 
 4 Kossel and Weiss, Zeitschr. f. physiol. Chem., 68. 
 
 5 Ber. d. k. sachs. Gesellsch. d. Wiss., 44 
 
 6 Drechsel, Arch. f. (Anat. u.) Physiol., 1891, and Ber. d. d. chem. Gesellsch., 25; 
 Siegfried, Arch. f. (Anat. u.) Physiol., 1891, and Ber. d. d. chem. Gesellsch., 24; Hedin, 
 Zeitschr. f. physiol. Chem., 21; Kossel, ibid., 25; Kossel and Mathews, ibid., 25; Kossel 
 
164 THE PROTEIN SUBSTANCES. 
 
 {0.07 and 0.15 per cent in two different fractions). The generally 
 accepted view that lysine is completely absent in gliadin is still doubtful. 
 They could not detect lysine in zein by the same method. 
 
 Lysine has been synthetically prepared by E. Fischer and Weigert. 1 
 This lysine was racemic, while that prepared from protein is always 
 optically active and dextrorotatory. The rotation depends upon the 
 concentration and degree of acidity; for the hydrochloride a rotation of 
 («)d = +14° to 17.25° has been found. . On heating with barium hydroxide 
 it is converted into the racemic modification. According to Ellinger 
 lysine yields cadaverine (pentamethylenediamine), CsHio(NH~2)2, on 
 putrefaction, and this base is formed from the lysine in the organism 
 of those with cystinuria and at the same time CO2 is split off (A. Loewy 
 and Neuberg). 2 
 
 Lysine is readily soluble in water but is not crystallizable. The aque- 
 -ous solution is precipitated by phosphotungstic acid, but not by silver 
 nitrate and baryta-water (differing from arginine and histidine). It 
 gives two hydrochlorides with hydrochloric acid, and with platinum 
 chloride a chloroplatinate which is precipitable by alcohol and has the 
 composition CoHn^C^.H^PtCle-r^HsOH. It gives two silver salts 
 with AgNOs; one has the formula AgN03+CeHi4N202 and the other 
 AgN03+C G Hi4N 2 02.HN03. With benzoyl chloride and alkali, lysine 
 forms an acid, lysuric acid, C6Hi2(C7H50)2N202 (Drechsel), which 
 is homologous with ornithuric acid, and whose difficultly soluble acid 
 barium salt may be used in the separation of lysine. 3 The rather 
 insoluble picrate, which is precipitated from a not too dilute solution 
 of the hydrochloride by sodium picrate, may also be used in the detec- 
 tion of lysine. 
 
 Kutscher and Lohmann 4 have found a lysine having somewhat different 
 properties in the final products of pancreas autolysis. 
 
 In the preparation of the so-called hexone bases we can first precipitate 
 all the bases by phosphotungstic acid, when the monamino-acids remain in 
 solution. The precipitate is then decomposed in boiling water by barium 
 hydroxide and the bases obtained as silver compounds from this filtrate. 
 In regard to further details and the methods of separating the various 
 
 and Kutscher, ibid., 31; Kutscher, ibid., 29; Schulze, ibid., 28; Winterstein, cited in 
 Schulze and Winterstein, Ergebnisse der Physiologic, I, Abt. 1, 1902; Kossel and 
 Dakin, Zeitschr. f. physiol. Chem., 40; Osborne and Leavenworth, Journ. of biol. 
 Chem., 14 
 
 1 Ber. «l. d. chem Gesellsch., 35. 
 
 2 See footnote 3, p. 163. 
 
 3 Drechsel, Ber. d. d. chem. Gesellsch., 28; see also C. Willdenow, Zeitschr. f. physiol. 
 Chem.. 2.V 
 
 * Zeitschr. f. physiol. Chem., 41. 
 
DIAMINO-ACIDS. 165 
 
 bases we will refer to Steudel in Abderhalden's Handbuch der biochem- 
 ischen Arbeitsmethoden, Bd. 2, II, s. 498. 
 
 We give below a tabulation of the amounts of the three hexone bases 
 found in certain protein substances (in weight per cent): 
 
 Arginine. Lysine. HLatidine. 
 
 Sturine 1 58.2 L2.0 12.9 
 
 Cyprinine (a) 6 1 9 is s 0.0 
 
 Other protamines » 62.5—87.4 0.0 0.0 
 
 Histories 1 14.36—15.52 7.7 —8.3 1.21—2.34 
 
 Casein- 4.70—4.84 1.92—5.80 2.53—2.59 
 
 Syntonin (from meat) « 5.06 3 26 2.66 
 
 Heterosyntonose * 8.53 3.08—7.03 0.37— 11 2 
 
 Protosyntonose 2 4.55 3.08 3.35 
 
 Edestin 3 11.0—14.07 1 .3 117 
 
 Proteid from conifera: seeds 3 10 . 9—11 .3 . 25—0 79 . 62—0 78 
 
 Gluten casein J 4 4 2.15 1.16 
 
 Gluten proteins « 2.75— 3.13 0.0 0.43—1.53 
 
 Gelatin ' and 2 7.62—9.3 2.49—6.0 0.40 
 
 Elastin 4 0.3 + 0.027 
 
 Of the oxydiamino-acids found on the hydrolysis of proteins we will 
 mention the following: 
 
 Oxydiaminosebacic acid, (?) CwHmNjOb, has been isolated by Wohlgemuth « 
 from a nucleoprotein of the liver. The free acid was obtained as small white 
 plates. It is soluble with difficulty in hot water, insoluble in cold water and 
 in alcohol. It was optically inactive in hydrochloric acid. The beautifully 
 crystalline phenylcyanate compound had a melting-point of 206°. 
 
 Dioxydiaminosuberic acid, C8H, 6 X>0 6 . has been obtained by Skraup 7 on the 
 hydrolysis of casein with hydrochloric acid. The copper salt crystallizes in 
 beautiful deep bluish-violet rosettes which are composed of long, irregular, right- 
 angled plates. It is quite soluble in cold water. The free acid crystallizes in 
 fern-like formations. Besides this acid Skraup obtained two other acids which 
 he calls caseanic acid, C9H16N2O7, and caseinic acid, C12H24N0O5. The caseanic 
 acid crystallizes, melts at 190-191°, is tribasic, and is probably an oxydiamino- 
 acid. The caseinic acid is dibasic and occurs in two modifications. The one 
 which melts at 228° is faintly dextrorotatory; the other modification melts 
 at 245° and is optically inactive. Both crystallize, but the inactive form does 
 not yield well-defined crystals. Caseinic acid seems also to be an oxydiamino- 
 acid. 
 
 Diaminotrioxydodecanoic acid, Ci 2 H 2 6N>0 5 , is an acid obtained by Fischer and 
 Abderhalden 8 on the hydrolysis of casein and seems to stand close to Skraup's 
 caseinic acid, but differs from it in its optical properties. This acid is faintly 
 levorotatory: (a) D =about— 9°. It crystallizes in plates, which grow into rosettes 
 
 1 Kossel and Kutscher, Zeitschr. f. physiol. Chem., 31. 
 * Hart, ibid., 33. 
 
 3 Schulze and Winterstein, ibid., 33; see also Kossel, Ber. d. d. chem. Gesellsch., 
 34, 3236. 
 
 4 Kossel and Kutscher, Zeitschr. f. physiol. Chem., 25, and Richards and Gies, 
 Amer. Journ. of Physiol., 7. 
 
 5 Kossel and Dakin, Zeitschr. f. physiol. Chem., 40. 
 
 6 Ber. d. d. chem. Gesellsch., 37, and Zeitschr. f. physiol. Chem., 44. 
 
 7 Zeitschr. f. physiol. Chem., 42. 
 
 8 Zeitschr. f. physiol. Chem.. 42. 
 
166 THE PROTEIN SUBSTANCES. 
 
 or spherical aggregations. It has a faint bitter taste, gives a crystalline hydro- 
 chloride which is slightly soluble in strong hydrochloric acid, and gives a crys- 
 talline copper salt. 
 
 After describing the different amino-acids it remains for us to call 
 attention to certain general reactions of the amino-acids. 
 
 By the action of formaldehyde the amino groups are changed into 
 methylene groups according to the scheme: 
 
 R.CH.NH 2 R.CH.N : CH 2 
 
 +HCOH = I +H 2 0. 
 
 COOH COOH 
 
 The amino-acids behave like neutral bodies while the methylene 
 combinations are acids and on this behavior is based Sorensen's 1 
 formoltitration which serves for the estimation of amino-acids in the 
 urine (Chapter XI V) as well as to follow the progress of proteolysis. 
 As the proteolysis progresses and imide bindings are loosened a large 
 number of atomic complexes with free NH2 and COOH groups are set 
 free. If now the NH2 groups are fixed as methylene groups by the addi- 
 tion of formol, the complex behaves like acids and the number of their 
 
 . . . N 
 
 COOH groups can be determined by titration with — barium or sodium 
 
 5 
 
 hydroxide solution, using phenolphthalein or thymolphthalein as in- 
 dicator. With the presumption that for every COOH group set free 
 there existed a free NH 2 group the extent of the proteolysis can also 
 be expressed in milligrams N by multiplying the number of cubic centi- 
 
 N 
 meters — alkali used by 2.8. 
 5 
 
 Siegfried has found that amino-acids in the presence of alkali or 
 
 alkaline earths de-ionize carbon dioxide and form salts of the type of 
 
 the carbamino salts, Siegfried's " carbamino-reaction." For example 
 
 glycocoll in the presence of lime yields with carbon dioxide, calcium 
 
 carbamino-acetic acid, CH2.NH.COO 
 
 I I • 
 
 COO Ca 
 
 If the nitrogen is determined and at the same time the combined carbon 
 
 dioxide estimated by means of the calcium carbonate split off on boil- 
 
 CO 
 ing the filtered solution, then the quotient — — gives the number of N 
 
 atoms for every molecule CO2 taken up. This quotient is equal to 1 
 for glycocoll and the aliphatic amino-acids because these go over quan- 
 
 1 Sorensen, Bioch. Zeitschr., 7; with Jessen Hansen, ibid., 7; with V. Henriques, 
 Zeitschr. f. physiol. Chem., 63 and 64; Henriques and Gjaldbak, Ibid., 67 and 75. 
 
COMPOUND PROTEINS. 167 
 
 titatively into carbaminoacids. With the diamino-acid arginine, which 
 contains 4 nitrogen atoms, it is on the contrary only one-fourth because 
 this acid reacts with only one amino group, that of the a-amino valeric 
 acid chain. 
 
 The reaction which has been developed and extensively used by 
 Siegfried 1 and his pupils is of great value in the characterization of pep- 
 tones, kyrines, and proteoses, for the separation and fractional pre- 
 cipitation and for the determination of their constitution. The binding 
 of the carbon dioxide as carbamino-salts seems also in many ways 
 to be of physiological importance, as for example, the solubility of cal- 
 cium carbonate in alkaline fluids and for the carbon dioxide binding in 
 blood, etc. 
 
 The amino-acids can by methylation form betaines, for example, 
 trimethyl glycocoll or betaine CH2 — N(CH3)3. Betaine occurs abundantly 
 
 CO 
 
 in the plant kingdom. In the animal kingdom such bodies have been 
 found under physiological conditions in cold blooded animals and they 
 belong to those groups of bodies which have been called " aporrhegmas" 
 by Ackermann and Kutscher. 2 As " aporrhegmas " they designate 
 all those fractions of amino-acids from the protein, which can be pro- 
 duced from the proteins in a physiological manner and indeed in the life 
 of animals as well as the plants. These bodies are essentially the same 
 as have been observed in the putrefaction of the amino-acids and which 
 have been specially mentioned with every amino-acid described. 
 
 The behavior of the amino-acids in yeast fermentation will be dis- 
 cussed in Chapter III. 
 
 In regard to the methods for separating and preparing, in a pure form, 
 the various amino-acids and other products of protein hydrolysis which 
 have not been given in the preceding pages, we must refer to Abderhal- 
 den's Handbuch der biochemischen Arbeitsmethoden, 1909-1910 Bd. 2. 
 
 II. Compound Proteins. 3 
 
 We designate as compound proteins those bodies which yield, on 
 cleavage, proteins (with their decomposition products) and other bodies 
 such as carbohydrates, nucleic acids, or pigments. 
 
 The compound proteins known at present can be divided into three 
 groups: glycoproteins, nucleoproleins and chromoproteins. Of these the 
 
 1 In regard to the literature see Siegfried in Ergebnisse d. Physiol. Bd. 9. 
 
 2 Zeitschr. f. physiol. Chem., 69. See also Engeland, ibid., 69. 
 
 3 Hoppe-Seyler has given the name proteide to these compound proteids, but as 
 this term is misleading in English we do not use it in English classifications in this 
 sense. 
 
168 THE PROTEIN SUBSTANCES. 
 
 last-mentioned group (haemoglobin and haemocyanine) will be discussed 
 in a subsequent chapter (Chapter V on the blood). 
 
 A. Glycoproteins (glucoproteins) . 
 
 Glycoproteins * are those compound proteins which on decomposi- 
 tion yield a protein on the one side, and a carbohydrate or derivatives 
 of this on the other, but no purine bodies. Some glycoproteids are free 
 from phosphorus (mucin substances, chondroproteins, and hyalogens), 
 and some contain phosphorus (phosphoglycoproteins). 
 
 The glycoproteins free from phosphorus may, as regards the nature 
 of the carbohydrate groups split off, be divided into two chief groups, 
 the mucin substances and the chondroproteins. The first yield on hydrolytic 
 cleavage an amino-sugar, which has been shown to be glucosamine in 
 all but a few exceptions. 2 In the chondroproteins, on the contrary, the 
 protein is united to chondroitin-sulphuric acid. 
 
 1. Mucin Substances. 
 
 Compared with the simple proteins the mucin substances are poorer 
 in nitrogen and as a rule also have considerably less carbon. The carbo- 
 hydrate complex, whose nature has been shown by the investigations 
 cf Fr. Muller 3 and his pupils, occurs, so it seems, in the mucin sub- 
 stances as a polysaccharide related to chitosan, which on hydrolytic 
 cleavage yields glucosamine (chitosamine), and, at least in most cases, 
 acetic acid also. The mucin substances differ very markedly among 
 themselves, hence we divide them into two groups, the mucins and the 
 mucoids. 
 
 The true mucins are characterized by the fact that their natural 
 
 1 Abderhalden (Lehrb d. physiol. Chem., 1909, p. 191) has proposed dropping the 
 name glycoproteids entirely and to consider these bodies as simple proteins, because 
 it has not been shown that the carbohydrate groups occupy the same relationship 
 to the protein component that the haemin or the nucleic acid bears to the haemo- 
 globin or the nuclooprotein molecule. It is possible that this proposition, which is 
 not applicable to the entire group (including the proteins containing chondroitin- 
 sulphuric acid) but applies only to the mucin group, will be found in the future to be 
 correct. It is the opinion of Hammarsten that it is better to wait for further research 
 in this direction before we drop the generally accepted nomenclature and the usual 
 subdivisions of the proteins. 
 
 2 See Schulz and Ditthorn, Zeitschr. f. physiol. Chem., 29; A. v. Ekenstein and 
 Blunksma, Chem. Centralbl., 1907. 2. When both carbohydrate groups are simul- 
 taneously combined in one body, then probably we are not dealing with a chemical 
 individual, but rather with a mixture. 
 
 3 See Fr. Muller, Zeitschr. f. Biologie, 42, which contains all the pertinent litera- 
 ture, and also L. Langstein, Die Bildung von Kohlenhydraten aus Eiweiss, Ergebnisse 
 der Physiologie, Jahr. I, Abt. 1. 
 
MUCINS. 1(39 
 
 solutions, or solutions prepared by the aid of a traee of alkali, arc mucilagi- 
 nous, ropy, and give a precipitate with acetic acid which is insoluble in 
 excess of acid or soluble only with great difficulty. The mucoids do not 
 show these physical properties, and have other solubilities and precipit- 
 ation properties. As we have intermediate steps between different pro- 
 tein 1 todies, so also we have such between true mucins and mucoids, and 
 a sharp line cannot be drawn between these two groups. 
 
 It is just as difficult at present to draw a sharp line between the pro- 
 teins and the mucins or mucoids, since we have been able to split off 
 carbohydrate complexes from several proteins, and as proteins have 
 been isolated from white of egg which yield more or less glucosamine. 
 The very variable amounts of glucosamine obtained under various con- 
 ditions from the crystalline ovalbumin seem to indicate that we are 
 dealing with a contamination with a glycoprotein. 
 
 True mucins are secreted by the larger mucous glands, by certain 
 mucous membranes, and by the skin of snails and other animals. True 
 mucin also occurs in the navel-cord. Sometimes, as in snails and in 
 the membrane of the frog-egg (Giacosa) and perch-eggs (Hammarsten l ), 
 a mother-substance of mucin, a mucinogen, has been found which may 
 be converted into mucin by alkalies. Mucoid substances are found in 
 certain cysts, in the cornea, the crystalline lens, white of egg, and in 
 certain ascitic fluids. ' The so-called tendon-mucin, which, according 
 to the investigations of Levene and of Cutter, and Gies, 2 contains 
 chondroitin-sulphuric acid or a related substance, cannot be classified 
 as a mucin, but must, like the chondromucoid and the osseomucoid, 
 be classified as chondroprotein. As the mucin question has not been 
 sufficiently studied, it is at the present time impossible to give any positive 
 statements in regard to the occurrence of mucins and mucoids, especially 
 as without doubt in many cases non-mucinous substances have been 
 described as mucins. 
 
 True Mucins. Thus far we have been able to obtain only a few 
 mucins in a pure and unchanged condition, because of the reagents used. 
 The elementary analyses of these mucins have given the following results: 
 
 c h n s 
 Mucin from mucous membrane (air- 
 passages) 48.26 6.91 10.70 1.40 (Fr. Muller «) 
 
 Mucin from submaxillary 48.84 6.80 12.32 0.84 (Hammarsten 5 ) 
 
 Mucin from snail 50 . 32 6 . 84 13 . 65 1 . 75 (Hammarsten j ) 
 
 Synovial mucin 51.05 6.53 13.01 1.34 (v. Holst *) 
 
 1 Giacosa, Zeitschr. f. physiol. Chem., 7; Hammarsten, Pfluger's Archiv., 36, and 
 Skand, Arch. f. Physiol., 17. 
 
 2 Levene, Zeitschr. f. physiol. Chem., 31; Cutter and Gies, Amer. Journ. of Physiol., 6. 
 
 3 Fr. Muller, Zeitschr. f. Biologie, 42; Hammarsten, Zeitschr. f. physiol. Chem.. 12, 
 .and Pfluger's Arch., 36. 
 
 4 Zeitschr. f. physiol. Chem., 43. 
 
170 THE PKOTEIN SUBSTANCES. 
 
 Muller obtained 35 per cent glucosamine from mucous-membrane 
 mucin and 23.5 per cent from the submaxillary mucin. 
 
 On boiling mucin with dilute mineral acids, acid albuminate and 
 bodies similar to proteoses are obtained, besides a reducing substance 
 which is not free glucosamine (Steudel 1 ). By the action of strong 
 acids upon mucins or mucoids Otori 2 obtained several of the cleavage 
 products of the proteins, such as leucine, tyrosine, glycocoll, glutamic 
 acid, oxalic acid, guanidine, arginine, lysine, and humus substances, 
 and also carbohydrate cleavage products, such as levulinic acid. Cer- 
 tain mucins, as the submaxillary mucin, are easily changed by very 
 dilute alkalies, as lime-water, while others, such as tendon-mucin, are 
 not affected. If a strong caustic-alkali solution, such as a 5-per cent 
 KOH solution, is allowed to act on submaxillary mucin, we obtain alkali 
 albuminate, bodies similar to proteoses and peptones and one or more 
 substances of an acid reaction which have strong reducing powers. 
 
 On peptic digestion proteoses and peptone-like bodies, still con- 
 taining the carbohydrate group, are produced. On tryptic digestion 
 still simpler cleavage products are formed, namely, leucine, tyrosine, 
 and tryptophane (Posner and Gies 3 ). The glucosamine, so far as we 
 know, is not split off by proteolytic enzymes, but only after strong 
 hydrolysis with acids. 
 
 In one or another respect the various mucins act somewhat dissimilarly. 
 For example, the snail and sputum mucins are insoluble in dilute hydro- 
 chloric acid of 1-2 p. m., while the mucin of the submaxillary gland and 
 the navel-cord is soluble. The former become flaky with acetic acid, 
 while the submaxillary mucin is precipitated in more or less fibrous, 
 tough masses. Still all the mucins have certain reactions in common. 
 
 In the dry state mucin forms a white or yellowish-gray powder. When 
 moist it forms, on the contrary, flakes or yellowish-white tough lumps 
 or masses. The mucins are acid in reaction. They give the color reac- 
 tions of the proteins. They are not soluble in water, but may give a 
 neutral solution with water with the aid of small amounts of alkali. Such 
 a solution does not coagulate on boiling, but acetic acid gives at the 
 normal temperature a precipitate which is nearly insoluble in an excess 
 of the precipitant. If 5-10 per cent NaCl be added to a mucin solution, 
 it can be carefully acidified with acetic acid without giving a pre- 
 cipitate. Such acidified solutions are copiously precipitated by tan- 
 nic acid; with potassium ferrocyanide they give no precipitate, but on 
 sufficient concentration they become thick or viscous. A neutral solu- 
 tion of alkali mucin is precipitated by alcohol in the presence of neutral 
 
 1 Zeitschr. f. physiol. Chem., 34. 
 
 2 Ibid., 42 and 43. 
 
 '■'■ Amer. Journ. of Physiol., 11. 
 
HYALOGENS. 171 
 
 salts; it is also precipitated by several metallic salts. If mucin is heated 
 on the water-bath with dilute hydrochloric acid of about 2 per cent, 
 the liquid gradually becomes a yellowish or dark brown, and reduces 
 copper salts in alkaline solutions. 
 
 The mucin most readily obtained in large quantities is the submax- 
 illary mucin, which may be prepared in the following way: The filtered 
 watery extract of the gland, free from form-elements and as colorless 
 as possible, is treated with 25 per cent hydrochloric acid, so that the 
 liquid contains 1.5 p. m. HC1. On the addition of the acid the mucin 
 is immediately precipitated, but dissolves on stirring. If this acid liquid 
 is immediately diluted with 2-3 vols, of water, the mucin separates and 
 may be purified by redissolving in 1-5 p. m. acid, and diluting with 
 water and washing therewith. The mucin of the navel-cord may be 
 prepared in the same way. As a rule the mucins can be prepared by 
 precipitation with acetic acid and repeated solution in dilute lime-water 
 or alkali, and reprecipitation with acetic acid. Finally they are treated 
 with alcohol and ether. In the preparation of sputum mucin the method 
 is very complicated (Fr. Muller). 
 
 Mucoids or Mucinoids. In this group we must include those non- 
 phosphorized glycoproteins which are neither true mucins nor chondro- 
 proteids, although they show among themselves such differences in 
 behavior that they can be divided into several subgroups of mucoids. 
 To the mucoids belong pseudomucin, the probably related body colloid, 
 ovomucoid, and other bodies, which on account cf their differences will be 
 best treated individually in their respective chapters. 
 
 Hyalogens. Under this name Krukenberg l has designated a number of 
 different bodies, which are characterized by the following: By the action of 
 alkalies they change, with the splitting off of sulphur and some nitrogen, into 
 soluble nitrogcnized products called by him hyalines, and which yield a pure car- 
 bohydrate by further decomposition. We find that very heterogeneous sub- 
 stances are included in this group. Certain of these hyalogens seem undoubtedly 
 to be glycoproteins. Neossin 2 of the Chinese edible swallow's-nest, membranin* 
 of Descemet's membrane and of the capsule of the crystalline lens, and spiro- 
 graphin 4 of the skeletal tissue of the worm Spirographs, seem to act as such. 
 Others, on the contrary, such as hyalin 5 of the walls of hydatid cysts, and arm- 
 phin* from the tubes of Onuphis tubicola, do not seem to be compound proteins. 
 The so-called mucin of the holothuria, 7 and chondrosin 8 of the sponge, Chondrosia 
 
 1 Verh. d. physik.-med. Gesellsch. zu Wurzburg, 1883; also Zeitschr. f. Biologie, 22. 
 
 2 Krukenberg, Zeitschr. f. Biologie, 22. 
 
 3 C. Th. Morner, Zeitschr. f. physiol. Chem., 18. 
 
 4 Krukenbere, Wurzburg, Verhandl., 1883: also Zeitschr. f. Biologie, 22. 
 
 5 A. Lucke, Virchow's Arch., 19; also Krukenberg, Vergleichende physiol. Stud., 
 Series 1 and 2, 1881. 
 
 6 Schmiedeberg, Mitth. aus d. zool. Stat, zu Neapel, 3, 1882. 
 
 7 Hilger. Pfliiger's Archiv, 3. 
 
 8 Krukenberg, Zeitschr. f. Biologie, 22. 
 
172 THE PROTEIN SUBSTANCES. 
 
 reniformis, and others may also be classed with the hyalogens. As the various 
 bodies designated by Krunkenberg as hyalogens are very dissimilar, it is not 
 of much advantage to arrange these in special groups. 
 
 2. Chondroproteins. 
 
 Chondroproteins are those glycoproteins which as primary cleav- 
 age products yield protein and an ethereal sulphuric acid, the chondroitin- 
 sulphuric acid. Chondromucoid, occurring in cartilage, is the best example 
 of this group. Amyloid occurring under pathological conditions also 
 belongs to this group. On account of the property of chondroitin-sul- 
 phuric acid of precipitating protein, it is also possible that under certain 
 circumstances combinations of this acid with protein may be precipitated 
 from the urine and be considered as chondroproteins. 
 
 The chondromucoid, the so-called tendon-mucin, and the osseomucoid 
 have greatest interest as constituents of cartilage, of the connective 
 tissues, and the bones, and on this account these bodies and their cleavage 
 product, chondroitin-sulphuric acid, will be treated in a following chap- 
 ter (IX). On the contrary, amyloid, which has always been considered 
 in connection with the protein substances, will be described here. 
 
 Amyloid, so called by Virchow, is a protein substance appear- 
 ing under pathological conditions in the internal organs, such as the spleen, 
 liver and kidneys, as infiltrations; and in serous membranes as granules 
 with concentric layers. It probably also occurs as a constituent of 
 certain prostate calculi. The chondroprotein occurring under physio- 
 logical conditions in the walls of the arteries is, perhaps, according to 
 Krawkow, very closely related to the amyloid substance, but not iden- 
 tical with it, as shown by Neuberg. 1 
 
 Recently O. Hanssen has studied the mechanically isolated amy- 
 loid obtained from the so-called " sago kernels " of an amyloid spleen, 
 and could not detect any conjugated sulphuric acid in it. According to 
 his investigations true amyloid is not a chondroprotein. Mayeda 2 
 has also prepared an amyloid substance free from chondroitin-sulphuric 
 acid. On the other hand, Hanssen has found that amyloid organs 
 (liver and spleen) are much richer in sulphuric acid that splits off than 
 normal organs, and it is not improbable that the amyloid formation goes 
 hand in hand with the formation of a chondroprotein. 
 
 The amyloid prepared by Krawkow and Neuberg had about the 
 same composition: C 49.0-50.1; H 7-7.2; N 14-14.1, and S 1.8-2.8 
 
 1 Krawkow, Arch. f. exp. Path. u. Pharm., 40, which contains the literature; Neu- 
 berg, Verhandl. d. d. Pathol. Gesellsch., 1004. 
 
 2 Hanssen, Bioch. Zeitschr., 13; Mayeda, Zeitschr. f. physiol. Chem., 58. 
 
AMYLOID. 173 
 
 per cent. The aorta amyloid of man and of the horse contained respect- 
 ively C 49.6 and 50.5; H 7.2; N 14.4 and 13.8; S 2.3 and 2.5 per cent. 
 As we cannot tell whether the amyloid analyzed was pure or not the 
 results are of questionable value. 
 
 According to older investigations amyloid splits, by the action of 
 alkali, into protein and chondroitin-sulphuric arid (see Chapter IX), 
 and according to Krawkow it is therefore a firm, perhaps ester-like 
 combination of this acid with protein. The protein, from the investiga- 
 tions of Neuberg, is of a basic nature and most comparable to the 
 histones. The investigations of Mayeda do not coincide with this view 
 as the amyloid protein obtained by him did not behave like a histone. 
 Its content of hexone bases was not greater than that of the proteins 
 of the normal organs and this amyloid protein did not yield any his- 
 tone-peptone. To all appearances, different investigators have worked 
 with different substances and it is possible that in the amyloid degenerated 
 organs partly chondroproteins and partly amyloid proteins may occur, 
 both of which give the color reactions. 
 
 Amyloid is an amorphous white substance, insoluble in water, alcohol, 
 ether, dilute hydrochloric and acetic acids. It is soluble in concen- 
 trated hydrochloric acid or caustic alkali with decomposition. On boil- 
 ing with dilute hydrochloric acid it yields sulphuric acid and a reducing 
 substance. It is not dissolved by gastric juice, according to Krawkow, 
 which agrees with most of the older reports. It is nevertheless changed 
 so that it is soluble in dilute ammonia, while the typical amyloid is 
 insoluble therein. Neuberg finds on the contrary that amyloid (from 
 liver) is digested by pepsin as well as by trypsin, although more slowly 
 than fibrin, and that it is also destroyed in autolysis, so that in life an 
 absorption is possible. The amyloid from the " sago " spleen studied 
 by Hanssen showed the same behavior with gastric juice as Krawkow 
 found, while trypsin, as well as autolysis for months, was without action. 
 Mayeda's amyloid was gradually dissolved by gastric juice. 
 
 Amyloid gives the xanthoproteic reaction and the reactions of Mil- 
 lon and Adamkiewicz-Hopkins. Its most important property is its 
 behavior with certain coloring matters. It is colored reddish-brown 
 or a dingy violet by iodine; a violet or blue by iodine and sulphuric 
 acid; red by methylaniline iodide, especially on the addition of acetic 
 acid; and red also by aniline green. Of these color reactions those with 
 aniline dyes are the most important. The iodine reaction appears less 
 constant and is greatly dependent upon the physical condition of the 
 amyloid. The color reactions are due to the presence cf the chondroitin- 
 sulphuric acid component, but this stands in opposition to the behavior 
 of the intact amyloid obtained by Hanssen from the " sago " spleen 
 and the amyloprotein of Mayeda. 
 
174 THE PROTEIN SUBSTANCES. 
 
 In preparing amyloid, extract the finely divided organs with very 
 dilute ammonia. The undissolved amyloid in the residue, if it does not 
 resist pepsin digestion, can be directly extracted by dilute barium hydrate 
 solution and then precipitated from the filtrate by hydrochloric acid. 
 Otherwise the above mentioned residue is digested for several days with 
 pepsin. The digestion residue is dissolved in dilute ammonia, filtered, 
 the amyloid precipitated by dilute hydrochloric acid, the precipitate 
 dissolved in baryta-water, when the nucleins remain behind, the barium 
 filtrate precipitated with hydrochloric acid and purified, if necessary 
 by repeated solution in ammonia and precipitating with hydrochloric 
 acid, washing and treating with alcohol and ether. 
 
 Phosphoglycoproteins. This group includes the phosphorized glycoproteins. 
 They yield no purine bases (nuclein bases) as cleavage products. They are not 
 nucleoproteins and therefore they must not be mistaken for them. On pepsin 
 digestion they may, like certain nucleoalbumins, yield pseudonuclein, but they 
 differ from the nucleoalbumins in that they yield a reducing substance on boil- 
 ing with dilute acid. They differ from the nucleoproteins, which also yield reduc- 
 ing carbohydrates, in, as above stated, not yielding any purine bases. 
 
 Only two phosphorized glycoproteins are known at the present time, namely, 
 ichthvlin, occurring in carp eggs and studied by Walter, 1 and which was con- 
 sidered as a vitellin for a time. Ichthulin has the following composition: C 53.52; 
 H 7.71; N 15.64; S 0.41; P 0.43; Fe 0.10 per cent. In regard to solubilities it 
 is similar to a globulin. Walter has prepared a reducing substance from the 
 pseudonuclein of ichthulin which gave a highly crystalline compound with 
 phenylhydrazine. 
 
 Another phosphoglycoprotein is helicoproteid, obtained by Hammarsten 2 
 from the glands of the snail Helix pomatia. It has the following composition: 
 C 46.99; H 6.78; N 6.08; S 0.62; P 0.47 per cent. It is converted into a gummy, 
 levorotatory carbohydrate, called animal sinistrin, by the action of alkalies. 
 On boiling with an acid it yields a dextrorotatory reducing substance. 
 
 The compound protein found by Shultz and Ditthorn 3 in the spawn of 
 the frog probably belongs to this group, but instead of glucosamine it gives 
 galactosamine on cleavage. 
 
 B. Nucleoproteins. 
 
 By this name we designate those compound proteins which yield 
 protein and nucleic acid on cleavage. The nucleoproteins seem to be 
 widely diffused in the animal body. They occur chiefly in the cell- 
 nuclei, but they also often occur in the protoplasm. They may pass 
 into the animal fluids on the destruction of the cells, hence nucleopro- 
 teins have also been found in blood serum and other fluids. 
 
 The nucleoproteins may be considered as combinations of a pro- 
 tein with a side chain, which Kossel calls the prosthetic group. This 
 side chain, which contains the phosphorus, may be split off as nucleic 
 acid on treatment with alkali. The protein may be of different kinds. 
 
 1 Zeitschr. f. physiol. Chem., 15. 
 
 2 IIammar.st.en, Pfltiger's Arch., 36. 
 
 3 Zeitschr. f. physiol. Chem., 29. 
 
NUCLE0PE0TEIN8. 175 
 
 In certain cases this is histone, and the combinations between nucleic 
 acid and protamines are also sometimes classified as nucleoproteins. 
 The combination between protamine and nucleic acid is, it seems, a 
 salt-like combination, and entirely different from the combination of the 
 proteins with nucleic acid in the nucleoproteins. The following facts, 
 given in connection with the nucleoproteins, do not apply to the nucleo- 
 protamines. The nucleoproteins differ not only according to the protein 
 component they contain, but also as to the nucleic acids, which vary 
 among themselves. There are essentially different nucleic acids, some 
 among which contain a pentose carbohydrate while others contain a 
 hexose carbohydrate. The nucleic acids also differ in regard to the 
 amount of purine and pyrimidine bases they contain (sec below). 
 
 The native nucleoproteins contain a variable, but not a high percentage 
 of phosphorus, which in most of the nucleoproteins investigated, ranges 
 between 0.5 and 1.6 per cent. They also regularly contain iron, and in 
 Octopodes, Henze i has observed an iron-free nucleoprotein with 0.96 
 per cent copper. The nucleoproteins behave like weak acids, especially 
 those having considerable protein in the molecule. They therefore 
 give the ordinary protein reactions and behave in this regard like the 
 proteins. The nucleoproteins prepared from organs rich in cell nuclei 
 seem to be characterized by containing more phosphorus and having a 
 stronger acid character. All nucleoproteins are bodies that are insoluble 
 in water, but whose alkali combination is soluble in water. From 
 such a solution the nucleoprotein can be precipitated by acetic acid, and 
 in an excess of the acid, the precipitate dissolves with more or less 
 difficulty and in some cases not at all. It dissolves, on the contrary, in 
 very dilute hydrochloric acid. In this respect nucleoproteins are similar 
 to the nucleoalbumins and the mucin substances, but differ from these 
 two groups in that they yield purine bases on hydrolysis. According 
 to Plimmer and Scott 2 the nucleoproteins differ from the nucleo-albu- 
 mins by the fact that with sodium hydroxide in 1 per cent solution the 
 nucleoalbumins split off phosphoric acid while the nucleoproteins do not. 
 The nucleoproteins give the color reactions of the proteins, but those 
 which have been investigated are dextrorotatory and not laevorotatory 
 (Gamgee and Jones 3 ). 
 
 The nucleoproteins are readily modified. The alkali combination 
 scluble in water suffers a decomposition on heating its solution, when 
 as neutral as possible, and coagulated protein separates while a protein 
 rich in phosphorus and poor in protein with strong acid character remains 
 
 1 Zeitschr. f. physiol. Chem., 55. 
 
 2 Plimmer and Scott, cited in Biochem. Centralbl., 8, p. 109. 
 
 3 Hofmeister's Beitrage, 4. 
 
176 THE PROTEIN SUBSTANCES. 
 
 in solution. By the action of weak acids and by gastric juice a similar 
 cleavage takes place, whereby the protein split off goes into solution 
 while the nucleoprotein rich in phosphorus, so-called nuclein (Miescher, 
 Hoppe-Seyler *) or true nuclein, remains undissolved. As the nuclein 
 is probably nothing but a partly modified nucleoprotein poorer in pro- 
 tein, having a composition varying with the intensity of the cleavage, 
 it seems unnecessary to give the name nuclein thereto. On the other 
 hand, the nucleins have other properties than the nucleoproteins, and 
 as the nucleins bear the same relation to the nucleoproteins that the 
 pseudonuclein does to the nucleoalbumins, we will here give a short 
 description of the nucleins as well as the pseud'o- or paranucleins. 
 
 Nucleins or true nucleins are formed, as above stated, from nucleo- 
 proteins in their peptic digestion or by treatment with dilute acids. It 
 must be remarked that the nucleins are not entirely resistant toward 
 gastric juice, and also that at least one nucleoprotein, namely, the one 
 obtained from the pancreas, completely dissolves, leaving no nuclein 
 residue on treatment with gastric juice (Umber, Milroy 2 ). The 
 nucleins are rich in phosphorus, containing in the neighborhood of 5 per 
 cent. According to Liebermann, 3 metaphosphoric acid can be split 
 off from true nucleins (yeast nuclein). The nucleins are decomposed 
 into protein and nucleic acid by caustic alkali, and as different nucleic 
 acids exist, so also there exist different nucleins. As previously stated 
 proteins may be precipitated in acid solutions by nucleic acids, and in 
 this way, as shown by Milroy, combinations of nucleic acid and pro- 
 teins may be prepared which behave quite like true nucleins. All nucleins 
 yield purine bases (so-called nuclein bases) on boiling with dilute acids. 
 They act like rather strong acids. 
 
 The nucleins are colorless, amorphous and insoluble or only slightly 
 soluble in water. They are insoluble in alcohol and ether. They are 
 more or less readily dissolved by dilute alkalies. The nucleins give the 
 biuret test and Millon's reaction. They show a great affinity for many 
 dyes, especially the basic ones, and take these up with avidity from watery 
 or alcoholic solutions. On burning they yield an acid residue which 
 is very difficult to incinerate and which contains metaphosphoric acid. 
 On fusion with saltpeter and soda the nucleins yield alkali phosphates. 
 
 To prepare nucleins from cells or tissues, first remove the chief mass 
 of proteins by artificial digestion with pepsin-hydrochloric acid, lixiviate 
 the residue with very dilute ammonia, filter, and precipitate Avith hydro- 
 chloric acid. The precipitate is further digested with gastric juice, 
 
 1 Hoppe-Seyler, Med. chem. Unters., 452. 
 
 5 ["ruber, Zeitsclir. f. klin. Med., 34; Milroy, Zeitschr. f. physiol. Chem., 22. 
 
 3 Pfluger's Arch., 47. 
 
FSEUDONUCLEINS. 177 
 
 washed and purified by alternately dissolving in very faintly alkaline 
 water and reprecipitating with an acid, washing with water, and treating 
 with alcohol and ether. A nuclein may be prepared more simply by the 
 digestion of a nucleoprotein. In the detection of nucleins we make use 
 of the above-described method, testing for phosphorus in the product 
 after fusing with saltpeter and soda. Naturally the phosphates and 
 phosphatides must first be removed by treatment with acid, alcohol, 
 and ether, respectively. No exact methods are known for the quanti- 
 tative estimation of nucleins in organs or tissues. 
 
 Pseudonucleins or Paranucleins. These bodies are obtained as 
 an insoluble residue on the digestion of certain nucleoalbumins or phospho- 
 glycoproteins with pepsin-hydrochloric acid. Attention is called to 
 the fact that the pseudonuclein may be dissolved by the presence of too 
 much acid or by a too energetic peptic digestion. If the relation between 
 the degree of acidity and the quantity of substance is not properly selected, 
 the formation of pseudonucleins may be entirely overlooked in the 
 digestion of certain nucleoalbumins. Pseudonucleins contain phosphorus, 
 which, as shown by Liebermann, 1 is split off as metaphosphoric acid 
 by mineral acids. 
 
 The pseudonucleins are amorphous bodies insoluble in water, alcohol, 
 and ether, but readily soluble in dilute alkalies and barium hydroxide 
 solution. They are readily split by barium hydroxide solution with the 
 splitting off of phosphoric acid, and according to Giertz 2 they differ 
 in this regard from the true nucleins, which are neither dissolved nor 
 decomposed by baryta. They are not soluble in very dilute acids, and 
 may be precipitated from their solution in dilute alkalies by adding 
 acid. They give the protein reactions very strongly, but do not yield 
 purine bases. 
 
 In preparing a pseudonuclein, dissolve the mother-substance in hydro- 
 chloric acid of 1-2 p. m., filter if necessary, add pepsin solution, and 
 allow the mixture to stand at the temperature of the body for about 
 twenty-four hours. The precipitate is filtered off, washed with water, 
 and purified by alternately dissolving in very faintly alkaline water and 
 reprecipitating with acid. 
 
 Cleavage Products of the Xucleoproteins. 
 
 1. The Nucleic Acids. 
 
 All nucleic acids are rich in phosphorus and yield phosphoric acid, 
 purine bases and a carbohydrate or carbohydrate derivative as cleavage 
 products; most of them also contain pyrimidine bases. The older 
 
 1 Ber. d. d. chem. Gesellsch., 21, and Centralbl. f. d. med. Wissensch., 1889. 
 
 2 Zeitschr. f. physiol. Chem., 28. 
 
178 THE PROTEIN SUBSTANCES. 
 
 statements as to the occurrence of more than two purine bases in a nucleic 
 acid are not correct and depend upon the fact that the two purine bases 
 xanthine and hypoxanthine can be secondarily formed from guanine and 
 adenine. There is no doubt that the most thoroughly studied nucleic 
 acids, such as the thvmus-nucleic acids, the closely related or perhaps 
 identical acids of the salmon sperm (salmo-nucleic acid), of the herring 
 sperm and burbot sperm, and of the pancreas, do not contain more than 
 two purine bases, namely, guanine and adenine. 
 
 Of the known nucleic acids we have two, the guanylic acid and inosinic 
 acid, which contain only one purine base, namely, guanine and hypoxan- 
 thine, respectively. These two acids do not contain any pyrimidine 
 bases, which are found thus far in all carefully investigated nucleic acids. 
 The occurrence of pyrimidine bases is somewhat different in the various 
 nucleic acids. In one group of animal nucleic acids (thymonucleic acids) 
 thymine, cytosine and uracil are found, the uracil being produced second- 
 arily from the cytosine. The plant nucleic acids (the triticonucleic acid 
 and the yeast nucleic acid, which may perhaps be identical with it) do 
 not contain any thymine and yields as nitrogenous cleavage products 
 besides the two purine bases only cytosine and uracil. 
 
 All nucleic acids, as above stated, contain a carbohydrate group. 
 In the plant nucleic acids and in two animal ones, the guanylic and inosinic 
 acids, the carbohydrate is a pentose. In the remaining animal nucleic 
 acids it is on the contrary a hexose or at least a hexacarbohydrate. 
 
 The nature of this hexacarbohydrate has not been determined and 
 the nature of the pentoses occurring in the nucleic acids is also a disputed 
 point. Based upon the investigations of Neuberg we have considered 
 the pentose of guanylic acid and of inosinic acid as /-xylose. The correct- 
 ness of this view is disputed by others. According to Levene and Jacobs 
 the pentose of all nucleic acids containing pentose, is d-ribose. Haiser 
 and Wenzel who for a time considered the pentose of inosinic acid as 
 '/-xylose are now of the view that it is probably d-ribose. The view of 
 Levene and .Jacobs, that the pentose of the guanylic acid is d-ribose has 
 received important support by the investigations of Schulze and Trier 
 on the identity of the plant guaninpentoside vcrnine with the guanin- 
 pentoside (see below) prepared by Levene and Jacobs. Still we have 
 no explanation why Neuberg and Rewald l obtained only /-xylose 
 from the pancreas on the hydrolysis of the entire organ, and Levene 
 and Jacobs on the contrary only r/-ribose. 
 
 1 Neuberg and Brahn, Bioch. Zeitschr., 5; see also Ber. d. d. chem. Gesellsch 
 41 and 42: Levene and Jacobs, Ber. d. d. chem. Gesellsch., 42 and 43; Haiser and 
 Wenzel, Monatah. f. Chern., 61; Schulze and Trier, Zeitschr. f. physiol. Chem., 70; 
 lid, Ber. d. d. chem. Gesellsch., 42. 
 
NUCLEIC ACIDS. 179 
 
 All nucleic acids contain phosphoric acid. The relation between ph< >- 
 phorus and nitrogen is as 1 : 1 in the inosinic acid and as 1:5 in the guanylic 
 acid. In the thymus- and the salmo-nucleic acids the relation accord- 
 ing to Schmibdeberg is 4:14 and according to Steudel 4:15. In the 
 triticonucleic acid, Osborne and Harris found the relation 4:10; in the 
 yeast nucleic acid, Lkyene and Jacobs found it was equal to 4:15. 
 
 According to the number of bases contained in the nucleic acids 
 we can differentiate between the simple nucleic acid with only one base 
 and the complex nucleic acids with several bases. Levene and Mandel ' 
 have called the first (inosinic acid, guanylic acid) nucleotides or mono- 
 nucleotides and the last polynucleotides. 
 
 The properties and the constitution of the nucleic acids, as far as we 
 know them, have been determined essentially by the work of Kossel 
 and his pupils, by Schmiedeberg, Steudel and Levene 2 and their 
 collaborators. 
 
 On complete acid hydrolysis the nucleic acids are split into the three 
 above mentioned components, phosphoric acid, carbohydrate and bases. 
 The purine bases are more readily split off than the pyrimidine bases 
 and on careful acid hydrolysis of thymus nucleic acid, a new acid, the 
 ihyminic acid of Steudel and Brigl is obtained. This acid is very 
 similar to the thyminic acid of Kossel and Neumann a with the barium 
 salt, CioH23N3P20i2Ba, and the nucleotinphosphoric acid of Schmiede- 
 berg. This acid differs probably from the original nucleic acid only 
 by the absence of purine bases. By the action of strong nitric acid in 
 the cold we can, according to the method suggested by Steudel, split 
 off the purine bases while nearly all the phosphoric acid remains in organic 
 combination with the carbohydrate complexes. 
 
 The hydrolyses of pentose containing nucleic acids as carried out by 
 Levene and Jacobs in neutral, or, if the pyrimidine complexes of the 
 plant nucleic acid were being studied, in ammoniacal reaction, by heating 
 to high temperatures in the autoclave or in sealed tubes, are of special 
 interest. In these cases the binding with the phosphoric acid was rup- 
 
 1 Ber. d. d. chem. Gesellsch., 41. 
 
 2 The work of Kossel and his pupils on the nucleic acids can be found in: Arch. 
 f. (Anat. u.) Physiol. 1892, 1893 and 1894; Sitz. Ber. d. Berl. Akad. d. Wiss., 18, 1S94; 
 Centralbl. f. d. med. Wiss. 1893; Ber. d. d. chem. Gesellsch., 26 and 27; Zeitschr. f. 
 physiol. Chem., 23 and 38; see also Neumann, Arch. f. (Anat. u.) Physiol., 1898 and 1899 
 Suppl.; Miescher, Hoppe-Seyler's Med. chem. Unters., p. 441 and Arch. f. exp. Path, 
 u. Pliann., 37; Schmiedeberg, ibid., 37, 43, and 57; Altnian. Arch. f. (Anat. u.) Physiol., 
 1889; Steudel, ibid., 42, 43, 46. 49, 50, 52, 53, 55, 56, 70, 77; Ascoli, Zeitschr. f. j 1 
 Chem., 28 and 31; Lev. tie, Orid., 52, 37, 38, 39, 43, 45; Levene and Mandel. ibid. 4*i. 
 47, 49, 50; Inouye and Kotake, ibid.. 46; Levene and Jacobs, Ber. d. d. i 
 Gesellsch., 42, 43, 44; with La Forge, ibid., 43 and 45. 
 
 3 Steudel and Brigl, Zeitschr. f. physiol. Chem., 70; Kossel and Neumann, ibid., 22. 
 
180 THE PROTEIN SUBSTANCES. 
 
 tared while the binding between the pentose and purine bases remained 
 intact. In this manner they obtained pentosides, i.e., glucoside-like^com- 
 bination between pentose and a purine base. These pentosides have 
 also been called nucleosides and such a nucleoside was the inosine, 
 which was first found by Haiser and Wenzel l and which is the pen- 
 toside of inosinic acid and is a combination of hypoxanthine with pentose 
 (f?-ribose). The other three nucleosides adenosine, guanosine and xantho- 
 sine have been prepared by Levene and Jacobs. 
 
 The nucleosides are crystalline bodies which give crystalline combina- 
 tions. Of special interest is guanosine because it is identical with the base 
 vernine, occurring in the plant kingdom and discovered by Schtjlze 2 
 and because of the identity of the pentose occurring in both has been 
 positively proved. The guanosine has also been found by Levene and 
 Jacobs 3 in the pancreas. On acid hydrolysis every nucleoside splits 
 into purine base and pentose. By the action of nitrite and glacial acetic 
 acid the guanosine is transformed into xanthosine and the adenosine 
 into inosine. 
 
 Mandel and Dunham have prepared, from acetone-yeast, a crystalline 
 adenine-hexose compound corresponding to the pentoside but whose 
 relation to the cleavage products of nucleic acids is not known. From 
 thymus nucleic acid Levene and Jacobs 1 have later isolated a guanine 
 hexoside. 
 
 The pyrimidine complexes corresponding to the nucleosides also 
 contain (in the plant nucleic acids) pentose, according to Levene and 
 La Forge 5 but in much firmer bondage. This is the reason why they 
 give only a faint orcin reaction, are much more resistant to enzymes 
 than the purine nucleosides and give off furfurol only very slowly on 
 distilling with hydrochloric acid. Still they contain pentose and pyrimi- 
 dine bases in equimolecular proportions. The pyrimidine complexes are 
 called cytidine and uridine, the first containing cytosine and the second 
 uracil. Uridine is crystalline; the cytidine has not been obtained in a crys- 
 talline form but it gives several crystalline salts. The uridine is claimed 
 to exist pre-formed in the yeast nucleic acid and not produced secondarily 
 from the cytidine. 
 
 Based upon the investigations carried out by Steudel, Levene and 
 Jacobs we can for the present represent the structure of the nucleic acids 
 in the following way: 
 
 1 Monatsh. f. Chem., 29. 
 
 ' E. Bchulze and Bosshard, Zeitschr. f. physiol. Chem., 10; with Trier, ibid., 70. 
 
 • Bioch. Zeitschr., 28. 
 
 4 Mandel and Dunham, Journ. of biol. Chem., 11; Levene and Jacobs, ibid., 12. 
 
 5 Ber. d. d. chem. Gesellseh., 45. 
 
NUCLEIC ACIDS. 181 
 
 The simple nucleic acids are ester-like combinations between phosphoric 
 acid and a purine base-pentoside. 
 
 The complex nuclei acids arc complex molecules each composed of 
 four simple nucleic acids (nucleotides). In regard to the complex nucleic 
 acids we differentiate between two groups. 
 
 The acids of the thymonucleic acid group are, according to Steudel, 
 tetrabasic phosphoric acid ester which corresponding to each phosphorus 
 atom, contains a hexose group and one of the four bases, guanine, 
 adenine, cystosinc and thymine. From the name of this group we infer 
 that these acids contain thymine. 
 
 The plant nucleic acid group differs from the preceding by the follow- 
 ing. They do not contain any thymine but uracil instead. They do not 
 contain any hexose but do contain pentose. In the acids of this group 
 for each atom of phosphor we have 1 mol. pentose and on each the 
 purine and pyrimidine bases are combined. 
 
 It must be remarked that the complex nucleic acids have not been 
 prepared from isolated component proteins but generally from organs, 
 namely perhaps from a mixture of different nucleoproteins and that for 
 this reason we do not know whether these acids are chemical individuals 
 or only a mixture of closely related simple nucleic acids. On the other 
 hand it is also possible that the simple nucleic acids originate from more 
 complex nucleic acid by cleavage because such cleavages are in fact 
 known. Such an assumption does not apply at least for the guanylic 
 acid from the pancreas as it is obtained from a compound protein with 
 only one base, namely guanine. 
 
 All nucleic acids are amorphous, white, and have an acid reaction. 
 They are readily soluble in ammoniacal or alkaline water. They also 
 dissolve in concentrated acetic acid and form insoluble salts with copper 
 chloride and salts of the heavy metals, and as a rule insoluble basic 
 salts with the alkaline earths. Their solubility in water is very different. 
 Inosinic acid, for example, is very readily soluble in cold water while 
 tf-guanylic acid is soluble with difficulty. The complex nucleic acids 
 are also soluble with difficulty in cold water. The solution of their 
 alkali combination is not as a rule precipitated by acetic acid but is 
 precipitated by a slight excess of hydrochloric acid, especially in the pres- 
 ence of alcohol. The nucleic acids soluble in dilute acids give in such 
 solution a precipitate with proteins, which are considered as nucleins. All 
 nucleic acids are insoluble in alcohol and ether. They do not give either 
 the biuret test or Millon's reaction. The nucleic acids are optically 
 active and, with the exception of inosinic acid (Gamgee and Jones) 
 and of guanylic acid (Levene and Jacobs l ), are dextro-rotatory. 
 
 1 Gamgee and Jones, Proc. Roy. Soc, 72; Levene and Jacobs, Journ. of biol. 
 Chem., 12. 
 
182 THE PJHOTEIN SUBSTANCES. 
 
 The proteolytic enzymes, such as pepsin and trypsin, decompose the 
 nucleoproteins more or less; the nucleic acids are apparently not 
 split by these enzymes or at least not as far as phosphoric acid and 
 purine bases. Such a cleavage can, on the contrary, be brought about 
 by erepsin (Nakayama) or by other closely allied enzymes found in 
 various organs which have been called nucleases. Micro-organisms can also 
 bring about a more or less deep cleavage of the nucleic acids (Schit- 
 
 TENHELM and SCHROTER 1 ). 
 
 Levene and Medigreceantt 2 differentiate between three kinds of 
 nucleases namely, nucleinases, nucleotidases and nucleosidases. The 
 nucleinases, which are found in the pancreatic juice and all organs 
 investigated, but not. in gastric juice, acts only upon the complex nucleic 
 acids and splits them into nucleotides. The nucleotidases, which, with 
 the exception of the gastric and pancreatic juices, occurs all over and 
 especially in the intestinal mucosa, split the simple nucleic acids (mono- 
 nucleotides) into phosphoric acid and the corresponding nucleoside 
 (purine pentoside). The nucleosidases, which are not found in the gastric, 
 pancreatic or intestinal juices, nor in the blood or the pancreas but in 
 other organs, split the nucleosides into purine base and pentose. It is 
 unknown how the cleavage of the pyrimidine and hexose complexes 
 of the nucleic acids is brought about. 
 
 According to W. Jones 3 the purine bases of the nucleic acids can be 
 deamidized without being previously split off as free base from the acid. 
 Thus the pig-pancreas contains an adencsin-deamidase which deami- 
 dizes the still combined adenine. On the contrary the same organ also 
 contains a guanase which deamiclizes the free guanine but does not 
 contain a guanosine deamidase. The pig liver, in which only traces of 
 guanase occur, contain on the contrary a guanosine-deamidase. Recent 
 investigations of Schittenhelm and K. Wiener 4 show that we must 
 also admit of nucleoside-deamidases besides purine deamidases. 
 
 Inosinic Acid, C10H13N4PO8 was first isolated by Liebig from the 
 flesh of certain animals and then closely studied by Haiser. It is obtained 
 from beef extracts, and according to the investigations of Neuberg and 
 Brahn, Fr. Bauer, and Levene and Jacobs it is a simple nucleic acid. 5 
 
 1 Nakayama, Zeitschr. f. physiol. Chem., 41; Iwanoff, iUd., 39; Fr. Sachs, " 1st die 
 Nuklease mit 'lorn Trypsin identisch? " Inaug.-Dissert, Heidelberg, 1905; Schitten- 
 helm and Schroter, f. physiol. Zeitschr. Chem., 41. 
 
 2 Journ. of biol. Chem., 9. 
 
 3 Journ. of bio!. Chem., 9. 
 
 4 Zeitschr. f. physiol. Chem., 77. 
 
 Liebig, Annul, d. chem. u. Pharm., 62; Haiser, Monatsh. f. chem., 16; Neuberg 
 and Brahn, Biochem. Zeitschr., 5 and Ber. d. d. chem. Gesellsr-h., 41, p. 3376; Bauer 
 Hofmeister's Beitriige, 10; Levene and Jacobs, Ber. d. d. chem. Gesellsch., 41, p. 2703. 
 
INOSINIC AND GUANYLIC ACIDS. 183 
 
 On hydrolysis it yields phosphoric acid, hypoxanthine and pentose, 
 according to the equation: 
 
 CiaH 18 N4POiB+2HjO=H3P04+C8H4N40+C5HioO«. 
 
 The pentose, whose somewhat disputed nature has been discussed 
 on page 178, is combined with hypoxanthine in a glucoside-like com- 
 bination forming the pentoside inosine, which, according to Levene and 
 Jacobs, is combined with the phosphoric acid, like an ester by means 
 of the 5-carbon atom of the pentose (ribose). 
 
 Inosinic acid is amorphous, syrupy, readily soluble in water and pre- 
 cipitable by alcohol. It is lavo-rotatory; for the Ba salt containing 
 hydrochloric acid Neuberg and Brahn found (a) D =— 18.5° at 10° C. 
 It gives several crystalline salts among which the barium salt, which is 
 soluble with difficulty in water, must be mentioned. 
 
 In regard to the preparation of this acid we must refer to the works 
 of Haiser, Neuberg and Erahn, Levexe and Jacobs mentioned in 
 footnote 5, page 182. 
 
 Guanylic acid. This acid, which was first prepared by Bang from 
 the pancreas has also been found by Jones and Rowxtree in the spleen 
 and by Levexe and Mandel ' in the liver. As cleavage products it yields 
 guanine, pentose and phosphoric acid and therefore its simplest formula 
 is ('10H14N5PO8. This formula is accepted also by Steudel and Brigl 
 and by Levexe and Jacobs, while Baxg basing his views on the results 
 of elementary analysis gives the formula C44H6GN20P4O34. Accord- 
 ing to this formula the acid would contain besides, guanine, pentose and 
 phosphoric acid also an unknown residue, C4H10O2, and according to Bang 
 is not a simple nucleic acid but would occupy an intermediary position 
 between the inosinic acid and the thymus nucleic acid. In opposition 
 to this it must be remarked that Levene and Jacobs 2 have recently 
 prepared the crystalline brucine salt of the acid and the analysis of this 
 salt as well as the barium salt substantiates the first mentioned, simple 
 formula. In regard to the pentose of guanylic acid see page 178. 
 
 The acid first described by Bang, the /3-acid, is soluble with great diffi- 
 culty in cold water and rather readily soluble in boiling water. It is easily 
 precipitated by acetic acid from the solution of the alkali combination 
 in water. The /3-acid may, according to Bang, be derived from another 
 guanylic acid, the a-guanylic acid, by the action of the alkali. The 
 
 1 Banc, Zeitschr. f. physiol. Chem., .26; with Raas-hou, Hofmeister's Beitrage. 4; 
 Jones and Rowntree, Journ. of biol. chem., 4; Levene and Mandel, Bioehem. Zeitschr. 
 10. 
 
 : Steudel and Brigl, Zeitschr. f. physiol. Chem., 68; Bang, ibid., 69 and Bioch. 
 Zeitschr., 26; Levene and Jacobs, Journ. of biol. Chem., 12. 
 
184 THE PROTEIN SUBSTANCES. 
 
 a-guanylic acid is readily soluble, even in cold water, and it is also similar 
 to thymus nucleic acid in other respects. It is precipitated from the 
 solution of its salts by hydrochloric acid but not by acetic acid, and its 
 solutions precipitate proteins. Steudel and Brigl believe that the 
 jS-acid is a potassium salt and that the a-acid is the actual acid, but this 
 view Bang disputes. Levene and Jacobs found that the acid con- 
 taminated with alkali does not gelatinize while the pure acid does. The 
 specific rotation of the latter was (o)d = —1-27° at 25° C. 
 
 In regard to the preparation of guanylic acid we refer, to the work 
 of Bang, Levene and Jacobs. 1 
 
 Thymonucleic Acids. A. Neumann has isolated two nucleic acids, 
 a- and /3-thymus nucleic acid, from the thymus gland. The a-acid is 
 soluble with difficulty, and in proper concentration gives a sodium salt 
 which gelatinizes in proper concentration, and a barium salt which is 
 precipitated by barium acetate in substance (Kostytschew) . The 
 barium salt of the /3-acid is not precipitated by barium acetate. The 
 a-acid is designated as anhydric by Schmiedeberg, 2 and the /3-acid as 
 hydrate, and the first can be transformed into the second by heating. 
 This transformation, according to Kostytschew, is a decomposition 
 whereby two-thirds of the purine bases are split off. 
 
 According to Schmiedeberg the thymus nucleic acid is identical 
 with the salmo-nucleic acid (from salmon sperm), and also according 
 to Steudel probably with the acid from the herring sperm. Other 
 nucleic acids, at least those very closely related to this nucleic acid, have 
 been prepared from the sperm of the burbot (Lota vulgaris) by Alsberg, 
 of the sturgeon (Noll) and of the sea-urchin (Mathews), also from 
 ox-sperm, brain, spleen (Levene), pancreas (Levene, v. Furth and 
 Jerusalem, Steudel), mammary glands and kidneys (Levene and Man- 
 del 3 ) and from other organs. 
 
 At the present time we arc not agreed as to the formula for the most 
 carefully studied thymonucleic acids (from the thymus, herring and sal- 
 mon sperms) . According to the numerous analyses of Schmiedeberg and 
 his collaborators for every 4 atoms of phosphorus there occur 14 atoms 
 of nitrogen. The relationship of C to P was 40 to 4 and the relationship of 
 C to N in 12 out of 15 preparations was 40 to 14, and only in 3 prepara- 
 tions 40 to 15. From these facts Schmiedeberg has given the acid 
 
 1 See footnotes 1 and 2, p. 183. 
 
 2 A. Neumann, Arch. f. (Anat. u.) physiol, 1898 and 1899; Kostytschew, Zeitschr. 
 f. physiol. Chem., 39; Schmiedeberg, 1. c. 
 
 3 Alsberg, Arch. f. exp. Path. u. Pharm., 51; Noll, Zeitschr. f. physiol. Chem., 25; 
 Mathews, ihvl., 23; v. Furth and Jerusalem Hofmeister's Birtrage, 10 and 11; Steudel, 
 Zeitschr. f. pi ysiol. Chem., 53; Levene and Mandel, ibid., 46, 47. See also footnote 2, 
 p. 179. 
 
PLANT NUCLEIC ACIDS. 185 
 
 the formula C40HMN14O16.2P2O8. According to Steudel for every 4 
 atoms of phosphorus we have 15 atoms nitrogen and from this he has 
 calculated fche formula ( UaHeiN 15P4O34+9H2O for the acid containing 
 
 water. 
 
 The probable constitution of the thymo-nucleic acids has been previ- 
 ously indicated and as positively known cleavage products we have at least 
 phosphoric acid, a hexose carbohydrate, guanine, adenine, thymine and 
 cytosine. 
 
 The thymo-nucleic acids have the reactions as given for the complex 
 nucleic acids. They are amorphous, dextro-rotatory, and soluble in cold 
 water with difficulty. They form soluble salts with alkalies and the acid 
 is precipitated from these solutions by mineral acid but not by acetic 
 acid. Tannic acid alone does not cause a precipitate but does in the 
 presence of sodium acetate. Proteins precipitate their solutions contain- 
 ing acetic acid. The two special thymo-nucleic acids differ from each 
 other by the different behavior of their salts (see above). 
 
 The preparation of the nucleic acids is based in the first place always 
 upon the cleavage of the nucleoprotein into protein and nucleic acid by 
 the action of alkali and then separating the nucleic acids from the 
 protein. The operations necessary for purifying the nucleic acids from 
 proteins are very complicated and we must refer to the works of 
 Schmiedeberg, Neumann, Levene, and others. 1 
 
 Plant Nucleic Acids. The two best known acids of this group are 
 the yeast nucleic acid and the triticonucleic acid isolated from the 
 wheat embryo. The identity of these two acids, as suggested by Osborne 
 and Harris has become more and more probable. According to Kowa- 
 lewskt 2 the yeast nucleic acid contain only adenine, guanine and cytosine, 
 the uracil is only formed secondarily from the cytosine. The yeast nucleic 
 acid may perhaps be a triphosphoric acid with three molecules of pentose 
 each with a molecule of adenine, guanine and cytosine. 
 
 This view stands in opposition to the observations of Levene and 
 Jacobs 3 that the yeast nucleic acid contains one molecule of pentose 
 combined with adenine and guanine, and besides this it contains two 
 pyrimidinehexose complexes, cytidine and uridine. 
 
 The triticonucleic acid yields also, as Osborne and Heyl, Wheeler 
 and Johnson and recently Levene and La Forge 4 have shown, the same 
 hydrolytic products as the yeast nucleic acid and both contain rf-ribose. 
 
 Schmiedeberg, Arch. f. exp. Path. u. Pharm., 43 and 57; Herlant, ibid., 44; Neu- 
 mann, Arch. f. (Anat. u.) Physiol. 1899 Supplb.; Levene, Zeitschr. f. physiol. Chem., 
 32 and 45; Kostytschew, 1. c. 
 
 2 Osborne and Harris, Zeitschr. f. physiol. Chem., 36; Kowalewsky, ibid., 69. 
 
 3 Ber. d. d. chem. Gesellsch., 44. 
 
 4 Osborne and Heyl, Amer. Journ. of Physiol., 21; Wheeler and Johnson, Amer. 
 Chem. Journ., 29; Levene and La Forge, Ber. d. d. Chem., Gesellsch., 43. 
 
186 THE PROTEIN SUBSTANCES. 
 
 The somewhat different results found on the elementary analysis of these 
 two acids do not seem to be of very great importance and we have strong 
 evidence for the identity of these acids. Osborne and his collaborators 
 found the formula C41H61N16P4O31 for triticonucleic acid. 
 
 The plant nucleic acids have the general reactions of the complex 
 nucleic acids but can be precipitated by an excess of acetic acid. They 
 are dextro-rotatory. 
 
 In regard to their preparation we refer to the works of Kossel, Osborne 
 and Harris and to Levene and co-workers. 1 
 
 Plasminic acid is an acid which was prepared by Ascoli and Kossel 2 by 
 the action of alkali upon yeast. It contains iron, and is soluble in very dilute 
 hydrochloric acid (1 p. m.). It is still a question whether it is a mixture or a 
 chemical individual. 
 
 2. Purine Bases. 
 
 The cleavage products obtained from the nucleic acids, the nuclein 
 bases, which are also called alloxuric bases by Kossel and Kruger, are 
 members of the larger group of purines, to which also belongs the uric 
 acid which is a substance occurring in the animal body. The constitu- 
 tion of these bodies has been explained by E. Fischer, 3 and he has 
 prepared many of the bodies synthetically. They can all be derived from 
 the synthetically prepared purine, C5H4N4, which has the formula given 
 below and which may be considered as a combination of a pyrimidine 
 ring with an imidazole ring. 
 
 N=CH N=CH 
 
 II II HC-NH> 
 
 HC C— NR HC CH | >CH 
 
 II II >CH || || HC N^ 
 
 N— C N^ N— CH 
 
 Purine Pyrimidine Imidazole 
 
 The different purine bodies are derived therefrom by the substitution of the 
 various hydrogen atoms by hydroxyl, amide, or alkyl groups. In order to signify 
 the different positions of substitution Fischer has proposed to number the nine 
 members of the purine nucleus in the following way: 
 
 1N=C6 
 
 I I 
 2C 5C— N7 
 
 > 
 
 .C8. 
 
 3N— C— N9 
 
 4 
 
 1 See footnote 2, p. 179, and footnote 3 and 4, p. 185. 
 
 2 Ascoli, Zeitschr. f. physiol. Chem., 28. 
 
 3 Sff ]■'.. Kisr-her, Untersuchungen in der Purinfjrwppe (1882-1906) Berlin, 1907. 
 
IT KINK BASES. 187 
 
 I IX— CO 
 
 I I 
 For example, uric acid, OC — NH\ , is 2, P>, S-trioxvpurine; adenine, 
 
 I II >co 
 
 HN-C— XJT 
 N=CHN, • IIX-CO 
 
 II M. 
 
 HC C — XI k , is G-aminopurine, and heteroxanthine, OC C — X.CH 3 , is 
 
 H II > CH I II >CH 
 
 X - ( ' X^ II x-c-x/ tn 
 
 7-methyl-2, 6-dioxypurine, etc. 
 
 The starting-point used by Fischer for the synthetical preparation of the 
 
 purine bases was 2, 6, S-triehlorpurine, which is obtained, with N-oxy-2, (i-dichlor- 
 
 purine as an intermediary product, from potassium urate and phosphorus oxychlo- 
 
 ride. 
 
 The purine bodies or alloxuric bodies, found in the animal body or its 
 
 excreta are as follows: Uric acid, xanthine, heteroxanthine, \-methylxan- 
 
 paraxanthine, guanine, epiguanine, hypoxanthine, episarkine, 
 
 adenine. The bodies theobromine, theophylline, and caffeine, occurring in 
 
 the vegetable kingdom, stand in close relation to this group. 
 
 The composition of the purine bodies most important from a physio- 
 logical standpoint is as follows: 
 
 Uric acid, C5H4X4O3 2, 6, 8-trioxypurine 
 
 Xanthine C5H4X4O2 2, 6-dioxypurine 
 
 l-methylxanthine, C6H 6 X 4 02 1-methyl " 
 
 Heteroxanthine, C 6 H 6 X 4 02 7 " " 
 
 Theophylline, C 7 H 8 X 4 () 2 1, 3-dimethyl 
 
 Paraxanthine, C7H8X4O2 1.7- " 
 
 Theobromine C7H8X4O2 3, 7- 
 
 Caffeine, C 8 H,oX 4 02 1,3, 7-trimethyl 
 
 Hypoxanthine, Cs^X^O 6-oxypurine 
 
 Guanine C 5 H 5 X 5 2-amino " " 
 
 Epiguanine, C 6 H 7 X 5 7-methyl " " " " 
 
 Adenine CsHsX'j 6-aminoourine 
 
 Episarkine, C4H 6 X 3 0(?) 
 
 After Salomon ! had shown the occurrence of xanthine bodies in 
 young cells, the importance of the purine bases as decomposition prod- 
 ucts of cell nuclei and of nucleins was shown by the pioneering researches 
 of Kossel, who discovered adenine and theophylline. In those tissues 
 in which, as in the glands, the cells have kept their original state, the 
 purine bases are not found free, but in combination with other atomic 
 groups (nucleic acids). In such tissues, on the contrary, as in muscles, 
 which are poor in cell nuclei, the purine bases are found in the free state. 
 Since the purine bases, as suggested by Kossel, stand in close relation- 
 ship to the cell nucleus, it is easy to understand why the quantity of 
 these bodies is so greatly increased when large quantities of nucleated 
 
 1 Sitzungsber. d. Bot. Verein der Provinz Brandenburg, 1880. 
 
188 THE PROTEIN SUBSTANCES. 
 
 cells appear in such places as were before endowed in a relatively poor 
 manner. As an example of this, the blood, in leucaemia, is extremely rich 
 in leucocytes. In such blood Kossel 1 found 1.04 p. m. purine bases, 
 against only traces in the normal blood. That these bases are also inter- 
 mediate steps in the formation of uric acid in the animal organism is 
 probable, and will be shown later (see Chapter XIV). 
 
 Only a few of the purine bases have been found in the urine or in the 
 muscles. Only four bases — xanthine, guanine, hypoxanthine, and ade- 
 nine — have been obtained, thus far, as cleavage products of nucleins, 
 and these do not always have a primary origin. In regard to the purine 
 bodies from other substances we refer the reader to their respective 
 chapters. Only the above four bodies, the real nuclein bases, will be 
 considered at this time. 
 
 Of these four bodies, xanthine and guanine form one special group 
 and hypoxanthine and adenine another. By the action of nitrous acid 
 guanine is converted into xanthine and adenine into hypoxanthine. 
 
 C5H4N40.NH+HN02 = C5H4N402+N2+H 2 0; 
 
 Guanine Xanthine 
 
 C5H4N4.NH + HNO2 + C5H4N4O + N2 + H2O. 
 
 Adenine Hypoxanthine 
 
 Similar transformation, where xanthine and hypoxanthine are pro- 
 duced secondarily, may also occur in the hydrolysis of nucleic acids as 
 well as in putrefaction and by the action of special enzymes. The 
 researches of Schittenhelm, Levene, Jones, Partridge, Winternitz, 
 and Burian have shown that in various organs deamination enzymes, 
 such as guanase and adenase, occur, which convert guanine and adenine 
 into xanthine and hypoxanthine respectively, and also oxidases which 
 oxidize hypoxanthine into xanthine and this then into uric acid. This 
 formation of uric acid from the purine bases, which will be discussed in 
 detail in a following chapter (XIV), is of very great interest. 
 
 The nuclein bases form crystalline salts with mineral acids, which, 
 with the exception of the adenine salts, are decomposed by water. They 
 are easily dissolved by alkalies, while with ammonia their action is some- 
 what different. They are all precipitated from acid solution by phos- 
 photungstic acid; they also separate as silver compounds on addition 
 of ammonia and ammoniacal silver-nitrate solution. These precipitates 
 are soluble in boiling nitric acid of 1.1 specific gravity. All purine bodies 
 are also precipitated by Fehling's solution (see Chapter III) in the pres- 
 ence of a reducing substance such as hydroxylamine (Drechsel and 
 Balke). Copper sulphate and sodium bisulphite may also be used to 
 
 1 Zeitschr. f. physiol. Chem.. 7. 
 
XANTHINE. 189 
 
 advantage in their precipitation (Kruger) 1 . This behavior of the 
 purine bases serves just as well as the behavior with the silver solution 
 for their precipitation and preparation. 
 
 HN— CO 
 
 I I 
 Xanthine, C 5 H 4 N40 2 , = OC C— NH V (2, 6-dioxypurine) i s f 0U nd 
 
 I II >CH 
 
 HN— C — W 
 
 in several cellular organs. It occurs in small quantities as a physio- 
 logical constituent of urine, and it occasionally has been found as a urinary 
 sediment, or calculus. It was first observed in such a stone by Marcet. 
 Xanthine is found in larger amounts in a few varieties of guano (Jarvis 
 guano) . 
 
 Xanthine can be prepared, according to E. Fischer, by boiling uric 
 acid with 25 per cent hydrochloric acid or, according to Sundvik 2 by 
 heating uric acid with anhydrous oxalic acid in glycerin to about 200° C. 
 
 Xanthine is amorphous, or forms granular masses of crystals, or may 
 also, according to Horbaczewski, 3 separate as masses of shining, thin, 
 large rhombic plates with 1 mol. water of crystallization. It is very 
 slightly soluble in water, in 14,151-14,600 parts at 16° C, and in 1300- 
 1500 parts at 100° C. (Almen 4 ). It is insoluble in alcohol or ether, but 
 is readily dissolved by alkalies and with difficulty by dilute acids. With 
 hydrochloric acid it gives a crystalline, difficultly soluble combination. 
 With very little caustic soda it gives a readily crystallizable compound, 
 which is easily dissolved by an excess of alkali. Xanthine dissolved in 
 ammonia gives with silver nitrate an insoluble, gelatinous precipitate 
 of silver xanthine. This precipitate is dissolved by hot nitric acid, and 
 by this means an easily soluble crystalline double compound is formed. 
 Xanthine in aqueous solution is precipitated on boiling with copper 
 acetate. At ordinary temperatures xanthine is precipitated by mercuric 
 chloride and by ammoniacal basic lead acetate. It is not precipitated 
 by basic lead acetate alone. 
 
 When evaporated to dryness in a porcelain dish with nitric acid, 
 xanthine gives a yellow residue, which turns, on the addition of caustic 
 soda, first red, and after heating, purple-red. If we place some chlorinated 
 lime with some caustic soda in a porcelain dish and add the xanthine 
 
 1 Balke, Zur Kenntnis der Xanthinkorper, Inaug.-Diss. Leipzig, 1893 ; Kruger 
 Zeitschr. f. physiol. Chem., 18. 
 
 2 E. Fischer, Ber. d. d. chem. Gesellsch, 43; Sundvik, Zeitschr. f. physiol.Chem. 
 76. In regard to the synthesis of xanthine and other purines see E. Fischer, footnote 
 3, p. 186. 
 
 5 Zeitschr. f. physiol. Chem., 23. 
 4 Journ. f. prakt. Chem., 96. 
 
190 THE PROTEIN SUBSTANCES. 
 
 to this mixture, at first a dark green and then quickly a brownish halo 
 forms around the xanthine grains and finally disappears (Hoppe-Seyler). 
 If xanthine is warmed in a small vessel on the water-bath with chlorine- 
 water and a trace of nit.'ic pcid, and evaporated to dryness, and the 
 residue is then exposed under a bell-jar to the vapors of ammonia, a 
 red or purple-violet color is produced (Weidel's reaction). E. Fischer x 
 has modified Weidel's reaction in the following way: He boils the xan- 
 thine in a test-tube with chlorine-water or with hydrochloric acid and a 
 little potassium chlorate, then evaporates the liquid carefully, and moistens 
 the dry residue with ammonia. 
 
 HN— CO 
 
 ! I 
 
 Guanine, C 5 H5N 5 0,=H2N.C C— NH\ (2-amino-6-oxypurine) . 
 
 II II >CH 
 
 N— C— W 
 
 Guanine is found in all organs rich in cells. It is further found in the 
 muscles (in very small amounts), in the scales and in the air-bladder of 
 certain fishes, as iridescent crystals of guanine-lime; in the retinal epithe- 
 lium of fishes, in guano, and in the excrement of spiders it is found as 
 chief constituent. It also occurs in human and pig urine. Under patholog- 
 ical conditions it has been found in leucsemic blood, and in the muscles, 
 ligaments, and articulations of pigs with guanine-gout. 
 
 Guanine is a colorless, ordinarily amorphous powder, which may be 
 obtained as small crystals by allowing its solution, in concentrated 
 ammonia, to evaporate spontaneously. According to Horbaczewski it may 
 under certain conditions appear in crystals similar to creatinine-zinc chlor- 
 ide. It is insoluble in water, alcohol, and ether. It is rather easily dissolved 
 by mineral acids and readily by alkalies, but it dissolves with great 
 difficulty in ammonia. According to Wulff 2 100 cc. of cold ammonia 
 solution containing 1, 3, or 5 per cent NH3 dissolve 9, 15, or 19 milli- 
 grams of guanine respectively. The solubility is relatively increased 
 in hot ammonia solution. The hydrochloride readily crystallizes, and 
 has been recommended by Kossel 3 for the microscopical detection of 
 guanine, on account of its behavior toward polarized light. The sul- 
 phate contains 2 molecules of water of crystallization, which is completely 
 expelled on heating to 120° C., and this fact, as well as the fact that 
 guanine yields guanidine on decomposition with chlorine-water, differ- 
 entiates it from 6-amino-2-oxypurine, which is considered as an oxida- 
 tion product of adenine and possibly occurs as a chemical metabolic 
 
 1 Ber (1 deutsch. chem. Gesellsch., 30, 2236. 
 
 2 Zeitschr f. physiol. Chem., 17. 
 
 3 Ueber die chem. Zusammensetz. der Zelle, Verh. d. physiol. Gesellsch. zu Berlin 
 1890-91, Nop. 5 and 6. 
 
HYPOXANTHINE. 191 
 
 product (E. Fischer). The G-amino-2-oxypurine sulphate contains 
 only 1 molecule of water of crystallization, which is not expelled at 120° C. 
 Very dilute guanine solutions arc precipitated by both picric acid and 
 metaphosphoric acid. These precipitates may be used in the quantita- 
 tive estimation of guanine. The silver compound dissolves with difficulty 
 in boiling nitric acid, and on cooling the double compound crystallizes 
 out readily. Guanine acts like xanthine in the nitric-acid test, but gives 
 with alkalies on heating a more bluish-violet color. A warm solution 
 of guanine hydrochloride gives with a cold saturated solution of picric 
 acid a yellow precipitate consisting of silky needles (Capranica). With 
 a concentrated solution of potassium bichromate a guanine solution 
 gives a crystalline, orange-red precipitate, and with a concentrated 
 solution of potassium ferricyanide a yellowish-brown, crystalline pre- 
 cipitate (Capranica). It also gives a compound with picrolonic acid 
 (Levene l ). Guanine gives Weidel's reaction. 
 
 HN— CO 
 
 I I 
 
 Hypoxanthine, Sarkine, C5H4N4O, =HC C — NPL = (6-oxypurine) . 
 
 II II >CH 
 N— C— N ^ 
 
 This body has been found in all cellular organs and in meat extracts, 
 and as a cleavage product of inosinic acid. It is especially abundant in 
 the sperm of the salmon and carp. Hypoxanthine occurs also in the mar- 
 row and in very small quantities in normal urine, and, as it seems, also in 
 milk. It is found in rather considerable quantities in the blood and urine 
 in leucsemia. 
 
 Hypoxanthine can be obtained according to Sundvik's 2 method 
 from uric acid or xanthine by heating with a formate or more simply 
 by heating with chloroform in alkaline solution. 
 
 Hypoxanthine forms very small, colorless, crystalline needles. It 
 dissolves with difficulty in cold water, but the claims concerning 
 solubility therein are very contradictory. 3 It dissolves more readily 
 in boiling water, in about 70-80 parts. It is almost insoluble in alcohol, 
 but is dissolved by acids and alkalies. The compound with hydrochloric 
 acid is crystalline, and is more soluble than the corresponding xanthine 
 derivative. It is easily soluble in ammonia. The silver compound 
 dissolves with difficulty in boiling nitric acid. On cooling, a mixture 
 of two hypoxanthine silver-nitrate compounds possessing an inconstant 
 composition separates out. On treating this mixture with ammonia 
 and an excess of silver nitrate and heating, a silver hypoxanthine is 
 
 1 Capranica, Zeitschr. f. physiol. Chem., 4; Levene, Bioch. Zeitsehr., 4. 
 
 5 1. c. and Skand, Arch. f. Physiol., 25. 
 
 'See E. Fischer, Ber. d. deutsch. chem. Gesellsch., 30. 
 
192 THE PROTEIN SUBSTANCES. 
 
 formed, which when dried at 120° C. has a constant composition, 
 2(C5HoAg2N40)H20, and is used in the quantitative estimation of 
 hypoxanthine. Hypoxanthine picrate is soluble with difficulty, but 
 if a boiling-hot solution of it is treated with a neutral or only faintly 
 acid solution of silver nitrate the hypoxanthine is almost quantitatively 
 precipitated as the compound C5H 3 AgN40.C6H 2 (N02)30H. Hypo- 
 xanthine does not yield an insoluble compound with metaphosphoric 
 acid. When treated, like xanthine, with nitric acid, it yields, an almost 
 colorless residue which, on warming with alkali, does not turn red. Hypo- 
 xanthine does not give Weidel's reaction. After the action of hydro- 
 chloric acid and zinc upon a hypoxanthine solution, followed by the 
 addition of an excess of alkali a ruby-red color develops, followed by 
 a brownish-red color (Kossel). According to E. Fischer 1 a red 
 coloration occurs even in the acid solution. 
 N— C.NH 2 
 
 Adenine, CsHsNs^HC C — NH\ (6-aminopurine), was first found 
 II II >CH 
 
 N— C— N ^ 
 
 by Kossel 2 in the pancreas. It occurs in all nucleated cells, but in 
 greatest quantities in the sperm of the carp and in the thymus. Adenine 
 has also been found in lucsemic urine (Stadthagen 3 ) . It may be obtained 
 in large quantities from tea-leaves. 
 
 Adenine crystallizes with 3 molecules of water of crystallization in 
 long needles which gradually become opaque in the air, but much more 
 rapidly when warmed. If the crystals are warmed slowly with a quan- 
 tity of water insufficient for solution, they suddenly become cloudy at 
 53° C, a characteristic reaction for adenine. It dissolves in 1086 parts 
 cold water, but is easily soluble in warm. It is insoluble in ether, but 
 somewhat soluble in hot alcohol and easily so in acids and alkalies. It 
 is more easily soluble in ammonia solution than guanine, but less soluble 
 than hypoxanthine. The silver compound of adenine is soluble with 
 difficulty in warm nitric acid, and deposits on cooling as a crystalline 
 mixture of adenine silver nitrates. With picric acid adenine forms a 
 compound, CsHsNs.Ce^CNC^sOH, which is very insoluble but 
 separates more readily than the hypoxanthine picrate, and can be 
 used in the quantitative estimation of adenine. We also have an adenine 
 mercury-picrate. Metaphosphoric acid with adenine gives a precipitate 
 which dissolves in an excess of the acid if the solution is not too dilute. 
 Adenine hydrochloride gives with gold chloride a double compound 
 
 1 Kossel, Zeitschr. f. physiol. Chem., 12, 252; E. Fischer, 1. c. 
 
 2 See Zeitschr. f . physiol. Chem., 10 and 12. 
 » Virchow's Arch., 109. 
 
PYRIMIDINE BASES. 193 
 
 which consists in part of leaf-shaped aggregations and in part of cubical 
 or prismatic crystals, often with rounded corners. This compound is 
 used in the microscopic detection of adenine. With the nitric-acid test 
 and with Weidel's reaction adenine acts in the same way as hypoxan- 
 thine. The same is true for i£s behavior with hydrochloric acid and 
 zinc with subsequent addition of alkali. 
 
 The procedure for the preparation and detection of the four above- 
 described purine bases is as follows: Boiling with 0.5-1 vol. per cent 
 sulphuric acid, saturating with baryta-water, removing the excess of 
 barium with CO2, precipitating all the bases as silver compounds 
 from the strongly ammoniacal filtrate and dissolving them in boiling 
 nitric acid when the xanthine compound remains in solution on cooling 
 while the combination with the other three bases precipitate. The silver 
 xanthine may be precipitated from the filtrate by the addition of ammonia 
 and the xanthine set free by means of sulphureted hydrogen. The 
 three above-mentioned silver-nitrate compounds are decomposed by sul- 
 phureted hydrogen and the guanine separated from the adenine and 
 hypoxanthine by treatment while hot with ammonia, in which the 
 guanine is soluble with difficulty. When the above filtrate containing the 
 adenine and hypoxanthine, which has been, if necessary, freed from 
 ammonia by evaporation, is allowed to cool, the adenine separates, 
 while the hypoxanthine remains in solution. According to Balke l — 
 we can advantageously precipitate the purine bases with copper sulphate 
 and hydroxylamine. Details for the above methods may be found in 
 complete hand-books. The same procedures are followed in the quan- 
 titative estimation of the purine bases in animal organs. 2 
 
 3. Pyrimidine Bases. 
 
 These bodies are closely related to the purines, and pyrimidine, 
 N=CH 
 
 C4H 4 N2, = HC CH, may be considered as the mother substance thereof. 
 
 li II 
 N— CH 
 
 The pyrimidine bases contained in the nucleic acids are cytosine, uracil 
 and thymine. 
 
 N=CNH 2 
 
 Cytosine, C4H5N3O, = OC CH (6 amino-2 oxypyrimidine), wag first 
 
 HN— CH 
 
 prepared by Kossel and Neumann from thymus nucleic acid, and then 
 by Kossel and Steudel and others from various nucleic acids. Wheeler 
 
 •See footnote 1, p. 190. 
 
 2 See Burian and Hall, Zeitschr. f. physiol. Chem., 38; Kossel ibid., 5-8, Bruhns, 
 ibid., 14; His and Hagen, ibid., 30. 
 
194 THE PROTEIN SUBSTANCES. 
 
 and Johnson l have also prepared it synthetically. It is transformed 
 into uracil by the action of nitrous acid. 
 
 The free base is soluble with difficulty in water {129 parts) and crystal- 
 lizes in thin leaves with a mother-of-pearl luster. It is insoluble in ether 
 and soluble with difficulty in alcohol. The double compound with platinum 
 chloride, the crystalline picrate, the nitrate, and the picrolonate are 
 of importance in the detection of cytosine. This base is precipitated 
 by phosphotungstic acid and by silver nitrate in the presence of an 
 excess of barium hydroxide, which fact is of importance in the detection 
 of cytosine (Kutscher). The double bismuth-potassium iodide gives 
 a brick-red precipitate. Cytosine gives the murexid reaction with 
 chlorine-water and ammonia (see Chapter XIV), and also the reaction 
 described by Wheeler and Johnson under uracil. In regard to 
 preDaration see Kossel and Steudel 2 and also Kutscher. 3 
 HN— CO 
 
 Uracil, C4H4N 2 02, = OC CH (2, 6 - dioxypyrimidine) , was first 
 
 HN— CH 
 
 obtained by Ascoli and Kossel from yeast nucleic acid and later from 
 various complex nucleic acids, perhaps secondarily from the cytosine as a 
 cleavage product. The synthetical preparation was first accomplished by 
 E. Fischer and Roeder. 4 
 
 Uracil crystallizes in needles which cluster in rosettes. On careful 
 heating it sublimes in part undecomposed, but develops red vapors and 
 decomposes in part. It is readily soluble in hot water, but less so in cold 
 water, and nearly insoluble in alcohol and in ether. Uracil is readily 
 soluble in ammonia. It is precipitated by mercuric nitrate, but not by 
 phosphotungstic acid. It is precipitated by silver nitrate only on the 
 careful addition of ammonia or baryta-water. Uracil gives the Weidel 
 reaction and the following reaction described by Wheeler and John- 
 son. 5 The uracil solution is treated with bromine-water until it is per- 
 manently cloudy and then treated with baryta-water, when a purple or 
 violet-colored precipitate appears almost immediately. The coloration 
 
 1 Amer. chem. Journ., 29. 
 
 2 Zeitschr. f. physiol. Chem., 37 and 38. 
 
 * Ibid., 38. As it is not excluded, but rather probable according to Wheeler, that 
 besides thymine also other related pyrimidine bases such as isocytosine, 6-amino 
 pyrimidine and 6-oxpyrimidine can be formed in the hydrolytic cleavage of the nucleic 
 acids, Wheeler has prepared salts and compounds of these bodies and described them 
 as a matter of comparison, Journ. of biol. Chem., 3. 
 
 4 Ascoli, Zeitschr. f. physiol. Chem., 31; Kossel and Steudel, ibid., 37; Levene, 
 HA'L, 3S, 39; Levene and Mandel, ibid., 49; E. Fischer and Roeder, Ber. d. d. chem. 
 Gesellsel,., 34. 
 
 5 Journ. of biol. Chem., 3. 
 
THYMINE. 195 
 
 varies with the dilution. This reaction which, as remarked above, is also 
 given by cytosine, depends upon the fact that dibromoxyhydrouraeil 
 us first formed, and from this, by the action of the barium hydroxide, 
 first isodialuric and then dialuric acid is produced, both of which give 
 the coloration. In regard to the preparation of uracil see Kossel and 
 Steudel. 1 
 
 HN— CO 
 
 I I 
 Thymine, C5HGX2O2. = OC C.CH 3 (5-methyluracil). This body, 
 
 HN— CH 
 
 which is identical with nucleosin obtained by Schmiedeberg from sal- 
 mo-nucleic acid, was first prepared by Kossel and Neumann from 
 thymus-nucleic acid, and then by other investigators, especially Levene 
 and Mandel, from other animal nucleic acids. Fischer and Roeder and 
 later Gerngross 2 have prepared it synthetically. 
 
 Thymine crystallizes in small leaves grouped in stellar or dendriform 
 clusters or, rarely, in short needles (Gulewitsch 3 ). It deflagrates at 
 318° C. and melts at about 321° and sublimes. It is soluble with diffi- 
 culty in cold water, mere soluble in hot water, and insoluble in alcohol. 
 It behaves like uracil toward ammonia or baryta-water and silver nitrate. 
 Thymine is precipitated by phosphotungstic acid, especially when impure. 
 Bromine-water is decolorized by thymine, producing bromthymine. 
 For its detection we make use of the sublimation, the behavior toward 
 silver nitrate, and its elementary analysis. 
 
 Meters * has prepared compounds of pyrimidine bases with several metals 
 such as K, Na, Pb, Hg and he considers it incorrect to call the pyrimidine bodies 
 bases. 
 
 In regard to the methods of preparation see Kossel and Neumann 
 and W. Jones. 
 
 The purine and pyrimidine bodies stand in close chemical and phys- 
 iological relation to each other and for this reason the question has 
 been repeatedly raised whether the pyrimidine bases might not be formed, 
 at least in part, from the purine bases by the action of acids. Thus 
 far no conclusive investigations have been made supporting this view, 
 while on the contrary the investigations of Steudel 6 seem to contradict 
 such a view. 
 
 1 Zeitschr. f. physiol. Chem., 37. 
 
 : Schmiedeberg, Arch. f. exp. Path. u. Pharm., 37; Kossel and Neumann, Ber. 
 d. d. chem. Gesellsch., 26 and 2"; Mandel and Levene, Zeitschr. f. physiol. Chem., 46 
 47, 49. 50; E. Fischer and Roeder, ibid., 34; Gerngross, ibid., 38. 
 
 3 Zeitsch:. f. physiol. Chem., 27. 
 
 4 Journ. of biol. Chem., 7. 
 
 6 Kossel and Neumann, 1. c, and W. Jones, Zeitschr. f. physiol. Chem., 29, 461. 
 
 6 Zeitschr. f. physiol. Chem., 51 and 53 (against Burian). 
 
CHAPTER III. 
 THE CARBOHYDRATES. 
 
 We designate by this name bodies which are especially abundant 
 in the plant kingdom. As the protein bodies form the chief portion 
 of the solids in animal tissues, so the carbohydrates form the chief por- 
 tion of the dry substance of the plant structure. They occur in the 
 animal kingdom only in proportionately small quantities, either free .or 
 in combination with more complex molecules, forming compound pro- 
 teins. Carbohydrates are of extraordinarily great importance as food 
 for both man and animals. 
 
 The carbohydrates contain only carbon, hydrogen, and oxygen. The 
 last two elements occur, as a rule, in the same proportion as they do in 
 water, namely, 2:1, and this is the reason why the name carbohydrates 
 has been given to them. This name is not quite pertinent, if strictly 
 considered, because we not only have bodies, such as acetic acid and 
 lactic acid, which are not carbohydrates and still have their oxygen and 
 hydrogen in the same proportion as in water, but we also have a sugar 
 (the methyl pentoses, C6H12O5) which has these two elements in another 
 proportion. At one time it was thought possible to characterize as 
 carbohydrates those bodies which contained 6 atoms of carbon, or a 
 multiple, in the molecule, but this is not considered tenable at the present 
 time. We have true carbohydrates containing less than 6, and also those 
 containing 7, 8, and 9 carbon atoms in the molecule. 
 
 The carbohydrates have no properties or characteristics in general 
 which differentiate them from other bodies; on the contrary, the various 
 carbohydrates are in many cases very different in their external prop- 
 erties. Under these circumstances it is very difficult to give a positive 
 definition for the carbohydrates. 
 
 From a chemical standpoint we can say that all carbohydrates are 
 aldehyde or ketone derivatives of polyhydric alcohols. The simplest 
 carbohydrates, the simple sugars or monosaccharides, are either alde- 
 hyde or ketone derivatives of such alcohols, and the more complex 
 carbohydrates seem to be derived from these by the formation of anhy- 
 drides. It is a fact that the more complex carbohydrates yield two 
 -or even more molecules of the simple sugars when made to undergo 
 hydrolytic splitting. 
 
 196 
 
MONOSACCHARIDES. 197 
 
 Correspondingly the carbohydrates can he divided into three chief 
 groups, namely, 1. Simple sugars or monosaccharides, 2. Complex sugars 
 or disaccharides, trisaccharides and crystalline polysaccharides, and 
 3. Non-crystalline or colloid polysaccharides. Of these groups the mono- 
 saccharides, disaccharides and colloid polysaccharides are of special 
 physiological importance. 
 
 Our knowledge of the carbohydrates and their structural relation- 
 ships has been very much extended by the pioneering investigations of 
 Killiani ' and especially those of E. Fischer. 2 
 
 As the carbohydrates occur chiefly in the plant kingdom it is naturally 
 not the place here to give a complete discussion of the numerous carbo- 
 hydrates known up to the present time. According to the plan of this 
 work it is only possible to give a short review of those carbohydrates 
 which occur in the animal kingdom or are of special importance as food 
 for man and animals. 
 
 1. Monosaccharides. 
 
 All varieties of sugars are characterized by the termination " ose," 
 to which a root is added signifying their origin or other relations. Accord- 
 ing to the number of carbon atoms contained in the molecule the mono- 
 saccharides are divided into, trioses, tetroses, pentoses, hexoses, heptoses, 
 and so on. 
 
 All monosaccharides are either aldehydes or ketones of polyhydric 
 alcohols. The former are termed aldoses and the latter ketoses. Ordinary 
 glucose is an aldose, while ordinary fruit sugar (fructose) is a ketose. 
 The difference may be shown by the structural formula? of these two 
 varieties of sugar: 
 
 Glucose = CH 2 (OH).CH(OH).CH(OH).CH(OH).CH(OH).CHO; 
 Fructose = CH 2 (OH).CH(OH).CH(OH).CH(OH).CO.CH 2 (OH). 
 
 A difference is also observed on oxidation. The aldoses can be con- 
 verted into oxyacids having the same quantity of carbon, while the ketoses 
 yield acids having less carbon. On mild oxidation the aldoses yield 
 monobasic oxyacids, and dibasic acids on more energetic oxidation. Thus 
 ordinary glucose yields gluconic acid in the first case and saccharic 
 acid in the second. 
 
 1 Ber. d. deutsch. chem. Gesellsch., 18, 19, and 20. 
 
 2 See E. Fischer's lecture, Synthesen in der Zuckergruppe, Ber. d. deutsch. chem. 
 Gesellsch., 23, 2114. Excellent works on carbohydrates are Tollen's Kurzes Hand- 
 buch der Kohlehydrate, Breslau, 2 (1895), and 1, 2. Auflage, 1898, which gives a 
 complete review of the literature, and E. O. v. Lippmann, Die Chemie der Zucker- 
 arten, Braunschweig, 1904. 
 
198 
 
 THE CARBOHYDRATES. 
 
 Gluconic acid = CH 2 (OH).[CH(OH)] 4 .COOH; 
 Saccharic acid = COOH.[CH(OH)] 4 .COOH. 
 
 The monocarboxylic acids are easily transformed into their anhydrides 
 (lactones), and these latter are of special interest because, as shown by 
 Fischer, they can be changed into the corresponding aldehyde, i.e., 
 the corresponding aldose, by nascent hydrogen. 
 
 The monosaccharides are converted into the corresponding poly- 
 hydric alcohol by nascent hydrogen. Thus arabinose, which is a 
 pentose, C5H10O5, is transformed into the pentatomic alcohol, arabite, 
 C5H12O5. The three hexoses, glucose, mannose, and galactose, 
 C6H12O6, are transformed into the corresponding three hexites, sorbite, 
 mannite, and dulcite, CeH^Oe- The ketoses, on the contrary, due 
 to their constitution, yield a mixture of two alcohols on the same treat- 
 ment. From d-fructose, for example, we obtain a mixture of d-sorbite 
 and Z-mannite. On careful oxidation of the polyhydric alcohols the cor- 
 responding sugar can be prepared. 
 
 Numerous isomers occur among the monosaccharides, and especially 
 in the hexose group. In certain cases, as, for instance, in glucose and 
 fructose, we are dealing with a different constitution (aldoses and ketoses), 
 but in most cases we have stereoisomerism due to the presence of asym- 
 metric carbon atoms. 
 
 As the monosaccharides from the trioses upward contain asymmetric 
 carbon atoms they occur as optically active bodies in an 1-, d r and racemic 
 form, r or d-l form, which is a mixture or a combination of the I- and 
 d-form in equal parts. As the number of asymmetric carbon atoms 
 increases so does the number of possible stereoisomeric forms enlarge. 
 As the number according to van't Hoff is 2 n , where n represents the num- 
 ber of asymmetric carbon atoms, then for the aldo-hexose, which con- 
 tains 4 asymmetric carbon atoms, 2 4 = 16 stereo-chemically different forms 
 can exist. In fact, of these, 12 have been prepared and their geometric 
 structure has been explained and for which Fischer has given configura- 
 tion formulae. 
 
 As these relations are readily conceived we will, for example, give only the 
 configuration formula? for the most important pentoses and hexoses occurring 
 in the animal body. 
 
 COH 
 
 COH 
 
 HOCH 
 
 HCOH 
 
 HCOH 
 
 HOCH 
 
 HCOH 
 
 HOCH 
 
 CH 2 OH 
 
 CH2OH 
 
 4-Arabmose 
 
 f-Arabinose 
 
 COH 
 HOCH 
 
 HCOH 
 HOCH 
 
 CH2OH 
 
 d-Xylose 
 
 COH 
 HCOH 
 HOCH 
 HCOH 
 CH 2 OH 
 
 i-Xylose 
 
SPECIFIC ROTATION. 
 
 199 
 
 1 
 
 • COH 
 
 COH 
 
 COH 
 
 COH 
 
 HCOH 
 
 HOCH 
 
 HCOH 
 
 HOCH 
 
 HCOH 
 
 HOCH 
 
 HOCH 
 
 HCOH 
 
 HCOH 
 
 HOCH 
 
 HCOH 
 
 HOCH 
 
 CHoOH 
 
 CH2OH 
 
 HCOH 
 
 HOCH 
 
 d-Ribose 
 
 l-Riboee 
 
 CH2OH 
 
 d-Glucose 
 
 CHoOH 
 
 /-Glucose 
 
 COH 
 
 COH 
 
 CH 2 OH 
 
 CH 2 OH 
 
 HCOH 
 
 HOCH 
 
 CO 
 
 CO 
 
 HOCH 
 
 HCOH 
 
 HOCH 
 
 HCOH 
 
 HOCH 
 
 HCOH 
 
 HCOH 
 
 HOCH 
 
 HCOH 
 
 HOCH 
 
 HCOH 
 
 HOCH 
 
 CH 2 OH 
 
 d-Galactose 
 
 CH2OH 
 
 /-Galactose 
 
 CH2OH 
 
 d-Fructose 
 
 CH2OH 
 
 Z-Fructose 
 
 We designate the optical activity of the carbohydrates with the 
 letter I- for levogyrate, d- for dextrogyrate, and r- for the racemic. 
 These are only partly indicative. Thus dextrorotatory glucose is 
 designated d-glucose, levorotatory Z-glucose, but Emil Fischer has 
 used these signs in another sense. He designates by these signs the 
 mutual relationship of the various kinds of sugars instead of their 
 optical activity. For example, he does not designate the .levorotatory 
 fructose Z-fructose, but d-fructose, showing its close relation in stereometric 
 structure to dextrorotatory d-glucose. This designation is generally 
 accepted, and the above-mentioned signs only show the optical proper- 
 ties in certain cases. 
 
 Specific rotation means the rotation in degrees produced by 1 gm. substance 
 dissolved in 1 cc. liquid placed in a tube 1 dcm. long. The reading is ordinarily 
 made at 20° C. and with the monochromatic sodium light. The specific rotation 
 with this light is represented by («) D , and is expressed by the following formula: 
 
 («) D = ±— r, in which a represents the reading of degrees, 1 the length of the 
 
 tube in decimeters, and p the weight of substance in 1 cc. of the liquid. Inversely 
 the per cent P of substance can be calculated, when the specific rotation is known, 
 
 by the formula P = — r- , in which s represents the known specific rotation. 
 
 In the determination of the change in specific rotation with various concen- 
 trations we must know also the amount of substance in grams in 1 gram of the 
 solution (p) and the specific gravity of the solution (d) at 20°. The rotation 
 
 is calculated according to the formula (a) D = ± — j-r, 
 
 A freshly prepared solution of a substance often shows a different rotation 
 from one that has been allowed to stand for some time (multirotation). The 
 correct values which are found on allowing the solution to stand for a sufficiently 
 long time can be obtained immediately by boiling or on the addition of very 
 little ammonia. 
 
200 THE CARBOHYDRATES. 
 
 In order to explain multirotation Hudson 1 believes that the aldoses exist in 
 two isomeric forms having different rotation, which on being dissolved pass* 
 into each other by means of a reversible reaction. The two forms can be derived 
 because a lactone-like binding exists between the end carbon atom in the alde- 
 hyde group and the 7-carbon atom according to the formula 
 
 CHo0HCHOH.CH.CHOH.CHOH.C( . In this way the end carbon becomes 
 
 1 XoH 
 
 asymmetric and according to whether the position of the atoms that are combined 
 with this carbon atom are: 
 
 C H 
 
 \ / 
 
 C or 
 
 C OH 
 
 \ / 
 C 
 
 / \ 
 OH 
 
 / \ 
 H 
 
 -we obtain the two forms. Tanret 2 has obtained two isomeric forms of glucose, 
 galactose, arabinose and lactose which pass from one form to the other. The 
 two forms of glucose correspond according to E. H. Armstrong 3 to the syntheti- 
 cally prepared a- and /3-methyl glucosides prepared by E. Fischer (see pages 
 61-62). 
 
 Like the ordinary aldehydes and ketones, the sugars may be made to 
 take up hydrocyanic acid. Cyanhydrins are thus formed. These addi- 
 tion products are of special interest in that they make possible the arti- 
 ficial preparation of sugars rich in carbon from sugars poor in carbon. 
 
 As an example, if we start from glucose we obtain glucocyanhydrin 
 on the addition of hydrocyanic acid: 
 
 CH 2 .(OH).[CH(OH)]4.COH+HCN = CH 2 (OH).[CH(OH)] 4 .CH(OH).CN. 
 
 On the saponification of glucocyanhydrin the corresponding oxyacid is 
 formed. 
 
 CH 2 (OH).[CH(OH)] 4 .CH(OH).CN+2H 2 
 
 = CH 2 (OH).[CH(OH)] 4 .CH(OH).COOH+NH 3 . 
 
 By the action of nascent hydrogen on the lactone of this acid we obtain 
 glucoheptose, C7H14O7 and according to this principle the construction 
 of sugars up to nine carbon atoms has been accomplished. 
 
 The monosaccharides give the corresponding oximes with hydroxyl- 
 amine: thus glucose yields glucosoxime, CH 2 (OH).|CH(OH)] 4 .CH:N.OH. 
 These compounds are of importance on account of the fact, as found by 
 Wohl, 4 that they are the starting-point in the formation of one class 
 
 Mourn. Amer. Chem. Boo., 31, 61, 955 (1909). 
 2 Bull. 80C. chim., 13, 728 (1895), 15, 195, 349 (1896). 
 'Journ. Chem. Soc, 83, 1305 (1903). 
 * Ber. d. d. chem. Gesellsch., 26, p. 730. 
 
DERIVATIVES. 201 
 
 of sugars from another class, namely, the preparation of sugars poor in 
 carbon from those rich in carbon, for example, pentoses from hexoses 
 (see Wohl). 
 
 According to Ruff, by the action of hydrogen peroxide and the cata- 
 lytic action of ferric salts upon the carbohydrate monocarboxylic acids 
 the carbon chain can be shortened by the splitting off of the elements 
 of formic acid, and with the formation of the next lower aldose. Nki - 
 berg 1 has accomplished the same result by electrolysis, and by this 
 method has split glucose step by step into formaldehyde. 
 
 By the action of alkalies, even in small amounts, as also of carbonates 
 and lead hydroxide, a reciprocal transformation of the sugars, such as 
 d-glucose, d-fructose, and d-mannose, may take place (Lobry de Bruyn 
 and Alberda van Ekenstein 2 ), and from each of these three varieties 
 of sugar the two others are produced so that after a certain time the 
 solution contains all three sugars. 
 
 The transformation of the different varieties of sugar into each other 
 also occurs in the animal body. Neuberg and Mayer 3 have shown by 
 experiments on rabbits the direct partial transformation of various 
 mannoses into the corresponding glucoses. Another example is, it 
 seems, the formation of galactose (or milk sugar) from glucose in the 
 mammary gland. 
 
 By the action of strong alkali the sugars are decomposed with the 
 formation of lactic acid and many other products. 
 
 With ammonia the glucoses may form compounds which have been 
 considered as osamines by Lobry de Bruyn, but to differentiate them 
 from the true osamines have been called osimines by E. Fischer. 4 The 
 corresponding osaminic acid can be obtained from such an osimine by 
 the action of ammonia and hydrocyanic acid, and from the hydrochloric- 
 acid lactone of this acid the osamine is obtained by reduction with sodium 
 amalgam. In this manner E. Fischer and Leuchs artificially prepared 
 first d-arabinosimine from d-arabinose, then d-glucosaminic acid and 
 finally from its lactone d-glucosamine, which occurs in the animal 
 body. In a similar manner they 5 obtained ^-glucosamine from 
 /-arabinose. 
 
 Knoop and Windaus 6 have obtained large amounts of methylimida- 
 
 1 Ruff, Ber. d. d. chem. Gesellsch., 31 and 32; Neuberg, Biochem. Zeitschr., 7. 
 
 2 Ber. d. d. chem. Gesellsch., 28, 3078; Bull. soc. chim. de Paris (3), 15; Chem. 
 Centralbl., 1896, 2, and 1897, 2. 
 
 3 Zeitschr. f. physiol. Chem., 37. 
 
 4 Lobry de Bruyn, Ber. d. d. chem. Gesellsch., 28; E. Fischer, ibid., 35. 
 6 Ibid., 35, p. 3787, and 36, 24 (1903). 
 
 8 Ibid., 38, and Hofmeister's Beitrage, 6. 
 
202 THE CARBOHYDRATES. 
 
 CH 3 
 
 I 
 zol, C — NH V , from glucose by the action of ammonium-zinc hydroxide 
 
 CH— N^ 
 at ordinary temperatures. This formation can be conceived as follows: 
 First methyl glyoxal is formed from the sugar, and then from this, or 
 from the sugar, formaldehyde is produced, which reacts with the meth\ 1 
 glyoxal with the formation of methylimidazole according to the following 
 equation: 
 
 CH3CO NH 3 H x H 3 C.C— NH X 
 
 I + + >CH= || >CH+3H 2 
 
 COH NH 3 0* CH— 1ST 
 
 Methylglyoxal Formaldehyde Methylimidazole 
 
 A genetic relationship of the carbohydrates to histidine and the purine 
 bodies is thus made probable by the imidazole formation. 
 
 As the sugars are derivatives of polyhydric alcohols, they also form 
 esters, among which the benzoyl ester is of special interest because it is 
 used in the detection and isolation of the sugars and also of other car- 
 bohydrates. The nucleic acids probably also belong to the acid esters 
 of the sugars, and thus may be considered as complex phosphoric acid 
 esters, and perhaps the chondroitin sulphuric acid and the glucothionic 
 acid are sulphuric acid esters. The nature of these two groups of 
 sulphuric acid esters is not yet thoroughly understood. 
 
 The sugars can also combine with other bodies and with each other, 
 forming ether-like combinations. By the action of hydrochloric acid 
 as catalyst, as shown by Fischer and collaborators, the sugars split off 
 water and unite with "other bodies, producing lactone-like combinations, 
 which have been called glucosides (see pages 61 and 200). These glucosides, 
 which are generally compounds with aromatic groups, occur widely dis- 
 tributed in the vegetable kingdom. The more complex carbohydrates 
 may be considered, according to Fischer, as glucosides of the sugars. 
 Thus maltose, for example, is the glucoside and lactose the galactoside 
 of glucose. The glucosides can be split into their components by chem- 
 ical agents, boiling with dilute mineral acids, as well as by the action 
 of enzymes. The complex sugars hereby yield simple sugars and the other 
 glucosides yield compounds which belong either to the aromatic or the 
 aliphatic series besides the sugar. A long-knoAvn example of a decom- 
 position of this kind is the splitting of amygdalin by the enzyme emulsin 
 (see page 60). 
 
 With phenylhydrazine or substituted phenylhydrazines, the sugars 
 first yield hydrazones with the elimination of water, and then on the fur- 
 ther action of hydrazine on warming in an acetic-acid solution we obtain 
 osazones. 
 
HYDRAZONES AND OSAZOXES. 203 
 
 The reaction takes place with the aldoses as follows: 
 
 <a)XH 2 (OH).[CH(OH)] 3 .CH(OH).CHO+H 2 X.XH.C 6 H 5 = 
 
 CH 2 (OH).[CH(OH)] 3 .CH(OH)CH:X.NH.C 6 H 5 +H 2 0. 
 
 Phenylglucosehydrazone 
 
 (b) CH,(OH)[CH(OH)]3.CH(OH).CH:X.NH.C 6 H 5 +H 2 N.NH.C 8 H 5 = 
 
 CH 2 (OH).[CH(OH)] 3 .C.CH:X.XH.C«H 5 
 
 N.XH.C 6 H 6 +H 2 0+H 2 . 
 
 Phenylglucosazone 
 
 and with the keteoses: 
 
 CH,fOH)[CH(OH)] 3 CO.CH 2 (OH)+H 2 N.NH.C 8 H 5 = 
 
 CH 2 (OH)[CH(OH)] 3 C.CH 2 OH 
 
 X.XH.C 6 H 5 +H 2 0, 
 and CH 2 (OH)[CH(OH)];.C.CH,(OH) 
 
 X.XH.C 6 H 5 +H 2 X.XH.C 6 H5 = 
 CH 2 (OH)[CH(OH)] 3 .C.CH :X.XH.C 6 H 5 +H 2 0+H 2 . 
 
 X.XH.C 6 H 3 
 
 The hydrogen is not evolved, but acts on a second molecule of phenylhy- 
 drazine and splits it into aniline and ammonia : 
 
 H 2 X.H.C 6 H 3 +H 2 =H 2 X.C 6 H 5 +XH 3 . 
 
 As seen from the above equations the aldoses and ketoses yield the 
 same osazones, while the hydrazones are different. 
 
 The osazones, which are more important than the hydrazones, are 
 generally yellow crystalline compounds which differ from each other in 
 melting-point, solubility, and optical properties, and hence have been of 
 great importance in the characterization of certain sugars. They have 
 also become of extraordinarily great interest in the study of the carbo- 
 hydrates for other reasons. Thus they are a very good means of pre- 
 cipitating sugars from solution in which they occur mixed with other 
 bodies, and they are of the greatest importance in the artificial prepara- 
 tion of sugars. On cleavage, by the brief action of gentle heat and fum- 
 ing hydrochloric acid (for disaccharides still better with benzaldehyde), 1 
 the osazones yield so-called osones, which on reduction yield aldoses or 
 more often ketoses. The hydrazones can be much more readily retrans- 
 formed into the corresponding sugar, especially by decomposition with 
 benzaldehyde (Herzfeld) or with formaldehyde (Ruff and Ollen- 
 dorff 2 ), whereby the sugar is replaced by the aldehyde used. 
 
 An important property, although not applicable to all sugars, is their 
 ability to undergo fermentation, especially their ability to undergo 
 alcoholic fermentation with alcohol-yeast. We must state, however, 
 that the power of fermentation with pure yeast has been shown only for 
 
 1 E. Fischer and Armstrong, Ber. d. d. chem. Gesellsch., 35. 
 
 2 Herzfeld, ibid., 2S; Ruff and Ollendorff, ibid., 32. 
 
204 THE CARBOHYDRATES. 
 
 the hexose group, and in fact all the hexoses do not ferment, and they 
 do not all ferment with the same readiness. d-Glucose and d-mannose 
 ferment readily, but d-galactose only with difficulty. The Worms of 
 the above-mentioned sugars do not ferment, and from the racemic forms 
 of these sugars the optical Z-antipode can be prepared by the fermenta- 
 tion of the d-sugar. Among the ketoses the d-fructose ferments while 
 the sorbose does not. Among the sugars containing nine atoms of car- 
 bon, the nonoses, the mannonose ferments while the glucononose does 
 not. In regard to the fermentability of the triose, dioxyacetone, see 
 page 205. The different behavior of the various sugars with yeast stands 
 in fixed relation to their configuration, and is not only of great importance 
 for the behavior of the sugar in lower organisms, but also for their behavior 
 in higher developed organisms. Thus the investigations of Neuberg 
 Wohlgemuth x upon arabinose and of Neuberg and Mayer 2 on man- 
 noses have shown that in rabbits the Z-arabinose and the d-mannose are 
 much better utilized than d- and r-arabinose or I- and r-mannose. See 
 also pages 61-62. 
 
 In the alcoholic fermentation the sugar is decomposed according 
 to the general equation C6Hi206 = 2C2H60-f2C02. The exact process 
 is not clear, and seems to be rather complicated. On page 52 it has 
 been mentioned that for the action of the fermentation enzymes the 
 presence of a dialyzable substance present in the press-juice is neces- 
 sary (Harden and Young 3 ). On the other hand the fermentation 
 power of the press-juice can also be considerably increased by the addi- 
 tion of secondary sodium phosphate. The phosphoric acid in the press- 
 juice after fermentation is only partly precipitable with magnesia mixture 
 (Harden and Young). The most favorable action of boiled press- 
 juice is inhibited by lipase (Buchner and Klatte). According to 
 Harden and Young we must consider the action of boiled press-juice 
 and of phosphate in that first a hexose-phosphoric acid ester is produced 
 with the simultaneous formation of carbon dioxide and alcohol, according 
 to the following formula: 
 
 2C6H 1 2O 6 +2R 2 HPO4 = 2CO2+2C2H6O+C6H 1 0O4(PO 4 R2)2+2H2O. 
 
 The hexose phosphate can then be split into hexose and phosphate 
 by a special enzyme. The hexose phosphoric acid has been isolated 
 as a lead salt by Young. Glucose, fructose and mannose produce in 
 their fermentation the same hexose phosphoric acid. According to 
 
 1 Zeitschr. f. physiol. Chem., 35. 
 *1M&., 37. 
 
 3 Literature in Harden and Young, Bioch. Zeitschr., 32, 173 (1911) as well as 
 Buchner and Klatte, ibid., 8, 520 (1908). 
 
ALCOHOLIC FERMENTATION. 205 
 
 Iwanoff ' the phosphoric acid combination is a triose phosphate which 
 is fermented, with the formation of carbon dioxide, alcohol and phos- 
 phoric acid, by the dead and not by the living yeast. On the contrary 
 Lebedew finds the same formula as Young 2 for the phosphoric acid 
 ester. Iwanoff as well as Euler and their collaborators admit that 
 the formation of phosphoric acid esters is brought about by a special 
 enzyme. 3 According to Iwanoff and to Lebedew the sugar is first 
 fermented after it has combined with the phosphoric acid. It seems, 
 according to all evidence, that phosphoric acid esters of carbohydrates 
 are formed and that these are in some way of importance for the accom- 
 plishment of the fermentation. It is not probable that in the fermenta- 
 tion the hexose does not directly break into alcohol and CO2. It is 
 generally admitted that the process takes place through intermediary 
 steps. Lactic acid is considered as one of these, although in fact, this 
 acid is not fermented with the formation of alcohol. Recently Buchner 
 and Meissenheimer 4 have proposed dioxyacetone (HOCH2.CO.CH2OH) 
 as a probable intermediary step. They found that dioxyacetone was 
 very readily fermented by press-juice in the presence of common salt 
 and indeed with the formation of alcohol and carbon dioxide. This has 
 been substantiated by Lebedew. 5 Harden and Young disputed the 
 possibility that dioxyacetone is an intermediary step in the alcoholic 
 fermentation of sugar because it is more slowly fermented than the 
 sugars. 6 
 
 Besides ethyl alcohol and carbon dioxide there are formed in the fer- 
 mentation of sugar, although in small amounts, several higher alcohols 
 which form the so-called fusel oil. The most important constituents 
 of fusel oil are isoamylalcohol, t/-amylalcohol, isobutylalcohol and normal 
 propylalcohol in varying proportions. The formation of fusel oil was 
 ascribed for a long time to the action of bacteria until F. Ehrlich 7 
 found that the higher alcohols could be produced from certain amino- 
 acids by the living activity of yeast. From an amino-acid probably 
 the corresponding oxyacid is formed first by the splitting off of ammonia, 
 
 1 Centralbl. f. Bakt. 24, 1 (1909). 
 2 Bioch. Zeitschr. 36, 248 (1911). 
 
 3 Euler and Kullberg, Zeitschr. f. physiol. Chem., 74, 15 (1911); 80, 175 (1912); 
 Bioch. Zeitschr., 37, 133 (1911). 
 
 4 In reference to the intermediary products in alcoholic fermentation see Buchner 
 and Meissenheimer, Ber. d. d. chem. Gesellsch., 43, 1773 (1910) which also contains 
 the literature. 
 
 5 Compt. Rend., 153, 136 (1911). 
 
 6 Bioch. Zeitschr., 40, 458 (1912). 
 
 7 Zeitschr. f. Ver. d. d. Zuckerind, 55, 539 (1905) also Ber. d. d. chem. Gesellsch, 
 40, 1027, 2538 (1907); Bioch. Zeitschr. 1, 8 (1906); 8, 438 (1908); 18, 391 (1909). 
 
206 THE CARBOHYDRATES. 
 
 and then from this by loss of CO2 the alcohol is derived. The ammonia 
 is assimilated by the yeast. If the amino-acid is in the racemic form 
 then only the one component occurring naturally is transformed into 
 alcohol while the other remains in great part unchanged. In this manner 
 leucine is converted into isoamylalcohol according to the following equa- 
 tion: 
 
 hoco.ch(nh 2 ).ch 2 .ch/ 3 +h 2 o= 
 
 Leucine X CH 3 
 
 /CH3 
 NH 3 +C0 2 +HOCH 2 .CH 2 .CH< 
 
 Isoamylalcohol ^P'TJo 
 
 Other examples of the same kind is the formation of d-amylalcohol 
 from d-isoleucine and of isobutylalcohol from a-amino-valeric acid. 
 The formation of higher alcohols takes place with yeast poor in nitrogen 
 and in the presence of large amounts of sugar. In an analogous man- 
 ner, under the influence of yeast in the presence of sugar and inor- 
 ganic nutritive salts, from tyrosine tyrosol (p-oxyphenylethyl alcohol) 
 HO.C6H 4 .CH 2 .CH 2 .OH is derived and from tryptophane we get tryptophol 
 (/3-indoxylethyl alcohol) 1 . 
 
 C.CH 2 .CH 2 OH 
 C 6 H 4 <f%CH 
 
 NH 
 
 Other fermentation processes which are brought about by yeast but without 
 the presence of sugar have been studied by Neuberg 2 and his collaborators. 
 Among these we will mention the decomposition of pyroracemic acid (pyruvic 
 acid) into carbon dioxide and acetaldehyde : 
 
 HO.CO.CO.CH3 =C0 2 +HOC.CH 3 . 
 
 The enzyme active in this fermentation is called carboxylase. If the pyro- 
 racemic acid exists in the form of an alkali salt then the cleavage takes place ac- 
 cording to the formula, 
 
 2KO.CO.CO,CH 3 +H 2 0=C0 2 +2HOC.CH 3 +K 2 C03 
 
 and alkali carbonate is formed from a neutral salt. In this case the aldehyde 
 is condensed by the alkali to aldol, the first polymerization product of acetaldehyde. 
 
 The previously mentioned (page 41) lactic acid fermentation of 
 various sugars is caused by the action of different varieties of bacteria. 
 The equation represents a cleavage of one hexose molecule into two 
 lactic acid molecules CV,H 12 G = 2HOCO.CH(OH).CH 3 . Nothing positive 
 
 1 Ber. d. d. chem. Gesellsch., 44, 139 (1910); 45, 883 (1912). 
 
 s Bioch. Zeitschr., 31, 170 (1910); 32, 323; 36, 60, 68, 76 (1911); 47, 405, 413 (1912). 
 
PENTOSES. 207 
 
 is known as to how this cleavage occurs. According to Buchner and 
 Meissenheimer 1 the fermentation with the enzymes contained in the 
 bacteria produces chiefly the racemic, inactive form of the acid. This 
 also occurs as a rule by the action of living bacteria. In reference to the 
 formation of lactic acid within the organism see Chapter X. 
 
 The monosaccharides are colorless and odorless bodies, neutral in 
 reaction, with a sweet taste, readily soluble in water, generally soluble 
 with difficulty in absolute alcohol, and insoluble in ether. Some of them 
 crystallize well in the pure state. They are strong reducing substances. 
 They reduce metallic silver from ammoniacal silver solutions and they 
 also reduce other metallic oxides such as copper, bismuth and mercury 
 oxides, on heating in alkaline solution. This behavior is of great 
 importance in the detection and quantitative estimation of the sugars. 
 
 The simple varieties of sugar occur in part in nature as such, already 
 formed, which is the case with both of the very important sugars, glucose 
 and fructose. They also occur in great abundance in nature as more 
 complex carbohydrates (di- and polysaccharides); also as ester-like 
 combinations with different substances, as so-called glucosides. 
 
 Among the groups of monosaccharides known at the present time, 
 those containing less than five and more than six carbon atoms in the 
 molecule have no great importance in biochemistry, although they are 
 of high scientific interest. Of the two groups the hexoses are the more 
 abundant and are cf special interest. The pentoses are becoming cf 
 greater importance, net only for the chemistry of plants, but also for 
 the chemical processes in the animal body. 
 
 4 
 
 Pentoses (C5H10O5). 
 
 As a rule the pentoses do not occur as such in nature. They are 
 obtained from animal tissues, organs and fluids as cleavage products 
 of the nucleic acids, or nucleoproteins. The pentoses are chiefly obtained 
 from the plant kingdom, where nucleic acids also occur, by the hydro- 
 lytic cleavage with dilute mineral acids, of more complex carbohydrates, 
 the so-called pentosans. The pentosans exist very widely distributed 
 in the plant kingdom, and are of especially great importance in the build- 
 ing up of certain plant constituents. Methyl pentosans and methyl 
 pentoses also occur in the plants, and of these, the methyl pentose, rham- 
 nose, which occurs in several glucosides, must be specially mentioned. 
 
 The pentoses were first found in the animal kingdom by Salkowski 
 and Jastrowitz in the urine of a person addicted to the morphine habit, 
 
 1 Ann. d. Chem. u. Pharm., 349, 125 (1906). 
 
208 THE CARBOHYDRATES. 
 
 and later by Salkowski and others in human urine. Small quantities 
 of pentoses have been detected by Kulz and Vogel x in the urine of 
 diabetics, as also in dogs with pancreas diabetes or phlorhizin diabetes. 
 Pentose has also been found by Hammarsten among the cleavage 
 products of a nucleoprotein obtained from the pancreas, or from the 
 corresponding guanylic acid, and seems also, according to the observa- 
 tions of Blumenthal, to be a constituent of nucleoproteins of various 
 organs, such as the thymus, thyroid, brain, spleen, and liver. Their 
 occurrence in the nucleic acids has been previously discussed. In regard 
 to the quantity of pentoses found in the various organs, we must refer 
 to the works of Grund and of Bendix and Ebstein and Mancini. 2 
 
 The pentosans (Stone, Slowtzoff) as well as the pentoses are of the 
 greatest importance as foods for herbivorous animals. In regard to the 
 value of the pentoses, the researches of Salkowski, Cremer, Neuberg, 
 and Wohlgemuth 3 upon rabbits and hens show that these animals 
 can utilize the pentoses. The question whether the pentoses are active 
 as glycogen-formers is still an open one (see Chapter VII). The pen- 
 toses seem to be absorbed by human beings and in part utilized, but 
 they pass in part into the urine even when small quantities are taken. 4 
 
 The natural pentoses are reducing aldoses, and as a rule do not belong 
 to the sugars fermentable by yeast. Still, the observations of Sal- 
 kowski, Bendlx, Schone and Tollens seem to indicate that the pen- 
 toses are fermentable. 5 They are readily decomposed by putrefaction 
 bacteria. With phenylhydrazine and acetic acid they give crystalline 
 osazones which are soluble in hot water, and whose melting-points and 
 optical behavior are important for the detection of the pentoses. On 
 heating with hydrochloric acid they yield furfurol, but no levulinic acid. 
 In this reaction furfuran is formed from the pentose molecule, and then 
 
 1 Salkowski and Jastrowitz, Centralbl. f. d. med. Wissensch., 1892, 337 and 593; 
 Salkowski, Berl. klin. Wochenscbr., 1895; Bial, Zeitschr. f. klin. Med., 39; Bial and 
 Blumenthal, Deutsch. med. Wochenschr., 1901, No. 2; Kulz and Vogel, Zeitschr. f. 
 Biologie, 32. 
 
 2 Hammarsten Zeitschr. f. physiol. Chem., 19; also Salkowski, Berl. klin. Wochen- 
 schr., 1895; Blumenthal, Zeitschr. f. klin. Med., 34; Grund, Zeitschr. f. physiol. Chem. 
 35; Bendix and Ebstein, Zeitschr. f. allgemein. Physiol., 2; Mancini, Chem. Centralbl., 
 1906. 
 
 3 Stone, Amer. Chem. Journ., 14; Slowtzoff, Zeitschr. f. physiol. Chem., 34; Sal- 
 kowski, ibid., 32; Cremer, Zeitschr. f. Biologie, 29 and 42; Neuberg and Wohlgemuth, 
 Zeitschr. f. physiol. Chem., 35. 
 
 4 See Ebstein, Virchow's Arch., 129; Tollens, Ber. d. deutsch. chem. Gesellsch., 29, 
 1208; Cremer, 1. c; Lindemann and May, Deutsch. Arch. f. klin. Med., 56; Salkowski, 
 Zeitschr. f. physiol. Chem., 30. 
 
 Salkowski, Zeitschr. f. physiol. Chem., 30; Bendix, see Chem. Centralbl., 1900, 1; 
 Sr-lione and Tollens, ibid., 1901, 1. 
 
PENTOSE REACTIONS. 209 
 
 HC— CH 
 
 II II 
 from this its aldehyde, the furfurol HC C.CHO. The furfurol pass- 
 
 O 
 ing over, on distilling with hydrochloric acid, can be detected by the aid 
 of aniline-acetate or xylidine acetate paper, which is colored a beautiful 
 red by furfurol. In the quantitative estimation we can use the method 
 suggested by Tollens, which consists in converting the furfurol in the 
 distillate into phloroglucide by means of phloroglucin and weighing (see 
 Tollens and Krober, Grund, Bendix and Ebstein), or according to 
 Jolles l by bisulphite and retitrating with iodine solution. In using 
 these methods it must be borne in mind that glucuronic-acid compounds 
 also yield furfurol under the same conditions. The two following pentose 
 reactions, as suggested by Tollens, are especially applicable. 
 
 The orcin-hydrochloric acid test. Mix with the solution or the substance 
 introduced into water an equal volume of concentrated hydrochloric acid, add 
 some orcin in substance, and heat. In the presence of pentoses the solution 
 becomes reddish-blue, then bluish-green, and on spectroscopic examination an 
 absorption-band is observed between C and D. If it is cooled and shaken with 
 amyl alcohol, a bluish-green solution which shows the same band is obtained. 
 
 The phloroglucin-hydrochloric acid test. This test is performed in the 
 same manner as the above, using phloroglucin instead of orcin. The solution 
 becomes cherry-red on heating and then becomes cloudy and hence a shaking out 
 with amyl alcohol is very necessary. The red amyl-alcohol solution shows an 
 absorption-band between D and E. The orcin test is better for several reasons 
 than the phloroglucin test (Salkowski and Neuberg 2 ). In regard to the use 
 of these tests in urine examination see Chapter XIV. 
 
 Many modifications of these tests have been suggested. Brat 3 performs 
 the crcin reaction by the addition of NaCl and heating to only 90-95°. Bial 4 
 uses a hydrochloric acid containing ferric chloride for the orcin test and claims 
 to get a greater delicacy. On too strong and too long heating (l§-2 minutes), 
 when using this modification, a confusion with sugars of the six carbon series may 
 occur (Bial, van Leersum). 5 According to R. Adler and 0. Adler the phlo- 
 roglucin and orcin tests can be made with glacial acetic acid and a few drops 
 hydrochloric acid instead of with the hydrochloric acid alone. These investigators 
 also use a mixture of equal volumes of aniline and glacial acetic acid as a reagent 
 for pentoses. On the addition of a little pentose to the boiling mixture a beautiful 
 red color of furfurol-aniline acetate is obtained. A. Neumann 6 performs the 
 orcin test with glacial acetic acid and adds concentrated sulphuric acid drop 
 by drop. On following the exact instructions not only do the pentoses give 
 this reaction, but also glucuronic acid, glucose, and fructose give characteristic 
 
 1 Bendix and Ebstein, 1. c, which contains the literature; Jolles, Ber. d. d. chem. 
 Gesellsch., 39 and Zeitschr. f. anal. Chem., 46. 
 
 2 Salkowski, Zeitschr. f. physiol. Chem., 27; Neuberg, ibid., 31. 
 
 3 Zeitschr. f. klin. Med., 47. 
 
 4 Deutsch. med. Wochenschr., 1902 and 1903, and Zeitschr. f. klin. Med., 50. 
 
 5 Bial, Zeitschr. f. klin. Med., 50; van Leersum, Hofmeister's Beitrage, 5. 
 
 6 R. and O, Adler, Pfliiger's Arch., 106; A. Neumann, Berl. klin. Wochenschr., 
 1904. 
 
210 THE CARBOHYDRATES. 
 
 colored solutions with special absorption-bands which can be made use of in 
 identifying the various sugars. Fr. Sachs has tested Bial's test and has given 
 special precautions to prevent confusion with glucuronic acid. Jolles * pre- 
 cipitates (from urine) the pentoses as osazones, distills the precipitate with 
 hydrochloric acid, and tests the distillate with Bial's reagent. 
 
 In performing the above two tests for pentose it must be borne in mind that 
 glucuronic acid gives the same reactions and also that the colors alone are not 
 sufficient. The spectroscopic examination must therefore never be omitted. 
 Both tests are to be considered as tests of detection rather than definite pentose 
 reactions, and therefore for a positive detection of pentoses we must prepare also 
 the osazones or other compounds. 
 
 Arabinoses. The pentose isolated by Neuberg from human urine 
 is r-arabinose. It can be isolated from the urine as the diphenylhydra- 
 zone, from which the arabinose can be separated by splitting with for- 
 maldehyde. The inactive r-arabinose seems to be the pentose regularly 
 occurring in pentosuria and thus far, in only one case, has /-arabinose 
 been found. /-Arabinose is said to pass into the urine after partaking of 
 certain fruits, such as plums, in large amounts (C. Barszczewski 2 ). 
 
 The r-arabinose is crystalline, has a sweetish taste, and melts at 
 163-164° C. Its diphenylhydrazone, which, according to Neuberg 
 and Wohlgemuth, 3 can be used in its quantitative estimation, melts 
 at 206° C, is insoluble in cold water and alcohol, but readily soluble 
 in pyridine. The osazone melts at 166-168° C. 
 
 The dextrorotatory /-arabinose is obtained by boiling gum arabic or 
 cherry gum with dilute sulphuric acid. The d-arabinose has been pre- 
 pared synthetically. The phenylosazone of /-arabinose melts at 160°. 
 The /-arabinose which crystallizes in plates or prisms melts at about 
 164°. The specific rotation is (a) D = +104.5°. 
 
 Xyloses. The /-xylose occurs extensively in the plant kingdom and 
 is prepared from wood-gum by the action of dilute acid. Xylose is 
 crystalline, melts at 150-153° C, dissolves very readily in water but 
 with difficulty in alcohol, is faintly dextrorotatory, (a) D = +18.1°, and 
 gives a phenylosazone which melts at 155-158° C, and according to 
 Tollens and Muther a diphenylhydrazone which melts at 107-108°. 
 According to Bertrand xylose can be transformed into xylonic acid, 
 CH2(OH)[CH(OH)]3COOH, by bromine-water and the brom-cadmium 
 compound or the brucine salt (Neuberg) of this acid is well suited for 
 the detection and isolation of /-xylose. On oxidation with nitric acid 
 the optically inactive trioxyglutaric acid, with a melting-point of 152° C. 
 is obtained. 
 
 1 Fr. Sachs, Biochem. Zeitschr., 1 and 2; Jolles, ibid., 2, Centralbl. f. inn. Med., 
 1907. and Zeitschr. f. anal. Chem., 46. 
 
 2 Neuberg, Ber. d. d. chem. Gesellsch., 33; Barszczewski, Maly's Jahrsb., 27, 733. 
 •Zeitschr. f. physiol. Chem., 35. 
 
HEXOSES: 211 
 
 According to Neitberg and to Rewald 1 the pentose obtained from a 
 pancreas nucleoprotein and the pentose isolated by Neuberg and Bkahn 
 from inosinic acid is identical with /-xylose. 
 
 Ribose. This pentose has been prepared synthetically by E. Fischer. 
 The phenylhydrazone melts at 154-155° C, the p-bromphenylhydr:iz<>n<- 
 at 164-165° C. The osazone is identical with arabinosazone. On oxida- 
 tion it yields an optically inactive trioxyglutaric acid, which melts at 170- 
 171° C. d-ribose is", according to Levene and Jacobs, the pentose of 
 inosinic acid, guanylic acid and yeast nucleic acid. According to these 
 workers the pentose exists in these nucleic acids in a glucoside-like com- 
 bination with the purine bases, as so-called nucleosides. It must be 
 remarked that Neuberg adheres to his claim that /-xylose exists at 
 least in the pancreas. 2 
 
 Hexoses (C0H12O6). 
 
 The most important and best-known simple sugars belong to this 
 group, and most of the other bodies which have been considered as car- 
 bohydrates in the past are anhydrides of this group. Certain hexoses, 
 such as glucose and fructose, either occur in nature already formed 
 or are produced by the hydrolytic splitting of other more complicated 
 carbohydrates or glucosides. Others, such as mannose or galactose, 
 are formed by the hydrolytic cleavage of other natural products, while 
 some, on the contrary, such as gulcse, talose, and others, are obtained 
 only by artificial means. 
 
 All hexoses, as also their anhydrides, yield levulinic acid, C5H8O3, 
 besides formic acid and humus substances on boiling with dilute min- 
 eral acids. Oxymethyl furfurol, CqHqO^, occurs here as an intermediary 
 step and this then quantitatively decomposes into levulinic acid and 
 formic acid. 3 Some of the hexoses, as above stated, are fermentable 
 with yeast. 
 
 Some hexoses are aldoses, while others are ketoses. Belonging to the 
 first group we have mannose, glucose, and galactose, and to the other 
 fructose, and also sorbinose. 
 
 The most important syntheses of the carbohydrates have been made 
 by E. Fischer and his pupils, chiefly within the members of the hexose 
 group. A short summary of the syntheses of hexoses will be given. 
 
 1 Tollens and Miither, Ber. d. d. chem. Gesellsch., 37; Bertrand, Bull. soc. chim. 
 (3), 5; Neuberg, Ber. d. d. chem. Gesellsch., 35; Neuberg and Brahn, Biochem. Zeitchr., 
 5; Rewald, Ber. d. d. chem. Gesellsch., 42 (1909). 
 
 2 E. Fischer, Ber. d. d. chem. Gesellsch, 24, Levene and Jacobs, ibid, 42, 2102, 2469 
 2474, 3247 (1909); 43, 3147 (1910); Neuberg, ibid., 42, 2806 (1909); 43, 3501 (1910). 
 
 1 Kiermeyer, Chem. Zeitung., 1895; v. Ekenstein and Blanksma, Ber. d. d. chem. 
 Gesellsch., 43, 2355 (1910). 
 
212 THE CARBOHYDRATES. 
 
 The first artificial preparation of a sugar was made by Butlerow. On 
 treating trioxymethylene, a polymer of formaldehyde, with lime-water he obtained 
 a faintly sweetish syrup called methylenitan. Loew l later obtained a mixture 
 of serveral sugars, from which he isolated a fermentable sugar, called methose, 
 by condensation of formaldehyde in the presence of bases. The most important 
 and comprehensive syntheses of sugar have been performed by E. Fischer 2 
 
 The starting-point of these syntheses is a-acrose, which occurs as a condensa- 
 tion product of formaldehyde. The name a-acrose has been given to this body 
 because it is obtained from acrolein bromide by the action of bases (Fischer). 
 It is also obtained admixed with fi-acrose on the oxidation of glycerin with 
 bromine in the presence of sodium carbonate and treating the resulting mixture 
 with alkali. On the oxidation with bromine a mixture of glyceric aldehyde, 
 CH 2 OH.CH(OH).CHO, and dioxyacetone, CH 2 (OH).CO.CH 2 OH, is obtained. 
 These two bodies may be considered as true sugars, namely, glyceroses or trioses. 
 It seems as if a condensation to hexoses takes place on treatment with alkalies. 
 
 a-acrose may be isolated from the above mixture and obtained pure by first 
 converting it into osazone and then retransforming this into the sugar, a-acrose 
 seems to be identical with r-fructose. With yeast one-half, the levogyrate 
 rf-fructose ferments, while the dextrogyrate Z-fructose remains. The r- and 
 Z-fructose may be prepared in this way. 
 
 On the reduction of a-acrose we obtain a-acrite, which is identical with r- 
 mannite. On oxidation of r-mannite we obtain r-mannose, from which only 
 Z-mannose remains on fermentation. On further oxidation of r-mannose it 
 yields r-mannonic acid. The two active mannonic acids may be separated from 
 each other by the fractional crystallization of their strychnine or morphine salts. 
 The two corresponding mannoses may be obtained from these two acids, d- and 
 Z-mannonic acids, by reduction. 
 
 rf-fructose can be obtained from cZ-mannose with the osazone as an inter- 
 mediary step and it remains now to speak of .the formation of glucoses. The 
 d- and Z-mannonic acids are partly converted into d- and Z-gluconic acids on heat- 
 ing with quinoline, and d- or Z-glucose is obtained on the reduction of these acids; 
 Z-glucose is best prepared from Z-arabinose by means of the cyanhydrin reaction, 
 using Z-gluconic acid as the intermediate step. The combination of Z- and d- 
 gluconic acids, forming r-gluconic acid, yields r-glucose on reduction. 
 
 The artificial preparation of sugars by means of the condensation of formalde- 
 hyde has received special interest because, according to Baeyer's assimilation 
 hypothesis, in plants formaldehyde is first formed by the reduction of carbon 
 dioxide, and the sugars are produced by the condensation of this formaldelvyde. 
 Bokorny 3 has shown, by special experiments on algse Spirogyra, that formalde- 
 hyde sodium sulphite was split by the living algse cells. The formaldehyde set 
 free is immediately condensed to carbohydrate and precipitated as starch. 4 
 
 Among the hexoses known at the present time only glucose, fructose, 
 and galactose are really of physiological-chemical interest; therefore of 
 the other hexoses only mannose will be incidentally mentioned. 
 
 rf-Glucose (grape sugar) also called dextrose and diabetic sugar — 
 occurs abundantly in the grape, and also, often accompanied with fructose 
 
 1 Butlerow, Ann. d. Chem. u. Pharm., 102; Compt. rend., 53; O. Loew, Journ. f. 
 prakt. Chem. (N. F.), 33, and Ber. d. deutsch. chem. Gesellsch., 20, 21, 22. 
 
 2 Ber. d. d. chem. Gesellsch., 21, and 1. c, p. 197. 
 
 3 Biolog. Centralbl. 12, pp. 321 and 481. 
 
 * In regard to the syntheses of sugar see also W. Lob and Pulvermacher Bioch. 
 Zeitschr, 23, 10 (1909), 26, 231 (1910). 
 
GLUCOSE. 213 
 
 (levulose), in honey, sweet fruits, seeds, roots, etc. It occurs in the 
 human and animal intestinal tract during digestion, also in small quan- 
 tities in the blood and lymph, and as traces in other animal fluids and 
 tissues. It occurs only as traces in urine under normal conditions, 
 while in diabetes the quantity is very large. It is formed in the hydro- 
 lytic cleavage of starch, dextrin, and other compound carbohydrates, 
 as also in the splitting of glucosides. The question whether glucose 
 can be formed in the body from proteins or from fats is disputed and will 
 be discussed in a following chapter (VII). 
 
 Properties of Glucose. Glucose crystallizes sometimes with 1 mole- 
 cule of water of crystallization in warty masses consisting of small leaves 
 or plates, and sometimes when free from water in fine needles or prisms. 
 The sugar containing water of crystallization melts even below 100° C. 
 and loses its water of crystallization at 110° C. The anhydrous sugar 
 melts at 146° C, and is converted into glucosan, CeHioOs, at 170° C. 
 with the elimination of water. On strongly heating it is converted into 
 caramel and then decomposes. 
 
 Glucose is readily soluble in water. This solution, which is not as 
 sweet as a cane-sugar solution of the same strength, is dextrogyrate and 
 shows strong birotation. The specific rotation is dependent upon the 
 concentration of the solution, as it increases with an increase in the con- 
 centration. A 10 per cent solution of anhydrous glucose can be taken as 
 +52.5° at 20° C. 1 Glucose dissolves sparingly in cold, but more freely 
 in boiling alcohol. One hundred parts alcohol of sp. gr. 0.837 dissolves 
 1.95 parts anhydrous glucose at 17.5° C. and 27.7 parts at the boiling 
 temperature (Anthon 2 ). Glucose is insoluble in ether. 
 
 If an alcoholic caustic-potash solution is added to an alcoholic solu- 
 tion of glucose, an amorphous precipitate of insoluble sugar-potash 
 compound is formed. On warming this compound it decomposes easily 
 with the formation of a yellow or brownish color, which is the basis of 
 Moore's test. Glucose also forms compounds with lime and baryta. 
 
 Moore's Test. If a glucose solution is treated with about one 
 quarter of its volume of caustic potash or soda and warmed, the solution 
 becomes first yellow, then orange, yellowish-brown, and lastly dark 
 brown. It has at the same time a faint odor of caramel, and this odor 
 is more pronounced on acidifiying. 3 
 
 Glucose forms several crystallizable combinations with NaCl of 
 which the easiest to obtain is (CeHioOG^-NaCl-fH^O, which forms 
 
 1 For further information see Tollens' Handbuch der Kohlehydrate, 2. AuflL, 44. 
 
 2 Cited from Tollens' Handbuch. 
 
 3 In regard to the products formed in this reaction, see Framm, Pfluger's Arch., 64; 
 Neff, Annal. d. Chem. u. Pharm., 357; Buchner and Meisenheimer, Ber. d. d. chem. 
 Gesellsch., 39; Meisenheimer, ibid., 41. 
 
214 THE CARBOHYDRATES. 
 
 large colorless six-sided double pyramids or rhomboids with 13.52 per 
 cent NaCl. 
 
 Glucose in neutral or very faintly acid (organic acid) solution under- 
 goes alcoholic fermentation with beer-yeast: CeHi206= : 2C2H5.0H+2C02. 
 In the presence of acid milk or cheese the glucose undergoes lactic-acid 
 fermentation, especially in the presence of a base such as ZnO or CaC03. 
 The lactic acid may then further undergo butyric-acid fermentation: 
 2C 3 H 6 03 = C4H 8 02+2C0 2 +4H. 
 
 Glucose reduces several metallic oxides, such as copper, bismuth, 
 and mercuric oxide, in alkaline solutions, and the most important 
 reactions for sugar are based on this fact. 1 
 
 Trommer's test is based on the property that glucose possesses of 
 reducing cupric hydroxide in alkaline solution into cuprous oxide. Treat 
 the glucose solution with about \- \ vol. caustic soda and then carefully 
 add a dilute copper-sulphate solution. The cupric hydroxide is thereby 
 dissolved, forming a beautiful blue solution, and the addition of copper 
 sulphate is continued until a very small amount of hydroxide remains 
 undissolved in the liquid. This is now warmed, and a yellow hydrated 
 suboxide or red suboxide separates even below the boiling temperature. 
 If too little copper salt has been added, the test will be yellowish-brown 
 in color, as in Moore's test; but if an excess of copper salt has been added, 
 the excess of hydroxide is converted on boiling into a dark-brown hydrate 
 which interferes with the test. To prevent these difficulties the so- 
 called Fehling's solution may be employed. This solution is obtained 
 by mixing just before use equal volumes of an alkaline solution of Rochelle 
 salt and a copper-sulphate' solution (173 grams Rochelle salt and about 
 50-60 grams NaOH per liter and 34.65 grams crystalline copper sulphate 
 per liter). This solution is not reduced or noticeably changed by boiling. 
 The tartrate holds the excess of cupric hydroxide in solution, and an excess 
 of the reagent does not interfere in the performance of the test. In 
 the presence of sugar this solution is reduced. 
 
 According to Benedict 2 this test is more delicate if sodium carbonate is 
 used instead of sodium hydroxide in the preparation of Fehling's solution. 
 
 B6ttger-Alm£n's test is based on the property glucose possesses 
 of reducing bismuth oxide in alkaline solution. The reagent best adapted 
 for this purpose is obtained, according to Nylander's 3 modification of 
 Almex's original test, by dissolving 4 grams of Rochelle salt in 100 parts 
 of 10 per cent caustic-soda solution and adding 2 grams of bismuth 
 subnitrate and digesting on the water-bath until as much of the bismuth 
 
 1 In regard to the products produced see Neff, Annal. d. Chem. u. Pharm., 357. 
 
 2 Journ. of biol. Chem., 3. 
 
 3 Zeitschr. f. physiol. Chem., 8. 
 
GLUCOSE. 215 
 
 salt is dissolved as possible. If a glucose solution \s treated with about 
 tV vol., or with a larger quantity of the solution when large quantities 
 of sugar are present, and boiled for a few minutes, the solution becomes 
 first yellow, then yellowish-brown, and finally nearly black, and after a 
 time a black deposit of bismuth (?) settles. 
 
 The property that glucose has of reducing an alkaline solution of 
 mercury on boiling is the basis of Knapp's reaction with alkaline mercuric 
 cyanide, and of Sachsse's reaction witli an alkaline potassium-mercuric 
 iodide solution. 
 
 On heating with phenylhydrazine acetate a glucose solution 
 gives a precipitate consisting of fine yellow crystalline needles which are 
 almost insoluble in water, but soluble in boiling alcohol, and which separate 
 again, on treating the alcoholic solution with water. The crystalline 
 precipitate consists of phenylglucosazone (see page 203). This com- 
 pound melts when pure at 205° C. It must be borne in mind that 
 the melting-point of this and other osazones is somewhat variable, depend- 
 ing upon the rapidity of the heating, the diameter of the tube and the 
 thickness of the sides of the tube. 1 The osazone dissolves readily in 
 pyridine (0.25 gram in 1 gram), and precipitates again from this solu- 
 tion as crystals on the addition of benzene, ligroin, or ether. According 
 to Neuberg 2 this behavior can be used in the purification of the osazone. 
 The diphenylhydrazone and the methyl phenylhydrazone are also of 
 interest. 
 
 Glucose is not precipitated by a lead-acetate solution, but is almost 
 completely precipitated by a solution of ammoniacal basic lead acetate. 
 On warming, the precipitate becomes flesh-color or rose-red (Rubner's 
 reaction 3 ) . 
 
 If a watery solution of glucose is treated with benzoylchloride and 
 an excess of caustic soda, and shaken until the odor of benzoylchloride 
 has disappeared, a precipitate of benzoic-acid ester of glucose will be 
 produced which is insoluble in water or alkali (B aumann 4 ) . 
 
 If \-\ cc. of a dilute watery solution of glucose is treated with a few 
 drops of a 10 per cent alcoholic solution (free from acetone) of a-nayhthol , 
 on the slow addition of 1-2 cc. of concentrated sulphuric acid a beautiful 
 reddish-violet ring forms at the juncture of the liquids, or on shaking, the 
 entire mixture becomes a beautiful reddish-violet color (Molisch 5 ). 
 
 1 See E. Fischer, Ber. d. d. ehem. Gesellsch., 41. 
 
 2 Ber. d. d. chim. Gesellsch., 32, 3384. 
 
 3 Zeitschr. f. Biologie, 20. 
 
 4 Ber. d. deutsch. chem. Gesellsch., 19; also Kueny, Zeitschr. f. physiol. Chem., 14, 
 and Skraup, ^Vien. Sitzungsber., 98 (1888). 
 
 5 Molisch, Monatshefte f . Chem., 7, and Centralbl. f. d. med. Wissensch., 1887, 
 pp. 34 and 49. 
 
216 THE CARBOHYDRATES. 
 
 This reaction depends, according to Ville and Derrien, as well as to 
 V. Ekenstein and Blanksma 1 upon the formation of oxymethylfurfurol 
 which reacts with the a-naphthol. As oxymethylfurfurol is formed from 
 all hexoses, hence Molisch's reaction is a general reaction for hexoses. 
 
 Diazobenzenesulphonic acid gives with a glucose solution made alkaline 
 with a fixed alkali a red color, which after 10-15 minutes gradually changes to 
 violet. Orthonitrophenylpropiolic acid yields indigo when boiled with a 
 small quantity of glucose and sodium carbonate, and this is converted into 
 indigo-white by an excess of sugar. An alkaline solution of glucose is colored 
 deep red on being warmed with a dilute solution of picric acid. The behavior 
 of glucose toward certain pentose reactions has been given on page 209. 
 
 A more complete description as to the performance of these several 
 tests will be given in detail in a subsequent chapter (on the urine). 
 
 Glucose is prepared, pure, by inverting cane-sugar by the follow- 
 ing simple method of Soxhlet and Tollens, which is a modification of 
 Schwarz's 2 method: 
 
 Treat 12 liters 90 per cent alcohol with 480 cc. fuming hydrochloric 
 acid and warm to 45-50° C; gradually add 4 kilos of powdered cane- 
 sugar, and allow to cool after two hours, when all the sugar will have 
 dissolved and been inverted. To incite crystallization, some crystals of 
 anhydrous glucose are added, and after several days the crystals are 
 sucked dry by the air-pump, washed with dilute alcohol to remove 
 hydrochloric acid, and crystallized from alcohol or methyl alcohol. 
 According to Tollens it is best to dissolve the sugar in one-half its 
 weight of water on the water-bath and then add double this volume of 
 90-95 per cent alcohol. 
 
 In detecting glucose in animal fluids or extracts of tissues we may 
 make use of the above-mentioned reduction tests, the optieal deter- 
 mination, fermentation, and phenylhydrazine tests. For the quantitative 
 estimation the reader is referred to the chapter on the urine. Those 
 liquids containing proteins must first have these removed by coagulation 
 with heat and addition of acetic acid, or by precipitation with alcohoL 
 or metallic salts, before testing for glucose. In regard to the difficulties 
 of operating with blood and serous fluids we refer the student to larger 
 works. 
 
 Mannoses. d-Mannose, also called seminose, is obtained with d-fructose on 
 the careful oxidation of d-mannite. It is also obtained on the hydrolysis of 
 natural carbohydrates, such as salep slime and reserve cellulose (especially 
 from the shavings of the ivory-nut). It is dextrorotatory, readily ferments, 
 with beer-yeast, gives a hydrazone not readily soluble in water, and an osazone 
 which is identical with that from d-glucose. 
 
 d-Galactose (not to be mistaken for lactose or milk-sugar) is obtained on 
 the hydrolytic cleavage of milk-sugar, and by the hydrolysis of many other 
 
 1 Bull. soc. chim. (4), 5, 895 (1909); Ber. d. d. ohem. Gesellsch., 43, 2358 (1910). 
 
 2 Tollens, Handbuch der Kohlehydrate, 2. Aufl. I, 39. 
 
FRUCTOSE. 217 
 
 carbohydrates, especially varieties of gums and mucilaginous bodies. 
 It is also obtained on heating cerebrin, a nitrogenized glucoside prepared 
 from the brain, with dilute mineral acids. 
 
 It crystallizes in needles or leaves which melt at 1G8° C. It is some- 
 what less soluble in water than glucose. It is dextrogyrate, and according 
 to Neuberg l has a rotation (a) D =+81°. With ordinary yeast galac- 
 tose is slowly, but nevertheless completely, fermented. It is fermented 
 by a great variety of yeasts (E. Fischer and Thierfelder), but not by 
 Saccharomyces apiculatus, 2 which is of importance in physiological- 
 chemical investigations. Galactose reduces Fehling's solution to a 
 less extent than glucose, and 10 cc. of this solution are reduced, accord- 
 ing to Soxhlet, by 0.0511 gram galactose in 1 per cent solution. Its 
 phenylosazone melts according to Neuberg at 196-197° C, and is soluble 
 with difficulty in hot water, but with relative ease in hot alcohol. Its 
 solution in glacial acetic acid is optically inactive. In the test with 
 hydrochloric acid and phloroglucin galactose gives a color similar to that 
 of the pentoses, but the solution does not give the absorption spectrum. 
 On oxidation it first yields galactonic acid and then mucic acid, and 
 these serve in the detection of galactose. 
 
 d-Fructose (levulose) also fruit-sugar, occurs, as above stated, mixed 
 with glucose, extensively distributed in the vegetable kingdom and 
 also in honey. It is formed in the hydrolytic cleavage of cane-sugar 
 and several other carbohydrates, but it is very readily obtained by the 
 hydrolytic splitting of inulin. In extraordinary cases of diabetes mellitus 
 we find fructose in the urine. Neuberg and Strauss 3 have detected 
 fructose with positiveness in human blood-serum, and exudates in cer- 
 tain cases. 
 
 Fructose crystallizes with comparative difficulty in coarse crusts 
 or warts or in fine needles. C. Morner 4 has obtained crystals 2-3 mm. 
 long which belonged to the rhombic system, and neither melted nor lost 
 in weight on heating to 100° C. The melting-point is 110° C. Fructose 
 is readily soluble in water, but almost insoluble in cold absolute alcohol, 
 though rather readily in boiling alcohol. Its aqueous solution is levogy- 
 rate. C. Morner found the rotation for a 10 and 20 per cent solution 
 was (a) D =— 93° and —94.1° respectively. Fructose ferments with 
 yeast, and gives the same reduction tests as glucose, and also the same 
 osazone. It gives a compound with lime which is less soluble than the 
 corresponding glucose compound. Fructose is not precipitated by 
 sugar of lead or basic lead acetate. 
 
 1 See C. Oppenheimer, Handb. d. Biochem. 1, p. 197. 
 
 2 See F. Voit, Zeitschr. f. Biol., 28 and 29. 
 
 3 Zeitschr. f. physiol. Chem., 36, which also contains the older literature. 
 
 * Svensk. Farmac. Tidskr, No. 6, 1907. See also Maly's Jahresb., 37, p. 95. 
 
218 THE CARBOHYDRATES. 
 
 Fructose does not reduce copper to the same extent as glucose. 
 Under similar conditions the reduction relationship is 100 : 92.08. 
 
 In detecting fructose and those varieties of sugar which yield fructose on 
 cleavage we make use of the following reaction, suggested by Seliwanoff which 
 consists in heating with hydrochloric acid and resorcinol. This depends upon 
 the formation of oxymethylfurfurol and is therefore obtained by all hexoses. 
 As the ketoses give about 20 per cent oxymethylfurfurol and the aldoses only 
 1 per cent the reaction is more readily obtained with the ketohexoses than with 
 the aldohexoses (v. Ekenstein and Blanksma, page 216). To a few cubic 
 centimeters of fuming hydrochloric acid add an equal volume of water and a small 
 quantity of the sugar solution or of the solid substance and a few crystals of 
 resorcinol, and apply heat. The liquid becomes a beautiful red, and gradually 
 a substance precipitates which is red in color and soluble in alcohol. According 
 to Ofnek 1 the mixture must not contain more than 12 per cent HC1, and the 
 boiling must not be continued longer than twenty seconds, if it is boiled for 
 a longer time and with more hydrochloric acid this reaction is also given with 
 the aldoses. R. and 0. Adler 2 perform the test with glacial acetic acid and a 
 drop of hydrochloric acid and some resorcinol, in which case a reaction with 
 aldoses is not obtained. Seliwanoff' s reaction, according to Rosin, 3 may 
 be made more delicate by a combination with the spectroscopic examination. 
 In regard to its use in urine examinations see Chapter XIV. 
 
 The naphtho-resorcinol reaction as suggested by B. Tollens and Rorive 4 
 can be carried out as follows: A few particles of the sugar and about the same 
 quantity of naphthoresorcinol are treated with about 10 cc. of a mixture of equal 
 volumes of water and concentrated hydrochloric acid of sp. gr. 1.19. This is 
 slowly heated to boiling over a low flame, and is continued for 1-3 minutes. 
 The fluid becomes more purple or violet than with Seliwanoff's resorcin test. 
 The spectroscopic examination shows a faint band in the green. 
 
 According to Neuberg, 5 methylphenlhydrazine is an excellent substance 
 to use for the separation and detection of fructose, as it gives a characteristic 
 fructose methylphenylosazone. This osazone when recrystallized from alcohol 
 melts at 153°. It shows a dextrorotation of 1° 40' when 0.2 gram of the osazone 
 is dissolved in 4 cc. pyridine and 6 cc. absolute alcohol. 
 
 Ofner has made objections to the use of methylphenylhydrazine in the detec- 
 tion of fructose. He has obtained the osazone from glucose and methylphenylhy- 
 drazine, although the osazone is formed much more quickly with fructose than 
 with glucose. Only when the separation of the osazone crystals with methyl- 
 phen} r lhydrazine after the addition of acetic acid takes place within five hours 
 at ordinary temperatures is the presence of fructose positively proven (Ofner 6 ). 
 
 The use of secondary asymmetric hydrazines as a general reagent for ketoses 
 and as a means of separation from aldoses is objected to by Ofner. 
 
 (/-Sorbinose (sorbin) is a ketose obtained from the juice of the berry of the 
 mountain ash under certain conditions. It is crystalline and levogyrate, and 
 is converted into c/-sorbite by reduction. 
 
 1 Monatshefte f. Chem., 25. 
 
 2 See footnote 6, p. 209. 
 
 3 Ber. d. d. chem. Gesellsch., 38. 
 
 * Ibid., 41, p. 1783 and Tollens, ibid., 41, p. 1788. See also Mandel and Neuberg, 
 Biochem. Zeitschr., 13. 
 
 1 Ibid., 35; also Neuberg and Strauss, ibid., 36. 
 6 Ibid., 37, and Zeitschr. f. physiol. Chem., 45. 
 
GLUCOSAMINE. 219 
 
 Appendix to the Monosaccharides, 
 a. Amino-sugars. 
 
 The most important amino-sugar is the already mentioned glucosamine. 
 
 CH 2 OH 
 
 (/-Glucosamine (chitosamine), C6H13NO5, = /,„ " TTJ , whose synthet- 
 
 Cn.1N.ri2 
 
 COH 
 ical preparation has been given on page 201 was first prepared by 
 Ledderhose J from chitin by the action of concentrated hydrochloric 
 acid. Recently it has been obtained as a cleavage product of several 
 mucin substances and proteins (see pages 84 and 168). Glucosamine is, 
 as E. Fischer and Leuchs 2 have shown, a derivative of glucose or of 
 d-mannose (probably glucose), and is an a-amino-sugar. 
 
 The free base, which can crystallize in needles, is readily soluble in 
 water giving an alkaline reaction, and quickly decomposes. The charac- 
 teristic hydrochloride forms colorless crystals which are stable in the air 
 and readily soluble in water, soluble with difficulty in alcohol, and insoluble 
 in ether. The solution is dextrorotatory, (a) D = +70.15° to 74.64°, at vari- 
 ous concentrations. 3 Glucosamine has a reducing action similar to that of 
 glucose, and gives the same osazone, but is not fermentable. With 
 benzoyl-chloride and caustic soda it gives a crystalline ester. In alkaline 
 solution it gives with phenylisocyanate a compound which can be con- 
 verted into its anhydride by acetic acid, and is used in the separation and 
 detection of glucosamine (Steudel). 4 On oxidation with nitric acid it 
 yields norisosaccharic acid, whose lead salt can be separated, and whose 
 salts with cinchonine or quinine are soluble with difficulty in water and can 
 also be used very successfully in the detection of glucosamine (Neuberg and 
 Wolff 5 ). On oxidation with bromine, chitaminic acid (rf-glucosaminic 
 acid) is produced, and this is converted into chitaric acid, CgHioOg, 
 by nitrous acid. On treatment with nitrous acid glucosamine yields 
 a non-fermentable sugar called chitose. 
 
 Ehrlich 6 has suggested a test which does not respond with the free glucos- 
 amine, but with the mucins and other protein bodies containing an acetylated 
 glucosamine. It consists in warming the substance, which has been previously 
 
 1 Zeitschr. f. physiol. Chem., 2 and 4. 
 
 2 Ber. d. d. chem. Gesellsch., 36. 
 
 3 See Hoppe-Seyler-Thierfelder's Handbuch, 8, Aufl.; Sundvik, Zeitschr. f. physiol 
 Chem., 34. 
 
 * Zeitschr. f. physiol. Chem., 34. 
 6 Ber. d. d. chem. Gesellsch., 34. 
 p Mediz. Woche, 1901, No. 15; see Langstein, Ergebnisse der Physiol., I, Abt. 1, 88. 
 
220 THE CAKBOHYDRATES. 
 
 treated with alkali, with a hydrochloric-acid solution of dimethylaminobenzalde- 
 hyde, when a beautiful red color is obtained. 
 
 Glucosamine is best prepared from decalcified lobster-shells by treat- 
 ing with hot concentrated hydrochloric acid. 1 In regard to its prepara- 
 tion from protein substances we must refer to the works cited on page 
 84, footnote 5. 
 
 Albamine (diglucosamine), (CeHnC^N^+H^O, is the name given by S. Fran- 
 kel - to a body which he isolated from the products of the hydrolysis of ovalbumin 
 with baryta, as well as in its digestion. Albamine is amorphous, dextrogyrate, 
 and reduces after boiling with acids. As hydrolytic cleavage product it yields 
 ^-glucosamine. 
 
 Galactosamine is claimed to have been found by Schulz and Ditthorn in 
 a glycoprotein of the spawn of the frog. This claim is not generally accepted, 
 v. Ekexstein and Blanksma 3 obtained galactose on the hydrolysis of the slimy 
 envelope of frog eggs. 
 
 According to Offer, 4 pentosamine occurs in the liver of the horse. Accord- 
 ing to Offer, the pentose derivative, which he calls dipentosamine (CsHyOs.NH^o-l- 
 H 2 and a second, perhaps a diacetyl-pentosamine 2(CH 3 CO)CioHi8N207 (?), 
 also occur in the liver. The first gives pentose reactions and reduces Fehling's 
 solution after boiling with acid. The only amino-sugar positively detected in 
 the animal organs is glucosamine. 
 
 The amino-sugars, as intermediary bodies between the carbohydrates 
 and oxyamino-acids, are of great physiological interest, and this interest 
 has become still more important since Netjberg was first able to pre- 
 pare the corresponding amino-aldehyde from glycocoll and then also 
 from other amino-acids. From the ethyl ester of glycocoll in acid solu- 
 tion Neuberg 5 obtained the amino-acetaldehyde. NH2.CH2.CHO, by 
 treatment with sodium amalgam. This aldehyde is very unstable and 
 has a tendency to condensation with ring formation, and Neuberg 
 obtained therefrom by oxidation with corrosive sublimate and caustic 
 soda, pyrazine according to the equation: 
 
 NH 2 
 
 
 1 
 
 N 
 
 CH2+CHO 
 
 y\ 
 
 1 1 
 
 HC CH 
 
 CHO CH 2 4-0 = 
 
 = | || +3H 2 
 
 1 
 
 HC CH 
 
 NH 2 
 
 \/ 
 
 
 N 
 
 1 See Hoppe-Seyler-Thierfelder's Handbuch, 8. Aufl. 
 
 2 Monatsh. f. Chem., 19. 
 
 -'•hulz and Ditthorn, Zeitsohr. f. physiol. Chem., 29; v. Ekenstein and Blanksma, 
 Chem. Centralis, 1907, 2, p. 1001. 
 4 Hofmeister's Beitrage, 8. 
 6 Ber. d. d. chem. Gesellsch., 41. 
 
GLUCURONIC ACIDS. 221 
 
 On account of this tendency to ring-formation the amino-acetalde- 
 hyde as well as the amino-aldehydes as a group, stand, according to Neu- 
 berg, in close relationship to many ring systems, such* as imidazole, 
 piperazine, pyrazine, pyridine and others, and also to the alkaloids. 
 
 The amino-sugars, like the amino-aldehydes, can also unite, form- 
 ing ring compounds, and this seems to be the case on the decomposi- 
 tion of free glucosamine in aqueous solution, which occurs with access 
 cf air (Lobry de Bruyn). As found by Stolte l 2,5-ditetraoxybutyl 
 pyrazine (= fructosazine) is hereby produced according to the following 
 equation: 
 
 NH 2 N 
 
 / y\ 
 
 O.1H9C4.CH+CHO O4H9C4.C CH 
 
 +0= I || +3H 2 
 
 CHO CH.C4H9O4 HC C.C4H9O4 
 
 / V 
 
 NH 2 N 
 
 Fructosazine 
 
 The 2,5-ditetraoxybutyl pyrazine, which Stolte obtained by Lobry 
 de Bruyn's 2 method from fructose in methyl alcohol solution and 
 ammonia, and which he calls fructosazine, can be oxidized outside of the 
 body into 2,5-pyrazine dicarboxylic acid. 
 
 The same acid can be formed in the animal body (rabbits), although not 
 constantly, after introducing fructosazine. It also passes into the urine of 
 rabbits after intravenous injection of d-fructose and glycocoll (Spiro), a behavior 
 which Spiro claims indicates that carbohydrates in metabolism react with the 
 cleavage products of proteins. Stolte's experiments to decide the question 
 whether in the animal body the glucosamine in its decomposition passes into 
 fructosazine did not at first yield conclusive results. His more recent investiga- 
 tions 3 show on the contrary that in rabbits 2-oxymethylpyrazine-5-carboxylic 
 acid is formed as an oxidation product, and this can be oxidized outside of the 
 body into pyrazine-2, 5-dicarboxyIic acid. 
 
 b. Glucuronic Acids. 
 
 The glucuronic acids occurring in the animal body either physiolog- 
 ically or pathologically, are conjugated acids which will be described in 
 detail in a subsequent chapter (XIV). We will here describe only the 
 d-glucuronic acid in connection with the carbohydrates. 
 
 CHO 
 d-Glucuronic acid (glycuronic acid), C6Hio07 = (CH.OH) 4 , is a deriva- 
 
 COOH 
 tive of glucose, and has been synthetically prepared by E. Fischer and 
 
 1 Hofmeister's Beitriige, 11. 
 
 2 Cited by Stolte, Hofmeister's Beitrage, 11. 
 
 3 Spiro, Hofmeister's Beitriige, 10, p. 283; Stolte, Biochem. Zeitschr., 12. 
 
222 THE CARBOHYDRATES. 
 
 Piloty l by the reduction cf the lactone of saccharic acid. On oxidation 
 with bromine it forms saccharic acid, and on reduction it yields gulonic- 
 acid lactone. -Salkowski and Neuberg 2 have obtained i-xylcse from 
 glucuronic acid by splitting off CO2 by means of putrefaction bacteria. 
 
 Glucuronic acid has not been found in the free state in the animal 
 body. It occurs to a slight extent in normal urine as a conjugated acid 
 (Mayer and Neuberg). It occurs to a much greater extent in urine as 
 conjugated acid after the ingestion of certain aromatic and also aliphatic 
 substances, especially camphor and chloral hydrate. It was obtained 
 first by Schmiedeberg and Meyer from camphoglucuronic acid, and 
 then by v. Mering 3 from urochloralic acid by cleavage with dilute acids. 
 According to P. Mayer, 4 on the oxidation of glucose a partial forma- 
 tion of glucuronic acid and oxalic acid takes place, and therefore, according 
 to him, an increased elimination of conjugated glucuronic acids shows 
 in certain cases an incomplete oxidation of glucose. Conjugated glucu- 
 ronic acids may also occur in the blood (P. Mayer, Lepine and Boulud 5 ), 
 in the feces, and in the bile. 6 Neuberg and Neumann 7 have prepared 
 certain conjugated glucuronic acids (see Chapter XIV) synthetically, 
 among them being euxanthic acid. The most abundant source of glucu- 
 ronic acid is the artist's pigment " Jaune indien," which contains the 
 magnesium salt cf euxanthic acid (euxanthon-glucuronic acid). 
 
 Glucuronic acid is not crystalline, but is only obtainable as a syrup. 
 It dissolves in alcohol and is readily soluble in water. If the aqueous 
 solution is boiled for an hour the acid is partly (20 per cent) converted 
 into the crystalline lactone, glucurone, CaHsOo, which is soluble in water 
 and insoluble in alcohol, and which has a melting-point of 175-178° C. 
 The alkali salts of the acid are crystalline. If a concentrated solution 
 of the acid is saturated with barium hydroxide the basic barium salt is 
 obtained as a precipitate. The neutral lead salt is soluble in water, 
 while the basic salt is insoluble. The readily crystallizable cinchonine 
 salt can be used in isolating glucuronic acid (Neuberg 8 ). Glucuronic 
 acid is dextrorotatory, while the conjugated acids are levorotatory; 
 they behave like glucose with the reduction tests, and do not ferment 
 
 1 Ber. d. d. chem. Gesellsch., 24. 
 
 J Zeitschr. f. physiol. Chem., 36. 
 
 3 Mayer and Neuberg, Zeitschr. f. physiol. Chem., 29; Schmiedeberg u. Meyer, 
 ibid. ,3; v. Mering, ibid, 6. 
 
 * Zeitschr. f. klin. Med., 47. See Chapter XIV. 
 
 5 Mayer, Zeitschr. f. physiol. Chem., 32; Lepine and Boulud, Compt. rend.., 133, 
 134, 138. 
 
 8 See Bial, Hofmeister's Beitrage, 2, and v. Leersum, ibid., 3. 
 
 7 Zeitschr. f. physiol. Chem., 44. 
 
 8 Ber. d. d. chem. Gesellsch., 33. 
 
DISACCHAKIDES. 223 
 
 with yeast. With the phcnylhydrazine test it gives crystalline emu- 
 pounds which are not sufficiently characteristic (Thierfelder, P. 
 Mayer l ). By the action of 3 mol. phenylhydrazine and the necessary 
 amount of acetic acid upon 1 mol. glucuronic acid at 40° for a few days, 
 Neuber<; and Neimann obtained the glucuronic-acid osazone, which 
 was very similar to glucosazone and melted at 200-205°. With p-brom- 
 phenylhydrazine hydrochloride and sodium acetate, glucuronic acid gives 
 p-bromphenylhydrazine glucuronate, which is characterized by its insolu- 
 bility in absolute alcohol and by a very prominent levorotatory action. 
 This compound is very well suited for the detection of glucuronic acid. 2 
 Dissolved in a mixture of alcohol and pyridine (0.2 gram substance in 4 cc. 
 pyridine and 6 cc. alcohol) the rotation is 7° 25', which corresponds to 
 (a)o=— 309°. On distillation with hydrochloric acid, glucuronic acid 
 yields furfurol and also carbon dioxide, and on this behavior Tollens 
 and Lefevre 3 have based their quantitative method for the estimation 
 of glucuronic acid. 
 
 They give the pentose reactions with phloroglucin or orcin and hydrochloric 
 acid, and also a good reaction with naphthoresorcinol and hydrochloric acid 
 (see page 218). The product produced herewith is soluble in ether with a blue, 
 bluish-violet or reddish-violet color, and the solution shows an absorption band 
 somewhat to the right and on the D-line. According to Maxdel and Xeuberg 4 
 this reaction is not characteristic of glucuronic acid, as many aldehyde and ketone 
 acids give the same reaction; still, it is important in the differentiation of the 
 pentoses. 
 
 Glucuronic acid is best prepared from euxanthic acid, which decom- 
 poses on heating it with water to 120° C. for several hours. The nitrate 
 from the euxanthon is concentrated at 40° C, when the anhydride 
 gradually crystallizes out. On boiling the mother-liquor for some time 
 and evaporating further, the crystals of the lactone are obtained. In 
 regard to the quantitative estimation of glucuronic acid we must refer 
 the reader to the works of Tollens and his collaborators and of Neuberg 
 and Neimann. 5 
 
 2. Disaccharides. 
 
 Some of the varieties of sugar belonging to this group occur ready 
 formed in nature. Thus we have saccharose and lactose. Some, on the 
 contrary, such as maltose and isomaltose, are produced by the partial 
 
 1 Thierfelder, Zeitschr. f. physiol. Chem., 11, 13, 15; P. Mayer, ibid., 29. 
 
 2 See Neuberg, Ber d. d. chem. Gesellsch., 32; and Mayer and Neuberg, Zeitschr. 
 f. physiol. Chem., 29. 
 
 3 Ber. d. d. chem. Gesellsch., 40. 
 
 4 Bioch. Zeitschr. 13. 
 
 6 Tollens, Zeitschr. f. physiol. Chem., 41, which cites also the older work; Neuberg 
 and Neimann, ibid., 44; Neuberg, ibid., 45. 
 
224 THE CARBOHYDRATES. 
 
 hydrolytic cleavage of complex carbohydrates. Isomaltose is also obtained 
 from glucose by reversion (see page 225). 
 
 The disaccharides or hexobioses are to be considered as glucosides, 
 each of which is derived from two monosaccharides with the exit of 1 
 molecule of water. Corresponding to this, their general formula is 
 C12H22O11. On hydrolytic cleavage and the addition of water they 
 yield 2 molecules of hexoses, either 2 molecules of the same hexose or 
 one each of two different hexoses. Thus 
 
 Saccharose -j- H2O = glucose + fructose ; 
 Maltose -f- H2O = glucose + glucose ; 
 Lactose +H2O = glucose + galactose. 
 
 The configuration of the disaccharides has not been positively determined. 
 
 The fructose turns the polarized ray more to the left than the glucose 
 does to the right; hence the mixture of hexoses obtained on the cleavage 
 of saccharose has an opposite rotation to the saccharose itself. On 
 this account the mixture is called invert-sugar, and the hydrolytic 
 splitting is designated as inversion. This term, "inversion," is not only 
 used for the splitting of saccharose, but is also used for the hydrolytic 
 cleavage of compound sugars into monosaccharides. The reverse reaction, 
 whereby monosaccharides are condensed into complex carbohydrates, is 
 called reversion. 
 
 We subdivide the disaccharides into two groups, first, the group to 
 which saccharose belongs, where the members do not have the property 
 of reducing certain metallic oxides; and the second group, to which the 
 two maltoses and lactose belong, the members acting like monosaccharides 
 in regard to the ordinary reduction tests. The members of the latter 
 group have the character of aldehyde alcohols, and in milk-sugar the 
 aldehyde characteristics are connected with the glucose fraction. 
 
 Saccharose, or cane-sugar, occurs extensively distributed in the 
 plant kingdom. It occurs to the greatest extent in the stalk of the sugar- 
 millet and sugar-cane, the roots of the sugar-beet, the trunks of certain 
 varieties of palms and maples, in carrots, etc. Cane-sugar is of extraor- 
 dinary great importance as a food and condiment. 
 
 Saccharose forms large, colorless monoclinic crystals. On heating it 
 melts in the neighborhood of 160° C, and on heating more strongly it 
 turns brown, forming so-called caramel. It dissolves very readily in 
 water, and according to Herzfeld, 1 100 parts of saturated saccharose 
 solution contain 67 parts of sugar at 20° C. It dissolves with difficulty 
 in strong alcohol. Cane-sugar is strongly dextrorotatory. The specific 
 rotation is only slightly modified by concentration, but is markedly 
 
 1 See Tollens' Handbuch der Kohlehydrate, 2. Aufl. 1, 154. 
 
MALTOSE. 225 
 
 changed by the presence of other inactive substances. The specific 
 rotation is (a) D = +66.5°. 
 
 Saccharose acts indifferently toward Moore's test and to the ordinary 
 reduction tests. On continuous boiling it may reduce an alkaline copper 
 solution, perhaps on account of a partial inversion. It does not ferment 
 directly, but only after inversion, which can be brought about by an 
 enzyme (invertin) contained in the yeast. An inversion of cane-sugar also 
 takes place in the intestinal canal. Cane-sugar does not combine with 
 hydrazines. Concentrated sulphuric acid blackens cane-sugar very 
 quickly even at the ordinary temperature, and anhydrous oxalic acid 
 does the same on warming on the water-bath. Various products are 
 obtained on the oxidation of cane-sugar, dependent upon the variety of 
 oxidizing agent and also upon the intensity of the action. Saccharic acid 
 and oxalic acid are the most important products. 
 
 The reader is referred to complete text-books on chemistry for' the 
 preparation and quantitative estimation of cane-sugar. 
 
 Maltose (malt-sugar) is formed in the hydrolytic cleavage of starch 
 by malt diastase, saliva, or pancreatic juice. It is obtained from glyco- 
 gen under the same conditions (see Chapter VII). Maltose is also pro- 
 duced transitorily in the action of sulphuric acid on starch. Maltose 
 forms the fermentable sugar of the potato or grain mash, and also of the 
 beerwort. 
 
 Maltose crystallizes with one molecule water of crystallization in fine 
 white needles. It is readily soluble in water, rather easily in alcohol, 
 but insoluble in ether. Its solutions are dextrorotatory; and the specific 
 rotation is variable, depending upon the concentration and temperature, 
 but is considerably stronger than glucose, 1 and is generally given as 
 (o0d = +137 to 138°. Maltose ferments readily and completely with 
 yeast, and acts like glucose in regard to the reduction tests. It yields 
 phenylmaltosazone on warming with phenylhydrazine for 1^ hours. 
 This phenylmaltosazone melts at 205° C, and is more soluble in hot 
 water than the glucosazone. Maltose differs from glucose chiefly in the 
 following: It does not dissolve as readily in alcohol, has a stronger dex- 
 trorotatory power, and has a feebler reducing action on Fehling's solu- 
 tion; 10 cc. Fehling's solution are, according to Soxhlet, 2 reduced 
 by 77.8 milligrams anhydrous maltose in approximately 1 per cent solution. 
 
 Isomaltose. This variety of sugar, as has been shown by Fischer, 3 
 is produced, as are dextrin-like products, by reversion, and by the action 
 of fuming hydrochloric acid on glucose. A re-formation of isomaltose 
 
 1 See Hoppe-Seyler-Thierfelder's Handbuch, S. Aufl. 
 
 2 Cited from Tollens' Handbuoh. der Kohlehydrate, 2. Aufl. 1, 154. 
 5 Ber. d. deutsch. ehem. Gesellsch., 23 and 28. 
 
226 THE CARBOHYDRATES. 
 
 and other sugars from glucose can also Le Lrought about by means of 
 yeast maltase (Hill and Emmerling, see page 58). It is also formed, 
 besides ordinary maltose, in the action of diastase on starch paste, and 
 occurs in beer and in commercial starch-sugar. It is produced, with 
 maltose, by the action of saliva or pancreatic juice (Kulz and Vogel) 
 or blood-serum (Rohmann *) on starch. The formation of isomaltose 
 in the hydrolysis of starch has been denied by many investigators because 
 they considered isomaltose only as contaminated maltose. 2 
 
 Isomaltose dissolves very readily in water, has a pronounced sweetish 
 taste, and does not ferment, or, according to some, only very slowly. 
 It is dextrorotatory, and has very nearly the same power of rotation as 
 maltose. Isomaltose is characterized by its osazone. This forms fine 
 yellow needles, which begin to form drops at 140° C. and melt at 150- 
 153° C. These are rather easily soluble in hot water and dissolve in hot 
 absolute alcohol much more readily than the maltosazone. Isomaltose 
 reduces copper as well as bismuth solutions. 
 
 Lactose (milk-sugar). As this sugar occurs exclusively in the animal 
 world, in the milk of human beings and animals, it will be treated in a 
 following chapter (on milk). 
 
 3. Colloid Polysaccharides. 
 
 If we exclude the not well known trisaccharides and the tetrasaccharide 
 stachyose this group includes a great number of very complex carbo- 
 hydrates which occur only in the amorphous condition, or at least not 
 as crystals in the ordinary sense. Unlike the bodies belonging to the 
 other groups, these have no sweet taste. Some are soluble in water, 
 while others swell up therein, especially in warm water, and finally 
 some are neither dissolved nor visibly changed. Polysaccharides are 
 ultimately converted into monosaccharides by hydrolytic cleavage. 
 
 The polysaccharides are ordinarily divided into the following groups: 
 starches with the dextrins, plant gums and mucilages, and the celluloses. 
 
 Starch Group. 
 
 Starch, amylum (CeHic-C^x. This substance occurs in the plant 
 kingdom very extensively distributed in the different parts of the plant, 
 especially as reserve food in the seed, roots, tubers, and trunks. 
 
 Starch is a white, odorless, and tasteless powder, consisting of small 
 
 1 Kulz and Vogel, Zeitschr. f. Biologie, 31; Rohmann, Centralbl. f. d. med. Wis- 
 eh., 1893, 849. 
 
 2 Brown and Morris, Journ. of Chera. Soc, 1895; Chem. News, 72. See also Ost 
 Ulrich, and Jalowetz, Ref. in Ber. d. deutsch. chem. Gesellsch., 28; Ling and Baker, 
 Journ. of Chem. Soc, 1895; Pottevin, Chem. Centralbl., 1899, II, 1023. 
 
STARCH. 227 
 
 granules which have ;i stratified structure and different shape and size 
 in different plants. Starch is considered insoluble in cold water. The 
 grains swell up in warm water and hurst, yielding a paste. 
 
 According to the ordinary opinion the starch granules consist of two 
 different substances, starch granulose and starch cellulose (v. 
 Nageli), the first of which turns blue with iodine and forms the chief 
 part of the granule. According to Maquenne and Roux 1 this is 
 not the fact. According to them the starch granule consists of two 
 constituents, of which the .first, amylase, forms the chief mass (80-85 
 per cent) and the other, amijlopectin, forms only 15-20 per cent of the 
 granule. Amylopectin is not identical with v. Nageli's starch cellulose, 
 and the above investigators consider starch cellulose as only an insoluble 
 form of amylase. The amy lose can occur in two forms; one, which is 
 soluble, is colored blue by iodine and is immediately transformed into 
 sugar by malt, the other is a solid substance, which is not colored 
 with iodine and resists the action of malt infusion. One modification 
 can be transformed into the other. 
 
 In the paste, besides amylopectin, we also have soluble amylose, and 
 this can, by a process called retrogradation by Maquenne and Roux, be 
 transformed into the solid modification, " artificial starch." This 
 solid form occurs in the starch granule, and is identical with v. Nageli's 
 starch cellulose. As the starch granules are directly colored blue by 
 iodine they must, besides this, also contain soluble amylose. If the author 
 understands the above investigators correctly the starch granules con- 
 tain three constituents, namely, soluble amylose, which is colored blue 
 by iodine ( = starch granulose), insoluble amylose, which is not colored 
 by iodine ( = starch cellulose), and amylopectin. 
 
 In the formation of paste the amount of amylose is not the essential 
 but rather the quantity of amylopectin. The amylopectin is a slime- 
 like substance, insoluble in boiling water and dilute alkalies, only becom- 
 ing pasty therein, and not colored blue by iodine. Accordingly the 
 paste is a solution cf amylose made thick by amylopectin. The amylo- 
 pectin, unlike the amylose, is only slowly transformed into sugar with 
 dextrin formation. Starch is insoluble in alcohol and ether. On heat- 
 ing starch with water alone, or heating with glycerin to 190° C, or on 
 treating the starch grains with 6 parts dilute hydrochloric acid of sp. gr. 
 1.06 at ordinary temperature for six to eight weeks, 2 it is converted into 
 soluble starch (amylodextrin, amidulin). Soluble starch is also 
 
 *v. Nageli, Botan. Mitteil., 1863; Maquenne and Roux, Compt. rend., 138, 140, 
 142, 146, and Bull. Soc. chim. de Paris (3), 33 and 35. 
 
 2 See Tollens' Handb., 191. In regard to other methods, see Wr6blewsky, Ber. d. 
 deutsch. chem., Gesellsch., 30; Syniewski, ibid. 
 
228 THE CARBOHYDRATES. 
 
 formed as an intermediate step in the conversion of starch into sugar 
 by dilute acids or diastatic enzymes. Soluble starch may be precipitated 
 from very dilute solutions by baryta-water. 1 
 
 Starch granules swell up and form a pasty mass in caustic potash or 
 soda. This mass gives neither Moore's nor Trommer's test. Starch 
 paste does not ferment with yeast. The most characteristic test for starch 
 is the blue coloration produced by iodine in the presence of hydriodic 
 acid or alkali iodides. 2 This blue coloration disappears on the addition of 
 alcohol or alkalies, and also on warming, but reappears again on cooling. 
 
 On boiling with dilute acids starch is converted into glucose. In 
 the conversion by means of diastatic enzymes we have, as a rule, besides 
 dextrin, maltose, and isomaltose, onl}- very little glucose. We are 
 considerably in the dark as to the kind and number of intermediate 
 products produced in this process (see Dextrins) . 
 
 Starch may be detected by means of the microscope and by the 
 iodine reaction. Starch is quantitatively estimated, according to Sachsse's 
 method, 3 by converting it into glucose by hydrochloric acid and then 
 determining the glucose by the ordinary methods. 
 
 Inulin (C6Hio05)x+H20, occurs in the underground parts of many 
 Composite, especially in the roots of the Inula helenium, the tubers 
 of the Dahlia, the varieties of Helianthus, etc. It is ordinarily obtained 
 from the tubers of the Dahlia. 
 
 Inulin forms a white powder similar to starch, consisting of spheroid 
 cr} r stals which are readily soluble in warm water without forming a paste. 
 It separates slowly on cooling, but more rapidly on freezing. Its solu- 
 tions are levogyrate and are precipitated by alcohol, and are colored 
 only yellow with iodine. Inulin is converted into the levogyrate mono- 
 saccharide d-fructose on boiling with dilute sulphuric acid. Diastatic 
 enzymes of higher animals have no, or only a ver} r slight, action on inulin. 4 
 
 According to Dean 5 inulin occurs in combination with other substances, 
 levulins, which are more soluble and have less rotation. He suggests that we 
 limit the name inulin to that carbohydrate (or mixture of carbohydrates), which 
 is readily precipitable by 60 per cent alcohol and shows a specific rotation of 
 («) D =-3Sto40°. 
 
 Lichenin (moss-starch) occurs in many lichens, especially in Iceland moss. 
 It is not soluble in cold water, but swells up into a jelly. It is soluble in hot 
 water, forming a jelly on allowing the concentrated solution to cool. It is colored 
 yellow by iodine and yields glucose on boiling with dilute acids. Lichenin is 
 not changed by diastatic enzymes such as ptyalin or amylopsin (Nilson 6 ). 
 
 1 In regard to the compounds of soluble starch and dextrins with barium hydroxide, 
 Bee Biilow, Pfliiger's Arch., 62. 
 
 2 See Mylius, Ber. d. deutsch. chem. Gesellsch., 20, and Zeitsch. f. physioL Chem., 11. 
 
 3 Tollens' Handb., 2. Aufl., 1, 187. 
 * To] lens' Handbuch, 208. 
 
 6 Amer. Chem. Journ., 32. 
 c Upsala Lakaref. Forh., 28. 
 
DEXTRINS. 229 
 
 Glycogen. This carbohydrate, which stands to a certain extent 
 between starch and dextrin, is principally found in the animal kingdom, 
 hence it will be considered in a subsequent chapter (on the liver). 
 
 Dextrins and Gums. 
 
 The dextrins stand in close relation to the starches, and are formed 
 therefrom as intermediate products by the action of acids or diastatic 
 enzymes. They yield as final products only hexoses, indeed only glu- 
 cose, on complete hydrolysis. The vegetable gums, the vegetable 
 mucilages and the pectin bodies, which all stand close to the hemicellu- 
 loses, yield, on the contrary, abundance of pentose and, among the hex- 
 oses. galactose is very often found. 
 
 Dextrin (starch-gum, British gum), is produced on heating starch to 
 200-210° C., or by heating starch, which has previously been moistened 
 with water containing a little nitric acid, to 100-110° C. Dextrins are 
 also produced by the action of dilute acids and diastatic enzymes on 
 starch. There have been numerous investigations as to the steps 
 involved in the last-mentioned process, but they have led to conflicting 
 views. One of these, which used to be generally accepted, is as follows: 
 The first product, which gives a blue color with iodine, is soluble starch 
 or amylodextrin, which on further hydrolytic cleavage yields sugar and 
 erythrodextrin, which is colored red by iodine. On further cleavage of 
 this erythrodextrin more sugar and a dextrin, achroodextrin, which is 
 not colored by iodine, is formed. From this achroodextrin after suc- 
 cessive splittings Ave have sugar and dextrins of lower molecular weights 
 formed, until finally we have sugar and a dextrin, maltodextrin, which 
 refuses to split further, as final products. The views are rather contra- 
 dictory in regard to the number of dextrins which occur as intermediate 
 steps. The sugar formed is maltose (or in first place isomaltose), and 
 only very little glucose is produced. Another view is that first several 
 dextrins are formed consecutively in the successive splittings, by hydra- 
 tion, and then finally the sugar is formed by the splitting of the last 
 dextrin. According to Moreau, in the first stages of saccharification 
 amylodextrin, erythrodextrin, achroodextrin and sugar are formed sim- 
 ultaneously. Other investigators, especially Syniewski, have recently 
 suggested other views on the subject. 1 
 
 This question has taken another direction by the investigations c£ 
 
 1 In regard to the various views on the theories of the saccharification of starch, 
 see Musculus and Gruber, Zeitschr. f. physiol. Chem., 2; Lintner and Dull, Ber. d. d. 
 chem. Gesellsch., 26 and 28; Brown and Heron, Journ. of Chem. Soc, 1S79; Brown 
 and Morris, ibid., 1885 and 1S89; Moreau, Biochem. Centralbl., 3, 648; Syniewski, 
 Annal. d. Chem. u. Pliarm., 309, and Chem. Centralbl., 1902, 2. 
 
230 THE CARBOHYDRATES. 
 
 Maquenne, mentioned above. According to him the amylose passes 
 directly into maltose without the formation of dextrin by the action of 
 malt infusion. The dextrins produced are only formed from the amylo- 
 pectin, which does not undergo saccharification with freshly prepared 
 malt infusions, but only with older or especially active infusions. This 
 also explains why in the older investigations the saccharification was 
 only about 80 per cent while Maquenne has been able to completely 
 convert the starch into sugar by enzymotic action. 
 
 The various dextrins are very hard to isolate as chemical individuals 
 and to separate from each other. Young i has tried their separation 
 by means of neutral salts, especially ammonium sulphate, and Moreau 
 by the aid of a baryta-alcohol method. We cannot enter into the dif- 
 ferences as to the dextrins so separated, and only the characteristic 
 properties and reactions will be given for the dextrins in general. 
 
 The dextrins appear as amorphous, white or yellowish-white powders 
 which are readily soluble in water. Their concentrated solutions are 
 viscid and sticky, like gum solutions. The dextrins are dextrogyrate. 
 They are insoluble or nearly so in alcohol, and insoluble in ether. Watery 
 solutions of dextrins are not precipitated by basic lead acetate. Dex- 
 trins dissolve cupric hydroxide in alkaline liquids, forming a beautiful 
 blue solution, which, as is generally admitted, is reduced by pure dex- 
 trins. According to Moreau pure dextrin has no reducing action. The 
 dextrins are not directly fermentable. 
 
 Schardinger has discovered a bacillus which forms acetone from 
 starch and which is especially useful for the perparation of crystalline 
 cleavage products from starch. He obtained two crystalline substances, 
 dextrin a and f3, which are not fermentable by yeast and on hydrolysis 
 with acid yield glucose. For the a-dextrin Pringsheim and Langhans 
 have determined the formula (CeHinOs^ while Biltz and Truthe 2 
 found the formula (CgHioCs^g for the /3-dextrin. 
 
 The vegetable gums are soluble in water, forming solutions which are viscid 
 but may be filtered. We designate, on the contrary, as vegetable mucilages 
 those varieties of gum which do not or only partly dissolve in water, and which 
 swell up therein to a greater or less extent. The natural varieties of gum and 
 mucilage, to which belong several generally known and important substances, 
 such as gum arabic, wood-gum, cherry-gum, salep, and quince mucilage, and 
 probably also the little-studied pectin substances, will not be treated in detail, 
 because of their unimportance from a physiological standpoint. 
 
 1 Journ. of Physiol., 22, which contains the older researches of Nasse, Kriiger 
 Neumeister, Pohl, and Halliburton. Moreau, 1. c. 
 
 - Schardinger, Centralbl. f. Bak. u. Parasitenkunde, II, 22, 98 (1909); 29, 118 (1911); 
 Pringsheim and Langhans, Ber. d. d. Chem. Gesellsch., 45, 2533 (1912); Biltz and 
 Truthe, ibid., 46, 1377 (1913). 
 
CELLULOSE. 231 
 
 The Cellulose Group (C 6 Hi O 5 )x. 
 
 Cellulose is that carbohydrate, or perhaps more correctly, mixture 
 of carbohydrates, which forms the chief constituent of the walls of the 
 plant-cells. This is true for at least the walls of the young cells, while 
 in the walls of the older cells the cellulose is extensively incrusted with a 
 substance called lignin, and with many other cellulose derivatives and 
 compounds. 
 
 The true celluloses are characterized by their great insolubility. They 
 are insoluble in cold or hot water, alcohol, ether, dilute acids, and alkalies. 
 We have only one specific solvent for cellulose, and that is, an ammo- 
 niacal solution of copper oxide called Schweitzer's reagent. The 
 cellulose may be precipitated from this solvent by the addition of acids, 
 and obtained as an amorphous powder after washing with water. 
 
 Cellulose is converted into a substance, so-called amyloid, which 
 gives a blue coloration with iodine, by the action of concentrated sul- 
 phuric acid. With oxidizing agents (nitric acid, etc.) oxycelluloses are 
 produced. By the action of strong nitric acid or a mixture of nitric 
 acid and concentrated sulphuric acid, celluloses are converted into nitric- 
 acid esters or nitrocelluloses, which are highly explosive and have found 
 great practical use. 
 
 The ordinary celluloses when treated at the ordinary temperature 
 with strong sulphuric acid and then boiled for some time after diluting 
 with water are coverted into glucose. In this case it must be observed, 
 according to Maqtjenne, that it is not maltose that is produced as an 
 intermediate step, but another disaccharide, called cellose or cellobiose. 
 
 The cellulose, at least in part, undergoes decomposition in the intestinal 
 tract of man and animals. A closer discussion of the nutritive value 
 of cellulose will be given in a future chapter (on digestion). The great 
 importance of the carbohydrates in the animal economy and to animal 
 metabolism will also be given in the following chapters. 
 
 Hemicelluloses are, according to E. Schulze, 1 thoseconstituents of the cell- 
 wall related to cellulose which differ from the ordinary cellulose by dissolving 
 on heating with strongly diluted mineral acids, such as 1.25 per cent sulphuric 
 acid, and of yielding arabinose, xylose, galactose, and mannose instead of glucose. 
 Those hemicelluloses which serve partly as reserve food and partly as support- 
 substance, are very widely distributed in the plant kingdom. It must be recalled 
 that according to Bierry and Giaja 2 the digestive organs of different inverte- 
 brates (Helix, Astacus, Maja. Hommarus) contain enzymes which have an 
 energetic splitting action upon such polysaccharides as well as on the natural 
 celluloses. 
 
 1 E. Schulze, Zeitschr. f. physiol. Chem., 16 and 19, with Castro, ibid., 36. 
 2 Bioch. Zeitschr., 40, 370 (1912). 
 
CHAPTER IV. 
 ANIMAL FATS AND PHOSPHATIDES. 
 
 1. Neutral Fats and Fatty Acids. 
 
 The fats form the third chief group of the organic food of man and 
 animals. They occur very widely distributed in the animal and plant 
 kingdoms. Fat occurs in all organs and tissues of the animal organism, 
 though the quantity may be so variable that a tabular exhibit of the 
 amount of fat in different organs is of little interest. The marrow con- 
 tains the largest quantity, having over 96 per cent. The three most 
 important deposits of fat in the animal organism are the intermuscular 
 connective tissue, the fatty tissue in the abdominal cavity, and the 
 subcutaneous connective tissues. In plants, the seeds and fruit and in 
 certain instances also the roots, are rich in fat. Fat also occurs deposited, 
 during the winter's rest, in the trunks of trees. 
 
 The fats consist almost entirely of so-called neutral fats, with only 
 very small quantities of fatty acids. The neutral fats are esters of the 
 triatomic alcohol, glycerin, with monobasic fatty acids. These esters 
 are triglycerides; that is, the hydrogen atoms of the three hydroxyl 
 groups of the glycerin are replaced by the fatty-acid radicals, and their 
 general formula is therefore, C3H5.O3.R3- The animal fats consist 
 chiefly 'of esters of the three fatty acids, stearic, palmitic, and oleic acids. 
 In certain fats, especially in milk-fat, glycerides of fatty acids such as 
 butyric, caproic, caprylic, and capric acids also occur in considerable 
 amounts. Besides the above-mentioned ordinary fatty acids, stearic, 
 palmitic, and oleic acids, we also find in human and animal fat, exclusive 
 of certain fatty acids only little studied, the following non-volatile fatty 
 acids, as glycerides, namely, lauric acid, C12H24O2, myristic acid, C14H28O2, 
 and arachidic acid, C20H40O2. Of the unsaturated fatty acids, besides 
 oleic acid, we probably also have in small quantities glycerides of acids 
 of the linolic acid series C„H 2a -402 and of the linolenic acid series, 
 C„H2 O -G-02. In this case the question can be raised whether or not 
 tin -c acids are not derived from the phosphatides mixed with the fats, 
 in the plant kingdom triglycerides of other fatty acids, such as lauric 
 acid, myristic acid, linoleic acid, erucic acid, etc., sometimes occur 
 
 232 
 
NEUTRAL FATS. 233 
 
 abundantly. Besides these, oxyacids and high molecular alcohols have 
 been found in many plant fats. The extent to which traces of I 
 oxyacids occur in the animal kingdom has not been thoroughly inves- 
 tigated, but the occurrence of monoxystearic acid seems to have been 
 proved. 1 The occurrence of high molecular alcohols, although ordinarily 
 only in small amounts, has on the contrary been positively shown in 
 animal fat. 
 
 The animal fats are of the greatest interest and consist of a mixture of 
 varying quantities of tristearin, tripalmitin, and triolein, having 
 an average elementary composition of C 76.5, H 12.0, and O 11.5 per 
 cent. It must be remarked that in animal fat (mutton and beef tallow) 
 as well as in plant fat (olive-oil) mixed triglycerides, such as dipalmityl- 
 olein, distearyl-palmitin and distearyl-olein, occur, and that these mixed 
 glycerides may also be prepared synthetically. 2 
 
 Fats from different species of animals, and even from different parts 
 of the same animal, have an essentially different consistency, depending 
 upon the relative amounts of the different individual fats present. In 
 solid fats — as tallow — tristearin and tripalmitin are in excess, while 
 the less solid fats are characterized by a greater abundance of triolein. 
 This last-mentioned fat is found in greater quantities proportionally 
 in cold-blooded animals, and this accounts for the fact that the fat of 
 these animals remains fluid at temperatures at which the fat of warm- 
 blooded animals solidifies. Human fat from different organs and tissues 
 contains, in full numbers, 67-85 per cent triolein. 3 The melting-point 
 of different fats depends upon the composition of the mixtures, and it 
 not only varies for fat from different tissues of the same animal, but also 
 for the fat from the same tissues in various kinds of animals. 4 
 
 Neutral fats are colorless or yellowish, and, when perfectly pure, 
 ordorless and tasteless. They are lighter than water, on which they 
 float when in a molten condition. They are insoluble in water, dissolve 
 in boiling alcohol, but separate on cooling — often in crystals. They are 
 easily soluble in ether, benzene, chloroform, carbon disulphide and petro- 
 leum ether. The fluid neutral fats give an emulsion when shaken with 
 a solution of gum or albumin. With water alone they give an emulsion 
 
 1 Erben, Zeitschr. f. physiol. Chem., 30; Bernert, Arch, f . exp. Path. u. Pharm., 40. 
 2 Guth, Zeitschr. f. Biologie, 44; W. Hansen, Arch. f. Hygiene, 42; Holde and 
 Stange, Ber. d. d. chem. Gesellsch., 34; Kreis and Hafner, ibid., 36. 
 
 3 See Knopfelmacher, "Untersuch. iiber das Fett im Sauglingsalter," etc., Jarhbuch 
 f. Kinderheilkunde (X. F.), 45, which also contains the older literature; Jaeckle, 
 Zeitschr. f. physiol. Chem., 36. 
 
 4 According to Gilkin (Ber. d. d. chem. Gesellsch., 41) the fat from bone-marrow 
 and also other fats of animal and plant origin contain iron, which cannot be removed 
 by water containing hydrochloric acid. 
 
234 ANIMAL FATS AND PHOSPHATIDES. 
 
 only after vigorous and prolonged shaking, but the emulsion is not per- 
 sistent. The presence of some soap causes a very fine and permanent 
 emulsion to form easily. Fat produces spots on paper which do not 
 disappear; it is not volatile; it boils at about 300° C. with partial decom- 
 position, and burns with a luminous and smoky flame. The fatty acids 
 have most of the above-mentioned properties in common with the neutral 
 fats, but differ from them in being soluble in alcohol-ether, in having 
 an acid reaction, and by not giving the acrolein test. The neutral fats 
 generate a strong irritating vapor of acrolein, due to the decomposition 
 of glycerin, C 3 H 5 (OH)3— 2H 2 = C 2 H 3 .CHO, when heated alone, or 
 more easily when heated with potassium bisulphate or with other dehy- 
 drating substances. 
 
 The neutral fats may be split by the addition of the constituents of 
 water according to the following equation: 
 
 C 3 H 5 (OR) 3 4-3H 2 = CVH 5 (OH) 3 4-3HOR. 
 
 This splitting may be produced by the pancreatic enzjine and other 
 enzymes occurring in the animal and vegetable kingdoms, 'for example, 
 the castor lipase. The reverse action, namely, the synthesis of fatty acid 
 esters, can be brought about by enzymes, such as pancreatic lipase (see 
 page 60). The cleavage of the neutral fats can also be accomplished 
 by superheated steam or by dilute acids. We most frequently decompose 
 the neutral fats by boiling them with not too concentrated caustic alkali, 
 or. still better (in biochemical researches), with an alcoholic potash solu- 
 tion or with sodium alcoholate. By this procedure, which is called sapon- 
 ification, the alkali salts of the fatty acids (soaps) are formed. If the 
 saponification is made with lead oxide, then lead plaster, the lead salt of 
 the fatty acids is produced. By saponification is to be understood not 
 only the cleavage of neutral fats by alkalies, but also the splitting of neutral 
 fats into fatty acids and glycerin in general. 
 
 On keeping fats for a long time in contact with air they undergo a 
 change, becoming yellow in color and acid in reaction, and they develop 
 an unpleasant odor and taste, becoming rancid. In this change a part 
 of the fat is split into fatty acids and glycerin, and then an oxidation 
 of the free fatty acids takes place, producing volatile bodies of an 
 unpleasant odor. 
 
 The three most important fats of the animal kingdom are stearin, 
 
 valmitin, and olein. 
 
 CH 2 .O.Ci 8 H 35 
 
 Stearin, or tristearin, CsyHnoOe^CH.O.CisHssO, occurs especially in 
 
 CH 2 .O.CisH 35 
 
 the solid varieties of tallow but also in the vegetable fats. Stearic acid, 
 
 C 18 H:jf;0 2 , is found in the free state in decomposed pus, in the expectora- 
 
PALM IT IN. 235 
 
 tions in gangrene of the lungs, and in cheesy tuberculous masses. It 
 occurs as lime soap in excrement and adipocere, and in this last product 
 also as an ammonium soap. It also exists as alkali soap in the blood, 
 bile, transudations and pus, and in the urine to a slight extent. 
 
 Stearin is the hardest and most insoluble of the three ordinary neutral 
 fats. It is nearly insoluble in cold alcohol, and soluble with great dif- 
 ficulty in cold ether (225 parts). It separates from warm alcohol on 
 cooling as rectangular, and less frequently as rhombic plates. The opinions 
 regarding the melting-point are somewhat varied. Pure stearin, ac- 
 cording to Heintz, 1 melts transitorily at 55° and permanently at 71.5°. 
 The stearin from the fatty tissues (not pure) melts at 63° C. 
 CH 3 
 
 Stearic acid, (CH 2 )i6, crystallizes (on cooling from boiling alcohol) in 
 COOH 
 large, shining, long rhombic scales or plates. It is less soluble than the 
 other fatty acids and melts at 68.2° C. 2 Its barium salt contains 19.49 
 per cent barium, and its silver salt contains 27.59 per cent silver. 
 
 CH 2 .O.C 16 H 3 iO 
 
 Palmitin, or tripalmitin, C5iH 9 s0 6 , = CH.O.Ci6H 31 0. Of the two solid 
 
 CH 2 .O.Ci 6 H 3 iO 
 varieties of fats, palmitin is the one which occurs in predominant quan- 
 tities in human fat (Langer 3 ). Palmitin is present in all animal fats 
 and in several kinds of vegetable fat. A mixture of stearin and palmitin 
 ■was formerly called margarin. As to the occurrence of palmitic acid, 
 C16H32O2, about the same remarks apply as to stearic acid. The mixture 
 of these two acids has been called margaric acid, and this mixture occurs 
 — often as very long, thin, crystalline plates — in old pus, in expectora- 
 tions from gangrene of the lungs, etc. 
 
 Palmitin crystallizes, on cooling from a warm saturated solution in 
 ether or alcohol, in starry rosettes of fine needles. The mixture of pal- 
 mitin and stearin, called margarin, crystallizes, on cooling from a solu- 
 tion, as balls or round masses which consist of short or long, thin plates 
 or needles which often appear like blades of grass. Palmitin, like stearin, 
 has a variable melting- and solidifying-point, depending upon the way it 
 has been previously treated. The melting-point is often given as 62° C, 
 but some investigators 4 claim that it melts at 50.5° C, solidifies on 
 further heating, and melts again at 66.5° C. 
 
 1 Annal. d. Chem. u. Pharm., 92. 
 
 2 According to Carlinfanti and Levi-Malvano, Chem. Centralbl., 1910. 
 
 3 Monatshefte f. Chem., 2; see also Jaeckle, Zeitschr. f. physiol. Chem., 36. 
 * R. Benedikt, Analyse der Fette, 3. Aufl., 1897, p. 44. 
 
236 ANIMAL FATS AND PHOSPHATIDES. 
 
 CH 3 
 Palmitic acid, (CHo)i4, crystallizes from an alcoholic solution in tufts 
 COOH 
 of fine needles. It melts at 61° C.; 1 still the admixture with stearic acid, 
 essentially changes them elting- and solidifying-points according to th 
 relative amounts of the two acids. Palmitic acid is somewhat more 
 soluble in cold alcohol than stearic acid; but they have about the same 
 solubility in boiling alcohol, ether, chloroform, and benzene. Its barium 
 salt contains 21.17 per cent barium, and silver salt contains 29.72 per 
 cent silver. 
 
 CH2.O.C18H33O 
 Olein, or triolein, C57Hi04O6, = CH.O.Ci8H 33 O, is present in all animal 
 
 CH2.O.C18H33O 
 fats, and in greater quantities in vegetable fats. It is a solvent for 
 stearin and palmitin. The oleic acid (elaic acid), C18H34O2, as soaps, 
 probably has about the same occurrence as the other fatty acids. 
 
 Olein is, at ordinary temperatures, a nearly colorless oil of a specific 
 gravity of 0.914, without odor or marked taste, and solidifies in crystalline 
 needles at -6° C. It becomes rancid quickly if exposed to the air. It 
 dissolves with difficulty in cold alcohol, but more easily in warm alcohol 
 or in ether. It is converted into its isomer, elaidin, by nitrous acid. 
 CH 3 
 
 (CH 2 ) 7 
 r*vx 
 Oleic acid, =,„, is an unsaturated acid of the series C n H„-202, and 
 
 (CH 2 ) 7 
 COOH 
 correspondingly takes up two halogen atoms, i.e., iodine, at the double 
 bondage, a factor which is the basis of v. Hubl's method for determining 
 the iodine equivalent. On taking up hydrogen, which can be accomplished 
 by heating with hydroiodic acid and amorphous phosphorus, it is trans- 
 formed into the corresponding saturated acid, namely, stearic acid. On 
 oxidation the double bonds are satisfied by 2HO groups, and dioxystearic 
 acid, CH3(CH 2 )7CHOH.CHOH(CH 2 )7COOH, is formed. Oleic acid 
 readily undergoes oxidation in the air with the formation of acid products, 
 and the occurrence of monoxystearic acid, found in animal fats in certain 
 instances, can be explained by this oxidation. Oleic acid on heating 
 yields, besides volatile fatty acids, sebacic acid, C10H18O4, which melts 
 at 127°C; and with nitrous acid it is transformed into its isomer, solid 
 elaidic acid, which melts at 45° C. 
 
 Oleic acid forms at ordinary temperature a colorless, tasteless, and 
 
 1 Carlinfanti and Levi-Malvano. Chem. Centralbl. 1910. 
 
OLEIC ACID. 237 
 
 odorless oily liquid which solidifies in crystals at about 4° C, which 
 latter melt at 14° C. Oleic acid is insoluble in water, but dissolves 
 in alcohol, ether, chloroform and petroleum ether. With concentrated 
 sulphuric acid and some cane-sugar it gives a beautiful red or reddish- 
 violet liquid whose color is similar to that produced in Pettenkofer's 
 test for bile-acids. If a solution of oleic acid in glacial acetic acid is treated 
 with a little chromic acid (in glacial acetic acid) and then with concen- 
 trated sulphuric acid, the green solution gradually becomes violet or 
 cherry-red, and shows two characteristic absorption bands in the green, 
 one a broad band near the blue and a second but fainter band near the 
 yellow (Lifschutz). 1 The barium salt of oleic acid contains 19. G5 per 
 cent barium and the silver salt 27.73 per cent silver. 
 
 If the watery solution of the alkali compounds of oleic acid is pre- 
 cipitated with lead acetate, a white, tough, sticky mass of lead oleate is 
 obtained, which is not soluble in water and only slightly in alcohol, but is 
 soluble in ether. This salt is more easily soluble in benzene than the lead 
 salts of stearic and palmitic acids, and this behavior of the lead salts 
 toward ether and benzene is made use of in separating oleic acid from 
 the other fatty acids. 
 
 An acid related to oleic acid, doeglic acid, which is solid at 4° C, liquid at 
 16° C, and soluble in alcohol, is found in the blubber of the Balcena rostrata. 
 According to Bull this acid is probably only a mixture of oleic acid and another 
 acid — gadoleic acid, C^oH.-sOo, having a melting-point of +24.5° C, and occurring 
 in cod-liver oil, herring oil and in whale blubber. In addition to this acid Bull 
 found in cod-liver oil, besides myristic, palmitic, oleic and erucic acids, another 
 acid, having the formula CieEUCK. According to Ellmer 2 the most abundant 
 acid (80-90 per cent) in cod-liver oil is therapinic acid, Ci 8 H 2 802 which is changed 
 into stearic acid by reduction and jecoleic acid, which seems to be identical with 
 Bull's gadoleic acid. Kurbatoff has demonstrated the presence of linoleic 
 acid in the fat of the silurus, sturgeon, seal, and certain other animals. Drying 
 fats have also been found by Amthor and Zink 3 in hares, wild rabbits, wild 
 boar, and mountain-cock. 
 
 To detect the presence of fat in an animal fluid or tissue the fat must 
 first be shaken out or extracted with ether. After the evaporation of 
 the ether the residue is tested for fat and fatty acids. The neutral 
 fats are differentiated from the fatty acids by the acrolein test, and the 
 fatty acids by the fact that their solution in a mixture of alcohol and 
 ether has an acid reaction. In separating the fats from cholesterin 
 and other non-saponifiable substances, as well as for the determination 
 of the kind of the various fatty bodies, they are saponified with caustic 
 alkali, alcoholic potash, or with sodium alcoholate. In regard to these 
 operations, as well as the further investigation and the separation of the 
 
 1 Zeitschr. f. physiol. Chem., 56. 
 
 2 Bull, Ber. d. d. chem., Gesellsch., 39; Ellmer, Bioch. Zeitschr., 9. 
 
 3 Kurbatoff, Maly's Jahresb., 22; Amthor and Zink, Zeitschr. f. anal. Chem., 36. 
 
238 ANIMAL FATS AND PHOSPHATIDES. 
 
 various fatty acids from each other, we must refer to more complete 
 hand-books. 
 
 In addition to the methods already suggested there are other chemical methods 
 which are important in investigating fats. Besides ascertaining the melting- 
 and congealing-point we also determine the following: 1. The acid equivalent, 
 which is a measure of the amount of fatty acids in a fat, is determined by titrat- 
 ing the fat dissolved in alcohol-ether with N/10 alcoholic caustic potash, using 
 phenolphthalein as indicator. 2. The saponification equivalent, which gives 
 the milligrams of caustic potash uniting with the fatty acids in the saponification 
 of 1 gram fat with N/2 alcoholic caustic potash. 3. Reichert-Meissl's equivalent, 
 which gives the quantity of volatile fatty acids contained in a given amount 
 of neutral fat (5 grams). The fat is saponified, then acidified with mineral acid, 
 and distilled, whereby the volatile fatty acids pass over; the distillate is then 
 titrated with alkali. 4. Iodine equivalent is the quantity of iodine absorbed by 
 a certain amount of the fat by addition. It is chiefly a measure of the quantity 
 of unsaturated fatty acids, principally oelic acid or olein, in the fat. Other bodies, 
 such as cholesterin, may also absorb iodine or halogens. The iodine equiva- 
 lent is generally determined according to the method suggested by v. Hubl. 
 5. The acetyl equivalent measures the quantity of those constituents of fats which 
 contain OH groups, and' is found by converting these bodies (oxyfatty acids, 
 alcohols and others) into the corresponding acetyl ester by boiling them with 
 acetic acid anhydride. 
 
 In the quantitative estimation of fats, the finely divided dried tissues 
 or the finely divided residue from an evaporated fluid is extracted with 
 ether, alcohol-ether, benzene, or any other proper extraction medium. 
 The lecithin (phosphatides) and other bodies are dissolved by the various 
 extraction media, hence the results for fats are too high. The most 
 exact method for the estimation of fat seems to be the method sug- 
 gested by Ktjmagawa and Suto, 1 who give a complete review of the 
 literature of the subject. 
 
 The fats are poor in oxygen, but rich in carbon and hydrogen. They 
 therefore represent a large amount of chemical energy, and yield correspond- 
 ingly large quantities of heat on combustion. They take first rank 
 among the foods in this regard, and are therefore of very great impor- 
 tance in animal life. We will speak more in detail of this significance, 
 also of fat formation and of the behavior of the fats in the body, in the 
 following chapters. 
 
 Cholesterin and isocholesterin ester, which will be discussed in a sub- 
 sequent chapter, as well as the following bodies, are closely related to 
 the fats. 
 
 Spermaceti. In the living spermaceti or white whale there is found, in a large 
 cavity in the skull, an oily liquid called spermaceti, which on cooling, after death, 
 separates into a solid crystalline part ordinarily called spermaceti, and into a 
 liquid, spermaceti-oil. This last is separated by pressure. Spermaceti is also 
 found in other whales and in certain species of dolphin. 
 
 The purified, solid spermaceti, which is called cetin, is a mixture of esters of 
 fatty acids. The chief constituent is the cetyl-palmitic ester mixed with small 
 
 1 Biochem. Zeitschr., 8. See also y. Schimidzu, ibid., 28. 
 
PHOSPHATIDES. 230 
 
 quantities of compound esters of Iauric, myristic, and stearic acid-; with radicals 
 of the alcohols, lethal. CuH«.OH, methal, CmH».OH, and stethal, CuHit.OH. 
 Cetin is a snow-white mass shining like mother-of-pearl, crystallizing in plates, 
 brittle, fatty to the touch, and which has a varying melting-point of 30 to 50° C., 
 depending upon its purity. Cetin is insoluble in water, hut dissolves easily 
 in cold ether or volatile and fatly oils. It dissolves in boiling alcohol, but crys- 
 tallizes on cooling. It is saponified with difficulty by a solution of caustic potash 
 in water, but with an alcoholic solution it saponifies readily, and the above-men- 
 tioned alcohols are set free. 
 
 CH 3 
 
 Ethal or cetyl alcohol, Ci 6 H 31 0. = (CHj) H , which occurs in smaller quantities 
 
 CH..OH 
 in beeswax and was found by Ludwig and v. Zeyxek in the fat from dermoid 
 Cysts — though this is denied by Ameseder, 1 — forms white, transparent, odorless, 
 and tasteless crystals which are insoluble in water but dissolve easily in alcohol 
 and ether. Ethal melts at 4!ho° C. 
 
 Spermaceti-oil yields on saponification valeric acid, small amounts of solid 
 fatty acids, and PHY6ETOLEIC ACID. This acid, which has. like hypogffiic acid, 
 the composition CuH&Os, occurs also, as found by Ljubarsky, 2 in considerable 
 amounts in the fat of the seal. It forms colorless and odorless needle-shaped 
 crystals which easily dissolve in alcohol and ether and melt at 34° C. 
 
 Beeswax may be treated here as concluding the subject of fats. It con- 
 tains three chief constituents: (1) cerotic acid, CseHo-.Oi, 3 which occurs as cetyl 
 ether in Chinese wax and as free acid in ordinarv wax. It dissolves in boiling 
 alcohol and separates as crystals on cooling. The cooled alcoholic extract of 
 wax contains (2) ceroleix, which is probably a mixture of several bodies, and 
 (3) mvricix, which forms the chief constituent of that part of wax which is 
 insoluble in warm or cold alcohol. Myricin consists chiefly of palmitic-acid 
 ester of nielissyl (myricyl) alcohol, C 3 oH 6 i.OH. This alcohol is a silky, shining, 
 crystalline body melting at So C. Dunham * has found camaubic acid, C^J^sO-- 
 in a phosphatide from the ox kidney. 
 
 2. Phosphatides. 
 
 In close relaticn to the fats stands a group of esters containing 
 nitrogen, phosphoric acid and fatty acid radicals. The representative 
 of this group longest known is lecithin. This latter is an ester combina- 
 tion of a nitrogenous base, choline, with a fatty acid-glycerophosphoric 
 acid, and Thudichum 5 has shown that a large number of more or less 
 analogous bodies occur in the animal body, especially in the brain. 
 All of these bodies have received the name phosphatides. 
 
 Those phosphatides which contain only one phosphoric acid radical 
 in the molecule are called monophosphatides; those with two such radicals 
 di phosphatides. The monophosphatides may contain one, two or more 
 
 1 Ludwig and v. Zeynek, Zeitschr. f. physiol. Chem. 23; Ameseder, ibid., 52. 
 ; Journ. f. prakt. Chem. (N. F.), 57. 
 
 1 See Henriques, Ber. d. deutsch. chem. Gesellsch., 30, 1415. 
 4 Journ. of biol. Chem., 4. 
 
 s J. L. W. Thudichum, Die chemische Konstitution des Gehirns des Menschen, 
 etc., Tubingen, 1901. 
 
240 ANIMAL FATS AND PHOSPHATIDES. 
 
 atoms of nitrogen in the molecule, and hence we differentiate between 
 monamido- (P : N = 1 : 1) , diamido- (P : N = 1 : 2) , triamido- (P : N = 1 : 3) 
 monophosphatides, etc. 
 
 So also may the diphosphatides contain 1, 2 or 3 atoms of nitrogen 
 for every 2 atoms of phosphorus (mono- di- or triaminodiphosphatides) . 
 Phosphatides with 4 or more atoms of nitrogen for every atom of phos- 
 phorus are also claimed to occur, but these statements seem to be uncer- 
 tain. On the other hand, according to Thudichum, non-nitrogenous 
 phosphatides occur in the brain; but if such be true these bodies must 
 not, for the present at least, be classified as phosphatides. 
 
 The phosphatides thus far investigated seem to be chiefly ester com- 
 binations between nitrogenous bases and fatty acid-glycerophosphoric 
 acid. According to Thudichum phosphatides exist which contain no 
 glycerin group and the carnaubon obtained by Dunham * from beef 
 kidneys seems to be such a phosphatide. The fatty acids occurring in 
 the phosphatides may be of different kinds. It seems that at least 
 one oleic acid radical, or another still less saturated fatty acid, occurs 
 in most of the phosphatides; still we know of phosphatides that con- 
 tain only saturated fatty acids. On this account the phosphatides may. 
 be divided into saturated and unsaturated phosphatides. The unsaturated 
 add iodine, take up oxygen from the air and are auto-oxidizable and 
 are changed readily. They also give a beautiful reaction with Petten- 
 kofer's bile-acid test. 
 
 Choline has generally been obtained as a basic constituent of the 
 phosphatides. Still other not sufficiently studied bases, have been found 
 in the plant as well as animal phosphatides and according to Trier 2 
 aminoeihyl alcohol is a probable generally distributed component of the 
 lecithins (phosphatides). 
 
 The phosphatides are very widely distributed in the plant as well as 
 the animal kingdom and they must undoubtedly exist as primary cell 
 constituents. We differentiate between such cell constituents which seem 
 to be absolutely necessary for the life of the cells, and those which are 
 stored up as reserve material, or are products of metabolism. The first, 
 which seem to occur in all developing cells, have been called 'primary 
 by Kossel 3 while he calls the others secondary. The question as to 
 the division of the known cell constituents into the primary or secondary 
 groups in the above sense, cannot be answered positively in many cases. 
 In the primary group besides water and mineral bodies we include pro- 
 teins of various kinds, nucleic acids and the so-called lipoids (see below) 
 to which the phosphatides belong. 
 
 l Zeitschr. f. physiol. Chem., 64, 303 (1910). 
 2 Ibid., 73, 76 and 80. 
 8 Verhandl. d. physiol. Gesellsch. zu Berlin, 1890-91. 
 
PHOSPHATIDES. 241 
 
 Attention has been called in Chapter I to the importance of the phos- 
 phatides (lipoids) for the limiting layer of the cells as well as for the 
 osmotic processes and for the metabolism of the cells. The unsat- 
 urated, readily oxidizable phosphatides also play a possible role as oxygen 
 carriers and the phosphatides are undoubtedly of great importance as 
 constituents of the food-stuffs. There is also no doubt that they are 
 very important for development and growth. It has been found that 
 the amount of phosphatides is especially abundant in the new-born, 
 and that these latter, to a certain extent, bring into the world a store of 
 phosphatides and this store diminishes during growth. 1 
 
 The phosphatides seem to be closely related to one another; they 
 influence the solubility and precipitation properties of one another, and 
 are generally precipitated as mixtures which are extremely difficult 
 to separate into individual constituents. They are also amorphous, 
 and readily oxidized, and it is easy to understand why their preparation 
 in a pure state is so extremely difficult. Under these circumstances 
 we have no sufficient guarantee as to their chemical individuality, and 
 the description of their properties and composition must be accepted 
 with some reservation. 
 
 The phosphatides are generally amorphous, colorless or yellowish; 
 they melt, on warming, and burn. As a rule they are insoluble in water 
 and swell up therein forming colloidal solutions, which are precipitated by 
 certain salts. The phosphatides as above stated, belong to the lipoids 
 and it is for this reason that each phosphatide is dissolved by at least one 
 of the solvents for fats (alcohol, ether, benzene, petroleum ether, etc.). 
 The lipoid group cannot be otherwise characterized. Originally we 
 included in this group, bodies similar to fat or in certain respects related 
 to the fats such as phosphatides, cholesterin and cerebrosides, but later 
 the conception has been developed and now we consider as lipoids 
 those bodies that are soluble in ether or equivalent. Under these 
 circumstances, as the diverse known and unknown bodies, such as lactic 
 acid, phenols, alkaloids and extractive bodies of various kinds may 
 belong to the lipoid group there does not seem to be any sense in speaking 
 of a special lipoid group, and especially from a chemical standpoint it 
 would be better to drop the name entirely. 
 
 The various phosphatides show a different behavior toward the 
 solvents for lipoids, namely some are soluble in ether while others 
 are insoluble therein, etc., and these differences are important for their 
 
 1 In regard to the quantity and importance of the phosphatides (the lecithins) 
 see Siwertzow, Bioch. Centralbl., 2, 310; Glikin, Bioch. Zeitschr., 4 and 7; Nerking, 
 ibid., 10; Stoklasa, Ber. d. d. chem. Gesellsch., 29, Wien. Sitz. Ber., 104; Zeitschr. 
 f. physiol. Chem. 25; Danilewsky, Compt. Rend., 121 and 123 and \Y. Koch., Zeitschr. 
 f. physiol. Chem., 37; Kyes, ibid., 41 and Berl. klin. Wochenschr., 1904. 
 
242 ANIMAL FATS AND PHOSPHATIDES. 
 
 preparation. They are generally all precipitated from their solution 
 by acetone although not completely, and this behavior is also of especial 
 importance in their preparation. The phosphatides are also nearly all 
 precipitated by metallic salts, especially by platinum chloride and cad- 
 mium chloride, and this method is also often used in their preparation. 
 The usefulness of this method has been questioned at least for certain 
 phosphatides, since Erlandsen * showed that a decomposition occurs. 
 
 Erlandsen has also found that when finely divided heart-muscle, dried in 
 the air, is completely extracted with ether and then with alcohol, the first extract 
 contains the monophosphatides, and the alcohol extract contains the diamino 
 phosphatides which were not free in the tissues, but existed in the combined state. 
 Whether this observation is of general importance in the preparation of pure 
 phosphatides remains to be seen. 
 
 As the phosphatides among themselves are rather difficult to char- 
 acterize and as there is a question whether a pure phosphatide has thus far 
 been prepared it seems of little interest to give a review here of the division 
 of the isolated phosphatides among the different groups. In this chapter 
 we will only discuss the three most studied phosphatides, namely, lecithin, 
 cephalin and cuorin; the others will be treated of in the respective chapters. 
 Lecithins. In correspondence with the generally accepted view 
 lecithin is a monoaminomonophosphatide, which' forms an ester com- 
 pound of glycerophosphoric acid substituted by two fatty-acid radicals 
 with a base called choline, 2 hence there must exist several groups of 
 lecithins. According to the kind of fatty acid contained in the lecithin 
 molecule it is possible to have various lecithins, such as stearyl-, palmityl-, 
 and oleyl-lecithins. According to Thudichum 3 every true lecithin always 
 contains at least one oleic-acid radical. According to the investigations 
 of Henriques and Hansen, Cousin and Erlandsen, 4 there is no ques- 
 tion that the so-called lecithin of the egg-yolk and muscles must contain 
 a fatty acid, still less saturated than oleic acid. All lecithins are mon- 
 aminophosphatides, according to the following type: 
 
 CH 2 — — fatty-acid radical. 
 
 CH — — fatty-acid radical. 
 
 CH 2 — (X 
 
 hcApo 
 
 /C2H4— O/ 
 N£-(CH 8 ) 3 
 MDH 
 
 1 Zeitschr. f. physiol. Chem., 51. 
 
 2 Strecker, Annal. d. Chem. u. Pharm., 148; Hundeshagen, Journ. f. prakt. Chem. 
 X. V.), 28; Gilson, Zeitschr. f. physiol. Chem., 12. A different view is held by 
 
 Malengreau and Pridgent, ibid., 77. 
 
 * Thudichum, Die chemische ^Constitution des Gehirns des Menschen, etc., Tubingen, 
 IfiOl. 
 
 4 Henriques and Hansen, Skand. Arch. f. Physiol., 14 (1903); Cousin, Compt. 
 Piend., 137; Erlandsen, Zeitschr. f. physiol. Chem., 51. 
 
LECITHINS. 243 
 
 The various lecithins stand close to each other in regard to constitu- 
 tion. The amount of phosphorus varies between 3.7-3.97 per cent and 
 the amount of nitrogen between 1.7-1.9 per cent. The so-called 
 di-stearyl-lecithin studied by Hoppe-Seyler and Diacoxow, 1 which 
 probably has a different structure, has the formula C44H90XPO9. Erlaxd- 
 sex gives the formula C^HsoNPOj for the lecithin isolated by him from 
 the heart muscles. 
 
 On saponification with alkalies or baryta-water, lecithin yields fatty 
 acids, glycerophosphoric acid, and choline. It is remarkable that in 
 the cleavage of lecithins a smaller amount of nitrogen than corresponds 
 to the choline is obtained. Mac Lean 2 who has especially investigated 
 this could not re-obtain the total nitrogen in the lecithins as choline but 
 only a part thereof — from heart muscle lecithin, 42 per cent, and from egg- 
 yolk lecithin, G5 per cent. He is therefore of the opinion that the choline 
 group is not the only nitrogenous group in the lecithins and that there- 
 fore the generally accepted formula for lecithin is incorrect. Trier 3 
 has indeed obtained aminoethyl alcohol as a cleavage product from sev- 
 eral phosphatides, which he calls lecithins, but because of the difficulty 
 in preparing phosphatides in a pure condition we are not sure that he was 
 "working with pure substances. Lecithin is slowly decomposed by 
 dilute acids. Besides small quantities of glycerophosphoric acid we 
 have large quantities of free phosphoric acid split off. The lecithins 
 are also decomposed by enzymes (lipase) with the splitting off of fatty 
 acids. 
 
 Lecithin is optically active, and as the glycerophosphoric acid which 
 can be split off is also active, Willstatter and Ludecke 4 claim that the 
 phosphoric acid is not bound on the middle unsymmetric CH group, but 
 rather at the end CH2 group of glycerin. 
 
 Lecithin, according to Hoppe-Seyler, 5 is found in nearly all animal 
 and vegetable cells thus far studied, and also in nearly all animal fluids. 
 It is especially abundant in the brain, nerves, fish eggs, yolk of the egg, 
 electrical organs of the Torpedo electricus, semen, and pus, and also in 
 the muscles and blood-corpuscles, blood-plasma, lymph, milk, especially 
 woman's milk, and bile. Lecithin is also found in different pathological 
 tissues or liquids. As the presence of lecithin is only indirectly deter- 
 mined by the detection of phosphorus in organic combinations, it must 
 be borne in mind that the above assertions relate chiefly to the occur- 
 rence of phosphatides. 
 
 1 Hoppe-Seyler, Med. chem. Unters., Heft 2 and 3. 
 
 2 Zeitschr. f. physiol. Chem., 59 and Bioch. Centralbl., 9. 
 3 1. c, Zeitschr. f. physiol. Chem., 73, 76 and 80. 
 
 4 Ber. d. d. chem. Gesellsch., 37. 
 
 5 Physiol. Chem. Berlin, 1877-81, p. 57. 
 
244 ANIMAL FATS AND PHOSPHATIDES. 
 
 The same also applies to the claims as to the quantity of lecithin 
 in various organs and tissues as well as in different ages. In these cases 
 the lecithin has not been prepared in a pure state, and the determina- 
 tions represent only the approximate quantity of phosphatides. These 
 determinations of Siwertzow, Glikin and Nerking, 1 show that lecithins 
 (phosphatides) occur abundantly in the bone marrow, suprarenal capsule, 
 heart and lungs, besides in the spinal marrow, brain, and egg, and also 
 that the quantity varies strikingly in different varieties of animals. 
 Nerking found 41.7 per cent lecithin in the bone marrow and 21.33 
 per cent in the suprarenal capsule of the sea-urchin when calculated on 
 the living organs, while the corresponding results in the rabbit were 2.71 
 and 2.39 per cent, respectively. 
 
 The statements as to the properties of the lecithins apply chiefly to 
 the lecithin of the hen's egg, which since Hoppe-Seyler and Diaconow's 
 time has been considered as distearyl-lecithin without any positive founda- 
 tion. Other lecithin preparations correspond essentially with this, and 
 certain differences between the various lecithins may be possibly due 
 to decomposition products or to admixture with other phosphatides. 
 It is still questioned whether the so-called distearyl-lecithin is a unit 
 body or not. 
 
 Lecithin may be obtained in grains or warty masses composed of small 
 crystalline plates by thoroughly cooling its solution in strong alcohol. In 
 the dry state it has a waxy appearance, is plastic, but forms pulverizable 
 masses when dried in vacuum, and is soluble in alcohol, especially on 
 heating (to 40-50° C.) ; it is less soluble in ether. It is dissolved also by 
 chloroform, carbon disulphide, benzene, and fatty oils. The solution 
 of lecithin from egg-yolk is dextrorotatory (Ulpiani 2 ). P. Mayer 3 
 claims to have prepared racemic lecithin from ordinary lecithin, and 
 Z-lecithin from the r-lecithin by cleavage with lipase. As he did not 
 make use of pure lecithin it is difficult to judge his results. The solu- 
 tion of lecithin in alcohol-ether or chloroform is precipitated by acetone, 
 although not completely. It swells in water to a pasty mass which shows 
 under the microscope slimy, oily drops and threads, so-called myelin 
 forms (see Chapter XI). On warming this swollen mass or the concen- 
 trated alcoholic solution, decomposition takes place with the produc- 
 tion of a brown color. On allowing the solution or the swollen mass to 
 stand, decomposition takes place and the reaction becomes acid. Accord- 
 ing to the investigations of Long 4 the lecithins seem to be much more 
 
 1 Siwertzow, see Biochem. Zeitschr., 2, p. 310; Glikin, Biochem. Zeitschr., 4 and 
 7; Nerking, ibid., 10. 
 
 2 Chem. Centralbl., 1901, 2, 30 and 193. 
 
 3 Biochem. Zeitschr., 1. 
 
 4 Journ. of Arner. chem. Soc, 30, 1908. 
 
LECITHINS. 245 
 
 resistant than was generally believed, and further investigations with 
 pure lecithin are desirable. 
 
 With considerable water the lecithin gives an emulsion or colloidal 
 solution which is not only precipitated by salts with divalent cations. 
 Ca, Mg, and others as claimed by W. KOCH, but is also precipitated accord- 
 ing to Long and F. Gephart 1 by salts with monovalent cations, although 
 slowly. In putrefaction, lecithins yield dvcerophosphoric acid and choline; 
 the latter further decomposes with the formation of methylamine, ammonia, 
 carbon dioxide, and marsh gas (Hasebiioek 2 ). If dry lecithin be heated 
 it decomposes, takes fire, and burns, leaving a phosphorized ash. On 
 fusing with caustic alkali and saltpetre it yields alkali phosphates. 
 
 Lecithins combine with acids and bases. The compound with hydro- 
 chloric acid gives with platinum chloride a double salt which is insoluble 
 in alcohol, soluble in ether, and which contains 10.2 per cent platinum 
 (for distearyl-lecithin). The cadmium-chloride compound, whose com- 
 position has been found somewhat variable by different investigators 
 is soluble with difficulty in alcohol, but dissolves in a mixture of carbon 
 disulphide and ether or alcohol. A solution of lecithin in alcohol is not 
 precipitated by lead acetate and ammonia. 
 
 Lecithins (and the same applies to the phosphatides in general) are 
 easily carried down during the precipitation of other compounds, such 
 as the protein bodies, and may therefore very greatly change the solubil- 
 ilities of other bodies. It is not known whether we are here dealing 
 with an adsorption or a chemical combination, and the conditions are 
 not the same in all cases. The combination with protein, the vitellines 
 and lecithalbumins have been discussed in a previous chapter, and 
 attention is there called to the necessity for more thorough investigation 
 of this subject. Further investigations of the so-called lecithin-sugar 
 (Bixg) is also desirable, as we know nothing definite as to its nature. 
 According to the investigations of Winterstein, Hiestand and E. 
 Schulze, lecithins (phosphatides) containing carbohydrates occur in 
 the plant kingdom, and contain about 20 per cent carbohydrate. We 
 are still not decided whether we are here dealing with combinations 
 or admixtures. 3 The same is true for the iron content of the lecithins or 
 phosphatides as observed by Glikin. 4 
 
 1 W. Koch., Zeitschr. f. physiol. Chem., 37; Long and Gephart, Journ. of Amer. 
 Chem. Soc, 30; see also Porges and Neubauer, Biochem. Zeitschr., 7. 
 - Zeitschr. f. physiol. Chem., 12. 
 
 3 Winterstein and Hiestand, Zeitschr. f. physiol. Chem., 47 and 54; Schulze, ibid. t 
 52 and 55; V. Xjegovan, ibid., 76. 
 
 4 Ber. d. d. chem. Gesellsch., 41. 
 
246 ANIMAL FATS AND PHOSPHATIDES. 
 
 Various methods have been suggested by Strecker, Hoppe-Seyler 
 and Diaconow, Thudichum, Gilson, Zuelzer and Bergell x for the 
 preparation of the lecithins. As none of these yield a positively pure 
 product we will here only mention them. According to Erlandsen's 
 experience all methods which are based upon the precipitation of the 
 lecithin as a metallic compound should be avoided. The best method 
 depends upon the solubility of the lecithin in alcohol and in ether in 
 the cold and its precipitation by acetone (Erlandsen, H. E. Roaf and 
 E. Edie 2 ). The work of Erlandsen is especially referred to in the 
 preparation of lecithins. 
 
 For the present we have no quantitative method for estimating 
 lecithin. The methods used in the past, when the amount of lecithin 
 was calculated from the amount of phosphorus contained in the alcohol- 
 ether extract is useless, as in this case the phosphorous content of all 
 the phosphatides is determined and not alone of the lecithins. Even 
 the detection of choline is not evidence, as this base probably occurs also 
 in other phosphatides. In the detection of choline the double platinum 
 compound is ordinarily prepared, and this can be done as described 
 below. In special determinations of lecithin and cephalin Koch used 
 to heat with hydroiodic acid, and determined the methyl groups split 
 off below 240° and those at about 300°. Instead of this he recommends 
 with Woods 3 to separate the two by precipitation in alcoholic solution, 
 while boiling, with alcoholic lead acetate solution and a little ammonia, 
 which precipitates only the cephalin. 
 
 Of the cleavage products of the lecithins choline is of especially 
 great interest. 
 
 Choline (trimethyloxyethyl ammonium hydroxide), 
 
 /CH 2 .CH 2 .(OH) 
 C 5 H 15 N0 2 , = HO.N< 
 
 X (CH 3 ) 3 , 
 
 stands in close "relation to the poisonous base neurine (trimethylvinyl 
 
 /(CH 3 ) 3 
 ammonium hvdroxide), HO.N<^ , which according to Brieger 
 
 X CH:CH 2 
 
 can be formed from choline by the action of bacteria, and also to mus- 
 
 /(CH 3 ) 3 
 carine, HO.N<y , which is the aldehyde of choline and occurs in 
 
 XH 2 CHO 
 
 / ° \ 
 the fly agaric, and also to betaine, trimethyl glycocoll, (CH 3 ) 3 N<( /CO, 
 
 X CH-/ 
 
 1 Strecker, Annal. d. Chem. u. Pharm., 148; Hoppe-Seyler and Diaconow, 1. c; 
 Thudichum, 1. c; Gilson, Zeitschr. f. physiol. Chem., 12; Zuelzer, ibid., 27; Bergell, 
 Per. d. d. chem. Gesellsch., 33. 
 
 2 Erlandsen. 1. c; Roaf and Edie, Thompson Yates Laboratory Reports, Vol. 6 
 part I, 1905. 
 
 3 Koch and Woods, Journ. of biol. Chem., 1. 
 
CHOLINE. 247 
 
 which may be considered as the anhydride of the arid corresponding to 
 choline. Muscarine and betaine can he obtained from choline on oxida- 
 tion. Choline yields trimethylamine as a decomposition product, and this 
 seems to be formed in the transformation of choline in the animal body. 
 
 ( holine occurs in the plant kingdom as well as in the animal kingdom. 
 Mott and Halliburton have repeatedly found choline in the blood in 
 degenerative diseases of the nervous system. It was first shown also in 
 normal blood by Marino Zuco, l and this investigator first found it in 
 tbe suprarenal capsule, but designated it neurine. Lohmann found it 
 later in this organ, and recently it has been found in various organs by 
 other investigators, especially by C. Schwarz and v. Furth. The fact 
 that choline is a cleavage product of lecithin in the animal, and that it is 
 antagonistic to adrenalin (of the suprarenal capsule) by its depressing 
 action upon the blood pressure, and that it has an exciting action upon 
 certain secretions (Lohmann, Theissier and Thevenot, v. Furth 
 and Schwarz 2 ), gives choline great physiological importance. The 
 physiological action of choline is still very much disputed. 
 
 Choline is a syrupy fluid, readily miscible with absolute alcohol. 
 Hydrochloric acid gives with it a compound which is very soluble in water 
 and alcohol, but insoluble in ether, chloroform, and benzene. This com- 
 pound forms a double combination with platinum chloride, is soluble in 
 water, insoluble in absolute alcohol and ether, and crystallizing from 
 water in monoclinic system and this form is strongly double-refractive. 
 From a mixture of water and alcohol it crystallizes in the regular form 
 (octahedral). Both forms can be changed from one to the other 
 and are used according to Kauffmann and Vorlander 3 in the detec- 
 tion of choline. Choline also forms a crystalline double compound 
 with mercuric chloride and with gold chloride. Choline is precipitated 
 by potassium iodide and iodine (Gulewitsch), and potassium triiodide 
 can be used for the quantitative estimation of this base (Stanek 4 ). 
 On heating the free base it decomposes into trimethylamine, ethylene 
 oxide, and water. 
 
 In preparing choline from lecithins, and also for the detection of 
 lecithin in an alcohol-ether extract, proceed as follows: The residue from 
 
 1 Mott and Halliburton, Philos. Trans., Ser. B, 191 (1899) and 194 (1901); Marino 
 Zuco, see Maly's Jahresber., 24, pp. 181 and 698. 
 
 2 Lohmann, Pfluger's Arch., 118 and 122; v. Furth and Schwarz, ibid., 124, which 
 also contains the literature. 
 
 3 See Gulewitsch, Zeitschr. f. physiol. Chem., 24; Kauffmann and Vorlander, Ber. 
 d. d. chem. Gesellsch., 43. 
 
 4 Gulewitsch, Zeitschr. f. physiol. Chem., 24; Stanek, ibid., 56. In regard to the 
 quantitative estimation see also Kiesel, ibid., 53; Stanek, ibid., 54; Moruzzi, ibid., 
 55; and MacLean, ibid., 55. 
 
248 ANIMAL FATS AND PHOSPHATIDES. 
 
 the above, or the solid lecithin is boiled one hour with baryta-water, 
 filtered, and the excess of baryta precipitated by CO2; filter while hot, 
 concentrate to a syrup, and extract with absolute alcohol, when the 
 insoluble barium glycerophosphate remains; then precipitate the filtrate 
 with an alcoholic platinum chloride solution. 
 
 CH2.OH 
 Glycerophosphoric acid, C 3 H 9 P0 6 =CH.OH , is a bibasic acid which prob- 
 
 CH 2 — 0\ 
 OH->PO 
 OH/ 
 abfy occurs in the animal fluids and tissues only as a cleavage product of lecithins. 
 According to Willstatter and Ludecke 1 the glycerophosphoric acid split off 
 from lecithins is optically active. Its barium and potassium salts are levorotatory, 
 and behave in certain respects differently from the corresponding salts of syn- 
 thetically prepared glycerophosphoric acid. The Ba and Ca salts of glycero- 
 phosphoric acid are crystalline and are more soluble in cold than in warm water. 
 The acid itself is a syrupy fluid. 
 
 Cephalin is also a monoaminophosphatide whose formula, based upon 
 the investigations of Thudichum, Koch, Thierfelder and Stern, 2 is 
 probably C42H82NPO13. The views of these investigators as to the con- 
 stitution of this body, which is difficult to purify, differ very considerably. 
 According to Thudichum, on cleavage it yields neurine, glycerophosphoric 
 acid, stearic acid, and a specific fatty acid, cephalic acid. According to 
 Koch it contains, on the contrary, only one methyl group attached to 
 nitrogen, and is therefore probably dioxystearylmonomethyl lecithin. 
 Frankel and Dimitz found no choline, while according to Cousin it 
 yields, like lecithin, stearic acid, an unsaturated fatty acid, glycero- 
 phosphoric acid and choline as decomposition products. The glycero- 
 phosphoric acid from brain cephalin gives, according to Frankel and 
 Dimitz, a dextrorotatory Ba salt and is therefore not identical with the 
 glycerophosphoric acid from lecithin. According to these investigators 
 the cephalin of the human brain is a mixture of palmityl and stearyl- 
 cephalin. Besides these two fatty acids cephalin also contains an unsat- 
 urated fatty acid, cephalinic acid, which according to Parnas 3 is related 
 to leinoleic acid or perhaps identical therewith. 
 
 From the investigations carried on thus far we can conclude that 
 cephalin differs from lecithin in that it contains cephalinic acid, another 
 glycerophosphoric acid and probably no choline but a monomethyl 
 base. Cephalin has probably never been obtained in a pure form. 
 
 1 Ber d. d. chem. Gesellsch., 37. 
 
 * Thudichum, 1. c; Koch, Zeitschr. f. physiol. Chem., 36; Thierfelder and Stern, 
 ibid. 53. 
 
 3 Frankel and Dimitz, Bioch. Zeitschr., 21; Parnas, ibid., 22; Cousin, Compt. 
 Rend. soc. biol., 62. 
 
CEPHALIN AND CUOBIN. 249 
 
 The cephalin from the brain has, according to Falk, 1 a different composi- 
 tion than that of the nerves and certain observations indicate that there 
 are several cephalins. 
 
 Cephalin occurs quite abundantly in the brain and also in nerves 
 and in the egg-yolk. The statements as to its further occurrence in the 
 animal kingdom require substantiation. 
 
 Cephalin is amorphous, not very plastic, and more easily triturated 
 than lecithin. It is readily soluble in cold ether, in chloroform and 
 benzene but differs from lecithin by being insoluble or soluble with difficulty 
 in alcohol. As unsaturated phosphatide it gives, like lecithin, a positive 
 reaction with Pettenkofer's bile-acid test. The cadmium- and plat- 
 inum chloride combinations are soluble in ether. Cephalin is obtained 
 from the brain, after dehydration with acetone, by extracting with ether 
 and precipitating the concentrated ethereal extract with alcohol. In 
 regard to the preparation and detection of cephalin we must refer to more 
 extensive hand-books. 
 
 The purest phosphatide prepared thus far seems to be cuorin, dis- 
 covered by Erlandsen. 
 
 Cuorin, C71H125NP2O21, is a monaminodiphosphatide prepared by 
 Erlandsen 2 from the heart muscle of the ox, and which has an iodine 
 equivalent of 101. It yields as cleavage products 3 molecules fatty acids 
 of unknown nature, partly or entirely belonging to the series C n H2 n -402 
 and C n H2 n -602; also glycerin, phosphoric acid and a base which is 
 not well known, but it is not choline. Cuorin is autooxidizable, and gives 
 Pettenkofer's bile-acid test. 
 
 Cuorin is amorphous, yellowish-brown and similar to rosin. It 
 gives a neutral solution with water which is like an emulsion. Cuorin 
 does not reduce Fehling's solution, even after boiling with acids. It is 
 soluble in ether, chloroform, petroleum ether and carbon disulphide. 
 It dissolves with difficulty in benzene ; it is insoluble in ethyl and methyl 
 alcohol and in acetone. Cuorin is precipitated from its alcohol-ether 
 solution by cadmium or platinum chloride. 
 
 1 Bioch. Zeitschr., 13 and 16. 
 
 2 Zeitschr. f. physiol. Chem., 51, where the method of preparation is described. 
 
CHAPTER V. 
 
 THE BLOOD 
 
 The blood is to be considered from a certain standpoint as a fluid 
 tissue; it consists of a transparent liquid, the blood-plasma, in which 
 a vast number of solid particles, the red and white blood-corpuscles (and 
 the blood-plates), are suspended. 
 
 Outside of the organism the blood, as is well known, coagulates more 
 or less quickly; but this coagulation is accomplished generally in a 
 few minutes after leaving the body. All varieties of blood do not coagulate 
 with the same degree of rapidity. Some coagulate more quickly, others 
 more slowly. In vertebrates with nucleated blood-corpuscles (birds, 
 reptiles, batrachia, and fishes) Delezenne has shown that the blood 
 coagulates very slowly if it is collected under such precautions that it 
 does not come in contact with the tissues. On contact with the tissues or 
 with their extracts it coagulates in a few minutes. The blood with 
 non-nucleated blood-corpuscles (mammals), on the contrary, coagulates 
 very rapidly. The coagulation of the blood in these cases may also be 
 somewhat retarded by preventing the blood from coming in contact 
 with the tissues (Spangaro, Arthus 1 ). Among the varieties of blood 
 of mammals thus far investigated the blood of the horse coagulates most 
 slowly. The coagulation may be more or less retarded by quickly cool- 
 ing; and if we allow equine blood to flow directly from the vein into a 
 glass cylinder which is not too wide and which has been cooled, and let it 
 stand at 0° C, the blood may be kept fluid for several days. An upper 
 amber-yellow layer of plasma gradually separates from a lower red layer 
 composed of blood-corpuscles with only a little plasma. Between these 
 is observed a whitish-gray layer which consists of white blood-corpuscles. 
 
 The plasma thus obtained and filtered is a clear amber-yellow alkaline 
 (toward litmus) liquid which remains fluid for some time when kept 
 at 0° C, but soon coagulates at the ordinary temperature. 
 
 The coagulation of the blood may be prevented in other ways. After 
 the injection of peptone, or, more correctly, proteose solutions into 
 
 1 Drlezenne, Corr.pt. rend. soc. de biol., 49; Spangaro, Arch. ital. de Biol., 32; 
 Arthus, Journ. de Physiol, et Pathol., 4. 
 
 250 
 
PREVENTION OF COAGULATION. 251 
 
 the blood (in the living dog), it does not coagulate on leaving the veins 
 (Fano, Schmidt-Mulheim 1 ). The plasma obtained from such blood 
 by means of centrifugal force is called peptone-plasma. According to 
 Arthus and Htjber 2 the caseoses and gelatoses act like fibrin proteose 
 in dogs. Eel serum and certain lymph-forming extracts of organs 
 (see Chapter VI) have an analogous action. The coagulation of the 
 blood of warm-blooded animals is prevented by the injection of an 
 effusion of the mouth of the officinal leech or a solution of the active 
 substance of such an infusion, hirudin (Franz), into the blood current 
 (Haycraft 3 ). If the blood is allowed to flow directly, while stirring 
 it, into a neutral salt solution — best a saturated magnesium-sulphate 
 solution (1 vol. salt solution and 3 vols, blood) — we obtain a mixture 
 of blood and salt which remains uncoagulated for several days. The 
 blood-corpuscles, which, because of their adhesiveness and elasticity, 
 would otherwise easily pass through the pores of the filter-paper, are 
 made solid and stiff by the salt, so that they may be easily filtered 
 off. The plasma thus obtained, which does not coagulate spontaneously, 
 is called salt-plasma. 
 
 An especially good method of preventing coagulation of blood con- 
 sists in drawing the blood into a dilute solution of potassium oxalate, 
 so that the mixture contains 0.1 per cent oxalate (Arthus and Paces 4 ). 
 The soluble calcium salts of the blood are precipitated by the oxalate, 
 and hence the blood loses its coagulability. On the other hand, Horne 5 
 found that chlorides of calcium, barium, and strontium, when present 
 in large amounts (2-3 per cent), may prevent coagulation for several 
 days. According to Arthus 6 a non-coagulable blood-plasma may be 
 obtained by drawing the blood into a sodium-fluoride solution until it 
 contains 0.3 per cent NaFl. 
 
 On coagulation there separates in the previously fluid blood an insoluble 
 or a very difficultly soluble protein substance, fibrin. When this separa- 
 tion takes place without stirring, the blood coagulates in a solid mass, 
 which, when carefully severed from the sides of the vessel, contracts, 
 and a clear, generally yellow-colored liquid, the blood-serum, exudes. 
 The solid coagulum which encloses the blood-corpuscles is called the 
 blood-clot (placenta sanguinis). If the blood is beaten during coagula- 
 tion, the fibrin separates in elastic threads or fibrous masses, and the 
 
 ^ano, Arch f.. (anat. u.) Physiol., 1881; Schmidt-Mulheim, ibid., 1880. 
 
 2 Arch, de Physiol. (5), 8. 
 
 3 Haycraft, Proc. Physiol. Soc, 1SS4, 13, and Arch. f. exp. Path. u. Pharm., 18; 
 Franz, Arch. f. exp. Path. u. Pharm., 49. 
 
 4 Archives de Physiol. (5), 2, and Compt. Rend., 112. 
 
 5 Journ. of Physiol., 19. 
 
 6 Journ. de Physiol, et Path., 3 and 4. 
 
252 THE BLOOD. 
 
 djibrinated blood which separates is sometimes called cruor l and con- 
 sists of blood-corpuscles and blood-serum, while uncoagulated blood 
 consists of blood-corpuscles and blood-plasma. The essential chemical 
 difference between blood-serum and blood-plasma is that the blood- 
 serum does not contain even traces of the mother-substance of fibrin, 
 the fibrinogen, which exists in the blood-plasma, while the serum is pro- 
 portionally richer in another body, the fibrin ferment (see below). 
 
 I. BLOOD-PLASMA AND BLOOD-SERUM. 
 The Blood-plasma. 
 
 In the coagulation of the blood a chemical transformation takes 
 place in the plasma. A part of the proteins separate as insoluble fibrin. 
 The albuminous bodies of the plasma must therefore be first described. 
 They are, as far as we know at present, fibrinogen, nucleoprotein, ser- 
 globulins, and ser albumins. 
 
 Fibrinogen occurs in blood-plasma, chyle, lymph, certain transudates 
 and exudates, in bone-marrow (P. Muller), and perhaps also in other 
 lymphoid organs. The seats of formation of fibrinogen are, according 
 to Mathews, the leucocytes, especially of the intestine, according to 
 Muller, the bone-marrow and probably other lymphoid organs such 
 as the spleen and lymph glands, and according to Doyon and Nolf, 
 the liver. The statement that the intestinal wall is a seat of formation 
 of fibrinogen, a view that had been held by Dastre, is substan- 
 tiated not only by the direct researches of Mathews, but also by the 
 older and substantiated opinion that the blood from the mesentery vein 
 is richer in fibrinogen than the arterial blood. This origin of fibrinogen 
 has been shown to be improbable by the recent researches of Doyon, Cl. 
 Gautier and Morel. The occurrence of fibrinogen in the bone-marrow 
 and other lymphoid organs as shown by Muller, and an increase of 
 fibrinogen in the blood as well as in the bone-marrow of animals immunized 
 with certain bacteria, especially pus-staphylococci, indicates the forma- 
 tion of fibrinogen in this tissue. The relation between the quantity of 
 fibrin and leucocytosis as shown by many investigators such as Lang- 
 stein and Mayer, Morawitz and Rehn, also indicate such a formation 
 of fibrinogen. The observations of Doyon, Gautier and Mawas that 
 a rapid re-formation of fibrinogen takes place in splenectomized animals 
 
 1 The name cruor is used in different senses. We sometimes mean thereby only 
 the blood when coagulated in a red solid mass, in other cases the blood-clot after the 
 separation of the serum, and again the sediment consisting of red blood-corpuscles 
 which is obtained from defibrinated blood by means of centrifugal force or by letting 
 it stand. 
 
FIBRINOGEN. 253 
 
 without any changes in the bone-marrow speak against the especially 
 great importance of the spleen and bone-marrow for the formation of 
 fibrinogen. That the liver takes part in the formation of fibrinogen 
 is implied by the fact that the quantity of fibrinogen in the blood 
 strongly diminishes after the extirpation of the liver (Nolf), and that 
 fibrinogen may indeed be entirely absent in the blood in phosphorus 
 poisoning (Corin and Ansiaux, Jacoby, Doyon, Morel, and Kareff l ), 
 and that the blood of the hepatic vein, according to Doyon, Morel 
 and Kareff, is richer in fibrinogen than the blood from other vessels, 
 and finally according to Whipple and Hurwitz 2 in chloroform poison- 
 ing the fibrinogen content of the blood diminishes with the injury to 
 the liver and rises again with restitution of the organ. 
 
 Fibrinogen has the general properties of the globulins, but differs 
 from other globulins as follows: In a moist condition it forms white 
 flakes which are soluble in dilute common salt solutions, and which 
 easily conglomerate into tough, elastic masses or lumps. The solution 
 in 5-10 per cent NaCl coagulates on heating at 52-55° C, and the faintly 
 alkaline or nearly neutral weak salt solution coagulates at 56° C, or at 
 exactly the same temperature at which the blood-plasma coagulates. 
 Fibrinogen solutions are precipitated by an equal volume of a saturated 
 common salt solution, and are completely precipitated by adding an excess 
 of NaCl in substance (thus differing from serglobulin) . A salt-free 
 solution of fibrinogen in as little alkali as possible gives with CaCb 
 a precipitate which contains calcium and soon becomes insoluble. In 
 the presence of NaCl or by the addition of an excess of CaCk the precipitate 
 does not appear. 3 A neutral solution of fibrinogen is precipitated by 
 a concentrated solution of sodium fluoride when added in a sufficient quan- 
 tity. Fibrinogens from different kinds of blood behave somewhat dif- 
 ferently in this regard. According to Huiskamp 4 fibrinogen from horse- 
 blood hardly dissolves in NaCl of 3-5 per cent at ordinary temperatures, 
 while it does dissolve at 40-45°. It also dissolves in ammonia of 0.05 
 
 1 P. Miiller, Hofmeister's Beitrage, 6; Mathews, Amer. Journ. of Physiol., 3; Nolf, 
 Bull. Acad., Roy. Belg., 1905, and Arch, intern, de Physiol., 3, 1905; Langstein, and 
 Mayer, Hofmeister's Beitrage, 5; Morawitz and Rehn, Arch. f. exp. Path. u. Pharm., 
 58; Corin and Ansiaux, Mary's Jahresber., 24; Jacoby, Zeitschr. f. physiol. Chem., 
 30; Doyon, Morel and Kareff, Compt. Rend., 140; Do3'on, Morel, and P6ju, Comp. 
 rend. soc. biolog., 58; Doyon, CI. Gautier, and Morel ibid., 62; Doyon, Gautier 
 and Mawas, ibid., 64. 
 
 2 Doyon, Morel and Kareff, Journ. de Physiol., 8 (1906); Whipple and Hurwitz, 
 Journ. of exp. Med. 13. See also Meek, Amer. Journ. of Physiol., 30. 
 
 3 See Hammarsten, Zeitschr. f. physiol. Chem., 22; Cramer, ibid., 23. 
 
 4 Huiskamp, ibid., 44 and 46. In regard to fibrinogen the reader is referred to 
 the author's investigations. Pfluger's Archiv., 19 and 22, and Zeitschr. f. physiol. 
 Chem., 28. 
 
254 THE BLOOD. 
 
 per cent, and on the addition of 3-5 per cent NaCl this solution can be 
 neutralized. The fibrinogen prepared by Huiskamp in this way retained 
 its typical properties. Fibrinogen differs from the myosin of the muscles, 
 which coagulates at about the same temperature, and from other pro- 
 tein bodies, in the property of being converted into fibrin under certain 
 conditions. Fibrinogen has a strong decomposing action on hydrogen 
 peroxide. It is quickly made insoluble by precipitation with water or 
 with dilute acids. Its specific rotation is (a) D =— 52.5° according to 
 
 MlTTELBACH. 1 
 
 Fibrinogen may be easily separated from the salt-plasma or oxalate- 
 plasma by precipitation with an equal volume of a saturated NaCl solu- 
 tion. It must be observed that the oxalate-plasma can only be employed 
 after the precipitate, containing proenzymes, and produced by exposure 
 to cold, has settled and been filtered off. If this is not done then the 
 fibrinogen is always impure. For further purification the precipitate 
 is pressed, redissolved in an 8-per cent salt solution, the filtrate pre- 
 cipitated by a saturated salt solution as above, and after being treated 
 in this way three times, the precipitate at last obtained is pressed between 
 filter-paper and finely divided in water. The fibrinogen dissolves with 
 the aid of the small amount of NaCl contained in itself, and the solution 
 may be made salt-free by dialysing with very faintly alkaline water. The 
 fibrinogen can be almost freed from fibrin-globulin, -which will be spoken 
 of later, by precipitating with double the volume of saturated sodium- 
 fluoride solution, redissolving in water with 0.05-per cent ammonia, 
 and then neutralizing this solution, treated with NaCl, and repeating 
 this several times. Fibrinogen may also, according to Reye, 2 be prepared 
 by fractionally precipitating the plasma with a saturated solution of 
 ammonium sulphate. We have no knowledge as to the purity of the 
 fibrinogen so prepared. The methods for the detection and quantitative 
 estimation of fibrinogen in a liquid were formerly based on its property 
 of yielding fibrin on the addition of a little blood, of serum, or of fibrin 
 ferment. Reye has suggested the fractional precipitation with ammonium 
 sulphate as a quantitative method. The value of this method has not 
 been sufficiently tested. 
 
 Fibrinogen stands in close relation to its transformation product, 
 fibrin. 
 
 Fibrin is the name of that protein body which separates on the so- 
 called spontaneous coagulation of blood, lymph, and transudates as well 
 as in the coagulation of a fibrinogen solution after the addition of serum 
 or fibrin ferment (see below). 
 
 If the blood is beaten during coagulation, the fibrin separates in 
 elastic, fibrous masses. The fibrin of the blood-clot may be beaten to 
 
 1 Zcitschr. f. physiol. Chem., 19. 
 
 2 W. Reye, Ueber Nachweifl und Bestimmung des Fibrinogens, Inaug-Diss. Strass- 
 burg, 1898. 
 
FIBRIN. 255 
 
 small, less elastic, and not particularly fibrous, lumps. The typical 
 fibrous and elastic white fibrin, after washing, stands, in regard to its 
 solubility, close to the coagulated proteins. It is insoluble in water, 
 alcohol, or ether. It expands in hydrochloric acid of 1 p. m., as also in 
 caustic potash or soda of 1 p. m., to a gelatinous mass, which dissolves at 
 the ordinary temperature only after several days; but at the temperature 
 of the body it dissolves more readily, although still slowly. Fibrin may 
 be dissolved by dilute salt solutions, after a long time, at the ordinary 
 temperature, or much more readily at 40° C, and this solution takes place, 
 according to Arthus and Hubert and also Dastre, 1 without the aid 
 of micro-organisms. This action is due to proteolytic enzymes car- 
 ried down by the fibrin or enclosed within the leucocytes (Rulot 2 ). 
 According to Green and Dastre 3 two globulins are formed in the solu- 
 tion of fibrin in neutral salt solution, and according to Rulot also pro- 
 teoses (and peptones) on the solution of fibrin containing leucocytes. 
 Fibrin, like fibrinogen, decomposes hydrogen peroxide, due to a con- 
 tamination with catalases, but this property is destroyed by heating or 
 by the action of alcohol. 
 
 What has been said of the solubility of fibrin relates only to the typical 
 fibrin obtained from the arterial blood of oxen or man by whipping 
 and washing first with water and with common salt solution, and then 
 with water again. The blood of various kinds of animals yields fibrin 
 with somewhat different properties, and according to Fermi 4 pig-fibrin 
 dissolves much more readily than ox-fibrin in hydrochloric acid of 5 p. m. 
 Fibrins of varying purity or originating from blood from different parts 
 of the body have unlike solubilities. 
 
 The fibrin obtained by beating the blood, and purified as above 
 described, is alwaj'S contaminated by secluded blood-corpuscles or 
 remains thereof, and also by lymphoid cells. It can be obtained pure 
 only from filtered plasma or filtered transudates. For the preparation 
 of pure fibrin, as well as for the quantitative estimation of it, the spon- 
 taneously coagulating liquid is at once, or the non-spontaneously coagu- 
 lating liquid only after the addition of blood-serum or fibrin ferment, 
 thoroughly beaten with a whale-bone, and the separated coagulum is 
 washed first in water and then with a 5-per cent common salt solution, 
 and again with water, and finally extracted with alcohol and ether. If 
 the fibrin is allowed to stand for some time in contact with the blood 
 from which it was formed, it partly dissolves (fibrinolysis — Dastre. 5 ). 
 This fibrinolysis must be prevented in the exact quantitative estimation 
 
 Arthus and Hubert, Arch, de Physiol. (5) 5; Dastre, ibid., (5) 7. 
 
 2 Arch, intern, de Physiol., 1. 
 
 3 Green, Journ. of Physiol., 8; Dastre, 1. c. 
 
 4 ZeiHcl.r. f. Biologie, 28. 
 
 5 Archives, de Physiol. (5), 5 and 6. 
 
256 THE BLOOD. 
 
 of fibrin (Dastre). The blood constituents that are active in fibrinolysis 
 are still unknown, but they are without doubt of enzymotic nature. 
 It must be mentioned that a strong fibrinolysis takes place in blood 
 after acute phosphorus poisoning (Jacoby and others), after extirpation 
 of the liver (Nolf), and also when the coagulability of the blood has been 
 reduced by the injection of proteoses (Nolf, Rulot 1 ). 
 
 A pure fibrinogen solution may be kept at the ordinary temperature 
 until putrefaction begins without showing a trace of fibrin coagula- 
 tion. But if to this solution is added a water-washed fibrin-clot or a 
 little blood-serum, it immediately coagulates, and may yield a perfect 
 typical fibrin. The transformation of the fibrinogen into fibrin requires 
 the presence of another body contained in the blood-clot and in the serum. 
 This body, whose importance in the coagulation of fibrin was first observed 
 by Buchanan 2 , was later rediscovered by Alexander Schmidt, 3 and 
 designated as fibrin ferment or thrombin. The nature of this enzymotic 
 body has not been ascertained with certainty. Even after careful 
 purification it gives very faint protein reactions and it is a much disputed 
 question whether it is a globulin or a nucleoprotein. It is a fact that 
 powerfully active solutions of thrombin can be obtained that do 
 not give either the reactions for globulins or nucleoproteins. Fibrin fer- 
 ment is produced, according to Pekelharing, 4 by the influence of soluble 
 calcium salts on a preformed zymogen existing in the non-coagulated 
 plasma. Schmidt admits the presence of such a mother-substance 
 of the fibrin ferment in the blood, and calls it prothrombin. The con- 
 version of this mother-substance into thrombin is a very complicated 
 process, which will be discussed under the coagulation of the blood. 
 Thrombin behaves like other enzymes in that the very smallest amount of 
 it produces an action, and its solution becomes inactive on heating. The 
 velocity of coagulation is dependent upon the quantity of thrombin, 
 and indeed a time law has been proposed for the action of thrombin. 
 According to Fuld the action of thrombin, at least within certain limits, 
 follows Schutz's law, and according to Stromberg the thrombin follows 
 in its action a time law, which at least in the beginning, corresponds to 
 
 1 Jacoby, Zeitschr. f. physiol. Chem., 30; Nolf, Arch, intern, de Physiol., 3, 1905; 
 Rulot, 1. c. 
 
 2 London Med. Gazette, 1845, 617. Cit. by Gamgee, Journal of Physiol, 1879. 
 
 3 Pfliiger's Arch., 6; see also Zur Blutlehre, 1892, and Weitere Beitriige zur Blut- 
 lehre, 1895. 
 
 * Pekelharing, Verhandl. d. Kon. Akad. d. Wetensch. te Amsterdam, 1892, Deel 1; 
 ibid., 1895, and Centralbl. f. Physiol., 9; Wright, Proc. Roy. Irish Acad. (3), 2; The 
 Lancet, 1892, and On Wooldridge's Method, etc., British Med. Journal, 1891; Lilien- 
 feld, Hamatol. Untersuch. Arch. f. (Anat. u.) Physiol., 1892; Ueber Leukocyten und 
 Blutgerinnung, ibid.; Halliburton and Brodie, Journal of Physiol., 17 and 18; Huis- 
 kamp, Zeitschr. f. physiol. Chem., 32; Pekelharing and Huiskamp, ibid., 39. 
 
FIBRIN FORMATION. 257 
 
 ScHtJTz's law while on increasing dilution deviates more and more 
 and finally shows a proportionally slow, and more irregular procedure. 
 Martin l has found another law from experiments with plasma and snake- 
 poisons containing thrombin. According to him the behavior is as 
 follows: As in the casein coagulation with rennin, the celerity of coagula- 
 tion is inversely proportional to the quantity of ferment; and Loeb 
 has observed a similar conduct with invertebrates. The optimum of the 
 thrombin action lies at about 40° C; at 70-75° C, in neutral solution, 
 the enzyme is destroyed. According to Howell and Rettger 2 throm- 
 bin, under proper conditions, can withstand boiling for a short time. The 
 question as to whether the thrombin found in different animals is the 
 same substance or whether we have several thrombins, has not been 
 decided. The latter is not improbable; nevertheless a definite specificity 
 of different thrombins has not been observed with certainty. 
 
 The isolation of thrombin has been tried in several ways. Ordinarily, 
 it may be prepared by the following method, proposed by Alex. Schmidt: 
 Precipitate the serum or defibrinated blood with 15-20 vols, of alcohol 
 and allow it to stand a few- months. The precipitate is then filtered 
 off and dried over sulphuric acid. The ferment may be extracted from 
 the dried powder by means of water. Other methods have been suggested 
 by Hammarsten, Pekelharing, and Howell. 3 According to a method 
 suggested by Hammarsten a solution of thrombin so poor in lime salts 
 that it contains only 0.3-0.4 p. m. solids and about 0.0007 p. m. CaO 
 can be prepared. 
 
 If a fibrinogen solution containing salt, as above prepared, is treated 
 with a solution of thrombin, it coagulates at the ordinary temperature 
 more or less quickly and yields a typical fibrin. Besides the thrombin, 
 the presence of neutral salts is necessary, for Alex. Schmidt has shown 
 that fibrin coagulation does not take place without them. The presence 
 of soluble calcium salts is not, as is generally assumed, a positive con- 
 dition for the formation of fibrin, because, thrombin can transform 
 fibrinogen into typical fibrin in the absence of lime salts precipitable by 
 oxalate. 4 The fibrin is not richer in lime than the fibrinogen used in its 
 preparation if the fibrinogen and thrombin solutions are employed as lime- 
 free as possible, and the view that the fibrin formation is connected with 
 a taking up of lime has been shown to be untenable (Hammarsten). 
 The quantity of fibrin obtained on coagulation is always smaller than 
 
 1 Martin, Journ. of Physiol., 32; Fuld, Hofmeister's Beitrage, 2; Loeb, ibid., 9; 
 Stromberg, Biochem. Zeitschr., 37. 
 
 2 Howell, Amer. Journ. of Physiol., 26; Rettger, ibid., 24. 
 
 3 Hammarsten, ibid., 18; Pekelharing, 1. c; Howell, 1. c. 
 
 * See Hammarsten, Zeitschr. f. physiol. Chem., 22, which also cites the works of 
 Schmidt and Pekelharing, and ibid., 28. 
 
258 THE BLOOD. 
 
 the amount of fibrinogen from which the fibrin is derived, and we always 
 find a small amount of protein substance in the solution. It is therefore 
 not improbable that the fibrin coagulation, in accordance with the views 
 first proposed by Denis, is a cleavage process in which the soluble fibrinogen 
 is split into an insoluble protein, the fibrin, which forms the chief mass, 
 and a soluble protein substance which is produced only in small amounts. 
 We find a globulin-like substance which coagulates at about 64° C. in 
 blood-serum as well as in the serum from coagulated fibrinogen solutions. 
 This substance is called fibrin-globulin by Hammarsten. The investiga- 
 tions of Huiskamp have shown that this substance is not formed as a 
 cleavage product from pure fibrinogen, but occurs in plasma or in fibrinogen 
 solutions not purified from sodium fluoride or perhaps in loose com- 
 bination with fibrinogen. The view that a cleavage takes place in the 
 coagulation of the fibrinogen has not been supported by these investi- 
 gations. 1 
 
 Opinions are not unanimous in regard to the enzyme nature of throm- 
 bin and the enzymotic formation of fibrin, and there are, indeed, investiga- 
 tors who consider the coagulation as another process. A more thorough 
 discussion of this subject can take place only in connection with the 
 coagulation of the blood. 
 
 Nucleoprotein. This substance, which, as above-mentioned, is considered 
 by Pekelharing and Huiskamp as identical with the prothrombin or thrombin, 
 occurs in the blood-plasma as well as in the serum, and is precipitated from the 
 latter with the globulin. It is similar to the globulin in that it is readily soluble 
 in neutral salt solution, and can be completely salted out on saturation with 
 magnesium sulphate, and separates only incompletely on dialysis. It is much 
 less soluble than serglobulin in an excess of dilute acetic acid, and coagulates 
 at 65-69° C. C. G. Liebermeister 2 found only 0.08-0.09 per cent phosphorus 
 in the nucleoprotein, which indicates that the nucleoprotein was contaminated 
 with other proteins. He also found that the substance was soluble in acetic acid 
 with difficulty, a property which is used by Pekelharing as an important means 
 of separating the compound proteins from the globulins. 
 
 Serglobulins, also called paraglobulin (Kuhne), fibrinoplastic substance 
 (Alex. Schmidt), serum-casein (Panum 3 ), occur in the plasma, serum, 
 lymph, transudates and exudates, in the white and red corpuscles, and 
 probably in many animal tissues and form-elements, though in small 
 quantities. They are also found in the urine in many diseases. 
 
 The so-called serglobulin is without doubt not an individual sub- 
 stance, but consists of a mixture of two or more protein bodies which 
 
 1 See Hammarsten, Zeitschr. f. physiol. Chem., 28; Heubner, Arch. f. exp. Path, 
 u. Pharm., 4'J, ami Zeitschr. f. physiol. Chem., 45; Huiskamp, ibid., 44 and 40. 
 
 - Hofineister'.s Beitnijie, 8; Pekelharing and Huiskamp, 1. c. footnote 1, page 256. 
 
 :t Kii!, no, Lehrbucb d. physiol. Chem., Leipzig., 1866-68; Alex. Schmidt, Arch. f. 
 (Anat. n. ) Physiol., 1861-62; Panum, Virchow's Arch., 3 and 4. 
 
SEKGLOBULINS. 259 
 
 cannot be completely and positively separated from each other. The 
 mixture of globulins obtained from blood-plasma or blood-serum hy 
 
 saturation with magnesium sulphate or half-saturation with ammonium 
 sulphate consists of nucleoprotein, fibrin-globulin, and the true serglobulin 
 or mixture of globulins. 
 
 The nucleoprotein has been previously discussed. The fibrin-globulin, 
 which occurs in the serum only in small amounts, can be completely pre- 
 cipitated by NaCl. It has the general properties of the globulins, but 
 differs from the serglobulins by a lower coagulation temperature, 64- 
 66° C, and also in that it is precipitated by (NH^SCU even in 28 per 
 cent solution. 
 
 Serglobulins. If the globulin obtained by saturation with magnesium 
 sulphate is dialyzed, then, as has been known for a long time and further 
 substantiated by Marcus, only a part of the globulin separates out, 
 while a portion remains in solution and cannot be precipitated by the 
 addition of acid. For this reason Marcus 1 also differentiates between 
 a water-soluble globulin and one insoluble in water. According to the 
 later investigations of Hofmeister and Pick 2 the part insoluble in 
 water corresponds chiefly to a globulin fraction readily precipitated by 
 (NH4)2S04 (by 28-36 vols, per cent saturated solution), and the part 
 soluble in water corresponds to a fraction difficult to precipitate (by 
 36-44 vols, per cent saturated solution). The first fraction is called 
 euglobulin and the second pseudoglobulin. According to Porges and 
 Spiro 3 the serglobulins can be separated by (NHU^SO-t into three 
 fractions whose precipitation limits are 28-36, 33-42, and 40-46 vols, 
 per cent saturated solution. All three fractions contain globulin insoluble 
 in water. Freund and Joachim 4 have found that the euglobulin as 
 well as the pseudoglobulin fraction is a mixture of globulin soluble in 
 water and globulin insoluble in water, and consequently the number 
 of different globulins in the serum may be still greater. 
 
 It follows from all these investigations that either the difference between 
 the globulin soluble in water and that insoluble is not sufficient or that the frac- 
 tional precipitation with ammonium sulphate is not suited for the separation 
 of the various globulins. This latter seems to be the case, as shown by Mellanby 
 Haslam and recently by Wiener. 5 It must not be forgotten that the globulin 
 fractions are always contaminated with other serum constituents, and that these 
 may influence the solubility and precipitability. As Hammarsten has shown, 
 a water-soluble globulin, can be transformed into a globulin insoluble in water 
 
 1 Zeitschr. f. physiol. Chem., 28. 
 
 2 Hofmeister's Beit rage, 1. 
 ■ Hofmeister's Beit rage, 3. 
 
 4 Zeitschr. f. physiol. Chem., 36. 
 
 5 Mellanby, Journ. of Physiol., 36; Haslam, ibid., 32; Wiener, Zeitschr. f. physiol 
 Chem., 74. 
 
260 THE BLOOD. 
 
 by careful purification, and also the reverse, namely, a globulin insoluble in 
 water can sometimes be converted into one soluble in water by allowing it to lie 
 in the air. An insoluble protein like casein can also, according to Hammarsten, 1 
 have the solubilities of a globulin due to contamination with constituents of the 
 serum, and K. Morner - has also shown that a contamination of the serum- 
 globulins with soap can essentially modify the precipitation of these globulins. 
 Under these circumstances the above assumptions in regard to the different 
 globulin fractions must be accepted with great caution. 
 
 The investigations made thus far upon the so-called serglobulin, 
 have not led to any positive results. That this globulin, with the 
 exception of the enzymes, antienzymes, immune bodies, and other . 
 unknown substances which are carried down by the various fractions, is 
 a mixture of globulins there seems to be no doubt. The serglobulin or 
 the globulin mixture which is obtained from the serum by the methods 
 to be described has the following properties: 
 
 In a moist condition it forms snow-white flaky masses, neither tough 
 nor elastic, which always contain thrombin and hence can bring about 
 coagulation in a fibrinogen solution. The neutral solution is only incom- 
 pletely precipitated by NaCl added to saturation, and is not precipitated 
 by an equal volume of a saturated salt solution. It is only partly 
 precipitated by dialysis or by the addition of acid. On saturation with 
 magnesium sulphate, or one-half saturation with ammonium sulphate 
 a complete precipitation is obtained. The coagulation temperature 
 is, w r ith 5-10 per cent NaCl in solution, 69-76°, but more often 75° C. 
 The specific rotation of the solution containing salt is (a) D =— 47.8° 
 for the serglobulin from ox-blood (Fredericq 3 ). The various globulin 
 fractions do not differ essentially from each other in their coagulation 
 temperatures, specific rotation, refraction coefficient (Reiss 4 ), and their 
 elementary composition. The average composition is, according to 
 Hammarsten, C. 52.71, H 7.01, N 15.85, S 1.11 per cent. K. Morner 5 
 found 1.02 per cent sulphur and 0.67 per cent lead-blackening sulphur. 
 All the sulphur seems to exist as cystine. 
 
 Serglobulin contains, as K. Morner first showed, a carbohydrate 
 group wdiich can be split off. Langstein 6 has obtained several car- 
 bohydrates from the blood-globulin, namely, glucose, glucosamine, 
 
 1 See Hammarsten, Ergebnisse, d. Physiol., 1, Abt. 1. 
 
 2 Zeitschr. f. physiol. Chem., 34. 
 
 3 Bull. Acad. Roy. de Belg. (2), 50. In regard to paraglobulin, see Hammarsten, 
 Pfliiger's Arch., 17 and 18, and Ergebnisse d. Physiol, 1, Abt. 1. 
 
 4 Hofmeister's Beitrage, 4. 
 
 6 Zeitsrhr. f. physiol. Chem., 34. 
 
 8 Morner, Centralbl. f. Physiol., 7; Langstein, Munch, med. Wochenschr., 1902, 
 1876, and Wien. Sitzungsber., 112, Abt. lib, 1903; Monatsheft f. Chem., 25; Hof- 
 meister's Beitrage, 6; see also footnote 5, p. 84. 
 
SERALBUMIN*. 261 
 
 and carbohydrate acids of unknown kinds. It has not boon shown 
 whether these small amounts of carbohydrate are derived from the globulin 
 or from other contaminating bodies. According to Zanetti and also 
 Bywaters, the blood-serum contains a glucoproteid, seromucmd, and 
 the investigations of Eichholz * seem to show that the globulins are 
 contaminated by a glucoproteid. According to Langstein the sugar 
 is not only mixed with the globulin, but it exists in a combined form, 
 probably in loose combination. 
 
 Serglobulin (the euglobulin) may be easily separated as a fine floc- 
 eulent precipitate from blood-serum by neutralizing or making faintly 
 acid with acetic acid and then diluting with 10-20 vols of water. For 
 further purification this precipitate is dissolved in dilute common salt 
 solution, or in water with the aid of the smallest possible amount of 
 alkali, and then reprecipitated by diluting with water or by the addition 
 of a little acetic acid. All the serglobulin may also be separated from 
 the serum by means of magnesium or ammonium sulphate; in these 
 cases it is difficult to completely remove the salt by dialysis. As long 
 as we are not agreed as to the number of globulins in the serum, it is 
 not necessary to give a method of separating the various globulins in this 
 mixture. Thus far the fractional precipitation with (NH^SCU has 
 chiefly been used. The serglobulin from blood-serum is always contam- 
 inated with lecithin and thrombin. A serglobulin free from thrombin 
 may be prepared from ferment-free transudates, as sometimes from 
 hydrocele fluids, and this shows that serglobulin and thrombin are dif- 
 ferent bodies. For the detection and the quantitative estimation of 
 serglobulin we may use the precipitation by magnesium sulphate added 
 to saturation (Hammarsten), or by an equal volume of a saturated 
 neutral ammonium-sulphate solution (Hofmeister and Kauder and 
 Pohl 2 ). In the quantitative estimation the precipitate is collected on a 
 weighed filter, washed with the salt solution employed, dried with the 
 filter at about 115° C, then washed with boiling-hot water, so as to 
 completely remove the salt, extracted with alcohol and ether, dried, 
 weighed, and incinerated to determine the ash. This method, according 
 to the investigations of Wiener, 3 can only be used when the serum is 
 sufficiently diluted with water. 
 
 Seralbumins are found in large quantities in blood-serum, blood- 
 plasma, lymph, transudates, and exudates. Probably they also occur 
 in other animal fluids and tissues. The proteins which pass into the 
 urine under pathological conditions consist largely of seralbumin. 
 
 The seralbumin, like the serglobulin, seems also to be a mixture of 
 at least two protein bodies. The preparation of crystalline seralbumin 
 
 1 Zanetti, Chem. Centralbl., 189S, I, p. 624; Bywaters, Journ. of Physiol., 35, and 
 Biochem. Zeitschr., 15; Eichholz, Journ. of Physiol., 23. 
 
 2 Hammarsten, 1. c; Hofmeister, Kauder and Pohl, Arch. f. exp. Path. u. Pharm., 20. 
 
 3 Zeitschr. f. physiol. Chem., 74. 
 
262 THE BLOOD. 
 
 (from horse-serum) was first performed by Gurber. It crystallizes 
 with difficulty from other blood-sera (Gruzewska). Even from horse- 
 serum only a portion, according to Robertson 1 not more than 40 per 
 cent, of the albumin can be obtained as crystals, and it is also pos- 
 sible that the amorphous albumin, which is precipitated by ammo- 
 nium sulphate with difficulty, represents two seralbumins (Maximo- 
 witsch). According to Gurber and Michel it would seem that the 
 crystalline seralbumin is also a mixture, but this is disproved by the obser- 
 tions of Schulz, Wichmann, and Krieger 2 . We know nothing as 
 to the behavior of the amorphous fraction of the seralbumin in this respect. 
 Because of the different coagulation temperatures, Halliburton claims 
 the existence of three different albumins in the blood-serum, a view 
 which has been disputed by several experimenters, and recently by 
 Hougardy. Oiv. the other hand, the earlier investigations of Kauder, 
 as well as the more recent work of Oppenheimer, 3 seem to indicate a 
 non-unit nature of the seralbumins, but this question is still an open 
 one. 
 
 The crystalline seralbumin may perhaps be a combination with 
 sulphuric acid (K. Morner, Inagaki). The coagulated albumin obtained 
 from the aqueous solution of the crystals with the aid of alcohol has 
 almost the same elementary composition (Michel) as the amorphous 
 mixture of albumin prepared from horse-serum (Hammarsten and 
 K. Starke 4 ). The average composition was C 53.06, H 6.98. N 15.99, 
 S 1.84 per cent. K. Morner, after the removal of the sulphuric acid 
 from crystalline albumin, found 1.73 per cent total sulphur, which prob- 
 ably exists only as cystine. Langstein 5 has been able to split off a nitrog- 
 enous carbohydrate (glucosamine) from crystalline seralbumin. The 
 quantity was so small that the question is still undecided whether or 
 not the carbohydrate was a contamination. The fact that Abder- 
 halden, Bergell, and Dorpinghaus 6 were able to prepare a seral- 
 bumin entirely free from carbohydrate and which did not respond to 
 Molisch's very delicate reaction, seems to be decisive on this point. 
 The specific rotation of crystalline seralbumins from horse-serum was 
 found by Michel to be (a) D =— 61 to 61.2°, and by Maximowitsch on 
 the contrary (o:) D = —47.47°. 
 
 1 Journ. of biol. Chem., 13. 
 
 2 In regard to the literature on the crystalline seralbumins, see Schulz, Die Kristal- 
 lisation von Eiweissstoffen, Jena, 1901; Maximowitsch, Maly's Jahresber., 31, 35. 
 
 3 Halliburton, Journ. of Physiol., 5 and 7; Hougardy, Centralbl. f. Physiol., 15, 
 665; Oppenheimer, Verhandl. d. physiol. Gesellsch., Berlin, 1902. 
 
 4 Michel, Verhandl. d. phys-med. Gesellsch. zu Wiirzburg, 29, No. 3; K. Starke,. 
 Maly's Jahresber., 11; K. Morner, 1. c; Inagaki, Biochem, Centralbl., 4, p. 515. 
 
 6 K. Morner, 1. c; Langstein, Hofmeister's Beitrage, 1. 
 • Zeitschr. f. physiol. Chem., 41. 
 
SERUM PROTEINS. 2G3 
 
 The crystalline and amorphous seralbumin in aqueous solution give 
 the ordinary albumin reactions. The coagulation temperature of a 
 1-per cent solution, poor in salts is about 50° C, but rises with the quan- 
 tity of salt. The coagulation of the mixture of albumins from serum 
 generally takes place at 70-85° C, but is essentially dependent upon 
 the reaction and the amount of salt present. Up to the present time no 
 seralbumin solution has been prepared free from mineral bodies. A 
 solution as free from salts as possible does not coagulate either on boil- 
 ing or on the addition of alcohol. On the addition of a little common 
 salt it coagulates in both cases. 1 
 
 Seralbumin differs from the albumin of the white of the hen's egg in 
 the following particulars: It is more levogyrate; the precipitate formed 
 by hydrochloric acid easily dissolves in an excess of the acid; it is rendered 
 less insoluble by alcohol. 
 
 In preparing the seralbumin mixture, first remove the globulins, 
 according to Johansson, by saturating with magnesium sulphate at 
 about 30° C. and filtering at the same temperature. The cooled filtrate 
 is separated from the crystallized salt and is treated with acetic acid so 
 that it containes about 1 per cent. The precipitate formed is filtered 
 off, pressed, dissolved in water with the addition of alkali to neutral 
 reaction, and the solution freed from salt by dialysis. The mixture of 
 albumins may be obtained in a solid form from the dialyzed solution, 
 either by evaporating the solution at a gentle temperature or by pre- 
 cipitating with alcohol, which must be quickly removed. Starke 2 
 has suggested another method, which is also to be recommended. The 
 crystalline seralbumin may be prepared from serum freed from globulin 
 by half saturating with ammonium sulphate, by the addition of more 
 salt until a cloudiness appears, and then proceeding according to the 
 suggestion of Gurber and Michel. On acidifying with acetic acid 
 or sulphuric acid the crystallization may be considerably accelerated. 3 
 The quantity of seralbumin is best calculated as the difference between 
 the total proteins and the globulins. A method for the quantitative 
 estimation of globulins and albumins in blood serum by refractometric 
 means has been suggested by Robertson. 4 
 
 Summary of the elementary composition of the above-mentioned and described 
 proteins (from horse-blood): 
 
 c h n s 
 
 Fibrinogen 52 . 93 6 . 90 16 . 66 1 . 25 22 . 26 (Hammarsten) 
 
 Fibrin 52.68 6.83 16.91 1.10 22.48 
 
 Fibrin-globulin 52.70 6.98 16.06 
 
 Serglobulin 52.71 7.01 15.85 1.11 23.32 
 
 Seralbumin 53 . CS 7.10 15 . 93 1 . 90 21 .86 (Michel) 
 
 'In regard to the relationship of neutral salts to heat coagulation,, see J. Starke, 
 Sitzungsber. d. Gesellsch. f. Mori>h. u. Physiol, in Munehen, 1897. 
 
 2 Johansson, Zeitschr. f. fhvsiol. Chem., 9; K. Staike, Maly's Jahresber., 11. 
 
 3 See Hopkins and Pinkus, Journ. of Physiol., 23; Krieger, I'eber die Darstellung. 
 krystallinscher tierischer Eiweissstoffe, Inaug. -Dissert. Strassburg, 1899. 
 
 * Journ. of biol. Chem., 11. 
 
204 THE BLOOD. 
 
 Proteose-like substances have been found in blood-serum by several 
 investigators, and Nolf ! has shown that after the abundant introduc- 
 tion of proteoses into the intestine, they pass into the blood. Bor- 
 chardt 2 has also been able to show that not only after the introduction 
 of elastin-proteose per os, but also after feeding dogs with not over- 
 abundant quantities of elastin, a proteose, hemielastin, passes into the 
 blood and can indeed be eliminated in the urine. The question whether 
 the proteoses are normal constituents of the blood under ordinary con- 
 ditions is still much disputed. The difficulty in deciding this ques- 
 tion lies in the fact that in the removal of the proteins a small amount 
 of proteose-like substance is formed from other proteins (namely from the 
 globin of the blood pigment), and on the other hand the proteoses can be 
 precipitated with the other bodies. The question as to the physiological 
 occurrence of proteoses in the blood or plasma must be considered as 
 still undecided. 3 
 
 In close relation to the proteoses stands perhaps the above-men- 
 tioned seromucoid, which was discovered by Zanetti and especially 
 studied by Bywaters. It is a glycoprotein which is soluble in water, 
 and precipitated by alcohol. Seromucoid contains, according to By- 
 waters, 4 11.6 per cent N, 1.8 per cent S, and yields approximately 25 
 per cent glucosamine. The quantity in the blood is 0.2-0.9 p. m. 
 
 The Blood-serum. 
 
 As above stated, the blood-serum is the clear liquid which is pressed 
 out by the contraction of the blood-clot. It differs chiefly from the 
 plasma in the absence of fibrinogen and in containing an abundance 
 of fibrin ferment. Otherwise considered qualitatively, the blood-serum 
 contains the same chief constituents as the blood-plasma. 
 
 Blood-serum is a sticky liquid which is more alkaline toward litmus 
 than the plasma. The specific gravity in man is 1.027 to 1.032, average 
 1.028. The color is more or less yellow; in human blood-serum it is 
 pale yellow with a shade toward green, and in horses it is often amber- 
 yellow. The serum is ordinarily clear; after a meal it may be opales- 
 cent, cloudy, or milky white, according to the amount of fat contained 
 in the food. 
 
 Besides the above-mentioned bodies, the following constituents are 
 found in the blood-plasma or blood-serum: 
 
 1 Bull. Acad. Roy. Belg., 1903 and 1904. 
 
 2 Zeitschr. f. physiol. Chem., 51 and 57. 
 
 3 See especially Abderhalden, Zeitschr. f. physiol. Chem., 51, and Biochem. Zeitschr., 
 8 and 10, and E. Freund, ibid., 7 and 9, which also contains the literature. 
 
 4 Biochem. Zeitschr., 15. 
 
BLOOD SERUM. 265 
 
 Fat occurs from 1-7 p. m. in fasting animals. After partaking of 
 food the amount is increased to a great extent. Fatty acids, or soaps, 
 glycerin (Nicloux, Fr. Tangl, and St. Weiser l ) phosphatides and 
 cholesterin are also found. Cholesterin occurs, according to Hurthle 2 , 
 at least in part, as fatty-acid esters (serolin according to Boudet). Accord- 
 ing to Letsche 3 free cholesterin probably also occurs in the serum. 
 
 Sugar seems to be a physiological constituent of the plasma and 
 serum. According to the investigations of many workers 4 the sugar 
 found is glucose. Strauss 5 has also detected fructose in blood-serum 
 and in transudates and exudates. The question as to the occurrence 
 of other varieties of sugar, such as isomaltose (Pavy and Siau) and pen- 
 tose (Lepine and Boulud 6 ), in blood serum is still undecided. Asher 
 and Rosenfeld and Michaelis and Rona in a more conclusive manner, 
 have shown that at least a considerable part of the sugar can 1 le removed 
 from the blood by dialysis, hence it must exist in solution in the free 
 state. These observations do not exclude the possibility of the existence 
 of another part of the sugar which is in combination with protein. 
 Lepine and Boulud 7 could only obtain a diffusion of the sugar by a 
 short dialysis from serum 12 hours old, but not from perfectly fresh 
 serum, an observation which somewhat diminishes the conclusiveness 
 of Michaelis and Rona's experiment with 24-hour dialysis. A fur- 
 ther testing of this question is therefore very desirable. 
 
 The quantity of sugar in the serum or plasma is for man 0.6-1 p. m. 
 calculating the total reduction as glucose, and in animals about the same 
 but in rabbits considerably higher or 2.2 p. m. 8 Besides the sugar, the 
 blood contains, as first shown by J. Otto, also another or perhaps several 
 reducing substances, a part existing in the serum and another part in the 
 blood-corpuscles. We will discuss the nature of these bodies as well 
 as the so-called virtual sugar and glycolysis in speaking of the division 
 of the sugar in the blood-corpuscles and plasma in connection with 
 
 1 Nicloux, Compt. Rend. soc. biol., 55; Tangl and St. Weiser, Pfluger's Arch., 115. 
 
 2 Hurthle, Zeitschr. f. physiol. Chem., 21, where Boudet is also cited. In regard 
 to the quantity of these esters in bird-serum, see Brown, Amer. Journ. of Physiol., 2. 
 
 3 Zeitschr. f. physiol. Chem., 53. 
 
 4 See v. Mering, Arch. f. (Anat. u.) Physiol., 1877 (this article contains numerous 
 references); Seegen, Pfluger's Arch., 40; Miura, Zeitschr. f. Biologie, 32. 
 
 8 Fortschritte d. Mediz., 1902. 
 
 6 Pavy and Siau, Journ. of Physiol., 26; Lepine and Boulud, Compt. Rend., 133, 
 135, and 136. 
 
 7 Rosenfeld, Centralbl. f. Physiol., 19, p. 449; Lupine and Boulud, Compt. Rend., 
 143; Asher, Biochem. Zeitschr., 3; Michaelis and Rona, ibid., 14. 
 
 8 See E. Frank, Zeitschr. f. physiol. Chem., 70; Lyttkens and Sandgren, Bioch. 
 Zeitschr., 21 and 26. 
 
266 THE BLOOD. 
 
 the total blood. The same applies to the conjugated glucuronic acids, 
 which it seems, originate from the form-elements. 
 
 The blood-plasma and the serum, as well as the lymph also contain 
 enzymes of various kinds. According to Rohmann, Bial, Hamburger, 1 
 and others, diastases, which convert starch and glycogen into maltose or 
 isomaltose, as well as a maltase, are found in the blood. The diastase, 
 whose quantity is very variable in the blood of different animals, seems 
 at least in great part, to originate in the pancreas but can also come 
 from other organs and according to Haberlandt also from the leucocytes. 2 
 Hanriot and others have detected, in the serum, lipases or esterases which 
 decompose butyrin and neutral fats and other esters. The occurrence 
 of butyrinases which split mono- as well as tributyrin has been recently 
 substantiated by Rona and Michaelis, while the property of this lipase 
 of splitting olein and other neutral fats is not generally acknowledged 
 (Arthus, Doyon and Morel 3 ). 
 
 This lipolytic property, if it exists to the extent that Hanriot ascribes to 
 it, must not be confounded with the transformation of fat into unknown sub- 
 stances soluble in water, a phenomenon first observed by Connstein and Michae- 
 lis and further studied by Weigert. The occurrence of such a body is positively 
 denied by G. Mansfeld. 4 
 
 Besides the above-mentioned enzymes and thrombin and the gly- 
 colytic enzymes that will be discussed later, several other enzymes have 
 been found in the blood-serum, namely, oxidases, catalases, proteolytic 
 enzymes, among which we must mention the polypepiide-splitting 
 enzymes studied by Abderhalden and collaborators, 5 also rennin and 
 several antienzymes. We cannot enter into the discussion of these, nor 
 of the many not chemically characterized bodies which have been called 
 toxines and antitoxines, immune bodies, alexines, Iwmoly sines, cytotoxines, 
 etc., and which have been discussed in Chapter I. The various enzymes 
 and antienzymes, and the above mentioned bodies are as a rule pre- 
 cipitated with the globulin, but differ among each other in that some are 
 
 'Rohmann: Rohmann and Hamburger, Ber. d. deutsch. chem. Gesellsch., 25 
 and 27; Pfliiger's Arch., 52 and 60; Bial. Ueber das diast. Ferm., etc., Inaug.-Diss. 
 Breslau, 1892 (older literature). See also Pfliiger's Arch., 52, 54, and 55; Wohlgemuth, 
 Bioch. Zeitschr., 21. 
 
 2 Wohlgemuth, 1. c; Moeckel and Rost, Zeitschr. f. physiol. Chem., 67; Clerk 
 and Loeper, Compt. Rend. soc. biol.. 66: Haberlandt, Pfliiger's Arch.. 132. 
 
 3 Hanriot, Compt. Rend. soc. biol.. 48 and 54. Compt. Rend. 123 and 132; Rona, 
 and Michaelis, Bioch. Zeitschr., 31; Rona, ibid.. 33; Arthus, Journ. de Physiol, et 
 de Pathol., 4; Doyon and Morel, Compt. Rend. soc. biol., 54; Achard and Clerc 
 (Lipase in Disease), Compt. Rend., 129, and Arch. d. med. expe>., 14. 
 
 * Connstein and Michaelis, Pfliiger's Arch., 65 and 69: Weigert, ibid., 82; Mansfeld, 
 Centralbl. f. Physiol, 21. 
 
 s Zeitschr. f. physiol. Chem., 51, 53, 55. 
 
BLOOD SERUM. 207 
 
 carried down by the euglobulin, while the others are carried down by 
 the pseudoglobulin fraction. 
 
 The non-protein organic constituents of the serum have been given 
 especial and careful study by E. Letsche l and he has found, besides the 
 previously known bodies, that the serum contains several acids, among 
 which there are tw r o nitrogeneous acids whose nature has not been studied. 
 These, including other nitrogenous substances found by him, represent 
 a part of the so-called rest nitrogen, i.e., that nitrogen which remains in 
 the serum after the complete removal of the coagulable proteins. As 
 representatives of the bodies occurring as rest nitrogen in the serum we 
 must in the first place mention area, also creatine, carbarriic acid, ammonia, 
 hippuric acid, phosphocarnic acid (Panella), traces of indol (Hervieux), 
 perhaps also uric acid found by Abeles 2 in human blood, while Letsche 
 could not find any in horse-blood. 
 
 According to Browtnski proteic acids (see Chapter XIV) occur in 
 the serum and Czernecki 3 has investigated the quantity of proteic 
 acid nitrogen in serum and transudates under different conditions. The 
 occurrence of proteoses is, as above mentioned, somewhat disputed. 
 We have several investigations on the occurrence of amino-acids (v. 
 Bergmann, Howell, Letsche, Abderhalden and others) which make 
 the occurrence of these very probable, and recently Bingel has been able 
 to show the presence of glycocoll in normal ox-blood. Otherwise the 
 amino-acids have often been sought for in normal blood but in vain; 
 still recently certain investigators like van Slyke and Meyer 4 have 
 show r n the presence of amino-acids in the blood under normal con- 
 ditions. In dog blood after 24 hours' starvation they found 3-5 milli- 
 grams of amino-acid nitrogen in 100 parts blood. Under pathological 
 conditions lysine (Neuberg and Richter 5 ), leucine and tyrosine have 
 been found. Also purine bases and bile acids have been found in the 
 serum under pathological conditions. That the quantity of rest nitro- 
 gen is larger during digestion than in starvation requires further con- 
 firmation. 6 
 
 1 Zeitschr. f. physiol. Chem., 53. 
 
 2 Panella, cited in Virehow's Jahresb., 1902, 150, Hervieux Compt. Rend. soc. 
 biol., 56; Abeles, Wien. med. Jahrb., 1887. 
 
 3 Browinski, Zeitschr. f. physiol. Chem., 54 and 58; Czernecki, Mary's Jahresb., 
 39 and 40. 
 
 4 v. Bergmann, Hofmeister's Beitrage, 6; Howell, Amer. Journ. of Physiol., 17; 
 Letsche, 1. c; Abderhalden, Zeitschr. f. physiol. Chem., 72; Bingel, ibid., 57; D. 
 v. Slyke and Meyer, Journ. of biol. Chem., 12. 
 
 5 Deutsch. med. Wochenschr., 1904. 
 
 6 v. Bergmann and Langstein, Hofmeister's Beitrage, 6; Hohlweg and Meyer, 
 ibid., 11. 
 
268 THE BLOOD. 
 
 As rest-carbon. Mancini x designates that carbon which is not precipitated 
 by phosphotungstic acid. It originates in great part from the urea and sugar 
 and amounts to 0.076-0.089 gram in 100 cc. 
 
 The pigments of the blood-serum are very little known. Besides 
 other pigments horse-serum contains, as first shown by Hammarsten, 
 bilirubin, which, according to Ranc, is the only pigment of the serum of 
 this animal. This pigment occurs, although in small amounts, sometimes 
 in the serum of other animals and, according to Biffi and Galli, 2 is 
 especially abundant in the blood of new-born. 
 
 Urobilin is not, according to Auche, Roth and. Herzfeld, a physi- 
 ological serum-pigment. Urobilinogen may occur in extraordinary 
 cases according to Hildebrandt, 3 and on allowing the blood in such 
 cases to stand urobilin may be formed therefrom. The yellow coloring- 
 matter of the serum seems to belong to the group of luteins, which 
 are often called lipochromes or fat-coloring matters. From ox-serum 
 Krukenberg 4 was able to isolate with amyl alcohol a so-called lipo- 
 chrome whose solution shows two absorption-bands, of which one encloses 
 the line F and the other lies between F and G. 
 
 The mineral bodies in serum and plasma are qualitatively, but not 
 quantitatively, the same. A part of the calcium, magnesium, and 
 phosphoric acid is removed on the coagulation of the fibrin. By means 
 of dialysis, the presence of sodium chloride, which forms the chief mass 
 or 60-70 per cent of the total mineral bodies, lime-salts, sodium car- 
 bonate, and traces of sulphuric and phosphoric acids and of potassium, 
 may be directly shown in the serum. 5 Traces of silicic acid, fluorine, 
 copper, iron, and manganese, are claimed to have been found in the serum. 
 As in most animal fluids, the chlorine and sodium are in the blood- 
 serum in excess of the phosphoric acid and potassium (the occurrence 
 of which in the serum is even doubted). The acids present in the ash 
 are not sufficient to saturate the bases found, a condition which shows 
 that a part of the bases is combined with organic substances, perhaps 
 proteins. This also coincides with the fact that the great part of 
 the alkalies does not exist in the serum as diffusible alkali compounds, 
 carbonate and phosphate, but as non-diffusible compounds, protein 
 combinations. According to Hamburger 37 per cent of the alkali of 
 the serum from horse-blood was diffusible and 63 per cent non-diffusible. 
 
 1 Bioch. Zeitschr., 26 and 32. 
 
 2 Hammarsten, see Maly's Jahresb., 8 (1878); Ranc, Compt. Rend. soc. biol., 62; 
 Biffi and Galli, Journ. de Physiol, et Path., 9 (1907). 
 
 ' Auch6, Compt. Rend. soc. biol., 67; Roth and Herzfeld, Deutsch. med. Wochenschr., 
 37; Hildebrandt, Munch. Med. Wochenschr., 57. 
 * Sitz.-Ber. d. Jen. Gesellsch. f. Med., 1885. 
 6 See Giirber, Verhandl. d. phys.-med. Gesellsch. zu Wurzburg, 23. 
 
BLOOD SERUM. 269 
 
 According to Rona and Takahashi 1 25-30 per cent of the calcium is 
 non-diffusible, probably combined with proteins. 
 
 Iodine, which seems to be habitually found, is also considered as 
 a mineral constituent of the plasma or serum (Gley and Bourcet), 
 while arsenic, although not found in all blood, occurs in human blood 
 (Gautier, Bourcet 2 ). Iodine occurs to a greater extent in menstrual 
 blood than in other blood and does not exist as a salt, but as an organic 
 compound (Bourcet). 
 
 The gases of the blood-serum, which consists chiefly of carbon dioxide 
 with only a little nitrogen and oxygen, will be described when treating 
 of the gases of the blood. 
 
 We have only a few analyses of blood-plasma. As an example the 
 results of the analyses of the blood-plasma of the horse will be given 
 below. The analysis No. 1 was made by Hoppe-Seyler. 3 No. 2 is the 
 average of the results of three analyses made by Hammarsten. The 
 figures are given for 1000 parts of the plasma. 
 
 No. 1. No. 2. 
 
 Water 908.4 917.0 
 
 Solids 91.0 82.4 
 
 Total proteins 77 . 09 . 5 
 
 Fibrin 10.1 0.5 
 
 Globulin 38 . 4 
 
 Seralbumin 24 . 
 
 Fat 1.2] 
 
 Extractive substances 4.01 , on 
 
 Soluble salts 0.4 12 - 9 
 
 Insoluble salts 1 . 7 J 
 
 Lewinsky 4 has determined the total proteins and the individual 
 proteins in the blood-plasma of man and animals with the following 
 results : 
 
 Total Protein. Albumin. Globulin. Fibrinogen. 
 
 Man 72.0 40.1 28.3 4.2 
 
 Dog 00.3 31.7 22.0 0.0 
 
 Sheep 72.9 38.3 30.0 4.0 
 
 Horse 80.4 28.0 47.9 4.5 
 
 Pig 80.5 44.2 29.8 0.5 
 
 Abderhalden has made complete analyses of the blood-serum of 
 several domestic animals. From these analyses, as well as from those 
 made by Hammarsten of the serum from human, horse, and ox-blood, 
 it follows that the amount of solids ordinarily varies between 70-97 
 p. m. The chief mass of the solids consists of proteins, about 55-84 
 p. m. In hens Hammarsten found much lower values, namely, 54 
 
 1 Hamburger, Arch. f. (Anat. u.) Physiol., 1898; Rona and Takahashi, Bioch. 
 Zeitschr., 31. 
 
 2 Gley and Bourcet, Compt. Rend., 130; Bourcet, ibid., 131; Gautier, ibid., 131. 
 
 3 Cit. from v. Gorup-Besanez's Lehrbuch der physiol. Chem., 4. Aufl., 340. 
 * Pfluger's Arch., 100. 
 
270 THE BLOOD. 
 
 p. m. solids, with only 39.5 p. m. protein, and Halliburton found only 
 25.4 p. m. protein in frog's blood. The relation between globulin and 
 seralbumin is, as shown by the analyses of Hammarsten, Hallibur- 
 ton, and Rubbrecht, 1 very different for various animals, but may also 
 vary considerably in the same species of animal. In human blood- 
 serum Hammarsten found more seralbumin than globulin, and the 
 relation of serglobulin to seralbumin was as 1:1.5. Lewinsky found the 
 relationship in man greater than 1, indeed 1:1.39-2.13. In regard to the 
 quantity of the remaining organic constituents of the serum we refer 
 the reader to Abderhalden's complete analyses. 
 
 In starvation it seems, as first found by Burckhardt and then sub- 
 stantiated by other investigators, that the quantity of globulins relative 
 to that of albumin in dogs and also in rats (Robertson 2 ), is increased. 
 According to Robertson, in the horse, ox and rabbit the reverse exists, 
 namely, the amount of albumin relative to the globulin increases in 
 starvation. A change in the relation with a decrease in the albumin 
 and an increase in the globulin may also occur in animals which have 
 been made sick or in part immune by inoculation with pathogenic 
 micro-organisms (Langstein and Mayer 3 ) . The total protein content 
 is raised in nearly all cases. The amount of fibrinogen in the plasma 
 is especially increased by pneumococci, streptococci, and pus-staphy- 
 lococci (P. Muller 4 ). 
 
 The quantity of mineral bodies in the serum has been determined by 
 many investigators. The conclusion drawn from the analyses is that 
 there exists a rather close correspondence between human and animal 
 blood-serum, and it is therefore sufficient to here give the analysis of C. 
 Schmidt 5 of (1) human blood, and Bunge and Abderhalden's analyses 
 (2) of serum of ox, bull, sheep, goat, pig, rabbit, dog, and cat. The results 
 correspond to 1000 parts by weight of the serum. 
 
 1 2 
 
 K 2 0.387-0.401 0.226-0.270 
 
 Na 2 4.290-4.290 4.251-4.442 
 
 CI 3.565-3.659 3.627-4.170 
 
 CaO 0.155-0.155 0.119-0.131 
 
 MgO 0.101 0.040-0046 
 
 P,0 5 (inorg.) 0.052-0.085 
 
 1 Abderhalden', Zeitschr. f. physiol. Chem., 25; Hammarsten, Pfliiger's Arch., 17; 
 Halliburton, Journ. of Physiol., 7; Rubbrecht, Travaux du laboratoire de l'institut 
 de physiologie de Liege, 5, 1896. 
 
 2 Burckhardt, Arch. f. exp. Path. u. Pharm., 16; Githens, Hofmeister's Beitrage, 
 5; see also Morawitz, ibid., 7, and Inagaki, Zeitschr. f. Biol., 49; Robertson, Journ. 
 of biol. Chem., 13. 
 
 3 Hofmeister's Beitrage, 5. 
 ' Ibid., 8. 
 
 * Cit. from Hoppe-Seyler, Physiol. Chem., 1881, p. 439. 
 
BLOOD SERUM. 271 
 
 A Macallum l has determined the quantity of mineral bodies in 
 the serum of certain , cold-blooded animals (fishes, shark, lobster and 
 others). The amount of sodium and chlorine in the serum of these animals 
 living in sea-water was much greater than in warm-blooded animals. 
 
 Even if we bear in mind that certain bodies, such as carbon dioxide, 
 arc driven oft" during incineration, and that other bodies, such as sul- 
 phuric acid and phosphoric acid, are formed from sulphurized and 
 phosphorized organic substances, still quantitative analyses like the 
 above are not sufficient for the scientific demands of to-day. They 
 do not show the true composition, and especially do not give an explana- 
 tion of the number of different ions present in the serum or in other fluids, 
 a question which is of the greatest physiological importance. An 
 answer to these questions is obtainable only by physico-chemical investiga- 
 tions, which have thus far been used chiefly in determining the molecular 
 concentration, the amount of electrolytes and non-electrolytes, and the 
 degree of dissociation. 
 
 The average depression of the freezing-point of mammalian blood 
 corresponds, as given in Chapter I, closely to a 9 p. m. (A = about 
 —0.56°) solution of common salt, and at the present time such a solution 
 is considered as a physiological salt solution for man and other mammalia. 
 In lower animals and fish the conditions are otherwise, as shown in 
 the above-mentioned chapter. 
 
 There are recorded a great number of investigations on the changes 
 in the osmotic pressure or the molecular concentration of the blood- 
 serum under various physiological conditions as w r ell as in disease, but 
 still it is no doubt too early to draw any definite conclusions from these 
 observations. 
 
 The degree of dissociation (see Chapter I) of sera has been determined 
 by several investigators, and according to Hamburger 2 it lies between 
 0.65 and 0.82. The molecular concentration, which represents the 
 total number of molecules and ions per liter, is according to Bvrgarsky 
 and Tangl, on an average about 0.320 mol. per liter. They also 
 found that about three-fourths of the total number of dissolved mole- 
 cules in blood-serum were electrolytes, although the serum contained 
 about 70-80 p. m. protein and 10 p. m. inorganic bodies, and also that 
 three-fourths of the quantity of electrolytes consisted of NaCl. 
 
 In the determination of the alkalinity of blood and blood-serum, 
 up to the present time, we have estimated the amount of alkali by titra- 
 tion with an acid. We cannot dispense v ith such d< terminations, although 
 
 iProc. Roy. Soc, ser. B., 82. 
 
 2 Osmotisher Druck und Ionenlehre, Wiesbaden, 1902-1904, where the literature 
 on the physical chemistry of the blood can be found. 
 
272 THE BLOOD. 
 
 they do not yield any information as to the true alkalinity, apart from 
 the fact that the results are dependent upon the indicator used, because 
 we understand as true alkalinity the concentration of the hydroxyl 
 ions. The Na2C03 is in aqueous solution more or less dissociated into 
 2Na + and C03 = , depending upon the dilution. The C03° ions com- 
 bine partly with the H + ions of the dissociated water, forming HCO3-, 
 and the corresponding HO~ ions produce the alkaline reaction. If now 
 by the addition of a little acid, a few of the HO" ions are removed, 
 the equilibrium is then disturbed, a new quantity of Na2C03 is dissociated, 
 and this process is repeated every time a new quantity of acid is added 
 until all the carbonate is dissociated. The dissociation of the carbonate 
 existing in the original concentration, upon which the number of HO~ 
 ions is dependent, cannot therefore be determined by titration. 
 
 For these reasons we generally determine the quantity of HO 
 and H ions in the serum and blood by methods based upon Nernst's 
 theory for the electromotive force of gas-chains. According to these 
 investigations it has been found that the concentration of the hydroxyl 
 ions in blood-serum and blood is only a little higher than in distilled 
 water (see Chapter I page 76, and the reaction of the blood below). 
 
 H. THE FORM-ELEMENTS OF THE BLOOD. 
 The Red Blood-corpuscles. 
 
 The blood-corpuscles are round, biconcave disks without membrane 
 and nucleus, in man and mammalia (with the exception of the llama, 
 the camel, and their congeners). In the latter animals, as also in birds, 
 amphibia, and fish (with the exception of the Cyclostoma) the cor- 
 puscles have in general a nucleus, are biconvex and more or less ellip- 
 tical. The size varies in different animals. In man they have an average 
 diameter of 7 to 8 ju (/x = 0.001 mm.) and a maximum thickness of 1.9 /x. 
 They are heavier than the blood-plasma or serum, and therefore sink 
 in these liquids. In the discharged blood they may sometimes lie with 
 their fiat surfaces together, forming a cylinder like a roll of coin (rouleaux). 
 The reason for this phenomenon, which is considered as an agglutination, 
 has not been sufficiently studied, but as it may be observed in defibrinated 
 blood it seems probable that the formation of fibrin has nothing to 
 do with it. 
 
 The number of red blood-corpuscles is different in the blood of various 
 animals. In the blood of man there are generally 5 million red cor- 
 puscles in 1 c.mm., and in woman 4 to 4.5 million. 
 
 The blood-corpuscles consist principally of two chief constituents, 
 the stroma, which forms the real protoplasm, and the intraglobular 
 contents, whose chief constituent is haemoglobin. We cannot state 
 
RED BLOOD-CORPUSCLES. 273 
 
 anything positive for the present in regard to a more detailed arrange- 
 ment, and the views on this subject are somewhat divergent. The two 
 following views are more or less related to each other. According to 
 one view the blood-corpuscles consist of a membrane which encloses a 
 haemoglobin solution, while the other view considers the stroma as a proto- 
 plasmic structure soaked with haemoglobin. This latter view is in accord 
 with the assumption as to an outside boundary-layer. Thus accord- 
 ing to Hamburger the stroma forms a protoplasmic net in whose meshes 
 there exists a red fluid or semi-fluid mass which consists in great meas- 
 ure of haemoglobin. This mass represents the water-attracting force 
 of the blood-corpuscles, and besides this it is also considered that the 
 outer protoplasmic boundary is semi-permeable, i. e., permeable to water 
 but not permeable to certain crystalloids. The researches of Koppe, 
 Albrecht, Pascucci, Rywosch, 1 and others indicate the presence of a 
 special envelope or boundary-layer, and there is no doubt that the outer 
 layer contains so-called lipoids, such as cholesterin, lecithin, and similar 
 bodies. 
 
 The red blood-corpuscles retain their volume in a salt solution which 
 has the same osmotic pressure as the serum of the same blood, although 
 they may change their form in such solutions, becoming more spherical, 
 and may also undergo a chemical change. Such a salt solution is iso- 
 tonic with the blood-serum, and its concentration for a NaCl solution is 
 approximately 9 p. m. for human and mammalian blood. A solution 
 of greater concentration, a hyperisotonic solution, abstracts water from 
 the blood-corpuscles until osmotic equilibrium is established, hence the 
 corpuscles shrink and their volumes become smaller. In solutions of 
 less concentration, hypisotonic solutions, the corpuscles swell, due to 
 the taking up of water, and this swelling may be so great, on diluting 
 the blood with water, that the haemoglobin is separated from the stroma 
 and passes into the watery solution. This process is called haemolysis 
 (see Chapter I). 
 
 A haemolysis may also be brought about by alternately freezing and 
 thawing the blood, as well as by the action of various chemical substances, 
 which act as protoplasmic poisons. These bodies are ether, chloroform 
 alkalies, bile-acids, solanin, saponin, and also the saponin substances, 
 which have a very strong haemoloytic action, also metabolic products of 
 bacteria, higher plants and animals (snakes, toads, bees, spiders and 
 others) and also bodies occurring in blood serum of normal or immunized 
 animals. 
 
 x See Hamburger, Osmotischer Druck und Ionenlehre, 1902; Koppe, Pfluger's 
 Arch., 99 and 107; Albrecht, Centralbl. f. Physiol., 19; Pascucci, Hofmeister's 
 Beitrage, 6; Rywosch, Centralbl. f. Physiol., 19. 
 
274 THE BLOOD. 
 
 When the haemoglobin is separated from the so-called stroma by a 
 sufficiently strong dilution with water the stroma is found in the solution 
 in a swollen condition. By the action of carbon dioxide, by the careful 
 addition of acids, acid salts, tincture of iodine, or certain other bodies, 
 this residue, rich in proteins, condenses, and in many cases the form of 
 the blood-corpuscles may be again obtained. This residue, the so- 
 called ghosts or stromata of the blood-corpuscles, can also be directly 
 colored in dilute blood by methyl violet and in this way detected, and 
 attempts have been made to isolate it for chemical investigation. In 
 the following pages we mean by the name stroma only that residue which 
 remains after the removal of haemoglobin and other bodies soluble in 
 water. 
 
 To isolate the stromata from the blood-corpuscles, they are washed 
 first by diluting the blood with 10-20 vols, of a 1-2 per cent common 
 salt solution and then separating the mixture by centrifugal force or 
 by allowing it to stand at a low temperature. This is repeated a few 
 times until the blood-corpuscles are freed from serum. These purified 
 blood-corpuscles are, according to Wooldridge, mixed with 5-6 vols, 
 of water and then a little ether is added until complete solution is obtained. 
 The leucocytes gradually settle to the bottom, a movement which may 
 be accelerated by centrifugal force, and the liquid which separates there- 
 from is very carefully treated with a 1-per cent solution of KHSO4 until 
 it is about as dense as the original blood. The separated stromata are 
 collected on a filter and quickly washed. Pasctjcci, 1 on the contrary, 
 treats the mass of corpuscles with 15-20 vols, of a one-fifth saturated 
 ammonium-sulphate solution, allows the corpuscles to settle, siphons 
 off the fluid, repeatedly centrifuges, allows the residue to dry quickly 
 (on porcelain plates) at the ordinary temperature, and then washes 
 with water until the blood-pigments and the other soluble bodies are 
 dissolved out. 
 
 Wooldridge found as constituents of the stromata lecithin, choles- 
 terin, nucleoalbumin, and a globulin which, according to Halliburton, 
 is probably a nucleoproteid which he calls cell-globulin. No nuclein 
 substances or seralbumin or proteoses could be detected by Hallibur- 
 ton and Friend. According to Pascucci, the stromata (from horse- 
 blood) consists of one-third cholesterin and lecithin (besides a little 
 cerebroside), and two-thirds protein substances and mineral bodies. 
 The nucleated red blood-corpuscles of the bird contain, according to 
 Plosz and Hoppe-Seyler, 2 a protein (nucleoprotein) which swells to a 
 slimy mass in a 10-per cent common salt solution, and which seems to 
 
 1 Hofmeister'fi Beitrage, 0. 
 
 * Wooldridge, Arch. f. (Anat. u.) Physiol., 1881,387; Halliburton and Friend, 
 Journal of Physiol., 10; Halliburton, ibid., 18; P16sz, Hoppe-Seyler's Med. chem. 
 Untersuch., 510. 
 
RED BLOOD-CORPUSCLES. 275 
 
 be closely related to the hyaline substance (hyaline substance of Rovida), 
 occurring in the lymph-cells. In the mass extracted by alcohol from 
 the blood-corpuscles of the hen, Ackermann found 3.93 per cent phos- 
 phorus and 17.2 per cent nitrogen, which on calculation gave 42.10 per 
 cent nucleic acid and 57.82 per cent histone. Piettre and Vila * found, 
 in the stromata, 0.3 per cent phosphorus in the horse and 2.3-2.6 per 
 cent in birds (ducks and hens), calculated on the ash-free substance. They 
 found the quantity of nitrogen to be 11.7 and 13.21 per cent for the horse 
 and dog respectively. The non-nucleated red blood-corpuscles are, 
 as a rule, very poor in protein, but rich in haemoglobin; the nucleated 
 corpuscles are richer in protein and poorer in haemoglobin than the 
 non-nucleated. The reducing substances, and in certain animal sugars, 
 probably also conjugated glucuronic acids and several enzymes, among 
 which occurs the proteolytic enzyme studied by Abderhalden and col- 
 laborators, 2 belong to the stromata. It is difficult to decide in many 
 cases whether the enzymes found in the blood belong to the fluid or to 
 the various kinds of form-elements. 
 
 A gelatinous, fibrin-like protein body may be obtained from the red 
 blood-corpuscles under certain circumstances. This fibrin-like mass 
 has been observed on freezing and then thawing the sediment of the 
 blood-corpuscles, or on discharging the spark from a large Ley den jar 
 through the blood, or on dissolving the blood-corpuscles of one kind of 
 animal in the serum of another (Landois, stroma-fibrin) ; i.e., in the so- 
 called hemagglutination, a clumping of the red blood-corpuscles into 
 clusters takes place. This agglutination can be brought about by bodies 
 similar to the haemolysines and also by serum constituents produced 
 normally or by immunization. It has not been shown that a fibrin for- 
 mation from the stroma takes place, nor is it probable. Fibrinogen 
 has been detected only in the red corpuscles of frog's blood (Alex. Schmidt 
 and Semmer 3 ). *•« 
 
 Closely related to the anatomical and chemical structure of the erythro- 
 cytes is the question, which is important for the metabolism in the blood, 
 as to the permeability of the erythrocytes, that is, their power of tak- 
 ing up substances of different kinds. This question as well as the per- 
 meability of the blood-corpuscles for anions under the influence of carbon 
 dioxide has been discussed in Chapter I, pages 7 and 8. 
 
 The mineral bodies of the red corpuscles will be treated in connection 
 with their quantitative constitution. 
 
 1 Ackermann, Zeitschr. f. physiol. Chem., 43; Piettre and Vila, Compt. Rend., 143. 
 - Zeitschr. f. physiol. Chem., 51, 53 and 55. 
 
 3 Landois, Centralbl. f. d. med. Wissensch., 1874, 421; Schmidt, Pfliiger's Arch., 
 11, 550-559. 
 
276 THE BLOOD. 
 
 The constituent of the blood-corpuscles existing in greatest quantity 
 is the red pigment haemoglobin. 
 
 Blood-pigments. 
 
 According to Hoppe-Seyler the coloring-matter of the red blood- 
 corpuscles is not in a free state, but combined with some other sub- 
 stance. The crystalline coloring-matter, the haemoglobin or oxyhaemo- 
 globin, which may be isolated from the blood, is considered, according 
 to Hoppe-Seyler, as a cleavage product of this compound, but it acts 
 in many ways unlike the questionable compound itself. This compound 
 is insoluble in water and uncrystallizable. It strongly decomposes 
 hydrogen peroxide without being oxidized itself; it shows a greater resist- 
 ance to certain chemical reagents (as potassium ferricyanide) than the 
 free coloring-matter; and, lastly, it gives off its loosely combined oxygen 
 much more easily in vacuum than the free pigment. To distinguish 
 between the cleavage products, the haemoglobin, and the oxyhaemoglobin, 
 Hoppe-Seyler calls the compound of the blood-coloring matter of the 
 venous blood-corpuscles phlebin, and that of the arterial arterin. Other 
 investigators, such as H. U. Kobert and Bohr, 2 the latter calling the 
 pigment of the blood-corpuscles hcemochrom, are of a similar opinion. 
 Since the above-mentioned combinations of the blood-coloring matters 
 with other bodies, for example (if they really do exist) with lecithin, have 
 not been closely studied, the following statements will apply only to the 
 free pigment, the haemoglobin. 
 
 The color of the blood depends in part on haemoglobin and in part 
 on a molecular combination of this substance with oxygen, the oxy- 
 hemoglobin. We find in blood after asphyxiation almost exclusively 
 haemoglobin, in arterial blood disproportionately large amounts of 
 oxyhemoglobin, and in venous blood a mixture of both. Blood-color- 
 ing matters are also found in striated as well as in certain smooth muscles, 
 and lastly in solution in different invertebrates, although this pigment 
 is not quite identical with that from higher animals. The quantity of 
 haemoglobin in human blood may indeed be somewhat variable under 
 different circumstances, but amounts to about 14 per cent on an average, 
 or 8.5 grams for each kilo of the weight of the body. 
 
 Haemoglobin belongs to the group of compound proteins, and yields 
 as cleavage products, besides very small amounts of volatile fatty acids 
 and other bodies, chiefly a protein globin, and a coloring-matter, hcemo- 
 
 2 Hoppe-Seyler, Zeitschr. f. physiol. Chem., 13, 479; H. U. Kobert, Das Wirbeltier- 
 blut in mikro-kristallogr. Hinsicht, Stuttgart, 1901; Bohr, Centralbl. f. Physiol., 17, 
 p. 688. 
 
BLOOD-PIGMENTS. 
 
 277 
 
 chromogen (about 4 per cent), containing iron, which in the presence cf 
 oxygen is easily oxidized into hcematin. 
 
 As first shown by Schunck and Marchlewski, and especially by the 
 work of the latter, a close relation exists between chlorophyll and the 
 blood-pigment, because a derivative of the first, phylloporphyrin, 
 stands very close in certain respects to a derivative of the blood-pigment 
 haematoporphyrin. By the investigations of Nencki in conjunction 
 with Marchlewski and Zaleski, 1 it was shown that haemopyrrol could 
 be prepared from the derivatives of both the leaf-pigment and the blood- 
 pigments by reduction, and also the investigations of Piloty and Will- 
 statter on chlorophyll and blood pigments have further developed 
 the interesting biological fact that the chlorophyll and blood pigments 
 are closely related bodies. 
 
 The haemoglobin prepared from different kinds of blood has not 
 exactly the same composition, which seems to indicate the presence 
 of different haemoglobins. The analyses by different investigators of 
 the haemoglobin from the same kind of blood do not always agree with 
 one another, which probably depends upon the somewhat varying methods 
 of preparation. The following analyses are given as examples of the 
 constitution of different haemoglobins: 
 
 Hemoglobin from the C H N S Fe 
 
 Dog 53.85 7.32 16.17 0.390 0.430 21.84 (Hoppe-Seyler) 
 
 " 54.57 7.22 16.38 0.568 0.336 20.93 (Jaquet) 
 
 Horse 54.87 6.79 17.31 0.650 0.470 19.73 (Kossel) 
 
 " 51.15 6.76 17.94 0.390 0.335 23.43 (Zinoffsky) 
 
 Ox 54.66 7.25 17.70 0.447 0.400 19.543 (Hufner) 
 
 Pig 54.17 7.38 16.23 0.660 0.430 21.360 (Otto) 
 
 " 54.71 7.38 17.43 0.479 0.399 19.602 (Hufner) 
 
 Guinea-pig 54.12 7.36 16.78 0.580 0.480 20.680 (Hoppe-Seyler) 
 
 Squirrel 54.09 7.39 16.09 0.400 0.590 21.440 
 
 Goose 54.26 7.10 16.21 0.540 0.430 20.690 
 
 Hen 52.47 7.19 16.45 0.857 0.335 22.500 (Jaquet) 
 
 That the repeatedly observed quantity of phosphorus in the haemo- 
 globin of birds (Inoko and others) is due to a contamination has been 
 proved by Abderhalden and Medigreceanu. In the haemoglobin 
 from the horse (Zinoffsky), the pig, and the ox (Hufner), we have 
 1 atom of iron to 2 atoms of sulphur, while in the haemoglobin from the 
 dog (Jaquet) the relation is 1 to 3. From the data of the elementary 
 analysis, as also from the amount of loosely combined oxygen, Hufner ' 
 has calculated the molecular weight of dog-haemoglobin as 14.129, and 
 
 Schunck and Marchlewski, Annal. d. Chem. u. Pharm., 278, 284, 288, 290; 
 Nencki, Ber. d. deutsch. chem. Gesellsch., 29; Marchlewski, and Nencki, Ber. d. d. 
 chem., Gesellsch., 34; Nencki and Zaleski, ibid., Marchlewski, Chem. Centralbl., 
 1902, I, 1016; Zaleski, Zeitschr. f. physiol. Chem., 37. The literature and the works 
 of Willstatter and Piloty will be given under haemopyrrol, page 297. 
 
278 THE BLOOD. 
 
 the formula Ce 3 6Hi625Ni64FeS3Cisi. According to the more recent 
 determinations of Hufner and Jaquet, ox-haemoglobin contains an 
 average of 0.336 per cent iron, and the human haemoglobin, according 
 to Butterfield 1 contains 0.334 per cent iron. From the iron a molec- 
 ular weight of 16,669 may be calculated. Barcroft and Hill have 
 arrived at exactly the same value by using an entirely different method 
 and Hufner and Gansser 2 have attempted to learn the size of the molec- 
 ular weight of haemoglobin by means of osmotic pressure determinations, 
 and they found the following approximate results: for horse-haemoglobin 
 15,115 and for ox-hsemoglobin 16,321. The haemoglobin from various 
 kinds of blood not only shows a diverse constitution, but also a different 
 solubility and crystalline form, and a varying quantity of water of crys- 
 tallization; hence we infer that there are several kinds of haemoglobin. 
 Bohr is a very zealous advocate of this supposition. He has been able 
 to obtain haemoglobins from clog- and horse-blood, by fractional crystalliza- 
 tion, which had different powers of combining with oxygen and contained 
 different quantities of iron. Hoppe-Seyler had already prepared two 
 different forms of haemoglobin crystals from horse-blood, and Bohr 
 concludes from all these observations that the ordinary haemoglobin 
 consists of a mixture of different haemoglobins. In opposition to this 
 statement, Hufner 3 has shown that only one haemoglobin exists in ox- 
 blood, and that this is probably true for the blood of many other animals. 
 Oxyhemoglobin, which has also been called h^ematoglobulin or 
 HjEMatocrystallin, is a molecular combination of haemoglobin and 
 oxygen. For each molecule of haemoglobin 1 molecule of oxygen is 
 present, as shown by the investigations of Hufner as well as Hufner 
 and Gansser, and the amount of loosely combined oxygen which is united 
 to 1 gram of haemoglobin (of the ox) has been determined by Hufner 4 
 as 1.34 cc. (calculated at 0° C. and 760 mm. mercury). 
 
 According to Bohr, the facts are different. He differentiates between four 
 oxyhemoglobins, according to the quantity of oxygen which they absorb, namely 
 
 1 Hoppe-Seyler, Med. chem. Untersuch., 370; Jaquet, Zeitschr. f. physiol. Chem., 
 14, 296; Kossel, ibid,. 2, 150; ZinofTsky, ibid., 10; Hufner, Beitr. z. Physiol., Festschr. 
 f. C. Ludwig, 1887, 74-81, Journ. f. prakt. Chem. (N. F.), 22; Otto, Zeitschr. f. physiol. 
 Chem., 7; Inoko, ibid., 18; Abderhalden and Medigreceanu, ibid., 59; Hufner and 
 Jaquet, Arch. f. (Anat. u.) Physiol., 1894; E. Butterfield, Zeitschr. f. physiol. Chem., 
 02. 
 
 •-' Barcroft and Hill, Journ. of Physiol. 30; Hufner and Gansser, Arch. f. (Anat. u.) 
 Physiol., 1907. 
 
 : ' Bohr, "Sur les combinaisons de l'h6moglobine avec I'oxygene," Extrait du 
 Bulletin de I'Academie Royale Danoise des sciences, 1890; also Centralbl. f. Physiol. 
 1890, '-'4'.). Boppe-Seyler, Zeitschr. f. physiol. Chem., 2; Hufner, Arch. f. (Anat. u.) 
 Physiol., L894 
 
 1 Arch. f. (Anat. u.) physiol., 1901, Suppl. 
 
OXYHEMOGLOBIN. 279 
 
 a . t fi. ) y. and 6-oxyhsemoglobin, all having the same absorption-spectrum, and 1 
 pram combining with respectively 0.4, 0.8, 1.7, and 2.7 cc. oxygen at the tem- 
 perature of the room and with an oxygen pressure of 150 mm. mercury. The 
 7-oxyha>moglobin is the ordinary one obtained by the customary method of 
 preparation. Bohk designates as a-oxyhaunoglobin the crystallin powder 
 obtained by drying Y-oxyhsemoglobin in the air. On dissolving a-oxyhaemo- 
 globin in water it is converted into j3-oxylurmoglobin without decomposition, 
 and the quantity of iron is increased. On keeping a solution of 7-oxyhaimoglobin 
 in a sealed tube it is transformed into 5-oxhyaemoglobin, although the exact 
 conditions under which this change takes place are not known. According to 
 Hufner ' these are nothing but mixtures of genuine and partly decomposed 
 haemoglobins. 
 
 The ability of haemoglobin to take up oxygen seems to be a function 
 of the iron it contains, and when this is calculated as about 0.33-0.40 
 per cent, then 1 atom of iron in the haemoglobin corresponds to about 
 2 atoms or 1 molecule of oxygen. By increasing the partial pressure as 
 well as by increasing the quantities of oxygen, the haemoglobin in solu- 
 tion takes up more oxygen, until it is completely saturated, when 1 mole- 
 cule of haemoglobin is combined with 1 molecule of oxygen. With reduced 
 oxygen pressure a dissociation must naturally take place and oxygen 
 is given off, and a re-formation of haemoglobin takes place, and this makes 
 it possible to expel completely the oxygen from an oxyhaemoglobin solu- 
 tion or blood by means of vacuum, or by passing an indifferent gas 
 through the solution. The equilibrium between oxyhaemoglobin, haemo- 
 globin, and oxygen depends, therefore, according to Hufner, upon 
 a mass action, corresponding to the formula Hb-f-02<=^Hb02. Bohr 2 
 has arrived at the conclusion that not only a double dissociation takes 
 place, in which a dissociation of the oxygen-iron combination in the 
 oxyhaemoglobin occurs, but also a dissociation of the haemoglobin 
 into a ferruginous as well as into a non-ferruginous part. Correspond- 
 ingly he has suggested another formula and hence the dissociation curves 
 for oxyhaemoglobin given by Hufner and Bohr are different. 
 
 Important investigations have recently been carried out on this 
 question by Barcroft and his co-workers Camis and Roberts from 
 which it follows that a generally valid dissociation curve cannot be given, 
 as the curve direction is dependent upon the nature and concentration 
 of the salts present in the solution. A haemoglobin solution with the salts 
 of the blood-corpuscles of the dog gives a dissociation curve of dog-blood 
 while with the salts from human blood-corpuscles it gives a curve like 
 human blood. In the presence of salts the dissociation follows Bohr's 
 formula, and on the contrary while a salt-free haemoglobin solution follows 
 the oxygen combination according to the mass-action law of Hufner. 
 
 1 Arch. f. (Anat. u.) physiol., 1894. 
 
 2 Bohr, Centralbl. f. physiol., 17, pp. 682 and 688. 
 
280 THE BLOOD. 
 
 Thus far there does not seem to be any necessity for considering the 
 gas combination in the blood and in haemoglobin solutions to be adsorp- 
 tion processes as suggested by W. Ostwald. 1 
 
 That the gas combining ability of an isolated pure haemoglobin cannot 
 be compared with the gas combining ability of the so-called native haemo- 
 globin of the blood has been suggested by many experimenters. In this 
 connection we must mention the observations of Manchot 2 who found 
 that the combining ability of the blood for gases such as O2, CO,NO,C2H4 
 could be increased at least to a certain limit by increasing the dilution 
 so that at 8-10 times the dilution the combining power was close to the 
 limit value of 2 mol. gas for each atom of iron. 
 
 The elucidation of these mentioned conditions is of the greatest 
 importance, as the knowledge of the various conditions which influence 
 the taking up and the giving up of oxygen by the haemoglobin is of the 
 greatest importance for our knowledge of the taking up of oxygen in the 
 lungs and the giving up of the same to the tissues. 
 
 Oxyhaemoglobin which is generally considered as a weak acid, is 
 according to Gamgee, 3 dextrorotatory. The specific rotation for light 
 of medium wave-length of C is (a) C = about +10°, which corresponds 
 also for carbon-monoxide haemoglobin. The haemoglobin is also, like 
 carbon-monoxide haemoglobin (COHb) and methaemoglobin (MHb), 
 diamagnetic, while the haematin, which is richer in iron, is strongly mag- 
 netic (Gamgee 4 ). On passing an electric current through an oxyhaemo- 
 globin solution, the pigment first separates unchanged at the anode in a 
 colloidal but still soluble form, and is then gradually transferred to the 
 cathode in the colloidal state (Gamgee 5 ) . According to Gamgee, the 
 haemoglobin probably exists in such a colloidal condition in the blood- 
 corpuscles. 
 
 Oxyhaemoglobin has been obtained in crystals from several varieties 
 of blood. These crystals are blood-red, transparent, silky, and may 
 be 2-3 mm. long. The oxyhaemoglobin from squirrel's blood crystallizes 
 in six-sided plates of the hexagonal system; the other varieties of blood 
 yield needles, prisms, tetrahedra, or plates which belong to the rhombic 
 system. 6 The quantity of water of crystallization varies between 
 
 1 Barcroft with Camis, Journ. of Physiol., 39; with Roberts, ibid.; W. Ostwald, 
 Kolloid-Zeitschr., 2, cited in Maly's Jahresb., 38, 187. 
 
 2 Annal. d. Chem. u. Pharm., 370 and Zeitschr. f. physiol. Chem., 70. 
 
 3 Hofmeister's Beitrage, 4. 
 
 4 Proceedings of Roy. Society, 68. 
 « Itnd., 70. 
 
 6 The observation of Uhlik (Pfliiger's Arch., 104) that the haemoglobin from horse- 
 blood can also crystallize in hexagonal plates seems to be due to the fact that he had 
 haemoglobin and not oxyhaemoglobin. 
 
OXYHEMOGLOBIN. 281 
 
 3-10 per cent for the different oxyhemoglobins. When completely 
 dried at a low temperature over sulphuric acid the crystals may be 
 heated to 110-115° C. without decomposition. At higher temperatures, 
 somewhat above 160° C, they decompose, giving an odor of burned horn, 
 and leave, after complete combustion, an ash consisting of oxide of 
 iron. The oxyhemoglobin crystals from difficultly crystallizable blood, 
 for example from such as ox's, human, and pig's blood, are easily 
 soluble in water. The oxyhemoglobins from easily crystallizable 
 blood, as from that of the horse, dog, squirrel, and guinea-pig, are soluble 
 with difficulty in the order above given. The oxyhemoglobin dissolves 
 more easily in a very dilute solution of alkali carbonate than in pure water, 
 and this solution may be kept. The presence of a little too much alkali 
 causes the oxyhemoglobin to decompose quickly. The crystals are 
 insoluble in absolute alcohol without decolorization. According to 
 Nencki l it is converted into an isomeric or polymeric modification, 
 called by him parahoemoglobin. Oxyhemoglobin is insoluble in ether, 
 chloroform, benzene, and carbon disulphide. 
 
 A solution of oxyhemoglobin in water is precipitated by many metallic 
 salts, but is not precipitated by sugar of lead or basic lead acetate. On 
 heating the watery solution it decomposes at about 70° C, and splits 
 off protein and hematin when sufficiently heated. It is also readily 
 decomposed by acids, alkalies, and many metallic salts. It gives the 
 ordinary reactions for proteins with those protein reagents which first 
 decompose the oxyhemoglobin with the splitting off of protein. Oxy- 
 hemoglobin, like the other blood-pigments, has a direct oxidizing action 
 upon tincture of guaiacum. It has, on the other hand, like all blood- 
 pigments containing iron, the property of an " ozone transmitter " in 
 that it turns tincture of guaiacum blue in the presence of reagents con- 
 taining peroxide, such as old turpentine. 
 
 A sufficiently dilute solution of oxyhemoglobin or arterial blood 
 shows a spectrum with two absorption-bands between the Fraun- 
 hofer lines D and E (spectrum Plate 1). The one band, a, which is nar- 
 rower but darker and sharper, lies on the line D; the other, broader, 
 less defined and less dark band, /3, lies at E. The middle of the first 
 band corresponds to a wave-length X = 579 and the second X = 542. On 
 dilution the band /3 first disappears. By increased concentration of the 
 solution the two bands become broader, the space between them smaller 
 or entirely obliterated, and at the same time the blue and violet part 
 of the spectrum is darkened. Besides these two bands we can also observe 
 
 1 Nencki and Sieber, Ber. d. d. chem. Gesellsch., 18. According to Kriiger (see 
 Biochem. Centralbl., I, 40, 463) haemoglobin is somewhat changed by alcohol as well 
 as by chloroform. 
 
282 THE BLOOD. 
 
 by the aid of special appliances (L. Lewin, Miethe, and Stenger) the 
 band first described by Soret and then by Gamgee in the ultra-violet 
 portion. This violet band, X = 415, is of importance in the detection 
 of very small quantities of blood. While the two oxyhemoglobin bands 
 are still detectable in a dilution of 1 : 14, 700 the violet band may be seen, 
 according to Lewin, Miethe and Stenger l in a dilution of 1 :40,000. 
 
 The observation of Piettre and Vila that so-called laky blood and oxyhsemo- 
 globin solutions in thick layers also show a third band in the red (X = 634) depends 
 in all probability, as also claimed fey Ville and Derrien, upon a partial forma- 
 tion of niethsemoglobin winch according to Aron 2 exists preformed in all blood. 
 
 A great many methods have been proposed for the preparation of 
 oxyhemoglobin crystals, but in their chief features they all agree with 
 the following one suggested by Hoppe-Seyler: The washed blood- 
 corpuscles (best those from the dog or the horse) are stirred with 2 
 vols, of water and then shaken with ether. After decanting the ether and 
 allowing the ether which is retained by the blood solution to evaporate 
 in an open dish in the air, cool the filtered blood solution to 0° C, add 
 while stirring one-fourth vol. of alcohol also cooled, and allow to stand 
 a few days at —5° to —10° C. The crystals which separate may be 
 repeatedly recrystallized by dissolving in water of about 35° C, cooling, 
 and adding cooled alcohol as above. Lastly, they are washed with 
 cooled water containing alcohol (one-quarter vol. alcohol) and dried 
 in vacuum at 0° C. or a lower temperature. 3 
 
 For the preparation of oxyhemoglobin crystals in small quantities 
 from easily crystallizable blood, it is often sufficient to stir a drop of 
 blood with a little water on a microscope slide and allow the mixture 
 to evaporate so that the drop is surrounded by a dried ring. After 
 covering with a cover-glass, the crystals gradually appear radiating from 
 the ring. These crystals are formed more surely if the blood is first 
 mixed with some water in a test-tube and shaken with ether, and a drop 
 of the lower deep-colored liquid treated as above on the slide. 
 
 Haemoglobin, also called reduced haemoglobin or purple cruorin 
 (Stokes 4 ), occurs only in very small quantities in arterial blood, in 
 larger quantities in venous blood, and is almost the only blood-coloring 
 matter after asphyxiation. 
 
 Hemoglobin is much more soluble than the oxyhemoglobin, and 
 it can therefore be obtained as crystals only with difficulty. These 
 
 1 Soret, cited in Maly's Jaresb., 8; Gamgee, Zeitschr. f. Biol., 34; Lewin, Miethe 
 and Stenger, Pfliiger's Arch., 118; Lewin and Miethe, ibid., 121. 
 
 2 Piettre and Vila, Compt. Rend., 140; Ville and Derrien, ibid., 140; Aron, Biochem. 
 Zeitschr., 3. 
 
 3 In regard to the preparation of oxyhemoglobin, see also Hoppe-Seyler-Thier- 
 felder's Handbuch, 8. Aufl.; also the works cited in footnote 1, p. 278; also Schuur- 
 manns-Stekhoven, Zeitschr. f. physiol. Chem., 33, 296; see also Bohr, Skand. Arch, 
 f. Physiol., 3; J. Offringa, Bioch. Zeitschr., 28. 
 
 * Philosophical Magazine, 28, No. 190, Nov., 1864. 
 
METHEMOGLOBIN. 283 
 
 crystals are as a rule isomorphous with the corresponding oxyhemo- 
 globin crystals, but are darker, having a shade toward blue or purple, 
 and are decidedly more pleochromatic. The haemoglobin from horse- 
 blood has also been obtained by Uhlik x in hexagonal plates. Its 
 solutions in water are darker and more violet or purplish than solu- 
 tions of oxyhemoglobin of the same concentration. They absorb the 
 blue and the violet rays of the spectrum in a less marked degree, but 
 strongly absorb the rays lying between C and D. In proper dilution 
 the solution shows a spectrum with one broad, not sharply defined band 
 between D and E, whose darkest part corresponds to the wave-length 
 X = 559 (spectrum Plate, 2). This band does not lie in the middle 
 between D and E, but is toward the red end of the spectrum, a little 
 over the line D. This pigment also gives a band in the ultra-violet, 
 X = 429. A haemoglobin solution actively absorbs oxygen from the air 
 and is converted into an oxyhemoglobin solution. 
 
 A solution of oxyhemoglobin may be easily converted into a solution 
 having the spectrum of hemoglobin by means of a vacuum, by passing 
 an indifferent gas through it, or by the addition of a reducing substance, 
 as, for example, an ammoniacal ferrous-tartrate solution (Stokes' reduc- 
 tion liquid). If an oxyhemoglobin solution or arterial blood is kept in 
 a sealed tube, we observe a gradual consumption of oxygen and a reduc- 
 tion of the oxyhemoglobin into hemoglobin. If the solution has a 
 proper concentration, a crystallization of hemoglobin may occur in the 
 tube at lower temperatures (Hufner 2 ). 
 
 Methaemoglobin. This name has been given to a coloring-matter 
 which is easily obtained from oxyhemoglobin as a transformation prod- 
 uct and which has been correspondingly found in transudates and cystic 
 fluids containing blood, in urine in hematuria or hemoglobinuria, and 
 also in urine and blood on poisoning with potassium chlorate, amyl 
 nitrite or alkali nitrite, and many other bodies. 
 
 Methemoglobin does not contain any oxygen in molecular or dis- 
 sociable combination, but still the oxygen seems to be of importance in 
 the formation of methemoglobin, because it is formed from oxyhemo- 
 globin and not from hemoglobin in the absence of oxygen or oxidizing 
 agents. If arterial blood be sealed up in a tube, it gradually consumes 
 its oxygen and becomes venous, and by this absorption of oxygen a little 
 methemoglobin is formed. The same occurs on the addition of a small 
 quantity of acid to the blood. By the spontaneous decomposition 
 of blood some methemoglobin is formed, and by the action of ozone, 
 potassium permanganate, potassium ferricyanide, chlorates, nitrites, 
 
 1 Pfluger's Arch., 104. 
 
 2 Zeitschr. f. physiol. Chem., 4; see also Uhlik, 1. c. 
 
284 THE BLOOD. 
 
 nitrobenzene, pyrogallol, pyrocatechin, acetanilide, and certain other 
 bodies on the blood an abundant formation of methsemoglobin takes place. 
 By the action of light, Hasselbach, 1 especially by the use of rays hav- 
 ing a wave-light below 310 n n, obtained methsemoglobin from oxyhemo- 
 globin, but not from haemoglobin in the absence of oxygen, and by this 
 behavior pure methsemoglobin can be prepared. 
 
 According to the investigations of Hufner, Kulz, and Otto 2 
 methsemoglobin contains just as much oxygen as oxyhsemoglobin, but 
 it is more strongly combined, a view which is accepted by most investiga- 
 tors. According to Hufner and v. Zeynek we can admit in the methsemo- 
 globin formation of an expulsion of oxygen and a combination of two 
 
 ,OH 
 hydroxyl groups; methsemoglobin would then be Hb<^ . Accord- 
 
 N OH 
 ing to others, Hoppe-Seyler, Kuster, Letsch the methsemoglobin 
 contains less oxygen than the oxyhsemoglobin and is HbO or HbOH. 
 A methsemoglobin solution is converted into a haemoglobin solution 
 by reducing substances. The reaction taking place in the forma- 
 tion of methsemoglobin from oxyhsemoglobin by the action of potassium 
 ferricyanide has been quantitatively followed by v. Reinbold. 3 He 
 found that one molecule of K3Fe(CN)6 was necessary to transform 1 
 molecule oxyhsemoglobin or to drive off 1 molecule of oxygen from the 
 oxyhsemoglobin. The reaction takes place according to the equation: 
 
 /° 
 Hb< | +K 3 Fe(CN) 6 -|-H20 = Hb.OH+K3HFe(CN)6+02 
 
 x O 
 
 and from his investigations he gives the formula Hb.OH to methsemo- 
 globin, in correspondence to the views of Kuster. 
 
 According to Hufner and Reinbold 4 1 gram methsemoglobin can 
 take up 2.685 cc. nitric oxide. 
 
 Methsemoglobin crystallizes, as first shown by Hufner and Otto, 
 in brownish-red needles, prisms, or six-sided plates. It dissolves easily 
 in water; the solution has a brown color and becomes a beautiful red 
 on the addition of alkali. The solution of the pure substance is not 
 precipitated by basic lead acetate alone, but by basic lead acetate and 
 
 1 Bioch. Zeitschr., 19. 
 
 *See Otto, Zeitschr. f. physiol. Chem., 7; v. Zeynek, Arch. f. (Anat. u.) Physiol., 
 1899; Hufner, ibid. 
 
 3 Kuster, Zeitschr. f. physiol. Chem., 66; Letsche, ihid., 80; v. Reinbold, Zeitschr. 
 f. physiol. Chem., 85. 
 
 4 Arch. f. (Anat. u.) Physiol., 1904, Suppl. 
 
METH.EMOGOBLV OYANMETHiEMOGLOBIN. 285 
 
 ammonia. The a l»<>rpt ion-spectrum of a watery or acidified solution 
 of met haemoglobin is, according to Jaderholm and Bertin-Sans, very 
 similar to that of haematin in acid solution, but is easily distinguished 
 from the latter since, on the addition of a little alkali and a reducing 
 substance, the former passes over to the spectrum of reduced haemoglobin, 
 while a haematin solution under the same conditions gives the spectrum 
 of an alkaline haemochromogen solution (see below). According to 
 Araki and Dittrich, a neutral or faintly acid methaemoglobin solution 
 shows only one characteristic band, a, between C and D, whose middle 
 corresponds to about X = 634. The two bands between D and E are 
 only due to contamination with oxyhsemoglobin (Menzies, Lewin, 
 Miethe and Stenger. According to Hasselbach's l experience a 
 pure neutral solution of methaemoglobin gives four absorption bands cor- 
 responding to a maxima X = 630, 580, 540 and 500. Methaemoglobin 
 in alkaline solution shows two absorption-bands which are like the 
 two oxy haemoglobin bands, but they differ from these in that the band 
 /S is stronger than a. By the side of the band a and united with it by a 
 shadow lies a third fainter band between C and D, near to D. (Spec- 
 trum Plate, 4.) 
 
 The claims as to the action of sodium fluoride upon haemoglobin and methaemo- 
 globin are somewhat contradictory. 2 
 
 Crystallized methaemoglobin may be easily obtained by treating a 
 concentrated solution of oxyhemoglobin with a sufficient quantity of 
 concentrated potassium-ferricyanide solution to give the mixture a porter- 
 brown color. After cooling to 0° C. add cne-fourth vol. cooled alcohol 
 and allow the mixture to stand a few days in the cold. The crystals 
 may be easily purified by recrystallizing from water by the addition 
 of alcohol. According to Hasselbach this method ordinarily gives 
 imnure products while a pure preparation can be obtained by the action 
 of i^ht (see above). 
 
 Cyanmethaemoglobin (cyanhaemoglobin) is, according to Haldaxe. identical 
 with photomethaemoglobin (Bock), which is produced by the influence of sun- 
 light upon a methaemoglobin solution containing potassium ferricyanide. It 
 wu- first carefully described by R. Robert and obtained in a crystalline form 
 by v. Zeyxek. 3 It is immediately formed in the cold by the action of a hydro- 
 cyanic-acid solution upon methaemoglobin, but is formed by its action upon oxy- 
 
 1 Jaderholm, Zeitschr. f. Biol. 16; Bertin-Sans. Comp. Rend., 106; Araki, Zeitschr. f. 
 physiol. Chem., 14; Dittrich. Arch. f. exp. Path. u. Pharm.. 29: Menzies, Journ. of 
 Physiol., 17; Lewin and collaborators, footnote 1, page 282: Hasselbach, Bioch. 
 Zeitschr., 19, and Proceedings of the 7th Internat. Congr. of Appl. Chem., London, 
 1909. Important references on methaemoglobin are given by Otto, Pfluger's Arch., 31. 
 
 2 Piettre and Vila, Compt. Rend., 140; Ville and Derrien, ibid., 140. 
 
 3 Haldane, Journ. of Physiol., 25; Bock, Skand. Arch. f. Physiol., 6: Robert, 
 Pfluger's Arch.. 82; v. Zeynek, Zeitschr. f. physiol. Chem., 33. See also Leers, 
 Biochem. Zeitschr., 12. 
 
286 THE BLOOD. 
 
 hsemoglobin only at the body temperature. The neutral or faintly alkaline 
 solutions show a spectrum which is very similar to the hsemoglobin spectrum. 
 The question as to a special cyanmethsemoglobin is still disputed. 
 
 Acid haemoglobin is a coloring-matter produced by the action of very weak 
 acids upon oxyhemoglobin, which according to Harnack * is not, as used to be 
 admitted, identical with methsemoglobin. 
 
 Carbon-monoxide Haemoglobin 2 is the molecular combination between 
 
 1 molecule of hsemoglobin and 1 molecule of CO, according to Hufner, 3 
 which contains 1.34 cc. of carbon monoxide (at 0° and 760 mm. Hg) 
 for 1 gram haemoglobin. This combination is stronger than the oxygen 
 combination of hsemoglobin. The oxygen is for this reason easily driven 
 out of oxyhsemoglobin by carbon monoxide, and this explains the poison- 
 ous action of this gas, which kills by the expulsion of the oxygen of the 
 blood. In regard to the division of the blood-pigments between the car- 
 bon monoxide and oxygen under different partial pressures of both gases 
 in the air, we must refer to the investigations of Hufner, Douglas and 
 Haldane. 4 
 
 The carbon monoxide can be driven out by a vacuum as well as by 
 passing an indifferent gas, or oxygen, or nitric oxide, through the solu- 
 tion for a long time, and in these cases hsemoglobin, oxyhsemoglobin, 
 or nitric-oxide hsemoglobin are formed. The carbon-monoxide is also 
 expelled by potassium ferricyanide and methaemoglobin is formed 
 (Haldane 5 ) . The above-mentioned behavior found by Manchot for 
 the absorption of oxygen, namely, that the amount of gas taken up 
 increases w r ith the dilution of the blood so that for every atom of iron 
 
 2 mol. of gas are absorbed applies also for the carbon-monoxide hsemo- 
 globin as well as for the nitric-oxide hsemoglobin, which will be discussed 
 further on. 
 
 Carbon-monoxide hsemoglobin is formed by saturating blood or 
 a hsemoglobin solution with carbon monoxide, and may be obtained 
 as crystals by the same means as oxyhsemoglobin. These crystals are 
 isomorphous with the oxyhsemoglobin crystals, but are less soluble and 
 more stable, and their bluish-red color is more marked. For the detec- 
 
 1 Zeitschr. f. physiol. Chem., 26. 
 
 2 In reference to carbon-monoxide haemoglobin, see especially Hoppe-Seyler, Med.- 
 chem. Untersuch., 201; Centralbl. f. d. med. Wissensch., 1S64 and 1865; Zeitschr. 
 f. physiol. Chem., 1 and 13. 
 
 3 Arch. f. (Anat. u.) Physiol., 1894. On the dissociation constant of carbon- 
 monoxide hsemoglobin, see ibid., 1895. In regard to the contradictory statements 
 of Saint-Martin and others and their disapproval, see Hufner, Arch. f. (Anat. u.) 
 Physiol., 1903. 
 
 4 Hufner, Arch. f. exp. Path. u. Pharm., 48; Douglas and Haldane, Journ. of 
 Physiol., 44. 
 
 6 Journ. of Physiol., 22. 
 
CARBON-MONOXIDE HAEMOGLOBIN. 287 
 
 tion of carbon-monoxide haemoglobin, its absorption-spectrum is of the 
 
 greatest importance. This spectrum shows two bands which are very 
 similar to those of oxyhemoglobin, hut they occur more toward the violet 
 part of the spectrum. The middle of the first hand corresponds to 
 X = 570, and the second to X = 542 (Lewix, Miethe and Stenger). 
 These bands do not change noticeably on the addition of reducing 
 substances; this constitutes an important difference between carbon- 
 monoxide haemoglobin and oxyhemoglobin. If the blood contains oxy- 
 hemoglobin and carbon-monoxide haemoglobin at the same time, we 
 obtain on the addition of a reducing substance (ammoniacal ferro-tar- 
 trate solution) a mixed spectrum originating from the haemoglobin and 
 carbon-monoxide haemoglobin. Carbon-monoxide haemoglobin also gives 
 a band in the violet X = dd6. 
 
 A great many reactions have been suggested for the detection of 
 carbon-monoxide haemoglobin in medico-legal cases. A simple and at 
 the same time a good one is Hoppe-Seyler's alkali test. The blood is 
 treated with double its volume of caustic-soda solution of 1.3 sp. gr., 
 by which ordinary blood is converted into a ding}' brownish mass, which 
 when spread out on porcelain is brown with a shade of green. Carbon- 
 monoxide blood gives under the same conditions a red mass, which if 
 spread out on porcelain shows a beautiful red color. Several modifica- 
 tions of this test have been proposed. Another very good reagent is tan- 
 nic acid, which gives with dilute normal blood a brownish-green precip- 
 itate and with carbon-monoxide blood a pale crimson-red precipitate. 1 
 
 As according to Bohr there are several oxyhemoglobins, so also accord- 
 ing to Bohr and Bock,'- there are several carbon-monoxide hemoglobins, with 
 different amounts of carbon monoxide. As haemoglobin can unite with oxygen 
 and carbon dioxide simultaneously, as shown by Bohr and Troup, so also can it 
 unite with carbon monoxide and carbon dioxide simultaneously and independently 
 of each other. 
 
 Carbon-monoxide methaemoglobin has been prepared by Weil and v. Axrep 
 by the action of potassium permanganate on carbon-monoxide haemoglobin, 
 but this is contradicted by Bertix-Saxs and Moitessier. 3 Sulphur methaemo- 
 globin is the name given by Hoppe-Seyler to that coloring-matter which is 
 formed by the action of sulphureted hydrogen upon oxyhemoglobin and which 
 is generally designated sidphcemoglobin. The solution has a greenish-red, dirty 
 color, and shows two absorption-bands between C and D. This coloring-matter 
 is claimed to be the greenish color seen on the surface of putrefying flesh. Accord- 
 ing to Harxack the conditions are different when H>S is passed through an 
 oxygen-free solution of hemoglobin (or carbon-monoxide haemoglobin). The 
 
 1 In regard to this test (as suggested by Kunkel) and others we refer to Kostin, 
 Pfliiger's Arch., S4, which contains a very excellent summary of the literature on the 
 subject. See also de Domenicis, Chem. Centralbl., 1908, 2, p. 06. 
 
 2 Centralbl. f. Physiol., 8, and Mary's Jahresber., 25. 
 
 3 v. Anrep, Arch. f. (Anat. u.) Physiol., 1880; Sans and Moitessier, Compt. Rend., 
 113. 
 
288 THE BLOOD. 
 
 sulphsemoglobin thus formed shows one band in the red between C and D. 
 According to Clarke and Hurtley ' the formation of sulphsemoglobin takes 
 place after the reduction to haemoglobin. 
 
 Carbon-dioxide Haemoglobin, Carbohcemoglobin. Haemoglobin, accord- 
 ing to Bohr and Torup, 2 also forms a molecular combination with 
 carbon dioxide whose spectrum is similar to that of haemoglobin. Accord- 
 ing to Bohr there are three different carbohsemoglobins, namely, a-, 
 /3-, and 7-carbohsemoglobin, in which 1 gram combines with respectively 
 1.5, 3, and 6 cc. CO2 (measured at 0° C. and 760 mm.) at 18° C. and a 
 pressure cf 60 mm. mercury. If a hsemoglobin solution is shaken with a 
 mixture of oxygen and carbon dioxide, the hsemoglobin combines loosely 
 with the oxygen as well as with the carbon dioxide, independently of 
 each other, just as if each gas existed alone (Bohr). He considers 
 that the two gases are combined with different parts of the hsemoglobin, 
 that is, the oxygen with the pigment nucleus and the carbon dioxide 
 with the protein component. Attention must be called to the fact that, 
 as observed by Torup, hsemoglobin is in part readily decomposed by 
 the carbon dioxide with the splitting off of some protein. 
 
 Nitric-oxide Haemoglobin is also a crystalline molecular combina- 
 tion which is even stronger than the carbon-monoxide hsemoglobin. 
 Its solution shows two absorption-bands, which are paler and less sharp 
 than the carbon-monoxide hsemoglobin bands, and they do not dis- 
 appear on the addition of reducing bodies. Hsemoglobin also forms a 
 molecular combination with acetylene and ethylene. 
 
 Haemorrhodin is the name given by Lehmann to a beautiful red pigment 
 soluble in alcohol and ether, which is extracted from meat and meat products 
 by boiling alcohol and which seems to be produced by the action of small amounts 
 of nitrites. Another pigment isolated by Lewin 3 from the blood of animals 
 poisoned by phenylhydrazine, has been called hcenioverdin. By heating a solu- 
 tion of blood-pigment treated with caustic potash and mixed with alcohol to 
 60° C. we obtain, according to v. Klaveren, a pigment which he calls kathoemo- 
 globin, but called by Arnold, 4 who first obtained it, neutral hopmatin, which is 
 produced by the splitting off of a ferruginous complex. This pigment still con- 
 tains protein, but is poorer in iron than the hsemoglobin or methsemoglobin and 
 probably forms an intermediary product in the conversion of the above into 
 hsematin. 
 
 Decomposition products of the blood-pigments. By its decomposi- 
 tion, hsemoglobin yields, as previously stated, a protein which has Leon 
 
 1 Hoppe-Seyler, Med. -chem. Untersuch., 151. See Araki, Zeitschr. f. physiol. 
 Chem., 14; Harnack, 1. c; Clarke and Hurtley, Journ. of Physiol., 36. 
 
 2 Bohr, Extrait, du Bull, de l'Acad. Danoise, 1890; Centralbl. f. Physiol., 4 and 
 17; Torup, Maly'e Jahresber., 17. 
 
 3 K. B. Lehmann, Sitzungsber. d. phys.-med. Gesellsch. Wiirzburg, 1899; Lewin, 
 Compt. Rend., 133. 
 
 * v. Klaveren, Zeitschr. f. physiol. Chem., 33; Arnold, ibid, 29. 
 
HSEMOCHROMOGEN. 289 
 
 called globin (Preyer, Schulz), and a ferruginous pigment as chief prod- 
 ucts. According to Lawrow 94.09 per cent protein, 4.47 per cent 
 hsematin, and 1.44 per cent other bodies are produced in this decom- 
 position. The globin, which was isolated and studied by Schulz, 1 
 differs from most other proteins by containing a high amount of carbon, 
 54.97 per cent., with 16.98 per cent of nitrogen. It is insoluble in water, 
 but very easily soluble in acids or alkalies. It is not dissolved by ammonia 
 in the presence of ammonium chloride. Nitric acid precipitates it in 
 the cold, but not when warm. It may be coagulated by heat, but the 
 coagulum is readily soluble in acids. Because of these reactions it is 
 considered as a histone by Schulz. 
 
 On hydrolytic cleavage globin (from horse-blood) yields, accord- 
 ing to Abderhalden, 2 the ordinary cleavage products of the proteins 
 and especially leucine, 29 per cent. It is also important to call attention 
 to the large amount of histidine, 10.96 per cent, while the quantities of 
 arginine and lysine were only 5.42 and 4.28 per cent respectively. 
 
 The pigment split off is different, depending upon the conditions 
 under which the cleavage takes place. If the decomposition takes place 
 in the absence of oxygen, a coloring-matter is obtained which is called 
 by Hoppe-Seyler hcemochromogen, by other investigators (Stokes) 
 reduced hcematin. In the presence of oxygen, hsemochromogen is quickly 
 oxidized to hsematin, and there is therefore obtained in this case hcematin 
 as a colored decomposition product. As hsemochromogen is easily 
 converted by oxygen into hsematin, so this latter may be reconverted 
 into hsemochromogen by reducing substances. 
 
 Heemochromogen was discovered by Hoppe-Seyler. 3 It is, accord- 
 ing to Hoppe-Seyler, the colored atomic group of hsemoglobin and of 
 its combinations with gases, and this atomic group is combined with 
 proteins in the pigment. The characteristic absorption of light depends 
 on the hsemochromogen, and it is also this atomic group which binds, in 
 the oxy hsemoglobin, 1 molecule of oxygen and, in the carbon-monoxide 
 hsemoglobin, 1 molecule of carbon monoxide with 1 atom of iron. Hsemo- 
 chromogen is produced in an alkaline solution of hsematin by the action 
 of reducing bodies. By the reduction of hsematin in alcoholic ammoniacal 
 solution by means of hydrazine v. Zeynek 4 was able to obtain the solid 
 brownish-red ammonia combination. A crystalline combination between 
 pyridine and hsemochromogen can be obtained according to Kalmus 
 
 1 Lawrow, ibid., 26; Schulz, ibid., 24; Preyer, Die Blutkristalle, Jena, 1871. 
 
 2 Zeitschr. f. physiol. Chem., 37; with Baumann, ibid., 51. 
 
 3 Ibid., 13. 
 
 * Zeitschr. f. physiol., Chem., 25. 
 
290 THE BLOOD. 
 
 and v. Zeynek 1 from haemoglobin and pyridine by boiling, or from 
 haematin and haemin and pyridine after the addition of hydrazin-hydrate. 
 
 Haemochromogen also combines, as Hoppe-Seyler first showed, with 
 carbon monoxide. This compound, which in aqueous solution gives 
 a spectrum similar to oxyhemoglobin, has been obtained by Pregl 2 
 in the solid condition as a deep-violet powder which is insoluble in 
 absolute alcohol. In opposition to haemoglobin the haemochromogen 
 combines with oxygen more firmly than with carbon monoxide. The 
 assumption of Hoppe-Seyler, that this compound is a combination of 1 
 molecule haemochromogen and therefore contains 1 molecule carbon 
 monoxide for 1 molecule of iron has been experimentally substantiated 
 by Hufner and Ruster and by Pregl. 3 
 
 An alkaline haemochromogen solution has a beautiful cherry-red 
 color. It shows two absorption-bands, first described by Stokes (spec- 
 trum Plate, 6), one of which is dark and whose center corresponds to 
 X = 556.4 between D and E, and a second broader band, less dark, which 
 covers the Fraunhofer lines E and b. The middle of this band cor- 
 responds to X = 526 to 530 according to Lewin, Miethe and Stenger. 
 In acid solution haemochromogen shows four bands, which, according 
 to Jaderholm, 4 depend on a mixture of haemochromogen and haemato- 
 porphyrin (see below), this last formed by a partial decomposition 
 resulting from the action of the acid. 
 
 Milroy, 5 from an alcoholic solution of haematin containing oxalic 
 acid, after driving out the air by means of hydrogen gas, gradually obtained 
 an acid solution of reduced haematin (haemochromogen) by means of 
 zinc dust. This solution showed one absorption-band between D and E. 
 
 Haemochromogen may be obtained as crystals by the action of caustic 
 soda on haemoglobin at 100° C. in the absence of oxygen (Hoppe-Seyler). 
 By the decomposition of haemoglobin by acids (of course in the absence 
 of air) we obtain haemochromogen contaminated with a little haemato- 
 porphyrin. An alkaline haemochromogen solution is easily obtained by 
 the action of a reducing substance (Stokes' reduction liquid) on an 
 alkaline haematin solution. An ammoniacal solution of haematin on 
 reduction with hydrazine yields haemochromogen very easily. An alco- 
 holic, alkaline hydrazine solution is also recommended by Riegler 6 
 as a reagent for blood-pigments, converting them into haemochromogen. 
 
 Haematin, also called Oxyh^ematin, is sometimes found in old transu- 
 dates. It is formed by the action of the gastric or pancreatic juices on 
 
 1 E. Kalmue, Zeitschr. f. Chem., 70; v. Zeynek, ibid., 70. 
 
 2 Ibid., 44. 
 
 • Hufner and Kiister, Arch. f. (Anat. u.) Physiol., 1904, Suppl. Pregl, 1. c. 
 
 * Nord. Med. Arkiv., 16. 
 5 Journ. of Physiol., 32. 
 
 8 Zeitschr. f. anal. Chem., 43. 
 
HiEMATIN. 291 
 
 oxyhemoglobin, and is, therefore, found in tlio feces after hemorrhage 
 in the intestinal canal, and also after a meat diet and food rich in blood. 
 It is stated that hsematin may occur in urine after poisoning with arseniu- 
 reted hydrogen. As shown above, the haematin is formed by the decom- 
 position of oxyhemoglobin, or at least of haemoglobin, in the presence 
 of oxygen. 
 
 The views in regard to the composition of haematin are rather con~ 
 tradictory, which seems to be due to the fact that the substance haemin 
 (see below), from which the formula of haematin is derived, has a some- 
 what different composition, dependent upon various conditions. Accord- 
 ing to Hoppe-Seyler haematin has the formula C34H34N4FeOs, and 
 from the recent investigations upon haemin, which will be mentioned 
 below, this formula seems to be now generally accepted. According 
 to this formula 1 atom of iron occurs with every 4 atoms of nitrogen. 
 According to Cloetta, and also Rosenfeld, 1 hsematin has the formula 
 CaoH34N3Fe03, with 1 atom of iron for every 3 atoms of nitrogen. 
 
 v. Zeynek has prepared a hsematin by the digestion of an oxyhemoglobin 
 solution with pepsin-hydrochloric acid, from which he then prepared haemin. 
 As this hsematin of v. Zeynek was readily convertible into haemin, and while the 
 ordinarily prepared haematin from haemin cannot be retransformed into haemin, 
 Kuster considers that these two forms of haematin are not identical. The 
 first he calls a-haanatin and the ordinary which is a polymeric body, he calls 
 /3-haematin. That a retransformation of haemin is possible from ordinary haematin 
 is still admitted by Piloty and Ellinger. 2 
 
 Haematin contains at least three hydroxy 1 groups, one of which acts 
 as hydroxyl ion and seems to be united with the iron, and is replaced 
 in the haemin formation (see below) by the chlorine. By means of the 
 two others, salts with metals as well as alkyl derivatives may be formed, 
 which latter (as haemin derivatives) have been especially studied by Nencki 
 and Zaleski and Kuster. 3 Haematin dissolves in concentrated sulphuric 
 acid and is converted into haematoporphyrin, with the splitting off of 
 iron. On heating dry hsematin it yields an abundance of pyrrol. The 
 products produced on the oxidation and reduction of haematin and the 
 question as to the constitution of haematin will be discussed in connec- 
 tion with haematoporphyrin. 
 
 Haematin is amorphous, dark brown or bluish-black. It may be 
 heated to 180° C. without decomposition; on burning it leaves a residue 
 
 1 Hoppe-Seyler, Med.-chem. Untersuch., p. 525; Cloetta, Arch. f. exp. Path. u. 
 Pharm., 36; Rosenfeld, ibid., 40. 
 
 2 v. Zeynek, Zeitschr. f. physiol. Chem., 30 and 49; Kuster. ibid., 66 and Ber. 
 d. d. chem. Gesellsch., 43; Piloty, Annal. d. Chem. u. Pharm., 377; Eppinger, Unters. 
 uber den Blutfarbstoff. Dissert. Munchen, 1907. 
 
 5 Xencki and Zaleski, Zeitschr. f. physiol. Chem., 30; Kuster, Ber. d. d. Chem. 
 Gesellsch. 43 and 45, and Zeitschr. f. physiol. Chem., 82. 
 
292 THE BLOOD. 
 
 consisting of iron oxide. It is insoluble in water, dilute acids, alcohol, 
 ether, and chloroform, but it dissolves slightly in warm glacial acetic acid. 
 Haematin dissolves in acidified alcohol or ether. It easily dissolves in 
 alkalies, even when very dilute. The alkaline solutions are dichroic; 
 in thick layers they appear red by transmitted light and in thin layers 
 greenish. The alkaline solutions are precipitated by lime- and baryta- 
 water, as also by solutions of neutral salts of the alkaline earths. The 
 acid solutions are always brown. 
 
 An acid haematin solution (spectrum Plate, 4), absorbs the red part 
 of the spectrum only slightly and the violet parts strongly. The solu- 
 tion shows a rather sharply defined band between C and D, whose posi- 
 tion may change with the variety of acid used as a solvent. Between 
 D and F a second, much broader, less sharply defined band occurs, which 
 by proper dilution of the liquid is converted into two bands. The one 
 between b and F, lying near F, is darker and broader; the other, between 
 D and E, lying near E, is lighter and narrower. Also by proper dilution 
 a fourth very faint band is observed between D and E, lying near D. 
 Haematin may thus in acid solution show four absorption-bands; ordi- 
 narily one sees, distinctly, only the bands between C and D and the broad, 
 dark band — or the two bands — between D and F. In alkaline solution, 
 hsematin (spectrum Plate, 5), shows a broad absorption-band, which 
 lies in greatest part between C and D, but reaches a little over the line 
 D toward the right in the space between D and E. As the position of 
 the haematin bands in the spectrum is quite variable, the exact wave- 
 lengths corresponding thereto cannot be given exactly. 
 
 Haemin, Haemin Crystals, or Teichmann's Crystals. Haemin is 
 formed, as generally admitted, by the replacement of an HO group by 
 chlorine in the haematin, and is the starting point in the preparation of 
 the latter. 
 
 The statements as to the composition of haemin differ quite considerably, 
 and various haemins have been accepted, which is partly due to the fact, 
 as first shown by Nencki and Zaleski, that haemin combines with acid 
 and alkyl radicals and can also give addition products with other 
 bodies. Thus for example the methylhaemins, carefully studied by 
 Kuster, especially monomethylhaemin, is produced in the preparation 
 of haemin according to Morner's method (see below) by means of methyl 
 alcohol. These behaviors have been further explained by the work 
 of numerous investigators, especially by Kuster, and most investigators 
 generally admit that only one haemin exists whose general formula is 
 C34H3 3 4 N4FeCl. According to Piloty the formula is C34H 3 204N4FeCl 
 while Piettre and Vila l deny this formula and claim to have 
 
 1 Nencki and Zaleski, Zeitschr. f. physiol. Chem., 30; Nencki and Sieber, Arch. 
 f. exp. Path. u. Pharm., 18 and 20, and Ber. d. d. chem. Gesellsch., 18; Schalfejeff 
 
H/EMTN. 293 
 
 prepared a haemin free from chlorine, from pure crystalline oxy- 
 hemoglobin. 
 
 Ha>min crystals form, in large masses, a bluish-black powder, but are 
 so small that they can be seen only by aid of the microscope. They 
 consist of dark-brown or nearly brownish-black long, rhombic, or spool- 
 like crystals, isolated or grouped as crosses, rosettes, or stellar forms. 
 Cubical crystals may also occur, according to Cloetta. They are 
 insoluble in water, dilute acids at the normal temperature, alcohol, ether, 
 and chloroform. They are slightly soluble in glacial acetic acid with heat. 
 They dissolve in acidified alcohol, as also in dilute caustic alkalies or 
 carbonates; and in the last case they form, besides alkali chlorides, 
 soluble haematin alkali, from which the haematin may be precipitated by 
 an acid. As shown by Piloty and Eppinger and then also by v. Sie- 
 wert, 1 crystalline haemin can be reobtained from the haematin. 
 
 On shaking with cold aniline and treating first with acetic acid and then 
 with ether, Kuster obtained a product, dehydrochloride haemin, which was 
 poor in the elements of hydrochloric acid, and which again took up HC1 and was 
 converted into haemin. By the action of boiling aniline, hydrogen is driven out 
 and a combination with aniline, without loss of iron, takes place. 
 
 The principle of the preparation of haemin crystals in large quan- 
 tities is as follows: The washed sediment from the blood-corpuscles 
 is coagulated with alcohol or by boiling after dilution with water and 
 the careful addition of acid. The strongly pressed but not dry mass 
 is rubbed with 90-95 per cent alcohol which has been previously treated 
 with oxalic acid or |-1 per cent concentrated sulphuric acid, and this 
 is allowed to stand several hours at the temperature of the room. The 
 filtrate is warmed to about 70° C, treated with hydrochloric acid (for 
 each liter of filtrate add 10 cc. 25 per cent hydrochloric acid diluted 
 with alcohol — Morner), and allowed to stand in the cold. The crystals, 
 which separate in one or two days, are first washed with alcohol and then 
 "with water. On dissolving the haemin in chloroform containing quinine 
 and treating the filtrate with alcoholic hydrochloric or acetic acid we can 
 recrystallize the haemin according to Schalfejefp. By adding glacial 
 acetic acid saturated with salt to a solution of haematin in chloroform 
 containing quinine Piloty and Eppinger obtained crystalline haemin. 
 For particulars as to the various methods of preparation and purification 
 we refer the reader to the above-cited works of Nencki and Sieber, 
 Morner, Nencki and Zaleski (ScHALFEjEFF),and especially to Kuster. 2 
 
 Haematin is obtained on dissolving the haemin crystals in very dilute 
 caustic alkali and precipitating with an acid. 
 
 with Nencki and Zaleski, 1. c; Bialobrzeaki, Arch, des scienc. biol. de St. P6tersbourg- 
 5; K. Morner, Nord. Med. Arkiv. Festband, 1897, Nos. 1 and 26, and Zeitschr. f. 
 physiol. Chem., 41; Zaleski, ibid., 37; Hetper and Marchlewski, ibid., 41 and 42; 
 Kuster, ibid., 40 and 82 and footnote 1, page 292; Piettre and Vila, Compt. Rend., 141, 
 p. 734; Piloty, 1. c. 
 
 1 Piloty and Eppinger, 1. c; v. Siewert, Arch. f. exp. Path. u. Pharm., 58. 
 
 J Kuster, Zeitschr., f . physiol. Chem., 40. 
 
294 THE BLOOD. 
 
 In preparing haemin crystals in small quantities proceed in the fol- 
 lowing manner: The blood is dried after the addition of a small quantity 
 of common salt, or the dried blood may be rubbed with a trace of the 
 same. The dry powder is placed on a microscope slide, moistened 
 with glacial acetic acid, and then covered with the cover-glass. Add, 
 by means of a glass rod, more glacial acetic acid by applying the drop 
 at the edge of the cover-glass until the space between the slide and the 
 cover-glass is full. Now warm over a very small flame, with the pre- 
 caution that the acetic acid does not boil and pass with the powder from 
 under the cover-glass. If no crystals appear after the first warming 
 and cooling, warm again, and if necessary add some more acetic acid. 
 After cooling, if the experiment has been properly performed, a number 
 of dark-brown or nearly black haemin crystals of varying forms will 
 be seen. 
 
 In regard to the preparation and properties of the iodine-, bromine-, 
 and acetone-haemin we refer to the work of Strzyzowski, Meruno- 
 wicz and Zaleski. 1 
 
 By the action of acids upon haemochromogen, haematin, or haemin, a 
 new iron-free pigment, which was first closely studied by Hoppe-Seyler 
 and called hcematoporphyrin, is produced. According to the method of 
 preparation, hsematoporphyrins having different solubilities, and whose 
 relation to each other is not perfectly clear, are produced, but all show 
 the same characteristic absorption-spectrum. The best-studied haema- 
 toporphyrin is the one obtained according to Nencki and Sieber's 
 method, by the action of glacial acetic acid saturated with hydrobromic 
 acid upon haemin crystals, best at the temperature of the body (Nencki 
 and Zaleski). Another porphyrin is the mesoporphyrin obtained by 
 Nencki and Zaleski 2 by the reduction of haemin in glacial acetic acid 
 by hydriodic acid and iodophosphonium. 
 
 Haematoporphyrin, C34H38N4O6, which, according to recent molec- 
 ular weight determinations must perhaps be doubled (Piloty) occurs 
 according to Mac Munn 3 as a physiological pigment in certain animals. 
 A porphyrin occurs, as shown by Garrod and Saillet, as a normal con- 
 stituent in human urine, although only as traces and it has also been 
 observed several times in large amounts in the urine after the use of 
 sulphonal (see Chapter XIV). This urine porphyrin is generally con- 
 sidered as haematoporphyrin. 
 
 In the production of haematoporphyrin from haemin or haematin the iron is 
 split off. Opinions are not unanimous in regard to this process. According 
 
 1 Strzyzowski, Therap. Monatsh., 1901 and 1902; Merunowicz and Zaleski, Bull, 
 de 1'Acad. d. Scienc. de Cracovie, 1907. 
 
 2 Hoppe-Seyler, Med.-chem. Untersuch., 528; Nencki and Sieber, Monatshefte f. 
 Chem., 9, and Arch. f. exp. Path. u. Pharm., 18, 20, and 24; Nencki and Zaleski, 
 Zeitschr. f. physiol. Chem., 30. 
 
 3 Piloty, Annal. d. Chem. u. Pharm., 388; MacMunn, Journ. of Physiol., 7. 
 
ILfflMATOPOKPHYRIN. 295 
 
 to Piloty two carboxyl groups axe formed with the taking up of water and 
 these occur to a certain extent latent in lactam combination in the baemin. 
 
 According to Kusteh, 1 who admits of two already funned carboxylfl in the ha-min, 
 two bydroxyls are produced secondarily in the fuematoporphyrin, in that (by the 
 action of the glacial acetic acid and hydrobromie acid) primarily an attachment 
 of hydrobromic acid takes place and then from this as intermediary product, 
 by tiie action of water, bromine is split off ami is replaced by hydroxyl. In the 
 formation of mesoporphyrin the procedure is still different because among others, 
 mesoporphyrin contains 2 oxygen atoms less than the luematoporphyrin. 
 
 On the gentle reduction of hsemin with glacial acetic acid, bydriodic acid 
 and red phosphorus, Piloty and Fink obtained besides mesoporphyrin a second 
 body, phonoporphyrin, which differs from the mesoporphyrin by containing 
 more oxygen, a brown color and almost complete insolubility in dilute hydro- 
 chloric acid. It is not reduced to mesoporphyrin by hydriodic acid but yields 
 hsematinic acid and methyl-ethyl maleic imide on oxidation with chromic acid. 
 They obtained no other cleavage products from hsemin under the above men- 
 tioned experimental conditions. The two porphyrins were produced in about 
 equal quantities and they formed about 90 per cent of the calculated cleavage 
 products. They each represent one-half of the ha?min, whose formula corresponds 
 to CesH^XgOsFciCla which must be doubled. As these two porphyrins yield 
 methyl ethylmaleic imide, while this is not the case with either the haemin or the 
 hsematoporphyrin, it is believed that both are combined together in the haemin 
 or ha?matoporphyrin with that part of their molecules which allow of the maleic 
 imide formation. 
 
 By the action of glacial acetic acid, and hydriodic acid upon hsemin in the 
 cold (room temperature) in the presence of iodophosphonium, H. Fischer and 
 Bartholomaus have obtained a beautiful crystalline, colorless product, por- 
 phyrinogen, whose formation and behavior have been further studied by Rose. 2 
 Porphyrinogen, C.^H^NjO^ is formed from hsemin, the meso- and the hsemato- 
 porphyrin in acid, and from the meso- or hsematoporphyrin also in alkaline 
 reduction. Porphyrinogen can be transformed into mesoporphyrin by oxidative 
 action of various kinds. Like the latter it yields hsematinic acid as well as methyl 
 ethyl maleic imide as oxidation products. 
 
 Haematoporphyrin is closely related to the bile pigment bilirubin 
 (see Chapter VII) and also stands in close relation with the urinary 
 pigment, urobilin. By action of reducing substances several investiga- 
 tors (Hoppe-Seyler, Nencki and Sieber, Le Nobel, MacjMunn and 
 others) have obtained pigments similar to urobilin, and by experiments 
 with rabbits, Nencki and Rotschy 3 have proved that haematoporphyrin 
 introduced into the animal body may in part be transformed into a 
 urobilin substance. 
 
 In connection with the question of the behavior of haematoporphyrin in the 
 animal body, the poisonous action of this body, as discovered by Hausmann, 
 and which manifests itself as a photobiological sensibilisation, is of interest. 
 Hausmann has found that white mice that have had fuematoporphyrin injected 
 
 1 Piloty, 1. c; Kiister, Ber. d. d. ehem. Gesellsch., 45. 
 
 2 Piloty and Fink, Ber. d. d. chem. Gesellsch., 46, 2021; H. Fischer and Bartholo- 
 maus, ibid., 46, 511; Rose, Zeitschr. f. physiol. Chem., 84. 
 
 3 Hoppe-Seyler, Med. Chem. Unters. p. 533; Le Xobel, Pfluser's Arch., 40; 
 Nencki and Sieber, 1. c; MacMunn, Proc. Roy. Soc, 30, and Journ. of Physiol., 10; 
 Nencki and Rotschy, Monatsh. f. chem., 10. 
 
29G THE BLOOD. 
 
 subcutaneously and then exposed to bright light die very quickly with character- 
 istic symptoms, while control animals kept in the dark show no symptoms of disease. 
 H. Fischer and Meyer-Betz ' have also shown that in this regard a certain 
 difference exists between the hsematoporphyrin and the mesoporphyrin. The 
 perfectly pure crystalline mesoporphyrin does not show the photobiological 
 action which occurs with crystalline hamaatoporphyrin. 
 
 Of especial interest is the close relationship of the hsematoporphyrin 
 to certain chlorophyll derivatives, especially to phylloporphyrin, 
 C32H36N4O2. Phylloporphyrin is similar to the above-mentioned meso- 
 porphyrin, C34H^sN404, and the absorption spectrum of the brom- 
 porphyrins, bromphylloporphyrin and brommesoporphyrin as prepared 
 by Schunck and Marchlewski, seems to be almost identical. Just as 
 from mesoporphyrin, with sodium chloride, glacial acetic acid and an 
 iron salt we can regenerate a product very similar to haemin (Zaleski) 
 so Marchlewski 2 has been able under similar conditions to prepare from 
 phylloporphyrin a pigment, phyllohcemin, which contained iron and was 
 similar to hgemin. A comparison of the cleavage products gives still 
 more conclusive and important proofs of the close relationship of the 
 "blood and leaf pigments. 
 
 We have important investigations of Kuster, 3 Piloty, Willstatter, 
 H. Fischer 4 and their collaborators upon the constitution of hsemin and 
 hgematoporphyrin. The constitution of chlorophyll has been explained 
 by the pioneering researches of Willstatter. 
 
 On the oxidation of hsematin in glacial acetic acid by potassium 
 dichromate or chromium trioxide Kuster obtained the imide of the 
 
 /CO— C.CH0.CH2.COOH 
 tribasic hcematinic acid, HN<^ , which is a deriva- 
 
 X CO— C.CH 3 
 tive of maleic acid, and from which methylethylmaleic acid anhydride, 
 
 .CO— C.CH2CH3 
 0<f , can be readily obtained. The same heematinic 
 
 X CO— C.CH 3 
 
 1 Hausmann, Bioch. Zeitschr., 30; Hans Fischer and Meyer-Betz. Zeitschr. f. 
 physiol. Chem., 82. 
 
 2 The pertinent literature will be found in L. Marchlewski, Die Chemie der Chlor- 
 ophylle und ihre Beziehung zur Chemie des Blutfarbstoffes, 1909 and Ber. d. d. chem. 
 Gesellsch., 45. 
 
 3 Beitrage zur Kenntnis. des Hamatins. Tubingen, 1896; Ber. d. d. chem. Gesellsch., 
 27, 30, 32, 35, 43, and 15, Annal. d. Chem. u. Pharm., 315, arid Zeitschr. f. physiol. 
 Chem., 28, 40, 44, 54, 55, 61, 62, and 82. 
 
 * The work of Piloty and collaborators may be found in Annal. d. Chem. u. Pharm., 
 360, 37", 388, 390, and 392 and Ber. d. d. chem. Gesellsch., 42, 43, and 45. In regard 
 to the work of Willstatter and the literature on chlorophyll (to 1911) see Willstatter 
 in Abderhalden's Bioch. Handlexicon, Bd. VI and Annal. d. Chem. u. Pharm., 378; 
 380, 382, and 385. Hans Fischer and collaborators, Zeitschr. f. physiol. Chem., 82, 
 and footnote 2, p. 297, and the literature on bilirubin Chapter VII. 
 
CONSTITUTION OF THE BLOOD-PIGMENTS. 297 
 
 acid imide, which also haemato- and mesoporphyrin give, were obtained by 
 Marchlewski on the oxidation of phylloporphvrin and by Willstatter, 
 
 XX)— C.C 2 H 5 
 besides methylclhylmaleic imide, HX<^ , on the oxidation of 
 
 x CO— C.CH 3 
 certain chlorophyll derivatives. The sarne two products were obtained 
 1 \ Ivuster l on the oxidation of mesoporphyrin while haemin and haema- 
 toporphyrin gave no methylethylmaleic imide. 
 
 It has bem known for a long time that haemin and hacmatoporphyrin 
 gave an abundance of pyrrol on heating, and that phylloporphyrin has a 
 similar behavior was first shown by Schunck and Marchlewski. 
 That at least one pyrrol, of the pyrrol mixture, the so-called hcemopyrrol 
 is common to both the blood and leaf pigments has been shown by the 
 investigations of Marchlewski and his collaborators, and from haemo- 
 pyrrol Kuster was the first to obtain methylethylmaleic imide on oxida- 
 tion, showing that haemopyrrol was probably a dimethylethylpyrrol. 
 This behavior has been further developed by the investigations of 
 Piloty and Willstatter on the reduction products of the blood and 
 leaf pigments and by H. Fischer and Bartholomaus 2 on the sub- 
 stituted pyrrols. 
 
 Willstatter obtained a pyrrol mixture from hsemin and haemato- 
 porphyrin, as well as from chlorophyll derivatives, by reduction, from 
 which he isolated three different pyrrols. The first, which he calls hcemo- 
 pyrrol, was perhaps not perfectly pure, consisted at least in great part 
 of the cryptopyrrol (Fischer and Bartholomaus, c-haemopyrrol of 
 Piloty and Stock) which is identical with the 2, 4-dimethyl-3-ethyl 
 
 H 3 C.C C.C2H5 
 
 pvrrol = prepared svnthetically bv Knorr and Hess. 3 
 
 HC— NH— C.CH 3 
 The second haemopyrrol, which he calls isohocmopyrrol ( = haemopyrrol 
 of Fischer and Bartholomaus, B-haemopyrrol of Piloty and Stock) 
 is also a trisubstituted pyrrol, namely 2, 3-diinethyl-l-ethylpyrrol 
 
 C2H5.O C.CH3 
 
 HC— NH-C.CH3 
 
 These two dimethylethylpyrrols give with nitrous acid the correspond- 
 
 1 Ber. d. d. chem. Gesellsch., 45. 
 
 2 Piloty, Annal. d. Chem. u. Pharm., 377, 388, 390, and 392; Willstatter and 
 
 Asahina, ibid., 385. In this article will be found on pages 1S9 and 190 the references 
 to the literature on the investigations of Marchlewski and others on ha?mopyrrol. 
 H-lFischer and Bartholomaus, Zeitschr. f. physiol. Chem., 77 and 80 and Ber. d. d. 
 chem. Gesellsch., 45. 
 
 3 Piloty and Stock, Annal. d. Chem. u. Pharm., 392; Knorr and Hess, Ber. d. 
 d. chem. Gesellsch., 44 and 45. 
 
29S THE BLOOD. 
 
 ing oxime of methylethylmaleic imide. The third pyrrol found by 
 Willstatter, which he calls phyllopyrrol, is a tetra-substituted pyrrol, 
 
 H 3 C.C C.C2H5 
 
 namelv, 2, 3, 5 trimethyl-4-ethvlpyrrol = 
 
 H3C.C— NH— C.CH 3 
 
 The statement that the haemopyrrol of Willstatter is in part derived 
 from cryptopyrrol is not correct, and must be changed because of the 
 investigations of Piloty and Stock, who find that the haemopyrrol of 
 Willstatter (and Asahina) undoubtedly contains cryptopyrrol, but 
 consists chiefly of the B-haemopyrrol, consequently isohaemopyrrol. Ac- 
 cording to more recent investigations of Piloty and Stock l the haemo- 
 pyrrol question is even more complicated than was expected. 
 
 In the crude pyrrol obtained by the reduction of the blood pigments 
 several other pyrrol bodies have been found, for example the phonopyrrol 
 of Piloty which has not been sufficiently explained. According to 
 Grabowski and Marchlewski 2 as well as to Piloty and Stock, the 
 crude haemopyrrol contains also disubstituted pyrrol, namely, jSi, /3-methyl- 
 ethylpyrrol. On fusing haematoporphyrin or haematopyrrolidinic acid 
 (see below) with caustic alkali we obtain, according to Piloty, a mixture 
 of pyrrols among which we will mention 2, 3-dimethylpyrrol 
 
 HC- C.CH 3 
 
 , which has been studied by Piloty and Wilke. 3 
 HC— NH— C.CH 3 
 
 By the reduction of haematoporphyrin and haemin by various methods, 
 Piloty and co-workers have obtained, besides haemopyrrol, several 
 acids namely haematopyrrolidinic acid, phonopyrrolcarboxylic acid 
 (isophonopyrrolcarboxylic acid) and xanthopyrrolcarboxylic acid. The 
 haematopyrrolidinic acid seems from the most recent investigations 
 not to be a unit substance. Piloty obtained from it phonopyrrolcar- 
 
 H3C.C C.CH 2 .CH 2 .COOH 
 
 boxylic acid, C 9 Hi 3 N0 2 = which on 
 
 H3C.C— NH— CH 
 treatment with nitrous acid lost a methyl group and was converted into 
 
 H 3 C.C CCH2CH2.COOH. 
 
 the oxime of haematinic acid, The acid 
 
 HONC— NH— CO 
 received this name because, according to Piloty, it yields a special 
 dimethylethylpyrrol, called phonopyrrol by him. The question as to the 
 nature of xanthopyrrolcarboxylic acid and its occurrence has not been 
 
 1 Piloty and Stock, Annal. d. Chem. u. Pharm., 392 and Ber. d. d. chem. Gesellsch., 
 46, 1008. 
 
 '-' Grabowski and Marchlewski, Zeitschr. f. physiol. Chem., 81. 
 ' Piloty and Wilke, Ber. d. d. chem. Gesellsch., 45. 
 
PYRROLE DERIVATIVES. 299 
 
 answered; still there does not seem to be any doubt that there exists 
 an isophonopyrrolcarboxylic acid, which can be obtained from the blood 
 as from the bile pigments. From the mixture of acid cleavage products 
 obtained by the reduction of haemin with hydriodic acid, and glacial acetic 
 acid Piloty and Dormann 1 have obtained as well characterized prod- 
 ucts, phonopyrrolcarboxylic acid and isophonopyrrolcarboxylic acid and 
 also xarithopyrrolcarboxylic acid, C10H15NO2 and they consider the 
 existence of this acid as positively proved. The melting-point of the 
 crystalline acid was 108°, the picrate 143°, and the oxime 208°. The 
 corresponding values for isophonopyrrolcarboxylic acid was 122°, 146° 
 and 210° respectively. An isomeric xanthopyrrolcarboxylic acid, called 
 D-phonopyrrolcarboxylic acid, seems also to occur. 
 
 It is extremely difficult to correlate the somewhat contradictory 
 statements of the various authors in this subject and to draw quite 
 positive conclusions from these statements. It is nevertheless positive 
 that from the haemopyrrol mixture the three pyrrols, cryptopyrrol, 
 isohaemopyrrol and phyllopyrrol can be obtained and also that there 
 are two haemopyrrolcarboxylic acids (phonopyrrol- and isophonopyrrol- 
 carboxylic acids), of which one possibly is related to the crypto- 
 pyrrol and the other to the isohaemopyrrol. On account of the uncer- 
 tainty of the experimental foundation it is difficult to enter into a 
 discussion of the variously proposed hypothetical constitutional formulae 
 for the derivatives of the blood pigments. The same is true for the 
 disputed question as to the form of binding of the iron in haematin and 
 in hsemin. It is generally admitted that the iron here is trivalent. The 
 views are different in regard to the valence of the iron in haemoglobin, 
 namely, Maxchot considers that haemoglobin is a ferric combination 
 while Kuster 2 on the contrary considers it a ferrous combination. 
 
 Haematoporphyrin gives with hydrochloric acid a compound which 
 crystallizes in long brownish-red needles. If the solution in hydrochloric 
 acid is nearly neutralized with caustic soda and then treated with sodium 
 acetate, the pigment separates out as amorphous, brown flakes not 
 readily soluble in amyl alcohol, ether, or chloroform, but readily soluble 
 in ethyl alcohol, alkalies, and dilute mineral acids. The compound 
 with sodium crystallizes as small tufts of brown crystals and several 
 other salts of haematoporphyrin are known such as the methyl and ethyl 
 esters. The acid alcoholic solutions have a beautiful purple color, 
 which become violet-blue on the addition of large quantities of acid. 
 The alkaline solution has a beautiful red color, especially when too much 
 alkali is not present. 
 
 1 Piloty and Dormann, Ber. d. d. chem. Gesellsch., 46. 
 
 2 Manchot, Zeitschr. f. physiol. Chem., 70; Kuster, ibid., 71. 
 
300 THE BLOOD. 
 
 An alcoholic solution of heematoporphyrin, acidulated with hydro- 
 chloric or sulphuric acid, shows two absorption-bands (spectrum Plate, 
 7), one of which is fainter and narrower and lies between C and D, near 
 D. The other is much darker, sharper, and broader, and lies midway 
 between D and E. An absorption extends from these bands toward 
 the red, terminating with a dark edge, which may be considered as a 
 third band between the other two. 
 
 A dilute alkaline solution shows four bands, namely, a band between 
 C and D; a second, broader band surrounding D and with the greater 
 part between D and E; a third, between D and E, nearly at E; and 
 lastly, a fourth, broad and dark band between b and F. On the addi- 
 tion of an alkaline zinc-chloride solution the spectrum changes more 
 or less rapidly, 1 and finally a spectrum is obtained with only two bands, 
 one of which surrounds D and the other lies between D and E. If an 
 acid haematoporphyrin solution is shaken with chloroform, a part of the 
 pigment is taken up by the chloroform, and this solution often shows a 
 five-banded spectrum with two bands between C and D. The position 
 of the hsematoporphyrin bands in the spectrum differ with the various 
 methods of preparation and other conditions, so that they do not cor- 
 respond to the same wave length. These facts coincide well with the 
 recent investigations of A. Schulz; 2 according to which the appearance 
 of the spectrum is not only dependent upon the reaction but also upon 
 the character of the solvent and the method of preparation. 
 
 In regard to the preparation of hsematoporphyrin, see Hoppe-Seyler- 
 Thierfelder's Handbuch, 8. AufL, and the works cited on page 294. 
 
 Mesoporphyrin, C34H38N4O4, has the same spectrum as haematoporphyrin. 
 It has two oxygen atoms less, and further differs from it in that on oxidation it yields 
 hsematinic acid as well as methylethylmaleic imide, and does not show the 
 above-mentioned biological action of haematoporphyrin. 
 
 Haematinogen is a ferruginous pigment so named by Freund, 3 which he 
 obtained by carefully extracting blood with alcohol containing hydrochloric 
 acid. It is closely related to haematin, but is not sufficiently characteristic and is 
 not considered as a cleavage product. 
 
 A question of great interest is whether it is possible to produce the 
 blood-pigment from its cleavage products. In this respect certain recent 
 investigations are interesting. Zaleski obtained from mesoporphyrin 
 hydrocloride dissolved in 80 per cent acetic acid saturated with NaCl 
 and heated to 50°-70°, a hsemin-like pigment by the addition of a solu- 
 tion of iron in acetic acid, and this pigment had a spectrum in acid 
 solution very similar to that of haematin, although not identical with it. 
 
 1 See Hammarsten, Skand. Arch. f. Physiol., 3, and Garrod, Journ. of Physiol., 13. 
 
 2 Arch. f. (Anat. u.) Physiol., 1904, Suppl. 
 
 3 Wien. klin. Wochenschx., 1903. 
 
KLEMATOIDIN. 301 
 
 Zaleski considers this pigment as a hydrogcnized haemin. A regenera- 
 tion of haematin from haematoporphyrin has been performed by Laid- 
 law. If ha'matoporphyrin is dissolved in dilute ammonia and warmed 
 with Stokes' solution and hydrazine hydrate, iron is taken up again 
 and haemochromogen is produced, which is changed into hsematin by 
 shaking with air. According to Ham and Balean, 1 it is possible to pro- 
 duce haemoglobin from haemochromogen and globin, and it is indeed 
 possible that other proteins can replace globin in this formation. 
 
 Haematoidin, thus called by Virchow, is a pigment which crystallizes 
 in orange-colored rhombic plates, and which occurs in old blood extrav- 
 asations, and whose origin from the blood-coloring matters seems to 
 be established (Langhans, Cordua, Quincke, and others 2 ). A solu- 
 tion of haematoidin shows no absorption-bands, but only a strong absorp- 
 tion from the violet to the green (Ewald 3 ). According to most observers, 
 haematoidin is identical with the bile-pigment bilirubin. It is not 
 identical with the crystallizable lutein from the corpora lutea of the ovaries 
 of the cow (Piccolo and Lieben, 4 Kuhne and Ewald). 
 
 In the detection of the above-described blood-coloring matters the 
 spectroscope is the only entirely trustworthy means of investigation. 
 If it is only necessary to test for blood in general and not to determine 
 definitely whether the coloring-matter is haemoglobin, methaemoglobin 
 or haematin, then the preparation of haemin crystals is an absolutely 
 positive test. In regard to the detection of blood in urine, see Chapter 
 XIV, and for the detection of blood in intestinal contents, in pathological 
 fluids and in chemico-legal cases we must refer the reader to more extended 
 text-books. 
 
 The methods proposed for the quantitative estimation of the blood- 
 coloring matters are partly chemical and partly physical. 
 
 Among the chemical methods to be mentioned is the incineration of the 
 blood and the determination of the amount of iron contained in the ash from 
 which the amount of haemoglobin may be calculated. We must refer to works 
 on chemical methods of investigation in regard to these methods. 
 
 The physical methods consist either of colorimetric or of spectroscopic 
 investigations. 
 
 The principle of Hoppe-Seyler's colorimetric method is that a measured 
 quantity of blood is diluted with an exactly measured quantity of water 
 until the diluted blood solution has the same color as a pure oxy haemo- 
 globin solution of a known strength. The amount of coloring-matter 
 present in the undiluted blood may be easily calculated from the degree 
 of dilution. In the colorimetric testing we use a glass vessel with parallel 
 
 1 Zaleski, Zeitsehr. f. physiol. Chem., 43; Laidlaw, Journ. of Physiol., 31; Ham 
 and Balean, ibid., 32. 
 
 2 A comprehensive review of the literature pertaining to haematoidin may be found 
 in Stadelmann, Der Icterus, etc., Stuttgart, 1891, pp. 3 and 45. 
 
 » Zeitsehr. f. Biologie, 22, 475. 
 
 4 Cit. from Gorup-Besanez, Lehrbuch d. physiol. Chem., 4. Aufl., 1878. 
 
302 THE BLOOD. 
 
 sides containing a layer of liquid 1 cm. thick (Hoppe-Seyler's hsematinom- 
 eter). The use of Hoppe-Seyler's colorimetric double pipette is more 
 advantageous. Other good forms of apparatus have been constructed 
 by Giacosa and Zangermeister. 1 Instead of an oxyhemoglobin solu- 
 tion we now generally use a carbon-monoxide haemoglobin solution as 
 a standard liquid because it may be kept for a long time. The blood 
 solution in this case is saturated with carbon monoxide. 2 
 
 The quantitative estimation of the blood-coloring matters by means 
 of the spectroscope may be done in different ways, but at the present 
 time the spectrophotometric method is chiefly used, and this seems to be 
 the most reliable. This method is based on the fact that the extinction 
 coefficient of a colored liquid for a certain region of the spectrum is directly 
 proportional to the concentration, so that C : E = Ci : E\, when C and 
 C\ represent the different concentrations and E and E\ the corresponding 
 
 C C. 
 coefficients of extinction. From the equation —=-=r 7 it follows that for 
 
 hi tii\ 
 
 one and the same pigment this relation, which is called the absorption 
 ratio, must be constant. If the absorption ratio is represented by A, 
 the determined extinction coefficient by E, and the concentration (the 
 amount of coloring-matter in grams in 1 cc.) by C, then C = AE. 
 
 Different forms of apparatus have been constructed (Vierordt and 
 Hufner 3 ) for the determination of the extinction coefficient, which is 
 equal to the negative logarithm of those rays of light which remain 
 after the passage of the light through a layer 1 cm. thick of an absorbing 
 liquid. In regard to this apparatus the reader is referred to other text- 
 books. 
 
 For purposes of control the extinction coefficients are determined in two dif- 
 ferent regions of the spectrum. Hufner has selected (a) the region between the 
 two absorption-bands of oxyhemobglobin, especially between the wave-lengths 
 554 nn and 565 n^ and (b) the region of the second band, especially the inter- 
 val between the wave-lengths 531.5 mm and 542.5 mm- The constants or the 
 absorption ratio for these two regions of the spectrum are designated by Hufner 
 by A and A'. Before the determination the blood must be diluted with water, 
 and if the proportion of dilution of the blood be represented by V, then the con- 
 centration or the amount of coloring-matter in 100 parts of the undiluted blood 
 is 
 
 C = 100. V. A. E and 
 
 C = 100. V.A'.E'. 
 
 The absorption ratio or the constants in the two above-mentioned regions 
 of the spectrum have been determined for oxyhemoglobin, haemoglobin, carbon- 
 monoxide hemoglobin, and methemoglobin, as follows: 
 
 Oxyhemoglobin A =0.002070 and A' =0.001312 
 
 Hemoglobin A r =0.001354 and A'r =0.001778 
 
 Carbon-monoxide hemoglobin. .A<- =0.001383 and A'c =0.001263 
 
 Methemoglobin Am =0.002077 and A'm = 0.001754 
 
 1 F. Hoppe-Seyler, Zeitschr. f. physiol. Chem., 16; G. Hoppe-Seyler, ibid., 21; 
 Winternitz, ibid.; Giacosa, Maly's Jahresber., 26; Zangermeister, Zeitschr. f. Biol- 
 ogic, 88. 
 
 - See Haldane. Journ of Physiol., 26. 
 
 3 See Vierordt. Die Anwondung des Spektralapparates zu Photometrie, etc. (Tubin- 
 gen, 187 3), and Hufner. Arch. f. (Anat. u.) Physiol., 1894, and Zeitschr. f. physiol 
 Chern , 3, v. Noorden, ibid . 4; Otto, Pfluger's Arch, 31 and 36. 
 
QUANTITATIVE ESTIMATION OF BLOOD-PIGMENTS. 303 
 
 From what has been said above about the absorption behavior) 
 
 the concentration and the extinction coefficient it follows that the quo- 
 
 E' 
 tient of the extinction coefficient — measured at two different parts of 
 
 E 
 
 the spectrum, independently of the concentration, is a characteristic 
 
 constant for the respective pigments. According to Hufner's figures 
 
 this quotient for oxyhemoglobin is 1.58, for haemoglobin 0.76, for 
 
 carbon-monoxide haemoglobin 1.10 and for methemoglobin 1.19. But- 
 
 terfield l who has made a thorough investigation on this, finds the 
 
 figure 1.58 for normal and pathological human blood as well as for 
 
 crystalline human, horse and ox oxyhemoglobin. 
 
 The quantity of each coloring-matter may be determined in a mixture 
 of two blood-coloring matters by this method; this is of special impor- 
 tance in the determination of the quantity of oxyhemoglobin and 
 haemoglobin present in blood at the same time. 
 
 In order to facilitate these determinations, Hufner 2 has worked 
 out tables which give the relation between the two pigments existing 
 in a solution containing oxyhemoglobin and another pigment (hemo- 
 globin, methemoglobin, or carbon-monoxide hemoglobin), and thus 
 allowing of the calculation of the absolute quantity of each pigment. 
 
 Among the many apparati constructed for clinical purposes fcr the 
 quantitative estimation of hemoglobin, Fleischl's hcemometer, which has 
 undergone numerous modifications, Henoccue's hwmatoscope, and 
 Sahli's hcemometer, are to be specially mentioned. In regard to these 
 apparati we must refer to larger hand-books and text-books on clinical 
 methods. 
 
 Many other pigments are found besides the often-occurring hemoglobin 
 in the blood of invertebrates. In a few Arachnide, Crustacea, Gasteropoda? 
 and Cephalopoda 1 a body analogous to hemoglobin, containing copper, hcemo- 
 cyanin, has been found by P'redericq. By the taking up of loosely bound oxygen 
 this body is converted into blue oxyhecmocyanin, and by the escape of the oxygen 
 becomes colorless again. According to Henze 1 gram haunocyanin combines 
 with about 0.4 cc. oxvgen. It is crvstalline and has the following composition: 
 C 53.66; H 7.33; N 16.09; S 0.86; Cu 0.38; O 21.67 per cent. On hydrolytic 
 cleavage with hydrochloric acid Henze found the following division of the nitro- 
 gen in ha>mocyanin: Of the total nitrogen 5.78 per cent was split off as ammonia, 
 2.67 per cent as humus nitrogen, 27.65 per cent as diamino nitrogen, and 63.39 
 per cent as monamino nitrogen. He found no arginine in the cleavage products, 
 but could detect histidine. lysine, tyrosine, and glutamic acid. A coloring- 
 matter called chlorocruorin by Lankester is found in certain Chaetopodffi. 
 Hamcrylhrin, so called by Krukenberg but first observed by Schwalhe. is a 
 red coloring-matter from certain Gephyrea. Besides luemocyanin we find in the 
 blood of certain Crustacea the red coloring-matter tetronerythrin (Halliburton), 
 which is also widely spread in the animal kingdom. Echinochrom, so named 
 
 1 Zetischr. f. physiol. Chem.,'62. 
 
 2 Arch. f. (Anat. u.) Physiol., 1900. 
 
304 THE BLOOD. 
 
 by MacMunn, 1 is a brown coloring-matter occurring in the perivisceral fluid of 
 a variety of echinoderms. According to Henze 2 the Ascidia contain a brown 
 pigment which contains vanadium but does not hold any oxygen in a dissociable 
 form. 
 
 The quantitative constitution of the red blood-corpuscles. The amount 
 of water varies in different varieties of blood-corpuscles between 570- 
 644 p. m., with a corresponding amount, 430-356 p. m., of solids. The 
 chief mass, about tV-jV °f the dried substance consists of haemoglobin 
 (in human and mammalian blood). 
 
 According to the analyses of Hoppe-Seyler 3 and his pupils, the red 
 corpuscles contain in 1000 parts of the dried substance : 
 
 Haemoglobin. Protein. Lecithin. Cholesterin. 
 
 Human blood 868-944 122-51 7.2-3.5 2.5 
 
 Dog's " 865 126 5.9 3.6 
 
 Goose's " 627 364 4.6 4.8 
 
 Snake's " 467 525 
 
 Abderhalden found the following composition for the blood- 
 corpuscles from the domestic animals investigated by him: Water, 
 591.9-644.3 p. m.; solids 408.1-335.7 p. m.; haemoglobin, 303.3-331.9 
 p. m.; protein, 5.32 (dog)-7.85 p. m. (sheep); cholesterin, 0.388 (horse) 
 -3.593 p. m. (sheep) ; and lecithin, 2.296 (dog)-4.855 p. m. 
 
 Of special interest is the varying proportion of the haemoglobin to the 
 protein in the nucleated and in the non-nucleated blood-corpuscles. These 
 last are much richer in haemoglobin and poorer in protein than the 
 former. 
 
 The amount of mineral bodies in various species of animals is different. 
 According to Bunge and Abderhalden the red corpuslces from the pig, 
 horse, and rabbit contain no soda, while those from man, the ox, sheep, 
 goat, dog, and cat are relatively rich in soda. In the five last-mentioned 
 species the amount of soda was 2.135-2.856 p. m. The quantity of potash 
 was 0.257 (dog) -0.744 p. m. (sheep). In the horse, pig, and rabbit 
 the quantity of potash was 3.326 (horse)-5.229 p. m. (rabbit). Human 
 blood-corpuscles contain, according to Wanach, about five times as 
 much potash as soda, on an average 3.99 p. m. potash and 0.75 p. m. 
 soda. The nucleated erythrocytes of the frog, toad, and turtle also 
 
 1 Fredericq, Extrait des Bulletins de l'Acad. Roy. de Belgique (2), 46, 1878; Lan- 
 kester, Journ. of Anat. and Physiol., 2 and 4; Henze, Zeitschr. f. physiol. Chem., 
 33 and 43; Krukenberg, see Vergl. physiol. Studien Reihe 1, Abt. 3, Heidelberg, 
 1880; Halliburton, Journal of Physiol., 6; MacMunn. Quart. Journ. Microsc. Science, 
 1885. 
 
 2 Zeitschr. f. physiol. Chem., 72 and 79. 
 
 3 Med.-chem. Untersuch., 390 and 393. 
 
WHITE BLOOD-CORPUSCLES. 305 
 
 contain, according to Bottazzi and Cappelli, 1 considerably more 
 potassium than sodium. Lime is claimed to be absent in the blood- 
 corpuscles, but according to Hamburger 2 this is not true for at least 
 ox-blood, and magnesia occurs only in small amounts: 0.016 (sheep) 
 -0.150 p. m. (pig). The blood-corpuscles of all animals investigated 
 contain chlorine, 0.460-1.949 p. m. (both in horse), generally 1 to 2 
 p. m., and also phosphoric acid. The amount of inorganic phosphoric 
 acid shows great variation: 0.275 (sheep)-1.916 p. m. (horse). All 
 of the above figures are calculated on the fresh, moist blood-corpuscles. 
 
 By quantitative determinations of the swelling and shrinking of the cells 
 under the influence of NaCl solutions of various concentration, or of serum of 
 various dilutions, Hamburger has attempted to determine for the erythrocytes, 
 as well as the leucocytes, the percentage relationship between the two chief con- 
 stituents of the cells (the frame and the intracellular fluid). He found that the 
 volume of the frame-substance for both varieties of blood-corpuscles of the horse 
 was equal to 53-56.1 per cent. The volume for the red blood-corpuscles was 
 for the rabbit 48.7-51; hen, 52.4-57.7, and for the frog, 72-76.4 per cent. 
 Koeppe has raised objections to these determinations. 3 
 
 The White Blood- corpuscles and the Blood-plates. 
 
 The White Blood-corpuscles, also called Leucocytes or Lymphoid 
 Cells, are of different kinds, and ordinarily we differentiate between 
 the small forms poor in protoplasm, called lymphocytes, and the larger, 
 granular, often more nucleated forms, called leucocytes. The poly- 
 nuclear leucocytes occur in greater abundance in the blood than the 
 lymphocytes. In human and mammalian blood, most of the white 
 blood-corpuscles are larger than the red blood-corpuscles. They also 
 have a lower specific gravity than the red corpuscles, move in the circulat- 
 ing blood nearer to the walls of the blood-vessels, and also have a slower 
 motion. 
 
 The number of white blood-corpuscles varies not only in the different 
 blood-vessels, but also under different physiological conditions. On 
 an average there is only 1 white corpuscle for 350-500 red corpuscles. 
 According to the investigations of Alex. Schmidt 4 and his pupils, 
 the leucocytes are destroyed in great part on the discharge of the blood 
 before and during coagulation, so that discharged blood is much poorer 
 in leucocytes than the circulating blood. The correctness of this state- 
 ment has been denied by other investigators. 
 
 1 Bunge, Zeitschr. f. Biologie, 12, and Abderhalden, Zeitschr. f. physiol. Chem., 
 23 and 25; Wanach, Maly's Jahresber., 18, 88; Bottazzi and Cappelli, Arch. Ital. 
 de Biologie, 32. 
 
 2 Zeitschr. f. physik. Chem. 69. 
 
 3 Hamburger, Arch. f. (Anat. u ) Physiol., 1898; Koeppe, ibid., 1899 and 1900. 
 
 4 Pfluger's Arch., 11 and Kriiger, Arch. f. exp. Path. u. Pharm., 51. 
 
306 THE BLOOD. 
 
 From a histological standpoint we generally, as above indicated, 
 discriminate between the different kinds of colorless blood-corpuscles. 
 Chemically considered, however, there is no known essential difference 
 between them, and what little we do know chemically is chiefly in con- 
 nection with the leucocytes. With regard to their importance in the 
 coagulation of fibrin, Alex. Schmidt and his pupils distinguish between 
 the leucocytes which are destroyed in the coagulation and those which 
 are not. The last mentioned give with alkalies or common-salt solutions 
 a slimy mass; the first do not show such behavior. 
 
 The protoplasm of the leucocytes has, during life, amoeboid move- 
 ments which serve partly to make possible the wandering of the cells, 
 and partly 'to aid in the absorption of smaller grains or foreign bodies 
 and make the phagocytosis possible. The action of various agents 
 such as hyper- and hypotonic ' salt solutions, of foreign ions, such as 
 iodine, bromine, and salts of the alkaline earths upon the chemotaxis 
 and the phagocytic activity of the leucocytes has been thoroughly 
 studied by Hamburger and de Haan, 1 and among other things they have 
 shown that the Ca causes an accellerating influence upon phagocytosis 
 which is peculiar for Ca and does not depend upon its properties as a 
 divalent ion. Because of the contractibility of the leucocytes, the 
 occurrence of myosin in them has been admitted even without any 
 special proof therefore. We know nothing positively whether in the 
 leucocytes, or in the cells, in general, globulins occur with traces 
 of albumins, because cell constituents which used to be called globulins 
 have on more careful investigation been found to be nucleoalbumins 
 or nucleoproteins. The substance observed by Halliburton, 2 and 
 occurring in all cells, which coagulates at 47 to 50° C, is considered as 
 a true globulin. Alex. Schmidt claims to have found serglobulin in 
 equine-blood leucocytes which have been washed with ice-cold water. 
 
 The proteins of the leucocytes as well as the cells in general are prin- 
 cipally compound proteins. For the present it is impossible to state to 
 what extent the nucleoalbumins occur in leucocytes or cells, because in the 
 past no careful differentiation was made between the nucleoalbumins 
 and nucleoproteins. The nucleoproteins are without any doubt the 
 principal constituents of the protoplasm of the white blood-corpuscles, 
 and one of these it seems is identical with the so-called hyaline substance 
 of Rovida, which yields a slimy mass when treated with alkalies or 
 NaCl solutions and which occur in pus-cells. 
 
 On digesting the leucocytes with water, a solution of a protein body 
 
 1 Bioch. Zeitschr., 24 and 20. 
 
 2 See Halliburton, On the chem. Physiol, of the animal cell. King's College, London, 
 Physiol. Labor. Collected papers, 1893. 
 
LEUCOCYTE-. 307 
 
 ia obtained which ran be precipitated by acetic acid and which forms the 
 chief mass of the leucoeytes. This substance, which is undoubtedly 
 concerned in the coagulation of the blood, has been described under 
 different names, such as tissue fibrinogen (Wooldridge) cytoglobin and 
 prdglobulin (Alex. Schmidt) or nucleohistone (Kossel and Lilienfeld ') 
 and consists, chiefly at least, of nucleoprotein. The ordinary view that 
 this is nucleohistone does not seem to be correct, according to the invest- 
 igations of Bang, 2 and further proof is necessary. 
 
 Besides these constituents of the protoplasm of the leucocytes we 
 must also include lecithin and especially phosphatides, cholesterin, glu- 
 cotkiorHc acid (in pus-corpuscles, Mandel and Levene 3 ), purine bodies 
 derived from the nuclein substances and glycogen. According to Hoppe- 
 Seyler glycogen is a constant constituent of all cells having amoeboid 
 movement, and he found it in the colorless blood-corpuscles but not in 
 the non-mobile pus-cells. Nevertheless glycogen has also been found 
 in pus-cells by Salomon 4 and by others. The glycogen found by 
 Huppert, Czerny, Dastre, 5 and others in blood and lymph probably 
 originated from the leucocytes. Enzymes also occur in the leucocytes 
 and the proteolytic enzymes are of special importance. According to 
 Opie and Barker two proteolytic enzymes occur in the leucocytes, 
 one of which is active in alkaline solution and occurs in the polynuclear 
 cells while the other is active in acid solution and occurs in the large 
 mononuclear cells. According to Fiessinger and Marie, the leucocytes 
 contain a proteolytic enzyme which forms peptone, leucine and tyrosine 
 from protein and which is probably identical with the proteolytic enzyme 
 discovered earlier by Achalme in pus. It acts best in faintly alkaline 
 solution, but also in weak acid reaction, and is destroyed at 75-80° C. 
 It occurs in the polynuclear leucocytes but principally in those which have 
 a medullary origin, while it is absent in the leucocytes of the lymph series. 
 The lipase occurring in pus and in blood seems, according to the above 
 experimenters, to originate in the lymphocytes. Tschernoruzki 6 has 
 
 1 See Wooldridge, Die Gerinnung des Blutes (published by M. v. Frey, Leipzig, 1S91); 
 A. Schmidt, Zur Blutlehre, Leipzig, 1892; Lilienfeld, Zeitsehr. f. physiol. Chem., 18. 
 2 1. Bang, Studier over Xukleoproteider, Kristiania, 1902. 
 
 3 Bioehem. Zeitsehr., 4. 
 
 4 In regard to the literature on Cdycogen, see Chapter VII. 
 
 5 Huppert, Centralbl. f. Physiol., 6, 394; Czerny, Arch. f. exp. Path. u. Pharm., 
 31; Dastre, Compt. Rend., 120, and Arch, de Physiol. (5), 7. See also Hirschberg, 
 Zeitsehr. f. klin. Med.. 64. 
 
 6 In regard to the enzymes see Erbcn, Jochmann and E. Midler, Jochmann and 
 Lockemann, Hofmeister*s Beitriige, 11, which contains the literature. Opie. Journ. 
 of exper. Medicine, 8; with Barker, ibid., 9; Fiessinger, and Marie, Journ. de physiol. 
 et de pathol. generate, 11, which also contains the literature and Compt. rend' soc. 
 biol., 66, 6"; Tschernoruzki, Zeitsehr. f. physiol. Chem., 73. 
 
308 THE BLOOD. 
 
 also shown the presence of amylase (diastase), catalase, nuclease and per- 
 oxidase in the polynuclear leucocytes. 
 
 The blood-plates (Bizzozero), hsematoblasts (Hayem), whose nature, 
 preformed occurrence, and physiological importance have been much 
 questioned, are pale, colorless, gummy disks, round or somewhat oval 
 in shape, generally with a diameter one-half or one-third that of the 
 blood-corpuscles. In mammalia their number, according to Aynaud, 
 is on an average 500,000 in 1 c.mm. They change their shape readily, 
 attack foreign bodies and agglutinate under conditions which Aynaud 
 has carefully studied. Human blood-plates consist, according to Deetjen, 1 
 of a nucleus and a hyaline protoplasm. They are very sensitive toward 
 alkalies and much more so than the plates from other mammalia. They 
 are destroyed in a concentration of hydroxyl ions, CoH = i XlO~ 5 and 
 in a concentration of H ions, C H = 2X10 -4 . 
 
 According to the researches of Kossel and of Lilienfeld 2 the blood- 
 plates consist of a chemical combination between protein and nuclein, 
 and hence they are also called nuclein-plates by Lilienfeld, and are 
 considered as derivatives of the cell nucleus. It seems certain that the 
 blood-plates have some connection with the coagulation of blood. The 
 views on this question, especially in regard to the manner in which these 
 plates act in coagulation, are unfortunately very divergent. 
 
 HI. THE BLOOD AS A MLXTURE OF PLASMA AND BLOOD-CORPUSCLES. 
 
 The blood in itself is a thick, sticky, light or dark red liquid, opaque 
 even in thin layers, having a salty taste and a faint odor differing in 
 different kinds of animals. On the addition of sulphuric acid to the 
 blood the odor is more pronounced. In adult human beings the specific 
 gravity ranges between 1.045 and 1.075. It has an average of 1.058 
 for grown men and a little less for women. Lloyd Jones found that the 
 specific gravity is highest at birth and lowest in children until about 
 two years old, and in pregnant women. The determinations of Lloyd 
 Jones, Hammerschlag, 3 and others show that the variation of the specific 
 gravity, dependent upon age and sex, corresponds to the variation in 
 the quantity of haemoglobin. 
 
 The determination of the specific gravity is accurately obtained 
 
 1 Aynaud, Maly's Jahresb., 39; Deetjen, Zeitschr. f. physiol. Chem., 63. 
 
 2 In regard to the literature of the blood-plates, see Lilienfeld, Arch. f. (Anat. u.) 
 Physiol.. 1892, and "Leukocyten und Blutgerrinnung," Verhandl. d. physiol. Gesellsch. 
 zu Berlin, 1892; and also Mosen, Arch. f. (Anat. u.) Physiol., 1893, and Maly's Jahres- 
 ber., 30 and 31. 
 
 3 Lloyd Jones, Journ. of Physiol., 8; Hammerschlag, Wien. klin. Wochenschrift, 
 1880, and Zeitschr. f. klin. Med., 20. 
 
ALKALINITY OF THE BLOOD. 309 
 
 by means of the pyknometer. For clinical purposes, where only small 
 amounts are available, it is best to proceed by the method as suggested 
 by Hammerschlag. Prepare a mixture of chloroform and benzene of 
 about 1.050 sp. gr. and add a drop of the blood to this mixture. If the 
 drop rises to the surface then add benzene, and if it sinks add chloroform. 
 Continue this until the drop of blood suspends itself midway and then 
 determine the specific gravity of the mixture by means of an areometer. 
 This method is not strictly accurate and must be performed quickly. 
 In regard to the necessary details refer to Zuntz and A. Levy. 1 
 
 The reaction of the blood is alkaline toward litmus, and various bodies 
 such as alkali carbonates, the phosphates, alkali-protein combinations, 
 the amino-acids and carbon dioxide all take part in bringing about the 
 normal reaction. According to Henderson 2 the normal reaction is 
 also partly brought about by ammonia formation and partly by the 
 phosphates, in that the kidneys secrete acid salts (phosphates) and return 
 alkali to the blood and regulate the reaction of the blood. 
 
 In considering the alkalinity of the blood we must, as previously 
 remarked, differentiate between the amount of titratable alkali in the blood 
 and the true alkalinity, i.e., the amount of hydroxyl or hydrogen ions 
 in the blood. 
 
 We have a large number of determinations of the quantity of titratable 
 alkali, calculated as Na2C03, in fresh as well as defibrinated blood of 
 animals and man, and in the latter case under healthy and diseased con- 
 ditions. As these determinations have been carried out with dif- 
 ferent methods which were not without error they cannot be given any 
 great importance. The results found generally vary between 3 and 6 
 p. m. Na2CC>3 and for man the figures below 3.3 p. m. and above 5.3 p. m. 
 are considered as pathological. The alkaline reaction diminishes out- 
 side of the body, and indeed the more quickly the greater the original 
 alkalinity of the blood. This depends on the formation of acid in the 
 blood, in which the red-blood corpuscles seem to take part in some way or 
 another. After excessive muscular activity the alkalinity is diminished 
 (Peiper, Cohnstein), and it is also decreased after the continuous 
 ingestion of acids (Lassar, Freudberg, 3 ) and others. 
 
 1 Zuntz, Pfliiger's Arch., 66; Levy, Proceed. Roy. Soc, 71. 
 
 2 Amer. Journ. of Physiol., 21, and Journ. of biol. Chem., 9; see also Robertson, 
 ibid., 6 and 7. 
 
 3 Peiper, Virchow's Arch.. 116; Cohnstein, ibid., 130, which also cites the works 
 of Minkowski, Zuntz, and Geppert; Freudberg, ibid., 125 (literature); in regard to the 
 methods for the estimation of the alkalinity see, besides the above-mentioned authors, 
 v. Jaksch, Klin. Diagnostik; v. Limbeck, Wien. med. Blatter, 18; Wright, The Lancet, 
 1897; Biernacki, Beitrage zur Pneumatologie, etc., Zeitschr. f. klin. Med.. 31 and 
 32; Hamburger, Eine Methode zur Trennung, etc., Arch., f. (Anat, u.) Physiol., 
 
310 THE BLOOD. 
 
 The methods for the determination of the true reaction of animal 
 fluids, also the blood, have been given in Chapter I. For the true 
 alkalinity of the blood, as first shown by Hober and especially by 
 Hasselbalch and Lundsgaard, the carbon dioxide is of the greatest 
 importance in that with an increasing carbon-dioxide tension the con- 
 centration of the H ions increase. Thus Hasselbalch and Lunds- 
 gaard J found that a rise in the carbon-dioxide tension of 30-50 mm., 
 that is a rise of 20 mm., increased the concentration of the H ions about 
 36 per cent. 
 
 For the determination of the true reaction the temperature at which 
 the measurement is made is of the greatest importance. As the dis- 
 sociation constant of water strongly rises with the temperature, the 
 HO ion concentration of the blood must rise with the temperature, and 
 we can believe that the alkalinity of the blood at body temperature 
 must be 2-3 times greater than when measured at 18° and that this 
 alkalinity increases 15-20 per cent when the normal temperature of the 
 body (38°) rises to that of a high fever (42°). 
 
 The true alkalinity of the blood is somewhat variable under different 
 conditions. In this connection it must be remarked that also age and 
 other conditions have an action upon the alkalinity. As the determin- 
 ations are made with different, and not always exact methods, and some- 
 times without consideration of the action of carbon dioxide and tem- 
 perature, it is extremely difficult to give satisfactory average results. 
 Under these circumstances it is perhaps sufficient to refer to the figures 
 given in Chapter I (page 76). 
 
 The alkali of the blood as above mentioned exists in part as alkaline 
 salts, carbonate and phosphate, and partly in combination with protein 
 or hsemoglobin. The first are often spoken of as readily diffusible alkalies, 
 while the others are not or are only diffusible with difficulty (see page 
 268). The quantity of the first, in human blood, is about one-fifth of the 
 total alkali (Brandenburg). The readily as well as the difficultly 
 diffusible alkali is divided between the blood-corpuscles and plasma, and 
 the blood-corpuscles seem to be richer in difficultly diffusible alkali than 
 the plasma or serum. This division may be changed by the influence of 
 even very small amounts of acid, even of carbonic acid, and also, as shown 
 by Zuntz, Loewy and Zuntz, Hamburger, Limbeck, and Gurber, 2 
 
 189*1. See also Maly'fl Jahresber., 2d, 30, and 81; Salaskin and Pupkin, Zeitschr. f. 
 physiol. Chem., 42, and O. Folin, ibid., 43; Laitinen, Hammarsten's Festschr., 1903; 
 Westenrijk, Arch. f. exp. Path. u. Pharm. Suppl., 190S, Schmiedeberg-Festschrift. 
 
 1 The literature may be found in Sorenssen, Messung und Bedeutung der Wasser- 
 Btoff-ionkonzentrationen, Ergbn. d. Physiol, 12; Hasselbalch and Lundsgaard, Bioch. 
 Zeitsrhr., 38, and Skand. Arch. f. Physiol., 27; Hasselbalch, Bioch. Zeitschr., 30; Lunds- 
 gaard, ibid., 41. 
 
 2 Zuntz, in Hermann's Handbuch der Physiol., 4, Abt. 2; Loewy and Zuntz, 
 
BLOOD-CORPUSCLES AND GASES. VISCOSITY. 3U 
 
 by the influence of the respiratory exchange of gas. The blood-corpuscles 
 give up a part of the alkali united with protein to the serum by the action 
 of carbon dioxide, hence the serum becomes more alkaline. The equilib- 
 rium of the osmotic tension in the blood-corpuscles and in the serum 
 is thus disturbed; the blood-corpuscles swell up because they take up 
 water from the serum, and this then becomes more concentrated and richer 
 in alkali, protein, and sugar. Under the influence of oxygen, the cor- 
 puscles take their original form again and the above changes are reversed. 
 The blood-corpuscles for this reason are less biconcave in their small 
 diameter in venous than in arterial blood (Hamburger). 
 
 These conditions have been further studied by v. Koranyi and 
 Bence, 1 and they have investigated the relation between the changes of 
 the volume of the blood-corpuscles and the electrical conductivity, the 
 refractivity of the serum and the viscosity of the blood. The refrac- 
 tion coefficient of the serum is highest with an increase in the amount of 
 carbon dioxide, while it is lowest when the blood is rich in oxygen and 
 poor in carbon dioxide. They consider this as an action of acid, as a 
 similar rise is observed after the addition of acid, while after the addition 
 of alkali a fall in the refraction coefficient of the serum takes place, and 
 these same changes can be brought about by CO2 or by a current of oxygen. 
 With an increase in the amount of carbon dioxide, the conductivity 
 of the blood diminishes; the viscosity is, on the other hand, highest 
 when the blood is richest in carbon dioxide. If the CO2 is driven off 
 by O the viscosity diminishes to a minimum, and on leading in more 
 oxygen it rises again. The changes in viscosity of the blood runs parallel 
 with the volume changes of the blood-corpuscles, and changes in the 
 viscosity, which can be brought about by the removal of carbon dioxide, 
 cause a change in the electric charge of the blood-corpuscles (v. Koranyi 
 and Bence). The viscosity of the blood is a variable quantity which, 
 besides the gas content of the blood, is also dependent upon many other 
 circumstances (Adam 2 ) and which is different at various ages and under 
 unequal physiological and pathological conditions. 
 
 The color of the blood is red — light scarlet-red in the arteries and dark 
 bluish-red in the veins. Blood free from oxygen is dichroic, dark red 
 by reflected light and green by transmitted light. The blood-coloring 
 matters occur in the blood-corpuscles. For this reason blood is opaque 
 
 Pfliiger's Arch., 58; Hamburger, Arch. f. (Anat. u.) Physiol., 1894 and 1898, and 
 Zeitschr. f. Biologie, 28 and 35; v. Limbeck, Arch. f. exp. Path. u. Pharm., 35; Giirber, 
 Sitzungsber. d. phys. med. Gesellsch. zu Wiirzburg, 1895. 
 
 1 Pfliiger's Arch., 110. 
 
 - In regard to the viscosity of the blood and the literature of the subject, see R. 
 Hober in Oppenheimer's Handb. der Bioch., 2, p. 12-18. See also Adam Zeitschr 
 f. klin. Med., 68. 
 
312 THE BLOOD. 
 
 in thin layers. If the haemoglobin is removed from the stroma and 
 dissolved by the blood liquid by any of the above-mentioned means 
 (see page 273), the blood becomes transparent and has then a " lake 
 color." 1 Less light is now reflected from its interior, and this laky 
 blood is therefore darker in thicker layers. On the addition of salt 
 solutions to the blood-corpuscles they shrink, more light is reflected, 
 and the coJor appears lighter. A great abundance of red corpuscles 
 makes the blood darker, while by diluting with serum or by a greater 
 abundance of white corpuscles the blood becomes lighter in appearance. 
 The different colors of arterial and of venous blood depend on the vary- 
 ing quantities of gas contained in these two varieties of blood, or, bet- 
 ter, on the different amounts of oxyhemoglobin and haemoglobin they 
 
 contain. 
 
 The most striking property of blood consists in its. coagulating within 
 a shorter or longer time, but as a rule very shortly after leaving the veins. 
 Different kinds of blood coagulate with varying rapidity; in human 
 blood the first marked sign of coagulation is seen in two to three minutes, 
 and within seven to eight minutes the blood is thoroughly converted into 
 a gelatinous mass. If the blood is allowed to coagulate slowly, the red 
 corpuscles have time to settle more or less before the coagulation, and 
 the blood-clot then shows an upper yellowish-gray or reddish-gray layer 
 consisting of fibrin enclosing chiefly colorless corpuscles. This layer 
 has been called crusta inflammatoria or phlogistica, because it has been 
 especially observed in inflammatory processes and is considered one 
 of the characteristics of them. This crusta f or " huffy coat,'" is not 
 characteristic of any special disease, and it occurs chiefly when the blood 
 coagulates slowly or when the blood-corpuscles settle more quickly 
 than usual. A buffy coat is often observed in the slowly coagulating 
 equine blood. The blood from the capillaries is not supposed to have the 
 power of coagulating. 
 
 Coagulation is retarded by cooling, by diminishing the oxygen, and by 
 increasing the amount of carbon dioxide, which is the reason that venous 
 blood and to a much higher degree blood after asphyxiation coagulates 
 more slowly than arterial blood. The coagulation may be retarded or 
 prevented by the addition of acids, alkalies, or ammonia, even in small 
 quantities; by concentrated solutions of neutral alkali salts and alkaline 
 earths, alkali oxalates and fluorides; also by egg-albumin, solutions of 
 sugar or gum, glycerin, or much water; also by receiving the blood in 
 oil. Coagulation may be prevented by the injection of a proteose solu- 
 tion or of an infusion of the leech into the circulating blood, but this 
 
 1 R. Du Bois-Reymond presents objections to the general use of the above terms, 
 in Centralbl. f. Physiol., 19, p. 65. 
 
COAGULATION OF THE BLOOD. 313 
 
 infusion also acts in the same way on blood just drawn. Coagulation 
 is also hindered by snake poison (cobra-poison), and bacterial toxines. 
 The coagulation may be facilitated by raising the temperature; by con- 
 tact with foreign bodies, to which the blood adheres; by stirring or beat- 
 ing it; by admission of air; by diluting with very small amounts of 
 water; by the addition of platinum-black or finely powdered carbon; 
 by the addition of laky blood, which does not act by the presence of 
 dissolved blood-coloring matters, but by the stromata of the blood-corpus- 
 cles; and also by the addition of the leucocytes from the lymphatic 
 glands, or of a watery saline extract of the lymphatic glands, testicles, or 
 thymus and various other organs (Delezenne, Wright, Arthus, 1 and 
 others) . 
 
 An important question to answer is why the blood remains fluid 
 in the circulation, while it quickly coagulates when it leaves the circula- 
 tion. The reason why blood coagulates on leaving the body is therefore 
 to be sought for in the influence which the walls of the living and unin- 
 jured blood-vessels exert upon it. These views are derived from the 
 observations of many investigators. From the observations of Hewson, 
 Lister, and Fredericq it is known that when a vein full of blood is 
 ligatured at the two ends and removed from the body, the blood may remain 
 fluid for a long time. Brucke 2 allowed the heart removed from a tortoise 
 to beat at 0° C, and found that the blood remained uncoagulated for 
 some days. The blood from another heart quickly coagulated when 
 collected over mercury. In a dead heart, as also in a dead blood-vessel, 
 the blood soon coagulates, and also when the walls of the vessel are changed 
 by pathological processes. 
 
 What then is the influence which the walls of the vessels exert on 
 the liquidity of the circulating blood? Freund found that the blood 
 remains fluid when collected by means of a greased canula under oil or 
 in a vessel smeared with vaseline. If the blood collected in a greased 
 vessel be beaten with a glass rod previously oiled, it does not coagulate, 
 but it quickly coagulates on beating it with an unoiled glass rod or when 
 it is poured into a vessel not greased. The non-coagulability of blood 
 collected under oil was confirmed later by Haycraft and Carlier. 
 Freund found on further investigation that the evaporation of the 
 upper layers of blood or their contamination with small quantities of dust 
 causes a coagulation even in a vessel treated with vaseline. According 
 
 1 Delezenne, Arch, de Physiol. (5), 8; Wright, Journ. of Physiol., 28; Arthus, 
 Journ. de Physiol, et Pathol., 4. 
 
 2 Hewson's works, edited by Gulliver, London, 1876, cited from Gamgee, Text- 
 book of Physiol. Chem., 1, 1880; Lister, cited from Gamgee, ibid.; Fredericq, Recher- 
 ches sur la constitution du plasma sanguin, Gand, 1878; Brucke, Virchow's Arch. 12. 
 
314 THE BLOOD. 
 
 to Frevnd 1 it is this adhesion between the blood and a foreign substance 
 — and the diseased walls of the vessel also act as as such — that gives the 
 impulse toward coagulation, while the lack of adhesion prevents the 
 blood from coagulating. Bordet and Gengou 2 have also shown that 
 the plasma obtained by centrifuging blood collected in a paraffined 
 vessel, and perfectly free from form-elements, can be kept without coagulat- 
 ing in a paraffined vessel, and that it does coagulate on being transferred 
 to an unparaffined vessel. The adhesion of the plasma to a foreign 
 body may also, in the absence of form-elements, give the impulse to 
 coagulation. That this adhesion of the form-elements is of great impor- 
 tance cannot be denied and is also generally accepted. By this adhesion 
 the form-elements undergo certain changes which seem to stand in a 
 certain relation to the coagulation of the blood. 
 
 The views in regard to these changes are, unfortunately, very diver- 
 gent. According to Alex. Schmidt 3 and the Dorpat school an abun- 
 dant destruction of the leucocytes, especially polynuclear leucocytes, 
 takes place in coagulation, and important constituents for the coagula- 
 tion of the fibrin pass into the plasma. A direct relation between the 
 destruction of leucocytes and coagulation is denied by many investigators, 
 while according to other experimenters the essential factor is not a 
 destruction of the leucocytes, but an elimination of constituents from 
 the cells into the plasma. This process is called plasmoschisis by Lowit. 4 
 The passage of cell constituents into the plasma before coagulation must 
 not necessarily be considered as a phenomenon of death, as it may just 
 as well be a secretory process (Arthus, Morawitz, Dastre 5 ). 
 1 ' Great importance has also been ascribed to the blood-plates in coagula- 
 tion as certain investigators (Bizzero, Lilienfeld, Schwalbe, Mora- 
 witz, Burker, Deetjen, Le Sourd and Pagniez) found that they 
 induce, accelerate or make coagulation possible. According to Vinci and 
 Chistoni they are not necessary as they are absent in the blood of 
 birds, which coagulates rapidly, and also in the lymph, of the dog, rabbit 
 and cat. They may nevertheless accelerate coagulation and they are 
 necessary for the contraction of the clot. According to Aynaud they 
 
 1 Freund, Wien. med. Jahrb., 1886; Haycraft and Carlier, Journ. of Anat. and 
 Physiol., 22. 
 
 1 Annal. de l'Institute Pasteur, 17. 
 
 3 Pfliiger's Arch., 11. The works of Alex. Schmidt are found in Arch. f. Anat. 
 und Physiol., 1861, 1862; Pfluger's Arch., 6, 9, 11, 13. See especially Alex. Schmidt, 
 Zur Hlutlehre (Leipzig, 1892), which also gives the work of his pupils, and Weitere 
 Beitnige zur Blutlehre, 1895. 
 
 4 Wien. Sitzungsber., 89 and 90, and Prager med. Wochenschr., 1889, referred 
 to in Centralbl. f. d. med. Wissensch., 28, 265. 
 
 6 Morawitz, Hofmeister's Beitriige, 5; Arthus, Compt. rend. soc. biolog., 55; 
 Dastre, ibid., 55. 
 
FORM-ELEMENTS AND COAGULATION. 315 
 
 arr nol necessary for the contraction of the clot nor for the coagulation 
 ae a whole, and they are absent in the lymph and serous fluids. Accord- 
 ing to Petrone ' they indeed have a function in retarding coagulation. 
 
 WoOLDBTOGl 2 takes a very peculiar position in regard to this question: he 
 considers the form-elements as only of secondary importance in coagulation. 
 Afl he has found, a peptone-plasma which has been freed from all form-con- 
 stituents by means of centrifugal force yields abundant fibrin when it is not 
 separated from a substance which precipitates on cooling. This substance, 
 which WOOLDRIDGE has called A-fibrinogen, seems to all appearances to be a 
 nucleoproteid. which, according to the unanimous view of several investigators, 
 originates from the form-elements of the blood, either the blood-plates or the 
 leucocytes and the generally accepted view as to the great importance of the 
 form-elements in the coagulation of the blood is not really contrary to Wool- 
 dridge's experiments. 
 
 There is great diversity of opinion in regard to those bodies which 
 are eliminated from the form-elements of the blood before and during 
 coagulation. 
 
 According to Alex. Schmidt the leucocytes, like all cells, contain 
 two chief groups of constituents, one of which accelerates coagulation, 
 while the other retards or hinders it. The first may be extracted from the 
 cells by alcohol, while the other cannot be extracted. Blood-plasma 
 contains only traces of thrombin, according to Schmidt, but does con- 
 tain its antecedent, prothrombin. The bodies which accelerate coagu- 
 lation are neither thrombin nor prothrombin, but they act in this wise 
 in that they split off thrombin from the prothrombin. On this account 
 they are called zymoplastic substances by Alex. Schmidt. The nature 
 of these bodies is unknown, and Schmidt has given no opinion as to 
 their relation to the lime salts, which have been found to have zymoplastic 
 activity by other investigators. 
 
 The constituents of the cells which hinder coagulation and which are insoluble 
 in alcohol-ether are compound proteins, and have been called eytoglobin and 
 preglobidin by Schmidt. The retarding action of these bodies may be sup-, 
 pressed by the addition of zymoplastic substances, and the yield of fibrin on coagula- 
 tion in this case is much greater than in the absence of the compound protein 
 retarding coagulation. This last supplies the material from which the fibrin is 
 produced. The process is, according to Schmidt, as follows: The preglobulin 
 first splits, yielding serglobulin, then from this the fibrinogen is derived, and from 
 this latter the fibrin is produced. The object of the thrombin is two-fold. The 
 thrombin first splits the fibrinogen from the paraglobulin, and then converts the 
 
 »See footnote 2, p. 308. Also Schwalbe, ntcrs. z. Blutgerinnung, etc., Braun- 
 schweig, 1900; Morawitz, Deutsch. Arch. f. klin. Med.. 79, and Hofmeister's Beitrage, 
 4 and 5; Biirker, Pflliger's Arch., 102, and Centralbl. f. Physiol., 21; Deetjen, 1. c; 
 Le Sourd and Pagniez, Journ. de Physiol., 11; Vinci and Chistoni, Chem. Centralbl., 
 1909, 2, 838, and Maly's Jahresb., 39; Aynaud, Maly's Jahresb., 39; 165; Petrone, 
 Mary's Jahresber., 31, p. 170. 
 
 2 Die Gerinnung des Blutes (published by M. v. Frey, Leipzig, 1891). 
 
316 THE BLOOD. 
 
 fibrinogen into fibrin. The assumption that fibrinogen can be split from para- 
 globulin has not sufficient foundation and is even improbable. 
 
 According to Schmidt the retarding action of the cells is prominent 
 during life, while the accelerating action is especially pronounced out- 
 side of the body or by coming in contact with foreign bodies. The paren- 
 chymous masses of the organs and tissues, through which the blood flows 
 in the capillaries, are those cell-masses which serve to keep the blood 
 fluid during life. 
 
 Lilienfeld has given further proof as to the occurrence, in the form- 
 elements of the blood, of bodies which accelerate or retard the coagula- 
 tion. According to this author the nature of these bodies is very markedly 
 different from Schmidt's idea. While, according to Schmidt, the coagula- 
 tion accelerators are bodies soluble in alcohol, and the compound proteins 
 exhausted with alcohol act only retardingly on coagulation, Lilienfeld 
 states that the substance which acts acceleratingly and retardingly 
 on coagulation are contained in a nucleoprotein, namely, nucleohistone. 
 Nucleohistone readily splits into leuconuclein and histone, the first 
 of which acts as a coagulation-excitant, while the other, introduced 
 into the blood-vascular system, either intravascular or extravascular, robs 
 the blood of its property of coagulating. Introduced into the circulatory 
 system the nucleohistone splits into its two components. It therefore 
 causes extensive coagulation on one side and makes the remainder of 
 the blood uncoagulable en the other. This theory as well as that of 
 Schmidt is not based upon sufficiently demonstrated facts. 
 
 Brucke showed long ago that fibrin left an ash containing calcium 
 phosphate. The fact that calcium salts may facilitate or even cause a 
 coagulation, in liquids poor in ferment, has been known for several years, 
 through the researches of Hammarsten, Green, Ringer and Sains- 
 bury. The necessity of the lime salts for the coagulation of blood and 
 plasma was first shown positively by the important investigations of 
 Arthus and Pages. Recent investigations of Sabbatani 1 have also 
 shown the importance of calcium salts or the free calcium ions for 
 coagulation without explaining the mode of their action. 
 
 According to the generally accepted tow of Arthus and Pages the soluble 
 lime salts precipitable by oxalate are necessary requisites for the fermentive 
 transformation of fibrinogen, because thrombin remains inactive in the absence 
 of soluble lime salts. This view is untenable, as shown by the researches of 
 Alex. Schmidt, Pekelharing, and Hammarsten. 2 Thrombin acts as well in 
 the absence as in the presence of precipitable lime salts. 
 
 1 Hammarsten, Nova Acta, reg. Soc. Scient. Upsala, (3), 10, 1879; Green, Journ. 
 of Physiol., 8; Ringer and Hainsbury, ibid., 11 and 12; Arthus et Pages and Arthus, 
 see footnote 4, p. 251; Hammarsten, Zeitschr. f. physiol. Chem., 22; Sabbatani, 
 rited, Centralbl. f. Physiol., 16, 665. 
 
 2 Hammarsten, Zeitschr. f. physiol. Chem., 22, where the other investigators are 
 cited. 
 
COAGULATION OF THE BLOOD. 317 
 
 According to Pekelhalixg ' thrombin is the lime compound of 
 prothrombin, and the process of coagulation consists, according to him, 
 in the thrombin transferring the lime to the fibrinogen, which is thereby 
 converted into an insoluble lime compound, fibrin. Among the objec- 
 tions to this theory can be mentioned, the fact that fibrin has not 
 been obtained absolutely free from lime, but still so poor in lime 
 (Hammarsten 2 ) that if the lime belongs to the fibrin, its molecule 
 must be more than ten times greater than the haemoglobin molecule, 
 which is not probable. These as well as many other observations 
 indicate that the lime is carried down by the fibrinogen only as a 
 contamination. 
 
 If, as it seems, the lime is not of importance in the transformation 
 of fibrinogen into fibrin in the presence of thrombin, still this does not 
 contradict the above-mentioned observations of Arthus and Pages that 
 the lime salts are necessary for coagulation of blood and plasma. It 
 is very probable that the lime salts, as admitted by Pekelharing, are 
 a requisite for the transformation of prothrombin into thrombin. 
 
 If we attempt to summarize the more or less contradictory investi- 
 gations and views as given in the preceding pages, we can consider the 
 following facts as conclusive: In the first place, two bodies, the fibrin- 
 ogen and the thrombin, are necessary for the coagulation. The fibrinogen 
 exists preformed in the plasma. The thrombin, on the contrary, does 
 not occur in living blood, at least not in appreciable amounts as such, 
 but is formed from another substance, the prothrombin. The presence 
 of calcium salts is necessary for the formation of this thrombin, while 
 the calcium salts are not necessary for the enzymotic transformation 
 of fibrinogen into fibrin. Besides the calcium salts also other substances, 
 the zymoplastic active substances, are active in the formation of thrombin 
 from its mother-substance, and these zymoplastic substances stand in 
 some relation to the form-elements of the blood. 
 
 The formation of thrombin and the relation of the form-elements 
 therewith are still unexplained and disputed questions. 
 
 It is a question whether the mother-substance of thrombin exists in 
 the plasma of the circulating blood or whether it is a body eliminated 
 from the form-elements before coagulation. We have two opposing 
 views on this question, namely, those of Alex. Schmidt and of Pekel- 
 haring. According to Schmidt prothrombin occurs preformed in the 
 circulating plasma, and it is transformed into thrombin by the zymo- 
 plastic substances which pass out from the form-elements. Pekel- 
 haring, on the contrary, holds the view that the plasma does not contain 
 
 1 See footnote 4, p. 256, and especially Virchow's Festschrift, 1, 1891. 
 
 2 Zeitschr. f. physiol Chem., 28. 
 
318 THE BLOOD. 
 
 appreciable amounts of prothrombin. This body, according to him, 
 passes before coagulation from the form-elements into the plasma, and 
 is there converted into thrombin by the calcium salts. The observa- 
 tion that uncoagulated leech-plasma does not coagulate on the addition 
 of calcium salts, while it does coagulate on the addition of prothrombin 
 solutions, seems to support this view; still it is not quite conclusive. 
 Leech-extract contains a body, hirudin, which, seems to be an anti- 
 body toward thrombin and quantitatively neutralizes it. On the addi- 
 tion of prothrombin, new thrombin may be formed, which may act 
 if the hirudin is not present in too great an excess. 
 
 Other observations which dispute the occurrence of prothrombin in 
 the circulating plasma can be explained in various ways and it is the 
 general view at present that the prothrombin is a preformed constituent 
 of the plasma. : 
 
 Although the opinions are rather united as to the occurrence of at 
 least three bodies, fibrinogen, prothrombin (thrombogen) and lime salts 
 in the plasma, still the question arises how the thrombin is formed from 
 the thrombogen. The zymoplastic substances must be here considered, 
 and the starting-point in these new investigations is the accelerat- 
 ing action upon coagulation, of different tissue extracts, an action which 
 has been known for a long time and was especially studied by Dele- 
 zenne on the plasma from bird's blood. Unfortunately we are not in 
 accord as to the nature and manner of action of the active constituents 
 of these extracts. According to Morawitz the active body is not 
 thrombin, but another substance called thrombokinase, besides lime- 
 salts, which are necessary for the transformation of prothrombin (throm- 
 bogen according to Morawitz). The production of thrombokinase is, 
 according to Morawitz, a general property of the protoplasm, and also 
 occurs in the leucocytes (and blood-plates). Three substances are nec- 
 essary, according to his view, for the formation of thrombin, namely: 
 thrombogen, thrombokinase and lime salts. Thrombogen is, he claims, 
 not quite identical with the prothrombin (other investigators), which 
 he calls a-prothrombin, but is a mother-substance of it. The process 
 of thrombin formation can be given as follows: The kinase first trans- 
 forms the thrombogen into a-prothrombin, which latter then is converted 
 into thrombin (a) by the lime salts. 
 
 'Arthus, Journ. de Physiol, et Pathol., 3 and 4, and Compt. rend. soc. biol., 56. 
 The works of Morawitz may be found in Hofmeister's Beitrage, 4 and 5, Deutsch. 
 Arch. f. klin. Med., 79 and 80, and in Oppenheimer's Handb. der Bioch., 2; Fuld, 
 Centralbl. f. Physiol., 17, p. 529; with Spiro, Hofmeister's Beitrage, 5; Schittenhelm 
 and Bodong, Arch. f. exp. Path. u. Pharm., 54; Bordet and Gengou, Annal. Institut 
 Pasteur, 18. For more recent literature see Loeb, Biochem. Centralbl., 6, p. 907. 
 P. Xolf, Arch internat. de Physiol., 6, 1908. 
 
COAGULATION OF THE BLOOD. 319 
 
 The thrombokinase does not occur to any appreciable extent in 
 the circulating blood, but is supplied by the form-elements. The 
 accelerating action upon coagulation of tissues or parts of tissues depends, 
 as above stated, upon their content of kinase; but it also in part depends 
 upon the fact that the tissue fluids excite the secretory activity of the form- 
 elements. 
 
 Fuld l has arrived at about the same results independently of Mora- 
 witz, but he has selected other names. The three substances, throm- 
 bogen, kinase, and thrombin are called by him plasmozym, cytozym, 
 and holozym. The principal reason why circulating blood remains fluid is, 
 according to Fuld, because the cytozym is only slowly formed therein 
 and the ferment (holozym) produced thereby is quickly changed into an 
 inactive form. Another reason is that the blood contains an antibody 
 for the fibrin ferment. The assumption of Alexander Schmidt that 
 the blood contains substances retarding coagulation (anti-thrombins) 
 has recently also received support by the observations of Fuld and 
 Spiro, Morawitz, Loeb, Nolf, Pugliese, Howell 2 and others. Accord- 
 ing to Howell the non-coagulability of circulating blood depends on 
 the fact that the antithrombin prevents the activation of the prothrombin 
 into thrombin. 
 
 According to the theory of Morawitz, Fuld and Spiro, which is the 
 most accepted, of those substances necessary for coagulation, only 
 the thrombokinase (the cytozym) is absent in the circulating blood, 
 and this is the reason why the circulating blood remains fluid. The 
 reason why the plasma does not contain any thrombokinase lies in the 
 fact that the healthy endothelium of the vessels does mot have any irritat- 
 ing action upon the form-elements, and therefore no mentionable quan- 
 tity of kinase is given off under these circumstances. Such an elimina- 
 tion occurs first outside of the blood vessels, and indeed very quickly 
 in contact with foreign bodies. The formation of thrombin from the 
 thrombogen takes place in an unknown manner by the action of the 
 kinase only in the presence of lime salts (in the plasma), and this throm- 
 bin then transforms the fibrinogen into fibrin. 
 
 A serum poor in ferment and having a weak action can be reactivated by the 
 addition of acid or alkali (Alex. Schmidt, Morawitz), and in this action, accord- 
 ing to Morawitz, a thrombin (/3) is produced which is somewhat different from 
 a-thrombin. The ^-thrombin is produced from a special ^-prothrombin which 
 never occurs in the plasma, but only in the serum. Fuld explains this by 
 affirming that the a-thrombin is changed in the serum into metazym (/?-pro- 
 
 ^entralbl. f. Physiol., 17. See also Fuld and Spiro, Hofmeister's Beitrage, 5. 
 
 2 Fuld and Spiro, 1. c; 'Morawitz, 1. c; Loeb, Hofmeister's Be ; trage, 5; Xolf, Arch, 
 internat. de Physiol., 6; Pugliese, Biochem. Centralbl., 5, p. 930; Howell, Amer. 
 Journ. of Physiol., 29. 
 
320 THE BLOOD. 
 
 thrombin), which is then transformed by the alkali or acid into neozym (=/3- 
 thrombin). Nevertheless it is a fact that the quantity of thrombin in the serum 
 diminishes after coagulation, and that the thrombin action is considerably increased 
 by the addition of alkali or acid as well as by zymoplastic substances. The above 
 view as to the occurrence of different thrombins has not sufficient basis, and 
 Pekelharing x has also raised objections thereto. 
 
 The theories of Morawitz, Fuld and Spiro at least stand in accord 
 with several known facts but do not take sufficient account of the action 
 of the zymoplastic substances of Alex. Schmidt. Thrombokinase is 
 precipitated, by alcohol and is not thermostabile, while the zymoplastic 
 substances, of Schmidt are thermostabile and soluble in alcohol. The 
 thrombokinase cannot therefore be identical with these zymoplastic 
 substances, and hence this theory does not explain the action of these 
 latter. Further, the mode of action of tissue extracts is unexplained, 
 and is a much disputed subject. It can be said that these two views are 
 in the main opposed to each other. According to one (Alex. Schmidt, 
 Arthtjs, Morawitz and others) they do not act like fibrin ferment, 
 but have an indirect action. According to the other (Pekelharing, 
 Huiskamp, Delezenne and Loeb 2 ) they are thrombin, or at least bodies 
 having an analogous action. 
 
 Cramer and Pringle 3 have made the important observation that a 
 carefully prepared oxalate plasma when filtered through a Berkefeld 
 filter does not coagulate on adding calcium chloride, while the unfiltered 
 but centrifuged plasma does coagulate. The reason for this lies in the 
 fact that centrifuged plasma contains blood-plates, which are absent 
 in the filtered plasma. By means of these blood-plates, which yield 
 thrombokinase, the coagulation is produced on the addition of calcium 
 chloride. The points in Nolf's theory of coagulation that are difficult 
 to understand as well as the observations of Freund (page 313) and of 
 Bordet and Gengou (page 314) are explained by this observation. 
 
 L. Loeb, 4 who has carried out complete investigations on the coagulation of 
 blood, especially of Crustaceae, has arrived at the following view: The coagula- 
 tion in the Crustacea? can, according to him, be of two kinds. It may in part 
 be an agglutination of the amcebocytes and in part a fibrin formation from a fibrino- 
 gen of the plasma. This latter coagulation is essentially the same as occurs in 
 vertebrates. The substance acting here as the excitant for the coagulation is 
 also active in the absence of lime salts, and behaves therefore like a thrombin. 
 The tissues contain constituents which accelerate coagulation, which Loeb calls 
 coagnlins, which are not identical with the coagulins of the clot or the blood serum, 
 
 1 Bioch. Zeitschr., 11. 
 
 2 Huiskamp. Zeitschr. f. physiol. Chem., 34, 39; Delezenne, Arch. de. physiol., 
 1897; Loeb, Biochem. Centralbl., 6, pages 829 and 889. 
 
 • Quarterly Journ. of exp. Physiol., 6. 
 
 4 Medical News, New York, 1903, and Virchow's Arch., 176; Hofmeister's Beitrage, 
 5, 6, 8. 9, and Biochem. Centralbl., 6, pages 829 and 889. 
 
COAGULATION OF THE BLOOD. 321 
 
 and these have also, although only in the presence of lime salts (if the author 
 understands Loeb), a direct coagulating action upon fibrinogen. According to 
 Loeb the tissue coagulina do not act as kinases in the invertebrates, and he also 
 finds it improbable that they would act as kinases in the vertebrates. Under 
 favorable conditions the combined blood and tissue coagulins are more active 
 than the Bum of the individual action. That this is due to an activation by a 
 kinase, which is a possible explanation, has, in Loeb's opinion, not been proved. 
 
 The coagulins of the blood are, as above stated, according to Loeb, different 
 from the tissue coagulins. The latter are for different classes of animals so 
 adapted that they bring about a quicker coagulation in the blood of certain classes 
 of animals than do the other class. The erythrocytes of mammalia (cat, dog, ral )1 tit ) 
 contain, on the contrary, according to Loeb and Fleisher ' coagulins of such a 
 specific adaptability that it is possible to differentiate between the blood corpuscles 
 of different kinds of mammalia or, if the erythrocytes are known, to detect an 
 unknown plasma. 
 
 Opinions are strikingly at variance in regard to the mode cf action 
 of the tissue constituents which accelerate coagulation, and their nature 
 also is entirely unknown, hence great confusion exists on the whole in 
 this subject. 
 
 If we accept the fact that thrombokinase does not occur in the plasma, 
 but is produced under the influence of a foreign body acting as an excitant, 
 it is rather difficult to understand why the plasma obtained from blood 
 collected in a paraffined vessel and quickly and strongly centrifuged, 
 and which is perfectly free from form-elements, should remain fluid for 
 a long time in a paraffined vessel while it coagulates in an ordinary glass 
 vessel. Nolf has tried by his theory to explain this difficulty, as well 
 as the action of the alcohol-soluble zymoplastic substances (Alex. 
 Schmidt). 
 
 According to Nolf 2 the following bodies take direct part in the 
 coagulation of the blood, namely: Fibrinogen, thrombogen (formerly 
 called hepatothrombin by him) thrombozym ( = thrombokinase of Mora- 
 witz) and lime salts. The coagulation of the blood, according to him, 
 is a different process from the coagulation of a fibrinogen solution by 
 thrombin. While in this last case the thrombin is the substance exciting 
 coagulation, in the other case the thrombin is a product of the coagu- 
 lation, as suggested by Wooldridge. In the coagulation of the plasma, 
 according to Nolf we have a mutual precipitation of the three above- 
 mentioned colloids — fibrinogen, thrombogen and thrombozym, all three 
 of wliich are contained in the fibrin clot. This latter has correspondingly 
 no constant composition, but varies according to the relative propor- 
 tions of these three colloids. In the presence of only a little fibrinogen 
 thrombin is produced from the three colloids (in the presence of lime 
 salts) ; in the presence of abundance of fibrinogen, on the contrary, fibrin 
 
 1 Loeb and Fleisher, Bioch. Zeitchr. 28. 
 
 2 Arch, internat. de Physiol., 6, Fasc, 1, 2, and 3 and 7 and 9. 
 
322 THE BLOOD. 
 
 is formed. Thrombin is a fibrin incompletely saturated with fibrinogen, 
 and in the coagulation of fibrinogen with thrombin the still unsatisfied 
 affinities of the latter are saturated. ("La thrombine d'A. Schmidt n'est- 
 pas autre chose que de la fibrine insuffisamment pourvue de fibrinogene. 
 Dans la coagulation du fibrinogene par la thrombine les affinites restees 
 libres de celle-ci peuvent s'assouvir; le compose moins sature se trans- 
 forme en un compose plus satureV') The formation of fibrin from 
 fibrinogen is not, according to Nolf, an enzymotic process, and the 
 thrombin is only a residue of the fibrin remaining in solution. 
 
 In Nolf's opinion the thrombogen is probably formed in the liver 
 and found to a large extent in all plasma. The thrombozym is secreted 
 by the leucocytes and the endothelial cells, and in opposition to Mora- 
 witz is not secreted by other cells. It is also a normal constituent of the 
 blood-plasma circulating in the living body. Most tissues, on the con- 
 trary, contain no thrombozym. The tissue extracts, Nolf believes, 
 also contain no substances absolutely necessary for the coagulation, 
 but only bodies which can have a powerful accelerating action, the 
 thromboplastic substances which are mixed with the thrombokinase of 
 Morawitz. The circulating blood-plasma contains all the bodies directly 
 necessary in the coagulation, namely, fibrinogen, thrombogen, throm- 
 bozym and lime salts. Besides these it also contains a substance that 
 inhibits coagulation, antithrombin, which is formed in the liver. There 
 exists, if the author understands the work of Nolf, a labile equilibrium 
 between the various constituents of the plasma, and this equilibrium is 
 destroyed in coagulation. The first impulse to coagulation is given by 
 the thromboplastic substances. 
 
 Nolf considers as thromboplastic active any influence of a physical 
 or chemical nature which, be it produced by the walls of the vessel, a 
 suspended body, a solvent or a dissolved body, a colloid or crystalloid, 
 a molecule or an ion, makes the combination of the three above colloids 
 possible. To the thromboplastic agents belong the walls of a glass 
 vessel, finely powdered glass, the precipitates of calcium oxalate or 
 calcium fluoride, also living protoplasm, aqueous tissue extracts, the 
 alcohol soluble zymoplastic substances of Alex. Schmidt, and other 
 substances. All these agents in some way or other may serve as points 
 of precipitation. That a plasma free from form-elements coagulates 
 for example on contact with the walls of a glass vessel depends upon the 
 fact that the inhibitory action of the antithrombin is retarded by the 
 thromboplastic action of the foreign surface. Unfortunately we are 
 ox 1 certain as to how this thromboplastic action is brought about. 
 
 An important side of Nolf's theory of coagulation is also the fibrinol- 
 ysis whifli is brought about by the thrombin. The proteolytic action 
 of the thrombin is due only to the thrombozym contained therein, and 
 
COAGULATION OF THE BLOOD. 323 
 
 it has a proteolytic action only upon fibrin and not upon fibrinogen. 
 According to 'Nolf, coagulation is merely a preparation for the prote- 
 olysis, ;ui<l is a nutrition phenomenon, and in addition is of special 
 importance, In arresting hemorrhage. In order to prevent a rapid 
 fibrinolysis, the plasma also contains one or more antifibrinolytic sub- 
 stances, which are secreted by the liver. 
 
 What has been given contains the chief points in Nolf's theory 
 of coagulation, and it is impossible in a text-book to enter more into 
 detail in regard to his remarkable investigations or the foundations 
 on which he bases his theory and the objections which can be raised 
 against it. 
 
 Recently other investigators as Rettger and Howell have raised 
 objections to the view that the coagulation of the blood is an enzymotic 
 process. Stromberg 1 also leans toward such a conception and they 
 all raise the objection that the quantity of fibrin increases with the quan- 
 tity of thrombin. This behavior, which has been known for a long time, 
 is of such a complicated nature, that no positive conclusions can be drawn 
 therefrom. 
 
 The belief of Mellanbt 2 that the plasma originally only contains one 
 globulin, fibrinogen, from which by enzymotic cleavage the fibrin and serglobulin 
 are formed, is untenable and is based upon the imperfect methods of preparing 
 fibrinogen that he used. 
 
 From the above description of the various theories of coagulation 
 it at least follows that in the study of the coagulation of the blood there 
 are many contradictor}' statements and observations, and so many obscure 
 points, that for the present it is impossible to give a clear, comprehensive 
 summary of the different views and to deduce a theory of the process 
 of coagulation which would embrace all the factors. In spite of this 
 confusion and all contradictions, still we are sure that certain bodies such 
 as fibrinogen and thrombin, even though this latter be an enzyme or a 
 colloid combination, are directly concerned in the formation of fibrin, 
 while other bodies act indirectly as accelerators or inhibitors of coagulation. 
 
 The bodies accelerating coagulation, with the exception of gelatin, 
 whose action in this regard has not been positively proved, have been 
 mentioned several times above. The mode of action of the bodies retard- 
 ing coagulation is not clear and is much disputed. Their action may, 
 it seems, also be more of a direct or indirect kind. Thus, for example, 
 the oxalate and fluoride may prevent the formation of thrombin by 
 precipitation of the lime. The cobra-poison seems to prevent the forma- 
 
 1 Rettger, Amer. Journ. of Physiol., 24; Howell, ibid., 26; Stromberg, Bioch. 
 Zeitschr., 37. 
 
 2 Journ. of Phvsiol., 38. 
 
324 THE BLOOD. 
 
 tion of thrombin by the action upon the thrombokinase ; the hirudin l 
 may, it is generally believed, as antithrombin make the thrombin inactive, 
 and the normal constituents of the plasma retarding coagulation perhaps 
 act in a similar manner. In other cases the retarding bodies act indirectly, 
 for they may, like the proteoses and others, cause the body to produce 
 special bodies which stand in close relation to intravascular coagulation. 
 
 Intravascular Coagulation. It has been shown by Alex. Schmidt 
 and his students, as also by Wooldridge, Wright, 2 and others, that an 
 intravascular coagulation may be brought about by the intravenous 
 injection into the circulating blood of a large quantity of a thrombin 
 solution, as also by the injection of leucocytes or tissue fibrinogen (impure 
 nucleoprotein) . Intravascular coagulation may also be brought about 
 under other conditions, such as after the injection of snake-poison 
 (Martin 3 and others) or certain of the protein-like colloid substances, 
 synthetically prepared according to Grimaux's method (Halliburton 
 and Pickering 4 ). If too little of the above-mentioned bodies be injected, 
 then we observe only a marked retarding tendency in the coagulation 
 of the blood. According to Wooldridge it can generally be maintained 
 that after a short stage of accelerated coagulability, which may lead to a 
 total or partial intravascular coagulation, a second stage of a diminished 
 or even arrested coagulability of the blood follows. The first stage is 
 designated (Wooldridge) as the positive and the other as the negative 
 phase of coagulation. These statements have been confirmed by several 
 investigators. 
 
 There is no doubt that the positive phase is brought about by an 
 abundant introduction of thrombin, or by a rapid and abundant for- 
 mation of the same. The explanation of the production of the negative 
 phase, which can easily be brought about by pepsin proteoses, by various 
 bodies such as extracts of crabs' muscles and other organs, eel-serum, 
 enzymes, bacterial toxines, certain snake-poisons, etc., has been attempted 
 in different ways. The best studied is the action of proteoses, but no 
 conclusive results have been obtained thus far. The assertion of Pick 
 and Spiro that the action of the proteoses does not depend upon the 
 proteoses themselves, but upon a contaminating substance, the protozym, 
 is claimed to' be incorrect by Underhill, while the recent investigations 
 of Popielski indicate that this is correct. The bodies retarding coagu- 
 
 1 The action of hirudin is somewhat doubtful. See Schittenhelm and Bodong, 1. e. 
 
 2 A study of the Intravascular Coagulation, etc., Proceed, of the Roy. Irish Acad. 
 (3), 2. See also Wright, Lecture on Tissue or Cell Fibrinogen, The Lancet, 1892; 
 and Wooldridge's Method of Producing Immunity, etc., Brit. Med. Journ., Sept., 1891. 
 
 3 Journ. of Physiol., 15. 
 * Uriel, 18. 
 
RETARDATION OF COAGULATION. 325 
 
 lation, obtained by Conradi l in autolysis, which are probably antithrom- 
 bins, seem to act in a different way from the proteoses, and cannot for 
 the present be made use of in explaining this question. 
 
 There are a large number of researches on the action of proteoses 
 and of other similar retarding substances by a great number of different 
 investigators, especially by Gley and Pachon, Spiro, Morawitz, Nolf, 
 Delezenne, Doyon and collaborators. 2 We can say with certainty 
 that the action is indirect, and that the liver is important for the process. 
 The non-coagulability of " peptone-blood " seems to be due to several 
 reasons, but it has not been thoroughly explained. On the one hand 
 such blood contains an antithrombin, and on the other it seems as if the 
 formation of thrombin is not sufficient, although the plasma contains 
 the necessary conditions for the thrombin formation, as it coagulates as 
 a rule on dilution with water or the addition of a little acid. This last 
 behavior speaks, according to Mellanby, 3 for the assumption that the 
 liver, because of the proteose injection, gives up an excess of alkali to the 
 blood thus preventing the coagulation of the peptone-blood. Opinions 
 in regard to the occurrence of an antithrombin in the peptone- 
 plasma seem to be unanimous, and we have gained considerable 
 experience in regard to the formation of this antithrombin. According 
 to Nolf, the peptones (more correctly the proteoses) cause an altera- 
 tion in the leucocytes and the walls of the vessels, and a substance is 
 secreted which brings about, in the liver, the formation of antithrombin. 
 According to Delezenne the proteoses bring about a destruction of 
 leucocytes, and thereby a substance accelerating coagulation and another 
 having a retarding action is set free. The first is destroyed by the liver, 
 and hence the action of the retarding substance (the antithrombin) is 
 obtained. Doyon and co-workers have also shown that the isolated 
 washed liver on transfusing normal arterial blood, gives off a thermo- 
 stable antithrombin, which behaves like a nucleoprotein. That the liver 
 takes part in the retardation of coagulation is positively known. 
 
 1 Pick and Spiro, Zeitschr. f. physiol. Chem., 31; Underhill, Amer. Journ. of 
 Physiol., 9; Popielski, Arch. f. expt. Path. u. Pharm., Suppl. 1908, Schmiedeberg's 
 Festschrift; Conradi, Hofmeister's Beitrage, 1. 
 
 2 Grosjean, Travaux du laboratoire de L. Fredericq, 4, Liege, 1892; Ledoux, ibid., 
 5, 1896; Nolf, Bull. l'Acad. roy. de Belgique, 1902 and 1905, and Biochem. Centralbl., 
 3; and footnote 1, p. 318; Spiro and Ellinger, Zeitschr. f. physiol. Chem., 23; Fuld 
 and Spiro, 1. c; Morawitz, 1. c. The works of the above-mentioned French investi- 
 gators can be found in Compt. rend. soc. biol., 46, 47, 48, 50, and 51, and Arch. d. 
 Physiol. (5), 7, 8, 9, and 10; see also especially Delezenne, Arch. d. Physiol. (5), 10; 
 Compt. rend. soc. biol., 51, and Compt. Rend., 130; Doyon, Compt. rend. soc. biol., 
 68, with Morel and Policard, ibid, 70. 
 
 3 Journ. of Physiol., 38. 
 
326 THE BLOOD. 
 
 The reason of the slow coagulation of the blood in hcemophilia is not 
 well known. Recent investigations of Morawitz and Lossen, Sahli, 
 Nolf and Henry x make it very probable that the thrombokinase plays 
 an important part. According to Sahli the quantity of kinase is dimin- 
 ished, while according to Nolf and Henry, it is qualitatively changed 
 so that it is less active. Both cases explain the repeatedly observed 
 relation of the vessel-walls to haemophilia as, according to Nolf, the 
 thrombokinase (his thrombozym) is also secreted by the endothelial cells. 
 
 The non-coagulability of cadaver blood depends usually, according to Mora- 
 witz, 2 upon the fact that it contains no fibrinogen, due to a fibrinolysis. 
 
 The gases of the blood will be treated in Chapter XVI (on respiration) 
 
 IV. THE QUANTITATIVE COMPOSITION OF THE BLOOD. 
 
 The quantitative analyses of the blood are of little value. We must 
 ascertain on one side the relation of the plasma and blood-corpuscles to 
 each other, and on the other the constitution of each of these two chief 
 constituents. The difficulties which stand in the way of such a task, 
 especially in regard to the living, non-coagulated blood, have not been 
 removed. Since the constitution of the blood may differ not only in 
 different vascular regions, but also in the same region under different 
 circumstances, which renders a number of blood analyses necessary, it 
 can hardly appear remarkable that our knowledge of the constitution of 
 the blood is still relatively limited. 
 
 The relative volume of blood-corpuscles and serum in blood has been 
 determined by various methods. Of these methods that of L. and M. 
 Bleibtreu, 3 against which important objections have been raised by 
 several investigators, such as Eykman, Biernacki and Hedin, 4 must 
 be especially mentioned. In regard to this as well as to the method 
 of St. Bugarsky and Tangl, which is based upon a difference in the 
 electrical conductivity of the blood and the plasma, and Stewart's 5 
 colorimetric method, we must refer to the original publications. 
 
 For clinical purposes the relative volume of corpuscles in the blood 
 may be determined by the use of a small centrifuge called a hcematocrit, 
 constructed by Blix and described and tested by Hedin. A measured 
 quantity of blood is mixed with a known volume (best an equal volume) 
 
 1 Morawitz and Lossen, Deutsch. Arch. f. klin. Med., 94; Sahli, ibid., 99; Nolf 
 and Henry, Revue de medicine, 29, 1909. 
 
 2 Hofmeister's Beitrage, 8. 
 
 3 Pfliiger's Arch., 51, 55, and 60. 
 
 4 Biernacki, Zeitschr. f. physio! . Chem., 19; Eykman, Pfliiger's Arch.. 60; Hedin, 
 ibid., and Skand. Arch. f. Physiol., 5. 
 
 6 Bugarsky and Tangl, Centralbl. f. Physiol., 11; Stewart, Journ. of Physiol., 24. 
 
QUANTITATIVE COMPOSITION OF THE BLOOD. 327 
 
 of a fluid which prevents coagulation. This mixture is introduced into 
 a tube and then centrifuged. According to Hedin it is best to treat the 
 blood, which is kept fluid by 1 p. m. oxalate, with an equal volume of 
 a 9 p. m. NaCl solution. After complete centrifugalization, the layer of 
 blood-corpuscles is read off on the graduated tube and the volume of 
 blood-corpuscles (or more correctly the layer of blood-corpuscles) in 100 
 vols, of the blood calculated therefrom. By means of comparative counts, 
 Hedin and Daland have found that an approximately constant relation 
 exists between the volume of the layer of blood-corpuscles and the number 
 of red corpuscles under physiological conditions, so that the number of 
 corpuscles may be calculated from the volume. Daland x has shown 
 that such a calculation gives approximate results also in disease, when 
 the size of the blood-corpuscles does not essentially deviate from the 
 normal. In certain diseases, such as pernicious anaemia, this method 
 gives such inaccurate results that it cannot be used. 
 
 Koppe 2 has showTi that in centrifuging blood very rapidly, more 
 than 5000 turnes per minute, the blood-corpuscles may be so completely 
 separated that all intermediate fluid is removed. Because of the absence 
 of this intermediate fluid the refraction is changed; the outer layers of 
 the erythrocytes containing fat become transparent, and the column 
 of blood-corpuscles becomes transparent and laky. If the volume of 
 the separated column of blood-corpuscles is determined and the number 
 of red blood-corpuscles counted, the absolute volume of these latter 
 can be determined by this method. 
 
 In determining the relation between the weight of blood-corpuscles and 
 the weight of blood-fluid, we generally proceed in the following manner: 
 
 If any substance is found in the blood which belongs exclusively to 
 the plasma and does not occur in the blood-corpuscles, then the amount of 
 plasma contained in the blood may be calculated if we determine the 
 amount of this substance in 100 parts of the plasma or serum respectively 
 on the one side, and in 100 parts of the blood on the other. If we repre- 
 sent the amount of this substance in the plasma by p and that in the 
 blood by b, then the amount of x in the plasma from 100 parts of blood is 
 
 100.6 
 
 x = . 
 
 P 
 
 Such a substance, which occurs only in the plasma, is fibrin according 
 to Hoppe-Seyler, sodium according to Bunge (in certain kinds of blood). 
 The experimenters just named have tried to determine the amount of the 
 plasma and blood-corpuscles, respectively, in different kinds of blood, 
 starting from the above-mentioned substances. 
 
 Another method suggested by Hoppe-Seyler is to determine the 
 total amount of haemoglobin and proteins in a portion of blood, and on 
 the other hand the amount of haemoglobin and proteins in the blood- 
 corpuscles (from an equal portion of the same blood) which have been 
 sufficiently washed with common-salt solution by centrifugal force. The 
 figure obtained, as a difference between these two determinations, corre- 
 sponds to the amount of proteins which was contained in the serum of 
 
 1 Hedin, Skand. Arch. f. Physiol., 2, 134 and 361, and 5; Pflliger's Arch., t>0; 
 Daland, Fortschritte, d. Med., 9. 
 
 2 Pfluger's Arch., 107. 
 
328 
 
 THE BLOOD. 
 
 Pig-blood. 
 
 32 
 
 Water l272.20518.36 
 
 46.54 
 
 162.89 
 
 142.20 
 
 8.35 
 
 Solids 
 Haemoglobin. . . . 
 
 Protein 
 
 Sugar 
 
 Cholesterin 
 
 Lecithin. ...... 
 
 Fat 
 
 Fatty acids . ... 
 
 Phosphoric ac ! d \ 
 
 as nuclein J 
 
 Soda 
 
 Potash [ ? 
 
 Iron oxide J. 696 
 
 Lime. . 
 
 Magnesia. . . . 
 Chlorine. ... 
 Phosphoric acid 
 
 Inorganic P2O5 0.7194 
 
 38.26 
 0.684 
 0.231 
 0.805 
 1.104 
 0.027 ,0.448 
 
 0455 0.0123 
 
 '2.401 
 0.152 j 
 
 '0.0689 1 
 
 0.0233 
 
 2.048 
 
 0.1114 
 
 0.0296 
 
 0.213 
 
 1.504 
 
 57 
 
 0.0656 
 
 0.642 
 
 0.8956 
 
 Ox-b'o >d. 
 
 
 192.65 616.25 
 
 132.85,58.249 
 
 103.10 — 
 
 20.89 48.901 
 
 — 0.708 
 
 1.100 
 1.220 
 
 0.835 
 1.129 
 0.625 
 
 0.0178.0.0089 
 
 0.7266 
 0.2351 
 0.544 
 
 2.9084 
 0.1719 
 
 0.0056 
 0.5901 
 0.2392 
 0.1140 
 
 0.0805 
 0.0300 
 2.4889 
 0.1646 
 0.0571 
 
 Horse-blood. Dog-blood 
 
 -3 ~f- 
 
 30 
 
 243.86 551.14 
 
 153.84 51.15 
 
 125.8 
 
 20.05 
 
 0.26, 
 1.93 
 
 0.02, 
 0.05 
 
 1.32 
 0.59; 
 
 0.04 
 0.18 
 0.98, 
 
 0.76 1 
 
 42.65 
 0.90 
 0.31 
 1.05 
 0.50 
 0.36 
 
 0.01 
 2.62 
 0.15 
 
 0.07 
 0.03 
 2.20 
 0.15 
 0.05 
 
 o w* 1 
 
 s 
 
 
 277.71 514.30 
 
 165.10 
 
 145.6 i 
 
 2.36 
 
 0.56 
 1.02 
 
 0.05 
 
 1.27 
 0.11 
 0.71 
 
 0.03 
 0.60 
 0.67 
 0.54 
 
 Bull-blood. 
 
 42.89 
 
 34.05 
 0.74 
 0.37 
 0.98 
 0.91 
 0.70 
 
 0.01 
 
 2.39 
 0.14 
 
 0.06 
 0.03 
 2.31 
 0.14 
 0.05 
 
 206.81 608.03 
 127.50 57.66 
 106.40 — 
 15.38; 46.41 
 
 — !0.679 
 0.610 0.599 
 0.953 1.244 
 
 — 12.357 
 
 — 0.494 
 
 0.01940.0089 
 
 0.839 
 0.233 
 0.562 
 
 0.009 
 0.628 
 0.236 
 0.133 
 
 i2.873 
 0.174 
 
 0.073 
 0.027 
 2.453 
 [0.156 
 0.041 
 
 Sh?ep-blood. 
 
 200.03 624.16 
 
 118.82 56.63 
 
 102.80 — 
 
 12.80, 46.56 
 
 — 0.708 
 
 1.147 
 1.329 
 
 0.891 
 1.088 
 ■ 0.859 
 
 — 0.4908 
 
 0.0235 0.0109 
 
 0.760 2.917 
 
 0.236 10.172 
 
 0.545 I — 
 
 — !0.089 
 0.006 0.027 
 0.575 !2.516 
 0.228 |0.163 
 0.088 10.057 
 
 Goat-blood. 
 
 SUM 
 
 5 
 
 E<N 
 
 Water 
 
 Solids 
 
 Hamoglobin. . . . 
 
 Protein 
 
 Sugar 
 
 Cholesterin 
 
 Lecithin 
 
 Fat 
 
 Fatty acids 
 
 Phosphoric acid \ 
 as nuclein / 
 
 Soda 
 
 Potash 
 
 Iron oxide 
 
 Lime 
 
 Magnesia 
 
 Chlorine 
 
 Phosphoric acid. . 
 Inorganic PiOs. . . 
 
 211.35 
 
 135.86 
 
 112.50 
 
 18.76 
 
 0.601 
 1.339 
 
 0.028 
 
 0.755 
 0.236 
 0.547 
 
 0.014 
 0.514 
 0.243 
 0.097 
 
 592.54 
 60.25 
 
 50.96 
 0.822 
 0.698 
 1.127 
 0.0407 
 0.398 
 
 0.0117 
 
 2.824 
 0.160 
 
 0.078 
 0.026 
 2.409 
 0.154 
 0.045 
 
 Cat-blood. 
 
 270.90 
 163.11 
 143.2 
 11.62 
 
 0.556 
 1.354 
 
 0.063 
 
 1.174 
 0.112 
 0.694 
 
 0.035 
 0.455 
 0.697 
 0.515 
 
 -3 
 
 Esi 
 3 '-2 
 
 524.17 
 41.35 
 
 33.16 
 0.860 
 0.339 
 0.971 
 0.446 
 0.282 
 
 0.009 
 
 2.512 
 0.148 
 
 0.062 
 0.024 
 2.360 
 0.133 
 0.040 
 
 Rabbit-blood. 
 
 235.74 
 
 136.37 
 
 123.50 
 
 4.55 
 
 0.268 
 1.722 
 
 0.040 
 
 1.946 
 0.615 
 
 0.029 
 0.460 
 0.835 
 0.645 
 
 Sr>: 
 
 3?N 
 
 518.18 
 46.71 
 
 33.63 
 1.036 
 0.343 
 1.105 
 0.749 
 0.507 
 
 0.015 
 
 2.789 
 0.162 
 
 0.072 
 0.028 
 2.438 
 0.151 
 0.040 
 
 Human Blood, 
 Man. 
 
 'a 
 B<N 
 
 o o-< 
 o oO 
 
 s 
 
 349.69 
 163.33 
 
 i Organic 
 y bodies 
 I 159.59 
 
 Inorg. 
 3.74 
 
 0.24 
 
 1.59 
 
 Eo 
 
 439.02 
 47.96 
 
 43.82 
 
 4.14 
 
 1.66 
 0.15 
 
 1.72 
 
 Human Blood, 
 Woman. 
 
 o on 
 
 2 
 
 272.56 
 123.68 
 
 120.13 
 
 3.55 
 
 0.65 
 1.41 
 
 0.36 
 
 551.99 
 51.77 
 
 46.70 
 
 5.07 
 
 -1.92 
 0.20 
 
 0.14 
 
 the first portion of blood. If we now determine the proteins in a special 
 portion of serum of the same blood, then the amount of serum in the 
 blood is easily determined. The usefulness of this method has been 
 confirmed by Bunge by the control experiments with sodium determina- 
 t ions. If the amount of serum and blood-corpuscles in the blood is known, 
 and we then determine the amount of the different blood-constituents in 
 the blood-serum on one side and of the total blood on the other, the dis- 
 tribution of these different blood-constituents in the two chief components 
 
SUGAR IN THE BLOOD. 329 
 
 of the blood, plasma and blood-corpuscles may be ascertained. In the 
 table on page 328 are given analyses of the blood of various animals by 
 Abderhalden 1 according to Hoppe-Seyler's and Bunge's methods. 
 The analyses of human blood by C. Schmidt 2 are older and were made 
 according to another method, hence the results for the weights of the 
 corpuscles are perhaps a little too high. All the results are in parts per 
 1000 parts of blood. 
 
 The relation between blood-corpuscles and plasma may vary con- 
 siderably under different circumstances even in the same species of animal. 
 In animals, in most cases considerably more plasma is found, some- 
 times two-thirds of the weight of the blood. 3 For human blood Arronet 
 has found 478.8 p. m. blood-corpuscles and 521.2 p.m. serum (in defibrinated 
 blood) as an average of nine determinations. Schneider 4 found 349.6 
 and 650.4 p. m. respectively in women. 
 
 The sugar was considered as occurring only in the serum and not with 
 the blood-corpuscles. According to the investigations of Rona and 
 Michaelis the blood-corpuscles of the dog contain considerable amounts 
 of sugar; and the quantity of sugar in the blood, in the blood-corpuscles 
 as well as in the plasma, is increased in man with diabetes mellitus. 
 Hollinger 5 also found that in man, with normal quantity of sugar 
 in the blood, the sugar was distributed almost equally between the 
 blood-corpuscles and the plasma. 
 
 The amount of sugar in the blood-corpuscles, which was shown by 
 Lepixe and Boulud before Michaelis and Rona, has been the sub- 
 ject of numerous investigations by Bang and his pupils, Lyttkens and 
 Sandgren on the one hand and by Rona, Michaelis, Takahashi, 
 Frank and others on the other hand 6 . The results of these investiga- 
 tions are so contradictory that it is hardly possible for the present to 
 draw any positive conclusions. It seems to follow from them, nevertheless, 
 that the dog blood-corpuscles always contain sugar, while for the corpuscles 
 of the rabbit and man the conditions are somewhat doubtful and may 
 be variable (Frank and Bretschneider). According to Lyttkens 
 and Sandgren the blood-corpuscles of man contain as maximum 0.06 
 
 1 Zeitschr. f. physiol. Chem., 23 and 85. 
 
 1 Cited and in part recalculated from v. Gorup-Besanez, Lehrb. d. physiol Chem., 
 4. Aufl., 345. 
 
 'See Sacharjin in Hoppe-Seyler's Physiol. Chem., 447; Otto, Pfliiger's Arch., 
 35; Bunge. Zeitschr. f. Biol., 12; L. and M. Bleibtreu, Pfliiger's Arch., 51. 
 
 4 Arronet, Maly's Jahresber., 17; Schneider, Centralbl, f. Physiol.. 5, 362. 
 
 5 Rona and Michaelis, Bioch. Zeitschr. 16 and 18; Hollinger, ibid.. 17. 
 
 6 Lupine and Boulud, Bioch. Zeitschr., 32; Lyttkens and Sandgren, ibid., 26, 31. 
 36: Rona with Doblin, ibid., 31, with Michaelis, ibid., 37, with Takahashi, ibid., 30; 
 Takahashi, ibid., 37; E. Frank, Zeitschr. f. physiol. Chem., 70, with Bretschneider, 
 ibid., 71 and 76; see also Oppler, ibid., 64 and 75. 
 
330 THE BLOOD. 
 
 p. m. sugar. The blood-corpuscles of the ox, sheep, horse, pig, cat and 
 guinea-pig do not contain any sugar according to these last-mentioned 
 investigators. On the contrary the blood-plasma as well as the blood- 
 corpuscles contain a non-fermentable reducing substance. The quan- 
 tity of this in the human blood-corpuscles is 0.6 p. m. according to 
 Lyttkens and Sandgren and in the blood-corpuscles of different 
 animals an average of 0.44-0.8 p. m. calculated as glucose. The quan- 
 tity of the non-fermentable bodies in the blood-plasma of the animals 
 investigated by them was 0.3 to 0.5 p. m. 
 
 The quantity of glucose in the blood cannot be exactly determined. 
 As the blood also contains other reducing substances besides glucose the 
 total reduction naturally cannot be used as an exact value for the glucose 
 content; and it must also be added that the different methods do not 
 give uniform results. Thus on using the methods of Knapp and Bang, 
 which give the total reduction, higher values are obtained than with 
 Allihn's or Bertrand's methods, in which the quantity of precipitated 
 cuprous oxide is determined. The polarization method cannot give 
 exact results because of the presence of other optically active substances 
 and objections can also be raised against the fermentation method. 1 
 On using this last method Otto 2 first observed, and was substantiated 
 later by others, namely Bang and his co-workers, that the blood contained 
 non-fermentable bodies which reduced Knapp's (and also Bang's) solu- 
 tion. The remaining reduction "rest reduction" after the fermenta- 
 tion cannot be detected according to Bertrand's titration method. 
 
 The nature of this reducing but not fermentable substance occurring 
 in the plasma as well as in the blood-corpuscles is not known. The 
 assumption of Jacobsen, Bing, and Henri ques 3 that this question- 
 able substance is jecorin or lecithin sugar does not have sufficient founda- 
 tion, and the question of the identity with jecorin is doubtful and is con- 
 nected with the question as to the existence of jecorin at all. The 
 conjugated glucuronic acids have also been considered and according 
 to the investigations of Mayer, Lepine and Boulud 4 they occur in 
 blood and originate in the form-elements. For these assumptions we 
 do not have sufficient support, and especially we have no explanation 
 
 1 In regard to methods see Bang, Der Blutzucker, Wiesbaden, 1913 which also 
 describes a new method suggested by him for the determination of sugar in very small 
 amounts of blood. 
 
 2 Pfluger's Arch., 35. 
 
 3 Jacobson, Centralbl. f. physiol. 6; Bing, Skand. Arch. f. physiol., 9; Henriques, 
 Zeitechr. f. physiol Chem., 23. See also P. Mayer, Bioch. Zeitschr., 1 and 4. 
 
 4 Mayer, Zeitschr. f. physiol. Chem., 32; Lepine and Boulud, Compt. Rend., 133, 
 135, 136, 138, 141 and Journ. de Physiol., 7 (cited from Bioch. Centralbl., 4, page 421). 
 
SUGAR IN THE BLOOD. 331 
 
 for the total rest reduction. Frank and Bretschneider l have, never- 
 theless, shown that the reducing substance or mixture that occurs in the 
 blood-corpuscles, and which does not reduce Bertrand's solution, but 
 does reduce Bang's solution, yields a reduceable sugar on boiling with acid 
 which now reduces Bertrand's solution. The corresponding substance 
 in the blood-plasma has a similar behavior. If, as in the experiments 
 of Frank and Bretschneider, the extent of reduction after acid hydrol- 
 ysis is about the same as the original substance (titrated according to 
 Bang) we cannot here be dealing with dextrins and the nature of this 
 body in question (or mixture) is quite unknown. 
 
 In close relation to what has been given above is the question of 
 " sucre immediat " and the " sucre virtuel " of Lupine and Boulud. 2 
 They designate as " sucre immediat " the reduction, calculated as 
 sugar, of the blood immediately after leaving the blood vessels and as 
 " sucre virtuel " the increase in the reducing power brought on in part 
 by allowing the blood to stand after leaving the body, in part by the 
 action of invertase or emulsin at 39° C. and in part by boiling with hydro- 
 fluoric acid. The quantity of "sucre virtuel " in clogs amounts to an average 
 of 70 per cent of the "sucre immediat." The nature of the "sucre virtuel" 
 is not well known ; from what was said above we are probably dealing here 
 to all appearances with very different bodies. 
 
 From what has been presented above it can be understood why the 
 exact sugar content of the blood is not known. In consideration of the 
 above mentioned difficulties and sources cf error attempts have been made 
 to determine the sugar content of the blood and we will give the results 
 of some of these. 
 
 The quantity of actual sugar in the blood, amounts according to Lytt- 
 kens and Sandgren, in man to 0.63, in sheep 0.64, pig 0.82, ox 0.86, 
 horse 0.98, rabbit 2.22, guinea-pig 2.48 and in the cat 2.91 p. m. Small 
 animals with an active metabolism contain more sugar in the blood 
 than larger animals. According to Frank the amount of sugar in the 
 blood-plasma of man lies between 0.8 and 1.1 p. m. and according to 
 Frank and Cobliner 3 it is 1.19-1.26 p. m. in new-born. 
 
 The amount of blood sugar seems to be almost independent of the 
 character of the food. After feeding with large amounts of sugar or dex- 
 trin, Bleile, nevertheless, has observed a considerable increase in the 
 sugar. The amount of sugar is not only somewhat different with 
 various animals but it also varies with the same animal under different 
 
 1 Zeitschr. f. physiol. Chem., 71 and 76. 
 
 2 Compt. Rend., 137, 144, 147, and Journ de Physiol, et d. Path., 11 and 13. 
 
 3 Lyttkens and Sandgren, Bioch. Zeitschr., 36; Frank and Cobliner, Zetischr. f. 
 physiol. Chem., 70. 
 
332 THE BLOOD. 
 
 external conditions. When it amounts to more than 3 p. m., according 
 to a statement of CI. Bernard, 1 sugar appears in the urine and a gly- 
 cosuria occurs, a view that has not been substantiated. On the one 
 hand a glycosuria may occur at a lower sugar content in the blood and 
 on the other hand a glycosuria may be absent for a time with a higher 
 sugar content. An increase in the sugar content occurs, as first shown 
 by Bernard and subsequently proved by others, after drawing blood. 
 In this case not alone is the quantity of sugar increased but also the other 
 reducing substances. According to certain investigators the quantity 
 of these latter is especially increased (Henriques, N. Anderson, Lyttkens 
 andSANDGREN, Lepine and Boulud 2 ). 
 
 Bernard 3 has shown that the quantity of sugar in the blood 
 diminishes more or less rapidly on leaving the veins. Lepine, associated 
 with Barral, has specially studied this decrease in the quantity of 
 sugar, and calls it glycolysis. Lepine and Barral, as well as Arthus, 
 have shown that this glycolysis takes place in the complete absence of 
 micro-organisms. It seems to be due to a soluble glycolytic enzyme whose 
 activity is destroyed by heating to 54° C. This enzyme is derived, 
 according to the above investigators, from the leucocytes and, accord- 
 ing to Arthus as well as to Doyon and Morel 4 it occurs only in the 
 serum but not in the plasma. According to Lepine, 5 it has some con- 
 nection with the pancreas. The glycolysis is, according to Rohmann 
 and Spitzer and Sieber, an oxidation which is produced, according 
 to the two last-mentioned investigators, by an oxidation ferment. Accord- 
 ing to Rona and Doblin it takes place in an atmosphere of hydrogen, 
 which does not speak for the above view. The recent investigations of 
 Slosse, of Embden and collaborators Kraske, Kondo and K. v. Noorden 6 
 
 1 Bleile, Arch. f. (Anat. u.) Physiol., 1879; Bernard, Lecons sur le diabete. 
 
 2 Henriques, Zeitschr. f. physiol. Chem., 23, N. Anderson, Bioch. Zeitschr., 12; 
 Lyttkens and Sandgren, ibid., 26; Lepine and Boulud, Journ. de Physiol., 13. 
 
 8 Lecons sur le diabete, Paris, 1877. 
 
 4 Arthus, Arch, de Physiol. (5), 3; Doyon and Morel, Compt. rend soc. biol., 55. 
 
 5 In regard to the numerous memoirs of L6pine and Lepine and Barral, see Lyon 
 medical., 62 and 63 ; Compt Rend. 110, 112, 113, 120 and 139; Lepine, Le ferment 
 glycolytique et la pathog6nie du diabete (Paris, 1891), and Revue analytique et 
 critique des travaux, etc., in Arch, de m6d. exper. (Paris, 1892); Revue de m^decine 
 1895; Etat actuel de la question de la glycolyse, Semaine m^dicale, 1911; Arthus, 
 Arch, de Physiol (5), 3, 4; Nasse and Framm, Pfliiger's Arch., 63, Paderi, Maly's 
 Jahresber., 26; see also Cremer, Physiologie des Glykogens in Ergebnisse d. Physiol., 
 1, Abt. 1. 
 
 • Rohmann and Spitzer, Ber. d. d. chem. Gesellsch., 28; Spitzer, Pfliiger's Arch., 
 60 and 67; Sieber, Zeitschr. f. physiol Chem., 39 and 44; Rona and Doblin, Bioch. 
 Zeitschr., 32; Slosse, Arch, internat. de Physiol., 11; Kraske, Kondo, and v. Noorden 
 Bioch. Zeitschr., 45. 
 
LACTIC ACID FORMATION FROM SUGAR. 333 
 
 speak positively for the statement that in glycolysis a formation of lactic 
 acid from the sugar occurs. 
 
 That a formation of lactic acid from glucose, and indeed by means of 
 the leucocytes, takes place in glycolysis was shown by Levene and Meyer 
 before Embden and collaborators. On continuing these investigations 
 Levene and Meyer found that fructose as well as mannose and galactose 
 under the same conditions with leucocytes, yield d-lactic acid while with 
 the investigated pentoses, arabinose and xylose, this is not the case. Accord- 
 ing to Embden and co-workers, this formation of lactic acid takes place 
 probably with glyceric aldehyde, and perhaps also with small amounts 
 of dioxyacetone, as intermediary steps, and a formation of lactic acid from 
 glyceric aldehyde (and dioxyacetone) can in fact, as A. Loeb and Gries- 
 bach l have shown, be brought about by enzymotic means by the form- 
 elements of the blood. It seems as if several enzymes were active in the 
 formation of lactic acid from glucose. According to Loeb those varieties 
 of blood which show no glycolysis with the formation of lactic acid, or 
 none worth mentioning, can form lactic acid from glyceric aldehyde 
 and according to Griesbach in this last -mentioned process an enzyme 
 is active which is soluble in water and resistant toward the haemolysis 
 of the blood with water, while the action of the blood upon glucose is 
 destroyed in the destruction of the form-elements by haemolysis. In 
 regard to the formation of lactic acid from methyl glyoxal see page 584. 
 According to Lepine and Boulud a double process takes place in the 
 glycolysis. On one side the sugar is destroyed and on the other side 
 a re-formation of sugar from the "sucre virtuel" takes place. Hereby the 
 actual glycolysis may be greater than the visible, and the mentioned 
 investigators have therefore suggested a method for determining the 
 extent of the actual glycolysis. 2 
 
 The quantity of urea, which, according to Schondorff, is equally 
 divided between the blood-corpuscles and the plasma, is greater on tak- 
 ing food than in starvation (Grehant and Quinquatid, Schondorff) 
 and varies between 0.2 and 1.5 p. m. In dogs Schondorff found in 
 starvation a minimum of 0.348 p. m. and a maximum of 1.529 p. m. at 
 the point of highest urea formation. Gottlieb obtained much lower 
 results by another direct method, namely, in starvation 0.1-0.2, and 
 after meat feeding 0.28-0.56 p. m., Folin and Denis found 0.3-0.77 
 p. m. in the Mood of the cat. In man v. Jaksch 3 found 0.5-0.6 p. m. 
 
 1 Levene and Meyer, Journ. of biol. Chem., 11 and 14; A. Loeb, Bioch. Zeitschr. 
 49 and 50; Griesbach, ibid., 50. 
 
 2 Lepine and Boulud, Journ. de Physiol., et de Path. gene>ale, 13. 
 
 8 Grehant et Quinquaud, Journ. de l'anatomie et de la physiol., 20, and Compt. 
 Rend., 98; Schondorff, Pfliiger's Arch., 54 and 63; Gottlieb, Arch. f. exp. Path. u. 
 Pharm., 42; Folin and Denis, Journ. of biol. Chem., 11 and 12; v. Jaksch, Leyden- 
 Fetschr., I, 1901. 
 
33i THE BLOOD. 
 
 urea in normal blood. The quantity of urea is somewhat increased in 
 fever, and in general in augmented protein metabolism the increased 
 urea formation is dependent upon this. A more important increase in the 
 quantity of urea in the blood occurs in a retarded elimination of urea, 
 as in cholera, also in cholera infantum, and in infections of the kidneys 
 and urinary passages. After ligaturing the ureters or after extirpation 
 of the kidneys of animals, an accumulation of urea takes place in the 
 blood. 
 
 v. Schroder first showed that the blood of the shark was very rich 
 in urea, and the quantity indeed amounted to 26 p. m. Baglioni l 
 has recently shown that this large quantity of urea is of the greatest 
 importance, as the presence of urea in these animals is a necessary life- 
 condition for the heart and very probably for all organs and tissues. 
 
 The blood also contains traces of ammonia. According to Horodyn- 
 ski, Salaskin, and Zaleski, 2 the quantity in arterial dog-blood was 
 0.41 milligram in 100 grams of blood. According to Winterberg, 3 the 
 blood from healthy persons contains on an average 0.90 milligram per 
 100 cc. 3 The quantity of uric acid may be 0.1 p. m. in bird's 
 blood (v. Schroder 4 ). Uric acid has only recently been positively 
 detected under normal conditions, while it has been found, earlier, 
 in the blood in gout, croupous pneumonia, and certain other diseased 
 conditions. Folin and Denis 5 have determined the uric acid in the 
 blood of certain animals as well as in man by a colorimetric method 
 suggested by Folin. Normal human blood contains not less than 1 
 to 2-2.5 milligrams uric acid per 100 grm.; in gout they found 5.5 
 milligrams as maximum. They also determined the quantity of total 
 non-protein nitrogen and urea nitrogen in human blood. In normal 
 blood the first was equal to 22-26 milligrams and the last equal to 11-13 
 ( = 24-28 urea) milligrams in 100 grams of blood. In disease great varia- 
 tions were found. Lactic acid was first found in human blood by 
 Solomon and then by Gaglio, Berlinerblau, and Irisawa. The 
 quantity of lactic acid may vary considerably. Berlinerblau found 
 0.71 p. m. as maximum, in dog's blood. Saito and Katsuyama 6 found 
 on an average 0.269 p. m. in hen's blood, and after carbon-monoxide 
 poisoning the quantity increased to 1.227 p. m. Fat and fatty acids occur 
 
 1 v. Schroder, Zeitschr. f. physiol. Chem., 14; Baglioni, Centralbl. f. Physiol., 19. 
 
 2 Zeitschr. f. physiol. Chem., 35, which also gives the older literature. 
 
 3 Wien. klin. Wochenschr., 1897, and Zeitschr. f. klin. Med., 35. 
 
 4 Lud wig's Festschrift, 1887. 
 
 6 Journ. of biol. Chem., 13 and 14. 
 
 8 Irisawa, Zeitschr. f. physiol. Chem., 17, which also gives the older literature; 
 Saito and Katsuyama, ibid., 32. 
 
BLOOD IN DIFFERENT VASCULAR REGIONS. 335 
 
 perhaps only in the serum. The small traces of bile acids occurring in 
 normal blood, according to Ckoftan, 1 are contained in the leucocytes. 
 
 The calcium occurs, with the exception perhaps of the blood cor- 
 puscles of the ox, only in the plasma and the same applies at least for 
 the principal part of the magnesium. The division of the alkali between 
 the blood-ccrpuscles and the plasma is very different, namely, the blood- 
 corpuscles of the pig, horse and rabbit contain no sodium, the human 
 corpuscles are richer in potassium and those of the ox, sheep, goat, dog 
 and cat are much richer in sodium than potassium. Chlorine occurs in 
 greater abundance in the serum of all animals than in the blood-corpuscles. 
 The iodine only occurs in serum, while iron regularly, almost without ex- 
 ception occurs in the form-elements, especially in the erythrocytes. As the 
 nucleoproteins contain iron, some iron occurs in the leucocytes and traces 
 of iron also occur in the serum. This quantity is very small under normal 
 conditions while in disease the relationship between the haemoglobin- 
 iron and the other blood-iron may, it seems, changes very distinctly. 
 Manganese has also been found in the blood, as well as traces of lithium 
 copper, lead, silver, and also arsenic in menstrual blood. The entire 
 blood contains in ordinary cases 770-820 p. m. water with 180-230 
 p. m. solids, among these 173-220 p. m. are organic and 6-10 p. m., 
 inorganic. The organic consist, after substracting 6-12 p. m. extractives, 
 of protein and haemoglobin. The quantity of the latter in man is 130- 
 150 p. m. In the dog, cat, pig and horse the haemoglobin content is 
 about the same; in ox, bull, sheep, gcat and rabbit blood it is lower 
 (Abderhalden). 
 
 The Composition of the Blood in Different Vascular Regions and under 
 
 Different Conditions. 
 
 Arterial and Venous Blood. The most striking difference between 
 these two kinds of blood is the variation in color caused by their con- 
 taining different amounts of gas and different amounts of oxyhaemoglobin 
 and haemoglobin. The arterial blood is light red; the venous blood is 
 dark red, dichroic, greenish by transmitted light through thin layers. 
 The arterial coagulates more quickly than the venous blood. The latter, 
 on account of the transudation which takes place in the capillaries, was 
 formerly said to be somewhat poorer in water but richer in blood-cor- 
 puscles and haemoglobin than the arterial blood; but this is denied by 
 modern investigators. According to Krtjger 2 and his pupils the quan- 
 
 1 Pfliiger's Arch., 90. 
 
 2 Zeitschr. f. Biologie, 26. This also gives the literature on the composition of 
 the blood in different vascular regions. 
 
336 THE BLOOD. 
 
 tity of dry residue and haemoglobin in blood from the carotid artery and 
 from the jugular vein (in cats) is the same. Rohmann and Muhsam 
 could not detect any difference in the quantity of fat in arterial and venous 
 blood. The serum from dog's blood has, according to Wiener, 1 a rela- 
 tively higher globulin content relative to the albumin in the venous blood 
 as compared with the arterial blood. 
 
 Blo»d from the Portal Vein and the Hepatic Vein. In consequence of 
 the small quantities of bile and lymph formed relatively to the large 
 -quantity of blood circulating through the liver in a given time, we can 
 hardly expect to detect by chemical analysis a positive difference in the 
 composition between the blood of the portal and hepatic veins. The 
 •statements in regard to such a difference are in fact contradictory. For 
 example, Drosdoff found more haemoglobin in the hepatic than in 
 the portal vein, while Otto found less. Kruger finds that the quantities 
 cf haemoglobin, as well as of the solids, in the blood from the vessels 
 passing to and from the liver are different, but a constant relation can- 
 not be determined. The hepatic vein, according to Doyon and collab- 
 orators, 2 is richer in fibrinogen than the blood from the portal vein. 
 The disputed question as to the varying quantities of sugar in the por- 
 tal and hepatic veins will be discussed in a following chapter (see Chap- 
 ter VII, on the formation of sugar in the liver). After a meal rich in 
 carbohydrates, the blood of the portal vein not only becomes richer 
 in glucose, but may also contain dextrin and other carbohydrates (v. 
 Mering, Otto 3 ). The amount of urea in the blood from the hepatic 
 vein is greater than in other blood (Grehant and Quinquaud). In 
 portal blood Folin and Denis found about the same amount of urea 
 as in the carotid blood. Like Horodjnski, Salaskin and Zaleski, 4 
 they found that the portal blood was richer in ammonia than the carotid 
 Hood. The largest amount of ammonia was always found in the blood 
 from the mesentery vessels of the large intestine. 
 
 Blood of the Splenic Vein is decidedly richer in leucocytes than the 
 blood from the splenic artery. The red blood-corpuscles of the blood 
 from the splenic vein are smaller than the ordinary, are less flattened, and 
 show a greater resistance to water. The blood from the splenic vein is 
 also claimed to be richer in water, fibrin, and protein than the ordinary 
 venous blood. According to v. Middendorff, it is richer in haemoglobin 
 
 1 Rohmann and Miihsam, Pfliiger's Arch., 46; Wiener, Zeitschr. f. physiol. Chem., 
 $2. 
 
 2 See footnote 2, page 253. 
 
 1 Drosdoff, Zeitschr. f. physiol. Chem., 1; Otto, Maly's Jahresber, 17; v. Mering, 
 Arch. f. (Anat. u.) Physiol., 1877, 214. 
 
 4 Gr6hant et Quinquaud, 1. c. footnote 3, page 333; Folin and Denis, Journ. of biol. 
 Chem., 11 and 12; Horodjnski, Salaskin and Zaleski, Zeitschr. f. physiol. Chem., 35. 
 
BLOOD OF THE TWO SEXES. 337 
 
 than arterial blood. Kruger 1 and his pupils found that the blood 
 from the vana lienalis is generally richer in haemoglobin and solids than 
 arterial blood; still the contrary is often found. The blood from the 
 splenic vein coagulates slowly. 
 
 The Blood from the Veins of the Glands. The blood circulates with 
 greater rapidity through a gland during activity (secretion) than when 
 at rest, and the outflowing venous blood has therefore during activity a 
 lighter red color and a greater amount of oxygen. Because of the secre- 
 tion, the venous blood also becomes somewhat poorer in water and richer 
 in solids. 
 
 The blood from the Muscular Veins shows an opposite behavior, for 
 during activity it is darker and more venous in its properties because 
 of the increased absorption of oxygen by the muscles and still greater 
 production of carbon dioxide than when at rest. 
 
 Menstrual Blood, according to an old belief, has not the power of 
 coagulating. This statement, is nevertheless, false, and the apparent 
 uncoagulability depends in part on the retarding action of the mucous 
 membrane of the uterus upon coagulation (Cristea and Denk 2 ) and in 
 part on a contamination with vaginal mucus, which disturbs the coagula- 
 tion. Menstrual blood, according to Gautier and Bourcet, contains 
 arsenic and is also richer in iodine than other blood (see Blood-serum, 
 page 269). 
 
 The Blood of the Two Sexes. Women's blood coagulates somewhat 
 more quickly, has a lower specific gravity, a greater amount of water, 
 and a smaller quantity of solids than the blood of man. The amount 
 of blood-corpuscles and haemoglobin is somewhat smaller in woman's 
 blood. The amount of haemoglobin is 146 p. m. for man's blood and 133 
 p. m. for woman's. 
 
 During pregnancy Nasse has observed a decrease in the specific gravity, 
 with an increase in the amount of water, until the end of the eighth month. 
 From then the specific gravity increases, and at delivery it is again 
 normal. The amount of fibrin is somewhat increased (Becquerel and 
 Rodier, Nasse). The number of blood-corpuscles seems to decrease. 
 In regard to the amount of haemoglobin the statements are somewhat 
 contradictory. Cohnstein found the number of red corpuscles diminished 
 in the blood of pregnant sheep as compared with non-pregnant, but 
 the red corpuscles were larger and the quantity of haemoglobin in the blood 
 was greater in the first case. Mollenberg found in most cases an 
 increase in the amount of haemoglobin in pregnancy in the last months, 
 
 1 v. Middendorff, Centralbl. f. Physiol., 2, 753; Kriiger, 1. c. 
 - Cristea and Denk, Maly's Jahresb., 40, 181. 
 
338 THE BLOOD. 
 
 and according to Hermann and Naumann l an increase in the cholesterin 
 ester and the neutral fats occurs in the blood during pregnancy. 
 
 The Blood at Different Periods of Life. Fetal and infant blood is 
 richer in erythrocytes and haemoglobin than the blood of the mother. 
 In animals this is true at least for the haemoglobin while the number 
 of erythrocytes in growing or adult animals may be greater than in new- 
 born animals. The highest percentage of haemoglobin in the blood has 
 been observed by several investigators, such as Cohnstein and Zuntz, 
 Otto, Winternitz, Abderhalden, Schwinge, and others, immediately 
 or very soon after birth or at least within the first few days. In man 
 two or three days after birth the haemoglobin reaches a maximum (200- 
 210 p. m.) which is greater than at any other period of life. This is the 
 cause of the great abundance of solids in the blood of new-born infants, 
 as observed by several investigators. The quantity of haemoglobin and 
 blood-corpuscles sinks gradually from this first maximum to a minimum 
 of about 110 p. m. haemoglobin, which minimum appears in human beings 
 between the fourth and eighth years. The quantity of haemoglobin then 
 increases again until about the twentieth year, when a second maximum 
 of 137-150 p. m. is reached. The haemoglobin remains at this point 
 only to about the forty-fifth year, and then gradually and slowly 
 decreases (Leichtenstern, Otto 2 ) . According to earlier reports, the 
 blood at old age is poorer in blood-corpuscles and protein bodies, but 
 richer in water and salts. 
 
 The Influence of Food on the Blood. In complete starvation no 
 decrease in the amount of solid blood-constituents is found to take place 
 (Panum and others). The amount of haemoglobin is increased a little, 
 at least in the early period (Subbotin, Otto, Hermann and Groll, 
 Luciani and Bufalini), and also the number of red blood-corpuscles 
 increases (Worm Muller, Buntzen 3 ), which probably depends partly 
 on the fact that the blood-corpuscles are not so quickly transformed as 
 the serum and partly on a greater concentration due to loss of water. 
 
 1 Nasse, Maly's Jahresber., 7; Becquerel and Rodier, Traitc de chim. pathol., 
 Paris, 1854; Cohnstein, Pfliiger' Arch., 34, 233; Mollenberg, Maly's Jahresber., 31, 
 185. See also Payer, Arch. f. Gynak., 71; Herrmann and Naumann, Bioch. Zeitschr., 
 43. 
 
 2 Cohnstein and Zuntz, Pfluger's Arch., 34; Winternitz, Zeitschr. f. physiol. Chem., 
 22; Leichtenstern, Untersuch. liber, den Hamoglobingehalt des Blutes, etc., Leipzig, 
 1878; Otto, Maly's Jahresber., 15 and 17; Abderhalden, Zeitschr. f. physiol. Chem., 
 34; Schwinge, Pfluger's Arch., 73 (literature). See also Fehrsen, Journ. of Physiol., 
 30. 
 
 3 Panum, Virchow's Arch., 29; Subbotin, Zeitschr. f. Biologie, 7; Otto. 1. c, 
 Worm Muller, Transfusion und Plethora, Christiania, 1875; Buntzen, see Maly's 
 Jahresber., 9; Hermann and droll, Pfluger's Arch., 43; Luciani and Bufalini, Maly's 
 Jahresber., 12. 
 
INFLUENCE OF FOOD ON THE BLOOD. 339 
 
 In rabbits and to a less extent in dogs, Popel found that complete absti- 
 nence had a tendency to increase the specific gravity of the blood. The 
 amount of fat in the blood may be somewhat increased in starvation 
 because the fat is taken up from the fat deposits and carried to the various 
 organs by the blood (N. Schulz, Daddi '). 
 
 After a rich meal, or after secretion of digestive juices or absorption 
 of nutritive liquids, the relative number of blood-corpuscles may be 
 increased or diminished (Buntzen, Leichtenstern). The number of 
 white blood-corpuscles may be considerably increased after a diet rich 
 in proteins. After a diet rich in fat the plasma becomes, even after a 
 short time, more or less milky-white, like an emulsion. According to 
 Just, in rabbits, on the contrary, the various food-stuffs such as carbohy- 
 drate, fat and protein or peptone has no influence on the number of red 
 and white corpuscles, which he considers as a proof for the difference 
 between the digestive processes in carnivora and herbivora (rabbits). 
 The composition of the food acts essentially on the amount of haemo- 
 globin in the blood. Subbotin has observed in dogs after a one-sided 
 feeding with food rich in carbohydrates that the amount of haemoglobin 
 sank, from the physiological average of 137.5 p. m. to 103.2-93.7 p. m. 
 Tsuboi 2 has also shown in experiments on rabbits and dogs that with 
 an insufficient diet of bread and potatoes, where the body gave up pro- 
 tein and contained relatively considerable carbohydrate, the amount 
 of haemoglobin decreased and the blood became richer in water. Accord- 
 ing to Leichtenstern, a gradual increase in the amount of haemoglobin 
 is found to take place in the blood of human beings on enriching the food, 
 and according to the same investigator the blood of lean persons is gen- 
 erally somewhat richer in haemoglobin than blood from fat ones of the 
 same age. The addition of iron salts to the food greatly influences 
 the number of blood-corpuscles and especially the amount of haemoglobin 
 they contain. The action of the iron salts is obscure. 3 There does not 
 seem to be any doubt that the iron contained in the food in the form 
 of organic compounds is active, but also iron salts and therapeutic iron. 
 According to Bunge and his pupils the iron preparations act indirectly 
 only. They may combine with the sulphureted hydrogen of the intes- 
 tinal canal and thereby prevent the iron associated in the food as assim- 
 
 1 Popel, Arch, des scienc. biol. de St. P€tersbourg, 4, 354; Schulz, Pfluger's Arch., 
 65; Daddi, Maly's Jahresber., 30. 
 
 2 Just, Centralbl. f. Physiol., 23; Subbotin, 1. c; Tsuboi, Zeitschr. f. Biologie, 44. 
 
 3 See Bunge, Zeitschr. f. physiol. Chem., 9; Hausermann, ibid., 23, where the 
 works of Woltering, Gaule, Hall, Hochhaus, and Quincke are cited (the same work 
 contains a table of the quantity of iron in various foods); Kunkel, Pfluger's Arch., 
 61; Macallum, Journal of Physiol., 16; Abderhalden, Zeitschr. f. Biologie, 39. 
 
340 THE BLOOD. 
 
 ilal le protein compounds from being eliminated as iron sulphide (Bunge), 
 a view which is now generally discarded. 
 
 An increase in the number of red corpuscles, takes place after trans- 
 fusion of blood of the same species of animal. According to the observa- 
 tions of Panum and Worm Muller, 1 the blood-liquid is quickly eliminated 
 and transformed in this case — the water being eliminated principally 
 by the kidneys and the protein burned into urea, etc. — while the blood- 
 corpuscles are preserved longer and cause a " polycythcemia." A relative 
 increase in the number of red corpuscles is found after abundant transuda- 
 tion from the blood, as in cholera and heart-failure with considerable 
 congestion. An increase in the number of red blood-corpuscles has 
 also been observed under the influence of diminished pressure or in high 
 altitudes. Viault first called attention to the fact that the number of 
 red corpuscles was very great in the blood of man and animals living 
 in high regions. According to him the llama has about 16 million blood- 
 corpuscles per cubic millimeter. By observations on himself and others, 
 as well as on animals, Viault found the first effect of sojourning in high 
 altitudes was a very considerable increase in the number of red corpuscles, 
 in his own case 5-8 millions. In a young man residing for four weeks 
 in 2900 meters altitude, Lacqueur observed an increase in the erythro- 
 cytes as well as the haemoglobin in the unit volume of the blood. The 
 maximum, which appeared first after 15 days, was 15 per cent increase 
 for the erythrocytes and 16 per cent for the haemoglobin. He also 
 found that dogs from whom about one-half of the blood was drawn required 
 at 2900 meters altitude, on an average of 16 days to replace the same, 
 while at the normal level this requires on an average of 27 days or in 
 round numbers an increase of 70 per cent. Both observations show 
 a re-formation of blood under the influence of high altitude. Cohnheim 
 and Weber 2 found in 23 men engaged on the Jungfrau railroad, who had 
 lived for a long time in high altitudes that the number of erythrocytes 
 (5.2-6.3 million) as well as the haemoglobin (generally 87-94 as com- 
 pared to 80 per cent as normal) was higher than under normal conditions, 
 and they consider this as a proof for the actual formation of blood- 
 corpuscles in high altitudes. A similar increase of the red blood-cor- 
 puscles, as also an increase in the quantity of haemoglobin under the 
 influence of diminished pressure, has been observed by many other inves- 
 tigators, in human beings as well as in animals. Investigators are not 
 united as to how this increase is brought about. The increase in the 
 blood-corpuscles is not absolute, but is only relative, and it is considered 
 by several observers that there is neither a new formation nor a dimin- 
 
 1 Panum, Yin-how's Arch., 29; Worm Muller, 1. c. 
 I : iquim , Deutscb. Arch. f. klin. Med., 110; Cohnheim and Weber, ibid., 110. 
 
VARIATIONS IN THE NUMBER OF RED-CORPUSCLES. 341 
 
 ished destruction of the blood-corpuscles. A relative increase may be 
 brought about in different ways. For example, another division of the 
 blood-corpuscles in the vascular system has been supposed, whereby 
 the blood-corpuscles accumulate in the capillaries, from which region 
 the blood has been examined most often (Zuntz). It is also claimed 
 that a concentration of the blood takes place by increased evaporation 
 (Grawitz), and finally an increase in the blood-corpuscles has also been 
 explained by assuming a contraction of the vascular system with the 
 pressing out of plasma (Bunge, Abderhalden 1 ). In connection with 
 these experiments, it must be remarked that several trustworthy observa- 
 tions show that under the influence of diminished blood-pressure an 
 actual increase in the red blood-corpuscles takes place. These and 
 especially those of Zuntz and his co-workers have shown that under 
 these conditions an increased activity occurs in the red bone-marrow. 
 This question is still not clear. Cohnheim and collaborators 2 have 
 observed in man and dogs, that no essential increase in the blood-corpuscles 
 and haemoglobin occurs in high altitudes after 12 days. They do not 
 dispute the action of a continued residence in high altitudes, and they also 
 do not dispute such an action upon rabbits and mice. The}' explain 
 this in these animals by a concentration of the blood due to a loss of water 
 which is not replaced. In man and dogs on the contrary the loss of 
 water brought about by perspiration is immediately replaced and the 
 concentration of the blood prevented and the increase in the number 
 of blood-corpuscles and of haemoglobin is not observed. 
 
 A decrease in the number of red corpuscles occurs in anaemia from differ- 
 ent causes. Every excessive hemorrhage causes an acute anaemia, or, more 
 correctly, oligaemia. Even during the hemorrhage, the remaining blood 
 becomes by diminished secretion and excretion, as also by an abundant 
 absorption of parenchymous fluid, richer in water, somewhat poorer in 
 proteins, and strikingly poorer in red blood-corpuscles. The oligaemia 
 soon passes into an hydraemia. The amount of protein then gradually 
 increases again; but the re-formation of the red blood-corpuscles is slower, 
 and after the hydraemia follows also an oligocythaemia. After a little 
 time the number of blood-corpuscles rises to normal. Inagaki 3 has 
 made thorough investigations on the changes which the number, volume 
 and haemoglobin content of the erythrocytes undergo after drawing blood 
 as well as during regeneration. It is impossible here to enter more in 
 
 1 The literature on this subject may be found in Abderhalden, Zeitschr. f. Biologie, 
 43; van Voornveld, Pfliiger's Arch., 92. 
 
 2 Hohenklima und Bergwanderungen, by N. Zuntz, A. Loewy, Franz Muller, and 
 W. Caspari, Berlin, 1906; Otto Cohnheim, G. Kreglinger, L. Tobler, O. H. Weber, 
 Zeitschr. f. physiol. Chem., 78 and Cohnheim, Ergebn. d. Physiologie, 1912, 12. 
 
 3 Zeitschr. f. Biol., 49. 
 
342 THE BLOOD. 
 
 detail as to the results, but simply to state that they substantiate the 
 previously known observation that, during regeneration, irregularties 
 may occur in the relation between the quantity of haemoglobin and the 
 number of erythrocytes. A considerable decrease in the number of red 
 corpuscles also occurs in chronic anaemia and chlorosis; still in such cases 
 an essential decrease in the amount of haemoglobin occurs without an 
 essential decrease in the number of blood-corpuscles. The decrease in the 
 amount of haemoglobin is more characteristic of chlorosis than a decrease 
 in the number of red corpuscles. The opinions on the changes in the 
 blood in anaemia and chlorosis differ very considerably. 1 
 
 A very considerable decrease in the number of red corpuscles (300,000- 
 400,000 in 1 c.mm.) and diminution in the amount of haemoglobin 
 ( | - jfo) occurs in pernicious anaemia (Hayem, Laache, and others 2 ). 
 On the contrary, the individual red corpuscles are larger and richer in 
 haemoglobin than they ordinarily are, and the number stands in an inverse 
 relation to the amount of haemoglobin (Hayem). Besides this the red 
 corpuscles often, but not always, show in pernicious anaemia remarkable 
 and extraordinary irregularities of form and size, which has been termed 
 poikilocytosis. 
 
 The number of leucocytes may, as stated above, be increased under 
 physiological conditions as well as after a meal rich in protein (physiological 
 leucocytosis). Under pathological conditions a high leucocytosis may 
 occur, and this is especially found in leucaemia, which is characterized 
 by a very great abundance of leucocytes in the blood. The number of 
 leucocytes is markedly increased in this disease, and indeed, not only 
 absolutely, but also in relation to the number of red blood-corpuscles, 
 which are diminished to a considerable extent in leucaemia. Leucaemic 
 Hood has a lower specific gravity than the ordinary blood (1035-1040), 
 and a paler color, as if it were mixed with pus. The reaction is alkaline, 
 but after death it is frequently acid, probably due to a decomposition 
 of lecithin, which is often considerably increased in leucaemia. Volatile 
 fatty acids, lactic acid, glycero-phosphoric acid, large amounts of purine 
 bases, and so-called Charcot's crystals (see Semen, Chapter XII) have 
 also been found in leucaemic blood. The peptone (proteose) which is 
 found in the leucaemic blood after death, and which does not exist in 
 the fresh blood, is, according to Erben, 3 a digestive product which is 
 
 1 Complete analyses of chlorotic blood may be found in Erben, Zeitschr. f. klin. 
 Med., 47. 
 
 2 Laache, Die Anamie (Christiania, 1883), which also contains the older liter- 
 ature. A complete chemical analysis of the blood has been made by Erben, Zeitschr. 
 f. klin. Med., 40. 
 
 Erben, Zeitschr. f. Heilkunde, 24, and Hofmeister's Beitrage, 5. See also Schumm, 
 ibid., 4 and 5. See also footnote 3, page 342. 
 
QUANTITY OF BLOOD. 343 
 
 produced by a tryptic enzyme which originates from the leucocytes 
 as well as by traces of a peptic enzyme. A chemical analysis of leucrcmic 
 blood has been made by Erben. 1 
 
 A great number of investigations have been made on the chemical 
 composition of blood in disease. But as we have only a few analyses 
 of the blood of healthy individuals, and as the possible variations under 
 physiological conditions are little known, it is difficult to draw any pos- 
 itive conclusions from the analyses of pathological blood. Unfortunately, 
 on account of the large number of contradictory deductions concern- 
 ing the composition of the blood of diseased human beings, it is impossible 
 to give a brief summary of the results, still the changes in the blood in 
 disease must be of the greatest importance. 
 
 The quantity of blood is indeed somewhat variable in different species 
 of animals and in different conditions of the b©dy; in general we consider 
 the entire quantity of blood in adults as about tV-tV (; f the weight of the 
 body, and in new-born infants about tV- Haldane and Lorrain Smith, 2 
 who have determined the quantity of blood by a new method, find in 
 fourteen persons that it varies between -fg- and -£$ of the weight of the body. 
 According to the same method Oerum 3 has determined the quantity 
 of blood in men as about iV and in woman 2 V of the weight of the body. 
 Fat individuals are relatively poorer in blood than lean ones. During 
 inanition the quantity of blood decreases less quickly than the weight 
 of the body (Panum 4 ), and it may therefore be also proportionally greater 
 in starving individuals than in well-fed ones. 
 
 By careful bleeding, the quantity of blood may be considerably dimin- 
 ished without any dangerous symptoms. A loss of blood amounting 
 to one-fourth of the normal quantity has as a sequence no lasting sink- 
 ing of the blood-pressure in the arteries, because the smaller arteries 
 accommodate themselves to the small quantities of blood by contract- 
 ing (Worm Muller 5 ). A loss of blood amounting to one-third of 
 the quantity reduces the blood-pressure considerably, and a loss of 
 one-half of the blood in adults is dangerous to life. The more rapid the 
 bleeding the more dangerous it is. New-born infants are very sensitive 
 to loss of blood, and likewise fat, old, and weak persons cannot stand 
 much loss of blood. Women can stand loss of blood better than men. 
 The quantity of blood may be considerably increased by the injection 
 of blood from the same species of animal (Panum, Landois, Worm 
 
 1 Zeitschr. f. klin. Med., 66 (1908). 
 
 2 Journ. of Physiol., 25. 
 
 3 Deutsch. Arch. f. klin. Med., 93 (1908). 
 
 4 Yirchow's Arch., 29. 
 
 5 Transfusion und Plethora, Christiania, 1875. 
 
344 THE BLOOD. 
 
 Muller, Ponfick). According to Worm Muller the normal quantity 
 of blood may indeed be increased as much as 83 per cent without pro- 
 ducing any abnormal conditions or lasting high blood-pressure. An 
 increase of 150 per cent in the quantity of blood may, with a considerable 
 variation in the blood-pressure, be directly dangerous to life (Worm 
 Muller). If the quantity of blood of an animal is increased by trans- 
 fusion with blood of the same kind of animal, an abundant formation 
 of lymph takes place. The water in excess is eliminated by the urine; 
 and as the protein of the blood-serum is quickly decomposed, while the 
 red blood-corpuscles are destroyed much more slowly (Tschirjew, Fors- 
 ter, Panum, Worm Muller 1 ), a polycythemia is gradually produced. 
 The quantity of blood in the different organs depends essentially on 
 their activity. During work the exchange of material in an organ is 
 more pronounced than during rest, and the increased metabolism is 
 connected with a more abundant flow of blood. Although the total 
 quantity of blood in the body remains constant, the distribution of the 
 blood in the various organs may be different at different times. As a 
 rule the quantity of blood in an organ is an approximate measure of the 
 more or less active metabolism going on in it, and from this point of 
 view the distribution of the blood in the different organs is of interest. 
 According to Ranke, 2 to whom we are especially indebted for our knowl- 
 edge of the relation of the activity of the organs to the quantity of blood 
 contained therein, of the total quantity of blood (in the rabbit) about 
 one-fourth comes to the muscles in rest, one-fourth to the heart and the 
 large blood-vessels, one-fourth to the liver, and one-fourth to the other 
 organs. 
 
 1 Panum, Xord. med. Ark., 7; Virchow's Arch., 63; Landois, Centralbl. f. d. 
 med. Wissensch., 1875, and Die Transfusion des Blutes, Leipzig, 1875; Worm Muller, 
 Transfusion und Plethora; Ponfick, Virchow's Arch., 62; Tschirjew, Arbeiten aus 
 der physiol. Anstalt zu Leipzig, 1874, 292; Forster, Zeitschr. f. Biologie, 11; Panum, 
 Virchow's Arch., 29. 
 
 2 Die Blutvertheilung und der Thatigkeitswechsel des Organe, Leipzig, 1871. 
 
CHAPTER VI. 
 CHYLE, LYMPH, TRANSUDATES AND EXUDATES. 
 
 I. CHYLE AND LYMPH. 
 
 The lymph is at least in part the mediator in the exchange of con- 
 stituents between the blood and the tissues. The bodies necessary for 
 the nutrition of the tissues pass from the blood into the lymph, and the 
 tissues deliver water, salts, and products of metabolism to the lymph. 
 The lymph, therefore, originates partly from the blood and partly from 
 the tissues. From a purely theoretical standpoint one can, according 
 to Heidehain, differentiate between blood-lymph and tissue-lymph 
 according to origin. It is impossible at the present time to separate 
 completely that which comes from the one or the other source. 
 
 The lymph formed in the different organs and tissues has a different 
 composition, and as the lymph is not obtained directly but only from the 
 large lymph vessels, hence the lymph that we use for investiga- 
 tions is generally a mixture, whose composition may vary under certain 
 conditions. The most easily obtained and best studied is the lymph 
 from the thoracic duct. In starving individuals this lymph, which is 
 called starvation lymph, does not essentially differ from other lymphs. 
 After fatty food the lymph, which is called digestion lymph or chyle, 
 differs from other lymphs by its great richness in very finely divided fat, 
 which gives it a milky appearance, and which has led to the old name 
 " lacteal fluid." 
 
 Chemically the lymph is the same as plasma, and contains, at least 
 to a great extent, the same bodies. The observation of Asher and Bar- 
 bera, 1 that the lymph contains poisonous metabolic products, does 
 not contradict such an assumption, as no doubt these products are trans- 
 ferred to the blood with the lymph. Although the blood does not show 
 the same poisonous action as the lymph, still this can be explained by the 
 great dilution these bodies undergo in the blood, and the difference 
 between blood-plasma and lymph is no doubt of a quantitative nature. 
 This difference consists chiefly in that the lymph is poorer in proteins. 
 
 1 Zeitschr. f . Biologie 36. 
 
 345 
 
346 CHYLE, LYMPH, TRANSUDATES AND EXUDATES. 
 
 Lymph, like the plasma, contains seralbumin, serglobulins, fibrinogen, 
 and fibrin ferment. The two last-mentioned bodies occur only in very 
 small amounts; therefore the lymph coagulates slowly (but spontaneously) 
 and yields but little fibrin. Like other liquids poor in fibrin ferment, 
 lymph does not at once coagulate completely, but repeated coagula- 
 tions take place. 
 
 The extractive bodies seem to be the same as in plasma. Sugar (or 
 at least a reducing substance) is found in about the same quantity as in 
 the blood-serum, namely, about 1 p. m. The glycogen detected by Dastre * 
 in the lymph occurs only in the leucocytes. According to Rohmann and 
 Bial, lymph contains a diastatic enzyme similar to that in blood-plasma, 
 and Lepine 2 found that the chyle of a dog during digestion has great gly- 
 colytic activity. Lipases may also occur in lymph. The amount of urea 
 has been determined by Wurtz 3 as 0.12-0.28 p. m. The mineral bodies 
 appear to be the same as in plasma. 
 
 As form-elements, leucocytes and in certain cases red blood-corpuscles 
 are common to both chyle and lymph. Chyle in fasting animals has the 
 appearance of lymph. After fatty food it is, on the contrary, milky, 
 due partly to small fat-globules, as in milk, and partly, indeed, mostly 
 to finely divided fat. The nature of the fat occurring in chyle depends 
 upon the kind of fat in the food. By far the greater part consists of 
 neutral fat, and even after feeding with large quantities of free fatty 
 acids, Munk 4 found that the chyle contained chiefly neutral fat with 
 only small amounts of fatty acids or soaps. 
 
 The gases of the entirely normal human lymph have not thus far been 
 investigated. The gases from dog-lymph contain, according to Hammar- 
 sten, only traces of oxygen, and consist of 37.4-53.1 per cent CO2 and 
 1.6 per cent N, calculated at 0° C, and 760 mm. mercury. The chief mass 
 of the carbon dioxide of the lymph seems to be in firm chemical com- 
 bination. Comparative analyses of blood and lymph have shown that the 
 lymph contains more carbon dioxide than arterial, but less than venous 
 blood. The tension of the carbon dioxide of lymph is, according to 
 Pfluger and Strassburg, 5 smaller than in venous, but greater than in 
 arterial, blood. 
 
 The quantitative composition of the chyle must evidently be very 
 variable. The specific gravity varies between 1.007 and 1.043. As an 
 
 1 Compt. rend, de soc. biol., 47, and Compt. Rend., 120; Arch, de Physiol. (5), 7. 
 
 2 Rohmann and Bial, Pfluger's Arch., 52, 53, and 55; Lepine, Compt. Rend., 110. 
 
 3 Compt, Rend., 49. 
 
 * Virchow's Arch., 80 and 123. In regard to the analysis of the fat of chyle, see 
 Erben, Zeitschr. f. physiol. Chem., 30. 
 
 5 Hammarsten, Die Gase der Hundelymphe, Arbeiten aus d. physiol. Anstalt zu 
 Leipzig, 1871 ; Strassburg, Pfluger's Archiv, 6. 
 
CHYLE AND LYMPH. 347 
 
 example of the composition of human chyle two analyses will be given. 
 The first is by Owen-Rees, of the chyle of an executed person, and the 
 second by Hoppe-Seyler, 1 of the chyle in a case of rupture of the thoracic 
 duct. In the latter case the fibrin had previously separated. The results 
 are in parts per 1000. 
 
 No. 1. No. 2. 
 
 Water 904.8 940.72 water 
 
 Solids 95.2 59.28 solids 
 
 Fibrin Traces 
 
 Albumin 70.8 36.67 albumin 
 
 Fat 9.2 7.23 fat 
 
 Remaining organic bodies. . . 10.8 
 Salts 4.4 
 
 2 . 35 soaps 
 0.83 lecithin 
 
 1 . 32 cholesterin 
 
 3 . 63 alcohol extractives 
 . 58 water extractives 
 
 J 6.80 soluble salts 
 i . 35 insoluble salts 
 
 The quantity of fat is very variable and may be considerably increased 
 by partaking of food rich in fats. I. Munk and A. Rosenstein 2 have 
 investigated the lymph or chyle obtained from a lymph fistula at the 
 end of the upper third of the leg of a girl eighteen years old and weigh- 
 ing 60 kg., and the highest quantity of fat in the chylous lymph was 47 
 p. m. after partaking of fat. In the starvation lymph from the same 
 patient they found only 0.6-2.6 p. m. fat. The quantity of soaps was 
 always small, and on partaking of 41 grams of fat the quantity of soaps was 
 only about 2 V of the neutral fats. Schumm 3 found in the creamy 
 contents of a chylous cyst of the mesentery, 357.8 p. m. fat and compara- 
 tively large amounts of calcium soaps. 
 
 A great many analyses of chyle from animals have been made, and 
 they chiefly show the fact that the chyle is a liquid with a very changeable 
 composition which stands closely related to blood-plasma, but with the 
 principal difference that it contains more fat and less solids. The reader 
 is referred to special works for these analyses, as, for example, to v. Gorup- 
 Besanez's " Lehrbuch der physiologischen Chemie," 4th edition. 
 
 The composition of the lymph is also very changeable, and its specific 
 gravity shows about the same variation as the chyle. In the following 
 analyses, 1 and 2, made by Gubler and Quevenne, are the results 
 obtained from lymph of the upper part of the thigh of a woman aged 
 thirty-nine; and 3, made by v. Scherer, is an analysis of lymph from 
 
 'Owen-Rees, cited from Hoppe-Seyler's Physiol. Chem., 595; Hoppe-Seyler, 
 ibid., 597. See also Carlier, Brit. Med. Journ., 1902, 175, and T. Sollmann, Amer. 
 Journ. of Physiol., 17. 
 
 • Yirchow's Arch., 123. 
 
 3 Zeitschr. f. physiol. Chem., 49. 
 
348 CHYLE, LYMPH, TRANSUDATES AND EXUDATES. 
 
 the sac-like dilated lymphatic vessels of the spermatic cord. No. 4 
 was made by C. Schmidt 1 the data being obtained from lymph from the 
 neck of a colt. The results are expressed in parts per 1000. 
 
 12 3 4 
 
 Water 939.9 934.8 957.6 955.4 
 
 Solids 60.1 65.2 42.4 44.6 
 
 Fibrin 0.5 0.6' 0.4 2.2 
 
 Albumin 42.7 42.8 34.71 
 
 Fat, cholesterin, lecithin 3.8 9.2 Y 35.0 
 
 Extractive bodies 5.7 4.4 J 
 
 Salts 7.3 8.2 7.2 7.5 
 
 The salts found by C. Schmidt in the lymph of the horse have the 
 following composition, calculated in parts per 1000 parts of the lymph: 
 
 Sodium chloride 5 . 67 
 
 Soda 1.27 
 
 Potash 0. 16 
 
 Sulphuric acid . 09 
 
 Phosphoric acid united with alkalies . 02 
 
 Earthy phosphates . 26 
 
 In the cases investigated by Munk and Rosenstein the quantity of 
 solids in the fasting condition varied between 35.7 and 57.2 p. m. This 
 variation was essentially dependent upon the extent of secretion, so that 
 the low amount coincides with a more active secretion, and the reverse 
 in the other case. The chief portion of the solids consisted of proteins, 
 and the relation between globulin and albumin was as 1 :2.4 to 4. The 
 mineral bodies in 1000 parts lymph (chylous) were: NaCl 5.83; Na2CC>3 
 2.17; K 2 HPO 4 0.28; Ca 3 (P0 4 ) 2 0.28; Mg 3 (P0 4 ) 2 0.09; and Fe(PO 4 )0.025. 
 The quantity of titratable alkali in the lymph is much smaller than in 
 the blood. Carlson, Greer and Luckhardt 2 have recently made com- 
 parative estimations of NaCl in blood-serum and lymph of the same 
 individual (horse and dog) and find that the lymph is regularly richer in 
 chlorides, a condition which, according to them, is difficult to reconcile 
 with the view of the filtration and transudation processes in the forma- 
 tion of lymph. 
 
 In this connection it must be recalled that according to many inves- 
 tigators the lymph has a somewhat higher osmotic pressure and therefore 
 a somewhat greater molecular concentration than the serum. Carlson, 
 Greer and Becht 3 found, nevertheless, that the osmotic pressure of 
 neck-lymph in the dog is often lower than the serum. 
 
 1 Gubler and Quevenne, cited from Hoppe-Seyler's Physiol. Chem., 591; v. Scherer, 
 ibid., 591; C. Schmidt, ibid., 592. See also Zaribnicky, Zeitschr. f. physiol. Chem., 
 78. 
 
 2 Amer. Journ. of Physiol., 22 (1908). 
 ' Ibid., 19 (1907). 
 
CHYLE AND LYMPH. 340 
 
 Under special conditions the lymph may be so rich in finely divided fat that 
 it appears like chyle. Such lymph has been investigated by Hensen in a case of 
 lymph fistula in a ten-year-old boy, and by Lang l in a case of lymph fistula in 
 the upper part of the left thigh of a girl of seventeen. The lymph investigated 
 by Hensen varied in the quantity of fat, as an average of nineteen analyses, 
 between 2.8 and 36.9 p. m.; while that investigated by Lang contained 24.85 
 p. m. of fat. 
 
 The quantity of lymph secreted must naturally change considerably 
 under various conditions, and there are no means of measuring it. The 
 size of the flow of lymph is, as Heidenhain suggests, no measure of the 
 abundance of supply of nutritive material to the organs, and the lymph- 
 tubes act according to him as " drain-tubes," removing the excess of 
 fluid from the lymph fissures as soon as the pressure therein rises to a 
 certain height. Attempts have been made to determine the quantity 
 of lymph flowing in 24 hours through the thoracic duct of animals. Ac- 
 cording to Heidenhain the quantity averages 640 cc. for a dog weighing 
 10 kilos. 
 
 Detei-minations of the quantity of lymph in man have also been 
 attempted. Noel-Paton 2 obtained 1 cc. of lymph per minute from the 
 severed thoracic duct of a patient weighing 60 kilos. The quantity in 
 the 24 hours cannot be calculated from this amount. In the case of 
 Munk and Kosenstein, 1134-1372 grams of chyle were collected within 
 12-13 hours after partaking of food. In the fasting condition or after 
 starving for 18 hours they found 50 to 70 grams per hour, sometimes 120 
 grams and above, especially in the first few hours after powerful muscular 
 exercise. 
 
 Several circumstances have a marked influence on the extent of lymph 
 secretion. During starvation less lymph is secreted than after partak- 
 ing of food. Nasse 3 has observed that the formation of lymph in dogs 
 is increased 36 per cent more after feeding with meat than after feeding 
 with potatoes, and about 54 per cent more than after 24 hours' depriva- 
 tion of food. In this connection mention must be made of the important 
 observations of Asher and Barbera 4 that with pure protein diet the 
 lymph current is increased in the thoracic cavity, and also that the increase 
 in the lymph secretion runs parallel with the elimination of nitrogen in 
 the urine, i.e., with the absorption of the protein from the digestive tract. 
 
 An increase in the total quantity of blood, as by transfusion of blood, 
 also especially in preventing the flow of blood by means of ligatures, 
 
 1 Hensen, Pfliiger's Arch., 110; Lang, see Maly's Jahresber., 4. 
 
 2 Journ. of Physiol., 11. 
 
 3 Cited from Hoppe-Seyler, Physiol. Chem., 593. 
 
 4 The works of Asher and collaborators, Barbera, Gies, and Busch, upon lymph 
 fonration may be found in Zeitschr. f. Biologie, 36, 37, 40. 
 
350 CHYLE, LYMPH, TRANSUDATES AND EXUDATES, 
 
 causes an increase in the quantity of lymph. According to Heidenhain, 
 on the contrary, a very considerable change in the pressure in the aorta 
 causes only a little change in the abundance of the lymph flow. The 
 quantity of lymph may be raised by powerfully active and passive move- 
 ments of the limbs (Lesser). Under the influence of curare, an increase 
 of the lymph secretion is observed (Paschutin, Lesser 1 ), and the quan- 
 tity of solids in the lymph is also increased. 
 
 The bodies inciting lymph flow, the so-called lymphagogues, are of 
 especially great interest, and they may, according to Heidenhain, 2 be 
 divided into two different chief groups. The lymphagogues of the first 
 series — extracts of crab-muscles, blood-leech, anodons, liver and intestine 
 of dogs, as well as peptone and egg albumin, strawberry extracts, meta- 
 bolic products of bacteria and others — cause a greatly increased secre- 
 tion of lymph without raising the blood-pressure, and in this way the 
 blood-plasma becomes poorer in proteins and the lymph richer than 
 before. For the formation of this lymph, which Heidenhain designates 
 blood-lymph, we must admit with him that a special secretory activity 
 of the capillary-wall endothelium exists. The lymphagogues of the second 
 series, such as sugar, urea, sodium chloride, and other salts, also cause 
 an abundant lymph formation. The blood, as well as the lymph, thereby 
 becomes richer in water. This increased amount of water depends, 
 according to Heidenhain, upon an increased delivery of water by the 
 tissue-elements, and this lymph is chiefly tissue-lymph, in his opinion. 
 Diffusion is no doubt of great importance in the formation of this lymph, 
 but the secretory activity of the endothelium is also of importance, 
 at least for certain bodies, such as sugar. 
 
 In the past, the formation of lymph was explained in a purely phj'sical 
 way by the united action of filtration from the blood and the osmosis 
 between the blood and tissue-fluid. Later Heidenhain and also Ham- 
 burger ascribed a special activity to the capillary endothelium, assum- 
 ing that they take part in the formation of lymph in a secretory manner. 
 The above-mentioned observations on the greater NaCl content in the 
 lymph as compared to the plasma as well as the regularly found higher 
 osmotic pressure of the lymph speak for such a view. 
 
 According to Asher and his collaborators (Barbera, Gies and 
 Busch) the lymph is a product of the work of the organs. Its amount 
 is dependent upon an increased or diminished activity of the organs, 
 
 1 Lesser, Arbeiten aue der physiol. Anstalt zu Leipzig, Jahrgang, 6; Paschutin, 
 ibid., 7. 
 
 2 Heidenhain, Pfliiger's Arch., 49; Hamburger, Zeitschr. f. Biologie, 27 and 30. 
 See especially Ziegler's Beitr. zur Path. u. zur allg. Pathol., 14, 443; also Arch. f. 
 (Anat. u.) Physiol., 1895 and 1896. 
 
CHYLE AND LYMPH. 351 
 
 and the lymph is therefore a measure of the work in these. The close 
 relation between lymph formation and the work of organs has also been 
 show r n for several of them, especially for the liver. Starling has 
 shown that after the introduction of lymphagogues of the first series, 
 chiefly liver lymph is secreted, which he claims is a proof against Heiden- 
 hain's view, and he explains the increased permeability of the vessel 
 wall by the fact that these bodies have an irritating, poisonous action. 
 On the contrary, Ashek explains this increased lymph flow by the state- 
 ment that the substance in question — as well as those influences which 
 incite the activity of the liver — produces an increased formation of lymph 
 in these organs. This view is supported by experiments upon the action 
 of lymphagogues on blood coagulation and liver activity (Delezenne 
 and others), for, according to Gley, these bodies have at the same time 
 a lymphagogue action and an action upon the secretion of the glands. 
 We have no direct evidence of the action of the lymphagogues of the 
 first series upon the organs, but we know from Kusmine's work that 
 peptone, leech extract, and the extractives of the crab-muscles act directly 
 upon the liver-cells and bring about morphological changes. The con- 
 nection betw r een organ activity and lymph formation has also been 
 shown upon muscles and glands by others besides the above-mentioned 
 investigators (Hamburger, Bainbridge 1 ). 
 
 The extent of organ work essentially influences the quantity and 
 properties of the lymph. Still from this we cannot draw any posi- 
 tive conclusions as to whether the lymph formation is brought about 
 by physico-chemical processes alone or whether in this process a specific, 
 not closely definable secretory force is at work at the same time. In 
 regard to this much-disputed question, attention must be called in the 
 first place to the fact that the important works of Heidenhain, Ham- 
 burger, Lazarus-Barlow, and others, as well as the investigations of 
 Asher and Gies and of Mendel and Hooker 2 upon the lengthy post- 
 mortem lymph flow, have shown that the older filtration hypothesis is 
 untenable. 
 
 That osmotic processes play an important role in the lymph formation 
 is generally admitted and that the work of the glands and tissue cells 
 must cause a difference in the osmotic pressure on both sides of the capillary 
 w r alls, has been shown by the researches of many investigators (Koranyi, 
 Starling, Roth, Asher and others). That this is so follows from 
 several circumstances, and especially from the fact that, in disassimila- 
 
 1 In regard to the works cited, as well as the literature upon lymph formation, see 
 Ellinger, "Die Bildung der Lymphe," Ergebnisse der Physiol., I, Abt. 1, 1902, and 
 Asher, Biochem. Centralbl., 4. 
 
 2 Amer. Journ. of Physiol., 7. See also footnote 1. 
 
352 CHYLE, LYMPH, TKANSUDATES AND EXUDATES. 
 
 tion in the cells, bodies of high molecular weight are split into a number 
 of smaller molecules, which latter, either directly, if they leave the cells 
 and pass into the tissue-fluid, or indirectly, when they remain in the cells, 
 produce an increase in the osmotic tension within the cells, and in this 
 way cause a taking up of water from the fluid, and must therefore increase 
 the osmotic pressure of the tissue-fluids. As the cells can by synthesis 
 build up highly complex constituents from simple molecules, and as the 
 chief products of catabolism are carbon dioxide and water, it is difficult 
 to explain these intricate conditions. Still, irrespective of whatever 
 view, a change in one or the other direction in the osmotic pressure 
 upon both sides of the capillary wall must be produced thereby. Whether 
 this and other physico-chemical processes are alone sufficient to explain 
 the lymph formation (Cohnstein, Ellinger) remains an open and 
 disputed question. 1 
 
 H. TRANSUDATES AND EXUDATES. 
 
 The serous membranes are normally kept moistened by liquids whose 
 quantity is sufficient only in a few instances, as in the pericardial cavity 
 and the subarachnoidal space, for a complete chemical analysis to be 
 made of them. Under diseased conditions an abundant transudation 
 may take place from the blood into the serous cavities, into the sub- 
 cutaneous tissues, or under the epidermis; and in this way pathological 
 transudates are formed. Such true transudates, which are similar to lymph, 
 are generally poor in form-elements and leucocytes, and yield only very 
 little or almost no fibrin, while the inflammatory transudates, the so-called 
 exudates, are generally rich in leucocytes and yield proportionally more 
 fibrin. As a rule, the richer a transudate is in leucocytes the closer it 
 stands to pus, while a diminished quantity of leucocytes renders it more 
 nearly like a real transudate or lymph. 
 
 It is ordinarily accepted that filtration is of the greatest importance 
 in the formation of transudates and exudates. The facts coincide with 
 this view that all these fluids contain the salts and extractive bodies 
 occurring in the blood-plasma in about the same quantity as the blood- 
 plasma, while the amount of proteins is habitually smaller. While the 
 different fluids belonging to this group have about the same quantities 
 of salts and extractive bodies, they differ from one another chiefly in 
 containing differing quantities of protein and form-elements, as well as 
 varying quantities of transformation and decomposition products of 
 these latter — changed blood-eoloring matters, cholesterin, etc. The 
 
 x On this question see Ellinger, "Die Bildung der Lymphe," Ergebnisse der Phys- 
 iologic, I, Abt. 1, 355, and Asher, Biochem. Centralbl., 4, pp. 1 and 45. 
 
TRANSUDATES AND EXUDATES. 353 
 
 correspondence in the amount of salts and extractive bodies present in 
 the blood and in transudates supplies just as little proof for a filtration 
 as it does for the formation of lymph; but still it cannot be doubted for 
 other reasons that filtration is often of great importance in the forma- 
 tion of a transudate. To what extent filtration is active in the perfectly 
 normal vascular wall cannot be answered. 
 
 The altered permeability of the capillary walls in disease is a second 
 important factor in the formation of transudates. The circumstance 
 that the greatest quantity of protein occurs in transudates in inflammatory 
 processes, to which is also due the abundant quantity of form-elements 
 in such transudates, has been explained by this hypothesis. The greater 
 quantity of protein in the transudates in formative irritation is in great 
 part explained by the large amount of destroyed form-elements. The 
 interesting observation made by Paijkull, 1 that in those cases in which 
 an inflammatory irritation has taken place the fluid contains nucleoal- 
 bumin (or nucleoprotein?), while this substance does not occur in 
 transudates in the absence of inflammatory processes, can be explained 
 by the presence of form-elements. Still, such a phosphorized protein 
 substance does not occur in all inflammatory exudates. 
 
 As the secretory importance of the capillary endothelium has been 
 made probable by the investigations of Heidenhain, it is a priori to be 
 expected that an abnormally increased secretory activity of the endothe- 
 lium is a cause of transudates. Those observations which substantiate 
 such an assumption can also be explained just as well by assuming a 
 changed permeability of the capillary walls. 
 
 The varying quantities of protein observed by C. Schmidt 2 in the 
 tissue-fluids in different vascular regions can perhaps be explained by the 
 different condition of the capillary endothelium. For example, the 
 amount of protein in the pericardial, pleural, and peritoneal fluids 
 is considerably greater than in those fluids which are found in the sub- 
 arachnoidal space, in the subcutaneous tissues, or in the aqueous 
 humor, which are poor in protein. The condition of the blood also 
 greatly affects the transudates, for in hydrsemia the amount of protein in 
 the transudate is very small. With the increase in the age of a transudate, 
 of a hydrocele fluid for instance, the quantity of protein is increased, 
 probably by resorption of water, and indeed exceptional cases may occur 
 in which the amount of protein, without any previous hemorrhage, is 
 even greater than in the blood-serum. 
 
 The proteins of transudates are chiefly seralbumin, serglobulin, and 
 a little fibrinogen. Proteoses and peptones do not occur, excepting 
 
 1 See Maly's Jahresber., 22. 
 
 2 Cited from Hoppe-Seyler, Physiol. Chera., 607. 
 
354 CHYLE, LYMPH, TRANSUDATES AND EXUDATES. 
 
 perhaps in the cerebrospinal fluid, and in those cases where an autolysis 
 has taken place in the liquid. 1 The non-inflammatory transudates do 
 not as a rule undergo spontaneous coagulation or do so only very slow- 
 ly. On the addition of blood or blood-serum they coagulate. Inflam- 
 matory exudates coagulate spontaneously, and Paijkull has shown 
 that these often contain nucleoprotein (or nucleoalbumin) . In inflam- 
 matory exudates a protein substance has been habitually observed which 
 is precipitated by acetic acid, but which does not occur in transudates, 
 or only in very small quantities. This substance, which has been observed 
 and studied by Moritz, Staehelin, Umber, and Rivalta, is claimed 
 by the first three observers to be free from phosphorus, while Rivalta 
 considers it to be a phosphorized pseudoglobulin. Umber calls it sero- 
 samucin, although it yields only very little reducing carbohydrate. 
 According to Joachim 2 it is only a part of the globulin, a view which can- 
 not be correct for all cases, v. Holst 3 has so far substantiated Umber's 
 observation in that he has isolated a mucin substance from an ascitic 
 fluid in carcinoma of the stomach and the peritoneum, which seemed to 
 be identical with Umber's serosamucin, as well as with the synovial 
 mucin. There does not seem to be any doubt that in transudates and 
 exudates, different protein substances may occur under different cir- 
 cumstances, although the globulins form besides seralbumin the principal 
 mass of the protein bodies. Mucoid substances, which were first 
 observed by Hammarsten in certain cases of ascites without complica- 
 tions with ovarial tumors, and which are cleavage products of a more 
 complicated substance, seem according to Paijkull 4 to be regular 
 constituents of transudates and are closely related to the above-men- 
 tioned serosamucin. The occurrence of the above-mentioned substances 
 precipitable by acetic acid, the globulins (Rivalta) and the nucleo- 
 proteins, in puncture fluids, has been recognized as of very great impor- 
 tance in the differential diagnosis between transudates and exudates. 
 There are numerous investigations on the relation between glob- 
 ulin and seralbumin, and Joachim has determined the relation between 
 euglobulin and the total globulin. No conclusive results can be drawn 
 from these determinations. The relation between globulin and seral- 
 bumin varies very much in different cases, but, as Hoffmann and 
 
 1 Umber, Munch, med. Wochenschr., 1902, and Berlin, klin. Wochenschr., 1903. 
 In regard to the autolysis in transudates, see also Galdi, Biochem. Centralbl., 3; 
 Eppinger, Zeitsehr. f. Heilkunde, 25, and Zak, Wien. klin. Wochenschr., 1905. 
 
 - Paijkull, 1. c; Moritz, Munch, med. Wochenschr., 1903; Staehelin, ibid., 1902, 
 Qmber, Zeitsehr. f. klin. Med., 48; Rivalta, Biochem. Centralbl., 2 and 5; Joachim; 
 Pfltlger'e Arch., 93. 
 
 3 Zeitsehr. f. physiol. Chem., 43. 
 
 4 Hammarsten, ibid., 15; Paijkull, 1. c. 
 
TRANSUDATES AND EXUDATES. 355 
 
 Pigeand l have shown, the variation is in each case the same as in t he 
 blood-serum of the individual. 
 
 The specific gravity runs almost parallel with the quantity of protein. 
 The varying specific gravity has been suggested as a means of differentia- 
 tion between transudates and exudates by Reuss, 2 as the first often show 
 a specific gravity below 1015-1010, while the others have a specific gravity 
 of 1018 or above. This rule holds good in many, but not in all cases. 
 
 The gases of the transudates consist of carbon dioxide besides small 
 amounts of nitrogen and traces of oxygen. The tension of the carbon 
 dioxide is greater in the transudates than in the blood. When mixed 
 with pus, the amount of carbon dioxide is decreased. 
 
 The extractives are, as above stated, the same as in the blood-plasma. 
 Urea seems to occur in very variable amounts. Sugar also occurs in 
 transudates, but it is not known to what extent the reducing power is 
 due to other bodies, as in blood-serum. A reducing, non-fermentable 
 substance has been found by Pickardt in transudates. The sugar is 
 generally glucose, but fructose seems to have been found 3 in several 
 cases. Sarcolactic acid has been found by C. Kulz in the pericardial 
 fluid from oxen. Succinic acid has been found in a few cases in hydrocele 
 fluids, while in other cases it is entirely absent. Leucine and tyrosine 
 have been found in transudates from diseased livers and pus-like trans- 
 udates which have undergone decomposition, and after autolysis. Among 
 other extractives found in transudates must be mentioned allantoin 
 (Moscatelli 4 ), uric acid, purine bases, creatine, inosite, and pyrocate- 
 chin (?). 
 
 The division of the nitrogenous substances in human transudates 
 and exudates has so far been little studied. Otori found that no 
 essential difference exists between serous exudates and transudates in 
 regard to the quantity of urea and amino-acids. The amount of total 
 nitrogen and proteins runs parallel with the specific gravity, and the 
 same is generally true for the absolute values for ammonia nitrogen and 
 purine nitrogen. According to the investigations of Czernecki, 5 in 
 pathological puncture fluids, also oxyproteic acids (see Chapter XIV 
 on the urine) occur and which represent 13.3 — 25.9 per cent of the total 
 nitrogen of the protein free filtrate. The question as to the amount of 
 
 1 Joachim, 1. c; Hoffmann, Arch. f. exp. Path. u. Pharm., 16; Pigeand, see Maly's 
 Jahresber., 16. 
 
 2 Reuss, Deutsch. Arch. f. klin. Med., 28. See also Otto, Zeitschr. f. Heilkunde, 17. 
 8 Pickardt, Berl. klin. Wochenschr., 1897. See also Rotmann, Munch, med. Woch- 
 
 enschr., 1898; Neuberg and Strauss, Zeitschr. f. physiol. Chem., 36; Sittig, Bioch. 
 Zeitschr. 21. 
 
 4 C. Kulz, Zeitschr. f. Biologie, 32; Moscatelli, Zeitschr. f. physiol. Chem. 13. 
 
 6 Otori, Zeitschr. f. Heilk. 25; Czernecki, Maly's Jahresb., 39. 
 
356 CHYLE, LYMPH, TRANSUDATES AND EXUDATES. 
 
 urea nitrogen and amino-acid nitrogen in such fluids must, under these 
 circumstances, require further study. 
 
 The investigations upon the molecular concentration have shown 
 that no essential and constant difference exists between exudates and 
 transudates. The osmotic concentration and the concentration of the 
 electrolytes are as a rule the same as in blood-serum, although some- 
 times rather divergent results have been found. The concentration of 
 the electrolytes shows, according to Bodon, 1 like the blood-serum, much 
 less variation than the total concentration. The alkalinity determined 
 by titration is about the same in transudates and exudates, and is equal 
 to that of the blood-serum. The determination of the HO ion concen- 
 tration has shown that the transudates and exudates in this regard are 
 about as neutral as the blood-serum (Bodon). 
 
 As above stated, irrespective of the varying number of form-elements 
 contained in the different transudates, the quantity of protein is the most 
 characteristic chemical distinction in the composition of the various 
 transudates; therefore a quantitative analysis is of importance only 
 in so far as it considers the quantity of protein. On this account, in the 
 following, relative to the quantitative composition, stress will be put on 
 the quantity of protein. 
 
 Pericardial Fluid. The quantity of this fluid is, even under physio- 
 logical conditions, so large that a sufficient quantity for chemical inves- 
 tigation has been obtained (from persons who had been executed). This 
 fluid is lemon-yellow in color, somewhat sticky, and yields more fibrin 
 than other transudates. The amount of solids, according to the analyses 
 performed by v. Gorup-Besanez, Wachsmuth, and Hoppe-Seyler, 2 
 is 37.5-44.9 p. m., and the amount of protein is' 22.8-24.7 p. m. The 
 analysis made by Hammarsten of a fresh pericardial fluid from a young 
 man who had been executed yielded the following results, calculated in 
 1000 parts by weight. 
 
 Water 960.85 
 
 Solids 39.15 
 
 f Fibrin 0.31 
 
 Proteins 28 . 60 Globulin .... 5 .95 
 
 (Albumin 22.34 
 
 Soluble salts 8.60 NaCl 7.28 
 
 Insoluble salts 0.15 
 
 Extractive bodies 2 . 00 
 
 Friend 3 found almost the same composition for a pericardial fluid 
 from a horse, with the exception that this liquid was relatively richer 
 
 1 Pflviger's Arch., 104, where literature on this subject may be found. 
 
 2 v. Gorup-Besanez, Lehrbuch d. physiol Chem., 4. Aufl., 401; Wachsmuth, Vir- 
 chow's Arch., 7; Hoppe-Seyler, Physiol. Chem., 605. 
 
 •Halliburton, Text-book of Chem. Physiol., etc., London, 1891. 
 
PLEURAL FLUID. 357 
 
 in globulin. The ordinary statement that pericardial fluids are richer 
 in fibrinogen than other transudates is hardly based on sufficient proof. 
 In a case of chylopericardium, which was probably due to the rupture 
 of a chylous vessel, or caused by a capillary exudation of chyle because 
 of stoppage, Hasebroek l found in 1000 parts of the fluid 103.61 parts 
 solids, 73.79 parts proteins, 10.77 parts fat, 3.34 parts cholesterin, 1.77 
 parts lecithin, and 9.34 parts salts. 
 
 The pleural fluid occurs under physiological conditions in such small 
 quantities that a chemical analysis of it cannot be made. Under patho- 
 logical conditions this fluid may show very variable properties. In 
 certain cases it is nearly serous, in others again sero-fibrinous, and in others 
 similar to pus. There is a corresponding variation in the specific gravity 
 and the properties in general. If a pus-like exudate is kept enclosed for 
 a long time in the pleural cavity, a more or less complete maceration 
 and solution of the pus-corpuscles is found to take place. The ejected 
 yellowish-brown or greenish fluid may then be as rich in solids as the 
 blood-serum; and an abundant flocculent precipitate of a nucleoalbumin 
 or nuceloprotein (the pyrin of early writers) may be obtained on the 
 addition of acetic acid. This precipitate is soluble with difficulty in 
 an excess of acetic acid. 
 
 Numerous analyses, by many investigators, 2 of the quantitative 
 composition of pleural fluids under pathological conditions have been 
 published. From these analyses we learn that in hydrothorax the 
 specific gravity is lower and the quantity of protein less than in pleuritis. 
 In the first case the specific gravity is generally less than 1.015, and the 
 quantity of protein 10-30 p. m. In acute pleuritis the specific gravity 
 is generally higher than 1.020, and the quantity of protein 30-65 p. m. 
 The quantity of fibrinogen, which in hydrothorax is about 0.1 p. m., 
 may amount to more than 1 p. m. in pleuritis. In pleurisy with an 
 abundant accumulation of pus, the specific gravity may rise even to 1.030 
 according to the observations of Hammarsten. The quantity of solids 
 is often 60-70 p. m., and may be even more than 90-100 p. m. (Ham- 
 marsten). Mucoid substances have also been detected in pleural fluids 
 by Paijkull. Cases of chylous pleurisy are also known; in such a 
 case M£hu 3 found 17.93 p. m. fat and cholesterin in the fluid. 
 
 The quantity of peritoneal fluid is very small under physiological 
 conditions. The investigations refer only to the fluid under diseased 
 
 1 Zeitschr. f. physiol. Chem., 12. 
 
 2 See the works of M6hu, Runeberg, F. Hoffmann, Reuss, all of which are cited in 
 Bernheim's paper in Virchow's Arch., 131, 274. See also Paijkull, 1. c, and Halli- 
 burton's Text-book, 346; Joachim, 1. c. 
 
 1 Arch. gen. de meYl., 1886, 2, cited from Maly's Jahresber., 16. 
 
358 CHYLE, LYMPH, TRANSUDATES AND EXUDATES. 
 
 conditions (ascitic fluid). The color, transparency, and consistency of 
 these may vary greatly. 
 
 In cachectic conditions or a hydrsemic condition of the blood the fluid 
 has little color, is milky, opalescent, watery, does not coagulate spon- 
 taneously, has a very low specific gravity, 1.006-1.010-1.015, and is 
 almost free from form-elements. The ascitic fluid in portal stagnation, 
 or in general venous congestion, has a low specific gravity and contains or- 
 dinarily less than 20 p. m. protein, although in certain cases the quantity 
 of protein may rise to 35 p. m. In carcinomatous peritonitis it may have a 
 cloudy, dirty-gray appearance, due to its richness in form-elements of 
 various kinds. The specific gravity is then higher, the quantity of solids 
 greater, and it often coagulates spontaneously. In inflammatory proc- 
 esses it is straw- or lemon-yellow in color, somewhat cloudy or reddish, 
 due to leucocytes and red blood-corpuscles, and from great richness in 
 leucocytes it may appear more like pus. It coagulates spontaneously 
 and may be relatively richer in solids. It contains regularly 30 p. m. 
 or more protein (although exceptions with less protein occur), and may 
 have a specific gravity of 1.030 or above. On account of the rupture 
 of a chylous vessel, the ascitic fluid may be rich in very finely emulsified 
 fat (chylous ascites). In such cases 3.86-10.30 p. m. fat has been 
 found in the ascitic fluid (Guinochet, Hay 1 ), and even 17-43 p. m. 
 has been found by Minkowski. 
 
 As first shown by Gross, an ascitic fluid may have a chylous appearance 
 without the presence of fat, i.e., pseudochylous. The cause of the chylous 
 properties of a transudate is not known, although numerous investigators, 
 such as Gross, Bernert, Mosse, and Strauss, have studied the sub- 
 ject; several observations, however, seem to show that it is connected 
 with the amount of lecithin contained therein. In a case investigated 
 by H. Wolff 2 the oleic-acid ester of cholesterin was combined either 
 chemically or molecularly with the euglobulin. 
 
 By admixture of ascitic fluid with that from an ovarian cyst the 
 former may sometimes contain pseudomucin (see Chapter XII). There 
 are also cases in which the ascitic fluid contains mucoids which may be 
 precipitated by alcohol after removal of the proteins by coagulation at 
 boiling temperature. Such mucoids, which yield a reducing substance 
 on boiling with acids, have been found by Hammarsten in tuberculous 
 peritonitis and in cirrhosis hepatis syphilitica in men. According to the 
 investigations of Paijkull, these substances seem to occur often and 
 perhaps habitually in the ascitic fluids. 
 
 1 Guinochet, see Strauss, Arch, de Physiol., 18. See Maly's Jahresber., 16, 475. 
 
 2 Gross, Arch. f. exp. Path. u. Pharm., 44; Bernert, ibid., 49; Mosse, Leyden's 
 Festschrift, 1901; Strauss, cited in Biochem. Centralbl., 1, 437; Wolff, Hofmeister's 
 Beitrage, 5. 
 
HYDROCELE AND SPERMATOCELE FLUIDS. 359 
 
 As the quantity of protein in ascitic Quids ie dependent upon the same 
 factors as in other transudates and exudates, it is sufficient to give the 
 following example of the composition, taken from Bernheim's l treatise. 
 The results arc expressed in 1000 parts of the fluid: 
 
 Max. Min. Mean. 
 
 Cirrhosis of the liver 34.5 5.6 9.69—21.06 
 
 Bright's disease 16.11 10.10 5.6—10.36 
 
 Tuberculous and idiopathic peritonitis. . . 55.8 18.72 30.7 — 37.95 
 
 Carcinomatous peritonitis 54.20 27.00 35.1—58.96 
 
 Joachim found the highest relative globulin amounts and lowest albumin 
 percentages in cirrhosis; in carcinoma, on the contrary, the lowest globulin and 
 the highest albumin. The values in cardiac stagnation stand between the cirrhosis 
 and carcinoma percentages. 
 
 Urea has also been found in ascitic fluids, sometimes only as traces, some- 
 times in larger quantities (4 p. m. in albuminuria), also uric acid, allantoin in 
 cirrhosis of the liver (Moscatelli), xanthine, creatine, cholesterin, sugar, diastatic 
 and proteolytic enzymes, and according to Hamburger 2 also a lipase. 
 
 Hydrocele and Spermatocele Fluids. These fluids differ essentially 
 from each other in various ways. The hydrocele fluids are generally 
 colored light or dark yellow, sometimes brownish with a shade of green. 
 They have a relatively higher specific gravity, 1.016-1.026, with a variable 
 but generally higher amount of solids, an average of 60 p. m. They 
 sometimes coagulate spontaneously, sometimes only after the addition of 
 fibrin ferment or blood. They contain leucocytes as chief form-elements. 
 Sometimes they contain smaller or larger amounts of cholesterin crystals. 
 
 The spermatocele fluids, on the contrary, are as a rule colorless, 
 thin, and cloudy like water mixed with milk. They sometimes have an 
 acid reaction. They have a lower specific gravity, 1.006-1.010, a lower 
 amount of solids — an average of about 13 p. m. — and do not coagulate 
 either spontaneously or after the addition of blood. They are, as a rule, 
 poor in protein and contain spermatozoa, cell-detritus, and fat-globules as 
 form constituents. To show the unequal composition of these two kinds 
 of fluids we will give the average results (calculated in parts per 1000 
 parts of the fluid) of seventeen analyses of hydrocele fluids and four 
 of spermatocele fluids made by Hammarsten. 3 
 
 Hydrocele. Spermatocele. 
 
 Water 938.85 986.83 
 
 Solids 61.15 12.17 
 
 Fibrin . 59 
 
 Globulin 13.25 0.59 
 
 Seralbumin. . 35.94 1 .82 
 
 Ether extractive bodies 4 . 02 
 
 Soluble salts 8.60 \ 10.76 
 
 Insoluble salts . 66 
 
 1 1. c. As it was impossible to derive mean figures from those given by Bernheim, 
 the author has given the maximum and minimum of the averages given by him. 
 'Arch. f. (Anat. u.) Physiol., 1900, 433. 
 * Upsala Lakaref. Forh., 14, and Mary's Jahresber., 8, 347. 
 
360 CHYLE, LYMPH, TRANSUDATES AND EXUDATES. 
 
 In the hydrocele fluid traces of urea and a reducing substance have been 
 found, and in a few cases also succinic acid and inosite. A hydrocele fluid may, 
 according to Devillard, 1 sometimes contain paralbumin or metalbumin (?). 
 Cases of chylous hydrocele are also known. 
 
 Cerebrospinal Fluid. The cerebrospinal fluid is thin, water-clear, 
 of low specific gravity, 1.007-1.008. The spina bifida fluid is very poor 
 in solids, 8-10 p. m. with only 0.19-1.6 p. m. protein. The fluid of 
 chronic hydrocephalus is somewhat richer in solids (13-19 p. m.) and 
 proteins. The amount of protein in the cerebrospinal fluid seems to be 
 rather variable under diseased conditions and Frenkel-Heiden 2 found 
 0.875-3 p. m. protein in the lumbar fluid in progressive paralysis and 
 0.7-2.8 p. m. protein in tuberculous meningitis. In the perfectly fresh 
 fluid from healthy calves Nawratzki found an average of 0.22 p. m. 
 protein. 
 
 According to Halliburton the protein of the cerebrospinal fluid 
 is a mixture of globulin and proteose; occasionally some peptone occurs, 
 and more rarely, in special cases, seralbumin appears. The conclusions 
 of Halliburton on the occurrence of proteose do not coincide with the 
 observations of other investigators (Panzer, Salkowski 3 ) . In general 
 paralysis, Halliburton and Mott obtained a nucleoprotein in the 
 cerebrospinal fluid. Choline occurs in several diseases, as in general 
 paralysis, brain-tumors, tabes dorsalis, and epilepsy (Halliburton 
 and Mott, Donath, Rosenheim). According to Kaufmann 4 we 
 are not here dealing with choline but with another base. Glucose, or at 
 least a fermentable sugar, occurs habitually in the cerebrospinal fluid, 
 while the claims of Halliburton as to the occurrence of a substance 
 similar to pyrocatechin could not be substantiated in calves and men 
 by Nawratzki, 5 and hence this substance does not exist in all cerebro- 
 spinal fluids. Urea occurs in cerebrospinal fluids, but not always. In 
 the cases investigated by Frenkel-Heiden indeed all the rest-nitrogen 
 occurred as urea and the urea-nitrogen varied in different pathological 
 cases between 0.196-1.12 p. m. Lactic Acid has been found by Lehnt- 
 dorff and Baumgarten 6 in many pathological cases. The quantity 
 of NaCl is regularly much greater than the KC1, 6-7 p. m. NaCl against 
 
 1 Bull. Soc. chim., 42, 617. 
 
 2 Bioch. Zeitschr., 2. 
 
 'Halliburton's Text-book; Panzer, Wein. klin. Wochenschr., 1899; Salkowski, 
 Jaffe" Festschrift, 265. 
 
 * Halliburton and Mott, Phil. Transact. Roy. Soc. London, Series B, 191; Donath, 
 Zeitschr. f. physiol Chem., 39 and 42; see also Mansfield, ibid., 42; Rosenheim, Journ. 
 of Physiol., 35; Kaufmann, Zeitschr. f. physiol. Chem., 66. 
 
 6 Zeitschr. f. physiol. Chem., 23. See also Rossi, ibid., 39 (literature). 
 
 "Zeitschr. f. exp. Path. u. Therap., 4 (literature). 
 
AQUEOUS HUMOR AND BLISTER-FLUID. 361 
 
 about 0.4 p. m. KC1, and the variable relation between potassium and 
 sodium is probably due, according to Salkowski, 1 to the absence or pres- 
 ence of fever during the formation of the exudate; the amount of potas- 
 sium is high in the acute cases and low in the chronic ones. According 
 to Landau and Halpern 2 a certain antagonism seems to exist between 
 nitrogen and sodium chloride, as the highest results of the first correspond 
 to the lowest results of the other. According to Cavazzani, 3 who has 
 especially studied the cerebrospinal fluids, the alkalinity of these fluids 
 is considerably less than that of the blood and independent of this last 
 fluid. For this and several other reasons Cavazzani draws the con- 
 clusion that the cerebrospinal fluid is formed by a true secretory process. 
 
 A large number of investigations on the cerebrospinal fluid have been made 
 on the fluid obtained from cadavers and in consideration of this it must be remarked 
 that this fluid quickly changes after death and that the results obtained therefore 
 are not comparable with the fluid during life. 
 
 Aqueous Humor. This fluid is clear, alkaline toward litmus, and has 
 a specific gravity of 1.003-1.009. The amount of solids is on an average 
 13 p. m., and the amount of proteins only 0.8-1.2 p. m. The protein 
 consists of seralbumin and globulin and very little fibrinogen and mucin. 
 According to Gruenhagen it contains paralactic acid, another dextro- 
 gyrate substance, and a reducing body which is unlike sugar or dextrin. 
 Pautz 4 found urea and sugar in the aqueous humor of oxen. 
 
 Blister-fluid. The content of blisters caused by burns, and of vesi- 
 catory blisters and the blisters of the 'pemphigus chronicus, is generally a 
 fluid rich in solids and proteins (40-65 p. m.). This is especially true 
 of the contents of vesicatory blisters. In a burn-blister K. Morner 5 
 found 50.31 p. m. proteins, among which were 13.59 p. m. globulin and 
 0.11 p. m. fibrin. The fluid contains a substance which reduces copper 
 oxide, but no pyrocatechin. The fluid of the pemphigus is alkaline in 
 reaction. A wound secretion collected by Lieblein 6 under aseptic 
 conditions was alkaline in reaction, and contained less protein than the 
 blood-serum. It formed a slight fibrin clot, and contained proteoses 
 only at first or at the beginning of the abscess formation. As the wound 
 healed, the relation between the globulin and albumin changed, and on 
 
 1 See Salkowski, 1. c. New quantitative analyses of cerebrospinal and hydrocephalus 
 fluids may be found in the cited works of Nawratzki, Panzer, and Salkowski. 
 
 2 Bioch. Zeitschr., 9. 
 
 s See Maly's Jahresber., 22, 346, and Centralbl. f. Physiol., 15, 216. 
 * Gruenhagen, Pfluger's Arch., 43; Pautz. Zeitschr. f. Biologie, 31. 
 8 Skand. Arch. f. Physiol., 5. 
 8 Habilitationsschrift Prag. 1902, printed by H. Laupp, Tubingen. 
 
3132 CHYLE, LYMPH, TRANSUDATES AND EXUDATES. 
 
 the third day of the healing the quantity of albumin was at least nine- 
 tenths of the total protein. 
 
 The fluid of subcutaneous oedema. This is, as a rule, very poor in 
 solids, purely serous, does not contain fibrinogen, and has a specific 
 gravity of 1.005-1.013. The quantity of proteins is in most cases lower 
 than 10 p. m. — according to Hoffmann 1-8 p. m. — and in serious 
 affections of the kidneys, generally with amyloid degeneration, less than 
 1 p. m. has been shown (Hoffmann *). The cedematous fluid also habit- 
 ually contains urea, 1-2 p. m., and sugar. 
 
 The fluid of the echinococcus cyst is related to the transudates, and is 
 poor in proteins. It is thin and colorless, and has a specific gravity of 1.005- 
 1.015. The quantity of solids is 14-20 p. m. The chemical constituents are 
 sugar (2.5 p. m.), inosite, traces of urea, creatine, succinic acid, and salts (8.3-9.7 
 p. m.). Proteins are found only in traces, and then only after an inflammatory 
 irritation. In the last-mentioned case 7 p. m. proteins have been found in the fluid. 
 
 The Synovial Fluid and Fluid in Synovial Cavities around Joints, 
 etc. The synovia is hardly a transudate, but it is often discussed in an 
 appendix to the transudates. 
 
 The synovia is an alkaline, sticky, fibrous, yellowish fluid which 
 is cloudy, from the presence of cell-nuclei and the remains of destroyed 
 cells, but is also sometimes clear. Besides proteins and salts, it also 
 contains a mucin substance, synoviamucin (v. Holst 2 ). In pathological 
 synovia, Hammarsten found a mucin-like substance which is not mucin. 
 It behaves like a nucleoalbumin or a nucleoprotein, and gives no reducing 
 substance on boiling with acids. Salkowski 3 also found a mucin-like 
 substance in a pathological synovial fluid, which was neither mucin nor 
 nucleoalbumin. He called the substance synovin. 
 
 The composition of synovia is not constant, but is different in rest 
 and in motion. In the last-mentioned case the quantity of fluid is less, 
 but the amount of the mucin-like body, of proteins, and of the extractive 
 bodies is greater, while the quantity of salts is diminished. This may 
 be seen from the following analyses by Frerichs. 4 The figures repre- 
 sent parts per 1000. 
 
 I. Synovia from II. Synovia from 
 
 __ a Stall-fed Ox. a Field-fed Ox. 
 
 ^ater 969.9 948.5 
 
 Solids 30.1 51.5 
 
 Mucin-like body 2.4 5.6 
 
 Albumin and extractives 15.7 35.1 
 
 Fat 0.6 -0.7 
 
 Salts 11.3 9.9 
 
 1 Deutsch. Arch. f. klin. Med., 44. 
 
 2 Zeitschr. f. physiol. Chem., 43. 
 
 ' Hammarsten, Maly's Jahresber., 12; Salkowski, Virchow's Arch., 131. 
 4 Wagner's Handwdrterbuch, 3, Abt. 1, 463. 
 
PUS. 363 
 
 The synovia of new-born babes corresponds to that of resting animal-. 
 The fluid of the bursae mucosae, as also the fluid in the synovial cavities 
 around joints, etc., is similar to synovia from a qualitative standpoint. 
 
 III. PUS. 
 
 Pus is a yellowish-gray or yellowish-green, creamy mass of a faint 
 odor and an unsavory, sweetish taste. It consists of a fluid, the pus- 
 serum, in which solid particles, the pus-cells, swim. The number of these 
 cells varies so considerably that the pus may at one time be thin and at 
 another time so thick that it scarcely contains a drop of serum. The 
 specific gravity, therefore, may also greatly vary, namely, between 
 1.020 and 1.040, but ordinarily it is 1.031-1.033. The reaction of fresh 
 pus is generally alkaline, but it may become neutral or acid from a decom- 
 position in which fatty acids, glycerophosphoric acid, and also lactic 
 acid are formed. 
 
 In the chemical investigation of pus, the pus-serum and the pus- 
 corpuscles must be studied separately. 
 
 Pus-serum. Pus does not coagulate spontaneously nor after the 
 addition of defibrinated blood. The fluid in which the pus-corpuscles 
 are suspended is not to be compared with the blood-plasma, but rather 
 with the serum. The pus-serum is pale yellow, yellowish-green, or brownish- 
 yellow, and has an alkaline reaction toward litmus. It contains, for the 
 most part, the same constituents as the blood-serum; but sometimes 
 besides these — when, for instance, the pus has remained in the body 
 for a long time — it contains a nucleoalbumin or a nucleoprotein which 
 is precipitated by acetic acid and is soluble with great difficulty in an 
 excess of the acid (pyin of the earlier authors). This nucleoalbumin 
 seems to be formed from the hyaline substance cf the pus-cells by macera- 
 tion. The pus-serum contains, moreover, at least in many cases, no 
 fibrin ferment. According to the analyses of Hoppe-Seyler l the pus- 
 serum contains in 1000 parts: 
 
 i. ii. 
 
 Water 913.70 905.65 
 
 Solids 86 . 30 94 35 
 
 Proteins 63.23 77 21 
 
 Lecithin 1 . 50 . 56 
 
 Fat 0.26 0.29 
 
 Cholesterin . 53 . 87 
 
 Alcohol extractives 1 . 52 . 73 
 
 Water extractives 11 . 53 6 . 92 
 
 Inorganic salts 7 . 73 7 . 77 
 
 1 Med.-Chem. Untersuch., 490. 
 
364 CHYLE, LYMPH, TRANSUDATES AND EXUDATES. 
 
 The ash of pus-serum has the following i composition, calculated to 
 
 1000 parts of the serum: 
 
 i. ii. 
 
 NaCl 5.22 5.39 
 
 Na 2 SO< 0.40 0.31 
 
 Na 2 HP0 4 0.98 0.46 
 
 Na 2 C0 3 0.49 1.13 
 
 Ca 3 (P0 4 ) 2 0.49 0.31 
 
 Mg 3 (P0 4 ) 2 0.19 0.12 
 
 P0 4 (in excess) 0.05 
 
 The pus-corpuscles are generally thought to consist chiefly of emi- 
 grated white blood-corpuscles, and their chemical properties have there- 
 fore been given in discussing these. The molecular granules, fat- 
 globules, and red blood-corpuscles are considered rather as casual form- 
 elements. 
 
 The pus-cells may be separated from the serum by centrifugal force, 
 or by decantation directly or after dilution with a solution of sodium 
 sulphate in water (1 vol. saturated sodium-sulphate solution and 9 vols, 
 water) and then washed by this same solution in the same manner as 
 the blood-corpuscles. 
 
 The chief constituents of the pus-corpuscles are proteins, of which 
 the largest portion seems to be a nucleoprotein which is insoluble in 
 water and which expands into a tough, slimy mass when treated with a 
 10-per cent common-salt solution. This protein substance, which is 
 soluble in alkali but is quickly changed thereby, is called Rovida's hyaline 
 substance, and the property of the pus of being converted into a slime- 
 like mass by a solution of common salt depends on this substance. Besides 
 this substance, to which the nucleoprotein of the pus-cells investigated 
 by Strada 1 seems to stand in close relation, we also have a globulin 
 which coagulates at 48-49° C, as well as serglobulin (?), seralbumin t 
 a substance similar to coagulated protein (Miescher), and lastly peptone 
 or proteose (Hofmeister 2 ). It is very remarkable that no nucleo- 
 histone or histone has been detected in the pus-cells, although histone 
 occurs in the cells of the lymph glands. 
 
 There are also found in the protoplasm of the pus-cells, besides the 
 proteins, lecithin, cholesterin, glucolhionic acid, 3 purine bodies, fat, and soaps. 
 Hoppe-Seyler has found cerebrin, a decomposition product of a pro- 
 tagon-like substance, in pus (see Chapter XI). Kossel and Freytag 4 
 have isolated from pus two substances, pyosin and pyogenin, which 
 
 1 Bioch. Zeitschr., 16. 
 
 2 Miescher in Hoppe-Seyler's Med.-Chem. Untersuch., 441; Ch. Pone. Maly's Jahresb., 
 39; Hofmeister, Zeitschr. f. physiol. Chem., 4. 
 
 3 Mandel and Levene, Bioch. Zeitschr., 4. 
 ♦Zeitschr. f. physiol. Chem., 17, 452. 
 
PUS. 365 
 
 belong to the cerebrin group (see Chapter XI). Hoppe-Seyler x 
 claims that glycogen appears only in the living, contractile white blood- 
 cells and not in the dead pus-corpuscles. Several other investigators 
 have, nevertheless, found glycogen in pus. The cell-nucleus contains 
 nuclein and nucleoproteins. 
 
 In regard to the occurrence of enzymes in the pus-cells it must be 
 remarked that neither thrombin nor prothrombin is found therein, 
 although these bodies are generally considered as being derived from 
 the leucocytes, and also obtainable from the thymus leucocytes. The 
 occurrence in the pus-cells, besides catalases and oxidases, of a proteolytic 
 enzyme, is of great interest. It is not only important for the intracellular 
 digestion and for the amount of proteoses in the pus-cell, but also for 
 the solution of the fibrin clot and pneumonic infiltrations (Fr. Muller, 
 O. Simon 2 ). A lipase, which splits neutral fats, also occurs, according to 
 Fiessinger and Marie, in pus. 
 
 The mineral constituents of the pus-corpuscles are potassium, sodium, 
 calcium, magnesium, and iron. A part of the alkalies exists as chlorides, 
 and the remainder, as well as the chief part of the other bases, exists 
 as phosphates. 
 
 The quantitative composition of the pus-cells from the analyses of 
 Hoppe-Seyler is as follows, in parts per 1000 of the dried substance: 
 
 i. ii. 
 
 Proteins 137 . 62 ] 
 
 Nuclein 342.57^685.85 673.69 
 
 Insoluble bodies 205 . 66 J 
 
 Lecithin \ . . Q „ Q 75.64 
 
 Fat / 14d8d 75.00 • 
 
 Cholesterin 74.00 72.83 
 
 Cerebrin 51 .99 
 
 Extractive bodies 44 . 33 
 
 102.84 
 
 MINERAL SUBSTANCES IN 1000 PARTS OF THE DRIED SUBSTANCE. 
 
 NaCl 4.34 
 
 Ca 3 (P0 4 ) 2 2.05 
 
 Mg 3 (P0 4 ) 2 1 . 13 
 
 FePO-4 1.06 
 
 P0 4 9.I6 
 
 Na 0.68 
 
 K Traces (?) 
 
 Miescher obtained other results for the alkali compounds, namely, potas- 
 sium phosphate 12, sodium phosphate 6.1, earthy phosphate and iron phos- 
 phate 4.2, sodium chloride 1.4, and phosphoric acid combined with organic sub- 
 stances 3.14-2.03 p. m. 
 
 In pus from congested abscesses which has stagnated for some time 
 there occur peptone (proteose), leucine and tyrosine, free fatty acids and 
 
 1 Hoppe-Seyler, Physiol. Chem., 790. 
 
 2 Fr. Muller, Verhandl. Nat. Gesellsch. zu. Basel, 1901; O. Simon, Deutsch. Arch, 
 f. klin. Med., 70. 
 
366 CHYLE, LYMPH, TRANSUDATES AND EXUDATES. 
 
 volatile fatty acids, such as formic acid, butyric acid and valeric acid. 
 There are also found urea, glucose (in diabetes), bile-pigments, and bile- 
 acids (in catarrhal icterus). 
 
 As more specific but not constant constituents of the pus must be 
 mentioned the following: pyin, which seems to be a nucleoprotein pre- 
 cipitable by acetic acid, and also pyinic acid and chlorrhodinic acid, which 
 have been so little studied that they cannot be more fully treated here. 
 
 In many cases a blue, more rarely a green, color, has been observed 
 in the pus. This depends on the presence of micro-organisms (Bacillus 
 pyocyaneus). From such pus Fordos and Lucke x have isolated a crys- 
 talline blue pigment, pyocyanin, and a yellow pigment, pyoxanthose, 
 which is produced from the first by oxidation. 
 
 Appendix. 
 Lymphatic Glands, Spleen and Endocrinic Glands. 
 
 The Lymphatic Glands. The cells of the lymphatic glands are 
 found to contain the protein substances generally occurring in cells 
 (Chapter V). According to Bang 2 they also contain histone nucleates 
 (nucleohistone), but in smaller amounts and of a different variety from 
 the better-studied nucleohistone from the thymus gland. Proteoses 
 occur as products of autolysis. By a lengthy autolysis of lymph glands 
 Reh 3 found ammonia, tyrosine, leucine (somewhat scanty), thymine, 
 and uracil among the cleavage products. Besides the other ordinary 
 tissue constituents, such as collagen, reticulin, elastin, and nuclein, there 
 occur in the lymphatic glands also cholesterin, fat, glycogen, sarcolactic 
 acid, purine bases, and leucine. In the inguinal glands of an old woman 
 Oidtmann found 714.32 p. m. water, 284.5 p. m. organic and 1.16 p. m. 
 inorganic substances. In the cells of the mesenteric lymphatic glands 
 of oxen, Bang 4 found 804.1 p. m. water, 195.9 p. m. solids, 137.9 total 
 proteins, 6.9 p. m. histone nucleate, 10.6 p. m. nucleoprotein, 47.6 p. m. 
 bodies soluble in alcohol, and 10.5 p. m. mineral constituents. 
 
 The Thymus. The cells of this gland are very rich in nuclein bodies 
 and relatively poor in the ordinary proteins, but their nature has not been 
 closely studied. The chief interest is attached to the nuclein substances. 
 Kossel and Lilienfeld first prepared from the watery extract of the 
 gland, by precipitating with acetic acid and then further purifying, a 
 
 1 Fordos, Compt. Rend., 51 and 56; Lucke, Arch. f. klin. Chirurg., 3; Boland, Cen- 
 tralbl. f. Bakt. u. Parasit., I., 25. 
 
 2 Studio- over Nucleoproteider, Krietiania, 1902, and Hofmeister'e Beitrage, 4. 
 
 3 Hofmeister'a Beitrage, 3. 
 M. c. 
 
THYMUS. 367 
 
 protein substance which has been generally called nucleohislone. By the 
 action of dilute hydrochloric acid upon nucleohistone it splits, according 
 to these investigators, into histone and leuconuclein. The leuconuclein 
 is a true nuclein; hence it is a nucleic-acid compound with protein which 
 is relatively poor in protein and rich in phosphorus. The more recent 
 investigations of Bang, Malengreau, Huiskamp and Gouban * upon 
 nucleohistone all show that this nucleoprotein is not a unit substance, but 
 a mixture of at least two bodies. The views of the investigators men- 
 tioned differ quite essentially from one another as to the nature of these 
 bodies, but this is partly due to the different methods used by them and 
 partly to the ready changeability of the substances in question. 
 
 Besides the real nucleohistone, B-nucleoalbumin of Malengreau, 
 Lilienfeld's histone contains a second nucleoprotein which Bang and 
 Huiskamp call simple nucleoprotein, while Malengreau designates 
 it A-nucleoalbumin. This protein, which contains only about 1 per cent 
 phosphorus and which is possibly identical with the nucleoprotein found 
 by Lilienfeld in the thymus, yields a nuclein, but no free nucleic acid, 
 on cleavage. As a second cleavage product it yields, according to Mal- 
 engreau, the A-histone, which can be readily precipitated by magnesium 
 and ammonium sulphates from the ordinary B-histone of the thymus 
 gland. The occurrence of A-histone in the gland has been verified by 
 Bang, and according to Bang and Huiskamp the A-histone is not derived 
 from the nucleoprotein, as these investigators claim that it yields no his- 
 tone. According to Bang the nucleoprotein yields only an albuminate, 
 besides the nuclein, as cleavage products. According to Gouban we 
 have been dealing with three substances, namely a nucleoprotein which 
 does not yield any histone, and two nucleohistones, which correspond 
 to the nucleoalbumins A and B of Malengreau and form the mixture 
 of lime-nucleohistone of Huiskamp. They occur in this last mentioned 
 mixture in a somewhat modified form due to the method of preparation. 
 
 The true nucleohistone, which is much richer in phosphorus (the- 
 calcium salt containing, according to Bang, on an average 5.23 per cent 
 P), yields ordinary histone (or 2 histones) as one cleavage product and 
 free nucleic acid as the other. According to Bang, whose statements 
 on this point have been substantiated by Malengreau, it splits on saturat- 
 ing with NaCl into nucleic acid and histone without yielding any other 
 protein. On this account Bang does not consider this body as nucleo- 
 histone in the ordinary sense, i.e., not as a nucleoprotein, but as a histone 
 
 1 Lilienfeld, Zeitschr. f. physiol. Chem., 18; Kossel, ibid., 30 and 31; Bang., ibid. 
 30 and 31. See also Arch. f. Math, og Naturvidenskab, 25, Kristiania, 1902, and 
 Hofmeister's Beitrage, 1 and 4; Malengreau, La Cellule, 17 and 19; Huiskamp, Zeit- 
 schr. f. physiol. Chem., 32, 34 and 39; Gouban, Bioch. Centralbl., 9, 803. 
 
36S CHYLE, LYMPH, TRANSUDATES AND EXUDATES. 
 
 nucleate. We cannot say whether this applies to the two nucleohistones 
 (if there are two). The nucleohistone or mixture of nucleohistones 
 behave like an acid, whose salts, especially the calcium salt, have been 
 closely studied by Huiskamp. On the electrolysis of a solution of alkali 
 nucleohistone in water Huiskamp also found that the nucleohistone 
 collected in traces at the anode, and that the sodium compound is there- 
 fore ionized in the solution. The nucleic acid-calcium histone-com- 
 pound has been prepared, it seems, in a pure state by Bang, and he found 
 the following average composition: C 43.69; H 5.60; N 16.87; S 0.47; 
 P 5.23; Ca 1.71 per cent. 
 
 The nucleohistone prepared by Huiskamp' s method of precipitating with 
 CaCb is, according to him, a mixture of two nucleohistones, of which one, the 
 a-nucleohistone, contains 4.5 per cent phosphorus, and the other, |3-nucleohistone 
 contains, on the contrary, only in round numbers 3 per cent phosphorus. 1 As 
 the two nucleohistones are poorer in phosphorus than the nucleic acid-histone 
 compound analyzed by Bang, and as Huiskamp on cleavage of his preparation 
 did not, like Bang and Malengreau, obtain pure nucleic acid, it is still a ques- 
 tion whether Huiskamp was working with sufficiently pure substances. 
 
 In regard to the methods used by the above investigators in the 
 isolation of the bodies in question we must refer to the original publications. 
 
 In connection with the so-called nucleohistone, attention must be called to 
 tissue fibrinogen and cell fibrinogen, which are compound proteins, and are claimed 
 by certain investigators to stand in close relation to the coagulation of the blood. 
 These may be in part nucleoproteins and in part also nucleohistones. To this same 
 group belong also the important cell constituents described by Alex. Schmidt 2 
 and called cytoglobin and preglobulin. The cytoglobin, which is soluble in water, 
 may be considered as the alkali compound of preglobulin. The residue of the 
 cells left after complete extraction with alcohol, water, and salt solution has 
 been called cytin by Alex. Schmidt. 
 
 Besides the above-mentioned and the ordinary bodies belonging to 
 the connective-tissue group, small quantities of fat, leucine, succinic 
 acid, lactic acid, sugar, and traces of iodothyrin are present. According 
 to Gautier 3 arsenic also occurs in very small amounts, and no doubt 
 here as well as in other organs it is related to the nuclein substances. The 
 richness in nuclein bodies explains the occurrence of large quantities 
 of purine bases, chiefly adenine, whose quantity, according to Kossel 
 and Sciiindler, 4 is 1.79 p. m. in the fresh organ and 19.19 p. m. in the 
 dry substance, and guanine. The bodies thymine and (uracil?) obtained, 
 besides lysine and ammonia, by Kutscher, as products of autodiges- 
 tion of the gland, probably have a similar origin. Among the enzymes, 
 
 1 Zeitschr. f. phy.siol. Chem., 39. 
 
 2 See footnote 1, p. 307. 
 8 Compt. Rend., 129. 
 
 * Zeitschr. f. physiol. Chem., 13; Kutacher, Zeitachr. f. phyaiol. Chem., 34. 
 
THYMUS AND SPLEEN. 369 
 
 besides argimue, guanase, adenase, and proteolytic enzyme we must 
 especially mention the enzyme studied by Jones, 1 which acts like a nu- 
 clease, splitting off phosphoric acid and purine bases, from the nucleo- 
 proteins. This enzyme, contrary to trypsin, acts best in acid liquids, and 
 is readily destroyed by alkalies at body temperature. The quantitative 
 composition of the lymphocytes from the thymus of a calf is, according to 
 Lilienfeld's analysis, as follows. The results are given in 1000 parts 
 of the dried substance: 
 
 Proteids 17.7 
 
 Leuconuclein 687 . 9 
 
 Histone 86 . 7 
 
 Lecithin 75 . 1 
 
 Fat 40.2 
 
 Cholesterin 44 . 
 
 Glycogen 8.0 
 
 The dried substance of the leucocytes amounted to an average of 
 114.9 p. m. Potassium and phosphoric acid are prominent mineral 
 constituents. Lilienfeld found KH2PO4 among the bodies soluble in 
 alcohol. 
 
 Attention must be called to the analyses of Bang, 2 which show that 
 the thymus contains about the same quantity of nucleoprotein, but about 
 five times as much histone nucleate as the lymphatic glands — calculated 
 in both cases upon the same amount of dry substance. Oidtman 3 found 
 807.06 p. m. water, 192.74 p. m. organic and 0.2 p. m. inorganic sub- 
 stances in the gland of a child two weeks old. 
 
 In regard to the functions of the thymus it seems to be the general 
 view that this gland takes part in the recruiting of the blood lympho- 
 cytes and correspondingly belong to the lymphoid organs. On the other 
 hand also certain other observations indicate that it may belong to the 
 endocrinic organs. It is generally admitted that the extirpation of the 
 thymus leads to a reduction and change in the formation of bone. A 
 certain relation also exists with the organs of generation and perhaps 
 a reciprocal action also exists between it and other organs with internal 
 secretion. 
 
 The Spleen. The pulp of the spleen cannot be freed from blood. 
 The mass which is separated from the spleen capsule and the structural 
 tissue by pressure, and which ordinarily serves as material for chemical 
 investigations is, therefore a mixture of blood and spleen constituents. 
 For this reason the proteins of the spleen are little known. The nucleo- 
 protein isolated by Levene and Mandel 4 is to be considered as a true 
 
 1 Zeitschr. f. physiol. Chem., 41. 
 2 1. c, Arch. f. Math., etc. 
 
 3 Cited from v. Gorup-Besanez, Lehrb. d. physiol. Chem., 4. Aufl., p. 732. 
 
 4 Bioch. Zeitschr., 5. 
 
370 CHYLE, LYMPH, TRANSUDATES AND EXUDATES. 
 
 spleen constituent, and this nucleoprotein yields 25 per cent glutamic 
 acid on hydrolysis. Histone has not been directly detected in the spleen; 
 but its presence is to be admitted because Krasnosselsky x was able 
 to isolate a histone-peptone as sulphate from the spleen. The ferruginous 
 albuminate has been considered as a spleen constituent for a long time, 
 and especially also a protein substance which does not coagulate on boil- 
 ing and which is precipitated by acetic acid and yields an ash contain- 
 ing much phosphoric acid and iron oxide. This substance is probably 
 identical with the nucleoproteins which later investigators such as Sato 
 and Capezzuoli 2 have prepared from the spleen. These nucleoproteins, 
 which are modified products, contain iron in variable amounts and more 
 or less firmly combined. 
 
 The pulp of the spleen, when fresh, has an alkaline reaction, but 
 quickly turns acid, due partly to the formation of free paralactic acid 
 and partly perhaps to glycerophosphoric acid. Besides these two acids 
 there are found in the spleen also volatile fatty acids, as formic, acetic, 
 and butyric acids, as well as succinic acid, neutral fats, cholesterin, traces 
 of leucine, inosite (in ox-spleen), scyllite, a body related to inosite (in the 
 spleen of Plagiostoma) , glycogen (in dog-spleen), uric acid, purine bases, 
 and jecorin. Levene found a glucothionic acid in the spleen, i.e., an 
 acid which is related to chondroitin-sulphuric acid but not identical 
 therewith, and which gives a beautiful violet coloration with orcin and 
 hydrochloric acid. The question whether this glucothionic acid originates 
 from the above-mentioned nucleoprotein or from the mucoid substance 
 has not been decided (Levene and Mandel). In regard to the question 
 whether this acid is a unit body or not we refer to the work of Mandel 
 and Neuberg and Levene and Jacobs. 3 
 
 In the human and ox-spleen Burow 4 has found three phosphatides 
 which all contain iron in organic combination. Among these one is a 
 saturated diaminomonophosphatide and the other two are unsaturated 
 phosphatides. 
 
 Many enzymes are found in the spleen also, and certain of these 
 are of special interest. To these belong the uric-acid-forming enzyme, 
 the xanthine oxidase (Burian), which occurs in the spleen of many 
 animals, but not in man, and which transforms the oxypurines, 
 hypoxanthine, and xanthine into uric acid; also the deamidizing enzymes 
 
 1 Zeitschr. f. physiol. Chem., 49. 
 
 2 Sato, Bioch. Zeitschr., 22; Capezzuoli; Zeitschr. f. physiol. Chem., 60. 
 
 8 Levene, Zeitschr. f. physiol. Chem., 37; Levene and Mandel, ibid., 45 and 47; 
 Mandel and Neuberg, Bioch. Zeitschr., 13; Levene, ibid., 16; Neuberg, ibid., 16; Levene 
 and Jacobs, Joum. of experim. Medic, 10. 
 
 * Bioch. Zeitschr., 25. 
 
SPLEEN. 371 
 
 guanase and adenase (Levene, Schittenhelm, Jones and Partridge, 
 Jones and Winternitz), by the first of which the guanine is transformed 
 into xanthine, and by the latter the adenine into hypoxanthine. The 
 guanase also occurs in the spleen of the ox and horse, but not (Jones), 
 or only in small amounts (Schittenhelm), in the pig-spleen. 1 The 
 spleen also contains two enzymes, lienases, as shown by Hedin (and 
 Rowland), one of which, the a-lienase, acts chiefly in alkaline solution, 
 while the other, 0-lienase, is active only in acid reaction. These enzymes, 
 which without doubt stand in close relation to the leucocytes, not only 
 act autolytically upon the proteins of the spleen, but they also dissolve 
 fibrin and coagulated blood-serum. The spleen also contains nucleases 
 and besides, as Tanaka 2 has found for the pig-spleen, diastase, invertin, 
 lipase, urease, trypsin and an erepsin like enzyme. 
 
 Among the constituents of the spleen the deposit rich in iron, which 
 consists of ferruginous granules or conglomerate masses of them, and 
 which is derived from a transformation of the red blood-corpuscles, is of 
 special interest. It was closely studied by Nasse. This deposit does 
 not occur to the same extent in the spleen of all animals. It is found 
 especially abundant in the spleen of the horse. Nasse 3 on analyzing 
 the grains (from the spleen of a horse) obtained 840-630 p. m. organic 
 and 160-370 p. m. inorganic substances. These last consisted of 566- 
 726 p. m. Fe 2 3 , 205-388 p. m. P 2 5 , and 57 p. m. earths. The organic 
 substances consisted chiefly of proteins (660-800 p. m.), nuclein (52 p. m. 
 maximum), a yellow coloring-matter, extractive bodies, fat, cholesterin, 
 and lecithin. 
 
 In regard to the mineral constituents, it is to be observed that the 
 amount of iron in new-born and young animals is small (Lapicque, 
 Kruger, and Pernou), in adults more appreciable, and in old animals 
 sometimes very considerable. Nasse found nearly 50 p. m. iron in the 
 dried pulp of the spleen of an old horse. Guillemonat and Lapicque 4 
 have determined the iron in man. They find no regular increase with 
 growth, but in most cases 0.17-0.39 p. m. (after subtracting the blood- 
 iron) calculated on the fresh substance. A remarkably high amount of 
 iron is not dependent upon old age, but is a residue from chronic diseases. 
 Magnus-Levy found 0.72 p. m. iron in the fresh human spleen. 
 
 1 See Chapter XIV for the literature. 
 
 2 Hedin and Rowland, Zeitschr. f. physiol. Chem., 32, and Hedin, Journ. of Physiol., 
 30, and Hammarsten's Festschr., 1906; Tanaka, Bioch. Zeitschr, 37. 
 
 3 Maly's Jahresber., 19, p. 315. 
 
 4 Lapicque, ibid., 20; Lapicque and Guillemonat, Cornpt, rend, de soc. biol., 48, 
 and Arch de Physiol. (5) 8; Kruger and Pernou, Zeitschr. f. Biologie, 2"; Nasse, cited 
 from Hoppe-Seyler, Physiol. Chem., 720. 
 
372 CHYLE, LYMPH, TRANSUDATES AND EXUDATES. 
 
 On the analysis of the human spleen Magnus-Levy found 784.7 
 parts water, 215.3 parts solids, 27.7 parts fat and 27.9 parts nitrogen in 
 1000 parts of the fresh organ. In the dog spleen, Corper 1 found 750 
 to 770 p. m. water, and 120-150 p. m. ether soluble substances, of which 
 one-fourth consisted of cholesterin and three-fourths of lecithin. As 
 purine bases he found 1.1 p. m. guanine, 0.6 p. m. adenine, 0.15 p. m. 
 hypoxanthine and 0.04 p. m. xanthine. 
 
 In regard to the pathological processes going on in the spleen we must 
 specially recall the abundant re-formation of leucocytes in leucaemia and 
 the appearance of amyloid substance (see page 172) . 
 
 The physiological functions of the spleen are little known, with the 
 exception of its importance in the formation of leucocytes. Some 
 consider the spleen as an organ for the dissolution of the red blood- 
 corpuscles, and the occurrence of the above-mentioned deposit rich in 
 iron seems to confirm this view, but this iron could in part have another 
 origin. Asher and his collaborators Grossenbacher, Zimmermann and 
 H. Vogel have found that the spleen is an organ for the iron metabolism, 
 as they found in a splenectomized dog that the iron elimination was much 
 greater than in a dog with its spleen. R. Bayer 2 has made a similar 
 observation on a splenectomized human being, and the spleen it seems 
 has the purpose of retaining for the organism the iron set free in the 
 metabolism and also in starvation metabolism. 
 
 The spleen has also been claimed to play a certain part in digestion 
 especially in pancreatic digestion. This organ is said by Schiff, Herzen, 
 and others to be of importance in the production of trypsin in the pan- 
 creas. The investigations of Herzen seem to confirm this relation, but 
 the recent work of Prym 3 has made the assumption doubtful. 
 
 Splenectomized dogs require according to Richet 4 for their mainte- 
 nance more food, about one-third more, than normal dogs. The spleen 
 makes a complete utilization of the food possible or diminishes its con- 
 sumption. 
 
 An increase in the quantity of uric acid eliminated in splenic leucaemia 
 has been observed by many investigators (see Chapter XIV), while the 
 reverse has been observed under the influence of quinine in large doses, 
 which produces an enlargement of the spleen. These facts give a rather 
 positive proof that there is a close relation between the spleen and the 
 
 1 Magnus-Levy, Bioch. Zeitschr., 24; H. J. Corper, Journ. of biol. Chem., 11. 
 
 2 Asher and Grossenbacher, Centralbl. f. Physiol.. 22, 375, and Bioch. Zeitschr, 17 ; 
 Zimmermann, Bioch. Zeitschr., 17; R. Bayer, Bioch. Centralbl., 9, 815. 
 
 :; Schiff, cited by Herzen, Pfliiger's Arch., 30, 295, 308, and 84, and Maly's Jahr- 
 esber., 18; Prym, Pfliiger's Arch., 104 and 107; see also Chapter VIII. 
 4 Journ. de Physiol, et de Pathol, gen., 14 and 15. 
 
THYROID GLAND. 373 
 
 formation of uric acid. This relation has been studied by Horbac- 
 zewski. He has shown that when the spleen-pulp and blood of calves 
 arc allowed to act on each other, under certain conditions and certain tem- 
 perature, in the presence of air, large quantities of uric acid are formed, 
 and he has also shown that the uric acid originates from the nucleins of 
 the spleen. 1 This behavior is explained by the above-mentioned inves- 
 tigations of Burian, Schittenhelm, Jones, and others on the enzymotic 
 formation of uric acid, and the deamidization of the purine bodies, and a 
 relation between the spleen and uric-acid formation is indisputable. 
 Still we cannot say that the spleen shows a special relation to the uric-acid 
 formation as compared with other organs (see Chapter XIV). 
 
 The spleen has the same property as the liver of retaining foreign 
 bodies, metals and metalloids. 
 
 The Thyroid Gland. The nature of the different protein substances 
 occurring in the thyroid gland has not been sufficiently studied, but at 
 present, through the researches of Oswald, there are known at least two 
 bodies which are constituents of the so-called secretion of the glands, 
 the colloids. One of these, iodothyreoglobulin, behaves like a globulin, while 
 the other is a nucleoprotein (see also Gotjrlay 2 ). The iodine present 
 in the gland occurs chiefly in the first body, while the arsenic, which has 
 been shown to be a normal constituent by Gautier and Bertrand,* 
 seems to be related to the nuclein substances. 
 
 According to Oswald the iodothyreoglobulin occurs only in those 
 glands which contain colloid, while the colloid-free glands, the parenchyma- 
 tous goitre, and the glands of the new-born contain thyreoglobulin free 
 from iodine. The thyreoglobulin first becomes iodized into iodothyreo- 
 globulin on passing from the follicle-cells. Besides these mentioned 
 bodies leucine, xanthine, hypoxanthine, choline, iodothyrine, lactic and 
 succinic acids occur in the thyreoidea. Like certain other organs, sub- 
 stances also occur in the thyroid which act upon the blood pressure and 
 indeed partly as vasodilator and partly depressing but whose chemical 
 nature has not been positively established. Among the enzymes we 
 •find lipases and catalases which, according to Juschtschenko, 4 are 
 related to the corresponding enzymes of the blood. Magnus-Levy 5 
 found 757 parts water, 243 parts solids, 43.8 parts fat, 26.8 parts nitrogen, 
 and 0.058 parts iron in 1000 parts of the human thyroid gland. 
 
 1 Monatshefte f. Chem., 10, and Wein. Sitzungsber. Math. Nat. Klasse, 100, Abt. 3. 
 
 2 Gourlay, Journ. of Physiol., 16; Oswald, Zeitschr. f. physiol. Chem., 32, and 
 Biochem. Centralbl., 1, 249. 
 
 3 Gautier, Compt. Rend., 129. See also ibid., 130, 131, 134, 135; Bertrand, ibid., 
 134, 135. 
 
 4 Juschtschenko, Bioch. Zeitschr., 25 and Arch, scienc. biol. de St. Petersbourg, 15. 
 
 5 Bioch. Zeitschr., 24. 
 
374 CHYLE, LYMPH, TRANSUDATES AND EXUDATES. 
 
 In " strumacystica " Hoppe-Seyler found hardly any protein in the smaller 
 glandular vessels, but an excess of mucin, while in the larger he found a great 
 deal of protein l 70-80 p. m. 1 Cholesterin is regularly found in such cysts, some- 
 times in such large quantities that the entire contents form a thick mass of cho- 
 lesterin plates. Crystals of calcium oxalate also occur frequently. The contents 
 of the struma cysts are sometimes of a brown color, due to decomposed coloring- 
 matter, methcemoglobin (and haematin?). Bile-coloring matters have also been 
 found in such cysts. (In regard to the paralbumins and colloids which have been 
 found in struma cysts and colloid degeneration, see Chapter XII.) 
 
 Those substances which bear a close relation to the functions of the 
 gland seem to be of special interest. 
 
 The complete extirpation, as also the pathological destruction, of the 
 thyroid gland causes great disturbances, ending finally in death. In 
 dogs, after the total extirpation, a disturbance of the nervous and muscular 
 systems occurs, such as trembling and convulsions, ^and death generally 
 supervenes shortly after, most often during such an attack. The researches 
 of Gley, Vassale and Generalt 2 upon various animals have shown 
 that for the success cf the operation it is of the greatest importance 
 whether the parathyroids, discovered by Sandstrov, 3 are removed at 
 the same time or not. In herbivora (rabbits) because of the anatomical 
 relations, the parathyroids are seldom extirpated in the operation of 
 the removal of the thyroid, the tetany does not regularly occur and 
 the disturbance in metabolism is most striking. If these glands are 
 not extirpated in dogs, the tetany also does not appear, and the dis- 
 turbances in metabolism occur. In human beings, after the removal of 
 the gland by operation, different disturbances appear, such as nervous 
 symptoms, diminished intelligence, dryness of the skin, falling out of 
 the hair, and, on the whole, those symptoms which are included under 
 the name cachexia thyreopriva, death coming gradually. Among these 
 symptoms must be mentioned the peculiar slimy infiltration and exuber- 
 ance of the connective tissue called myxedema. 
 
 All these conditions indicate that the thyroids belong to those glands 
 with internal secretion, so called endocrinic glands. The most con- 
 vincing proof of this is the fact that the ordinary symptoms do not occur 
 if a small piece of the gland is allowed to remain in the body, or even 
 when a piece of the gland is transplanted in any part of the body. The 
 observations of Asher and Flack 4 that the irritation of the nerves of the 
 thyroid causes an internal secretion from the thyroid gland into the 
 blood, is of great interest in this connection. A further proof of practical 
 
 ^Physiol. Chem., p. 721. 
 
 J Gley, Compt. rend. soc. biol., 1891, and Arch, de Physiol (5), 4; Vassale and 
 Generali, Arch. Ital. d. Biol., 25 and 2fi. 
 •Upaala L&karef. Fdrh., 15 (1880). 
 * Asher and Flack, Zeitscbr. f. Biol., 55. 
 
THYROID GLAND. 375 
 
 importance is that the injurious results from removal of the thyroids 
 can be counteracted by the introduction of artificial extracts of the 
 thyroid gland into the body or by feeding with thyroid glands. 
 
 Of the disturbances in metabolism which occur on the extirpation 
 or reduction of the thyroid function (athyreoidismus or hypothyreoid- 
 ismus) we must especially mention the reduction in the protein catabolism 
 which in a starving dog without thyroids may fall to about one-half of 
 the starvation protein metabolism in a normal dog of the same size 
 (Falta and collaborators J ). The reverse is observed when large quan- 
 tities of the thyroid gland substance is fed, namely, a strong increase in 
 the protein metabolism, besides certain other symptoms. Basedow's 
 disease is also considered as a form of hyperthyreoidismus which, by an 
 increased activity of the glands, brings about an overproduction of the 
 specific secretion. There does not seem to be any doubt that the thyroid 
 glands stand in close relation to other endocrinic glands although for the 
 present we are unable to survey this very complicated condition. One 
 side of this reciprocal action with other organs, which is of special impor- 
 tance, is the relation of the thyroids to glycosuria, which will be discussed 
 in a following chapter. 
 
 The glands with internal secretion, the so-called endocrinic glands, 
 to which the adrenals belong, which will be discussed below, and the 
 hypophysis, are of especially great interest because of the reciprocal 
 action which they exert among each other and with other organs. A 
 chemical correlation exists between different organs, of a kind, that 
 bodies which are formed in one organ can awaken or regulate the func- 
 tions of another organ or other organs. These chemically active sub- 
 stances, which awaken or regulate the activity of other organs have 
 been given the group name hormone (6pfLaw = I awaken or excite) by Star- 
 ling and to this group belong the specifically active constituents of the 
 endocrinic glands. 
 
 It is impossible for the present to state anything about the kind of 
 bodies having a specific action in the thyroid gland or anything about the 
 importance of the bases found by certain investigators, such as S. Frank- 
 el, Drechsel, and Kocher, 2 as these bodies have not been characterized 
 sufficiently. It seems proved that the specifically active substance is, as 
 first shown by Notkin 3 and Oswald, 4 a protein substance: Notkin's 
 thyreoproteid, Oswald's thyreoglobulin . This does not conflict with the views 
 
 1 Eppinger, Falta and Rudinger, Zeitschr. f. klin. Med., 66. 
 
 5 Frankel, Wein. med. Blatter, 1895 and 1896; Drechsel and Kocher, CentralbL 
 f. Physiol., 9, 705. 
 
 3 Wien. med. Wochenschr., 1895, and Virehow's Arch., 144, Suppl., 224. 
 * Zeitschr. f. physioi. Ohem., 32, and Bioch. CentralbL, 1, 249. 
 
376 CHYLE, LYMPH, TRANSUDATES AND EXUDATES. 
 
 of Baumann and Roos that the active substance is iodothyrin, as thk 
 can be produced as a cleavage product from the iodothyreoglobulin. In 
 fact Oswald has found in the tryptic digestion of iodothyreoglobulin 
 that a substance 'similar to iodothyrin is produced; important inves- 
 tigations x nevertheless make it probable that the thyreoglobulin is 
 the active substance and not the iodothyrin. There are several reasons 
 why the action of the thyroid gland substance is not due to one substance, 
 but to several. 
 
 Iodothyrin is considered by Baumann, who first showed that the thyroid con- 
 tained iodine and who with Roos 2 proved the importance of this substance for the 
 physiological activity of the gland, as the only active substance. By boiling the 
 finely divided gland with dilute sulphuric acid Baumann obtained iodothyrin 
 as an amorphous, brown mass, nearly insoluble in water but readily soluble in 
 alkali and precipitated again by the addition of acid. The iodothyrin, which 
 is not a unit bod}', has a variable content of iodine and is not a protein substance. 
 According to v. Furth and Schwarz it is probably a melanoid-like transforma- 
 tion product of the iodized protein of the gland produced by the action of the 
 acid. 
 
 Thyreoglobulin or iodothyreoglobulin was obtained by Oswald from 
 the watery extract of the gland by half saturating with ammonium sul- 
 phate. It has the properties of the globulins and with the exception of 
 the iodine content it has about the same composition as the proteins. 
 The amount of iodine varies: 0.46 per cent in pigs, 0.86 per cent in oxen, 
 and 0.34 per cent in man. In the iodothyreoglobulin of the ox, Nuren- 
 berg 3 found 0.59-0.86 per cent iodine and 1.83-2.0 per cent sulphur. 
 In young animals, whose glands contain no iodine, the thyreoglobulin 
 is iodine-free. Thyreoglobulin on taking up iodine is converted into 
 iodothyreoglobulin. By introducing iodine salts the iodine content of the 
 iodothyreoglobulin can be raised in living animals and thus the physiological 
 activity increased (Oswald). The amount of iodine in the gland is 
 markedly dependent upon the food. 
 
 Jolin has examined a large number of thyroid glands from healthy and 
 diseased persons (in Sweden), for their iodine content. In 28 children, ages 
 
 1 See Oswald. Arch. f. exp. Path. u. Pharm., 60; Pick and Pineles, Zeitschr. f. 
 exp. Path. u. Therap., 7. 
 
 2 In regard to this subject, see Baumann and Roos, Zeitschr. f. physiol. Chem., 21 
 and 22; also Baumann, Miinch. med. Wochenschr., 1896; Baumann and Goldmann, 
 ibid.; Roos., ibid.; v. Furth and Schwarz, Pfliiger's Arch., 124. An extensive review 
 of the literature on the action of iodothyrin and the thyroid preparations can be found 
 in Roos, Zeitschr. f. physiol. Chem., 22, 18. In regard to their action in protein catabo- 
 lism and in metabolism, see F. Voit, Zeitschr. f. Biologie, 35; Schondorff, Pliiger's Arch., 
 67, and Andersson and Bergman, Skand. Arch. f. Physiol., 8; Magnus-Levy, Zeitschr. 
 f. klin. Med., 32. In regard to the function of the thyroid gland see also Sw. Vincent^ 
 Innere Sckretion etc. Ergebnisse d. Physiol., 11, 218-302. 
 
 3 Bioch. Zeitschr., 16. 
 
ADRENAL BODIES. 377 
 
 varying between 1 and 10 years, he found an average of 0.28 p. m. iodine in the 
 glands. In 108 normal glands above 10 years old or adults the iodine content 
 varied with an average of 1.56 p. in. iodine. In glands from persons after using 
 iodine preparations (o4 rases) the iodine content was 2.56 p.m. The amount 
 of silicic acid in normal thyroid glands was found by 11. Schulz ' to be on an 
 average 0.084 |». m., calculated on the dry substance. In goitres from Grbifswald 
 and ZURICH he found 0.17") and 0.434 p. m., respectively. There does not seem 
 to be any connection between the silicic acid content of the drinking water and 
 the occurrence of goitre. 
 
 YYe cannot enter into a discussion as to the various hypotheses and 
 theories in regard to the mode of action of the constituents of the thyroids. 
 In the tetany appearing after parathyroidectomy many investigators 
 find an increased elimination of calcium, nitrogen and ammonia and the 
 hypothesis has been suggested that the tetany depends upon an increased 
 irritability of the nervous system due to lack of calcium. The fact as 
 found by several experimenters that a diminished calcium content of the 
 organs in question does not occur, speaks against this theory. On the 
 contrary, it seems to be generally admitted that lime salts reduce or 
 prevent the tetany and, according to Frouin, 2 this depends upon the lime 
 combining with the carbonic acid produced, which is the cause of the 
 tetany. The tetany is produced at least from a poison which is formed 
 only on the removal of the parathyroids or if it is regularly produced 
 it is made harmless by these organs. 
 
 G. Mansfield and Fr. Muller 3 have made investigations in regard 
 to the action of the thyroids upon protein metabolism which indicate 
 that lack of oxygen acts as an excitant upon the thyroids and that the 
 increased protein catabolism, which occurs to a mean degree with lack 
 of oxygen, depends upon a hyperfunction of the thyroid glands brought 
 on by this condition. With greater lack of oxygen besides this a general 
 damage to the protoplasm of the bod}' cells may occur. 4 
 
 The Adrenal Bodies. Besides proteins, substances of the connect- 
 ive tissue, and salts, there occur in the suprarenal capsule inosite, purine 
 bases, especially xanthine (Oker-Blom), phosphatides and glycerophos- 
 phoric acid, which is probabty a decomposition product of the latter. 
 The earlier accounts of the occurrence of benzoic acid, hippuric acid, 
 and bile-acids are, on the contrary, doubtful, and are not substantiated 
 by recent investigations (Stadelmann 5 ). The medullary substance 
 
 1 John, Hammarsten's Festschr., 1906; H. Schulz, Bioch. Zeitschr., 46. 
 
 2 Compt. Rend., 148. 
 
 3 Pfluger's Arch., 143. 
 
 4 A very complete discussion of the physiology of the thyroid gland and the pertinent 
 literature may be found in Sw. Vincent, Ergebnisse der Physiologie, 11, 218-302. 
 
 5 Oker-Blom, Zeitschr. f. physiol. Chein., 28; Stadelmann, ibid., 18, which also 
 contains the literature on this subject. 
 
378 CHYLE, LYMPH, TRANSUDATES AND EXUDATES. 
 
 contains the so-called chromaffine tissue, i.e., cells, whose substance is 
 colored brown by chromic acid or chromates. 
 
 Earlier investigators, like Vulpian and Arnold, have found, in the 
 medulla, a chromogen which has been considered as connected with the 
 abnormal pigmentation of the skin in Addison's disease. This chromogen, 
 which is transformed by air, light, alkalies, iodine, and other bodies into 
 a red pigment, seems, on the contrary, to be related to the substance 
 adrenalin, of the gland which produces an increase in the blood-pressure. 
 Choline has been shown to have a reverse effect upon this blood-pressure 
 raising action, and Lohmann has shown that it is formed in the cortical 
 substance of the adrenals. In the cortical this last-mentioned exper- 
 imenter x has found besides neurin, another not known base. That 
 the watery extract of the adrenals has a blood-pressure raising action 
 was shown by Oliver and Schafer, Cybulski and Szymonowicz. 2 
 The substance which is here active was formerly called sphygmogenin 
 and has also other actions besides bringing about a marked increase 
 in blood-pressure by the strong contraction of the muscles of the periphery 
 vessels; for instance, it can bring about glycosuria and mydriasis, espe- 
 cially in the frog's eye, has been chemically investigated by numerous 
 experimenters. 3 v. Furth calls it suprarenin, Abel epinephrin. and 
 Takamine adrenalin. This last name seems to be the most generally 
 accepted one. 
 
 Adrenalin (suprarenin epinephrin) (methylaminoethanolpyrocatechin) 
 
 CH 
 
 (HO)C C.CH(OH).CH 2 .NHCH 3 
 
 C 9 H 13 N0 3 ,= I || 
 
 (HO)C CH 
 
 V 
 
 CH 
 
 The constitution of adrenalin has been essentially proved by Friedmann, 4 
 and he has shown the correctness of the above formula, which was given by 
 Pauly. The synthesis of adrenalin, which was first performed by Stolz, 5 is 
 also in accordance with this formula. By the action of methylamine upon 
 chloracetopyrocatechin we obtain methylaminoaceto-pyrocatechin: 
 
 C 6 H 3 (OH) 2 .COCH,Cl +XH.CH 3 = C 6 H 3 (OH) 2 .COCH,.NHCH 3 .HCl, 
 
 which yields adrenalin on reduction. 
 
 'Centralbl. f. Physiol., 21, and Pfliiger's Arch., 118 and Zeitschr. f. Biol., 56. 
 
 2 Oliver and Schafer, Proceed, of Physiol. Soc, London, 1895. Further literature 
 on the function of the adrenals may be found in Sw. Vincent, Innere Sekretion, etc. 
 Ergebnisse d. Physiol., 9, 505-585. 
 
 3 The literature on this subject may be found in Abderhalden's Bioch. Handlexikon 
 Bd. 5, s. 454-495. 
 
 4 Hofmeister's Beitrage, 8. 
 
 s Ber. d. d. chem. Gesellsch., 37. 
 
ADRENALIN. 379 
 
 The synthetically prepared adrenalin is optically inactive r/-/-adrcnalin, 
 while that from the adrenals is optically active /-adrenalin. FLACHEB has 
 divided the racemic adrenalin into the two optically active components, 
 and the identity of the so-obtained synthetical adrenalin with the natural 
 has been shown by Abderhalden and Fr. Muller. 1 These last inves- 
 tigators also found that the /-adrenalin had at least 15 times as strong 
 an action upon the blood-pressure as the (/-adrenalin, and later Abder- 
 halden with Thies and Slavu found that the /-adrenalin had also in 
 other respects a much stronger action than d-adrenalin. 
 
 Adrenalin crystallizes in masses of needles or rhombic leaves. It is 
 soluble in water, and can be precipitated from its solution by ammonia 
 as a crystalline substance. Its aqueous solution containing hydrochloric 
 acid is levorotatory : (c*) D = —50.72° (Abderhalden and Guggenheim 2 ). 
 On heating adrenalin it turns yellowish-brown at about 205° and decom- 
 poses at about 218° C. Its solution turns emerald green with ferric chlor- 
 ide in acid solution and carmine red in alkaline solution. Adrenalin 
 reduces Fehling's solution and ammoniacal silver solution. 
 
 Among the reactions for adrenalin in solution we must especially 
 mention the red coloration which is obtained on the addition of an oxidizing 
 medium such as iodine or bi-iodate and dilute phosphoric acid and warm- 
 ing (Frankel and Allers), or of mercuric chloride in the presence of a 
 catalyst such as the lime salts in tap-water (Comesatti). These reactions 
 are extremely delicate, 1 : 1000000-2000000. A still more delicate reaction 
 (1:5000000) is the one suggested by Ewins, 3 namely a characteristic 
 red coloration is obtained on adding a 0.1 per cent solution of potassium 
 persulphate and w r arming gently in a boiling water-bath. 
 
 As above stated, it has been considered for some time that the color 
 of the skin in Addison's disease was connected with the adrenals or their 
 chromogen. We know nothing positive in regard to this relation, 
 but it is nevertheless of interest that pigments, and finally melanins or 
 at least dark-brown substances, can be produced from adrenalin by the 
 action of enzymes. Neuberg has brought about such melanin forma- 
 tion by the extract from the metastases of a melanoma of the adrenals 
 and also with the extract of the ink-sac of the sepia, and Abderhalden 
 and Guggenheim 4 with tyrosinase. This would indicate a close relation 
 
 1 Flacher, Zeitschr. f. physiol. Chem., 58; Abderhalden and Franz Muller, ibid. 
 58; with Thies, ibid., 59; with Slavu, ibid., 59; with Kautsch and Muller, ibid., 61 and 
 62; see also Frohlich, Centralbl. f. physiol., 23 and Waterman, Zeitschr. f. physiol. 
 Chem., 63. 
 
 2 Zeitschr. f. physiol. Chem., 57. 
 
 * Frankel and Allers, Bioch. Zeitschr., 18; Comesatti, Munch, med. Wochenschr. 
 1908 and Physiol. Centralbl., 23; Ewins, Journ. of Physiol., 40. 
 
 4 Neuberg, Bioch. Zeitschr., 8; Abderhalden and Guggenheim, Zeitschr. f. physiol. 
 Chem., 57. 
 
380 CHYLE, LYMPH, TRANSUDATES AND EXUDATES. 
 
 between adrenalin and tyrosine, which also gives melanin with the sepia 
 enzyme, and indeed tyrosine has been considered as the probable mother- 
 substance of adrenalin (Halle). The investigations of Ewin's and 
 Laidaw x to prove this last-mentioned possibility have not given any 
 support thereto. 
 
 Besides the action of producing a rise in the blood-pressure, adrenalin 
 is also of special interest because, as first shown by Blum, 2 it also has a 
 glycosuric action. We will discuss the question of adrenalin glycosuria 
 and the relation which seems to exist between the internal secretions 
 of the thyroids, the adrenals and the pancreas, when we treat of the 
 formation of sugar and pancreas diabetes. We cannot here enter into 
 the question of the reciprocal action between the adrenals and the other 
 organs. 
 
 The hypophysis or pituitary gland has been little studied from a chemical 
 standpoint. An extract of the gland shows, by its action, a certain similarity to an 
 extract of the adrenals in that it causes a rise in blood pressure and by causing a 
 dilation of the pupils of the frog's eye. Still no adrenalin could be detected in the 
 gland. Also no iodine occurs in the glands (Wells, Denis 3 ) . 
 
 The gland consists essentially of two parts, one an outside formation of vascular- 
 glandular epithelium and a lower nervous part the infundibular part. The out- 
 side part seems to have a relation to the growth of the tissues and skeleton and 
 acromegalie and gigantism are claimed by many investigators to be related to this 
 part. The infundibular part, on the contrary, contains the specific bodies which 
 raises the blood -pressure and stimulates the smooth muscles of the uterus and 
 upon the kidney secretion. The relation of the hypophysis to other endocrinic glands 
 is still very much disputed. 
 
 1 Halle, Hofmeister's Beitrage 8; Ewins and Laidaw, Journ. of Physiol., 40. 
 
 2 Deutsch. Arch. f. klin. Med., 91 and Pfliiger's Arch., 90. 
 
 3 H. G. Wells, Journ. of biol. Chem., 7; W. Denis, ibid., 9. 
 
CHAPTER VII. 
 THE LIVER. 
 
 The liver, which is the largest gland of the body, stands in close 
 relation to the glands mentioned in Chapter VI. The importance 
 of this organ for the assimilation of the food-stuffs and for the phys- 
 iological composition of the blood is evident from the fact that the 
 blood coming from the digestive tract, laden with absorbed bodies, must 
 circulate through the liver before it is driven by the heart through the 
 different organs and tissues. An assimilation of food-stuffs in the liver 
 .has been positively shown in the first place for carbohydrates in that the 
 liver constructs a polysaccharide glycogen from hexoses, which according to 
 the needs is then again retransformed into glucose. The liver is a storage 
 organ for fats and takes up food fat as well as fat from depots (in 
 starvation) and as it seems, at least in part, prepares them so that they can 
 be further used in the animal body. 
 
 We are not clear as to what extent an assimilation of products of pro- 
 tein digestion takes place in the liver. The subject will be discussed in detail 
 under absorption in Chapter VIII. It is claimed that the liver can serve 
 as a storage organ for proteins, 1 and it is at least certain that it retains 
 alien protein which is brought to it by the blood. 2 The retention 
 of alien protein stands probably in close relationship to the ability of 
 the liver to take up and retain foreign substances as a group from the blood. 
 This is not only true for different metals but also, as shown by several 
 investigators. 3 alkaloids which perhaps are also partly decomposed in the 
 liver. Toxins are also withheld by the liver and hence this organ has 
 a protective action against poisons. 
 
 The formation of glycogen from glucose is one of the numerous syn- 
 theses occurring in the liver and this is no doubt the one which takes 
 place to the greatest extent. Other syntheses in the liver are, for example, 
 
 1 See Seitz, Pfliiger's Arch., Ill and Asher and Boehm, Zeitschr. f. Biol. 51. 
 
 2 See Reach, Bioch. Zeitschr., 16 and Pacchioni and Carlini, Maly's Jahresb., 39. 
 
 3 Roger, Action du foie sur les poisons (Paris, 1887), which quotes the works of 
 Schiff, Heger and others; also W. N. Woronzow, Maly's Jahresb., 40 and Z. Vamossy, 
 Orid., 40. 
 
 381 
 
382 THE LIVER. 
 
 the formation of urea or uric acid (in birds) from ammonium salts, the 
 formation of etheral sulphuric acids and conjugated glucuronic acids 
 from the phenols produced in intestinal putrefaction and the recently 
 shown syntheses of amino-acids in the liver. On the other hand a 
 deamidation of amino-acids and purine bodies, hydrolyses, oxidations, 
 reductions and fermentative processes of various kinds occur in the liver. 
 Because of these diverse processes, the results of which we must espe- 
 cially mention the formation of bile as well as the fact that the liver is 
 introduced between the intestine and the general circulation, makes the 
 liver a central organ for metabolism. 
 
 Among the numerous chemical processes which take place in the liver 
 there are especially two which give special interest to this organ, namely, 
 the formation of glycogen or the carbohydrate metabolism in the liver, 
 and the formation of bile. For this reason only these two processes will 
 be discussed in this chapter while the others will be discussed in other 
 chapters and in other connection. Before we begin to discuss these two 
 processes a short review of the constituents and the chemical com- 
 position of the liver seems to be appropriate. 
 
 The reaction of the liver-cells is alkaline toward litmus during life, 
 but becomes acid after death, due to a formation of lactic acid, chiefly 
 fermentation lactic acid and other organic acids (Morishima, Magnus- 
 Levy J ). A coagulation of the protoplasmic proteins in the cells probably 
 takes place. A positive difference between the proteins of the dead 
 and the living, non-coagulated protoplasm has not been observed. 
 
 The proteins of the liver were first carefully investigated by Pl6sz. 
 He found in the watery extract of the liver an albuminous substance 
 which coagulates at 45° C. (globulin, Halliburton), also a globulin 
 which coagulates at 75° C, a nucleoalbumin which coagulates at 70° C, 
 and lastly a protein body which is closely related to the coagulated albumi?is 
 and which is insoluble in dilute acids or alkalies at the ordinary tem- 
 perature, but dissolves on the application of heat, being converted into 
 an albuminate. Halliburton 2 found two globulins in the liver-cells, 
 one of which coagulates at 68-70° C, and the other at 45-50° C. He 
 also found, besides traces of albumin, a nucleoprotein which possessed 
 1.45 per cent phosphorus and a coagulation-point of 60° C. Pohl has 
 obtained an '" organ plasma " by extracting the finely divided liver 
 which had previously been entirely freed from blood by washing with 
 8 p. m. NaCl solution, in which he was able to detect a globulin having 
 a low coagulation temperature. The very variable phosphorus, content 
 
 1 Morishima, Arch. f. exp. Path. u. Pharm., 43; Magnus-Levy, Hofmeister's Bei- 
 trage, 2. 
 
 2 P16sz, Pfliiger's Arch., 7; Halliburton, Journ. of Physiol., 13, Suppl. 1892. 
 
PROTEINS OF THE LIVER. 383 
 
 (0.28-1.3 per cent) of this globulin as well as the insolubility of the pre- 
 cipitates produced by little acid, in an excess of acid, and in neutral salt- 
 seem to indicate that we here have a mixture which consists chiefly of 
 nucleoproteins and not of globulins. The almost complete digestibility 
 with pepsin-hydrochloric acid does not controvert this assumption, 
 because, as is known, nucleoproteins may on digestion yield no residue 
 (see Chapter II). Nor can we be positive concerning the nature of 
 the liver-globulin found by Dastre, 1 having a coagulation temperature 
 of 56° C. The proteins extractable from the liver without modification 
 must be thoroughly investigated. 
 
 Besides the above-mentioned proteins, which are very soluble, the 
 liver-cells contain large quantities of difficultly soluble protein bodies 
 (see Pl6sz). The liver also contains, as first shown by St. Zaleski 
 and later substantiated by several other investigators, ferruginous 
 proteins of different kinds. 2 . The chief portion of the protein- substances 
 in the liver seems in fact to consist of ferruginous nucleoproteins. On 
 boiling the liver with water, such a nucleoprotein or perhaps several 
 are split, and a solution is obtained containing a nucleic-acid-rich nucleo- 
 protein or a mixture of these which are precipitable by acids. This 
 protein or protein mixture, which has been called ferratin by Schmiede- 
 berg, 3 has been studied by Wohlgemuth. 4 The quantity of phos- 
 phorus was 3.0fi per cent. As cleavage products on hydrolysis he found 
 /-xylose, or at least a pentose, the four nuclein bases, and also arginine, 
 lysine (and histidine?), tyrosine, leucine, glycocoll, alanine, a-proline, 
 glutamic acid, aspartic acid, phenylalanine, oxyaminosuberic acid, and 
 oxydiaminosebacic acid (see Chapter II). The /-xylose depends, no 
 doubt, at least in part, upon the guanvlic acid isolated from the liver, 
 by Levene and Mandel, 5 and the finding of adenine among the cleavage 
 products also indicates the presence of a thymonucleic acid. There does 
 not seem to be any doubt that the ferratin, as above stated, is a mixture, 
 and the correctness of this assumption is shown by the recent investiga- 
 tions of Scaffidi and Salkowski. 6 
 
 1 Pohl, Hofmeister's Beitrage, 7; Dastre, Compt. rend. soe. biolog.. 58. 
 
 2 St. Zaleski, Zeitschr. f. physiol. Chem., 10, 486; Woltering, ibid., 21; Spitzer, 
 Pniiger's Arch., 67. 
 
 ' Arch. f. exp. Path. u. Pharm., 33; see also Vay, Zeitschr. f. physiol. Chem.. 20. 
 
 ♦Wohlgemuth, Zeitschr. f. physiol. Chem., 37, 42, and 44, and Ber. d. d. chem. 
 Gesellsch., 37. See on liver nucleoproteins also Salkowski, Berl. klin. Wochenschr., 
 1895; Hammarsten, Zeitschr. f. physiol. Chem., 19; Blumenthal, Zeitschr. f. klin. Med., 
 34. 
 
 5 Bioch. Zeitschr., 10. 
 
 •Scaffidi, Zeitschr. f. physiol. Chem., 58; Salkowski, ibi>1., 58. 
 
384 THE LIVER. 
 
 The yellow or brown pigment of the liver has been little studied. Dastre 
 and Floresco l differentiate, in vertebrates and certain invertebrates, between a 
 ferruginous pigment soluble in water, ferrine, and a pigment soluble in chloroform 
 and insoluble in water, chlorochrome. They have not isolated these pigments in a 
 pure condition. In certain invertebrates chlorophyll originating from the food also 
 occurs in the liver. 
 
 The fat of the liver occurs partly as very small globules and partly 
 (especially in nursing children and suckling animals, as also after food 
 rich in fat) as rather large fat-drops. The occurrence of a fatty infiltra- 
 tion, i.e., a transportation of fat to the liver, may not only be produced 
 by an excess of fat in the food (Noel-Paton), but also by a migration 
 from other parts of the body under abnormal conditions, such as poison- 
 ing with phosphorus, phlorhizin, and certain other bodies (Leo, Lebedeff, 
 Rosenfeld, and others 2 ). The fatty infiltration occurring in poisoning, 
 and which is accompanied with degenerative changes in the cells, may 
 cause a diminution in the amount of protein and a rise in the water con- 
 tent. If the amount of fat in the liver is increased by an infiltration, the 
 water decreases correspondingly, while the quantity of the other solids 
 remains little changed. Changes of a kind may occur, so that, because 
 of the antipathy (Rosenfeld, Bottazzi 3 ) existing between glycogen 
 and fat, a liver rich in fat is habitually poor in glycogen. The reverse 
 occurs after feeding with carbohydrate-rich food, namely, the liver is 
 rich in glycogen and poor in fat. 
 
 The composition of the liver-fat seems to vary not only in different 
 animals, but is variable with differing conditions. Thus Noel-Paton 
 found that the liver-fat in man and several animals was poorer in oleic 
 acid and had a correspondingly higher melting-point than the fat from 
 the subcutaneous connective tissue, while Rosenfeld 4 observed the 
 opposite condition on feeding dogs with mutton-fat. 
 
 Several investigators, Hartley, Leathes and Mottram suggested 
 as a difference between the fat of the liver and the connective tissues, 
 the great amount in the first of unsaturated, higher fatty acids. Accord- 
 ing to Hartley 5 the fat of the pig liver contains palmitic acid, stearic 
 
 1 Arch, de Physiol. (5), 10. 
 
 2 Noel-Paton, Journ. of Physiol., 19; Leo, Zeitschr. f. physiol. Chem., 9; Lebedeff, 
 PfluRer's Arch., 31; Athanasiu, Pfliiger's Arch., 74; Taylor, Journ. of Exp. Med., 4; 
 Kraus u. Sommer, Hofmeister's Beitrage, 2; Rosenfeld, Zeitschr. f. klin. Med., 36. 
 See also Rosenfeld, Erfiebnisse der Physiologie, 1, Abt. 1, and Berl. klin. Wochenschr. 
 1904; Schwalbe, Centralbl. f. Physiol., 18, p. 319; Shibata, Bioch. Zeitschr., 37. 
 
 3 Arch. Ital. d. Biol., 48 (1908), cited in Bioch. Centralbl., 7, p. 833. 
 
 * Cited by Lummert, Pfluger's Arch., 71. In regard to the liver-fat of children, 
 see Thiemich, Zeitschr. f. physiol. Chem., 26. 
 
 6 Hartley, Journ. of Physiol., 38; Leathes and Meyer-Wedell, ibid., 38; Mottram,, 
 ibid., 38. 
 
PHOSPHATIDES OF THE LIVER. 385 
 
 acid, and oleic acid which is not identical with the ordinary oleic acid, also 
 linoleic acid and an acid having the formula C20H32O2. A part of 
 these unsaturated fatty acids are contained in the phosphatides but as 
 the unsaturated acids are about one-half of the fatty acids they must 
 also occur in the fats. The abundant occurrence of unsaturated fatty 
 acids is considered by the above-mentioned investigators as the first 
 step in the cleavage of the transportable fat from the fat tissues to the 
 liver and destined for use in the body. There is no doubt that the phos- 
 phatides are of great importance for this transformation of the fat. 
 
 Phosphatides, which were formerly designated lecithin, and whose 
 quantity is generally calculated as such, also belong to the normal con- 
 stituents of the liver. The quantity (as lecithin) amounts to over 23.5 
 p. m. according to Noel-Paton. 1 In starvation the lecithin, according 
 bo Xoel-Paton, forms the greater part of the ethereal extract, while 
 with food rich in fat, on the contrary, it forms the smaller part. In 
 the liver of a healthy dog Baskoff 2 found 84 p. m. phosphatides (cal- 
 culated as lecithin) in the dry substance. The phosphatides are undoubt- 
 edly of various kinds, but they have not been closely studied. Among 
 others, Ave have lecithin and the so-called jecorin. Cholesterin is also a 
 constituent of the liver, although only in small quantities, and Kondo 3 
 finds that cholesterin ester occurs in the liver. 
 
 Jecorin was first found by Drechsel in the liver of horses, and also in the 
 liver of a dolphin, and later by Baldi in the liver and spleen of other animals, in 
 the muscles and blood of the horse, and in the human brain. It contains sul- 
 phur and phosphorus, but its constitution is not positively known. Jecorin dis- 
 solves in ether, but is precipitated from this solution by alcohol. It reduces 
 copper oxide, and gives a wine-red coloration with an ammoniacal silver-solution. 
 ( hi boiling with alkali and then cooling it solidifies to a gelatinous mass. Manasse 
 has detected glucose as osazone in the carbohydrate complex of jecorin. 
 
 The statement by Bing that jecorin is a combination of lecithin and glucose 
 does not follow from the analyses of jecorin thus far known. Jecorin contains 
 sulphur, even as much as 2.75 per cent, and further the relation of P:N in lecithin is 
 1:1, while in jecorin it is quite different, 1: 2 to 1: 0. According to the investiga- 
 tions of Baskoff the liver jecorin, prepared according to Drechsel's sugges- 
 tion, and when it is so pure that it is completely soluble in ether, and quantitatively 
 precipitated by alcohol from this solution, is a rather constant compound at 
 least in regard to the X, P and glucose content. Baskoff found as average 2.55 
 per cent X, 2.87 per cent P, and about 14 per cent glucose. The relation P:X 
 was nearly 1 :2 and therefore jecorin is correspondingly a diaminomonophosphatide. 
 
 The variable composition and divergent properties of the jecorin isolated and 
 analyzed by various investigators 4 depends, according to Baskoff, upon imper- 
 
 1 1. c. See also Heffter, Arch. f. exp. Path. u. Pharm., 28. 
 
 2 Zeitschr. f. physiol. Chem., 62. 
 
 3 Bioch. Zeitschr., 26. 
 
 * Drechsel, Ber. d. sachs. Gesellsch. d. Wissensch., 1886, p. 44, and Zeitsch. f. Biol- 
 ogie, 33; Baldi, Arch. f. (Anat. u.) Physiol., 1887, Suppl., 100; Manasse, Zeitschr. f. 
 physiol. Chem., 20; Bing, Centralbl. f. Physiol., 12, and Skand. Arch. f. Physiol., 9; 
 
386 THE LIVER. 
 
 I 
 
 feet purification. His investigations do not give any explanation for the quantity 
 of sulphur and it is very probable that jecorin is only a mixture of several bodies 
 among which a sulphurized and a phosphorized substance occurs. According 
 to Baskoff it is very probable that the jecorin is a decomposition product of lecithin 
 (or other phosphatides). 
 
 Another phosphatide, which does not reduce directly or after boiling with acid, 
 has been called heparphosphatide by Baskoff. In certain respects this body is 
 similar to cuorin, and the relation P:N =1.45:1, although it was not pure. 
 
 Among the extractive substances besides glycogen, which will be treated 
 later, rather large quantities of the purine bases occur. Kossel 1 found 
 in 1000 parts of the dried substance 1.97 p. m. guanine, 1.34 p. m. hypo- 
 xanthine, and 1.21 p. m. xanthine. Adenine is also contained in the 
 liver. In addition there are found urea and uric acid (especially in 
 birds), and indeed in larger quantities than in the blood, paralactic acid, 
 choline, leucine, taurine, and cystine. In pathological cases inosite and 
 amino-acids have been detected. The occurrence of bile-coloring matters 
 in the liver-cell under normal conditions is doubtful; but in retention of 
 the bile the cells may absorb the coloring-matter and become colored 
 thereby. 
 
 A large number of enzymes are found in the liver, such as catalases, 
 oxidases, aldehydases and hydrolytic enzymes of various kinds; the dias- 
 tase acting upon glycogen, the lipases and the different proteolytic enzymes. 
 Nucleases and the nucleic acid splitting enzymes of different kinds men- 
 tioned in Chapter II have been formed in the liver and deamidases for 
 amino-acids as well as purine bodies also occur in the liver. The last 
 group of deamidases show a different behavior in regard to their occurrence 
 in different animals and the same is true for the uric acid forming and 
 uric acid destroying enzymes (Chapter XIV). We must also mention the 
 arginase which splits off urea from arginine. 
 
 The proteolytic enzymes of the liver are of special interest, especially 
 in regard to the study of the autolysis of this organ. The processes 
 in the liver in phosphorus poisoning and in acute yellow atrophy of the 
 liver are considered as an intravitally increased autolysis. In these 
 cases a softening of the organ takes place, and proteoses, mono- and 
 diamino-acids, and other bodies are produced, which may also be found 
 in the urine, and although they may not all be derived from the liver 
 (X Ei; berg and Richter), they are at least in part derived from this organ. 
 Wakeman has found in phosphorus poisoning that not only is the quan- 
 tity of nitrogen markedly diminished in the liver (of dogs), but also 
 that the quantity of nitrogen of the hexone bases is diminished, and 
 
 Meinertz, Zeitschr. f. physiol. Chem., 46; Siegfried and Mark, ibid.; Paul Mayer, 
 Bioch. Zeitschr., 1, and Baskoff, Zeitschr. f. physiol. Chem., 57, 61, 62. 
 1 Zeitschr. f. physiol. Chem., 8. 
 
IRON IN THE LIVER. 387 
 
 that the part of the protein molecule richer in nitrogen is first removed 
 and eliminated under these conditions. A similar condition has been 
 observed by Wells in the idiophatic, acute yellow atrophy of the liver. 
 In consideration of the variable results for the diamino-nitrogen even 
 under normal conditions (Glikin and A. Loewy 1 ), it is desirable that 
 a greater number of observations be made on this subject. The increased 
 consumption of glycogen under the above-mentioned pathological con- 
 ditions may also be considered as an increased autolysis, while the claim 
 of certain observers that fat is formed in the autolysis of the liver is, 
 according to Saxl, 2 to be considered only as a more pronounced appear- 
 ance of the fat previously occurring in the organ. 
 
 Besides the above-mentioned organic constituents in the liver we must 
 mention the glucothionic acid found by Mandel and Levene, 3 whose 
 chemical individuality is doubted. 
 
 The mineral bodies of the liver consist of phosphoric acid, potassium, 
 sodium, alkaline earths, and chlorine. The potassium is in excess of 
 rhe sodium. Iron is a regular constituent of the liver, but it occurs 
 in very variable amounts. Bunge found 0.01-0.355 p. m. iron in 
 the blood-free liver of young cats and dogs. This was calculated on the 
 liver substance freshly washed with a 1-per cent XaCl solution. Cal- 
 culated on 10 kilos bodily weight, the iron in the liver amounted to 3.4- 
 80.1 mg. Recent determinations of the quantity of iron in the liver of 
 the rabbit, dog, hedge-hog, pig, and man have been made by Guille- 
 monat and Lapicque, and in rabbits by Scaffidi. The variation was 
 great in human beings. In men the quantity of iron in the blood-free 
 liver (blood-pigment subtracted in the calculation) was regularly 0.23 
 p. m., and in women 0.09 p. m. (calculated on the fresh moist organ), 
 and this relation was not changed after the twentieth year. Above 
 0.5 p. m. is considered as pathological. According to Bielfeld, 4 who 
 worked with another method, an even greater quantity of iron occurs in 
 men. 
 
 The quantity of iron in the liver can be increased by drugs contain- 
 ing iron. The quantity of iron may also be increased by an abundant 
 destruction of red blood-corpuscles, which will result from the injection 
 
 1 Neuberg and Richter, Deutsch. rned. Wochenschr., 1904; Wakeman, Zeitschr. f. 
 physiol. Chem., 44; Wells, Journ. of Exper. Med., 9; Glikin and Loewy, Bioch. Zeitschr., 
 10. 
 
 2 Hofmeister's Beitrage, 10. 
 
 3 Mandel and Levene, Zeitschr. f. physiol. Chem., 45. 
 
 4 Bunge, Zeitschr. f. physiol. Chem., 17, 78; Guillemonat and Lapicque, Compt. 
 rend, de soc. bid., 48, with Bailie, ibid., 68; and Arch de Physiol. (5) 8; Biefeld, Hof- 
 meister's Beitrage, 2; see also Schmey, Zeitschr. f. physiol. Chem., 39; Scaffidi, ibid., 
 54. 
 
388 THE LIVER. 
 
 of dissolved haemoglobin, in which process the iron combinations derived 
 from the blood-pigments in other organs, such as the spleen and marrow, 
 also seem to take part. 1 A destruction of blood-pigments, with a splitting 
 off of compounds rich in iron, seems to take place in the liver in the for- 
 mation of the bile-pigments. Even in invertebrates, which have no 
 haemoglobin, the so-called liver is rich in iron, from which Dastre and 
 Floresco 2 conclude that the quantity of iron in the liver of inverte- 
 brates is entirely independent of the decomposition of the blood-pigment, 
 and in vertebrates it is in part so. According to these authors the liver 
 has, on account of the quantity of iron, a specially important oxidizing 
 function, which they call the " fonction martiale " of the liver. 
 
 The richness in iron of the liver of new-born animals is of special 
 interest — a condition which was shown by the analyses of St. Zaleski, 
 but was especially studied by Kruger and Meyer. In oxen and cows 
 they found 0.246-0.276 p. m. iron (calculated on the dry substance), 
 and in the cow-foetus about ten times as much. The liver-cells of a calf 
 a week old contain about seven times as much iron as the adult animal; 
 the quantity decreases in the first four weeks of life, when it reaches 
 about the same amount as in the adult. Lapicque 3 also found that in 
 rabbits the quantity of iron in the liver steadily diminishes from the 
 eighth day to three months after birth, namely, from 10 to 0.4 p. m., 
 calculated on the dry substance. " The fcetal liver-cells bring an abun- 
 dance of iron in the world to be used up, within a certain time, for a pur- 
 pose not well known." A part of the iron exists as phosphate, but the 
 greater part is in combination in the ferruginous protein bodies (St. 
 Zaleski). 
 
 The quantity of calcium oxide in the fresh, moist liver of the horse, 
 ox, and pig, according to Toyonaga, amounts to 0.148-0.193 p. m., or 
 more than the human liver (0.101 p. m. according to Magnus-Levy). 
 The amount of magnesium oxide was remarkably high, namely, 0.168, 
 0.198 and 0.158 p. m., in the livers of the horse, ox, and pig, respectively, 
 but considerably less than the human liver in which Magnus-Levy found 
 0.292 p. m. Kruger 4 found the quantity of calcium in the livers of 
 adult cattle and of calves to be respectively 0.71 p. m. and 1.23 p. m. 
 of the dried substance. In the foetus of the cow it is lower than in calves. 
 During pregnancy the iron and calcium in the foetus are antagonistic; 
 
 1 See Lapicque, Compt. Rend., 124, and Schurig, Arch. f. exp. Path. u. Pharm., 41. 
 
 2 Arcb.de Physiol. (5), 10. 
 
 4 St. Zaleski, 1. c; Kruger and collaborators, Zeitschr. f. Biologie, 27; Lapicque, 
 Maly'fi Jahresber., 20. 
 
 4 Kruger, Zeitschr. f. Biologie, 31; Toyonaga, Bull, of the College of Agriculture, 
 Tokio, 6; A. Magnus-Levy, Bioch. Zeitschr., 24. 
 
STORAGE OF PROTEIN IN THE LIVER. 389 
 
 that is, an increase in the quantity of calcium in the liver causes a diminu- 
 tion in the iron, and an increase in the iron causes a decrease in the calcium. 
 Copper seems to be a physiological constituent, and occurs to a considerable 
 extent in Cephalopods (Henze l ). Foreign metals, such as lead, zinc, 
 arsenic, and others (also iron), are easily taken up and combined by the 
 liver (Slowtzoff, v. Zeynek, and others 2 ). 
 
 v. Bibra 3 found in the liver of a young man who had suddenly died 
 762 p. m. water and 238 p. m. solids, consisting of 25 p. m. fat, 152 p. m. 
 protein, gelatin-forming and insoluble substances, and 61 p. m. extract- 
 ive substances. 
 
 Magnus-Levy 4 found in the liver of a healthy suicide 606 p. m. 
 water, 394 p. m. solids among which 212.8 p. m. fat occurred. If the 
 total nitrogen, 27 p. m., is calculated as protein the amount would be 
 approximately 169 p. m. 
 
 Profitlich 5 found 68.2-75.17 per cent water in the dog liver and 70.76- 
 72. S6 per cent in the ox liver. The relation N : C in the fat and glycogen-free 
 dried substance was 1:3.21 in dogs and 1:3.13 in oxen or about the same as in 
 flesh (see Chapter XI). 
 
 The quantitative composition of the liver may show great varia- 
 tion, depending upon the kind and amount of the food supplied. The 
 amount of carbohydrate (glycogen) and fat may vary considerably, 
 which is due to the fact that the liver is a storage-organ for these bodies, 
 especially for the glycogen. 
 
 Based upon special experiments, Seitz claims that the liver is a 
 storehouse for protein also. In experiments on hens and ducks which 
 had previously been starved, he found that the liver took up abundant 
 protein on feeding meat, and that its weight as compared with the weight 
 after starvation was doubled or quadrupled. As it is characteristic of 
 storage or reserve bodies that their amount in the storage-organs on 
 feeding with such bodies strongly increases in percentage, it is remarkable 
 in Seitz's feeding experiments that the percentage of protein in the liver 
 did not increase, but rather diminished slightly. In this case we did not 
 have a higher percentage of protein, but an increase in the weight of the 
 total cell mass of the organ, probably brought about by increased work 
 of the liver due to the protein feeding. The investigations of Grund 6 
 have shown that with protein feeding in dogs, the relation P:N in the 
 
 1 Zeitschr. f . physiol. Chem., 33. 
 
 2 Slo\vtzoff, Hofmeister's Beitrage, 1; v. Zeynek, see Centralbl. f. Physiol., 15. 
 
 3 See v. Gorup-Besanez, Lehrbuch d. physiol. Chem., 4. Aufl., p. 711. 
 
 4 Bioch. Zeitschr., 24. 
 
 5 Pfluger's Arch., 119. 
 
 6 Seitz, Pfluger's Arch., Ill; Grund, Zeitschr. f. Biol., 54. 
 
390 THE LIVER. 
 
 liver was not essentially changed which speaks against a simple storage 
 of food protein. 
 
 Glycogen and its Formation. 
 
 Glycogen was first discovered by Bernard. It is a carbohydrate 
 closely related to the starches or dextrins, with the general formula 
 m(C6Hio05). Its molecular weight is unknown, but seems to be very 
 large (Gatin-Gruzewska and v. Knaffl-Lenz 1 ). The largest quan- 
 tities are found in the liver of adult animals, and smaller quantities 
 in the muscles (Bernard, Nasse). It is found in very small quantities 
 in nearly all tissues of the animal body. Its occurrence in lymphoid 
 cells, blood, and pus has been mentioned in a previous chapter, and it 
 seems to be a regular constituent of all cells capable of development. 
 Glycogen was first shown to exist in embryonic tissues by Bernard 
 and Kuhne, and it seems on the whole to be a constituent of tissues 
 in which a rapid cell formation and cell development are taking place. 
 It is also present in rapidly forming pathological tumors (Hoppe-Seyler). 
 Some animals, as certain mussels (Bizio), Taenia and Ascaridae (Wein- 
 land 2 ), are very rich in glycogen. Glycogen also occurs in the vegetable- 
 kingdom, especially in many fungi. 
 
 The quantity of glycogen in the liver, as also in the muscles, depends 
 essentially upon the food. In starvation it disappears almost com- 
 pletely after a short time, but more rapidly in small than in large animals, 
 and it disappears earlier from the liver than from the muscles. As 
 shown by C. Voit, Kulz and especially by Pfluger, 3 it never entirely 
 disappears in starvation, as a re-formation of glycogen always takes 
 place. After partaking of food, especially such as is rich in carbo- 
 hydrates, the liver becomes rich again in glycogen, the greatest incre- 
 ment occurring 14 to 16 hours after eating (Kulz). The quantity of 
 liver-glycogen may amount to 120-160 p. m. after partaking of large 
 quantities of carbohydrates, and in dogs which had been especially 
 fed for glycogen Schondorff and Gatin-Gruzewska found still higher 
 results, even more than 180 p. m. Ordinarily it is considerably less, 
 namely, 12-30 to 40 p. m. The highest amount of glycogen in the liver 
 thus far observed was 201.6 p. m., found by Mangold 4 in the frog. The 
 
 1 Gatin-Gruzewska, Pfluger's Arch., 103; v. Knaffl-Lenz, Zeitschr. f. physiol. Chem., 
 4«. 
 
 2 Zeitschr. f. Biologie, 41. The extensive literature on glycogen may be found in 
 E. Pfliiger, Glykogen, 2. Aufl., Bonn, 1905; and in Cremer, " Physiol, des Glykogens," 
 in Ergebnisse der Physiologic, 1, Abt. 1. In the following pages we shall refer to these^ 
 works. 
 
 1 Pfluger's Arch., 119, which contains the literature. 
 *Itnd., 121 
 
GLYCOGEN. 391 
 
 shark, whose liver is very rich in fat, even though well nourished, only 
 has comparatively low values for the glycogen in the liver, 9.3-23.8 
 p. m. (Bottazzi J ). According to Cbemeb the quantity of glycogen in 
 plants (yeast-cells) is, as in animals, dependent upon the food. He 
 finds that the yeast-cells contain glycogen, which disappears from the 
 cells in the auto-fermentation of the yeast, but reappears on the intro- 
 duction of the cells into a sugar solution. 
 
 The quantity of glycogen of the liver (and also of the muscles) is 
 also dependent upon rest and activity, because during rest, as in hiberna- 
 tion, it increases, and during work it diminishes. Iyulz has shown that 
 by hard work the quantity of glycogen in the liver (of dogs) is reduced 
 to a minimum in a few hours. The muscle-glycogen does not diminish 
 to the same extent as the liver-glycogen. Kulz, Ztjntz and Vogelius, 
 Frentzel, and others have been able to render rabbits and frogs nearly 
 glycogen-free by suitable strychnine poisoning. The same result is pro- 
 duced by starvation followed by hard work. According to Gatin- 
 Gruzewska, 2 the liver and muscles in rabbits can be made glycogen- 
 free after 36-40 hours by first starving one day and then injecting 
 adrenalin. 
 
 Glycogen forms an amorphous, white, tasteless, and non-odorous powder. 
 When perfectly pure, and by proper alcohol precipitation, it can be obtained 
 as rods or prisms which look like crystals (Gatin-Gruzewska). It 
 gives an opalescent solution with water which, when allowed to evaporate 
 on the water-bath, forms a pellicle over the surface that disappears again 
 on cooling. It is undecided whether we here have a true solution or 
 not. Like other colloids, glycogen in water under the influence of the 
 electric current migrates to the anode, on which it collects (Gatin- 
 Gruzewska). According to Bottazzi, 3 who obtained the same results, 
 a little acid or a little alkali modify the results so that the glycogen becomes 
 isoelectric. Its aqueous solution is dextrorotatory, and Htjppert found 
 it to be (tt) D = +196.63°. Gatin-Grtjzewska has recently obtained 
 the same result by using a perfectly pure solution of glycogen. A 
 solution of glycogen, especially on the addition of NaCl, is colored wine- 
 red by iodine. It may hold cupric hydroxide in solution in alkaline 
 liquids, but does not reduce it. A solution of glycogen in water is not 
 precipitated by potassium-mercuric iodide and hydrochloric acid, but is 
 precipitated by alcohol (on the addition of NaCl when necessary), or 
 
 1 Arch. Ital. d. Biol., 48; cited in Bioch. Centralbl., 7, 833. 
 
 2 Compt. Rend., 142. 
 
 3 Bottazzi, Chem. Centralbl. 1009 p. 1423; Bottazzi and d'Errico (Pfluger's Arch., 
 115) have investigated the viscosity, the electrical conductivity and the freezing-point 
 of glycogen solutions at different concentrations. 
 
392 THE LIVER. 
 
 ammoniacal basic lead acetate. An aqueous solution of glycogen made 
 alkaline with caustic potash (15 per cent KOH) is completely precipitated 
 by an equal volume of 96 per cent alcohol. Tannic acid also precipitates 
 glycogen. It gives a Avhite granular precipitate of benzoyl-glycogen 
 with benzoyl chloride and caustic soda. Glycogen is completely pre- 
 cipitated by saturating its solution at ordinary temperatures with magne- 
 sium or ammonium sulphate. It is not precipitated by sodium chloride, 
 or by half saturation with ammonium sulphate (Nasse, Neumeister, 
 Halliburton, Young 1 ). On boiling with dilute caustic potash (1-2 
 per cent) the glycogen may be more or less changed, especially if it has 
 been previously exposed to the action of acid or to Brucke's reagent 
 (see below) (Pfluger). On boiling with stronger caustic potash (even 
 of 36 per cent) it is not injured (Pfluger). By diastatic enzymes 
 glycogen is converted into maltose or glucose, depending upon the nature 
 of the enzyme. It is transformed into glucose by dilute mineral acids. 
 According to Tebb 2 various dextrins appear as intermediary steps in 
 the saccharification of glycogen, depending on whether the hydrolysis 
 is caused by mineral acids or enzymes. The glycogen from various 
 animals and different organs is the same according to Pfluger. 3 Nor has 
 it been decided whether all the glycogen in the liver occurs as such or 
 whether it is in part combined with protein (Pfluger-Nerking). The 
 investigations of Loeschcke 4 have shown that we have no positive 
 reasons for this assumption. 
 
 The preparation of pure glycogen (most easily from the liver) is 
 generally performed by the method suggested by Brucke, of which the 
 main points are the following: Immediately after the death of the animal 
 the liver is thrown into boiling water, then finely divided and boiled 
 several times with fresh water. The filtered extract is now sufficiently 
 concentrated, allowed to cool, and the proteins removed by alternately 
 adding potassium-mercuric iodide and hydrochloric acid. The glycogen 
 is precipitated from the filtered liquid by the addition of alcohol until 
 the liquid contains 60 vols, per cent. By repeating this and precipitating 
 the glycogen several times from its alkaline and acetic-acid solution it 
 is purified on the filter by washing first with 60 per cent and then with 
 95 per cent alcohol, then treating with ether, and drying over sulphuric 
 acid. It is always contaminated with mineral substances. To be able 
 to extract the glycogen from the liver or, especially, from muscles and 
 other tissues completely, which is essential in a quantitative estimation, 
 these parts must first be warmed for two hours with strong caustic potash 
 (30 per cent) on the water-bath. As the glycogen changes in this purifica- 
 
 1 Young, Journ. of Physiol., 22, citing the other investigators. 
 
 2 Jonrri. of Phyeiol., 22. 
 
 * Pfluger's, Arch. 129. 
 
 * Ibid., 102. 
 
GLYCOGEN FORMERS. 393 
 
 tion, as suggested by BrxJcke, it is better, for quantitative determinations 
 of glycogen, to precipitate it directly from the alkaline solution by alcohol 
 (Pfluger 1 ). 
 
 The quantitative estimation is best performed according to Pfluger's 
 method, which is as follows: The finely divided organ is heated on 
 the water-bath for 2-3 hours in the presence of 30 per cent KOH; after 
 diluting with water and filtering, the glycogen is precipitated with 
 alcohol, and the redissolved glycogen estimated in part by the polar- 
 iscope and in part as sugar after inversion. One part by weight of sugar 
 equals 0.927 part glycogen. As in the estimation the prescribed direc- 
 tions must be exactly followed, we must refer to the original work of 
 Pfluger for the details of the method. Other methods of estimating 
 glycogen, such as those of Brucke-Kulz, Pavy, and Austin, are described 
 in Pfluger's Archiv. 96. Also compare the recent works of Pfluger. 2 
 
 Numerous investigators have endeavored to determine the origin 
 of glycogen in the animal body. It is positively established by the 
 unanimous observations of many investigators 3 that the varieties of 
 sugars and their anhydrides, dextrins and starches, have the property of 
 increasing the quantity of glycogen in the body. The action of inulin 
 seems to be somewhat uncertain. 4 The statements are questioned in 
 regard to the action of the pentoses. Cremer found that in rabbits and 
 hens various pentoses, such as rhamnose, xylose, and arabinose, have a 
 positive influence on the glycogen formation, and Salkowski obtained 
 the same result on feeding /-arabinose. Frentzel, on the contrary, 
 found no glycogen formation on feeding xylose to a rabbit which had 
 previously been made glycogen-free by strychnine poisoning, and Neu- 
 berg and Wohlgemuth 5 obtained similar negative results on feed- 
 ing rabbits with d- and r-arabinose. In general we can for the present 
 accept the view that the pentoses are not direct glycogen formers. 
 
 The hexoses, and the carbohydrates derived therefrom, do not all 
 possess the ability cf forming or accumulating glycogen to the same 
 extent. Thus C. Voir 6 and his pupils have shown that glucose has a 
 more powerful action than cane-sugar, while milk-sugar is less active 
 (in rabbits and hens) than glucose, fructose, cane-sugar, or maltose. 
 
 The following substances when introduced into the body also increase the 
 quantity of glycogen in the liver: Glycerin, gelatin, arbutin, and likewise, accord- 
 
 *See also the method suggested by Gautier, Compt. Rend., 129. 
 
 2 Pfluger's Arch., 103, 104, 121 and especially 129. 
 
 J In reference to the literature on this subject, see E. Kiilz, Pfluger's Arch., 24, 
 and Ludwig, Festschrift, 1891; also the cited works of Pfluger and Cremer, foot-note 
 2, p. 390. 
 
 * See Miura, Zeitschr. f. Biologie, 32, and Nakaseko, Amer. Journ. of Physiol., 4. 
 
 6 Salkowski, Zeitschr. f. physiol. Chem., 32; Neuberg and Wohlgemuth, ibid., 35. 
 See also Pfluger, 1. c, and Cremer, 1. c. 
 
 6 Zeitschr. f. Biologie, 28. 
 
394 THE LIVER. 
 
 ing to the investigations of Kulz, erythrite, quercite, dulcite, mannite, inosite, 
 ethylene and propylene glycol, glucuronic anhydride, saccharic acid, mucic acid, 
 sodium tartrate, saccharin, isosaccharin, and urea. Ammonium carbonate, gly- 
 cocoll; and asparagine may similarly, according to Rohmann, cause an increase 
 in the amount of glycogen in the liver. Nebelthau finds that other ammonium 
 salts and some of the amides, as well as certain narcotics, hypnotics, and antipyretics, 
 produce an increase in the glycogen of the liver. This action of the antipyretics 
 (especially antipyrine) had been shown by Lepine and Porteret. 1 
 
 The fats, according to Bouchard and Desgrez, increase the glycogen 
 content of the muscles but not of the liver, while Cotjvreur believes that 
 the glycogen is increased at the expense of the fat in the silkworm larva, 
 but these statements have been shown to be incorrect by the recent 
 investigations of Kotake and Sera. 2 In general it is believed that fat 
 does not increase the amount of glycogen in the liver or in the animal 
 body, although a carbohydrate formation from glycerin, but not a gly- 
 cogen formation, is probable. 
 
 The question whether the proteins have the ability to increase the 
 glycogen content of the liver or the animal body has been long disputed. 
 The feeding experiments with meat or with pure proteins by older exper- 
 imenters, such as Naunyn, v. Mering and E. Kulz seem to show an 
 ability. But the proof of these investigations has been strongly dis- 
 puted by Pfluger and later investigations of Schondorff, Blumenthal 
 and Wohlgemuth, as also those of Bendix and Stookey 3 yield contradic- 
 tory results. These investigations have really only historical interest, 
 since now a carbohydrate formation as well as a glycogen formation from 
 proteins have been positively observed. 
 
 If the question is raised as to the action of the various bodies on 
 the accumulation of glycogen in the liver, it must be recalled that a forma- 
 tion of glycogen takes place in this organ, as well as a consumption of the 
 same. An accumulation of glycogen may be caused by an increased 
 formation of glycogen, but also by a diminished consumption, or by both. 
 
 It is not known how the various bodies above mentioned act in this 
 regard. Certain of them probably have a retarding action on the trans- 
 formation of glycogen in the liver, while others perhaps are more com- 
 bustible, and in this way protect the glycogen. Some probably excite 
 the liver-cells to a more active glycogen formation, while others yield 
 material from which the glycogen is formed, and are glycogen-formers 
 
 1 Rohmann, Pfliiger's Arch., 39; Nebelthau, Zeitschr. f. Biologie, 28; Lepine and 
 Porteret, Compt. Rend., 107. 
 
 2 Bouchard et Desgrez, Compt. Rend., 130; Couvreur, Compt. rend, de soc. biol., 
 47; Kotake and Sera Zeitschr. f. physiol. Chem., 62. 
 
 Bee the work on glycogen by Pfluger and also Schondorff, Pfliiger's Arch., 82 and 88; 
 Blumenthal and Wohlgemuth, Berl. klin. Wochenschr., 1901; Bendix, Zeitschr. f. physiol. 
 Chem., 32 and 34; Stookey, Amer. Journ. of Physiol., 9. 
 
FORMATION OF GLYCOGEN. 395 
 
 in the strictest sense of the word. The knowledge of these last-mentioned 
 bodies is of the greatest importance in the question as to the origin of 
 glycogen in the animal body, and the chief interest attaches to the 
 question: To what extent are the two chief groups of food, the proteins 
 and carbohydrates, glycogen-formers? 
 
 The great importance of the carbohydrates in the formation of gly- 
 cogen has given rise to the opinion that the glycogen in the liver is pro- 
 duced from sugar by a synthesis in which water separates with the for- 
 mation of an anhydride (Luchsinger and others). This theory (anhy- 
 dride theory) has found opponents because it neither explains the forma- 
 tion of glycogen from such bodies as proteins, carbohydrates, gelatin, 
 and others, nor the circumstance that the glycogen is always the same, 
 independent of the properties of the carbohydrate introduced, whether 
 it is dextrogyrate or levogyrate. This last circumstance does not now 
 present any special difficulty, since we know that the simple sugars can 
 easily be transformed into each other. It was formerly the opinion of 
 many investigators that all glycogen is formed from protein, and that 
 this splits into two parts, one containing nitrogen and the other being 
 free from nitrogen; the latter is the glycogen. According to these 
 views, the carbohydrates act only in that they spare the protein and 
 the glycogen produced therefrom (sparing theory of Weiss, Wolffberg, 
 and others 1 ). 
 
 In opposition to this theory C. and E. Voit and their pupils have 
 shown that the carbohydrates are " true " glycogen-formers. After par- 
 taking of large quantities of carbohydrates, the amount of glycogen stored 
 up in the body is sometimes so great that it cannot be covered by the 
 protein? decomposed during the same time, and in these cases a gly- 
 cogen formation from the carbohydrates must be admitted. According 
 to Cremer only the fermentable sugars of the six carbon series or their 
 di- and polysaccharides are true glycogen-formers. For the present, only 
 glucose, fructose, and to a much less degree galactose (Weinland 2 ), 
 and perhaps also e?-mannose (Cremer) are designated as true glycogen- 
 formers. Other monosaccharides may indeed, according to Cremer, 
 influence the formation of glycogen, but they are not converted into 
 glycogen, and hence are called only pseudoglycogen-formers. 
 
 The poly- and disaccharides may, after a cleavage into the cor- 
 responding fermentable monosaccharides, serve as glycogen-formers. 
 This is true for at least cane-sugar and milk-sugar, which must first 
 
 1 In regard to these two theories, see especially Wolffberg, Zeitschr. f . Biologie, 16. 
 
 1 E. Voit, Zeitschr. f. Biologie, 25, 543, and C. Voit, ibid., 2S. See also Kausch 
 and Socin, Arch. f. exp. Path. u. Pharm., 31; Weinland, Zeitschr. f. Biologie, 40 and 
 38; Cremer, ibid., 42, and Ergebnisse der Physiol., 1. 
 
39G THE LIVER. 
 
 be inverted in the intestine. These two varieties of sugar, therefore, 
 cannot, like glucose and fructose, serve as glycogen-formers after sub- 
 cutaneous injection, but reappear almost entirely in the urine (Dastre, 
 Fr. Voit). Maltose, which is inverted by an enzyme present in the blood, 
 passes only to a slight extent into the urine (Dastre and Bourquelot 
 and others), and it can, like the monosaccharides, even after subcuta- 
 neous injection, be used in the formation of glycogen (Fr. Voit). 1 Of 
 the disaccharides the maltose and the cane-sugar are strong glycogen- 
 formers while milk-sugar has only a weak action. 
 
 The ability of the liver to form glycogen from monosaccharides has 
 also been shown by K. Grube in a very interesting and direct manner, 
 by perfusion experiments with solutions of various carbohydrates. In 
 these perfusion experiments on tortoise livers, glucose produced an 
 abundant glycogen formation, while with fructose and galactose it was 
 less abundant. Pentoses, disaccharides, casein and amino-acids (gly- 
 cocoll, alanine and leucine) were inactive while on the contrary glycerin 
 and also formaldehyde acted as glycogen-formers. The formation of 
 glycogen from formaldehyde is disputed by Schondorff and Grube. 2 
 
 After Pavy 3 first showed the occurrence of carbohydrate groups in 
 ovalbumin, other investigators were able to split off glucosamine from 
 this and other protein substances (see Chapter II), and the question 
 arose whether the amino-sugar could serve in the formation of glycogen. 
 The investigations carried out in this direction by Fabian, Frankel 
 and Offer, Cathcart and Bial, have shown that the glucosamine 
 introduced into the organism is in part eliminated unchanged in the 
 urine and has no glycogen-forming action. No definite conclusions 
 can be drawn from this on the behavior of the carbohydrate groups, 
 which exist not as free groups but combined with the protein molecules. 
 The investigations of Forschbach on the behavior of glucosamine 
 chained to an acid-group in an amide-like combination, as well as the 
 investigations of Kurt Meyer and Stolte, 4 have yielded no proofs for 
 the theory that glycogen is formed from glucosamine. 
 
 Whether or not, or to what extent, the glucoproteins by their glucosa- 
 mine component take part in the sugar or glycogen formation in the animal 
 
 'Dastre, Arch, de Physiol. (5) 3, 1891; Dastre and Bourquelot, Compt. Rend., 98; 
 Fritz Voit, Verhandl. d. Gesellsch. f. Morph. u. Physiol, in Miinchen, 1896, and Deutsch. 
 Arch. f. klin. Med., 58. In regard to the glycogen formation after intravenous injection 
 of sugar see Freund and Popper, Bioch. Zeitschr., 41. 
 
 2 Pfluger's Arch., 138; Grube, ibid., 118, 121, 122, 126 and 139. 
 
 3 The Physiology of the Carbohydrates, London, 1894. 
 
 * Fabian, Zeitschr. f. physiol. Chem., 27; Frankel and Offer, Centralbl. f. Physiol., 
 13; Cathcart, Zeitschr. f. physiol. Chem., 39; Bial, Berl. klin. Wochenschr., 1905; 
 Forschbach, Hofmeister's Beitrage, 8; Meyer, ibid., 9; Stolte, ibid., 11. 
 
FORMATION OF GLYCOGEN. 397 
 
 body is difficult to answer for the present, as but little is known of the 
 quantity of these substances in the body, and our knowledge of the amount 
 of carbohydrate which can be split off from the various protein substances 
 is also very meager. 
 
 If the proteins are to be counted, and this is in agreement with the 
 generally accepted view, among those bodies which increase the glycogen 
 of the body, then Ave must ask the question: Do the proteins act only 
 indirectly as pseudoglycogen-formers, or are they direct glycogen- 
 formers which can serve as material for the formation of glycogen or 
 sugar? This question stands in close relation to the sugar formation 
 and sugar elimination in the various forms of glycosuria, and will be best 
 discussed below in connection with the question of diabetes. 
 
 Glycogen is a reserve-food deposited, in the liver and which, like other 
 carbohydrates can be transformed into fat, and it is generally admitted 
 that such a fat formation from glycogen also takes place in the liver. 
 There is no doubt that the glycogen deposited in the liver is formed in 
 the liver-cells from the sugar; but where does the glycogen existing in 
 the other organs, such as the muscles, originate? Is the glycogen of the 
 muscles formed on the spot cr is it transmitted to the muscles by the blood? 
 These questions cannot at present be answered with certainty, and the 
 investigations on this subject by different experimenters have given 
 varying results. The experiments of Iyulz, 1 in which he studied the 
 glycogen formation by passing blood containing cane-sugar through 
 the muscle, have led to no conclusive results, while the perfusion exper- 
 iments of Hatcher and Wolff with glucose seem to indicate a glycogen 
 formation from sugar in the muscles. The investigations of de Filippi 2 
 on dogs with so-called Eck's fistula also show a glycogen formation from 
 sugar in the muscles. In the Eck fistula operation the portal vein is 
 ligated near the liver hilus and sewed to the inferior vena cava and an 
 opening established between the two veins so that the portal blood flows 
 directly into the vena cava without passing through the liver. In 
 well-nourished animals, operated upon in this manner, the livers had the 
 same properties as those from starving animals, while, on the contrary, 
 the muscles contained quantities of glycogen which corresponded to 
 those found in a normal over-fed dog. 
 
 If it be true that the blood and lymph contain a diastatic enzyme 
 which transforms glycogen into sugar, and also that the glycogen regularly 
 occurs in the form-elements and is not dissolved in the fluids, it seems 
 probable that the glycogen in solution is not transmitted by the blood to 
 
 1 See Minkowski and Laves, Arch. f. exp. Path. u. Pharra., 23; Kiilz, Zeitschr. f. 
 Biologie, 27; Hatcher and Wolff, Journ. of Biol. Chern., 3. 
 
 2 Zeitschr. f. Biol., 49 and 50. 
 
398 THE LIVER. 
 
 the organs, but perhaps more likely, if the leucocytes do not act as car- 
 riers, it is formed on the spot from the sugar. 1 The glycogen formation 
 seems to be a general function of the cells. In adults, the liver, which 
 is very rich in cells, has the property, on account of its anatomical posi- 
 tion, of transforming large quantities of sugar into glycogen. 
 
 This glycogen, which is deposited in the liver as reserve-food, in order 
 that it can be useful to the body, must at least in greater part be trans- 
 formed into sugar and supplied to the various organs by the blood. The 
 question now arises whether there is any foundation for the statement 
 that the liver glycogen is transformed into sugar. 
 
 As first shown by Bernard and redemonstrated by many inves- 
 tigators, the glycogen in a dead liver is gradually changed into sugar, 
 and this sugar formation is caused, as Bernard supposed and then shown 
 by numerous investigators by a diastatic enzyme whose relation to the 
 diastatic enzyme of the blood is not quite clear. 2 
 
 This post-mortem sugar formation led Bernard to the assump- 
 tion of the formation of sugar from glycogen in the liver during life. 
 Bernard suggested the following arguments for this theory: The liver 
 always contains some sugar under physiological conditions, and the 
 blood from the hepatic vein is always somewhat richer in sugar than the 
 blood from the portal vein. Bernard's views found in Seegen an active 
 supporter, as he tried to show by numerous experiments the physio- 
 logical sugar content of the liver as well as the high sugar content of the 
 blood of the liver veins. On the other hand the correctness of the 
 observations of Bernard and Seegen is disputed by many investigators 
 such as Pavy, Ritter, Schiff, Eulenberg, Lussana, Mosse, N. Zuntz 
 and others, 3 and in regard to the sugar content in the two kinds of 
 blood we have come to the general conclusion that when only the stasis 
 and other disturbing influences of the operation are prevented, the blood 
 of the liver veins, if at all, is only slightly richer in sugar than the blood 
 of the portal vein. 4 
 
 The circumstance that the blood-sugar rapidly sinks to |-f of its 
 original quantity, or even disappears when the liver is cut out of the 
 circulation, indicates a vital formation of sugar in the liver (Seegen, 
 Bock and Hoffmann, Kaufmann, Pavy and others). In geese whose 
 
 1 See Dastre, Compt. rend, de soc. biol., 47, 280, and Kaufmann, ibid., 316. 
 2 R6hmann, Verh. d. Ges. deutsch. Naturf. u. Aerzte. Breslau, 1903; Borchardt, 
 Pfluger's Arch., 100; Zegla, Bioch. Zeitschr., 16; E. Starkenstein, ibid., 24. 
 
 3 In regard to the literature on sugar formation in the liver see Bernard, Lecons sur 
 le diabete, Paris, 1877; Seegen, Die Zuckerbildung im Tierkorper, 2. Aufl. Berlin, 
 1900; M. Bial, Pfluger's Arch., 55, 434. 
 
 4 Seegen, Die Zuckerbildung, etc., and Centralbl. f. Physiol., 10, 497 and 822; 
 Zuntz, ibid., 561; Mosse, Pfluger's Arch., 63; Bing, Skand. Arch. f. Phy.siol., 9. 
 
SUGAR FORMATION FROM GLYCOGEN. 39$ 
 
 livers wire removed from the circulation, Minkowski found no sugar 
 in the blood after a few hours. On removing the liver from the 
 circulation by tying all the vessels to and from the organ, the quan- 
 tity of sugar in the blood is not increased (Schenck 1 ). An important 
 proof of the possibility of a vital formation of sugar from the liver gly- 
 cogen lies in the fact that we shall learn below of certain poisons and 
 operative changes which may cause an abundant elimination of sugar, 
 but only when the liver contains glycogen. 
 
 A vital formation of sugar from the liver glycogen is now generally 
 accepted. Most investigators consider this as an enzymotic transforma- 
 tion of the glycogen by means of the liver diastase, while certain inves- 
 tigators such as Dastre, Noel-Paton, E. Cavazzani, McGuigan and 
 Brooks 2 and others explain it by a special activity of the protoplasm. 
 Bang 3 has studied the formation of sugar in frogs' livers, which had not 
 appreciably changed in weight in a Ringer's solution which was isotonic 
 with the frog blood and which correspondingly had retained their vital 
 properties. This sugar formation does not depend upon a protoplasmic 
 activity but is of an enzymotic nature. It is caused by a diastase^ 
 which in Rana esculenta occur in great part in a latent, inactive form 
 due to the inhibitory action of the liver lipoids. Common salt is espe- 
 cially important as an activator for this enzyme. The surviving frog 
 liver is stimulated to a strong sugar production by adrenalin, and this 
 sugar formation is also of an enzymotic nature. The action of the 
 adrenalin consists in an activation of the liver diastase, brought about 
 in various ways. 
 
 The relation of the sugar eliminated in the urine under certain 
 conditions, such as in diabetes mellitus, certain intoxications, lesions 
 of the nervous system, etc., to the glycogen of the liver is also an important 
 question. 
 
 It does not enter into the plan and scope of this book to discuss in 
 detail the various views in regard to glycosuria and diabetes. The 
 appearance of glucose in the urine is a symptom which may have essen- 
 tially different causes, depending upon different circumstances. Only 
 a few of the most important points will be mentioned. 
 
 The blood always contains about the average of 1 p. m., while the 
 urine has in it at most only traces of glucose. When the quantity of 
 
 'Seegen, Bock, and Hoffmann, see Seegen, 1. c; Kaufmann, Arch, de Physiol. (5), 
 8; Tangl and Harley, Pfluger's Arch., 61; Pavy, Journ. of Physiol., 29, Minkowski, 
 Arch. f. exp. Path. u. Pharm., 21; Schenck, Pfluger's Arch., 57. 
 
 2 See Dastre, Noel-Paton, Cavazzani and their work cited in Pick, Hofmeister's- 
 Beitrage, 3, and McGuigan and Brooks, Amer. Journ. of Physiol., 18; R. G. Pearce,. 
 ibid., 25. 
 
 8 Bioch. Zeitschr., 49. 
 
400 THE LIVER. 
 
 sugar in the blood rises above this average, sugar passes into the urine, 
 sometimes even with slight rise and in other cases with stronger rise. 
 The kidneys have the property to a certain extent of preventing the 
 passage of blood-sugar into the urine; and it follows from this that an 
 elimination of sugar in the urine may be caused partly by a reduction 
 or suppression of this above-mentioned activity, and partly also by an 
 abnormal increase of the quantity of sugar in the blood. 
 
 The first seems, according to v. Mering and Minkowski, and others 
 to be the case in phlorhizin diabetes, v. Mering found that a strong 
 glycosuria appears in man and animals on the administration of the 
 glucoside phlorhizin. The sugar eliminated is not derived from the 
 glucoside alone. It is formed in the animal body, and in fact from the 
 carbohydrates, or as generally admitted on prolonged starvation, from 
 the protein substances of the body (Lusk). The quantity of sugar in 
 the blood is not increased, but rather diminished, in phlorhizin diabetes 
 (Minkowski), which does not indicate increase in the sugar production 
 but rather an increased excretion of the sugar by the kidneys. The 
 fact that after extirpation of the kidney in phlorhizin diabetes no rise 
 in the blood-sugar is observed, and that after the injection of phlorhizin 
 in the renal artery of one side the urine secreted by this kidney contains 
 sugar sooner and more abundantly than the urine from the other kidney 
 (Ztjntz), tends to favor this view. The experiments especially performed 
 by Pavt, Brodie, and Siau upon blood containing phlorhizin and sur- 
 viving kidneys also indicate the same, namely, that the phlorhizin acts 
 upon the kidneys and the researches of Erlandsen also lead to the same 
 conclusion. He found that on combining the phlorhizin action with 
 bleeding that the glycosuria was increased while after bleeding alone 
 without phlorhizin poisoning the hyperglycemia was absent. While 
 v. Mering and others believe in an increased permeability of the kidneys 
 for sugar, produced by the phlorhizin Lepine * is of the view that the 
 phlorhizin causes a formation of glucose from the virtual sugar in the 
 kidneys. Pavy is, on the contrary, of the opinion that the kidneys, 
 under the influence of the phlorhizin, split off sugar from a substance 
 
 1 In regard to the literature on phlorhizin diabetes see v. Mering, Zeitschr. f. klin. 
 Med., 14 and 16; Minkowski, Arch. f. exp. Path. u. Pharm., 31; Moritz and Prausnitz, 
 Zeitschr. f. Biologie, 27 and 29; Kiilz and Wright, ibid., 27, 181; Cremer and Rioter, 
 ihvL, 28 and 29; Contejean, Compt. rend, de soc. biol., 48; Lusk, Zeitschr. f. Biologie, 
 36 and 42; Levene, Journal of Physiol., 17; Pavy, ibid., 20, and with Brodie and Siau, 
 29; Arteaga, Amer. Journ. of Physiol., 6; O. Loewi, Arch. f. exp. Path. u. Pharm., 47; 
 X. Zuntz, Arch. f. (Anat. u.) Physiol., 189.5; Stiles and Lusk, Amer. Journ. of Physiol., 
 10: Lusk, ibid., 22; Cremer, Ergebnisse der Physiol., 1, Abt. 1; Erlandsen, Bioch. 
 Zeitschr., 23 and 24; I.epine, Compt. rend. soc. biol., 68; Lusk, Ergebnisse der Physiol., 
 Bd. 12, 315-392, and the monographs upon diabetes. 
 
GLYCOSURIAS. 401 
 
 •circulating in the blood, perhaps from a protein with loosely combined 
 •carbohydrate groups. 
 
 Grube from experiments upon the surviving tortoise liver has made the 
 suggestion that it is not the kidneys which are first attacked by the phlorhizin 
 action in phlorhizin glycosuria but the liver. Important experimental evidence 
 against this view has been raised by Schondorff and Suckrow. 1 
 
 Another form of glycosuria which according to certain investigators 
 is to be connected with a changed permeability of the kidneys (Under- 
 hill and Closson) is the glycosuria first observed by Bock and Hoff- 
 mann after the intravascular injection of large quantities of a 1-per 
 cent salt solution, which is also of great interest because, as shown by 
 Martin Fischer, 2 it can be again arrested by an injection of a salt solu- 
 tion containing CaCUj. There are investigators who attempt to connect 
 this glycosuria with the adrenals and a hyperglycemia. 
 
 With the exception of these two forms of glycosuria, the phlorhizin 
 diabetes and the salt-glycosuria, and also the glycosuria produced by 
 certain kidney poisons, all other forms of glycosuria or diabetes, as far 
 as known at present, depend on a hyperglycemia. 
 
 A hyperglycemia may be caused in various ways. It may be caused, 
 for example, by the introduction of more sugar than the body can destroy. 
 
 The ability of the animal body to assimilate the different varieties 
 of sugar has naturally a limit. If too much sugar is introduced into the 
 intestinal tract at one time, so that the so-called assimilation limit 
 (see Chapter VIII, on absorption) is overreached, then the excess of 
 absorbed sugar passes into the urine. This form of glycosuria is called 
 alimentary glycosuria, 3 and is caused by the passage of more sugar into 
 the blood than the liver and other organs can destroy. 
 
 As the liver cannot transform into glycogen all the sugar which comes 
 to it in these, to a certain extent physiological, alimentary glycosurias, 
 it is possible that a glycosuria may also be produced under pathological 
 conditions, even by a moderate amount of carbohydrate (100 grams 
 glucose), which a healthy person could overcome. This is true, among 
 other cases, in various affections of the cerebral system and in certain 
 chronic poisonings. Certain observers include the lighter forms of 
 
 1 Grube, Pfltiger's Arch., 128; Schondorff and Suckrow, ibid., 138. See also the 
 opposed view of Underhill, Journ. of biol. Chem., 13. 
 
 2 Bock and Hoffmann, Arch. f. (Anat. u.) Physiol., 1871; M. Fischer, University 
 of California publications Physiol., 1903 and 1904, and Pfliiger's Arch., 106 and 109; 
 Underhill and Closson, Amer. Journ. of Physiol., 15, and Journ. of Biol. Chem., 4. 
 
 3 In regard to alimentary glycosuria see Moritz, Arch. f. klin. Med., 46, which also 
 contains the earlier literature; B. Rosenberg, Ueber das Vorkommen der alimentaren 
 Glykosurie, etc. (Inaug.-Dissert. Berlin, 1897); van Oondt, Munch, med. Wochen- 
 schr., 1898; v. Noorden, Die Zuckerkrankheit, 3. Aufl., 1901. 
 
402 THE LIVER. 
 
 diabetes, where the sugar disappears from the urine when the carbohy- 
 drates are cut off as much as possible from the food, in this class of gly- 
 cosuria. 
 
 A hyperglycemia which passes into a glycosuria may also be brought 
 about by an excessive or sudden formation of sugar from the glycogen 
 and other substances within the animal body. 
 
 To this group of glycosurias belongs, it seems, the adrenalin glycosuria, 
 in which an increased mobilization of the carbohydrate occurs, espe- 
 cially the liver glycogen. Several circumstances indicate this origin 
 of the sugar. Thus, after adrenalin injection the glycogen disappears 
 from the liver and, according to Michaud, 1 adrenalin is without action 
 in dogs with Eck fistula. The activity of the adrenalin in starving 
 animals whose livers are very poor in glycogen speaks for the possibility 
 that the sugar also may in part have another origin than that from the 
 liver glycogen. 
 
 Adrenalin glycosuria takes, to a certain degree, a central position and 
 as such a glycosuria we consider also several other forms of gl}'cosuria 
 caused by hyperglycemia. This is for example the case with the gly- 
 cosuria after Bernard's sugar puncture or piqUre. That the glycosuria, 
 produced after piqure is due to an increased transformation of the gly- 
 cogen, follows from the fact that no glycosuria appears, under the above- 
 mentioned circumstances, when the liver has been previously made free 
 from glycogen by starvation or other means. The close relation of 
 this form of hyperglycemia and glycosuria to the adrenals follows from 
 the fact that the sugar puncture is without action after the extirpation 
 of the two adrenals. In rats, Schwarz found, after such a double extirpa- 
 tion of the adrenals, that the liver was glycogen free and he considers 
 this lack of glycogen as the cause for the inaction of the piqure under 
 these conditions. According to Kahn and Starkenstein 2 the conditions 
 must be different, as they found in rabbits who remained alive a year 
 after the total extirpation of the adrenals, that the liver had a normal 
 amount of glycogen and that the sugar puncture nevertheless was with- 
 out action. Adrenalin caused glycosuria in such animals. 
 
 It is generally admitted that the stimulation which the sugar center 
 in the fourth ventricle exerts, through the sympathetic nerve reaches to 
 the adrenals and causes a secretion of adrenalin, which increases the sugar 
 formation. Certain circumstances, for example, that a glycosuria can be 
 brought about in starving animals, in which the piqure is without action, 
 by adrenalin, make the mechanism of this glycosuria somewhat uncer- 
 
 1 Verhandl. d. deutsch. Kongr. f. inn. Med. Wiesbaden, 1911. 
 * Schwarz, Pfliiger's Arch., 134; Kahn and Starkenstein, ibid., 139; Kahn, ibid. T 
 140; Starkenstein, Arch. f. exp. Path. u. Therap., 10. 
 
GLYCOSURIAS. 403 
 
 tain. Under all circumstances the sugar puncture glycosuria stands in 
 close relation to the adrenals and is generally considered as an adrenalin- 
 glycosuria. The same is true for the glycosuria after splanchnic stimula- 
 tion and probably for several other forms of glycosuria. In the gly- 
 cosuria produced by stimulation of the central vagus, according to 
 Bang, Ljungdahl and Bohm, 1 the hyperglycemia (in rabbits) depends 
 upon an increased destruction of the glycogen of the muscles and not of 
 the liver. 
 
 Many investigators consider the glycosuria appearing after the occur- 
 rence of dyspncc, 2 produced in various ways, and also after certain poisons 
 such as carbon monoxide, curare, ether, chloroform, strychnine, morphine, 
 piperidin and others as adrenalin glycosurias. That also in many of 
 such cases the glycosuria is brought about by an increased glycogen 
 destruction is not doubted. In certain cases, as in carbon monoxide 
 poisoning, a formation of sugar has been claimed from protein, because 
 Straub and Rosenstein 3 found that this glycosuria only occurred in 
 those animals that had a sufficient quantity of protein at their disposal. 
 Protein starvation and simultaneous abundant carbohydrate supply cause a 
 disappearance of this glycosuria. 
 
 A hyperglycemia and glycosuria may also be caused by a decreased 
 ability of the animal to consume or to utilize the sugar or to transform 
 it into glycogen. In this case the sugar must accumulate in the blood, 
 and the formation of severe cases of diabetes mellitus is now generally 
 explained by this process. 
 
 The inability of diabetics to destroy .or consume the sugar does not 
 seem to be connected with any decrease in the oxidative energy of the 
 cells. The oxidative processes are not generally diminished in diabetes 
 (Schultzen, Nencki and Sieber), and this has recently been sub- 
 stantiated by Baumgarten. 4 This latter investigator made experiments 
 with several bodies which on account of their aldehyde nature were 
 closely related to sugar or were cleavage or oxidation products of it, 
 namely, glucuronic acid, d-gluconic acid, d-saccharic acid, glucosamine, 
 
 1 Hofmeister's Bietrage, 10. 
 
 2 On the importance of the oxygen and the carbon dioxide content of the blood 
 for the non-appearance or appearance of glycosuria see Underhill, Journ. of biol. Chem., 
 1; Penzoldt and Fleischer, Virchow's Arch., 87; Sauer, Pfluger's Arch., 49, 425, 426; 
 Macleod, Amer. Journ. of Physiol., 19, with Briggs, Cleveland Med. Journ., 1907; 
 Eddie, Bioch. Journ., 1, with Moore and Roaf, ibid., 5; Henderson and Underhill, 
 Amer. Journ. of Physiol., 28. 
 
 3 Straub, Arch. f. exp. Path. u. Pharm., 38; Rosenstein, ibid., 40. 
 
 4 Schultzen, Berl. klin. Wochenschr., 1872; Nencki and Sieber, Journ. f. prakt. 
 Chem. (N. F.), 26, 35; Baumgarten, " Ein Beitrag zur Zenntniss des Diabetes mel- 
 litus," Habilitationschrift, also Zeitschr. f. exp. Path. u. Therap., 2, 1905. 
 
404 THE LIVER. 
 
 mucic acid, and others, and he found that diabetics destroyed or burned 
 these bodies to the same extent as healthy individuals. Besides this 
 it must be remarked that the two varieties of sugar, glucose and fructose, 
 which are oxidized with the same readiness, act differently in diabetics. 
 According to Rulz and other investigators fructose is, contrary to 
 glucose, utilized to a great extent in the organism, but this in man is, 
 not always the case or at least to a less extent than in certain animals. 
 In animals with pancreas diabetes (see below) fructose x may cause 
 a deposition of glycogen in the liver while with glucose this does not 
 occur. The combustion of protein and fat takes place as in healthy 
 subjects, and the fat is completely burned into carbon dioxide and water. 
 In this diabetes the ability of the cells to utilize the glucose suffers diminu- 
 tion, and the explanation of this has been sought in the fact that the glu- 
 cose is not previously split before combustion. 
 
 , ■ c °2 
 The variation in the respiratory quotient, i.e., the relation — — , seems 
 
 to show an insufficiency of the glucose combustion in the tissues in 
 diabetes. As will be thoroughly explained in a subsequent chapter, this 
 quotient is greater the more carbohydrates are burned in the body, and 
 it is correspondingly smaller when protein and fat are chiefly burned. 
 The investigations of Leo, Hanriot, Weintraud and Laves, 2 and 
 others have shown that in severe cases of diabetes, in the starving con- 
 dition, the low quotient is not raised after partaking of glucose, as in 
 healthy individuals, but that it is raised after feeding fructose, which is 
 also of value to diabetics. 
 
 The poverty of the organs and tissues of diabetics in glycogen indicates 
 that the glycogen in them is more abundantly transformed into sugar. 
 From what has been said above in regard to the different behavior of 
 fructose and glucose in the glycogen formation in diabetes, indicates that 
 in diabetes, also an inability of the body to transform glucose into glycogen 
 exists and that the lack of glycogen may come about in this way. 
 
 Indeed it has been suggested that a preliminary transformation of 
 glucose into glycogen is necessary before it can be burned in the animal body. 
 This assumption is without foundation, at least for the glycogen for- 
 mation in the liver, as the animal body as is shown with experiments 
 on dogs, can assimilate and burn considerable quantities of carbohy- 
 drates even after the liver is excluded (Wehrle, Verz ar 3 ) . The admitted 
 
 1 Kiilz, Beitrage zur Path. u. Therap. des Diabetes mellitus (Marburg, 1874), 1; 
 Weintraud and Laves, Zeitsehr. f. physiol. Chem., 19; Haycraft, ibid.; Minkowski, 
 Arch. f. exp. Path. u. Pharm., 31. 
 
 2 See v. Noorden, Die Zuckerkrankheit, 3. Aufl., 1901. 
 
 3 Wehrle, Bioch. Zeitsehr., 34; Verzar, ibid., 34. 
 
PANCREAS DIABETES. 405 
 
 ability of the liver in diabetes to use fructose and not glucose in the 
 formation of glycogen is, according to E. Neubauer, 1 not characteristic 
 for diabetes, because it also occurs in phosphorus poisoning. Whether 
 the different behavior of the two kinds of sugar actually depends upon 
 a diminished ability of the liver in diabetes to form glycogen from glucose 
 or to another unknown circumstance has not been sufficiently proved. 
 In experiments on tortoise livers, by perfusion of Ringer's solution con- 
 taining sugar, Nishi 2 found that the livers of diabetic animals formed 
 as much glycogen as the livers of normal animals. These results, which 
 cannot be applied to other animals, require at least further investiga- 
 tion. 
 
 The relation of the pancreas to diabetic glycosuria is of the greatest 
 importance for its proper understanding. 
 
 The investigations of Minkowski, v. Mering, Dominicis, and later 
 of many other investigators, 3 show that a true diabetes of a severe 
 kind is caused by the total or almost total extirpation of the pancreas 
 of many animals, especially dogs. As in man in severe forms of diabetes, 
 so also in dogs with pancreatic diabetes, an abundant elimination of 
 sugar takes place even on the complete exclusion of carbohydrates from 
 the food. 
 
 Artificial pancreas diabetes may indeed also in other respects present 
 the same picture as diabetes in man, but there exist important differences 
 between these two. 4 It is generally accepted that in pancreas diabetes 
 a diminished consumption exists, i.e., diminished utilization, which 
 does not exclude an increased sugar formation from other bodies not 
 carbohydrates. 
 
 Many important observations show that a close relation exists between 
 the liver and pancreas diabetes. Pfluger has also especially shown 
 that in diabetes produced by Sandmeyer's method (partial extirpa- 
 tion with subsequent destruction of the remains of the gland in the abdom- 
 inal cavity, when the animal remains alive for a longer time than after 
 total extirpation) the liver does not lose weight, although the total weight 
 of the animal diminishes greatly, while in starvation without diabetes 
 
 1 Arch. f. exp. Path. u. Pharm., 61. 
 
 2 Ibid., 62. 
 
 3 See Minkowski, Untersuchungen liber Diabetes mellitus nach Exstirpation des 
 Pankreas (Leipzig, 1S93); v. Noorden, Die Zuckerkrankheit (Berlin, 1901), which 
 contains a very complete index of the literature. In regard to diabetes see also CI. 
 Bernard, I.econs sur le diabete (Paris), Seegen, Die Zuckerbildung im Thierkorper 
 (Berlin, 1890), and Pfluger, Des Glykogen, 2. Aufl., 1905, and especially v. Xoorden's 
 Hanb. d. Pathol, des Stoffwechsels, 2. Aufl., 1907, Bd. 2, Chapter I. 
 
 4 See Falta " Ueber den Eiweissumsatz beim Diabetes mellitus." Berl. klin. Woch- 
 enschr., 1908, and Zeitschr. f. klin. Med., 66; Gigon, Deutsch. Arch. f. klin. Med., 97. 
 
406 THE LIVER. 
 
 the liver loses weight more than the other parts of the body. Pfluger 
 concludes from this that the liver in diabetes works actively, and is the 
 most important seat of production of diabetic sugar. 
 
 Pfluger has found that in frogs the total extirpation of the duodenum 
 causes a strong and continuous glycosuria and based upon his investigations 
 and those of other investigators, he believes that a certain relation exists between 
 the duodenum and pancreas diabetes. The question as to the occurrence of a 
 duodenal diabetes has been the subject of numerous investigations but the works 
 -of Ehrmann, Minkowski and Rosenberg x show that such a view is untenable. 
 
 There does not seem to be any doubt as to the existence of a certain 
 relationship between the pancreas to the adrenals and adrenalin gly- 
 cosuria. The glycosuric action of adrenalin could be prevented by 
 Zuelzer by the injection of pancreas extracts, and this statement is 
 confirmed by Frugoni by experiments with pancreatic juice or pancreatic 
 extracts, v. Furth and Schwarz 2 have confirmed the correctness of 
 .Zuelzer's statement but dispute the fact that we are here dealing with 
 an antagonistic hormone action as they have obtained similar results also 
 with other bodies, for example with turpentine. 
 
 Very stimulating views on the relationship of pancreas diabetes 
 to the adrenals and the thyroids have been given by. Falta, Eppinger 
 and Rudinger. 3 According to these investigators a reciprocal retarda- 
 tion exists between the pancreas and thyroid as between the pancreas 
 and the adrenals while a mutual accelerating action exists between the 
 thyroids and the adrenals. Tn depancreatized dogs the retarding action 
 of the pancreas upon the thyroids is removed, and in this way we explain 
 the strong increase in the protein, fat (Mohr) and salt-metabolism 
 (Falta and Whitney 4 ) observed in pancreas diabetes. By the removal 
 of the retarding action of the pancreas upon the adrenals, the mobiliza- 
 tion of the carbohydrates by means of the adrenalin is increased, and 
 herein, as well as the diminished sugar utilization, lies the reason for the 
 strong elimination of sugar. The relations between the above three 
 glands is still further described by the above-mentioned authors, but we 
 cannot enter more into detail in regard to the interesting question, 
 which requires further study. 
 
 The conditions in pancreas diabetes are certainly very complicated, 
 and the reasons for this are still very uncertain. Most investigators are of 
 
 1 Rosenberg, Bioch. Zeitschr., 18, which contains the literature. 
 
 2 Frugoni, Berl. klin. Wochenschr., 45, 1908; v. Furth and Schwarz, Bioch. Zeitschr., 
 31. 
 
 3 Eppinger, Falta and Rudinger, Zeitschr. f. klin. Med., 66, which also contains the 
 literature on adrenalin diabetes. 
 
 4 Mohr, Zeitschr. f. exp. Path. u. Therap., 4; Falta and Whitney, Hofmeister's 
 Beitrage, 11. 
 
PANCREAS DIABETES. GLYCOLYSIS. 407 
 
 the opinion that we arc here dealing with the abolition of one or more 
 bodies which are considered as products of the internal secretion of the 
 glands (hormones according to Starling) and which in an unknown 
 manner regulate the sugar destruction or carbohydrate metabolism. 
 
 The assumption of an internal secretion is based on the investiga- 
 tions of Minkowski, H£don, Lanceraux, Thiroloix, and others l 
 upon the action of the subcutaneous transplantation of the gland. 
 According to these investigations a subcutaneously transplanted piece 
 of the gland can completely perform the functions of the pancreas as 
 to the sugar exchange and the sugar elimination, because on the removal 
 of the intra-abdominal piece of gland, the animal in this case does not 
 become diabetic, but if the subcutaneously embedded piece of pancreas 
 is subsequently removed, an active elimination of sugar appears immedi- 
 ately. As this occurs also on completely cutting off the nerve supply, 
 it is explained by the assumption of a formation of a special product 
 in the gland, which passes into the blood; on the other hand Zuelzer, 
 Dohm and Marxer 2 have made preparations from the pancreas which, 
 in dogs as well as in man, cause a diminution in the elimination of sugar 
 (and acetone bodies) in diabetes and an improvement in the general con- 
 dition. 
 
 This internal secretion of the pancreas has in recent times been sup- 
 posed to be connected with the so-called islands of Langerhans; but no 
 positive results have been obtained in this connection. Nor are we 
 acquainted with the kind of active substance here formed. 
 
 The glycolytic property of the blood as shown by Lupine was con- 
 sidered for a time to be due to a glycolytic enzyme formed in the pancreas, 
 and pancreas diabetes used to be explained by the fact that the action 
 of this enzyme was removed when the gland was extirpated. This 
 glycolysis is net sufficient, even if it is derived from the pancreas, to 
 explain the transformation of the large quantity of sugar in the body, 
 and for the destruction of sugar we are also obliged to accept a glycolysis 
 in the organs and tissues. Opinions in regard to this glycolysis differ 
 in certain points. According to one view (Siitzer and others) special 
 oxidases are active in the glycolysis, while another (Stoklasa 3 ) con- 
 considers the glycolysis as analogous to alcoholic fermentation, where we 
 have processes brought on by special tissue zymases, in which lactic 
 
 1 See Minkowski, Arch. f. exp. Path. u. Pharm., 31; Hedon, Diabete pancreatique, 
 Travaux de Physiologic (Laboratoire de Montpellier, 1898), and the works on diabetes. 
 
 2 Deutsch. med. Wochenschr., 1908. 
 
 3 Hofmeisters Beitrage, 3, Centralbl. f. Physiol., 16, 17, 18; Ber. d. d. chem. Ge- 
 sellsch., 38; also with Czerny, ibid., 36; with Jelinek, Simacgk and Vitek, Pfluger's 
 Arch., 101. 
 
408 THE LIVER. 
 
 acid is an intermediary step. Many x objections have been advanced 
 against the view of Stoklasa that in animal as well as in plant tissues, 
 in anaerobic respiration, an alcoholic fermentation may occur as this 
 observed action of the tissues could only be brought about by the presence 
 of micro-organisms. 
 
 That lactic acid can be an intermediary step in the destruction of 
 sugar in the animal body cannot be denied. On the contrary it follows 
 from several circumstances which will be mentioned in Chapter 
 X. (muscle) on the origin of lactic acid that such a condition exists 
 and the following observations of A. R. Mandel and Lusk 2 on the 
 relation of lactic acid to diabetes indicate the same. These exper- 
 imenters showed after phosphorus poisoning in dogs, that the blood and 
 urine contained abundance of lactic acid, and on producing phlorhizin- 
 diabetes it disappeared from these fluids, and also that phosphorus poison- 
 ing does not cause a lactic acid formation in dogs with phlorhizin- 
 diabetes. Although it is difficult to give a satisfactory interpretation 
 of these observations, it is still very probable that in the elimination 
 of the sugar in phlorhizin-diabetes a mother-substance of the lactic acid 
 is lost. 
 
 We do not agree as to the ways and means which bring about 
 the so-called glycolysis, and another disputed question is whether the 
 glycolysis can be produced by one organ or only by the combined action 
 of several organs. Cohnheim 3 found that a cell-free fluid can be obtained 
 from a mixture of pancreas and muscle, which destroys glucose, while 
 the pancreas alone does not have this action, and the muscle only to a 
 slight extent. The pancreas does not contain, according to Cohnheim, 
 a glycolytic enzyme, but a substance resistant to boiling temperatures, 
 which is soluble in water and alcohol, and which, like an amboceptor, 
 activates a glycolytic proenzyme which exists in the muscle fluid, but 
 which is inactive alone and which retards glycolysis when it exists in 
 excess. 
 
 The statements of Cohnheim have been disputed, and recently Levene 
 and Meyer 4 have shown that we are not here dealing with a disap- 
 pearance of glucose by glycolysis, but more likely with a disappearance 
 
 1 See the works of O. Cohnheim, Zeitschr. f. physiol. Chem., 39, 42, 43; Batelli, 
 Compt, rend., 137; Portier, Compt. rend. soc. biol., 57; Harden and Maclean, Journ. 
 of Physiol., 42 and 43. 
 
 2 Amer. Journ. of Physiol., 16. 
 
 3 Cohnheim, Zeitschr. f. physiol. Chem., 39, 42, 43, and 47. 
 
 4 Stocklasa and collaborators, Centralbl. f. Physiol., 17, and Ber. d. d. chem. 
 Gesellsch., 36 and 38; Feinschmidt, Hofmeister's Beitrage, 4; Hirsch, ibid.; Claus and 
 Embden, ibid., 6; Arnheim and Rosenbaum, Zeitschr. f. physiol. Chem., 40; Braun- 
 stein, Zeitschr. f. klin. Med., 51; Levene and Meyer, Journ. of biol. Chem., 9. 
 
ORIGIN OF THE SUGAR. 409 
 
 due to synthesis, where a disaccharide is formed. According to J. de 
 Mkyku ■ neither the pancreas nor the tissues as a whole contain any 
 glycolytic enzymes. According to him only the blood has a glycolytic 
 action, and this action is supported by a body acting as an amboceptor 
 and produced in the pancreas. Our knowledge as to the existence of the 
 glycolysis and the mode of action of the pancreas in the metabolism of 
 sugar in the animal body is very meager and incomplete. 
 
 Where does the sugar eliminated in diabetes originate? Does it 
 depend entirely upon the carbohydrates of the food or the store of car- 
 bohydrates in the body, or has the body the power of producing sugar 
 from other material? To Luthje belongs the credit for positively 
 deciding this question. He has made experiments on dogs with pan- 
 creas diabetes, in which on a protein diet free from carbohydrates so much 
 sugar was eliminated that it could not possibly be accounted for by 
 the store of glycogen or other carbohydrate-containing substances in the 
 body. Similar experiments were also performed later by Pfluger, 2 
 with the results that the power of the animal body to produce sugar from 
 non-carbohydrate material is now definitely proved. 
 
 Is this sugar produced from protein or fat, or from both? This ques- 
 tion so far has not been answered, and it is the subject of continuous 
 dispute. It is not possible to enter into an exhaustive and detailed 
 discussion of the question in a text-book, and we will only mention, 
 briefly, certain of the most important observations and historical points. 
 
 The largest amount of sugar which we can obtain theoretically from 
 protein is 8 grams of sugar from 1 gram of protein nitrogen, if we admit 
 that all the carbon of the protein, with the exception of that necessary 
 to form ammonium carbonate, is used for the formation of sugar. These 
 results are still somewhat too high for the average carbon and nitrogen 
 content of the proteins and the values D:N = 6.6 is probably more correct. 3 
 The actual relation between glucose and nitrogen in the urine, i.e., 
 the quotient D: N, has been repeatedly determined in various forms of 
 diabetes, and in depancreatized dogs it is generally 2.8 and in starving 
 dogs or dogs fed with protein and poisoned with phlorhizin it is equal to 
 3.65 (Lusk). It may undergo considerable variation, and in certain 
 cases it may indeed be lower than 1 as well as higher than 8, and high 
 results have been repeatedly obtained in cases of human diabetes. From 
 these quotients conclusions have been drawn as to the amount of sugar 
 
 1 Cited from Centralbl. f. Physiol., 20 and 23. See also Lepine, Etat actuel de la 
 question de la Glycolyse, La semaine medicale, 1911. 
 
 'Luthje, Deutsch. Arch. f. klin. Med., "9, and Pfluger's Arch., 106; Pfluger, Pflii- 
 ger's Arch., 10S. 
 
 * See Falta, Zeitschr. f. klin. Med., 65; see also Gigon, Deutsch. Arch. f. klin. Med., 97. 
 
410 THE LIVER. 
 
 formed, as well as the origin of the sugar, but according to the views of 
 Hammarsten such conclusions are mostly very uncertain. The sugar 
 eliminated by the urine represents the difference between the total sugar 
 production of the body and the quantity of sugar burned or utilized. 
 Only under the supposition that the body cannot burn or utilize any 
 sugar, is the sugar of the urine a measure of the quantit}^ produced, 
 and this seems to be the case in phlorhizin diabetes ; but it is 
 difficult to decide how these suppositions apply to the different forms 
 of diabetes. Still several observations seem to show that in the different 
 forms of diabetes variable amounts of the sugar are burned, and only 
 in special cases can we draw approximately accurate conclusions. 
 
 The property of protein of increasing the elimination of sugar is 
 considered as an important proof of the formation of sugar from protein. 
 In this regard those experiments are of special interest in which the 
 diabetic animal is allowed to starve until the urine is poor in sugar or 
 indeed free from sugar, and then on feeding with protein, an abundant 
 elimination of sugar is produced. If we do not accept the view in this 
 case that the protein, but rather the fat, was the material from which 
 the sugar was produced, still we must admit either of a sugar-sparing 
 action due to protein or of a strong sugar formation from fat, incited 
 by the protein. 
 
 A sparing in the sense that the protein is oxidized instead of the sugar, 
 and in this manner protects it, is naturally possible only under the sup- 
 position that the body can burn &t least a part of the sugar, otherwise 
 there would be nothing to spare and nothing to protect from burning. 
 The assumption of such an indirect action of proteins is difficult to recon- 
 cile with the common view of the inability of the body to burn sugar 
 in diabetes. Luthje l has communicated one experiment among others, 
 in which a dog with pancreas diabetes, ariose weight before starvation 
 was 18 kilos, with nineteen days' starvation eliminated an average of 
 10.4 grams sugar for the last six days of starvation. By exclusive pro- 
 tein feeding the quantity of sugar per day could be raised to a maximum 
 of 123.6 grams, and as average it was 97.5 grams for the ten protein 
 days. The protein, therefore, had protected daily an average of 87 
 grams sugar from burning, which is hardly possible; and if in the diabetic 
 animal we admit of this considerable power of burning sugar, the quotient 
 D : N becomes valueless as a measure of the quantity of sugar formed. 
 
 If, on the contrary, we admit of an indirect action of proteins in 
 that they incite a sugar formation from fat, perhaps by a certain very 
 important increase in the activity of the liver, we are opposed by the 
 great difficulty that, according to known laws; of metabolism, the pro- 
 
 1 Deutsch. Arch. f. klin. Med., 79. 
 
SUGAR FORMATION FROM PROTEINS. 411 
 
 triiis do not raise the fat metabolism, but rather diminish it. The pro- 
 tein displaces a corresponding quantity of fat from the metabolism, 
 and if the fat were the only source of sugar then in this case we would 
 expect a diminished elimination of sugar instead of an increased one. 
 Nevertheless the above action of protein upon sugar elimination is much 
 more easily explained by the assumption of a sugar formation from pro- 
 tein than from fat. 
 
 The action of monamino-acids upon the carbohydrate metabolism 
 has also given important ground for the assumption cf a sugar forma- 
 tion from protein. That a deamidation occurs in the animal body was 
 shown by the earlier observations of Baumann and Blendermann. 
 Further proofs of this were furnished by the investigations of Neuberg 
 and Langstein, where in feeding experiments with alanine they found 
 abundance of lactic acid in the urine, and P. Mayer * observed glyceric 
 acid in the urine after the subcutaneous injection of diaminopropionic 
 acid. As from amino-acids by deamidation ketone acids or oxyacids 
 may be formed (see Chapter XIV) it would be of interest to test the action 
 of amino-acids upon the carbohydrate metabolism. Several investiga- 
 tions have been carried on with this in view, such as those of Langstein 
 and Neuberg, R. Cohn and F. Kraus, which have shown a very prob- 
 able formation of carbohydrate under the influence of amino-acids; but 
 the investigations of Embden and Salomon, and of Embden and Almagia 
 have positively shown, in a dog without a pancreas, that the amino- 
 acids can bring about a re-formation of carbohydrate. Lusk alone 
 and with Ringer 2 have shown the same for several amino-acids by 
 experiments on dogs poisoned with phlorhizin. According to the exper- 
 iments and calculations of the two last mentioned investigators glycocoll 
 and alanine can be completely transformed into glucose. Of the four 
 carbon atoms of aspartic acid and of the five carbon atoms of glutamic 
 acid three appear as glucose. 
 
 The investigations of Weinland 3 tend to prove a sugar formation 
 from protein He studied the formation of sugar in the chrysalis pulp 
 of the Calliphora and showed that the sugar formed thereby did not orig- 
 inate from the fat, but that the protein was the only material from 
 
 1 Baumann, Zeitschr. f. physiol. Chem., 4; Blendermann, ibid., 6; Neuberg and 
 Langstein, Arch. f. (Anat. u.) Physiol., 1903, Suppl.; Mayer, Zeitschr. f. phvsiol. Chem., 
 42. 
 
 2 Langstein and Neuberg, 1. c; Cohn, Zeitschr. f. physiol. Chem., 28; F. Kraus, 
 Berl. klin. Wochenschr., 1904; Embden and Salomon, Hofmeister's Beitrage, 5 and 
 6, and with Almagia, ibid., 7; Lusk, Amer. Journ. of Physiol., 22; Ringer and Lusk, 
 Zeitschr. f. physiol. Chem., 66. 
 
 3 Zeitsflir. f. Biol., 49 (N. F., 31); with Krummacher, ibid., 52. 
 
412 THE LIVER. 
 
 which the sugar was formed. The formation of sugar from protein is 
 now generally considered as positively proved. 
 
 Darin l has found with experiments with phlorhizinized dogs that 
 serine, cysteine, proline, ornithine and arginine yield abundant sugar in 
 glycosuric animals. Valine, leucine, isoleucine, lysine, histidine, phenyl- 
 alanine and tryptophane gave relatively little sugar or none at all. 
 The amino-acids with straight chains (with the exception of lysine) give 
 sugar while those with branched chains do not. Proline is the only 
 cyclic amino-acid, which yields abundance of sugar. Arginine is the 
 only one with more than five carbon atoms which yields sugar and the 
 sugar comes in this case from the ornithine components. 
 
 If we assume a formation of sugar from fat, we must differentiate 
 between the two components of neutral fats, that is, between the glyc- 
 erin and the fatty acids. A formation of sugar from glycerin can 
 be considered as proved by the investigations of Cremer, and especially 
 those of Luthje 2 and in the following we will discuss only the forma- 
 tion of sugar from the fatty acids. 
 
 The formation of sugar from fat seems to occur in the plant king- 
 dom, and as the chemical processes in the animal and plant life are in 
 principle the same, it makes the possibility of a sugar formation from 
 fat very probable. Such an origin of sugar in the animal body is accepted 
 by many investigators, especially by Pfuuger and several French observ- 
 ers, among whom we must specially mention Chauveau and Kauf- 
 mann. 3 
 
 When food as free from carbohydrate as possible is taken, the quo- 
 tient D:N is high, i.e., higher than 8, as well as when the quantity of 
 sugar is so large that it cannot be accounted for by the calculated 
 protein (and carbohydrate) metabolism, then if the observations are 
 otherwise free from error we can admit of a formation of sugar from fat. 
 Several such cases of diabetes in man have been published (Rumpf, 
 Rosenqvist, Mohr, v. Noorden, Allard, Falta and co-workers and 
 others), and also in animals (Hartogh and Schumm 4 ). Although these 
 researches are not fully conclusive, still certain of them indicate a prob- 
 able formation of su«ar from fat. We also have several conditions which 
 
 1 Journ. of biol. Chem., 14, 321. 
 
 2 Cremer, Sitzungsber. d. Ges. f. Morph. u. Physiol. Munchen, 1902; Luthje, Deutsch. 
 Arch. f. klin. Med., 80. 
 
 3 Kaufmann, Arch. f. Physiol. (5), 8, where Chauveau's work is cited. 
 
 4 Rumpf, Berl. klin. Wochenschr., 1899; Rosenqvist, ibid.; Mohr, ibid., 1901; v. 
 Noorden, Die Zuckerkrankheit, 3. Aufl. Berlin, 1901; Allard, Arch. f. exp. Path. u. 
 Pharm., 57; Falta and co-workers, Zeitschr. f. klin. Med., 66; Hartogh and Schumm, 
 Arch. f. Path. u. Pharm., 45. See also the works of O. Loewi, ibid., 47, and Lusk, 
 Zeitschr. f. Biologie, 42. 
 
SUGAR FORMATION FROM FATS. 413 
 
 indicate the same, namely, that in phlorhizin diabetes after the disap- 
 pearance of the liver-glycogen the fat which migrates to the liver serves 
 as material for the formation of sugar (Pfluger). These observations 
 make the formation of sugar from fat highly probable and the same is true 
 for the observations of Junkersdorf. 1 He found that in an animal made 
 glycogen free, by starvation and with phlorhizin poisoning, that toward 
 death, the nitrogen as well as the sugar elimination increased but that 
 the D : N ratio was higher than with the sugar formation from protein 
 alone. His calculations are not free from exception. 
 
 On the other hand there are many observations on animals and 
 also clinical observations which oppose the theory of the formation 
 of sugar from fat in diabetes. Lusk found in a dog with phlorhizin 
 diabetes that the quotient D:N = 3.65:1 was not changed on feeding fat, 
 and he has published further results of experiments 2 which show that 
 active muscular work, which strongly increases the fat decomposition, 
 does not change the quotient in dogs with phlorhizin diabetes. It is 
 difficult to draw positive conclusions from these experiments, still Lusk 
 seems to deny the formation of sugar from fat. 
 
 Attempts have been made to solve the question as to the material 
 from which sugar is formed by the determination of the respiratory 
 quotient and comparing this with the quotient D:N. The calculations 
 in this direction have not led to positive results. 3 As the quotient D : N 
 is not an accurate measure of the quantity of sugar formed, and as we, 
 as yet, do not know the quantity of oxygen necessary to form sugar 
 from protein, Hammarsten believes that it is just as impossible to con- 
 clude from the respiratory quotient that sugar is formed from the fats 
 as from the proteins. 
 
 We have no complete proofs for the formation of sugar from fat, still 
 we can indicate the probable proofs therefor. There is really no objec- 
 tion from a theoretical standpoint to the assumption that the body has 
 the power of producing sugar from protein as well as from fat, and such 
 a power does not seem improbable. 
 
 As a formation of sugar from protein is now generally considered as 
 proved, it follows that the protein can yield material for the formation 
 of glycogen and that it is a true glycogen-former. Pfluger and Junkers- 
 dorf 4 have given direct proof for this. They fed a dog, which had 
 previously been made glycogen-free by starvation and phlorhizin injec- 
 
 1 Pfliiger's Arch., 137. 
 
 2 Amer. Journ. of Physiol., 22. 
 
 3 Magnus-Levy, Zeitschr. f. klin. Med., 56; Pfliiger's Arch., 108; Mohr, Zeitschr. 
 I. exp. Path. u. Therap., 4. 
 
 * Pfliiger's Arch., 131. 
 
414 THE LIVER. 
 
 tions, with abundance of codfish and then found so much glycogen (6.46 
 per cent in the liver and 1 per cent in the muscle) that a re-formation of 
 glycogen must have undoubtedly occurred. By special control exper- 
 iments with fat feeding they also showed that the glycogen did not orig- 
 inate from the fat but must unquestionably have come from the protein. 
 Carbohydrates and proteins are without question true glycogen-formers, 
 while the question in regard to fats is still open. 
 
 The Bile and Its Formation. 
 
 By the establishment of a biliary fistula, an operation which was 
 first performed by Schwann in 1844 and which has been improved lately 
 by Dastre and Pawlow, 1 it is possible to study the secretion of the bile. 
 This secretion is continuous, but with varying intensity. It takes 
 place under a very low pressure; therefore an apparently unimportant 
 hindrance in the outflow of the bile, namely, a stoppage of mucus in the 
 exit, or the secretion of large quantities of viscous bile, may cause stagna- 
 tion and absorption of the bile by means of the lymphatic vessels (absorp- 
 tion icterus). 
 
 The quantity of bile secreted in the twenty-four hours in dogs can be 
 exactly determined. The quantity secreted by different animals varies, 
 and the limits are 2.9-36.4 grams. of bile per kilo of weight in the twenty- 
 four hours. 2 
 
 The reports as to the extent of bile secretion in man are few and 
 not to be depended on. Noel-Pa yton, Mayo-Robson, Hammarsten, 
 Pfaff and Balch, and Brand 3 found a variation between 514 and 
 1083 cc. per twenty-four hours. Such determinations are of doubtful 
 value, because in most cases it follows from the composition of the 
 collected bile that the fluid is not the result of a secretion of normal liver 
 bile. 
 
 The quantity of bile secreted is, however, as shown by Stadel- 
 mann, 4 subject to such great variation, even under physiological con- 
 ditions, that the study of those circumstances which influence the secre- 
 tion is very difficult and uncertain. The contradictory statements 
 by different investigators may probably be explained by this fact. 
 
 1 Schwann, Arch. f. (Anat. u.) Physiol., 1844; Dastre. Arch, de Physiol. (5i) 2; 
 Pawlow, Ergebnisse der Physiol., 1, Abt. 1. 
 
 2 In regard to the quantity of bile secreted in animals see Heidenhain, Die Gallenab- 
 eonderung, in Hermann's Handbuch der Physiol., 5, and Stadelmann, Der Icterus und 
 seine verschiedenen Formen (Stuttgart, 1891). 
 
 3 Noel-Payton, Rep. Lab. Roy. Coll. Edinburgh, 3; Mayo-Robson, Proc. Roy. Soc, 
 47; Hammarsten, Nova Act. Reg. Soc. Scient. Upsala (3), 16; Pfaff and Balch, Journ, 
 of Exp. Med., 1897; Brand, Pfluger's Arch., 90. 
 
 4 Stadelmann, Der Icterus, etc., Stuttgart, 1891. 
 
BILE SECRETION. 415 
 
 In starvation the secretion diminishes. According to Lukjanow 
 and Albertoni, 1 under these conditions the absolute quantity of solids 
 decreases, while the relative quantity increases. After partaking of 
 food the secretion increases again. The findings are very contradictory 
 in regard to the time necessary, after partaking of food, before the 
 secretion reaches its maximum. After a careful examination and com- 
 pilation of all the existing reports, Heidenhain 2 has come to the con- 
 clusion that in dogs the curve of rapidity of secretion shows two maxima, 
 the first at the third to fifth hour and the second at the thirteenth to 
 fifteenth hour after partaking of food. According to Barbera the 
 time when the maximum occurs is dependent upon the kind of food. 
 With carbohydrate food it is two to three hours, after protein food three 
 to four hours, and with fat diet it is five to seven hours, after feeding. 
 According to Loeb 3 the maximum occurs in dogs one to two hours after 
 feeding with meat, casein or gliadin. 
 
 According to earlier observations, the proteins of all the various 
 foods cause the greatest secretion of bile, while the carbohydrates dimin- 
 ish the secretion, or at least excite it much less than the proteins. This 
 coincides with the recent observations of Barbera. The authorities 
 by no means agree as to the action of the fats. While many older 
 investigators have not observed any increase, but rather the reverse 
 in the secretion of bile after feeding with fats, the researches of Barbera 
 show an undoubted increase in the secretion of bile on fat feeding, greater 
 even than after carbohydrate feeding. According to Rosenberg olive- 
 oil is a strong cholagogue, a statement which, according to other inves- 
 tigators — Mandelstamm, Doyon and Dufourt ' — has not been proved. 
 
 As Barbera has shown, a close relation exists between the bile 
 secretion and the quantity cf urea formed, as an increase in the first 
 goes hand in hand with an increase of the latter. The bile is, therefore, 
 according to him, a product of disassimilation, whose quantity rises and 
 falls with the degree of activity of the liver. 
 
 The question whether there exists special medicinal bodies, so-called 
 cholagogues, which have a specific excitant action on the secretion of 
 
 lukjanow, Zeitschr. f. physiol. Chem., 16; Albertoni, Recherches sur la s£cr6tion 
 biliaire, Turin, 1893. 
 
 2 Hermann's Handb., 5, and Stadelmann, Der Icterus, etc. 
 
 3 Barbera, Centralbl. f. Physiol., 12 and 16; A. Loeb, Zeitschr. f. Biol., 55. 
 
 4 Barbera, Bull, della scienz. med. di Bologna (7), 5, Maly's Jahresber., 24, and 
 Centralbl. f. Physiol., 12 and 16; Rosenberg, Pfluger's Arch., 46; Mandelstamm. Ueber 
 den Einfluss einiger Arzneimittel auf Sekretion und Zusammensetzung der Galle (Dis- 
 sert. Dorpat, 1890); Doyon and Dufourt, Arch, de Physiol, (o), 9. In regard to the 
 action of various foods on the secretion of bile see also Heidenhain, 1. c; Stadelmann, 
 Der Icterus; and Barbera, 1. c. 
 
416 THE LIVER. 
 
 bile, has been answered in very different ways. Many, especially the 
 older investigators, have observed an increase in the bile secretion after 
 the use of certain therapeutic agents, such as calomel, rhubarb, jalap, 
 turpentine, olive-oil, etc.; while others, especially the more recent inves- 
 tigators, have arrived at quite opposite results. From all appearances 
 this contradiction is due to the great irregularity of the normal secretion, 
 which might readily cause mistakes in tests with therapeutic agents. 
 
 Schiff's view, that the bile absorbed from the intestinal canal increases 
 the secretion of bile and hence acts as a cholagogue, seems to be a pos- 
 itively proved fact by the investigations of several experimenters. 1 
 Sodium salicylate is also perhaps a cholagogue (Stadelmann, Doyon 
 and Dufourt, Winogradow) and according to Petrowa 2 in dogs sodium 
 benzoate, thymole, phenol, menthol and all such bodies which are 
 conjugated to ethereal sulphuric acid in the animal body, increase the 
 secretion of bile. 
 
 Acids, and especially, under normal conditions, hydrochloric acid, 
 seem to be physiological excitants for bile secretion. According to 
 Falloise and Fleig the acids act upon the duodenum and the upper 
 part of the jejunum, and the action is brought about by a secretin forma- 
 tion similar to the action of acids upon the secretion of pancreatic juice 
 (see Chapter VIII). According to Falloise 3 chloral hydrate introduced 
 into the duodenum causes a secretion of bile in an analogous manner, by 
 the aid of a special chloral secretin. 
 
 The bile is a mixture of the secretion of the liver-cells and the so- 
 called mucus which is secreted by the glands of the biliary passages 
 and by the mucous membrane of the gall-bladder. The secretion of the 
 liver, which is generally poorer in solids than the bile from the gall- 
 bladder, is thin and clear, while the bile collected in the gall-bladder 
 is more ropy and viscous on account of the absorption of water and the 
 admixture of " mucus," and cloudy because of the presence of cells, 
 pigments, and the like. The specific gravity of the bile from the gall- 
 bladder varies considerably, being in man between 1.010 and 1.040. 
 Its reaction is alkaline to litmus. The color changes in different animals: 
 golden-yellow, yellowish-brown, olive-brown, brownish-green, grass-green 
 or bluish-green. Bile obtained from an executed person immediately 
 after death is golden-yellow or yellow with a shade of brown. Still cases 
 
 1 SchifF, Pfliiger's Arch., 3. See Stadelmann, Der Icterus, and the dissertations of 
 his pupils, especially Winteler, " Experimentelle Beitrage zur Frage des Kreislaufes 
 der Galle " (Inaug.-Diss. Dorpat, 1892), and Gartner, " Experimentelle Beitrage zur 
 Physiol, und Path, der Gallensekretion " (Inaug.-Dis. Jurjew, 1893); also Stadelmann, 
 " Ueber den Kreislauf der Galle," Zeitschr. f. Biologie, 34. 
 
 2 Zeitschr. f. physiol. Ghem., 74 (literature). See also footnote 4, page 415. 
 J Falloise, Bull. Acad. Roy. de Belg., 1903; Fleig, ibid., 1903. 
 
BILE-SALTS. 417 
 
 occur in which fresh human bile from the gall-bladder lias a green color. 
 The ordinary post-mortem bile has a variable color. The bile of cer- 
 tain animals has a peculiar odor; for example, ox-bile has an odor of 
 musk, especially on warming. The taste of bile is also different in 
 different animals. Human as well as ox-bile has a bitter taste, with a 
 sweetish after-taste. The bile of the pig and rabbit has an intensely 
 persistent bitter taste. On heating bile to boiling it does not coagulate. 
 It contains (in the ox) only traces of true mucin, and its ropy properties 
 depend, it seems, chiefly on the presence of a nucleoalbumin similar to 
 mucin (Paijkull). The bile from the animals investigated by Ham- 
 marstex showed a similar behavior. Hammarstex x has, on the con- 
 trary, found a true mucin in human bile. To all appearances this mucin 
 originates from the billiary passages, as he found it in the bile flowing 
 from the hepatic duct, and also because the mucous membrane of the 
 gall-bladder, according to Wahlgrex, 2 does not in man secrete any 
 mucin, but a mucin-like nucleoalbumin. 
 
 The specific constituents of the bile are bile-acids combined with alkalies, 
 bile-pigments, and, besides small quantities of lecithin and phosphatides, 
 cholesterin, soaps, neutral fats, urea, ethereal sulphuric acid, traces cf 
 conjugated glucuronic acids, enzymes and mineral substances, chiefly chlorides, 
 besides phosphates of calcium, magnesium, and iron. Traces of copper 
 also occur. 
 
 Bile-salts. The bile-acids, which thus far have best been studied, 
 may be divided into two groups, the glycocholic and taurocholic acid 
 groups. As found by Hammarstex 3 a third group of bile-acids occurs 
 in the shark, which are rich in sulphur, and like the ethereal sulphuric 
 acids they split off sulphuric acid on boiling with hydrochloric acid. 
 All glycocholic acids contain nitrogen, but are free from sulphur and 
 can be split, with the addition of water, into glycocoll (amino-acetic acid) 
 and a nitrogen-free acid, a cholic acid. All taurocholic acids contain 
 nitrogen and sulphur and are split, with the addition cf water, into 
 taurine and a cholic acid. The reason for the existence of different glyco- 
 cholic and taurocholic acids depends on the fact that there are several 
 cholic acids. 
 
 The conjugated bile-acid found in the shark, and called scynuwl-sulphuric acid 
 by Hammarstex, yields as cleavage products sulphuric acid and a non-nitrogenous 
 substance, scymnol (C27H46O5), which gives the characteristic color reactions of 
 cholic acid. 
 
 1 Paijkull, Zeitschr. f. physiol. Chem., 12; Hammarsten, 1. c, Nova Act. (3), 16, 
 and Ergebnisse der Physiol., Bd. 4. 
 
 2 Maly's Jahresber., 32. 
 
 * Hammarsten, Zeitschr. f. physiol. Chem., 24. 
 
41S THE LIVER. 
 
 The different bile-acids occur in the bile as alkali salts, generally 
 the sodium compounds, even in sea-fishes, although this is contrary to 
 the earlier observations (Zanetti 1 ). In the bile of certain animals we 
 find almost solely glycocholic acid, in others only taurocholic acid, and 
 in still others a mixture of both (see below). 
 
 All alkali salts of .the biliary acids are soluble in water and alcohol, 
 but insoluble in ether. Their solution in alcohol is therefore precipitated 
 by ether, and this precipitate, with proper care in manipulation, gives, 
 for nearly all kinds of bile thus far investigated, rosettes or balls of fine 
 needles, or four- to six-sided prisms (Plattner's crystallized bile). Fresh 
 human bile also crystallizes readily. The bile-acids and their salts 
 are optically active and dextrorotatory. The salts of the different bile- 
 acids act somewhat differently toward neutral salts. The alkali salts 
 of the ordinary and best-studied bile-acids from man, ox, and dog are, 
 according to Tengstrom, 2 precipitated by ammonium and magnesium 
 sulphates, and also, in pure form, by sodium nitrate and sodium chloride 
 (added to saturation). Potassium and sodium sulphates do not precip- 
 itate them. The alkali salts cannot be directly precipitated from the 
 bile by NaCl, on account of the presence of bodies retarding precipita- 
 tion, among which we find oil-soaps. 
 
 The bile-acids are dissolved by concentrated sulphuric acid at the 
 ordinary temperature, forming a reddish-yellow liquid which has a beautiful 
 green fluorescence. According to Pregl an oxidation with a reduction 
 of the sulphuric acid into sulphur dioxide takes place. The fluorescent 
 substance has been called dehydrocholan (see below) by Pregl. 3 On 
 carefully warming with concentrated sulphuric acid and a little cane- 
 sugar, the bile-acids give a beautiful cherry-red or reddish-violet liquid. 
 Pettenkofer's reaction for bile-acids is based on this behavior. 
 
 Pettenkofer's test for bile-acids is performed as follows: A small 
 quantity of bile in substance is dissolved in a small porcelain dish in con- 
 centrated sulphuric acid and warmed, or some of the liquid contain- 
 ing the bile-acids is mixed with concentrated sulphuric acid, taking special 
 care in both cases that the temperature does not rise higher than 60- 
 70° C. Then a 10-pcr-cent solution of cane-sugar is added, drop by 
 drop, continually stirring with a glass rod. The presence of bile is indi- 
 cated by the production of a beautiful red liquid, whose color does not 
 disappear at the ordinary temperature, but becomes more bluish-violet 
 in the course of a day. This red liquid shows a spectrum with two absorp- 
 tion-bands, the one at F and the other between D and E, near E. 
 
 iSee Chem. Centralbl., 1903, 1, 180. 
 
 2 Zeitschr. f. physiol. Chem., 41. 
 
 3 Zeitschr. f. physiol. Chem., 45. 
 
GLYCOCHOLIC ACID. 419 
 
 This extremely delicate test fails, however, when the solution is 
 heated too high, or if an improper quantity — generally too much — of 
 the sugar is added. In the last-mentioned case the sugar easily car- 
 bonizes and the test becomes brown or dark brown. The reaction fails 
 if the sulphuric acid contains sulphurous acid or the lower oxides of 
 nitrogen. Many other substances, such as proteins, oleic acid, amyl 
 alcohol, and morphine, give a similar reaction, and therefore in doubt- 
 ful cases the spectroscopic examination of the red solution must not be 
 forgotten. '< 
 
 Pettenkofer's test for the bile-acids depends essentially on the 
 fact that furfurol is formed from the sugar by the sulphuric acid (Mylius). 
 According to Mylius and v. Udranszky ' a 1 p. m. solution of furfurol 
 should be used. Dissolve the bile, which must first be decolorized by 
 animal charcoal, in alcohol. To each cubic centimeter of alcoholic 
 solution of bile in a test-tube add 1 drop of the furfurol solution and 
 1 cc. concentrated sulphuric acid, and cool when necessary, so that the 
 test does not become too warm. This reaction, when performed as 
 described, will detect -fa to fa milligram cholic acid (v. Udranszky). 
 Other modifications of Pettenkofer's test have been proposed. 
 
 The reaction with furfurol is not identical with that obtained with 
 cane-sugar, according to Ville and Derrien, and the absorption-bands 
 do not occur in the same place in the two cases. The reaction with cane- 
 sugar does not depend, according to these investigators, upon a furfurol 
 formation from the sugar. The acid hydrolyzes the sugar, and from the 
 fructose produced, 4-methyl-2-oxyfurfurol is formed by the further action 
 of the acid, and this gives the color reaction with the cholic acid. Instead 
 of furfurol other aldehydes such as vanillin and anisaldehyde can be 
 used according to Ville and Derrien. 2 
 
 Glycocholic Acid. The constitution of the glycocholic acid occurring 
 in human and ox-bile, and which has been most studied, is represented 
 by the formula C26H43NO6. Glycocholic acid is absent, or nearly so, 
 in the bile of carnivora. On boiling with acids or alkalies this acid, 
 which is analogous to hippuric acid, is converted into cholic acid and 
 glycocoll. 
 
 By the action of hydrazine hydrate upon the ethyl ester of cholic acid 
 Bondi and Muller 3 prepared first cholic-acid hydrazide, and then, by 
 the action of nitrous acid upon this, they obtained the cholic-acid azide, 
 C23H39O3CO.N3, and finally from this last in alkaline solution with glyco- 
 
 1 Mylius, Zeitschr. f. physiol. Chem., 11; v. Udranszky, ibid., 12. 
 
 2 Ville and Derrien, Chem. Centralbl. 1909, 2, 1699 and Compt. rend, eoc, biol. 64 
 and 66. 
 
 3 Zeitschr. f. physiol. Chem., 47. 
 
420 THE LIVER. 
 
 coll they synthetically prepared the alkali salt of glycocholic acid, at 
 the same time splitting off nitrogen. 
 
 Glycocholic acid crystallizes in fine, colorless needles or prisms. It 
 is soluble with difficulty in water (in about 300 parts cold and 120 parts 
 boiling water), and is easily precipitated from its alkali-salt solution 
 by the addition of dilute mineral acids. According to Bondi j glyco- 
 cholic acid is a rather strong acid, about as acid as lactic but much 
 stronger than acetic acid. This last-mentioned acid precipitates gly- 
 cocholic acid from the solution of its alkali salts in water. It is readily 
 soluble in strong alcohol, but with great difficulty in ether. The solu- 
 tions have a bitter but at the same time sweetish taste. The acid melts 
 between 132-152°, depending upon the method of preparation. Accord- 
 ing to Letsche, the acid containing water of crystallization (1| mol.) 
 deflagrates on heating rapidly at 126°, and at 130° an active frothing is 
 observed. The acid free from water of crystallization deflagrates at 
 130-132°, and decomposes at 154-155° C. with frothing. The salts of 
 the alkalies and alkaline earths are soluble in alcohol and water. 
 
 The solution of the alkali salt in water can be salted out by NaCl, 
 but not by KC1. The salts of the heavy metals are mostly insoluble or 
 soluble with difficulty in water. The solution of the alkali salts in water 
 is precipitated by sugar of lead, cupric and ferric salts, and silver nitrate. 
 
 On boiling with water glycocholic acid is probably transformed into its 
 physical isomer paraglycocholic acid, according to Letsche, 2 and this 
 crystallizes in long leaves which, when containing water of crystalliza- 
 tion, show ready deflagration at 186° and decompose with frothing at 
 198° C. On solution in alcohol or dilute alkalies the paraglycocholic 
 acid passes into the ordinary glycocholic acid. 
 
 Glycocholeic Acid is a second glycocholic acid, first isolated by Wahl- 
 gren 3 from ox-bile, and has the formula C26H43NO5 or C27H45NO5. 
 This acid, which on hydrolytic cleavage yields glycocoll and choleic 
 acid, has also been detected in human bile and the bile of the musk-ox 
 (Hammarsten 4 ). 
 
 Glycocholeic acid may, like glycocholic acid, crystallize in tufts of 
 fine needles, but is often obtained as short thick prisms. It is much more 
 insoluble in water, even on boiling, than glycocholic acid, and it melts 
 at 175-176° C. The alkali salts are soluble in water, have a pure bit- 
 ter taste, and are more readily precipitated by neutral salts (NaCl) than 
 the glycocholates. The solution of the alkali salts is not only precipitated 
 
 1 Zeitschr. f. physiol. Chem., 53. 
 
 2 Ibid., 60 and 73. 
 
 3 Ibid., 36. 
 * Ibid., 43. 
 
TAUROCHOLIC ACID. 421 
 
 by the salts of the heavy metals, but also by the salts of barium, cal- 
 cium and magnesium. 
 
 The principle in the preparation of the pure glycocholic acids con- 
 sists in treating a 2-'.\ per cent solution of bile free from mucus, when 
 rich in glycocholic acid (so-called Hefner's bile l ), with ether, and then 
 with 2 per cent hydrochloric acid. If the bile is not directly precipitable 
 with hydrochloric acid (bile relatively poor in glycocholic acid), then 
 precipitate the chief mass of the glycocholic acid with ferric chloride, 
 or better with lead acetate, decompose the precipitate with soda and treat 
 the 2 per cent solution as above stated with ether and hydrochloric acid. 
 The crystalline and washed mass is boiled with water, and on cooling 
 glycocholic acid crystallizes out, and then this is recrystallized from 
 water or from alcohol by the addition of water. The residue that remains 
 after boiling in water (paraglycocholic acid and glycocholeic acid) is 
 converted into their barium salts, and after a complicated method (see 
 Wahlgren) the glycocholeic acid is obtained. The reader is referred to 
 more exhaustive works for other methods of preparation. 
 
 Hyoglycocholic Acid, C27H43NO5, is the crystalline glycocholic acid obtained 
 from the bile of the pig. It is very insoluble in water. The alkali salts, whose 
 solutions have an intensely bitter taste, without any sweetish after-taste, are 
 precipitated by CaCb, BaCl 2 , and MgCl 2 , and may be salted out like a soap by 
 Na 2 S0 4 when added in sufficient quantity. According to Piettre it can be salted 
 out entirely, free from sulphur, by caustic alkali which is not possible by other 
 methods. By precipitation with NaCl in such quantity that the precipitate re- 
 dissolves on warming, Hammarsten 2 obtained the alkali salt, as macroscopic 
 crystals, on cooling. Besides this acid there occurs in the bile of the pig still 
 another glycocholic acid (Jolin 3 ). 
 
 The glycocholate in the bile of rodents is also precipitated by the above 
 mentioned earthy salts, but cannot, like the corresponding salt in human or ox- 
 bile, be directly precipitated on saturating with a neutral salt (Na 2 S0 4 ). Guano 
 bile-acid possibly belongs to the glycocholic-acid group, and is found in Peruvian 
 guano, but has not been thoroughly studied. 
 
 Taurocholic Acid. This acid, which is found in the bile of man, car- 
 nivora, oxen, and a few other herbivora, such as sheep and goats, has the 
 constitution C26H45NSO7. On boiling with acids and alkalies it splits 
 into cholic acid and taurine. Taurocholic acid has also been prepared 
 synthetically by Bondi and Muller, using the same method as they used 
 for glycocholic acid. 
 
 Taurocholic acid can be readily obtained, by the method suggested 
 by Hammarsten, 4 as groups of fine needles or as beautiful prisms on 
 slow crystallization. The crystals do not change in the air, but they 
 decompose above 100°. They are soluble in alcohol but insoluble in 
 
 1 Hiifner, Journ. f. prakt. Chfem. (N. F.i, 10, 10, and 25. 
 
 2 Not published. M. Piettre, Recherches sur la bile, Laval, 1910. 
 
 3 Zeitschr. f. physiol. Chem., 12 and 13. 
 1 Ibid., 43. 
 
422 THE LIVER. 
 
 ether, benzene, and acetone. Taurocholic acid is very soluble in water, 
 and the solution has a very sweet taste, with only a slight bitter taste. 
 It can hold the difficultly soluble glycocholic acid in solution. This is 
 the reason why a mixture of glycocholate with a sufficient quantity of 
 taurocholate, which often occurs in ox-bile, is not precipitated by a dilute 
 acid. Its salts are, as a rule, readily soluble in water, and the solutions 
 of the alkali salts are not precipitated by copper sulphate, silver nitrate 
 or lead acetate. Basic lead acetate gives, on the contrary, a precipitate 
 which is soluble in boiling alcohol. The alkali salts are not only pre- 
 cipitated from their solution by the same neutral salts that precipitate 
 glycocholic acid, but also by potassium chloride, and by sodium and 
 potassium acetates. 
 
 Taurocholeic Acid is a second taurocholic acid, detected by Hammar- 
 sten in dog-bile and isolated by Gullbring 1 from ox-bile, and has the 
 formula C26H45NSO6 or C27H47NS06- Thus far it has been obtained 
 only in the amorphous form. It is readily soluble in water, and has a 
 disagreeably bitter taste. It is also readily soluble in alcohol, but insoluble 
 in ether, acetone, chloroform, and benzene. The alkali salt, soluble in 
 water, can be salted out by NaCl as a pasty mass. The solutions of the 
 salts can be precipitated by ferric chloride. The cleavage products are 
 taurine and choleic acid. 
 
 The taurocholic acids are most simply prepared from bile, free from 
 plycocholic acid or poor therein, such as fish- or dog-bile, easiest from the 
 latter. The aqueous solution of the mucus-free bile is almost completely 
 precipitated by ferric chloride. The precipitate is worked for tauro- 
 choleic acid and the filtrate for taurocholic acid. The iron is first removed 
 from the filtrate by Na2COs, and then the faintly alkaline filtrate satu- 
 rated with Nad. The taurocholate separates out and after further 
 purification is decomposed by alcohol containing hydrochloric acid. The 
 taurocholic acid is precipitated from the alcoholic filtrate by ether and 
 recrystallized from alcohol containing water by the addition of ether. 
 The taurocholeic acid is obtained from the above iron precipitate by treat- 
 ing it with soda, and decomposing the alkali salt of the taurocholeic acid 
 with alcohol, containing HC1, and precipitating the acid from the alcoholic 
 solution with ether and repeating this precipitation from alcohol by ether. 
 
 Cheno-taurocholic Acid. This is the most essential acid of goose-bile and has 
 the formula GaEUNSOe. This acid, but little studied, is amorphous and solu- 
 ble in water and alcohol. 
 
 The taurocholic acids differ from the glycocholic acids in being 
 readily soluble in water. In the bile of the walrus, on the contrary, a 
 relatively insoluble, readily crystallizable taurocholic acid occurs, which 
 
 Hammarsten, Zeitschr. f. physiol. Chem., 43; Gullbring, ibid., 45. 
 
CHOLIC ACID. 423 
 
 can be precipitated from the solution of the alkali salts by the addition 
 of mineral acids, like glycocholic acid (Hammarsten x ). 
 
 As repeatedly mentioned above, the two bile-acids split on boiling 
 with acids or alkalies into non-nitrogenous cholic acids and into glycocoll 
 or taurine. Of the various cholic acids the following have been best 
 studied. 
 
 Cholic Acid or Cholalic Acid. The ordinary cholic acid obtained as 
 a decomposition product of human and ox-bile, which occurs, regularly 
 in the contents of the intestine, and also in the urine in icterus, has, accord- 
 int to Strecker and nearly all recent investigators, the constitution 
 
 f CHOH 
 C24H40O5, = C2oH3i j (CH 2 OH)2. According to Mylius, 2 cholic acid is a 
 
 LCOOH 
 monobasic alcohol-acid with one secondary and two primary alcohol 
 groups. Curtius 3 has shown by preparing the cholamine, C23H39O3.NH2, 
 from the above-mentioned (p. 419) cholic-acid azide, with cholic-acid 
 urethane as an intermediary step, that the carboxyl group is not imme- 
 diately connected with the CHOH group, but is combined with the chief 
 nucleus without the neighboring secondary alcohol group. On oxida- 
 tion it first yields dehydrocholic acid, C24H34O5 (Hammersten) from 
 which by electric reduction, Schenck obtained the reducto-dehydro- 
 cholic acid, C24H36O5. On further oxidation bilianic acid, C24H34OS 
 (Cleve), is obtained, or, more correctly, according to Latschinoff, 
 Lassar-Cohn and Pregl, a mixture of bilianic and isobilianic acids 
 discovered by Latschinoff. On oxidation, bilianic acid yields cilianic 
 acid (Lassar-Cohn), whose formula, according to Pregl, 4 is C20H28O8. 
 
 The products formed on a more active oxidation are of great interest. 
 If we discard the still somewhat problematic cholesterinic acid, we 
 find in these products in the first place choloidanic acid which has also 
 been called cholecamphoric acid and has the formula, Cis^sOs, accord- 
 ing to Pregl. This acid, as well as the acid obtained by Letsche s 
 on the oxidation of cholic acid and with the formula, C19H28O10, have 
 been obtained by Pregl 6 from the three most closely studied cholic acids, 
 
 1 Zeitschr. f. physiol. Chem., 61. 
 
 2 The important researches of Strecker on the bile-acids may be found in Annal. d. 
 Chem. u. Pharm., 65, 67, and 70; Mylius, Ber. d. deutsch. chem. Gesellsch., 19. 
 
 • Ibid., 39. 
 
 4 Hammarsten, Ber. d. deutsch. chem. Gesellsch., 14; Schenck, ibid., 63 and 69; 
 Clevc, Bull. Soc. chim., 35; Latschinoff, Ber. d. d. chem. Gesellsch., 15; Lassar-Cohn, 
 Ber. d. d. chem. Gesellsch., 32; Pregl, Wein. Sitzungsber., Ill, 1902. 
 
 5 Zeitschr. f. physiol. Chem., 61. 
 
 6 Ibid., 65. 
 
424 THE LIVER 
 
 namely from cholic acid, choleic acid and desoxycholic acid, and these 
 three acids are identically constructed in regard to their 19 carbon atoms. 
 
 The choloidanic acid is interesting in several respects. Panzer 1 has obtained 
 from it by distillation with soda-lime, a hydrocarbon, CuHi 6 , a homologue of 
 benzene, and on the oxidation of the cholic acid he has obtained an acid with the 
 formula, CgO^Os, which he considers as an oxyhexahydro-benzene-l-4-dicarboxylic 
 acid and from which he obtained paraoxybenzaldehyde. Pregl has obtained 
 from choloidanic acid, by heating, pyrocholoidanic acid, C15H20O4 which he con- 
 siders as parabcnzoic acid d-methyl-n-capric acid, and is produced from the 
 hexahydrobcnzene derivative by total dehydrogenation of a benzene derivative. 
 
 v. Furth and collaborators have investigated the products obtained on the 
 dry distillation of cholic acid at ordinary pressure, and Wieland and Weil 2 on 
 such distillation in vacuum. In the first case chiefly hydrocarbons with 12 to 
 17 carbon atoms were obtained, and in the second instance chiefly an unsaturated 
 acid, C24H34O2, was obtained, and in both cases these products and their double 
 bindings have been carefully investigated. We must wait for further developments 
 in these investigations before we attempt to draw any positive conclusions from 
 them. 
 
 From the investigations on the cholic acids carried out thus far we 
 are not able to draw any positive conclusions on their constitution, but 
 that they are derivatives of hexahydrobenzene, is very probable for sev- 
 eral reasons. 
 
 Cholic acid crystallizes partly in rhombic plates or prisms with one 
 molecule of water, and partly in larger rhombic tetrahedra or octahedra 
 with one molecule of alcohol of crystallization (Mtlius). These crystals 
 quickly become opaque and porcelain-white in the air. They are quite 
 insoluble in water (in 4000 parts cold and 750 parts boiling), rather 
 soluble in alcohol, but soluble with difficulty in ether. The amorphous 
 cholic acid is less insoluble. The solutions have a bitter-sweetish taste. 
 The crystals lose their alcohol of crystallization only after a lengthy 
 heating to 100-120° C. The acid free from water and alcohol melts at 
 195-196° C. According to Bondi and Muller the melting-point of the 
 perfectly pure acid is 198° C. It forms a characteristic blue compound 
 with iodine (Mylius). If finely powdered cholic acid is added to 25 
 per cent hydrochloric acid at the ordinary temperature, a beautiful violet- 
 blue coloration gradually appears, and this color is permanent for some 
 time and then becomes gradually green and yellow. The blue solution 
 shows an absorption band in the neighborhood of the D line (Hammar- 
 sten 3 ) . 
 
 The alkali salts are readily soluble in water, but when treated with a 
 concentrated caustic or carbonated alkali solution, they may then be 
 
 1 Panzer, Zeitschr. f. physiol. Chem., 48 and 60. 
 
 2 v. Furth with Link, Bioch. Zeitschr., 26, with Ishihara, ibid., 43; Wieland and 
 Weil, Zeitschr. f. physiol. Chem., 80. 
 
 3 Zeitschr. f. physiol. Chem., 61. 
 
CHOLEIC ACID. 425 
 
 separated as an oily mass which becomes crystalline on cooling. The 
 
 alkali salts are not readily soluble in alcohol, and on the evaporation of 
 alcohol they may crystallize. The specific rotatory power of the 
 Ljdium salt l is (a) D = +30.01° (2.29 per cent concentration) to +27.40° 
 (7..")',) per cent concentration). The watery solution of the alkali salts, 
 when not too dilute, is precipitated immediately or after some time by 
 Lead acetate or by barium chloride. The barium salt crystallizes in fine, 
 silky needles, and is rather insoluble in cold, but somewhat easily soluble 
 in warm water. The barium salt, as well as the lead salt, which is 
 insoluble in water, is soluble in warm alcohol. 
 
 Choleic Acid (C25H42O4, Latschinoff) is another cholic acid which, 
 according to Lassar-Cohn, 2 has the formula, C24H40O4. This acid, 
 which occurs in varying but always small quantities in ox-bile, and also 
 in gall-stones (H. Fischer and P. Meyer 3 ) yields dehydrocholeic acid, 
 C24H34O4, and then cholanic acid, C24H34O7, and isocholanic acid on 
 oxidation. 
 
 Choleic acid crystallizes when free from water in hexagonal vitreous 
 prisms with pointed ends, melting at 185-187° C. The crystalline acid 
 containing water melts at 135-140° C. (Latschinoff). The acid 
 dissolves in water with difficulty and is also relatively difficultly soluble 
 in alcohol. It has an intensely bitter taste and gives the Mylius iodine 
 reaction for cholic acid, and also the color reaction of cholic acid with 
 'hydrochloric acid. The specific rotation is (a) D = +48.87° (Vahlen). 
 The barium salt which crystallizes from the hot alcoholic solution as 
 spherical aggregations of radial needles is more difficultly soluble in 
 water than the corresponding cholate. 
 
 Desoxycholic Acid, C24H40O4, is the name given by Mylius 4 to a 
 cholic acid isolated by him from putrid ox-bile, also in gall-stones (Ktis- 
 ter) and in faeces (Fischer 5 ), and which is formed from the cholic acid 
 (on the putrefaction of the bile) by reduction. This last is still very 
 improbable, and the investigations of Ekbom do not support such an 
 assumption. On using perfectly pure cholic acid he was able to regain 
 it almost quantitatively after the action of metallic sodium on the alcoholic 
 solution of the acid, or of zinc and alkali. By treatment with zinc 
 and glacial acetic acid a reaction took place, but the product was a 
 mixture of mono- and diacetyl derivatives. The observation of Pregl 
 
 1 See Vahlen, Zeitschr. f. physiol. Chem., 21. 
 
 2 Latschinoff, Ber. d. deutsch. chem. Gesellsch., 18 and 20; Lassar-Cohn, ibid., 
 and Zeitschr. f. physiol. Chem., 17. See also Vahlen, Zeitschr. f. physiol. Chem., 23. 
 
 3 Zeitschr. f. physiol. Chem., 76. 
 
 4 Ber d. d. chem. Gesellsch, 19 and 20. 
 
 5 Kuster, Zeitschr. f. physiol. Chem., 69; H. Fischer, ibid., 73. 
 
426 THE LIVER. 
 
 that desoxycholic acid, like choleic acid, yields dehydrocholeic acid and 
 cholanic acid as oxidation products, makes the formation of desoxycholic 
 acid from cholic acid by reduction very improbable. The conclusion 
 of Latschinoff that both choleic and desoxj^cholic acids are identi- 
 cal, is not to be accepted on account of the different properties of the 
 two acids, and as shown by Langheld and also found by Hammarsten, 1 
 both acids can be detected in the same perfectly fresh ox-bile. Pregl 2 
 has given important proofs that we are here dealing with two different, 
 probably, isomeric acids. He found that the two acids yielded dehydro- 
 choleic acid on oxidation but that the dehydro-acid was not the same 
 in both cases. The choleic acid yielded a dehydro-acid with a lower 
 melting-point and a weaker specific rotation than the desoxycholic acid. 
 The desoxycholic acid crystallizes from glacial acetic acid in needles 
 with 1 molecule acetic acid, having a melting-point of 144-145°. The 
 melting-point of the acid crystallized from alcohol-ether is 153-155°, 
 and for the anhydrous acid or crystallized from acetone it is 172-173°. 
 It is soluble with difficulty in water, more readily soluble in alcohol, but 
 somewhat less soluble in glacial acetic acid than choleic acid. It has an 
 intensely bitter taste. The acid does not give a blue iodine compound, 
 and no color reaction with hydrochloric acid. Its barium salt is soluble 
 with difficulty in cold water, but dissolves in boiling alcohol and crys- 
 tallizes on cooling. 
 
 The cholic acids are best prepared from ox-bile, which is boiled for 
 24 hours with 5-10 per cent caustic soda. The crude acid is precipitated 
 by hydrochloric acid, dissolved in ammoniacal water and precipitated 
 by BaCb. The precipitate contains essentially choleic and desoxy- 
 cholic acids, while the nitrate contains a part of these and the chief part 
 of the cholic acid. In regard to the further rather complicated method 
 of separating the various acids, as also in regard to the many methods 
 suggested for the preparation of the pure cholic acids, we must refer to 
 more extensive hand-books. 3 
 
 Fellic Acid, C23H40O4 is a cholic acid, so called by Schotten, which he obtained 
 from human bile, along with the ordinary acid. This acid is crystalline, is insolu- 
 ble in water, and yields barium and magnesium salts, which are very insoluble. 
 It does not respond to Pettenkofer's reaction easily and gives a more reddish- 
 blue color. The existence of this acid is still doubtful. 
 
 The conjugate acids of human bile have not been sufficiently investi- 
 gated. To all appearances human bile contains under different circum- 
 
 1 Ekbom, Zeitschr. f. physiol. Chem., 50; Pregl, Wien. Sitz-Ber. Bd., Ill, Math. 
 Naturw. Kl., 1902; Latschinoff, Ber. d. d. Chem. Gesellsch., 20; Langheld, ibid., 
 41; Hammarsten in Abderhalden's Handbuch d. bioch. Arbeitsmethoden Bd. 2, 2. 
 
 2 Zeitschr f. physiol. Chem., 65. 
 
 Vbderhalden's Handbuch d. bioch. Arbeitsmethoden Bd. II. 2; also Pregl and 
 Buchtala, Zeitsfhr. f. physiol. Chem., 74 and Schryver, Journ. of Physiol., 44. 
 
SPECIAL CHOLIC ACIDS. 427 
 
 stances various conjugate 1 bile-acids. In some cases the bile-Baits of 
 human bile arc precipitated by BaCta and in others not. According to 
 the statements of Lassar-Cohn l three cholic acids may be prepared 
 from human bile, namely, ordinary cholic acid, choleic acid, and 
 fellic acid. 
 
 Lithofellic Acid, CsoHseCb, is the acid related to cholic acid which occurs in 
 the oriental bezoar stones, which is insoluble in water, comparatively easily solu- 
 ble in alcohol, but only slightly soluble in ether. 2 
 
 Lithocholic Acid, C^EUOs, is a cholic acid found by H. Fischer 3 in gall- 
 stones. It melts at 184-186° and is tasteless. 
 
 The hyo-glycocholic and cheno-taurocholic acids, as well as the 
 glycocholic acid of the bile of rodents, yield corresponding cholic acids. 
 This also seems to be the case with the glycocholic acid of the hippopota- 
 mus-bile, which stands very close to the pig-bile (Hammarsten 4 ). In- the 
 polar bear a third cholic acid exists besides cholic and choleic acids. 
 It is called ursocholeic acid, C19H30O4 or C18H28O4 (Hammarsten 5 ). 
 Also in the bile of other animals (walrus* seal) Hammarsten 6 has found 
 special cholic acids, phoccecholic acids, of which one, the cx-acid crystallizes 
 from benzene or petroleum ether in six-sided thin plates which melt at 
 152-154° C. Its formula seems to be C22H36O5. The other, tf-phocse- 
 cholic acid has the formula C24H40O5 and is isomeric w r ith cholic acid. 
 The isocholic acid melts at 220-222° C. 
 
 On boiling with acids, on putrefaction in the intestine, or on heating, 
 cholic acids lose water and are converted into anhydrides, the so-called 
 dyslysins. The dyslysin, C24H36O3, corresponding to ordinary cholic 
 acid, which occurs in faeces, is amorphous, insoluble in water and alkalies. 
 Choloidic acid, C24H38O4, is called the first anhydride or an intermediary 
 product in the formation of dyslysin. On boiling dyslysins with caustic 
 alkali they are reconverted into the corresponding cholic acids. 
 
 The Detection of Bile-acids in Animal Fluids. To obtain the 
 bile-acids pure so that Pettenkofer's test can be applied to them, the 
 protein and fat must first be removed. The protein is removed by 
 making the liquid first neutral and then adding a great excess of alcohol, 
 so that the mixture contains at least 85 vols, per cent of water-free alcohol. 
 Now filter, extract the precipitated protein with fresh alcohol, unite all 
 filtrates, distil the alcohol, and evaporate to dryness. The residue is 
 
 1 Schotten, Zeitschr. f. physiol. Chem., 11; Lassar-Cohn, Ber. d. deutsch. chem. 
 Gesellsch., 27. 
 
 2 See Junger and Klages, Ber. d. deutsch. chem. Gesellsch. 28 (older literature). 
 
 3 Zeitschr. f. physiol. Chem., 73. 
 
 4 Ibid., 74. 
 8 Ibid., 36. 
 
 6 Ibid., 61 and 68. 
 
428 THE LIVER. 
 
 completely exhausted with strong alcohol, filtered, and the alcohol entirely 
 evaporated from the filtrate. The residue is extracted with ether and 
 dissolved in water, and filtered if necessary, and the solution precipitated 
 by basic lead acetate and ammonia. The washed precipitate is dissolved 
 in boiling alcohol, filtered while warm, and a few drops of soda solution 
 added. Then evaporate to dryness, extract the residue with absolute 
 alcohol, filter, and add an excess of ether. The precipitate now formed 
 may be used for Pettenkofer's test. It is not necessary to wait for 
 cystallization; but one must not consider the crystals which form in the 
 liquid as being positively crystallized bile. It is also possible for needles 
 of alkali acetate to be formed. In this connection it must be remarked 
 that a confusion with phosphatides, which also give Pettenkofer's 
 reaction, is not excluded, and a further testing and separation are advisable. 
 
 Bile-pigments. The bile-coloring matters known thus far are rela- 
 tively numerous, and in all probability there are still more of them. Most 
 of the known bile-pigments are not found in the normal bile, but occur 
 either in post-mortem bile or principally in the bile concrements. The 
 pigments which occur under physiological conditions in human bile are 
 the reddish-yellow bilirubin, the green biliverdin, and sometimes also 
 urobilin (and urobilinogen) or a closely related pigment. The pigments 
 found in gall-stones are (besides the bilirubin and biliverdin) choleyrasin, 
 bilifuscin, biliprasin, bilihumin, bilicyanin and (choletelinf). Besides 
 these, others have been noticed in human and animal bile by various 
 observers. The two above-mentioned physiological pigments, bilirubin 
 and biliverdin, are those which serve to give the golden-yellow or orange- 
 yellow or sometimes greenish color to the bile; or when, as is most fre- 
 quently the case in ox-bile, the two pigments are present in the bile at 
 the same time, they produce the different shades between reddish-brown 
 and green. 
 
 Bilirubin. This pigment has the formula, C1GH18N2O3, or according 
 to Orndorff and Teeple and Kuster, 1 more correctly C32H36N4O6, 
 and is designated by the names cholepyrrhin, biliph^ein, bilifulvin, 
 and h^ematoidin. It occurs chiefly in the gall-stones as calcium bilirubin. 
 Bilirubin is present in the liver-bile of all vertebrates, and in the bladder- 
 bile especially in man and carnivora; sometimes, however, the latter 
 may have a green bile when fasting or in a starving condition. It also 
 occurs in the contents of the small intestine, in the blood serum of the 
 horse, in old blood extravasations (as haematoidin) , and in the urine and 
 the yellow-colored tissue in icterus. 
 
 On reduction with sodium amalgam Maly obtained a reduction 
 product, which he called hydrobilirubin, with the formula, C32H40N4O7, 
 
 1 Orndorff and Teeple, Salkowski's Festschrift, Berlin, 1904; Kuster, Zeitschr. f. 
 Physiol. Chem., 59. 
 
BILIRUBIN. 429 
 
 and which shows great similarity to tho urinary pigment, urobilin, as well 
 as to siercobilin found in the contents of the intestine (Masius and 
 Vanlaib j ). The reduction products have been carefully investigated 
 by H. Fischer and then by Paul Meter and F. Meyek-Betz. They 
 have found that hy< In (bilirubin is a mixture of bodies, among which there 
 is one which forms at least one-half and therefore, called henribilirvbin, 
 gives colorless crystals, and according to Fischer and Meyer-Betz 
 is identical with the urobilinogen of the urine. The formula of this 
 body is, C32H44N4O6 or C33H44N4OG. The other body is amorphous 
 but in properties and composition shows great similarity to the hemi- 
 bilirubin. The analyses correspond closely to the formula, C32H40X4O6. 
 This body as well as the hemibilirubin yields hsematinic acid and met Inl- 
 et hylmaleic imide on oxidation. As Kuster 2 first showed, bilirubin 
 yields hsematinic acid as oxidation product. It does not on the con- 
 trary yield methylethyl maleic imide. 
 
 Piloty and Thannhauser 3 obtained bilinic acid, C17H2GN2O3 
 from bilirubin on reduction with hydriodic acid and iodophosphonium. 
 Tliis acid corresponded to the hsematopyrrolidine carboxylic acid obtained 
 from haematoporphyrin. This bilinic acid is identical with the bilirubinic 
 acid described below and hence has this name. 4 They also obtained an 
 isomeric acid to phonopyrrolic acid, the isophonopyrrol carboxylic acid 
 and in the potash fusion they found partly a dimethyl- and partly a 
 trimethylpyrrol. From bilinic acid they later obtained on mild oxida- 
 tion an intensely yellow colored acid, the dehydrobilinic acid. 
 
 From bilirubin and hemibilirubin, on heating with sodium methylate, 
 H. Fischer and Rose 5 have obtained 2, 4, 5- tri methyl pyrrol-S-propionic 
 acid which was previously obtained by H. Fischer and Bartholomai s 
 from phonopyrrolcarboxylic acid. From bilirubinic acid on the con- 
 trary, with the same procedure they did not obtain this acid but another, 
 xanthobilirubinic acid, C17H22X2O3, which is probably identical with 
 dehydrobilinic acid, and which contains two atoms of hydrogen less than 
 bilirubinic acid, and which can be retransformed into the latter by glacial 
 acetic acid and hydriodic acid. As bilirubin, as well as hemibilirubin, 
 yields xanthobilirubinic acid as a side product with sodium methylate, 
 these experimenters consider this as a proof that the bilirubinic acid con- 
 
 1 Maly, Wien. Sitzungsber., 57, and Annal. d. Chem., 163; Masius and Vanlair, 
 Centralbl. f. d. med. Wissenseh., 1871, 369. 
 
 2 Hans Fischer, Zeitschr. f. physiol. Chem., 73, with Paul Meyer, ibid., 75, with. 
 Meyer-Betz, ibid., 75; Kuster, ibid., 26 and Ber. d. d. chem. Gesellsch, 32 and 35. 
 
 3 Piloty and Thannhauser, Annal. d. chem. u. Pharm., 390 and Ber d. d. chem. 
 Gesellsch., 45. 
 
 4 Piloty, Ber. d. d. chem. Gesellsch., 46, 1000; H. Fischer, ibid., 46, 1574. 
 s Ber. d. d. chem. Gesellsch., 46, 439. 
 
430 THE LIVER. 
 
 figuration exists already formed in these two bodies, and that the above- 
 mentioned tetrasubstituted acid, which is not obtained from bilirubinic 
 acid, must come from a special third pyrrol nucleus in the bilirubin and 
 hemibilirubin. Hsematinic acid (Kuster from bilirubin) and methyl- 
 ethylmaleic imide (H. Fischer and Meyer from hemibilirubin) have 
 been obtained from the two other pyrrol nuclei. Hsematinic acid as 
 well as methylethylmaleic imide have also been obtained from biliru- 
 binic acid. 
 
 Fischer and Rose * have earlier obtained, from hemibilirubin as 
 well as from the above-mentioned bodies and from bilirubin, by reduc- 
 tion with hydricdic acid, glacial acetic acid, a new crystalline acid, 
 the bilirubinic acid, C1-H24N2O3. This acid, to which Pilot y and 
 Thannhauser's bilinic acid stands in close relation, yields hsematinic 
 as well as methylethylmaleic imide on oxidation. By changing the 
 method of reduction Fischer and Rose 2 have obtained cryptopyrrol 
 and isophonopyrrolcarboxylic acid from bilirubin. The bilirubinic acid 
 also yielded the same products. 
 
 The close relation of the blood pigments to the bile pigments was 
 first shown by Kuster when he obtained the two hsematinic acids (as 
 imide) as oxidation products of these. This close relation is further 
 shown by the investigations given above although it is perhaps too 
 early to draw positive conclusions in regard to the structure of the two 
 groups of pigments and the differences existing between them. 
 
 Bilirubin is sometimes amorphous and sometimes crystalline. The 
 amorphous bilirubin is a reddish-yellow or reddish-brown powder; the 
 crystals have a reddish-yellow, reddish-brown, or more reddish color, 
 and sometimes they have nearly the color of crystalline chromic acid. 
 The crystals, which can easily be obtained by allowing a solution of bili- 
 rubin in chloroform to evaporate spontaneously, are reddish-yellow, 
 rhombic plates, whose obtuse angles are often rounded. On crystalliz- 
 ing from hot dimethylaniline it forms, on cooling, broad columns with 
 both ends sharply cut (Kuster 3 ). On dissolving in chloroform both 
 kinds of crystals are converted into long needles or whetstones. 
 
 Bilirubin is insoluble in water, behaves like an acid, and occurs in 
 animal fluids as soluble alkali bilirubin. It is very slightly soluble in 
 ether, benzene, carbon disulphide, amyl alcohol, fatty oils, and glyc- 
 erin. It is somewhat more soluble in alcohol. In cold chloroform it 
 dissolves with difficulty, and is much more readily soluble in warm chloro- 
 form. Its solubility varies, and supersaturated solutions are readily 
 formed (Orndorpf and Teeple). The varying solubility of bilirubin 
 
 1 Zeitschr. f. physio]. Choin., 82. 
 
 2 Ber. d. d. chem. Gesellsch, 45. 
 
 ' Ibid., 30 and 35, and Zeitschr. f. physiol. Chem., 47. 
 
BILIRUBIN. 431 
 
 in chloroform depends, according to Kuster, on the fact that in its 
 preparation, derivatives which are readily soluble and contain chlorine 
 or other transformation products are formed, or perhaps the bilirubin 
 goes over into polymeric modifications having different solubilities. In 
 cold dimethylaniline it dissolves in the proportion of 1:100, and in hot 
 dimethylaniline much more readily. Its solutions show no absorption- 
 Viands, but only a continuous absorption from the red to the violet end 
 of the spectrum, and they have a decided yellow color, even on diluting 
 greatly (1:500000), in .a layer 1.5 cm. thick. The combinations of 
 bilirubin with alkali are insoluble in chloroform, and the bilirubin in solu- 
 tion in chloroform can be removed from this solution by shaking with 
 dilute alkali (differing from lutein). Solutions of bilirubin-alkali in 
 water are precipitated by the soluble salts of the alkaline earths and also 
 by metallic salts. If a dilute solution of alkali bilirubin in water is 
 treated with an excess of ammonia and then with a zinc-chloride solution, 
 the liquid is first colored deep orange and then gradually olive-brown 
 and then green. This solution first gives a darkening of the violet and 
 blue part of the spectrum, and then the bands of alkaline cholecyanin 
 (see below), or at least the bands of this pigment in the red between 
 C and D, close to C. This is a good reaction for bilirubin. The fol- 
 lowing reaction has been suggested by Auche l . Treat 5 cc. of an alcoholic 
 solution of bilirubin (1:20000) which contains 1 drop of ammonia in 
 100 cc, with 5 to 6 drops of an alcoholic zinc acetate solution (1:1000) 
 and then 1 drop alcoholic iodine solution (1:100) when a beautiful bluish- 
 green coloration with a beautiful garnet-red fluorescence is obtained on 
 shaking. The spectrum shows a dark band between B and C, and a 
 pale band at D. If a few drops of hydrochloric acid are added to the 
 solution the color becomes violet, the fluorescence disappears and the 
 two Jaffa's cholecyanin bands appear. This reaction is extremely 
 delicate. 
 
 As Ehrlich first showed, bilirubin forms combinations with diazo 
 compounds, which have been closely studied by Proscher, Orndorff 
 and Teeple. 2 A test suggested by Ehrlich for bilirubin is based upon 
 this behavior with sulphodiazobenzene. 
 
 If an alkaline solution of bilirubin be allowed to stand in contact 
 with the air, it gradually absorbs oxygen, and green biliverdin is formed. 
 This process is accelerated by warming. According to Kuster, in this 
 case the alkali also has a splitting action upon the pigment, and among 
 the products formed we find hsematinic acid. Biliverdin is formed only 
 
 1 Compt. rend. soc. biol., 64. 
 
 2 Ehrlich, Zeitschr. f. anal. Chem., 23; Proscher, Zeitschr. f. physiol. Chem., 39; 
 Orndorff and Teeple, 1. c. 
 
432 THE LIVER. 
 
 from bilirubin by oxidation under special conditions (Kuster). A green 
 coloring-matter similar in appearance is formed by the action of other 
 reagents such as CI, Br, and I. According to Jolles, 1 , biliverdin is 
 produced by the use of Hubl's iodine solution, while according to others 
 (Thudichum, Malt 2 ) substitution products of bilirubin are formed. 
 
 Gmelin's Reaction for Bile-pigments. If one carefully pours nitric 
 acid, containing some nitrous acid, under an aqueous solution of alkali 
 bilirubin, there is obtained a series of colored layers at the juncture of the 
 two liquids in the following order from above downward: Green, blue, 
 violet, red, and reddish-yellow. This color reaction, Gmelin's test, 
 is very delicate, and serves to detect the presence of one part bilirubin 
 in 80,000 parts liquid. The green ring must never be absent; and also the 
 reddish-violet must be present at the same time, otherwise the reaction 
 may be confused with that for lutein, which gives a blue or greenish ring. 
 The nitric acid must not contain too much nitrous acid, for then the reac- 
 tion takes place too quickly and it does not become typical. Alcohol 
 must not be present in the liquid, because, as is well known, it gives a 
 play of colors, in green or blue, with the acid. 
 
 Hammarsten's Reaction. An acid is first prepared consisting of 1 
 vol. nitric acid and 19 vols, hydrochloric acid (each acid being about 
 25 per cent). One volume of this acid mixture, which can be kept for 
 at least a year, is, when it has become yellow by standing, mixed with 
 4 vols, alcohol. If a drop of bilirubin solution is added to a few cubic 
 centimeters of this colorless mixture a permanent beautiful green color 
 is obtained immediately. On the further addition of the acid mixture 
 to the green liquid all the colors of Gmelin's scale, as far as choletelin, 
 can be produced consecutively. 
 
 Huppert's Reaction. If a solution of alkali bilirubin is treated with 
 milk of lime or with calcium chloride and ammonia, a precipitate is 
 produced consisting of calcium bilirubin. If this moist precipitate, which 
 has been washed with water, is placed in a test-tube and the tube half 
 filled with alcohol which has been acidified with hydrochloric acid, and 
 heated to boiling for some time, the liquid becomes emerald-green or 
 bluish-green in color. 
 
 In regard to the modifications of Gmelin's test and certain other 
 reactions for bile-pigments, see Chapter XIV (Urine). 
 
 That the characteristic play of colors in Gmelin's test is the result 
 of an oxidation is generally admitted. The first oxidation step is the 
 
 1 Kuster, Ber. d. d. chem. Gesellsch., 35 and 59; Jolles, Journ. f .prakt. Chem. (N.F.), 
 59, and Pfluger's Arch., 75. 
 
 2 Thudichum, Journ. of Chem. Soc. (2),' 13, and Journ. f. prakt. Chem. (N.F.), 
 53; Maly, Wien. Sitzungsber., 72. 
 
BILIRUBIN, BILIVEKDIN. 433 
 
 green hiliverdin. Then follows a blue coloring-matter which Heinsm B 
 and Campbell call bilicyanin, and Stokvis calls cholecyanin, and which 
 shows a characteristic absorption-spectrum. The neutral solutions of 
 this coloring-matter are, according to Stokvis, bluish-green or steel-blue 
 with a beautiful blue fluorescence. The alkaline solutions are green 
 and have no marked fluorescence, and show three absorption-bands: 
 one, sharp and dark, in the red between C and D, nearer to C; a second, 
 less well defined, covering D; and a third between E and F, near E. 
 The strongly acid solutions are violet-blue and show two bands, described 
 by Jaffe between the lines C and E, separated from each other by a 
 narrow space near D. A third band between b and F is seen with dif- 
 ficulty. The next oxidation step after these blue coloring-matters is 
 a red pigment, and lastly a yellowish-brown pigment, called choletelin, 
 by Malt, which in neutral alcoholic solutions does not give any absorp- 
 tion-spectrum, but in acid solution gives a band between b and F. On 
 oxidizing cholecyanin with lead peroxide, Stokvis 1 obtained a "product 
 which he calls choletelin, which is quite similar to urinary urobilin, to 
 be discussed later. 
 
 Bilirubin is best prepared from gall-stones of oxen, these concretions 
 being very rich in calcium bilirubin. The finely powdered concrement 
 is first exhausted with ether and then with boiling water, so as to remove 
 the cholesterin and bile-acids. In order to .remove the mineral con- 
 stituents it is better to use 10 per cent acetic acid instead of hydrochloric 
 acid (Kuster 2 ). A green pigment is now removed by extraction with 
 alcohol, and the choleprasin is extracted with hot glacial acetic acid. 
 After washing with water it is dried, and extracted repeatedly with boil- 
 ing chloroform. The bilirubin separates from the chloroform as crusts, 
 which are treated once or twice in the above manner. It is then extracted 
 with alcohol and precipitated from its chloroform solution by alcohol, or 
 crystallized from boiling dimethylaniline. Further details are given by 
 Kuster. 3 
 
 The quantitative estimation of bilirubin may be made by the spectro- 
 photometric method, according to the steps suggested for the blood- 
 coloring matters. 4 
 
 Biliverdin, Ci 6 Hi8N 2 04 or C32H36N4O8. This body, which is formed 
 by the oxidation of bilirubin, occurs in the bile of many animals, in 
 vomited matter, in the placenta of the bitch (?), in the shells of birds' 
 
 1 Heinsius and Campbell, Pfluger's Arch., 4; Stokvis, Centralbl. f. med. Wis- 
 sensch., 1872, 785; ibid., 1873, 211 and 449; Jaffe\ ibid., 1868; Maly, Wien. Sitzungs- 
 ber., 59. 
 
 2 Zeitschr. f. physiol. Chem., 47. 
 
 3 Ibid., 59. 
 
 4 See also Herzfeld, Zeitschr. f. physiol. Chem., 77 and 78. 
 
434 THE LIVER. 
 
 eggs, in the urine in icterus, and sometimes in gall-stones, although in 
 very small quantities. 
 
 Biliverdin is amorphous; at least it has not been obtained in well- 
 defined crystals. It is insoluble in water, ether, and chloroform (this is 
 true at least for the artificially prepared biliverdin) but is soluble in 
 alcohol or glacial acetic acid, showing a beautiful green color. It is dis- 
 solved by alkalies, giving a brownish-green color, and this solution is 
 precipitated by acids, as well as by calcium, barium, and lead salts. 
 Biliverdin gives Huppert's, Gmelin's, and Hammarsten's reactions, 
 commencing with the blue color. It is converted into hydrobilirubin 
 by nascent hydrogen. On allowing the green bile to stand, also by the 
 action of ammonium sulphide, the biliverdin may be reduced to bilirubin 
 (Haycraft and Scofield *). 
 
 Biliverdin is most simply prepared by allowing a thin layer of an 
 alkaline solution of bilirubin to stand exposed to the air in a dish until 
 the color is brownish-green. The solution is then precipitated by hydro- 
 chloric acid, the precipitate washed with water until no HC1 reaction 
 is obtained, then dissolved in alcohol and the pigment again separated by 
 the addition of water. Any contaminating bilirubin may be removed 
 by means of chloroform. Kuster has shown that the biliverdin is only 
 formed by the oxygen of the air from bilirubin under certain conditions: 
 The presence of 2 molecules caustic alkali with the addition of water so 
 that the solution contains 0.2 per cent and, a temperature not above 5° C. 
 Hugounenq and Doyon 2 prepared biliverdin from bilirubin by the 
 action of sodium peroxide and a little hydrochloric acid. 
 
 Choleprasin is a green pigment isolated by Kuster 3 from gall-stones,, which 
 is soluble in glacial acetic acid but insoluble in alcohol. It differs from the other 
 bile-pigments by containing sulphur. On distillation with zinc powder it gives, 
 the pyrrol reaction, and on oxidation with chromic acid, Kuster could not 
 observe any formation of hsematinic acid. 
 
 Bilifuscin, so named by Stadeler, 4 is an amorphous brown pigment soluble 
 in alcohol and alkalies, almost insoluble in water and ether, and soluble with great 
 difficulty in chloroform (when bilirubin is not present at the same time). Pure 
 bilifuscin does not give Gmelin's reaction. This is also true for the bilifuscin 
 prepared by v. Zumbusch, 5 which is more like a humin substance, and the formula 
 of which is, Cc^H^NyOu. Bilifuscin has been found in gall-stones. Biliprasin 
 is a green pigment prepared by Stadeler from gall-stones, and is generally con- 
 sidered as a mixture of biliverdin and bilirubin. Dastre and Floresco, 6 on the 
 
 1 Centralbl. f. Physiol., 3, 222, and Zeitschr. f. physiol. Chem., 14. 
 
 2 Hugounenq et Doyon, Arch, de Phyisol. (5), 8; Kuster, Zeitschr. f. physiol. Chem.,, 
 59. 
 
 'Zeitschr. f. physiol. Chem., 47. 
 
 4 Cited from Hoppe-Seyler, Physiol, u. Path. chem. Analyse, 6. Aufl., p. 225. 
 
 6 Zeitschr. f. physiol. Chem., 31. 
 
 •Arch, de Physiol. (5), 9. 
 
SPECIAL BILE PIGMENTS. 435 
 
 contrary, consider biliprasin as an intermediate step between bilirubin and bili- 
 verdin. According to them it occurs as a physiologica] pigmenl in the bladder- 
 bile of several animals, and is derived from bilirubin by oxidation. Thia oxida- 
 tion is brought about by an oxidative ferment existing in the bile. Bilihumin 
 is the name given by Stadeler to that brownish amorphous residue which is left 
 after extracting gall-stones, with chloroform alcohol, and ether. It does not 
 give Gmelin's test. Bilicyani/i is also found in human gall-stones (Heinsics and 
 Campbell). Cholohcematin, so-called by MacMunn, is a pigment often occurring 
 in sheep- and ox-bile and characterized by four absorption-bands, which is 
 formed from hsematin by the action of sodium amalgam. In the dried condition, 
 as when obtained by the evaporation of the chloroform solution, it is green, and in 
 alcoholic solution olive-brown. This pigment, which has also been found by 
 Hammarsten in the bile from the musk-ox and hippopotamus, is, according to 
 Marchlewski, identical with the crystalline bilipurvurin isolated by Loebisch 
 and Fischler from ox-bile. This latter pigment, according to Marchlewski, 
 is not a bile-pigment, but phjlloerythrin, a transformation product of chlorophyll. 
 1 hvlloervthrin has been detected by Marchlewski l in the excrement of cows 
 fed on green grass. 
 
 Gmelin's and Huppert's reactions are generally used to detect the 
 presence of bile-pigments in animal fluids or tissues. The first, as a rule, 
 can be performed directly, and the presence of proteins does not interfere 
 with it, but, on the contrary, it brings out the play of colors more strik- 
 ingly. If blood-coloring matters are present at the same time, the bile- 
 coloring matters are first precipitated by the addition of sodium phos- 
 phate and milk of lime. This precipitate containing the bile-pigments 
 may be used directly in Huppert's reaction, or a little of the precipitate 
 may be dissolved in Hammarsten's reagent. Bilirubin is detected in 
 blood, according to Hedenius, by precipitating the proteins with alcohol, 
 filtering and acidifying the filtrate with hydrochloric or sulphuric acid, 
 and boiling. The liquid becomes of a greenish color. Serum and serous 
 fluids may be boiled directly with a little acid after the addition of alcohol. 
 According to Obermeyer and Popper 2 the alcoholic filtrate from the 
 protein precipitation can be tested with an alcoholic solution of iodine 
 or ferric chloride. 
 
 Besides the bile-acids and the bile-pigments, there occur in the bile 
 also cholesterin, lecithin, jecorin or other phosphatides (Hammarstex), 
 palmitin, stearin, olein, myristic acid (Lassar-Cohn 3 ), soaps, ethereal 
 sidphuric acids, conjugated glucuronates, diastatic and proteolytic enzymes, 
 oxidases and catalases. Choline, and glycerophosphoric acid, when they 
 are present, may be considered as decomposition products of lecithin. 
 Urea occurs, though only in traces, as a physiological constituent of human, 
 
 1 MacMunn, Journ. of Physiol., 6; Loebisch and Fischler, Wien. Sitzungsber., 112 
 (1903); Marchlewski, Zeitschr. f. physiol. Chem., 41, 43, and 45; Hammarsten, ibid., 
 43, and investigations not published. 
 
 2 Hedenius Upsala Lakaref. Forh., 29 and Maly's Jahresber., 24; Obermeyer and 
 Popper, Wien. med. Wochenschr., 60. 
 
 3 Zeitschr. f. physiol. Chem., 17; Hammarsten, ibid., 32, 36 and 43. 
 
436 THE LIVER. 
 
 ox-, and dog-bile. Urea occurs in the bile of the shark and ray in such 
 large quantities that it forms one of the chief constituents of the bile. 1 
 The mineral constituents of the bile are, besides the alkalies, to which the 
 bile-acids are united, sodium and potassium chloride, calcium and 
 magnesium phosphate, and iron — 0.04-0.115 p. m. in human bile, 
 chiefly combined with phosphoric acid (Young 2 ). Traces of copper 
 are habitually present, and traces of zinc are often found. Sulphates are 
 entirely absent, or occur only in very small amounts. 
 
 The quantity of iron in the bile varies greatly. According to Novi 
 it is dependent upon the kind of food, and in dogs it is lowest with a bread 
 diet and highest with a meat diet. According to Dastre this is not the 
 case. The quantity of iron in the bile varies even though a constant 
 diet is maintained, and the variation is dependent upon the forma- 
 tion and destruction of blood. According to Beccari 3 the iron does 
 not disappear from the bile in inanition, and the percentage shows no 
 constant diminution. The question as to the extent of elimination by 
 the bile of the iron introduced into the body has received various answers. 
 There is no doubt that the liver has the property of collecting and retain- 
 ing iron, as well as other metals, from the blood. Certain investigators, 
 such as Novi and Kunkel, are of the opinion that the iron introduced 
 and transitorily retained in the liver is eliminated by the bile, while 
 others, such as Hamburger, Gottlieb, and Anselm, 4 deny any such 
 elimination of iron by the bile. 
 
 Quantitative Composition of the Bile. Complete analyses of human 
 bile have been made by Hoppe-Seyler and his pupils. The bile was 
 removed from the gall-bladder of cadavers, hence these analyses can 
 be of little interest. Older and less complete analyses of perfectly fresh 
 human bile have been made bjr Frerichs and v. Gorup-Besanez. 5 
 The bile analyzed by them was from perfectly healthy persons who 
 had been executed or accidentally killed. The two analyses of 
 Frerichs are, respectively, of (I) an 18-year-old and (II) a 22-year- 
 old male. The analyses of v. Gorup-Besanez are of (I) a man of 49 
 and (II) a woman of 29. The results are, as usual, in parts per 1000. 
 
 1 Hammarsten, ibid., 24. 
 
 2 Journ. of Anat. and Physiol., 5, 158. 
 
 3 Novi, see Maly's Jahresber., 20; Dastre, Arch, de Physiol. (5), 3; Beccari, Arch, 
 ital. de Biol., 28. 
 
 4 Kunkel, Pfliiger's Arch., 14; Hamburger, Zeitschr. f. physiol. Chem., 2 and 4; 
 Gottlieb, ibid., 15; Anselm, " Ueber die Eisenausscheidung der Galle," Inaug.-Diss. 
 Dorpat, 1891 . See also the works cited in footnote 3, p. 339. 
 
 5 ,See Hoppe-Seyler Physiol. Chem., 301; Socoloff, Pfliiger's Arch., 12; Trifanow- 
 Bki, ibid., 9; Frerichs in Hoppe-Seyler's Physiol. Chem., 299; v. Gorup-Besanez, ibid. 
 
v. Gone 
 
 p-Besanez. 
 
 I. 
 
 II. 
 
 822 . 7 
 
 898.1 
 
 177.3 
 
 101.9 
 
 107.9 
 
 56.5 
 
 22.1 
 
 14.5 
 
 47.3 
 
 30.9 
 
 10.8 
 
 6.2 
 
 COMPOSITION OF THE BILE. 437 
 
 Frerichb. 
 
 I. II. 
 
 Water 800. 850 2 
 
 Solids 140.0 140.8 
 
 Biliary salts 72.2 91.4 
 
 Mucus and pigments 26.6 29.8 
 
 Cholesterin 1.6 2.6 
 
 Fat 3.2 9.2 
 
 Inorganic substances 6.5 7.7 
 
 Human liver-bile is poorer in solids than the bladder-bile. In 
 several cases it contained only 12-18 p. m. solids, but the bile in these 
 cases is hardly to be considered as normal. Jacobsen found 22.4-22.8 
 p. m. solids in a specimen of bile. Hammarsten, who had occasion to 
 analyze the liver-bile in seven cases of biliary fistula, has often 
 found 25-28 p. m. solids. In a case of a corpulent woman the quantity 
 of solids in the liver-bile varied between 30.10-38.6 p. m. in ten days. 
 Brand ' observed still higher figures, more than 40 p. m., in two 
 cases. This investigator suggests that the bile from an imperfect 
 fistula, when it is partly absorbed, is richer in solids than when it comes 
 from a perfect fistula. 
 
 The molecular concentration of human bile, according to Brand, 
 Bonanni, and Strauss, 2 is generally identical with that of the 
 blood, although the amount of water and solids varies. The freezing- 
 point varies only between —0.54° and —0.58°. This constancy of the 
 osmotic pressure is explained by the fact that in concentrated biles with 
 larger amounts of organic substances (with larger molecules) the amount 
 of inorganic salts is lower. 3 
 
 Human bile, sometimes, but not always, contains sulphur in an ethereal 
 sulphuric-acid-like combination (Hammarsten, Oerum, Brand). The 
 quantity of such sulphur may even amount to |-| of the total sulphur. 
 We do not know the nature of these ethereal sulphuric acids. According 
 to Oerum 4 they are not precipitated by lead acetate, but are precipitated 
 by basic lead acetate, especially with ammonia. Human bile is habitually 
 richer in glycocholic than in taurocholic acid. In six cases of liver-bile 
 analyzed by Hammarsten the relation of taurocholic to glycocholic acid 
 varied between 1 : 2.07 and 1 : 14.36. The bile analyzed by Jacobsen 
 contained no taurocholic acid. 
 
 As an example of the composition of human liver-bile the following 
 
 1 Jacobsen, Ber. d. deutsch. chem. Gesellsch., 6; Hammarsten, Nova Acta Reg. 
 Soc. Scient. Upsala. 16; Brand, Pfluger's Arch., 90. 
 
 2 Brand, 1. c, Bonanni, Biochem. Centralbl., 1; Strauss, Berl. klin. Wochenschr., 
 1903. 
 
 s See Brand, 1. c; Hammarsten, 1. c. 
 4 Skand. Archiv. f. Physiol., 16. 
 
438 THE LIVER. 
 
 results of three analyses made by Hammarsten are given. The results 
 are calculated in parts per 1000 : l 
 
 Solids 25.200 35.260 25.400 
 
 Water 974.800 964.740 974.600 
 
 Mucin and pigments 5 . 290 4 . 290 5 . 150 
 
 Bile-salts 9.310 18.240 9.040 
 
 Taurocholate 3.034 2.079 2.180 
 
 Glycocholate 6.276 16.161 6.860 
 
 Fattv acids from soaps 1 . 230 1 . 360 1 . 010 
 
 Cholesterin 0.630 1.60J 1.500 
 
 Lecithin \ n 990 0574 °- 650 
 
 Fat J 0.956 0.610 
 
 Soluble salts 8.070 6.760 7.250 
 
 Insoluble salts 0.250 0.490 0.210 
 
 Among the mineral constituents the chlorine and sodium occur to 
 the greatest extent. The relation between potassium and sodium 
 varies considerably in different samples, Sulphuric acid and phosphoric 
 acid occur only in very small quantities. 
 
 Baginskt and Sommerfeld 2 found true mucin, mixed with some 
 nucleoalbumin, in the bladder-bile of children. The bile contained 
 on an average 896.5 p. m. water; 103.5 p. m. solids; 20 p. m. mucin; 
 9.1 p. m. mineral substances; 25.2 p. m. bile-salts (of which 16.3 p. m. 
 were glycocholate and 8.9 p. m. taurocholate); 3.4 p. m. cholesterin; 
 6 p. m. lecithin; 6.7 p. m. fat, and 2.8 p. m. leucine. 3 
 
 The quantity of pigment in human bile is, according to Noel-Paton, 
 0.4-1.3 p. m. (in a case of biliary fistula). The method used in deter- 
 mining the pigments in this case was not quite trustworthy. Accurate 
 results for dog-bile obtained by spectrophotometric methods are on 
 record. According to Stadelmann 4 dog-bile contains on an average 
 0.6-0.7 p. m. bilirubin. At the most only 7 milligrams of pigment are 
 secreted per kilo of body in the twenty-four hours. 
 
 In animals the. relative proportion of the two acids varies con- 
 siderably. It has been found, on determining the amount of sulphur, 
 that, so far as the experiments have gone, taurocholic acid is the pre- 
 vailing acid in carnivorous mammals, birds, snakes, and fishes. Among 
 the herbivora, sheep and goats have a predominance of taurocholic 
 acid in the bile. Ox-bile sometimes contains taurocholic acid in excess, 
 in other cases glycocholic acid predominates, and in a few cases the 
 latter occurs almost alone. The bile of the rabbit, hare, kangaroo, 
 
 decent quantitative analyses may be found in Brand, 1. c; v. Zeynek, Wien. 
 klin. Wochensehr., 1899; Bonanni, 1. c. 
 
 2 Verhandl. d. physiol. Gesellsch. zu Berlin, 189-95. 
 
 3 Analyses of bile from children may be found in Heptner, Maly's Jahresber., 30. 
 
 4 Noel-Paton, Rep. Lab. Roy. Soc. Coll. Phys. Edinburgh, 3; Stadelmann,. Der 
 Icterus. 
 
CONSTITUTION OF THE BILE. 439 
 
 hippopotamus, and orang-utang (Hammarsten ! ) contains, like the 
 bile of the pig, almost exclusively glycocholic acid. A distinct influence 
 on the relative amounts of the two bile-acids exerted by differences in 
 diet has not been detected. Hitter 2 claims to have found a* decrease 
 in the quantity of taurocholic acid in calves when they pass from the 
 milk to the vegetable diet. 
 
 In the above-mentioned calculation of the taurocholic acid from the 
 quantity of sulphur in the bile-salt, it must be remarked that no definite 
 conclusion can be drawn from such a determination, since it is known 
 that other kinds of bile (e. g., human and shark bile) contain sulphur in 
 compounds other than taurocholic acid. 3 
 
 The phosphorized constituents of bile are not well known; never- 
 theless, there is no doubt that bile contains other phosphatides besides 
 lecithin (Hammarsten). These phosphatides are in part precipitated in 
 the precipitation of the bile-salts and they in part keep the bile-salts in 
 solution, preventing their complete precipitation, and hence they have a 
 double disturbing action in the quantitative analysis of bile. Those biles 
 richest in phosphatides, so far as known, are the following, in the order of 
 their amount: Polar bear, man (in special cases), dog, black bear, orang- 
 utang. The bile of certain fishes contains but little phosphatides 
 (Hammarsten 4 ). 
 
 The cholesterin, which, according to several investigators, originates 
 not only from the liver but also from the biliary passages, occurs in 
 larger quantities in the bladder-bile than in the liver-bile, and is present 
 to a greater extent in the non-filtered than in the filtered bile (Doyon 
 and Dufourt). The quantity seems to be very variable and in patients 
 with bile fistulas Bacmeister 5 found 0.24-0.59 p. m. The gases 
 of the bile consist of a large quantity of carbon dioxide, which increases 
 with the amount of alkalies, only traces of oxygen, and a very small 
 quantity of nitrogen. 
 
 Little is known in regard to the composition of the bile in disease. The quantity 
 of urea is found to be considerably increased in uraemia. Leucine and tyrosine are 
 observed in acute yellow atrophy of the liver and in typhoid. Traces of albumin 
 (without regard to nucleoalbumin) have several times been found in the human 
 "bile. The so-called pigmentary acholia, or the secretion of a bile containing 
 bile-acids but no bile-pigments, has also been repeated!}' noticed. In all such 
 eases observed by Ritter he found a fatty degeneration of the liver-cells, in return 
 for which, even in excessive fatty infiltration, a normal bile containing pigments 
 was secreted. The secretion of a bile nearly free from bile-acids has been 
 
 1 See Ergebnisse der Physiol., 4. 
 
 1 Cited from Maly's Jahresber., 6, 195. 
 
 3 Hammarsten, Zeitschr. f. physiol. Chem., 32, and Ergebnisse, der Physiol., 4. 
 
 4 Zeitschr. f. physiol. Chem., 36, and Ergebnisse der Physiol., 4. 
 
 6 Doyon and Dufort, Arch, de Physiol. (5), 8; Bacmeister, Bioch. Zeitschr., 26. 
 
440 THE LIVER. 
 
 observed by Hoppe-Seyler : in amyloid degeneration of the liver. In animals,, 
 dogs, and especially rabbits, it has been observed that the blood-pigments pass 
 into the bile in poisoning and in other conditions, causing a destruction of the 
 blood-corpuscles, as also after intravenous haemoglobin injection (Wertheimer 
 and Meyer, Filehne, Stern 2 ). Albumin can pass into the bile after the intra- 
 venous injection of a foreign protein (casein) (Gurber and Hallauer), as well 
 as after poisoning with phosphorus or arsenic (Pilzecker), or after the irrita- 
 tion of the liver by the introduction of ethyl alcohol or amyl alcohol (Brauer). 
 Sugar occurs in bile only in exceptional cases. 3 
 
 The physiological secretion of the gall-bladder in man is, according 
 to Wahlgren 4 a viscous, alkaline fluid with 11.24-19.63 p. m. solids. 
 The mucilaginous properties are not due to mucin, but to a phosphorized 
 protein substance (nucleoalbumin or nucleoprotein) . 
 
 Instead of bile there is sometimes found in the gall-bladder under pathological 
 conditions a more or less viscous, thready, colorless fluid which contains pseudo- 
 mucins or other peculiar protein substances. 5 
 
 Chemical Formation of the Bile. The first question to be answered 
 is the following: Do the specific constituents of the bile, the bile-acids 
 and bile-pigments originate in the liver; and if this is the case, do they 
 come from this organ alone, or are they also formed elsewhere? 
 
 The investigations of the blood, and especially the comparative 
 investigations of the blood of the portal and hepatic veins under normal 
 conditions, have not given any answer to this question. To decide this, 
 therefore, it is necessary to extirpate the liver of animals or to isolate 
 it from the circulation. If the bile constituents are not formed in the 
 liver, or at least not alone in this organ, but are eliminated only from 
 the blood, then, after the extirpation or removal of the liver from the 
 circulation, an accumulation of the bile constituents is to be expected 
 in the blood and tissues. If the bile constituents, on the contrary, are 
 formed exclusively in the liver, then the above operation naturally would 
 give no such result. If the ductus choledochus is tied, then the bile 
 constituents will be collected in the blood or tissues whether they are 
 formed in the liver or elsewhere. 
 
 From these principles Kobner has tried to demonstrate by exper- 
 iments on frogs that the bile-acids are produced exclusively in the liver. 
 While he was unable to detect any bile-acids in the blood and tissues of 
 
 fitter, Compt. Rend., 74, and Journ. de l'anat. et de la physiol. (Robin), 1872; 
 Hoppe-Seyler, Physiol. Chem., 317. 
 
 2 Wertheimer and Meyer, Compt. Rend., 108; Filehne, Virchow's Arch., 121; Stern, 
 ibid, 123. 
 
 3 Gurber, and Hallauer, Zeitschr. f. Biologie, 45; Pilzecker, Zeitschr. f. physiol. 
 Chem., 41; Brauer, ibid., 40. 
 
 1 Sec Maly'.s Jahresber., 32. 
 
 ' Winternitz, Zeitschr. f. physiol. Chem., 21; Sollmann, Amer. Medicine, 5 (1903). 
 
FORMATION OF BILE PIGMENTS. 441 
 
 these animals after extirpation of the liver, he was able to discover them 
 on tying the ductus choledochus. The investigations of Ludwig and 
 Fleischl ' show that in the dog the bile-acids originate in the liver alone. 
 After tying the ductus choledochus, they observed that the bile constituents 
 were absorbed by the lymphatic vessels of the liver and passed into the 
 blood through the thoracic duct. Bile-acids could be detected in the 
 blood after such an operation, while they could not be detected in the 
 normal blood. But when the common bile and thoracic ducts were both 
 tied at the same time, then not the least trace of bile-acids could be 
 detected in the blood, while if they are also formed in other organs and 
 tissues they should have been present. 
 
 From earlier reports of Cloez and Vulpian, as well as Virchow, the bile- 
 acids also occur in the suprarenal capsule. These claims have not been confirmed 
 by later investigations of Stadelmann and Beier. 2 At the present time there 
 is no ground for supposing that the bile-acids are formed elsewhere than in 
 the liver. 
 
 It has been undoubtedly proved that the bile-pigments may be formed 
 in other organs besides the liver, for, as is generally admitted, the color- 
 ing-matter haematoidin, which occurs in old blood extravasations, is 
 identical with the bile-pigment bilirubin (see page 301). Latschen- 
 berger 3 also observed in horses, under pathological conditions, a 
 formation of bile-pigments from the blood-coloring matters in the tissues. 
 The occurrence of bile-pigments in the placenta also seems to depend 
 on their formation in that organ, while the occurrence of small quantities 
 of bile-pigments in the blood-serum of certain animals probably depends 
 on an absorption of these substances. 
 
 Although the bile-pigments may be formed in other organs besides 
 the liver, still it is of first importance to know what bearing this organ 
 has on the elimination and formation of bile-pigments. In this regard 
 it must be recalled that the liver is an excretory organ for the bile-pig- 
 ments circulating in the blood. Tarchanoff observed in a dog with 
 biliary fistula, that intravenous injection of bilirubin causes a very 
 considerable increase in the bile-pigments eliminated. This statement 
 has been later confirmed by the investigations of Vossius. 4 
 
 Numerous experiments have been made to decide the question whether 
 the bile-pigments are only eliminated by the liver, or whether they are 
 also formed therein. By experimenting on pigeons, Stern was able 
 
 1 Kobner, see Heidenhain, Physiologie der Absonderungsvorgange, in Hermann's 
 Handbuch, 5; Fleischl, Arbeiten aus der physiol. Anstalt zu Leipzig, Jahrgang, 9. 
 
 2 Zeitschr. f. physiol. Chem., 18, in which the older literature may be found. 
 
 • See Maly's Jahresber., 16, and Monatshefte f. Chem., 9. 
 
 * Tarchanoff, Pfliiger's Arch., 9; Vossius, cited from Stadelmann, Der Icterus. 
 
442 THE LIVER. 
 
 to detect bile-pigments in the blood-serum five hours after tying the 
 biliary passages alone, while after tying all the vessels of the liver and also 
 the biliary passages, no bile-pigments could be detected either in the 
 blood or the tissues of the animal, which was killed 10-24 hours after 
 the operation. Minkowski and Naunyn 1 also found that poisoning 
 with arseniureted hydrogen produces a liberal formation of bile-pig- 
 ments, and the secretion, after a short time, of a urine rich in biliverdin 
 in previously healthy geese. In geese with extirpated livers this does 
 not occur. 
 
 With experiments on dogs, Whipple and Hooper 2 found after intra- 
 venous injection of blood-corpuscles of the same animal hsemolyzed 
 with water, that a transformation of the haemoglobin into bile-pigments 
 occurred with the same rapidity in normal animals as with animals with 
 Eck fistulas, or with such a fistula and the hepatic artery ligatured. The 
 formation of bile-pigments also occurred on removing the liver, spleen 
 and abdomen from the circulation, as well as by circulation through the 
 head and thorax. A transformation of haemoglobin into bile-pigments, 
 at least in dogs, can take place easily without the medium of the liver 
 and these experimenters suggest the possibility that the endothelial 
 cells are here active. 
 
 No such experiments can be carried out on mammalia, as they do 
 not live long enough after the operation; still there is no doubt that this 
 organ is the chief seat of the formation of bile-pigments under physiolog- 
 ical conditions. 
 
 In regard to the materials from which the bile-acids are produced, 
 it may be said with certainty that the two components, glycocoll and 
 taurine, which are both nitrogenized, are formed from the protein bodies. 
 The close relation of taurine to the cystine group of the protein mole- 
 cule has been especially shown by the investigations of Freidmann, 
 (see Chapter III), and recently v. Bergmann 3 has shown by feeding 
 dogs with sodium cholate and cystine that the animal body can trans- 
 form cystine into taurine, and that the taurine of the bile originates 
 from the proteins of the food. In regard to the origin of the non-nitro- 
 genized cholic acid, which was formally considered as originating from 
 the fats, nothing is positively known; to all appearances it is from 
 proteins. 
 
 The blood-coloring matters are considered as the mother-substances 
 of the bile-pigments. If the identity of haematoidin and bilirubin was 
 
 1 Stern, Arch. f. exp. Path. u. Pharm., 19; Minkowski and Naunyn, ibid., 21. 
 
 2 Journ. of exp. Med., 17. 
 
 1 Hofmeister's Beitrage, 4. See also Wohlgemuth, Zeitschr. f. physiol. Chem., 40. 
 
FORMATION OF BILE PIGMENTS. 443 
 
 settled beyond a doubt, then this view might be considered as proved: 
 Independently, however, of this identity, which is not admitted by 
 all investigators, the view that the bile-pigments are derived from the 
 blood-coloring matters has strong arguments in its favor. It has been 
 shown by several experimenters that a yellow or yellowish-red pigment 
 can be formed from the blood-coloring matters, which gives Gmelin's 
 test, and which, though it may not form a complete bile-pigment, is 
 at least a step in its formation (Latschenberger). The previously 
 mentioned relationship between the blood and bile-pigments must be 
 recalled, and the formation of bilirubin from the blood-pigments is 
 shown, according to the unanimous observations of several investi- 
 gators, 1 bv the fact that the appearance of free haemoglobin in the plasma, 
 produced by the destruction of the red corpuscles by widely differing 
 influences (see below) or by the injection of haemoglobin solution, causes 
 an increased formation of bile-pigments. The amount of pigments in 
 the bile is not only considerably increased, but the bile-pigments may 
 even pass into the urine under certain circumstances (icterus). After 
 the injection of haemoglobin solution into a dog either subcutaneously 
 or in the peritoneal cavity, Stadelmann and Gorodecki 2 observed an 
 increase of 61 per cent in the secretion of pigments by the bile, which 
 lasted for more than twenty-four hours. Recently Brusch and Yosh- 
 imoto, 3 by quantitative estimations of the bile-pigments and urobilin 
 in animals with bile fistulas with ligated ductus choledochus, have 
 shown the increased formation of bile-pigments after the injection of 
 known amounts of haematin, and in this manner further proved the 
 genetic relationship between the bile-pigments and haematin. 
 
 If bilirubin, which contains no iron, is derived from haematin, which 
 contains iron, then iron must be split off. The question in what form or 
 combination the iron is split off is of special interest, and also whether 
 it is eliminated by the bile. This latter does not seem to be the case, 
 at least to any great extent. In 100 parts of bilirubin which are eliminated 
 by the bile there are only 1.4-1.5 parts iron, according to Kunkel, while 
 100 parts haematin contain about 9 parts iron. Minkowski and Base- 
 rin 4 also found that the abundant formation of bile-pigments occurring 
 in poisoning by arseniureted hydrogen does not increase the quantity 
 of iron in the bile. The quantity apparently does not seem to correspond 
 with that in the decomposed blood-coloring matters. It follows from the 
 
 1 See Stadelmann, Der Icterus, etc., Stuttgart, 1891. 
 1 See Stadelmann, ibid. 
 
 3 Zeitschr. f. exp. Path. u. Therap., 8. 
 
 4 Kunkel, Pfluger's Arch., 14; Minkowski and Baserin, Arch. f. exp. Path. u. 
 Pharm., 23. 
 
444 THE LIVER. 
 
 researches of several investigators * that the iron is, at least chiefly, 
 retained by the liver as a ferruginous pigment or protein substance. 
 
 What relation does the formation of bile-acids bear to the forma- 
 tion of bile-pigments? Are these two chief constituents of the bile derived 
 simultaneously from the same material, and can we detect a certain 
 connection between the formation of bilirubin and bile-acids in the liver? 
 The investigations of Stadelmann teach us that this is not the case. 
 With increased formation of bile-pigments the amount of bile-acids 
 is decreased, and the introduction of haemoglobin into the liver strongly 
 increases the formation of bilirubin, but simultaneously strongly decreases 
 the production of bile-acids. According to Stadelmann the formation 
 of bile-pigments and bile-acids is due to a special activity of the cells. 
 
 An absorption of bile from the liver, and the passage of the bile con- 
 stituents into the blood and urine occurs in retarded discharge of the 
 bile, and usually in different forms of hepatogenic icterus. But bile- 
 pigments may also pass into the urine under other circumstances, espe- 
 cially when a solution or destruction of the red blood-corpuscles takes 
 place in animals through injection of water or a solution of biliary salts, 
 through poisoning by ether, chloroform, ars3niureted hydrogen, phos- 
 phorus, or toluylenediamine, and in other cases. This also occurs in man 
 in severe infectious diseases where the red blood-corpuscles are dissolved 
 or destroyed. It has also been claimed many times that a transformation 
 of blood-pigments into bile-pigments occurs elsewhere than in the liver, 
 namely, in the blood. Such a belief has been made very improbable 
 and in some of the above-mentioned cases, as after poisoning with phos- 
 phorus, toluylenediamine, and arseniureted hydrogen, it has been dis- 
 proved by direct experiment. 2 In these cases we are also dealing with 
 an abundant working up of the blood-pigments in the liver. 
 
 Bile Concretions. 
 
 The concrements which occur in the gall-bladder vary considerably 
 in size, form, and number, and are of three kinds, depending upon the 
 kind and nature of the bodies forming their principal mass. One group of 
 gall-stones contains lime-pigment as chief constituent, another cholesterin, 
 and the third calcium carbonate and phosphate. The concrements 
 of the last-mentioned group occur very seldom in man. The so-called 
 cholesterin-stones are those which occur most frequently in man, while 
 
 < Naunyn and Minkowski, Arch. f. exp. Path. u. Pharm., 21; Latschenberger, 
 \.c; Neumann, Virchow's Arch., Ill, and the literature in footnote 2, p. 383. 
 
 2 The literature belonging to this subject is found in Stadelmann, Der Icterus, etc., 
 Stuttgart, 1891. 
 
BILE CONCRETIONS. CHOLESTERIN. 445 
 
 the lime-pigment stones are not found very often in man, hut often in 
 oxen. 
 
 The pigment-stones are generally not large in man, but in oxen and 
 pigs they are sometimes found the size of a walnut or even larger. In 
 most cases they consist principally of calcium-bilirubin with little or no 
 biliverdin, and they also often contain very small amounts of cholic acids. 
 Sometimes also small black or greenish-black, metallic-looking stones 
 are found, which consist chiefly of bilifuscin along with biliverdin. Iron 
 and copper seem to be regular constituents of pigment-stones. Man- 
 ganese and zinc have also been found in a few cases. The pigment-stones 
 are generally heavier than water. 
 
 The cholesterin-stones, whose size, form, color, and structure may vary 
 greatly, are often lighter than water. The fractured surface is radiated, 
 crystalline, and frequently shows crystalline, concentric layers. The 
 cleavage fracture is waxy in appearance, and the fractured surface when 
 rubbed by the finger-nail also becomes like wax. By rubbing against 
 each other in the gall-bladder they often become faceted or take other 
 remarkable shapes. Their surface is sometimes nearly white and wax- 
 like, but generally their color is variable. They are sometimes smooth, 
 in other cases they are rough or uneven. The quantity of cholesterin 
 in the stones varies from G42 to 981 p. m. (Ritter l ). The cholesterin- 
 stones sometimes contain variable amounts of lime-pigments, which may 
 give them a very changeable appearance. 
 
 Cholesterin. The formula for this body, although not positively 
 determined, is generally given as C27H46O (Obermuller) or C27H44O 
 (Mauthner and Suida). 
 
 Because of the fact that from cholesterin, hydrocarbons which 
 have been called cholesteriline, cholesterone and cholesterilene, can be 
 prepared in different ways, it was believed that a certain analogy exists 
 between the cholesterin and the terpenes. The color reactions as 
 well as the recent investigations on the constitution of cholesterin indicate 
 that this body belongs to the terpenes. 
 
 The constitution of cholesterin has not been completely determined, 
 although we have the very laborious and thorough investigations of 
 many workers of whom we especially mention Mauthner and Suida, 
 Windaus, Stein, Diels and Abderhalden. 2 From these investigations 
 we conclude that cholesterin is a monoatomic, unsaturated, secondary 
 alcohol whose hydroxyl group exists in a hydrogenized ring, between 
 
 1 Journ. de l'anat. et de la physiol. (Robin), 1872. 
 
 2 The literature on cholesterin can be found in Windaus, Arch. d. Pharm., 246, 
 Hft. 2, and in Abderhalden's Bioch. Handlexikon, Bd., 3, and also in Glikin, Bioch. 
 Centralbl., 7, 372-377. 
 
446 THE LIVER. 
 
 two methyl groups, and which also contains an isopropyl group. 
 It is also generally admitted that cholesterin contains only one double 
 bond, which occurs in a vinyl group, CH:CH2, at the end. No constitu- 
 tional formula for cholesterin can be given; still there is no doubt but 
 that it is a complex terpene which stands in close relation to retene as 
 well as to the cholic acids. 
 
 By the reduction of cholesterin by metallic sodium and amyl alcohol, Diels 
 and Abderhalden as well as Neuberg and Rauchwerger obtained a dihydro- 
 cholesterin, the a-cholestanol, C 2 7H 48 0. On treating cholestenon, the ketone of 
 cholesterin, Diels and Abderhalden obtained a second dihydrocholesterin, 
 the @<holesta?wl, which Willstatter and E. W. Mayer obtained directly from 
 cholesterin in ethereal solution by reduction with hydrogen and platinum-black. 
 According to Diels and Linn * /3-cholestanol is obtained from cholestenon by 
 the action of sodium and amyl alcohol, and a-cholestanol with sodium amy late. 
 The relation of these bodies to each other is still not understood. These dihydro- 
 cholesterins have a physiological interest in regard to the question whether they 
 are identical or not with koprosterin, which will be discussed below. 
 
 On heating cholesterin, when contaminated with iron, to 300-320°, according 
 to Diels and Linn, 2 it in part yields cholestenon and partly an isomeric cholesterin, 
 the fl-cholesterin. This last body can be retransformed into cholesterin by the 
 saponification of the cholesteryl benzoate. 
 
 Cholesterin occurs in small amounts in nearly all animal fluids and 
 juices. It occurs only rarely in the urine, and then in very small quanti- 
 ties. It is also found in the different tissues and organs, especially 
 abundant in the brain and the nervous system; further, in the yolk of 
 the egg, in semen, in wool-fat (together with isocholesterin), and in 
 sebum. It also appears in the contents of the intestine, in excrements, 
 and in the meconium. It especially occurs pathologically in gall-stones 
 as well as in atheromatous cysts, in pus, in tuberculous masses, old 
 transudates, cystic fluids, sputum, and tumors. It does not exist free 
 in all cases; for example, it exists in part as fatty-acid esters in wool- 
 fat, blood, lymph, brain, vernix caseosa and epidermis formations. Sev- 
 eral kinds of cholesterin, called phytosterines, have been found in the 
 vegetable kingdom. 
 
 Cholesterin which has been crystallized from warm alcohol on cooling, 
 and also that which is present in old transudates, contains one molecule 
 of water of crystallization, and melts at 148.5° C. when dried in a vacuum, 
 and forms colorless, transparent plates whose sides and angles frequently 
 appear broken, and whose acute angle is often 76° 30' or 87° 30'. In 
 large quantities it appears as a mass of white plates which shine like 
 mother-of-pearl and have a greasy touch. 
 
 Cholesterin is insoluble in water, dilute acids, and alkalies. It is 
 neither dissolved nor changed by boiline; caustic alkali. It is easily 
 
 1 Willstatter and Mayer, Ber d. d. chem. Gesellsch., 41; Diels and Linn, ibid., 41. 
 
 2 Ibid., 41. 
 
CHOLESTERIN. 447 
 
 soluble in boiling alcohol, and crystallizes on cooling. It dissolves readily 
 in'ether, chloroform, and benzene, and also in the volatile or fatty oils. 
 It is dissolved to a slight extent by alkali salts of the bile-acids, better 
 in the presence of oleic soap (Gerard l ). The solutions in ether and 
 chloroform are levorotatory, (a) D = —31.12° (2 per cent ethereal solu- 
 tion). 
 
 Among the many compounds of cholesterin the propionic ester 
 C2H5.CO.O.C27H45 is of special interest because of the behavior of the 
 fused compound on cooling, and it is used in the detection of choles- 
 terin. For the detection of cholesterin use is made of its reaction with 
 concentrated sulphuric acid, which gives colored products. 
 
 If a mixture of five parts sulphuric acid and one part water acts on 
 cholesterin crystals, they show colored rings, first a bright carmine-red 
 and then violet. This test is employed in the microscopic detection 
 of cholesterin. Another test, and one very good for the microscopical 
 detection of cholesterin, consists in treating the crystals first with the 
 above dilute acid and then with some iodine solution. The crystals 
 will be gradually colored violet, bluish-green, and a beautiful blue. 
 
 Salkowski's 2 Reaction. The cholesterin is dissolved in chloroform 
 and then treated with an equal volume of concentrated sulphuric acid. 
 The cholesterin solution becomes first bluish-red, then gradually more 
 violet-red, while the sulphuric acid appears dark red with a greenish 
 fluorescence. If the chloroform solution is poured into a porcelain dish 
 it becomes violet, then green, and finally yellow. 
 
 Liebermann-Burchard's 3 Reaction. Dissolve the cholesterin in 
 about 2 cc. chloroform and add first 10 drops of acetic anhydride and then 
 concentrated sulphuric acid drop by drop. The color of the mixture 
 will first be a beautiful red, then blue, and finally, if not too much 
 cholesterin or sulphuric acid is present, a permanent green. In the pres- 
 ence of very little cholesterin the green color may appear immediately. 
 
 Neuberg-Rauchwerger's 4 Reaction. With rhamnose, or better still 
 with 6-methylfurfurol and concentrated sulphuric acid, an alcoholic 
 solution of cholesterin gives a pink ring, or after mixing the liquids and 
 cooling, a pink solution. On proper dilution an absorption-band can 
 be seen just beginning before E and whose other side coincides with b. 
 This reaction is of interest because it is also given by bile-acids, some 
 camphor derivatives, abietinic acid, and a hydride of retene. 
 
 1 Compt. rend. soc. biol., 58. 
 
 2 Pfluger's Arch., 6. 
 
 3 C. Liebermann, Ber. d. deutsch. chem. Gesellsch., 18; 1804, H. Burchard, Bei- 
 trage zur Kenntnis der Cholesterine, Rostock, 1899. 
 
 4 Salkowski's Festschrift, 1904. 
 
448 THE LIVER. 
 
 LiFSCHtJTz's 1 Reaction. Dissolve a few milligrams of cholesterin 
 in 2-3 cc. glacial acetic acid, add a little benzoylsuperoxide thereto, and 
 boil once or twice. On adding 4 drops concentrated sulphuric acid to 
 the cooled solution and shaking, a pure green coloration is obtained, 
 which changes immediately into blue or with violet-red as an intermediary 
 color. An absorption-band is formed between C and d, and a broad band 
 at D. In this reaction an oxidation of the cholesterin occurs, and Lif- 
 schutz 2 therefore uses the glacial acetic acid-sulphuric acid reaction 
 (color and spectrum) for the detection of oxidation products of choles- 
 terin in blood and tissues. 
 
 Pure, dry cholesterin when fused in a test-tube over a low flame with two or 
 three drops of propionic anhydride yields a mass which on cooling is first violet, 
 then blue, green, orange, carmine-red, and finally copper-red. It is best to re-fuse 
 the mass on a glass rod and then to observe the rod on cooling, holding it against 
 a dark background (Obermuller). 3 
 
 Schiff's Reaction. If a little cholesterin is placed in a porcelain dish with 
 the addition of a few drops of a mixture of 2 or 3 vols, of concentrated hydrochloric 
 acid or sulphuric acid and 1 vol. of a rather dilute solution of ferric chloride, and 
 carefully evaporated to dryness over a small flame, a reddish-violet residue is. 
 first obtained and then a bluish- violet. 
 
 If a small quantity of cholesterin is evaporated to dryness with a drop of 
 concentrated nitric acid, one obtains a yellow spot which becomes deep orange-red 
 with ammonia or caustic soda (not a characteristic reaction). ' 
 
 Cholesterin combines with saponin (Windaus, Yagi) and when a solution 
 of cholesterin in boiling 95 per cent alcohol is treated with a warm 1 per cent 
 solution of digitonin in 90 per cent alcohol, a precipitate of digitonin-cholesteride 
 is obtained. If the amount of the washed and dried digitonin-cholesteride is 
 multiplied by 0.25 the quantity of cholesterin is obtained (Windaus 4 ). The 
 cholesterin esters are not precipitated by digitonin. 
 
 Koprosterin is the name given by Bondzynski to the cholesterin which was 
 isolated by him from human feces, although it was prepared earlier by Flint 
 and designated as stercorin. It dissolves in cold, absolute alcohol and very readily 
 in ether, chloroform, and benzene. It crystallizes in fine needles which melt at 
 95-96° C. (89-90° according to Hausmann), and is dextrorotatory (of) D = +24°. 
 It gives the same color reactions as cholesterin, with the exception that it does 
 not give a reaction with propionic anhydride. According to Bondzynski and 
 Humnicki it is a dihydrocholesterin, with the formula C27H48O, which is formed 
 in the human intestine by the reduction of ordinary cholesterin. According to 
 Kusumoto as well as Dorbe and Gardner, koprosterin also occurs in the intes- 
 tine of dogs. The koprosterin prepared by H. Fischer from human feees seems 
 to be identical with that prepared by Bondzynski. It is remarkable that Boehm 6 
 
 1 Ber. d. d. chem. Gesellsch., 41. 
 
 2 Zeitschr. f. physiol. Chem., 50, 53, 58, and Ber. d. d. chem. Gesellsch., 41. 
 
 3 Zeitschr. f. physiol. Chem., 15. 
 
 * Windaus, Zeitschr. f. physiol. Chem., 65; Yagi, Arch. f. exp. Path. u. Pharm., 64. 
 
 6 Bondzynski, Ber. d. deutsch. chem. Gesellsch., 20; Bondzynski and Humnicki, 
 Zeitschr. f. physiol. Chem., 22; Flint, ibid., 23, and Amer. Journ. Med. Sciences, 
 1862; Muller, Zeitschr. f. physiol. Chem., 20; Hausmann, Hofmeister's Beitrage, 6; 
 Kusumoto, Bioch. Zeitschr., 14; Dor6e and Gardner, Proc. Roy. Soc. London, 80, 
 Bet I' : II. Fischer, Zeitschr. f. physiol. Chem., 73; Boehm, Bioch. Zeitschr., 33. 
 
CH0LESTERIN8. 449 
 
 found a dihydrocholesterin in the contents in a part of the ileum which had been 
 disconnected from the other part of the intestine for 14 years. This had the 
 same optical rotation and the same melting-point, 142-143° C, as the dihydro- 
 cholesterin (j8-cholestanol) prepared by Diels and Abderhalden, Willstatter 
 and Mayer. 
 
 Hippokoprosterin is another cholesterin richer in hydrogen, which Bondzynbkj 
 and Hi M\i( Ki found in the feces of the horse. Its formula is C^Ho.O. According 
 to I>i>kki: and Gardner it is not an animal cleavage product, but a constituent 
 of the grass used as fodder. It melts at 78.5-79.5° C. 
 
 Isocholesterin is a cholesterin, so called by Schulze, 1 with the formula 
 ChHuO, which occurs in wool-fat, and is therefore found to a great extent in 
 so-called lanolin. It gives the Liebermann-Burchard reaction, but .does not 
 give Salkowski's reaction. It melts at 138-138.5° C. The specific rotation in 
 7 per ceni ethereal solution is (a) D = 4-59.1°. 
 
 Spongosterin, C^ILsO is themame given by Henze 2 to a cholesterin isolated by 
 him from a silicious sponge. It is very similar to cholesterin, but is not identical 
 with it or with phytocholesterins. It gives the Liebermann-Burchard reaction as 
 well as Salkowski's reaction, but the last test is not quite so beautiful a red. 
 Obermuller's reaction is negative. Melting-point 123-124°. 
 
 Bombicesterin is the name given by Menozzi and Moreschi 3 to a cholesterin 
 isolated by them from the chrysalis of the silkworm, which has a melting-point 
 of 148° and a specific rotation of (<*)d = —34°. 
 
 The cholesterin occurring in the intestine is derived in part from the 
 food, in part from the bile and part, as shown from the contents of a 
 ligatured portion of the intestine (see Chapter VIII), from the epithelium 
 or the secretion of the intestinal mucosa. That a part of the cholesterin 
 of the intestine disappears has been shown by Kusumoto, although 
 it remains undecided whether this takes place by bacterial decomposi- 
 tion or by absorption. Levites 4 on the contrary, recovered the cho- 
 lesterin introduced into dogs almost quantitatively. The behavior of 
 cholesterin in metabolism is not well known; Lifschutz believes that he 
 has detected by his color-reaction the oxidation products of cholesterin 
 in the blood and in bone-fat. 
 
 The cholesterins belong to the so-called lipoids, which have been 
 mentioned in previous chapters (I and VI), and are of the greatest 
 importance as constituents of the outer envelope of erythrocytes and 
 the cells in general. Cholesterin is also of great interest because it inhibits 
 or prevents the haemolysis produced by certain bodies, and therefore 
 acts as a certain protective power in the animal bod}'. This action of 
 the cholesterins in regard to inhibiting the haemolytic action of saponin, 
 
 1 Ber. d. deutsch. chem. Gesellsch., 6; Journal f. prakt. Chem. (N. F.), 25; and 
 Zeitschr. f. physiol. Chem., 14, 522. See also E. Schulze and J. Barbieri, Journal f. 
 prakt. Chem. (N. F.) ( 25, 159. In regard to the formula for isocholesterin, see 
 Darmstiidter and Lifschutz, Ber. d. deutsch. chem. Gesellsch., 31, and E. Schulze, 
 ibid., 1200. 
 
 2 Zeitschr. f. physiol. Chem., 41 and 55. 
 
 s Cited from Chem. Centralbl., 1908, 1377 and 1910, 872. 
 4 Zeitschr. f. physiol. Chem., 57. 
 
450 THE LIVER. 
 
 as first discovered by Ransom, is destroyed, as shown by Hausmann, by 
 replacing the hydroxyl groups. These combinations between cholesterin 
 and saponins have been studied by Madsen and Noguchi and by 
 Windaus. 1 
 
 The so-called cholesterin-stones are best employed in the preparation 
 of cholesterin. The powder is first boiled with water and then repeatedly 
 boiled with alcohol. The cholesterin which on cooling separates from the 
 warm filtered solution is boiled with a solution of caustic potash in alcohol 
 so as to saponify any fat. After the evaporation of the alcohol the choles- 
 terin is extracted from the residue with ether, by which the soaps are 
 not dissolved; filter, evaporate the ether, and purify the cholesterin by 
 recrystallization from alcohol-ether. The cholesterin may be extracted 
 with fat from tissues and fluids by first extracting with ether and then 
 proceeding as suggested by Ritter. 2 The essential points in his method 
 consist in saponifying the fat with sodium alcoholate, removing the alcohol 
 by evaporating to dryness with NaCl, and finally extracting the dried 
 pulverized mass with ether. After evaporating the ether the residue is 
 dissolved in as little alcohol as possible and the cholesterin precipitated 
 by the addition of water. It is ordinarily easily detected in transudates 
 and pathological formations by means of the microscope. In regard to 
 the methods of preparation, detection and quantitative estimation of 
 cholesterin we refer to the larger text-books. 
 
 Ransom, Deutsch. med. Wochenschr., 1901; Hausmann, Hofmeister's Beitrage, 
 6; Madsen and Noguchi, Kgl. Dansk. Vidensk. Selskabs. Forh., 1904; Windaus, Ber. 
 d. d. chem. Gesellsch., 42. 
 
 2 Zeitschr. f. physiol. Chem., 34. See also Corper, Journ. of biol. Chem., 11. 
 
CHAPTER VIII. 
 DIGESTION. 
 
 The purpose of digestion is to separate those constituents of the 
 food which serve as nutriment for the body from those which are use- 
 less, and to separate each in such a form that it may be taken up by the 
 blood from the alimentary canal and employed for various purposes in 
 the organism. This demands not only mechanical, but also chemical, 
 action. The first action, which is essentially dependent upon the physical 
 properties of the food, consists in a tearing, cutting, crushing, or grinding 
 of the food, while the second serves chiefly in converting the nutritive 
 bodies into a soluble and easily absorbable form, or in splitting them into 
 simpler compounds for use in the animal syntheses. The solution of the 
 nutritive bodies may take place in certain cases by the aid of water alone, 
 but in most cases a chemical metamorphosis or cleavage is necessary; 
 this is effected by means of the acid or alkaline fluids secreted by the 
 glands. The study of the processes of digestion from a chemical stand- 
 point must therefore begin with the digestive fluids, their qualitative 
 and quantitative composition, as well as their action on the nutriments 
 and foods. 
 
 I. THE SALIVARY GLANDS AND THE SALIVA. 
 
 The salivary glands are partly albuminous glands (as the parotid 
 in man and mammals, and the submaxillary in rabbits), partly mucous 
 glands (as some of the small glands in the buccal cavity and the sub- 
 lingual and submaxillary glands of many animals), and partly mixed 
 glands (as the submaxillary gland in man). The alveoli of the albuminous 
 glands contain cells which are rich in protein but which contain no mucin. 
 The alveoli of the mucin-glands contain cells rich in mucin but poor in 
 protein. Cells arranged in different ways, but rich in proteins, also occur 
 in the submaxillary and sublingual glands. According to the analyses 
 of Magnus-Levy 1 the human salivary glands contain 274 p. m. solids, 
 of which 114 p. m. was fat and 154 p. m. was protein. Among the 
 solids we find mucin, proteins, nucleoproteins, nuclein, enzymes and theix 
 
 1 Bioch. Zeitschr., 24. 
 
 451 
 
452 DIGESTION. 
 
 zymogens, besides extractive bodies, leucine, purine bases, and mineral 
 substances. 
 
 The occurrence of a mucinogen has not been proved. On the complete removal 
 of all mucin E. Holmgren 1 found no mucinogen in the submaxillary gland of 
 the ox, but a mucin-like gluconucleoproteid. 
 
 1 he saliva is a mixture of the secretion of the above-mentioned groups 
 of glands; therefore it is proper that a study be made of each of the dif- 
 ferent secretions by itself and then of the mixed saliva. 
 
 The submaxillary saliva in man may be easily collected by intro- 
 ducing a canula through the papillary opening into Wharton's duct. 
 
 The submaxillary saliva has not always the same composition or 
 properties; this depends essentially, as shown by experiments on animals, 
 upon the conditions under which the secretion takes place. That is to 
 say, the secretion is partly dependent on the cerebral system, through 
 the facial fibers in the chorda tympani, and partly on the sympathetic 
 nervous system, through the fibers entering the vessels in the gland. In 
 consequence of this dependence the two distinct varieties of submaxillary 
 secretion are distinguished as chorda- and sympathetic saliva. A third 
 kind of saliva, the so-called paralytic saliva, is secreted after poisoning 
 with curare or after the severing of the glandular nerves. 
 
 The difference between chorda- and sympathetic saliva (in dogs) 
 consists chiefly in their quantitative constitution; the less abundant 
 sympathetic saliva is more viscous and richer in solids, especially in 
 mucin, than the more abundant chorda-saliva. The specific gravity of 
 the chorda-saliva of the dog is 1.0039-1.0056, and contains 12-14 p. m. 
 solids (Eckhard 2 ) . The sympathetic has a specific gravity of 1 .0075-1 .018, 
 with 16-28 p. m. solids. The freezing-point of the chorda-saliva obtained 
 from dogs on electric stimulation varies, according to Nolf, 3 between 
 A = -0.193° and -0.396°, with a content of 3.3-6.5 p. m. salts and 4.1- 
 11.5 p. m. organic substances. The osmotic pressure is on am average a 
 little higher than one-half the osmotic pressure of the blood-serum. The 
 spontaneously secreted submaxillary saliva is ordinarily somewhat diluted. 
 On changing the osmotic pressure of the blood the osmotic pressure 
 of the saliva, according to Jappelli, 4 changes in the same direction. 
 According to Demoor, Locke's solution with some dog serum is well 
 suited by transfusion to keep the submaxillary gland of the dog in 
 activity, while ox serum is unsuited. 5 The gases of the chorda-saliva 
 
 1 Upsala Lakaref. F6rb. (N. F.), 2; also Maly's Jahresber., 27. 
 
 2 Cited from Kiihne'a Lebrb. d. physiol. Chem., 7. 
 <<■<-. Maly's Jahresber., 81, 494. 
 
 4 .Jappelli, ibid., 48 and 51. 
 
 5 Arch, intern, de Physiol., 10 (1911). 
 
SALIVA. 453 
 
 have been investigated by PflUgeb. 1 He found 0.5-0.8 per cent oxygen, 
 0.9-1 per cent nitrogen, and 64.73-85.13 per cent carbon dioxide — 
 all results calculated at 0° C. and 760 mm. pressure. The greater part 
 of the carbon dioxide was chemically combined. 
 
 The two kinds of submaxillary secretion just named have not thus 
 far been separately studied in man. The secretion may be excited by an 
 emotion, by mastication, and by irritating the mucous membrane of the 
 mouth, especially with acid-tasting substances. The submaxillary saliva 
 in man is ordinarily clear, rather thin, a little ropy, and froths easily. 
 Its reaction is alkaline toward litmus. The specific gravity is 1.002- 
 1.003, and it contains 3.6-4.5 p. m. solids. 2 As organic constituents 
 are found mucin, traces of protein and diastatic enzyme, which latter is 
 absent in several species of animals. The inorganic bodies are alkali 
 chlorides, sodium and magnesium phosphates, and bicarbonates of the 
 alkalies and calcium. Potassium sulphocyanide occurs in this saliva. 
 
 The Sublingual Saliva. The secretion of this saliva is also influenced 
 by the cerebral and the sympathetic nervous system. The chorda-saliva, 
 which is secreted only to a small extent, contains numerous salivary 
 corpuscles, but is otherwise transparent and very ropy. Its reaction 
 is alkaline, and it contains, according to Heidenhain, 3 27.5 p. m. solids 
 (in dogs). 
 
 The sublingual secretion in man is clear, mucilaginous, more alka- 
 line than the submaxillary saliva, and contains mucin, diastatic enzyme, 
 and potassium sulphocyanide. 
 
 Buccal mucus can be obtained pure, from animals only, by the method 
 suggested by Bidder and Schmidt, which consists in tying the exit to 
 all the large salivary glands and cutting off their secretion from the mouth. 
 The quantity of liquid secreted under these circumstances (in dogs) was 
 so very small that the investigators named were able to collect only 2 
 grams of buccal mucus in the course of one hour. It is a thick, ropy, 
 sticky liquid containing mucin; it is rich in form-elements, above all 
 in flat epithelium cells, mucous cells, and salivary corpuscles. The 
 quantity of solids in the buccal mucus of the dog is, according to 
 Bidder and Schmidt, 4 9.98 p. m. 
 
 Parotid Saliva. The secretion of this saliva is also partly dependent 
 on the cerebral nervous system (n. glossopharyngeus) and partly on the 
 sympathetic. The secretion may be excited by emotions and by irri- 
 
 1 Pfluger's Arch., 1. 
 
 2 See Maly's "Chemie der Verdauungssafte und der Verdauung," in Hermann's 
 Handb., 5, part II, 18. This article contains also the pertinent literature. 
 
 3 Studien. d. physiol. Instituts zu Breslau, Heft 4. 
 
 * Die Verdauungssafte und der Stoffwechsel (Mitau and Leipzig, 1852), p. 5. 
 
454 DIGESTION. 
 
 tation of the glandular nerves, either directly (in animals), or reflexly, 
 by mechanical or chemical irritation of the muccus membrane of the 
 mouth. Among the chemical irritants the acids take first place. Mas- 
 tication also exercises a strong influence upon the secretion of parotid 
 saliva, which is specially marked in certain herbivora. 
 
 Human parotid saliva may be readily collected by the introduction 
 of a canula into Stenson's duct. This saliva is thin, less alkaline than 
 the submaxillary saliva (the first drops are sometimes neutral or acid), 
 without special odor or taste. It contains a little protein but no mucin, 
 which is to be expected from the construction of the gland. It also con- 
 tains a diastatic enzyme, which, however, is absent in many animals. 
 The quantity of solids varies between 5 and 16 p. m. The specific gravity 
 is 1.003-1.012. Potassium sulphocyanide seems to be present, though 
 it is not a constant constituent. Kulz * found a maximum of 1.46 per 
 cent oxygen, 3.8 per cent nitrogen, and in all 66.7 per cent carbon dioxide 
 in human parotid saliva. The quantity of firmly combined carbon dioxide 
 was 62 per cent. 
 
 The quantity and composition of the saliva, from the mucin glands 
 as well as from the albuminous glands, show differences in the various 
 classes of animals but these cannot be entered into here. According 
 to Pawlow 2 and his pupils the quantity as well as the composition of 
 the saliva of the various glands and the mixed saliva in dogs is to a great 
 degree dependent upon the psychical stimulation, but also upon the 
 kind of substances introduced into the mouth, and an adaptation of 
 the glands for various mechanical and chemical irritants is found to occur. 
 
 Popielski 3 disputes the existence of such an accommodation (in 
 dogs) to the kind of food and to the kind of stimulation. In man an 
 accommodation of the salivary glands, to the needs, has also been sug- 
 gested but the statements are still not unanimous. 1 See also Chapter 
 I (page 53). 
 
 The mixed buccal saliva in man is a colorless, faintly opalescent, 
 slightly ropy, easily frothing liquid without special odor or taste. It 
 is made turbid by epithelium cells, mucous and salivary corpuscles, 
 and often by food residues. Like the submaxillary and parotid saliva, 
 on exposure to the air it becomes covered with an incrustation consist- 
 ing of calcium carbonate and a small quantity of an organic substance, 
 
 1 Zeitschr. f . Biologie, 23. 
 
 2 Arch, internat. de Physiol., 1, 1904. See also Boos, Maly's Jahresber., 36. 390, 
 and Neilson and Terry, Amer. Journ. of Physiol., 15, as well as the work of Mendel 
 and Underbill, Journ. of biol. Chem., 3. 
 
 'Popielski, Pfluger's Arch., 127; Zebrowski, Pfluger's Arch., 110; Neilson and 
 Lewis, Journ. of biol. Chem., 4, with Scheele, ibid., 5; Carlson and Chittenden, Amer.. 
 Journ. of Physiol., 2(J. 
 
MIXED SALIVA. 455 
 
 or it gradually becomes cloudy. Its reaction is generally alkaline to 
 litmus. The degree of alkalinity varies considerably not only in dif- 
 ferent individuals but also in the same individual during different parts 
 of the day, so that it is difficult to state the average alkalinity. Accord- 
 ing to Chittenden and Ely it corresponds to the alkalinity of 0.8 p. m. 
 Na2C03 solution, or to 0.2 p. m. solution according to Cohn. According 
 to Foa the actual alkalinity (OH-ion concentration) is always consider- 
 ably less than that found by titration, and the reaction determined 
 electrometrically is very nearly neutral. The reaction may also be acid, 
 as found to be the case by Sticker some time after a meal, but this is 
 not true, at least for all individuals. The specific gravity varies between 
 1.002 and 1.008, and the quantity of solids between 5 and 10 p. m. 
 According to Cohn, 1 A= —0.20° on an average, and the amount of NaCl 
 is 1.6 p. m. The solids, irrespective of the form-constituents men- 
 tioned, consist of protein, mucin, oxidases, 2 two enzymes, ptyalin and 
 maltase, as well as a dipeptid and a tripeptid splitting enzyme 3 and 
 mineral bodies. It is also claimed that urea is a normal constituent of 
 the saliva. The mineral bodies are alkali chlorides, bicarbonates of 
 the alkalies and calcium, phosphates, and traces of sulphates, nitrites, 
 ammonia, and sulphocyanides, which latter average about 0.1 p. in. 
 (Munk and others). Smaller quantities, 0.03-0.04 p. m., are found in 
 the saliva of non-smokers (Schneider and Kruger), while from ordin- 
 ary smokers the quantity of sulphocyanides may rise to 0.2 p. m. 
 (Fleckseder 4 ). 
 
 Sulphocyanides, which, although not constant, occur in the saliva of 
 man and certain animals, may be easily detected by acidifying the saliva 
 with hydrochloric acid and treating with a very dilute solution of ferric 
 chloride. As control, especially in the presence of very small quantities, 
 it is best to compare the test with another test-tube containing an equal 
 amount of acidulated water and ferric chloride. Other methods have 
 been suggested by Gscheidlen, Solera, and Ganassini. The quantita- 
 tive estimation can be done according to Munk's 5 method. 
 
 Chittenden and Ely, Amer. Chem. Journ., 4, 1883; Chittenden and Richards, 
 Amer. Journ. of Physiol., 1; Foa, Compt. rend. soc. biol., 58; Sticker, cited from 
 Centralbl. f. Physiol., 3, 237; Cohn, Deutsch. med. Wochenschr., 1900. 
 
 2 Bogdanow-Beresowski, cited from Biochem. Centralbl., 2, 653; Herlitzka, Maly's 
 Jahresber., 40; Spanjer-Herford, Virchow's Arch., 205. 
 
 MVarfield, Johns Hopkins Hosp. Bull. 22 (1911); Koelker, Zeitschr. f. physiol. 
 Chem., 76, (1911). 
 
 4 Munk, Virchow's Arch., 69; Schneider, Amer. Journ. of Physiol., 5; Kruger, 
 Zeitschr. f. Biologie, 37; Fleckseder, Centralbl. f. innere Med., 1905. In regard to 
 the variation in the amount of various constituents in saliva see Fleckseder, 1. c, and 
 Tezner, Arch, internat. de Physiol., 2. 
 
 5 Gscheidlen, Maly's Jahresber., 4; Solera, see ibid., 7 and 8; Munk, Virchow's 
 Arch., 69; Ganassini, Biochem. Centralbl., 2, p. 361. 
 
456 DIGESTION. 
 
 Ptyalin, or salivary diastase, is the amylolytic enzyme of the saliva. 
 This enzyme is found in human saliva, 1 but not in that of all animals, 
 especially not in the typical carnivora. It occurs not only in adults, 
 but also in new-born infants. In opposition to Zweifel's views, Ber- 
 ger 2 claims that it is present not only in the parotid gland of children, 
 but also in the mucin glands. 
 
 According to H. Goldschmidt 3 the saliva (parotid saliva) of the horse does 
 not contain ptyalin, but a zymogen of the same, while in other animals and man 
 the ptyalin is formed from the zymogen during secretion. In horses the zymogen 
 is transformed into ptyalin during mastication, and bacteria seem to give the 
 impulse to this change. During precipitation with alcohol the zymogen is changed 
 into ptyalin. 
 
 Ptyalin has not been isolated in a pure form up to the present time. 
 It can be obtained purest by Cohnheim's 4 method, which consists in 
 carrying the enzyme down mechanically with a calcium-phosphate 
 precipitate, and washing the precipitate with water, which dissolves the 
 ptyalin, and from which it can be obtained by precipitating with alcohol. 
 For the study or demonstration of the action of ptyalin one employs a 
 watery or glycerin extract of the salivary glands, or simply the saliva 
 itself. 
 
 Ptyalin, like other enzymes, is characterized by its action. This 
 consists in converting starch into dextrins and sugar. Our knowledge 
 as to the process going on here is just as uncertain as our knowledge 
 on the formation of sugar from starch (see page 229). The nature of 
 the sugar thus produced is known with certainty. For a long time it 
 was considered that glucose was the sugar formed from starch and 
 glycogen, but Seegen and O. Nasse have shown that this is not true. 
 Muculus and v. Mering have shown that the sugar formed by the 
 action of saliva, amylopsin, and diastase upon starch and glycogen is 
 for the most part maltose. This has been substantiated by Brown 
 and Heron. E. Kulz and J. Vogel 5 have also demonstrated that 
 in the saccharification of starch and glycogen, isomaltose, maltose, and 
 some glucose are formed, the varying quantities depending upon the 
 amount of ferment and the length of its action. The formation of 
 
 1 In regard to the variation in the quantity of ptyalin in human saliva see Hof- 
 bauer, Centralbl. f. Physiol., 10, and Chittenden and Richards, Amer. Journ. of Physiol., 
 1; Schiile, Maly's Jahresber., 29; Tezner, 1. c. 
 
 2 Zweifel, Untersuchungen iiber den Verdauungsapparat der Neugeborenen (Berlin, 
 1874); Berger, see Maly's Jahresber., 30, 399. 
 
 1 Zeitschr. f. physiol. Chem., 10. 
 
 * Virchow's Arch., 28. 
 
 6 Seegen, Centralbl. f. d. med. Wissensch., 1876, and Pfliiger's Arch., 19; Nasse, 
 ibid., 14; Musculus and v. Mering, Zeitschr. f. physiol. Chem., 2; Brown and Heron, 
 Liebig's Annal., 199 and 204; Kulz and Vogel, Zeitschr. f. Biologie, 31. 
 
PTYALIN. 457 
 
 glucose is claimed by Tebb, Rohmann, and Hamburger 1 to be only a 
 product of the inversion of the maltose by the maltase. 
 
 The action of ptyalin in various reactions has been the subject of 
 1 umerous investigations. 2 Natural alkaline saliva is very active, but 
 it is not so active as when made neutral. It may be still more active 
 under certain circumstances in faintly acid reaction, and according to 
 Chittenden and Smith it acts better when enough hydrochloric acid 
 is added to saturate the proteins present than when only neutralized. 
 When the acid-combined protein exceeds a certain amount, then the 
 diastatic action is diminished. The addition of alkali to the saliva 
 decreases its diastatic action; on neutralizing the alkali with acid or 
 carbon dioxide the retarding or preventive action of the alkali is arrested. 
 According to Schierbeck, carbon dioxide has an accelerating action in 
 neutral liquids, while Ebstein claims that it has, as a rule, a retarding 
 action. Organic as well as inorganic acids, when added in sufficient 
 quantity, may stop the diastatic action entirely. The degree of acidity 
 necessary in this case is not always the same for a certain acid, but is 
 dependent upon the quantity of ferment. The same degree of acidity 
 in the presence of large amounts of ferment has a weaker action than in 
 the presence of smaller quantities. Hydrochloric acid is of special 
 physiological interest in this regard, for it prevents the formation of 
 sugar even in very small amounts (0.03 p. m.). Hydrochloric acid has 
 not only the property of preventing the formation of sugar, but, as 
 shown by Langley, Nylen, and others, may entirely destroy the 
 enzyme. This is important in regard to the physiological significance 
 of the saliva. 
 
 Foreign substances, such as metallic salts, 3 have different effects. 
 Certain salts, even in small quantities, completely arrest the action; 
 for example, HgCl2 accomplishes this result completely in the presence 
 of only 0.05 p. m. Others have an accelerating action, and this seems 
 to apply to the salts of the saliva. According to Guyenot the saliva 
 has a weaker action the more it is freed from salts by dialysis. On the 
 
 1 Tebb, Journ. of Physiol., 15; Rohmann, Ber. d. deutsch. chem. Gesellsch., 27; 
 Hamburger, Pfluger's Arch., 60. 
 
 2 See Hammarsten, Maly's Jahresber., 1; Chittenden and Griswold, Amer. Chem. 
 Journ., 3; Langley, Journal of Physiol., 3; Nylen, Maly's Jahresber., 12, 241; Chit- 
 tenden and Ely, Amer. Chem. Journ., 4; Langley and Eves, Journal of Physiol., 4: 
 Chittenden and Smith, Yale College Studies, 1, 1885, 1; Schlesinger, Virchow's Arch. 
 125; Schierbeck, Skand. Arch. f. Physiol., 3; Ebstein and C. Schulze, Virchow's 
 Arch., 134; Kubel, Pfluger's Arch., 56. 
 
 3 See O. Nasse, Pfluger's Arch., 11, and Chittenden and Painter, Yale College 
 Studies, 1, 1885, 52; Kubel, Pfluger's Arch., 76; Patten and Stiles, Amer. Journ. of 
 Physiol., 17. 
 
458 
 
 DIGESTION. 
 
 addition of salts the dialyzed saliva becomes active again, especially 
 on the addition of calcium or potassium chloride (see also page 71). 
 Roger 1 believes that the presence of phosphates is a necessity for the 
 action of saliva. The amount of salts added is of special importance 
 for the action of the saliva, and one salt, which in small quantities has 
 an accelerating action, may in large quantities have a retarding action. 
 The presence of peptone has an accelerating action on the sugar forma- 
 tion (Chittenden and Smith and others). 
 
 To show the action of saliva or ptyalin on starch the three ordinary 
 tests for glucose may be used, namely, Moore's or Trommer's test or 
 the bismuth test (see Chapter III). It is also necessary, as a control, 
 to first test the starch-paste and the saliva for the presence of glucose. 
 The steps in the transformation of starch into amidulin, erythrodextrin, 
 and achroodextrin may be shown by testing with iodine. 
 
 Maltase occurs in saliva to only a slight extent. It converts maltose 
 into glucose. According to Sticker, 2 saliva also has the power of split- 
 ting sulphureted hydrogen from the sulphur oils of radishes, onions, 
 and certain other vegetables. 
 
 The quantitative composition of the mixed saliva must vary consider- 
 ably, not only because of individual differences, but also because under 
 varying conditions there may be an unequal division of the secretion 
 from the different glands. We give herewith a few analyses of human 
 saliva as examples of its composition. The results are in parts per 1000. 
 
 
 as 
 D 
 
 B 
 M 
 PS 
 
 W 
 
 pa 
 
 a 
 
 m 
 
 O 
 
 m 
 P 
 o 
 < 
 
 m 
 W 
 
 o 
 
 5 
 s 
 a 
 
 13 
 
 a 
 a 
 
 z . 
 
 s J 
 "a 
 sO 
 H 
 
 a 
 
 H 
 
 « 
 B 
 W 
 
 55 
 Z 
 <! 
 
 a 
 « 
 
 H 
 
 i « 
 
 K a 
 S W 
 
 < « 
 
 Water 
 
 992.9 
 7.1 
 
 1.4 
 3.8 
 
 995 . 16 
 4.84 
 
 1.62 
 1.34 
 
 994.1 
 5.9 
 
 2.13 
 1.42 
 
 988.3 
 11.7 
 
 994.7 
 5.3 
 
 3.5-8.4 
 
 in 
 filtered 
 saliva. 
 
 994.2 
 
 Solids. 
 
 5.8 
 
 Mucus and epithelium .... 
 Soluble organic substances. 
 
 2.2 
 
 
 3.27 
 
 
 1.4 
 
 (Ptyalin of early investigators.) 
 
 
 0.06 
 
 0.10 
 
 
 
 0.064 
 
 0.04 
 
 Salts 
 
 1.9 
 
 1.82 
 
 2.19 
 
 
 1.30 
 
 to 
 0.090 
 
 2.2 
 
 
 
 1 Guyenot, Compt. rend. soc. biol., 63; Roger, ibid., 65; see also Bang, Bioch. 
 Zeitechr., 32 (1911). 
 
 2 Miinch. med. Wochenschr., 43. 
 
 » Zeitechr. f. physiol. Chem., 5. The other analyses are cited from Maly, Chemie 
 der Verflauvmgssafte, Hermann's Handbuch d. Physiol., 5, Part II, 14. 
 
SECRETION OF SALIVA. 459 
 
 Hammerbacher found in 1000 parts of the ash from human saliva: potash 
 457.2, soda 95.9, iron oxide 50.11, magnesia 1.55, sulphuric anhydride (S0 3 ) G3.8, 
 phosphoric anhydride (PiO») 188.48, and chlorine 183.52. 
 
 The quantity of saliva secreted during twenty-four hours cannot 
 be exactly determined, but has been calculated by Bidder and Schmidt 
 to be 1400-1500 grams. The most abundant secretion occurs during 
 meal-times. According to the calculations and determinations of 
 Tuczek, 1 in man 1 gram of gland yields 13 grams of secretion in the course 
 of one hour during mastication. These figures correspond fairly well 
 with those representing the average secretion from 1 gram of gland in 
 animals, namely, 14.2 grams in the horse and 8 grams in oxen. The 
 quantity of secretion per hour may be 8 to 14 times greater than the entire 
 mass of glands, and there is probably no gland in the entire body, so far 
 as is known at present — the kidneys not excepted — whose ability of 
 secretion under physiological conditions equals that of the salivary glands. 
 But as the secretion of saliva is so very variable under different con- 
 ditions no positive results can be given as to the extent. A remark- 
 ably abundant secretion of saliva is induced by pilocarpine, while 
 atropine, on the contrary, inhibits it. 
 
 That the secretion of saliva, even if we do not consider such sub- 
 stances as ptyalin, mucin, and the like, is not a process of filtration, 
 follows for many reasons, especially the following: The salivary glands 
 have a specific property of eliminating certain substances, such as 
 potassium salts (Salkowpki 2 ), iodine, and bromine compounds, but 
 not others, for example, iron compounds and glucose. It is also notice- 
 able that the saliva is richer in solids when it is eliminated quickly by 
 gradually increased stimulation, and in larger quantities than when the 
 secretion is slower and less abundant (Heidenhain). The amount of 
 salts increases also to a certain degree by an increasing rapidity of 
 elimination (Heidenhain, Werther, Langley and Fletcher, Novi 3 ). 
 
 Like the secretion processes in general, the secretion of saliva is 
 closely connected with the processes in the cells. The chemical processes 
 going on in these cells during secretion are still unknown. 
 
 The Physiological Importance of the Saliva. — The quantity of water 
 in the saliva renders possible the action of certain bodies on the organs 
 of taste, and it also serves as a solvent for a part of the nutritive sub- 
 stances. The importance of the saliva in mastication is especially 
 marked in herbivora, and there is no question as to its importance in 
 
 1 Bidder and Schmidt, 1. c, 13; Tuczek, Zeitschr. f. Biologie, 12. 
 
 2 Virchow's Arch., 53. 
 
 1 Heidenhain, Pfluger's Arch., 17; Werther, ibid., 38; Langley and Fletcher, 
 Proc. Roy. Soc, 45, and especially Phil. Trans. Roy. Soc. London, 180; Novi, Arch, 
 f. (Anat. u.) Physiol., 1888. 
 
460 DIGESTION. 
 
 facilitating the act of swallowing. The saliva containing mucin is espe- 
 cially important in this regard, and Pawlow's school has shown that the 
 secretion also regulates itself in this regard. The saliva is also of import- 
 ance, as it serves in washing out the mouth and thereby acts as a pro- 
 tection against destructive substances or bodies foreign to the mouth. 
 The power of converting starch into sugar is not inherent in the saliva 
 of all animals, and even when it possesses this property the intensity 
 varies in different animals. In man, whose saliva forms sugar rapidly, 
 a production of sugar from (boiled) starch undoubtedly takes place in the 
 mouth, but how far this action proceeds after the morsel has entered the 
 stomach depends upon the rapidity with which the acid gastric juice mixes 
 with the swallowed food, and also upon the relative amounts of the 
 gastric juice and food in the stomach. The large quantity of water which 
 is swallowed with the saliva must be absorbed and pass into the blood, 
 and it must in this way go through an intermediate circulation in the 
 organism. Thus the organism possesses in the saliva an active medium 
 by which a constant stream, conveying the dissolved and finely divided 
 bodies, passes into the blood from the intestinal canal during digestion. 
 The relation of the saliva or the salivary glands to the secretion of gastric 
 juice will be mentioned in the next section. 
 
 Salivary Concrements. The so-called tartar is yellow, gray, yellowish-gray, 
 brown or black, and has a stratified structure. It may contain more than 200- 
 p. m. organic substances, which consist of mucin, epithelium, and leptothrix- 
 chains. The chief part of the inorganic constituents consists of calcium car- 
 bonate and phosphate. The salivary calculi may vary in size from that of a 
 small grain to that of a pea or still larger (a salivary calculus has been found 
 weighing 18.6 grams), and they contain variable quantities of organic substances 
 (50-380 p. m.), which remain on extracting the calculus with hydrochloric acid. 
 The chief inorganic constituent is calcium carbonate. 
 
 H. THE GLANDS OF THE MUCOUS MEMBRANE OF THE STOMACH, AND 
 
 THE GASTRIC JUICE. 
 
 The glands of the mucous coat of the stomach have long been 
 divided into two distinct classes. Those which occur in the greatest 
 abundance and which have the greatest size in the fundus are called 
 fundus, rennin or pepsin glands, and the others, which occur onl} r in 
 the neighborhood of the pylorus, have received the name of pyloric 
 glands, sometimes also, though incorrectly, called mucous glands. The 
 division of these two forms of glands in the mucous membrane of the 
 stomach is essentially different in various animals. The mucous coat- 
 ing of the stomach is covered throughout with a layer of columnar 
 epithelium, which is generally considered as consisting of goblet cells that 
 produce mucus by a metamorphosis of the protoplasm. 
 
GASTRIC JUICE. 461 
 
 The fundus glands contain two kinds of cells: adelomorphic or chief 
 cells, and delomorphic or cover cells, the latter formerly called rennin 
 or pepsin cells. Both kinds consist of protoplasm rich in proteins; 
 but their relation to coloring-matters seems to show that the protein 
 substances of both are not identical. The nucleus must consist princi- 
 pally of nuclein. Besides the above-mentioned constituents, the fundus 
 glands contain as more specific constituents several enzymes or their 
 zymogens, besides a little fat and cholesterin. 
 
 The pyloric glands contain cells which are generally considered as 
 related to the above-mentioned chief cells of the fundus glands. As 
 these glands were formerly thought to contain a larger quantity of 
 mucin, they were also called mucous glands. According to Heiden- 
 hain, independent of the columnar epithelium of the excretory ducts, 
 they take no part worthy of mention in the formation of mucus, which 
 according to his views is effected by the epithelium covering the mucous 
 membrane. The pyloric glands also seem to contain zymogens. Alkali 
 chlorides, alkali phosphates, and calcium phosphates are found in the 
 mucous coating of the stomach. 
 
 The Gastric Juice. The observations of Helm and Beaumont on 
 persons with gastric fistula led to the suggestion that gastric fistulas 
 be made on animals, and this operation was first performed by Basso w l 
 in 1842 on a dog. Verneuil performed the same on a man in 1876 with 
 successful results. Pawlow 2 has recently improved the surgery of 
 gastric fistula and has added much to the study of gastric secretion. 
 
 As most investigations upon gastric digestion, and also upon diges- 
 tion as a whole, are based on observations upon dogs and then upon man, 
 and for this reason, when not otherwise stated, in this chapter on the 
 study of digestion we give the conditions in dogs and man. 
 
 The secretion of gastric juice is not continuous, at least in man and 
 in the mammals experimented upon. It only occurs under psychic 
 influence, and also by stimulation of the mucous membrane of the stomach 
 or the intestine. The most exhaustive researches on the secretion of 
 gastric juice (in dogs) have been made by Pawlow and his pupils. 
 
 In order to obtain gastric juice free from saliva and food residues, they arranged 
 besides a gastric fistula also an oesophageal fistula from which the swallowed food 
 could be withdrawn with the saliva without entering the stomach, and in this 
 manner an apparent or sham feeding was possible. In this way it was possible to 
 
 1 Helm, Zwei Krankengeschichten, Wien, 1803, cited from Hermann's Handbuch, 
 5, part II, 39; Beaumont, "The Physiology of Digestion," 1833; Bassow, Bull, de 
 la eoc. des natur. de Moscou, 16, cited from Maly in Hermann's Handbuch, 5, 38; 
 Verneuil, see Ch. Richet, "Du sue gastrique chez l'homme," etc. (Paris, 1878), 158. 
 
 2 Pawlow, The Work of the Digestive Glands, (translated by Thompson, Phila- 
 delphia, 1910), where the works of his pupils are also mentioned. See also Ergebnisse 
 der Physiologie, 1, Abt. 1. 
 
462 DIGESTION. 
 
 study the influence of psychical moments on one side and the direct action of food 
 on the mucous membrane on the other. After a method suggested by Heidenhain 
 and later improved by Pawlow and Chigin, they have succeeded in preparing 
 a blind sac by partial dissection of the fundus part of the stomach, and the secre- 
 tion processes could be studied in this sac while the digestion in the other parts 
 of the stomach was going on. In this way they were able to study the action of 
 different foods on the secretion. 
 
 The most essential results of the investigations of Pawlow and his 
 pupils are as follows: Mechanical stimulation of the mucosa does not 
 produce any secretion. Mechanical irritation of the mucous membrane 
 of the mouth causes no reflex excitation of the secretory nerves of the 
 stomach. There are two moments which cause a secretion, namely, 
 the psychical moment — the passionate desire for food and the sensa- 
 tion of satisfaction and pleasure on partaking it — and the chemical 
 moment, the action of certain chemical substances on the mucous mem- 
 brane of the stomach. The first moment is the most important. The 
 secretion occurring under its influence by the vagus fibers appears earlier 
 than that produced by chemical irritants, but only after an interval of 
 at least 4^ minutes. This secretion is more abundant but less contin- 
 uous than the " chemical." It yields a more acid and active juice than 
 the latter. As chemical excitants which cause a secretion reflexively 
 through the stomach mucosa we include water (slight action) and cer- 
 tain unknown extractive substances contained in meat and meat extracts, 
 in impure peptone, and also, it seems, in milk. According to Herzen 
 and Radzikowski 1 and others, alcohol is also a strong agent in produc- 
 ing a flow of juice. The claims in regard to the action of sodium chloride 
 and alkali carbonates are somewhat disputed. That the alkali carbonates 
 retard or inhibit secretion is the opinion of many, but from more 
 recent determinations 2 it would seem as if the concentration of the car- 
 bonate as well as of sodium chloride exercises a certain influence, so that 
 a weaker concentration is indifferent or retarding, while somewhat stronger 
 concentration has an accelerating action upon secretion, though inves- 
 tigators are not agreed as to results. Bitter substances partaken of in 
 small amounts a certain time before a meal increase the secretion, while 
 larger amounts have a retarding action (Bomssow, Strashesko 3 ). 
 Fats have a retarding action on the appearance of secretion and diminish 
 the quantity of juice secreted as well as the amount of enzyme. The 
 substances, such as egg-albumin, which do not act as chemical stimulants, 
 
 1 Pfluger's Arch., 84, 513. 
 
 2 See Rozenblatt, Bioch. Zeitschr, 4; Mayeda, ibid., 2; Pimenow, Bioch, Centralbl., 
 6; Lonnquist, Maly's Jahresb., 36. 
 
 ' Borissow, Arch. f. exp. Path. u. Pharm., 51; Strashesko, see Biochem. Centralbl., 
 4, 148. 
 
GASTRIC JUICE. 463 
 
 may be digested by the " psychical " secretion, and then perhaps cause 
 a chemical secretion by their decomposition products. 
 
 The secretion in the stomach may also be influenced by the small 
 intestine, and in this way, as shown by the investigations of Pawlow 
 and his pupils, the fats have a retarding action upon the secretion of 
 juice and upon digestion by acting reflexly upon the duodenal mucosa. 
 In dogs on feeding fat (oil) with food containing starch, the secretion of 
 gastric juice remains reduced during the entire period of feeding, and 
 Cat in connection with protein food has a similar action, with the excep- 
 tion that in this case the retarding action is observed only in the first 
 hours of digestion. According to Piontkowski x the oil-soaps differ from 
 the neutral fats by having a strong action on the flow of juice, and this 
 is the reason why about five to six hours after a meal with fat the secre- 
 tion of juice is stopped, as just at this time the soaps are being formed. 
 According to Frouin the food in the intestine produces a secretion of 
 gastric juice which continues after the action of the psychic moment 
 has ceased. Lecoxte - arrived at similar results, and he ascribes a less 
 subordinate importance to the chemical secretion as compared with the 
 psychic secretion, than Pawlow does. 
 
 The behavior of the different parts of the stomach in secretion is 
 also of interest. The work of Pawlow and his pupils Gross and 
 Krshyschkowsky, has shown that meat and its extractives as well as 
 the digestion products and milk especially act upon the pyloric part r 
 although not entirely, while they are inactive upon the fundus. Alcohol 
 also acts upon the fundus part. Popielski 3 found that meat extracts 
 had an exciting action upon the secretion of gastric juice, even when intro- 
 duced subcutaneously. In close relation to what has been said above 
 stands the observation of Edkins that the pylorus part of the stomach 
 contains a substance, a prosecreti?i, which by acids and certain other 
 substances is transformed into a secretin, which when introduced into 
 the blood circulation causes a secretion of gastric juice. Hemmeter, 
 claims that a secretin for the secretion of gastric juice is also produced 
 in the salivary glands. The extirpation of all the salivary glands in 
 dogs causes a marked diminution in the secretion of gastric juice, while 
 the intravenous or peritioneal injection of an extract of the salivary 
 glands of dogs produces a secretion of gastric juice. Emsmann 4 has 
 also obtained bodies having a similar action, from the mucosa of the 
 
 1 See Biochem. Centralbl., 3, 660. 
 
 2 Frouin, Compt. rend. soe. biol., 53; Leconte, La Cellule, 17. 
 
 3 Gross, Bioch. Centralbl., 5, 669; Krshyschkowsky, Maly's Jahresb., 36, 403; 
 Popielski, ibid.. 39. 
 
 * Edkins, Journ. of Physiol., 34; Hemmeter, Bioch. Zeitschr., 11; Emsmann r 
 Intern. Beitr. zu. Path. u. Ther. d. Ernahrungs storungen 3 (1911). 
 
464 DIGESTION. 
 
 duodenum, jejunum, and ileum as well as from the liver and pancreas 
 by hydrochloric acid. 
 
 We know very little, positively, in regard to the gastric secretion in 
 man. According to the earlier authorities the irritants may be mechan- 
 ical, thermic, and chemical. Among the chemical excitants we include 
 alcohol and ether, which in too great a concentration bring about no 
 physiological secretion, but rather the transudation of a neutral or 
 faintly alkaline fluid. Certain acids, such as carbonic acid, neutral 
 salts, meat extracts, spices, and other bodies also belong to this group. 
 The reports on this subject are unfortunately very uncertain and con- 
 tradictory. 
 
 The question as to how far the observations made by PAWLOwand 
 his school can be applied to man is of special interest. Many observa- 
 tions on this question have been collected 1 and they compare favor- 
 ably with the observations made upon dogs. Thus in man a psychic 
 secretion of gastric juice can be brought about, and it has also fceen 
 observed that it can be stopped by emotions. As in dogs, so also in man, 
 after sham feeding, a secretion takes place after a pause, the duration 
 of which varies in different cases. In some cases, as in dogs after meat 
 feeding, the pause was about five minutes. The chewing of indifferent 
 bodies did not affect the glands, while bodies acting upon the organs of 
 smell and taste had an exciting action. Umber observed besides this, that 
 after the introduction of a nutritive enema into the rectum, a secretion 
 of gastric juice was produced by reflex action. 
 
 From these observations of Hornborg and Umber, as well as from 
 some earlier observations of Schule, Troller, Riegel, and Scheuer, 2 
 we conclude that in man the psychic secretion is much less than that 
 produced by the introduction of food or bodies having a pleasant taste. 
 That the preparation of the food in the mouth has an essential influence 
 upon the secretion is proved without doubt, but we do not agree as to 
 how this action takes place. Certain experimenters consider the secreted 
 and swallowed saliva as the most essential factor in this action, while 
 others believe that the act of chewing, and still others that the chemical 
 action and the sense of taste, are the most important. 
 
 In regard to the action of saliva, Hemmeter finds that after the 
 extirpation of the salivary glands, the introduction into the stomach 
 of chewed food soaked with dog-saliva, has no special action upon the 
 
 1 Hornborg, Maly's Jahresb., 33, 547; Umber, Berl. klin. Wochenschr., 1905; 
 Cade and Latarjet, Compt. rend. soe. biol., 57; Kaznelson, Pfliiger's Arch., 118; 
 Bogen, ibid., 117; Bickel, Deutsch. med. Wochenschr., 32, and Maly's Jahresb., 36, 
 411. See also Maly's Jahresb. 39, 40, and Bioch, Centralbl. 12. 
 
 'The literature may be found in Umber's v.ork, 1. c. 
 
COMPOSITION OF THE GAS1RIC JUICE. 465 
 
 secretion of juice. On the other hand Frouin 1 observed that the intro- 
 duction of saliva into the large stomach of dogs, acts favorably upon 
 the secretion in the small stomach (see page 462), and the acidity as well 
 as the digestive activity of the juice is increased. This action does not 
 depend, according to Frouin, upon the alkali of the saliva. 
 
 The Qualitative and Quantitative Composition of the Gastric Juice. 
 The human gastric juice, which can seldom be obtained pure and free 
 from residues of the food or from mucus and saliva, is a clear, or only 
 very faintly cloudy, and nearly colorless fluid of an insipifcl, acid taste 
 and strong acid reaction. It contains, as form-elements, glandular cells 
 or their nuclei, and more or less changed columnar epithelium. 
 
 The acid reaction of the gastric juice depends on the presence of free 
 acid, whith, as has been learned from the investigations of C. Schmidt, 
 Richet, and others, consists, when the gastric juice is pure and free 
 from particles of food, chiefly or in large part of hydrochloric acid. Con- 
 tejean 2 regularly found traces of lactic acid in the pure gastric juice 
 of fasting dogs. After partaking of food, especially after a meal rich in 
 carbohydrates, lactic acid occurs abundantly, and sometimes acetic 
 and butyric acids. In new-born dogs the acid of the stomach is lactic 
 acid, according to Gmelin. 3 The quantity of free hydrochloric acid in 
 the gastric juice is, according to Pawlow and his pupils, in dogs 5-6 
 p. m., and in cats an average cf 5.20 p. m. HO. In man the results 
 obtained are variable but regularly much lower. Since it has been 
 possible to obtain pure human gastiic juice for investigation it has been 
 found (Umber, Hornborg, Bickel, Sommerfeld 4 ) that the amount 
 of hydrochloric acid is about 4-5 p. m. There is hardly any doubt that 
 at least a part of the hydrochloric acid of the gastric juice does not 
 exist free in the ordinary sense, but combined Avith organic substances. 
 The results obtained in testing for the acidity of gastric juice by phys- 
 ical methods are almost identical with those obtained by titration (P. 
 Franc kel 5 ). 
 
 The specific gravity of gastric juice is low, 1.001-1.010. It is corre- 
 spondingly poor in solids. Earlier analyses of gastric juice from man, 
 the dog, and the sheep were made by C. Schmidt. 6 As these analyses 
 
 1 Compt. rend. soc. biol., 62. 
 
 2 Bidder and Schmidt, Die Verdauungssafte, etc., 44; Richet, 1. c; Contejean, Con- 
 tributions a l'etude de la physiol. de l'estomac, Theses, Paris, 1892. 
 
 3 Pfliiger's Arch., 90 and 103. 
 
 4 See Richet, 1. c; Contejean, 1. c; Verhaegen, "La Cellule," 1896 and 1897; 
 Sommerfeld, Bioch, Zeitschr, 9, and also footnote 1, page 464, and the literature on 
 the estimation of hydrochloric acid in the gastric juice contents (p. 489) ; see also 
 Cohnheim and Dreyfus, Zeitschr. f. physiol. Chem. 58 (1908). 
 
 5 Zeitschr, f. exp. Path. u. Therap., 1. 
 6 i. c. 
 
466 DIGESTION. . 
 
 refer only to impure gastric juice they are of little value. Rosemann, 1 
 who has investigated the gastric juice secreted by a dog after sham 
 feeding, found an average of 4.22 p. m. solids, among which 1.32 p. m. 
 were mineral bodies and about 2.90 p. m. organic substance. The 
 amount of nitrogen in one case was 0.36 p. m., in another 0.54 p. m. and 
 the quantity of HC1 was about 5.6 p. m. The ash consisted chiefly of 
 potassium chloride, namely 980-990 p. m. of the inorganic part. Nencki 
 and Sieber 2 found 3.06 p. m. solids in the pure gastric juice of a dog. 
 Nencki 3 found 5 milligrams sulphocyanic acid per liter of gastric juice 
 of a dog. 
 
 In the ash of human gastric juice after sham-feeding Albu 4 found 
 356.2 p. m. K 2 0; 226.5 p. m. Na 2 0, and 497.3 p. m. CI. The amount 
 of salts insoluble in water was 23.9 p. m. In hyperacidity he found 
 almost the same composition. 
 
 Besides the free hydrochloric acid, pepsin, rennin, and a lipase are 
 the other physiologically important constituents of gastric juice. 
 
 Pepsin. This enzyme is found, with the exception of certain fishes, 
 in all vertebrates thus far investigated. 
 
 Pepsin occurs in adults and in new-born infants. This condition 
 is different in new-born animals. While in a few herbivora, such as the 
 rabbit, pepsin occurs in the mucous coat before birth, this enzyme is 
 entirely absent at the birth of those carnivora which have thus far been 
 examined, such as the dog and cat. 
 
 In various invertebrates enzymes have also been found which have 
 a proteolytic action in acid solutions. It has been shown that these 
 enzymes, nevertheless, are not in all animals identical with ordinary 
 pepsin. According to Klug and Wr6blewski 5 the pepsins found 
 in man and various higher animals are somewhat different, an observa- 
 tion which according to the experience of Hammarsten is very prob- 
 able. Enzymes also occur in various plants and animal organs, although 
 not identical with pepsin, but which act in acid reaction. The enzyme 
 obtained from the Nepenthes, which dissolves proteins only in acid 
 reaction, stands very close to pepsin. An enzyme more closely related 
 to 'trypsin or erepsin (see sections III and IV) is, on the contrary, 
 Glaessner's pseudopepsin, which according to him is the only peptic 
 enzyme in the pyloric end. Pseudopepsin, whose existence is disputed 
 by Klug, while others (Reach, Pekelharing) affirm its occurrence in 
 
 1 Pfliiger's Arch., 118. 
 
 2 Zeitschr. f. physiol. Chem., 32. 
 
 3 Ber. d. d. Chem. Gesellsch., 28. 
 
 4 Zeitschr. f. Path. u. Therap., 5. 
 
 6 Klug. Pfliiger's Arch. 60; Wr6blewski, Zeitschr. f. physiol. Chem., 21. 
 
PEPSIN. 467 
 
 the mucous membrane, cannot, according to Hammarsten, either be the 
 only or the most prominent peptic enzyme of the pyloric part. According 
 to Glaessner, it also acts in neutral and alkaline reaction and yields 
 tryptophane among other cleavage products. According to Bergmann l 
 it is identical with erepsin (see below). Among the enzymes of the 
 mucosa of the stomach belongs the so-called antipepsin discovered by 
 Weinland, 2 which has a retarding action upon pepsin digestion and, 
 as some claim, prevents the self-digestion of the mucous membrane. 
 
 Pepsin is as difficult to isolate in a pure condition as are other 
 enzymes. The pepsin prepared by Brucke and Sundberg gave negative 
 results with most reagents for proteins, and showed nevertheless a 
 powerful action, which seems to indicate that it was very pure. Schou- 
 mow-Simanowski, Nencki and Sieber, have designated as the true 
 enzyme the substance containing chlorine, which they obtain by strongly 
 cooling the gastric juice. That this precipitate is not a chemical indi- 
 vidual, and hence cannot be pepsin, follows from the investigations of 
 Pekelharing. While pepsin, according to Nencki and Sieber, was 
 rich in phosphorus and contained nucleoprotein, Pekelharing's pepsin 
 was free from phosphorus and yielded no nucleoprotein. Friedenthal 
 and Miyamota 3 have also shown that the pepsin is still active after 
 the splitting off of the nuclein complex (and also the protein). As pepsin 
 is readily precipitated with the proteins and combines therewith, it is 
 difficult to decide whether pepsin is a protein substance or not, and the 
 question as to its nature is still undecided, just as is the case with other 
 enzymes. As ordinarily known, pepsin, at least in an impure form, is 
 soluble in water and glycerin. It is precipitated by alcohol, but is only 
 slowly destroyed thereby. In aqueous solution its action is quickly 
 destroyed on heating to boiling. According to Biernacki 4 pepsin 
 in neutral solutions is destroyed by heating to 55° C. In the dry state 
 it can be heated to over 100° C. without losing its activity. In the 
 presence of 2 p. m. HC1 a temperature of 55° C. is not injurious, and the 
 compound with acid is more resistant than the free pepsin (Grober 5 ). 
 Pepsin in acid solution is destroyed by heating to 65° C. for five minutes. 
 
 'Glaessner, Hofmeister's Beitrage, 1; Klug, Pfluger's Arch., 92; Reach, Hofmeis- 
 ter's Beitrage, 4; Pekelharing, Arch, des scienc, biolog., St. P6tersbourg 11; Pawlow- 
 Festband, 1904; Bergmann, Skand. Arch. f. Physiol., 18. 
 
 2 Zeitschr. f. Biologie, 44. 
 
 3 Brucke, Wien. Sitzungsber,. 43; Sundberg, Zeitschr. f. physiol. Chem., 9; Schou- 
 mow-Simanowski, Arch. f. exp. Path. u. Pharm., 33; Pekelharing, Zeitschr. f. physiol. 
 Chem., 22 and 35; Xencki and Sieber, ibid., 32; Friedenthal and Miyamota, Centrabl. 
 f. Physiol., 15, 785. 
 
 « Zeithschr. f . Biologie, 28. 
 
 6 Arch. f. exp. Path. u. Pharm., 51. 
 
468 DIGESTION. 
 
 On adding peptone or certain salts the pepsin may be heated to 70 c C. 
 for the same time without destruction. 
 
 The behavior of pepsin on heating its acid solution is influenced not 
 only by the degree of acidity, but by the duration of heating and also 
 by the amount of other bodies in the solution. If an acid (0.2 per cent 
 HC1) infusion of the calf's stomach be warmed for several days to about 
 40 or 45° C, a part of the pepsin is destroyed, but we obtain in this 
 manner an infusion which still dissolves proteins but has no rennin action 
 (Hammaesten 1 ). The pepsin from different animals acts differently 
 in this regard and the pepsin of the pike stomach is very quickly destroyed 
 at 37-40° C. 
 
 Pepsin is extraordinarily sensitive to the action of alkalies, not only 
 caustic, and carbonated, but also against the hydroxides of the alka- 
 line earths. It is easily made inactive by these substances. If the 
 action of the alkali is not too strong then, as shown by Pawlow and 
 Tichomieow, 2 the enzyme can in part be reactivated by the addition 
 of acid if the greater part (about four-fifths), of the alkalinity be neutral- 
 ized by the addition of acid and then after some hours more acid be added. 
 If the entire quantity of acid be added at one time the reactivation does 
 not take place. 
 
 The only property which is characteristic of pepsin is that it dissolves 
 protein bodies in acid but not in neutral or alkaline solutions, with the 
 formation of proteoses, peptones, and other products. 
 
 The methods for the preparation of relatively pure pepsin depend, 
 as a rule, upon its property of being thrown down with finely divided 
 precipitates of other bodies, such as calcium phosphate or cholesterin. 
 The rather complicated methods of Beucke and Sundbeeg are based 
 upon this property. Pekelhaeing makes use of a prolonged dialysis 
 and precipitation with 0.2 p. m. HC1. 
 
 Very permanent pepsin solutions, from which the enzyme with con- 
 siderable protein can be precipitated by alcohol, may be prepared by 
 extraction with glycerin. Solutions having a strong action may also 
 be prepared by making an infusion of the gastric mucosa of an animal 
 in acidified water (2-5 p. m. HC1). This is unnecessary, as we can obtain 
 pure gastric juice according to Pawlow's method, and also because very 
 active commercial preparations of pepsin can be bought in the market. 
 
 The Action of Pepsin on Proteins. Pepsin is inactive in neutral or 
 alkaline reactions, but in acid liquids it dissolves coagulated protein 
 bodies. The protein always swells and becomes transparant before 
 it dissolves. Unboiled fibrin swells up in a solution containing 1 p. m. 
 HO, forming a gelatinous mass, and does not dissolve at ordinary tem- 
 
 1 Zeitschr. f. physiol. Chem., 56. 2 Ibid., 54. 
 
PEPSIN. 469 
 
 perature within a couple of days. Upon the addition of a little pepsin, 
 however, this swollen mass dissolves quickly at ordinary temperatures. 
 Hard-boiled egg albumin, cut in thin pieces with sharp edges, is not 
 perceptibly changed by dilute acid (2-4 p. m. HC1) at the temperature 
 of the body in the course of several hours. But the simultaneous pres- 
 ence of pepsin causey the edges to become clear and transparent, blunt 
 and swollen, and the protein gradually dissolves. 
 
 From what has been said above in regard to pepsin, it follows that 
 proteins may be employed as a means of detecting pepsin in liquids. 
 Ox-fibrin may be employed as well as coagulated egg albumin, which 
 latter is used in the form of slices with sharp edges. As the fibrin is 
 easily digested at the normal temperature, while the pepsin test with 
 egg albumin requires the temperature of the body, and as the test with 
 fibrin is somewhat more delicate, it is often preferred to that with egg 
 albumin. When we speak of the " pepsin test " without further explana- 
 tion we ordinarily understand it as the test with fibrin. 
 
 This test, nevertheless, requires care. The fibrin used should be 
 ox-fibrin and not pig-fibrin, which last is dissolved too readily with dilute 
 acid alone. The unboiled ox-fibrin may be dissolved by acid alone 
 without pepsin, but this generally requires more time. In testing with 
 unboiled fibrin at normal temperature, it is advisable to make a control 
 test with another portion of the same fibrin with acid alone. Since at 
 the temperature of the body unboiled fibrin is more easily dissolved by 
 acid alone, it is best always to w T ork with boiled fibrin. 
 
 As pepsin has not thus far been prepared in a positively pure condi- 
 tion, it is impossible to determine the absolute quantity of pepsin in a 
 liquid. It is possible only to compare the relative amounts of pepsin 
 in two or more liquids, which may be done in several ways. 
 
 The older method, that of Brucke, consists in diluting the two pepsin solu- 
 tions to be compared with certain proportions of 1 p. m. hydrochloric acid, so 
 that when the amount of pepsin contained in the original solution is equal to 1, 
 each solution contains a degree of dilution, p, corresponding to 1, §, |, \, ^, 
 etc. A flock of fibrin or a piece of hard-boiled egg is added to each test and the 
 time noted when each test begins to digest and when it ends. The relative 
 amount of pepsin is calculated from the rapidity of digestion as follows: The tests 
 P=i, I) A, of one series is digested in the same time as tests p = l, \, \ of the 
 other series, hence the first solution contained four times as much pepsin. 
 Grutzner 1 has improved this test by using fibrin colored with carmine, and on 
 comparing with carmine solutions of known dilution he determines colorimetrically 
 the rapidity of digestion. 
 
 Mett's Method. Draw up white of egg in a glass tube 1-2 millimeters in 
 diameter, coagulate it by plunging it into water at 95° C, and cut the ends off 
 sharply; then add two tubes to each test-tube with a few cubic centimeters 
 of the acid pepsin solution; allow them to digest at body temperature, and after 
 a certain time, generally, after ten hours, measure the lineal extent of the digested 
 
 1 Grutzner, Pfliiger's Arch., 8 and 106. See also A. Korn, '' Ueber Methoden Pepsin 
 quantitativ zu bestimmen," Inaug.-Dissert., Tubingen, 1902. 
 
470 DIGESTION. 
 
 layer of albumin in the various tests, bearing in mind that the digested layer at 
 each end must not be longer than 6-7 millimeters. The quantity of pepsin in 
 the comparative tests is as the square of the millimeters of the albumin-column 
 dissolved in the same time. Thus if in one case 2 millimeters of albumin were 
 dissolved and in the other 3 millimeters, then the quantity of pepsin is as 4:9. 
 If the fluid removed from the stomach, which is rich in bodies having a disturb- 
 ing influence upon pepsin digestion, is to be tested, then the liquid must be first 
 properly diluted with hydrochloric acid (Nierenstein and Schiff 1 ). 
 
 Objections have been raised against these methods from several sides, and they 
 are in fact very uncertain. Huppert and E. Schutz measure the relative 
 quantities of pepsin from the amount of secondary proteoses formed under cer- 
 tain conditions. The proteoses were determined by the polariscope. J. Schutz 
 determines the total proteose-nitrogen, and Spriggs 2 finds that the change in 
 the viscosity is a measure of the amount of pepsin. 
 
 Volhard and Lohlein 3 use an acid casein solution for the pepsin determina- 
 tion, and determine, after precipitation with sodium sulphate, the acidity of the 
 nitrate of the digested test as well as of the original control solution. The casein 
 is precipitated as an acid compound by the sulphate, and the filtrate separated 
 from the precipitate contains less acid than the original solution. In propor- 
 tion as the digestion progresses less substance is precipitated by the sulphate, 
 and the acidity of the filtrate becomes correspondingly higher. The increase 
 in acidity in the different portions varies within certain limits as the square root 
 of the quantity of ferment. 
 
 Jacoby suggested a method which is based on the fact that a cloudy solution 
 of ricin becomes clear by the action of pepsin-hydrochloric acid, and indeed with 
 varying rapidity with different quantities of pepsin. This method, which requires 
 further testing, seems to be delicate and is of value, as is doubtless the following 
 method of Fuld and Levison. 4 This is based on the property that edestan can 
 be precipitated from acid solution by NaCl, but not the proteoses formed therefrom. 
 
 A solution of 1 p. m. edestin in hydrochloric acid (jro normal) is prepared 
 whereby the edestin is changed into edestan. The activity of a gastric juice (or 
 a pepsin-hydrochloric acid solution) is tested in the following manner: the solu- 
 tion to be tested is placed in decreasing quantities in a series of test-tubes and 
 allowed to act upon an equal quantity of the edestan solution, 2 cc., and the 
 minimum of juice determined which is necessary to digest the solution, within 
 one-half an hour and at room temperature, so that on the addition of solid 
 NaCl and shaking no precipitate occurs. Gross 5 suggested a similar method by 
 using an acid casein solution and precipitating with sodium acetate. 
 
 The rapidity of the pepsin digestion depends on several circumstances. 
 Thus different acids are unequal in their action; hydrochloric acid shows 
 in slight concentration, 0.8-1.8 p. m., a more powerful action than any 
 other acid, whether inorganic or organic. In greater concentration other 
 acids may have a powerful action; but no constant relation has been 
 found between the strength of various acids and their action in pepsin 
 digestion, and the reports of the action of different acids are contradic- 
 
 1 Mett, see Pawlow, I.e.; 28; Nierenstein and Schiff, Berl. klin. Wochenschr., 40; 
 Jastrowitz, Bioch. Zeitschr., 2. 
 
 2 Huppert and Schutz, Pfluger's Arch., 80; J. Schutz, Zeitschr. f. physiol. Chem., 
 30; Spriggs, ibid., 35. 
 
 3 Hofmeister's Beitrage, 7. 
 
 4 Jftcoby, Bioch. Zeitschr., 1; Fuld and Levison, ibid. G. 
 6 Berl. klin. Wbchensclir., 45. 
 
PEPSIN DIGESTION. 471 
 
 tory. 1 Sulphuric acid, it seems, has a weaker action than the other 
 inorganic acids. The degree of acidity is also of the greatest importance. 
 With hydrochloric acid the degree of acidity is not the same for differ- 
 ent protein bodies. For fibrin it is 0.8-1 p. m., for myosin, casein, and 
 vegetable proteins about 1 p. m., for coagulated egg albumin, on the 
 contrary, about 2.5 p. m. In regard to the dependence of the extent 
 of transformation upon the quantity of enzyme and the time of diges- 
 tion we refer to page 58. The kind of protein is of importance, for 
 example, for besides what was said above in regard to the fibrin, hard- 
 boiled egg albumin is much easier digested by an acidity of 1-2 p. m. 
 HC1 than liquid egg albumin, which is rather resistant to the action 
 of gastric juice. The accumulation of products of digestion has a retard- 
 ing action on digestion (page 65), although, according to Chittenden 
 and Amerman, 2 the removal of the digestion products by means of dialysis 
 does not essentially change the relation between the proteoses and true 
 peptones. Pepsin acts more slowly at low temperatures than it does at 
 higher ones. It is even active in the neighborhood of 0° C, but with 
 increasing temperature the rapidity of digestion also increases until 
 about 40° C, when the maximum is reached. If the swelling up of the 
 protein is prevented, as by the addition of neutral salts, such as NaCl, 
 in sufficient amounts, or by the addition of bile to the acid liquid, 
 digestion can be prevented to a greater or less extent. Foreign bodies 
 of different kinds produce dissimilar effects, in which naturally the 
 variable quantities in which they are added are of the greatest impor- 
 tance. Salicylic acid and carbolic acid, and especially sulphates 
 (Pfleiderer), retard digestion, while arsenious acid promotes it (Chit- 
 tenden), and hydrocyanic acid is relatively indifferent. Salts of the 
 alkali and alkaline earth metals have a strong retarding action in strong 
 concentration. By experiments with salt solutions so strongly diluted 
 that the action, on account of the strong dissociation, was brought about 
 by ions and not by the electrolytically neutral molecules (min. -fa and 
 max. I normal salt solutions), J. Schutz 3 found that the anions had a 
 much greater retarding action upon pepsin digestion than the cations. 
 Of these latter the sodium cation had the strongest retarding action. 
 Alcohol in large quantities (10 per cent and above) disturbs the digestion, 
 while small quantities act indifferently. Metallic salts in very small 
 quantities may indeed sometimes accelerate digestion, but otherwise 
 
 1 See Wr6blewski, Zeitschr. f. physiol. Chem., 21, and especially Pfleiderer, Pfliiger's 
 Arch., 66, which also gives references to other works; Larin, Biochem. Centralbl., 1, 
 484; and A. Pick, Wein. Sitzungsber., M. N. Klasse, 112. 
 
 * Journ. of Physiol. 14. 
 
 1 Hofmeister's Beitriige, 5. 
 
472 DIGESTION. 
 
 they tend to retard it. The action of metallic salts in different cases 
 can be explained in various ways, but they often seem to form with pro- 
 teins insoluble or difficultly soluble combinations. The alkaloids may 
 also retard the pepsin digestion (Chittenden and Allen 1 ). A very 
 large number of observations have been made in regard to the action 
 of foreign substances on artificial pepsin digestion, but as these observa- 
 tions have not given any direct result in regard to the action of the 
 same substances in natural digestion, as well as upon secretion and 
 absorption, we will not discuss them here. 
 
 The Products of the Digestion of Proteins by Means of Pepsin and Acid. 
 In the digestion of nucleoproteins or nucleoalbumins an insoluble residue 
 of nuclein or pseudonuclein always remains, although under certain 
 circumstances a complete solution may occur. Fibrin also yields an 
 insoluble residue, which consists, at least in great part, of nuclein, 
 derived from the form-elements inclosed in the blood-clot. This residue 
 which remains after the digestion of certain proteins was called dyspep- 
 tone by Meissner. This name is therefore not only unnecessary but 
 indeed erroneous, as this residue does not consist of bodies related to the 
 peptones. In the digestion of proteins, substances similar to acid albu- 
 minates, parapeptone (Meissner 2 ), antialbumate, and antialbumid 
 (Kuhne), may also be formed. On separating these bodies the filtered 
 liquid, neutralized at boiling-point, contains proteoses and peptones in 
 the old sense, while the so-called Kuhne true peptone and the other 
 cleavage products are obtained only after a longer and more intense 
 digestion. The relation, between the proteoses, changes very much in 
 different cases and in the digestion of the proteins. For instance, a larger 
 quantity of primary proteoses is obtained from fibrin than from hard- 
 boiled egg albumin or from the proteins of meat; and the different 
 proteins, according to the researches of Klug, 3 yield on pepsin diges- 
 tion unequal quantities of the various digestive products. In the diges- 
 tion of unboiled fibrin an intermediate product may be obtained in the 
 earlier stages of the digestion — a globulin which coagulates at 55° C 
 (Hasebroek 4 ). For information in regard to the different proteoses 
 and peptones which are formed in pepsin digestion see pages 127 to 136. 
 
 Action of Pepsin-Hydrochloric Acid on Other Bodies. The gelatin- 
 forming substances of the connective tissue, of the cartilage, and of the 
 
 1 .Studies from the Lab. Physiol. Chem. Yale University, 1, 76. See also Chitten- 
 den and Stewart, ibid. 3, 60. 
 
 2 Tlie works of Meissner on pepsin digestion are found in Zeitschr. f. rat. Med., 7, 
 8, 10, 12, and 14. 
 
 » Pfluger's Arch., 65. 
 
 4 Zeitschr. f. physiol. Chem., 11. 
 
PEPSIN DIGESTION. 473 
 
 bones, from which last the acid dissolves only the inorganic substances, 
 is converted into gelatin by digesting with gastric juice. The gelatin is 
 further changed so that it loses its property of gelatinizing and is con- 
 verted into gelatoses and peptone (see page 120). True mucin (from the 
 submaxillary) is dissolved by the gastric juice, yielding substances similar 
 to peptone, and a reducing substance similar to that obtained by boil- 
 ing with a mineral acid. • Mucoids from tendons, cartilage, and bones 
 dissolve, according to Posner and Gies, 1 in pepsin-hydrochloric acid, 
 but leave a residue which amounts to about 10 per cent of the original 
 material and which, as it seems, consists in great part, if not entirely, 
 of a combination of proteid with glucothionic acid (Chapters VI and 
 VII). The solution contains primary and secondary mucoproteoses 
 and mucopeptones. The former contain glucothionic acid, but the latter 
 do not. Elastin is dissolved more slowly and yields the previously men- 
 tioned substances (page 117). Keratin and the epidermal formations 
 are insoluble. The nucleins are dissolved with difficulty, and the cell 
 nuclei, therefore, remain in great part undissolved in the gastric juice. 
 According to London 2 and his collaborates the nucleic acids are not 
 attacked in the stomach. The animal cell-membrane is, as a rule, more 
 easily dissolved the nearer it stands to elastin, and it dissolves with 
 greater difficulty the more closely it is related to keratin. The mem- 
 brane of the plant-cell is not dissolved. Oxyhemoglobin is changed into 
 hsematin and protein, the latter undergoing further digestion. It is 
 for this reason that blood is changed into a dark-brown mass in the 
 stomach. The gastric juice does not act upon fat, but, on the contrary, 
 dissolves the cell-membrane of fatty tissue, setting the fat free. Gastric 
 juice has no action on starch or the simple varieties of sugar. The 
 statements in regard to the ability of gastric juice to invert cane-sugar 
 are very contradictory. At least this action of the gastric juice is not 
 constant, and if it is present at all, it is probably due to the action of the 
 acid. 
 
 Pepsin alone, as above stated, has no action on proteins, and an acid of the 
 intensity of the gastric juice can only very slowly, if at all, dissolve coagulated 
 albumin at the temperature of the body. Pepsin and acid together not only 
 act more quickly, but qualitatively they act otherwise than the acid alone, at 
 least upon dissolved protein. This has led to the assumption of the presence of 
 a pepsin-hydrochloric acid whose existence and action are only hypothetical. 
 As pepsin digestion, it seems, yields finally the same products as the hydrolytic 
 cleavage with acids, we can say for the present only that this enzyme acts like 
 other catalysts in very powerfully accelerating a process which would also pro- 
 ceed without the catalvte. 
 
 1 Amer. Journ. of Physiol., 11. 
 
 * Zeitschr. f. physiol. Chem. 70, 72. 
 
474 DIGESTION. 
 
 Rennin or (thymosin is the enzyme, which is especially character- 
 ized by the fact that it coagulates milk or casein solutions containing 
 lime in neutral or indeed faintly alkaline reaction. It must probably 
 be considered as a proteolytic enzyme. Rennin is habitually found in 
 the neutral, watery infusion of the fourth stomach of the calf and sheep, 
 especially in an infusion of the fundus part. In other mammals and in 
 birds it is seldom found, and in fishes hardly ever in the neutral infusion. 
 In these cases, as in man and the higher animals, a rennin-forming sub- 
 tance, a rennin zymogen, occurs, which is converted into rennin by the 
 action of an acid (Hammarsten) . Hedin has obtained a retarding 
 solution by treating a neutral infusion of the stomachs of various animals 
 with dilute ammonia and then neutralizing. These solutions entirely 
 or partly retard the action of the rennin from the same animal and is 
 destroyed by acid with the setting free of rennin. Hedin therefore 
 considers the rennin zymogen as a combination between rennin and an 
 inhibitory substance, in which combination the inhibitory body is 
 destroyed by treatment with acid; consequently the rennin appears in 
 an active form. 
 
 According to Bang the rennin of the human and pig stomachs differs 
 from that of the calf's stomach in being much more resistant to acids, 
 more easily destroyed by alkalies, and that its action is much more 
 accelerated by calcium chloride than that from the calf's stomach. 1 
 Active rennin occurs in the human stomach under physiological condi- 
 tions, but may be absent under special pathological conditions. 2 
 
 According to the experience of Hammarsten the rennin of the pike 
 and of the dog differs from that of the calf, and Hedin 3 finds in the specific 
 kind of inhibitory action of rennin produced by means of ammonia 
 treatment as well as by immune serum, a proof that the rennin enzyme 
 of different kinds of animals differ more or less from each other. In 
 regard to this inhibition see pages 62-64. 
 
 Enzymes having a rennin action has also been found in the blood 
 and several organs of higher animals as well as in invertebrates. Sim- 
 ilar enzymes are also very widely distributed in the plant kingdom and 
 numerous micro-organisms have the ability to produce rennin. 
 
 1 Deutsch. med. Wochenschr., 1899, and Pfliiger's Arch., 79. 
 
 2 Schumburg, Virchow's Arch., 97. A good review of the literature may be found 
 in Szydlowski, Beitrage zur Kenntnis des Labenzym nach Beobaehtungen an Saug- 
 lingen, Jahrb. f. Kinderheilkunde (N. !\), 34. See also Lorcher, Pfliiger's Arch., 69. 
 which also contains the pertinent literature. An excellent review of the literature 
 on rennin and its action may be found in E. Fuld, Ergebnisse der Physiol., 1, Abt. 1, 
 468. 
 
 3 Hammarsten, Upsala Liikaref. forh. 8, 78 (1872). Zeitschr. f. physiol. Chem. 
 56, 18 (1908), 68, 119 (1910); Hedin, ibid. 72, 187, 74, 242, 76, 355 (1911), 77, 229 (1912). 
 
REN N IN. 475 
 
 The law given on page 58 in which the time of coagulation is 
 inversely proportional to the amount of enzyme, is true for calf rennin 
 (Fuld ! ) and for sheep rennin (Hedin 2 ). The other rennins investi- 
 gated do not follow this law at 37° C, which, according to van Dam, is 
 due in the case of the pig rennin to its less resistance toward the alkali 
 of the milk. 3 
 
 Rennin is just as difficult to prepare in a pure state as the other 
 enzymes. The purest rennin enzyme thus far obtained did not give the 
 ordinary protein reactions. On heating its solution rennin is more or 
 less quickly destroyed, depending upon the length of heating and upon 
 the concentration. If an active and strong infusion of the gastric mucosa 
 of the calf's stomach in water containing 3 p. m. HC1 is heated to 40-45° 
 C. for 48 hours, the rennin or nearly all, is destroyed, while the pepsin 
 remains. A pepsin solution free from rennin can be obtained in this 
 way. 
 
 A much-discussed question is, whether the digestion of protein and 
 the rennet action are brought about by two special enzymes, or represent 
 two different enzyme actions, or whether there is only one enzyme, the 
 pepsin, which has both actions. . The supporters of this last view dispose 
 of the question in different ways. Some, like Pawlow and Parast- 
 schuk, consider the rennet action simply as the reverse of the synthetical 
 action of pepsin, a view which is improbable in the highest degree. 
 Others, such as Sawjalow 4 and Gewin, consider, on the contrary, that 
 the coagulation of milk is only a pepsin action and indeed as the first 
 step in the beginning of proteolysis, namely, the beginning of peptic 
 digestion of casein. Rokoczy 5 believes in the presence of two enzymes 
 in the calf's stomach, one of which, the rennin, disappears on the increas- 
 ing age of the animal. 
 
 The simultaneous occurrence in the animal and plant kingdoms of 
 enzymes having a proteolytic and rennet action and the parallelism of 
 the pepsin and rennet action indicates an identity of both enzymes and 
 enzyme actions. This parallelism in fact does not prove much, because 
 it has mostly been studied in acid reaction, while rennet is character- 
 istically active in neutral or faintly alkaline reaction. 
 
 At the same time Hammarsten 6 finds that in acid reactions no 
 
 1 Hofmeister's Beitrage 2. 
 1 Not published investigations. 
 8 Zeitschr. f. physiol. Chem. 64, 316 (1910). 
 
 4 The recent literature on this question can be found in Hammarsten, Zeitschr. 
 physiol. Chem., 56, 18 (1909). 
 *Ibid. 68, 421 (1910), 73, 453 (1911). 
 8 Zeitschr. f. physiol. Chem. 68, 119 (1910), which also contains the recent literature. 
 
470 DIGESTION. 
 
 parallelism exists in the two enzyme actions with extracts of the dog's 
 and calf's, stomach, and, also on testing the two enzyme actions upon the 
 same casein solution no parallelism was present. The pathological cases 
 in man, if the observations are reliable, where only one enzyme action 
 occurs, seems to dispute the identity of the action of these two enzymes. 
 This opposition is also shown by the fact that pepsin, so far as known, 
 only has a digestive action in the presence of free H ions, while the 
 coagulation of milk occurs in the absence of these and indeed in the 
 presence of HO ions. Among other facts which contravene the identity 
 is the fact that a pepsin solution can be prepared which has a digestive 
 action but cannot coagulate milk, and the reverse, namely, rennet solu- 
 tions can be made which coagulate milk but do not have digestive action 
 in acid reaction (Hammarsten 1 ). The observations of Ducceschi, 2 
 that pepsin but no rennin occurs in the stomach of the Didelphys, also 
 conflict with the identity of the two enzymes. 
 
 The views of Nencki and Sieber 3 take a certain reconciliary posi- 
 tion. According to them pepsin forms a gigantic molecule which has 
 various side-chains, one of which has digestive action in acid solution 
 while the others coagulate milk. This. view coincides well with most 
 of the observations made thus far. 
 
 In regard to the formation of plasteins under the influence of rennin 
 solutions and other enzyme solutions, see Chapters I and II. 
 
 Gastric Lipase (stomach steapsin). F. Volhard 4 made the dis- 
 covery that the gastric juice has a strong fat-splitting action only when 
 the fat is in a fine emulsion, as in the yolk of the egg, in milk or in cream. 
 Considerable controversy has arisen in regard to the importance of the 
 splitting of fat, and the occurrence of a special gastric lipase is indeed 
 disputed. From numerous observations it follows without question 
 that in man and many animals a gastric lipase occurs and is secreted 
 with the gastric juice. Nevertheless the extent of fat splitting in the 
 stomach is generally not very great. In its action this lipase follows 
 Schutz's rule and in its other properties it seems to vary in different 
 animals. 
 
 The question whether the cover cells, principally, or the chief cells 
 
 1 Zeitschr. f. physiol. Chem., 56. 
 
 2 Centralbl., f. Physiol. 22, 784. 
 J Zeitschr. f. physiol. Chem., 32. 
 
 4 Volhard, Munch, med. Wochenschr., 1900, and Zeitschr. f. klin. Med., 42, 43. 
 See also Stade, Hofmeister's Beitrage, 3; A. Fromme, ibid., 7; A Zinsser, ibid.; H. 
 Engel., ibid.; and Inouye, Arch. f. Verdauangskrank., 9; Falloise, Arch, internat. d. 
 Physiol., 3 and 4; London, Zeitschr. f. physiol, Chem., 50; Levites, ibid., 49; Laqueur. 
 Hofmeister's Beitrage, 8, 281; Heinsheimer, Deutsch. med. Wochenschr., 32, and 
 Arbeiten aus d. pathol. Institute, Berlin (Hirschwald, 1906). 
 
FORMATION OF HYDROCHLORIC ACID. 477 
 
 also, or both, take part in the formation of free acid is disputed. 1 There 
 can be no doubt that the hydrochloric acid of the gastric juice origin- 
 ates in the chlorides of the blood, because, as is well known, a secretion 
 of perfectly typical gastric juice takes place in the stomachs of fasting 
 animals or those which have starved for some time. As the chlorides 
 of the blood are derived from the food, it is easily understood, as shown 
 by Cahn, 2 that in dogs after a sufficiently long common-salt starva- 
 tion, the stomach secreted a gastric juice containing pepsin, but no free 
 hydrochloric acid. On the administration of soluble chlorides, a gastric 
 juice containing hydrochloric acid was immediately secreted. The 
 conditions are not so simple, because in the first case not only does the 
 amount of hydrochloric acid diminish but, as shown by Wohlgemuth 
 and then by Kudo, the quantity of juice diminishes greatly, and on the 
 introduction of NaCl the quantity of juice secreted increases. Accord- 
 ing to Pugliese 3 the gastric juice in starvation, after a certain time, 
 has a neutral reaction, and the introduction of NaCl does not now change 
 its properties. In the secretion of free acid it is assumed by Pugliese 
 that the gland cells, which decompose the chloride, have sufficient 
 amounts of protein at their disposal. On the introduction of alkali 
 iodides or bromides, Kulz, Nencki and Schoumow-Simanowski 4 have 
 shown that the hydrochloric acid of the gastric juice is replaced by HBr, 
 and to a less extent by HI. The secretion of free hydrochloric acid 
 from the alkaline blood has been explained in various ways, but as yet 
 no satisfactory theory has been suggested. 5 
 
 In regard to the secretion of pepsin we must recall that this last 
 is not already produced, but is formed from a preliminary step, a pep- 
 sinogen or propepsin. Langley 6 has positively shown the existence 
 of such a substance in the mucous coat. This substance, propepsin, 
 shows a comparatively strong resistance to dilute alkalies (a soda solu- 
 
 1 See Heidenhain, Pfliiger's Arch., 18 and 19, and Hermann's Handbuch, 5, part I, 
 " Absonderungsvorgange;" Klemensiewicz, Wien. Sitzungsber,. 71; Frankel, Pfliiger's 
 Arch., 48 and 50; Contejean. 1. c; Kranenburg, Archives Teyler, Ser. II, Haarlem, 
 1901; and Mosse, Centralbl. f. Physiol., 17, 217; Fitzgerald, Proc. Roy. Soc. B. 82, 
 83; L6pez-Suarez, Bioch. Zeitschr. 46, 490 (1912). 
 
 2 Zeitschr. f. physiol. Chem., 10. 
 
 1 Wohlgemuth, Arbeiten aus d. pathol. Institute, Berlin, 1906; Kudo, Bioch. 
 Zeitschr. 16, 217 (1909), Pugliese, Maly's Jahresb., 36, 394. 
 
 4 Kulz, Zeitschr. f. Biologie, 23; Nencki and Schoumow, Arch, des sciences biol. 
 de St. Petersboura, 3. 
 
 5 Koeppe, Pfliiger's Arch., 62; Benrath and Sachs, ibid., 109; Maly, see v. Bunge's 
 Lehrbuch der physiol. u. pathol. Chem., 4. Aufl., 1898; Schwarz, Hofmeister's Bei- 
 trage. 5, 
 
 6 SchilT, Lecons, sur la physiol. de la digestion, 1867, 2; Langley and Edkins, Journ. 
 of Physiol., 7. 
 
478 DIGESTION. 
 
 tion of 5 p. m.) which easily destroy pepsin (Langley). Pepsin, on the 
 other hand, withstands better than propepsin the action of carbon dioxide, 
 which quickly destroys the latter. The occurrence of a rennin zymogen 
 and possibly also of a steapsinogen, in the mucous coat has been men- 
 tioned above. 
 
 The question in what cells the two zymogens, especially the pro- 
 pepsin, are produced, has been extensively discussed for several years. 
 Formerly, it was the general opinion that the cover cells were pepsin 
 cells, but since the investigations of Heidenhain and his pupils, Langley 
 and others, the formation of pepsin has been attributed to the chief 
 cells. 1 
 
 The Pyloric Secretion. That part of the pyloric end of the dog's 
 stomach which contains no fundus glands was dissected by Klemensie- 
 wicz, one end being sewed together in the shape of a blind sac and the 
 other sewed into the stomach. From the fistula thus created he was 
 able to obtain the pyloric secretion of a living animal, later the secretion 
 from a pyloric fistula has been obtained in other ways. This secretion 
 is alkaline, viscous, jelly-like, rich in mucin, of a specific gravity of 
 1.009-1.010, containing 16.5-20.5 p. m. solids. It habitually con- 
 tains pepsin, which has been proved by Heidenhain by observations 
 on a permanent pyloric fistula, and the amount may sometimes be con- 
 siderable. Contejean investigated the pyloric secretion in other ways, 
 and finds that it contains both acid and pepsin. The alkaline reaction 
 of the secretions investigated by Heidenhain and Klemensiewicz 
 is due, according to Contejean, to an abnormal secretion caused by the 
 operation, because the stomach readily yields an alkaline juice instead 
 of an acid one under abnornal conditions. The reports of Heidenhain 
 and Klemensiewicz have nevertheless been substantiated by Aker- 
 mann, Kresteff, Schemiakine and others. 2 
 
 The secretion of gastric juice under different conditions may vary 
 considerably. The statements concerning the quantity of gastric juice 
 secreted in a certain time are therefore unreliable. Rosemann ob- 
 served, on sham feeding in dogs, a secretion of 917 cc. in the course of 
 Z\ hours — a considerable quantity. Kudo s found more pepsin in the 
 secreted juice when the quantity of juice was less. 
 
 The Chyme and the Digestion in the Stomach. By means of the 
 chemical stimulation caused by the food, a copious secretion of gastric 
 
 1 See footnote 1, p. 477. 
 
 'Heidenhain and Klemensiewicz, 1. c; Contejean, 1. c., Chapter II, and Skand. 
 Arch f. Physiol, 6; Akermann, ibid., 5; Kresteff, Maly's Jahresber., 30; Schemia- 
 kine Arch, des scienc. biolog. de St. P£tersbourg, 10. 
 
 1 Rosemann, Pfluger's Arch. 118; Kudo, Bioch. Zeitschr. 16. 
 
CHYME AND DIGESTION IN THE STOMACH. 479 
 
 juice occurs, which gradually mixes with the swallowed food, and digests 
 it more or less strongly. The material in the stomach during digestion, 
 which has a pasty or thick consistency, and is called chyme, is not a 
 homogeneous mixture of the ingesta with the various digestive fluids, 
 gastric juice, saliva, and gastric mucus, but the conditions seem to 
 be more complicated. 
 
 From the investigations of several workers, 1 on the movements of 
 the stomach, we conclude that this organ in carnivora and also in man 
 consists of two physiologically different parts, the pylorus and the 
 fundus. The greater fundus part, which serves essentially as a reservoir, 
 may be a rhythmic, strong contraction of the muscle, acting like a 
 sphincter between it and the pylorus part, be separated from the latter, 
 and according to some observers so completely so that during contrac- 
 tion scarcely anything passes from the fundus to the pylorus part. 
 Differing from the fundus part the pylorus is the seat of very powerful 
 contractions by which its contents are intimately mixed with gastric 
 juice and are also driven through the pyloric valve into the intestine. 
 
 The contents of the pylorus part have an acid reaction, and a strong 
 pepsin digestion takes place in the contents, which are thoroughly mixed 
 with gastric juice. The contents of the fundus, on the contrary, show 
 a different behavior, for here, as Ellenberger first showed, a special 
 stratification of the various solid food-stuffs takes place. 
 
 By very instructive investigations on different animals (frogs, rats, 
 rabbits, guinea-pigs, and dogs) Grutzner 2 later showed that when 
 the aminals are fed with food having different colors, and the stomach 
 removed after a certain time, and the contents frozen, the frozen sec- 
 tions show a regular stratification of the contents. These layers are 
 so arranged that the food first taken is found in direct contact with the 
 mucosa, while the food taken later is enclosed by that partaken of first, 
 and this prevents contact with the walls of the stomach. The empty 
 stomach, whose walls touch each other, is so filled that, as a rule, the 
 foodstuffs taken later are in the middle of the older food. 
 
 Because of this fact only the foodstuffs which lie close to the surface 
 of the mucous membrane undergo the process of peptic digestion, and 
 it is principally these ingesta, which lie on the surface and are laden with 
 pepsin and mixed with gastric juice, which are shoved to the pylorus 
 end, here mixed and digested, and finally moved into the intestine 
 
 1 Hofireister and Schutz, Arch. f. exp. Path. u. Pharm., 20; Moritz. Zeitschr. f. 
 Biologie, 32; Cannon, Amer. Journ. of Physiol., 1; Sehemiakine, 1. c; Cathcart, 
 Journ. of Physiol., 1911, 42. 
 
 1 See Ellenberger, Pfliiger'a Arch., 114, and Scheunert, ibid., 144; Grutzner, ibid., 
 106. 
 
480 DIGESTION. 
 
 The fundus part is therefore less a digestion-organ than a storage-organ, 
 and in the interior of the same, the food may remain for hours without 
 coming in contact with a trace of gastric juice. 
 
 What has been said above applies at least to solid food. We have 
 no extensive observations on the behavior of fluids or semifluid food. 
 According to Grutzner, in these cases, as well as in the above-mentioned 
 experiments, the swallowed foodstuffs are not irregularly mixed together. 
 Fluids quickly leave the stomach, which is also the case with a mixture 
 of solid and fluid food. 
 
 Milk is an exception because it coagulates and the clot remains in the 
 stomach while the whey quickly leaves the stomach. 
 
 The fact that only that part of the ingesta lying on the mucous 
 membrane is mixed with gastric juice, while the mass in the interior is 
 not acid in reaction, is of special importance for the digestion of starches 
 in the stomach. By this we can explain why the salivary diastase, 
 although sensitive toward acids, can continue its action for a long time 
 in the contents of the stomach. That this is true was first found by 
 Ellenberger and Hofmeister and then by Cannon and Day * by 
 special experiments upon animals. The occurrence of sugar and dex- 
 trin in the contents of the human stomach has been repeatedly observed. 
 In carnivora, whose saliva shows scarcely any diastatic action, it is 
 a 'priori not expected that there should be a diastatic action in the 
 stomach, but the conditions are different in herbivora, where an abun- 
 dant digestion of starch takes place in the various stomachs, according 
 to the different species. 
 
 The gastric contents which have been prepared in the pylorus part 
 are passed through the pylorus into the intestine intermittently. This' 
 material is generally fluid, but it is possible that pieces of solid food 
 may also occur, and this has often been observed. Thin or plastic 
 food leaves the stomach earlier than solid food, and it is obvious that the 
 time in which the stomach unburdens itself depends naturally upon 
 the coarseness or fineness of the food. This depends essentially upon 
 the reflex action of the stomach or intestine, causing an opening or 
 closing of the pylorus, which action is dependent upon the quantity and 
 character of the food, the amount of fat, and the degree of acidity in 
 the contents of the stomach and intestine. The emptying of the food 
 into the small intestine causes, as shown by Pawlow, a closing of the 
 pylorus by chemo-reflex in which the hydrochloric acid and the fat take 
 part, and we thus find in this regard an alternate action between the 
 stomach and duodenum. 
 
 1 EllenberRer and Hofmeister, Maly's Jahresb., 15 and 16; Cannon and Day, Amer. 
 Journ. of Physiol.. 9. 
 
DIGESTION IN THE STOMACH. 481 
 
 This alternate action, according to Cannon l is due to the fact that 
 the acid in the pylorus which acts upon the sphincter and makes pos- 
 sible the passage of the fluid chyme by the contraction of the muscles 
 of the stomach. In the intestine the acid has a reverse stimulation 
 upon the sphincter and causes a contraction of the same. As soon as 
 the acid is neutralized the contractions of the sphincter cease and the 
 passage of new portions of the chyme occur. If the flow of bile and pan- 
 creatic juice is prevented, and the neutralization of the acid contents 
 of the stomach in the intestine is retarded, then the stomach does not 
 eject its contents so often. The duration of gastric digestion varies 
 according to conditions, and in consequence the reports of observers are 
 widely divergent. Beaumont 2 found in his extensive observations 
 on the Canadian hunter St. Martin that the stomach, as a rule, is 
 emptied 1£-5| hours after a meal, depending upon the character of the 
 food. 
 
 The time in which different foods leave the stomach also depends 
 upon their digestibility. Respecting the unequal digestibility in the 
 stomach we must differentiate between the rapidity with which the food- 
 stuffs are chemically transformed and that with which they leave the 
 stomach and pass into the intestine. This distinction is especially 
 important, and it is evident that the main factors governing speed of 
 digestion and the time required before the food leaves the stomach are the 
 kind of food and the fineness of its subdivision, and its action upon the 
 gastric secretion, upon the pyloric reflexes, etc. 
 
 The observations of Boldyreff and others 3 on the action of fats 
 and fatty acids and not too dilute hydrochloric acid (stronger than 0.2 
 per cent) are conclusive concerning the manner in which the properties 
 of the food act upon the gastric secretion and upon the digestion in the 
 stomach as a whole. Irrespective of the reducing action of the fats 
 upon the extent and digestive power of the gastric juice Boldyreff 
 found after food very rich in fat that the bile, pancreatic juice and intes- 
 tinal juice migrate from the intestine into the stomach so that the diges- 
 tion in the stomach in these cases is essentially brought about by the 
 pancreatic juice. 
 
 We have numerous investigations on the rapidity with which the 
 food is digested in the stomach of dogs, but we must especially mention 
 
 1 Amer. Journ. of Physiol., 20. 
 
 2 The Physiology of Digestion, 1833. 
 
 3 Boldyreff, Pfliiger's Arch., 121, 140; Migay, Maly's Jahresb. 39; Best and 
 Cohnheim, Zeitschr. f. Physiol. Chem. 69; Cathcart, Journ. of Physiol. 42. See 
 also Abderhalden and Medigreeeanu, Zeitschr. f. physiol. Chem., 57. 
 
482 DIGESTION. 
 
 the researches of E. Zunz, 1 London 2 and his co-workers. London, 
 Polowzowa and Sagelmann have observed that all the foodstuffs do 
 not leave the stomach with the same rapidity, indeed, by feeding with 
 bread (Polowzowa), the carbohydrates leave more quickly than the 
 protein, and with a mixture of gliadin and beef-fat (Sagelmann) the 
 protein left the stomach more quickly than the fat. This is in accord 
 with the recent observations of London and Sivr^ which show that the 
 fats remain longest in the stomach, the starches the shortest and meat 
 takes a middle position. 3 According to these authors the stomach has 
 a sort of " selective capacity," but this is strongly disputed by Schetjn- 
 ert and Grimmer. 4 Nevertheless the researches of Cannon 5 on cats, 
 making use of another method, have shown that this is true. After 
 preliminary hunger the animals received different food, such as meat, 
 fat, and carbohydrate mixed with bismuth subnitrate and then with 
 the aid of the Rontgen rays the time was noted when the food passed 
 into the intestine. The carbohydrate leaves the stomach first, the 
 proteins next, and the fats last. If the carbohydrate is given before 
 the protein food, then it leaves the stomach with ordinary rapidity; 
 while if protein food and then carbohydrate is given the passage of the 
 carbohydrate is retarded. A mixture of protein food and carbohydrates 
 leaves the stomach more slowly than carbohydrates alone, but faster 
 than protein food alone. The fat, which remains in the stomach for a 
 long time and leaves the stomach only in amounts which are absorbed 
 or removed from the duodenum, retards the passage of the protein foods 
 as well as the carbohydrates. Tangl and Erdelyi 6 have found in 
 regard to the different kinds of fat, that a fat leaves the stomach the slower 
 according to the he : ght of its melting-point. According to London and 
 Schwarz, 7 with mixed protein feeding, the digestion in the stomach 
 is regulated by that kind of protein, which, when alone, is removed from 
 the stomach the slowest. 
 
 The reason why different foodstuffs leave the stomach with unequal rapidity 
 is explained by Cannon by the above-mentioned action of the hydrochloric acid 
 
 1 E. Zunz, Hofrr.eister's Beitrage, 3; Annal de la soc. roy. des scienc. med. Bruxelles 
 12, 13, and Memoires publ. par l'Acad. roy. Belg., 1906, 1907, and 1908. Intern. 
 Beitr. zu. Path. u. Ther. der Ernahrungsstorungen 2; Bull, de l'Acad. roy. de med. 
 de Belgique, 24 (1910). 
 
 1 The numerous works of London and co-workers will be found in Zeitschr. f. 
 physiol. Chem., 45-53, 55-58, 60-74. 
 
 1 London with Polowzowa, Zeitschr. f. physiol. Chem., 49, with Sagelmann, ibid., 
 62; London and SivTe, ibid. 60 (1909). 
 
 4 Scheunert, Zeitschr. f. physiol. Chem., 51; Grimmer, Bioch. Zeitschr., 3. 
 
 6 Amer. Journ. of Physiol., 12 and 20; Amer. Journ. Med. Sciences, 138, 504 (1909). 
 
 •Bioch. Zeitschr. 34, 94 (1911). 
 
 T Zeitschr. f. physiol. Chem. 68, (1910). 
 
DIGESTION IN THE STOMACH. 483 
 
 upon the pyloric sphincter. The proteins combine with the hydrochloric acid 
 and hence its action upon the sphincter becomes weaker, while this is not the 
 case with the carbohydrates. If the carbohydrates are moistened with alkali 
 tin y Leave the Btomach more slowly than usual, and the acid proteins, on the con- 
 trary, leave the stomach earlier than other proteins. 
 
 As our knowledge of the digestibility of the different foods in the 
 stomach is slight and uncertain, so also our knowledge of the action of 
 other bodies, such as alcoholic drinks, bitter principles, spices, etc., on 
 the natural digestion is very uncertain and imperfect. The difficulties 
 which stand in the way of this kind of investigation are very great, and 
 therefore the results obtained thus far are often ambiguous or conflict 
 with each other. For example, certain investigators have observed that 
 small quantities of alcohol or alcoholic drinks do not prevent but rather 
 facilitate digestion; others observed only a disturbing action, while 
 still others report having found that the alcohol first acts somewhat as 
 a disturbing agent, but afterward, when it is absorbed, produces and abun- 
 dant secretion of gastric juice, and thereby facilitates digestion. The 
 accelerating action of alcohol upon the flow of gastric juice has been 
 mentioned on page 464. 
 
 In regard to the importance of the stomach we used to be of the 
 general opinion that an abundant peptonization of protein does not 
 occur in the stomach, and that the food rich in protein is only 
 chiefly prepared in the stomach for the real digestion in the intestine. 
 That the stomach, at least the fundus, acts in the first place as a storage 
 chamber, follows from the shape of this organ, especially in certain 
 animals, and this function becomes especially prominent in certain new- 
 born animals, as dogs and cats. In these animals the gastric secretion 
 contains acid but no pepsin, and the casein of the milk is precipitated 
 by the acid alone as solid lumps or as a solid coagulum filling the 
 stomach. Gradually small quantities of this coagulum pass into the 
 intestine and an overburdening of the intestine is thus prevented. In 
 other animals, as the snake and certain fishes which swallow entire animals, 
 the major part of the digestive work goes on in the stomach. The 
 importance of the stomach for digestion cannot therefore be established 
 in all instances. It varies in different animals and differs even in indi- 
 vidual animals of the same species, depending upon the fineness or coarse- 
 ness of the food, upon the greater or less rapidity with which pepton- 
 ization takes place, and also upon the rapid or slow increment in the 
 quantity of hydrochloric acid, etc. 
 
 In regard to the extent of chemical digestive work, i.e., in the first 
 place the destruction of protein in the stomach, we have numerous 
 researches, some carried out by the use of older methods and others 
 by using newer and more reliable methods. Among these latter we 
 must mention those of Zunz, London and collaborators, Tobler, Lanq 
 
484 DIGESTION. 
 
 and Cohnheim. 1 These investigations refer to the conditions in dogs, 
 and as shown by Rosenfeld 2 in horses, and by Lotsch 3 in pigs, that 
 the conditions are different in other animals. The following description 
 applies only to dogs. 
 
 In the dog Abderhalden, London and co-workers 4 have shown 
 that in the stomach proteoses and peptones are formed, but no amino- 
 acids, or at least not in any mentionable quantity. The scanty occurrence 
 -of amino-acids is substantiated by the observations of Zunz and others 5 
 that the amount of amino-nitrogen titratable with formol in the stomach 
 contents, is only small. 
 
 In like manner we must agree in the belief that a part of the protein 
 always leaves the stomach undigested and that the principal mass, about 
 80 per cent, passes into the intestine more or less digested. Besides this 
 it also seems as if the peptones occur in the pylorus part to a greater 
 extent than the proteoses, while in the fundus part the reverse is the 
 case. Of the dissolved protein of the entire stomach-contents about 
 60 per cent exists as proteoses. Opinions are also contradictory in 
 regard to the absorption of the decomposition products of the proteins 
 in the stomach. While several investigators, like Tobler, Lang, Cohn- 
 heim, Zunz and others accept such an absorption, London and co-workers 
 positively deny this. 
 
 The digestion of sundry foods is not dependent on one organ alone, 
 but is divided among several. For this reason it is to be expected that 
 the various digestive organs can act for one another to a certain extent, 
 and that therefore the work of the stomach could be taken up more or 
 less by the intestine. This in fact is the case. Thus the stomachs of 
 dogs and cats have been completely extirpated or nearly so (Czerny, 
 Carvallo and Pachon, London and collaborators), or that part 
 necessary in the digestive process has also been eliminated by plugging 
 the pyloric opening (Ludwig and Ogata), and in both cases it was pos- 
 sible to keep the animal alive, well fed, and strong for a shorter or 
 longer time. The extirpation of the stomach has also been repeatedly 
 
 1 Tobler, Zeitschr. f. physiol. Chem., 45; Lang, Bioch. Zeitschr., 2; Cohnheim, 
 Munch, med. Wochenschr., 1907. In regard to the works of Zunz, London, and 
 collaborators, see footnotes 1, 2 and 3, p. 482. 
 
 2 Rosenfeld, Ueber die Eiweissverdauung im Magen des Pferdes, Inaug.-Dissert., 
 Dresden, 1908. 
 
 ' Lotsch. Zur Kenntnis der Verdauung von Fleisch im Magen und Dunndarm des 
 Schweines, Inaug.-Dissert. Freiburg i. Sa., 1908; see also Abderhalden, Klingemann 
 and Pappenhusen, Zeitschr. f. physiol. Chem. 71, 411 (1911). 
 
 4 Abderhalden and London, with Kautsch, Zeitschr. f. physiol. Chem., 48, with 
 L. Baurnann, ibid., 51, and with v. Korosy, ibid., 51. 
 
 : Intern. Beitr. zu Path. u. Ther. d. Ern.-Stor. 2; London and Rabinowitsch, 
 Zeitschr. f. physiol. Chem. 74. 
 
DIGESTION IN THE STOMACH. 485 
 
 performed on* human beings with the same results. 1 In these cases it is 
 evident that the digestive work of the stomach was taken up by the 
 intestine; but all food cannot be digested in these cases to the same 
 extent, and the connective tissue of meat especially is sometimes found 
 to a considerable extent undigested in the excrements. 
 
 It is a well-known fact that the contents of the stomach may be 
 kept without decomposing for some time by means of hydrochloric acid, 
 while, on the contrary, when the acid is neutralized a fermentation 
 commences by which lactic acid and other organic acids are formed. 
 According to Cohn, an amount of hydrochloric acid above 0.7 p. m. 
 completely arrests lactic-acid fermentation, even under otherwise favor- 
 able circumstances, and according to Strauss and Bialocour the limit 
 of lactic-acid fermentation lies at 1.2 p. m. hydrochloric acid united 
 to organic bodies. The hydrochloric acid of the gastric juice has unques- 
 tionably an antifermentative action, and also, like all dilute mineral 
 acids, an antiseptic action. This action is of importance, as many path- 
 ogenic micro-organisms may be destroyed by the gastric juice. The 
 common bacillus of cholera, certain streptococci, etc., are killed by the 
 gastric juice, while others, especially as spores, are unacted upon. The 
 fact that gastric juice can diminish or retard the action of certain tox- 
 albumins, such as tetanotoxine and diphtheria toxine, is also of great 
 interest (Nencki, Sieber, and Schoumowa 2 ). 
 
 Because of this antifermentative and antitoxic action of gastric juice 
 it is considered that the principal importance of this juice lies in 
 its antiseptic action. The fact that intestinal putrefaction is not 
 increased on the extirpation of the stomach, as derived from experi- 
 ments made on man and animal, 3 does not uphold this view. 
 
 Since the hydrochloric acid of the gastric juice prevents the con- 
 tents of the stomach from fermenting, with the generation of gas, those 
 gases which occur in the stomach probably depend, at least in great 
 measure, upon the swallowed air and saliva, and upon those gases gen- 
 erated in the intestine and returned through the pyloric valve. Planer 
 found in the stomach-gases of a dog 66-68 per cent N, 23-33 per cent 
 
 1 Czerny, cited from Bunge, Lehrbuch d. physiol. u. path. Chem. 4. Aufl., Theil 2, 
 173; Carvallo and Pachon, Arch. d. Physiol. (5), 7; Ogata, Arch. f. (Anat. u.) Physiol. 
 1883; Grohe, Arch. f. exp. Path. u. Pharm. 49; London and collaborators, Zeitschr. 
 f. physiol. Chem. 74, 328 (1911) ; in regard to the case in man, see Schlatter in 
 Wr6blewski, Centralbl. f. Physiol. 11, p. 665, and the surgical journals. 
 
 2 Cohn, Zeitschr. f. physiol. Chem., 14; Strauss and Bialocour, Zeitschr. f. klin. 
 Med., 28. See also Kiihne, Lehrb., 57; Bunge, I.ehrb. d. Physiol., 4. Aufl., 148 and 
 159; Hirschfeld, Pfliiger's Arch., 47; Nencki, Sieber, and Schoumowa, Centralbl. f. 
 Bacterid., etc., 23. In regard to the action of gastric juice upon pathogenic microbes 
 we must refer the reader to hand-books of bacteriology. 
 
 3 See Carvallo and Pachon. 1. c, and Schlatter in Wr6blewski, 1. c. 
 
486 DIGESTION. 
 
 CO2, and only a small quantity, 0.8-6.1 per cent, of oxygen. Schier- 
 beck ! has shown that a part of the carbon dioxide is formed by the 
 mucous membrane of the stomach. The tension of the carbon dioxide 
 in the stomach corresponds, according to him, to 30-40 mm. Hg in the 
 fasting condition. It increases after partaking food, independently of 
 the kind of food, and may rise to 130-140 mm. Hg during digestion. 
 The curve of the carbon-dioxide tension in the stomach is the same as 
 the curve of acidity in the different phases of digestion, and Schier- 
 beck also found that the carbon-dioxide tension is considerably increased 
 by pilocarpine, but diminished by nicotine. According to him, the 
 carbon dioxide of the stomach is a product of the activity of the secretory 
 cells. 
 
 After death, if the stomach still contains food, autodigestion goes 
 on not only in the stomach, but also in the neighboring organs, during 
 the slow cooling of the body. This leads to the question, Why does the 
 stomach not digest itself during life? Ever since Pavy has shown that 
 after tying the smaller blood-vessels of the stomach of dogs the cor- 
 responding part of the mucous membrane was digested, efforts have 
 been made to find the cause in the neutralization of the acid of the gas- 
 tric juice by the alkali of the blood. That the reason for the non- 
 digestion during life is to be sought for in the normal circulation of the 
 blood cannot be contradicted; but the reason is not to be found in the 
 direct neutralization of the acid. The investigations of Fermi and 
 Otte 2 show that the blood circulation acts in an indirect manner by the 
 normal nourishment of the cell protoplasm, and this is the reason why 
 the digestive fluids, the gastric juice as well as the pancreatic juice, act 
 differently upon the living protoplasm as compared with the dead. We 
 know nothing about this resistance of the living protoplasm. Some 
 claim that it is closely connected with occurrence of different inhibitory 
 substances in the gastric mucosa. Of these the substance found by 
 Weinland is thermolabile while that of Danilewsky, Hansel and 
 Schwarz is resistant toward heat. 3 Without mentioning the still un- 
 known nature of these bodies, the neutral gastric juice, as well as an 
 acid infusion of the mucosa, has such a strong digestive action that the 
 inhibiting action of the mentioned substances can only be shown under 
 special conditions, and it is therefore difficult to conceive how these sub- 
 stances could have a protective action in life. 
 
 1 Planer, Wien. Sitzungsber., 42; Schierbeck, Skand. Arch. f. Physiol., 3 and 5. 
 
 2 Pavy, Phil. Transactions, 153, Part I, and Guy's Hospital Reports, 13; Otte, 
 Travaux du laboratoire de l'lnstitut de Physiol, de Liege, 5, 1896, which also contains 
 the literature. 
 
 1 Wf-inland, Zeitschr. f. Biologie, 44; Hansel, Biochem. Centralbl., 1, p. 404, 
 and 2, p. 320; Schwartz, Hofmeister's Beitrage, 6. 
 
EXAMINATION OF THE GASTRIC CONTENTS. 487 
 
 Under pathological conditions irregularities in the secretion may 
 occur. The quantity of enzymes may be diminished and both enzymes 
 or, as found in certain cases, one (the rennin), may be absent. The 
 hydrochloric acid may also be absent or may exist in very small amounts. 
 A pathological high degree of acidity of the pure juice is not very prob- 
 able, while on the contrary a hypersecretion of gastric juice in different 
 forms does occur. 
 
 In testing the gastric juice or the filtered stomach contents, diluted 
 with digestive hydrochloric acid, for pepsin, we make use of the pepsin 
 tests given on pages 469, 470. In testing for rennin the liquid must be 
 first carefully neutralized, and 1-2 cc. of this liquid added to 10 cc. 
 milk. In the presence of appreciable quantities of rennin, the milk 
 should coagulate at room temperature within 10-20 minutes without 
 changing its reaction. The addition of lime salts is unnecessary, and 
 may readily lead to erroneous conclusions. 
 
 In many cases it is especially important to determine the degree of 
 acidity of the gastric juice. This may be done by the ordinary titration 
 methods. Phenolphthalein must not be used as an indicator, as too 
 high results are produced in the presence of large quantities of proteins. 
 Good results may be obtained, on the contrary, by using very delicate 
 litmus paper. Although the acid reaction of the contents of the stomach 
 may be caused simultaneously by several acids, still the degree of acidity 
 is here, as in other cases, expressed in only one acid, e.g., HO. Gen- 
 erally the acidity is designated by the number of cubic centimeters of 
 N/10 sodium hydroxide required to neutralize the several aeids in 
 100 cc. of the liquid of the stomach. An acidity of 43 per cent means 
 that 100 cc. of the liquid of the stomach required 43 cc. of N/10 sodium 
 hydroxide to neutralize it. 
 
 It is also important to be able to ascertain the nature of the acid or 
 acids occurring in the contents of the stomach. For this purpose, and 
 especially for the detection of free hydrochloric acid, a great number of color 
 reactions have been proposed which are all based upon the fact that the 
 coloring substance gives a characteristic color with very small quanti- 
 ties of hydrochloric acid, while lactic acid and the other organic acids 
 do not give these colorations, or only in a certain concentration, which 
 can hardly exist in the contents of the stomach. These reagents are a 
 mixture of ferric-acetate and potassium-sulphocyanide solutions 
 (Mohr's reagent has been modified by several investigators), methyl- 
 aniline-violet, TROPiEOLIN 00, CONGO RED, MALACHITE-GREEN, PHLORO- 
 
 glucinol-vanillin, dimethylaminoazobenzene, and others. As reagents 
 for free lactic acid, Uffelmann suggests a strongly diluted, amethyst-blue 
 solution of ferric chloride and carbolic acid, or a strongly diluted 
 nearly colorless solution of ferric chloride. These give a yellow 
 color with lactic acid, but not with hydrochloric acid or with volatile 
 fatty acids. 
 
 The value of these reagents in testing for free hydrochloric acid or lactic 
 acid is still disputed. Among the reagents for free hydrochloric acid it seems 
 Steensma's ' modification of Gunzburg's test with phloroglucinol-vanilhn, and 
 
 1 Bioch. Zeitschrift, 8. 
 
488 DIGESTION. 
 
 the test with tropseolin 00, performed at a moderate temperature as suggested by 
 Boas, and the test with dimethylamincazobenzene, which is the most delicate, 
 seem to be the most valuable. If these tests give positive results, then the presence 
 of hydrochloric acid may be considered as proved. A negative result does not 
 eliminate the presence of hydrochloric acid, as the delicacy of these reactions 
 has a limit, and also the simultaneous presence of protein, peptones, and other 
 bodies influences the reactions more or less. The reactions for lactic acid may 
 also give negative results in the presence of comparatively large quantities of 
 hydrochloric acid in the liquid to be tested. Sugar, sulphocyanides, and other 
 bodies may act with these reagents like lactic acid. 
 
 In testing for lactic acid it is safest to shake the material with ether and test 
 the residue after the evaporation of the solvent. On the evaporation of the ether 
 the residue may be tested in several ways. Boas utilizes the property possessed 
 by lactic acid of being converted into aldehyde and formic acid on careful oxida- 
 tion with sulphuric acid and manganese dioxide. The aldehyde is detected by 
 its forming iodoform with an alkaline iodine solution or by its forming aldehyde- 
 mercury with Nessler's reagent. Croner and Cronheim: x have suggested 
 another method. 
 
 The quantitative estimation consists in the formation of iodoform with N/10 
 iodine solution and caustic potash, adding an excess of hydrochloric acid and 
 titrating with a N/10 sodium-arsenite solution, and retitrating with iodine solu- 
 tion, after the addition of starch-paste, until a blue coloration is obtained. This 
 method presupposes the use of ether entirely free from alcohol. For details see 
 the original publication and the modification of this method suggested by 
 Jerusalem. 2 
 
 In order to be able to judge correctly of the value of the different 
 reagents for free hydrochloric acid, it is naturally of greatest importance 
 to be clear in regard to what we mean by free hydrochloric acid. It is 
 a well-known fact that hydrochloric acid combines with proteins, and a 
 considerable part of the hydrocholoric acid may therefore exist in the 
 contents of the stomach, after a meal rich in proteins, in combination 
 with them. This hydrochloric acid combined with proteins cannot 
 be considered as free, and it is for this reason that certain investigators 
 consider such methods as those of Sjoqvist, which will be described 
 below, as of little value. However, it must be remarked that, according 
 to the unanimous experience of many investigators, the hydrochloric 
 acid combined with proteins is physiologically active and in this regard 
 we must refer to the recent investigations of Alb. Muller and J. Schutz. 3 
 Those reactions (color reactions) which only respond to actually free 
 hydrochloric acid do not show the physiologically active hydrochloric 
 acid. The suggestion of determining the " physiologically active " 
 hydrochloric acid instead of the " free " seems to be correct in principle; 
 and as the conceptions of free and of physiologically active hydrochloric 
 acid are not the same, it must always be well defined whether one wishes 
 to determine the actually free or the physiologically active hydro- 
 
 1 Boas. Deutsch. med. Wochenschr., 1803, and Munchener med. Wochenschr. 1893, 
 Croner and Cronheim, Berl. klin. Wochenschr., 1905. See also Thomas, Zeitschr. f. 
 physiol. Chem., 50. 
 
 2 Bioch. Zeitschr., 12. 
 
 8 Alb. Muller, Deutsch. Arch. f. klin. Med., 88, and Pfluger's Arch., 116; J. Schiitz. 
 Wien, klin. Wochenschr., 20. and Wien. med. Wochenschr., 1900 (older literature). 
 
SECRETION OF BRUNNER'S GLANDS. 489 
 
 chloric acid before any conclusions are drawn as to the value of a certain 
 reaction. 
 
 The acid reaction may be partly due to free acid, partly to acid salts (mono- 
 phosphates), and partly. to both. According to Leo, 1 one can test for acid phos- 
 phates by calcium carbonate, which is not neutralized therewith, while the free 
 acids are. If the gastric content has a neutral reaction after shaking with cal- 
 cium carbonate, and the carbon dioxide is driven out by a current of air, it con- 
 tains only free acid; if it has an acid reaction, acid phosphates are present, 
 and if it is less acid than before, it contains both free acid and acid phosphate. 
 It must not be forgotten that a faint acid reaction may, after treatment with cal- 
 cium carbonate, also be due to the protein. This method can likewise be applied 
 in the estimation of free acid. 
 
 Various titration methods have been suggested for the estimation of the 
 free hydrochloric acid, but these cannot yield conclusive results for the reasons 
 given in Chapter I. For this determination physico-chemical methods (page 74), 
 are necessary, but they have not been used to any great extent for clinical pur- 
 poses. Holmgren 2 has suggested a method for estimating hydrochloric acid 
 based upon the adsorption phenomenon. 
 
 A great number of methods have been suggested for the quantitative estima- 
 tion of the total acidity, among which we must mention those of K. Morner and 
 Sjoqvist, which are extensively used. As the value of a special determination 
 of the free and total hydrochloric acid is doubtful, or at least disputed, and also as 
 the question is chiefly of clinical interest we must refer to the hand-books of clinical 
 investigations of v. Jaksch, Eulenburg, Kolle, and Weintraud and of Sahli. 
 The same applies to the tests for volatile fatty acids. 
 
 m. THE GLANDS OF THE MUCOUS MEMBRANE OF THE INTESTINE AND 
 
 THEIR SECRETIONS. 
 
 The Secretion of Brunner's Glands. These glands are partly con- 
 sidered as small pancreatic glands and partly as mucous or salivary 
 glands. Their importance is not the same in all animals. According 
 to Grutzner they are in dogs closely related to the pyloric glands and 
 contain pepsin. This also coincides with the observations of Glaessner 
 and of Ponomarew, which differ from each other only in that Pono- 
 marew finds that the secretion is inactive in alkaline reaction and con- 
 tains only pepsin, while Glaessner claims it is active in both acid and 
 alkaline reaction and that it contains pseudopepsin. According to 
 Abderhalden and Rona the pure duodenal secretion of the dog contains 
 a proteolytic enzyme which does not belong to the trypsin type but 
 rather to the pepsin variety. The statements as to the occurrence of a 
 diastatic enzyme in Brunner's glands are disputed. Scheunert and 
 Grimmer 3 indeed found diastatic enzyme in the duodenal glands of the 
 horse, ox, pig and rabbit, but no proteolytic or rennin enzyme. 
 
 Centralbl, f. d. med. Wissensch., 1889, p. 481; Pfliiger's Arch., 48, and Berlin, 
 klin. Wochenschr., 1905, p. 1491. 
 
 2 Deutsch. med. Wochenschr., 1911, p. 247. 
 
 3 Grutzner, Pfluger's Arch., 12; Glaessner, Hofmeister's Breitrage, 1; Ponomarew, 
 Biochem. Centralbl., 1, 351; Abderhalden and Rona, Zeitschr. f. physiol. Chem., 
 47; Scheunert and Grimmer, cited in Bioch. Centralbl., 5, 673. 
 
490 DIGESTION. 
 
 The Secretion of Lieberkuhn's Glands. The secretion of these glands 
 has been studied with the aid of a fistula in the intestine according to 
 the method of Thiry and Vella or of Pawlow. According to Boldy- 
 reff, 1 in dogs, with an empty stomach, a scanty secretion lasting about 
 15 minutes occurs at regular intervals for about two hours. According 
 to Boldyreff the intestinal juice is obtained from a Thiry- Villa fistula 
 outside of the digestion period without any apparent stimulation. Accord- 
 ing to this experimenter, during gastric digestion the juice is periodically 
 but less abundantly secreted as the time interval is much longer, namely 
 three, four or five hours. Otherwise it is generally admitted that the 
 partaking of food causes the secretion, or if this is continuous, as in 
 lambs (Pregl), it increases the secretion. The researches of Dele- 
 zenne and Frouin show without question that the passage of chyme 
 into the intestine increases the secretion of the intestinal juice. The 
 acid causes a formation of secretin (see below), and this produces, 
 according to the above investigators, a secretion of intestinal juice. 
 Among the chemically active substances causing a secretion we must 
 mention acids in general and gastric juice. Soaps, chloral, ether and 
 on intravenous injection, also intestinal juice or an extract of the intes- 
 tinal mucosa (Frouin), are chemical excitants of intestinal juice. 
 Several salts, NaCl, Na2SOi, and others, may cause an abundant secre- 
 tion of fluid into the intestine when injected intravenously or subcu- 
 taneously, as well as after direct application to the peritoneal surface 
 of the intestine. This action can be arrested by the antagonistic, 
 inhibiting action of a lime salt (MacCallum). Pilocarpine, which has 
 the power of increasing the activity of secretions, does not increase the 
 secretion in lambs, and in dogs it does not seem to be always active 
 (Gamgee 2 ). 
 
 Mechanical irritation of the intestinal mucosa increases the secre- 
 tion in dogs (Thiry) as well as in man (Hamburger and Hekma), but 
 it is still doubtful whether we here have a perfectly physiological juice. 
 In the cases observed by Hamburger and Hekma 3 the flow of fluid was 
 greatest at night as well as between five and eight o'clock in the after- 
 noon, and was lowest between two and five o'clock in the afternoon. 
 The quantity of this secretion in the course of twenty-four hours has 
 not been exactly determined. 
 
 1 Thiry, Wien, Sitz.-Ber., 50; Vella, Molleschott's Untersuch., 13; Boldyreff, 
 Zeitschr. f. physiol. Chem., 50, Centralbl. f.'Physiol. 24, 93 (1910). 
 
 2 Delezenne and Frouin, Compt. rend. soc. biol., 56; Frouin, ibid., 56 and 58; 
 MacCallum, University of California Publications, 1, 1904; Gamgee, Physiol. Chem- 
 istry, 2, 410 (literature). 
 
 3 Journ. de Physiol, et d. path, gen., 1902 and 1904. 
 
INTESTINAL JUICE. 491 
 
 According to Delezenne and Frouin, if any mechanical irritation 
 is prevented, the fluid flowing spontaneously from a fistula in a dog 
 is ten times more abundant in the duodenum than that in the middle 
 or lower part of the jejunum. In the upper part of the small intestine 
 of the dog, on the contrary, this secretion is scanty, slimy, and gelatin- 
 ous; in the lower part it is more fluid, with gelatinous lumps or flakes 
 (Rohmann). Intestinal juice has a strong alkaline reaction toward 
 litmus, generates carbon dioxide on the addition of an acid, and contains 
 (in dogs) nearly a constant quantity of NaCl and Na2CC>3, 4.8-5 and 4-5 
 p. m. respectively (Gumilewski, Rohmann 1 ). The intestinal juice 
 of the lamb corresponded to an alkalinity of 4.54 p. m. Na2C03. It 
 contains protein (Thiry found 8.01 p. m.), the quantity decreasing 
 with the duration of the elimination. The quantity of solids varies. 
 In dogs the quantity of solids is 12.2-24.1 p. m. and in lambs 29.85 p. m. 
 The specific gravity of the intestinal juice of the dog, according to the 
 observations of Thiry, is 1.010-1.0107, and in lambs 1.0143 (Pregl). 
 The intestinal juice from lambs contains 18.097 p. m. protein, 1.274 p. 
 m. proteoses and mucin, 2.29 p. m. urea, and 3.13 p. m. remaining organic 
 bodies. 
 
 We have the investigations of Demant, Turby and Manning, H. 
 Hamburger and Hekma and Nagano 2 on the human intestinal juice. 
 Human intestinal juice has a low specific gravity, nearly 1.007, about 
 10-14 p. m. solids, and is strongly alkaline toward litmus. The con- 
 tent of alkali calculated as sodium carbonate is 2.2 p. m., according 
 to Nagano, Hamburger and Hekma, and 5.8-6.7 p. m. Na CI. The 
 determination of the freezing-point was —0.62° (Hamburger and Hekma). 
 
 The intestinal juice of the dog contains, according to Boldyreff 
 a lipase which acts especially upon emulsified fat (milk), and is different 
 from pancreas lipase, in that its action is not accelerated by bile. 
 Jansen 3 found that the lipase was secreted from a Thiry- Vella fistula 
 especially under the influence of bile and acid. The intestinal juice of 
 animals and man also contains an enzyme, erepsin, discovered by O. 
 Cohnheim, which does not ordinarily have a splitting action upon native 
 proteins, but upon proteoses and peptones. It also possibly contains 
 a nuclease, and it also has a faint amylolytic action. The juice, and to 
 a high degree the mucous coat, contains iwertase and maltase, which 
 
 1 Gumilewski, Pfliiger's Arch., 39. Rohmann, ibid., 41. 
 
 2 Demant, Virchow's Arch., 75; Turby and Manning, Centralbl. f. d. med. Wis- 
 senschaft, 1892, 945; Hamburger and Hekma, 1. c; Nagano, Mitt, aus d. Grenzgeb. 
 d. Med. u. Chir., 9. 
 
 1 Boldyreff, Archiv. d. sciences biolog, de St. P6tersbourg, 11; Jansen, Zeitschr. 
 f. physiol. Chem. 58, 400 (1910). 
 
492 DIGESTION. 
 
 fact has been substantiated by the observations of Paschutin, Brown" 
 and Heron, Bastianelli, and Tebb. 1 A lactose-inverting enzyme, 
 a lactase, also occurs, as shown by Rohmann and Lappe, Patjtz and 
 Vogel, Weinland, and Orban, 2 in new-born infants and young ani- 
 mals, and also in grown mammals which were fed upon a milk diet (see 
 Chapter I, page 52). The lactase can be obtained more abundantly 
 from the mucosa than from the juice and according to some occurs only 
 in the cells. The claims as to the occurrence of a glucoside splitting 
 enzyme are disputed (Frouin, Omi 3 ) . 
 
 Besides erepsin and the other enzymes mentioned, the intestinal 
 mucosa also contains substances which have an inhibitory action upon 
 pepsin and trypsin. (Danilewsky and Weinland 4 ), also enterokinase 
 or a mother-substance of the same, and finally also the so-called 'pro- 
 secretin. These two last-mentioned bodies, which are closely connected 
 with the secretion of pancreatic juice, will be discussed in connection 
 with this digestive fluid. 
 
 The various enzymes are not formed in equal quantities in all parts 
 of the intestine. Diastase and invertase occur, according to Boldy- 
 reff, all through the intestine, while the lipase on the contrary does not 
 occur in the lower parts. The kinase occurs only in the upper part of 
 the intestine (Boldyreff, Bayliss and Starling, Delezenne). Ac- 
 cording to Hekma the kinase occurs in all parts of the intestine, but 
 most abundantly in the duodenum and the upper part of the jejunum. 
 The enzymes, Falloise claims, generally occur in greatest abundance 
 in the upper parts of the intestine; but the erepsin occurs to a greater 
 extent in the jejunum than in the duodenum. According to the investi- 
 gations of Vernon the behavior of erepsin is not the same in different 
 animals. In cats and hedge-hogs the duodenum is richer in erepsin than 
 the jejunum and ileum; in rabbits it is the reverse, namely, the ileum 
 is much richer than the duodenum. The secretin, according to Bay- 
 liss and Starling, is formed entirely in the upper part of the intestine.. 
 The epithelium-cells of the glands or the mucous membrane are generally 
 considered as the seat of formation of the enzymes, and the same is 
 true also for the enterokinase, according to Bayliss and Starling,. 
 
 Paschutin, Centralbl. f. d. med. Wissensch., 1870, 561; Brown and Heron, Annal. 
 d. Chem. u. Pharm., 204; Bastianelli, Moleschott's Untersuch, zur Naturlehre, 14 
 (this contains all the older literature). See also Miura, Zeitschr. f. Biologie, 32; Wid- 
 dicombe, Journ. of Physiol., 28; Tebb, ibid., 15. 
 
 2 Rohmann and Lappe, Ber. d. deutsch, chem. Gessellch., 28; Pautz and Vogel, 
 Zeitschr. f. Biologie. 32; Weinland, ibid., 38; Orban, Maly's Jahresber., 29. 
 
 3 Frouin and Thomas, Arch, internat. de Physiol., 7; Omi, Das Verhalten dea 
 Salizins irn tierschen Organisrnus, Inaug.-Dissert. Breslau, 1907. 
 
 * See footnote 3, p. 486. 
 
EREPSIN. 493 
 
 Hekma, Falloise, and others, which, however, Delezenne says. 1 is 
 formed in the leucocytes and Peyer's glands. 
 
 Erepsin. This enzyme, discovered by O. Cohnheim, has no direct 
 action upon native proteins with the exception of casein, but has the 
 power of splitting proteoses, peptones and certain polypeptides. In 
 this change mono- as well as diamino-acids are produced. Erepsin 
 occurs in the mucous membrane and in the intestinal juice of man as 
 well as of dogs; the mucous membrane seems to be richer than the juice 
 (Salaskin, Kutscher and Seemann 2 ). An enz3me like erepsin also 
 occurs in the pancreas (Bayliss and Starling, Vernon), and this has 
 the power of acting upon casein, but not, or only faintly, upon fresh 
 fibrin. This erepsin is probably identical with the enzyme nvclease, 
 discovered by F. Sachs in the pancreas, which acts upon nucleic acids, 
 while Nakayama claims that erepsin differs from trypsin in having a 
 cleavage action upon nucleic acids. Intestinal erepsin is not inhibited, 
 according to Glaessner and Stauber, by blood-serum and differs in 
 this regard from trypsin. Erepsin shows a great similarity to the intra- 
 cellular enzymes active in autolysis, and according to Vernon and others 
 erepsins occur in the various tissues of invertebrates as well as verte- 
 brates. These tissue erepsins, which are closely related to the auto- 
 lytic enzymes, if they are not identical with them, behave somewhat 
 differently from the intestinal erepsin and are not identical therewith. 
 Enzymes, having an action similar to erepsin, occur, Vines believes, 3 
 in all plants so far investigated. 
 
 Erepsin becomes inactive on heating to 59°. It works best in 
 alkaline solution, but has hardly any action in faint acid reaction. In 
 this regard, as well as by the fact that only a little ammonia is split off 
 by its action upon peptone substances, it differentiates itself from cer- 
 tain of the autolytic enzymes studied so far. The optimum of alkalinity 
 is, according to Euler, 4 at least in the splitting of a polypeptide, much 
 lower than the optimum for tryptic digestion. 
 
 The secretion of the glands in the large intestine seems to con- 
 sist chiefly of mucus. Fistulas have also been introduced into these 
 
 1 Boldyreff, Arch. d. scienc. biolog, de St. Petersbourg, 11; Bayliss and Starling, 
 Journ. of Physiol., 29, 30; Hekma, 1. c; Falloise, see Biochem. Centralbl., 4, p. 153; 
 Vernon, Journ. of Physiol., 33; Delezenne, Compt. rend. soc. biolog., 54 and 56. 
 
 2 Cohnheim, Zeitschr. f. Physiol. Chem., 33, 35, 36, and 47; Salaskin, ibid., 35; 
 Kutscher and Seemann, ibid., 35. 
 
 * Bayliss and Starling, Journ. of Physiol., 30; Vernon, ibid., 30 and 33. See also 
 Cohnheim and Pletnew, Zeitschr. f. physiol. Chem. 69; F. Sachs, Zeitschr. f. physiol. 
 Chem., 46; Nakayama, ibid., 41; Glaessner and Stauber, Bioch. Zeitschr. 25; Vines, 
 Annals of Botany, 18, 19, and 23. 
 
 4 Zeitschr. f. physiol. Chem., 51 
 
494 DIGESTION. 
 
 parts of the intestine, which are chiefly, if not entirely, to be considered 
 as absorption organs. The investigations on the action of this secretion 
 on nutritive bodies have not as yet yielded any positive results. 
 
 IV. THE PANCREAS AND PANCREATIC JUICE. 
 
 In invertebrates, which have no pepsin digestion and which also 
 have no formation of bile, the pancreas, or at least an analogous organ, 
 seems to be the essential digestive gland. On the contrary, an anatom- 
 ically characteristic pancreas is absent in certain vertebrates and in 
 certain fishes. Those functions which should be regulated by this organ 
 seem to be performed in these animals by the liver, which may be 
 rightly called the hepatopancreas. In man and in most vertebrates 
 the formation of bile, and of certain secretions, containing enzymes 
 important for digestion, is divided between the two organs, the liver and 
 the pancreas. 
 
 The pancreatic gland is similar in certain respects to the parotid 
 gland. The secreting elements of the former consist of nucleated cells 
 whose basis forms a mass rich in proteins, which expands in water and 
 in which two distinct zones exist. The outer zone is more homogene- 
 ous, the inner cloudy, due to a quantity of granules. The nucleus lies 
 about midway between the two zones, but this position may change 
 with the varying relative size of the two zones. According to Heiden- 
 hain * the inner part of the cells diminishes in size during the first stages 
 of digestion, in which the secretion is active, while at the same time 
 the outer zone enlarges owing to the absorption of new material. In 
 the later stage, when the secretion has decreased and the absorption 
 of the nutritive bodies has taken place, the inner zone enlarges at the 
 •expense of the outer, the substance of the latter having been converted 
 into that of the former. Under physiological conditions the glandular 
 cells are undergoing a constant change, at one time consuming from the 
 inner part and at another time growing from the outer part. The 
 inner granular zone is converted into the secretion, and the outer, more 
 homogeneous zone, which contains the repairing material, is then con- 
 verted into the granular substance. The so-called islands of Langer- 
 hans are related to the internal secretion or contain a substance taking 
 part in the transformation of the sugar of the animal body. 2 
 
 The chief portion of protein substances contained in the gland con- 
 sists, it seems, of a protein insoluble in water or neutral salt solution and 
 
 1 Pfluger's Arch., 10. 
 
 1 See Diamare and Kuliabko, Centralbl. f. Physiol., 18 and 19; Rennie, ibid., 18; 
 Sauerbeck, Yirchow's Arch., 177, Suppl. 
 
PANCREAS AND PANCREATIC JUICE. 495 
 
 of nucleoproteins, while the globulin and albumin occur only to a slight 
 extent as compared with the nucleoproteins. Among the compound 
 proteins is the substance studied and isolated by Umber but previously 
 discovered by Hammarsten l and called a-proteid. This nucleopro- 
 tein contains, as an average, 1.G7 per cent P, 1.29 per cent S, 17.12 per 
 cent N, and 0.13 per cent Fe. It yields, according to Hammarsten, 
 tf-proteid on boiling, which is much richer in phosphorus than the nucleo- 
 protein. The native proteid (a) is the mother-substance of guanylic 
 acid; according to Umber it dissolves on pepsin digestion without leaving 
 any residue, and yields on trypsin digestion guanylic acid on one side 
 and proteoses and peptones on the other. It can be extracted from the 
 gland by a physiological salt solution, and is precipitated by acetic acid. 
 Besides this compound protein the pancreas must contain at least one 
 other protein which is the mother-substance of the thymonucleic acid 
 obtainable from the pancreas. 
 
 Besides these protein substances the gland also contains several 
 enzymes, or more correctly zymogens, which will be discussed later. 
 Among the extractive bodies, which are probably in part formed by 
 post-mortem changes and chemical action, we must mention leucine- 
 tyrosine, purine bases in variable quantities, 2 inosite, lactic acid, volatile 
 fatty acids and fats. The mineral bodies vary considerably in quantity, 
 not only in animals and man but also in men and women (Gossmann). 
 'The calcium seems, according to Gossmann, to exist in much greater 
 amount than the magnesium. According to the investigations of Mag- 
 nus-Levy the human pancreas contains 278 p. m. solids with 106 p. m. 
 fat and 156 p. m. protein. Gossmann 3 found in man 17.92 p. m. ash 
 and in women 13.05 p. m. 
 
 Besides the previously-mentioned (Chapter VII) relation to the trans- 
 formation of sugar in the aminal body, the pancreas has the property 
 of secreting a juice especially important in digestion. 
 
 Pancreatic Juice. This secretion may be obtained by adjusting a 
 fistula in the excretory duct, according to the methods suggested by 
 Bernard, Ludwig, and Heidenhain, and perfected by Pawlow, 4 
 
 In herbivora, such as rabbits, whose digestion is uninterrupted, the 
 secretion of the pancreatic juice is continuous. In carnivora, it seems, 
 on the contrary, to be intermittent and dependent on the digestion. 
 
 1 Umber, Zeitschr. f. klin. Med., 40 and 43; Hammarsten, Zeitschr. f. physioL 
 Chem., 19. 
 
 2 See Kossel, Zeitschr. f. physiol. Chem., 8. 
 
 1 Magnus-Levy, Bioch Zeitschr. 24; Gossmann, Maly's Jahresb. 30. 
 
 * Bernard, Lecons de Physiol., 2, 190; Ludwig, see Bernstein, Arbeiten, ad. physioL 
 Anstalt zu Leipzig, 1869; Heidenhain, Pfluger's Arch., 10, 604; Pawlow, The Work 
 of the Digestive Glands, (translated by Thompson, Philadelphia, 1910), and Ergebnisse 
 der Physiologie, 1, Abt. 1. 
 
496 DIGESTION. 
 
 During starvation the secretion almost stops, but commences again 
 after partaking of food and reaches its maximum, it is claimed by 
 Bernstein, Heidenhain, and others, within the first three hours. 
 
 Pawlow and his pupils, especially Schepowalnikoff, have shown 
 that the above-mentioned (page 492) enterokinase activates the trypsino- 
 gen into trypsin. These observations were later confirmed by others, 
 by Delezenne and Frouin, Popielski, Camus and Gley, Bayliss and 
 Starling, Zunz, and have been further studied. The pure juice con- 
 tains, at least as a rule, only trypsinogen, and no trypsin. By mixing 
 with the intestinal juice, or by contact with the intestinal mucosa, the 
 trypsinogen is converted into trypsin by the kinase. Enterokinase, 
 which itself has no action upon proteins, and therefore is not a pro- 
 teolytic enzyme, is not well known. It is made inactive by heating and 
 is therefore considered by many (including Pawlow) as an enzyme. 
 Others, on the contrary, like Hamburger and Hekma, Dastre and 
 Stassano, deny the enzyme nature of enterokinase because they find 
 that a certain quantity of intestinal juice will activate only a certain 
 quantity of trypsin. Enterokinase has been found in man and all 
 mammals investigated. According to most investigators it is formed 
 in the glands or the cells of the intestinal mucosa, while according to 
 Delezenne it comes from Peyer's patches and from the lymph-glands 
 and leucocytes, hence impure fibrin containing leucocytes acts as a kin- 
 ase. These deductions of Delezenne are disputed by Bayliss and" 
 Starling, Hekma and others. 
 
 If we accept the view that the juice secreted after partaking food 
 is regularly free from trypsin, still under other circumstances the juice 
 may contain trypsin. Thus, according to Camus and Gley, the juice 
 secreted under the influence of secretin (see below) is not always free 
 from trypsin, and Zunz found that Witte's peptone or pilocarpine 
 causes a secretion of juice which often contained trypsin and was directly 
 active. According to Camus and Gley not only does an exterior activa- 
 tion of the trypsinogen in the juice take place, but also in the interior 
 of the gland. An auto-activation of the juice in certain cases is also 
 accepted by others (Sawitsch 1 ). 
 
 The activation of the trypsinogen into trypsin may, in life, be brought about 
 — as the researches of Herzen, which have been substantiated by Gachet and 
 Pachon, Bellamy, Mendel and Rettger, have shown — not only in the intestine, 
 but also in the gland itself. This activation of the trypsinogen in the gland itself 
 is caused in a still undiscovered manner by a body of unknown nature formed in the 
 spleen, which is congested during digestion. Such a " charging " of the pancreas 
 
 1 Camus and Gley, Journ. de Physiol, et de Pathol, gem, 1907; Zunz, Recherches 
 sur l'activation de sac pancreatique par les Sels., Bruxelles, 1907; Sawitsch, Zentralbl. 
 f. d. ges. Physiol, u. Path, des Stoffwechsels, 1909. 
 
TRYPSINOGEN. 497 
 
 by the sploon has beeti repeatedly suggested by Schiff, 1 but this has recently 
 been denied by Prym. According to this experimenter the extirpation of the 
 spleen causes no change in the properties of the pancreatic juice, and the intra- 
 venous injection of spleen infusion is also without action on a splenectomized 
 dog with permanent pancreatic fistula. The observations of Herzen that a 
 spleen infusion has a strong activating action upon a weak pancreas infusion 
 were substantiated by Prym, 2 but he claims that this is due essentially to micro- 
 organsims. Besides this the spleen itself contains proteolytic enzymes (page 
 -371). 
 
 The conversion of the trypsinogen into trypsin in the removed gland 
 or in an infusion under the influence of air and water and also by other 
 bodies has been known for a long time. According to Vernon the tryp- 
 sin itself has a strong activating action upon trypsinogen, and in this 
 regard it is more active than enterokinase. The correctness of this 
 statement is still denied by Bayliss and Starling and by Hekma. The 
 ordinary view of Heidenhain, that the transformation of trypsinogen 
 into trypsin is also brought about by acids, has been found to be incor- 
 rect by Hekma. 3 Besides the enterokinase and the micro-organisms, 
 there are other activators of the trypsinogen. As first shown by 
 Delezenne and then by Zunz, by further investigations the lime salts 
 have a special power in activating trypsinogen. 4 These last do not act 
 immediately, but only after some time, for example, a couple of hours, 
 and then they activate suddenly. The lime salts are not necessary 
 for the digestive action of the juice, and when the activation has once 
 taken place, they can be removed without any harm. They probably 
 have a similar action as in the coagulation of the blood. According 
 to Delezenne the lime salts have the same importance in the activa- 
 tion of the rennin-zymogen of the juice as in the activation of the 
 trypsinogen. This enzyme is also activated by enterokinase. The 
 erepsin of the pancreatic juice (page 493) occurs as an active enzyme. 
 
 We are not quite clear whether the two other enzymes, the diastase 
 and lipase, are secreted as such or as zymogens. It seems, nevertheless, 
 that both are in part secreted as complete enzymes. 
 
 In the human embryo the trypsinogen and the erepsin (as well as also the 
 pepsin) appear in the fourth and fifth fcetal month. The enterokinase appears 
 at the same time or shortly after the trypsinogen. 5 
 
 1 Bellamy, Journ. of Physiol., 27; Mendel and Rettger, Amer. Journ. of Physiol., 7. 
 A very complete reference to the literature may be found in Menia Besbokaia Du 
 rapport fonctionell entre le pankreas et la rate, Lausanne, 1901. 
 
 2 Pfliiger's Arch., 104., and 107. 
 
 3 Vernon, Journ. of Physiol., 28; Hekma, Kon. Akad. v. Wetenschappen te 
 Amsterdam, 1903, and Arch. f. (Anat. u) Physiol., 1904; Bayliss and Starling, Journ. 
 of Physiol., 30 
 
 4 Delezenne, Compt. rend. soc. biol., 59, 60, 62, 63; Zunz, footnote 1, p. 496. 
 s Ibrahim., Bioch. Zeitschr. 22, 24 (1909). 
 
498 DIGESTION. 
 
 The way in which the trypsinogen is converted into trypsin is still 
 unknown and is the subject of dispute. According to one view, proposed 
 by Pawlow and defended by Bayliss and Starling, the trypsinogen 
 is transformed into trypsin by the action of the kinase. In the opinion 
 of Delezenne, Dastre, and Stassano, and others, 1 the trypsin, on the 
 contrary, is a combination of the kinase and trypsinogen, analogous to 
 the cytotoxines, which, according to Ehrlich's side-chain theory, are 
 combinations between a complement and an amboceptor. (See page 
 69.) 
 
 The specific excitants for the secretion of pancreatic juice are, 
 according to Pawlow and his collaborators, acids of various kinds- 
 hydrochloric acid as well as lactic acid — and fats, the latter acting 
 probably by virtue of the soaps produced therefrom. Alkalies and 
 alkali carbonates have, on the contrary, a retarding action. It appears 
 that the acids act by irritating the mucosa of the duodenum. Accord- 
 ing to London and Schwarz the secretion can also be excited from the 
 entire jejunum and the upper part of the ileum. The secretion becomes 
 weaker the further away the exciting source is from the duodenum. 2 
 Water, which causes a secretion of acid gastric juice, likewise becomes, 
 indirectly, a stimulant for the pancreatic secretion, but may also be 
 an independent exciter. The psychical moment may, at least in the 
 first place, have an indirect action (secretion of acid gastric juice), 
 and the food seems otherwise to have an action on pancreatic secretion 
 by its action on the secretion of gastric juice. 
 
 The most important excitant for the secretion of juice is hydrochloric 
 acid, but opinions are not in unison as to the manner in which the acid 
 acts. Pawlow's school claims that the acid acts reflexly upon the 
 intestine, causing a secretion of juice. That a reflex action is here pro- 
 duced is not contradicted by the investigations of Popielski, Wert- 
 heimer and Lepage, Fleig, 3 and others. According to the researches 
 of Bayliss and Starling, which have been confirmed by Camus, Gley, 
 Fleig, Herzen, Delezenne, and others, a second factor must also be 
 active here. Bayliss and Starling have shown that a body which 
 they have called secretin can be extracted from the intestinal mucosa 
 by a hydrochloric-acid solution of 4 p. m., and this when introduced into 
 the blood produces a secretion of pancreatic juice, bile, and in the 
 opinion of some investigators also of saliva and intestinal juice. The 
 
 1 Bayliss and Starling, Journ. of Physiol., 30 and 32, which also cities the other 
 investigators and also O. Cohnheim, Bioch. Centralbl. 1, 169 and S. Rosenberg, ibid., 
 2, 708. 
 
 * Zeitschr. f. physiol. Chem. 68, 346 (1910) which also contains the literature. 
 
 ' Fleig, Centralbl. f. Physiol., 16, 681, and Compt. rend. soc. biol., 55. See also 
 footnote 1. 
 
SECRETION OF PANCREATIC JUICE. 499 
 
 secretin, which according to Bayliss and Starling, 1 is the same in all 
 vertebrates examined, is not destroyed by heat; it is therefore not 
 identical with ciitorokinase, and is not considered an enzyme. It is 
 formed from another substance, prosecretin, by the action of acids. 
 According to Delezenne and Pozerski secretin occurs as such in the 
 intestinal mucosa, and the acids act only by the elimination of certain 
 bodies having a retarding action. According to Popielski secretin 
 action is different from acid action; and the secretin action can also be 
 obtained by Witte's peptone. He believes that the secretin is not 
 a specific constituent of the intestine but a body widely distributed. 
 (Iizelt disputes the occurrence of a specific secretin and he compares 
 this body to peptone. Gley has obtained a solution which had a stronger 
 secreting action than secretin by macerating the mucosa with proteoses. 2 
 v. Furth and Schwarz 3 also call attention to the uncertainty of our 
 knowledge as to the nature of secretin. According to them secretin 
 is probably a mixture of bodies, among which probably the choline, found 
 by them in the intestinal walls, acts the role of an exciter of secretion. 
 
 A second means of causing secretion is the fat, which probably only 
 acts after it has been saponified. Oil-soap directly introduced into the 
 duodenum brings about a strong secretion of pancreatic juice (Sawitsch, 
 Babkin 4 ), and at the same time a flow of bile, gastric juice, and the 
 secretion of Brunner's glands occurs. The pancreatic juice secreted 
 under these circumstances has about the same amount of enzymes as 
 the juice secreted after partaking of food. 
 
 We know very little as to how the soaps act. Fleig 5 found that by macera- 
 tion of the mucosa of the upper part of the duodenum with soap solution, a sub- 
 stance goes into solution which he calls sapokrinin, and which when introduced 
 into the blood brings about a strong secretion of pancreatic juice. This sapok- 
 rinin, which is derived from a prosapokrinin. is not an enzyme and is not identical 
 with secretin. After the action of chloral hydrate an abundant secretion occurs 
 in the duodenum (Wertheimer and Lepage), which Falloise considers as pro- 
 duced by a special secretin, chloral secretin. The secretion of pancreatic juice 
 can also be increased by alcohol, and Fleig 6 claims to have obtained a secretin, 
 ethyl secretin, by macerating the intestinal mucosa with alcohol. Further investiga- 
 tions are necessary of all these so-called secretins. 
 
 1 Journ. of Physiol., 29. 
 
 2 Delezenne and Pozerski, Compt. rend. soc. biol., 56; Popielski, Centralbl. f. 
 Physiol., 19; Pfluger's Arch. 128; Gizelt, Pfluger's Arch. 123; Gley, Compt. Rend. 151, 
 345. 
 
 3 v. Furth and Schwarz, Pfluger's Arch. 124 (literature on secretin). 
 
 4 Arch des scienc. biol. de St. Petersbourg, 11, and Zeitschr. f. physiol. Chem., 56. 
 6 Compt. rend. soc. biol., 55, and Journ. de Physiol, et de Pathol, gen., 1904. 
 
 6 Wertheimer and Lepage, Compt. rend. soc. biol., 52; Fleig, ibid., 55; Falloise, 
 Bull. Acad. Roy. Belg., 1903. 
 
500 DIGESTION. 
 
 The estimation as to the quantity of pancreatic juice secreted in the 
 twenty-four hoars differs very much. According to the determina- 
 tions of Pawlow and his collaborators, Kuwschinski, Wassiliew, and 
 Jablonsky, 1 the average quantity (with normally acting juice) from a 
 permanent fistula in dogs is 21.8 cc. per kilo in the twenty-four hours. 
 
 The pancreatic juice of the dog is a clear, colorless, and odorless 
 alkaline fluid which when obtained from a temporary fistula is very 
 rich in proteins, sometimes so rich that it coagulates like the white of 
 the egg on heating. Besides proteins, the juice also contains the three 
 above-mentioned enzymes (or their zymogens), amylopsin, perhaps also 
 maltase, trypsin, steapsin, also an enzyme similar to erepsin, and besides 
 these a rennin, which was first observed by Ruhne. Besides the above- 
 mentioned bodies the pancreatic juice invariably contains small quan- 
 tities of leucine, fat, and soaps. As mineral constituents it contains 
 chiefly alkali chlorides and considerable alkali carbonate, some phos- 
 phoric acid, lime, magnesia, and iron. 
 
 The quantity of solids in the pancreatic juice of the dog varies, as 
 found by Mazurkiewicz, Babkine and Sawitsch, 2 according to the 
 rapidity of secretion and the kind of excitant. As a rule the amount 
 of solids is in inverse proportion to the rapidity of secretion. The juice 
 secreted after the action of acids has the lowest amount of solids, 9-37.4 
 p. m. The juice after taking food is more concentrated, about 60-70 
 p. m. and that after vagus stimulation often contains 90 p. m. solids. 
 The juice analyzed by C. Schmidt 3 from a temporary fistula contained 
 99-116 p. m. solids. The quantity of mineral bodies was 8.8 p. m. 
 
 The mineral constituents consisted chiefly of NaCl, 7.4 p. m., which is remark- 
 able because the juice contains such a large amount of alkali carbonate. In the 
 juice examined by De Zilwa 4 the quantity of alkali in the secretin juice was 
 5-7.9 p. m. and in the pilocarpin juice 2.9 -5.3 p. m. Na 2 C0 3 . 
 
 In the pancreatic juice of rabbits 11-26 p. m. solids have been found, and 
 in that from sheep 14.3-36.9 p. m. In the pancreatic juice of the horse 9-15.5 
 p. m. solids have been found; in that of the pigeon, 12-14 p. m. 
 
 The human physiological pancreatic secretion from a fistula has been 
 investigated by Glaessner. 5 The secretion was clear, foamed readily, 
 
 1 Arch, des sciences de St. Petersbourg, 2, 391. The previous- claims of Bidder and 
 Schmidt, and others may be found in Kiihne, Lehrbuch, 114. 
 
 2 Mazurkiewicz, 1. c; Babkin and Sawitsch, Zeitschr, f. physiol. Chem., 56. 
 8 Cited from Maly in Hermann's Handbuch der Physiol., 5, Theil II, 189. 
 
 4 Journ. of Physiol., 31. 
 
 6 Zeitschr. f. physiol. Chem., 40. See also Ellinger and Kohn, ibid., 45, and the 
 investigations upon cystic fluids from the pancreas by Schumm, ibid., 36, and Murray 
 and Gies, American Medicine, 4, 1902; Glaessner and Popper, Deutsch. Arch. f. klin. 
 Med. 04, 46; see also Wohlgemuth, Bioch. Zeitschr. 39; Bradley, Journ. of Biol. 
 Chem. 6. 
 
AMYLOPSIN. STEAPSIN. 501 
 
 had a strong alkaline reaction even toward phenolphthalein, and con- 
 tained globulin and albumin but no proteoses and peptones. The specific 
 gravity was 1.0075 and the freezing-point depression was A =—0.46- 
 0.49°. The solids were 12.44-12.71 p. m., the total protein 1.28-1.74 
 p. m., and the mineral bodies 5.66-6.98 p. m. The secretion contained 
 trypsinogen, which was activated by the intestinal juice. Diastase and 
 lipase were present; inverting enzymes, on the contrary, were not. The 
 daily quantity of juice was 500-800 cc. The quantity of secretion, of 
 ferments, and of alkalinity was lowest in starvation, but soon rose with 
 the taking of food, and reached its maximum in about four hours. 
 
 Amylopsin, or pancreatic diastase, which, according to Korowin 
 and Zweifel, is not found in new-born infants and does not appear 
 until more than one month after birth, seems, although not identical 
 with ptyalin, to be closely related to it. Amylopsin acts very energetic- 
 ally upon boiled starch, and according to Ivuhne also upon unboiled 
 gtarch, especially at 37 to 40° C, and according to Vernon 1 best at 
 35° C. It forms, similarly to the action of saliva, besides dextrin, chiefly 
 isomaltose and maltose, with only very little glucose (Musculus and 
 v. Mering, Kulz and Vogel 2 ). The glucose is probably formed by the 
 action of the invertin existing in the gland and juice. The pancreatic 
 juice of the dog in fact, contains, according to Bierry and Terroine, 3 
 maltase, its action becomes apparent only after very faint acidification 
 cf the juice. According to Rachford the action of the amylopsin is 
 not reduced by very small quantities of hydrochloric acid, but is dimin- 
 ished by larger amounts. Vernon, Grutzner, and Wachsmann find 
 that the action is indeed accelerated by very small quantities of hydro- 
 chloric acid, 0.045 p. m., while alkalies in very small amounts have a 
 retarding action. This retarding action of alkalies and hydrochloric 
 acid may be stopped by bile (Rachford). Wohlgemuth as well as 
 Minami 4 find that the action of diastase is increased to a high degree 
 by bile. The active constituent of the bile was soluble in water and 
 alcohol but was not identical with the bile salts or cholesterin. The 
 statements in regard to the action of lecithin are contradictory. 
 
 Steapsin, or Fat-splitting Enzyme. The action of the pancreatic 
 juice on fats is twofold. First, the neutral fats are split into fatty acids 
 
 1 Korowin, Maly's Jahresber., 3; Zweifel, footnote 2, p. 456, Kuhne, Lehrbuch, 
 117; Vernon, Journ. of Physiol., 27. 
 
 2 See footnote 5, p. 456. 
 
 3 See Tebb. Journ. of Physiol., 15; Bierry and Terroine, Compt. rend. soc. biolog., 
 58; Bierry, ibid., 62. 
 
 4 Rachford, Amer. Journ. of Physiol., 2; Vernon. 1. c; Grutzner, Pfliiger's Arch. 
 91; Wohlgemuth, Bioch. Zeitschr. 21, 447 (1909); Minami, ibid., 39, 339 (1912). 
 
502 DIGESTION. 
 
 -and glycerin, which is an enzymotic process; and secondly, it has also 
 the property of emulsifying the fats. 
 
 The action of the pancreatic juice in splitting the fats may be shown 
 in the following way: Shake olive-oil with caustic soda and ether, 
 siphon off the ether and filter if necessary, then shake the ether repeatedly 
 with water and evaporate at a gentle heat. In this way is obtained a 
 residue of fat free from fatty acids, which' is neutral and which dissolves 
 in acid-free alcohol and is not colored red by alkanet tincture. If such 
 fat is mixed with perfectly fresh alkaline pancreatic j uice or with a freshly 
 prepared infusion of the fresh gland and treated with a little alkali or 
 with a faintly alkaline glycerin extract of the fresh gland (9 parts glyc- 
 erin and 1 part 1 per cent soda solution for each gram of the gland), 
 and some litmus tincture added and the mixture warmed to 37° C, 
 the alkaline reaction will gradually disappear and an acid one take its 
 place. This acid reaction depends upon the conversion of the neutral 
 fats by the enzyme into glycerin and free fatty acids. A very much 
 used method consists in determining the acidity of the mixture by means 
 of titration before and after the action of the juice or the infusion. 
 
 The action of the pancreatic juice in splitting fats is a process analo- 
 gous to that of saponification, the neutral fats being decomposed, by 
 the addition of the elements of water into fatty acids and glycerin 
 according to the following equation. C3H5.O3.R3 (neutral fat)+3H20 = 
 C3H5.O3.H3 (glycerin) + 3 (H.O.R) (fatty acid). This depends upon 
 a hydrolytic splitting, which was first positively proved by Bernard 
 and Berthelot. The pancreas enzyme also decomposes other esters, 
 just as it does the neutral fats (Nencki, Baas, Loevenhart x and 
 others). The fat-splitting action of the lipase is, according to Paw- 
 low, Bruno and many others 2 aided in its action by the bile. Rosen- 
 heim and Shaw-Mackenzie found that the lipase action was accel- 
 erated by ha3molytic substances, as well as by normal serum; this 
 accelerating action was inhibited by cholesterin. The accelerating 
 substance of the serum was dialyzable and resistant to heat. Rosen- 
 heim was able to divide the lipase existing in a glycerin extract of the 
 pig pancreas into enzyme and co-enzyme (page 52); in diluting with 
 water a precipitate occurred which contained the real thermolabile 
 enzyme while the dialyzable, heat resisting co-enzyme remained in the 
 
 1 Bernard, Ann. de chim. et physique (3), 25; Berthelot, Jahresber, d. Chem., 
 1855, 733; Nencki, Arch. f. exp. Path. u. Pharm., 20; Baas, Zeitschr. f. physiol. Chem., 
 14, 416; Loevenhart, Journ. of Biol. Chem., 2; Terroine an 1 Z. Morel, Compt. rend. 
 80C. biol., 65, 66. 
 
 2 Bruno, Arch, des scienc biol. de St. P6tersbourg, 7; see also Loevenhart and 
 Souder, Journ. of biol. Chem., 2; v. Furth and Schutz, Hofmcister's Beitrage, 9; Ter- 
 roine, Bioch. Zeitschr. 23; Compt. rend. soc. biol. 68, 439, 518, 666, 754 (1910). 
 
TRYPSIN. 503 
 
 filtrate. 1 In regard to the synthetic action of pancreatic lipase see 
 page 60. 
 
 The fatty acids which are split off by the action of the pancreatic 
 juice combine in the intestine with the alkalies, forming soaps, which 
 have a strong emulsifying action on the fats, and thus the pancreatic 
 juice becomes of great importance in the emulsification and the absorp- 
 tion of the fats. 
 
 Trypsin. The action of the pancreatic juice in digesting proteins 
 was first observed by Bernard, but first proved by Corvisart. 2 It 
 depends upon a special enzyme called, by Kuhne, trypsin. This enzyme 
 as previously explained, does not occur in the gland as such, but as 
 trypsinogen. According to Albertoni 3 this zymogen is found in 
 the gland in the last third of the intra-uterine life. Enzymes more or 
 less like trypsin occur in other organs, and are very widely diffused in 
 the vegetable kingdom, 4 in yeast and in higher plants, and are also formed 
 by various bacteria. The enzymes similar to trypsin occurring in the 
 plant kingdom are, according to Vines, a mixture of peptases, which 
 transform the proteins into peptone, and ereptases, which split the pep- 
 tones into amino-acids. 
 
 As we know of so-called antienzymes for other enzymes, so we also have anti- 
 trypsins, and not only in the intestinal canal but also in the blood-serum (see page 
 63). The results as to the possibility of producing antitrypsins by immuniza- 
 tion, is still disputed. 
 
 Trypsin, like other enzymes, has not been prepared in a pure con- 
 dition. Nothing is positively known in regard to its nature, but as 
 obtained thus far it shows a variable behavior (Kuhne, Klug, Levene, 
 Mays, and others). At least it does not seem to be a nucleoprotein, and 
 trypsin has also been obtained which did not give the biuret test (Klug, 
 Mays, Schwarzschild). Trypsin dissolves in water and glycerin, while 
 Kuhne's trypsin was insoluble in glycerin. It is very sensitive to heat, 
 and even the body temperature gradually decomposes it (Vernon, Mays). 
 In neutral solution it becomes inactive at 45° C. In dilute soda solu- 
 tion of 3-5 p. m. it is still more readily destroyed (Biernacki, Vernon 5 ). 
 
 1 Journ. of Physiol. 40 (1910). 
 
 2 Gaz. hebciomadaire, 1857, Nos. 15, 16, 19, cited from Bunge, Lehrbuch, 4, Aufl., 
 185. 
 
 3 See Maly's Jahresber., 8, 254. 
 
 4 In this connection see Vines, Annals of Botany, 16, 17, 18, 19, 22, and 23, and 
 Oppenheimer, Die Fermente, 1910. 
 
 5 Kiihne, Verh. d. naturh.-med. Vereins zu Heidelberg (N. F.), 1, 3; Klug, Math, 
 naturw. Ber. aus Ungarn., 18, 1902; Levene, Amer. Journ. of Physiol., 5; Mays, 
 Zeitschr. f. Physiol. Chem., 38; Vernon, Journ. of Physiol., 28 and 29; Biernacki, 
 Zeitschr. f. Biologie, 28; Schwarzschild, Hofmeister's Beitrage, 4. 
 
504 DIGESTION. 
 
 The presence of protein or proteoses has, to a certain extent, a pro- 
 tective action on heating an alkaline trypsin solution, and this has 
 been substantiated by recent investigations of Bayliss and Vernon. 
 The simpler cleavage products have a still greater protective action 
 (Vernon 1 ). Trypsinogen, according to the unanimous statements 
 of several experimenters, is more resistant toward alkalies than trypsin. 
 Trypsin is gradually destroyed by gastric juice and even by digestive 
 hydrochloric acid alone. 
 
 The preparation of pure trypsin has been tried by various experimen- 
 ters. The most careful work in this direction was done by Kuhne and 
 [Mays. Various methods have been suggested by Mays, but we cannot 
 enter into a discussion of them. A very pure preparation can be obtained 
 by making use of the combined salting out with NaCl and MgSO-i. A 
 very active solution, and one that can be kept for a long time (for more 
 than twenty years according to Hammarsten), can be obtained by extract- 
 ing with glycerin (Heidenhain 2 ) . An impure but still very active 
 infusion can be obtained after a few days by allowing the finely divided 
 gland to stand with water which contains 5-10 cc. chloroform per liter 
 (Salkowski) at the temperature of the room. Such infusions can be 
 obtained, nearly free from proteins, by dialyzing with running water after 
 the addition of toluene. 
 
 Like other enzymes, trypsin is characterized by its action, and this 
 action consists in dissolving protein and in splitting it into simpler prod- 
 ucts, mono- and diamino-acids, tryptophane, etc., in alkaline, neutral, 
 and indeed in very faintly acid solutions. This action has been so far 
 considered as characteristic for trypsin. Recent investigations seem 
 to indicate that this action is not due to one enzyme alone, but to the 
 combined action of several enzymes. 
 
 Although contrary to May's statement, there is no question that 
 there occurs in the pancreas besides trypsin, an enzyme similar to erepsin 
 (Bayliss and Starling, Vernon 3 ). According to the latter this 
 erepsin has a strong action upon peptone, and he believes that the pep- 
 tone-splitting action of a pancreas infusion is in great part due to the 
 erepsin. The pancreas, besides these, also contains a nuclease (see page 
 493), whose relation to pancreas erepsin has not been determined. 
 
 The unity of trypsin has also been disputed from another point of view. 
 According to Pollak the trypsin (in the ordinary sense) contains a second 
 enzyme, which does not act upon protein, but only upon gelatin, and he calls 
 
 1 Bayliss, Arch, des scienc. biolog. de St. Petersbourg. 11, Suppl.; Vernon, Journ. 
 of Physiol., 31. 
 
 - PfluKer's Arch., 10. 
 
 * Bayliss and Starling, Journ. of Physiol., 30; Vernon, ibid., 30; and Zeitschr. . f.. 
 physiol. Chem., 50; Mays, ibid., 49 and 51. 
 
ACTION OF TRYPSIN. 505 
 
 this enzyme glutinase. This glutinasc is much more resistant toward acids 
 than trypsin, and by proper treatment with acids Pollak was able to change a 
 pancreas infusion so that it acted upon gelatin and not upon certain proteins. 
 The correctness of these observations has, indeed, not been generally accepted, 
 and it is disputed by Ascoli and Neppi. 1 According to them the action of the 
 trypsin is weakened by the acid, and indeed to such varying degrees for differ- 
 ent proteins that the action upon albumin is lost while the action upon gelatin 
 is noticeable. Nevertheless, we here have a warning to be careful as to the 
 conclusions drawn from results where impure infusions are used. For many 
 experiments it is undoubtedly advisable to use the natural pancreatic juice. 
 
 The following reports on the action of trypsin applies to the so- 
 called trypsin, with the reservation that it is perhaps not a unit enzyme. 
 
 The action of trypsin on proteins is best demonstrated by the use of 
 fibrin. Very considerable quantities of this protein body are dissolved 
 by a small amount of trypsin at 37-40° C. It is always necessary to 
 make a control test with fibrin alone, with or without the addition of 
 alkali. Fibrin is dissolved by trypsin without any putrefaction; the 
 liquid has a pleasant odor somewhat like bouillon. To completely 
 exclude putrefaction a little thymol, chloroform, or toluene should be 
 added to the liquid. Tryptic digestion differs essentially from peptic 
 digestion, irrespective of the difference in the digestive products, in that 
 the first takes place in neutral or alkaline reaction and not, as is neces- 
 sary for peptic digestion, in an acidity of 1-2 p. m. HC1, and further 
 by the fact that the proteins dissolve in trypsin digestion without pre- 
 viously swelling up. 
 
 As trypsin not only dissolves proteids, but also other protein sul - 
 stances such as gelatin, this latter body may be used in detecting tryp- 
 sin. The liquefaction of strongly disinfected' gelatin is, according to 
 Fermi, 2 a very delicate test for trypsin or tryptic enzymes. Various 
 suggestions for the use of gelatin in the trypsin test have been made. 
 In consideration of the observations of Ascoli and Neppi that a trypsin 
 may not act upon fibrin or other proteids but still digest gelatin, it is 
 advisable never to make use of gelatin or proteid alone in testing for 
 trypsin, but always the two. 
 
 For the quantitative estimation of trypsin by measuring the rapidity of 
 digestion we generally make use of the method of Mett, described under pepsin 
 digestion. Another method, suggested by Weiss, consists in determining the 
 nitrogen in the filtrate after coagulation with heat and acetic acid. Lohlein 
 recommends the titration method of Volhard as used in pepsin determinations, 
 and has given directions for its use. Jacoby recommends the use of (ricin, and 
 Gross suggests a method based upon the precipitation of casein by acid. Bay- 
 
 1 Pollak, Hofmeister's Beitrage, 6; contradictory statements are found in Ehren- 
 reich, cited in Bioch. Centralbl., 4; Ascoli and Neppi, Zeitschr. f. physiol. Chem., 56. 
 
 2 Arch. f. Hyg. 12 and 55. 
 
506 DIGESTION. 
 
 liss follows the digestion by the electrical conductivity, and F. Weiss l determines 
 the quantity of nitrogen not precipitated by tannic acid. The formol titration 
 can also be used with advantage for determining the decomposition (page 166). 
 
 The reaction has a great influence upon the rapidity of the trypsin 
 digestion. Trypsin acts energetically in neutral, or still better in alkaline, 
 solutions, and according to older statements, best in an alkalinity of 
 3-4 p. m. Na2C03| but the nature of the protein is also of importance. 
 The reports in regard to the action of trypsin in various reactions are 
 still somewhat disputed. 2 The action of the alkali depends upon the 
 number of hydroxyl ions (Dietze, Kanitz), and according to Kanitz 3 
 the digestion proceeds best in those solutions which are 1/70-1/200 
 normal in regard to hydroxyl ions. Free mineral acids, even in very 
 small quantities, completely prevent the digestion. If the acid is not 
 actually free, but combined with protein bodies, then the digestion 
 may take place quickly when the acid combination is not in too great 
 excess (Chittenden and Cummins). Organic acids act less disturbingly, 
 and in the presence of 0.2 p. m. lactic acid and the simultaneous presence 
 of bile and common salt, the digestion may indeed proceed more quickly 
 than in a faintly alkaline liquid (Lindberger). The assertion of Rach- 
 ford and Southgate, that the bile can prevent the injurious action 
 of the hydrochloric acid, and that a mixture of pancreatic juice, bile, 
 and hydrochloric acid digests better than a neutral pancreatic juice, 
 could not be substantiated by Chittenden and Albro. That bile has 
 an action tending to aid the tryptic digestion has been shown by many 
 investigators, and recently by Bruno, Zuntz and Ussow and others. 4 
 Carbon dioxide, according to Schierbeck, 5 has a retarding action 
 in acid solutions, but it accelerates the tryptic digestion in faintly 
 alkaline liquids. Foreign bodies, such as potassium cyanide, may pro- 
 mote tryptic digestion, while other bodies, such as salts of mercury, iron, 
 and others (Chittenden and Cummins), or salicylic acid in large quan- 
 tities, may have a preventive action. According to Weiss 6 the halogen 
 
 1 Weiss, Zeitschr. f. physiol. Chem., 40; Lohlein, Hofmeister's Beitrage, 7; Jacoby, 
 Bioch. Zeitschr., 10; Gross, Arch. f. exp. Path. u. Pharm., 58; Bayliss, Arch, des 
 scienc. biol. de St. Petersbourg, 11, Suppl.; and Journ. of Physiol., 36; Weiss, Zeitschr. 
 f. physiol. Chem. 31, 78 (1900). 
 
 2 See Kudo, Bioch. Zeitschr., 15. 
 
 3 Kanitz, Zeitschr. f. physiol. Chem., 37, who also cites Dietze. 
 
 4 Chittenden and Cummins, Studies from the Physiol. Chem. Laboratory of Yale 
 College, New Haven, 1885, 1, 100; Lindberger, Maly's Jahresber., 13; Rachford and 
 Southgate, Medical Record, «1895; Chittenden and Albro, Amer. Journ. of Physiol., 1, 
 1898; Rachford, Journ. of Physiol., 25; Bruno, 1. c; Zuntz and Ussow, Arch. f. 
 (Anat. u.) Physiol., 1900. 
 
 '■> Skand. Arch. f. Physiol., 3. 
 
 6 Weiss, Zeitschr. f. physiol. Chem., 40; See also Kudo, Bioch. Zeitschr. 15, 473 
 (1908). 
 
PRODUCTS OF TRYPTIC DIGESTION. 507 
 
 alkali salts disturb tryptic digestion only slightly, and NaCl seems to 
 have the strongest action. The sulphates have a much stronger retard- 
 ing action than the chlorides. The nature of the proteins is also of 
 importance. Unboiled fibrin is, relatively to most other proteins, dis- 
 solved so very quickly that the digestion test with raw fibrin gives an 
 incorrect idea of the power of trypsin to dissolve coagulated protein 
 bodies in general. Boiled fibrin is digested with much greater difficulty 
 and also requires a higher alkalinity: 8 p. m. Na2C03 (Vernon 1 ). 
 The resistance of certain native protein solutions, such as blood-serum 
 and egg-white, against the action of trypsin is remarkable. In regard 
 to the inhibition of the action of trypsin see Chapter I, page 63. 
 
 The Products of the Tryptic Digestion. In the digestion of unboiled 
 fibrin a globulin which coagulates at 55-60° C. may be obtained as an 
 intermediary product (Herrmann 2 ). Besides this, one obtains from 
 fibrin, as well as from other proteins, the products previously men- 
 tioned in Chapter II. In trypsin digestion the cleavage may proceed 
 so far that the mixture fails to give the biuret reaction. This does not 
 indicate, as E. Fischer and Abderhalden have shown, a complete 
 cleavage of the protein molecule into mono- and diamino-acids, etc. 
 In tryptic digestion, as shown by Abderhalden and Reinbold, using 
 the protein edestin, and by Abderhalden and Voegtlin 3 with casein, 
 a gradual cleavage of the protein takes place, and thereby certain amino- 
 acids, like tyrosine and tryptophane, are readily and completely split 
 off, while others, like leucine, alanine, aspartic acid, and glutamic acid, 
 are slowly and less readily split off, and others, such as a-proline, phenyl- 
 alanine, and glycocoll, stubbornly resist the cleavage action of the trypsin. 
 The polypeptide-like bodies discovered by Fischer and Abderhalden, 
 which are produced in digestion, and which do not give the biuret 
 reaction, are the atomic complexes which resist the action of trypsin. 
 These peptoids contain the pyrrolidine carboxylic acid and phenylal- 
 anine groups of the protein, but also yield other monamino-acids such 
 as leucine, alanine, glutamic acid, and aspartic acid. Among the above- 
 mentioned products we find on the autodigestion of the gland other 
 substances, such as oxyphenylethylamine (Emerson), which is pro- 
 duced from tyrosine by fermentive CO2 cleavage, also uracil (Levene), 
 guanidine (Kutscher and Otori), the purine bases, which originate 
 from the nuclein bodies, and choline, which latter is formed from lecithin 
 
 1 Journ. of Physiol., 28. 
 
 2 Hermann, Zeitschr. /. physiol. Chem., 11. 
 
 3 Abderhalden and Reinbold, Zeitschr. f. physiol. Chem., 44 and 46, with Voegtlin, 
 ibid. 53. 
 
508 DIGESTION. 
 
 (Kutscher and Lohmann 1 ). If putrefaction is not completely pre- 
 vented, still other bodies occur which will be considered later in con- 
 nection with the putrefactive processes in the intestine. 
 
 The Action of Trypsin upon other Bodies. The nucleoproteins and 
 nucleins are so digested that the protein complex is separated from the 
 nucleic acid and then digested. The nucleic acids may, nevertheless, 
 be somewhat changed (Araki), which is probably brought about by 
 another enzyme, the nuclease (Sachs). A cleavage of nucleic acids with 
 the setting free of phosphoric acid and purine bases is, according to 
 Iwanoff, 2 not brought about by trypsin. The splitting is first pro- 
 duced by the action of nuclease or erepsin (see page 493). Gelatin is 
 dissolved and digested by pancreatic juice. A cleavage with the sepa- 
 ration of glycocoll and leucine does not occur (Kuhne and Ewald), or 
 only to a trivial extent (Reich-Herzberge 3 ). 
 
 The gelatin-forming substance of the connective tissues is not directly 
 dissolved by trypsin, but only after it has been treated with acids or 
 soaked in water at 70° C. By the action of trypsin on hyaline cartilage 
 the cells dissolve, leaving the nucleus. The matrix is softened and 
 shows an indistinctly constructed network of collagenous substances 
 (Kuhne and Ewald). The elastic substance, the structureless membranes, 
 and the membrane of the fat-cells, are also dissolved. Parenchymatous 
 organs, such as the liver and the muscles, are dissolved all but the nuclei , 
 connective tissue, fat-corpuscles, and the remainder of the nervous 
 tissue. If the muscles are boiled, then the connective tissue is also 
 dissolved. Mucin is dissolved and split by trypsin, while chitin and horn 
 substance do not seem to be acted upon by the enzyme. Oxyhemoglobin 
 is decomposed by trypsin with the splitting off of hsematin. Trypsin 
 splits off large amounts of hydriodic acid from diiodotyrosine (Oswald 4 ) . 
 Trypsin has no action upon fats and carbohydrates. 
 
 The action of trypsin on simply constructed substances of known 
 constitution such as acid-amides, polypeptides, is of especially great 
 interest. In this regard we have the somewhat earlier investigations 
 of Gulewitsch, Gonnermann, and Schwarzschild, 5 but the investi- 
 
 1 Fischer and Abderhalden, Zeitschr. f. physiol. Chem., 39; Emerson, Hofmeister's 
 Beitrage, 1; Levene, Zeitschr. f. physiol. Chem., 37; Kutscher and Lohmann, ibid. 
 39; Kutscher and Otori, ibid., 43, and Centralbl. f. Physiol., 18. 
 
 2 Iwanoff, Zeitschr. f. physiol. Chem., 39, which also contains the literature; Sachs, 
 ibid., 46. 
 
 3 Kuhne and Ewald, Verh. d. naturh.-med. Vereins zu Heidelberg (N. F.), 1; Reich- 
 Herzberge, Zeitschr. f. physiol. Chem., 34. 
 
 4 Zeitschr. f. physiol. Chem. 62, 432 (1909). 
 
 5 Hofmeister's Beitrage, 4, where the other works are also cited. 
 
PANCREATIC EENNIN. 509 
 
 gations of Fischer and of Abderhalden and their co-workers, 1 are 
 much more complete and important. In this connection see page 62. 
 
 The behavior of the polypeptides with trypsin, or closely related 
 enzymes, is of the greatest interest and in many respects very im- 
 portant. Thus in the polypeptides we have a means of determining 
 the kind of enzyme, whether it belongs to the pepsin, trypsin, or erepsin 
 group. We know of no polypeptide which is split by pepsin; trypsin 
 splits only certain polypeptides, but not others, while the erepsin on the 
 contrary seems to split all polypeptides, occurring in nature, which 
 are composed of aminoacids. By the aid of the polypeptide reaction 
 Abderhalden and co-workers have also been able to show that the 
 trypsin-like enzyme, occurring in the blood-plasma, is not identical 
 with trypsin because it does not attack glycyl-Z-tyrosine, which is split 
 by trypsin. 
 
 Pancreatic rennin is an enzyme found in the gland and in the juice, 
 which coagulates neutral or alkaline milk (Kuhne and Roberts and 
 others). This enzyme, according to Pawlow's school, is identical 
 with trypsin. The similarity of action of these two enzymes and the 
 fact that both are activated simultaneously from the zymogens by enter- 
 okinase or lime salts (Delezenne, Wohlgemuth 2 ) seem to point to 
 this identity. On the other hand the optimum of the enzyme action 
 for the pancreatic rennin is 60-65° C. (Vernon), which is much higher 
 than for the trypsin, and Glaessner and Popper 3 have also observed 
 a case where the human pancreatic juice contained no rennin enzyme. 
 
 According to Halliburton and Brodie, 4 casein is converted by the pancreatic 
 juice of the dog into " pancreatic casein," a substance which, in regard to solubility, 
 stands to a certain extent between casein and paracasein (see Chapter XIII), 
 and which is converted into paracasein by rennin. Further investigations on 
 the action of this enzyme upon milk and especially upon pure casein solutions are 
 very desirable. 
 
 Pancreatic Calculi. The concrement from a cystic enlargement of Wirsung's 
 duct in a man, as analyzed by Baldoni, contained in 1000 parts as follows: 
 Water 34.4, ash 126.7, protein substances 34.9, free fatty acids 133, neutral fats 
 124, cholesterin 70.9, soaps and pigment 499.1, parts. Scheunert and Berg- 
 holz 8 have reported a pancreatic calculi in the ox. 
 
 1 Fischer and Bergell, Ber. d. d. chem. Gesellsch., 36 and 37; Fischer and Abder- 
 halden, Sitzungsber. der Kgl. Pr. Akad. d. Wissensch., Berlin, 1905. The works of 
 Abderhalden and co-workers cannot be specially cited, but may be found in Zeitschr. 
 f. physiol. Chem., 47, 48, 49, 51, 52, 53, 54, 55, and 57. 
 
 2 Kuhne and Roberts, Maly's Jahresber., 9; see also Edkins, Journ. of Physiol., 
 12 (literature); Delezenne, Compt. rend. soc. biol., 62 and 63; Wohlgemuth, Bioch. 
 Zeitschr., 2. 
 
 3 Vernon, Journ. of Physiol., 12; Glaessner and Popper, Deutsch. Arch. f. klin. 
 Med., 94. 
 
 * Journ. of Physiol., 20. 
 
 5 Baldoni, Maly's Jahresb., 29, 353; Scheunert and Bergholz., Zeitschr. f. physiol. 
 Chem., 52. 
 
510 DIGESTION. 
 
 Besides the enzymes which have been discussed in connection with 
 the pancreatic juice, the gland also contains others, among which can be 
 mentioned the enzyme which, according to Stoklasa and his collab- 
 orators, occurs principally in organs and tissues and which decomposes 
 sugar into alcohol and carbon dioxide, like zymase. Opinions as to the 
 importance of the pancreas for glycolysis are diverse, and we therefore 
 refer the reader to what has been previously stated on this subject in 
 Chapter VII, pages 407 and 408. 
 
 V. THE CHEMICAL PROCESSES IN THE INTESTINE. 
 
 The action which belongs to each digestive secretion may be essen- 
 tially changed under certain conditions by being mixed with other 
 digestive fluids for various reasons, and also by the action of the 
 enzymes upon each other; 1 and since the digestive fluids which flow 
 into the intestine are mixed with still another fluid, the bile, it will be 
 readily understood that the combined action of all these fluids in the 
 intestine makes the chemical processes going on therein very complicated. 
 
 As the acid of the gastric juice acts destructively on ptyalin, this 
 enzyme has no further diastatic action, even after the acid of the gastric 
 juice has been neutralized in the intestine. Roger and Simon 2 claim 
 to have observed in saliva made inactive by the gastric juice, a reac- 
 tivation caused by the pancreatic juice, but these investigations do 
 not seem to be fully conclusive. The bile has, at least in certain animals, 
 a slight diastatic action, which in itself can hardly be of any great 
 importance, but which shows that the bile has not a preventive, but 
 rather a beneficial influence on the energetic diastatic action of the pan- 
 creatic juice. Several experimenters 3 have observed a beneficial action 
 of the bile on the diastatic action of the pancreas infusion. To this 
 may be added that the micro-organisms which habitually occur in the 
 intestine and sometimes in the food have partly a diastatic action and 
 partly produce a lactic-acid and butyric-acid fermentation. The 
 maltose which is formed from the starch seems to be converted into 
 glucose in the intestine. It seems conclusively that the cellulose cannot 
 be digested in the organism of the dog. 4 Lohrisch found that on an 
 average of 50 per cent of the introduced cellulose and hemicellulose was 
 digested in human beings and yielded the corresponding sugar. That 
 
 1 See Wr6ble\vski and collaborators, Hofmcister's Beitrage, 1. 
 
 2 Compt. rend. soc. biol., 02. 
 
 3 Martin and Williams, Proceed, of Roy. Soc, 45 and 48; Bruno, footnote 2, p. 
 502; Buglia, Bioch. Zeitschr. 25. 
 
 * Scheunert, cited from Bioch. Centralbl. 10, 71; see also Lorhisch, Zeitschr. f. pbysiol. 
 Chem. 69, 143 (1910) as well as Bioch. Centralbl. 8, 334. 
 
CHEMICAL PROCESSES IN THE [NTESTINE. 511 
 
 cellulose undergoes a fermentation in the intestine by the action of micro- 
 organisms, producing marsh-gas, acetic acid, and butyric acid, has 
 been especially shown by Tappeiner; still it is not known to what extent 
 the cellulose is destroyed in this way. 1 
 
 The bile has, as shown by Moore and Rockwood 2 and then espe- 
 cially by Pfluger, the property to a high degree of dissolving fatty 
 acids, especially oleic acid, which itself is a solvent for other fatty acids, 
 and hence, as will be seen later, it is of great importance in the absorp- 
 tion of fat. It is also of great importance that the bile, as previously 
 stated, not only activates the steapsinogen, but that, as first shown by 
 Nencki and Rachford, 3 it accelerates the fat-splitting action of the 
 steapsin. According to v. Furth and Schutz 4 the bile-salts are the 
 active constituents of the bile in this cleavage, and the fatty acids set 
 free can combine with the alkalies of the intestinal and pancreatic juices 
 and the bile, producing soaps which are of great importance in the 
 emulsification of the fats. 
 
 If to a soda solution of about 1-3 p. m. pure, perfectly neutral 
 olive-oil is added in not too large a quantity, a transient emulsion is 
 obtained after vigorous shaking. If, on the contrary, one adds to the 
 same quantity of soda solution an equal amount of commercial olive- 
 oil (which always contains free fatty acids), the vessel need only be 
 turned over for the two liquids to mix, and immediately there appears 
 a very finely divided and permanent emulsion, making the liquid appear 
 like milk. The free fatty acids of the commercial oil, which is always 
 somewhat rancid, combine with the alkali to form soaps which act to 
 emulsify the fats (Brucke, Gad, Loewenthal 5 ). This emulsifying 
 action of the fatty acids split off by the pancreatic juice is undoubtedly 
 assisted by the habitual occurrence of free fatty acids in the food, as 
 well as by the splitting off of fatty acids from the neutral fats in the 
 stomach (see page 476). 
 
 Bile completely prevents peptic zymolysis in artificial digestion, 
 
 l On the digestion of cellulose see Henneberg and Stohmann, Zeitschr, f. Biologie, 
 21, 613; v. Knieriem, ibid., 67; Hofmeister, Arch. f. wiss. u. prakt. Thierheilkunde, 
 11; Weiske, Zeitschr. f. Biologie, 22, 373; Tappeiner, ibid., 20 and 24; Mallevre, 
 Pfliiger's Arch., 49; Omeliansky, Arch. d. scienc. biol. de St. Petersbourg, 7; E. Muller, 
 Pfliiger's Arch., 83; Lohrisch, Zeitschr. f. physiol. Chem., 47 (literature); Pringsheim, 
 ibid. 78, 266 (1912). 
 
 2 Proceedings of Roy. Soc, 60, and Journ. of Physiol., 21. In regard to Pfliiger's 
 work see Absorption. 
 
 3 Nencki, Arch. f. exp. Path. u. Pharm., 20; Rachford, Journal of Phvsiol., 12. 
 
 4 Centralbl. f. Physiol., 20. 
 
 * Brucke, Wien, Sitzungsber., 61, Abt. 2; Gad, Arch. f. (Anat. u.) Physiol., 1878; 
 Loewenthal, ibid., 1897. 
 
512 DIGESTION. 
 
 because it retards the swelling up of the proteins. The passage of bile 
 into the stomach during digestion on the contrary, seems, according, 
 to several investigators, especially Oddi and Dastre, 1 to have no dis- 
 turbing action on gastric digestion. According to Boldyreff, 2 after 
 continuous starvation, on feeding fat and food rich in fat, as well as after 
 large amounts of acid, a mixture of bile, pancreatic juice, and intestinal 
 juice pass readily into the stomach. After food rich in fat, which 
 retards the secretion of gastric juice and the motility of the stomach, 
 a digestion due to this alkaline mixture may take place in the stomach. 
 
 Bile itself has no solvent action on proteins in neutral or alkaline 
 reaction, but still' it may exert an influence on protein digestion in the 
 intestine. The acid contents of the stomach, containing an abundance 
 of proteins, give with the bile a precipitate of proteins and bile-acids. 
 This precipitate carries a part of the pepsin with it, and for this reason, 
 and also on account of the partial or complete neutralization of the 
 acid of the gastric juice by the alkali of the bile and the pancreatic 
 juice, the pepsin digestion cannot proceed further in the intestine. 
 According to Baumstark and Cohnheim 3 connective tissue is digested 
 on the other side of the pylorus in the intestine by the pepsin-hydrochloric 
 acid. On the contrary, the bile does not disturb the digestion of pro- 
 teins by the pancreatic juice in the intestine. The action of these diges- 
 tive secretions, as above stated, is not disturbed by the bile, not even 
 by the faintly acid reaction due to organic acids; but, on the contrary, 
 the action of trypsin is accelerated by the bile. In a dog killed while 
 digestion is going on, the faintly acid, bile-containing material of the 
 intestine shows regularly a strong digestive action on proteins. 
 
 The precipitate of protein and bile-salts formed on the meeting of the 
 acid contents of the stomach with the bile easily redissolves in an excess 
 of bile, and also in the NaCl formed in the neutralization of the hydro- 
 chloric acid of the gastric juice. This may take place even in faintly 
 acid reaction. Since in man the excretory ducts of the bile and the 
 pancreatic juice open near one another, in consequence of which the 
 acid contents of the stomach are probably immediately in great part 
 neutralized by the bile as soon as it enters, it is doubtful whether a pre- 
 cipitation of proteins by the bile occurs in the intestine. 
 
 Besides the previously mentioned processes caused by enzymes, 
 there are others of a different nature going on in the intestine, namely, 
 the fermentation and putrefaction processes caused by micro-organ- 
 isms. These are less intense in the upper parts of the intestine, but 
 
 » Oddi, in Centralbl. f. Physiol., 1, 312; Dastre, Arch, de Physiol. (5), 2, 316. 
 1 Centralbl. f. Physiol., 18, 457, and Pfluger's Arch., 121. 
 3 Zeitschr. f. physio). Chem., 65, 477 (1910). 
 
PUTREFACTION IN THE INTESTINE. 513 
 
 increase in intensity toward the lower part, and decrease in the large 
 intestine because of the consumption of fermentable material and by 
 the removal of water by absorption. Fermentation processes, but only 
 very slight putrefaction, occur in the small intestine of man. Mac- 
 fadyen, M. Nencki, and N. Seeber * have investigated a case of 
 human anus praeternaturlis, in which the fistula occurred at the lower 
 end of the ileum, and they were able to investigate the contents of the 
 intestine after it had been exposed to the action of the mucous mem- 
 brane of the entire small intestine. The mass was yellow or yellowish- 
 brown, due to bilirubin, and had an acid reaction which, on a mixed 
 but principally animal diet, calculated as acetic acid, amounted to 1 p. m. 
 The contents were nearly odorless, having an empyreumatic odor recall- 
 ing that of volatile fatty acids, and infrequently had a putrid odor 
 resembling that of indol. The essential acid present was acetic acid, 
 accompanied by fermentation and paralactic acid, volatile fatty acids, 
 succinic acid, and bile-acids. Coagulable proteins, peptone, mucin, 
 dextrin, sugar, and alcohol were present. Leucine and tyrosine could 
 not be detected. 
 
 According to the above-mentioned investigators, the proteins are 
 only to a very slight extent, if at all, decomposed by the microbes in 
 the small intestine of man. The organisms present in the small intestine 
 preferably decompose the carbohydrates, forming ethyl alcohol and the 
 above-mentioned organic acids. 
 
 Further investigations of Jakowsky and of Ad. Schmidt 2 lead to 
 the same result, namely, that in man the putrefaction of the proteins 
 takes place chiefly in the large intestine, and the conditions are the 
 same in carnivora. In these latter it has been possible to follow the 
 intestinal digestion by investigating the contents of the various parts 
 of the intestine as well as by forming fistulas along the intestine. Again 
 Pawlow and his pupils, especially London 3 and his collaborators, have 
 essentially advanced our knowledge on this subject. 
 
 In regard to the digestion of protein, it has been found that after 
 feeding meat, bread, or certain protein bodies, the digestion in the 
 stomach and small intestine is so complete that on the passage of the 
 contents into the caecum all the protein is digested and absorbed. 
 Unboiled white of egg is an exception and is digested with difficulty. 
 In experiments with unboiled white of egg, London and Suleima reob- 
 
 1 Arch. f. exp. Path. u. Pharm., 28. 
 
 2 Jakowsky, Arch, des scienc. biol. de St. Petersbourg, 1; Ad. Schmidt, Arch. f. 
 Verdauunskr., 4. 
 
 ' The works of London and collaborators cannot be cited in detail, but may be 
 found in Zeitschr. f. physiol. Chem., 46-57. 
 
514 DIGESTION. 
 
 tained 73 per cent of the coagulable protein from a fistula in the ileum 
 (2-3 cm. in front of the caecum). Elastin is, according to London, * 
 more slowly digested in the small intestine than other proteins. Kut- 
 scher and Seemann, Abderhalden, London and collaborators 2 have 
 also found that non-biuret giving products and amino-acids are regularly 
 split off, probably by the combined action of trypsin and erepsin. These 
 amino-acids occur to a slight extent only, but from this no conclusion 
 can be drawn as to the extent of amino-acid formation, because we do 
 not know the extent of their absorption. The digestion of protein 
 in the intestine, it seems, according to Abderhalden, London, Oppler 
 and Reemlin, 3 is similar to the artificial digestion with trypsin, nainely, 
 that the destruction takes place step-wise, that certain amino-acids, 
 such as tyrosine, are split off earlier than others. Zunz 4 found the same 
 end result in the protein cleavage in the small intestine, with bread as 
 with meat feeding. London, Schittenhelm and Wiener 5 found 
 that a cleavage of nucleic acids with the formation of nucleosides 
 occurred in the lower part of the jejunum and ileum. 
 
 The decomposition products of the proteins formed by the action of 
 gastric juice can, according to London, 6 be absorbed without further 
 cleavage by the pancreatic juice, and a further cleavage in the intestine 
 seems to be more necessary for assimilation than for absorption. 
 
 The carbohydrates and the fats (Levites 7 ) may be so completely 
 split in the stomach and small intestine that their absorption is com- 
 plete before the contents pass into the caecum. According to London 
 and Polowzowa 8 a strong cleavage of starch, dextrins and disaccharides 
 takes place, especially in the duodenum, while the absorption is less 
 strong here. The carbohydrates are here prepared for the absorption 
 taking place in the lower parts of the intestine, though the cleavage also 
 goes on in the other parts, namely in the jejunum and the upper part 
 of the ileum. 
 
 As above remarked, ordinarily no putrefaction takes place in the 
 small intestine, but occurs generally only in the large intestine. This 
 
 1 London and Suleima, Zeitschr. f. physiol. Chem., 46; London, ibid., 60. 
 
 2 Kutscher and Seemann. ibid., 34; Abderhalden and London, with Kautzsch, 
 ibid., with L. Baumann, ibid., 51, with v. Korosy, ibid., 53. 
 
 3 Zeitschr. f. physiol. Chem., 55 and 58. 
 
 4 Intern. Beitr. z. Pathol, u. Ther. d. Ernahrungsstorungen, 2, 195, 459 (1910 and 
 1911). 
 
 '■> Zeitschr. f. physiol. Chem., 72, 459 (1911). 
 • Ibid., 49. 
 7 Ibid., 49 and 53. 
 y Ibid., 56. 
 
PUTREFACTION IN THE INTESTINE. 515 
 
 putrefaction of the proteins is not the same as the pancreatic digestion. 
 In putrefaction the decomposition goes much further, and a mixture of 
 products is obtained which have become known through the labors 
 of numerous investigators, especially Nbncki, Baimanx, Bkieger, H. 
 and E. Salkowski, and their pupils. The products which are formed 
 in the putrefaetion of proteins are (in addition to proteoses, peptones, 
 amino-acids, and ammonia) indol, skatol, paracresol, phenol, phenylpro- 
 pioitic acid, and phenylacetic acid, also paraoxyphenylacetic acid and 
 hydroparacoumaric acid (besides paracresol, produced in the putrefaction 
 of tyrosin), volatile fatty acids, carbon dioxide, hydrogen, marsh-gas, 
 ■mcthylmercaptan, and sulphureted hydrogen. In the putrefaction of 
 gelatin neither tyrosine nor indol is formed, while glycocoll is produced 
 instead. 
 
 Among these products of decomposition a few are of special interest 
 because of their behavior within the organism and because after their 
 absorption they pass into the urine. A few, such as the oxyacids, pass 
 unchanged into the urine. Others, such as phenols, are directly trans- 
 formed into ethereal sulphuric acids by synthesis, and are eliminated as 
 such by the urine; on the contrary, others, such as indol and skatol, are 
 converted into ethereal sulphuric acids after oxidation only (for details 
 see Chapter XIV). The quantity of these bodies in the urine also varies 
 with the extent of the putrefactive processes in the intestine; at least 
 this is true for the ethereal sulphuric acids. Their quantity increases 
 in the urine with a stronger putrefaction, and the reverse takes place, 
 namely, a disappearance from the urine, or a great reduction in quantity, 
 as Baumann, Harley and Goodbody 1 have shown by experiments 
 on dogs, when the intestine is disinfected by various agents. 
 
 The gases which are produced by the decomposition processes are 
 mixed in the intestinal tract with the atmospheric air swallowed with 
 the saliva and food, and as the gas developed in the decomposition of 
 different foods varies, so the mixture of gases after various foods should 
 have a dissimilar composition. This is found to be true. Oxygen is 
 found only in very faint traces in the intestine; this may be accounted 
 for in part by the formation of reducing substances in the fermenta- 
 tion processes which combine with the oxygen, and partly, perhaps 
 chiefly, to a diffusion of the oxygen through the tissues of the walls of 
 the intestine. To show that these processes take place mainly in the 
 stomach, the reader is referred to page 486, on the composition of the 
 gases of the stomach. Xitrogen is invariably found in the intestine, 
 and it is probably clue chiefly to the swallowed air. The carbon dioxide 
 
 1 Baumann, Zeitschr. f. physiol. Chem., 10; Harley and Goodbody, Brit. Med. 
 Journ., 1899. 
 
516 DIGESTION. 
 
 originates partly from the contents of the stomach, partly from the 
 putrefaction of the proteins, partly from the lactic-acid and butyric- 
 acid fermentation of carbohydrates, and partly from the setting free of 
 carbon dioxide from the alkali carbonates of the pancreatic and intes- 
 tinal juices by their neutralization through the hydrochloric acid of 
 the gastric juice and by organic acids formed in the fermentation. 
 Hydrogen occurs in largest quantities after a milk diet, and in smallest 
 quantities after a purely meat diet. This gas seems to be formed 
 chiefly in the butyric-acid fermentation of carbohydrates, although it 
 may occur in large quantities in the putrefaction of proteins under certain 
 circumstances. There is no doubt that the methylmercaptan and sul- 
 phureted hydrogen which occur normally in the intestine originate from 
 the proteins. The marsh-gas undoubtedly originates in the putrefac- 
 tion of proteins. As proof of this Ruge *■ found 26.45 per cent marsh- 
 gas in the human intestine after a meat diet. He found a still greater 
 quantity of this gas after a vegetable (leguminous) diet; this coincides 
 with the observation that marsh-gas may be produced by a fermentation 
 of carbohydrates, but especially of cellulose (Tappeiner 2 ) . Such an 
 origin of marsh-gas, especially in herbivora, is to be expected. A small 
 part of the marsh-gas and carbon dioxide may also arise from the decom- 
 position of lecithin (Hasebroek 3 ). 
 
 Putrefaction in the intestine not only depends upon the composi- 
 tion of the food, but also upon the albuminous secretions and the bile. 
 Among the constituents of bile which are changed or decomposed, , there 
 are not only the pigments — the bilirubin yields urobilin and a brown 
 pigment — but also the bile-acids, especially taurocholic acid. Glyco- 
 cholic acid is more stable, and a part is found unchanged in the excre- 
 ment of certain animals, while taurocholic acid is so completely decom- 
 posed that it is entirely absent in the feces. In the fetus, on the con- 
 trary, in whose intestinal tract no putrefaction processes occur, undecom- 
 posed bile-acids and bile-pigments are found in the contents of the 
 intestine. The transformation of bilirubin into urobilin does not occur, 
 as previously stated, in the small, but in the large intestine in man. 
 
 As under normal conditions no putrefaction, or a,t least none worth 
 mentioning, occurs in the small intestine, and as often nearly all the pro- 
 tein of the food is absorbed, it follows that ordinarily it is the secretions 
 and cells rich in proteins which undergo putrefaction. That the secre- 
 tions rich in proteins are destroyed in putrefaction in the intestine 
 
 1 Wien. Sitzungsber., 44. 
 
 2 Zeitschr. f. Biologie., 20 and 24. 
 
 3 Zeitschr. f. phyaiol. Chem., 12. 
 
PUTREFACTION IN THE INTESTINE. 517 
 
 follows from the fact that putrefaction may also continue during com- 
 plete fasting. From the observations of Muller l upon Cetti it was 
 found that the elimination of indican during starvation rapidly de- 
 creased and after the third day of starvation it had entirely disappeared, 
 while the phenol elimination, which at first decreased so that it was 
 nearly minimum, increased again from the fifth day of starvation, and 
 on the eighth or ninth day it was three to seven times as much as in man 
 under ordinary circumstances. In dogs, on the contrary, the elimina- 
 tion of indican during starvation is considerable, but the phenol elimina- 
 tion is slight. Among the secretions which undergo putrefaction in the 
 intestine, the pancreatic juice, which putrefies most readily, takes first 
 place. 
 
 From the foregoing facts it must be concluded that the products 
 formed by the putrefaction in the intestine are in part the same as 
 those formed in digestion. The putrefaction may be of benefit to the 
 organism in so far as the formation of such products as proteoses, pep- 
 tones, polypeptides and amino-acids is concerned. The question has 
 indeed been asked (Pasteur), Is digestion possible without micro-organ- 
 isms? Nuttal and Thierfelder have shown that guinea-pigs, removed 
 from the uterus of the mother by Caesarian section, could with sterile 
 air digest well and assimilate sterile food (milk and crackers) in the 
 complete absence of bacteria in the intestine, and developed normally 
 and increased in weight. Schottelius 2 has arrived at other results 
 by experiments with hens. The chickens, hatched under sterile con- 
 ditions, kept in sterile rooms and fed with sterile food, had continuous 
 hunger and ate abundantly, but soon died, in about the same time as a 
 starving chicken. On mixing with the food, at the proper time, a vari- 
 ety of bacteria from hen feces, they gained weight again and recovered. 
 
 The bacterial action in the intestinal canal is, at least in certain cases, 
 as with food rich in cellulose, necessary, and it acts in the interest of the 
 organism. This action may, by the formation of further cleavage prod- 
 ucts, involve a loss of valuable material to the organism, and it is there- 
 fore important that putrefaction in the intestine be kept within certain 
 limits. If an animal is killed while digestion in the intestine is going 
 on, the contents of the small intestine give out a peculiar but not putres- 
 cent odor. Also the odor of the contents of the large intestine is far less 
 offensive than a putrefying pancreas infusion or a putrefying mixture 
 rich in protein. From this one may conclude that putrefaction in the 
 intestine is ordinarily not nearly so intense as outside of the organism. 
 
 1 Berlin, klin. Wochenschr., 1887. 
 
 2 Nuttal and Thierfelder, Zeitschr. f. physiol. Chem., 21 and 22; Schottelius, Arch, 
 f. Hygiene, 34, 42, and 67. 
 
518 DIGESTION. 
 
 It seems thus to be provided, under physiological conditions, that 
 putrefaction shall not proceed too far, and the factors which here come 
 into consideration are probably of different kinds. Absorption is 
 undoubtedly one of the most important of them, and it has been proved 
 by actual observation that the putrefaction increases, as a rule, as the 
 absorption is checked and fluid masses accumulate in the intestine. The 
 character of the food also has an unmistakable influence, and it seems 
 as if a large quantity of carbohydrates in the food acts against putre- 
 faction (Hirschler 1 ). It has been shown by Pohl, Biernacki, 
 Rovighi, Winternitz, Schmitz, and others 2 that milk and kephir 
 have a specially strong preventive action on putrefaction. This action 
 is not due to the casein, but chiefly to the lactose and also in part to the 
 lactic acid. 
 
 A specially strong preventive action on putrefaction has been 
 ascribed for a long time to the bile. This anti-putrid action does not 
 exist in neutral or faintly alkaline bile, which itself easily putrefies, but 
 to the free bile-acids, especially taurocholic acid (Maly and Emich, 
 Lindberger 3 ). There is no question that the free bile-acids have a 
 strong preventive action on putrefaction outside of the organism, and 
 it is therefore difficult to deny such an action in the acid reacting con- 
 tents of the intestine. Notwithstanding this, the anti-putrid action 
 of the bile in the intestine is not considered by certain investigators 
 (Voit, Rohmann, Hirschler and Terray, Landauer and Rosen- 
 berg 4 ) as of great importance. 
 
 Biliary fistulas have been established so as to study the importance 
 of the bile. in digestion (Schwann, Blondlot, Bidder and Schmidt, 5 
 and others). As a result it has been observed that with fatty foods an 
 imperfect absorption of fat regularly takes place and the excrement 
 contains, therefore, an excess of fat and has a light-gray or pale color. 
 The extent of deviation from the normal after the operation is essen- 
 tially dependent upon the character of the food. If an animal is fed 
 on meat and fat, then the quantity of food must be considerably increased 
 after the operation, otherwise the animal will become very thin, and 
 
 1 Hirschler, Zeitschr. f. physiol. Chem., 10; Zimnitzki, ibid., 39 (literature). 
 
 2 Schmitz, ibid., 17, 401, which gives references to the older literature, and 19. See 
 also Salkowski, Centralbl. f. d. med. Wiss., 1893, 467, and Seelig, Virchow's Arch., 
 126 (literature). 
 
 3 Maly and Emich, Monatshefte, f. Chem., 4; Lindberger, footnote 4, p. 506. 
 
 4 Voit, Beitr. zur Biologie, Jubilaumschrift, Stuttgart, 1882; Rohmann, Pfltiger's 
 Arch. 29; Hirschler and Terray, Maly's Jahresber., 26; Landauer, Math, u. Naturw. 
 Ber. aus Ungarn, 15; Rosenberg, Arch. f. (Anat. u.) Physiol., 1901. 
 
 'Schwann, Miiller's Arch. f. Anat. u. Physiol., 1844; Blondlot, cited from Bidder 
 and Schmidt, Verdauungssafte, etc., 98. 
 
PUTREFACTION IN THE INTESTINE. 519 
 
 indeed die with symptoms of starvation. In these cases the excrement 
 has the odor of carrion, and this was considered a proof of the action 
 of the bile in checking putrefaction. The emaciation and the increased 
 want of food depend, naturally, upon the imperfect absorption of the 
 fats, whose high calorific value is reduced and must be replaced by the 
 taking up of larger quantities of other nutritive bodies. If the quan- 
 tity of proteins and fats be increased, then the latter, which can be only 
 incompletely absorbed, accumulate in the intestine. This accumulation 
 of the fats in the intestine only renders the action of the digestive juices 
 on proteins more difficult, and thus increases the amount of putrefac- 
 tion. This explains th'e appearance of fetid feces, whose pale color is 
 not due to a lack of bile-pigments, but to a surplus of fat (Rohmann, 
 Voit). If the animal is, on the contrary, fed on meat and carbohy- 
 drates, it may remain quite normal, and the leading off of the bile does 
 not cause any increased putrefaction. The carbohydrates may be 
 uninterruptedly absorbed in such large quantities that they replace 
 the fat of the food, and this is the reason why the animal on such a diet 
 dots not become emaciated. As with this diet the putrefaction in the 
 intestine is no greater than under normal conditions even though the bile 
 is absent, it would seem that the bile in the intestine exercises no pre- 
 ventive action on putrefaction. 
 
 To this conclusion the objection may be made that the carbohy- 
 drates, which are capable of checking putrefaction, can, so to speak, 
 undertake the anti-putrid action of the bile. But as there are also cases 
 (in dogs with biliary fistula) where the intestinal putrefaction is not 
 increased with exclusive meat diet, 1 it is maintained that the absence 
 of bile in the intestine, even by exclusive carbohydrate food, does not 
 always cause an increased putrefaction. 
 
 Although the question as to the manner in which the putrefactive 
 processes in the intestine under physiological conditions are kept within 
 certain limits cannot le answered positively, still it may be asserted 
 that the faint acid reaction, and the absorption of water, and the rela- 
 tively rapid movement, of the contents of the small intestine and their 
 absorption, are important factors. 
 
 That the acid reaction in the intestine has a preventive influence on 
 the putrefactive processes follows from the existing relation between 
 the degree of acidity of the gastric juice and the putrefaction in the 
 intestine. Since the investigations and observations of Kast, Stadel- 
 mann, Wasbutzki, Biernacki and Mester had proved that an 
 increased putrefaction in the intestine occurred when the quantity of 
 hydrochloric acid in the gastric juice was diminished or deficient, 
 
 1 See Hirschler and Terray, 1. c. 
 
520 DIGESTION. 
 
 Schmitz 1 has shown in man that on the administration of hydro- 
 chloric acid, producing a hyperacidity of the gastric juice, the putrefac- 
 tion in the intestine may be checked. The question arises whether the 
 reaction in the small intestine is always acid and whether the acidity 
 is strong enough to prevent putrefaction. In this connection it must 
 be recalled that the acidity of the contents of the small intestine is not 
 due to hydrochloric acid, but chiefly to organic acids, acid salts, and 
 free carbon dioxide. There are several observations as to the reaction 
 of the intestinal contents, by Moore and Rockwood, Moore and 
 Bergin, Matthes and Maequardsen, I. Munk, Nencki and Zaleski, 
 Hemmeter, 2 although they are somewhat contradictory. From these 
 reports one can conclude that the reaction may vary not only among 
 different animals, but also in the same animals under varying conditions. 
 There is no doubt that the acid reaction in many cases is due to the pres- 
 ence cf organic acids. On testing with various indicators it has been 
 shown that sometimes the upper parts, and often the lower parts, are 
 acid, due to acid salts such as NaHCC>3 and free C0 2 , and finally that 
 in certain animals the intestinal contents are alkaline throughout. The 
 question how, under these conditions, putrefaction is excluded, and how 
 the acidity of the gastric contents influences the intestinal putrefaction, 
 cannot be explained. It is very probable that the bacterial flora of the 
 intestine is of very great importance and it is possible, as Bienstock 
 admits, that the explanation lies in an antagonistic bacterial action and 
 that the carbohydrates, especially lactose, which retard putrefaction, 
 form a good nutritive media for those bacteria which destroy the putre- 
 factive producers or retard their development. According to Horo- 
 witz an unequal division of the various bacteria occurs in dogs in the 
 different parts of the intestine and certain varieties of bacteria occur 
 in greater quantities than others, according to the kind of food taken. 
 The influence of the kind of food upon the intestinal flora has also been 
 studied by Kendall. Perhaps, also, agreeing with the experience of 
 Conradi and Kurpjuweit, 3 the toxins produced by the intestinal bacteria 
 may, by their antiseptic action, keep the putrefactive processes in the 
 intestine within bounds. 
 
 1 Zeitschr. f. physiol. Chem., 19, 401, which includes all the pertinent literature. 
 
 2 Moore and Rockwood, Journ. of Physiol., 21; Moore and Bergin, Amer. Journ. 
 of Physiol., 3; Matthes and Marquardsen, Maly's Jahresber., 28; Munk, Centralbl. f. 
 Physiol., 16; Nencki and Zaleski, Zeitschr. f. physiol. Chem., 27; Hemmeter, Pfluger's 
 
 Arch., 81. 
 
 3 Bienstock, Arch. f. Hygiene, 39; Horowitz. Zeitschr. f. physiol. Chem., 52; Ken- 
 dall, Journ. of biol. Chem. 6, 499 (1909); Conradi and Kurpjuweit, Munch, med. 
 Wochenschr., 1905. 
 
feci;-. 521 
 
 Feces. It is evident that the residue which remains after complete 
 digestion and absorption in the intestine must be different, both quali- 
 tatively and quantitatively, according to the variety and quantity of 
 the food. In man the quantity of excrement from a mixed diet is 
 120-150 grams, with 30-37 grams of solids, per twenty-four hours, while 
 the quantity from a vegetable diet, according to Voit, 1 was 333 
 grams, with 75 grams of solids. With a strictly meat diet the excre- 
 ment is scanty, pitch-like, and black. The scanty feces in starva- 
 tion have a similar appearance. A large quantity of coarse bread yields 
 a great amount of light-colored excrement. In these cases the feces 
 are also habitually poorer in nitrogen than after food rich in protein. 
 The individuality also plays an important role in the utility of the food 
 and the formation of feces (Schierbeck 2 ). If there is a large propor- 
 tion of fat, it takes a lighter clay-like appearance. The decomposi- 
 tion products of the bile-pigments seem to play only a small part in the 
 normal color of the feces. 
 
 The constituents of the feces are of different kinds. In the excre- 
 ment are found digestible or absorbable constituents of the food, such 
 as muscle fibers, connective tissues, lumps of casein, grains of starch, 
 and fat, which have not had sufficient time to be completely digested 
 or absorbed in the intestinal tract. In addition the excrement con- 
 tains indigestible bodies, such as the remains of plants, keratin sub- 
 stances, and others; also form-elements originating from the mucous coat 
 and the glands; constituents of the different secretions, such as mucin, 
 cholic acid, dyslysine, and cholesterin (koprosterin or stercorin), purine 
 bases, 3 and enzyro.es; mineral bodies of the food and the secretions; 
 and, lastly, products of putrefaction or of digestion, such as skatol, indol, 
 volatile fatty acids, purine bases, lime, and magnesia soaps. Occasion- 
 ally, also, parasites of different kinds occur; and lastly, the excrement 
 contains micro-organisms of various species. 
 
 That the mucous membrane of the intestine by its secretion and by 
 the abundant quantity of detached epithelium contributes essentially 
 to the formation of feces follows from the discovery first made by L. 
 Hermann and substantiated by others, 4 that a clean, isolated loop 
 
 1 Zeitschr. f . Biologie, 25, 264. 
 
 2 Arch, f . Hygiene, 51. 
 
 s In regard to the purine bases in feces, see Hall, Journ. of Path, and Bacteriol., 
 9; Schittenhelm, Arch. f. klin. Med., 81. Schittenhelm and Kriiger, Zeitschr. f. physiol. 
 Chem., 45. 
 
 4 Hermann, Pfliiger's Arch., 46. See also Ehrenthal, ibid., 48; Berenstein, ibid., 
 53; Klecki, Centralbl. f. Physiol., 7; 736, and F. Voit, Zeitschr. f. Biologie, 29; v. 
 Moraczewski, Zeitschr. f. physiol. Chem., 25; F. Lippich, Prager med. Wochenschr., 
 32. 
 
522 DIGESTION. 
 
 of intestine collects material similar to feces. These masses are rich 
 in mineral substances and especially rich in bodies soluble in alcohol- 
 ether, among which cholesterin occurs, as previously mentioned (Chap- 
 ter VII). With a mixed diet with an excess of meat, the human feces 
 consist only in small part of food residues and consist in great part, 
 or after meat or milk diet, almost entirely, of intestinal secretions. Many 
 foods, therefore, produce a large quantity of feces chiefly by causing an 
 abundant secretion. 1 
 
 The reaction of the feces is very variable, but in man with a mixed 
 diet it is neutral or faintly alkaline. It is often acid in the inner part, 
 while the outer layers in contact with the mucous coat have an alka- 
 line reaction. In nursing infants it is habitually acid. The odor is 
 perhaps chiefly due to skatol, which was first found in the feces by 
 Brieger, and so named by him. Indol and other substances also take 
 part in the production of odor. The color is ordinarily light or dark 
 brown, and depends above all upon the nature of the food. Medicinal 
 bodies may give the feces an abnormal color. The excrement is col- 
 ored black by bismuth, yellow by rhubarb, and green by calomel. This 
 last-mentioned color was formerly accounted for by the formation of a 
 little mercury sulphide, but now it is said that calomel checks the putre- 
 faction and the decomposition of the bile-pigments, so that a part of the 
 bile-pigments passes into the feces as biliverdin. In the yolk-yellow or 
 greenish-yellow excrement of nursing infants one can detect bilirubin. 
 Neither bilirubin nor biliverdin seems to exist in the excrement of mature 
 persons under normal conditions. In adults under normal conditions 
 the feces contain neither bilirubin nor biliverdin. On the contrary, there 
 is found stercobilin (Masitjs and Vanlair), which is identical with uro- 
 bilin (Jaff£ 2 ). Bilirubin may occur in pathological cases in the feces 
 of mature persons. It has been observed in a crystallized state (as 
 hsematoidin) in the feces of children as well as of grown persons. 
 
 The absence of bile (acholic feces) causes the feces to have, as above 
 stated, a gray color, due to large quantities of fat; this may, however, 
 be partly attributed to the absence of bile-pigments. In these cases 
 a large quantity of crystals has been observed which consist principally 
 of magnesium soaps or sodium soaps. Hemorrhage in the upper parts 
 of the digestive tract yields, when it is not very abundant, a dark-brown 
 excrement, due to haematin. 
 
 1 In regard to the constitution of feces with various foods, see Hammerl, Kermauner, 
 Moeller, and Prausnitz, Zeitschr. f. Biologie. 35, and Poda, Micko, Prausnitz and 
 Mtiller, ibid., 39. 
 
 2 See bile-pigments, Chapter VII, and urobilin, Chapter XIV. 
 
MECONIUM. INTESTINAL CONCREMENT8. 523 
 
 Excretin, so named by Marcet, 1 is a crystalline body occurring in human 
 excrement, but which, according to Hopi'e-Seylek, is perhaps only impure choles- 
 terin (koprosterin or stercorin?). Excretolic acid is the name given by Marcet 
 to an oily body with an excrementitious odor. 
 
 In consideration of the very variable composition of feces, quanti- 
 tative analyses are of little value and therefore will be omitted. 2 
 
 Meconium is a dark brownish-green, pitchy, mostly acid mass without any 
 strong odor. It contains greenish-colored epithelium cells, cell-detritus, numer- 
 ous fat-globules, and cholesterin plates. The amount of water is 720-800, 
 and solids 2S0-200 p. m. Among the solids there are mucin, bile-pigments, 
 and bile-acids, cholesterin, fat, soaps, traces of enzymes, calcium and magnesium 
 phosphates. Sugar and lactic acid, soluble protein bodies and peptones, also 
 leucine and tyrosine and the other products of putrefaction occurring in the 
 intestine, are absent. Meconium may contain undecomposed taurocholic acid, 
 bilirubin and biliverdin, but it does not contain any stercobilin. which is con- 
 sidered as proof of the non-existence of putrefactive processes in the digestive 
 tract of the fetus. 
 
 The coyitents of the intestine under abnormal conditions are perhaps less the 
 subject of chemical analysis than of an inspection and microscopical investiga- 
 tion or bacteriological examination. On this account the question as to the 
 properties of the contents of the intestine in different diseases cannot be thor- 
 oughly treated here. 3 
 
 Appendix. 
 
 INTESTINAL CONCREMENTS. 
 
 Calculi occur very seldom in the human intestine or in the intestine 
 of carnivora, but they are quite common in herbivora. Foreign bodies 
 or undigested residues of food may, when for some reason or other they 
 are retained in the intestine for some time, become incrusted with salts, 
 especially ammonium-magnesium phosphate or magnesium phosphate, 
 and these salts usually form the chief constituent of the concrements. 
 In man they are sometimes oval or round, yellow, yellowish-gray, or 
 brownish-gray, of variable size, consisting of concentric layers and 
 containing chiefly ammonium-magnesium phosphate and calcium phos- 
 phate, besides a small quantity of fat or pigment. The nucleus ordi- 
 narily consists of some foreign body, such as the stone of a fruit, a 
 fragment of bone, or something similar. Sjoqvist 4 has recorded an ex- 
 traordinary concrement consisting principally of fatty acids and a bile-acid. 
 In those countries where bread made from oat-bran is an important food, 
 
 1 Annal. de chim. et de phys., 59. 
 
 1 In regard to these analyses as well as to the feces under abnormal conditions 
 and to the pertinent literature, see Ad. Schmidt and J. Strassburger, Die Faeces des 
 Menschen, etc., Berlin, 1901 and 1902. 
 
 * See Schmidt and Strassburger, 1. c. 
 
 4 Hygiea, Festband, 1908. 
 
524 DIGESTION. 
 
 we often find in the large intestine, balls similar to the so-called hair- 
 balls (see below). Such calculi contain calcium and magnesium phos- 
 phate (about 70 per cent), oat-bran (15-18 per cent), soaps and fat (about 
 10 per cent). Concretions which contain very much fat (about 74 per 
 cent) occasionally occur, and those consisting of fibrin clots, sinews, 
 or pieces of meat incrusted with phosphates are also rare. 
 
 Intestinal calculi often occur in animals, especially in horses fed on bran. 
 These calculi, which attain a very large size, are hard and heavy (as much as 8 
 kilos) and consist in great part of concentric layers of ammonium-magnesium 
 phosphate. Another variety of concrements which occur in horses and cattle 
 consists of gray-colored, often very large, but relatively light stones which contain 
 plant residues and earthy phosphates. Stones of a third variety are sometimes 
 cylindrical, sometimes spherical, smooth, shining, brownish on the surface, con- 
 sisting of matted hairs and plant-fibers, and termed hair-balls. The so-called 
 " ^egagropil.e," which occur in the antilope rupicapra, belong to this group, 
 and are generally considered as nothing else than the hair-balls of cattle. 
 
 The so-called oriental bezoar-stone also belongs to the intestinal concrements, 
 and probably originates from the intestinal tract' of the capra .egagrus and ante- 
 lope dorcas. There may exist two varieties of bezoar-stones. One is olive- 
 green, faintly shining and formed of concentric layers. On heating it melts with 
 the development of an aromatic odor. It contains as chief constituent lithofellic 
 acid, C20H36O4, which is related to cholic acid, and besides this a bile-acid, litho- 
 bilic acid. The others are nearly blackish brown or dark green, very glossy, 
 consisting of concentric layers, and do not melt on heating. They contain as 
 chief constituent ellagic acid, a derivative of gallic acid, of the formula CuHeOs, 
 which, according to Graebe, 1 is the dilactone of hexaoxydiphenyldicarboxylic 
 acid, and which gives a deep-blue color with an alcoholic solution of ferric chlo- 
 ride. The last-mentioned bezoar-stone originates, to all appearances, from the 
 food of the animal. 
 
 Ambergris is generally considered an intestinal concrement of the sperm whale. 
 Its chief constituent is ambrain, which is a non-nitrogenous substance perhaps 
 related to cholesterin. Ambrain is insoluble in water and is not changed by boil- 
 ing alkalies. It dissolves in alcohol, ether, and oils. 
 
 VI. ABSORPTION. 
 
 The contents of the intestine are gradually pushed onward by the 
 peristalsis or rhythmical movement of the intestinal musculature, but 
 the mechanism is not well known. 2 By these processes the intestinal 
 contents are intimately mixed and the constituents of the food which 
 are valuable to the organism are transformed, in the manner previously 
 mentioned, so that they are adaptable for the processes of absorption. 
 In discussing the absorption processes we must treat of the form into 
 which the different foods are changed before absorption, of the man- 
 ner in which this is accomplished, and lastly, of the forces which act 
 in these processes. 
 
 1 Ber. d. d. chem. Gesellsch., 36. 
 
 2 See Cannon, Amer. Journ. of Physiol., 6, 12, 29; Magnus, Pfluger's Arch., 102, 
 103, 108, 111; Baumstark and Cohnheim, Zeitschr. f. physiol. Chem., 65. 
 
ABSORPTION OF PROTEINS. 525 
 
 Before we can answer the question as to the form in which the pro- 
 teins are absorbed from the intestinal canal, it is of interest to learn 
 whether the animal body can, perhaps, also utilize such proteins as are 
 introduced intravenously, Bubcutaneously, or into a body-cavity, i.e., 
 evading the intestinal canal, or, as it is called parenteral. 
 
 Since the first investigations of Zuntz and v. Mering on this sub- 
 ject, several experimenters x have shown, without any doubt, that the 
 animal body can more or less completely utilize different, parenterally 
 introduced proteins, although different varieties of animals show a differ- 
 ence in this regard. Still we do not know where and how these foreign 
 proteins are changed and assimilated, but Cramer ascribes great impor- 
 tance to the leucocytes in this regard. See Abderhalden's experiment 
 given on page 54. 
 
 That the animal body can also assimilate not previously digested 
 or split proteins introduced directly into the intestine has been shown 
 by Brucke, Bauer and Yoit, Eichhorst, Czerny and Latschen- 
 berger, Yoit and Friedlaxder, and others. 2 In the experiments 
 of the two last-mentioned investigators neither casein (as milk) nor 
 hydrochloric-acid myosin or acid albuminate (in acid solution) was 
 absorbed, while, on the contrary, about 21 per cent of ovalbumin or 
 seralbumin and 69 per cent of alkali albuminate (dissolved in alkali) 
 were absorbed. Mendel and Rockwood, on the contrary, in experi- 
 ments with casein and edestin in the living intestinal loop, could prove 
 only the slightest absorption en excluding digestion as completely as 
 possible, while the corresponding proteoses were abundantly absorbed. 
 
 It is difficult to decide in these experiments as to how far the pro- 
 teins were taken up in an actually unchanged or partly modified form. 
 The alimentary albuminaria, observed repeatedly after the introduction 
 of large quantities of protein into the intestinal canal, indicates an 
 absorption of undigested protein under certain circumstances. To decide 
 this question the biological method, using the precipitine reaction, has 
 been made use of, and Ascoli and Vigno, 3 using this method, claim to 
 
 1 Zuntz and v. Mering, Pfliiger's Arch., 32; Xeumeister, Verh. d. phys.-med. 
 Gesellsch. zu Wiirzburg, 18S9, and Zeitschr., f. Biologie, 27; Friedenthal and Lewan- 
 dowsky, Arch. f. (Anat. u.) Physiol., 1S99; Munk and Lewandowsky, ibid., 1S99, 
 Supp.; Oppenheimer, Hofmeisters Beitrage, 4; Mendel and Rockwood, Amer. Journ. 
 of Physiol., 12; Heilner, Zeitschr. f. Biol., 50, and Munch, med. Wochenschr., 49; 
 Cramer, Journ. of Physiol., 3", with Pringle, ibid.; Rona and Michaelis. Pfluuer's 
 Arch., 123 and 124; v. Korosy, Zeitschr. f. physiol. Chem. 62, 68 (1909), 69, 313 (1910). 
 
 2 Brucke, Wien. Sitzungsber., 59; Bauer and Voit, Zeitschr. f. Biologie, 5; Eich- 
 horst, Pfliiger's Arch., 4; Czerny and Latschenberger, Yirchow's Arch., 59; Voit and 
 Friedlander, Zeitschr. f. Biologie, 33. Contradictory observations can be found in 
 Keller, Beitr. z. Frage d. Resorption im Dickdarm. I naug. -Dissert. Breslau, 1909. 
 
 3 Zeitschr. f. Physiol. Chem., 39. 
 
526 DIGESTION. 
 
 have shown the passage of non-modified protein into the blood and 
 lymph (page 66). Based upon many investigations on this subject 
 we can consider it possible that under certain circumstances, as on flood- 
 ing the intestinal canal with protein, with a greater permeability of the 
 intestinal wall, as in new-born and sucking animals, and with a dimin- 
 ished modification by the gastric juice, a passage of non-modified pro- 
 tein may take place in the blood-vessels, but that under normal con- 
 ditions this is not the case, or at least does not take place to any men- 
 tionable degree. As a rule, the absorption of protein follows a modi- 
 fication of it. In this connection the experiments of Orni 1 are of interest 
 which show that the dog's intestine takes up the serum of the dog but 
 not that of the ox or horse. In regard to the previously split proteins 
 the question arises, whether the proteins are chiefly absorbed as pro- 
 teoses or peptones or as simpler atomic complexes. 
 
 According to the earlier investigations of Ludwig and Schmidt- 
 Mulheim, as well as those of Munk and Rosenstein, 2 it is generally 
 agreed that the products of protein digestion do not pass into the 
 blood through the lymph vessels, but through the intestinal capillaries. 
 The question of the absorption of these products resolves itself into 
 the form in which they are taken up by the intestine and the form in 
 which they pass into the blood. 
 
 It was mentioned above that proteoses and peptones as well as non- 
 biuret-giving products and amino-acids have been found in the con- 
 tents of the intestine. The amino-acids occur to a less extent than the 
 proteoses and peptones. This may indicate that the amino-acids are 
 more abundantly formed, but also more quickly absorbed, but it may 
 also indicate that the amino-acids are produced to a slight extent only, 
 in the intestinal contents. There is no doubt that the amino-acids can 
 be absorbed as such, but there is still another question, namely, whether 
 the proteoses and peptones are absorbed as such or only after a pre- 
 vious cleavage into amino-acids. 
 
 Nolf and Honof.e found, what was later substantiated by Zunz, 3 
 that the proteoses and peptones disappear more quickly from the 
 intestine than the non-biuret-giving products. This does not prove 
 that the proteoses are absorbed as such, but rather against such a view. 
 A more direct proof for the absorption of the non-split proteoses lies 
 in the fact, as shown by Nolf, that the proteoses when introduced in 
 
 1 Pfltiger's Arch. 126, 428 (1909). 
 
 * Schmidt-Mulheim, Arch. f. (Anat. u.) Physiol., 1877; Munk and Rosenstein. 
 Virchow'e Arch., 128. 
 
 3 Nolf and Honored Arch, internat. de Physiol., 1905; Nolf, Journ. de Physiol, et 
 Pathol, gdn., 1907; Zunz, Memoires cour., etc., Acad. Roy. Med., Belg., 20, Fasc. 1. 
 
ABSORPTION OF PROTEINS. 521 
 
 large quantities in the intestine pass in small amounts into the blood. 
 Another proof is the findings of Borchakdt, 1 that after feeding dogs 
 with not too large amounts of elastin, the passage of a proteose, the 
 hemielastose, could be detected in the blood. Attention must also be 
 called to the fact that according to Hofmeistek - the walls of the 
 stomach and intestine are the only parts of the body in which pro- 
 teoses and peptones occur during digestion. 
 
 We have reason for believing that the proteoses, as well as their 
 cleavage products, are taken up by the intestine, and if this is the case 
 the next question to be answered is, in what form do these bodies leave 
 the intestine and pass into the blood? 
 
 In order to decide this question the blood has been repeatedly 
 tested in regard to the quantity of proteoses. As seen on page 264 
 this has led to very contradictory results, and if we exclude those 
 exceptional cases where a large quantity of proteose was introduced 
 into the intestine at once, then we can say that the occurrence of pro- 
 teoses in the blood, or at least in the blood-plasma, has not been posi- 
 tively shown under physiological conditions. 3 It can also be said that 
 such investigations do not prove much because of the large quantity 
 of blood passing through the intestine for a given time, and the quan- 
 tity of proteose must be so small, so that when divided in the entire blood 
 it can hardly be detected. It is therefore of interest that neither amino- 
 acids nor proteoses were found in the blood after cutting out several 
 organs or groups of organs so that the blood circulated only through 
 the intestinal canal, heart, lungs, pancreas and intercostal muscles 
 (Kutscher and Seemann, v. Korosy 4 ). 
 
 We are therefore obliged to consider that the proteoses and amino- 
 acids are transformed in the intestinal walls in some manner or other. 
 Such a belief, especially applied to the proteoses, coincides with the 
 observations of Hofmeister, that the proteoses occurring in the mucous 
 membrane during digestion disappear at the temperature of the room 
 from the removed, but still apparently living, mucous membrane after a 
 certain time. This also coincides well with the observations of Ludwig 
 and Salvioli. 5 These investigators introduced a peptone solution into 
 a double-ligatured, isolated piece of the small intestine, which was kept 
 alive by passing defibrinated blood through it, and observed that the 
 
 1 In regard to the literature on proteoses in the blood see Chapter V, footnotes- 
 1, 2 and 3, p. 264. 
 
 2 Zeitschr f. physiol. Chem., 6, and Arch. f. exp. Path. u. Pharm., 19, 20, 22. 
 
 * See footnote 1. 
 
 * Kutscher and Seemann, Zeitschr. f. physiol. Chem., 34; v. Korosy, ibid., 57. 
 
 s Arch. f. (Anat. u.) Physiol., 1880, Supplbd. See also Cathcart and Leathee r 
 .'ourn. of Physiol., 33. 
 
528 DIGESTION. 
 
 peptone disappeared from the intestine, but that the blood passing 
 through did not contain any peptone. 
 
 What becomes of the amino-acids in the intestinal wall? Ktjtscher 
 and Seemann have shown that the crystalline cleavage products are 
 so transformed in the intestinal wall that they cannot be detected. 
 We have here to think of two possibilities: The amino-acids are either 
 further split or they are used in synthesis (of proteins?). 
 
 It is a long-known fact that with the digestion and absorption an 
 increased elimination of nitrogen in the urine goes hand in hand. The 
 •quantity of nitrogen eliminated in the urine after partaking of protein 
 corresponded, according to Asher and Haas, 1 to 65 per cent of the 
 nitrogen introduced. It is hardly credible that this elimination of 
 nitrogen depends upon an increased destruction of body protein, and 
 it is more probable that this represents decomposed food-protein. 
 But according to Nencki and Zaleski 2 an abundant formation of 
 ammonia occurs in the cells of the digestive apparatus after a rich 
 protein diet, so we must consider the possibility that a considerable part, 
 perhaps the very greatest part, of the amino-acids are deamidized in 
 the intestinal wall. The other part of the amino-acids may be used 
 in the syntheses to be mentioned below. Such a partial deamidization 
 of the digestive products has been shown by Cohnheim 3 in his absorp- 
 tion experiments with the fish intestine. 
 
 The proteoses taken up by the intestinal mucosa, if this does take 
 place, can naturally undergo a further conversion into amino-acids in 
 the walls of the intestine. Still there are other possibilities. A direct 
 utilization of the proteoses in the synthesis of the proteins in the intes- 
 tine is not very probable, but on the contrary it is more probable that 
 the proteoses, in order to undergo further cleavage or further utiliza- 
 tion, are taken up by the leucocytes and carried off. Hofmeister 
 has advocated such a possibility for a long time. Heidenhain raised 
 objections to this suggestion in which he called attention to the dis- 
 proportion between the number of leucocytes and the large quantity of 
 peptones (proteoses) to be absorbed, but at that time the deep cleavage 
 of a great part of the protein into amino-acids was not known. Recently 
 Pringle . and Cramer 4 urged the theory of the importance of the 
 leucocytes, and the observations of Inagaki 5 also show the possibility 
 
 1 Bioch. Zeitschr., 12. 
 
 2 Arch, des scienc. biol. de St. P6tersbourg, 4; Arch. f. exp. Path. u. Pharm., 37; 
 see also Salaskin, Zeitschr. f. Physiol. Chem., 25. 
 
 1 Zeitschr. f. physiol. Chem., 59. 
 
 ♦Hofmeister, 1. c; Heidenhain, Pfluger's Arch., 43; Pringle and Cramer, Journ. 
 of Physiol., 37. 
 
 6 Zeitschr. f. physiol. Chem., 50. 
 
ABSORPTION OF PROTEINS. 529 
 
 of the leucocytes taking up the proteoses and fixing them, it seems, in 
 the cell substance. 
 
 It is for the present impossible to say with certainty whether or 
 not and to what extent the proteoses, as such, are absorbed and to give 
 their further fate thereafter in the intestine. The present view is prob- 
 ably as follows: That they do not pass as such into the blood, and 
 that they are transformed into amino-acids in part in the intestinal 
 -contents and in part in the intestinal mucosa, and then from these amino- 
 acids the coagulable proteins are constructed by synthesis. In sup- 
 port of the theory of a protein synthesis from amino-acids we have a 
 series of experiments where deeply split or completely split proteins 
 were fed. In these experiments by Loewi, Henderson and Dean, 
 Henriques and Hansen, and especially by Abderhalden and his 
 co-workers l on dogs, mice and rats, it was possible to keep the animals 
 in nitrogenous equilibrium or indeed nitrogen retention for a long time 
 with the cleavage products of proteins besides non-nitrogenous food- 
 stuffs and salts. According to the recent experiments of Abderhalden 
 the organism can build up proteins from amino-acids when the indi- 
 vidual amino-acids are supplied in proportions as they exist in the cell 
 proteins. Certain, sometimes absent amino-acids seem to be capable 
 of being produced within the organism (for example, glycocoll, proline) 
 while other (tryptophane) cannot be produced. This explains why 
 gelatine which does not contain any tryptophane cannot replace protein 
 in the food. 
 
 The results of the experiments are generally considered as proof of 
 the ability of the animal body to construct proteins from amino-acids 
 by synthesis, and in the present state of our knowledge we can hardly 
 draw other conclusions from them or advance any simpler theory. 
 
 Where does the protein synthesis take place? If it were positively 
 sure that the amino-acids did not pass into the blood then we would 
 have transferred this synthesis to the intestinal walls. Otherwise we 
 must think in the first place of the liver; but this organ does not seem 
 to play an important role in this synthesis. Abderhalden and London 2 
 made an experiment on a dog with an Eck fistula (see page 397), feeding 
 the dog with decomposed protein, and they found that this animal 
 behaved exactly like a normal animal, as it was kept for eight days not 
 only in nitrogenous equilibrium but also in nitrogen retention. On 
 
 1 Loewi, Arch. f. exp. Path. u. Pharm., 48. See also Henderson and Dean, Amer. 
 Journ. of Physiol., 9; Abderhalden and Rona, Zeitschr. f. physiol. Chem., 42, 44, 47, and 
 52; Henriques and Hansen, ibid., 43, 49; Henriques, ibid., 54; Abderhalden with 
 Olinger, ibid., 57, with Messner and Windrath, ibid., 59; Abderhalden, ibid., 77, 22, 
 78, 1 (1912). 
 
 * Zeitschr. f. physiol. Chem., 54. 
 
530 DIGESTION. 
 
 the other hand it is not possible to deny the importance of the liver for 
 the protein syntheses. As Embden and his collaborators have shown on 
 perfusing the liver containing a large amount of glycogen, that d-alanine 
 was formed and Embden explains this formation by a destruction of 
 glucose or lactic acid and pyroracemic acid. With experiments with 
 blood perfusion of the liver, a-amino-acids are formed from the am- 
 monium salts of the corresponding a-keto-acids. The combination 
 NH4.O.CO.CO.R passes into HO.CO.CH(NH 2 ).R. The cleavage 
 products of the carbohydrates can be converted in the liver into char- 
 acteristic constituents of the protein molecule. 1 In this connection 
 we must here mention the experiments of Luthje 2 in which he found 
 a nitrogen retention after feeding only one amino-acid with abundance 
 of carbohydrate. 
 
 What kind of protein is formed in the synthesis? This we do not 
 know. Abderhalden's belief is that it is plasma protein, which, as 
 is well known, is the same in each animal independent of the kind of 
 protein introduced with the food and from which the cells of the body 
 then create the further protein material. Objections can be raised 
 against this hypothesis, but still it is worth consideration. In favor 
 of this we can also add that according to the investigations of Freund 
 and v. Korosy 3 the blood coming from the intestine during digestion 
 is richer in coagulable protein than other blood, and also that this 
 protein, Freund asserts, belongs to the globulin group. This globulin, 
 according to Freund and Toepfer, is not identical with the ordinary 
 serglobulin mixture, but is a pseudoglobulin formed in the. intestine 
 from the food protein by synthesis, and which is more easily decom- 
 posed or further utilized in the liver and other organs. Further research 
 in this direction is necessary, as we have other investigations which 
 are essentially different. If a re-formation of coagulable proteins takes 
 place from amino-acids during digestion, it is to be expected that a 
 relatively greater quantity of coagulable protein should occur in the 
 mucosa of the digesting intestine as compared with the non-digesting 
 intestine. Pringle and Cramer, by a method which requires con- 
 firmation, claim that in the digesting animal (cat), the blood, and to a 
 still higher degree the intestinal mucosa, and especially the lymph nodes 
 of the intestine, are richer in non-coagulable protein than the starving 
 animal, a condition which is related to the r61e of the leucocytes in the 
 
 iBioch. Zeitschr. 29, 423 (1910); 38, '393, 407, 414 (1911); 45, 1-207 (1912); 
 summary, 45, 201. 
 
 2 Pfliiger's Arch. 113, 547 (1906). 
 
 a v. Korosy, Zeitschr. f. physiol. Chem., 57; Freund, Zeitschr. f. exp. Path. u. 
 Therap., 4; G. Toepfer and Freund, and Toepfer, ibid., 3; Pringle and Cramer, Journ. 
 of Physiol., 37. 
 
ABSORPTION OF PROTEINS. 531 
 
 protein assimilation. This question of the absorption of proteins in 
 the intestine is still unexplained in many directions. 
 
 The extent of the protein absorption is dependent essentially upon 
 the kind of food introduced, since as a rule the protein substances from 
 an animal source are much more completely absorbed than from a 
 vegetable source. As proof of this the following observations are 
 given: In his experiments on the utilization of certain foods in the intes- 
 tinal canal of man, Rubner found that with an exclusively animal diet, 
 on partaking of an average of 738-884 grams of fried meat, or 948 grams 
 of eggs per day, the nitrogen deficit with the excrement was only 2.5-2.8 
 per cent of the total nitrogen introduced. With a strictly milk diet 
 the results were somewhat unfavorable, since after partaking of 4100 
 grams of milk the nitrogen deficit increased to 12 per cent. The con- 
 ditions are quite different with vegetable food, as shown by the re- 
 searches of Meyer, Rubner, Hultgren and Landergren, who made 
 experiments with various kinds of rye bread and found that the loss of 
 nitrogen through the feces amounted to 22-48 per cent. Experiments 
 with other vegetable foods, and also the investigations of Schuster, 
 Cramer, Meinert, Mori, 1 and others on the utilization of foods with 
 mixed diets, have led to similar results. With the exception of rice, 
 wheat bread, and certain very finely divided vegetable foods, it is found 
 in general that the nitrogen deficit by the feces increases with a larger 
 quantity of vegetable material in the food. 
 
 The reason for this is manifold. The large quantity of cellulose 
 frequently present in vegetable foods impedes the absorption of pro- 
 teins. The greater irritation produced by the vegetable food itself or 
 by the organic acids formed in the fermentation in the intestinal canal 
 causes a more violent peristalsis, which drives the contents of the intes- 
 tine faster than otherwise along the intestinal canal. Another and most 
 important reason is the fact that a part of the vegetable protein sub- 
 stances seems to be indigestible, and because of the difficultly digestible 
 vegetable food, a large quantity of digestion fluids containing nitrogen 
 is secreted. 
 
 In speaking of the functions of the stomach we stated that after 
 the removal or excision of this organ, an abundant digestion and absorp- 
 tion of proteins may take place. It is therefore of interest to learn how 
 the digestion and absorption of proteins go on after the extirpation of 
 the second protein-digesting organ, the pancreas. In this connection 
 
 1 Rubner, Zeitschr. f. Biologie, 15; Meyer, ibid., 7; Hultgren and Landergren, 
 Nord. med. Arch., 21; Schuster, in Voit's " Untersuch. d. Kost," etc., 142; Cramer, 
 Zeitschr. f. physiol. Chem., 6; Meinert, " Ueber Massennahrung," Berlin, 1SS5; Kell- 
 ner and Mori, Zeistchr. f. Biologie, 25. 
 
532 DIGESTION. 
 
 there are the observations on animals after complete or partial extirpa- 
 tion of the gland by Minkowski and Abelmann, Sandmeyer, V. Har- 
 lbt, after destroying the gland by Rosenberg, and also in man after 
 closing the pancreatic duct by Harley and Deucher. In all these 
 cases such discrepancy of figures has resulted for the utilization of the 
 proteins — between 80 per cent after the apparently complete exclusion 
 of pancreatic juice in man (Deucher) and 18 per cent after extirpa- 
 tion of the gland in dogs (Harley) — that one can hardly draw any 
 clear conception as to the extent and importance of the trypsin diges- 
 tion in the intestine. That on completely preventing the entrance of 
 pancreatic juice only a slight diminution in the protein absorption takes 
 place follows from the researches of Lombroso and Niemann. 1 In 
 order to understand, in these cases, why the digestion and absorption 
 took place so abundantly it would be of interest to know how other 
 digestion fluids act substitutingly. In this regard Zunz and Mayer 2 
 found that in dogs (meat digestion) the tying of the pancreatic passages 
 is essentially compensated for by an increased secretion of pepsin and 
 other proteolytic enzymes, and that in this case the demolition of the 
 protein in the stomach goes further than in a normal animal. 
 
 The carbohydrates are, it seems, chiefly absorbed as monosaccharides. 
 Glucose, fructose, and galactose are probably absorbed as such. The 
 two disaccharides, saccharose and maltose, ordinarily undergo an inver- 
 sion in the intestinal tract and are converted into glucose and fructose. 
 Lactose is also, at least in certain animals, inverted in the intestine. 
 In other mature animals, on the contrary, if the lactase formation is not 
 excited by milk food, the sugar is not inverted or only to a slight 
 extent (Voit and Lusk, Weinland, Portier, Rohmann and Nagano), 
 and it probably is absorbed as such in these animals if it does not under- 
 go fermentation, or, as Rohmann and Nagano 3 assumed, if it is not 
 transformed in the intestinal mucosa in some unknown way. An 
 absorption of non-inverted carbohydrates is not improbable, and accord- 
 ing to Otto and v. Mering the portal blood contains, after a carbo- 
 hydrate diet, besides glucose, a dextrin-like carbohydrate. Moscati 4 
 
 1 Abelmann, " Ueber die Ausniitzung der Nahrungsstoffe nach Pankreasexstirpa- 
 tion" (Inaug.-Dissert. Dorpat, 1890), cited from Maly's Jahresber., 20; Sandmeyer, 
 Zeitschr. f. Biologie, 31; Rosenberg, Pfliiger's Arch., 70; Harley, Journ. of Pathol, 
 and Bacterid., 1895; Deucher, Correspond. Blatt. f. Schweiz. Aerzte, 28; Lombroso, 
 Arch. f. exp. Path. u. Pharm., 60; Niemann, Zeitschr. f. exp. Path. u. Therap., 5; 
 See also Brugsch and Pletnew, Zeitschr. f. exp. Path. u. Therap. 6, 326. 
 
 2 Mem. de l'acad. roy. de m6dic. de Belg., 18. 
 
 J Voit and Lusk, Zeitschr. f. Biologie, 28; Rohmann and Nagano, Pfliiger's Arch., 
 95, which contains the references to the literature. 
 
 4 Otto, see Maly's Jahresber., 17; v. Mering, Arch. f. (Anat., 4.) Physiol., 1877; 
 Moscati, Zeitschr. f. physiol. Chem., 50 
 
ABSORPTION OF CARBOHYDRATES. 533 
 
 • 
 
 believes that when homogeneous starch solutions are injected intra- 
 venously or subcutaneously, the starch is taken up by the organs, namely 
 the spleen, liver and lungs, and is utilized as the starch can be changed 
 into glycogen. A part of the carbohydrates is destroyed by fermenta- 
 tion in the intestine, with the formation of lactic and acetic acids and 
 other bodies. 
 
 The different varieties of sugars arc absorbed with varying degrees 
 of rapidity, but as a general thing absorption occurs very quickly. This 
 absorption takes place more quickly in the upper part of the intestine 
 than in the lower part (Rohmann, Lannois and Lepine, Rohmann 
 and Nagano 1 ). It is generally admitted that the simpler sugars are 
 more quickly absorbed than the disaccharides, while the reports as to 
 the absorption of the disaccharides differ somewhat (Hedon, Alber- 
 toni, Waymouth Reid, Rohmann and Nagano). There seems to be 
 no doubt that lactose is absorbed more slowly than the two other disac- 
 charides. According to the extensive experiments of Rohmann and 
 Nagano, saccharose is absorbed more quickly than maltose. Nagano 2 
 contends that the pentoses are absorbed more slowly than hexoses. 
 
 On the introduction of starch even in very considerable quantities 
 into the intestinal tract no glucose passes into the urine, a condition 
 which probably depends in this case upon the absorption and assimila- 
 tion and the slow saccharification taking place simultaneously. If, 
 on the contrary, large quantities of sugar are introduced at one time, 
 then an elimination of sugar by the urine takes place, and this elimina- 
 tion of sugar is called alimentary glycosuria. In these cases the assimila- 
 tion of the sugar and the absorption do not take place together. 
 
 That quantity of sugar to which we must raise the ingested sub- 
 stance in order to produce an alimentary glycosuria gives, according 
 to Hofmeister, 3 the assimilation limit for that same sugar. This limit- 
 is different for various kinds of sugar; and it also varies for the same 
 sugar not only in different animals, but also in different members of the 
 same species, as also in the same individual under varying circum- 
 stances. In general it can be said that in regard to the ordinary varie- 
 ties of sugar, such as glucose, fructose, galactose, saccharose, maltose, 
 and lactose, the assimilation limit is highest for glucose and lowest for 
 lactose. It must be admitted that with an overabundant quantity of 
 sugars in the intestinal tract the disaccharides do not have sufficient 
 time for their complete inversion, and this has been directly shown by 
 
 'Lannois and Lupine, Arch, de physiol. (3), 1; Rohmann, Pfluger's Arch., 41; see 
 also footnote 3, p. 532. 
 
 2 In regard to the literature on the absorption of sugars, see footnote 3, p. 532. 
 1 Arch. f. exp. Path. u. Pharm., 25 and 26. 
 
534 DIGESTION. 
 
 • 
 Rohmann and Nagano. It is, therefore, not remarkable that disac- 
 eharides, as well, have been found in the urine in cases of alimentary 
 glycosuria. 1 
 
 The investigations of Ludwig and v. Mering and others have 
 explained how the sugars enter into the blood-stream, namely, that they 
 as well as other bodies soluble in water do not ordinarily pass over into 
 the chylous vessels in measurable quantities, but are chiefly taken up by 
 the blood in the capillaries of the villi, and in this way pass into the mass 
 of the blood. These investigations have been confirmed by observa- 
 tions of I. Munk and Rosenstein 2 on human beings. 
 
 The reason why the sugars and other soluble bodies do not pass 
 over into the chylous vessels in appreciable quantity is, according to 
 Heidenhain, 3 to be found in the anatomical conditions, in the arrange- 
 ment of the capillaries close under the layer of epithelium. Ordinarily 
 these capillaries find the necessary time for the removal of the water 
 and the solids dissolved in it. But when a large quantity of liquid, 
 such as a sugar solution, is introduced into the intestine at once, this is 
 not possible, and in these cases a part of the dissolved bodies passes into 
 the chylous vessels and the thoracic duct (Ginsberg and Rohmann 4 ). 
 
 The passage of sugar into the urine, when at one time large quanti- 
 ties of sugar are taken and the assimilation limit is exceeded, can be 
 best explained by the assumption that a part of the sugar escaped the 
 liver and passed into the large circulation, or that the liver did not have 
 time to retain the sugar and transform it into glycogen. According to 
 the observations of de Filippi 5 upon dogs with Eck fistula, it seems as if 
 the role of the liver in these cases is too highly estimated. An animal 
 with Eck fistula could take an unlimited quantity of starch without 
 glycosuria occurring. The assimilation limit was in these cases some- 
 what lower, but qualitatively they behave like normal animals and with 
 increasing sugar supply they could also retain increasing quantities of 
 sugar. 
 
 The introduction of larger quantities of sugar into the intestine at 
 one time can readily cause a disturbance with diarrheal evacuations 
 of the intestine. If the carbohydrate is introduced in the form of starch, 
 
 1 For the literature in regard to the passage of various kinds of sugars into the 
 urine, see C. Voit. Ueber die Glykogenbildung, Zeitschr. f. Biologie, 28, and F. Voit, 
 footnote 1, p. 396. See also Blumenthal, Zur Lehre von der Assimilationsgrenze 
 der Zuckerarten, Inaug.-Dissert. 1903, Strassburg and Brasch, Zeitschr. f. Biol., 50. 
 
 2 v. Mering. Arch. f. (Anat. u). Physiol., 1877; Munk and Rosenstein, Virchow's 
 Arch. 123. 
 
 3 Pfluger's Arch., 43, Suppl. 
 
 4 Ginsberg, Pfluger's Arch., 44; Rohmann, ibid., 41. 
 '• Zeitschr. f. Biol., 4!) and 50. 
 
ABSORPTION OF FATS. 535 
 
 then very large quantities may be absorbed without causing any dis- 
 turbance, and the absorption may be very complete. Rubner found 
 the following: On partaking 508-G70 grams of carbohydrates, as wheat 
 bread, per day, the part not absorbed amounted to only 0.8-2.6 per cent. 
 For peas, where 357-588 grams were eaten, the loss was 3.6-7 per cent, 
 and for potatoes (718 grams) 7.6 per cent. Constantinidi found on 
 partaking 367-380 grams of carbohydrates, chiefly as potatoes, a loss 
 of only 0.4-0.7 per cent. In the experiments of Rubner, as also of 
 Hultgren and Landergren, 1 with rye bread the utilization of car- 
 bohydrates was less complete, and the loss in a few cases rose even to 
 10.4-10.9 per cent. It at least follows from the experiments made thus 
 far that man can absorb more than 500 grams of carbohydrates per diem 
 without difficulty. 
 
 We generally consider the pancreas as the most important organ 
 in the digestion and absorption of amylaceous bodies, and it is a ques- 
 tion how these bodies are absorbed after the extirpation of the pan- 
 creas. As on the absorption of proteins, so also on the absorption of 
 starch, the observations have given variable results. In certain cases 
 the absorption was not impaired, while in others it was, on the contrary, 
 rather diminished, and with dogs devoid of pancreas it has been found 
 that the absorption was decreased to 50 per cent of the starch partaken 
 (Rosenberg, Cavazzani 2 ). 
 
 Em unification used to be considered as of the greatest importance 
 in the absorption of fats, and this emulsion occurs in the chyle on the 
 introduction into the intestine of not only neutral fats, but also of fatty 
 acids. The fatty acids do not exist as such in the emulsified fat of the 
 chyle. The investigations of I. Munk, later confirmed by others, have 
 shown that the fatty acids undergo in great part a synthesis into neutral 
 fats in the walls of the intestine, and are carried as such by the stream 
 of chyle into the blood. This synthesis seems to take place in the 
 mucous membrane (Moore and others 3 ). 
 
 The assumption that the fat is absorbed chiefly as an emulsion is 
 partly based on the abundance of emulsified fat in the chyle after feed- 
 ing with fat, and partly on the fact that a fat emulsion is often found 
 in the intestine after such food. As an abundant cleavage of neutral 
 
 1 Rubner, Zeitschr. f. Biologie, 15 and 19; Constantinidi, ibid., 23; Hultgren and 
 Landergren, Nord. med. Arch. 21. 
 
 2 Cavazzani, Centralbl. f. Physiol., 7. See footnote 1, p. 532; also Lombroso, 
 Hofmeister's Beitriige, 8. 
 
 3 Munk, Virchow's Arch., 80. See also v. Walther, Arch. f. (Anat, u.) Physiol., 
 1890; Minkowski, Arch. f. exp. Path. u. Pharm., 21; Frank, Zeitschr. f. Biologie, 
 36; Moore, see Biochem. Centralbl., 1, 741; Frank and Ritter, Zeitschr. f. Biologie, 
 47; Noll, Pfl tiger's Arch. 136. 
 
536 DIGESTION. 
 
 fats occurs in the intestinal canal, and also as the fatty acids do not 
 occur in the chyle as such, but as emulsified fat after a synthesis with 
 glycerin into neutral fats, it is to be doubted whether the emulsified 
 fat of the chyle originates from an absorption of emulsified fat in the 
 intestine or from a subsequent emulsification of neutral fats formed 
 synthetically. This doubt has greater warrant in the observation of 
 Frank l that the fatty-acid ethyl ester is extensively taken up from the 
 intestine, not as such, but as split-off fatty acids from which then the 
 neutral emulsified fats of the chyle are formed. 
 
 The assumption of an absorption of fats as an emulsion is inconsist- 
 ent with the fact that an emulsion produced by means of soaps is not 
 permanent in an acid liquid; hence we cannot consider as possible the 
 presence of an emulsion in the intestine so long as it is acid. This 
 difficulty is not too serious, as the reaction is often only due to carbonic 
 acid and bicarbonates, and besides as found by Kuhne and recently 
 shown by Moore and Krumbholz, 2 the proteins have a preserving 
 action upon fat emulsions. 
 
 The earlier opinions as to fat absorption were, that fat was absorbed 
 as soaps, soluble in water, as well as finely emulsified fat, and this last 
 form was considered as of the greatest importance. This view has 
 recently undergone essential modifications, due to the work of Moore 
 and Rockwood, and especially to the extensive work of Pfluger. 3 
 
 Moore and Rockwood have shown the great solvent action of the 
 bile for fatty acids, and on continuing these investigations further, 
 Moore and Parker have found that the bile increases the solubility 
 of soaps in water, and can prevent their gelatinization, a fact which is 
 of greater importance for the absorption of fats than the solubility of 
 the fatty acids in bile. The quantity of lecithin in the bile is of great 
 importance for the solubility therein of the fatty acids as well as the 
 soaps. According to the above-mentioned investigators, the absorption 
 of fat from the intestine is essentially dependent upon the solubility of 
 the soaps and free fatty acids in the bile. The neutral fats are split 
 and the free fatty acids are in part absorbed, dissolved as such by the 
 bile, and in part combined with alkalies, forming soaps. Neutral fats 
 are regenerated from the fatty acids, and the alkali set free from the 
 soaps is secreted again into the intestine and used for the re-formation 
 
 1 Zeitschr. f. Biologie, 36. 
 
 1 Kuhne, Lehr. der physiol. Chem., 122; Moore and Krumbholz, Journ. of Physiol., 
 22. 
 
 3 In regard to the recent literature on fat absorption, see the works of Pfluger, 
 Pfluger 's Arch., 80, 81, 82, 85, 88, 89, and 90, where the work of other investigators is 
 cited and discussed. See also Croner, Bioch. Zeitschr. 23; Lombroso, Arch, di Fisiql. 5. 
 
ABSORPTION OF FATS. 537 
 
 of soaps. According to Croner the absorption of soaps occurs only 
 in the lower parts of the small intestine. 
 
 The importance of the bile, the soaps, and the alkali carbonates has 
 been closely studied, principally in the very thorough investigations of 
 Pfluger. He has quantitatively determined the solvent power of 
 the above-mentioned bodies — each alone as well as different mixtures 
 of these — for the various fatty acids, and has closely studied the mode 
 of action of the bile. From his investigations he has arrived at the 
 conclusion that no unsplit fat is absorbed, that all fats, before their 
 absorption, must first be split into glycerin and fatty acids, and that the 
 bile, on account of its solvent power for soaps and fatty acids, is sufficient 
 for the absorption of large quantities of fat eaten. The object of the 
 formation of an emulsion is, according to this view, that the fat in this 
 condition forms such a large surface for the action of the steapsin or 
 the fat-splitting agents. The possibility that all the fat must be first 
 split and that no unsplit fat is absorbed is, according to these researches, 
 not to be denied. 
 
 The next question is whether all the fat or the greater part of it 
 passes into the .blood through the lymphatics and the thoracic duct. 
 According to the researches of Walther and Frank x on dogs, it seems 
 that only a small part of the fats, or at least of the fatty acids fed, 
 passes into the chylous vessels; but these observations can hardly be 
 applied to the absorption of neutral fats, or to the absorption in man 
 under normal circumstances. Munk and Rosenstein, 2 in their inves- 
 tigations on a girl with a lymph fistula, found 60 per cent of the fat 
 ingested in the chyle, and of the total quantity of fat in the chyle only 
 4-5 per cent existed as soaps. On feeding with a foreign fatty acid, 
 such as erucic acid, they found 37 per cent of the introduced body as 
 neutral fat in the chyle. Not all the fat introduced is found in the 
 chyle, and there is always a not inconsiderable part of the absorbed 
 fat whose fate we are not able to follow. 
 
 The completeness with which fats are absorbed depends, under nor- 
 mal conditions, essentially upon the kind of fat. In this regard it is 
 known, especially from the investigations of Munk and Arnschixk, 3 
 that the varieties of fat with high melting-points, such as mutton-tallow, 
 and especially stearin, are not so completely absorbed as the fats with 
 low melting-points, such as hog- and goose-fat, olive-oil, etc. The kind 
 of fat also has an influence on the rapidity of absorption, as Munk and 
 Rosenstein found that solid mutton-fat was absorbed more slowly 
 
 1 Walther, Arch. f. (Anat. u.) Physiol., 1S90; Frank. Qrid., 1S92. 
 
 2 Virchow's Arch., 123. 
 
 s Munk, Virchow's Arch., 80 and 95; Arnschink, Zeitschr. f. Biologie. 26. 
 
538 DIGESTION. 
 
 than fluid lipanin. The extent of absorption in the intestinal tract is, 
 under physiological conditions, very considerable. In the case of a 
 dog investigated by Voit it was found that out of 350 grams of fat 
 (butter) partaken, 346 grams were absorbed from the intestinal canal, 
 and according to the investigations of Rubner x the human intestine 
 can absorb over 300 grams of fat per diem. The fats are, according 
 to Rubner, much more completely absorbed when free, in the form of 
 butter or lard, than when inclosed in cell-membranes, as in bacon. 
 
 Claude Bernard showed long ago with experiments on rabbits in 
 which the ductus choledochus was made to open into the small intestine 
 above the pancreatic duct, that after food rich in fats the chylous vessels 
 of the intestine above the pancreas passages were transparent, while 
 below they were milk-white, and also that the bile alone cannot pro- 
 duce an absorption of the emulsified fat without the pancreatic juice. 
 Dastre 2 has performed the reverse experiment on dogs. He tied the 
 ductus choledochus and adjusted a biliary fistula so that the bile flowed 
 into the intestine below the mouth of the pancreatic passages. On 
 killing the animal after a meal rich in fat the chylous vessels were first 
 found milk-white below the discharge of the biliary fistula. From this 
 Dastre draws the conclusion that a combined action of the bile and pan- 
 creatic juice is important in the absorption of fats — a conclusion which 
 stands in accord with the experience of many others. 
 
 Through numerous observations of many investigators, such as 
 Bidder and Schmidt, Voit, Rohmann, Fr. Muller, I. Munk, 3 and 
 others, it has been shown that the exclusion of the bile from the intes- 
 tinal tract diminishes the absorption of fat to such an extent that only 
 one-seventh to about one-half of the quantity of fat ordinarily absorbed 
 undergoes absorption. In icterus with entire exclusion of the bile, a 
 considerable decrease in the absorption of fat is noticed. As under 
 normal conditions, so also in the absence of bile in the intestine, the lower 
 melting parts of the fat are more completely absorbed than those which 
 have a high melting-point. I. Munk found in his experiments on dogs 
 with lard and mutton-tallow that the absorption of the high-melting 
 tallow was reduced twice as much as the lard on the exclusion of the 
 bile from the intestine. 
 
 We also learn from the investigations of Rohmann and I. Munk 
 that in the absence of bile the relation between fatty acids and neutral 
 fats is changed, namely, about 80-90 per cent of the fat existing in the 
 
 1 Voit, Zeitschr. f. Biologie, 9; Rubner, tirid., lo. 
 
 2 Arch, de Physiol. (5), 2. 
 
 a F. Muller, Sitzungsber. der phys.-med. Gesellsch. zu Wiirzburg, 1SS5; I. Munk, 
 Virchow's Arch., 122. See also footnotes 4 and 5, p. 518. 
 
ABSORPTION OF FATS. 539 
 
 feces consists of fatty acid, while under normal conditions the feces 
 contain 1 part neutral fat to about 2-2£ parts free fatty acids. It ia 
 not possible to state how this increased quantity of fatty acids in the fat 
 of the feces is produced upon the exclusion of the bile from the intestine. 
 
 There is no doubt that the bile is of great importance in the absorp- 
 tion of fats. Still there is also no doubt that rather considerable quan- 
 tities of fat may be absorbed from the intestine in the absence of bile. 
 What relation does the pancreatic juice bear to this fact? 
 
 Upon this point a rather large number of observations on animals 
 have been made by Abelmann and Minkowski, Sandmeyer, Harley, 
 Rosenberg, Hedon and Ville, and also on man by Fr. Muller and 
 Deucher. 1 In all of these investigations a more or less diminished 
 absorption of fat was observed after the extirpation or destruction of 
 the gland, or the exclusion of the juice from the intestine. The results 
 are very diverse as to the extent of this diminution, as in certain cases 
 no absorption of fat was observed, while, in other cases, a considerable 
 absorption was noted in the same class of animal (dog) and even in the 
 same animal. According to Minkowski and Abelmann, after the total 
 extirpation of the pancreas, the fat of the food introduced is not absorbed 
 at all, with the exception of milk, of which 28-53 per cent of the fat is 
 absorbed. Other investigators have obtained different results, and Har- 
 ley has observed a case where in a dog an absorption of only 4 per cent 
 of the milk fat, or, on the complete exclusion of intestinal bacteria, even 
 no absorption, took place. The conditions may vary in the different 
 cases, and the behavior is not the same in different varieties of animals. 
 
 As shown by Lombroso, there exists an essential difference between 
 the action of the extirpation of the gland, or a prevented flow of the 
 secretion into the intestine. In the last case, as the experiments reported 
 by Niemann show, no essential disturbance of the absorption takes place, 
 while the total extirpation of the gland is followed by a marked dis- 
 turbance (Lombroso 2 ). This investigator is also of the opinion that 
 the pancreas, independent of the external secretion in any way (by 
 endocrinic bodies), influences the absorption of the foodstuffs and the 
 activity of the pancreas enzymes in the intestine. In order to judge 
 this view it would be of the greatest interest to know how the exclusion 
 of the pancreatic juice from the intestine acts upon the other factors 
 
 1 Muller, " Unters. liber den Icterus," Zeitschr. f. klin. Med., 12; H6don and 
 Ville, Arch, de Physiol. (5), 9; Harley, Journ. of Physiol., 18, Journ. of Pathol, and 
 Bacteriol., 1895, and Proceed. Roy. Soc, 61. In regard to the other authors see foot- 
 note 1, p. 532. 
 
 2 Lombroso, see Bioch. Centralbl., 3, 67 and 566, and 4, 73S; also Compt. rend, 
 soc. biol., 57; Hofmeister's Beitriige, 8, 11; Pfluger's^Arch., 112; and Arch. f. exp. 
 Path. u. Pharm., 56 and 60; Niemann, 1. c. 
 
540 DIGESTION. 
 
 of the digestion, such as upon the formation of the secretions and their 
 activity. As to this we know at present very little, but the work of 
 Zunz and Mayer (see page 532), indicates that such a reverse action is 
 possible. Under these circumstances it is not possible to give Lombroso's 
 views too great a prominence. 
 
 Lombroso has also found that after the extirpation of the pancreas 
 in the dog, sometimes more fat is eliminated than was contained in the 
 food; that this eliminated fat, which depends upon a fat secretion into 
 the intestinal canal, has a different composition from the introduced fat, 
 and that in these cases an absorption of fat also takes place. That some 
 fat can be absorbed in animals even in the absence of the bile as well 
 as pancreatic juice has been shown by the investigations of Hedon and 
 Ville and Cunningham. 1 
 
 The reason for the fact that the fat absorption is diminished in the 
 absence of bile from the intestine must be sought for in the above-men- 
 tioned rdle of this fluid. It is more difficult to state why the absence 
 of pancreatic juice causes a reduction in the absorption of fat. The most 
 natural view is that the neutral fats are here less completely split, but 
 this does not seem to be the case, because the non-absorbed fat of the 
 feces consists, on the exclusion of bile and pancreatic juice (Minkowski 
 and Abelmann, Harley, Hedon and Ville, Deucher), principally of 
 free fatty acids. A still unknown change caused by gastric or intestinal 
 lipase or by micro r organisms may produce a cleavage of the fat in these 
 cases. The imperfect fat absorption after the extirpation of the pan- 
 creas can possibly be explained by the removal of a considerable part 
 of the alkalies necessary for the formation of the emulsion and for the 
 solution of the fatty acids, but as Sandmeyer found in dogs deprived of 
 their pancreas, that the fat absorption was raised by giving chopped 
 pancreas with the fat, this can hardly be a sufficient explanation. The 
 reason for this is perhaps that after the extirpation of the pancreas the 
 splitting of the fat is chiefly brought about by bacteria in those parts of 
 the intestinal canal where the conditions for absorption are not favor- 
 able. 
 
 The soluble salts are also absorbed with the water. The proteins, 
 which can dissolve a considerable quantity of salts, such as earthy phos- 
 phates which are otherwise insoluble in alkaline water, are of great 
 ' importance in the absorption of such salts. 
 
 The soluble constituents of the digestive secretions can be absorbed 
 like the other soluble substances and toxines, and ferments may also be 
 absorbed, especially by a diseased change in the intestinal walls. 
 
 The occurrence of urobilin in urine attests the absorption of the bile- 
 
 1 Hedon and Ville, 1. c.; Cunningham, Journ. of Physiol., 32. 
 
ABSORPTION OF BILE. 541 
 
 constituents under physiological conditions despite the fact that the 
 occurrence of very small traces of bile-acids in the urine is disputed. 
 The absorption of bile-acids by the intestine seems to be positively proved 
 by other observations. Tappeineh ' introduced a solution of bile- 
 salts of a known concentration into an intestinal knot and after a time 
 investigated the contents. He found that in the jejunum and the ileum, 
 but not in the duodenum, an absorption of bile-acids took place, and 
 further that of the two bile-acids only the glycocholic acid was absorbed 
 in the jejunum. Further, Schiff long ago expressed the opinion that 
 bile undergoes an intermediate circulation, in such wise that it is 
 absorbed from the intestine, then carried to the liver by the blood, and 
 lastly eliminated from the blood by this organ. Although this view has 
 met with seme opposition, still its correctness seems to be established by 
 the researches of various investigators, and more recently by Prevost and 
 Binet, and specially by Stadelmann and his pupils. 2 After the intro- 
 duction of foreign bile into the intestine of an animal, the foreign bile- 
 acids appear again in the secreted bile. 
 
 How does the removal of large portions of the various parts of the 
 intestine affect absorption? Harley 3 has been able to perform a par- 
 tial extirpation of the large intestine and in another instance a com- 
 plete extirpation. This last condition increased the feces considerably, 
 especially because of the large increase in the water (five-fold). Fats 
 and carbohydrates were absorbed just as completely as in the normal. 
 The absorption of the proteins, on the contrary, was reduced to only 
 84 per cent as compared to 93-98 per cent in normal dogs. After extir- 
 pation, the feces sometimes did not contain any urobilin, or only traces 
 thereof, while bile-pigments existed in large amounts. 
 
 Erlanger and Hewlett found that dogs from which 70-83 per 
 cent of the total length of the jejunum and ileum had been removed, 
 could be kept alive, like other animals, if only the food was not too rich 
 in fat. When the food contained large amounts of fat then 25 per 
 cent was evacuated by the feces as compared to 4-5 per cent in the 
 normal animal. Under these same conditions the amount of nitrogen 
 in the feces was increased to twice the normal amount. London and 
 Stassow 4 found on resection of the ileum that the eliminated diges- 
 tion and absorption were performed by the parts of the intestine higher 
 
 1 Wien. Sitzungsber., 77. 
 
 2 Schiff, Pfliiger's Arch., 3; Prevost and Binet, Compt. Rend., 106; Stadelmann, 
 see footnote 1, p. 416. 
 
 3 Proceed. Roy. Soc, 64. 
 
 4 Erlanger and Hewlett, Amer. Journ. of Physiol. 6; London and Stassow. Zeitschr. 
 f.physiol. Chem. 74, 349 (1911). 
 
542 DIGESTION. 
 
 up; after resection of the jejunum the large intestine seems to have a 
 compensating action. 
 
 After the exclusion of the colon in rabbits, Bergmann and Hult- 
 gren x could find no definite action upon the availability of the cellu- 
 lose nor could any diminution in the utility of the other constituents 
 of the food be observed. Zuntz and Ustjanzew 2 also found that the 
 removal of the caecum had no influence on the utilization of nitrogen; 
 but in respect of other factors they arrive at different results. They 
 found, namely, that the caecum of the rodent is of great importance for 
 the digestion of crude fiber and the pentosans. On feeding hay and 
 wheat to rabbits after the removal of the csecum, the digestion coefficient 
 for crude fiber fell from 42.8 to 23.4-18.7 per cent, and for pentosans 
 from 50 to 40-28.7 per cent. 
 
 The question as to the forces which are active in the intestine during 
 absorption has not been satisfactorily answered. Attempts have been 
 made to explain absorption as a filtration, due to a certain difference 
 in the hydrostatic pressure between the intestinal contents and the 
 blood. A sufficiently great difference in pressure does not seem to 
 exist and besides this the absorbed solution on account of its composi- 
 tion cannot be considered as a filtrate from the intestinal contents. 
 Diffusion processes without doubt play a much more important role. 
 These attempt to keep the same concentration of all dissolved sub- 
 stances on both sides of the intestinal epithelium (in intestinal contents 
 and in the blood). Such processes must be influenced, as mentioned 
 in Chapter I on the osmotic pressure, to a high degree upon the perme- 
 ability of the intestinal membrane for dissolved solids and for water. 
 Nevertheless the diffusion stream does not give sufficient explanation 
 for the absorption, as, according to Cohnheim, 3 the result is different 
 according to whether the intestine is alive or is dead and in general a 
 streaming from the lumen of the intestine into the outside fluid is 
 noticeable in the living intestine quite independent of the differences 
 in concentration. How this streaming is brought about has not been 
 explained. 
 
 Other investigators have suggested the question whether surface- 
 tension forces (adsorption phenomenon) are active in absorption. 4 
 Still it has not been possible to bring the absorbability of a substance 
 in simple relation to its influence on the surface-tension of the water. 
 
 1 Skand. Arch. f. Physiol., 14. 
 
 2 Verhandl. d. physiol. Gesellsch. zu Berlin, 1904-1905. 
 
 3 Zeitschr. f. physiol. Chem. 36-39. 
 
 4 J. Traube, Bioch. Zeitschr. 24, 324 (1910) which also contains literature. 
 
THEORIES OF ABSORPTION. 543 
 
 Under these circumstances and as it is not within the scope of this 
 book to enter into details upon the numerous investigations as to the 
 theory of absorption, we must refer to larger works l and to text-books 
 on physiology for further information. 
 
 1 See Hober, Physikalische Chemie der Zelle, Leipzig, 190G, Koranyi and Richter, 
 Phyeikalische Chemie u. Medizin. Leipzig 1907, Bd. 1, 295. I. Munk, Ergebnirae 
 der Physiologie, I, Abt. 1; Hamburger, Osmotisher Druck und Ionealehre, Bd. 2, 
 Wiesbaden, 1904. 
 
CHAPTER IX. 
 TISSUES OF THE CONNECTIVE SUBSTANCE. 
 I. THE CONNECTIVE TISSUES. 
 
 The form-elements of the typical connective tissues are cells of 
 various kinds, of a not very well-known chemical composition, and 
 gelatin-yielding fibrils, which, like the cells, are imbedded in an interstitial 
 or intercellular substance. The fibrils consist of collagen, the interstitial 
 substance contains chiefly mucoid (tendon-mucoid) , besides serglobulin 
 and seralbumin, which, occur in the parenchymatous fluid (Loebisch 1 ). 
 
 The connective tissue also often contains fibers or formations con- 
 sisting of elastin, sometimes in such great quantities that the connective 
 tissue is transformed into elastic tissue. A third variety of fibers, the 
 reticular fibers, also occurs, and according to Siegfried these consist 
 of reticulin. 
 
 If finely divided tendons are extracted in cold water or NaCl solu- 
 tions, the protein bodies soluble in the nutritive fluid in addition to a 
 little mucoid are dissolved. If the residue is extracted with half- 
 saturated lime-water, then the mucoid is dissolved and may be precipi- 
 tated from the filtered extract by adding an excess of acetic acid. The 
 extracted residue contains the fibrils of the connective tissue together 
 with the cells and the elastic substance. 
 
 The so-called tendon mucin is not true mucin, but a mucoid, which, 
 as first shown by Levene and then by Cutter and Gies, contains a. 
 part of its sulphur as an acid related to chondroitin-sulphuric acid. 
 These mucoids, which, according to Cutter and Gies, are mixtures of 
 several glycoproteins, contain 2.2-2.33 per cent sulphur, as shown by 
 the analyses of Chittenden and Gies, as well as those of Cutter and 
 Gies. The quantity of sulphur split off as sulphuric acid was 1.33-1.62 
 per cent (Cutter and Gies). van Lier 2 has prepared a substance at least 
 closely related to tendon mucoid from the hard skin of man and certain 
 animals. ' This mucoid yielded an ethereal sulphuric acid, a glucothionic 
 acid with 1.58-3.03 per cent sulphur in the barium salt, and was variable 
 in different animals. It gives the orcin reaction for glucuronic acid. 
 
 1 Zeitschr. f. physiol. Chem., 10. 
 
 2 Levene, ibid., 31 and 39; Cutter and Gies. Amer. Journ. of Physiol., 6; Chitten- 
 den and Gies, Journ. of Exp. Med., 1; van Lier, Zeitschr. f. physiol. Chem. 61. 
 
 544 
 
CONNECTIVE TISSUES. 545 
 
 The fibrils of the connective tissue are elastic and swell slightly in 
 water, somewhat more in dilute alkalies or in acetic acid. On the other 
 hand, they shrink by the action of certain metallic salts, such as ferrous 
 sulphate or mercuric chloride, and tannic acid, which form insoluble 
 compounds with the collagen. Among these compounds, which prevent 
 putrefaction of the collagen, that with tannic acid has been found of 
 the greatest technical importance in the preparation of leather. In 
 regard to the collagens, gelatins, elastins, and reticulins, see pages 116 
 to 121. 
 
 The tissues described under the names mucous or gelatinous tissues 
 are characterized more by their physical than by their chemical prop- 
 erties, and have been but little studied. This much, however, is 
 known, that the mucous or gelatinous tissues contain, at least in certain 
 cases, as in the Acalephse, no mucin. 
 
 The umbilical cord is the most accessible material for the investiga- 
 tion of the chemical constituents of the gelatinous tissues. The mucin 
 occurring therein yields, according to van Lier, an ethereal sulphuric 
 acid (glucothionic acid) like the tendon mucoid. C. Th. Morner 1 
 has found a mucoid in the vitreous humor which contains 12.27 per 
 cent nitrogen and 1.19 per cent sulphur. 
 
 Young connective tissue is richer in mucoid than old. Halliburton 2 
 found an average of 7.66 p. m. mucoid in the skin of very young children 
 and only 3.85 p. m. in the skin of adults. In so-called myxcedema, 
 in which a re-formation of the connective tissue of the skin takes place, 
 the quantity of mucoid is also increased. 
 
 The connective tissue and also the elastic tissue are richer in water 
 and poorer in solids in young animals as compared with full-grown 
 animals. This may be seen from the following analyses of the Achilles 
 tendon (Buerger and Gies) and of the ligamentum nuchse (Vande- 
 grift and Gies 3 ): 
 
 Achilles tendon. Ligament. 
 
 Calf. Ox. Calf. Ox. 
 
 Water 675.1p.m. 628.7 p.m. 651.0p.m. 575.7 p.m. 
 
 Solids 324.9 " 371.3 " 394.0 " 424.3 
 
 < )r«anic bodies 318.4 " 366.6 " 342.4 " 419.6 
 
 Inorganic bodies 6.1 " 4.7 " 6.6 " 4.7 
 
 Fat 10.4 " 11.2 
 
 Proteid 2.2 " 6.16 
 
 Mucoid 12.83 " 5.25 
 
 Elastin 16.33 " 316.70 
 
 Collagen 315.88 " 72.30 
 
 Extractives, etc 8.96 " 7.99 
 
 1 Zeitschr. f. physiol. Chem., IS, 250. 
 
 2 Mucin in Myxcedema: Further Analyses. King's College Collected Papers 
 No. 1, 1893. 
 
 1 Buerger and Gies, Amer. Journ. of Physiol., 6; Vandegrift and Gies, ibid, 5. 
 
546 TISSUES OF THE CONNECTIVE SUBSTANCE. 
 
 In regard to the mineral bodies it must be remarked that according 
 to the determinations of H. Schulz the connective tissue is rich in silicic 
 acid. The greatest amount was found by him, in the crystalline lens 
 of the ox, namely, 0.5814 gram per kilo of dried substance. In man he 
 found 0.0637 gram in the tendons, 0.1064 gram in the fascia, and 0.244 
 gram in Wharton's jelly for every kilo of dried substance. The quantity 
 of silicic acid is higher in the young than in the old; in man it is highest 
 in the embryonic connective tissue of the umbilical cord. In the last- 
 named substance Schulz also found 0.403 gram FeoOs, 0.693 gram 
 MgO, 3.297 grams CaO, and 3.794 grams P2O5 for every kilo of dried 
 substance. The report of Schulz on the quantity of silicic acid does 
 not correspond with the investigations of Frauenberger 1 who found, 
 in Wharton's jelly, only a fraction of the quantity of silicic acid that 
 Schulz gives. 
 
 H. CARTILAGE. 
 
 Cartilaginous tissues consist of cells and an original hyaline matrix, 
 which, however, may become changed in such wise that. there appears 
 in it a network of elastic fibers or connective-tissue fibrils. 
 
 Those cells that offer great resistance to the action of alkalies and 
 acids have not been carefully studied. According to earlier opinions 
 the matrix was considered as consisting of a body analogous to colla- 
 gen, so-called chondrigen. The investigations of Morochowetz and 
 others, but especialty those of C. Morner, 2 have shown that the 
 matrix of the cartilage consists of a mixture of collagen with other 
 bodies. 
 
 The tracheal, thyroideal, cricoidal, and arytenoidal cartilages of 
 full-grown cattle contain, according to Morner, four constituents in 
 the matrix, namely, chondromucoid, chondroitin-sulphuric acid, collagen, 
 and the albumoid. 
 
 Chondromucoid. This body, according to C. Morner, has the com- 
 position C 47.30, H 6.42, N 12.58, S 2.42, O 31.28 per cent. Sulphur is 
 in part loosely combined and may be split off by the action of alkalies, 
 and a part separates as sulphuric acid when boiled with hydrochloric 
 acid. Chondromucoid is decomposed fay dilute alkalies and yields alkali 
 albuminate, peptone substances, chondroitin-sulphuric acid, alkali sul- 
 phides, and some alkali sulphates. On boiling with acids it yields acid 
 albuminate, peptone substances, chondroitin-sulphuric acid, and on 
 
 1 Schulz, Pfluger's Arch, 84 and 89, 131 and 144; Frauenberger, Zeitschr. f. physiol. 
 Chem., 57. 
 
 1 Morochowetz, Verhandl., d. naturh. med. Vereins zu Heidelberg, 1, Heft 5; Morner, 
 Skand. Arch. f. Physiol., 1. 
 
( 1 1 ON DROITI N -SULPHURIC ACID. 547 
 
 account of the further decomposition of this last body, sulphuric acid 
 and a reducing substance are forme 1. 
 
 Chondromucoid is a white, amorphous, acid-reacting powder which 
 is insoluble in water, but dissolves easily on the addition of a little 
 alkali. This solution is precipitated by acetic acid in great excess and 
 by small quantities erf mineral acids. The precipitation may be retarded 
 by neutral sails or by ehondroitin-sulphuric acid. The solution con- 
 taining NaCl and acidified with HC1 is not precipitated by potassium 
 ferrocyanide. Precipitants for chondromucoid are alum, ferric chloride, 
 sugar of lead, or basic lead acetate. Chondromucoid is not precipitated 
 by tannic acid, and it may by its presence prevent the precipitation 
 of gelatin by this acid. It gives the usual color reactions for proteins, 
 namely, with nitric acid, with copper sulphate and alkali, with Mil- 
 lion's and Adamkiewicz-Hopkins' reagents. 
 
 Chcaidroitin-sulphuric Acid, chondroitic acid. This acid, which 
 was first prepared pure, from cartilage, by C. Morner and identified 
 by him as an ethereal sulphuric acid, occurs, according to Morner, in 
 all varieties of cartilage and also in the tunica intima of the aorta and 
 as traces in the bone substance. ' K. Morner x has also found it in the 
 ox-kidney and in human urine as a regular constituent. Its occurrence 
 in amyloid, as mentioned on page 173, has been disputed by Hanssen. 
 In the opinion of Levene, 2 the glucothionic acid which is prepared from 
 tendon mucoid, and which gives the orcin reaction for glucuronic acid, 
 and yields furfurol on distillation with hydrochloric acid, is not identical 
 with the ehondroitin-sulphuric acid, but is probably related thereto. 
 
 Chondroitin-sulphuric acid has the formula C18H27NSO17, accord- 
 ing to Schmiedeberg. 3 As primary products this acid yields, on 
 cleavage, sulphuric acid and a nitrogenous substance, chondroitin, accord- 
 ing to the following equation: 
 
 C18H27NSO17+H2O = H 2 S04+Ci8H 27 NOi4. 
 
 Chondroitin, which is similar to gum arabic, and which is a monobasic 
 acid, yields acetic acid and a new nitrogenous substance, chondrosin, 
 as cleavage products, on decomposition with dilute mineral acids: 
 
 C 18 H 27 NOi4+3H20 = 3C2H 4 02+Ci2H2iN0 1 i. 
 
 Chondrosin, which is also a gummy substance soluble in water, is a 
 monobasic acid and reduces copper oxide in alkaline solutions even 
 
 1 C. Morner, 1. c, and Zeitschr. f. physiol. Chem., 20 and 23; K. Morner, Skand. 
 Arch. f. Physiol., 6. 
 
 2 Zeitschr. f. physiol. Chem., 39. 
 
 * Arch. f. exp. Path. u. Pharm., 28. 
 
548 TISSUES OF THE CONNECTIVE SUBSTANCE. 
 
 more strongly than glucose. It is dextrogyrate, and represents the 
 reducing substance obtained by previous investigators in an impure 
 form on boiling cartilage with an acid. The products obtained on decom- 
 posing chondrosin with barium hydroxide tend to show, according to 
 Schmiedeberg, that chondrosin contains the atomic groups of glucuronic 
 acid and glucosamine. This assumption does not seem to have sufficient 
 foundation. According to Orgler and Neuberg, chondrosin does not 
 give the orcin test nor does it yield furfurol. They claim that on 
 cleavage with baryta it yields, besides a carbohydrate complex which 
 has not been studied, an oxyamino-acid having the formula C6H13O6N, 
 a hexosamine acid or tetraoxyaminocaproic acid. In opposition to this 
 S. Frankel has found that the chondrosin gives the orcin as well as the 
 phloroglucin test with hydrochloric acid, and he has prepared an acid 
 with the formula CeHnNOe, which he calls aminoglucuronic acid, 
 which gives the above tests and also reduces. Among other investi- 
 gators, Pons and Kondo l have also found that chondroitin-sulphuric 
 acid gives the orcin test and yields furfurol, according to Pons 6.6-6.9 
 per cent. The chondrosin obtained after boiling with acid and distilling 
 off the furfurol does not, according to Pons, give furfurol, which agrees 
 with Orgler and Neuberg's statement. From the hydrolytic products 
 of chondroitin-sulphuric acid with hydrochloric acid, Pons obtained 
 with phenylhydrazin. a crystalline substance melting at 143° C. 
 
 Chondroitin-sulphuric acid appears as a white amorphous powder 
 which dissolves very easily in water, forming an acid solution and, when 
 sufficiently concentrated, a sticky liquid similar to a solution of gum 
 arabic. Nearly all of its salts are soluble in water. The neutralized 
 solution is precipitated by stannous chloride, basic lead acetate, neutral 
 ferric chloride, and by alcohol in the presence of a little neutral salt. 
 The solution, on the other hand, is not precipitated by acetic acid, 
 tannic acid, potassium ferrocyanide and acetic acid, sugar of lead, mer- 
 curic chloride, or silver nitrate. Acidified solutions of alkali chondroitin- 
 sulphates cause a precipitation when added to solutions of gelatin or 
 proteid. 
 
 The preparation of chondromucoid, and its separation from chondroitin- 
 sulphuric acid can be accomplished after the method of C. Morner, but for 
 details we refer to the original work. 
 
 The pre-existing chondroitin-sulphuric acid, or that formed by the 
 decomposition of chondromucoid, is obtained by lixiviating the cartilage 
 with a 5-per cent caustic-alkali solution. The alkali albuminate formed 
 by the decomposition of the chondromucoid can be removed from the 
 solution by neutralization, then the peptone precipitated by tannic acid, 
 
 1 Orgler and Neuberg, Zeitschr. f. physiol. Chem., 37; Frankel, Annal. d. Chem. u. 
 Pharm., 351; Pons. Arch, intern, de Physiol., 8 (1909); Kondo, Bioch. Zeitschr., 26. 
 
CARTILAGENEOUS TISSUE. 549 
 
 the excess of this acid removed with sugar of lead, and the lead removed 
 from the filtrate by H2S. If further purification is necessary, the acid is 
 precipitated with alcohol, the precipitate dissolved in water, this solu- 
 tion dialyzed and precipitated again with alcohol — this solution in water 
 and precipitation with alcohol being repeated a few times — and lastly 
 the acid is treated with alcohol and ether. Other methods for the prepara- 
 tion of the acid (from the septum narium of the pig) have been suggested 
 by Schmiedeberg and Kondo. 
 
 The collagen of the cartilage gives, according to C. Morner, a gelatin 
 which contains only 16.4 per cent N, and which can hardly be considered 
 identical with ordinary gelatin. 
 
 In the above-mentioned cartilages of full-grown animals the chon- 
 droitin-sulphuric acid and chondromucoid, perhaps also the collagen, 
 are found surrounding the cells as round balls or lumps. These balls 
 (Morner's chondrin-balls) , which give a blue color with methyl-violet, 
 lie in the meshes of a trabecular structure, which is colored when 
 brought in contact with tropseolin. 
 
 The albumoid is a nitrogenized body which contains loosely com- 
 bined sulphur. It is soluble with difficulty in acids and alkalies and 
 resembles keratin in many respects, but differs from it by being soluble 
 in gastric juice. In other respects it resembles elastin, but differs from 
 this substance in containing sulphur. This albumoid gives the color 
 reactions of the protein bodies. 
 
 Cartilage gelatin and the albumoid may be prepared according to 
 the folowing method of Morner: First remove the chondromucoid 
 and chondroitin-sulphuric acid by extraction with dilute caustic potash 
 (0.2-0.5 per cent), remove the alkali from the remaining cartilage by 
 water, and then boil with water in a Papin's digester. The collagen 
 passes into solution as gelatin, while the albumoid remains undissolved 
 (contaminated by the cartilage-cells). The gelatin may be purified by 
 precipitating with sodium sulphate, which must be added to saturation 
 in the faintly acidified solution, redissolving the precipitate in water, 
 dialyzing well, and precipitating with alcohol. 
 
 In Morner's experience no albumoid is found in young cartilage, 
 but only the three first-mentioned constituents. Nevertheless, the young 
 cartilage contains about the same amounts of nitrogen and mineral 
 substances as the old. The cartilage of the ray (Raja batis Lin.), which 
 has been investigated by Lonnberg, 1 contains no albumoid and only 
 a little chondromucoid, but a large proportion of chondroitin-sulphuric 
 acid and collagen. 
 
 According to Pfluger and Handel, 2 glycogen occurs to a slight 
 
 1 Maly's Jahresber., 19, 325. 
 
 1 Pfluger in Pfliiger's Arch., 92; Handel, ibid. 
 
550 TISSUES OF THE CONNECTIVE SUBSTANCE. 
 
 extent in all matrices, and of these it is richest in the cartilage. Ten- 
 dons, ligamentum nucha?, and cartilage of the ox contained 0.06, 0.07, 
 and 2.17 p. m. glycogen respectively (Handel). 
 
 Hoppe-Seyler found in fresh human rib-cartilage 676.7 p. m. water, 
 301.3 p. m. organic, and 22 p. m. inorganic substance, and in the cartilage 
 of the knee-joint 735.9 p. m. water, 248.7 p. m. organic, and 15.4 p. m. 
 inorganic substance. Pickardt found 402-574 p. m. water and 72.86 
 p. m. ash (no iron) in the laryngeal cartilage of oxen. The ash of car- 
 tilage contains considerable amounts (even 800 p. m.) of alkali sulphate, 
 which probably does not exist originally as such, but is produced in great 
 part by the incineration of the chondroitin-sulphuric acid and the chon- 
 dromucoid. The analyses of the ash of cartilage therefore cannot 
 give a correct idea of the quantity of mineral bodies existing in this sub- 
 stance. The cartilage is richest in sodium of all the tissues of the body, 
 and according to Bunge 1 the amount of Na and CI is greatest in young 
 animals. In 1000 parts of cartilage dried at 120° C, Bunge found 91.26 
 parts Na^O in the shark, 33.98 in the ox embryo, 32.45 in a fourteen-day- 
 old calf, and 26.4 in a ten-weeks-old calf. 
 
 Ochronose is the brown to black coloration of the cartilage which 
 sometimes occurs, and which has also been observed in several cases of 
 alcaptonuria (see Chapter XIV) or after lengthy treatment with carbolic 
 acid bandages (Poulsen, Adler 2 ). The nature of these melanine- 
 like pigments is unknown. 
 
 The Cornea. The corneal tissue, which, in a chemical sense, is con- 
 sidered by many investigators to be related to cartilage, contains traces 
 of proteid and a collagen as chief constituent, which C. Morner 3 claims 
 contains 16.95 per cent N. According to him it also contains a mucoid 
 which has the composition C 50.16, H 6.97, N 12.79, and S 2.07 per 
 cent. On boiling with dilute mineral acid this mucoid yields a reducing 
 substance. The globulins found by other investigators in the cornea 
 are not derived from the matrix, according to Morner, but from the 
 layer of epithelium. Morner believes that Descemet's membrane 
 consists of membranin (page 171), which contains 14.77 per cent N and 
 0.90 per cent S. 
 
 In the cornea of oxen His 4 found 758.3 p. m. water, 203.8 p. m. 
 gelatin-forming substance, 28.4 p. m. other organic substance, besides 
 8.1 p. m. soluble and 1.1 p. m. insoluble salts. 
 
 1 Hoppe-Seyler, cited from Kiihne's Lehrbuch d. physiol. Chem., 387; Pickardt,, 
 Centralbl. f. Physiol., 6, 735; Bunge, Zeitschr. f. physiol. Chem., 28. 
 
 2 See Maly's Jahresb., 40, 424, Adler, Zeitschr. f. Krebsforschung, 11. 
 ' Zeitschr. f. physiol. Chem., 18. 
 
 4 Cited from Camgee, Physiol. Chem., 1880, 451. 
 
BONE. 551 
 
 IU. BONE. 
 
 The bony structure proper, when free from other format inns occurring 
 in bones, such a- marrow, nerves, and blood-vessels, consists of cella and 
 
 a matrix. 
 
 The alls have not been closely studied in regard to their chemical 
 constitution. On boiling with water they yield no gelatin. They 
 contain no keratin, which usually should not be present in the bony 
 structure (Herbert Smith *). 
 
 The matrix of the bony structure contains two chief constituents, 
 namely, an organic substance, and the so-called bone-earths, lime-salts, 
 inclosed in or combined with it. If bones are treated with dilute hydro- 
 chloric acid at the ordinary temperature, the lime-salts are dissolved 
 and the organic substance remains as an elastic mass, preserving the 
 shape of the bone. 
 
 The organic matrix consists chiefly of ossein, which is generally 
 considered as identical with the collagen of the connective tissue. It 
 also contains, as Hawk and Gies 2 have shown, mucoid and albuminoid. 
 After the removal of the lime-salts by hydrochloric acid of 2-5 p. m. 
 these experimenters were able to extract the mucoid by one-half sat- 
 urated lime-water, and to precipitate it with 2 p. m. hydrochloric acid. 
 After the removal of the osseomucoid and collagen (by boiling with 
 water) they obtained the albuminoid as an insoluble residue. 
 
 The osseomucoid on boiling w r ith hydrochloric acid yielded a reduc- 
 ing substance and sulphuric acid; 1.11 per cent sulphur appearing in 
 this form. The osseomucoid stands close to the chondro- and tendon 
 mucoid in elementary composition, as may be seen from the follow- 
 ing analyses: 
 
 c h n s o 
 
 Osseomucoid 47.43 6.63 12.22 2.32 31 .40 (Hawk and Gies) 
 
 Chondromucoid. . .. 47.30 6.42 12.58 2.42 31.28 (C. Morner) 
 
 Tendon mucoid ... . 48.76 6.53 11.75 2.33 30.60 (Chittenden and Gies) 
 
 Corneal mucoid.. .. 50.16 6.97 12.79 2.07 28.01 (C. Morner) 
 
 The osseoalbuminoid is insoluble in 2 p. m. hydrochloric acid, and 
 in 5 p. m. Xa2CC>3, but dissolves in 10 per cent KOH with the formation 
 of albuminates. The composition of chondro- and osseoalbuminoid 
 is as follows: 
 
 c 
 
 Osseoalbuminoid 50 16 
 
 Chondroalbuminoid . . . 50 46 
 
 1 Zeitschr. f. Biologie, 19. 
 
 s Amer. Journ. of Physiol., 5 and 
 
 H 
 
 N 
 
 
 s 
 
 
 
 
 7.03 
 
 16. 
 
 17 
 
 1.18 
 
 25 
 
 46\ 
 
 Hawk and 
 
 7.05 
 
 14. 
 
 95 
 
 1.86 
 
 25 
 
 .68J 
 
 Gies 
 
552 TISSUES OF THE CONNECTIVE SUBSTANCE. 
 
 The inorganic constituents of the bony structure, the so-called 
 bone-earths, which, after the complete calcination of the organic sub- 
 stance, remain as a white brittle mass, consist chiefly of calcium and 
 phosphoric acid, but also contain carbonic acid and, in smaller amounts, 
 magnesium, chlorine, and fluorine. Iron, which has been found in bone- 
 ash, does not seem to belong exactly to the bony substance, but to the 
 nutritive fluids or to the other constituents of bones. The traces of 
 sulphate occurring in the bone-ash are derived, according to Morner, 
 from the chondroitin-sulphuric acid. According to Gabriel, potassium 
 and sodium are essential constituents of bone-earth, and this has been 
 substantiated by Aron l . 
 
 The opinions of investigators differ slightly as to the manner in 
 which the mineral bodies of the bony structure are combined with 
 each other. Chlorine is present in the same form as 7m apatite 
 3(Ca3P208)CaCl2- If we eliminate the magnesium, the chlorine, and 
 the fluorine, the last, Gabriel claims, occurring only as traces, the remain- 
 ing mineral bodies form the combination 3(Ca3P20s)CaC03. In his 
 opinion the simplest expression for the composition of the ash of bones 
 and teeth is (Ca 3 (P04)2+Ca5HP 3 0,3+Aq), in which 2-3 per cent of the 
 lime is replaced by magnesia, potash, and soda, and 4-6 per cent of the 
 phosphoric acid by carbonic acid, chlorine, and fluorine. Recently, on 
 the contrary, Gassmann has given important reasons for the follow- 
 ing complex combination in Werner's 2 sense. 
 
 /OP0 3 Ca\ 
 Cf( > Ca 3 
 
 . VoPOsCa/ - 
 
 C0 3 
 
 Analyses of bone-earths have shown that the mineral constituents 
 exist in rather constant proportions, which are nearly the same in dif- 
 ferent animals. As an example of the composition of bone-earth we here 
 give the analyses of Zalesky. 3 The figures represent parts per thousand: 
 
 Man. Ox. Tortoise. Guinea-pig. 
 
 Calcium phosphate, Ca 3 P 2 8 838 . 9 860 . 9 859 . 8 873 . 8 
 
 Magnesium phosphate, MgJPaOs 10.4 10.2 13.6 10.5 
 
 Calcium combined with C0 2 , Fl, and CI. . . 76 . 5 73 . 6 63 . 2 70 . 3 
 
 C0 2 57.3 62.0 52.7 
 
 Chlorine 1.8 2.0 1.3 
 
 Fluorine* 2.3 3.0 2.0 
 
 1 Morner, Zeitschr. f. physiol. Chem., 23; Gabriel, ibid., 18, which. also contains 
 the pertinent literature; Aron, Pfliiger's Arch., 106. 
 
 2 Gassmann, Zeitschr. f. physiol. Chem. 70 and 83; Werner, Ber. d. d. Chem. 
 Gesellsch., 40. 
 
 1 Hoppe-Seyler, Med.-chem. Untersuch., p. 19. 
 
 4 The reports as to the quantity of fluorine disagree; see Harms, Zeistchr. f. 
 Biologic, 38; Jodlbauer, ibid., 41. 
 
Ox. 
 
 Elephant. 
 
 Femur 
 
 Femur. 
 
 857.2 
 
 900.3 
 
 15.3 
 
 19.6 
 
 4.5 
 
 4.7 
 
 3.0 
 
 2.0 
 
 119.6 
 
 72.7 
 
 BONK. 553 
 
 Some of the ( '( )•_> is always lost on calcining, so that the bone-ash 
 does not contain the entire CO2 of the bony substance. 
 
 Gautier and Clausmann 1 have determined the fluorine in various 
 organs and tissues. In man the diaphysis end of the femur had 0.495 
 p. m. fluorine, and the epiphysis end 0.119 p. m. fluorine. In children 
 the diaphysis end of the long bones contained 0.156 p. m. fluorine 
 and the epiphysis end 0.037 p. m. A similar difference also occurs in 
 animals. Cartilage of man with 0.014 p. in. fluorine and tendons (calf) 
 with 0.0035 p. m. fluorine, are much poorer in fluorine than the bones. 
 The dentin (dog) contains 0.56 p. m. fluorine and the enamel (of a 
 young dog) contained i.66. p. m. fluorine, all results obtained from the 
 fresh substance. 
 
 Ad. Carnot 2 found the following composition for the bone-ash of 
 man, ox, and elephant: 
 
 Man. 
 Femur Femur 
 
 (body). (head). 
 
 Calcium phosphate 874 . 5 878 . 7 
 
 Magnesium phosphate 15.7 17.5 
 
 Calcium fluoride 3.5 3.7 
 
 Calcium chloride 2.3 3.0 
 
 Calcium carbonate 101.8 92.3 
 
 Iron oxide 1.0 1.3 1.3 1.5 
 
 The quantity of organic substance in the bones, calculated from the 
 loss of weight in burning, varies between 300 and 520 p. m. This 
 variation may in part be explained by the difficulty in obtaining the 
 bony substance entirely free from water, and partly by the very variable 
 amount of blood-vessels, nerves, marrow, and the like in different bones. 
 The unequal amounts of organic substance found in the compact and 
 in the spongy parts of the same bone, as well as in bones at different 
 periods of development in the same animal, probably depend upon 
 the varying quantities of these above-mentioned tissues. Dentin, which 
 is comparatively pure bony structure, contains only 260-280 p. m. 
 organic substance, and Hoppe-Seyler 3 therefore thinks it probable 
 that perfectly pure bony substance has a constant composition and 
 contains only about 250 p. m. organic substance. The question whether 
 these substances are chemically combined with the bone-earths or only 
 intimately mixed has not been decided. 
 
 The nutritive fluids which circulate through the bones have not been isolated 
 and we only know that they contain some protein and some NaCl and alkali 
 sulphate. 
 
 1 Compt. Rend., 156. 
 
 i Ibid., 114. 
 
 » Physiol. Chem., 102-104 
 
554 TISSUES OF THE CONNECTIVE SUBSTANCE. 
 
 Bone Marrow. We differentiate between the red and yellow mar- 
 row, to which also belongs the gelatinous marrow, poor in fat, found in 
 fat atrophy and in old age. The difference between the first two-men- 
 tioned kinds of marrow lies, essentially, in the fact that the red marrow 
 contains a greater quantity of erythrocytes besides a higher content of 
 protein and less fat. The fat of the yellow marrow is, according to 
 X erring, 1 richer in oleic acid and poorer in solid fats than the fat of the 
 red marrow. Besides the fat, lecithin also occurs in the bone-marrow 
 and this varies in amount in different animals and at various ages, as 
 mentioned on page 244. The protein consists of a globulin coagulating 
 at 47-50° C. (Forrest) and a nucleoprotein with 1.6 per cent phos- 
 phorus (Halliburton 2 ) besides fibrinogen (P. Muller 3 ), traces of 
 albumin and proteose. In the extractives are found lactic acid, inosite, 
 hypoxanthine, cholesterine and bodies of an unknown kind. The quan- 
 titative composition of both kinds of marrow varies considerably with 
 the fat content, and the reports of the different investigators are corre- 
 spondingly discrepant (Xerking, Hutchinson and Macleod 4 ) . 
 
 The diverse quantitative composition of the various bones of the 
 skeleton depends probably on the varying quantities of other tissues, 
 such as marrow, blood-vessels, etc., which they contain. The same 
 reason explains, to all appearances, the larger quantity of organic 
 substance in the spongy part of the bones as compared with the more 
 compact parts. Schrodt 5 has made comparative analyses of different 
 parts of the skeleton of the same animal (dog) and has found an essen- 
 tial difference. The quantity of water in the fresh bones varies between 
 138 and 443 p. m. The bones of the extremities and the skull contain 
 138-222, the vertebrae 168-443, and the ribs 324-356 p. m. water. The 
 quantity of fat varies between 13 and 269 p. m. The largest amount 
 of fat, 256-269 p. m., is found in the long tubular bones, while only 
 13-175 p. m. fat is found in the small short bones. The quantity of 
 organic substance, calculated from fresh bones, was 150-300 p. m., and 
 the quantity of mineral substances 290-563 p. m. Contrary to the 
 general supposition the greatest amount of bone-earths was not found 
 in the femur, but in the first three cervical vertebrae. In birds the 
 tubular bones are richer in mineral substances than the flat bones 
 (During), and the greatest quantity of mineral bodies has been found 
 in the humerus (Hiller, During 6 ). 
 
 1 Bioch. Zeitschr., 10. 
 
 2 Forrest, Journ. of Physiol., 17; Halliburton, ibid., 18. 
 'See footnote 1, p. 253. 
 
 4 Nerking, 1. c; Hutchinflon and Macleod, Journ. of Anat. and Physiol., 36. 
 
 'Cited from Maly's Jahresber., 6. 
 
 6 Hiller, eited from Maly's Jahresber., 14; During, Zeitschr. f. physiol. Chem., 23. 
 
DISEASES OF THE BONES. 555 
 
 We do not possess trustworthy information in regard to the compo- 
 sition of bones at different ages. The analyses by E. Voit of bones of 
 dogs, and by Brubacher of bones of children, apparently indicate that 
 the skeleton becomes poorer in water and richer in ash with increase 
 in age. Graffenberger 1 has found in rabbits, 6£-7£ years old, that 
 the bones contained only 140-170 p. m. water, while the bones of the 
 full-grown rabbit 2-4 years old contained 200-240 p. m. The bones of 
 old rabbits contain more carbon dioxide and less calcium phosphate. 
 
 The composition of bones of animals of different species is but little known. 
 The bones of birds contain, as a rule, somewhat more water than those of mam- 
 malia, and the bones of fishes contain the largest quantity of water. The bones 
 of fishes and amphibians contain a greater amount of organic substance. The 
 bones of pachyderms and cetaceans contain a large proportion of calcium carbo- 
 nate; those of granivorous birds always contain silicic acid. The bone-ash of 
 amphibians and fishes contains sodium sulphate. The bones of fishes seem to 
 contain more soluble salts than the bones of other animals. 
 
 A great many experiments have been made to determine the exchange 
 of material in the bones — for instance, w r ith food rich in l'me and with 
 food deficient in lime — but the results have always been doubtful or 
 contradictory. The attempts to substitute other alkaline earths or 
 alumina for the lime of the bones have also given conflicting results. 2 
 On feeding sufficient calcium and phosphorus in the food Aron 3 found, 
 by strongly reducing the sodium and at the same time giving a large 
 amount of potassium, that the development of the bones was below 
 normal. On the administration of madder, the bones of the animal are 
 found to be colored red after a few days or weeks; but these experiments 
 have not led to any positive conclusion in regard to the growth or 
 metabolism in the bones. 
 
 Under pathological conditions, as in rachitis and softening of the 
 bones, an ossein has been found which does not give any typical gelatin 
 on boiling with water. This finding is still uncertain as otherwise path- 
 ological conditions seem to affect chiefly the quantitative composition 
 of the bones, and especially the relation between the organic and the inor- 
 ganic substance. In rachitis the bones are poorer in solids and these 
 are poorer in mineral substances than under normal conditions. 
 Attempts have been made to produce rachitis in animals by the use of 
 food deficient in lime. From experiments on fully developed animals 
 opposing results have been obtained. In young, undeveloped animals 
 
 1 Voit, Zeitschr. f. Biologie, 16; Brubacher, ibid., 27; Graffenberger in Maly's 
 Jahresber., 21. 
 
 2 See H. Weiske, Zeitschr. f. Biologie, 31, and W. Stoeltzner, Pfliiger'a Arch., 122, 
 and H. Stoeltzner, Bioch. Zeitschr., 12. 
 
 1 Pfluger'a Arch., 106. 
 
556 TISSUES OF THE CONNECTIVE SUBSTANCE. 
 
 Erwin Voit, Aron and Sebatjer and others x produced, by lack of 
 lime-salts, a change similar to rachitis. In full-grown animals the 
 bones were changed after a long time because of the lack of lime-salts 
 in the food, but did not become soft, only thinner (osteoporosis). The 
 attempts to remove the lime-salts from the bones by the addition of 
 lactic acid to the food have led to no positive results (Heitzmann, 
 Heiss, Baginsky 2 ). Weiske, on the contrary, has shown, by admin- 
 istering dilute sulphuric acid or monosodium phosphate with the food 
 (presupposing that the food gave no alkaline ash) to sheep and rab- 
 bits, that the quantity of mineral bodies in the bones might be dimin- 
 ished. On feeding continuously for a long time with a food which yielded 
 an acid ash (cereal grains), Weiske observed a diminution in the min- 
 eral substances of the bones in full-grown herbivora. 3 A few investi- 
 gators are of the opinion that in rachitis, as in osteomalacia, in which 
 disease the calcium content of the bones is also diminished, a solution 
 of the lime-salts by means of lactic acid takes place. This was sug- 
 gested by the fact that O. Weber and C. Schmidt 4 found lactic acid, 
 in the cyst-like, altered bony substance in osteomalacia. 
 
 Well-known investigators have disputed the possibility of the lime- 
 salts being washed from the bones in osteomalacia by means of lactic 
 acid. They have given special prominence to the fact that the lime- 
 salts held in solution by the lactic acid must be deposited on neutraliza- 
 tion of the acid by the alkaline blood. This objection is not very impor- 
 tant, as the alkaline blood-serum has the property to a high degree of 
 holding earthy phosphates in solution, which fact has been recently 
 shown by Hofmeister. The investigations of Levy contradict the 
 claim as to the solution of the lime-salts by lactic acid in osteomalacia. 
 He found that the normal relation 6PC>4:10Ca is retained in all parts 
 of the bones in osteomalacia, which would not be the case if the bone- 
 earths were dissolved by an acid. The decrease in phosphate occurs in 
 the same quantitative relation as the carbonate, and according to Levy, 
 in osteomalacia the exhaustion of the bone takes place by a decalcifica- 
 tion in which one molecule of phosphate-carbonate after the other is 
 removed. This does not agree with the findings of MoCrudden 5 who 
 
 1 Zeitschr. f. Biologie, 16; Aron and Sebauer, Bioch. Zeitsohr., 8; A. Baginsky, 
 Arch. f. (Anat. u.) Physiol., 1881. 
 
 2 Heitzmann, Maly's Jahresber., 3, 229; Heiss, Zeitschr. f. Biolojrie, 12; Basrinsky;, 
 Virchow's Arch., 87. 
 
 3 See Maly's Jahresber., 22; also Weiske, Zeitschr. f. physiol. Chem., 20, and 
 Zeitschr. f. Biologie, 31. 
 
 * Cited from v. Corup-Besanez. Lehrb. de. physiol. Chem., 4. Aufl. 
 6 Hofmeister, Ergebn. d. Physiol. 10; Levy. Zeitschr. f. physiol. Chem. 19; McCrud- 
 den, Journ. of biol. Chem., 7. 
 
TOOTH-STRUCTURE. 557 
 
 found a changed relation between the Ca and phosphoric acid in osteo- 
 malacia. 
 
 Rachitic bones are always poorer in mineral substances than normal bones. 
 The relation between Ca,PO« and CO., was found by GASSMANN to be the same 
 as in normal bones while he found a pathological increase in the magnesium. The 
 organic substance was found in rachitis to be relatively as well as absolutely 
 increased, at least in certain cases (Gassmann). The statements differ in 
 regard to the water content. According to Brubacher this is larger while accord- 
 ing to Gassmann it is 10 p. m. smaller than in normal bones. In opposition to 
 rachitis, osteomalacia is often characterized by the considerable amount of fat 
 in the bones, 230-290 p. m., but as a rule the composition varies so much that 
 the analysts are of little value. In a case of osteomalacia, Chabrie l found a 
 larger quantity of magnesium than calcium in a bone. The ash contained 417 
 p. m. phosphoric acid, 222 p. m. lime, 269 p. m. magnesia, and 86 p. m. carbon 
 dioxide. McCrudden found more magnesium than calcium; other investigators 
 have on the contrary found more calcium than magnesium. 
 
 The tooth-structure is closely related, from a chemical standpoint, 
 to the bony structure. 
 
 Of the three chief constituents of the teeth — dentin, enamel, and 
 cement — the cement is to be considered as true bony structure, and as 
 such has already been discussed to some extent. Dentin has the same 
 composition as the bony structure, but contains somewhat less water. 
 The organic substance yields gelatin on boiling; but the dental tubes 
 are not dissolved, therefore they cannot consist of collagen. In dentin 
 2G0-280 p. m. organic substance has been found. Enamel is an epithe- 
 lium formation containing a large proportion of lime-salts. Correspond- 
 ing to its character and origin, the organic substance of the enamel 
 does not yield any gelatin. Completely developed enamel contains 
 the least water, the greatest quantity of mineral substances, and is the 
 hardest of all the tissues of the body. In full-grown animals it con- 
 tains hardly any water, and the quantity of organic substance amounts 
 to only 20-40-68 p. m. The relative amounts of calcium and phosphoric 
 acid are shown by the analyses of Hoppe-Seyler to be about the same 
 as in bone-earths. The quantity of chlorine according to him is remark- 
 ably high, 0.3-0.5 per cent, while Bertz 2 found that the ash of enamel 
 was free from chlorine and that dentin was very poor in chlorine. 
 
 Carnot, 3 who has investigated the dentin from elephants, has found 4.3 p. m. 
 calcium fluoride in the ash. In ivory he found only 2 p. m. Dentin from 
 elephants is rich in magnesium phosphate, which is still more abundant in ivory. 
 
 1 Gassmann, Zeitschr. f. physiol. Chem. 70; Brubacher, Zeitschr. f. biol. 27. See 
 also Cappezzuoli, Bioch. Zeitschr. 16; Chabrie, Les phenomenee chim. de 1' ossification, 
 Paris, 1895, 65. 
 
 2 See Maly's Jahresber., 30. 
 3 Compt. Rend., 114. 
 
558 TISSUES OF THE CONNECTIVE SUBSTANCE. 
 
 Gabriel found that the quantity of fluorine is very small and 
 amounts to 1 p. m. in ox-teeth. It is no greater in the teeth and enamel 
 than in the bones. 1 The same investigator found that the amount of 
 phosphates is strikingly small in the enamel, and in the teeth consider- 
 able lime is replaced by magnesia. This coincides with Bertz's find- 
 ings, that dentin contains twice as much magnesia as the enamel. 
 
 According to Gassmann, 2 the teeth among themselves have dif- 
 ferent composition, and in man the wisdom teeth are poorer in organic 
 substance and richer in lime than the canine teeth. The great tend- 
 ency of the first to caries is probably explained by this fact. The reason 
 for the degeneration of the teeth is considered by C. Rose 3 to be a lack 
 of earthy salts, and according to him one finds the best teeth in localities 
 where the drinking water has high permanent hardness. 
 
 IV. THE FATTY TISSUE. 
 
 The membranes of the fat-cells withstand the action of alcohol and 
 ether. They are not dissolved by acetic acid or by dilute mineral acids, 
 but are dissolved by artificial gastric juice. They may possibly con- 
 sist of a substance closely related to elastin. The fat-cells contain, 
 besides fat, a yellow pigment which in emaciation does not disappear 
 so rapidly as the fat; and this is the reason that the subcutaneous cel- 
 lular tissue of an emaciated corpse has a dark orange-red color. The 
 cells deficient in, or nearly free from fat, which remain after the complete 
 disappearance of the latter, seem to have an albuminous protoplasm 
 rich in water. Adipose tissue is rich in a fat-splitting enzyme and in 
 catalases. 
 
 The less water the fatty tissue contains the richer it is in fat. 
 Schulze and Reinecke 4 found in 1000 parts: 
 
 Water. Membrane. Fat. 
 
 Fatty tissue of oxen 99 . 7 16 . 6 883 . 7 
 
 Fatty tissue of sheep 104.8 16.4 878.8 
 
 Fatty tissue of pigs 64.4 13.6 922.0 
 
 The fat contained in the fat-cells consists mainly of triglycerides of 
 stearic, palmitic, and oleic acids. Besides these, especially in the less 
 solid kinds of fats, there are glycerides of other fatty acids (see Chapter 
 IV). In all animal fats there are besides these, as Fr. Hofmann 5 has 
 
 1 See footnote 4, p. 552. 
 
 2 Zeitschr. f. physiol. Chem., 55. 
 
 3 Deutech. Monatsh. f. Zahnheilk., 1908. 
 
 4 Annal. d. Chem. u. Pharm., 142. 
 
 6 Ludwig-Festsohrift, 1874, Leipzig. 
 
FATTY TISSUE. 559 
 
 shown, also free, non-volatile fatty acids, although in very small 
 amounts. 
 
 Human fat is relatively rich in olein, the quantity in the subcutaneous 
 fatty tissue being 70-80 per cent or more. 1 In new-born infants it is 
 poorer in oleic acid than in adults (Knopfelmacher, Siegert, Jaeckle) ; 
 the quantity of olein increases until the end of the first year, when it is 
 about the same as in adults. The composition of the fat in man as well 
 as in different individuals of the same species of animals is rather variable, 
 a fact which is probably dependent upon the food. According to the 
 researches of Henriques and Hansen the fat of the subcutaneous fatty 
 tissue is richer in olein than that of the internal organs; this has also been 
 observed by Leick and Winkler. 2 In animals with a thick subcutaneous 
 fat deposit the outer layers, according to Henriques and Hansen, are 
 richer in olein than the inner layers. The fat of cold-blooded animals 
 is especially rich in olein, The fat of domestic animals has, according 
 to Amthor and Zink, a less oily consistency and a lower iodine and 
 acetyl equivalent than the corresponding fat of wild animals. Under 
 pathological conditions the fat may have a markedly pronounced varia- 
 tion. The fat of lipoma seems, from Jaeckle's experience, to be poorer 
 in lecithin than other fats. 
 
 The fat stored up in the organs and tissues can be changed somewhat 
 by the composition of the fat of the food, still, according to Abderhalden 
 and Brahm, 3 the fat actually occurring in the cells (with the exception 
 of the real fat cells) is not dependent in its composition upon the kind 
 of food fat taken. 
 
 The properties of fats in general, and the three most important varieties 
 of fat in particular, have been considered in a previous chapter, hence 
 the formation of the adipose tissue is of chief interest at this time. 
 
 The formation of fat in the organism may occur in various ways. The 
 fat of the animal body may consist partly of fat absorbed from the food 
 and deposited in the tissues, and partly of fat formed in the organism 
 from other bodies, such as proteins (?) or carbohydrates. 
 
 That the fat from the food which is absorbed in the intestinal canal 
 may be retained by the tissues has been shown in several ways. Rad- 
 ziejewski, Lebedeff, and Munk have fed dogs with various fats, such 
 as linseed-oil, mutton-tallow, and rape-seed-oil, and have afterward 
 
 1 See Jaeckle, Zeitschr. f. physiol. Chem., 36 (literature). 
 
 2 Knopfelmacher, Jahrbuch f. Kinderheilkunde (X. F.), 45 (older literature); 
 Siegert, Hofmeister's Beitriige, 1; Jaeckle, Zeitschr. f. physiol. Chem., 36 (literature); 
 Henriques and Hansen, Skand. Arch. f. Physiol., 11; Leick and Winkler, Arch. f. Path, 
 u. Pharm., 48. 
 
 8 Zeitschr. f. physiol. Chem., 65. 
 
560 TISSUES OF THE CONNECTIVE SUBSTANCE. 
 
 found the administered fat in the tissues. Hofmann starved dogs 
 until they appeared to have lost their fat, and then fed them upon large 
 quantities of fat and only little proteins. When the animals were killed 
 he found so large a quantity of fat that it could not have been formed 
 from the administered proteins alone, but the greater part must have 
 been derived from the fat of the food. Pettenkofer and Voit arrived 
 at similar results in regard to the action of the absorbed fats in the organ- 
 ism, though their experiments were of another kind. Munk found 
 that on feeding with free fatty acids, these are deposited in the tissues, 
 not, however, as such; but they are transformed by synthesis with 
 glycerin into neutral fats on their passage from the intestine into the 
 thoracic duct. The connection between the fat of the food, and of the 
 body has also been shown by others, especially Rosenfeld. Coro- 
 nedi and Marchetti and in particular Winternitz 1 have shown that 
 iodized fat is taken up in the intestinal tract and deposited in the various 
 organs. 
 
 Proteins and carbohydrates are considered as the mother-substances 
 of the fats formed in the organism. 
 
 The formation of the so-called corpse-wax, adipocere, which consists 
 of a mixture of fatty acids, ammonia, and lime-soaps, from parts of the 
 corpse rich in proteins, is sometimes given as a proof of the formation 
 of fats from proteins. The accuracy of this view has, however, been dis- 
 puted, and many other explanations of the formation of this substance 
 have been offered. According to the experiments of Kratter and 
 K. B. Lehmann, it seems as if it were possible by experimental means 
 to convert animal tissue rich in proteins (muscles) into adipocere by the 
 continuous action of water. Irrespective of this, Salkowski has shown 
 that in the formation of adipocere, the fat itself takes part, in that the 
 olein decomposes with the formation of solid fatty acids, still it must 
 be considered that lower organisms undoubtedly take part in its forma- 
 tion. The production of adipocere as a proof of the formation of fat 
 from proteins is disputed by many investigators for this and other reasons. 
 
 Fatty degeneration has been considered as another proof of the 
 formation of fat from proteins. From the investigations of Bauer 
 on dogs, and Leo on frogs, it was assumed that, at least in acute poisoning 
 by phosphorus, a fatty degeneration, with the formation of fat from 
 proteins, takes place. Pfluger has raised such strong arguments against 
 the older researches as well as the more recent one of Polimanti, who 
 claims to have shown the formation of fat from proteins in phosphorus 
 
 1 Coronedi and Marchetti, cited by Winternitz, Zeitschr. f. physiol. Chem., 24, 
 A review of the literature on fat formation may be found in Rosenfeld, Fettbildung. 
 in Ergebnisse der Physiologie, 1, Abt. 1. 
 
FORMATION OF FATS. 561 
 
 poisoning, that we cannot consider the formation of fat as conclusively 
 proved. The investigations of Lebedeff, Athanasiu, Taylor, Schwalbe 
 and others, have shown that probably no new formation of fat from 
 protein took place, but rather a fat migration and that this is actually 
 the case has been especially shown by Rosenfeld and recently by Shi- 
 bata [ in a conclusive manner. 
 
 Another more direct proof of the formation of fat from proteins 
 has been given by Hofmann. He experimented with fly-maggots. 
 A number of these were killed and the quantity of fat determined. The 
 remainder were allowed to develop in blood whose proportion of fat 
 had been previously determined, and after a certain time they were killed 
 and analyzed. He found in them from seven to eleven times as much 
 fat as was contained in the maggots first analyzed and the blood taken 
 together. Pfluger 2 has made the objection that a considerable number 
 of lower fungi develop in the blood under these conditions, in whose 
 cell-body fats and carbohydrates are formed from the different con- 
 stituents of the blood and their decomposition products, and that these 
 serve as food for the maggots. 
 
 Weinland 3 has observed the formation of higher non-volatile fatty 
 acids in the Calliphora larvae when they were rubbed to a homogeneous 
 paste after the addition of Witte's peptone. This experiment shows a 
 formation of fat from protein, but cannot be considered as quite con- 
 clusive. 
 
 As a more convincing proof of fat formation from proteins, the 
 investigations of Pettenkofer and Voit are often quoted. These 
 investigators fed dogs with large quantities of meat containing the least 
 possible proportion of fat, and found all of the nitrogen in the excreta, 
 but only a part of the carbon. As an explanation of these conditions 
 it has been assumed that the protein of the organisms splits into a 
 nitrogenized and a non-nitrogenized part, the former changing into the 
 nitrogenized final product, urea, and like products, and the other part, 
 on the contrary, being retained in the organism as fat (Pettenkofer 
 and Voit). 
 
 Pfluger has arrived at the following conclusion by an exhaustive 
 criticism of Pettenkofer and Voit's experiments and a careful recal- 
 culation of their balance-sheet; that these very meritorious invest iga- 
 
 1 Bauer, Zeitschr. f. Biologie, 7; Leo, Zeitschr. f. physiol. Chera., 9; Polimanti, 
 Pfliiger's Arch., 70; Pfluger, ibid., 51 (literature on the formation of fat from protein) 
 and 71; Athanasiu, ibid., 74; Taylor, Journ. Exp. Medicine, 4; see also footnote 2, 
 p. 384; Shibata,Bioch. Zeitschr., 37, which contains the literature; Rosenfeld, Ergebn. 
 d. Physiol., 1. 
 
 2 See Rosenfeld, Fettbildung, Ergebnisse der Physiologie, 1, Abt. 1. 
 
 3 Zeitschr. f. Biol., 51 and 52. 
 
562 TISSUES OF THE CONNECTIVE SUBSTANCE. 
 
 tions, which were continued for a series of years, were subject to such 
 great defects that they are not conclusive as to the formation of fat 
 from proteins. He especially emphasizes the fact that these investigators 
 started from a wrong assumption as to the elementary composition of 
 the meat, and that the quantity of nitrogen assumed by them was too 
 low and the quantity of carbon too high. The relation of nitrogen to 
 carbon in meat poor in fat was assumed by Voit to be as 1:3.68, while 
 according to Pfluger it is 1:3.22 for fat-free meat after deducting the 
 glycogen, and according to Rubner 1:3.28 without deducting the gly- 
 cogen. On recalculation of the figures, using these coefficients, Pfluger 
 has arrived at the conclusion that the assumption as to the formation 
 of fat from proteins finds no support in these experiments. 
 
 In opposition to these objections, E. Voit and M. Cremer have made 
 new feeding experiments, to show the formation of fat from proteins, 
 but the proof of these recent investigations has been disputed by Pfluger. 
 On feeding a dog on meat poor in fat (containing a known quantity of 
 ether extractives, glycogen, nitrogen, water, and ash), Kumagawa 1 
 could not prove the formation of fat from protein. According to him 
 the animal body under normal conditions has not the power of forming 
 fat from protein. 
 
 Several French investigators, especially Chauveau, Gautier, and 
 Kaufmann, 2 consider the formation of fat from proteins as positively 
 proved. Kaufmann has recently substantiated this view by a method 
 which will be spoken of in detail in Chapter XVII, in which he studied 
 the nitrogen elimination and the respiratory gas exchange in conjunction 
 with the simultaneous formation of heat. 
 
 As we are agreed that carbohydrates and glycogen, as well as sugar, 
 can be formed from proteins, the fact cannot be denied that possibly 
 an indirect formation of fat from proteins, with a carbohydrate as an 
 intermediate step, can take place. The possibility of a direct fat for- 
 mation from proteins without the carbohydrate as intermediary must 
 also be generally admitted, although such a formation has not been 
 conclusively proved. 
 
 According to Chauveau and Kaufmann, in the direct formation of 
 fat from proteins, the fat is formed besides urea, carbon dioxide, and 
 water, as an intermediary product in the oxidation of the proteins, while 
 Gautier considers the formation of fat from proteins as a cleavage 
 without the taking up of oxygen. If fat is formed from protein in the 
 animal body, then such formation is not a splitting off of fat from the 
 
 <■ Rosenfeld, Fettbildung, Ergebnisse der Physiologre, 1, Abt. 1. 
 2 Kaufniann, Arch, de physiol., ('>) 8, where the works of Chauveau and Gautier 
 are cited. 
 
FORMATION OF FATS. 503 
 
 proteins, but rather a synthesis from primarily formed cleavage products 
 
 of proteins which are poor in carbon. 
 
 The formation "/ foi from carbohydrates in the animal body' was 
 first suggested by Liebig. This was opposed for some time, and until 
 lately it was the general opinion that a direct formation of fat from 
 carbohydrates not only had not been proved, but also that it was 
 improbable. The undoubtedly great influence of the carbohydrates on 
 the formation of fat as observed and proved by Liebig was explained 
 by the statement, that the carbohydrates were consumed instead of 
 the absorbed fat or that derived from the proteins, hence they have a 
 sparing action on the fat. By means of a series of nutrition experiments l 
 with different animals, with foods especially rich in carbohydrates it has 
 been apparently proved that a direct formation of fat from carbohydrates 
 does actually occur. The processes by which this formation takes place 
 are still unknown. As the carbohydrates do not contain such com- 
 plicated carbon chains as the fats, the formation of fat from carbohydrates 
 must consist of a synthesis, in which the group CHOH is converted into 
 CH2; hence a reduction must occur. 
 
 After feeding with very large quantities of carbohydrates the relation between 
 the inspired oxygen and the expired carbon dioxide, i.e., the respiratory quotient 
 
 —p, was found greater than 1 in certain cases (H.\xRiOTand Richet, Bleibtreu, 
 
 Kaufmaxx. Laclaxie -). This is explained by the assumption that the fat 
 is formed from the carbohydrate by a cleavage setting free carbon dioxide and water 
 without taking up oxygen. This increase in the respiratory quotient also depends 
 in part on the increased combustion of the carbohydrate. 
 
 When food contains an excess of fat, the superfluous amount is stored 
 up in the fatty tissue, and on partaking of food deficient in fat this 
 accumulation is quickly exhausted; and it is very probable that the 
 lipase is of importance here, as Loevenhart 3 has found that all over 
 the body where fat is deposited in large amounts lipase also occurs in 
 considerable amounts. There is perhaps not one of the various tissues 
 that decreases so much in starvation as the fatty tissue. The organism, 
 then, possesses in this tissue a depot where there is stored, during proper 
 
 1 Lawes and Gilbert, Phil. Transactions, 1859, part 2; Soxhlet, see Mary's Jahresber., 
 11, 51; Tscherwinsky, Landwirthsch. Versuchsstaat, 29 (cited from Maly's Jahresber., 
 13); Meissl and Stromer, Wien. Sitzungsber., 88, Abt. 3; Schultze, Maly's 
 Jahresber., 11, 47; Chaniewski, Zeitschr. f. Biologie, 20; Voit and Lehmann, see C. 
 v. Voit, Sitzungsber, d. k. bayer. Akad. d. Wissensch., 1885; I. Munk, Virchow's Arch., 
 101; Rubner, Zeitschr. f. Biologie, 22; Lummert, Pfliiger's Arch., 71. 
 
 2 Hanriot and Richet, Annal. de Chim. et de Phys. (6), 22; Bleibtreu, Pfliiger's 
 Arch., 56 and So; Kaufmann, Arch, de Physiol. (5), 8; Laulanic, ibid., 791. 
 
 3 Amer. Journ. of Physiol., 6. 
 
564 TISSUES OF THE CONNECTIVE SUBSTANCE. 
 
 alimentation, a nutritive substance of great importance in the develop- 
 ment of heat and vital force, which substance, on insufficient nutrition, 
 is given up as may be needed. On account of their low conducting 
 power, the fatty tissues become of great importance in regulating the 
 loss of heat from the body. They also serve to fill cavities and act as 
 a protection and support to certain internal organs. 
 
CHAPTER X. 
 MUSCLES. 
 
 STRIATED MUSCLES. 
 
 In the study of the muscles the chief problem for physiological chem- 
 istry is to isolate their different morphological elements and to investigate 
 each element separately. By reason of the complicated structure of 
 the muscles this has been thus far almost impossible, and we must be 
 satisfied at the present time with a few microchemical reactions in the 
 investigation of the chemical composition of the muscular fibers. 
 
 Each muscle-tube or each muscle-fiber consists of a sheath, the 
 sarcolemma, which seems to be composed of a substance similar to 
 elastin, and containing a large proportion of protein. This last, which 
 in life possesses the power of contractility, has in the inactive muscle 
 an alkaline reaction, or, more correctly speaking, an amphoteric reac- 
 tion with a predominating action on red litmus paper. Rohmann 
 found that the fresh, inactive muscle shows an alkaline reaction with 
 red lacmoid, and an acid reaction with brown turmeric. From the effect 
 of various acids and salts on these coloring-matters, he concludes that the 
 alkalinity of the fresh muscle with lacmoid is due to sodium bicarbonate, 
 diphosphate, and probably also to an alkaline combination of protein 
 bodies, and the acid reaction with turmeric, on the contrary, to chiefly 
 monophosphate. The dead muscle has an acid reaction, or, more cor- 
 rectly, the acidity with turmeric increases on the decease of the muscle, 
 and the alkalinity with lacmoid decreases. The difference depends on the 
 presence of a larger quantity of monophosphate in the dead muscle, and 
 according to Rohmann free lactic acid is found in neither the one case 
 nor the other. 1 
 
 If the somewhat disputed statements relative to the finer structure 
 of the muscles are disregarded, one can differentiate in the striated muscles 
 between the two chief components, the doubly refracting — anisotropous 
 — and the singly refracting — isotropous — substance. Both contain 
 abundance of protein, which form the chief part of the solids of the muscles. 
 
 1 The various reports in regard to the reaction of the muscles and the cause thereof 
 are conflicting. See Rohmann, Pfluger's Arch., 50 and 55; Heffter, Arch. f. exp. 
 Path. u. Pharm., 31 and 38. These references contain the pertinent literature. 
 
 065 
 
5€6 MUSCLES. 
 
 If the muscular fibers are treated with reagents which dissolve proteins, 
 such as dilute hydrochloric acid, soda solution, or gastric juice, they swell greatly 
 and break up into " Bowman's disks." By the action of alcohol, chromic acid, 
 boiling water, or in general such reagents as cause a shrinking, the fibers split 
 longitudinally into fibrils; and this behavior shows that several chemically dif- 
 ferent substances of various solubilities enter into the construction of the muscular 
 fibers. 
 
 The protein myosin is generally considered as the principal constituent of the 
 diagonal disks, while the isotropous substance contains the chief mass of the 
 other proteins of the muscles as well as the chief portion of the extractives. 
 According to the observations of Danilewsky, confirmed by J. Holmgren, 1 
 myosin may be completely extracted from the muscle without changing its struc- 
 ture, by means of a 5-per cent solution of ammonium chloride, which fact con- 
 flicts with the above view. Danilewsky claims that another protein-like sub- 
 stance, insoluble in ammonium chloride and only swelling up therein, enters essen- 
 tially into the structure of the muscles. The proteins, which form^the principal 
 part of the solids of the muscles, are of the greatest importance. 
 
 Proteins of the Muscles. 
 
 Like the blood which contains a fluid, the blood-plasma, which sponta- 
 neously coagulates, separating fibrin and yielding blood-serum, so also 
 the living muscle, at least of cold-blooded animals, contains, as first 
 shown by Kuhne, a spontaneously coagulating liquid, the muscle-plasma, 
 which coagulates quickly, separating a protein body, myosin, and yield- 
 ing also a serum. That liquid which is obtained by pressing the living 
 muicle is called muscle-plasma, while that obtained from the dead 
 muscle is called muscle-serum. These two fluids contain at least in part 
 different protein bodies. 
 
 Muscle-plasma was first prepared by Kuhne from frog-muscles, and later 
 by Halliburton, according to the same method, from the muscles of warm- 
 blooded animals, especially rabbits. The principle of this method is as follows: 
 The blood is removed from the muscles immediately after the death of the animal 
 by passing through them a strongly cooled common-salt solution of 5-6 p. m. 
 Then the muscles are quickly cut and immediately frozen thoroughly so that 
 they can be ground in this state to a fine mass — " muscle-snow." This pulp is 
 strongly pressed in the cold, and the liquid which exudes is called muscle-plasma. 
 According to v. Furth 2 this cooling or freezing is not necessary. It is sufficient 
 to extract the muscle free from blood, as above directed, with a 6 p. m. common 
 salt solution. 
 
 Muscle-plasma forms a yellow to brownish-colored fluid with an 
 alkaline reaction. It varies in different animals. Muscle-plasma from 
 the frog spontaneously coagulates, slowly, at a little above 0° C, but more 
 
 1 Danilewsky, Zeitschr. f. physiol. Chem., 7; J. Holmgren, Maly's Jahresber., 23. 
 
 2 See Kuhne, Untersuchungen iiber das Protoplasma, (Leipzig, 1864), 2; Hallibur- 
 ton, Journ. of Physiol., 8; v. Furth, Arch. f. exp. Path. u. Pharm., 36 and 37; Hof- 
 meister's Beftrage, 3, and Ergebnisse der Physiologie, 1, Abt. 1; Stewart and Soll- 
 mann, Journ. of Physiol., 24. 
 
• PROTEINS OF THE MUSCLES. 567 
 
 quickly at the temperature of the body. Muscle-plasma from mammals 
 coagulates slowly, according to vv. Furth, even at the temperature of 
 the room, though only slightly, and it can hardly be considered as a 
 process comparable with the coagulation of the blood. Indeed the ques- 
 tion may be asked whether a true muscle-plasma does exist in warm- 
 blooded animals, or whether the rluid obtained from such muscles 
 exactly represents the plasma of the living muscle. According to Kuhne 
 and v. Furth the reaction remains alkaline during coagulation, while 
 Halliburton, Stewart and Sollmann find that it becomes acid. 
 Earlier investigators held that the clot consists of a globulin called 
 myosin, while v. Furth claims that it consists of two coagulated pro- 
 teins, myosin-fibrin and myogen-fibrin. 
 
 The study of the proteins of the muscles, as well as their nomen- 
 clature, has changed markedly in the last few years, and it is questionable 
 whether an essential difference exists between the proteins of the muscle- 
 plasma and the muscle-serum of warm-blooded animals. Nevertheless 
 it is necessary to discuss separately the proteins of the dead muscle as 
 well as those of the muscle-plasma. 
 
 The proteins of the dead muscle are in part soluble in water or dilute 
 salt solutions, and in part are insoluble therein. Myosin and musculin 
 and also myoglobulin and myoalbumin, which exist to a very slight 
 extent and are perhaps only derived from the remaining lymph, belong 
 to the first group, and the stroma substances of the muscle-tubes belong 
 to the second group. 
 
 Myosin was first discovered by Kuhne, and constitutes the principal 
 mass of the soluble proteins of the dead muscle. It is generally considered 
 as the most essential coagulation product of muscle-plasma. The name 
 myosin, Kuhne also gives to the mother-substance of the plasma-clot, 
 and this mother-substance forms, according to certain investigators, 
 the principal mass of contractile protoplasm. The findings as to the oc- 
 currence cf myosin in other organs besides the muscles require further 
 confirmation. The quantity of myosin in the muscles of different animals 
 varies, according to Danilewsky, 1 between 30 and 110 p. m. 
 
 Myosin, as obtained from dead muscles, is a globulin whose elementary 
 composition, according to Chittenden and Cummins. 2 is, on an average, 
 the following: C 52.28, H 7.11, N 16.77, S 1.27, O 22.03 per cent. If 
 the myosin separates as fibers, or if a myosin solution with a minimum 
 quantity of alkali is allowed to evaporate to a gelatinous mass on a 
 microscope-slide, doubly refracting myosin may be obtained. Myosin 
 has the general properties of the globulins and is readily converted into 
 
 1 Zeitsohr. f. physiol. Chem., 7. 
 
 5 Studies from the Physiol. Chem. Laboratory of Yale College, New Haven, 3, 115. 
 
568 MUSCLES. 
 
 albuminates by dilute acids or alkalies. It is completely precipitated 
 upon saturation with NaCl, also by MgSO-i, in a solution containing 
 94 per cent of the salt with its water of crystallization (Halliburton). 
 The precipitated myosin readily becomes insoluble. Like fibrinogen it 
 coagulates at 56° C. in a solution containing common salt, but differs 
 irom it, since under no circumstances can it be converted into fibrin. 
 The coagulation temperature, according to Chittenden and Cummins, 
 not only varies for myosins of different origin, but also for the same 
 myosin in different salt solutions. 
 
 Myosin may be prepared in the following way, as suggested by Halli- 
 burton: The muscle is first extracted by a 5-per cent magensium- 
 sulphate solution, and by fractional precipitation with magnesium sul- 
 phate the musculin and then the myosin are precipitated (see Halli- 
 burton, 1. c). 
 
 The older and perhaps the usual method of preparation consists, 
 according to Danilewsky, 1 in extracting the muscle with a 5-10 per cent 
 ammonium-chloride solution, precipitating the myosin from the filtrate 
 by strongly diluting with water, and redissolving the precipitate in ammo- 
 nium-chloride solution, and the myosin obtained from this solution is 
 reprecipitated either by diluting with water or by removing the salt 
 by dialysis. 
 
 Musculin, 2 called paramyosinogen by Halliburton, and myosin 
 by v. Furth, is a globulin which is characterized by its low coagulation 
 temperature, in frogs below 40°, in mammalia 42-48°, and in birds about 
 51° C, and which may vary in different species of animals. It is more 
 easily precipitated than myosin by NaCl or MgSC>4 (50 per cent salt, 
 including water of crystallization). According to v. Furth it is precipi- 
 tated by ammonium sulphate with a concentration of 12-24 per cent 
 salt. If the dead muscle is extracted with water a part of the musculin 
 goes into solution, and may be precipitated therefrom by carefully 
 acidifying. It separates from a dilute salt solution on dialysis. Mus- 
 culin readily passes into an insoluble modification which v. Furth calls 
 myosin fibrin. Musculin is called myosin by v. Furth, as he considers 
 it nothing but myosin. As musculin has a lower coagulation temper- 
 ature and has other precipitating properties for neutral salts than the 
 older substance called myosin, it is difficult to accept this view. 
 
 Myoglobulin. After the separation of the musculin and the myosin from the 
 salt extract of the muscle by means of MgSO-i, the myoglobulin may be precipitated 
 
 1 Zeitschr. f. physiol. Chem., 5, 158. 
 
 2 As we have up to the present no conclusive basis for the identity of the globulins 
 railed myosin and paramyosinogen, and also as the use of the name myosin for the 
 last-mentioned substance may readily cause confusion, the author does not feel 
 kistified in dropping the old name musculin (Nasse). 
 
PROTEINS OF THE MUSCLES. 569 
 
 by saturating the filtrate with the salt. It is similar to serglobulin, but coagu- 
 lates at 63° C. (Halliburton). MyoaUmmin, or muscle-albumin, seems to 
 be identical with seralbumin (seralbumin a, according to Hallibi bton), and 
 
 probably originates only from the blood or the lymph. Proteoses and peptones 
 
 do not seem to exist in the fresh muscles. 
 
 Alter the complete removal from the muscle of all protein bodies which are 
 soluble in water and ammonium chloride, an insoluble protein remains which 
 only swells in ammonium-chloride solution, and which forms with the other insoluble 
 constituents of the muscular fiber the " muscle-stroma." According to Danilew- 
 sky the amount of such stroma substance is connected with the muscle activity. 
 He maintains that the muscles contain a greater amount of this substance, com- 
 pared with the myosin present, when the muscles are quickly contracted and 
 relaxed, the correctness of which report has recently been disputed by Saxl. 1 
 
 According to J. Holmgren, 2 this stroma substance does not belong to either 
 the Qucleoalbumin or the nucleoprotein group. It is not a glucoproteid, as it 
 does not yield a reducing substance when boiled with dilute mineral acids. It is 
 very similar to the coagulable proteins, and dissolves in dilute alkalies, forming 
 an albuminate. The elementary composition of this substance is almost the same 
 as that of myosin. There is no doubt that the insjluble substances, myofiorin 
 and myosin fibrin, which are formed, according to v. Fukth, in the coagulation of 
 the plasma, also occur among the stroma substances. When the muscles are 
 previously extracted with water, the stroma substances also contain a part of the 
 myosin hereby made insoluble. The observations of Saxl on rabbits' muscles 
 agree with this view that the fresh muscle after work contains 11.5-21.6 per cent 
 of the total protein in an insoluble form, while the muscle after rigor mortis con- 
 tains on the contrary 71.5-73.2 per cent. 
 
 To the proteins insoluble in water, and neutral salts, belongs the 
 nucleoprotein detected by Pekelharing, which occurs as traces and is 
 soluble in faintly alkaline water, and which probably originates from 
 the muscle nuclei. According to Bottazzi and Ducceschi 3 the heart 
 muscle is richer in nucleoprotein than the skeletal muscle. 
 
 Muscle-syntonin, which may be obtained by extracting the muscles with 
 hydrochloric acid of 1 p.m., and which, according to K. Morner, is less soluble 
 and has a greater aptitude to precipitate than other acid albumins, seems not 
 to occur preformed in the muscles. Heubner's 4 mytolin is modified muscle- 
 proteid, chiefly myosin, which has lost a part of its sulphur by the action of alkali. 
 
 Proteins of the Muscle-plasma. As above stated, myosin was ordi- 
 narily considered as the coagulated modification of a soluble protein 
 existing in the muscle-plasma. As in blood-plasma there is present 
 a mother-substance of fibrin, fibrinogen, so also there exists in the 
 muscle-plasma a mother-substance of myosin, a soluble myosin or a 
 myosinogen. This body has not thus far been isolated with certainty. 
 
 1 Hofmeister's Breitage, 9. 
 
 2 See footnote 1, p. 566. 
 
 3 Pekelharing, Zeitschr. f. physiol. Chem., 22; Bottazzi and Ducceschi, Centraibl. 
 f. Physiol., 12. 
 
 4 Arch. f. exp. Pathol, u. Pharm., 53. 
 
570 MUSCLES 
 
 Halliburton, who has detected in the muscles an enzyme-like substance, 
 11 myosin ferment," which is related to fibrin ferment but is not identical with it, 
 has also found that a solution of purified myosin, in dilute salt solution (5 per 
 cent MgS0 4 ), and sufficiently diluted with water, coagulates after a certain 
 time, and at the same time becomes acid, and a typical myosin-clot separates. 
 This coagulation, which is accelerated by warming or by the addition of myosin ' 
 ferment, is, according to Halliburton, a process analogous to the coagulation 
 of the muscle-plasma. According to this same investigator, myosin when dis- 
 solved in water by the aid of a neutral salt is reconverted into nvyosinogen, while 
 after diluting with water myosin is again produced from the nvyosinogen. The 
 musculin (paramyosinogen) is carried down, according to Halliburton, with the 
 myosin-clot, but has nothing to do with the coagulation, as the myosin-clot also 
 forms in the absence of musculin, and this last is not changed into myosin. 
 
 Besides the traces of globulin and albumin, which perhaps do not 
 belong to the muscle-plasma, there occur in mammals, according to 
 v. Furth, two proteins, namely, musculin (myosin according to v. Furth) 
 and myogen. 
 
 Musculin (Nasse) = paramyosinogen (Halliburton) = myosin (v. 
 Furth) forms about 20 per cent of the total proteins of the muscle- 
 plasma of rabbits. Its properties have already been given, and it is 
 sufficient to remark that its solutions beccme cloudy on standing, and 
 a precipitate of myosin fibrin occurs, which is insoluble in salt solutions. 
 
 Myogen, or myosinogen (Halliburton), forms the chief mass, 
 75-80 per cent, of the proteins of rabbit muscle-plasma. It does not 
 separate from its solutions on dialysis and is not a true globulin, but 
 a protein sui generis. It coagulates at 55-65° C. and is precipitated 
 in the presence of 26-40 per cent ammonium sulphate. Myogen solu- 
 tions are precipitated by acetic acid only in the presence of some salt. 
 It is converted into an albuminate by alkalies, this albuminate being 
 precipitable by ammonium chloride. Myogen passes spontaneously, 
 especially with higher temperatures as well as in the presence of salt, 
 into an insoluble modification, myogen fibrin. A protein, coagulating 
 at 30-40° C, soluble myogen fibrin, is produced as a soluble intermediate 
 step. This substance occurs to a considerable extent in native frog- 
 muscle plasma. It does not always occur in the muscle-plasma of 
 warm-blooded animals, and when it does it is present only to a slight 
 extent. It can be separated by precipitating with salt or by diffusion. 
 Halliburton's assumption as to the action of a special myosin ferment 
 has not sufficient basis, according to v. Furth, nor has the often-admitted 
 analogy with the coagulation of the blood. The difference between 
 the musculin and the myogen in their becoming insoluble is that the 
 musculin passes into myosin fibrin without any soluble intermediate 
 
 Step-. 
 
 Myogen may be prepared, according to v. Furth, by heating, for a 
 short time, the dialyzed and filtered plasma to 52° C., separating it in 
 
PROTEINS OF THE MUSCLES. 571 
 
 this way from the rest of the musculin. The myogen exists in the new 
 nitrate and can be precipitated by ammonium sulphate. The musculin 
 may also be removed by adding 28 per cent ammonium sulphate; and then 
 precipitating the myogen from the filtrate by saturating with the salt. 
 
 Stewart and Sollmann admit of only two soluble proteins in the muscles. 
 One is the paramyosinogen, which is the same as v. Furth's myosin+the soluble 
 myogen fibrin. The other they call myosinogen, which corresponds to v. Furth's 
 myogen or to Halliburton's myosinogen-)- my oglobulin. It is a typical globulin 
 which coagulates at 50-60° C. The paramyosinogen as well as the myosinogen 
 is readily converted into an insoluble modification, myosin. The myosin of the 
 above investigators is the same as v. Furth's myosin fibrin + myogen fibrin, and 
 corresponds, it seems, also to myosin mixed with paramyosinogen (Halliburton). 
 Stewart and Sollmann differ from Halliburton in considering that paramy- 
 osinogen also coagulates and is converted into myosin. According to them 
 myosin is also insoluble in a NaCl solution. 
 
 The views of the various investigators differ so essentially and the 
 nomenclature is so complicated (three different things are designated 
 by the name myosin) that it is extremely difficult to give any correct 
 review of the various opinions. 1 Thorough investigations on this subject 
 are very necessary. 
 
 Myoproteid is a protein found by v. Furth in the plasma from fish-muscles. 
 It does not coagulate on boiling, is precipitated by acetic acid, and is considered 
 as a compound protein by v. Furth. 
 
 In connection with v. Furth's work, Przibram has carried on ivestiga- 
 tions on the occurrence of muscle-proteins in various classes of animals. The 
 myosin (v. Furth) and myogen occur in all classes of vertebrates; the myogen 
 is always absent in the invertebrates. Myoproteid occurs, at least in considerable 
 quantity, only in fishes. In the muscle after cutting the nerve, Steyrer 2 found 
 somewhat more musculin and less myogen in the muscle-juice than in the normal 
 muscle. 
 
 Muscle-pigments. There is no question that the red color of the 
 muscles, even when completely freed from blood, depends in part on 
 haemoglobin. K. Morner has shown that muscle-haemoglobin is not 
 quite identical with blood-haemoglobin. The statement of MacMunn 
 that in the muscles another pigment occurs which is allied to haemo- 
 chromogen, and called myohcematifi by him, has not been substantiated, 
 at least for muscles of higher animals (Levy and Morner 3 ). MacMunn 
 claims that myohaematin occurs in the muscles of insects, which do not 
 contain any haemoglobin. The reddish-yellow coloring-matter of the 
 muscles of the salmon has been little studied. 
 
 1 For these reasons the author is not sure whether he has understood and correctly 
 given the work of the different investigators. 
 
 2 Przibram, Hofmeister's Beitrage, 2; Steyrer, ibid., 4. 
 
 'See MacMunn, Phil. Trans, of Roy. Soc, 177, part 1, Journ. of Physiol., 8 and 
 Zeitsehr. f. Physiol. Chem., 13; Levy, ibid., 13; K. Morner, Nord. Med. Archiv. Fest- 
 band., 1897, and Maly's Jahresber., 27. 
 
572 MUSCLES. 
 
 Various enzymes have been found in the muscles. To these belong 
 (besides traces of fibrin ferment and myosin ferment?) the catalases and 
 oxidases, which occur only to a slight extent and the glycolytic enzyme 
 (Chapter VII). An amylolytic and a proteolytic enzyme (Hedin and 
 Rowland x ) have also been found, and the hydrolytic and oxidizing 
 enzymes (Chapter XIV) active in the formation and destruction of uric 
 acid are also present. 
 
 Extractive Bodies of the Muscles. 
 
 The nitrogenous extractives in the muscles of higher animals con- 
 sist chiefly of creatine and creatinine (especially in fishes) and carnosine. 
 To these also belong inosinic acid (and the closely related carnine), phos- 
 phocarnic acid, carnitine and purine bases, especially hypoxanihine. The 
 purine bases occur partly free (which is especially the case with hypoxan- 
 thine) and partly combined. 
 
 Among the extractive substances is also found the acid noticed by Limpricht 
 in the flesh of certain cyprinidea, namely, the nitrogenized protic acid, while the 
 isocreatinine found by J. Thesen in fish-flesh is nothing but impure creatinine, 
 according to Poulsson, Schmidt and Korndorfer. 2 The following have also 
 been found in the muscles, in certain cases only, of a few varieties of animals: 
 uric acid (especially in alligators), taurine (in cephalopoda and oysters), glycocoll 
 (in gasteropoda), betaine and methyl guanidine, in fish meat, several monamino 
 acids and also the three hexone bases histidine, lysine and arginine. 3 Urea occurs 
 in large quantities in the muscle of the shark and ray. The reports are very 
 contradictory in regard to the occurrence of urea in the muscles of higher animals. 
 According to the investigations of Kaufmann and Schondorff, confirmed by 
 Brunton-Blaikie, 4 urea is a regular constituent of the muscles, although M. 
 Nencki and Kowarski dispute this. 
 
 In regard to the division of the nitrogenous extractives of the muscles, v. 
 Furth and Schwarz found the following in 1000 grams of the moist extremity 
 musculature of the horse and dog (after subtracting the proteoses derived by 
 secondary cleavage processes), 3.27-3.82 gram extractive nitrogen. Of this 
 4.5-7 per cent was ammonia, 6.1-11.1 per cent purine bodies, 26.5-37.1 per cent 
 creatine and creatinine, 30.3-36.3 per cent carnosine fraction, 8.2-15.3 per cent 
 base residue (carnitine, methylguanidine, etc.) and 6.3-16 per cent urea, poly- 
 peptides and amino-acids. The quantity of purine base nitrogen, according 
 to Burian and Hall in fresh meat of the horse, ox and calf, was 0.55 p. m., 0.63 
 
 1 Zeitschr. f. physiol. Chem., 32. 
 
 2 See Limpricht, Annal. d. Chem. u. Pharm., 127, and Thesen, Zeitschr. f. physiol. 
 Chem., 24; Poulsson, Arch. f. exp. Path. u. Pharm., 51; Schmidt and Korndorfer, 
 ibid., 51. 
 
 3 In regard to the extractives of the muscles see besides the specially cited works, 
 Kurkenberg and Wagner, Zeitschr. f. biol. 21; U. Suzuki and collaborators, Zeitschr. f. 
 physiol. Chen). 62 and Chem. Centralbl. 1913, 1; Suwa, Pfluger's Arch. 128 and 129; 
 Zunz, Centralbl. f. Physiol., 18. 
 
 * Kaufmann, Arch, de Physiol. (5), 6; Schondorff, Pfluger's Arch., 62; Nencki 
 and Kowarski., Arch. f. exp. Path. u. Pharm., 36; Brunton-Blaikie, Journ. of Physiol., 
 23, Supplement. 
 
CREATINE. 573 
 
 p. m. and 0.71 p. m. respectively, which corresponds closely to the results found 
 by Scaffidi, Buglia and Costantino for the striated muscle of the calf, namely, 
 ().">s Of is p. in. According to Hinaldi and Scaffidi ' the lowest values for the 
 purine nitrogen occur in the striated muscles of the covering of polype, 0.436 
 p. m., then in fishes 0.595-0.82 p. m. and the highest L.061 p. in. in birds. Bi QUA 
 and Costantino have determined the nitrogen titcatable with formol, and from 
 this determined the amount of monamino-acid nitrogen as well as diamino-acid 
 nitrogen in various animals. In oxen they found in the moist, striated muscle 
 0.18 p. in. inonamino- and 0.40 p. m. diamino-nitrogen. In the heart the cor- 
 responding figures were 0.18 and 0.18 p. m. In percentage of the total nitrogen 
 the total amino-acid nitrogen in the striated muscle was 1.70 per cent and in 
 the heart 1.48 per cent. 
 
 The most extensively occurring nitrogenous extractives in the muscle 
 are creatine and carnosine. 
 
 ( /NH 2 
 
 Creatine, C4H9N3O2, C=NH , or methyl-guanidine- 
 
 \N(CH 3 ).CH 2 COOH 
 acetic acid, occurs in the striated as well as smooth muscles. In the 
 striated muscle of vertebrates the amount varies between 2.5 and 7 p. m. 
 It is also found in the brain, blood, transudates, amniotic fluid, and 
 sometimes also in the urine. 
 
 Creatine may be prepared synthetically from cyanamide and sar- 
 cosine (methylglycocoll). On boiling with baryta-water it decomposes, 
 with the addition of water, and yields urea, sarcosine, and certain other 
 products. Because of this behavior several investigators consider 
 creatine as a step in the formation of urea in the organism. On boiling 
 with acids, creatine is easily converted, with the elimination of water 
 into the corresponding anhydride, creatinine, C4H7N3O, which is retrans- 
 formed into creatine by the action of alkali. 
 
 The question as to the mutual relation of creatine to creatinine in 
 metabolism will be treated in Chapter XIV (urine). In this chapter, 
 besides the properties and reactions, we will discuss the question as to 
 the origin of creatine and its relation to the metabolism of the muscles. 
 
 Of special interest in this regard, besides the relation between creatine 
 and muscle work which will be discussed below, is the question as to 
 the occurrence of free or combined creatine in the muscle. Urano by 
 the aid of dialysis experiments has shown the probability that the crea- 
 tine does not exist free in the muscle, but as a labile, non-dialyzable 
 combination. Nevertheless Gottlieb and Stangassinger claim by 
 various researches to have shown in the autolysis of muscles and other 
 organs,. that. creatine is first formed and then first changed into creatinine 
 by special bodies of an enzymotic nature, and then destroyed. Seemann 
 
 1 v. Furth and Schwarz, Bioch. Zetschr., 30; Scaffidi, ibid., 33; Burian and Hall, 
 Zeitschr. f. physiol. Chem. 38; Buglia and Costantino, ibid., 81 and 82; Rinaldi and 
 Scaffidi, Bioch. Zeitschr., 41. 
 
574 MUSCLES. 
 
 claims, by an autolysis of three months' duration, .to have obtained two 
 to three times as much creatinine, directly from the muscle, and after 
 the addition of creatinine-free-gelatin four times as much, which is an 
 argument against the enzymotic destruction of creatinine in autolysis, 
 and he admits of the formation of creatine (or creatinine) from protein. 
 The autolytic experiments of Rothmann also indicate the formation of 
 creatine from a preliminary body, and the recent experiments of van 
 Hoogenhuyze and Verploegh make the enzymotic transformation 
 of creatine and creatinine probable. Mellanby positively denies the 
 re-formation of creatine as well as its destruction in autolysis entirely 
 free from bacteria. It is hard to draw positive conclusions from exper- 
 iments with autolysis. The transfusion experiments of Gottlieb and 
 Stangassinger with the kidneys and livers of dogs, not only point to 
 the ability of these organs to decompose creatine, but also for a re-forma- 
 tion of creatine in the liver. Further investigations are still very nec- 
 essary, especially as the conditions are probably not the same in all animals. 
 Thus Noel-Paton and Mackie l found that the exclusion of the liver 
 in birds is without influence upon the creatine metabolism. 
 
 As will be discussed in Chapter XIV, no certain relationship 
 exists between the quantity of food protein and the extent of creatine 
 and creatinine elimination. On the contrary, several observations speak 
 for a relation between creatine formation and catabolism of organ pro- 
 tein, especially muscle protein and according to Noel-Paton 2 the 
 elimination of creatine in bird urine, which here corresponds to the 
 creatinine in the mammalian urine, is a measure of the protein catabo- 
 lism of the muscles. 
 
 Under all circumstances the proteins, and especially the guanidine 
 groups contained therein, are the mother-substance for the creatine or 
 creatinine. The guanidine occurs in the protein molecule as arginine; 
 but according to Otori it is not improbable that in the protein also 
 other guanidine groups exist. Nevertheless the observations of Jaffe 3 
 speak against the assumption as to a creatine formation from argin- 
 ine as he found that arginine subcutaneously injected did not cause 
 any increase in the elimination of creatine substances. But as the 
 introduced arginine was probably decomposed by the enzyme arginase, 
 because the urea elimination was greatly increased, 4 does not exclude 
 
 1 Urano, Hofmeister's Beitrage, 9; Gottlieb and Stangassinger, Zeitschr. f. physiol. 
 Chem., 52 and 55; Stangassinger, ibid., 55; Seemann, Zeitschr. f. Biol., 49; Roth- 
 mann, Zeitschr. f. physiol. Chem., 57; v. Hoogenhuyze and Verploegh, ibid., 57; 
 Mellanby, Journ. of Physiol., 36; Noel-Paton and Mackie, Journ. of Physiol. 45. 
 
 2 Journ. of Physiol.. 39. 
 
 •Otori, Zeitschr. f. physiol. Chem., 42, 43; Jaffe, ibid., 48. 
 
 4 See Thompson, Journ. of Physiol., 32 and 33. 
 
CREATINE. 
 
 575 
 
 the possibility that in the muscles, which according to Kossel and Dakin 
 contain only little arginase, the arginine was decomposed in other ways. 
 In autolysis as well as perfusion experiments with livers, Inouye ' has 
 recently shown that an increase in the creatine occurs at the expense of 
 the arginine added. 
 
 Starting with the observation of Jaff6 2 that glyeocyamine (guanidine acetic 
 acid) in rabbits is transformed with a niethylation into creatine, we can consider 
 the cleavage of arginine into creatine in the following manner, basing this con- 
 ception upon the ruling conception on the cleavage of amino-acids and fatty acids 
 in the animal body. 
 
 NH 2 
 
 HN=C 
 
 / 
 
 \ 
 
 HX=C 
 
 XH 
 
 / 
 
 CH, 
 
 XH 2 
 
 / 
 
 \ 
 NH 
 
 / 
 
 NH 2 
 HX=C^ 
 
 NH 
 
 / 
 CH 2 
 
 NH, 
 
 HX 
 
 =< 
 
 N(CH,) 
 / 
 
 (CH 2 ) 2 — 
 
 I 
 COOH 
 
 7-guanidine 
 butyric acid. 
 
 -> COOH 
 
 Ouanidine acetic 
 acid (glyeocyamine). 
 
 CH 2 
 
 I 
 COOH 
 
 Creatine. 
 
 CH 2 
 
 I 
 
 CH 2 - 
 
 CH, 
 
 I 
 CH(XH 2 ) 
 
 I 
 COOH 
 
 Arginine. 
 
 The opinions are not unanimous in regard to the organ producing 
 creatine or creatinine. Based upon several investigations it is generally 
 admitted that the liver here plays an important role. Several other 
 organs may also be considered and in the first place, the muscles. Accord- 
 ing to Mellanby the creatinine is probably formed in the liver, trans- 
 formed into creatine in the muscles and there stored up as such. Other 
 observations still speak for the fact that the creatine is formed in the 
 muscles and transformed into creatinine in the liver, while according 
 to Noel-Paton and Mackie the exclusion of the liver in birds is without 
 effect upon the creatinine metab< lism. 
 
 Creatine crystallizes in hard, colorless, monoclinic prisms which 
 lose their water of crystallization at 100° C. It is soluble in 7-4 parts 
 of water at the ordinary temperature, and in 9419 parts absolute alcohol. 
 It dissolves more easily with the aid of heat. Its watery solution has 
 a neutral reaction. Creatine is not dissolved by ether. If a creatine 
 solution is boiled with precipitated mercuric oxide, this is reduced, 
 especially in the presence of alkali, to mercury and oxalic acid, and the 
 foul-smelling methyluramine (methylguanidine) is developed. A solu- 
 
 1 Kossel and Dakin, Zeitschr. f. physiol. Chem., 41 and 42; Inouye, ibid., 81. 
 1 Zeitschr. f. physiol. Chem., 48; see also Dorner, ibid., 52. 
 
576 MUSCLES. 
 
 tion of creatine in water is not precipitated by basic lead acetate, but 
 gives a white, flaky precipitate with mercurous nitrate if the acid reac- 
 tion is neutralized. When boiled for an hour with dilute hydrochloric 
 acid, creatine is converted into creatinine, and may be identified by its 
 reactions. On boiling with formaldehyde it can be transformed into 
 dioxymethylenecreatinine, which crystallizes readily (Jaffe '). 
 
 The preparation and detection of creatine is best accomplished by the 
 following method of Neubauer, 2 which was first used in the preparation 
 of creatine from muscles: Finely cut meat is extracted with an equal 
 weight of water at 50-55° C. for 10-15 minutes, pressed, and extracted 
 again with water. The proteins are removed from the united extracts 
 so far as possible by coagulation at boiling heat, the filtrate precipitated 
 by the careful addition of basic lead acetate, the lead removed from this 
 filtrate by H2S, and the solution then carefully concentrated to a small 
 volume. The creatine, which crystallizes in a few days, is collected on a 
 filter, washed with alcohol of 88 per cent, and purified, when necessary, 
 by recrystallization. In the preparation of large quantities of creatine 
 we can especially start with meat extracts. The quantitative estimation 
 of creatine is performed by transforming it into creatinine (see Chapter 
 XIV). 
 
 Carnosine, C9H14N4O3, is a base first isolated by Gulewitsch and 
 Amiradzibi from meat extracts and which subsequently was also pre- 
 pared directly from meat. The quantity seems to be relatively consider- 
 able, as according to the above-mentioned determination of v. Furth* 
 and Schwarz, the carnosine fraction from the horse and dog muscles 
 was just as large or indeed greater than the creatine-creatinine fraction 
 of the extractive nitrogen. Krimberg found 1.3 p. m. and Skworzow, 3 
 1.76 p. m. (as nitrate) in fresh meat. 
 
 Carnosine, which according to Gulewitsch is identical with the base 
 ignotine isolated from meat extracts by Kutscher while both bases are 
 isomeric bodies according to Kutscher, 4 is a histidine derivative accord- 
 ing to Gulewitsch which on cleavage yields /3-alanine besides histidine. 
 
 Carnosine is a base readily soluble in water, which is precipitated 
 as stellar warts of short delicate needles from the concentrated watery 
 solution by the addition of alcohol. The specific rotation for the light 
 X = 546 is according to Gulewitsch in watery solution where c = 12.925 
 per cent and 20.1° C. = -r-25.3°. The base is precipitated by phospho- 
 
 1 Ber. d. d. Chem. Gesellsch., 35. 
 
 2 Zeitschr. f. analyt. Chem., 2 and 6. 
 
 'Gulewitsch and Amiradzibi, Zeitsdir. f. physiol. Chem., 30; Gulewitsch, ibid., 
 
 50, 51, 52 and 73; Krimberg. ihid., 48; Skworzow, ibid., 68. 
 
 4 Gulewitsch and Amiradzibi, Zeitschr. f. physiol. Chem., 30; Gulewitsch, ibid., 50, 
 
 51, 52 and 73; Krimberg, ibid., 08; Skworzow, ibid., 68; Kutscher, ibid., 50, 51. 
 
CARNITINE. CARNINE. 577 
 
 tungstic acid, by mercuric nitrate and by silver nitrate with an excess of 
 barium hydrate. Carnosine-silver is soluble with difficulty in cold water 
 but readily soluble in hot water. Carnosine nitrate melts at 211-212° C. 
 Carnosine also gives a crystalline copper salt. 
 
 The principle in preparing this base consists in precipitat'ng with 
 phosphotungstic acid, separating the free base with barium hydrate, 
 conversion into the nitrate, precipitating with silver nitrate and barium 
 hydrate, decomposing the salt with H 2 8 and conversion into nitrate. 
 From the latter, which is readily obtained as crystals, the base is precip- 
 itated by phosphotungstic acid and then set free by barium hydrate. 
 
 Carnitine, C7H15NO3 (or C;Hi G X0 3 ), another base isolated by Gulewitsch 
 and Krimberg from meat extracts, has a strong alkaline reaction, is very 
 readily soluble in water, and was also found by Krimberg in fresh meat. Skworzow 
 found 0.19 p. m. carnitine in calf's muscles. Carnitine according to Krimberg 
 is probably 7-trimethvl-/3-oxybutyrobetaine with the formula 
 
 X) CO 
 
 (CH 3 ) : N\ ! . According to Engeland it is on the contrarv 
 
 \CH 2 — CH(OH)— CH 2 
 
 a 7-trimethyl-o;-oxybutyrobetaine (CH 3 ) 3 -N~ V T . 
 
 CH 2 CH 2 CH(OH)— CO 
 
 according to Krimberg and Engeland 1 identical with novaine prepared by Kossel 
 from meat extracts. It gives crystalline double compounds with platinum, gold 
 and mercuric chlorides, among which the following, C 7 Hi 5 N0 3 2HgCl2, with a 
 melting-point of 196-197° C, is especially used in the isolation of the base. 
 The hydrochloride and the nitrate are readily soluble and the solution of the first 
 isla?vo-rotatory, about («) D = —21°. 
 
 The inosinic acid has been discussed in Chapter II. In close relation to this 
 stands probably the carnine. 
 
 Carnine, C 7 H 8 X iO ;i +H 2 0, is one of the substances found by Weidel in American 
 meat extract. It has also been found by Krukenberg and Wagner in frog 
 muscles and in the flesh of fishes, and by Pouchet in the urine. Carnine is, accord- 
 ing to Haiser and Wenzel, 2 probably only an equimolecular mixture of hypo- 
 xanthine and the crystalline pentoside (hypoxanthin-riboside) inosine, which is 
 readily split by acid into hypoxanthine and pentose. 
 
 Carnine has been obtained as a white crystalline mass. It dissolves with 
 difficulty in cold water, but more readily in warm. It is insoluble in alcohol 
 and ether. It dissolves in warm hydrochloric acid and yields a salt crystallizing 
 in shining needles, which gives a double compound with platinum chloride. Its 
 watery solution is precipitated by silver nitrate, but this precipitate is dissolved 
 neither by ammonia nor by warm nitric acid. Its watery solution is precipitated 
 by basic lead acetate; but the lead compound may lie dissolved on boiling. 
 
 Phosphocarnic acid 3 is a complicated substance, first isolated by Siegfried 
 
 Gulewitsch and Krimberg, Zeitschr. f. physiol. Chem. 45; Krimberg, ibid.; 49, 
 50, 53 and 56, Ber. d. d. Chem. Gesellsch. 42; Engeland, ibid., 42; Skworzow, 1. c. 
 
 2 Weidel, Annal. d. Chem. u. Pharm., 158; Krunkenberg and Wagner, SUzungsber. 
 d. Wiirzb. phys.-med. Gesselsch., 1883; Pouchet, cite! from Neubauer-Huppert, 
 Analyse des Harnes, 10. Aufl., 335; Haiser and Wenzel, Monatsch. f. Chem., 29. 
 
 3 In regard to carnic acid and phosphocarnic acid, see the works of Siegfried, Arch, 
 f. (Anat. u.) Physiol., 1894, Ber. d. deutsch. chem. Gesellsch., 28, and Zeitschr. f. 
 physiol. Chem., 21 and 28; M. Miiller, ibid., 22; Kriiger, ibid., 22 and 28; Balke and 
 
578 MUSCLES. 
 
 from meat extracts, which yields as cleavage products succinic acid, paralactic 
 acid, carbon dioxide, phosphoric acid, and a carbohydrate group, besides the 
 previously mentioned carnic acid, which is identical with or nearly related to 
 antipeptone. It stands, according to Siegfried, in close relation to the nucleins, 
 and as it yields peptone (carnic acid), it is designated as a nucleon by Siegfried. 
 Phosphocarnic acid may be precipitated as an iron compound, carniferrine, from 
 the extract of the muscles free from proteins. The quantity of phosphocarnic 
 acid, calculated as carnic acid, can be determined by multiplying the quan- 
 tity of nitrogen in the compound by the factor 6.1237 (Balke and Ide). In 
 this way Siegfried found 0.57-2.4 p. m. carnic acid in the resting muscles 
 of the dog, and AI. Muller 1-2 p. m. in the muscles of adults and a maximum 
 of 0.57 p. m. in those of new-born infants. According to Cavazzani nucleon 
 occurs to a much greater extent in oysters, namely, an average of 3.725 p. m. 
 It also occurs, as he and Manicardi found, in the plant kingdom. Phospho- 
 carnic acid has not been prepared in the pure state and possesses on this account 
 a variable composition; according to Siegfried it serves as a source of energy 
 in the muscles and is consumed during work. Besides, by means of its property 
 of forming soluble salts with the alkaline earths, as also an iron combination 
 soluble in alkalies, it acts as a means of transportation for these bodies in the 
 animal body. 
 
 Phosphocarnic acid is prepared from the extract free from protein by first 
 removing the phosphate by CaCl 2 and NH 3 . . The acid is precipitated as carnifer- 
 rine by ferric chloride from the filtrate while boiling. 
 
 From Liebig's extract of beef Kutscher has isolated besides the above- 
 mentioned ignotine and novaine, several other bodies, neosine, CeH^NCK, which 
 according to Kutscher and Ackermann is a homologue of choline, vitiatine 
 (as gold salt, CoHi 4 X6.2HC1.2AuCl s ), carnomuscarine, methylguanidine (also found 
 by Gulewitsch), oblitine, CisHssXoOs, which probably contains two novaine 
 groups, which corresponds well with Krimberg's view, and also choline and 
 murine. From dog muscles Ackermann 1 has isolated a platinum compound, 
 CnH3oX 2 04PtCl6, of a base called myocynine, which seems to be a hexamethyl- 
 ornithine. Micro 2 found in meat extracts small quantities of alanine, glutamic 
 acid, taurine and inosite, but no dipeptides. In crab extract Kutscher and Ack- 
 ermann found no creatine and creatinine, but among others betaine and two new 
 bases, crangitine, CY(H i0 X2O 4 , and crangonine, C13H26X2O3. In crab muscles Suzuki 3 
 and collaborators found a base, canirine which although it has the same composi- 
 tion. C6H14X2O2, as lysine, is not identical therewith. 
 
 The base musculamine, isolated by Etard and Vila on the hydrolysis of veal, 
 is nothing but cadaverine, according to Posternak. 1 
 
 We must also include among the nitrogenous extractives those bodies which 
 were first discovered by Gautier, 6 and which occur only in very small quantities, 
 namely, the leucomaines, xanthocreatinine, C5H10H4O, crusocreatinine, C5H8N4O, 
 amphicreatine, C9H19X7O4, and pseudoxanthine, C4H5X5O. 
 
 Ide, ibid., 21, and Balke, ibid., 22; Macleod, ibid., 28; E. Cavazzani, Centralbl. f. 
 Physiol., 18, 666; Panella, Maly's Jahresber., 34. 
 
 1 Kutscher, Zeitschr. f. Unters. d. Nahrungs- u. Genussmittel, 10, 11, Centralbl. 
 f. Physiol., 19 and 21, Zeitschr. f. physiol. Chem., 48, 49, 50, 51, with Ackermann, 
 ibid., 56; Gulewitsch. ibid., 47; Krimberg, ibid., 56; Ackermann (on myocynine). 
 Zeitschr. f. biol. 59. 
 
 2 Zeitsfhr. f. physiol. Chem., 56. 
 
 ■■ Kutscher and Ackermann, Zeitschr. f. Unters. d. Nahrungs- u. Genuarnittel, 13 
 and 14; Suzuki, Chem. Centralbl. 1913, 1. 
 
 4 Etard and Vila, Cornpt. Rend., 135; Posternak, ibid., 135. 
 8 See M:.ly's Jahresber., 16, 523. 
 
INOSITE. 579 
 
 In the analysis of moat, and for the detection and separation of the various 
 extractive bodies of meat, we make use of the systematic method as suggested 
 by Gautier, 1 for details of which the reader is referred to the original article 
 Bfl well as for the Kutscht r method for working the meat extracts. 
 
 The non-nitrogenous extractive bodies of the muscles are inosite, gly- 
 cogen, sugar, and lactic aval. 
 
 Inosite, CgHi20c+H20 = C 6 Hg(OH)6+H20. This body, discovered 
 by Scherer, is not a carbohydrate, but belongs to the hydroaromatic 
 compounds, and is a hexahydroxybenzene (Maquenne 2 ). That it 
 stands in certain relation to the carbohydrates follows from the fact that 
 Neuberg obtained some furfurol from inosite by distillation with phos- 
 phoric anhydride, and also that P. Meyer 3 found fermentation lactic 
 acid in the urine of rabbits after the introduction of inosite per os. It 
 has been known for some time that inosite undergoes lactic acid fermenta- 
 tion. The acid formed thereby is sarcolactic acid according to Hilger 
 and fermentation lactic acid according to Vohl. 4 
 
 Inosite is found in the muscles, liver, spleen, leucocytes, kidneys, 
 suprarenal capsule, lungs, brain, testicles, and in the urine in pathological 
 cases, and as traces in normal urine. It is found very widely dis- 
 tributed in the vegetable kingdom, especially in the unripe fruit of green 
 beans (Phaseolus vulgaris), and therefore it is also called phaseomannite. 
 In the plant kingdom another substance occurs which is called phytin 
 and which is the Mg and Ca compound of inosite and phosphoric acid 
 and which was first isolated by Posternak. Winterstein identified this 
 as an inosite-phosphoric acid. This inosite-phosphoric acid can be split 
 into phosphoric acid and inosite by the plant enzyme phytase (Suzuki, 
 Yoshimura and Takaishi) as well as by enzymes of the animal tissues 
 (Starkenstein). Inosite is found in plants, especially in the develop- 
 ing organs (Meillere), and according to Starkenstein 5 it occurs to a 
 greater extent in the organs of young animals as compared with those of 
 older animals. From this it follows that inosite is probably not a decom- 
 position product of metabolism, but rather a body necessary for the devel- 
 opment of the cells (Meillere); but according to Starkenstein the 
 facts are different. 
 
 1 Maly's Jahresb., 22. 
 
 2 Bull. soc. chem. (2), 47 and 48; Compt. Rend., 104. 
 1 Neuberg, Bioch. Zeitschr., 9; P. Meyer, ibid., 9. 
 
 * Hilger, Annal. d. Chem. u. Pharm., 160; Vohl, Ber. d. d. Chem. Gesellsch., 9. 
 
 5 Winterstein, Ber. d. d. chem. Gesellsch., 30; :>.nd Zeitschr. f. physiol. chem., 58; 
 Posternak, Contribution a l'dtude chim. de I'assimilation chlorophyllienne. Revue 
 cenerale botanique, Tome 12 (1900), and Compt. Rend., 137; Suzuki, Yoshimura and 
 Takaishi, Bull, agric. Univers. Tokio, 7; Starkenstein, Bioch. Zeitschr., 30. 
 
580 MUSCLES. 
 
 According to Starkenstein the free inosite is without importance 
 and is only a decomposition product of metabolism; of importance, 
 especially for young, growing individuals is according to this worker 
 only the phytin, which is decomposed in the intestine by bacteria, and in 
 the tissues by enzymes, and correspondingly supplies phosphoric acid and 
 lime to the organism while the inosite is excreted as a valueless cleavage 
 product. The free inosite in the animal body originates according to 
 Starkenstein from the inositephosphoric acid and in this sense the 
 assumption of Rosenberger 1 as to the occurrence of an inositogen in the 
 animal body, is substantiated. 
 
 Inosite, which almost without exception is inactive mesoinosite, 
 crystallizes in large, colorless, rhombic crystals of the monoclinic sys- 
 tem, or, if not pure and if only a small quantity crystallizes, it forms 
 groups of fine crystals similar to cauliflower. It loses its water of crys- 
 tallization at 110° C, also if exposed to the air for a long time. Such 
 exposed crystals are non-transparent and milk-white. The crystals 
 melt at 225° C. when dry. Inosite dissolves in 7.5 parts of water at 
 ordinary temperature, and the solution has a sweetish taste. It is insoluble 
 in strong alcohol and in ether. It dissolves cupric hydrate in alkaline 
 solutions, but does net reduce on boiling. It gives negative results with 
 Moore's test and with Bottger-Almen's bismuth test. It does not 
 ferment with beer-yeast, but may undergo lactic- and butyric-acid fer- 
 mentation. With an excess of nitric acid inosite is oxidized to rhodizonic 
 acid, and the following reaction depends upon this. 
 
 If inosite is evaporated to dryness on paltinum-foil with nitric acid 
 and the residue treated with ammonia and a drop of calcium chloride 
 solution and carefully re-evaporated to dryness, a beautiful rose-red 
 residue is obtained (Sherer's inosite test). If we evaporate an inosite 
 solution to incipient dryness and moisten the residue with a little mer- 
 curic nitrate solution, there is obtained a yellowish residue on drying 
 which becomes a beautiful red on strongly heating. The coloration 
 disappears on cooling, but it reappears on gently warming (Gallois' 
 inosite test) . Other inosite reactions have been suggested by Deniges 2 
 and others. 3 
 
 To prepare inosite from a liquid or from a watery extract of a tissue, 
 the proteins are first removed by coagulation at boiling heat. The filtrate 
 
 1 Meillere, Journ. d. Chim. et Pharm. (6) 28; Starkenstein, Zeitschr. f. exp. Path, 
 u. Therap. 5, Bioch. Zeitschr. 30 and Zeitschr. f. physiol. Chem. 58; Rosenberger, 
 ibid., 56, 57 and 58. 
 
 2 Compt. rend. soc. biol., 62. 
 
 3 In regard to the salts of phytin and compounds of inosite see Anderson, Journ. 
 of biol. Chem. 11 and 12. 
 
GLYCOGEN. 581 
 
 is precipitated by sugar of Lead, this filtrate boiled with basic lead acetate 
 and allowed to stand 24-48 hours. The precipitate thus obtained, 
 which contains all the inosite, is decomposed in water by HfeS. The 
 nitrate is strongly concentrated, treated with 2-4 vols, hot alcohol, and 
 the liquid removed as soon as possible from the tough or flaky masses 
 which ordinarily separate. If no crystals separate from the liquid within 
 twenty-four hours, then treat with ether until the liquid has a milky 
 appearance and allow it to stand. In the presence of a sufficient quantity 
 of ether, crystals of inosite separate within twenty-four hours. The 
 crystals thus obtained, as also those which are directly obtained from the 
 alcoholic solution, are recrystallized by redissolving in very little boiling 
 water and adding 2-A vols, of alcohol. Meillere l and others have 
 suggested mollifications in the methods for detecting and quantitatively 
 estimating inosite. 
 
 Scyllite is a body which is isomeric with inosite, according to Joh. Muller, 2 
 and which was found long ago in the kidneys, liver and spleen of Plagiostomata 
 and also in the plant kingdom as cocosite and quercinite. Scyllite crystallizes 
 in shining prisms, is soluble in water 1:100 at 18° C, is similar to inosite in its 
 reactions, but has a much higher melting-point, namely about 360° C. From 
 the adductor muscles of the Mytilus Janssen 3 has isolated a substance, called 
 mytilite which is crystalline, soluble with , difficulty in cold water and readily sol- 
 uble in hot water, and having the formula C6Hi 2 Oo.2H 2 0. He claims that it is 
 stereisometric with the alcohol quercite. 
 
 Glycogen is a constant constituent of the living muscle, while it may 
 be absent in the dead muscle. The quantity of glycogen varies in the 
 different muscles of the same animal and according to Maignon this 
 is not only true for the same muscles in both halves of the body but also 
 for different parts of the same muscle. Bohm found 10 p. m. glycogen 
 in the muscles of cats, and moreover he found a smaller amount in the 
 muscles of the extremities than in those of the rump. Moscati found an 
 average of 4 p. m. in human muscles, and Schondorff 4 has found a 
 maximum of 37.2 p. m. in the dog-muscle. Reports as to the quantity 
 of glycogen in the heart are conflicting; although the heart is considered 
 as somewhat poorer in glycogen than the other muscles, still this difference 
 is not very great, and can be explained by the ready disappearance of 
 glycogen from the heart after death, as well as after starvation and 
 after strong work (Boruttatj, Jensen 5 ). Work and food have a great 
 influence upon the quantity of glycogen. Bohm found 1-4 p. m. 
 glycogen in the muscles of fasting animals, and 7-10 p. m. after partak- 
 
 ^ompt. rend. soc. biol., 60, and Journ. d. Chim. et Pharni. (6), 24; see also 
 Starkenstein, Zeitschr. f. exp. Path. u. Ther., 5. 
 
 2 Ber. d. d. chem. Gesellsch., 40. 
 
 3 Zeitschr. f. physiol. Chem., 85. 
 
 4 Maignon, Journ. de physiol. et d. path. 10 Bohm, Pfluger's Arch., 23, 44; Schon- 
 dorff, ibid., 99; Moscati, Hofmeister's Beitrage, 10. 
 
 6 Boruttau, Zeitschr. f. physiol. Chem., 18; Jensen, ibid., 35. 
 
582 MUSCLES. 
 
 ing of food. As stated' in Chapter VII, work, starvation, and lack of 
 carbohydrates in the food cause the glycogen to disappear earlier from 
 the liver than from the muscles. 
 
 The sugar of the muscles, of which only traces occur in the living mus- 
 cle, and which is probably formed after the death of the muscle from 
 the muscle-glycogen, is, according to the investigations of Panormoff, in 
 part glucose, but consists principally of maltose (Osborne and Zobel l ) 
 with some dextrin. 
 
 Lactic Acids. Of the oxypropionic acids with the formula C3H6O3 
 there is one, ethylene lactic acid, CH 2 (OH).CH 2 .COOH, which is not 
 found in the animal body, and therefore has no physiological chemical 
 interest. 
 
 CH 3 
 Indeed only a-oxypropionic acid or ethylidene lactic acid, CH(OH), of 
 
 COOH 
 which there are two physical isomers, namely, the dextrorotatory par- 
 alactic or sarcolactic acid, and the levolactic acid obtained by 
 Schardinger by the fermentation of cane-sugar by means of a special 
 bacillus. This levolactic acid, which is formed by the typhoid bacillus 
 and various vibriones 2 need not be discussed here, and we will only treat 
 here the d-Z-lactic acid (the inactive fermentation lactic acid) and the 
 dextrolactic acid. 
 
 The fermentation lactic acid, which is formed from lactose by allow- 
 ing milk to sour, and by the acid fermentation of other carbohydrates, 
 is considered to exist in small quantities in the muscles (Heintz), in the 
 gray matter of the brain (Gscheidlen), and in diabetic urine. The 
 occurrence of fermentation lactic acid in the brain and other organs 
 is still very improbable and has been disputed by Moriya. 3 During 
 digestion this acid is also found in the contents of the stomach and intestine, 
 and as alkali lactate in the chyle. The parallactic acid, is at all events, 
 the true acid of meat extracts, and this alone has been found with certainty 
 in dead muscle. The lactic acid which is found in the brain, spleen,, 
 lymphatic glands, thymus, thyroid gland, blood, bile, pathological 
 transudates, osteomalacial bones, in perspiration in puerperal fever, 
 in the urine after fatiguing marches, in acute yellow atrophy of the liver, 
 
 1 Panormoff, Zeitschr. f. physiol. Chem., 17; Osborne and Zobel, Journ. of Physiol., 
 29. 
 
 1 See Schardinger, Monatshefte f. Chem., 11; Blachstein, Arch, des sciences biol. 
 de St. Petersbourg, 1, 199 ; Kuprianow, Arch. f. Hygiene, 19, and Gosio, ibid., 21; 
 Herzog and Horth, Zeitschr. f. physiol. Chem., 60. I 
 
 1 Heintz, Annal. d. Chem. u. Pharm., 157, and Gscheidlen, Pfliiger's Arch., 8,. 
 171; Moriya, Zeitschrift f. physiol. Chem., 43. 
 
LACTIC ACIDS. 583 
 
 in poisoning by phosphorus, and especially after extirpation of the liver 
 seems to be paralactic acid. 
 
 The origin of paralactic acid in the animal organism has been sought 
 by several investigators, who took for basis the researches of Gaglio, 
 Minkowski, and Araki, in a decomposition of protein in the tissues 
 Gaglio claims a lactic-acid formation by passing blood through the sur- 
 viving kidneys and lungs. He also found 0.3-0.5 p. m. lactic acid in the 
 blood of a dog after protein food, and only 0.17-0.21 p. m. after fast- 
 ing for forty-eight hours. According to Minkowski the quantity of lactic 
 acid eliminated by the urine in animals with extirpated livers is increased 
 with protein food, while the administration of carbohydrates has no 
 effect. Araki has also shown that if we produce a scarcity of oxygen 
 in animals (dogs, rabbits, and hens) by poisoning with carbon monoxide, 
 by the inhalation of air deficient in oxygen, or by any other means, a 
 considerable elimination of lactic acid (besides sugar and also often 
 albumin) takes place through the urine, an observation which has been 
 confirmed by Saito and Katsuyama. 1 As a scarcity of oxygen, accord- 
 ing to the ordinary statements, produces an increase of the protein 
 catabolism in the body, the increased elimination of lactic acid in these 
 cases must be due in part to an increased protein destruction and in part 
 to a diminished oxidation. 
 
 Araki has not drawn such a conclusion from his experiments, but 
 he considers the abundant formation of lactic acid to be due to a cleavage 
 of the sugar formed from the glycogen. He found that in all cases where 
 lactic acid and sugar appeared in the urine the quantity of glycogen 
 in the liver and muscles was always diminished. Without denying 
 the possibility of a formation of lactic acid from protein, he states that 
 with lack of oxygen we have to deal with an incomplete combustion 
 of the lactic acid derived by a cleavage of the sugar. Although the 
 abundant formation of lactic acid under these circumstances can be 
 explained in different ways, still there are other conditions which make 
 the formation of lactic acid from proteins very probable. To this 
 belongs the lactic acid formation from alanine, in the liver, as mentioned 
 in a previous chapter, and recently further substantiated by Embden 
 and F. Kraus. 2 
 
 The carbohydrates are also considered as the mother-substance 
 of the lactic acid, as it is now generally admitted that the cleavage of the 
 
 1 Gaglio, Arch f (Anat. u.) Physiol., 1886; Minkowski , Arch exp. Path, u. Pharm., 
 21 and 31; Araki, Zeitschr. f. physiol. Chem., 15, 16, 17, and 19; Saito and Katsuyama. 
 ibid., 32. 
 
 2 Neuberg and Langstein, Arch. f. (Anat. u.) Physiol. 1903; Embden and F. Kraus, 
 Bioch Zeitschr. 45. 
 
584 MUSCLES. 
 
 sugar in the animal body occurs, or at least can occur, with lactic acid as 
 an intermediary step The views are indeed different 1 as to the closer 
 mechanism of this cleavage, but there does not exist any doubt that a 
 formation of lactic acid, and in fact paralactic acid, can take place from 
 carbohydrates in the animal body. Hoppe-Seyler 2 held the view that 
 the formation of lactic acid, in the absence of free oxygen, from gly- 
 cogen or glucose was probably a function of all living protoplasm and in 
 the anaerobic metabolism of the animal cells, according to the investiga- 
 tions of Stoklasa 3 and his collaborators on alcoholic fermentation in 
 the tissues, a formation of alcohol and carbon dioxide takes place from 
 the sugar with lactic acid as intermediary step. The correctness of these 
 statements is now disputed from many sides, but we have direct observa- 
 tions which speak positively for a lactic acid formation from glycogen 
 or sugar. Thus Embden 4 and co-workers have found that on transfus- 
 ing blood through the liver rich in glycogen, a formation of lactic acid 
 takes place, and an abundance of lactic acid is formed when blood rich in 
 sugar is transfused through a glycogen free liver, while a blood poor in 
 sugar led only to a very inconsiderable formation of lactic acid. 
 
 Certain investigators (see page 333) admit of the occurrence of glyceric 
 aldehyde (and also dioxyacetone) as intermediary products in the forma- 
 tion of lactic acid from sugar. Another intermediary product in the 
 lactic acid formation has been shown by recent thorough investigations 
 to be methylglyoxal, CH3.CO.CHO. An abundant formation of lactic 
 acid from methylglyoxal has been obtained by certain investigators, 
 such as Dakin and Dudley, and by Neuberg, in experiments with 
 tissues, organ extracts and organ pulp, and by Levene and Meyer 5 in 
 experiments with leucocytes or kidney tissue. The process is of an 
 enzymotic nature and the active enzyme, which also converts phenyl- 
 glyoxal into mandelic acid has been called ghjoxylase by Dakin and 
 Dudley. The process is reversible according to these experimenters, 
 in that they have been able to show a retransformation of lactic acid 
 into methylglyoxal. They also found that lactic acid as well as methyl- 
 glyoxal could form glucose in diabetic animals. The detailed procedure 
 in the cleavage of sugar to lactic acid is still undecided. 
 
 The carbohydrates, as well as the proteins, it seems, must be con- 
 sidered as the material from which the lactic acid is formed in the body. 
 
 »See Embden und Oppenheimer, Bioch. Zeitschr., 45; Parnas and Baer, ibid., 41. 
 
 2 Yirchow's Festschrift, also Her. d. deutsch. chem. Gesellsch., 25, Referatb., 685. 
 
 3 Simdeek, Centralbl. f. Physiol., 1"; Stoklasa, Jelinek, and Cerny, ibid., 16. In 
 regard to opposed statements see Harden and Mac Lean, Journ. of Physiol., 42. 
 
 4 Embden and Almagia with F. Kraus, Bioch. Zeitschr. 45; S. Oppenheimer, ibid., 45. 
 
 5 Dakin and Dudley, Journ. of biol. Chem., 14; Neuberg, Bioch. Zeitschr., 49; 
 Levene and Meyer, Journ. of biol. Chem., 14. 
 
LACTIC ACIDS. 585 
 
 The phosphocarnic acid (Siegfried) and the inosite are also considered 
 as possible mother-substances for sarcolactic acid. Further research 
 will show whether also other mother-substances for this acid occur. The 
 autolytic experiments of Tuhkel x with livers and the formation of lactic 
 acid in the muscles, not from carbohydrates, inosite or alanine, as observed 
 by Embden 2 and his collaborators seem to indicate this. 
 
 The lactic acids are amorphous. They have the appearance of 
 colorless or faintly yellowish, acid-reacting syrups, which mix in all pro- 
 portions with water, alcohol, or ether. The salts are soluble in water, 
 and most of them also in alcohol. The two acids are differentiated from 
 each other by theit different optical properties — paralactic acid being 
 dextrogyrate, while fermentation lactic acid is optically inactive — also 
 by their different solubilities and the different amounts of water of crys- 
 tallization cf the calcium and zinc salts. The zinc salt of fermentation 
 lactic acid dissolves in 58-63 parts of water at 14-15° C, and contains 
 18.18 per cent water of crystallization, corresponding to the formula, 
 Zn(C3Hs03)2+3H20. The zinc salt of paralactic acid dissolves in 17.5 
 parts of water at the above temperature and contains ordinarily 12.9 
 per cent water, corresponding to the formula, Zn(C3H503)o+2H20. 
 The calcium salt of fermentation lactic acid dissolves in 9.5 parts water 
 and contains 29.22 per cent (=5 molecules) water of crystallization, 
 while calcium paralactate dissolves in 12.4 parts water and contains 24.83 
 or 26.21 per cent ( = 4 or 4^ molecules) water of crystallization. Both 
 calcium salts crystallize, not unlike tyrosine, in spears or tufts of very 
 fine microscopic needles. Hoppe-Seyler and Araki, who have closely 
 studied the optical properties of the lactic acids and lactates, consider 
 the lithium salt as best suited for the preparation and quantitative estima- 
 tion of the lactic acids. The lithium salt contains 7.29 per cent Li. For 
 further information as to the salts and specific rotation of the lactic acids 
 see Hoppe-Seyler-Thierfelder's Handbuch. 8. Aufl., 1909. 3 
 
 Lactic acids may be detected in organs and tissues in the following 
 manner: After complete extraction with water, the protein is removed 
 by coagulation at boiling temperature and the addition of a small quan- 
 tity of sulphuric acid. The liquid is then exactly neutralized, while 
 boiling, with caustic baryta, and then evaporated to a syrup after filtra- 
 tion. The residue is precipitated with absolute alcohol, and the pre- 
 cipitate completely extracted with alcohol. The alcohol is entirely 
 distilled from the united alcoholic extracts, and the neutral residue is 
 
 1 Tiirkel, Bioch. Zeitsehr., 20. The statements on the formation of lactic acid 
 in the muscle autolysis are rather conflicting; see Fletcher, Journ. of Physiol., 43. 
 
 2 Embden, Kalberlah and Engel, Bioch. Zeitsehr. 45; Kondo, Und., 45. 
 
 3 See also E. Jungfleisch, Compt. Rend., 139, 140, and 142; Herzog and Slansky, 
 Zeitsehr. f. physiol. Chem., 73. 
 
586 MUSCLES. 
 
 shaken with ether to remove the fat. The residue is dissolved in water 
 and phosphoric acid is added, and the solution repeatedly shaken with fresh 
 quantities of ether, which dissolves the lactic acid. The ether is new 
 distilled from the united ethereal extracts, the residue dissolved in water, 
 and this solution carefully warmed on the water-bath to remove the last 
 traces of ether and volatile acids. A solution of zinc lactate is prepared 
 from this filtered solution by boiling with zinc carbonate, and this is 
 evaporated until crystallization commences, and is then allowed to stand 
 over sulphuric acid. An analysis of the salts is necessary in careful 
 work. In regard to methods for the detection and quantitative estima- 
 tion of lactic acid we must refer to larger hand-books. 
 
 Fat is never absent in the muscles. Some fat is always found in the 
 intermuscular connective tissue; but the muscle-fibers themselves also 
 contain fat. The quantity of fat in the real muscle substance is always 
 small, usually amounting to about 10 p. m. or somewhat more. A con- 
 siderable quantity of fat in the muscle-fibers is found only in fatty degenera- 
 tion. A part of the muscle-fat can be readily extracted, while another 
 part can be extracted only with the greatest difficulty. This latter 
 part, it is claimed, exists finely divided in the contractile substance 
 itself and is richer in free fatty acids, standing, according to Zuntz and 
 Bogdanow, 1 in close relation to the activity of the muscles because 
 it is consumed during work. Lecithin is a regular constituent of the 
 muscles, and it is quite possible that the fat which is difficult of extrac- 
 tion and which is rich in fatty acids depends in part on a decomposition 
 of the lecithin and the phosphatides. Erlandsen has shown that 
 phosphatides of various kinds occur in the muscles, the quantities 
 varying in different muscles. According to him the ox-heart muscle 
 is richer in phosphatides than the muscle of the thigh, and Rubow 2 
 claims that the heart of the dog is richer in phosphatides than the striated 
 muscle. Erlandsen found lecithin and diamino-phosphatide in the 
 heart as well as the thigh-muscle, while the monoamido-phosphatide 
 cuorin, which occurs abundantly in the heart, is found as traces in the 
 thigh-muscle. Costantino 3 has carried on investigations on the divi- 
 sion of the inorganic and organic phosphorus in striated and smooth 
 muscles. 
 
 The Mineral Bodies of the Muscles. The ash remaining after burning 
 the muscle, which amounts to about 10-15 p. m., calculated on the moist 
 muscle, is acid in reaction. The largest constituent of the ash is potas- 
 sium, whose occurrence, according to Macallum, 4 is restricted to the dark 
 
 1 Arch. f. ( Anat. u.) Physiol., 1897. 
 
 2 Erlandsen, Zeitschr. f. physiol. Chem., 51; Rubow, Arch. f. exp. Path. u. Pharm., 
 52. 
 
 1 Bioch. Zeitschr., 43. 
 ♦Journ. of Physiol., 32. 
 
MINERAL BODIES. 587 
 
 diagonal bundles, and phosphoric acid. Next in amount we have sodium 
 and magnesium, and lastly calcium, chlorine, and iron oxide. Sulphates 
 exist only as traces in the muscles, but are formed by the burning of the 
 proteins of the muscles, and therefore occur in abundant quantities in the 
 ash. The muscles contain such a large quantity of potassium and phos- 
 phoric acid, that potassium phosphate seems to be, unquestionably, the 
 predominating salt. Chlorine is found in such insignificant quantities 
 that it is perhaps derived from a contamination with blood or lymph. 
 The quantity of magnesium is, as a rule, considerably greater than that 
 of calcium. Iron occurs only in very small amounts. The water of the 
 muscle occurs in part free and partly as imbibition water of the colloids. 
 According to the investigations of Jensen and Fischer 1 only a small part, 
 a few per cent, of the total water exists in this condition. 
 
 Urano 2 has removed the salts of the intermediary fluid (blood, 
 lymph) from frogs' muscles by treating them with an isotonic cane-sugar 
 solution (of 6 per cent) and in this manner found that the sodium did 
 not belong to the muscle substance itself, but to the intermediary fluid, 
 while at least a small part of the chlorine is a true muscle constituent. 
 He also calculated, from the quantity of sodium, that the intermediary 
 fluid, if it has about the same composition as the muscle plasma, makes 
 up about one-sixth of the volume of the muscle. According to further 
 investigations of Urano the possibility of a disturbance in the osmotic 
 propertiesof the muscle-fibers by the sugar solution is not entirely excluded, 
 and the question whether the muscle-fibers are free from sodium or not 
 has therefore not been positively decided. Fahr's 3 researches make 
 the absence of sodium in frog's muscle very probable. 
 
 The importance of the various mineral bodies for the function of the 
 muscles has been the subject of numerous investigations and by many 
 of these we have obtained further proof, as mentioned in a previous 
 chapter, of the ion action of the electrolytes and the antagonism of 
 different ions. These researches also indicate that each of the ions 
 Na, Ca, and K plays a certain part in the maintenance of the excitability, 
 in the contraction and in the fatigue of the muscle (heart); still these 
 investigations have not led to concordant results, so that we are not yet 
 clear as to the action of these ions. Nevertheless it seems to be estab- 
 lished that the combined action of various ions is a necessity for the nor- 
 mal function of the muscles. It has also been shown that it is possible 
 to maintain the muscle (the heart) in regular activity for a long time by 
 means of a transfusion of liquid saturated with oxygen, and which con- 
 
 Jensen and Fischer, Bioch. Zeitschr., 20. 
 
 * Zeitschr. f. Biol., 50. 
 
 3 Urano, ibid., 51; P'ahr., ibid., 52. 
 
588 MUSCLES. 
 
 tained about 7 p. m. NaCl, besides small amounts of Cad2 (0.2 p. m.), 
 KC1 (0.1 p. m.), and NaHC0 3 (0.1 p. m.). 
 
 The gases of the muscles consist of large quantities of carbon dioxide 
 besides traces of nitrogen. 
 
 In regard to the permeability of the muscles for various bodies there 
 are the complete investigations of Overton. 1 The different sheaths of 
 the muscles, the sarcolemma and perimysium internum, offer no very 
 great resistance to the diffusion of the most soluble crystalloid com- 
 pounds, while the muscle-fibers, on the contrary (exclusive of the sar- 
 colemma), are almost if not entirely impervious to most inorganic com- 
 pounds and to many organic compounds. The muscle-fibers themselves 
 are actually semipermeable structures which are permeable to water 
 but not to the molecules or ions of sodium chloride and of potassium phos- 
 phate. The muscle-fibers, as well as the various sheaths, are impermeable 
 to colloids. 
 
 The behavior of the numerous bodies investigated cannot be discussed 
 in this work. The general rule is as follows: All compounds which, 
 besides having a marked solubility in water, are readily soluble in ethyl 
 ether, in the higher alcohols, in olive-oil and in similar organic solvents, 
 or are not much less soluble in the last-mentioned solvents than in water, 
 pass through the living muscle-fibers with great ease. The greater the 
 difference between the solubility of a compound in water and in the other 
 solvents mentioned, the slower does the passage into the muscle-fibers 
 take place. The permeability changes essentially on the death of the 
 muscle. 
 
 The living muscle-fibers are readily permeable to oxygen, carbon 
 dioxide, and ammonia, while the hexoses and disaccharides do not readily 
 pass into them. It is very remarkable that a great portion of those 
 compounds which take part in the normal metabolism of plants and 
 animals belongs to those bodies to which the muscle-fibers (and also other 
 cells) are entirely or at least nearly impermeable. On the contrary, 
 derivatives can be prepared from these bodies which pass into the cells 
 very readily, and Overton finds that it is not impossible that the organ- 
 ism in part makes use of a similar artifice in order to regulate the concen- 
 tration of the nutritive bodies within the protoplasm. (See Chapter I.) 
 
 Rigor Mortis of the Muscles. If the influence of the circulating 
 oxygenated blood is removed from the muscles, as after the death of 
 the animal or by ligature of the aorta or the muscle-arteries (Stenson's 
 test), rigor mortis sooner or later takes place. The ordinary rigor 
 appearing under these circumstances is called the spontaneous or the 
 
 1 Pfliiger's Arch., 92. See also Hober, ibid., 100, and Hamburger, Osmotischer 
 Druck und Ionenlehre. Bd. 3. 
 
RIGOR MORTIS. 589 
 
 fermentative rigor, because it seems to depend in part on the action of 
 an enzyme. A muscle may also become stiff or other reasons. The 
 muscles may become momentarily stiff by warming, in the case of frogs 
 to 40°, in mammalia to 48-50°, and in birds to 53° C. Distilled water 
 may also produce a rigor in the muscles (water-rigor). Acids, even very 
 weak ones, such as carbon dioxide, may quickly produce a rigor (acid- 
 rigor), or hasten its appearance. A number of chemically different 
 substances, such as chloroform, ether, alcohol, ethereal oils, caffeine, 
 and many alkaloids, produce a similar effect. 
 
 When the muscle passes into rigor mortis it becomes shorter and 
 thicker, harder and non-transparent, and less ductile. The acid part 
 of the amphoteric reaction becomes stronger, which is explained by most 
 investigators by the assumption of a formation of lactic acid. There is 
 hardly any doubt that this increase in acidity may at least in part be 
 due to a transformation of a part of the diphosphate into monophosphate 
 by the lactic acid. The statements as to whether in the rigor mortis 
 muscles, besides acid phosphate also free lactic acid exists or not are 
 rather contradictory; 1 that an acid formation precedes the rigor is gen- 
 erally admitted and this acid formation is now accepted as being in close 
 relation to the rigor. While we used to consider the appearance of a clot 
 consisting of myosin (Kuhne) or cf myogen- and myosin fibrin (v. Furth) 
 as the essential moment for the rigor, we now admit, based upon the 
 investigations of Meigs, v. Furth and Lenk, 2 that the most essential 
 factor is the imbibition of the disdiaclasts, which become broader or 
 shorter, by their taking up of water from the sarcolemma fluid and this 
 action produced by the acid formation. This view stands in accord with 
 the experience on. the imbibition of colloids and muscles in water or calt 
 solutions, in the presence and absence of acid, as well as the fact that the 
 rigor can be retarded by the artificial circulation of blood or by the action 
 of salt solutions, namely by those which contain small amounts of 
 NaHCOs. This also agrees well with the old experience, that the muscle 
 work, which is also connected with a formation of acid, accelerates the 
 appearance of rigor. 
 
 On further post-mortal changes, namely by a further accumulation 
 of acid, a progressive coagulation of the proteins gradually occurs. In 
 this coagulation the ability of the colloid systems to imbibe water 
 
 1 It is impossible to enter into the details of the disputed theories as to the reac- 
 tion of the muscles, etc. We shall only refer to the works of Rohmann, Pfluger's 
 Arch., 50 and 55, and Heffter, Arch. f. exp. Path. u. Pharm., 31 and 38. These 
 works contain also the researches of the earlier investigators more or less completely. 
 
 2 Meigs, Journ. of Physiol. 39 and especially, Amer. Journ. of Physiol., 24 and 
 26; v. Furth and Lenk, Bioch. Zeitschr., 33, and Wien. klin. Wochenschr., 24 (1911). 
 
590 MUSCLES. 
 
 diminishes, water is given off, and a re-imbibition takes place and the 
 so-called " solution of the rigor " appears (v. Furth and Lenk). 
 
 The ordinary rigor is an acid rigor and the same applies, according 
 to Meigs, 1 to the water rigor as a shortening of the muscles takes place 
 when placed in distilled water, by a formation of lactic acid, and because 
 when such a muscle is placed in Ringer's solution the acid is removed 
 and the muscle again expands. 
 
 The views are rather contradictory in regard to the production of heat 
 rigor. According to v. Furth this rigor depends upon the coagulation 
 of certain proteins, and its occurrence at lower temperatures in cold- 
 blooded as compared with warm-blooded animals is due, according to 
 v. Furth, to the fact that in the first a soluble myogen fibrin occurs 
 preformed in the muscle which coagulates at 30-40° C, while in the 
 warm-blooded animals the coagulating substance is musculin (myosin 
 of v. Furth) which coagulates at a higher temperature. According 
 to Inagaki 2 the various stages in contractions occurring on heating a 
 muscle (frog) do not correspond to those of the coagulation of the pro- 
 tein which would occur on heating the muscle plasma, and Meigs has 
 arrived at a similar view. It must be remarked that also a lactic acid 
 formation takes place on heating a muscle, and this prevents an exact 
 comparison of the coagulation of the proteins within and outside 
 of the muscle. The observations of Vernon that the striated and 
 the smooth muscles on heating to between 40 and 50° behave differently, 
 in that the striated become shorter and the smooth become longer, 
 while both kinds become shorter at higher temperatures, indicates against 
 a coagulation at these low temperatures. According to Meigs 3 we must 
 here also admit of an imbibition rigor, due to the formation of lactic acid, 
 and the different behavior of the two kinds of muscle depends upon a 
 different arrangement of their anatomical elements. 
 
 The chemical rigor produced by different chemically active substances 
 is also produced, according to Meigs as well as to v. Furth and Lenk, 
 upon a formation of acid, causing a chemical damage of the muscles, and 
 is to be considered as an iml ibition rigor. 
 
 As it is now generally admitted that the formation of lactic acid dur- 
 ing the death of the muscle is the cause of the muscle rigor, the question 
 arises, from what constituents of the muscle is this acid derived? The 
 most probable explanation is that the lactic acid is produced from the 
 glycogen, as certain investigators, such as Nasse and Werther, have 
 observed a decrease in the quantity of glycogen in rigor of the muscle. 
 
 1 Journ. of Physiol., 39. 
 
 2 Inagaki, Zeitschr. f. Biol., 48; Meigs, Journ. of Physiol., 24. 
 
 3 Vernon, Journ. of Physiol., 24; Meigs, Amer. Journ. of Physiol., 24. 
 
METABOLISM IN THE MUSCLES. 591 
 
 On the other side, Eohm has observed cases in which no consumption 
 of glycogen took place in rigor of the muscle, and he also found that the 
 quantity of lactic acid produced is not proportional to the quantity of 
 glycogen. According to Moscati l the diminution in the glycogen is- 
 independent of the appearance of rigor. It is therefore possible that the 
 consumption of glycogen and the formation of lactic acid in the muscles 
 are two processes independent of each other, and, as above stated in regard 
 to the formation of paralactic acid, the origin of the lactic acid in the 
 muscle is still not positively known. The phosphocarnic acid must 
 also be considered as a mother-substance of the lactic acid, and of the 
 carbon dioxide, also formed in the rigor, as it yields lactic acid as well as 
 carbon dioxide on its cleavage. 
 
 Metabolism in the Inactive and Active Muscles. It is admitted 
 by a number of prominent investigators, Pfluger and Colasanti, 
 Zuntz and Rohrig 2 , and others, that the metabolism in the muscles 
 is regulated by the nervous system. When at rest, when there is no 
 mechanical exertion, there exists a condition which Zuntz and Rohrig 
 have designated " chemical tonus." This tonus seems to be a reflex 
 tonus, for it may be reduced by discontinuing the connection between the 
 muscles and the central organ of the nervous system by cutting through 
 the spinal cord or the muscle-nerves. The possibility of reducing the 
 chemical tonus of the muscles in various ways offers an important means 
 of deciding the extent and kind of chemical processes going on in the 
 muscles when at rest. In comparative chemical investigation of the 
 processes in the active and the inactive muscles several methods of pro- 
 cedure have been adopted. The same active and inactive muscles have 
 been compared after removal, also the arterial and venous muscle-blood 
 in rest and activity, and lastly the total exchange of material, the receipts 
 and expenditures of the organism, have been investigated under these 
 two conditions. 
 
 By investigations according to these several methods it was found 
 that the resting muscle takes up oxygen from the blood and returns to 
 it carbon dioxide, and also that the quantity of oxygen taken up is greater 
 than the oxygen contained in the carbon dioxide eliminated at the same 
 time. The muscle, therefore, holds in some form of combination a part 
 of the oxygen taken up while at rest. During activity the exchange of 
 material in the muscle, and therewith the exchange of gas, is increased. 
 
 1 Nasse, Beitr. z. Physiol, der kontrakt. Substanz, Pfluger's Arch., 2; Werther, 
 ibid., 46; Bohm, ibid., 23 and 46; Moscati, Hofmeister's Beitrage, 10. 
 
 2 See the works of Pfliiger and his pupils in Pfluger's Arch., 4, 12, 14, 16, and 18; 
 Rohrig, ibid., 4. See also Zuntz, ibid., 12. In regard to the metabolism after curare 
 poisoning, see also Frank and Voit, Zeitschr. f. Biologie, 42, and Frank and Geb- 
 hard, ibid., 43. 
 
592 MUSCLES. 
 
 The animal organism takes up much more oxygen in activity than 
 when at rest, and eliminates also considerably more carbon dioxide. The 
 quantity of oxygen which leaves the body as carbon dioxide during 
 activity is much larger than the quantity of oxygen taken up at the same 
 time; and the venous muscle-blood is poorer in oxygen and richer in 
 carbon dioxide during activity than during rest. The exchange of gases 
 in the muscles during activity is the reverse of that at rest, for the active 
 muscle gives up a quantity of carbon dioxide which does not correspond 
 to the quantity of oxygen taken up, but is considerably greater. It 
 follows from this that in muscular activity not only does oxidation take 
 place, but also splitting processes occur. This also results from the fact 
 that removed blood-free muscles when placed in an atmosphere devoid of 
 oxygen can labor for some time and still yield carbon dioxide (Hermann 1 ). 
 
 During muscular inactivity, in the ordinary sense, a consumption 
 of glycogen takes place. This is inferred from the observations of sev- 
 eral investigators, that the quantity of glycogen is. increased and its cor- 
 responding consumption reduced in those muscles whose chemical tonus 
 is reduced either by cutting through the nerve or for other reasons 
 (Bernard, Chandelon, Vay, 2 and others). In activity this consump- 
 tion of glycogen is increased, and it has been positively proved by the 
 researches of numerous investigators 3 that the quantity of glycogen 
 in the muscles in activity decreases quickly and freely. The sugar is 
 removed from the blood and consumed during activity. 4 The recent 
 investigations of Joh. Muller, Locke and Rosenheim and Camis 5 
 have given direct proof of the consumption of sugar during muscular 
 activity. In experiments on surviving hearts of different animals through 
 which was perfused a salt solution containing sugar, they could detect 
 an undoubted consumption of sugar which was quite considerable and 
 which to all appearances was used as material for muscle work. 
 
 The amphoteric reaction of the inactive muscles is changed during 
 
 1 L. Hermann, Unters. iiber d. Stoffwechsel der Muskeln, etc., Berlin, 1867. 
 In regard to gas exchange in removed muscles, see also J. Tissot, Arch, de Physiol. 
 (5), 6 and 7, and Compt. Rend.. 120. 
 
 1 Chandelon, Pfluger's Arch.. 13; Vay, Arch. f. exp. Path. u. Pharm., 34, which 
 also contains the pertinent literature. 
 
 * Nasse, Pfluger's Arch., 2; Weiss, Wien. Sitzungsber., 64; Kiilz, in Ludwig's 
 Festschrift, Marburg, 1890; Marcuse, Pfluger's Arch., 39; Manchd, Zeitschr. f. Bio- 
 logie, 25; Moratand Dufour, Arch dc Physiol. (5), 4. 
 
 4 Chauveau and Kaufmann, Compt. Rend., 103, 104, and 105; Quinquaud, Maly's 
 Jahresber., 16; Morat and Dufour, 1. c; Cavazzani, Centralbl. f. Physiol., 8; Seegen, 
 '' Die Zuckerbildung im Thicrkorpcr," Berlin, 1890, Centralbl. f. Physiol., 8, 9, and 
 10; Arch. f. < Anat. u.) Physiol . 1895 and 1896; Pfluger's Arch., 50. 
 
 6 Joh. Muller, Zeitschr. f allgem. Physiol., 3; Camis, ibid., 8; Locke and Rosen- 
 heim, Journ. of Physiol , 36. 
 
LACTIC ACID FORMATION IN ACTIVE MUSCLES. 593 
 
 activity to an acid reaction (Du Bois-Reymond and others), and the 
 acid reaction increases, to a certain point, with the work. The quickly 
 contracting pale muscles produce, according to Gleiss, 1 more acid dur- 
 ing activity than the more slowly contracting red muscles. Numerous 
 investigations have been carried out on the cause of this increased acid 
 reaction, using the muscles in situ and also upon removed muscles and 
 rather contradictory results have been obtained. Some have found a 
 diminution in the amount of lactic acid in the active muscle while others 
 have found an increase. 2 The work of Fletcher and Hopkins 3 is of 
 great importance in this disputed question, in which they show that in 
 the removal of the muscle, and in its preparation for the testing for lactic 
 acid several sources of error are possible. The mechanical irritation 
 as well as warming or treating the muscle with alcohol (not ice-cold) 
 can lead to a formation of lactic acid. It was also shown that the 
 absence of oxygen accelerated the formation or accumulation of lactic 
 acid, while an abundance of oxygen had the opposite effect. 
 
 It is evident that the experiments with the muscles in situ — in other 
 words, with muscles through which blood is passing — cannot yield any 
 conclusion to the above question, as the lactic acid formed during work 
 may perhaps be removed by the blood. The following objections can 
 be made against those experiments in which lactic acid has been found, 
 after moderate work, in the blood or the urine, as also especially against 
 the experiments with removed active muscles, namely, that in these cases 
 the supply of oxygen to the muscles was not sufficient, and that the 
 lactic acid formed thereby is not, in accordance with the views of Hoppe- 
 Seyler, a perfectly normal process. The same is probably true also for 
 the formation of lactic acid with excessive work during life, and Zillessen 4 
 has found that the artificial cutting off of the oxygen supply in the 
 muscles during life, that more lactic acid was formed than under normal 
 conditions. Other observations indicate a formation of lactic acid dur- 
 ing activity. Thus Spiro and recently also H. Fries 5 found an increase 
 in the quantity of lactic acid in the blood during work. Colasanti 
 and Moscatelli found small quantities of lactic acid in human urine 
 after strenuous marches, and Werther 6 observed an abundance of lactic 
 acid in the urine of frogs after tetanization. 
 
 1 Pfliiger's Arch., 41. 
 
 2 Astaschewsky, Zeitschr. f. physiol. Chem., 4; Warren, Pfliiger's Arch., 24, 
 Monari, Maly's Jahresber., 19; Heffter, Arch. f. exp. Path. u. Pharm., 31; Marcuse, 
 1. c; Werther, Pfliiger's Arch., 46; Spiro, Zeitschr. f. physiol. Chem., 1; Colasanti. 
 
 3 Journ. of Physiol., 35. 
 
 4 Hoppe-Seyler, 1. c. and Zeitschr. f. physiol. Chem., 19, 476; Zillessen, ibid., 15. 
 6 Spiro, Zeitschr. f. physiol. Chem. 1; Fries, Bioch. Zeitschr., 35. 
 
 •Colasanti and Moscatelli; Maly's Jahresb.. 17, 212; Werther, Pfliiger's Arch., 46. 
 
594 MUSCLES. 
 
 According to Siegfried the amount of phosphocarnic acid is dimin- 
 ished during activity. Macleod * claims that this is true only for 
 intense muscular activity, and the mother-substance of lactic acid can 
 at least in part be phosphocarnic acid. The question as to the forma- 
 tion of lactic acid during activity, and the origin of the phosphocarnic 
 acid is certainly in many points somewhat undecided; the general 
 view seems to be, that during work lactic acid is formed, which transforms 
 a part of the diphosphates into monophosphates. 
 
 The amount of proteins in the removed muscles is, according to the 
 earlier investigators, decreased by work. The correctness of this state- 
 ment is, however, disputed by other investigators. Earlier reports in 
 regard to the nitrogenous extractive bodies of the muscle in rest and 
 in activity are likewise uncertain. According to the recent researches 
 of Monari 2 the total quantity of creatine and creatinine is increased by 
 work, and indeed the amount of creatinine is especially augmented 
 by an excess of muscular activity. The creatinine is formed essentially 
 from the creatine. The investigations of Graham Brown and Cathcart 
 on removed nerve-muscle preparations of frogs, and those of S. Weber 3 
 on hearts, indicate an increase in the formation of creatine and creatinine 
 during work. Weber found that the working heart gave up creatine 
 (and creatinine) to Ringer's solution, and indeed much more when 
 strongly active than during a lesser activity. An increased creatinine 
 elimination after work does not occur according to several investigators 
 (see Chapter XIV) and according to Pekelharing and v. Hoogenhuyze 
 with ordinary muscle activity neither an increased creatine formation 
 nor an increased creatinine elimination takes place. In the tonic 
 contraction the creatine is formed from the proteins, and correspond- 
 ingly according to Pekelharing and Harkink 4 the creatinine elimina- 
 tion is increased under the influence of the muscle tonus. The purine 
 bases are produced, according to Burian, in the muscles themselves, 
 also in activity, and an increased formation takes place during work 
 due to a re-formation. Scaffidi 5 found on the contrary, with frogs and 
 tortoise, during work that a diminution of the total quantity of purine 
 bases occurred and indeed not the free but the combined purines. 
 
 Attempts have been made to solve the question relative to the 
 behavior of the nitrogenized constituents of the muscle at rest and during 
 
 1 Siegfried, Zeitschr. f. physiol. Chem., 21; Macleod, ibid., 28. 
 
 5 Maly's Jahresber., 19, 296. 
 
 » Cathcart and Graham Brown, Journ. of Physiol., 37; Weber, Arch. f. exp. Path, 
 u. Pharm., 58. 
 
 * Pekelharing and v. Hoogenhuyze, Zeitschr. f. physiol. Chem.. 64, with Harkink, 
 ibid., 75. 
 
 6 Burian, Zeitschr. f. physiol. Chem., 43; Scaffidi; Bioch. Zeitschr., 30. 
 
NITROGEN CATABOLI8M IN ACTIVE MUSCLES. 595 
 
 activity by determining the total quantity of nitrogen eliminated under 
 these different conditions of the body. While formerly it was held with 
 Liebig that the elimination of nitrogen by the urine was increased by 
 muscular work, the researches of several experimenters, especially those 
 of Voit on dogs, and Pettenkofer and Voit on men, have led to quite 
 different results. They have shown, as has also lately been confirmed 
 by other investigators, especially I. Munk and Hirschfeld, 1 that during 
 work no increase, or only a very insignificant increase, in the elimination 
 of nitrogen takes place. 
 
 We should not omit to mention the fact that a series of experiments 
 has been made showing a significant increase in the metabolism of pro- 
 teins during or after work. There are for example the observations 
 of Flint and of Pavy on a pedestrian, v. Wolff, v. Funke, Kreuzhage, 
 and Kellner on a horse, and Dunlop and his collaborators on working 
 human beings, and of Krummacher, Pfluger, Zuntz and his pupils, 2 
 and others. The researches on the elimination of sulphur during rest 
 and activity also belong to this category. The elimination of nitrogen 
 and sulphur runs parallel with the metabolism of proteins in resting and 
 active persons, and the quantity of sulphur excreted by the urine is there- 
 fore also a measure of the protein catabolism. The earlier researches 
 of Engelmann, Flint, and Pavy, as well as the more recent ones of Beck 
 and Benedict, 3 and Dunlop and his collaborators, show an increased 
 elimination of sulphur during or after work, and this indicates an increased 
 protein metabolism because of muscular activity. 
 
 That an increased destruction of protein is not necessarily produced 
 by work follows from the observations of Caspari, Bornstein, Kaup, 
 Wait, A. Loewy, Atwater and Benedict, 4 that a retention of nitrogen 
 and a deposition of protein occur during work. The discordant observa- 
 tions on the protein destruction during, and caused by, work are not 
 directly in opposition to each other, because the extent of protein 
 metabolism is dependent upon many conditions, such as the quantity 
 
 1 Voit, Untersuchungen iiber den Einfluss des Kochsalzes, des Kaffees und der 
 Muskelbewegungen auf den Stoffwechsel (Miinchen, 1860), and Zeitschr. f. Biologie, 
 2; J. Munk, Arch. f. (Anat. u.) Physiol., 1890 and 1896; Hirschfeld, Virchow's Arch., 
 121. 
 
 2 Flint, Journ. of Anat. and Physiol., 11 and 12; Pavy, The Lancet, 1876 and 1877; 
 v, Wolff, v. Funke, Kellner, cited from Voit, Hermann's Handb., 86, 197; Dunlop 
 Noel-Paton, Stockman, and Maccadam, Journ. of Physiol., 22; Krummacher, Zeitschr. 
 f. Biologie, 33; Pfluger, Pfliiger's Arch., 50; Zuntz, Arch. f. (Anat. u.) Physiol., 1894. 
 
 3 Engelmann. Arch. f. (Anat. u.) Physiol., 1871; Beck and Benedict, Pfliiger's 
 Arch., 54, and also footnote 2. 
 
 "Caspari, Pfluecr's Arch., 83; Bornstein, ibid.; Kaup, Zeitschr. f. Biologie, 43; 
 Wait, U. S. Depart Agricult. Bulletin, 89; (1901) Atwater and Benedict, ibid., Bull., 
 69 (1S99); Loewy, Arch. f. (Anat. u.) Physiol., 1901. 
 
596 MUSCLES. 
 
 and composition of the food, the condition of the adipose tissue of the 
 body, the action of the work upon the respiratory mechanism, etc., all 
 of which have an influence on the results of the experiments. 
 
 What has been said above in regard to the protein catabolism dur- 
 ing muscular activity only applies for the metabolism experiments 
 carried on in the generally accepted manner. Thomas 1 has made an 
 experiment, under Rubner's direction, on the action of work upon the 
 nitrogen elimination upon a person when the nitrogen minimum was 
 reduced to the w r ear and tear quota (see Chapter XVII), and this experi- 
 ment seems to indicate a small increase in the nitrogen elimination due 
 to work. 
 
 The older investigations on the amount of fat in muscles removed 
 after activity and after rest have not led to any definite results. Accord- 
 ing to the investigations of Zuntz and Bogdanow, 2 the fat belonging 
 to the muscle-fibers, which is extracted with difficulty, takes part in work. 
 Besides these there are several researches by Voit, Pettenkofer and 
 Voit, J. Frentzel, 3 and others which make an increased destruction 
 of fat during work probable or proved. 
 
 If the results of the investigations thus far made of the chemical 
 
 processes going on in the active and inactive muscles were collected, 
 
 we would find the following characteristics for the active muscle: The 
 
 active muscle takes up more oxygen and gives off more carbon dioxide 
 
 than the inactive muscle; still the elimination of carbon dioxide is increased 
 
 considerably more than the absorption of oxygen. The respiratory 
 
 COo 
 quotient, ~pr~, is found to be regularly raised during work; yet this rise, 
 
 which will be explained in detail in a following chapter on metabolism, 
 can hardly be conditioned on the kind of processes going on in the muscle 
 during activity with a sufficient supply of oxygen. In work a consump- 
 tion of carbohydrates, glycogen, and sugar takes place. The acid reac- 
 tion of the muscle becomes greater with work. In regard to the extent 
 of a re-formation of lactic acid opinion is divided. An increased con- 
 sumption of fat has occasionally been observed. On the behavior of 
 creatine Cor creatinine) and purine bodies the statements are some- 
 what divergent. Protein metabolism has been found increased in cer- 
 tain series of experiments and not in others; but an increased elimina- 
 tion of nitrogen as a direct consequence of muscular exertion has thus 
 far not been positively proved. 
 
 In close connection with the above-mentioned facts there is the 
 
 1 Arch. f. (Anat. u.) Physiol. 1910, Supplelbd- 
 
 *Ibid., 1897. 
 
 • Pfliiger's Arch., 68. 
 
MUSCLE WORK. 597 
 
 question as to the material basis of muscular activity so far as it has 
 its origin in chemical processes. In the past the generally accepted 
 opinion was that of Liebig, that the source of muscular action con- 
 sisted of a catabolism of the protein bodies; to-day another generally 
 accepted view prevails. FlCK and Wislicenus 1 climbed the Faulhorn 
 and calculated the amount of mechanical force expended in the attempt. 
 With this they compared the mechanical equivalent transformed in the 
 same time from the proteins, calculated from the nitrogen eliminated in 
 the urine, and found that the work really performed was not by any 
 means compensated by the consumption of protein. It was, therefore, 
 proved by this that proteins alone cannot be the source of muscular 
 activity and that this depends in great measure on the metabolism of 
 non-nitrogenous substances. Many other observations have led to the 
 same result, especially the experiments of Voit, of Pettenkofer and Voit, 
 and of other investigators, whose observations show that while the 
 elimination of nitrogen remains unchanged, the elimination of carbon 
 dioxide during work is very considerably increased. It is also gen- 
 erally considered as positively proved that muscular work is produced, 
 at least in greatest part, by the catabolism of non-nitrogenous substances. 
 Nevertheless there is no warrant for the statement that muscular activity 
 is produced entirely at the cost of the non-nitrogenous substances, 
 and that the protein bodies are without importance as a source of 
 energy. 
 
 The investigations of Pfluger 2 are of great interest in this connec- 
 tion. He fed a bulldog for more than seven months with meat which 
 alone did not contain sufficient fat and carbohydrates even for the pro- 
 duction of heart activity, and then let him work very hard for periods 
 of 14, 35, and 41 days. The positive result obtained by these series 
 of experiments was that " complete muscular activity may be effected 
 to the greatest extent in the absence of fat and carbohydrates," and the 
 ability of proteins to serve as a source of muscular energy cannot be 
 denied. 
 
 The nitrogenous as well as the non-nitrogenous nutriments may serve 
 as a source of energy; but the views are divided in regard to the relative 
 value of these. Pfluger claims that no muscular work takes place 
 without a decomposition of protein, and the living cell-substance prefers 
 always the protein and rejects the fat and sugar, contenting itself with 
 these only when proteins are absent. Other investigators, on the con- 
 trary, believe that the muscles first draw on the supply of non-nitrogenous 
 
 1 Vierteljahrsschr. d. Zurich, naturf. Gesellsch., 10, cited from Centralbl. f. d. med. 
 Wiss., 18G6, 309. 
 
 2 Pfliiger's Arch., 50. 
 
598 MUSCLES. 
 
 nutriments, and according to Seegen, Chauveau, and Laulani6 1 
 the sugar is the only direct source of muscular force. The last-men- 
 tioned investigator holds that the fat is net directly utilized for work, 
 but only after a previous conversion into sugar. Zuntz and his collabora- 
 tors have made strong objections to the correctness of such a view. If 
 according to Zuntz, the fat must be first transformed into sugar before 
 it can serve as the source of muscular work, a definite expenditure of 
 force must require about 30 per cent more energy with fatty food than 
 it does with carbohydrates; but this is not the case. The investiga- 
 tions of Zuntz (together with), Loeb, Heinemann, Frentzel and Reach 
 show that all foodstuffs have nearly the same power of serving as the 
 material for the work of the muscles. The extensive metabolism investi- 
 gations of Atwater and Benedict 2 have also led to similar results 
 as to the fats being a source of muscular energy. The law of the sub- 
 stitution of the foodstuffs, according to their combustion equivalents, 
 is also true for muscular work, and fat correspondingly acts with its full 
 amount of energy without previously being transformed into sugar. 
 The question which of the foodstuffs the muscle prefers is dependent upon 
 the relative quantities of the same at the disposal of the muscle. A 
 direct substitution of the body material by the bodies supplied as food 
 does not take place in the muscular activity in the ordinary nutritive 
 condition. According to Johansson and Koraen 3 the CO2 excretion 
 produced by certain work is not influenced by the supply of foodstuffs 
 (protein or sugar). 
 
 Siegfried considers, as above stated, the phosphocarnic acid as a source of 
 energy. According to his and Kruger's 4 researches, phosphocarnic acid, which 
 yields on cleavage, among other bodies, carbon dioxide, occurs in part preformed 
 in the muscle, and in part as a hypothetical aldehyde compound of the same — 
 a compound which forms phosphocarnic acid on oxidation. Siegfried therefore 
 makes the suggestion that in the resting muscle, which requires more oxygen 
 than exists in the carbon dioxide eliminated, this reducing aldehyde substance is 
 gradually oxidized to phosphocarnic acid, which is used in the activity of the 
 muscle with the splitting off of carbon dioxide. 
 
 Quantitative Composition of the Muscle. A large number of analyses 
 have been made of the flesh of various animals for purely practical 
 purposes, in order to determine the nutritive value of different varieties 
 
 1 See Seegen, footnote 4, page 592. The works of Chauveau and his collaborators 
 are found in Compt. Rend., 121, 122, and 123; Laulanie, Arch, de Physiol. (5), 8. 
 
 - Loeb, Arch. f. (Anat. u.) Physiol., 1894; Heinemann, Pfliiger's Arch., 83; Frentzel 
 and Reach, ibid.; Atwater and Benedict, U. S. Dept. of Agric, Bull. 136, and Ergeb- 
 nisse der Physiologic, 3. 
 
 • Skand. Arch. f. Physiol., 13. 
 
 * Zeitschr. f. physiol. Chera., 22. 
 
QUANTITATIVE COMPOSITION OF THE MUSCLES. 
 
 599 
 
 of meat; but their are no exacl scientific analyses with sufficient regard 
 to the quantity of different protein bodies and the remaining muscle 
 constituents, that is, these analyses are incomplete or of little value. 
 We will only give a few of the results of the work of various investigators. 
 The figures are parts per 1000. 
 
 Muscles of Muscles of Muscles of 
 
 Mammals. Birds. Cold-boolded 
 
 Animals. 
 
 Solids 217-278 225-282 200 
 
 Water 722-783 717-773 800 
 
 Organic bodies 207-263 217-263 180-190 
 
 Inorganic bodies 10-15 '0-19 10-20 
 
 Myosin 30-106 29 .8-1 10 29 . 7-87 
 
 Struma substance (D\nilewski) 78-161 88.0-184 70.0-121 
 
 Creatine 2-4.5 3-4.9 2.3-7 
 
 Carnosine 1 . 3-4 — — 
 
 Carnitine 0.19 — — 
 
 Purinebases 1.3-1.7 0.7-1.3 0.53-0.88 
 
 Inosinic acid (barium salt) 0.1 0.1-0.3 — 
 
 Phosphocarnic acid . 57-2 .4 — — 
 
 Inosite 0.03 — — 
 
 Glycogen 1-37 — — 
 
 lactic acid . 4-0 .7 — — 
 
 Of the mineral substances the largest part consists of phosphoric 
 acid 3.4-4.8 p. m. and potassium 3-4 p. m. The amount of sodium 
 is ordinarily only |-J of that of the potassium. Pork, according to 
 Katz, 1 who has carried out complete investigations as to the quantity 
 of mineral constituents of the human muscle and of other animals, is 
 considerably richer than other varieties of meat, in sodium than potas- 
 sium. The quantity of chlorine, which is also variable, was found by 
 Magnus-Levy to be 2.4 p. m. (calculated as NaCl) for the human heart 
 muscle and 1.004 p. m. in other muscles. The amount of Ca and Mg 
 was found by him to be equal to 0.019 and 0.174 p. m. respectively in the 
 heart muscle and 0.065 and 0.215 p. m. respectively in other muscles, 
 v. Moraczewski obtained higher results for the Ca content of the human 
 heart muscle, namely 0.07 p.m. Gley and Richaud 2 found 0.25-0.26 
 p. m. Ca in the heart muscle of the dog, and 0.089-0.248 p. m. Ca in that 
 from the rabbit. The magnesium content of the muscles seems, with 
 the exception of the haddock, eel and pike (Katz), to be greater than the 
 calcium content. The statements differ very considerably in regard 
 to the iron content. Thus Schmey 3 found 0.0793 p. m. iron in the human 
 muscle, while Magnus-Levy found 0.253 p. m., and in the human heart 
 
 1 Katz, Pfluger's Arch., 63; see also Schmey, Zeitschr. f. physiol. Chem., 39. 
 
 2 Magnus-Levy, Bioch. Zeitschr. 24; v. Moraczewski, Zeitschr. f. physiol. Chem. 
 23; Gley and Richaud, Journ. de Physiol, et de Path., 12. 
 
 3 Zeitschr. f. physiol. Chem. 39; Magnus-Levy, 1. c. 
 
600 MUSCLES. 
 
 muscle only 0.067 p. m. iron. Other investigators have only found 0.014- 
 0.035 p. m. iron in the muscle. 
 
 In the table which is given above, no results are given as to the estimates 
 of fat. Owing to the variable quantity of fat in meat it is hardly possible 
 to quote a positive average for this substance. After most careful 
 efforts to remove the fat from the muscles without chemical means, it 
 has been found that a variable quantity of intermuscular fat, which does 
 not really belong to the muscular tissue, always remains. The smallest 
 quantity of fat in the muscles from lean oxen is 6.1 p. m. according to 
 Grouven, and 7.6 p. m. according to Petersen. This last observer 
 also regularly found a smaller quantity of fat, 7.6-8.6 p. m., in the 
 fore quarters of oxen, and a greater amount, 30.1-34.6 p. m., in the hind 
 quarters of the animal, but this could not be substantiated by Steil. 1 
 A small quantity of fat has also been found in the muscle of wild animals. 
 B. Konig and Farwick found 10.7 p. m. fat in the muscles of the extrem- 
 ities of the hare, and 14.3 p. m. in the muscles of the partridge. The 
 muscles of pigs and fattened animals are, when all the adherent fat is 
 removed, very rich in fat, amounting to 40-90 p. m. The muscles of 
 certain fishes also contain a large quantity of fat. According to Almen, 
 in the flesh of the salmon, the mackerel, and the eel there are contained 
 respectively 100, 164, and 329 p. m. fat. 2 
 
 The quantity of water in the muscle is liable to considerable variation. 
 The quantity of fat has a special influence on the quantity of water, and 
 one finds, as a rule, that the flesh which is deficient in water is correspond- 
 ingly rich in fat. The quantity of water does not depend upon the amount 
 of fat alone, but upon many other circumstances, among which must 
 be mentioned the age of the animal. In young animals, the organs 
 in general, and therefore also the muscles, are poorer in solids and richer 
 in water. In man the quantity of water decreases until mature age, 
 but increases again toward old age. Different muscles have also a 
 different water content and the uninterruptedly active heart is the 
 richest muscle in water. In man, Magnus-Levy found 748 p. m. water 
 in the heart, and 722 p. m. in the other muscles. That the quantity 
 of water may vary independently of the amount of fat is strikingly shown 
 by comparing the muscles of different species of animals. In cold-blooded 
 animals the muscles generally have a greater quantity of water, in birds 
 a lower. The comparison of the flesh of cattle and fish shows very strik- 
 ingly the different amounts of water (independent of the quantity of fat) 
 
 1 See Steil, Pfluger's Arch., 61. 
 
 2 In regard to the literature and complete reports on the composition of flesh of 
 various animals, see Konig, Chemie der menschlichen Nahrungs- und Genussmittel, 
 5. Aufl. 
 
SMOOTH MUSCLES. 601 
 
 in the flesh of different animals. According to the analysis of Almen l 
 the muscles of lean oxen contain 15 p. m. fat and 707 p. m. water: the 
 flesh of the pike contains only 1.") p. m. fat and 839 p. m. water. 
 
 For certain purposes, as, for example, in experiments mi metabolism, it is 
 important to know the elementary composition of flesh. In regard t<> the quan- 
 tity of nitrogen we generally accept Vorr's figure, namely, 3.4 per cent, as an 
 average for fresh lean meat. According to Nowak and BuPPERT - this quantity 
 may vary about 0.6 per cent, and in nunc exact investigations it is therefore 
 necessary to specially determine the nitrogen. Complete elementary analyses 
 of flesh have been made with greal care by AaGUTiNSKy. The average for ox- 
 flesh dried in vacuo and free from fat and with the glvcogen deducted was as fol- 
 lows: C 49.G; H 6.9; X 15.3; O+S 23.0; and ash 5.2 per cent. Kohler 
 found as an average for water and fat-free beef C 49.86; H 6.78; N 15.68; O+S 
 2l'.;i per cent, which are very similar results. This investigator also made similar 
 analyses of the flesh of various animals and determined the calorific value of the 
 ash- and fat-free dried meat substance. This value was, per gram of substance, 
 5509-5677 cal. The relation of the carbon to nitrogen, which ARGUTixsKy calls 
 the "flesh quotient," is on an average 3.24 : 1. From Kohler's analyses the 
 average for beef is 3.15 : 1 and for horse-flesh 3.38: 1. Max Muller has shown 
 with experiments on dogs, that the flesh of the same individual shows some varia- 
 tion in this quotient after different foods. According to Salkowski, of the total 
 nitrogen of beef 77.4 per cent was insoluble proteins, 10.08 per cent soluble pro- 
 teins, and 12.52 per cent other soluble bodies. Frextzel and Schreuer 3 find 
 that about 7.74 per cent of the total nitrogen belongs to the nitrogenous 
 extractives. 
 
 Smooth Muscles. 
 
 The smooth muscles have a neutral or alkaline reaction (Du Bois- 
 Reymond) when at rest. During activity they are acid, which is inferred 
 from the observations of Bernstein, who found that the almost con- 
 tinually contracting sphincter muscle of the Anodonta is acid during 
 life. The smooth muscles may also, according to Heidenhain and 
 Kuhne, 4 pass into rigor mortis and thereby become acid. A spontaneous 
 but slowly coagulating plasma has also been observed in several cases. 
 
 In regard to the proteins of the smooth muscles we have the earlier 
 accounts of Heidenhain and Hell wig; 5 but they were, first carefully 
 studied according to newer methods by Munk and Velichi. 6 These 
 
 1 Nova Act. Reg. Soc. Scient. Upsal., Vol. extr. ord., 1877; also Maly's Jahresber., 7. 
 2 Voit, Zeitschr. f. Biologie, 1; Huppert, ibid., 7; Nowak, Wien. Sitzungsber., 64, 
 Abt. 2. 
 
 3 Argutinsky, Pfliiger's Arch., 55; Kohler, Zeitschr. f. physiol. Chem., 31; Sal- 
 kowski, Centralbl. f. d. mcd. Wissensch., 1S94; Frentzel and Schreuer, Arch. f. (Anat. 
 u.) Physiol., 1902; Muller, Pfliiger's Arch., 116. 
 
 4 Du Bois-Reymond in Xasse, Hermann's Handb., 1, 339; Bernstein, ibid., Heiden- 
 hain, ibid., 340, with Helhvig, ibid., 339; Kuhne, Lehrbuch, 331. 
 
 5 Heidenhain in Xasse, Hermann's Handb., 1, 340, with Helhvig, ibid., 339; Kuhne, 
 Lehrbuch, 331. 
 
 8 Munk and Velichi, Centralbl. f. Physiol., 12. 
 
602 MUSCLES. 
 
 experimenters prepared a neutral plasma from the gizzard of geese, 
 according to v. Furth's method. This plasma coagulated spon- 
 taneously at the temperature of the room, although slowly. It con- 
 tained a globulin, precipitated by dialysis, which coagulated at 55-60° 
 C. and also showed certain similarities with Kuhne's myosin. A spon- 
 taneously coagulating albumin, which differed from myogen (v. Furth) 
 by coagulating at 45-50° C, and which passes by spontaneous coagula- 
 tion into the coagulated modification without a soluble intermediate 
 product, exists in still greater quantities in this plasma. Alkali albu- 
 minates do not occur, but a nucleoprotein is found, which exists in about 
 five times the quantity as compared with striated muscles. Nucleon 
 is, according to Panella, 1 a normal constituent of smooth muscles and 
 occurs in larger amounts than in striated muscles. 
 
 Recent investigations of Bottazzi and Cappelli, VincENT and 
 Lewis, Vincent and v. Furth, 2 some on the muscles of warm-blooded 
 and some on those of lower animals, have led to dissimilar results, but 
 they substantiate, as a whole, the observations of Munk and Velichi. 
 Besides the nucleoproteins the smooth muscles contain two bodies 
 corresponding in coagulation temperature to musculin and myosinogen 
 (myogen, v. Furth), but they are not identical therewith. Haemo- 
 globin occurs in the smooth muscles of certain animals, but is absent in 
 others. In the smooth muscles (in certain varieties of animals) creatine, 
 creatinine, hypoxanthine, taurine, inosite, glycogen, and lactic acid have 
 been found. Purine bases, especially xanthine also occur accord- 
 ing to Buglia and Costantino but the quantity is smaller than in 
 striated muscles. This applies at least to the total quantity while the 
 amount of free purine bases, according to Scafpidi, 3 in the smooth 
 muscles is greater than in the striated muscles. Creatine and carnosine 
 are less abundant in the smooth muscles than in the striated muscles. 
 The first are richer in diamino-acid than in monamino-acid-nitrogen 
 than the striated muscles (Buglia and Costantino). 
 
 In regard to the mineral constituents, Costantino has found that 
 the smooth muscles are richer in chlorine, namely 0.84-1.3 p. m., than 
 the striated muscles with 0.25-0.46 p. m. According to older state- 
 ments the sodium compounds exceed the potassium compounds but 
 Costantino 4 could not substantiate this. He found, namely, no general 
 
 1 Maly's Jahresber., 34. 
 
 1 Bottazzi, Centralbl. f. Physiol., 15; Vincent and Lewis, Journ. of Physiol., 26; 
 Vincent, Zeitschr. f. physiol. Chern., 34; v. Furth, ibid., 31. 
 
 ' Scaffidi, Bioch. Zeitschr. 33; Buglia and Costantino, Zeitschr. f. physiol. Chem. 
 83, 81 and 82. 
 
 4 Costantino, Bioch. Zeitschr. 37; See also Meigs and Ryan, Journ. of biol. 
 Chem. 11. 
 
SMOOTH MUSCLES. 603 
 
 difference in the proportion K : Na in the smooth and striated muscles. 
 According to Saiki 1 magnesium does not occur to a greater extent 
 than calcium in the smooth muscles of the stomach or the bladder of 
 pigs. The same investigator found 801-811 p. m. water and 199-189 
 p. m. solids in these muscles. 
 
 Henze found abundance of taurine in the muscles of Octopods, 5 p. m., but 
 no creatine, which, according to Fre.my and Valenciennes, 2 occurs in the muscles 
 of Cephalopods. He also found no glycogen and no paralactic acid, but, on the 
 contrary, small amounts of fermentation lactic acid. The muscles of Octopods 
 are richer in mineral bodies than the muscles of vertebrates, and are nearly twice 
 as rich in sulphur as these. 
 
 1 Journ. of Biol. Chem., 4. 
 
 2 Henze, ibid., 43; Fremy and Valenciennes, cited from Kiihne's Lehrbuch, p. 333. 
 
CHAPTER XI. 
 BRAIN AND NERVES. 
 
 On account of the difficulty in making a mechanical separation 
 and isolation of the different tissue-elements of the central nervous 
 organ and the nerves, we must resort to a few microchemical reactions, 
 pr ncipally to qualitative and quantitative investigations of the different 
 parts of the brain, in order to study the varied chemical composition 
 of the cells and the nerve-axes. This study is accompanied with the 
 greatest difficulty, and although our knowledge of the chemical com- 
 position of the brain and nerves has been somewhat extended by the 
 investigations of modern times, still it must be admitted that this sub- 
 ject is as yet one of the most obscure and complicated in physiological 
 chemistry. 
 
 Proteins of different kinds have been shown to be chemical constit- 
 uents of the brain and nerves, and these are representatives of the same 
 chief groups as occur in the protoplasm. In the brain there occur some 
 proteins which are insoluble in water and neutral salt solutions, and 
 which resemble the stroma substances of the muscles and cells, while 
 other proteins are soluble in water and neutral salt solutions. Among 
 the latter we find mainly nucleoproteins and globulins. The nucleo- 
 protein found by Halliburton and also by Levene 1 in the gray substance 
 contains 0.5 per cent phosphorus and coagulates as 55-60°. Levene 
 obtained adenine and guanine but no hypoxanthine as cleavage prod- 
 ucts. According to Halliburton there are two globulins, namely, 
 the neuroglobulin a, which coagulates at 47°, or as in the case of 
 birds, 50-53°, and the neuroglobulin /3, whose coagulation temperature 
 is 70-75°, but which varies somewhat in different animals. In the frog 
 still another protein body occurs, which coagulates at a still lower tem- 
 perature, about 40°. It must be remarked that the coagulation tempera- 
 ture of a-globulin corresponds with the temperature of the first heat 
 contraction of the nerves of different classes of animals (Halliburton). 
 
 1 Halliburton, On the Chemical Physiology of the Animal's Cell, King's College, 
 London, Physiological Laboratory, Collected Papers No. 1, 1S93, and Ergebnisse der 
 Phyeiologie, 4; Levene, Arch, of Neurology and Psychopathology, 2 (1899). 
 
 604 
 
CONSTITUENTS OF THE NERVOUS SYSTEM. 605 
 
 The gray substance is only slightly richer in proteins than the white 
 substance; but as the neurokeratin, which forms the neurolgia, and as 
 a double sheath envelops the outside of the nerves, belongs in great 
 part, or according to Koch, entirely, to the white substance (Kuhne, 
 and Chittenden, Baumstark l ), the gray substance is actually richer 
 in protein. The same is true also for the nucleoprotein or at least for 
 the nucleins which v. Jaksch found in large amounts in the gray sub- 
 stance. The mixture of amino-acids obtained from the proteins of 
 the gray and white substances has about the same composition (Abder- 
 halden and Weil 2 ). Glycocoll could not be detected in this mixture. 
 
 The so-called protagon has been considered as one of the chief con- 
 stituents, perhaps the only constituent (Baumstark), of the white 
 substance. This protagon, according to most investigators, is only a 
 mixture of phosphatides with cerebron or with a mixture of cerebrosides 
 (see below). Protagon belongs to the so-called brain lipoids, which 
 include three chief groups, phosphatides, cerebrosides and cholesterin and 
 which are contained to a greater extent in the white than in the gray 
 substance. Among the closely studied phosphatides the cephalin 
 seems to occur to the greatest extent in the brain. The lecithin, accord- 
 ing to Frankel, 3 does not occur in the human brain and only in very 
 small quantities in other brains (of sheep and beef). Other brain 
 phosphatides especially described by Thudichum and by Frankel, 4 
 have not been positively proved as chemical individuals. The same 
 is true for the jecorin and the sulphurized lipoids isolated from the 
 human brain and from ox brains. Cholesterin occurs chiefly in the 
 white substance. Fatty acids and neutral fats may be prepared from 
 the brain and nerves; but as these may be readily derived from a decom- 
 position of phosphatides, which exist in the fatty tissue between the 
 nerve- axes, it is difficult to decide what part the fatty acids and neutral 
 fats play as constituents of the real nerve-substance. 
 
 By allowing water to act on the contents of the medulla, round or oblong 
 double-contoured drops or fibers, not unlike double-contoured nerves, are formea. 
 These remarkable formations, which can also be seen in the medulla of the dead 
 nerve, have been called " myelins forms," and they were formerly considered a;s 
 produced from a special body, " myeline." Myeline forms may, however, be 
 obtained from other bodies, such as impure protagon, lecithin, and impure choles- 
 terin, and they depend upon a decomposition of the constituents of the medulla. 
 
 1 Koch, Amer. Journ. of Physiol., 11; Kuhne and Chittenden, Zeitschr. f Biologie, 
 26; Baumstark, Zeitschr. f. physiol. Chem., 9. 
 
 2 v. Jaksch, Pfliiger's Arch. 13; Abderhalden and Weil, Zeitschr. f. physiol. Chem. 
 81 and 83. 
 
 3 Bioch. Zeitschr., 24. 
 
 4 Thudichum, Die chemische Konstitution des Gehirns des Menschen und der 
 Tiere, Tubingen, 1901; S. Frankel, and collaborators, Bioch. Zeitschr., 24 and 28. 
 
606 BRAIN AND NERVES. 
 
 The extractive bodies seem to be almost the same as in the muscles. 
 One finds creatine, which may, however, be absent (Baumstark), purine 
 bases, inosite, choline, paralactic acid (Moriya), phosphocarnic acid, uric 
 acid, and the diamine neuridine, C5H14N2, discovered by Brieger l 
 and which is most interesting because of its appearance in the putrefac- 
 tion of animal tissues or in cultures of the typhoid bacillus. Among 
 the enzymes we must mention catalases, peroxidases, lipases and amylases 
 (Wr6blewski). According to the autolytic experiments of Simon 2 
 a proteolytic enzyme, and an enzyme acting upon the organic phos- 
 phorized substance with the splitting off of phosphoric acid also occur. 
 Under pathological conditions leucine and urea have been found in the 
 brain. Urea is also a physiological constituent of the brain of cartilagi- 
 nous fishes. 
 
 Several of the lipoids occurring in the brain have been discussed in 
 previous chapters, and we will here only speak of the protagon and the 
 cerebrosides. 
 
 Protagon. Under this name Liebreich described a crystalline, 
 nitrogenous and phosphorized substance, which has been found in the 
 brain of man, mammalia and also birds (Argiris) but not in the brain 
 of fishes (Argiris). Its elementary composition, according to Gamgee 
 and Blankenkorn, is C 66.39, H 10.69, N 2.39 and P 1.07 per cent. 
 The results obtained by Cramer correspond well with these figures and 
 he found that protagon also contained sulphur which had previously been 
 found by Ruppel and by Kossel. Recently Wilson and Cramer 3 have 
 reported more recent analyses and they find for protagon, recrystallized 
 4-5 times, almost the same figures as Gamgee and Blankenhorn, namely, 
 C 66.53, H 10.97, N 2.37, P 0.95 and S 0.73 per cent. They consider 
 protagon as a unit substance. 
 
 Gies, Posner and Rosenheim and Tebb 4 dispute the unit nature 
 of protagon. They have found, on fractional precipitation or on recrys- 
 tallization, that protagons can be obtained from the various solvents, 
 having variable composition, especially different P and N contents. They 
 
 1 Brieger, Ueber Ptomaine, Berlin, 1885 and 1886. 
 
 2 \Yr6blewski, Compt. Rend., 152; Fr. Simon, Zeitschr. f. physiol. Chem., 72. 
 
 3 Liebreich, Annal. d. Chem. u. Pharm., 134; Argiris, Zeitschr. f. physiol. Chem., 
 57; Gamgee and Blankenhorn, ibid., 3; Kossel and Freytag, ibid., 17; Ruppel, 
 Zeitschr. f. Biol., 31; Cramer, Journ. of Physiol., 31, with R. A. Wilson, Journ. of 
 exp. Physiol., 1, with Lockhead, Bioch. Journ., 2; also Cramer, Quarterly Journ. of 
 exp. Physiol. 3, and Bioch. Handlexikon (Abderhalden) Bd. 3, which contains the 
 literature. 
 
 * Gies and Lesem, Amer. Journ. of Physiol., 8; Posner and Gies, Journ. of biol. 
 Chem., 1; Gies, ilid.. 3; Rosenheim and Tebb, Journ. of Physiol., 36 and 37, Quarterly 
 Journ. of exp. Physiol., 2, and Bioch. Zeitschr., 25. 
 
PROTAGON. 607 
 
 are, therefore, as are Lesem, Thudichum, WdBNEB and Thierfelder, 1 
 and others, of the opinion that protagon does not exist as a chemical 
 individual, but as a mixture of cerebrosides and phosphatides. It is 
 not easy to come to any decision on this disputed question. On the 
 one hand it must be recalled that several investigators call the impure 
 mixture of brain lipoids, protagon, which they obtain from the solution 
 in warm alcohol on cooking, and which is not purified, and this mixture 
 is claimed to be identical, without sufficient basis, with the substance 
 isolated and analyzed by Gamgee and Cramer. On the other hand 
 it cannot be denied that certain investigations, especially those of ROSEN- 
 HEIM and Tebb speak against the chemical individuality of protagon. 
 These investigations do not exclude the possibility that protagon is a 
 loose chemical combination between cerebroside and phosphatide, which 
 like other readily dissociable combinations, exist only under certain 
 conditions or in certain solvents. It is difficult to understand how a 
 mixture of amorphous or only difficultly crystallizable bodies can be 
 so easily crystallized and yield a product, which with proper care, can be 
 recrystallized repeatedly without changing its composition, and physical 
 properties. According to Rosenheim and Tebb if the proper quantity 
 is used in solution, a crystalline product can be obtained from the decom- 
 position products cf protagon, which has the same specific rotation as 
 protagon and can be repeatedly recrystallized without changing its 
 composition or its optical activity. 2 A further study of these con- 
 ditions would naturally be of great interest. 
 
 As we are not decided whether protagon is only a mixture or is a body 
 contaminated with other substances, it is difficult to decide as to how 
 far the so-called decomposition products exist as preformed constituents 
 of the mixture or whether they are true decomposition products. On 
 boiling with baryta-water protagon yields cerebrosides (see below) and 
 the decomposition products of lecithin, namely, fatty acids, glycerophos- 
 phoric acid, and choline. Kossel and Freytag found three cerebro- 
 sides, namely, cerebrin, kerasix (homocerebrin), and encephalix. 
 According to Koch 3 the protagon molecule contains cerebroside, lecithin 
 and sulphuric acid (in ester-like combination with the cerebroside) 
 besides excess of cerebroside. Of interest is the finding of Kitagawa 
 and Thierfelder 4 that protagon dissolved in methyl alcohol contain- 
 ing chloroform, deposits crusts of cerebron (not pure) after a time at 
 
 1 Lesem, 1. c; Thudichum, 1. c; Worner and Thierfelder, Zeitschr. f. physiol. 
 Chem., 30. 
 
 1 Journ. of Physiol., 3"; Proc. physiol. Soc, January, 1908, p. 3. 
 
 * Zeitschr. f. physiol. Chem., 53. 
 
 * Kitagawa and Thierfelder, ibid., 49; Rosenheim and Tebb, Journ. of Physiol. 37 
 341 and 348. 
 
608 BRAIN AND NERVES. 
 
 ordinary temperature, and that as shown by Rosenheim and Tebb, 
 on dissolving in pyridine at 30° C. and heating or cooling the solution 
 deposits a precipitate of a substance rich in phosphorus. Although we 
 generally consider the phosphorized component of protagon as lecithin, 
 still, according to Rosenheim and Tebb, it is probably a diamido- 
 phosphatide, called sphingomyelin by Thudichum. On boiling protagon 
 with dilute mineral acids it yields galactose, due to the decomposition 
 of the cerebrosides. 
 
 Protagon appears, when dry, as a loose white powder. It dissolves 
 in alcohol of 85 vols, per cent at 45° C, but separates on cooling as a 
 snow-white, flaky precipitate, consisting of globules or groups of fine 
 crystalline needles. On heating to 150° it becomes yellowish, softens 
 at 180° and melts sharply at 200° forming a brown, oily liquid (Cramer). 
 It is difficultly soluble in cold alcohol or ether, but dissolves, at least 
 when freshly precipitated, in ether on warming. It dissolves in methyl 
 alcohol containing chloroform and, as above stated, separates cerebron. 
 Protagon is soluble in pyridine at 30° C, yielding a clear solution, and 
 this solution has a specific rotation (a) D = +6.9 to 7.7° according to 
 the concentration of the solution (Wilson and Cramer). On warm- 
 ing or cooling according to Rosenheim and Tebb, the rotation changes 
 with the separation of sphingomyelin so that it first diminishes in rota- 
 tion, then is zero, and then becomes strongly levorotatory until it reaches 
 — 242°, and finally, when nearly all the sphingomyelin has separated 
 out it becomes constant at about -13.3°. The strong levorotation 
 depends upon the accumulations of doubly refracting spheroid crystals 
 of sphingomyelin. With little water protagon swells up and is partly 
 decomposed. With more water it forms a jelly or pasty-like mass which, 
 with the addition of considerable water, forms an opalescent liquid. 
 
 Protagon can be prepared in the following way: The finely ground 
 brain-mass, as free as possible from blood and membrane, is dehydrated, 
 which is best done by cold acetone or by grinding with burned plaster-of- 
 paris or anhydrous sodium sulphate, and then extracted with ether. 
 The mass is then extracted at 45° C. with 85 vol. per cent alcohol until 
 the filtrate when cooled to 0° C. gives no more precipitate. All the 
 precipitates obtained on cooling to 0° C. are extracted with ether and 
 recrystallized from alcohol. Further details can be found in the cited 
 works of Cramer, Wilson, Gies, Rosenheim and Tebb. 
 
 Among the phosphatides occurring in the brain we must mention besides the 
 lecithin and ccphalin, the following substances. 
 
 Myelin, C.,oII 7 :,XPOio, according to Thudichum, is not well known but is char- 
 acterized by the fact that itfl alcoholic solution is not precipitated by CdCl 2 or 
 PtCl 4 . On" the contrary an alcoholic solution of lead acetate gives a precipitate. 
 The existence of a second monaminomonophosphatide, paramyelin, C38H26NPO9, 
 according to Thudichum, is very improbable. 
 
• CEREBROSIDES. 609 
 
 Sphingomyelin, is a diaminomonophosphatide which Thudichum prepared 
 from the brain and is the chief phosphatide obtainable from the impure protagon 
 mixtures. Rosenheim and Tebb obtained it, as above mentioned, from the 
 protagon. It has been given the formula ( , J H l „ 1 Xd ) 09+H 2 0. As cleavage 
 products an alcohol, Bphingol, iieurin, cholin, according to ROSENHEIM and Tebb, 
 the base sphingosin (see cerebron) and sphingostearic acid have been obtained. 
 Sphingomyelin is soluble with difficulty in cold alcohol but readily soluble in hot 
 alcohol and crystallizes therefrom in needles. It is insoluble in ether. In regard 
 to the specific rotation see above in reference to protagon. Amidomyelin (Thud- 
 ichum) is another diaininomonophosphatide of an unknown constitution and of 
 an uncertain composition. Its existence is uncertain. 
 
 Sahidin was found by Fu.vxkel 1 in the brain, and is a triaminodiphosphatide, 
 whose cadmium compound has the formula CsoHic7X3P20i2.3CdCl 2 . It is a crys- 
 talline powder which is insoluble in water, cold ethyl or methyl alcohol and in 
 ether. It is soluble with difficulty in warm alcohol but readily soluble in chloro- 
 form and hot benzene. It yields saturated and unsaturated fatty acids, choline 
 and glycerophosphoric acid. 
 
 Leucopoliin is an unsaturated phosphatide found b} r Frankel and Elias 2 in 
 the brain and which is a decaminodiphosphatide or a pentaminomonophosphatide. 
 It crystallizes from boiling alcohol on cooling. It does not contain any methylated 
 base but does contain a carbohydrate group. 
 
 Sulphatide is the name given by Koch 3 to a sulphurized and phosphorized 
 product obtained from the human brain which separates from warm pyridine 
 on cooling as a crystalline, granular mass. It contains phosphatide, sulphuric 
 acid and cerebroside and is claimed to be phosphatidesulphuric acid cerebroside. 
 
 Cerebrosides. 
 
 On decomposing protagon (or the protagons), or the brain substance 
 by the gentle action of alkalies we obtain, as cleavage products, as above 
 stated, one or more bodies which Thudichum has embraced under the 
 name cerebrosides. The cerebrosides are nitrogenous substances free 
 from phosphorus, which yield galactose on boiling with dilute mineral 
 acids. With concentrated sulphuric acid they first give a yellow and 
 then a purple-red coloration. With sulphuric acid and cane-sugar 
 they give a purple coloration directly. The cerebrosides isolated from 
 the brain are cerebrin, homocerebrin, phrenosin, kerasin, encephalin, 
 and cerebron, but it must be remarked that there is no doubt that 
 sometimes the same body of varying purity has received different names. 
 According to Levene and Jacobs 4 it must be admitted that the cere- 
 brosides are mixtures of stereoisomeric substances. 
 
 Cerebrin. Under this name W. Muller 5 first described a nitrog- 
 enous substance, free from phosphorus, which he obtained by extracting, 
 with boiling alcohol, a brain-mass which had been previously boiled with 
 
 1 Bioch. Zeitschr. 24. 
 
 2 Frankel and Elias, Bioch. Zeitschr. 28. 
 
 3 Zeitschr. f. physiol. Chem. 70. 
 
 4 Journ. of biol. Chem. 12. 
 B Annal. d. Chem. u. Pharm., 105. 
 
610 BRAIN AND NERVES. 
 
 baryta-water. Following a method essentially the same, but differing 
 slightly, Geoghegan prepared, from the brain, a cerebrin with the 
 same properties as Muller's, but containing less nitrogen. Accord- 
 ing to Parous 1 the cerebrin isolated by Geoghegan, as well as by 
 Muller, consists of a mixture of three bodies, " cerebrin," " homo- 
 cerebrin," and " encephalin." Kossel and Freytag isolated two 
 cerebrosides from protagon which were identical with the cerebrin and 
 homocerebrin of Parous. According to these investigators, the two 
 bodies phrenosin and kerasin, as described by Thudichum, seem to be 
 identical with cerebrin and homocerebrin. 
 
 Cerebrin, according to Parous, has the following composition: C 
 69.08, H 11.47, N 2.13, 17.32 per cent, which corresponds with the 
 analyses made by Kossel and Freytag. No formula has been given to 
 this body. In the dry state it forms a pure white, odorless, and tasteless 
 powder. On heating it melts, decomposes gradually, smells like burned 
 fat, and burns with a luminous flame. Melting-point is 170-176° C. It 
 is insoluble in water, dilute alkalies, or baryta-water; also in cold alcohol 
 and in cold or hot ether. On the contrary, it is soluble in boiling alcohol 
 and separates as a flaky precipitate on cooling, and this is found to con- 
 sist of a mass of globules or grains on microscopical examination. Cere- 
 brin forms a compound with baryta, which is insoluble in water and is 
 decomposed by the action of carbon dioxide. The variety of sugar 
 split off on boiling with mineral acids — the so-called brain-sugar — is, 
 as Thierfelder 2 first showed, galactose. On cleavage with nitric acid 
 fatty acids (stearic acid) were obtained. 
 
 Kerasin (Thudichum), or homocerebrin (Parous), has the following 
 composition: C 70.06, H 11.60, N 2.23, and O 16.11 per cent. Enceph- 
 alin has the composition C 68.40, H 11.60, N 3.09, and O 16.91 per cent. 
 Both bodies remain in the mother-liquor after the impure cerebrin has 
 precipitated from the warm alcohol. These bodies have the tendency 
 of separating as gelatinous masses. Kerasin is similar to cerebrin, but 
 dissolves more easily in warm alcohol and also in warm ether. It may 
 be obtained as extremely fine needles. Encephalin is, Parous thinks, 
 a transformation product of cerebrin. In the perfectly pure state it 
 crystallizes in small lamellae. It swells in warm water into a pasty mass. 
 
 As the purity and the chemical individuality of the above-mentioned bodies 
 is questionable, it is perhaps sufficient in regard to their preparation to simply 
 call attention to the cited works of Muller, Geoghegan, Kossel and Freytag. 
 All these methods split with barium hydroxide and purify the cerebroside by 
 solution in hot alcohol and a precipitation by cooling. 
 
 1 Geoghegan, Zeitschr. f. physiol. Chem., 3; Parcus, Ueber einige neue Gehrinstoffe, 
 Inaug.-Diss. Leipzig, 1881. 
 
 1 Zeitschr. f. physiol. Chem., 14. 
 
CEREBRON. 611 
 
 Whether the above-described cerebrosides are chemical individuals or 
 mixtures, i. e., impure substances, is still undecided. The purest cere- 
 broside thus far investigated is undoubtedly Thierfelder's cerebron, 
 and there is hardly any doubt that the above-mentioned cerebrosides 
 consist essentially of this body. 
 
 Cerebron. This cerebrin, isolated by Thierfelder and Worner 
 and then especially studied by Thierfelder, was first isolated by Gam- 
 gee and called pseudocerebrin by him. Thudichum's phrenosin is, 
 according to Gies, 1 identical with cerebron. Cerebron can be prepared 
 directly from the brain without saponification with baryta, by treat- 
 ment with alcohol containing benzene or chloroform at a temperature 
 of 50°, and hence it is considered as existing preformed in the brain. 
 According to Thierfelder, cerebron has the formula C48H93XO9; it 
 melts at 212°, dissolves in warm alcohol, and separates out on cooling. 
 From proper solvents (acetone or methyl alcohol containing chloroform) 
 it may be separated as small needles or plates. If cerebron is suspended 
 in 85-per cent alcohol at a temperature of 50° C. it balls together in 
 amorphous masses, and from these needle- and leaf-shaped crystals 
 gradually form. It is dextrorotatory, and in about a 5-per cent solu- 
 tion in methyl alcohol (containing 75 per cent chloroform) is (o0d = +7.6 o 
 (Kitagawa and Thierfelder). According to Thierfelder it yields 
 as cleavage products, galactose, cerebronic acid (Thudichum " neuro- 
 stearic acid ") and sphingosin which is in part obtained as such and part 
 as dimethylsphingosin. The base sphingosin, C17H35XO2, discovered 
 by Thudichum, is, according to Thierfelder and to Levene and Jacobs, 2 
 an unsaturated, diatomic, monoamino-alcohol which is readily soluble 
 in alcohol, ether, acetone and petroleum ether but insoluble in water, 
 nas an alkaline reaction and has not been obtained in a crystalline 
 state. The sulphate of dimethylsphingosin crystallizes, on the contrary, 
 from alcohol. Cerebronic acid is an oxyacid with the formula C25H50O3, 
 which is crystalline and which gives a crystalline methyl ester which 
 melts at 65° C. It has been obtained by Levene and Jacobs 3 in part 
 in a dextrorotatory and in part as an inactive form. The first melts 
 at 106-108° and the other at 82-85° C. 
 
 Cerebron can best be prepared, according to Thierfelder and 
 Kitagawa, by decomposing the protagon in methyl alcohol containing 
 
 1 Thierfelder and Worner, Zeitschr. f. physiol. Chem., 30; Thierfelder, ibid., 43, 
 44, 4G, with Kitagawa, ibid., 49; with H. Loening, ibid., 68, 74, 77; Gamgee, Text- 
 book of Physiol. Chem., London, 1880; Thudichum, 1. c; Gies, Journ. of Biol. 
 Chem., 1 and 2. 
 
 2 Thierfelder and O. Riesser and K. Thomas, Zeitschr. f. physiol. Chem., 77; 
 Levene and Jacobs, Journ. of biol. Chem. 11. 
 
 5 Journ. of biol. Chem., 12. 
 
612 BRAIN AND NERVES. 
 
 chloroform (see page 607), and purifying the separated cerebron from 
 contaminating phosphatides by precipitating these with an ammoniacal 
 solution of zinc hydroxide in methyl alcohol, and recrystallizing the cere- 
 bron from methyl alcohol containing chloroform. Thierfelder and 
 Loening have devised another method of purification and at the same 
 time they have suggested another method for preparing cerebron. This 
 method is based upon the resistance of the cerebrosides to baryta and 
 their solubility in hot acetone. It consists in boiling the impure protagon 
 mixture with baryta-water and boiling the insoluble residue with acetone. 
 
 Neuridine, C 6 Hi 4 N2, is a non-poisonous diamine discovered by Brieger, and 
 obtained by him in the putrefaction of meat and gelatin, and from cultures of 
 the typhoid bacillus. It also occurs under physiological conditions in the brain, 
 and as traces in the yolk of the egg. 
 
 Neuridine dissolves in water and yields on boiling with alkalies a mixture 
 of dimethylamine and trimethylamine. It dissolves with difficulty in amyl 
 alcohol. It is insoluble in ether or absolute alcohol. In the free state, neuridine 
 has a peculiar odor, suggesting semen. With hydrochloric acid it gives a compound 
 crystallizing in long needles. With platinic chloride or gold chloride it gives 
 crystallizable double compounds which are valuable in its preparation and detec- 
 tion. 
 
 The so-called corpuscula amylacea, which occur on the upper surface of the 
 brain and in the pituitary gland, are colored more or less pure violet by iodine 
 and more blue by sulphuric acid and iodine. They perhaps consist of the same 
 substance as certain prostatic calculi, but they have not been closely investigated. 
 
 Quantitative Composition of the Brain. The quantity of water is 
 greater in the gray than in the white substance, and greater in new-born 
 or young individuals than in adults. The brain of the foetus contains 
 879-926 p. m. water. The observations of Weisbach 1 show that the 
 quantity of water in the several parts of the brain (and in the medulla) 
 varies at different ages. The following figures are in 1000 parts — A for 
 men and B for women: 
 
 20-30 years. 30-50 years. 50-70 years. 70-94 years. 
 
 A. B. A. B. A. B. A. B. 
 
 White brain-substance. . 695.6 682.9 683.1 703.1 701.9 689.6 726.1 722.0 
 
 Gray " 833.6 826.2 836.1 830.6 838.0 838.4 . 847. S 839.5 
 
 Gyri 784.7 792.0 795.9 772.9 796.1 796.9 802.3 801.7 
 
 Cerebellum 788.3 794.9 778.7 789.0 787.9 784.5 803.4 797.9 
 
 Pons Varolii 734.6 740.3 725.5 722.0 720.1 714.0 727.4 724.4 
 
 Medulla oblongata 744.3 740.7 732.5 729.8 722.4 730.6 736.2 733.7 
 
 The recent investigations of K. Linnert 2 correspond to the above 
 in that the pons and the medulla were found to be next to the white sub- 
 stance, the poorest in water, of the human brain. 
 
 Quantitative analyses of human brains at different ages, namely 
 6 weeks, 2 and 19 years, have been made by Koch and Mann. 3 These 
 analyses show that with increasing age the water, proteins, extractives 
 
 1 Cited from K. B. Hoffmann's Lehrbuch d. Zioch., Wien, 1877, p. 121. 
 
 2 Wien. klin. Wochenschr., 23. 
 
 • Journ. of Physiol., 36, Proc. physiol. Soc, 1907. 
 
COMPOSITION OF THE BRAIN. 613 
 
 and salts diminish relatively, while the phosphatides, cerebrosidea and 
 especially cholesterin strikingly increase. The sulphur of the lipoids 
 increased to the second year, but then existed in the same amounts as at 
 nineteen years. 
 
 Baumstark claims to have found that a part of the cholesterin in the brain 
 occurs in a combined state, perhaps as ester; this view has been found to be 
 incorrect by the recent investigations of Bunz. He obtained from the brain 
 neither esters of cholesterin with higher fatty acids nor other compounds of 
 cholesterin which split on saponification. Tebb "i has also found only free 
 cholesterin. 
 
 According to Frankel, 2 who has fractionally extracted the human 
 brain with various solvents, found 230 p. m. solids in the brain and this 
 consisted of § lipoids and § proteins. Of the lipoids about 17 per cent 
 was cholesterin, 34.482 per cent saturated and 48.293 per cent unsat- 
 urated compounds. The amount of cholesterin in the different parts 
 of the brain was as follows, according to Frankel, Kirschbaum and 
 Linnert. In the cortex 11.5 p. m., in the white substance 24.7 p. m., 
 in the cerebellum 13.1 p. m., and in the bridge and medulla 40.3 p. m., 
 all calculated in the moist substance. 
 
 The analysis of the brain of an epileptic made by Koch 3 is of very great 
 interest. As the protagon is considered by Koch as a mixture, no results for 
 the quantity of protagon are given. As no accurate methods for the estima- 
 tion of the little known bodies cephalin, myelin, phrenosin and kerasin are 
 available, the figures given for these are of little value. The following results 
 are calculated to 1000 parts: 
 
 Corpua Cortex 
 
 Calloaum (prefrontal). 
 
 Water 679.7 841.3 
 
 Protein 32.0 50.0 
 
 Nucleoproteins 37.0 30.0 
 
 Neurokeratin 27.0 (Chittenden) 4.0 (Chittenden) 
 
 Extractives (water-soluble) 15. 1 15.8 
 
 Lecithins 51.9 31.4 
 
 Cephalin and myelin 34.9 7.4 
 
 Phrenosin and kerasin 45.7 15.5 
 
 Cholesterin 48.6 7.0 
 
 Sulphurized substance 14.0 14.5 
 
 Mineral bodies 8.2 8.7 
 
 Pighini and Carbone found that the brains of paralytics were richer in water, 
 considerably richer in cholesterin, but poorer in cephalin than healthy brains. 
 This last corresponds, to the observations of Koch and Mann 4 that the quan- 
 tity of lipoid phosphorus was diminished in paralytics. 
 
 1 Baumstark, Zeitschr. f. physiol. Chem. 9; R. Bunz ibid., 46; Tebb, Joum. of 
 Physiol., 34. 
 
 2 Bioch. Zeitschr., 19, with Kirschbaum and Linnert, ibid., 46. 
 
 3 Amer. Joum. of Physiol., 11. 
 
 4 Pighini and Carbone, Bioch. Zeitschr., 46; Koch and Mann, Arch, of Neurol, 
 and Psychol., 1910. 
 
614 BRAIN AND NERVES. 
 
 According to Fr. Falk j the cerebrosides occur in the medullary 
 nerve fibers as well as in the nerves without medullas. These latter 
 yielded much less substance on extraction than the medullary, namely, 
 11.51 per cent extract as compared to 46.59 per cent. The extract of 
 the first was poorer in cerebrosides, but richer in cholesterin, cephalin 
 and lecithin, as shown by the following figures. 
 
 Non-medullary fibers Medullary fibers in 
 
 in p. m. of the total p. m. of the total 
 
 extract extract 
 
 Cholesterin 470 250 
 
 Cephalin 237 124 
 
 Cerebrosides 60 182 
 
 Lecithins 98 29 
 
 S. Frankel and L. Dimitz 2 find that the spinal marrow contains on 
 an average 740 p. m. water, 180 p. m. lipoids and 80 p. m. protein. The 
 quantity of cholesterin (in the fresh, spinal marrow containing water) 
 is 40 p. m., the unsaturated phosphatide 120 p. m., and the saturated 
 15 p. m. The spinal marrow is the richest part of the nervous system 
 in unsaturated phosphatides and it contains abundance of cephalin. 
 
 According to Noll the white substance of the spinal marrow is some- 
 what richer in protagon than the brain, and in nerve degeneration the 
 quantity of protagon diminishes. The method used by him would not 
 allow of an exact determination of the disputed substance protagon. 
 Mott and Halliburton 3 have also shown that in degenerative diseases 
 of the nervous system, the quantity of substances containing phosphorus 
 diminishes, and that in these cases, especially in general paralysis, choline 
 passes into the cerebrospinal fluid and the blood. In degenerated nerves, 
 the quantity of water increases, and the phosphorus decreases. On 
 comparative investigations of the central nervous system of normal 
 persons, and those afflicted with dementia prsecox (5 cases), Koch 4 found 
 that the variation from the normal composition was not great enough 
 nor so constant that positive conclusions could be drawn therefrom. 
 
 The quantity of neurokeratin in the nerves and the different parts 
 of the brain has been carefully determined by Kuhne and Chittenden. 5 
 They found 3.16 p. m. in the plexus brachialis, 3.12 p. m. in the cortex 
 of the cerebellum, 22.434 p. m. in the white substance of the cerebrum, 
 25.72-29.02 p. m. in the white substance of the corpus callosum, and 
 3.27 p. m. in the gray substance of the cortex of the cerebrum (when 
 
 1 Bioch. Zeitschr., 13. 
 
 * Ibid., 28. 
 
 1 Noll, Zeitschr. f. physiol. Chem., 27; Mott and Halliburton, Philos. Transactions. 
 Ser. B., 191 (1899), and 194 (1901). 
 4 Arch, of Neurology, 3. 
 
 * Zeitschr. f . Biologje, 26. 
 
VISUAL PURPLE. C15 
 
 free as possible from white substance). The white is decidedly richer 
 in neurokeratin than the peripheral nerves or the gray substance. ' Accord- 
 ing to Griffiths, 1 neurochitin replaces neurokeratin in insects and Crus- 
 tacea, the quantity of the first being 10.6-12 p. m. 
 
 The quantity of mineral constituents in the brain amounts to 2.95- 
 7.08 p. m. according to Geoghegan. He found in 1000 parts of the 
 fresh, moist brain 0.43-1.32 CI, 0.956-2.01G P0 4 , 0.244-0.796 C0 3 , 
 0.102-0.220 S0 4 , 0.01-0.098 Fe 2 (P0 4 ) 2 , 0.005-0.022 Ca, 0.016-0.072 
 Mg, 0.58-1.778 K, and 0.450-1.114 Na. The gray substance yields an 
 alkaline ash, the white an acid ash. Magnus-Levy 2 found in fresh brain 
 substance 1.305 p. m. CI, 0.166 p. m. Ca, 0.139 p. m. Mg, and 0.083 p.m. 
 Fe. 
 
 Appendix. 
 
 THE TISSUES AND FLUIDS OF THE EYE. 
 
 The retina contains in all 865-899.9 p. m. water, 57.1-84.5 p. m. 
 protein bodies — myosin, albumin, and mucin (?), 9.5-28.9 p. m. lecithin, 
 and 8.2-11.2 p. m. salts (Hoppe-Seyler and Cahn ;; ). The mineral bodies 
 consist of 422 p. m. Na 2 HPC>4 and 352 p. m. NaCl. The retina con- 
 tains, according to Barbieri, 4 also cholesterin but no cerebrosides and in 
 fact none of the specific constituents of the brain substance. 
 
 Those bodies which form the different segments of the rods and cones 
 have not been closely studied, and the greatest interest is therefore con- 
 nected with the coloring-matters of the retina. 
 
 Visual purple, also called rhodopsin, erythropsin, or visual red, is 
 the pigment of the rods. Boll, 5 in 1876, observed that the layer of rods 
 in the retina during life had a purplish-red color which was bleached 
 by the action of light. Kuhne 6 later showed that this red color might 
 remain for a long time after the death of the animal if the eye was pro- 
 tected from daylight or investigated by a sodium light. Under these 
 conditions it was also possible to isolate and closely study this substance. 
 
 Visual red (Boll) or visual purple (Kuhne) has become known mainly by 
 the investigations of Kuhne. The pigment occurs mainly in the rods and only 
 in their outer parts. In animals whose retina has no rods the visual purple is 
 
 ^ompt. Rend., 115. 
 
 2 Geoghegan, Zeitschr. f. physiol Chem.. 1; Magnus- Levy, Bioch. Zeitschr. 24. 
 
 * Zeitschr. f. physiol. Chem., 5. 
 
 4 Compt. Rend., 154. 
 
 5 Monatsber. d. Kgl. Preuss. Akad., 12. Nov., 1876. 
 
 6 The investigations of Kuhne and his pupils, Evvald and Ayres, on the visual purple 
 will be found in ..Untersuchungen aus dem physiol. Institut der Universitat Heidel- 
 berg, 1 and 2, and in Zeitschr. f. Biologie, 32. 
 
616 BRAIN AND NERVES. 
 
 absent, and is also necessarily absent in the macula lutea. In a variety of bat 
 (Rhinolophus hipposideros), in hens, pigeons and new-born rabbits, no visual 
 purple has been found in the rods. 
 
 A solution of visual purple in water which contains 2-5 per cent crys- 
 tallized bile, which is the best solvent for it, is purple-red in color, quite 
 clear, and not fluorescent. On evaporating this solution in vacuo we 
 obtain a residue similar to ammonium carminate which contains violet 
 or black grains. If the above solution is dialyzed with water, the bile 
 diffuses and the visual purple separates as a violet mass. Under all 
 circumstances, even when still in the retina, the visual purple is quickly 
 bleached by direct sunlight, and with diffused light with a rapidity cor- 
 responding to the intensity of the light. It passes from red and orange 
 to yellow. Red light bleaches the visual purple slowly; the ultra-red 
 light does not bleach it at all. A solution of visual purple shows no special 
 absorption bands, but only a general absorption which extends from the 
 red side, beginning at D and extending to the G line. The strongest 
 absorption is found at E. 
 
 Koettgen and Abelsdorf" r have shown that there are, in accordance with 
 Kuhne's views, two varieties of visual purple, the one occurring in mammals, 
 birds, and amphibians, and the other, which is more violet-red, in fishes. The 
 first has its maximum absorption in the green and the other in the yellowish- 
 green. 
 
 Visual purple when heated to 52-53° C. is destroyed after several 
 hours, and almost instantly when heated to 76° C. It is also destroyed 
 by alkalies, acids, alcohol, ether, and chloroform. On the contrary, 
 it resists the action of ammonia or alum solution. 
 
 As the visual purple is easily destroyed by light, it must therefore also be 
 regenerated during life. Kuhne has also found that the retina of the eye of the 
 frog becomes bleached when exposed for a long time to strong sunlight, and that 
 its color gradually returns when the animal is placed in the dark. This regenera- 
 tion of the visual purple is a function of the living cells in the layer of the pigment 
 epithelium of the retina. This may be inferred from the fact that a detached 
 piece of the retina which has been bleached by light may have its visual purple 
 restored if it is carefully laid on the choroid having layers of the pigment-epithe- 
 lium attached. The regeneration has, it seems, nothing to do with the dark 
 pigment, the melanin or fuscin, in the epithelium cells. A partial regeneration 
 seems, according to Kuhne, to be possible in the retina which has been completely 
 removed. On account of this property of the visual purple of being bleached 
 by light during life we may, as Kuhne has shown, under special conditions and 
 by observing special precautions, obtain after death, by the action of intense 
 light or more continuous light, the picture of bright objects, such as windows 
 and the like — so-called optograms. 
 
 The physiological importance of visual purple is unknown. It follows 
 that the visual purple is not essential to sight, since it is absent in certain 
 animals and also in the cones. 
 
 1 Centralbl. f. Physiol., 9; also Maly's Jahresber., 25, 351. 
 
CRYSTALLINE LENS. 617 
 
 Visual purple must always be prepared exclusively in a sodium light. It is 
 extracted from the net membrane by means of a watery solution of crystallized 
 bile. The filtered solution is evaporated in vacuo or dialyzed until the visual 
 purple is separated. To prepare a visual-purple solution perfectly free from 
 hemoglobin, the solution of visual purple in cholates is precipitated by saturating 
 with magnesium sulphate, washing the precipitate with a saturated solution of 
 magnesium sulphate, and then dissolving in water by the aid of the cholates sim- 
 ultaneously precipitated. 1 
 
 The Pigments of the Cones. In the inner segments of the cones of birds, rep- 
 tiles, and fishes a small fat-globule of varying color is found. Kuhne 2 has 
 isolated from this fat a green, a yellow, and a red pigment called respectively 
 chlorophan, xanthophan, and rhodophan. 
 
 The dark pigment of the epithelium-cells of the net membrane, which was 
 formerly called melanin, but has since been named fuscin by Kuhne and Mays, 3 
 contains iron, dissolves in concentrated caustic alkalies or concentrated sul- 
 phuric acid on warming, but, like the melanins in general, has been little studied. 
 The pigment occurring in the pigment-cells of the choroid will be discussed with 
 the melanins in Chapter XV. 
 
 The vitreous humor is often considered as a variety of gelatinous 
 tissue. The membrane consists, according to C. Morner, of a gelatin- 
 forming substance. The fluid contains a little proteid and a mucoid, 
 hyalomucoid, which was first shown by Morner, and which is precipitated 
 by acetic acid. This contains 12.27 per cent N, and 1.19 per cent S. 
 Among the extractives we find a little urea — according to Picard 5 p. m., 
 according to Rahlmann 0.64 p. m. Pautz 4 found besides some urea, 
 paralactic acid, and, in confirmation of the claims of Chabbas, Jesner, 
 and Kuhn, also glucose in the vitreous humor of oxen. The reaction 
 of the vitreous humor is alkaline, and the quantity of solids amounts 
 to about 9-11 p. m. The quantity of mineral bodies is about 6-9 p. m., 
 and the proteins 0.7 p. m. In regard to the aqueous humor see page 
 361. 
 
 The Crystalline Lens. That substance which forms the capsule of 
 the lens has been investigated by C. Morner. It belongs, according 
 to him, to a special group of proteins, called membranins. The mem- 
 branin bodies are insoluble at the ordinary temperature in water, salt 
 solutions, dilute acids, and alkalies, and, like the mucins, yield a reducing 
 substance on boiling with dilute mineral acids. They contain lead- 
 blackening sulphur. The membranins are colored a very beautiful red 
 by Millon's reagent, but give no characteristic reaction with concentrated 
 hydrochloric acid or Adamkiewicz's reagent. They are dissolved with 
 
 1 Kuhne, Zeitschr. f. Biologie, 32. 
 
 2 Kuhne, Die nichtbestiindigen Farben der Netzhaut, Untersuch. aus dem physiol. 
 Institut Heidelberg, 1, 341. 
 
 3 Kuhne, ibid., 2, 324. 
 
 4 Morner, Zeitschr. f. physiol. Chem., 18; Picard, cited from Gamgee, Physiol. 
 Chem., 1, 454; Rahlmann, Maly's Jahresber., 6; Pautz, Zeitschr. f. Biologie, 31. A 
 complete review of the literature will also be found here. 
 
618 BRAIN AND NERVES. 
 
 great difficulty by pepsin-hydrochloric acid or trypsin solution, but are 
 soluble in dilute acids and alkalies in the warmth. Membranin of the 
 capsule of the lens contains 14.10 per cent N and 0.83 per cent S, and is 
 a little less soluble than that from Descemet's membrane. 
 
 The principal mass of the solids of the crystalline lens consists of 
 proteins, whose nature has been investigated by C. Morner. 1 Some of 
 these proteins dissolve in dilute salt solution, while others remain 
 insoluble in this solvent. 
 
 The Insoluble Protein. The lens fibers consist of a protein sub- 
 stance which is insoluble in water and in salt solution and to which 
 Morner has given the name albumoid. It dissolves readily in very dilute 
 acids or alkalies. Its solution in caustic potash of 0.1 per cent is very 
 similar to an alkali-alb uminate solution, but coagulates at about 50° 
 C. on nearly complete neutralization and the addition of 8 per cent NaCl. 
 Albumoid has the following composition: C 53.12, H 6.8, N 16.62, and 
 S 0.79 per cent. The lens fibers themselves contain 16.61 per cent N 
 and 0.77 per cent S. The inner parts of the lens are considerably richer 
 in albumoid than the outer. The quantity of albumoid in the entire 
 lens amounts on an average to about 48 per cent of the total weight of 
 the proteins of the lens. 
 
 The Soluble Protein consists, exclusive of a very Ftrall quantity of 
 albumin, of two globulins, a- and fi-crystallin. These two globulins differ 
 from each other in this manner: a-crystallin contains 16.68 per cent N 
 and 0.56 per cent S; /3-crystallin, on the contrary, 17.04 per cent N and 
 1.27 per cent S. The first coagulates at about 72° C. and the other at 
 63° C. Besides this, /3-crystallin is precipitated from a salt-free solu- 
 tion with greater difficulty and less completely by acetic acid or carbon 
 dioxide. These globulins are not precipitated by an excess of NaCl at 
 either the ordinary temperature or 30° C. Magnesium or sodium sul- 
 phate in substance precipitates both globulins, on the contrary, at 30° C. 
 These two globulins are not equally divided in the mass of the lens. The 
 quantity of a-crystallin diminishes in the lens from without inward; 
 /3-crystallin, on the contrary, from within outward. 
 
 A. Jess 2 has found that the different proteins of the crystalline lens 
 behave differently with Arnold's protein reaction with sodium nitro- 
 prusside (page 100). The albumoid gives negative results with this 
 reagent. The a-crystallin gives it faintly, while the /3-crystallin gives a 
 strong reaction. The absence of this reaction, as observed by Weiss 
 in senile cataract, is connected with the fact as Jess has shown by his 
 investigations on the senile cataract in oxen, that the crystallin con- 
 
 1 Zeitschr. f. physiol. Chem., 18. This contains also the pertinent literature. 
 2 Zeit.srhr. f. Biol.. 01. 
 
PROTEINS OF THE LENS. 619 
 
 taining cysteine, disappears in part from the lens and is partly transformed 
 into albumoid. The relation between albumoid and crystallins is changed 
 with increasing age, so that the albumoid increases. In normal lens 
 the relation of the crystallins to the albumoid changes correspondingly 
 from 82:18 in youth to 41:59 in old age; in senile cataract the relation 
 can be changed to 25 : 75. The amount of fat, cholesterin and lecithin 
 is on the contrary not changed. 
 
 The average results of four analyses made by Laptschinsky * of the 
 lens of oxen are here given, calculated in parts per 1000: 
 
 . Proteins 349 . 3 
 
 Lecithin 2.3 
 
 Cholesterin 2.2 
 
 Fat 2.9 
 
 Soluble salts 5.3 
 
 Insoluble salts 2.4 
 
 In cataract the amount of proteins is diminished and the amount of 
 cholesterin increased. This statement requires further substantiation. 2 
 
 The quantity of the different proteins in the fresh moist lens of oxen 
 is, as follows, according to Morner: 
 
 Albumoid (lens fibers) 170 p. m. 
 
 0-Crystallin 110 " 
 
 a-Crystallin 68 " 
 
 Albumin 2 " 
 
 The corneal tissue has been previously considered (page 550). The 
 sclerotic has not been closely investigated, and the choroid coat is princi- 
 pally of interest because of the coloring-matter (melanin) it contains 
 (see Chapter XV). 
 
 Tears consist of a water-clear, alkaline fluid of a salty taste. Accord- 
 ing to the analyses of Lerch 3 they contain 982 p. m. water, 18 p. m. solids 
 with 5 p. m. albumin and 13 p. m. NaCl. 
 
 THE FLUIDS OF THE INNER EAR. 
 
 The perilymph and endolymph are alkaline fluids, which, besides 
 salts, contain — in the same amounts as in transudates — traces of protein, 
 and in certain animals (codfish) also mucin. The quantity of mucin 
 is greater in the perilymph than in the endolymph. 
 
 Otoliths contain 745-795 p. m. inorganic substance, which consists 
 chiefly of crystallized calcium carbonate. The organic substance is very 
 similar to mucin. 
 
 1 Pfluger's Arch., 13. 
 
 2 See Gross, Arch. f. Augenheilk., 55 and 58. 
 
 8 Cited from v. Gorup-Besanez, Lehrbuch d. physiol. Chem., 4 Aufl., 401. 
 
CHAPTER XII. 
 ORGANS OF GENERATION. 
 
 (a) Male Generative Secretions. 
 
 The testes have been little investigated chemically. In the testes of 
 animals we find protein bodies of different kinds — seralbumin, alkali 
 albuminate (?), and an albuminous body related to Rovida's hyaline 
 substance; also leucine, tyrosine, creatine, purine bases, cholesterin, lecithin, 
 inosite, and fat. In regard to the occurrence of glycogen the reports are 
 conflicting. Dareste 1 found, in the testes of birds, starch-like granules, 
 which were colored blue with difficulty by iodine. 
 
 In the autolysis of the testes Levene 2 found tyrosine, alanine, leucine,, 
 aminovaleric acid, aminobutyric acid, a-proline, phenylalanine, aspartic acid, 
 glutamic acid, and hypoxanthine. Pyrimidine and hexone bases could not be 
 detected. 
 
 • The semen as ejected is a white or whitish-yellow, viscous, sticky 
 fluid of a milky appearance, with whitish, non-transparent lumps. The 
 milky appearance is due to spermatozoa. Semen is heavier than water, 
 contains proteins, has a neutral or faintly alkaline reaction and a peculiar 
 specific odor. Soon after ejection semen becomes gelatinous, as if it 
 were coagulated, but afterward becomes more fluid. When diluted 
 with water white flakes or shreds separate (Henle's fibrin). According 
 to the analyses of Slowtzoff 3 human semen contains on an average 
 96.8 p. m. solids with 9 p. m. inorganic and 87.8 p. m. organic substance. 
 The amount of protein substances was, on an average, 22.6 p. m. and 1.69 
 p. m. of bodies soluble in ether. The protein substances consist of nucleo- 
 proteins, traces of mucin, albumin, and a substance similar to proteose 
 f found earlier by Posner). According to Cavazzani semen contains 
 relatively considerable nucleon, more than any organ, v. Hoffmann 4 
 
 1 Compt. Rend., 74. 
 
 2 Amer. Journ. of Physiol., 11. 
 
 J Zeitschr. f. physiol. Chem., 35. 
 
 * Posner, Berl. klin. Wochenschr., 1888, No. 21, and Centralbl. f. d. med. Wissensch., 
 1890; Cavazzani, Biochem. Centralbl., 1, 502, and Centralbl. f. Physiol., 19; v. Hoff- 
 mann, cited in Bioch. Centralbl., 9, 206. 
 
 620 
 
SEMEN. SPERMINE. 021 
 
 has found a protamine in human semen which yielded arginine and 
 perhaps also lysine on cleavage. The mineral bodies consist mainly of cal- 
 cium phosphate and considerable NaCl. Potassium occurs only in 
 smaller amounts. 
 
 The semen in the vas deferens differs chiefly from the ejected semen 
 in that it is without the peculiar odor. This last depends on the admixture 
 with the secretion of the prostate. This secretion, according to Iversen, 
 has a milky appearance and ordinarily an alkaline reaction, very rarely 
 a neutral one, and contains small amounts of proteins, especially nucleo- 
 proteins, besides a substance similar to fibrinogen and to mucin (Stern '), 
 and mineral bodies, especially NaCl. Besides this it contains an enzyme 
 vesicidase (see below), lecithin, choline (Stern), and a crystalline com- 
 bination of phosphoric acid with a base, C2H5N. This combination has 
 been called Bottcher's spermine crystals, and it is claimed that the 
 specific odor of the semen is due to a partial decomposition of these 
 crystals. 
 
 The crystals which appear on slowly evaporating the semen, and 
 which are also observed in anatomical preparations kept in alcohol, are 
 not identical with the Charcot-Leyden crystals found in the blood and 
 in the lymphatic glands in leucaemia (Th. Cohn, B. Lewy 2 ). They are, 
 according to Schreiner, 3 as above stated, a combination of phosphoric 
 acid with a I ase, spermine, C2H5N, which he discovered. 
 
 Spermine. Opinions in regard to the nature of this base are not unanimous 
 According to the investigations of Ladenburg and Abel, it is not improbable 
 that spermine is identical with ethylenimine; but this identity is disputed by 
 Majert and A. Schmidt, and also by Poehl. The compound of spermine with 
 phosphoric acid — Bottcher's spermine crystals — is insoluble in alcohol, ether, 
 and chloroform, soluble with difficulty in cold water, but more readily in hot 
 wqter, and easily soluble in dilute acids or alkalies, also alkali carbonates and 
 ammonia. The base is precipitated by tannic acid, mercuric chloride, gold 
 chloride, platinic chloride, potassium-bismuth iodide, and phosphotungstic acid. 
 Spermine has a tonic action, and, according to Poehl, 4 it has a marked action on 
 the oxidation processes of the animal body. 
 
 On the addition of a solution of potassium iodide and iodine to spermatozoa, 
 characteristic dark-brown or bluish-black crystals are obtained — Florence's 
 sperm reaction, which is considered by many as a reaction for spermine. Accord- 
 ing to Bocarius, 5 this reaction is due to choline. 
 
 1 Iversen, Xord. med. Ark., 6; also Maly's Jahresber., 4, 358; Stern, Biochem. 
 Centralbl., 1, 74S. 
 
 J Th. Cohn, Centralbl. f. allg^Path. u. path. Anat., 10 (1899), and Zeitschr.f.Urolog., 
 1908; B. Lewy, Centralbl f. d. med. Wissensch., 1899, 479. 
 
 8 Annal. d. Chem. u. Pharni., 194. 
 
 4 Ladenburg and Abel, Ber. d. deutsch. chem. Gesellsch., 21; Majert and A. 
 Schmidt, ibid., 24; Poehl, Compt. Rend., 115, Berlin, klin. Wochenschr., 1891 and 
 1893, Deutsch. med. Wochenschr., 1892 and 1895, and Zeitschr. f. klin. Med., 1894. 
 
 6 In regard to Florence's sperm reaction, see Posner, Berl. klin. Wochenschr., 
 1897, and Richter, Wien. klin. Wochenschr., 1897; Bocarius, Zeitschr. f. physioL 
 Chem., 34. 
 
622 ORGANS OF GENERATION. 
 
 Camus and Gley ' have found that the prostate fluid in certain rodents has 
 the property of coagulating the contents of the seminal vesicles. This property 
 is due to a special ferment substance (vesiculase) of the prostate fluid. 
 
 The spermatozoa show a great resistance to chemical reagents in 
 general. They do not dissolve completely in concentrated sulphuric 
 acid, nitric acid, acetic acid, or in boiling-hot soda solutions. They 
 are soluble in a boiling-hot caustic-potash solution. They resist putre- 
 faction, and after drying they may be obtained again in their original 
 form by moistening them with a 1-per cent common-salt solution. By 
 careful heating and burning to an ash the shape of the spermatozoa may 
 be seen in the ash. The quantity of ash is about 50 p. m. and consists 
 mainly (three-quarters) of potassium phosphate. 
 
 The spermatozoa show well-known movements, but the cause of this 
 is not known. These movements may continue for a very long time, 
 as under some conditions they may be observed for several days in the 
 body after death, and in the secretion of the uterus longer than a week. 
 Acid liquids stop these movements immediately; they are also destroyed 
 by strong alkalies, especially ammoniacal liquids, also by distilled water, 
 alcohol, ether, etc. The movements continue for a longer time in faintly 
 alkaline liquids, especially in alkaline animal secretions, and also in 
 properly diluted neutral salt solutions. 2 
 
 Spermatozoa are nucleus formations and hence are rich in nucleic 
 acid, which exists in the heads. The tails contain protein, and are besides 
 this rich in lecithin, chclesterin, and fat, which bodies occur only to a 
 small extent (if at all) in the heads. The tails seem by their composi- 
 tion to be closely allied to the non-medullated nerves or the axis-cylinders. 
 In the various kinds of animals investigated, the head contains nucleic 
 acid, which in fishes is partly combined with protamines and partly 
 with histones. In other animals, such as the bull and boar, protein- 
 like substances occur with the nucleic acid, but no protamine. 
 
 Our knowledge of the chemical composition of spermatozoa has 
 been greatly enhanced by the important investigations of Miescher 3 
 on salmon milt. The intermediate fluid of the spermatozoa of Rhine 
 salmon is a dilute salt solution containing 1.3-1.9 p. m. organic and 
 6.5-7.6 p. m. inorganic bodies. The last consist principally of sodium 
 chloride and carbonate, besides some potassium chloride and sulphate. 
 The fluid contains only traces of protein, but no peptone. The tails consist 
 of 419 p. m. protein, 318.3 p. m. lecithin, and 262.7 p. m. cholesterin and 
 
 1 Compt. rend, de soc. biolog., 48, 49. 
 1 See G. Gunther, Pfliiger's Arch., 118. 
 
 3 See Miescher, " Die histochemischen und physiologischen Arbeiten von Friedrich 
 Miescher, gesammelt und herausgegeben von seinen Freunden," Lefpzig, 1897. 
 
OVARIES. 623 
 
 fat. The heads extracted with alcohol-ether contain on an average 
 960 p. m. protamine nucleate, which nevertheless is not uniform, but is 
 so divided that the outer layers consist of basic protamine nucleate, 
 while the inner layers, on the contrary, consist of acid protamine nucleate. 
 Besides the protamine nucleate there are present in the heads, although 
 to a very slight extent, organic substances. Of these we must mention 
 a nitrogenous substance containing iron which gives Millon's reaction 
 and which Miescher calls karyogen. The unripe salmon spermatozoa, 
 while developing, also contain nucleic acid, but no protamine, with a 
 protein substance, " albuminose," which probably is a step in the forma- 
 tion of protamine. According to Kossel and Mathews, 1 in the herring 
 as in the salmon, the heads of the spermatozoa consist of protamine 
 nucleate but no free protein. 
 
 The chemical investigations on the spermatozoa have not given 
 us any information as to the condition for fertilization and the develop- 
 ment of the egg. 
 
 Spermatin is a name which has been given to a constituent similar to alkali 
 albuminate, but it has not been closely studied. 
 
 Prostatic concrements are of two kinds. One is very small, generally oval 
 in shape, with concentric layers. In young but not in older persons they are 
 colored blue by iodine (Iversen 2 ). The other kind is larger, sometimes the size 
 of the head of a pin, consisting chiefly of calcium phosphate (about 700 p.m.), with 
 only a very small amount (about 160 p. m.) of organic substance. 
 
 (b) Female Generative Organs. 
 
 The stroma of the ovaries is of little interest from a physiologico- 
 chemical standpoint, and the most important constituents of the ovaries, 
 the Graafian follicles with the ovum, have not thus far been the subject 
 of a careful chemical investigation. The fluid in the follicles (of the 
 cow) does not contain, as has been stated, the peculiar bodies, paral- 
 bumin or metalbumin, which are found in certain pathological • ovarial 
 fluids, but seems to be a serous liquid. The corpora lutea are colored 
 yellow. Earlier investigators (Piccolo and Lieben. Kuhne and Ewald 3 ) 
 have found a crystalline pigment in the corpora lutea. In recent 
 investigations Escher 4 has shown that this substance is a crystalline 
 hydrocarbon (C40-H50) which seems to be identical with the carotin of 
 the carrot and green leaves. The color of the crystals as well as the con- 
 centrated solution is reddish-orange. Carotin differs from the yellow 
 pigment of the yolk of the egg, the lutein, in having another formula 
 
 1 Zeitschr. f. physiol. Chem., 23. 
 
 1 Nord. med. Ark., 6. 
 
 • See Chapter V, p. 301. 
 
 ♦Zeitschr. f. physiol. Chem., 83, 198 (1912). 
 
624 ORGANS OF GENERATION. 
 
 (page 631) and being soluble with difficulty in alcohol and readily soluble 
 in petroleum ether. 
 
 The cysts often occurring in the ovaries are of special pathological 
 interest, and these may have essentially different contents, depending 
 upon their variety and origin. 
 
 The serous cysts (Hydrops folliculorum Graafii), which are 
 formed by a dilation of the Graafian follicles, contain a serous liquid 
 which has a specific gravity of 1.005-1.022. A specific gravity of 1.020 
 is less frequent. Generally the specific gravity is lower, 1.005-1.014, 
 with 10-40 p. m. solids. As far as is known, the contents of these cysts 
 do not essentially differ from other serous liquids. 
 
 The proliferous cysts (myxoid cysts, colloid cysts), which are 
 developed from Pfluger's epithelium-tubes, may have a content of a 
 decidedly variable composition. 
 
 We sometimes find in small cysts a semi-solid, transparent, or some- 
 what cloudy or opalescent mass which appears like solidified glue or 
 quivering jelly, and which has been called colloid because of its physical 
 properties. In other cases the cysts contain a thick, tough mass which 
 can be drawn out into long threads, and as this mass in the different 
 cysts is more or less diluted with serous liquids their contents may have 
 a variable consistency. In still other cases the small cysts may also 
 contain a thin, watery fluid. The color of the contents is also variable. 
 Sometimes they are bluish-white, opalescent, and again they are yellow, 
 yellowish-brown, or yellowish with a shade of green. They are often 
 colored more or less chocolate-brown or red-brown, due to the decom- 
 posed blood-coloring matters. The reaction is alkaline or nearly neutral. 
 The specific gravity, which may vary considerably, is generally 1.015- 
 1.030, but may occasionally be 1.005-1.010 or 1.050-1.055. The amount 
 of solids is very variable. In rare cases it amounts to only 10-20 p. m. ; 
 ordinarily it varies from 50-70-100 p. m. In a few instances 150-200 
 p. m. solids have been found. 
 
 As form-elements one finds red and white blood-corpuscles, granular 
 cells, partly fat-degenerated epithelium and partly large so-called Gluge's 
 corpuscles, fine granular masses, epithelium-cells, cholesterin crystals, and 
 colloid corpuscles — large, circular, highly refractive formations. 
 
 Though the contents of the proliferous cyst may have a variable 
 composition, still it may be characterized in typical cases by its slimy 
 or ropy consistency; by its grayish-yellow, chocolate-brown, sometimes 
 whitish-gray color; and by its relatively high specific gravity, 1.015- 
 1.025. Such a liquid does not ordinarily show a spontaneous fibrin 
 coagulation. 
 
 We consider colloid, metalbumin, and paralbumin as characteristic 
 constituents of these cysts. 
 
COLLOID. PSEUDOMUCIN. G25 
 
 Colloid. This name does not designate any particular chemical 
 substance, but is given to the contents of tumors with certain physical 
 properties similar to gelatin jelly. Colloid is found as a pathological 
 product in several organs. 
 
 Colloid is a gelatinous mass, insoluble in water and acetic acid; it is 
 dissolved by alkalies and gives a liquid which is not precipitated by 
 acetic acid or by acetic acid and potassium ferrocyanide. According to 
 Pfannenstiel ' such a colloid is designated /3-pseudomucin. Some- 
 times a colloid is found which, when treated with a very dilute alkali, 
 gives a solution similar to a mucin solution. Colloid is very closely 
 related to mucin and is considered by certain investigators as a modified 
 mucin. An ovarial colloid analyzed by Panzer contained 931 p. m. 
 water, 57 p. m. organic substance, and 12 p. m. ash. The elementary 
 composition was C 47.27, H 5.8G, N 8.40, S 0.79, P 0.54, and ash 6.43 
 per cent. A colloid found by Wurtz 2 in the lungs contained C 48.09, 
 H 7.47, X 7.00, and 0(+S) 37.44 per cent. Colloids of different origin 
 seem to be of varying composition. 
 
 Mctolbumin. This name Scherer 3 gave to a protein substance 
 found by him in an ovarial fluid. The metalbumin was considered by 
 Scherer to be an albuminous body, but it belongs to the mucin group, 
 and it is for this reason called pseudomucin by Hammarsten. 4 
 
 Pseudomucin. This body, w r hich, like the mucins, gives a reducing 
 substance when boiled with acids, is a mucoid of the following com- 
 position: C 49.75, H 6.98, N 10.28, S 1.25, O 31.74 per cent (Hammar- 
 sten). With water pseudomucin gives a slimy, ropy solution, and it is 
 this substance which gives the fluid contents of the ovarial cysts their 
 typical ropy property. Its solutions do not coagulate on boiling, but 
 only become milky or opalescent. Unlike mucin, pseudomucin solutions 
 are not precipitated by acetic acid. With alcohol they give a coarse 
 flocculent or thready precipitate which is soluble even after having been 
 kept under water or alcohol, for a long time. 
 
 Paralbumin is another substance discovered by Scherer, which occurs 
 in ovarial liquids, and also in ascitic fluids, with the simultaneous presence 
 of ovarial cysts and rupture of the same. It is therefore only a mixture 
 of pseudomucin with variable amounts of protein, and the reactions of 
 paralbumin are correspondingly variable. 
 
 1 Arch. f. Gynak., 38. 
 
 2 Panzer, Zeitschr. f. physiol. Chem., 28; Wiirtz, see Lebert, Beitr. zur Kenntnis 
 des Gallertkrebses, Virchow's Arch., 4. 
 
 3 Yerh. d. physik.-med. Gesellsch. in Wurzburg, 2, and Sitzungsber. der physik.- 
 rr.ed. Gesellsch. in Wurzburg fur 1864-1865; Wurzburg med. Zeitschr., 7, No. 6. 
 
 4 Zeitschr. f . physiol. Chem., 6. 
 
626 ORGANS OF GENERATION. 
 
 Mitjukoff l has isolated and investigated a colloid from an ovarial cyst. It 
 had the following composition: C 51.76, H 7.76, N 10.7 S 1.09, and 28.69 per 
 cent, and differed from mucin and pseudomucin by reducing Fehling's solu- 
 tion before boiling with acid. It must be remarked that pseudomucin, on boiling 
 sufficiently long with alkali, or by the use of a concentrated solution of caustic 
 alkali, also splits and causes a reduction. This reduction is nevertheless weak 
 as compared with that produced after boiling with an acid. The body isolated 
 by Mitjukoff is called paramucin. 
 
 The pseudomucin as well as colloid are mucoid substances, and the 
 carbohydrate obtained from them is glucosamine (chitosairine), as espe- 
 cially shown by Fr. Muller, Netjberg and Heymann. 2 From pseudo- 
 mucin Zangerle 3 obtained 30 per cent glucosamine, and Netjberg and 
 Heymann have shown that the glucosamine is the only carbohydrate 
 regularly taking part in the structure of these substances. Still there are 
 reports as to the occurrence of chondroitin-sulphuric acid (or an allied 
 acid) in pseudomucin or colloid (Panzer), but this is not constant 
 according to the experience of Hammarsten. 
 
 As hydrolytic cleavage products of pseudomucin Otori obtained, 
 besides carbohydrate derivatives such as levulinic acid and humus sub- 
 stances, leucine, tyrosine, glycocoll, aspartic acid, glutamic acid, valeric 
 acid, arginine, lysine, and guanidine. The quantity of guanidine, it 
 seems, was greater than that which could be derived from the arginine, 
 hence this body probably originated from another complex. Pregl 4 
 obtained on the hydrolysis of a colloid, which behaved like paramucin, 
 no glycocoll and only traces of diamino acids, but otherwise the same 
 amino-acids as Otori found, besides alanine, proline, phenylalanine and 
 tryptophane. 
 
 The detection of metalbumin and paralbumin is naturally connected 
 with the detection of pseudomucin. A typical ovarial fluid containing 
 pseudomucin is, as a rule, sufficiently characterized by its physical proper- 
 ties, and a special chemical investigation is necessary only in cases where a 
 serous fluid contains very small amounts of pseudomucin. The pro- 
 cedure is as follows: The protein is removed by heating to boiling with 
 the addition of acetic acid; the filtrate is strongly concentrated and pre- 
 cipitated by alcohol. The precipitate, a transformation product of 
 pseudomucin, is carefully washed, with alcohol and then dissolved in water. 
 A part of this solution is digested with saliva at the temperature of the 
 body and then tested for glucose (derived from glycogen or dextrin). 
 If glycogen is present, it will be converted into glucose by the saliva; 
 precipitate again with alcohol and then proceed as in the absence of 
 
 1 K. Mitjukoff, Arch. f. Gynakol., 49. 
 
 2 Muller, Verh. d. Naturf. Gesellsch. in Basel. 12, part 2; Neuberg and Heymaim; 
 Hofmeister's BeitriiKe, 2. See also Leathes, Arch. f. exp. Path. u. Pharm., 43. 
 
 1 Mfinch. med. Wochenschr., 1900. 
 
 4 Otori, Zeitschr. f. physiol. Chem., 42 and 43; Pregl, ibid., 58. 
 
OVARIAL CYSTS. 627 
 
 glycogen. In this last-mentioned case, first add acetic acid to the solu- 
 tion of the alcohol precipitate in water so as to precipitate a/iy existing 
 mucin. The precipitate produced is filtered off, the filtrate treated with 
 2 per cent HC1 and wanned on the water-bath until the liquid is deep 
 brown in color. In the presence of pseudomucin this solution gives 
 Trommer's test. 
 
 The other protein bodies which have been found in cystic fluids are 
 serglobulin and seralbumin, peptone (?), mucin, and mucin-peptone (?). 
 Fibrin occurs only in exceptional cases. The quantity of mineral bodies 
 on an average amounts to about 10 p. m. The amount of extractive 
 bodies (cholesterin and urea) and fat is ordinarily 2-4 p. m. The remaining 
 solids, which constitute the chief mass, are protein bodies and pseudo- 
 mucin. 
 
 The intraligamentary, papillary cysts contain a yellow, yellowish- 
 green, or brownish-green, liquid which contains either no pseudomucin 
 or very little. The specific gravity is generally rather high, 1.032-1.036, 
 with 90-100 p. m. solids. The principal constituents are the simple 
 proteins of blood-serum. 
 
 The rare tubo-ovarial cysts contain as a rule a watery, serous fluid 
 containing no pseudomucin. 
 
 The parovarial cysts or the cysts of the ligamenta lata may attain 
 a considerable size. In general, and when quite typical, the contents are 
 watery, mostly very pale-yellow-colored, water-clear or only slightly 
 opalescent liquids. The specific gravity is low, 1.002-1.009, and the 
 solids only amount to 10-20 p. m. Pseudomucin does not occur as a 
 typical constituent; protein is sometimes absent, and when it does occur 
 the quantity is very small. The principal part of the solids consists of 
 salts and extractive bodies. In exceptional cases the fluid may be rich 
 in protein and may show a higher specific gravity. 
 
 In regard to the quantitive composition of the fluid from ovarial 
 cysts we refer the reader to the work of Oerum. 1 
 
 E. Ludwig and R. v. Zeynek have investigated the fat from dermoid cysts. 
 Besides a little arachidic acid, they found oleic, stearic, palmitic, and myristic 
 acids, cetyl alcohol, and a cholesterin-like substance. In regard to the occurrence 
 of cetyl alcohol see the work of Ameseder, 2 page 239. 
 
 The colloid from a uterine fibroma analyzed by Stollmann 3 contained a 
 pseudomucin soluble in water, and a colloid (paramucin) insoluble in water, both 
 of which behaved differently with alcohol as compared with the corresponding 
 substances from ovarial cysts. 
 
 1 Kemiske Studier over Ovariecystevaedsker, etc., Koebenhavn, 1S84. See also 
 Maly's Jahresber., 14, 450. 
 
 2 Ludwig and v. Zeynek, Zeitschr. f. physiol. Chem., 23; Ameseder, ibid., 52; 
 Salkowski, Bioch. Zeitschr., 32. 
 
 1 Amer. Gynecology, 1903. 
 
628 ORGANS OF GENERATION. 
 
 The Ovum. 
 
 The small ova of man and mammals eannot, for evident reasons, be 
 the subject of a searching chemical investigation. Up to the present 
 time the eggs of birds, amphibians, and fishes have been investigated, 
 but above all the hen's egg. We will here occupy ourselves with the con- 
 stituents of this last. 
 
 The Yolk of the Hen's Egg. In the so-called white yolk, which 
 forms the germ with a process reaching to the center of the yolk (latebra), 
 and forming a layer between the yolk and yolk-membrane, there occurs 
 protein, nuclein, lecithin, and potassium (Liebermann l ). The occur- 
 rence of glycogen is doubtful. The yolk-membrane consists of an albu- 
 minoid similar in certain respects to keratin (Liebermann). 
 
 The principal part of the yolk — the nutritive yolk or yellow — is a 
 viscous, non-transparent, pale-yellow or orange-yellow alkaline emulsion 
 of a mild taste. The yolk contains vitellin, lecithin, cholesterin, fat, color- 
 ing-matters, traces of neuridine (Brieger 2 ), purine bases (Mesernitzki 3 ), 
 glucose in very small quantities, and mineral bodies. The occurrence of 
 cerebrin and of granules similar to starch (Dareste 4 ) has not been posi- 
 tively proved. 
 
 Several enzymes have been found in the yolk, especially a diastatic 
 enzyme (Muller and Masuyama), a glycolytic enzyme (Stepanek) 
 which in the absence of air brings about an alcoholic fermentation of 
 sugar and in the presence of air forms carbon dioxide and lactic acid, 
 and finally a proteolytic, a lipolytic, and a chromolytic (?) enzyme 
 (Wohlgemuth 5 ). 
 
 Ovovitellin. This body, which is often considered as a globulin 
 is in reality a nucleoalbumin. The question as to what relation other 
 protein substances, which are related to ovovitellin, like the aleuron 
 grains of certain seeds, and the yolk spherules of the eggs of certain fishes 
 and amphibians, bear to this substance is one which requires further 
 investigation. 
 
 The ovovitellin which has been prepared from the yolk of eggs is not a 
 pure protein body, but always contains lecithin. Hoppe-Seyler found 
 25 per cent lecithin in vitellin. The lecithin may be removed by boiling 
 alcohol, but the vitellin is changed thereby, and it is therefore probable 
 
 1 Pfluger's Arch., 43. 
 1 Ueber Ptomaine, Berlin, 1885. 
 a Mesernitzki, Bioehem. Centralbl., 1, 739. 
 * Compt. Rend., 72. 
 
 6 Muller and Masuyama, Zeitschr. f. Biologie, 39; Stepanek, Centralbl. f. Physiol., 
 18, 188; Wohlgemuth in Salkowski's Festschrift and Zeitschr. f. physiol. Chem., 44. 
 
OVOVITELLIN. 629 
 
 that the lecithin is chemically ui.ited with the vitellin (Hoppe-Seyler l ). 
 According to Osborne and Campbell, the so-called ovovitellin is a mix- 
 ture of various vitellin-lecithin combinations, with 15 to 30 per cent of 
 lecithin. The protein substance freed from lecithin is the same in all 
 these compounds and has the following composition: C 51.24, H 7.16, 
 N 16.38, S 1.04, P 0.94, O 23.24 pur cent. These figures differ somewhat 
 from those obtained by Gross for vitellin prepared by another method 
 (precipitation with [NH 4 ] 2 S0 4 ), namely, C 48.01, H 6.35, N 14.91-16.97, 
 P 0.32-0.35, S 0.88, and the composition of ovovitellin is therefore not 
 positively known. Besides the vitellin Gross found a globulin coagulat- 
 ing at 76-77° C. in a solution containing salt, and Pllmmer 2 found a 
 protein which he calls livetin which only contained 0.1 per cent phos- 
 phorus and which gave more monamino acids but less amide and diamino 
 nitrogen than vitellin. 
 
 On the pepsin digestion of ovovitellin, Osborne and Campbell 
 obtained a pseudonuclein with varying amounts of phosphorus, 2.52- 
 4.19 per cent. Bunge 3 prepared a pseudonuclein by digesting the yolk 
 with gastric juice, and his pseudonuclein, . he claims, is of great impor- 
 tance in the formation of the blood, and on these grounds he called it 
 hcematogen. This hsematogen has the following composition: C 42.11, 
 H 6.08, N 14.73, S 0.55, P 5.19, Fe 0.29, and O 31.05 per cent. The 
 composition of this substance may vary considerably even on using the 
 same method of preparation. 
 
 Vitellin is similar to the globulins in that it is insoluble in water, but 
 on the contrary soluble in dilute neutral-salt solutions (although the solu- 
 tion is not quite transparent). It is also soluble in hydrochloric acid of 
 1 p. m. and in very dilute solutions of alkalies or alkali carbonates. It 
 is precipitated from its salt solution by diluting with water, and when 
 allowed to stand some time in contact with water the vitellin is gradually 
 changed, forming a substance more like the albuminates. The coagu- 
 lation temperature for the solution containing salt (NaCl) lies between 
 70 and 75° C, or, when heated very rapidly, at about 80° C. Vitellin 
 differs from the globulins in yielding pseudonuclein by peptic digestion. 
 It is not always completely precipitated by NaCl in substance. The 
 ovovitellin isolated by Gross gave Molisch's reaction. Neuberg 4 
 has also split off glucosamine from the yolk and has identified it as nori- 
 
 1 Med. ehem. Untersuch., 216. 
 
 2 Osborne and Campbell, Connecticut Agric. Exp. Station, 23d Ann. Report, New 
 Haven, 1900; Gross, Zur Kenntn. d. Ovovitellin, Inaug.-Diss. Strassburg, 1S99; 
 Plimmer, Journ. Chem. Soc, London, 93. 
 
 3 Zeitschr. f. physiol. Chem., 9, 49. See also Hugounenq and Morel, Compt. Rend., 
 140 and 141. 
 
 * Ber. d. d. chem. Gesellsch., 34. 
 
630 OPUAXS OF GENERATION. 
 
 sosaccharic acid. It is difficult to state whether this glucosamine was 
 derived from the vitellin or from some other constituent of the yolk. 
 
 The principal points in the preparation of ovovitellin are as follows: 
 The yolk is thoroughly agitated with ether; the residue is dissolved in 
 a 10-per cent common-salt solution, filtered, and the vitellin precipitated 
 by adding an abundance of water. The vitellin is now purified by repeat- 
 edly redissolving in dilute common-salt solutions and precipitating with 
 water. 
 
 Ichthulin, which occurs in the eggs of the carp and other fishes is, accord- 
 ing to Kossel and Walter, an amorphous modification of the crystalline body 
 tchthidin, which occurs in the eggs of the carp. Ichthulin is precipitated on 
 diluting with water. It was formerly considered as a vitellin. According to 
 Walter it yields a pseudonuclein on peptic digestion; and this pseudonuclein 
 gives a reducing carbohvdate on boiling with sulphuric acid. Ichthulin has the 
 following composition; C 53.42, H 7.63, N 15.63, 22.19, S 0.41, P 0.43 percent. 
 It also contains iron. The ichthulin investigated from codfish eggs by Levene 
 had the composition C 52.44, H 7.45, N 15.96, S 0.92, P 0.65, Fe+O 22.58 
 per cent, and yielded no reducing substances on boiling with acids. The pure 
 vitellin isolated by Hammarsten from perch eggs had a similar behavior and 
 was very readily changed by a little hydrochloric acid so that it was converted 
 into a typical pseudonuclein. The codfish ichthulin yielded a pseudonucleic acid 
 with 10.34 per cent phosphorus, but this acid still gave the protein reactions. 
 McClenden 1 has prepared a vitellin from frogs' eggs which he calls batrachiolin. 
 
 The yolk also contains albumin, besides vitellin and the above-men- 
 tioned proteins. 
 
 The fat of the yolk of the eg?, Liebermann 2 claims, is a mixture of 
 a solid and a liquid fat. The solid fat consists principally of tripalmitin 
 with some tristearin. On the saponification of the egg-oil Liebermann 
 obtained 40 per cent oleic acid, -38.04 per cent palmitic acid, and 15.21 
 per cent stearic acid. The fat of the yolk of the egg contains less carbon 
 than other fats, which may depend upon the presence of monoglycerides 
 and diglycerides, or upon a quantity of fatty acid deficient in carbon 
 (Liebermann). The composition of yolk fat is dependent upon the 
 food, as Henriques and Hansen 3 have shown that the fat of the food 
 passes into the egg. 
 
 The phosphatides of the yolk seem to be of various kinds. Thier- 
 felder and Stern have found three different phosphatides. One of 
 these, which was soluble in alcohol-ether, behaved like lecithin. The 
 second was soluble with difficulty in alcohol, but readily soluble in ether, 
 contained 1.37 per cent N and 3.96 per cent P. The third was a diamino 
 
 1 Walter, Zeitsfhr. f. physiol. Chem., 15; Levene, ibid., 32; Hammarsten, Skand. 
 Arch. f. Physiol., 17; McClenden, Amer. Journ. of Physiol. 25; see also Plimmer 
 and Scott, Journ. Chem. Soc, 1)3. 
 
 2 PfluKer's Arch., 4:5. 
 
 •Skand. Arch. f. Physiol., 14. 
 
LUTEIN. 631 
 
 phosphatide, soluble with difficulty in ether, but obtained in crystalline 
 needles from hot alcohol, and contained 2.77 per cent N and 3.22 per cent 
 P, and had a melting-point of 100-170° C. Frankel and Bolaffio 1 
 also found a substance crystallizing from hot alcohol and insoluble in 
 ether with 2.78 per cent N and 2.18 per cent P. They call this body 
 neottin and claim that it is a triamino-monophosphatide having the formula 
 C84H172N3PO15. Barbieri has obtained a sulphurized phosphatide 
 called ovin, containing 1.35 per cent P, 3.06 per cent N and 0.4 per cent S. 
 The relation of all these bodies to each other must be further studied. 
 
 Lutein. With the name lutein we in the past have included several 
 yellow or orange-red amorphous coloring-matters which occur in the 
 yellow of the egg, and in several other places in the animal organism; 
 for instance, in the blood-serum and serous fluids, fatty tissues, milk- 
 fat, corpora lutea, and in the fat-globules of the retina as well as in dif- 
 ferent plants (Thudichum). Among these bodies belong the crys- 
 talline substance obtained by Escher from the corpora lutea (page 623). 
 It was difficultly soluble in alcohol but readily soluble in petroleum ether 
 and showed itself isomeric or perhaps identical with the plant pigment 
 carotin (CioHsc) analyzed by Willstatter and Mieg. The lutein of 
 the egg yolk, which is more readily soluble in alcohol and less soluble in 
 petroleum ether than carotin has also been obtained by Willstatter and 
 Escher in a pure, crystalline form. On analysis it gave the formula 
 C40H56O2. As shown by C. A. Schtjnck the yolk lutein stands in 
 close relation to the yellow plant pigment, xanthophyll. The formula 
 given by Willstatter and Escher for lutein was in fact the same as 
 for the xanthophyll, as previously found by Willstatter and Mieg. 
 These two substances are also similar in other respects; still the melting- 
 points of the two are different. The carotin and the yolk lutein differ 
 also by the absorption spectra, which is different in different solvents 
 as well as by their formulae and different solubilities. 2 
 
 The relation of the other substances called luteins to each other and 
 to the yolk lutein is unknown. All are soluble in alcohol, ether, and chloro- 
 form. They differ from the bile-pigment, bilirubin, in that they are not 
 separated from their solution in chloroform by water containing alkali, 
 and also in that they do not give the characteristic play of colors with 
 nitric acid containing a little nitrous acid, but give a transient blue color. 
 The luteins withstand the action of alkalies so that they are not changed 
 when we remove the fats present by means of saponification. 
 
 1 Thierfeldcr and Stern, Zeitschr. f. physiol. Chem., 53; Frankel and Bolaffio, 
 Bioch. Zeitschr., 9; Barbieri, Compt. Rend., 145. 
 
 2 Thudichum, Centralbl. f. d. ined. Wiss. 1869; Willstatter and Mieg. Ann. d. 
 Chem., 355 (1907); Willstatter and Escher, Zeitschr. f. physiol. Chem., 64 1909); 
 76 (1911); Schunck, see Chem. Centralbl., 1903. 
 
632 ORGANS OF GENERATION. 
 
 Maly ' found two pigments free from iron in the eggs of a water-spider (Maja 
 squinado) — one a red (riteUorubin) and the other a yellow pigment (vitellolutein) . 
 Both of these pigments are colored blue by nitric acid containing nitrous acid 
 and a beautiful green by concentrated sulphuric acid. 
 
 The mineral bodies of the yolk of the egg consist, according to Poleck, 2 
 of 51.2-65.7 parts soda, 80.5-89.3 potash, 122.1-132.8 lime, 20.7-21.1 
 magnesia, 11.90-14.5 iron oxide, 638.1-667.0 phosphoric acid, and 5.5- 
 14.0 parts silicic acid in 1000 parts of the ash. We find phosphoric acid, 
 and lime the most abundant, and then potash, which is somewhat greater 
 in quantity than the soda. These results are not, however, quite cor- 
 rect: first, because no dissolved phosphate occurs in the yolk (Lieber- 
 mann), and secondly, in burning, phosphoric and sulphuric acids are 
 produced, and these drive away the chlorine, which is not accounted 
 for in the above analyses. 
 
 The yolk of the hen's egg weighs about 12-18 grams. The quan- 
 tity of water and solids amounts, according to Parke, 3 to 471.9 p. m. 
 and 528.1 p. m. respectively. Among the solids he found 156.3 p. m. 
 protein, 3.53 p. m. soluble and 6.12 p. m. insoluble salts. The quantity 
 of fat, according to Parke, is 228.4 p. m.; the lecithin, calculated from 
 the amount of phosphorus in the organic substance of the alcohol-ether 
 extract, was 107.2 p. m. and the cholesterin 17.5 p. m. 
 
 The white of the egg is a faintly yellow albuminous fluid inclosed 
 in a framework of thin membranes; and this fluid is in itself very liquid, 
 but seems viscous because of the presence of these fine membranes. That 
 substance which forms the membranes, and of which the chalaza con- 
 sists, seems to be a body closely related to horn substances (Lieber- 
 mann) . 
 
 The white of egg has a specific gravity of 1.038-1.045, and always 
 has an alkaline reaction toward litmus. It contains 850-880 p. m. water, 
 100-130 p. m. protein bodies, and 7 p. m. salts. Lehmann found a fer- 
 mentable variety of sugar which Salkowski showed was glucose. C. Th. 
 Morner could not find any other sugar in egg-white; the quantity of 
 glucose as found by Morner 4 was 3-5 p. m. Besides these one finds 
 in the white of egg traces of fats, soaps, lecithin and cholesterin. 
 
 The white of egg of the Insessores becomes transparent on boiling and acts 
 in many respects like alkali albuminate. This albumin Tarchanofp 6 called 
 " tatalbumin." 
 
 1 Monatshefte f. Chem., 2. 
 
 2 Cited from v. Gorup-Besanez, Lehrbuch d. physiol. Chem., 4, Aufl., 740. 
 1 Hoppe-Seyler, Med. chem. Untersuch., Heft 2, 209. 
 
 * Lehmann, Lehrb. d. physiol. Chem. 2 Aufl. 1855, Bd. 1, s. 271; Bd. 2, s. 312. 
 Salkowski, Centralbl. f. d. med. Wiss., 31 (1893); Morner. Zeitschr. f. physiol. Chem; 
 80 (1912). 
 
 * Pfliiger's Arch., 31, 33, and 39. 
 
OVOGLOBULIN. OVALBUMIN. G33 
 
 The protein substances of the white of egg behave like glycoproteins, 
 as they all yield glucosamine. For the globulin and albumin it has not 
 been proved, nor is it probable? that the glucosamine belongs to the pro- 
 tein molecule (sec page 84). According to the solution and precipita- 
 tion properties they arc similar to the globulins, albumins or proteoses. 
 The representatives of the first two groups, are ovoglobulin and ovalbumin. 
 The proteose-like body is ovomucoid. 
 
 Ovoglobulin separates in part on diluting the egg-white with water. 
 It is precipitated upon saturation with magnesium sulphate, or upon 
 one-half saturation with ammonium sulphate, and coagulates at about 
 75° C. By repeated solution in water and precipitation with ammonium 
 sulphate a part of the globulin becomes insoluble (Langstein). This 
 also occurs on precipitation by diluting with water or by dialysis, and 
 it is quite possible that the globulin is a mixture. That portion which 
 readily becomes insoluble seems to be identical with Eichholz's gly- 
 coprotein or Osborne and Campbell's ovomucin. Langstein obtained 
 11 per cent of glucosamine from the soluble ovoglobulin. The total 
 quantity of globulins, according to Dillner, is about 6.7 per cent of 
 the total protein substances, and this corresponds with the recent deter- 
 minations of Osborne and Campbell. In regard to the probable occur- 
 rence of several globulins in the white of the egg there are the determina- 
 tions of Corin and Berard as well as of Langstein, 1 but they have 
 not led to any positive conclusions. 
 
 Ovalbumin. The so-called albumin of the egg-white is undoubtedly 
 a mixture of at least two albumin-like proteins. Opinions differ con- 
 siderably in regard to the number of these proteins (Bondzynski and 
 Zoja, Gautier, Bechamp, Corin and Berard, Panormoff, and others). 
 Since Hofmeister has been able to prepare ovalbumin in a crystalline 
 form, and since Hopkins and Pinkus 2 have shown that not more than 
 one-half of the ovalbumin can be obtained in such a form, Osborne and 
 Campbell have isolated two different ovalbumins or principal fractions; 
 the crystallizable they call ovalbumin and the non-cry stallizable con- 
 albumin. The two fractions have only a slight variation in elementary 
 composition; the conalbumin coagulates between 50-60° C, nearer to 
 60° C, and the ovalbumin at 64° C or at a higher temperature. There 
 are no conclusive investigations as to whether the non-crystallizal le 
 
 1 Langstein, Hofmeister's Beitrage, 1; Eichholz, Journ. of Physiol., 23; Osborne 
 and Campbell, Connecticut Agric. Exp. Station., 23d Ann. Report, New Haven, 1900; 
 Dillner, Maly's Jahresber., 15; Corin and Berard, ibid., 18. 
 
 2 Hofmeister, Zeitschr. f. physiol. Chem., 14, 16, and 24, Gabriel, ibid., 15; Bond- 
 zynski and Zoja, ibid., 19; Gautier, Bull. Soc. chim., 14; Bechamp, ibid., 21; Corin 
 and Berard, 1. c.; Hopkins and Pinkus, Ber. d. d. ehem. Gesellsch., 31, and Journ. of 
 Physiol., 23; Osborne and Campbell, 1. c.; Panormoff, Maly's Jahresber., 27 and 28. 
 
634 ORGANS OF GENERATION. 
 
 conalbumin is a mixture or not, and the question concerning the unity 
 of the crystallizable ovalbumin is also disputed. According to Bond- 
 zynski and Zoja, crystallizable ovalbumin is a mixture of several albumins 
 having somewhat different coagulation temperatures, solubilities, and 
 specific rotations, while Hofmeister and Langstein on the contrary 
 believe that crystallizable ovalbumin is a unit. The reports as to the 
 specific rotation of the different fractions unfortunately differ, and the 
 elementary analyses have also given no positive results, as a variation 
 of 1.2-1.7 per cent has been observed in the quantity of sulphur. Accord- 
 ing to the consistent analyses of Osborne and Campbell and of Lang- 
 stein, the conalbumin contains about 1.7 per cent sulphur and about 
 16 per cent nitrogen, while the ovalbumin contains on an average about 
 15.3 per cent nitrogen. Langstein 1 obtained 10-11 per cent glucosa- 
 mine from ovalbumin and about 9 per cent from conalbumin. The 
 ovalbumin, like the conalbumin, has the properties of the albumins in 
 general, but differs from seralbumin in that the specific rotation 
 is lower. It is quickly made insoluble by alcohol and is precipitated 
 by a sufficient quantity of HC1, but dissolves in an excess of acid with 
 greater difficulty than the seralbumin. The products isolated by 
 Abderhalden and Pregl 2 on the hydrolysis of ovalbumin do not show 
 anything of special interest. 
 
 As in the past certain doubts have existed as to the purity and chem- 
 ical unity of the ovalbumins, or also of the crystalline ovalbumin, so now 
 this doubt has become still stronger since ovalbumin has been pre- 
 pared partly free from phosphorus and partly with a variable phos- 
 phorus content of 0.1-3.06 per cent (Kaas, Willcock and Hardy 3 ). 
 
 In preparing .crystalline ovalbumin, mix, according to Hofmeister, 
 the beaten white of egg free from foam with an equal volume of a saturated 
 ammonium-sulphate solution, filter off the globulin, and allow the nitrate 
 to evaporate slowly in thin layers at the temperature of the room. After 
 a time the masses which separate out are dissolved in water, treated 
 with ammonium sulphate-solution until they begin to get cloudy, and 
 are allowed to stand. After repeated recrystallization the mass is either 
 treated with alcohol, which makes the crystals insoluble, or they are 
 dissolved in water and purified by dialysis. From these solutions the 
 proteid does not crystallize again on spontaneous evaporation. (See also 
 page 633, footnote 2, for the Hopkins and Pinkus method.) Will- 
 cock 4 has recently found that* magnesium sulphate can also be used in 
 the crystallization of ovalbumin. 
 
 1 Zeitsrhr. f. physiol. Chem., 31. 
 - Ibid., 4«. 
 
 3 Kaas, Monatsh. f. Chem., 27; Willcock and Hardy, cited from Chem. Centralbl., 
 1907, 2, 821. 
 
 4 Journ. of Physiol., 37. 
 
OVOMUCOID. 635 
 
 Conalbumin can ho removed from the filtrate, after the complete 
 crystallization of the ovalbumin, by removing the sulphate by means of 
 dialysis and coagulating by heat. 
 
 Gautier ' found a fibrinogen-like substance in the white of egg, which was 
 changed into a fibrin-like body by the action of a ferment. 
 
 Ovomucoid. This substance, first observed by Neumeister and 
 considered by him as a pseudopeptone, and then later studied by Salkow- 
 ski, is, according to C. Th. Morner, 2 a mucoid with 12.65 per cent 
 nitrogen and 2.20 per cent sulphur. Ovomucoid exists in hens' eggs 
 to the extent of about 12 per cent of the total solids. 
 
 A solution of ovomucoid is not precipitated by mineral acids nor by 
 organic acids, with the exception of phosphotungstic acid and tannic 
 acid. It is not precipitated by metallic salts, but basic lead acetate and 
 ammonia render it insoluble. Ovomucoid is thrown down by alcohol, 
 but sodium chloride, sodium sulphate, and magnesium sulphate give 
 no precipitates cither at the ordinary temperature or when the salts are 
 added to saturation at 30° C. Its solutions are not precipitated by an 
 equal volume of a saturated solution of ammonium sulphate, but are 
 precipitated on adding more salt thereto. The substance is not pre- 
 cipitated on boiling, but the part which has become insoluble in cold 
 water and which has been dried, is dissolved by boiling water. Zanetti 
 has prepared glucosamine on splitting ovomucoid with concentrated 
 hydrochloric acid, and Seemann found that the quantity of glucosamine 
 in ovomucoid was 34.9 per cent. 3 
 
 Ovomucoid may be prepared by removing all the proteins by boil- 
 ing with the addition of acetic acid, and then concentrating the filtrate 
 and precipitating with alcohol. The substance is purified by repeated 
 solution in water and precipitation with alcohol. 
 
 Paxormow believes that the eggs of other birds, such as the pigeon and duck, 
 contain a special protein in the egg-white, which is not identical with that of the 
 hen's egg. Worms 4 has prepared a crystalline albumin from the white of the 
 turkey eggs which contained 15.37 per cent N, 1.6 per cent S and had a specific 
 rotation of (o;)d = —34.9°. 
 
 The mineral bodies of the white of egg have been analyzed by 
 Poleck and Weber. 5 They found in 1000 parts of the ash: 276.6- 
 
 iCompt. Rend., 135. 
 
 2 R. Neumeister, Zeitschr. f. Biologic, 27; Salkowski. Centralbl. f. d. med. Wis- 
 senseh., 1893, 513 and 706; C. Morner, Zeitsch. f. physiol. Chem., 18 and 80. See 
 also Langstein, Hofmeister's Beitrage, 3 (literature). 
 
 3 Zanetti, Chem. Centralbl., 1898, 1; Seemann, cited from Langstein, Ergebnisse 
 der Physiol., 1, Abt. 1, 86. 
 
 4 Panormow, see Bioch. Centralbl., 5; Worms, cited from Chem. Centralbl., 1906, 
 2, 1508. 
 
 5 Cited from Hoppe-Seyler, Physiol. Chem., 778. 
 
636 ORGANS OF GENERATION. 
 
 284.5 grams potash, 235.6-329.3 soda, 17.4-29.0 lime, 17-31.7 magnesia, 
 4.4-5.5 iron oxide, 238.4-285.6 chlorine, 31.6-48.3 phosphoric acid (P 2 5 ), 
 13.2-26.3 sulphuric acid, 2.8-20.4 silicic acid, and 96.7-116.0 grams carbon 
 dioxide. Traces of fluorine have also been found (Nickles 1 ). The 
 white of egg contains, as compared with the yolk, a greater amount of 
 chlorine and alkalies and a smaller amount of lime, phosphoric acid, and 
 iron. 
 
 The Shell-membrane and the Egg-shell. The shell-membrane con- 
 sists, as above stated (page 112), of a keratin substance. The shell con- 
 tains very little organic substance, 36-65 p. m. The principal mass, more 
 than 900 p. m., consists of calcium carbonate; besides this there are very 
 small amounts of magnesium carbonate and earthy phosphates. 
 
 The diverse coloring of birds' eggs is due to several different coloring-matters. 
 Among these we find a red or reddish-brown pigment called " oorodein " by 
 Sorby, 2 which is perhaps identical with hsematoporphyrin. The green or blue 
 coloring-matter, Sorby's oocyan, seems, according to Liebermann 3 and Kruken- 
 berg, 4 to be partly biliverdin and partly a blue derivative of the bile-pigments. 
 
 The eggs of birds have a space at their blunt end filled with gas; this 
 gas contains on an average 18.0-19.9 per cent oxygen (Hufner 5 ). 
 
 The weight of a hen's egg varies between 40-60 grams and may some- 
 times reach 70 grams. The shell and shell-membrane together, when 
 carefully cleaned, but still in the moist state, weigh 5-8 grams. The 
 yolk weighs 12-18 and the white 23-34 grams, or about double. The 
 entire egg contains 2.8-7.5, or average 4.6, milligrams of iron oxide, and 
 the quantity of iron can be increased by food rich in iron (Hartung 6 ) . 
 
 The white of the egg of cartilaginous and bony fishes contains only traces of 
 true albumin, but consists, at least in many fishes, of mucin substance; and the 
 cover of the frog's egg also consists, according to Giacosa, of mucin. The eggs 
 of the river-perch contain, Hammarsten 7 claims, mucin in the envelope in the 
 unripe state and only mucinogen in the ripe state. The crystalline formations 
 (yolk-spherules, or dotterpldttchen) which have been observed in the egg of the 
 tortoise, frog, ray, shark, and other fishes, and which are described by Valen- 
 ciennes and Fremy under the names emydin, ichthin, ichthidin, and ichthulin, 
 seem, as above stated in connection with ichthulin, to consist mainly of phos- 
 phoglycoproteins. The klupeovin obtained by Hugounenq 8 from the herrings' 
 eggs and from which he obtained the three so-called hexone bases and abundant 
 
 1 Compt. Rend., 43. 
 
 2 Cited from Krukenberg, Verb. d. phy.s.-cbem. Gessellsch. in Wurzburg, 17. 
 8 Ber. d. deutsch. chem. Gesellsch., 11. 
 
 •lie. 
 
 6 Arch. f. (Anat. u). Physiol., 1892. 
 »Zeitechr. f. Biol., 43. 
 
 7 Giacos;i, Zeitschr. f. physiol. Chem., 7; Hammarsten, Skand. Arch. f. Physiol., 17. 
 8 Valenciennes and Fremy. eited from Hoppe-Seyler, Physiol. Chem., p. 77; 
 Hugounenq, Bull. soc. chim. (3), 33, and Compt. Rend., 143. 
 
THE EGG. G37 
 
 monamino-acids, especially leucine, hut not glycocoll or glutamic acid, is to 
 all appearances not a unit body. The eggs of the river-crab and the lobster 
 contain the same pigment as the shell of the animal. This pigment, called 
 cyanocrystallin, becomes red on boiling in water. 
 
 C. Morner l has isolated a substance which he calls pcrcnglobulin, from the 
 unripe eggs of the river-perch. It is a globulin and has a strong astringent taste. 
 Especially striking is its property of precipitating certain glycoproteins, such as 
 ovomucoid and ovarial mucoids, and polysaccharides, such as glycogen, gum, 
 tragacanth and starch-paste, and of being precipitated by them. Percaglobulin 
 could not be obtained by Morner from the eggs of the sea-bass. 
 
 In fossil eggs (of apetnodytes, pelecanus, and hall,eus) in old guano deposits, 
 a yellowish-white, silky, laminated compound has been found which is called 
 guanovulit, (NH4)2S04-r-2K2S04+3KHS04-f-4H 2 0, and which is easily soluble in 
 water, but is insoluble in alcohol and ether. 
 
 Those eggs which develop outside of the mother-organism must con- 
 tain all the elements necessary for the young animals. One finds, there- 
 fore, in the yolk and white of the egg an abundant quantity of protein 
 bodies of different kinds, and especially phosphorized proteins in the 
 yolk. Further, we also find abundance of phosphatides in the yolk, 
 which seem to occur habitually in all developing cells. Kato and Bleib- 
 treu 2 found glycogen in the eggs of the frog which during the spawning 
 season increased at the cost of the liver glycogen. Besides this the egg 
 is very rich in fat, which doubtless is important as a source of supply 
 for nourishment and in maintaining respiration for the embryo. The 
 cholesterin or at least the lutein can hardly have a direct influence on 
 the development of the embryo. The egg also seems to contain the 
 mineral bodies necessary for the development of the young animal. 
 The lack of phosphoric acid is compensated by an abundant amount of 
 phosphorized organic substance, and the nucleoalbumin containing 
 iron, from which the haematogen (see page 629) is formed, is doubtless, as 
 Bunge claims, of great importance in the formation of the haemoglobin 
 containing iron. The silicic acid, necessary for the development of the 
 feathers, is also found in the egg. 
 
 During the period of incubation the egg loses weight, due chiefly to 
 loss of water. The quantity of solids, especially the fat and the proteins, 
 diminishes, and the egg gives off carbon dioxide, but Tangl disproves 
 the older claim of Liebermann 3 that nitrogen or a nitrogenous substance 
 is given off. On the contrary a corresponding absorption of oxygen 
 takes place, and it is found that during incubation a respiratory exchange 
 of gases occurs. 
 
 As Bohr and Hasselbalch have shown by exact investigations, the 
 elimination of carbon dioxide is very small in the first days of incuba- 
 
 1 Zeitschr. f. physiol. Chem., 40 and 58. 
 
 »Kato, Pfliiger's Arch. 132; Bleibtreu, ibid., 132 (1010). 
 
 * Tangl and v. Mituch, Pfluger's Arch., 121 ; Liebermann, ibid., 43. 
 
638 ORGANS OF GENERATION. 
 
 tion; on the fourth day the carbon-dioxide production gradually increases, 
 and after the ninth day it augments in the same proportion as the weight 
 of the foetus. Calculated upon 1 kilogram weight for one hour it is,, 
 from the ninth day on, about the same as in the full-grown hen. Hassel- 
 balch l has also shown that the fertilized hen's egg not only gives off 
 nitrogen the first five or six hours of incubation, but also some oxygen, 
 and that we are here dealing with an oxygen production which runs 
 parallel with the cell-division. It is not known whether this oxygen 
 formation connected with the life of the cell is a fermentative or a so- 
 called vital process. 
 
 While the quantity of dry substance in the egg during this period 
 always decreases, the quantity of mineral bodies, protein, and fat always 
 increases in the embryo. The increase in the amount of fat in the 
 embryo depends, in great part upon a taking up of the nutritive yolk 
 in the abdominal cavity. Plimmer and Scott 2 have observed in the incu- 
 bation of the hen's egg, that a rapid diminution of phosphorized substances 
 soluble in ether takes place, while at the same time an increase in the 
 inorganic phosphorus is found in the chick. 
 
 The weight of the shell and the quantity of lime-salts contained therein 
 do not remain unchanged, according, to the recent investigations of 
 Tangl. 3 The egg-shell (lime shell, and shell-membrane) of a hen's egg 
 weighing 60 grams loses (calculated on the dry) during incubation about 
 0.4 gram, of which 0.15 gram is calcium and 0.2 gram is organic substance. 
 
 A very complete and careful chemical investigation on the develop- 
 ment of the embryo of the hen has been made by Liebermann. 4 From 
 his researches we may quote the following: In the earlier stages of the 
 development, tissues very rich in water are formed, but upon the con- 
 tinuation of the development the quantity of water decreases. The 
 absolute quantity of the bodies soluble in water increases with the develop- 
 ment, while their relative quantity, as compared with the other solids, 
 continually decreases. The quantity of the bodies soluble in alcohol 
 quickly increases. A specially important increase is noticed in the fat, 
 whose quantity is not very great even on the fourteenth day, but after 
 that it becomes considerable. The quantity of protein bodies and albu- 
 minoids soluble in water grows continually and regularly in such a way 
 that their absolute quantity increases, while their relative quantity 
 remains nearly unchanged. Liebermann found no gelatin in the 
 
 1 Bohr and Hasselbaleh, Maly's Jahresber., 2i); Hasselbalch, Skand. Arch. f. 
 Physiol., 13. 
 
 2 Journ. of Physiol., .'{S. 
 
 a Tangl with Hammerschlag, Pfliiger's Arch.. 121. 
 
 * 1. c. 
 
DEVELOPMENT OF THE CHICK EMBRYO. 639 
 
 embryo of the hen. The embryo doea not contain any gelatin-forming 
 substance until the tenth day, and from the fourteenth da}' on it contains 
 a body which, when boiled with water, gives a BUDStance similar to chon- 
 drin. A body similar to mucin occurs in the embryo when about six 
 days old, but then disappears. The quantity of haemoglobin shows a 
 continual increase compared with the weight of the body. Liebermann 
 found that the relation of the haemoglobin to the body weight was 1:728 
 on the eleventh day and 1:421 on the twenty-first day. 
 
 By means of Berthelot's thermometric methods Tangl x has 
 determined the chemical energy present at the beginning and end of 
 the development of the embryo of the sparrow's and hen's eggs. The 
 difference was considered as work of development. He found that the 
 chemical energy necessary for the development of each gram of ripe hen's 
 embryo (Plymouth) was equal to 0.805 Cal. This energy originated 
 chiefly from the fat. Of the total chemical energy utilized, about 70 
 per cent was used for the embryo and about 30 per cent remained in the 
 yolk. Of the utilized energy about two-thirds was used in the con- 
 struction of the embryo and about one-third transformed into other 
 forms of energy as work of development. 
 
 By their investigations on the development of the trout egg, Tangl 
 and Farkas 2 have found that the loss in weight of each egg which had 
 an average weight of 88 milligrams was 4.9 milligrams during the 42 
 days of incubation, of which 4.11 milligrams was water and 0.722 milli- 
 gram dry substance with 0.367 milligram C. The eggs lose no nitro- 
 gen and no fat. The fat content increases a little, and indeed, as these 
 authors believe, at the expense of the proteins. The chemical energy 
 used during development was 6.68 gram-calories. 
 
 The highly interesting investigations made by Loeb upon the fer- 
 tilization of the eggs of lower sea-animals will be discussed in this con- 
 nection. According to these experiments after the fertilization of the 
 egg by means of a sort of cytolysis small drops of a colloid substance 
 form on the surface of the egg. These drops enlarge in volume and 
 conglomerate to a continuous mass, while its surface hardens to a tight, 
 continuous membrane — the fertilization membrane. The process of 
 membrane formation is in fact the essential step in the fertilization. 
 Besides, by spermatozoa, the membrane formation is caused by different 
 actions. For many eggs all that is necessary is the artificial calling 
 forth of the processes for the membrane formation in order that the 
 egg shall develop to normal larvae (for example the eggs of the star 
 fish and of certain worms). In other cases, for example the sea-urchin, 
 Strongylocentrotus, a second action is necessary for the production of 
 
 1 Pfluger's Arch., 93 ani 121. ! Ibid., 104. 
 
640 ORGANS OF GENERATION. 
 
 normal larva?. The principal points in the treatment of such eggs are 
 the following. 
 
 The formation of the fertilization membrane can be brought about 
 by placing the eggs in sea water which has been faintly acidified with 
 a fatty acid, for example with butyric acid, and after If to 2 minutes 
 placed again in sea-water. The formation of the membrane now takes 
 place. The oxy acids and especially the inorganic acids are less active, 
 than the fatty acids. The H-ions are without effect in this acid action 
 and Loeb explains the action by the introduction of the undissociated 
 molecules into the egg. Parallel with the membrane formation chemical 
 processes begin, among which we must especialty mention oxidations. 
 These processes, if they proceed undisturbed, especially at 15° or above, 
 lead quickly to the death of the egg. This can, nevertheless, be prevented 
 if the oxidation processes are inhibited 40-60 minutes after the mem- 
 brane formation by removing the oxygen or by the addition of some 
 potassium cyanide. In this process probably certain injurious substances 
 for the egg are destroyed. If eggs treated in this way are placed in 
 sea-water after 2-3 hours they develop in a normal manner. 
 
 The membrane formation can also be brought about in other ways 
 besides by the action of acids, for example by treating the egg with saponin, 
 solanin, digitalin, soaps and fat dissolving substances such as amylene, 
 benzene, toluene, chloroform, ether and alcohol. The sea-urchin egg is 
 also excited to membrane formation by the serum of certain animals. 
 Alkalies and elevation of temperature can also cause the formation of 
 membrane. 
 
 On the other hand the chemical processes, which, when not prevented, 
 lead to the death of the egg, can also be inhibited by placing the eggs in 
 a hypertonic solution (50 ccl sea-water and 8 cc. 2.5 normal NaCl) 
 about one hour after the artificial membrane has been formed and then 
 after 20-50 minutes placing them in sea-water again. 
 
 According to Loeb the artificial fertilization of the sea-urchin's egg 
 depends upon two special actions, of which the first brings about the for- 
 mation of membrane with oxidation processes by means of cytolysis 
 while the second gives the direction of these oxidation processes necessary 
 for the maintenance of life. 
 
 The non-fertilized, ripe egg, as the investigations of Loeb on star- 
 fish have shown, dies in 4-6 hours at sufficiently high temperatures. The 
 death of the egg can, nevertheless, be prevented if oxygen is removed 
 from the egg or the oxidation inhibited by the addition of traces of potas- 
 sium cyanide. If the ripe egg is fertilized by spermatozoa then it remains 
 alive although the process of fertilization, as Warburg l found, causes 
 
 1 Zeitschr. f. physiol. Chem., 57, 60, 66. 
 
PLACENTA. 64L 
 
 a considerable rise in the oxidation. For this reason Loeb believes that 
 the spermatozoa save fche life of the egg by bringing membrane forming 
 substances to the egg, but also other substances, which remove or make 
 inert a harmful substance or condition complex of the unfertilized egg, 
 so that even now the increased oxidation cannot have any harmful effect. 1 
 
 The enzymes of the sea-urchin suffer an increase in natural as well 
 as in artificial fertilization as Jacoby 2 has shown that glycyltryptophane 
 is split after fertilization but not before. 
 
 The placenta has recently been the subject of several investigations. 
 This tissue contains a protein which coagulates at 60-65° C. (Bottazzi 
 and Delfino) whose relation to the nucleoprotein, found by others, is 
 not clear. The protein found by Savare contained 0.45 per cent phos- 
 phorus. The nucleic acid studied by Kikkoji, 3 which is very similar to 
 the thymus nucleic acid, originates from this nucleoprotein. Glycogen 
 occurs regularly in the placenta, and Moscati believes the human pla- 
 centa contains 5 p. m. glycogen. After removal the glycogen diminishes, 
 and after 24 hours it has disappeared. According to Lochhead and 
 Cramer 4 the quantity of glycogen in the placenta is not increased by 
 food rich in carbohydrate. In the fcetus (rabbits) the above authors 
 found that the placenta is a storage organ for glycogen until the second 
 half of the gestation period, when the liver begins to functionate in this 
 direction. From this time on the quantity of glycogen in the placenta 
 diminishes. 
 
 Enzymes of various kinds, proteolytic as well as lipolytic (mono- 
 butyrase), amylases and oxidases have been found in the placenta. 5 
 In the edges of the placenta of the bitch and of cats, an orange-colored, 
 crystalline pigment (bilirubin) and a green, amorphous pigment, whose 
 relation to biliverdin is not clear, have been found. 6 
 
 From the cotyledons of the placenta in ruminants a white or faintly rose-colored 
 creamy fluid, the uterine milk, can be obtained by pressure. It is alkaline in 
 
 1 A complete review of the investigations of Loeb and his collaborators, with the 
 literature can be found in Vorlesungen iiber die Dynamik der Lebenserscheinungen, 
 Leipzig, 1906, s. 239. See also Uber den chemischen Charakter des Befruchtungsvor- 
 ganges, Leipzig, 1908; Zeitschr. f. physik. Chem. 70, 220 (1910), Arch. f. Entwickelungs- 
 mech., 31, 658 (1910). 
 
 2 Bioch. Zeitschr., 26, 333 (1910). 
 
 3 Bottazzi and Dcltino, Centralbl. f. Physiol., 18, 114; Savare, Hofmeister's, Beitrage, 
 11; Kikkoji, Zeitschr. f. physiol. Chem., 53. 
 
 4 Moscati, Zeitschr, f. physiol. Chem., 53; Lochhead and Cramer, Proc. Roy. Soc, 
 80 B. (1908). 
 
 6 Ascoli, Centralbl. f. Physiol., 16; Raineri, Bioch. Centralbl.. 4, 428; Bergell and 
 Liepmann, Munch, med. Wochenschr., 1905; Savare, Hofmeister's Beitrage, 9; Bergell 
 and Falk, Munch, med. Wochenschr., 55. 
 
 6 See Etti, Maly's Jahresber., 2, 287, and Preyer, Die Blutkristalle, Jena, 1871. 
 
642 ORGANS OF GENERATION. 
 
 reaction, but quickly becomes acid. Its specific gravity is 1.033-1.040. It con- 
 tains as form-elements fat-globules, small granules, and epithelium-cells. There 
 have been found 81.2-120.9 p. m. solids, 61.2-105.6 p. m. protein, about 10 p. m. 
 fat, and 3.7-8.2 p. m. ash in the uterine milk. 
 
 The fluid occurring in the so-called grape-mole (Mola racemosa) has a low 
 specific gravity, 1.009-1.012, and contains 19.4-26.3 p. m. solids with 9-10 p. m. 
 protein bodies and 6-7 p. m. ash. 
 
 The amniotic fluid in women is thin, w r hitish, or pale yellow; some- 
 times it is somewhat yellowish-brown and cloudy. AVhite flakes separate. 
 The form-elements are mucus-corpuscles, epithelium-cells, fat-drops, and 
 lanugo hair. The odor is stale, the reaction neutral or faintly alkaline. 
 The specific gravity is 1:002-1.028. 
 
 The amniotic fluid contains the constituents of ordinary transudates. 
 The amount of solids at birth is scarcely 20 p. m. In the earlier stages of 
 pregnancy the fluid contains more solids, especially proteins. Among 
 the protein bodies, Weyl found one substance similar to vitellin, and with 
 great probability also seralbumin, besides small quantities of mucin. 
 Enzymes of various kinds (pepsin, diastase, thrombin, lipase) occur, 
 according to Bondi. Sugar is regularly found in the amniotic fluid of 
 cows, but not in human beings. In the ox, pig, and goat Gurber and 
 Grunbaum also found fructose. The human amniotic fluid also contains 
 some urea, uric acid, allantoin and creatinine (Amberg and Rowntree). 
 The quantity of these may be increased in hydramnion (Prochownick, 
 Harnack), which depends on an increased secretion by the kidneys and 
 skin of the foetus. Lactates are doubtful constituents of the amniotic 
 fluid. The quantity of urea in the amniotic fluid, is, according to Pro- 
 chownick, 0.16 p. m. In the fluid in hydramnion Prochownick and 
 Harnack found, respectively, 0.34 and 0.48 p. m. urea. The principal 
 mass of the solids consists of salts. The quantity of chlorides (NaCl) is 
 5.7-6.6 p. m. The molecular concentration of the amniotic fluid is some- 
 what lower than that of the blood, which is no doubt due to a dilution 
 by the fcetal urine (Zangemeister and Meissl 1 ). 
 
 iWeyl, Arch. f. (Anat. u.) Physiol., 187G; Bondi, Centralbl. f. Gynakol., 1903; 
 Prochownick, Arch. f. Gynak., 11, also Maly's Jahresber., 7, 155; Harnack, Berlin, 
 klin. Wochenschr., 1888, No. 41; Zangemeister and Meissl, Munch, med. Wochenschr. 
 1903; Gurber and Grunbaum, ibid., 1904; Amberg and Rowntree, cited from Bioch. 
 Centralbl., 10, 237. 
 
CHAPTER XIII. 
 MILK. 
 
 The chemical constituents of the mammary glands have been little 
 studied. The cells are rich in protein and nucleoproteins. Among the 
 latter we have one that yields pentose and guanine, on boiling with dilute 
 mineral acids, but no other purine base. This compound protein, inves- 
 tigated by Odenius, contains as an average the following: 17.28 per 
 cent N, .0.89 per cent S, and 0.277 per cent P. Besides this compound 
 proteid we have at least one other, as Mandel and Levene and Loebisch l 
 have isolated a nucleic acid from the mammary gland, which, like the 
 thymonucleic acids, yielded adenine, guanine, thymine, and cytosine. 
 This nucleic acid also gave the pentose reactions and yielded an abundance 
 of levulinic acid. Besides this nucleic acid, Mandel and Levene isolated 
 from the glands a glucothionic acid with 2.65 per cent S and 4.38 per 
 cent N. Among the cleavage products of the nucleoprotein Mandel 2 
 obtained no glycocoll, and the products of hydrolysis show a great cor- 
 respondence with those of casein. We cannot state what relation the 
 above-mentioned nucleic acids and the glucothionic acid bear to the 
 not well-known constituent of the glands fcund by Bert and by Thier- 
 felder and which yields a reducing substance when boiled with dilute 
 acids. 
 
 It is to be expected that these bodies are steps in the formation of 
 milk-sugar; still we have no point of support for such an assumption, 
 and recent investigations seem to indicate that the milk-sugar is produced 
 in the glands by a transformation of the sugar of the blood. Fat seems, 
 at least in the secreting glands, to be a never-failing constituent of the 
 cells, and this fat may be observed in the protoplasm as large or small 
 globules similar to milk-globules. The extractive bodies of the mam- 
 mary glands have been little investigated, but among them are found 
 considerable amounts of purine bases. The mammary glands also 
 contain enzymes, among which we especially mention : catalase, 
 
 Odenius, Maly's Jahresber., 30; Mandel and Levene, Zeitschr. f. physiol. Chem., 
 46; Loebisch, Hofineister'sBeitriige, 8. 
 
 * Mandel and Levene, Zeitschr. f. physiol. Chem., 45, Mandel, Bioch. Zeitschr , 23 
 
 643 
 
644 MILK. 
 
 peroxidase and a proteolytic enzyme which, according to Hildebrandt, 1 
 occurs to a much greater extent in the active gland as compared with 
 the inactive one. 
 
 As human milk and the milk of animals are essentially of the same 
 constitution, it seems best to speak first of the one most thoroughly 
 investigated, namely, cow's milk, and then of the essential properties 
 of the remaining important kinds of milk. 2 
 
 Cow's Milk. 
 
 Cow's milk, like every other kind, forms an emulsion which consists 
 of very finely divided fat suspended in a solution consisting principally 
 of protein bodies, milk-sugar, and salts. Milk is non-transparent, white, 
 whitish-yellow, or in thin layers somewhat bluish-white, of a faint, insipid 
 odor and mild, faintly sweetish taste. The specific gravity is 1.028 
 to 1.0345 at 15° C. The freezing-point is -0.54-0.59° C., average 
 — 0.563° C, and the molecular concentration 0.298. 
 
 The reaction of perfectly fresh milk is generally amphoteric toward 
 litmus. The extent of the acid and alkaline part of this amphoteric 
 reac:ion has been determined by different investigators, especially 
 Thorner, Sebelien, and Cotjrant. 3 The results differ with the indi- 
 cators used, and moreover the milk from different animals, as well as 
 that from the same animal at different times during the lactation period, 
 varies slightly. Courant determined the alkaline part by N/10 sul- 
 phuric acid, using blue lacmoid as indicator, and the acid part by N/10 
 caustic soda, using phenolphthalein as indicator. He found, as an average 
 for the first and last portions of the milking of twenty cows, that 100 
 cc. milk had the same alkaline reaction toward blue lacmoid as 41 cc. 
 N/10 caustic soda, and the same acid reaction toward phenolphthalein 
 as 19.5 cc. N/10 sulphuric acid. The actual reaction of cow's milk, 
 which follows from the electrometric estimation, is, on the contrary, 
 Foa 4 claims, nearly neutral, like the reaction of animal fluids and 
 tissues in general. 
 
 Milk gradually changes when exposed to the air, and its reaction 
 becomes more and more acid. This depends on a gradual transforma- 
 tion of the milk-sugar into lactic acid, caused by micro-organisms. 
 
 'Bert, Compt. Rend., 98; Thierfelder, Pfliiger's Arch., 34, and Maly's Jahresber., 
 13; Hildebrandt, Hofmeister's Beitrage, 5. 
 
 * A very complete reference to the literature on milk may be found in Raudnitz's 
 "Die Bestandteile der Milch," in Er{r''bnisse der Physiol., 2, Abt. 1. The literature 
 of the last few years may be found in the references by Raudnitz, Monatsschrift f. 
 Kinderheilkunde. 
 
 ' Thorner, Maly's Jahresber., 22; Sebelien, ibid., Courant; Pfliiger's Arch., 50. 
 
 * Compt. rend. soc. biolog. (58), 59, 51. 
 
COW'S MILK. 645 
 
 Perfectly fresh amphoteric milk does not coagulate on boiling, but 
 forms a pellicle consisting of coagulated casein and lime-salts, which 
 rapidly re-forms after being removed. After a sufficiently strong spon- 
 taneous formation of acid it coagulates on boiling, and lastly, when the 
 formation of lactic acid is sufficient, it coagulates spontaneously at the 
 ordinary temperature, forming a solid mass. It may also happen, espe- 
 cially in the warmth, that the casein-clot contracts and a yellowish or 
 yellowish-green acid liquid (acid whey) separates. 
 
 Milk may undergo various fermentations. Lactic-acid fermentation, brought 
 about by Huppe's lactie-acid bacillus and also other varieties, takes first place. 
 In the spontaneous souring of milk we generally consider the formation of lactic 
 acid as the most essential product, but a formation of succinic acid may also take 
 place, and in certain bacterial decompositions of milk, succinic acid and no lactic 
 acid is formed. The materials from which these two acids are formed are lactose 
 and lactophosphocarnic acid. Besides the lactic acids, the optically inactive 
 as well as the dcxtro and levo acids, and succinic acid, volatile fatty acids, such 
 as acetic acid, butyric acid, and others, may be formed in the bacterial decompo- 
 sition of milk. 
 
 Milk sometimes undergoes a peculiar kind of coagulation, being converted 
 into a thick, ropy, slimy mass (thick milk). This conversion depends upon a 
 peculiar change in which the milk-sugar is made to undergo a slimy transforma- 
 tion. This transformation, which requires further investigation, is caused by 
 special micro-organisms. 
 
 If the milk is sterilized by heating, and contact with micro-organisms 
 prevented, the formation of lactic acid may be entirely stopped. The 
 production of acid may also be prevented, at least for sometime, by many 
 antiseptics, such as salicylic acid, thymol, boric acid, and other bodies. 
 
 If freshly drawn amphoteric milk is treated with rennet, it coagulates 
 quickly, especially at the temperature of the body, to a solid mass (curd) 
 from which a yellowish fluid (sweet whey) is gradually pressed out. This 
 coagulation occurs without any change in the reaction of the milk, and 
 therefore it is distinct from the acid coagulation. 
 
 In cow's milk we find as form-elements a few colostrum corpuscles 
 {see Colostrum) and a few pale nucleated cells. The number of these 
 form-elements is very small compared with the immense amount of the 
 most essential form-constituents, the milk-globules. 
 
 The Milk-globules. These consist of extremely small drops of fat 
 whose number is, according to Woll, 1 1.06-5.75 millions in 1 c.mm., 
 and whose diameter is 0.0024-0.0046 mm. and 0.0037 mm. as an average 
 for different kinds of animals. It is unquestionable that the milk-globules 
 contain fat, and we consider it as positive that all the milk-fat exists in 
 them. Another disputed question is whether the milk-globules consist 
 entirely of fat or whether they also contain protein. 
 
 ^n the Conditions Influencing the Number and Size of Fat-globules in Cow's 
 Milk, Wisconsin Exp. Station, 6, 1892. 
 
646 MILK. 
 
 The observations of Ascherson 1 show that drops of fat, when dropped in an 
 alkaline protein solution, are covered with a fine albuminous coat, a so-called haptogen- 
 membrane. As milk on shaking with ether does not give up its fat, or only very 
 slowly in the presence of a great excess of ether, and as this takes place very readily 
 after the addition of acids or alkalies, which dissolve proteins, it was formerly 
 thought that the fat-globules of the milk were enveloped in a protein coat. A 
 true membrane has not been detected; and since, when no means of dissolving 
 the protein is resorted to — for example, when the milk is precipitated by carbon 
 dioxide after the addition of very little acetic acid, or when it is coagulated by 
 rennet — the fat can be very easily extracted by ether, the theory of a special albu- 
 minous membrane for the fat-globule has been generally abandoned. The observa- 
 tions of Quincke 2 on the behavior of the fat-globules in an emulsion prepared 
 with gum have led, at the present time, to the conclusion that each fat-globule 
 in the milk is surrounded by a stratum of casein solution held by molecular attrac- 
 tion, and this prevents the globules from uniting with each other. Everything 
 that changes the physical condition of the casein in the milk or precipitates it 
 must necessarily help the solution of the fat in ether, and it is in this way that the 
 alkalies, acids, and rennet act. 
 
 V. Storch has shown, in opposition to these views, that the milk- 
 globules are surrounded by a membrane of a special slimy substance. 
 This substance is very insoluble, contains 14.2-14.79 per cent nitrogen, 
 and yields a sugar, or at least a reducing substance, on boiling with 
 hydrochloric acid. It is neither casein nor lactalbumin, but it seems to 
 all appearances to be identical with the so-called " stroma substance " 
 detected by Radenhausen and Danilewsky. Storch was able to 
 show, by staining the fat-globules with certain dyes, that this substance 
 enveloped them like a membrane. Recently Voltz has given further 
 proofs of the view that the fat-globules probably have a membrane, 
 which in his opinion is a very labile formation of variable composition, 
 and Bauer has also given further proofs for the assumption of a mem- 
 brane. Droop-Richmond and Bonnema, 3 on the other hand, present 
 several deductions conflicting with Storch's theory. If Storch's observa- 
 tion that the purified fat-globules contain a special protein substance 
 differing from the dissolved proteins of the milk is correct, then the 
 assumption as to a special body forming a membrane or stroma of the 
 fat-globules becomes very probable. The correctness of Storch's view 
 has been substantiated very recently by Abderhalden and Voltz. 4 
 On the acid hydrolysis of the fat-globules they obtained glycocoll, which 
 is absent in the casein as well as in the lactalbumin, and this shows that the 
 
 1 Arch. f. Anat. u. Physiol., 1840. 
 
 * Pfluger's Arch., 19. 
 
 1 V. Storch, see Maly's Jahresber., 27; Radenhausen and Danilewsky, Forschungen 
 auf dem Gebiete der Viehhaltung (Bremen, 1880), Heft 9; Voltz, Pfluger's Arch., 102; 
 Bauer, Bioch. Zeitecbf. 32; Droop-Richmond, see Chem. CentralbL, 1094, 2, 356;. 
 Bonnema, ibid., 1243. 
 
 4 Zeitschr. f. physiol. Chem., 59. 
 
MILK FAT. CASEIN. 647 
 
 fat-globules at least cannot contain these two proteins alone. They 
 must contain another protein, and it is still a question whether besides this 
 they also contain casein and lactalbumin. 
 
 The milk-fat which is obtained under the name of butter consists 
 mainly of olcin and palmitin. Besides these it contains, as triglycerides, 
 myristic acid, stearic acid, small amounts of lauric acid, arachidic acid, 
 and dioxystearic acid,, besides butyric acid and caproic acid, traces of 
 caprylic acid and capric acid. Riegel claims that triglycerides of vola- 
 tile fatty acids do not occur, but rather mixed triglycerides of volatile 
 and non-volatile fatty acids. Milk-fat also contains small quantities 
 of phosphatides (lecithin), and cholesterin and a yellow coloring-matter. 
 The quantity of volatile fatty acids in butter is, according to Duclaux, 
 on an average about 70 p.m., of which 37-51 p.m. is butyric acid and 
 30-33 p. m. is caproic acid. The non-volatile fat consists of iV- & olein 
 and the remainder is principally palmitin. The composition of butter 
 is not constant, but varies considerably under different circumstances. 1 
 The question whether the small fat-globules have a different composition 
 from the large ones is still disputed. 
 
 The milk-plasma, or that fluid in which the fat-globules are suspended, 
 contains several different proteins, the statements as to the number and 
 nature of which are somewhat at variance. The three following, casein, 
 lactalbumin, and lactoglobulin, have been most closely studied and are 
 well characterized. The milk-plasma contains at least two carbohy- 
 drates, of which the one, lactose, is of great importance. It also contains 
 extractive bodies, traces of urea, creatine, creatinine, orotic acid, hypoxan- 
 thine (?), cholesterin, citric acid (Soxhlet and Henkel 2 ), and lastly also 
 mineral bodies and gases. 
 
 Casein. This protein substance, which thus far has been detected 
 positively only in milk, belongs to the nucleoalbumins, and differs from 
 the albuminates chiefly by its content of phosphorus and by its behavior, 
 with the rennet enzyme. Casein from cow's milk has about the follow- 
 ing composition: C 53.0, H 7.0, N 15.7, S 0.8, P 0.85, and O 22.65 per 
 cent. Its specific rotation is, according to Hoppe-Seyler, rather variable; 
 in neutral solution it is (a) D =— 80°; its faintly alkaline solution has a 
 stronger rotation, namely, — 97.8 to —111.8°, in a solution of N/10-N/5 
 
 'Riegel, Maly's Jahresber., 34; Duclaux, Compt. Rend., 104. Various statement, 
 as to the composition of milk-fat can be found in Koefoed, Bull. d. l'Acad. Roys 
 Danoise, 1891, and Wanklyn, Chemical News, 63; Browne, Chem. Centralbl., 1899, 
 2, 883. In regard to the elementary composition of milk-fat see Fleischmann and 
 Warmbold, Zeitschr., f. Biol., 50. 
 
 2 Cited from Soldner, Die Salze der Milch, etc., Landwirthsch. Versuchsstation, 
 35, Separatabzug, 18. 
 
648 MILK. 
 
 NaOH (Long l ). The question whether the casein from different kinds 
 of milk is identical or whether there are several caseins cannot be decided 
 by the elementary analysis. According to Tangl and Csokas, 2 mare's 
 and ass's casein seem to be somewhat richer in nitrogen (16.44 and 16.28 
 per cent, respectively) but poorer in sulphur (0.528 and 0.588 per cent) 
 and carbon (52.36 and 52.27 per cent) than the casein from cud chewers. 
 The ass's casein was richer in phosphorus (1.057 per cent) than the mare's 
 or cow's casein (both with 0.887 per cent). 
 
 Casein when dry appears like a fine white powder, which has no 
 measurable solubility in pure water (Laqueur and Sackur). Casein 
 is only very slightly soluble in the ordinary neutral-salt solutions. Accord- 
 ing to Arthus it dissolves rather easily in a 1-per cent solution of sodium 
 fluoride, ammonium or potassium oxalate. Robertson thinks that it 
 is more soluble in potassium cyanide and the alkali salts of certain vola- 
 tile fatty acids such as butyric acid and valeric acid, than in solutions 
 of the ordinary neutral salts. It is at least a tetrabasic acid, whose 
 equivalent weight is 1135, according to Laqueur and Sackur, and 1250 
 according to Robertson. The statements as to the molecular weight 
 are disputed (Laqueur and Sackur, L. and D. van Slyke 3 ). 
 
 It dissolves readily in water with the aid of alkali or alkaline earths, 
 also calcium carbonate, from which it expels carbon dioxide ana it 
 thus forms caseinates of variable composition. If casein is dissolved 
 in lime-water and the solution carefully treated with very dilute phos- 
 phoric acid until it is neutral in reaction (to litmus), the casein appears 
 to remain in solution, but is probably only swollen as in milk, and the 
 liquid contains at the same time a large quantity of calcium phosphate 
 without any precipitate or any suspended particles being visible. The 
 casein solutions containing lime are opalescent, and have on warming 
 the appearance of milk deficient in fat (which is also true for the salts 
 of casein with the alkaline earths). Therefore it is not impossible that 
 the white color of the milk is due partly to the casein and calcium phos- 
 phate. Soldner and others have prepared two calcium compounds 
 of casein with 1.5 p. c. CaO (the neutral caseinate according to Soldner) 
 and 2.4 p. c. CaO (the basic caseinate). The first is neutral to litmus 
 while the other is neutral to phenolphthalein. 
 
 According to Robertson 4 the alkali equivalent of casein at neutrality 
 toward litmus =53 XlO -5 equivalent-grm.-mol. per gram and at neutrality toward 
 
 1 Hoppe-Seyler, Handb. d. physiol. u. pathol. chem. Analyse, 8. Aufl., 489; Long, 
 Journ. Amer. Chem. Soc, 27. 
 
 2 Pfluger'a An.-h., 121. 
 
 3 Laqueur and Sackur, Hofmeister's Beitrage, 3; M. Arthus, Theses presentees 
 a la faculty des sciences de Paris, 1893; Robertson, Journ. of biol. Chem., 2; L. an 1 
 D. van Slyke, Amer. Chem. Journ., 38. 
 
 4 See Ergebnis. d. Physiol. 10 and Journ. of physical Chem., 13. 
 
CASEIN. 649 
 
 phenolphthalein = S0xl0 -5 equivalent-gnu. -mol. per gram. On BaturatioD (with 
 monacidic liases) the alkali equivalent is = llxi0~ 5 grm.-mol. per gram. Od 
 saturating (with monobasic acids) the acid equivalent is=32X10 -5 grm.-mol. 
 per gram. 
 
 Besides the rather earlier investigations on the salts of casein by Sold- 
 ner, Courant, Rohmanx. Laqieur, Raudnitz ! and others we have 
 the recent observations and theoretical discussion of Robertson 2 on the 
 composition, nature and dissociation of the caseinates. We can here 
 only refer to this and the earlier investigations. 
 
 Casein solutions do not coagulate on boiling, but solutions of casein- 
 lime are covered, like milk, with a pellicle. They are precipitated by 
 very little acid, but the presence of neutral salts retards the precipitation. 
 A casein solution containing salt or ordinary milk requires, therefore, 
 more acid for precipitation than a salt-free solution of casein of the same 
 concentration. The precipitated casein dissolves very easily again in 
 a small excess of hydrochloric acid, but less readily in an excess of acetic 
 acid. The combination between casein and acid, like other protein 
 and acid compounds, is precipitated by neutral salts. These acid solu- 
 tions are precipitated by mineral acids in excess. 3 Casein is precipitated 
 from neutral solutions or from milk by common salt containing calcium, 
 or magnesium sulphate in substance, without changing its properties. 4 
 Metallic salts, such as alum, zinc sulphate and copper sulphate, com- 
 ply precipitate the casein from neutral solutions. 
 
 On drying at 100° C, casein, according to Laqieur and Sackur, decomposes 
 and splits into two bodies. One of these, called caseid. is insoluble in dilute alkalies, 
 while the other, the isocasein, is soluble therein. The isocasein is a stronger acid 
 and has other precipitation limits and a rather lower equivalent weight than the 
 casein. 
 
 The property which is the most characteristic of casein is that it 
 coagulates with rennet in the presence of a sufficiently large amount 
 of lime-salts. In solutions free from lime-salts the casein does not coagu- 
 late with rennet, but it is changed so that the solution (even if the enzymes 
 are destroyed by heating) yields a coagulated mass, ha ring the properties 
 of a curd, if lime-salts are added. The rennet enzyme, rennin, has there 
 
 1 Soldner, Die Salze der Milch, etc., and Maly's Jahresber., 25; Courant, 1. c. ; 
 Rohmann, Berlin, klin. Wochenschr., 1895; Laqueur, L c; and Hofmeister's Beitrage, 
 7; Raudnitz, Ergebn. d. Physiol., 2, Abt. 1. 
 
 2 Journ. of physical Chem., 11 and 12; Journ. of biol. Chem., 5. 
 
 3 In regard to the acid combinations of casein and the ability to take up acid, see 
 Laxa. Milch win hsoh. Centralbl., 1905; Long, Journ. Amer. Chem. Soc, 29; L. and 
 D. van Slyke, Amer. Chem. Journ., 38; Robertson, Journ. of biol. Chem., 4. 
 
 4 See the works of Hammarsten and Schmidt-Nielsen, Hammarsten's Festschrift, 
 1906. 
 
650 MILK. 
 
 fore an action on casein even in the absence of lime-salts. These last 
 are only necesary for the coagulation or the separation of the curd, 
 and the process of coagulation is hence a two-phase process. The first 
 phase is the transformation of the casein by the rennin, the second is 
 the visible coagulation caused by the lime-salts. This fact, which was 
 first proved by Hammarsten, was later confirmed by Arthus and Pages 
 and recently closely studied by Fuld, Spiro, and Laqueur and others. 1 
 
 The curd formed on the coagulation of milk contains large quantities of 
 calcium phosphate. According to Soxhlet and Soldner, the soluble lime-salts 
 are of essential importance only in coagulation, while the calcium phosphate 
 is without importance. Cocrant believes that the calcium-casein on coagula- 
 tion may earn' down with it, if the solution contains dicalcium phosphate, a part 
 of this as tricalcium phosphate, leaving mono-calcium phosphate in the solution. 
 A solution of calcium casein is not coagulated by rennin alone but only when soluble 
 lime-salts are added. Contrary to the generally accepted view that the soluble 
 lime-salts are of importance in the coagulation, van Dam 2 claims that it is the 
 quantity of lime combined with the casein which is of importance in the coagula- 
 tion process. The role of the lime-salts in coagulation is not clear, and this fol- 
 lows from the chemical procedure in rennin coagulation. 
 
 If one makes use of a pure solution of casein and as pure rennin as 
 possible, then after coagulation it is always found that the filtrate con- 
 tains very small amounts of a protein, the whey protein, which is probably 
 formed in the coagulation. This behavior, which was first shown by 
 Hammarsten, has been substantiated by many others and recently by 
 Fuld, Spiro and Schmidt-Nielsen. Whey protein is generally con- 
 sidered as a proteose substance, and Koster 3 found 13.2 per cent nitro- 
 gen therein. In correspondence with these observations casein coagula- 
 tion with rennin is considered as a cleavage process, in which the principal 
 mass of the casein, sometimes more than 90 per cent, is split off as para- 
 casein* a body closely related to casein, and in the presence of sufficient 
 
 1 See Maly's Jahresber., 2 and 4; also Hammarsten, Zur Kenntniss des Kaseins und 
 der Wirkung des Labfermentes, Nova Acta Reg. Soc. Scient. Upsala, 1877, Fest- 
 schrift; Zeitschr. f. physiol. Chem., 22; Arthus et Pages, Arch, de Physiol. (5), 2, 
 and M6m. soc. biol., 43; Fuld, Hofmeister's Beitrage, 2, and Ergebnisse der Physiol., 
 1, Abt. 1, where a good review of the literature may be found, Spiro, Hofmeister's 
 Beitrage, 6 and 7, with Reichel, ibid., 7 and 8; Laqueur, ibid., 7. 
 
 2 Zeitschr. f. physiol. Chem., 58. 
 
 'Hammarsten, 1. c; Fuld. Bioch. Zeitschr., 4, and Hofmeister's Beitrage, 10; 
 Spiro, Hofmeister's Beitrage, 8; Schmidt-Nielsen, Hammarsten's Festschrift, 1906; 
 Koster, see Maly's Jahresber., 11, 14. 
 
 * It has been proposed to designate the ordinary casein as caseinogen and the curd 
 as casein. Although such a proposition is theoretically correct, it leads in practice 
 to confusion. On this account the author calls the curd paracasein, according to 
 Schulze and Rose (Landwirthsch. V'ersuchsstat., 31). A summary of the literature on 
 the casein coagulation may he found in E. Fuld, Ergebnisse der Physiol., 1; Raudnitz, 
 ibid.. 2; and Laqueur, Biochem. Centralbl., 4, 344. 
 
CASEIN. 651 
 
 amounts of lime-salts the paraeasein-lime precipitates out while the 
 proteose-like substance (whey protein) remains in solution. In the 
 coagulation in an acid medium the conditions are entirely different and 
 proteoses and peptones are hereby formed to a considerable extent. 
 
 The paracasein is very similar to casein, but cannot be recoagulated 
 by rennin. A solution of alkali-paracaseinate is much more readily 
 precipitated by CaCb than an alkali-caseinate solution of the same con- 
 centration, and the precipitation limits for saturated ammonium-sul- 
 phate solution, the upper as well as the lower limit, lie, according to 
 Laqueur, lower with paracasein than with casein. The internal friction 
 of paracasein solutions is also, in his opinion, less than that of casein 
 solutions and indeed even to 20 per cent. 
 
 By continued action of rennin upon paracasein a further transformation 
 has been found in many cases (Petky, Slowtzoff, v. Hekwerden l ). This 
 is explained by the presence of another proteolytic enzyme in the (impure) rennin 
 preparation. This assumption seems to be plausible, and we are here probably 
 dealing only with a secondary process which has nothing whatever to do with the 
 true formation of paracasein. Whey protein is also formed after the very short 
 action of rennin, and the continued cleavage occurs with varying .speed. Thus 
 Schmidt-Nielsen found that the quantity of whey protein was even 3 per cent 
 of the casein nitrogen after the action of rennet for 15 minutes, and only 4.25 
 per cent after 6 hours' action. These and other recent investigations favor 
 the assumption that the casein coagulation by rennet is a hydrolytic cleavage, 
 but the conditions are not so clear that this can be considered as proved. 2 
 
 Frtsh, unchanged milk does not, as is known, coagulate on boiling; but in 
 not too rapid action of rennin a state may be observed in which the milk coagu- 
 lates on heating (metacasein reaction). A solution of paracasein lactate, accord- 
 ing to Laxa, 3 coagulates with rennin the same as a solution of casein lactate, 
 which indicates, he believes, that the paracasein is transformed into casein again 
 by the lactic acid. But as a precipitation of the paracasein from the acid solu- 
 tion is perhaps a pepsin action, the transformation of the paracasein into casein 
 by the lactic acid must not be considered as proved. 
 
 In the digestion of casein with pepsin-hydrochloric acid primarily a 
 phosphorized proteose is formed, from which then the pseudonuclein is 
 split off (Salkowski). The quantity thus split off is variable, as shown 
 by the researches of Salkowski, Hahn, Moraczewski, Sebelien, 
 and Zaitschek. 4 The amount of phosphorus in the pseudonucleins 
 obtained also varies considerably. Salkowski considers that the quan- 
 tity of pseudonuclein split off is dependent upon the relation between 
 the casein and the digestion fluid, e.g., the quantity of the pseudonu- 
 
 ^etry, Hofmeister's Beitriige, 8; Slowtzoff, ibid., 9; v. Herwerden, Zeitschr. f. 
 physiol. Chem., 52; \V. van Dam, ibid., 61. 
 
 2 See also Werncken, Zeitschr., f. Biol., 52. 
 
 3 Laxa, 1. c. 
 
 4 Salkowski, Zeitschr. f. physiol. Chem., 27; Salkowski and Hahn, Pflii^er's Arch., 
 59; Salkowski, ibid., 63; v. Moraczewski, Zeitschr. f. physiol. Chem., 20; Sebelien 
 ibid., 20; Zaitschek, Pfluger's Arch., 104. 
 
652 MILK. 
 
 cleins diminishes as the pepsin-hydrochloric acid increases. In the 
 presence of 500 grams of pepsin-hydrochloric acid to 1 gram of casein, 
 Salkowski digested the latter completely without obtaining any 
 pseudonuclein. 
 
 In peptic as well as tryptic digestion a part of the organic phosphorus 
 is split off as orthophosphoric acid, the quantity increasing as the" diges- 
 tion progresses. Another part of the phosphorus is retained in organic 
 combination in the proteoses as well as in the true peptones (Salkowski, 
 Biffi, Alexander, Aders-Plimmer and Bayliss '). 
 
 From the products of peptic digestion of casein, after the separation of the 
 pseudonuclein, Salkowski 2 has isolated an acid rich in phosphorus. He con- 
 siders this a paranucleic acid. This acid which gives the biuret test and a 
 faint xanthoproteic reaction, contains 4.05-4.31 per cent phosphorus. A still 
 richer product in phosphorus, with 6.9 per cent P, has been isolated by Reh 
 from the peptic digestive products of casein. He calls this body polypeptid 
 phosphoric acid. This product, which also gives the above-mentioned protein re- 
 actions, and is not comparable with the nucleic acids, is characterized by a remark- 
 ably high content of amino-nitrogen, namely, 23. S per cent. Among the products 
 obtained by Reh, Dietrich 3 found a mixture of at least four different lime-salts 
 of a peptone character, and which he considers as polypeptide-like combination 
 with P 2 5 , caseonphosphoric acids. The amount of phosphorus was, respectively, 
 10.0, 4.1, 3.84 and 3.88 per cent. 
 
 Casein may be prepared in the following way: The milk is diluted 
 with 4 vols, of water and the mixture treated with acetic acid to 0.75- 
 1 p. m. Casein thus obtained is purified by repeatedly dissolving in water 
 with the aid of the smallest quantity of alkali possible, by filtering and 
 reprecipitating with acetic acid and thoroughly washing with water. 
 Most of the milk-fat is retained by the filter on the first filtration, and the 
 casein contaminated with traces of fat is purified by treating with alcohol 
 and ether. 
 
 Lactoglobulin was obtained by Sebelien from cow's milk by saturating 
 it with NaCl in substance (which precipitated the casein) and saturating 
 the filtrate with magnesium sulphate. As far as it has been investigated 
 it had the properties of serglobulin; the globulin isolated by Tiemann 4 
 from colostrum had, nevertheless, a markedly low content of carbon, 
 namely, 49.83 per cent. 
 
 Lactalbumin was first prepared in a pure state from milk by Sebelien. 
 He gives its composition as, C 52.19, H 7.18, N 15.77, S 1.73, O 23.13 
 per cent. Lactalbumin has the properties of the albumins, and Wich- 
 
 1 Salkowski, 1. c; Biffi, Virchow's Arch., 152; Alexander, Zeitschr. f. physiol. 
 Chem., 25; Plimmer and Bayliss, Journ. of Physiol., 33; See also Kuttner, Pfluger'a 
 Arch. 129. 
 
 2 Zeitschr. f.. physiol. Chem., 32. 
 
 1 Reh. Hofmeister's Beitrage 11; Dietrich, Bioch. Zeitschr. 22. 
 4 Zeitschr. f. physiol. Chem., 25. 
 
LACTALBUMIN. ENZYMES. 653 
 
 mann found that it crystallizes in forms similar to ser- or ovalbumin. 
 It coagulates, depending on the concentration and the amount of salt 
 in solution, at 72-84° C. It is similar to seralbumin, but differs from 
 it in having a considerably lower specific rotatory power: (a) D =— 37°. 
 According to Fasal 1 it is especially rich in tryptophane, namely, 3.07 
 per cent. 
 
 The principle of the preparation of lactalbumin is the same as for the 
 preparation of seralbumin from serum. The casein and the globulin 
 are removed by MgSC>4 in substance, and the filtrate treated as previously 
 stated (page 263). 
 
 The occurrence of other proteins, such as proteoses and peptones, in milk has 
 not been positively proved. These bodies are easily produced as laboratory 
 products from the other proteins of the milk. Such a laboratory product is 
 Millox's and Comatlle's lactoprotein, which is a mixture of a little casein with 
 changed albumin, and proteose * which is formed by chemical action. In regard 
 to opalisin, see Human Milk, p. G62. 
 
 Milk also contains, Siegfried 3 claims, a nucleon related to phos- 
 phocarnic acid, which yields fermentation lactic acid (instead of para- 
 lactic acid) and a special carnic acid, orylic acid (instead of muscle carnic 
 acid), as cleavage products. Lactophosphocarnic acid may be precipitated 
 as an iron compound from the milk freed from casein and coagulable 
 proteins as well as from earthy phosphates. 
 
 Milk also contains enzymes of various kinds. Of these we must men- 
 tion catalases, peroxidases, and reductases, but the statements as to their 
 occurrence in the milk from different animals as well as the question 
 how much of their action is due to micro-organisms are conflicting. 
 Among these enzyme actions a special interest has been given to the 
 Schardinger reaction, which consists in the fact that milk at 70° C. 
 in the presence of formaldehyde or acetaldehyde reduces certain dyes, 
 such as methylene blue, to leucobases. An amylolytic enzyme which 
 converts starch into maltose occurs, especially, in human milk, while 
 it is absent in cow's milk or occurs only to a slight extent. A fermenta- 
 tion enzyme which in the absence of micro-organisms decompose^ the 
 lactose into lactic acid, alcohol, and CO2, occurs, according to Stoklasa 4 
 and his co-workers, in cow's milk as well as in human milk. Human 
 milk, as well as cow's milk, contains a lipase which has the property 
 at least of acting upon monobutyrin. Babcock and Russel have 
 found in these two kinds of milk, as well as certain others, a proteolytic 
 
 ^ebelien, Zeitschr., f. physiol. Chem., 9; Wichmann, ibid., 27; Fasal, Bioch. 
 Zeitschr., 44. 
 
 * See Hammarsten, Mary's Jahresber., 6, 13. 
 8 Zeitschr. f. physiol. Chem., 21 and 22. 
 ♦See Chem. Centralbl., 1905, 1, 107. 
 
654 MILK. 
 
 enzyme which they call galactase, which is allied to trypsin, but differs 
 therefrom in that it develops ammonia from milk even in the early stages 
 of digestion. The occurrence of such an enzyme is denied by Zaitschek 
 and v. Szontagh, but on the other hand Vandevelde, de Waele, and 
 Sugg * confirm the occurrence of a proteolytic enzyme in milk. 
 
 Orotic acid, C 5 HnN,04.2H 2 0, is the name given by Biscaro and Belloni l 
 to a new constituent of milk which they have discovered. This acid, which can 
 be precipitated by basic lead acetate from whey free from protein, is slightly 
 soluble in water," crystalline, and gives several crystalline salts. The mono- 
 methyl and ethyl esters of this acid are also known. It yields urea on treatment 
 with potassium permanganate. 
 
 Lactose, milk-sugar, Ci2H220n+H<20. This sugar, on hydrolysis, 
 can be split into two hexoses, glucose and galactose. It yields mucic acid 
 besides other organic acids, by the action of dilute nitric acid. Levulinic 
 acid is formed, besides formic acid and humin substances, by the stronger 
 action of acids. By the action of alkalies, among other products we 
 find lactic acid and pyrocatechin. 
 
 Milk-sugar occurs, as a rule, only in milk, but it has also been found 
 in the urine of pregnant women, on stagnation of milk, as well as in the 
 urine after partaking of large quantities of the same sugar. 
 
 Lactose occurs ordinarily as colorless rhombic crystals with 1 mole- 
 cule of water of crystallization, which is driven off by slowly heating to 
 100° C, but more easily at 130-140° C. On quickly boiling down a milk- 
 sugar solution, anhydrous milk-sugar separates out. Milk-sugar dissolves 
 in 6 parts cold or in 2.5 parts boiling water; it has a faintly sweetish 
 taste. It does not dissolve in ether or absolute alcohol. Its solutions 
 are dextrogyrate. The rotatory power, which on heating the solution to 
 100° C. becomes constant, is («);>= +52.5°. Milk-sugar combines with 
 bases; the alkali combinations are insoluble in alcohol. 
 
 Milk-sugar is not fermentable with pure yeast. It undergoes, on the 
 contrary, alcoholic fermentation by the action of certain schizomycetes, 
 and E. Fischer 3 found that the milk-sugar is first split into glucose 
 and galactose by an enzyme, lactase, existing in the yeast. The prep- 
 aration of milk-wine, " kumyss," from mare's milk and " kephir " and 
 " yoghurt " from cow's milk is based upon this fact. Other micro-organ- 
 isms also take part in this change, causing a lactic-acid fermentation of 
 the milk-sugar. 
 
 1 Babcock and Russel, Centralbl. f. Bakt. u. Parisitenkunde (II), 6, and Maly's 
 Jahresber., 31; Zaitsrhek and v. Szontagh, Pfliiger's Arch., 104; Vandevelde, de 
 Waele, and Sugg, Hofmeister's Beitrage, 5. 
 
 * See Chem. Centralbl., 1905, 2, 63. 
 
 * Ber. d. d. Chem. Gesellsch., 27. 
 
LACTOSE. 655 
 
 Lactose responds to the reactions of glucose, such as Moore's, 1 
 Trommer's and Rubner's, and the bismuth test. It also reduces mer- 
 curic oxide in alkaline solutions. After warming with phenylhydrazine 
 acetate it gives on cooling a yellow crystalline precipitate of phenyl 
 lactosazone, C24H32N4O9. It differs from cane-sugar by giving positive 
 reactions with Moobe's or Trommer's and the bismuth test, and also in 
 that it does not darken when heated to 100° C with anhydrous oxalic- 
 acid. It differs from glucose and maltose by its solubility and crystalline 
 form, but especially, by its not fermenting with yeast, and by yielding 
 mucie acid with nitric acid. 
 
 The osazone obtained with phenylhydrazine acetate, which melts at 
 200° C, differs from the other osazones by being inactive when 0.2 gram 
 is dissolved in 4 cc. of pyridine and G cc. of absolute alcohol and viewed 
 through a layer 10 centimeters long (Xeuberg 2 ). 
 
 For the preparation of milk-sugar we make use of the by-product 
 in the preparation of cheese, the sweet whey. The protein is removed 
 by coagulation with heat, and the filtrate evaporated to a syrup. The 
 crystals which separate after a certain time are recrystallized from water 
 after decolorizing with animal charcoal. A pure preparation may be 
 obtained from the commercial milk-sugar by repeated recrystallization. 
 The quantitative estimation of milk-sugar may be performed either by 
 the polaristrobometer or by means of titration with Fehling's solution. 
 Ten cc. of Fehling's solution are reduced by 0.0G7G gram of milk-sugar 
 in 0.5-1.5 per cent solution after boiling for six minutes. (In regard to 
 Fehling's solution and the titration of sugar see larger hand-books.) 
 
 From the non-correspondence between the quantity of sugar in the 
 milk as determined by polarization and gravimetrically, when the polar- 
 ization results are always higher, Sebelien 3 has concluded that the 
 milk must contain a second reducing substance which polarizes stronger 
 than lactose. This substance is probably a pentose and occurs to a very 
 slight extent in ordinary milk, 0.25-0.3") p. m. (Sebelien and Sunde), 
 and more in colostrum, 0.5 p. m. 
 
 Ritthausen found another carbohydrate in milk which is soluble in water, 
 non-crystallizable, which has a faint reducing action, and which yields, on boiling 
 with an acid, a body having a greater reducing power. Becha.mp 4 considers 
 this as dextrin. 
 
 1 The well-known beautiful red color, which milk produces after the addition of 
 alkali, at the room temperature and to which attention has been called recently by 
 Gautier, Morel, and Monod (Compt. rend. soc. biol., 60 and C»2), and Kriiger (Zeitschr. 
 f. Physiol. Chem., 50) is a Moore's reaction modified by the presence of protein and 
 perhaps also other milk constituents. 
 
 2 Ber. (1. d. Chem. Gesellsch., 32. 
 
 'Sebelien, Hammarsten's Festschrift, 1906; with Sunde, Zeitschr. f. angew. 
 Chem., 21. 
 
 4 Ritthausen, Journ. f. prakt. Chem. (N. F.), 15; Beehamp, Bull. Soc. Chim. (3), 6. 
 
656 MILK. 
 
 The mineral bodies of milk will be treated in connection with its quan- 
 titative composition. 
 
 The methods for the quantitative analysis of milk are very numerous, 
 and a* all cannot be treated here, we will give the principal points of a 
 few of the methods considered most trustworthy and most frequently 
 employed. 
 
 In determining the solids a carefully weighed quantity of milk is mixed with 
 an equal weight of heated quartz sand, fine glass powder, or asbestos. The 
 evaporation is first done on the water-bath and finished in a current of carbon 
 dioxide or hydrogen not above 100° C. 
 
 The mineral bodies are determined by incinerating the milk, using the pre- 
 cautions mentioned in the text-books. The results obtained for the phosphoric 
 acid are incorrect on account of the burning of phosphorized bodies, such as 
 casein and lecithin. We must, therefore, according to Soldner, subtract in round 
 numbers 25 per cent from the total phosphoric acid found in the milk. The 
 quantity of sulphate in the ash also depends on the combustion of the proteins. 
 
 In the determination of the total amount of proteins Ritthausen's method 
 is employed, namely, the precipitation of the milk with copper sulphate according 
 to the modification suggested by Munk. 1 He precipitates all the proteins by 
 means of cupric l)3 r droxide at boiling heat, and determines the nitrogen in the 
 precipitate by means of Kjeldahl's method. This modification gives more 
 exact results. 
 
 According to Sebelien's method, three to four grams of milk are diluted 
 with an equal volume of water, a little common-salt solution added, and the 
 proteins precipitated with an excess of tannic acid. The precipitate is washed 
 with cold water, and then the quantity of nitrogen determined by Kjeldahl's 
 method. The total nitrogen found when multiplied by 6.37 (casein and lactal- 
 bumin contain both 15.7 per cent nitrogen) gives the total quantity of proteins. 
 This method, which is readily performed, gives very good results. I. Munk 
 used this method in the analysis of woman's milk. In this case the quantity 
 of nitrogen found must be multiplied by 6.34. G. Simon 2 found that the 
 precipitation with tannic acid, also with phosphotungstic acid, is the simplest 
 and most accurate. The objection to this and other methods in which the pro- 
 teins are precipitated is that perhaps other bodies (extractives) may be carried 
 down at the same time (Camerer and Soldner 3 ). It is not known to what 
 extent this takes place. 
 
 A part of the nitrogen in the milk exists as extractives, and this nitrogen is 
 calculated as the difference between the total nitrogen and the protein nitrogen. 
 According to Mink's analyses about r$ of the total nitrogen belongs to the extract- 
 ives in cow's milk. Camerer and Soldner determine the nitrogen in the filtrate 
 from the tannic-acid precipitate by Kjeldahl's method, and also according to 
 Hufner's method (hypobromite). In this way they found 18 milligrams of 
 nitrogen according to Hufner (urea, etc.) in 100 grams of cow's milk. 
 
 To determine the casein and albumin separately we may make use of the 
 method first suggested by Hoppe-Seyler and Tolmatscheff, 4 in which the casein 
 is precipitated by magnesium sulphate. According to Sebelien the milk is diluted 
 with its own volume of a saturated magnesium-sulphate solution, then saturated 
 with the salt in substance, and the precipitate then filtered and washed with a 
 saturated magnesium-sulphate solution. The nitrogen is determined in the pre- 
 
 1 Ritthausen, Journ. f. prakt. Chem. (N. F.), 15; I. Munk, Virchow's Arch., 134. 
 
 ' Sebelien, Zeitschr. f. physiol, Chem., 13; Simon, ibid., 33. 
 
 1 Zeitschr. f. Biologie, 33 and 36. 
 
 4 Hoppe-Seyler, Med. chem. Untersuch., 272. 
 
MILK ANALYSIS. 657 
 
 cipitate by Kjeldahl's method, and the quantity of casein (+globulin) drtormined 
 by multiplying the result by 0.37. The quantity of lactalbumin may be calculated 
 as the difference between the casein and the total proteins found. The lactal- 
 bumin may also be precipitated by tannic acid from the filtrate from the casein 
 precipitate containing MgS0 4 , after diluting with water, the nitrogen determined 
 by Kjeldahl's method and the result multiplied by 0.37. 
 
 Schlossmann • suggests an alum solution, which precipitates the casein, 
 in order to separate the casein from the other proteins, and the albumin is then 
 precipitated from the filtrate by tannic acid. The nitrogen in the precipitate 
 is determined by the Kjeldahl method. This method has recently been tested 
 by Simon and he recommends it highly. 
 
 The fat is gravimetrically determined by thoroughly extracting the dried 
 milk with ether, evaporating the ether from the extract, and weighing the residue. 
 The fat may be determined by aerometric means by adding alkali to the milk, 
 shaking with ether, and determining the specific gravity of the fat solution by means 
 of Soxhlet's apparatus. In determining the amount of fat in a large number of 
 samples the lactocrit of De Laval may be used with success. There are numer- 
 ous other methods for estimating milk-fat, but they cannot be considered here. 
 
 In determining the milk-sugar the proteins are first removed. For this pur- 
 pose we precipitate either with alcohol, which must he evaporated from the filtrate, 
 or by diluting with water, and removing the casein by the addition of a little acid, 
 and the lactalbumin by coagulation at boiling heat. The sugar is determined by 
 titration with Fehling's or Knapp's solution (see Chapter XIV). The principle 
 of the titration is the same as for the titration of sugar in the urine; 10 cc. of 
 Fehling's solution correspond to 0.0070 gram of milk-sugar; 10 cc. of Knapp's 
 solution correspond to 0.0311-0.0310 gram of milk-sugar, when the saccharine 
 liquid contains about |-1 per cent of sugar. In regard to the modus operandi 
 of the titration we must refer the reader to more extensive works. 
 
 Instead of these volumetric determinations other methods of estimation, such 
 as Allihn's method, the polariscope method, and others, may be used. In calcu- 
 lating the analysis or in determining the solids it is of importance to remember, 
 as suggested by Camerer and Soldner, that the milk-sugar in the residue is 
 anhydrous. Many other methods for determining the milk-sugar have been 
 suggested and recommended. 
 
 The quantitative composition of cow's milk is naturally very variable. 
 The average obtained by Konig 2 is as follows in 1000 parts: 
 
 Water. Solids. Casein. Albumin. Fats. Sugar. Salts. 
 
 871.7 128.3 30.2 5.3 36.9 48.8 7.1 
 
 35.5 
 
 The quantity of mineral bodies in 1000 parts of cow's milk is, accord- 
 ing to the analyses of Soldner, as follows: K2O 1.72, Na20 0.51, CaO 
 1.98, MgO 0.20, P2O5 1.82 (after correction for the pseudonuclein) , 
 C1J1.98 grams. Bunge found 0.0035 gram Fe 2 3 , and Edelstein and 
 Csonka 3 found 0.0007-0.001 gm. Fe20?. According to Soldner the 
 K, Na, and CI are found in the same quantities in whole milk as in milk- 
 serum. Of the total phosphoric acid 36-56 per cent, and of the lime 
 
 1 Zeitschr. f. physiol. Chem., 22. 
 
 2 Chemie der menschlichen Nahrungs- und Genussmittel, 4. Aufl. 
 
 3 Bunge, Zeitschr. f. Biol. 10; Edelstein and Csonka, Bioch. Zeitschr., 38. 
 
658 MILK. 
 
 53-72 per cent is not in simple solution. A part of this lime is combined 
 with the casein; the remainder is found united with the phosphoric acid 
 as a mixture of dicalcium and tricalcium phosphates which is kept disr 
 solved or suspended by the casein. Rona and Michaelis x found that 
 about 40-50 per cent of the total quantity of lime was diffusable ; accord- 
 ing to them nearly one-half of the calcium is contained in the milk as a 
 non-dissociable casein compound, while the milk only contains the very 
 smallest amounts of suspended calcium phosphate. 
 
 The bases are in excess of the mineral acids in the milk-serum. 
 The excess of the first is combined with organic acids, which correspond 
 to 2.5 p. m. citric acid (Soldner). 
 
 The gases of the milk consist mainly of CO2, besides a little N and 
 traces of 0. Pfluger 2 found 10 vols, per cent CO2 and 0.6 vol. per cent 
 N calculated at 0° C. and 760 mm. pressure. 
 
 The variation in the composition of cow's milk depends on several 
 circumstances. 
 
 The colostrum, or the milk which is secreted before calving and in 
 the first few days after, is yellowish, sometimes alkaline, but often acid, 
 of higher specific gravity, 1.046-1.080, and richer in solids than ordinary 
 milk. The colostrum contains, besides fat-globules, an abundance of 
 colostrum-corpuscles — nucleated granular cells 0.005-0.025 mm. in di- 
 ameter with abundant fat-granules and fat-globules. The fat of colos- 
 trum has a somewhat higher melting-point and is poorer in volatile fatty 
 acids than the fat from ordinary milk (Nilson 3 ). The iodine equivalent 
 of the colostrum-fat is higher than that of milk-fat. The quantity of 
 cholesterin and lecithin is generally greater. The most apparent dif- 
 ference between it and ordinary milk is that colostrum coagulates on heat- 
 ing to boiling because of the absolutely and relatively greater quantities 
 of globulin and albumin that it contains. 4 The composition of colostrum 
 varies considerably. Konig gives as average the following figures in 
 1000 parts: 
 
 Water. 
 
 Solids. 
 
 Casein. 
 
 Albumin and Globulin. 
 
 Fat. 
 
 Sugar. 
 
 Salts. 
 
 740.7 
 
 253.3 
 
 40.4 
 
 130.0 
 
 35.9 
 
 20.7 
 
 15.0 
 
 The influence which food exercises upon the composition of milk will 
 be discussed in connection with the chemistry of the milk secretion. 
 
 'Bioch. Zeitschr., 21. 
 
 2 Pfliiger'sArch., 2. 
 
 * See Maly's Jahresber., 21. See also Engel and Bode, Zeitschr. f. physiol. Chem., 
 74. 
 
 ♦See Sebelien, Maly's Jahresber., 18, and Tiemann, Zeitschr. f. physiol. Chem., 
 25. See also Simon, ibid., 33; Winterstein and Strickler, ibid., 47. 
 
MILK OF OTHER ANIMALS. 059 
 
 In the following table is given the average composition of skimmed milk and 
 certain other preparations of milk: 
 
 Water f'r.itc-ins. Fat. Sugar. Lactic Acid. Salts. 
 
 Skimmed milk 906.6 31.1 7.4 47:, 7 | 
 
 Cream 655.1 :ti 1 267.5 35.2 6 I 
 
 Buttermilk <»<>-> 7 40.6 9.3 37 3 3 4 6 7 
 
 Whey 032 4 8.5 2.3 47.0 3.3 6.5 
 
 Kumyss, kephir and yoghurt are obtained, as above stated, by the alcoholic 
 and laetic-acid fermentation of the milk-sugar, the first from mare's milk and 
 the other from cow's milk. Large quantities of carbon dioxide are formed thereby, 
 and besides this the protein bodies of the milk are partly converted into proteoses 
 and peptones, which increase the digestibility. The quantity of lactic acid in 
 these preparations may be about 10-20 p. m. The quantity of alcohol varies 
 from 10 to 35 p. m. 
 
 Milk of Other Animals. Goat's milk has a more yellowish color and a 
 more specific odor than cow's milk. The coagulum obtained by acid or rennet 
 is more solid and is harder than that from cow's milk. Sheep's milk is similar 
 to goat's milk, but has a higher specific gravity and contains a greater amount 
 of solids. 
 
 Mare's milk is alkaline and contains a casein which is not precipitated, by 
 acids, in lumps or solid masses, but, like the casein from woman's milk, in fine 
 flakes. This casein is only incompletely precipitated by rennet, and it is very 
 similar also in other respects to the casein of human milk. In Beil's « opinion 
 the casein from mare's and cow's milk is the same, and the different behavior 
 of the two varieties of milk is due to varying amounts of salts and to a different 
 relation between the casein and the albumin. This does not agree with the 
 analyses of casein by Tangl and Csoa'ks given above nor with the investiga- 
 tions of Zaitschek and v. Szontagh, who find that the casein from mare's milk, 
 like that from human and ass's milk, is digested by pepsin-hydrochloric acid 
 without leaving a residue. According to Engel and Dennemark 2 the colostrum 
 from the mare differs from that from the ass by being richer in casein than the milk. 
 The milk of the ass is claimed by earlier authorities to be similar to human milk' 
 but Schlossmann finds it considerably poorer in fat. The researches of Ellen- 
 berger give similar results, and show great similarity between ass's milk and 
 human milk. The average results were 15 p. m. protein with 5.3 p. m. albumin 
 and 9.4 p. m. casein. This latter, like human casein, does not yield any pseudo- 
 nuclein on pepsin digestion, which agrees well with the above-mentioned investiga- 
 tions of Zaitschek. The quantity of nucleon was about the same as in woman's 
 milk. The quantity of fat was 15 p. m., and the sugar was 50-60 p. m. Reindeer 
 milk is characterized, according to Werenskiold, 3 by being very rich in fat, 
 144.6-197.3 p. m., and casein, 80.6- 86.9 p. m. 
 
 The milk of carnivora (the bitch and cat) is acid in reaction and very rich 
 in solids. The composition of the milk of these animals varies with the com- 
 position of the food. 
 
 To illustrate the composition of the milk of other animals the following figures, 
 the compilation of Konig, are given. As the milk of each kind of animal may 
 have a variable composition, these figures should only be considered as examples 
 of the composition of milk of various kinds: 4 
 
 1 Studein iiber die Eiweissstoffe des Kumys und Kefirs, St. Petersburg, 1S86 (Ricker). 
 
 2 Zeitschr. f. physiol. Chem., 76. 
 
 5 Zaitschek, 1. c; Schlossmann, Zeitschr. f. physiol. Chem., 22; Ellenberger, Arch. 
 f. (Anat. u.) Physiol., 1S99 and 1902; Werenskiold, Maly's Jahresber., 25. 
 
 4 Details in regard to the milk of different animals may be found in Proscher, 
 Zeitschr. f. physiol. Chem., 24; Abderhalden, ibid., 27. In regard to pig milk, see 
 Zuntz and Ostertag, Landw. Jahresb., 37. 
 
Solids 
 
 Proteins. 
 
 Fat. 
 
 Sugar. 
 
 Salts. 
 
 245.6 
 
 99.1 
 
 95.7 
 
 31.9 
 
 7.3 
 
 183.7 
 
 90.8 
 
 33.3 
 
 49.1 
 
 5.8 
 
 130.9 
 
 36.9 
 
 40.9 
 
 44.5 
 
 8.6 
 
 165.0 
 
 57.4 
 
 61.4 
 
 39.6 
 
 6.6 
 
 128.3 
 
 35.5 
 
 36.9 
 
 4S.8 
 
 7.1 
 
 99.4 
 
 18.9 
 
 10.9 
 
 66.5 
 
 3.1 
 
 100.0 
 
 21.0 
 
 13.0 
 
 63.0 
 
 3.0 
 
 176.3 
 
 60.9 
 
 64.4 
 
 40.4 
 
 10.6 
 
 321.5 
 
 30.9 
 
 195.7 
 
 88.5 
 
 6.5 
 
 513.3 
 
 
 437.6 
 
 
 4.6 
 
 302.0 
 
 luman 
 
 94.3 
 Milk. 
 
 194.0 
 
 
 9.9 
 
 660 MILK. 
 
 Milk of the Water. 
 
 Dog 754.4 
 
 Cat 816.3 
 
 Goat 869 . 1 
 
 Sheep 835.0 
 
 Cow 871.7 
 
 Horse 900 . 6 
 
 Ass 900.0 
 
 Pig 823.7 
 
 Elephant 678.5 
 
 Dolphin 486 . 7 
 
 Whale 1 698.0 
 
 Woman's milk is amphoteric in reaction. According to Cotjrant 
 its reaction is relatively more alkaline than cow's milk, but it has, never- 
 theless, a lower absolute reaction for alkalinity as well as for acidity. He 
 found between the tenth day and the fourteenth month after confinement 
 practically constant results. The alkalinity, as well as the acidity, was 
 a little lower than in childbed. One hundred cc. of the milk had the 
 same average alkalinity as 10.8 cc. N/10 caustic soda, and the same 
 acidity as 3.6 cc. N/10 acid. The relation between the alkalinity and the 
 acidity in woman's milk was as 3:1, and in cow's milk as 2.1:1. The 
 actual reaction determined electrometrically is, according, to FoA, 2 still 
 nearly neutral, like the other kinds of milk. Allaria has also arrived 
 at similar results, according to whom the tendency of human milk toward 
 alkaline reaction even in the most prominent cases never corresponds 
 
 N 
 
 to a NaOH solution. 
 
 1,000,000 
 
 Human milk also contains fewer fat-globules than cow's milk, but 
 they are larger in size. The specific gravity of woman's milk varies 
 between 1.026 and 1.036, generally between 1.028 and 1.034. It is highest 
 in well-fed and lowest in poorly-fed women. The freezing-point is lowered 
 on an average 0.589° C, according to Winter and Parmentier 3 con- 
 stant at 0.55°, and the molecular concentration is 0.318. 
 
 The fat of woman's milk has been investigated by Ruppel. It forms 
 a yellowish-white mass, similar to* ordinary butter, having a specific gravity 
 of 0.966 at 15°. It melts at 34.0° C. and solidifies at 20.2° C. The fol- 
 lowing fatty acids can be obtained from the fat, namely, butyric, caproic, 
 capric, myristic, palmitic, stearic, and oleic acids. The fat from woman's 
 milk is, according to Ruppel and Laves, 4 relatively poor in volatile 
 fatty acids. The non-volatile fatty acids consist of one-half oleic acid, 
 
 1 Scheibe, cited in Bioch. Centralbl., 7, 553. 
 
 2 Cornpt. rend. soc. biol. 58; Allaria, Maly's Jahresb., 39, 242. 
 
 ' .See Maly's Jahresber., 34. 
 
 * Ruppel, Zeitschr. f. Biologie, 31; Laves, Zeitschr. f. physiol. Chem., 19. 
 
HUMAN MILK. 661 
 
 while among the solid fatty acids myristic and palmitic acids are found 
 to a greater extent than stearic acid. 
 
 The essential qualitative difference between woman's and cow's milk 
 seems to lie in the proteins or in the more accurately determined casein. 
 A number of both the earlier and more recent investigators ' claim that 
 the casein from woman's milk has other properties than that from cow's 
 milk. The essential differences are the following: The casein from 
 woman's milk is precipitated with greater difficulty with acids or salts. 
 It does not coagulate uniformly in the milk after the addition of rennet, 
 which depends, essentially, upon the low amount of lime-salts and casein 
 contained in the milk. 2 It may be precipitated by gastric juice, but 
 dissolves completely and easily in an excess of gastric juice; the casein pre- 
 cipitate produced by an acid is more easily soluble in an excess of the acid; 
 and lastly, the clot formed from the casein of woman's milk does not 
 appear in such large and coarse masses as in the casein from cow's 
 milk, but is more loose and flocculent. This last-mentioned fact is of 
 great importance, since it explains the generally admitted fact of the 
 easy digestibility of the casein from woman's milk. 
 
 The question as to whether the above-mentioned variations depend 
 on a decided difference in the two caseins, or only on an unequal relation 
 between the casein and the salts in the two kinds of milk, or upon other 
 circumstances, has not as yet been decided. According to Szontagh 
 and Zaitschek and also Wr6blewsky, the casein from human milk 
 does not yield any pseudonuclein on peptic digestion, and hence it cannot 
 be a nucleoalbumin. According to Kobrak, woman's casein yields 
 some pseudonuclein, and with repeated solution in alkali and precipitation 
 by an acid it becomes more and more like cow's casein. He therefore 
 suggests the possibility that woman's casein is a compound between a 
 nucleoalbumin and a basic protein. Wroblewsky found the follow- 
 ing for the composition of casein from woman's milk: C 52.24, H 7.32, 
 N 14.97, P 0.6S, S 1.117 per cent. Langstein and Bergell obtained 
 much lower figures for N, S and especially P, namely, 14.34, 0.85 and 0.27 
 per cent, respectively. According to Langstein and Edelstein the 
 phosphorus content is only 0.22-0.29 per cent. On hydrolysis Abder- 
 halden and Langstein 3 could not find any difference between cow 
 and human casein. 
 
 1 See Biedert, Untersuchungen iiber die chemischen Unterschiede der Menschen- 
 und Kuhmilch (Stuttgart), 1884; Langgaard, Virchow's Arch., 64; Makris, Studien 
 iiber die Eiweisskorper der Frauen- und Kuhmilch. [naug.-Diss. Strassburg, 1876. 
 
 2 See among others Bienenfeld, Bioch. Zeitschr., 7, an 1 Full and Wohlgemuth, 
 ibid., 8. 
 
 s Szontagh, Maly's Jahresber., 22; Zaitschek, 1. c. ; Wr6blewsky, Beitrage zur 
 Kenntniss des Frauenkaseins, Inaug.-Diss. Bern. 1894, and Ein neuer eiweissartiger 
 
662 MILK. 
 
 Woman's milk also contains lactalbumin, besides the casein, and a protein 
 substance, very rich in sulphur (4.7 per cent) and relatively poor in carbon, which 
 Wroblewsky calls opalisin. The statements as to the occurrence of proteoses and 
 peptones are conflicting as in many other cases. No positive proof as to the 
 occurrence of proteoses and peptones in fresh milk has been given. 
 
 Because of the properties and low amount of casein in human milk 
 it is often difficult to precipitate it, with acid, and to prepare it, but 
 this can easily be accomplished by dialysis. A number of methods 
 have been suggested for the preparation of human casein. Fuld and 
 Wohlgemuth recommend the freezing of the milk previous to pre- 
 cipitation, so that the casein masses become larger to a certain extent 
 and the precipitation becomes easier. Engel : recommends dilution 
 with water to 5 volumes, and the addition of 60-80 cc. N/10 acetic acid 
 for each 100 cc. milk. The mixture is first cooled for 2-3 hours and then, 
 after shaking, warmed on the water-bath to 40° for a few minutes. 
 
 Even after those differences are eliminated which depend on the imper- 
 fect analytical methods emploj'ed, the quantitative composition of woman's 
 milk is variable to such an extent that it is impossible to give any average 
 results. The numerous analyses, especially those made on a large number 
 of samples by Pfeiffer, Adriance, Camerer and Soldner, 2 have posi- 
 tively shown that woman's milk is essentially poorer in proteins but 
 richer in sugar than cow's milk. The quantity of protein varies between 
 10-20 p. m., often amounting to only 15-17 p. m. or less, and is dependent 
 upon the length of lactation (see below). The quantity of fat also varies 
 considerably, but ordinarily amounts to 30-40 p. m. The quantity of 
 sugar should not be below 50 p. m., but may rise to even 80 p. m. About 
 60 p. m. may be considered as an average, but it should be borne in mind 
 that the quantity of sugar is also dependent upon the length of lactation, 
 as it increases with duration. The amount of mineral bodies varies 
 between 2 and 4 p. m. 
 
 The division of the total nitrogen in human milk is, according to A. 
 Frehn, 3 very variable. As approximate average figures we can say 
 that 40-45 per cent of the total nitrogen is casein, 35-40 per cent remain- 
 
 Bestandteil der Milch, Anzeiger der Akad. d. Wiss. in Krakau, 1898; Kobrak, Pfluger's 
 Arch., 80; Langstein and Bergell, cited in Bioch. Centralbl., 8, 323; Langstein and 
 Edelstein, Maly's Jahresber, 40, 254; Abderhalden and Langstein. Zeitschr., f. physiol. 
 Chem.. 06. 
 
 1 Fuld and Wohlgemuth, Bioch. Zeitschr., 5; Engel, ibid., 14. 
 
 2 Pfeiffer, Jahrb. f. Kinderheilkundc, 20, also Maly's Jahresber., 13; V. Adriance 
 and J. Adriance, A Clinical Report of the Chemical Examination, etc., Archives of 
 Pediatrics, 1897; Camerer and Soldner, Zeitschr. f. Biologie, 33 and 36. In regard 
 to the composition of Woman's milk, see also Biel, Maly's Jahresber., 4; Christenn, 
 ibid., 7; Mendes de Leon, ibid., 12; Cerber, Bull. soc. chim., 23; Tolmatscheff, 
 Hoppe-Seyler'fl Med. -chem. Untersuch., 272. 
 
 Zeitschr. f. physiol. Chem., 65; see also Engel and Frehn, Maly's Jahresber., 40. 
 
HUMAN MILK. 663 
 
 ing proteins and about 20 per cent for rest nitrogen. The principal part 
 of the rest nitrogen is considered as urea. 
 
 From a quantitative standpoint, the most essential differences between 
 woman's and cow's milk are the following: As compared with the quan- 
 tity of albumin, the quantity of casein is not only absolutely but also 
 relatively smaller in woman's milk than in cow's milk, while the latter is 
 poorer in milk-sugar. Human milk is richer in lecithin, at least relatively 
 to the amount of protein. Burow found 0.49-0.58 p. m. lecithin in cow's 
 milk and 0.58 p. m. in woman's milk, which corresponds to 1.40 per cent 
 for the first milk and 3.05 per cent for the second, calculated on the per- 
 centage of protein. Nerking and Haensel found as average for lecithin 
 in cow's milk 0.63 p. m. and in woman's milk 0.50 p. m. Glikin found 
 0.765 p. m. lecithin (phosphatides) as average for cow's milk and 1.329 
 p. m. for human milk. Koch found that both human milk and cow's 
 milk contain lecithin as well as cephalin. The total quantity of both 
 bodies in human milk was 0.78 p. m. and in cow's milk 0.72-0.86 p. m. 
 The quantity of nucleon is greater in woman's milk. Wittmaack claims 
 that cow's milk contains 0.566 p. m. nucleon, and woman's milk 1.24 
 p. m., and according to Valenti the quantity of nucleon in human milk 
 is indeed still higher. Siegfried finds that the nucleon phosphorus 
 amounts to 6.0 per cent of the total phosphorus in cow's milk and 41.5 
 per cent in woman's milk, and also that in human milk the phosphorus 
 is almost all in organic combination. This does not agree with the results 
 of Sikes who found on an average of only 42 per cent of the total P2O5 
 in organic combination. Because of the large amount of casein (and 
 calcium phosphate) cow's milk is much richer in phosphorus than human 
 milk. The relation PoOs:N, according to Schlossmann, 1 is equal to 
 1:5.4 in human milk and 1:2.7 in cow's milk. Woman's milk is poorer 
 in mineral bodies, especially lime, and it contains only one-sixth of the 
 quantity of lime as compared with cow's milk. The mineral constituents 
 of human milk are better assimilated by the organism of the nursing 
 child than those of cow's milk. Human milk is also claimed to be poorer 
 in citric acid (Scheibe 2 ), although this is not an essential difference. 
 
 Another difference between woman's milk and other varieties of milk is 
 Umikoff's reaction, which seems to depend upon the quantitative composition, 
 especially the relation between the milk-sugar, citric acid, lime, and iron (Sieber 3 ). 
 This reaction consists in treating 5 cc. of woman's milk with 2.5 cc. ammonia 
 
 1 Burow, Zeitschr. f. physiol. Chem., 30; Koch, ibid., 47; Wittmaack, ibid., 22; 
 Siegfried, ibid., 22; Nerking and Haensel, Bioch. Zeitschr., 13; Glikin, ibid., 21; Valenti, 
 Biochem. Centralbl., 4; Schlossmann, Arch. f. Kinderheilkunde, 40; Sikes, Journ. 
 of Physiol., 34. 
 
 2 Maly's Jahresber., 21. 
 'Zeitschr. f. physiol. Chem., 30. 
 
664 MILK. 
 
 (10 per cent) and heating to 60° C. for 15 —20 minutes, when the mixture becomes 
 violet-red. Cow's milk gives a yellowish-brown color when thus treated. 
 
 According to Rubner woman's milk contains about 3 p. m. soaps, but this 
 could not be substantiated by Camerer and Soldner. They conclude that 
 woman's milk contains no soaps, or at least only very small amounts. They also 
 found the quantity of urea nitrogen in woman's milk to be 0.11-0.12 p. m., 
 although Schondorff l found nearly twice this amount, namely, 0.23 p. m. 
 
 In regard to the quantity of mineral bodies in woman's milk we have 
 the analyses of several investigators, especially of Btjnge (analyses A 
 and B) and of Soldner and Camerer (analysis C). 2 Btjnge analyzed the 
 milk of a woman, fourteen days after delivery, whose diet contained 
 very little common salt for four days previous to the analysis {A), and again 
 three days later after a daily addition of 30 grams of NaCl to the food 
 (B). The figures are in 1000 parts of the milk- 
 
 ABC 
 
 K,0 0.780 0.703 0.884 
 
 Na,0 0.232 0.257 0.357 
 
 CaO 0.328 0.343 0.378 
 
 MgO 0.064 0.065 0.053 
 
 Fe>0 3 0.004 0.006 0.002 
 
 P 2 6 0.473 0.469 0.310 
 
 CI 0.438 0.445 0.591 
 
 The relation of the two bodies potassium and sodium to each other 
 may, Btjnge believes, vary considerably (1.3-4.4 equivalents of potash 
 to 1 of soda). By the addition of salt to the food, the quantity of 
 sodium and chlorine in the milk increases, while the quantity of potas- 
 sium decreases. De Lange found more Na than K in the milk at the 
 beginning of lactation. Jolles and Friedjung found on an average 
 5.9 milligrams of iron per liter of woman's milk. Camerer and Soldner 3 
 find about the same amount, namely, 10-20 milligrams Fe203 = 3.5-7 
 milligrams iron in 1000 grams human milk. 
 
 The gases of woman's milk have been investigated by Kulz 4 He 
 found 1.07 -1.44 cc. of oxygen, 2.35-2.87 cc. of carbon dioxide, and 3.37- 
 3.81 cc. of nitrogen in 100 cc. of milk. 
 
 The proper treatment of cow's milk by diluting it with water and by 
 certain additions in order to render it a proper substitute for woman's 
 milk in the nourishment of children cannot be determined before the 
 difference in the protein bodies of these two kinds of milk has been com- 
 pletely studied. 
 
 Rubner, Zeitschr. f. Biologie, 36; Camerer and Soldner, ibid., 39; Schondorff, 
 Pfliiger's Arch., 81. 
 
 2 Bunge, Zeitschr. f . Biologie, 10; Camerer and Soldner, ibid., 39 and 44. 
 
 3 De Lange, Maly's Jahresber., 27; Jolles and Friedjung, Arch. f. exp. Path. u„ 
 Pharm., 4(5; Camerer and Soldner, Zeitschr. f. Biologie, 46. 
 
 4 Zeitschr. f. Biologie, 32. 
 
HUMAN COLOSTRUM. GG5 
 
 The colostrum has a higher specific gravity, 1.040-1.060, a greater 
 quantity of coagulablo proteins, and a deeper yellow color than ordinary 
 woman's milk. Even a few days after delivery the color becomes Leefl 
 yellow, the quantity of albumin less, and the Dumber of colostrum-cor- 
 puscles diminishes. 
 
 We have the older analyses of Clemm 1 and the recent investigations 
 of Pfeiffer, V. and J. Adriance, Camerer and Soldner on the changes 
 in the composition of milk after delivery. It follows, as a unanimous 
 result from these investigations, that the quantity of protein, which 
 amounts to more the first two days, sometimes to more than 30 p. m. 
 at first, rather qu'ckly and then more generally diminishes as long as the 
 lactation continues, so that in the third week it equals about 10-18 p. m. 
 Like the protein substances, the mineral bodies also gradually decrease. 
 The quantity of fat shows no regular or constant variation during lacta- 
 tion, while the lactose, especially according to the observations of V. 
 and J. Adriance (120 analyses), increases rather quickly the first days 
 and then only slowly until the end of lactation. The analyses of Pfeiffer, 
 ( amerer and Soldner also show an increase in the quantity of milk-sugar. 
 
 The two mammary glands of the same woman may yield somewhat different 
 milk, as shown by Sourdat and later by Brunner. 2 Likewise the different 
 portions of milk from the same milking may have varying composition. The 
 first portions are always poorer in fat. 
 
 According to l'Heritier and to Vernois and Becquerel, the milk of blondes 
 contains less casein than that of brunettes, a difference which Tolmatscheff 3 
 could not substantiate. Women of delicate constitutions yield a milk richer in 
 solids, especially in casein, than women with strong constitutions (V. and B.). 
 
 According to Vernois and Becquerel, the age of the woman has an effect on 
 the composition of the milk, so that we find a greater quantity of proteins and 
 fat in women 15-20 years old and a smaller quantity of sugar. The smallest 
 quantity of proteins and the greatest quantity of sugar are found at 20 or from 
 25 to 30 years of age. Vernois and Becquerel, consider that the milk with the 
 first-born is richer in water — with a proportionate diminution of casein, sugar, 
 and fat — than after several deliveries. 
 
 The influence of menstruation seems to diminish slightly the milk-sugar and 
 to increase considerably the fat and casein (Vernois and Becquerel). 
 
 Witch's milk is the secretion of the mammary glands of new-born children 
 of both sexes immediately after birth. This secretion has from a qualitative 
 standpoint the same constitution as milk, but may show important differences and 
 variations from a quantitative point of view. Schlossberger and Hauff, 
 Gubler and Quevenne, and v. Genser, 4 have made analyses of this milk and 
 give the following results: 10.5-28 p. m. proteins, 8.2-14.6 p. m. fat, and 9-60 
 p. m. sugar. 
 
 1 See Hoppe-Seyler, Physiol. Chem., 734. 
 
 2 Sourdat, Compt. Rend., 71; Brunner, Pfluger's Arch., 7. 
 
 8 l'Heritier, cited from Hoppe-Seyler, Physiol. Chem., 738; Vernois and Becquerel, 
 Du lait chez la fen.n.e dans l'etat de sante, etc., (Paris, 1853); Tolmatscheff, Hoppe- 
 Seyler, Med.-chem. Untersuch., 272. 
 
 4 Schlossberger and Hauff, Annal. d. Chem., u. Pharm., 96; Gubler and Quevenne, 
 cited from Hoppe-Seyler 's Physiol. Chem., 723; v. Genser, ibid. 
 
666 MILK. 
 
 As milk is the only form of nourishment during a certain period of 
 the life of man and mammals, it must contain all the nutriment necessary 
 for life. This fact is shown by the milk containing representatives of 
 the three principal groups of organic nutritive substances — proteins, 
 carbohydrates, and fat, and the last two groups can here also in part 
 mutually substitute each other. Besides this all milk seems to contain, 
 without doubt, some lecithin and nucleon. The mineral bodies in milk 
 must also occur in proper proportions, and on this point the experiments 
 of Bunge on dogs are of special interest. He found that the mineral 
 bodies of the milk occur in about the same relative proportion as they 
 do in the body of the sucking animal. Bunge l found in 1000 parts of 
 the ash the following results (A represents results from the new-born 
 dog, and B the milk from the bitch) : 
 
 A B 
 
 K,0 114.2 149.8 
 
 Na 2 106.4 88.0 
 
 CaO 295.2 272.4 
 
 MgO 18.2 15.4 
 
 Fe 2 3 7.2 1.2 
 
 P„0 5 394.2 342.2 
 
 CI 83.5 169.0 
 
 Bunge explains the fact that the milk-ash is richer in potash and 
 poorer in soda than the new-born animal by saying that in the growing 
 animal the ash of the muscles rich in potash relatively increases and the 
 cartilage rich in soda relatively decreases. In regard to the amount 
 of iron we find an unexpected condition, the ash of the new-born animal 
 containing six times as much as the milk-ash. This condition Bunge 
 explains by the fact founded on his and Zalesky's experiments, that the 
 quantity of iron in the entire organism is highest at birth. The new-born 
 has therefore its own supply of iron for the growth of its organs even at 
 birth. 
 
 The investigations of Hugounenq, de Lange, Camerer and Soldner 2 
 have shown that in man the conditions are different from those in animals, 
 as the ash of the child has an entirely different composition as compared 
 with the milk. As an example the following analyses are given (of 
 Camerer and Soldner). (A, the ash of the sucking infant, and B, the 
 ash of the milk.) The results are in 1000 parts of the ash. 
 
 A B 
 
 K 2 78 314 
 
 Na 2 91 119 
 
 CaO 361 164 
 
 MgO 9 26 
 
 FfcO, 8 6 
 
 p.,Q 5 389 135 
 
 CI 77 200 
 
 1 Zeitschr. f. physiol. Chem., 13. 
 
 2 Hugounenq, Compt. Rend., 128; de Lange, Zeitschr., f. Biologie, 40; Camerer 
 and Soldner, ibid., 39, 40, and 44. 
 
INFLUENCE OF THE FOOD. 667 
 
 We cannot therefore state as a definite fact that the composition of 
 the ash of the sucking young and the ash of the corresponding milk coin- 
 cide. Binge l nevertheless claims that the composition of the ash of 
 the sucking young of various mammals is nearly the same, but that the 
 ash of the milk differs from the ash of the young in bo far as the slower 
 the young grows the richer it is in alkali chlorides and relatively poorer 
 in phosphates and lime-salts. The constituents of the ash have two 
 functions to perform, namely, the building up of the tissues and secondly 
 the preparation of the excreta, especially the urine. The faster the 
 young grows the more is the first in evidence, while the slower it develops, 
 the more prominent is the second. 
 
 The quantity of mineral l;odies in the milk, and especially the amount 
 of lime and phosphoric acid, as shown by Bunge and Proscher and 
 Pages, stands in close relation to the rapidity of growth, because the 
 amount of these mineral constituents in the milk is greater in animals 
 which grow and develop quickly than in those which grow only slowly. 
 A similar relation also exists, as shown by the researches of Proscher, 
 and especially of Abderhalden, 2 between the quantity of protein in 
 the mill: and the rapidity of development of the sucking young. The 
 amount of protein is greater in the milk the quicker the animal develops. 
 
 The influence of the food on the composition of the milk is of interest 
 from many points of view and has been the subject of many investigations. 
 From these we learn that in human beings as well as in animals an insuffi- 
 cient diet decreases the quantity of milk and the quantity of solids, while 
 abundant food increases both. From the observations of Decai^ne '•' 
 on nursing women during the siege of Paris in 1871, the amount of casein, 
 fat, sugar, and salts, but especially the fat, was found to decrease with 
 insufficient food, while the quantity of lactalbumin was found to be some- 
 what increased. Food rich in proteins increases the quantity of milk, 
 and also the solids contained, especially the fat, according to most 
 reports. The quantity of sugar in woman's milk is found by certain 
 investigators to be increased after food rich in proteins, while others 
 claim it is diminished. A diet rich in fat may, as the researches of Soxhlet 
 and many others 4 have shown, cause a marked increase in the fat of 
 the milk when the fat partaken is in a readily digestible and assimilable 
 form. The presence of large quantities of carbohydrates in the food 
 
 1 Bunge, " Die zunehmende (Tnfuhigkeit der Frauen ihre Kinder zu stillen," Miin- 
 chen, 1900, cited by Camerer, Zeitschr. f. Biologie, 40. 
 
 2 Proscher, Zeitschr. f. physiol. Chem., 24; Abderhalden, il>id., 27; Pages. Arch, 
 de Physiol. (5), 7. 
 
 3 Cited from Hoppe-Seyler, 1. c, 739. 
 
 4 See Maly's Jahresber., 26. See also Basch, Ergebnisse der Physiologie, 2, Abt. 1. 
 
668 MILK. 
 
 seems to cause do constant, direct action on the quantity of the milk 
 constituents. 1 From feeding experiments with different foods we come 
 to the conclusion that the character of the food is of comparatively little 
 influence, while the race and other conditions play an important role. 
 Watery food gives a milk containing an excess of water and having little 
 value. In the milk from cows which were fed on distillers' grain Com- 
 maille 2 found 906.5 p. .m. water, 26.4 p. m. casein, 4.3 p. m. albumin, 
 18.2 p. m. fat, and 33.8 p. m. sugar. Such milk has sometimes a peculiar 
 sharp after-taste, although not always. Tangl and Zaitschek 3 could 
 not find any difference in the average composition of the milk produced 
 after feeding with dry and with moist fodder. 
 
 Chemistry of Milk-secretion. That the constituents which occur 
 actually dissolved in milk pass into the secretion and not alone by filtra- 
 tion or diffusion, but more likely are secreted by a specific secretory 
 activity of the granular elements, is shown by the fact that milk-sugar, 
 which is not found in the blood, is to all appearances formed in the glands 
 themselves. A further proof lies in the fact that the lactalbumin is not 
 identical with seralbumin; and lastly, as Bunge 4 has shown, the mineral 
 bodies secreted by the milk are in quite different proportions from those 
 in the blood-serum. 
 
 Little is known in regard to the formation and secretion of the specific 
 constituents of milk. The older theory, that the casein was produced 
 from the lactalbumin by the action of an enzyme, is incorrect, and prob- 
 ably originated from mistaking an alkali albuminate for casein. Better 
 founded is the theory that the casein originates from the protoplasm 
 of the gland-cells. According to Basch's researches, the casein is formed 
 in the mammary gland by the nucleic acid of the nucleus being set 
 free and uniting intra-alveolar with the transudated serum, thus form- 
 ing a nucleoalbumin, the casein. The untenableness of this view has 
 been shown by Lobisch, and the investigations, of Hildebrandt 5 
 upon the proteolytic enzyme of the mammary gland, and the autolysis 
 
 1 In regard to the literature on the action of various foods on woman's milk, see 
 Zalesky, " Ueber die Einwirkung der Nahrung auf die Zusammensetzung und Nahr- 
 haftigkeit der Frauenmilch," Berlin, klin. Wochenschr., 1888, which also contains the 
 literature on the importance of diet on the composition of other kinds of milk. In 
 regard to the extensive literature on the influence of various foods on the milk pro- 
 duction of animals, see Konig, Chem. d. menschl. Nahrungs und Genussmittel. 3. Aufl., 
 1. 298. See also Maly's Jahresber., 29-40, and Morgen, Beger and Fingerling, Landw. 
 Versuchsst., 61, and Raudnitz, Monatschr. f. Kinderheilk. 
 
 'Cited from Konig, 2, 235. 
 
 • Landwirt. Vers. .St. 1911. 
 
 * Lehrbuch d. physiol. und pathol. Chem., 3. Aufl., 93. 
 
 ■ Basch, Jahrb. f. Kinderheilkunde, 1898; Hildebrandt, Hofmeister'a Beitrage, 5; 
 Lobisch. ibid., 8. 
 
CHEMISTRY OF MILK-SECRETION. 669 
 
 of the gland have not given any clue as to the mode of formation of 
 casein. The findings of Mandbl l that the hydrolytic cleavage products 
 of the nucleoprotein from the mammary glands occur approximately 
 quantitatively in the same proportions as in casein, are important in this 
 connection. 
 
 That the milk-fat is produced by a formation of fat in the protoplasm, 
 and that the fat-globules are set free by their destruction, is a generally 
 admitted opinion, which, however, does not exelude the possibility that 
 the fat is in part taken up by the glands from the blood and eliminated 
 with its secretion. That the fats of the food can pass into the milk 
 follows from the investigations of Winternitz, as he has been able to 
 detect the passage of iodized fats in the milk, and these observations 
 have been substantiated by the investigations of Caspari and Parascht- 
 schuk. 2 The abundant quantities of iodized fat which were eliminated 
 with the milk in these cases without doubt depend, at least in great part, 
 upon the iodized fat of the food, hence it cannot be said that all of the 
 milk-fat containing iodine was unchanged iodized fat of the food. The 
 previously-mentioned older investigations of Lebedeff and Rosenfeld 
 and also the recent ones of Spampani and Daddi, Paraschtschuk, Gogi- 
 tidse and others on the passage of foreign fats into the milk also indicate 
 the passage of the fat of the food into the milk, although we are still uncer- 
 tain on this point. According to Soxhlet the fat of the food does not 
 pass into the milk directly, but is destroyed in place of the body-fat, 
 which then becomes available and is, as it were, pushed into the milk. 
 Hexriques and Hansen could not detect any mentionable quantity of 
 linseed-oil in the milk after feeding with this oil; the milk-fat was not 
 normal, but had a higher iodine equivalent and a higher melting-point, 
 from which they also concluded that a transformation of the food-fat 
 in the glandular cells is possible. The results of the experiments of 
 Gogitidse 3 with soaps also indicate that the mammary glands have the 
 property of forming fats by synthesis from their components. As a 
 formation of fat from carbohydrates in the animal organism is at the 
 present day considered as positively proved, it is likewise possible that 
 the milk-glands also produce fats from the carbohydrates brought to 
 them by the blood. It is a well-known fact that an animal gives off 
 for a long time, daily, considerably more fat in the milk than it receives 
 
 1 Bioch. Zeitschr., 22. 
 
 : Winternitz, Zeitschr. f. physiol. Chem., 24; Caspari, Arch. f. (Anat. u.) Physiol., 
 1899, Supplbd. and Zeitschr. f. Biologie, -46; with Winternitz, ibid., 49; Paraschtschuk, 
 Chem. Centralbl., 1903, 1. 
 
 3 Lebedeff, Pfluger's Arch. 31; Rosenfeld, Ergebn. d. Physio!. 1 and 2; Spampani 
 and Daddi, Maly's Jahresber., 26; Hennques and Hansen, ibid., 29; Gogitidse, Zeitschr. 
 f. Biologie, 45, 46, and 47. See also Basch, Ergebnisse d. Physiol., 2, Abt. 1. 
 
670 MILK. 
 
 as food, and this proves that at least a part of the fat secreted by the 
 milk is produced from proteins or carbohydrates, or perhaps from both. 
 The question as to how far this fat is produced directly in the milk- 
 glands, or from other organs and tissues, and brought to the gland by 
 means of the blood, cannot be decided. 
 
 The origin of milk-sugar is not known. Muntz calls attention to the 
 fact that a number of very widely diffused bodies in the vegetable king- 
 dom — vegetable mucilage, gums, pectin bodies — yield galactose as a 
 product of decomposition, and he believes, therefore, that milk-sugar 
 may be formed in herbivora by a synthesis from glucose and galactose. 
 This origin of milk-sugar does not apply to carnivora, as they produce 
 milk-sugar when fed on food consisting entirely of lean meat. The 
 observations of Bert and Thierfelder * that a mother-substance of 
 the milk-sugar, a saccharogen, occurs in the glands, does not explain 
 the formation of milk-sugar, as the nature of this mother-substance 
 is still unknown. As the animal body has undoubtedly the power of 
 converting one variety of sugar into another, the origin of the milk- 
 sugar can be sought simply in the glucose introduced as food or formed 
 in the body. Certain observations of Porcher indicate such an origin 
 as he found in sheep, cows, and goats whose mammary glands "were 
 extirpated, that glucose appeared in the urine after delivery. He also 
 found that milk secreting animals became glycosuric on the removal 
 of the mammary glands, and he explains this glycosuria by the fact that 
 the lactose-forming action of the gland was removed at the time of delivery, 
 when large amounts of glucose were being produced. The experiments 
 of Kaufmann and Magne upon cows also indicate a formation of lactose 
 from glucose. They found that during secretion the glands took sugar 
 from the blood, so that the venous gland-blood was poorer in sugar than 
 otherwise. Noel-Paton and Cathcart 2 have carried on experiments 
 on phlorhinized dogs which show a lactose formation from glucose. 
 
 The passage of foreign substances into the milk stands in close connec- 
 tion with the chemical processes of milk secretion. 
 
 It is a well-known fact that milk acquires a foreign taste from the 
 food of the animal, which is in itself a proof that foreign bodies pass into 
 the milk. This fact becomes of special importance in reference to such 
 injurious substances as may be introduced into the organism of the nurs- 
 ing child by means of the milk. 
 
 Among these substances may be mentioned opium and morphine, 
 which after large doses pass into the milk and act on the child. Alcohol 
 
 1 Muntz, Compt. Rend., 102; Bert and Thierfelder, footnote 1, p. 644. 
 
 2 Porcher, Compt. Rend. 138 and 141 and Bioch. Zeitschr., 23; Kaufmann and 
 Magne, Compt. Rend., 143; Noel-Paton and Cathcart, Journ. of Physiol., 42. 
 
MILK IN DISEASES. 671 
 
 may also pass into the milk, but probably not in such quantities as to 
 have any direct action on the nursing child. 1 Alcohol is claimed to have 
 been detected in the milk after feeding cows with brewer's grains. 
 
 Among inorganic bodies, iodine, arsenic, bismuth, antimony, zinc, 
 lead, mercury, and iron have been found in milk. In icterus neither 
 bile-acids nor bile-pigments pass into the milk. 
 
 Under diseased conditions no constant change has been found in woman's 
 milk. In isolated cases Schlossberger, Joly and Filhol - have indeed observed 
 a markedly abnormal composition, but no positive conclusion can be derived 
 therefrom. 
 
 The changes in cow's milk in disease have been little studied. In tuber- 
 culosis of the udder, Storch 3 found tubercle bacilli in the milk, and he also noted 
 that the milk became more and more diluted, during the disease, with a serous 
 liquid similar to blood-serum, so that that the glands finally, instead of yielding 
 milk, gave only blood-serum or a serous fluid. Husson 4 found that milk from 
 murrain cows contained more proteins but considerably less fat and (in severe 
 cases) less sugar than normal milk. 
 
 The milk may be blue or red in color, due to the development of micro-organisms. 
 
 The formation of concrements in the exit-passages of the cow's udder is often 
 observed. These consist chiefly of calcium carbonate, or of carbonate and phos- 
 phate with only a small amount of organic substances. 
 
 x See Klingemann, Virehow's Arch., 126, and Rosemann, Pfliiger's Arch., 78. 
 * Schlossberger, Annal. d. Chem. u. Pharm., 96; Joly and Filhol, cited from v. 
 Gorup-Besanez, Lehrb., 4, Aufl., 438. 
 
 3 See Bang, Om Tuberkulose i Koens Yver og om tuberkulos Malk, Xord. Med. 
 Arkiv, 16, and also Maly's Jahresber., 14, 170; Storch, Maly's Jahresber., 14. 
 
 4 Compt. Rend., 73. 
 
CHAPTER XIV. 
 URINE. 
 
 Urine is the most important excretion of the animal organism; it 
 is the means of eliminating the nitrogenous metabolic products, also 
 the water and the soluble mineral substances; and in many cases it 
 furnishes important data relative to the metabolism, quantitatively 
 by its variation, and qualitatively by the appearance of foreign bodies 
 in the excretion. Moreover, in many cases we are able, from the chemical 
 or morphological constituents which the urine abstracts from the kidneys, 
 ureter, bladder, and urethra, to judge of the condition of these organs; and 
 lastly urinary analysis affords an excellent means of deciding the question 
 as to how certain medicinal agents or other foreign substances intro- 
 duced into the organism are absorbed and chemically changed. In this 
 respect, urinary analysis has furnished very important particulars especially 
 in regard to the nature of the chemical processes taking place within 
 the organism, and it is therefore not only an important aid to the 
 physician in diagnosis, but it is also of the greatest importance to the 
 toxicologist and the physiological chemist. 
 
 In studying the secretions and excretions, the relation must be 
 sought between the chemical structure of the secreting organ and the 
 chemical composition of its secreted products. Investigations with 
 respect to the kidneys and the urine have led to very few results from 
 this standpoint. Although the anatomical relation of the kidneys has 
 been carefully studied, their chemical composition has not been the sub- 
 ject of thorough analytical research. In cases in which a chemical 
 investigation of the kidneys has been undertaken, it has been in general 
 only of the organ as such, and not of the different anatomical parts. 
 An enumeration of the chemical constituents of the kidneys known at 
 the present time can, therefore, only have a secondary value. 
 
 In the kidneys we find proteins of different kinds. According to 
 Halliburton the kidneys do not contain any albumin, but only a 
 globulin and a nucleoprotein. The globulin coagulates at about 52° C, 
 and the nucleoprotein contains 0.37 per cent phosphorus. Lieber- 
 mann claims that the kidneys contain a lecithalbwnin, and he ascribes 
 to this body a special importance in the secretion of acid urines. The 
 
 672 
 
THE KIDNEYS. 673 
 
 kidneys also contain, according to Lonnberg, a mucin-like substance. 
 This substance yields no reducing body on boiling with acids, and belongs 
 chiefly to the papillae, and is, this author says, a nucleoalbumin 
 (nucleoproteid?). The cortical substance is richer in another nucleoal- 
 bumin (nucleoproteid) unlike mucin. It has not been decided what 
 relation this last substance bears to Halliburton's nucleoprotein. 
 Chondroitin sulphuric acid also occurs as traces. Mandel and Levenb 
 have also obtained glncothionic acid from the kidneys, and the question 
 as to the relation of this to the renosulphuric acid described by Mandel 
 and Neuberg l is still undecided. This renosulphuric acid to all appear- 
 ances is not a unit substance but a sulphuric acid ester, and a com- 
 ponent related to glucuronic acid which contained 2.63 p. c. S., 4.53 
 p. c, N., and 1.34 p. c. P. 
 
 Fat occurs only in very small amounts and this fat, like the organ 
 fat in general, is relatively rich in unsaturated fatty acids. The phos- 
 phatides seem to be of different kinds. Frankel and Nogueira 2 found 
 a cephalin-like substance, a triaminodiphosphatide and a diamino- 
 monophosphatide. Dunham and Jacobson 3 found in beef-kidneys a 
 substance which they called carnaubon which is soluble in alcohol but 
 insoluble in ether, and which is a triaminomonophosphatide with the 
 formula C74H150N3PO13. Carnaubon does not contain any glycerin 
 but an amino-sugar, two choline groups and a molecule of each of the 
 following acids: stearic, palmitic and carnaubic (C24H4SO2) acids. Among 
 the extractive bodies of the kidneys one finds purine bases, betaine, 4 urea, 
 uric acid (traces), glycogen, leucine, inosite, taurine, and cystine (in ox- 
 kidneys). The quantitative analyses of the kidneys thus far made 
 possess little interest. In the kidney of a healthy suicide Magnus- 
 Levy 5 found in 1000 parts of the fresh substance 756 p. m. water, 244 
 p. m. solids, 52.7 p. m. fat, 2.08 p. m. CI., 0.192 p. m. Ca., 0.207 p. m. 
 Mg and 0.158 p. m. Fe. 
 
 The fluid collected under pathological conditions, as in hydronephrosis, is 
 thin with a variable but generally low specific gravity. Usually it is straw-yellow 
 or paler in color, and sometimes colorless. Most frequently it is clear, or only 
 faintly cloudy from white blood-corpuscles and epithelium-cells; in a few cases 
 it is so rich in form-elements that it appears like pus. Protein generally occurs 
 in small amounts; occasionally it is entirely absent, but in a few rare cases the 
 
 1 Halliburton, Journ. of Physiol., 13, Suppl., and 18; Liebermann, Pfliiger's Arch., 
 50 and 54; Lonnberg, see Maly's Jahresber., 20; Mandel and Levene, Zeithschr. f. 
 physiol. Chem., 47; Mandel and Neuberg, Bioch. Zeitschr., 13; Morner, Skand. 
 Arch. f. Physiol., 6. 
 
 2 Bioch. Zeitschr., 16. 
 
 3 Zeitschr. f. physiol. Chem., 64. 
 
 4 Bebeschin, Zeitschr., f. physiol. Chem., 72. 
 
 5 Bioch. Zeitschr., 24. 
 
674 URINE. . 
 
 amount is nearly as large as in the blood-serum. Urea occurs sometimes in 
 considerable amounts when the parenchyma of the kidneys is only in part atro- 
 phied; in complete atrophy the urea may be entirely absent. 
 
 I. PHYSICAL PROPERTIES OF URINE. 
 
 Consistency, Transparency, Odor, and Taste of Urine. Under 
 physiological conditions urine is a thin liquid and gives, when shaken 
 with air, a froth which quickly subsides. Human urine, or urine from 
 carnivora, which is habitually acid, appears clear and transparent, often 
 faintly fluorescent, immediately after voiding. When allowed to stand for 
 a little while human urine shows a light cloud (nubecula), which consists of 
 the so-called " mucus," and generally also contains a few epithelium 
 cells, mucus-corpuscles, and urate-granules. The presence of a larger 
 quantity of urates renders the urine cloudy, and a clay-yellow, yellowish- 
 brown, rose-colored, or often brick-red precipitate (sedimentum lateri- 
 tium) settles on cooling, because of the greater insolubility of the urates 
 at the ordinary temperature than at the temperature of the body. 
 This cloudiness disappears on gently warming. In new-born infants 
 the cloudiness of the urine during the first 4-5 days is due to epithelium, 
 mucus-corpuscles, uric acid, and urates. The urine of herbivora, which 
 is habitually neutral or alkaline in reaction, is very cloudy on account 
 of the carbonates of the alkaline earths present. Human urine may 
 sometimes be alkaline under physiological conditions. In this case it 
 is cloudy, due to the earthy phosphates, and this cloudiness does not 
 disappear on warming, differing in this respect from the sedimentum 
 lateritium. Urine has a salty and faintly bitter taste produced by sodium 
 chloride and urea. The odor of urine is peculiarly aromatic; the bodies 
 which produce this odor are unknown. 
 
 The color of urine is normally pale yellow when the specific gravity 
 is 1.020. The color otherwise depends on the concentration of the urine 
 and varies from pale straw-yellow, when the urine contains small amounts 
 of solids, to a dark reddish-yellow or reddish-brown in stronger con- 
 centration. As a rule the intensity of the color corresponds to the con- 
 centration, but under pathological conditions, exceptions occur such as 
 are found in diabetic urine, which contains a large amount of solids and 
 has a high specific gravity and a pale-yellow color. 
 
 The reaction of urine depends essentially upon the composition of the 
 food. The carnivora, as a rule, void an acid, the herbivora, a neutral 
 or alkaline urine. If a carnivore is put upon a vegetable diet, its urine 
 may become less acid or neutral, while the reverse occurs when an herbi- 
 vore is starved, that is, when it lives upon its own tissues, as then the 
 urine voided is acid. 
 
 The urine of a healthy man on a mixed diet has an acid reaction, 
 
PHYSICAL PROPERTIES OF THE URINE. G75 
 
 and the sum of the acid equivalents is greater than the sum of the basic 
 equivalents. This depends upon the fact that in the physiological 
 combustion of neutral substances (proteins and others) within the 
 organism, acids are produced, chiefly sulphuric acid, hut also phosphoric 
 and organic acids, such as hippuric, uric, and oxalic acids, aromatic 
 oxyacids, oxyproteic acids and others. From this it follows that the 
 acid reaction is not due to one acid alone. The various acids take part 
 in the acid reaction in proportion to their dissociation, since, according 
 to the ion theory, the acid reaction of a mixture is dependent upon the 
 number of hydrogen ions present. Hence the theory that the acidity 
 is due entirely to dihydrogen phosphate is incorrect although this salt 
 takes such a great part in the acid reaction that its quantity is often 
 taken as a measure of the degree of acidity of the urine. 1 
 
 The composition of the food is not the only influence which affects the degree 
 of acidity of human urine. For example, after taking food at the beginning of 
 digestion, when a larger amount of gastric juice containing hydrochloric acid 
 is secreted, the urine may be neutral or even alkaline. 2 As to the time of the 
 appearance of the maximum and minimum of acidity, the various investigators 
 do not agree, which may in part be explained by the varying individuality and 
 conditions of life of the persons investigated. It has not infrequently been 
 observed that perfectly healthy persons in the morning void a neutral or alkaline 
 urine which is cloudy from earthy phosphates. The effect of muscular activity 
 on the acidity of urine has not been positively determined. According to Hoff- 
 mann, Ringstedt, Oddi, and Tarulli and Vozarik muscular work raises the 
 degree of acidity, but Aducco 3 claims that it decreases it. Abundant perspira- 
 tion reduces the acidity (Hoffmann). 
 
 In man and especially in carnivora it seems that the degree of acidity 
 of the urine cannot be increased above a certain point, even though 
 mineral acids or organic acids which are burned up with difficulty are 
 ingested in large quantities. Under such conditions a different behavior 
 has been repeatedly observed between carnivora and herbivora. In the 
 first (and also in man) it has been found that the acids are in part neu- 
 tralized by the alkalies and alkaline earths of the body, but that the 
 excess of acid is combined with ammonia, split off from the proteins or 
 their cleavage products, and eliminated in the urine as ammonium salt. 
 In herbivora such a combination of the excess of acid with ammonia 
 
 1 In regard to the acidity of the urine see the recent works of Ringer, Zeitschr. f. 
 physiol. Chem. 60; Henderson, Bioch. Zeitschr. 24, with Spiro, ibid., 15; De Jager, 
 Maly's Jahresb. 39 and Bioch. Zeitschr. 38; v. Skramlik, Zeitschr. f. physiol. Chem. 
 71; Klein and Moritz, Deutsch. Arch. f. klin. Med. 99; Quagliariello, Chem. Cen- 
 tralbl. 1912, 1, 506. 
 
 2 Contradictory statements are found in Linossier, Maly's Jahresber., 27. 
 
 3 Hoffmann, see Maly's Jahresber., 14; Ringstedt, ibid., 20; Oddi and Tarulli, 
 ibid., 24; Aducco, ibid., 17; Vozarik, Pfliiger's Arch., 111. 
 
676 URINE. 
 
 seems not to take place, or not to the same extent, 1 and this is given as 
 a reason why herbivora soon die when acids are given. This is true at 
 least for rabbits, while according to Baer this power of increasing the 
 elimination of ammonia exists also in the goat, monkey, and pig, hence 
 no definite difference in this regard exists between herbivora and carnivora. 
 The differences which have been observed are, according to Eppinger, 
 not of a special kind, and they may be caused, he says, from a different 
 amount of protein in the food which yields ammonia. Thus dogs with 
 food poor in protein behave like rabbits while, according to Eppinger, 
 in herbivora (rabbits) a de-toxification of the acid can be brought about 
 by the abundant supply of proteins or their cleavage products. The 
 correctness of this statement is still disputed (Pohl) or has only been 
 partly confirmed (Bostock). The point is disputed and it must not be 
 forgotten that, as A. Loewy 2 found, the sensitiveness toward the action 
 of acids varies very much in different individuals. 
 
 Although one cannot raise the degree of acidity of the urine above a 
 certain limit by the introduction of acid, still it may be easily diminished, 
 so that the reaction becomes neutral or alkaline. This occurs after the 
 taking of carbonates of the fixed alkalies or of such alkali salts of vege- 
 table acids — citric acid, and malic acid — as are easily burned into car- 
 bonates in the organism. Under pathological conditions, as in the 
 absorption of alkaline transudates, or the alkaline fermentation within 
 the bladder, the urine may become alkaline. 
 
 A urine with an alkaline reaction caused by fixed alkalies has a very 
 different diagnostic value from one whose alkaline reaction is caused by 
 the presence of ammonium carbonate. In the latter case we have to 
 deal with a decomposition of the urea of the urine by the action of micro- 
 organisms. 
 
 If one wishes to determine whether the alkaline reaction of the urine 
 is due to ammonia or to fixed alkalies, a piece of red litmus paper is dipped 
 into the urine and allowed to dry exposed to the air or to a gentle heat. 
 If the alkaline reaction is due to ammonia, the paper becomes red again; 
 but if it is caused by fixed alkalies, it remains blue. 
 
 Determination of the Acidity. As the quantity of phosphoric acid 
 present as dihydrogen salt, as above stated, cannot be used as a measure 
 of the acidity, none of the older methods suggested for the estimation 
 of this portion of the phosphoric acid is suited for acidity determinations. 
 
 1 See Winterberg, Zeitschr. f. phyaiol. Chem., 25, and J. Baer, Arch. f. exp. Path. u. 
 Pharm., 54. 
 
 2 Eppinger, Zeitschr. f. exp. Path. u. Therap., 3; with Tedesko, Bioch. Zeitschr., 
 16; Pohl. ibid., 18; Staal, Zeitschr. f. physiol. Chem., 58; Bostock, ibid., 84; A. 
 Loewy, Centralbl. f. Physiol., 20; 337. 
 
ACIDITY OF THE URINE. 677 
 
 We now determine the acidity simply by acidimetric methods, titrat- 
 ing with N/10 caustic alkali, using phenolphthalein as an indicator 
 (Naegeli, Hober, Folin). On account of the color of the urine and the 
 presence of ammonium salts and alkaline earths, this method cannot 
 yield entirely exact results. The greatest error is due to the alkaline 
 earths, which, on titration with caustic alkali, precipitate as earthy 
 phosphates in variable amounts and of variable composition. This 
 error can be prevented, according to Folin, by the addition of neutral 
 potassium oxalate, which precipitates the lime, and in this way the dis- 
 turbing action of the ammonium salts is also inhibited. Perfectly 
 accurate results are not obtained by this method, but it is the best of 
 those which have been suggested. 
 
 It is performed as follows: 25 cc. of urine are placed in an Erlenmeyer 
 flask (about 200 cc. capacity), treated with 1-2 drops of |-per cent 
 phenolphthalein solution, and shaken with 15-20 grams of powdered 
 potassium oxalate and immediately titrated with N/10 caustic soda 
 with constant shaking until a pronounced pale-rose color appears. 
 Vozarik 1 titrates the diluted urine without the addition of oxalate 
 and uses phenolphthalein as indicator. 
 
 The acidity, as determined by titration, varies considerably under 
 physiological conditions, but calculated as hydrochloric acid it amounts 
 in man to about 1.5-2.3 grams in the twenty-four hours. 
 
 By titration we learn the amount of hydrogen present which can 
 be substituted by a metal, i.e., the acidity in the ordinary older sense, 
 but not the true acidity, the ion acidity, which is given by the concentra- 
 tion of the hydrogen ions of the urine. For similar reasons, as previously 
 indicated in treating of the alkalinity of the blood-serum (page 272), 
 the ion acidity cannot be determined by titration, while it can be deter- 
 mined according to the principle of the electrometric gas-chain method 
 as there given. Such estimations have been made by v. Rhorer and by 
 Hober. For normal urine v. Rhorer found as a minimum 4X10 -7 , 
 as a maximum 76X10 -7 , and as an average 30X10 -7 . Hober found 
 4.7X10" 7 , 100X10" 7 , and 49X10" 7 , respectively. On an average the 
 urine therefore contains 30-50 grams of hydrogen ions in 10 million 
 liters. Henderson 2 has obtained much lower values, namely 10.10 ~ 7 
 as the average of 50 investigations, and has rather great differences 
 for different persons. From the comparative estimation of the titration 
 
 1 In regard to the degree of acidity and its estimation see Naegeli, Zeitschr. f . 
 physiol. Chem., 30; Hober, Hofmeister's Beitrage, 3; Folin, Amer. Journ. of Physiol., 
 9; Vozarik, 1. c; de Jager, Zeitschr. f. physiol. Chem., 55; and Ringer, ibid., 60; Grim- 
 bert and Morel, Compt. Rend., 154. 
 
 2 v. Rhorer, Pfliiger's Arch., 86; Hober, 1. c. See also Jolles, Bioch. Zeitschr., 
 13; Henderson, Bioch. Zeitschr., 24. 
 
678 UKINE. 
 
 acidity and the ion acidity it follows that no direct relation exists between 
 these and that the extent of these two acidities may be independent of 
 each other. 
 
 The osmotic pressure of the urine varies considerably even under 
 physiological conditions. The limit for the freezing-point depression 
 has been found by a number of investigators to be A 1.3° to 2.3° C. After 
 partaking of considerable water it may be markedly lower, and on 
 diminished supply of water it may be considerably higher. 
 
 In regard to the further physical-chemical imvestigations of the urine 
 and as to the conclusions drawn from a combination of the chemical 
 and the physico-chemical investigations of the urine, we must refer to 
 the extensive work of Carl Neuberg. 1 
 
 The specific gravity of urine, which is dependent upon the relation 
 existing between the quantity of water secreted and the solid urinary 
 constituents, especially the urea and sodium chloride, may vary con- 
 siderably, but is generally 1.017-1.020. After drinking large quantities 
 of water it may fall to 1.002, while after profuse perspiration or after 
 drinking very little water it may rise to 1.035-1.040. In new-born 
 infants the specific gravity is low, 1.007-1.005. The determination 
 of the specific gravity is an important means of learning the average 
 amount of solids eliminated from the organism in the urine, and on this 
 account the determination becomes of true value only when at the same 
 time the quantity of urine voided in a given time is determined. The 
 different portions of urine voided in the course of the twenty-four hours 
 are collected, mixed together, the total quantity measured, and then the 
 specific gravity taken. 
 
 The determination of the specific gravity is most accurately obtained 
 with the pycnometer. For ordinary cases the specific gravity may be 
 determined with sufficient accuracy by means of areometers. The 
 areometers found in the trade, or urinometers, are graduated from 1.000 
 to 1.040; for exact observations it is better to use two urinometers, one 
 graduated from 1.000 to 1.020, and the other from 1.020 to 1.040. 
 
 To determine the specific gravity of urine, if necessary filter the 
 urine, or if it contains a urate sediment, first dissolve it by gentle heat, 
 then pour the clear urine into a dry cylinder, avoiding the formation of 
 froth. Air bubbles or froth, when present, must be removed with a glass 
 rod or filter-paper. The cylinder, which should be about four-fifths full, 
 must be wide enough to allow the urinometer to swim freely in the liquid 
 without touching the sides. The cylinder and urinometer should both 
 be dry or previously washed with the urine. On reading, the eye is 
 brought on a level with the lower meniscus — which occurs when the sur- 
 face of the liquid and the lower limb of the meniscus coincide; the read- 
 
 1 Der Ham sowie die iibrigen Ausscheidungen und Korperfliissigkeiten von Mensch 
 und Tier. Teil. 2, Berlin, 1911. 
 
ORGANIC PHYSIOLOGICAL CONSTITUENTS. 679 
 
 ing is then made from the point where this curved line coincides with 
 the scale of the urinometer. If the eye is not in the same horizontal 
 plane with the convex line of the meniscus, hut is too high or too low, 
 the surface of the liquid assumes the shape of an ellipse, and the reading 
 in this position is incorrect. Before reading, press the urinometer gently 
 down into the liquid and then allow it to rise, and wait until it is at rest. 
 
 Each urinometer is graduated for a certain temperature, which, 
 at least in the case of the better ones, is marked on the instrument. 
 If the urine is not at the proper temperature, the following corrections 
 must be made: For every three degrees above the normal temperature 
 one unit of the last order is added to the reading, and for every three 
 degrees below the normal temperature one unit (as above) is subtracted 
 from the specific gravity observed. For example, when a urinometer 
 graduated for 15° C. shows a specific gravity of 1.017 at 24° C, then the 
 specific gravity at 15° C. = 1.017+0.003 = 1.020. 
 
 When great exactitude is required, as, for instance, a determina- 
 tion to the fourth decimal point, we make use of a urinometer constructed 
 by Lohnstein. 1 Jolles 2 has also devised a small urinometer for the 
 determination of the specific gravity of small amounts of urine, 20-25 
 cc. The specific gravity may also be determined by the Westphal 
 hydrostatic balance. 
 
 II. ORGANIC PHYSIOLOGICAL CONSTITUENTS OF URINE. 
 
 + /NH 2 
 
 Urea, Ur, CON2H4 = CO^ , has been synthetically prepared in sev- 
 
 N NH 2 
 eral ways, especially, as Wohler showed in 1828, by the metameric 
 transformation of ammonium isocyanate: CO.N.NH4 = CO(NH2)2- It 
 is also produced by the decomposition or oxidation of certain bodies 
 found in the animal organism, such as purine bodies, creatine, arginine, 
 other amino-acids, and other substances. 
 
 Urea is found most abundantly in the urine of carnivora and man,, 
 but in smaller quantities in that of herbivora. In carnivora (dog) the 
 urea nitrogen by abundant protein feeding may amount to 97-98 per cent 
 of the total nitrogen of the urine (Schondorff 3 ) . The quantity in human 
 urine is ordinarily 20-30 p. m. It has also been found in small quantities 
 in the urine of amphibians, fishes, and certain birds. Urea occurs in 
 the perspiration in small quantities, and as traces in the blood and in 
 most of the animal fluids. It also occurs in rather large quantities in the 
 blood, liver, muscle, 4 and bile 5 of sharks, even in rather large quantities. 
 Urea is also found in certain tissues and organs of mammals, especially 
 
 1 Pfliiger's Arch., 59; Chem. Centralbl., 1895, 1, and 1896, 2. 
 
 ! Wien. med. Presse, 1897, No. 8 
 
 s Pfluger's Arch., 117. 
 
 4 v. Schroeder, Zeitschr. f. physiol. Chem., 14. 
 
 6 Hammarsten, ibid., 24. 
 
€80 URINE. 
 
 in the liver, spleen, muscles and others, although only in small amounts. 
 Under pathological conditions, as in obstructed excretion, urea may 
 appear to a considerable extent in the animal fluids and tissues. 
 
 The quantity of urea which is voided in twenty-four hours on a mixed 
 diet is in a grown man about 30 grams, in women somewhat less. While 
 children void less, the excretion relative to their body weight is greater 
 than in grown persons. The physiological significance of urea lies in 
 the fact that this body forms in man and carnivora, from a quantitative 
 standpoint, the most important nitrogenous end-product of the metabolism 
 of protein bodies. On this account the elimination of urea varies to a 
 great extent with the catabolism of the protein, and above all with the 
 quantity of absorbable proteins in the food ingested. The elimination 
 of urea is greatest after an exclusive meat diet, and lowest, indeed less 
 than during starvation, after the consumption of non-nitrogenous sub- 
 stances, since these diminish the metabolism of the proteins of the body. 
 
 If the consumption of the proteins of the body is increased, then 
 the elimination of nitrogen is correspondingly increased. This is found to 
 be the case in fevers, after poisoning with arsenic, antimony, phosphorus, 
 and other protoplasmic poisons, and when there is a diminished supply 
 of oxygen — as in severe and continuous dyspncea, poisoning with carbon 
 monoxide, hemorrhage, etc. In these cases it used to be considered that 
 the rise in the excretion of nitrogen was due to an increased elimination 
 of urea, because no exact difference was made between the quantity 
 of urea and of total nitrogen in the urine. Recent researches have con- 
 clusively demonstrated Ihe untrustworthiness of these observations. 
 Since Pfluger and Bohland have shown that 16 per cent of the total 
 nitrogen of the urine exists under physiological conditions in other com- 
 pounds, not urea, attention has-been called to the relation of the dif- 
 ferent nitrogenous constituents of the urine to each other, and it has 
 been found, under pathological conditions, that this relation may vary 
 considerably, especially in regard to the urea. We have numerous 
 determinations by different investigator's, 1 on the relation of the different 
 nitrogenous constituents to each other in the normal urine of adults. 
 
 1 Pfluger and Bohland, Pfluger' s Arch., 38 and 43; Bohland, ibid., 43; Schultze, 
 ibid., 45; Camerer, Zeitschr. f. Biologie, 24, 27, and 28; Voges, Ueber die Mischung 
 der stickstoffhaltigen Bestandtheile im Ham. etc. (Inaug.-Diss. Berlin. 1892), cited 
 from Maly's Jahresber., 22; K. Morner and Sjoqvist, Skand. Arch. f. Physiol., 2. 
 See also Sjoqvist, Nord. Med. Arkiv., 1892, No. 36, and 1894, No. 10; Gumlich, Zeitschr. 
 f. physiol. Chem., 17; Bodtker, see Maly's Jahresber., 26; Folin, Amer. Journ. of 
 Physiol., 13; Osterberg and Wolff, Journ. of biol. Chem., 3; Haskins, ibid., 2; Donze 
 et Lambling, Journ. de Physiol, et de Path., 5; Bouchet, ibid., 14; Lambling et 
 Bouchet, Compt. rend. soc. biol., 71; Long and Gephart, Journ. Amer. Chem. 
 
UREA. 681 
 
 Thus Long and Gephart found in the urine of six healthy men to whom 
 the same qualitative diet was fed for a long time, the following division 
 of the nitrogen in percentage of the total nitrogen: urea 79.87-84. ill, 
 creatinine 5.21-6.87, ammonia 3.6-4.74, uric acid 1.57-1.99, purine 
 0.33-0.96 and rest nitrogen 4.23-6.01 per cent. Sjoqvist has made 
 similar determinations on new-born babes from 1 to 7 days old. From 
 all these analyses we obtain the following figures (A for adults and B for 
 new-born babes). Of the total nitrogen there exists: 
 
 A. B. 
 
 Per Cent. Per Cent. 
 
 Urea 84-91 73-76 
 
 Ammonia 2-5 7.8-9.6 
 
 Uric acid 1-3 3.0-8.5 
 
 Remaining nitrogenous substances 7-12 7.3-14.7 
 
 The variable relation between uric acid, ammonia, and urea nitro- 
 gen in children and adults is remarkable, since the urine of children 
 is considerably richer in uric acid and ammonia, and considerably poorer 
 in urea, than the urine of adults. A much larger number of analyses 
 of children's urine is necessary to explain the division of the nitrogen 
 .therein. The absolute quantity of urea nitrogen in adults amounts to 
 about 10-16 grams per day. In disease the proportion of the nitroge- 
 nous substances may be markedly changed, and a decrease in the quan- 
 tity of urea and an increase in the quantity of ammonia have been observed 
 in certain diseases of the liver. This will be considered in detail in 
 connection with the formation of urea in the liver. It is natural that 
 there should be a diminished formation of urea after a decrease in the 
 ingestion of proteins or in a lowered catabolism. In diseases of the 
 kidneys which disturb or destroy the integrity of the epithelium of 
 the convoluted urinary tubules, the elimination of urea is considerabl}'' 
 diminished. 
 
 Recently by means of Pfaundler's l method, by precipitating the urine with 
 phosphotungstic acid and closely studying the precipitate as well as the filtrate, 
 it has been possible to learn further about the division of the nitrogen of the urine. 
 We determine a, the total nitrogen; b, the nitrogen of the phosphotungstate pre- 
 cipitate; and c, the nitrogen in the filtrate from the phosphotungstate pre- 
 cipitate. This last contains the urea, hippuric acid, o'xyproteie acids, and other 
 bodies whose nitrogen is ordinarily designated as monamino-acid nitrogen. The 
 urea nitrogen is especially determined. The bodies precipitated by phospho- 
 tungstic acid are not all known; but uric acid and purine basest ammonia, 
 creatinine, pigments, diamino-acids, diamines and ptomaines (if they occur), sul- 
 phocyanides, carbamic acid, urine mucoid, and proteid belong to this group. 
 Special methods have been suggested for the determination of several of these 
 substances (see below). 
 
 The urea nitrogen is always the greatest part of the total nitrogen, 
 but otherwise the division of the nitrogen undergoes considerable varia- 
 
 1 Zeitschr. f. physiol. Chem., 30. 
 
682 URINE. 
 
 tion and very great variations seem to occur not only in the healthy 
 individual, but also and to a greater degree in diseased conditions. 1 
 
 Formation of Urea in the Organism. The older statements of Bechamp 
 that urea is directly formed from proteins by oxidation has been denied 
 by several investigators but according to recent statements of Fosse 2 
 this is correct. On the hydrolysis of proteins arginine is found among 
 other products, and as it is also produced in tryptic digestion, it is possible 
 that a small portion of the urea is produced in this manner, varying 
 according to the kind of protein. Drechsel claims that about 10 per 
 cent of the urea can be accounted for in this way. 
 
 The possibility of a formation of urea from arginine has gained in 
 interest since Kossel and Dakin have discovered the presence of an 
 enzyme, arginase, in the liver and other organs, which has the power 
 of splitting arginine with the formation of urea. Thompson 3 has given 
 a direct proof for the formation of urea from arginine. The introduc- 
 tion of arginine into the body of a dog either per os or subcutaneously 
 has in his experiments led t© an elimination of urea. While outside of 
 the body only one-half of the nitrogen of arginine is split off as urea 
 and the other half as ornithine, in the above experiments the increase 
 in urea in several instances corresponded to the greater part if not the 
 whole of the nitrogen of the arginine introduced. This increased forma- 
 tion of urea makes it probable that also ornithine is deamidized and the 
 urea is formed from the ammonia split off. 
 
 By the action of alkalies, as above mentioned (Chapter X), urea may 
 be formed from creatinine ; still such an origin of urea in the animal body 
 has not thus far been proved. 
 
 The amino-acids are considered as special mother-substances of urea. 
 By numerous, generally older experiments with these acids, it has been 
 proved that the amino-acids of the animal body are transformed in part 
 into urea. The investigations by Salaskin with the three amino-acids, 
 glycocoll, leucine, and aspartic acid, have unmistakably shown that the 
 surviving dog-liver, supplied with arterial blood, has the property of 
 transforming the above amino-acids into urea or a closely allied sub- 
 stance. 4 Like the amino-acids the polypeptides are also transformed into 
 
 1 See Satta, Hofmeister's Beitrage, 6, which also gives the literature, and Erben, 
 Zeitschr. f. Heilkunde, 25. 
 
 2 Compt. Rend., 154. 
 
 3 Kossel and Dakin, Zeitschr. f. physiol. Chem., 41; Thompson, Journ. of Physiol., 
 32 and 33. 
 
 4 Schultzen and Nencki, Zeitschr. f. Biologie, 8; v. Knieriem, ibid., 10; Salkowski, 
 Zeitschr. f. physiol. Chem., 4; Salaskin, ibid., 25; Stolte, Hofmeister's Beitrage, 5; 
 Levene and Meyer, Arner. Journ. of Physiol., 25; see also Loewi, Zeitschr. f. physiol. 
 Chem., 25; Richet, Compt. Rend., 118, and Compt. rend. Soc. biol., 49; Ascoli, 
 Pfliiger's Arch., 72. 
 
FORMATION OF UREA. 683 
 
 urea in the animal body, as shown by the investigations of Abderhalden 
 
 and his collaborators. 1 
 
 There is no doubt that the ammonia formation is of great importance 
 in the production of urea in the animal body. 
 
 A great number of older investigations 2 on the behavior of ammo- 
 nium salts in the animal body have shown that not only ammonium car- 
 bonate, but also those ammonium salts which are burned into carbonate 
 in the organism, are transformed into urea by carnivora as well as her- 
 bivora. v. Schroeder, 3 by irrigating the surviving dog's liver with 
 blood treated with ammonium carbonate or ammonium formate, has 
 shown that the formation of urea takes place, at least in part, in this organ. 
 Nencki, Pawlow, Zaleski and Salaskin 4 have also found that, in dogs, 
 the quantity of ammonia in the blood from the portal vein is considerably 
 greater than that from the hepatic vein, and they claim that the liver 
 retains in great part the ammonia thus supplied. The formation of urea 
 from ammonia in the liver is a positively proved fact. 
 
 The assumption of a splitting off of ammonia from amino-acids 
 stands in agreement with the experience that a deamidation of the amino- 
 acids takes place in the animal body. The ammonia split off finds, in 
 the blood and tissues, the carbon dioxide necessary for the formation 
 of carbonate, and the investigations of Nolf, as well as those of Macleod 
 and Haskins, 5 on the equilibrium of carbonate and carbamate solutions 
 and the conditions for the formation of both salts, must also be abundant 
 evidence of a carbamate formation. 
 
 Important observations have been made which give support to the 
 views of Schultzen and Nencki, 6 namely, that the amino-acids are 
 transformed into urea with ammonium carbamate, H4N.O.CO.NH2, 
 as an intermediate step. Drechsel has shown that the amino-acids 
 yield carbamic acid by oxidation in alkaline fluid outside of the organism, 
 and he obtained urea from ammonium carbamate by alternate oxidation 
 and reduction. Carbamate has also been found in the blood (Drechsel) 
 as well as in the urine (Drechsel, Abel and Muirhead) 7 and Nexckt 
 
 1 Abderhalden with Teruuchi and with Babkin, Zeitschr. f. physiol. Chem., 47, 
 with Schittenhelm, ibid., 51. 
 
 2 v. Knieriem, Zeitschr. f. Biologie, 10; Feder, ibid., 13; Salkowski, Zeitschr. f. 
 Biologie, 1; Munk, ibid., 2; Coranda, Arch. f. exp. Path. u. Pharm., 12; Schmiede- 
 berg and Walter, ibid., 7; Hallervorden, ibid., 10; Pohl and Miinzer, Arch., f. exp. 
 Path. u. Pharm., 43. 
 
 3 Arch. f. exp. Path. u. Pharm., 15. See also Salomon, Virchow's Arch., 97. 
 
 4 Arch, des sciences biol. de St. Petersbourg, 4; see also Chapter V, p. 336. 
 
 6 Nolf, Zeitschr. f . physiol. Chem., 23; Macleod and Haskins, Journ. of biol. Chem., 1. 
 
 6 Zeitschr. f. Biologie, 8. 
 
 7 Drechsel, Ber. d. sachs. Gesellsch. d. Wissensch., 1875. See also Journ. f. prakt. 
 Chem. (N. F.), 12, 16, and 22; Abel, Arch. f. (Anat. u.) Physiol., 1891; Abel and 
 Muirhead, Arch. f. exp. Path. u. Pharm., 31. 
 
684 URINE. 
 
 and Hahn have made further observations on dogs with Eck's fistula r 
 which substantiate this view. In such fistula dogs, they observed that 
 when meat was fed, violent poisonous symptoms developed which were 
 almost identical with those produced when carbamate was introduced 
 into the blood. The same symptoms also appeared on the introduction 
 of carbamate into the stomach of the fistula animal, while the intro- 
 duction of carbamate into the stomach of a normal dog had no action. 1 
 As these observers also found that the urine of the dog on which the 
 operation was made was richer in carbamate than that of the normal 
 dog, they concluded that the symptoms were due to the non-transforma- 
 tion of the ammonium carbamate into urea in the liver, and they consider 
 the ammonium carbamate as the substance from which the urea is derived 
 in the mammalian liver. 
 
 Besides the above view of the formation of urea from ammonium 
 carbonate and carbamate, which has been called the anhydride theory, 
 we also have the oxidation theory of Hofmeister. 
 
 F. Hofmeister 2 found in the oxidation of different members of 
 the fatty series, as well as in amino-acids and proteins, that urea was 
 formed in the presence of ammonia, and he therefore suggests the pos- 
 sibility that urea may be formed by an oxidation-synthesis. Accord- 
 ing to him, in the oxidation of nitrogenous substances a radical CONH2, 
 containing the amide group, unites at the moment of formation with the 
 radical NH2 remaining on the oxidation of ammonia, forming urea. 
 
 Besides the above-mentioned theories as to the formation of urea, 
 there are others which will not be given, because the only theory which 
 has thus far been positively demonstrated is the formation of urea in 
 the liver from ammonium compounds and amino-acids. 
 
 The liver is the only organ in which, up to the present time, a forma- 
 tion of urea has been directly detected; 3 and the question arises, what 
 importance has this urea formation which takes place in the liver? Is 
 the urea wholly or chiefly formed in the liver? 
 
 If the liver is the only organ capable of forming urea, it is to be 
 expected, on the extirpation or atrophy of that organ, that a reduced 
 
 1 Hahn, Massen, Nencki et Pawlow, La fistule d'Eck de la veine cave inferieure et 
 de la veine porte, etc. Arch, des sciences biol. de St. P<5tersbourg, 1, No. 4, 1892. 
 In regard to certain differences between the symptoms with carbamate poisoning and 
 after meat feeding with Eck fistula dogs, see Rothberger and Winterberg, Zeitschr. 
 f . exp. Path. u. Therap., 1 ; Hawk, Amer. Journ. of Physiol., 21. 
 
 2 Arch. f. exp. Path. u. Pharm., 37. 
 
 3 In regard to the investigations of Prevost and Dumas, Meissner, Voit, Grehant, 
 Gscheidlen and Salkowski, and others, on the r61e of the kidneys in the formation of 
 urea, see v. Schroeder, Arch. f. exp. Path. u. Pharm., 15 and 19, and Voit, Zeitschr. 
 f. Biologie, 4. 
 
FORMATION OF UREA. 685 
 
 or, in short experiments, at least a strongly diminished elimination of 
 urea should occur. As at hast a part of the urea is formed in the liver 
 from ammonium compounds, a simultaneous increase in the elimination 
 of ammonia is to be expected. 
 
 The extirpation and atrophy experiments made on animals by dif- 
 ferent methods l have shown that sometimes a rather marked increase 
 of ammonia and a diminished elimination of urea takes place after the 
 operation, but that there are also cases in which, irrespective of the pro- 
 nounced atrophy, an abundant formation of urea occurs, and no appre- 
 ciable. ; f any, change in the proportion of ammonia to the total nitrogen 
 and urea is observed. After shutting out from the circulation the organs 
 of the posterior part of the body, especially the liver and kidneys, Kauf- 
 maxx 2 also found an important increase in the urea of the blood, and these 
 different observations show that the liver is not the only organ, in the 
 various animals experimented upon, in which urea is formed. 
 
 The observations made by numerous investigators 3 on human beings 
 with cirrhosis of the liver, acute yellow atrophy of the liver, and phos- 
 phorus poisoning have led to the same result. These investigations 
 teach that in certain cases the proportion of the nitrogenous substances 
 may be so changed that urea is only 50-60 per cent of the total nitrogen, 
 while in other cases, on the contrary, even in very extensive atrophy 
 of the liver-cells, the formation of urea is not diminished, neither is the 
 proportion between the total nitrogen, urea, and ammonia essentially 
 changed. Even in the cases in which the formation of urea was relatively 
 diminished and the elimination of ammonia considerably increased, fur- 
 ther investigation must be instituted before it will be possible to assume 
 a reduced ability of the organism to produce urea. An increased elimi- 
 nation of ammonia may, as shown by Munzer in the case of acute 
 phosphorus poisoning, be dependent upon the formation of abnormally 
 large quantities of acids, caused by abnormal metabolism, and these acids 
 require a greater quantity of ammonia for their neutralization according 
 to the law of elimination of ammonia. That an abnormal formation 
 
 1 Xencki and Hahn, 1. c; Slosse, Arch. f. (Anat. u.) Physiol., 1890; Lieblein, Arch, 
 f. exp. Path. u. Pharm., 33; Nencki and Pawlow, Arch, des science, biol. de St. Peters- 
 bourp, 5. See also v. Meister, Maly's Jahresber., 25; Salaskin and Zaleski, Zeitschr. f . 
 physiol. Chem., 29; Fischler and Bardach, ibid., 78. 
 
 2 Compt. rend. soc. biol., 46, and Arch, de Physiol. (5), 6. 
 
 3 See Hallervorden, Arch. f. exp. Path. u. Pharm., 12; Weintraud, ibid., 31; Miinzer 
 and Winterberg, ibid., 33; Stadelmann, Deutsch. Arch. f. klin. Med., 33; Fawitzki, 
 ibid., 45: Miinzer, ibid., 52; Frankel, Berlin, klin. Wochenschr., 1878; Richter, ibid., 
 1896; Morner and Sjoqvist, Skand. Arch. f. Physiol., 2, and Sjoqvist, Nord. Med. 
 Arkiv. 1892; Guinlich. Zeitschr. f. physiol. Chem., 17; v. Xoorden, Lehrb. d. Pathol, 
 des Stoffwechsels, 2. Aufl., Bd. 1, 104. 
 
686 URINE. 
 
 of acid occurs after the cutting out of the liver has been especially shown 
 by Salaskin and Zaleski. 1 
 
 For the present we are not justified in the statement that the liver 
 is the only organ in which urea is formed, and only continued investiga- 
 tion can yield further information as to the extent and importance of the 
 formation of urea, from ammonium compounds, in the liver. 
 
 Properties and Reactions of Urea. Urea crystallizes in needles or in 
 long, colorless, four-sided, often hollow, anhydrous rhombic prisms. 
 It has a neutral reaction, and produces a cooling sensation on the tongue 
 like saltpeter. It melts at 132° C. At ordinary temperatures it dis- 
 solves in an equal weight of water and in five parts alcohol; it requires 
 one part boiling alcohol for solution: it is insoluble in alcohol-free anhy- 
 drous ether, and also in chloroform. If urea in substance is heated in a 
 test-tube, it melts, decomposes, gives off ammonia, and finally leaves a 
 non-transparent white residue which, among other substances, contains 
 cyanuric acid and biuret, which latter dissolves in water, giving a beautiful 
 reddish-violet liquid with copper sulphate and alkali (biuret reaction). 
 On heating with baryta-water or caustic alkali, also in the so-called 
 alkaline fermentation of urine caused by micro-organisms, urea splits 
 into carbon dioxide and ammonia with the addition of water. The 
 same decomposition products are produced when urea is heated with 
 concentrated sulphuric acid. An alkaline solution of sodium hypo- 
 bromite decomposes urea into nitrogen, carbon dioxide, and water accord- 
 ing to the equation 
 
 CON 2 H4+3NaOBr = 3NaBr-r-C02+2H 2 0+N 2 . 
 
 With a concentrated solution of furfuroi and hydrochloric acid, urea 
 in substance gives a coloration passing from yellow, green, blue, to violet, 
 and then after a few minutes beautiful purple-violet (Schiff's reaction). 
 According to Huppert 2 the test is best performed by taking 2 cc. of a 
 concentrated furfuroi solution, 4-6 drops of concentrated hydrochloric 
 acid, and adding to this mixture, which must not be red, a small crystal 
 of urea. A deep violet coloration appears in a few minutes. 
 
 Urea forms crystalline compounds with many acids. Among these 
 the one with nitric acid and the one with oxalic acid are the most 
 important. 
 
 Urea Nitrate, CO(NH2)2-HN03. On crystallizing quickly this 
 compound forms thin rhombic or six-sided overlapping tiles, or colorless 
 
 1 Zeitschr. f. physiol. Chem., 29. 
 
 1 Huppert-Neubauer, Analyse des Harns, 10. Aufl., 296. 
 
PROPERTIES AND REACTIONS OF UREA. C87 
 
 plates, with an angle of 82°. When crystallizing slowly, larger and 
 thicker rhombic pillars or plates are obtained. This compound is rather 
 easily soluble in pure water, but is considerably less soluble in water 
 containing nitric acid; it may be obtained by treating a concentrated 
 solution of urea with an excess of strong nitric acid free from nitroufl 
 acid. On heating this compound it volatilizes without leaving a residue. 
 
 This compound may bo employed with advantage in detecting small amounts 
 of urea. A drop of the concentrated solution is placed on a microscope slide and 
 the cover-glass placed upon it; a drop of nitric acid is then placed on the side 
 of the cover-glass and allowed to flow under. The formation of crystals begins 
 where the solution and the nitric acid meet. Alkali nitrates may crystallize 
 very similarly to urea nitrate when they are contaminated with other bodies; 
 therefore, in testing for urea, the crystals must be identified as urea nitrate by 
 heating and by other means. 
 
 Urea Oxalate, 2.CO(NH2)2-H2C204. This compound is nu re 
 sparingly soluble in water than the nitric-acid compound. It is obtained 
 in rhombic or six-sided prisms or plates on adding a saturated oxalic- 
 acid solution to a concentrated solution of urea. 
 
 Urea also forms combinations with mercuric nitrate in variable 
 proportions. If a very faintly acid mercuric-nitrate solution is added 
 to a 2 per cent solution of urea and the mixture carefully neutralized, 
 a compound is obtained of a constant composition which contains for 
 every 10 parts of urea 72 parts of mercuric oxide. This compound serves 
 as the basis of Liebig's titration method. Urea also combines with 
 salts, forming mostly crystallizable combinations, as, for instance, with 
 sodium chloride, with the chlorides of the heavy metals, etc. An alka- 
 line but not a neutral solution of urea is precipitated by mercuric chloride. 
 
 If urea is dissolved in dilute hydrochloric acid and then an excess of formal- 
 dehyde is added, a thick, white, granular precipitate is obtained which is dif- 
 ficultly soluble and whose composition is somewhat disputed. 1 With phenyl- 
 hydrazine, urea in strong acetic acid gives a colorless crystalline compound of 
 phenylsemicarbazid, C\Jd 6 NH.NH:CONH 2 , which is soluble with difficulty in cold 
 water and melts at 172° C. (Jaffe 2 ). 
 
 The method of preparing urea from urine is in the main as follows: 
 Concentrate the urine, which has been faintly acidified with sulphuric 
 acid, at a low temperature, add an excess of nitric acid, at the same time 
 keeping the mixture cool, press the precipitate well, decompose it in 
 water with freshly precipitated barium carbonate, dry on the water- 
 bath, extract the residue with strong alcohol, decolorize when necessary 
 with animal charcoal, and filter while warm. The urea which crystallizes 
 
 1 See Tollens and his pupils, Ber. d. deutsch. chem. Gesellsch., 29, 2751; Gold- 
 schmidt, ibid., 29; and Chem. Centralbl., 1897, 1, 33; Thorns, ibid., 2, 144 and 737. 
 
 2 Zeitschr. f. physiol. Chem., 22. 
 
<C8S URINE. 
 
 on cooling is purified by recrystallization from warm alcohol. A fur- 
 ther quantity of urea may be obtained from the mother-liquor by con- 
 centration. The urea is purified from contaminating mineral bodies 
 by redissolving in alcohol-ether. If it is only necessary to detect the 
 presence of urea in urine, it is sufficient to concentrate a little of the 
 urine on a watch-glass and, after cooling, treat it with an excess of nitric 
 acid. In this way we obtain crystals of urea nitrate. 
 
 Quantitative Estimation of the Total Nitrogen and Urea in Urine. 
 Among the various methods proposed for the estimation of the total 
 nitrogen, that suggested by Kjeldahl is to be recommended. Liebig's 
 method for the estimation of urea is really a method for determining 
 the total nitrogen, but as it is very seldom used now, we can refer to 
 larger works in regard to details. 
 
 Kjeldahl 's method consists in transforming all the nitrogen of the 
 organic substances into ammonia by heating with a sufficiently con- 
 centrated sulphuric acid. The ammonia is distilled off, after super- 
 saturating with alkali, and collected in standard sulphuric acid. The 
 following reagents are necessary: 
 
 1. Sulphuric Acid. Either a mixture of equal volumes of pure con- 
 centrated and fuming sulphuric acid, or else a solution of 200 grams 
 phosphoric anhydride in 1 liter of pure concentrated sulphuric acid. 
 2. Caustic soda free from nitrates, 30-40 per cent solution. The quantity 
 of this caustic-soda sohition necessary to neutralize 10 cc. of the acid 
 mixture must be determined. 3. Metallic mercury or pure yellow mercuric 
 oxide. (The addition of this facilitates the destruction of the organic 
 substances.) 4. A potassium-sulphide solution of 4 per cent, whose 
 object is to decompose any mercuric amide combination which might 
 not evolve its ammonia completely during the distillation with caustic 
 :soda. 5. 1/5 normal sulphuric acid and 1/5 normal caustic soda solution. 
 
 In performing the determination 5 cc. of the carefully measured and 
 filtered urine are placed in a long-necked Kjeldahl flask, a drop of mercury 
 or about 0.3 gram of mercuric oxide added, and then treated with 10-15 
 cc. of the strong sulphuric acid. The contents are heated very care- 
 fully, placing the flask at an angle, until they just begin to boil gently; 
 this is continued for about half an hour after the mixture becomes color- 
 less. On cooling, the contents are transferred to a voluminous distilling- 
 flask, carefully washing the Kjeldahl flask with water and the greater 
 part of the acid is neutralized by caustic soda. A few zinc shavings are 
 added to prevent too rapid ebullition on distillation, and then an excess 
 of caustic-soda solution which has previously been treated with 30-40 
 cc. of the potassium-sulphide solution. The flask is quickly connected 
 with the condenser-tube and all the ammonia distilled off. In order 
 to prevent loss of ammonia it is best to lower the end of the exit-tube 
 below the surface of the acid, the regurgitation of the acid being prevented 
 by having a bulb blown on the exit-tube. Not less than 25-30 cc. of 
 the standard acid is used for every 5 cc. of urine, and on completion 
 [of the distillation the acid is retitrated with 1/5 normal caustic soda 
 u.-ing rosolic acid, tincture of cochineal, or lacmoid as indicator. Each 
 cubic centimeter of the acid corresponds to 2.8 milligrams nitrogen. As a 
 control and in order to test the purity of the reagents, or to eliminate 
 any error caused by an accidental quantity of ammonia in the air, we 
 always make a blank determination with the reagents. 
 
METHODS FOR THE DETERMINATION OF UREA. 089 
 
 Recently Folin and Farmer 1 have suggested a method for the estima- 
 tion of the total nitrogen in very small quantities of urine, 1 cc. dilute 
 urine. After hydrolysis with acid the ammonia formed is colorimetrically 
 determined by means of Nessler's reagent. 
 
 Among the methods suggested f<<r the special estimation of urea, 
 that of Morner-Sjo ovist, in combination with Folin's method, is the 
 one that is generally used. 
 
 Principle of M&rner-Sjoqvist's Method. 2 According to this method 
 the nitrogenous constituents of the urine, with the exception of urea, 
 ammonia, hippuric acid, creatinine, and traces of allantoin, 3 are pre- 
 cipitated by a mixture of alcohol and ether after the addition of a solu- 
 tion of barium chloride and barium hydroxide, or in the presence of sugar 
 with solid barium hydroxide. The urea is determined in the concentrated 
 filtrate, after driving off the ammonia, by Kjeldahl's nitrogen estima- 
 tion. The slight error due to the presence of hippuric acid and creatinine 
 can be prevented according to Morner by a combination of his method 
 with Folin's method. 
 
 Principle of Folin's Method.* On heating urea with hydrochloric 
 acid and crystalline magnesium chloride, which melts in its water of 
 crystallization at 112-115° C. and then boils at about 150-155° C, the 
 urea is completely decomposed, while no appreciable decomposition 
 of the hippuric acid and creatinine takes place. The ammonia produced 
 from the urea is distilled off and determined by titration. The amount 
 of ammonia previously existing in the urine must be specially determined. 
 
 Determination of Urea by the Mdrner-Sjoqvist and Folin Method. 5 
 Five cc. of the urine are treated with 1.5 grams of powdered barium 
 hydroxide, and when as much of this is dissolved as possible by gently 
 mixing, it is precipitated by 100 cc. of the alcohol and ether mixture 
 (I vol. ether). On the following day it is filtered and the precipitate 
 washed with the alcohol and ether mixture. The alcohol and ether 
 are distilled off from the filtrate at about 55° C. (not above 60° C). The 
 remaining liquid is treated with 2 cc. of hydrochloric acid of sp.gr. 
 1.124 (for 5 cc. urine), and carefully transferred to a flask of 200 cc. 
 capacity, and evaporated to dryness on the water-bath. Then add 20 
 grams of crytalline magnesium chloride to the contents of the flask 
 and 2 cc. of concentrated hydrochloric acid, and boil on a wire gauze 
 over a small flame for two hours, making use of a proper return cooler. 
 
 1 Journ. of biol. Chem., 11. 
 
 2 Skand. Arch. f. Physiol., 2, and Morner, ibid., 14, where the recent literature 
 may also be found. 
 
 3 According to Wiechowski, Hofmeister's Beitriige, 11, the quantity of allantoin 
 is so great in urine that it must be considered in this method. 
 
 4 Zeitschr. f. physiol. Chem., 32, 3<> and 37. 
 8 See Morner, Skand. Arch. f. Physiol., 14. 
 
690 URINE. 
 
 After cooling it is diluted to about f to 1 liter with water, the ammonia 
 completely distilled off, after making it alkaline with caustic soda, and 
 the ammonia collected in standard acid. After boiling in order to drive 
 off the CO2 and cooling, the acid is retitrated. 
 
 In recent years objections of various kinds have been made against 
 these methods, which are directed towards their exactness and which have 
 led to changes in several directions (Benedict and Gephart, Levene 
 and Meyer, Gill, Allison and Grindley). These changes are: 
 precipitation of the other nitrogenous substances (nearly all the ammonia) 
 with phosphotungstic acid, decomposition of the urea in the nitrate by 
 heating with acid in an autoclave to 150,° and distilling off the ammonia 
 from the solution, made alkaline, not by boiling with alkali, but by the 
 aid of a vacuum or by means of a current of air. These changes have 
 been carefully studied by Henriques and Gammeltoft * and they have 
 suggested the following method: 
 
 Henriques and Gammeltoft Method. First determine in 5 cc. urine 
 how much of a 10 per cent phosphotungstic acid solution (in N/2 H2SO4) 
 is necessary to exactly cause a complete precipitation. Then place 10 cc. 
 of the urine in a 100 cc. flask, add the determined quantity of phospho- 
 tungstic acid solution and fill the flask up to the 100 cc. mark with N/2 
 H2SO4. The liquid is allowed to stand after mixing until it has settled 
 and it is then filtered. Two portions of 10 cc. each are placed in test- 
 tubes of Jena glass, covered with tin-foil and placed in the autoclave at 
 150° C. for 1^ hours. The contents of the test-tubes are now placed in a 
 flask, and the ammonia determined either by passing a current of air 
 through it (after the addition of sodium carbonate) or by distillation in a 
 vacuum (after the addition of barium hydrate dissolved in methyl alcohol). 
 Folin and Pettibone 2 have suggested a method, according to which the 
 ammonia is determined colorimetrically with Nessler reagent. 
 
 Kxop-Hupner's method 3 is based on the fact that urea, by the action of 
 sodium hypobromite, splits into water, carbon dioxide (which dissolves in the 
 alkali), and nitrogen, whose volume is measured (see page 686). This method 
 is less accurate than the preceding ones, and therefore in scientific work it is dis- 
 carded. It is of value to the physician and for practical purposes, because of 
 the ease and rapidity with which it may be performed, even though it may not 
 give very accurate results. For practical purposes a number of different appa- 
 ratus have been constructed to facilitate the use of this method. 
 
 1 Benedict and Gephart, Journ. of Amer. Chern. Soc, 30; Levene and Meyer, 
 ibid., 81; Gill, Allison and Grindley, ibid., 31; Henriques and Gammeltoft, Skand. 
 Arch. f. Physiol., 25. 
 
 :in and Pettibone, Journ. of Biol. Chem., 11. 
 
 ' Knop, Zeitechr. f. analyt. Chern., 9; Hiifner, Journ. f. prakt. Chem. (N. F.), 3. 
 In regard to the extensive literature, see Huppert-Neubauer, 10. Aufl., 304, and follow- 
 ing. See also Keogh, Zeitechr. f. physiol. Chern., 84. 
 
CARBAMIC ACID. G91 
 
 In regard to other met lux Is such as Bunben's method with its man. 
 modifications as suggested by Pfluger, Bohland and Bleibtreu, 
 we refer to more complete handbooks. 
 
 For the quantitative estimation of urea in blood or other animal 
 fluids, as well as in the tissues. ScHONDOBFF has proposed a method 
 where the proteins and extractives are first precipitated by a mixture 
 of phosphotungstic acid and hydrochloric acid, and then the filtrate 
 made alkaline with lime. The quantity of ammonia formed on heating 
 a part of this filtrate to 150° C. with phosphoric acid and the amount 
 of carbon dioxide produced by heating the other part to 150° C. are 
 determined. In regard to the principles of this method, as well a- to 
 the details, we refer to the original article (Pfluger's Arch., (>2). Sal- 
 cowski ' has recently suggested a method for estimating the urea in 
 tissues. 
 
 Urein is the name given by Ovid Moor to a product which he obtained by 
 extracting urine, which had been evaporated to a syrup, with absolute alcohol 
 and precipitating the urea with alcohol containing oxalic acid, or by cooling and 
 treatment with alcohol. Urein is a golden-yellow oil which is poisonous; it 
 reduces permanganate in the cold, and it forms the chief portion of the nitro- 
 genous extractives of urine. There is no doubt that urein is a mixture of several 
 substances. According to Moor, 2 the amount of urea in the urine is only about 
 one-half that ordinarily given, and he has suggested a new method for the deter- 
 mination of the true quantity of urea. The possibility that in the urine we have 
 other bodies besides urea which have been determined with the urea cannot be 
 denied a priori. From the investigations published so far it must be said that 
 Moor's assertions are not sufficientlv grounded. 3 
 
 /XH 2 
 
 Carbamic Acid, CH 3 XOj=CO< . This acid is not known in the free 
 
 x OH 
 state, but only as salts. Ammonium carbamate is produced by the action of 
 dry ammonia on dry carbon dioxide, but also after the addition of Xa.-C'O, to a 
 solution which contains an ammonium salt (Macleod and Haskixs). Carbamic 
 acid is also produced by the action of potassium permanganate on protein and 
 several other nitrogenous organic bodies. 
 
 The occurrence of carbamic acid in human and animal urines has already 
 been considered in connection with the formation of urea. The calcium salt 
 which is soluble in water and ammonia but insoluble in alcohol, is the most impor- 
 tant in the detection of this acid. The solution of the calcium salt in water 
 becomes cloudy on standing, but much more quickly on boiling, and calcium car- 
 bonate separates. Xolf, Macleod and Haskixs have made experiments as to 
 the method of formation of carbamic acid. The latter have indicated a new 
 method for the quantitative estimation of carbamates. 4 
 
 1 Arbeiten aus deni pathol. Institute, Berlin, 1906. 
 
 2 0. Moor, BuM. Acad, de St. Petersbourg, 14 (also Maly's Jahresber., 31, 415), 
 and Zeitschr. f. Biologie, 44 and 45, and Zeitschr. f. physiol. Cheai., 41) and 48. 
 
 3 See Kuliabko. Maly's Jahresber., 31, 415; Erben, Zeitschr. f. physiol. Chem., 
 38; Folin, ibirl., 37; Gies, Journ. Amer. Chem. Soc, 25; Haskins, Anier. Journ. of 
 Physiol., 12; Lippich, Zeitschr. f. physiol. Chem., 48 and 52. 
 
 4 Xolf, Zeitschr. f. physiol. Chem., 23; Macleod and Haskins, Amer. Journ. of 
 Physiol., 12, and Journ. of biol. Chem., 1. 
 
692 URINE. 
 
 Carbamic-acid ethylester (urethane), as shown by Jaffe, 1 may pass, by the 
 mutual action of alcohol and urea, into the alcoholic extract of urine when one 
 is working with large quantities. 
 
 Folin 2 claims that all human urine contains a body which is probably methyl- 
 urea. 
 
 7 NH CO 
 
 Creatinine, C4H7N3O, or NH : C\ , is the anhydride of 
 
 \N(CH 3 ).CH 2 
 
 /NH 2 
 Creatine, NH:C\ , which occurs in the muscles, 
 
 x N(CH 3 ).CH 2 .COOH 
 bird urine and sometimes also in human urine. 
 
 Creatinine occurs in human urine and in that of certain mammalia. 
 It has also been found in ox-blood, milk, though in very small amounts, 
 in meat extracts, and in the flesh of certain fishes. 
 
 The quantity of creatinine in human urine is, in a grown man voiding 
 a normal quantity of urine in the course of a day, 0.6-1.3 grams (Netj- 
 bauer), or on an average i gram. Johnson 3 found 1.7-2.1 grams per 
 day, and similar results have been obtained by v. Hoogenhuyze and 
 Verploegh. 4 The quantity of creatinine with a diet free from meat 
 is, Folin 5 says, variable for different individuals, but is constant for the 
 same person. He never found the quantity below 1 gram and often 
 between 1.3 and 1.7 grams. Nurslings also eliminate creatinine, although 
 the quantity is small (v. Hoogenhuyze and Verploegh). The quantity 
 of creatinine nitrogen in per cent of the total nitrogen varies under 
 different conditions, but is on an average about 4.5-6.9 per cent, as 
 determined by several experimenters. 
 
 Creatine occurs especially in the urine of birds and also in the urine 
 of nurslings, but also in older children (Rose, Folin and Denis). It 
 has also been found in the urine of pregnant women (Kratjse and Cramer) 
 but otherwise only in starvation, in diabetes, diseases of the liver, fevers 
 and diseases accompanied by a destruction of the body proteins, espe- 
 cially muscle-proteins. Between creatine and creatinine elimination a 
 relation exists it seems, at least for certain cases, namely with a decrease 
 in the quantity of creatinine eliminated the quantity of creatine increases 
 (Levene and Kristellerj. 6 
 
 1 Zeitschr. f. physiol. Chem., 14. 
 
 7 Journ. of biol. Chem., 3. 
 
 J Huppert-Neubauer, Harnanalyse, 10. Aufl., 387. 
 
 * Zeitschr. f . physiol. Chem., 4f>. 
 
 * Amer. Journ. of Physiol. 13; af. Klercker, Hofmeister's Beitrage, 8. 
 
 6 Rose. Journ. of biol. Chem., 10; Folin and Denis, ibid., 11; Krauee and Cramer, 
 Journ. of Physiol., 40 (Proc. physiol. Soc, July, 1910, LXI); Schaffer, Amer. Journ. 
 of Physiol., 23; Levene and Kristeller, ibid., 24. 
 
CREATININE. 693 
 
 As the two bodies, creatine and creatinine, can easily be transformed 
 into each other, it has been considered for a long time that the urinary 
 creatinine is formed from the creatine of the muscles and other organs. 
 Unfortunately the authorities disagree on this question. Folin in his 
 investigations found that about 80 per cent of the creatinine intro- 
 duced was again eliminated, while the creatine taken did not appear in 
 the urine as creatinine, but was partly retained by the body and in 
 part eliminated, as such. An intravital transformation of creatine into 
 creatinine is disputed by v. Klercker, Mellanby and Lefmann, 1 while 
 it is accepted by Gottlieb, Stangassinger, S. Weber, v. Hoogen- 
 huyze and Verploegh and Rothmann. The observations of Myers 
 and Fine indicate a production of urinary creatinine from creatine, that is 
 they found that the creatinine elimination by the urine in rabbits was 
 greater according to the total creatine content of the respective animal. 
 The investigations of Pekelharing and v. Hoogenhuyze on the behavior 
 of parenterally introduced creatine in rabbits and dogs, show without 
 any doubt that a part of the creatine is actually transformed into creatinine. 
 Towles and Voegtlin 2 have also observed that the subcutaneously 
 injected creatine increases somewhat the creatinine elimination, while this 
 is not the case with creatine taken per os. The condition of the digestive 
 apparatus also seems to be of importance here. Pekelharing and v. 
 Hoogenhuyze found that in dogs of the parenterally introduced creatine 
 always a smaller part (as creatine and creatinine) passed into the urine 
 during the digestion than during rest of the digestive organs. They 
 explain this by the accepted ability of the liver to partly destroy the 
 creatine and partly by an anhydride formation of transforming the creatine 
 into creatinine. 
 
 As mentioned in Chapter X the proteins and the guanidine groups 
 therein are considered as the mother-substance of these two bodies. 
 If the creatinine (creatine) originates from the protein it is evident that 
 we must differentiate between food-protein and body-protein. The quan- 
 tity of creatinine is, inasmuch as it is increased by meat diet, dependent 
 upon the food; but otherwise, as found by Folin and in chief substantiated 
 by others, is rather independent of the food. Its elimination does not 
 run parallel with the urea and the total nitrogen, and consequently is 
 not in general greater with food rich in protein than with food poor therein. 
 On the contrary, its extent, as shown by other conditions, is dependent 
 upon the intensity of the metabolism in the cells, especially the muscle 
 
 1 Folin, Hammarsten's Festschrift, 1906; v. Klercker, Bioch. Zeitschr., 3; Mellanby, 
 Journ. of Physiol., 36; Lefmann, Zeitschr. f. physiol. Chem., 57. 
 
 2 See footnote 1, page 574, and v. Hoogenhuyze and Verploegh, Zeitschr. f. physiol. 
 Chem., 59; Pekelharing and v. Hoogenhuyze, ibid., 69; Towles and Voegtlin, Journ. 
 of biol. Chem., 10; Myers and Fine, ibid., 14. 
 
694 URINE. 
 
 tissue, and the creatinine, according to Folin, is a product of the endo- 
 genous protein metabolism. 
 
 Reports as to the behavior of the creatinine elimination with work 
 are conflicting, v. Hoogenhuyze and Verploegh, who made use of 
 a much more trustworthy method of quantitative estimation than their 
 predecessors, find that muscular activity as a rule does not cause any 
 rise in the creatinine elimination, and that in man such a rise with work 
 occurs only when the body is obliged to live upon its own tissues. S. 
 Weber l also finds an absolute increase in the elimination of creatinine 
 onty in starving dogs. Other investigators could not find any increase 
 in the elimination of creatinine by work, although such a rise was found 
 as shown by Pekelharing and Harkink, 2 by the muscle tonus. 
 
 In starvation, a decrease in the creatinine but a simultaneous increase 
 in the elimination of creatine has been found in man (v. Hoogenhuyze 
 and Verploegh, Cathcart, Benedict and Myers 3 ). Such an increase 
 in the creatinine elimination only occurs in those conditions which are 
 accompanied by acidosis, and correspondingly it can be prevented by 
 the introduction of carbohydrates (Cathcart, Mendel and Rose) 
 The creatinine elimination in certain cases has therefore been explained 
 by a disturbed carbohydrate metabolism. This is neverthless on the 
 other hand disputed by Wolf and Oesterberg 4 who find that the crea- 
 tinine elimination in starvation can be arrested by the introduction of 
 proteins alone. 
 
 Little is known about the behavior of creatinine in disease, nor are 
 the observations in accord. In anaemia and cachexia the elimination 
 of creatinine is diminished, and when the metabolism is increased the 
 elimination is also increased. That this is the case, at least in fevers, 
 seems to be borne out by several concurrent observations. 5 In diseases 
 of the liver a diminished elimination of creatinine may occur, and in cases 
 of carcinoma of the liver considerable creatine has been found in the urine 
 (v. Hoogenhuyze and Verploegh, Mellanby). The role of the liver 
 in the creatine-creatinine metabolism, is, as has already been mentioned 
 in Chapter X, not clear. The exclusion of the liver from the metabolism 
 of a dog with Eck fistula had no result in the experiments of Towles 
 
 1 Arch. f. exp. Path. u. Pharm., 58. Further literature may be found in v. Hoog- 
 enhuyze and Verploegh, Zeitschr. f. physiol. Chem., 46. 
 
 2 Maillard and Clausmann, Journ. de Physiol, et de Path., 12; Prayon, Maly's 
 Jahresb., 40; Pekelharing and Harkink, Zeitschr. f. physiol. Chem., 75. 
 
 3 v. Hoogenhuyze and Verploegh, Zeitschr. f. physiol. Chem., 57; Cathcart, Bioch. 
 Zeitschr., 6; Benedict and Myers, Amer. Journ. of Physiol., 18; Jaffe, 1. c. 
 
 4 Cathcart, Jour, of Physiol., 39: Mendel and Rose, Journ. of biol. Chem., 10; 
 Wolf and Osterberg, Bioch. Zeitschr., 35; Wolf, Journ. of biol. Chem., 10. 
 
 6 See 0. af Klercker, Zeitschr. f. klin. Med., 68. 
 
PROPERTIES OF CREATININE. 895 
 
 and Voegtlin. The dogB, after feeding with creatine and creatinine, 
 
 I ehaved like normal d< gs, and the observations of other investigators 
 such as London and Boljabski, Foster and Fisher 1 upon dogs with 
 Eck fistula have not had any unanimous results or they are hard to explain. 
 Properties of Creatinine. Creatinine crystallizes in colorless, Bhinin; 
 monoclinic prisms which differ from creatine crystals in not becoming 
 white with loss of water when heated to 100° C. It dissolves in 11 parts 
 cjld water, but more easily in warm water. It is difficultly soluble in 
 cold alcohol, but the reports in regard to its solubility differ widely. 2 
 It is more soluble in warm alcohol and nearly insoluble in ether. In 
 alkaline solution creatinine is very easily converted into creatine on 
 warming. 
 
 Creatinine gives an easily soluble crystalline compound with hydro- 
 chloric acid. A solution of creatinine acidified with mineral acids gives 
 crystalline precipitates with phosphotungstic and phosphomolybdic 
 acids even in very dilute solutions (1:10000), (Kerner, Hofmeister 3 ). 
 It is precipitated, like urea, by mercuric-nitrate solution and also by 
 mercuric chloride. On treating a dilute creatinine solution with sodium 
 acetate and then with mercuric chloride a precipitate of glassy globules 
 having the composition 4(C4H7N30.HCl.HgO)3HgCl2 separates on 
 standing some time (Johnson). Among the compounds of creatinine, 
 that with zinc chloride, creatinine-zinc chloride, (C^H^NsO^ZnClo. is 
 of special interest. This combination is obtained when a sufficiently 
 concentrated solution of creatinine in alcohol is treated w r ith a concentrated, 
 faintly acid solution of zinc chloride. Free mineral acids dissolve the com- 
 pound, hence they must not be present; this, however, may be prevented 
 by an addition of sodium acetate. In the impure state, as from urine, 
 creatinine-zinc chloride forms a sandy, yellowish powder which under 
 the microscope appears as fine needles, forming concentric groups, 
 mostly complete rosettes or yellow balls or tufts, or grouped as brushes. 
 On slowly crystallizing or when very pure, more sharply defined prismatic 
 crystals are obtained. The compound is slightly soluble in water. 
 
 Creatinine acts as a reducing agent. Mercuric oxide is reduced to 
 metallic mercury, and oxalic acid and methylguanidine (methyluramine) 
 are formed. Creatinine also reduces cupric hydroxide in alkaline solution, 
 forming a colorless soluble compound, and only after continued boiling 
 with an excess of copper salt is free suboxide of copper formed. Creat- 
 
 1 Towles and Voegtlin, 1. c; London and Boljarski, Zeitschr. f. physiol. Chem., 
 62; Foster and Fischer, Journ. of biol. Chem., 9. 
 
 2 See Huppert-Xeubauer, 10. Aufl., and Hoppe-Sevler-Thierfelder's Handbuch. 
 8. Aufl. 
 
 3 Kerner, Pfliiger's Arch., 8; Hofmeister, Zeitschr. f. physiol. Chem., o. 
 
696 . URINE. 
 
 inine interferes with Trommer's test for sugar, partly because it has a 
 reducing action, and partly by retaining the copper suboxide in solution. 
 The compound with copper suboxide is not soluble in a saturated soda 
 solution, and if a little creatinine is dissolved in a cold saturated soda 
 solution and then a few drops of Fehling's reagent added, a white flocculent 
 compound separates after heating to 50-60° C. and then cooling (v. 
 Maschke's 1 reaction). An alkaline bismuth solution (see Sugar Tests) 
 is not reduced by creatinine. 
 
 An aqueous solution of creatinine is precipitated by picric acid. 
 The precipitate consists on recrystallization from hot water, of thin, 
 silky, pale yellow needles (Jaffe). If the urine is treated with picric 
 acid (20 cc. of a 5 per cent solution in alcohol for each 100 cc. urine), 
 then a double picrate of creatinine and potassium is precipitated (Jaffe). 
 If a solution of creatinine in water (or urine) is treated with a watery 
 solution of picric acid and a few drops of a dilute caustic-soda solution, 
 a red coloration, lasting several hours, immediately occurs at the ordinary 
 temperature, which turns yellow on the addition of acid (Jaffa's 2 reac- 
 tion). Acetone gives a more reddish-yellow color. Glucose gives 
 with this reagent a red coloration only after heating. If we add a few 
 drops of a freshly prepared very dilute sodium-nitrcprusside solution 
 (sp.gr. 1.003) to a dilute creatinine solution (or to the urine) and then a 
 few drops of caustic soda, a ruby-red liquid is obtained which quickly 
 turns yellow again (Weyl's 3 reaction). If the cold yellow solution is 
 neutralized and treated with an excess of acetic acid, a crystalline pre- 
 cipitate of a nitroso-compound (C4H6N4O2) of creatinine separates on 
 stirring (Kramm) or creatininoxim (Schmidt 4 ). If, on the contrary, 
 the yellow solution is treated with an excess of acetic acid and heated, 
 the solution becomes first green and then blue (Salkowski 5 ) ; finally 
 a precipitate of Prussian blue is obtained. 
 
 A reaction which in description is similar and which, although not solely 
 (Arnold) but at least partially (Holobut), appears after partaking of protein 
 food or meat soup is Arnold's reaction. 6 This reaction is due to an unknown 
 endogenous metabolism product. If 10-20 cc. urine are treated with a few drops 
 of a 4 per cent sodium nitroprusside solution and then with 5-10 cc. of a 5 per 
 cent sodium or potassium hydroxide solution, at first a strong and pure violet 
 color is obtained with an absorption band between D and E, then it becomes 
 purple-red and then brown-red and finally yellow. On the addition of acetic 
 
 1 Zeitschr. f. analyt. Chem., 17. 
 
 2 Zeitschr. f. physiol. Chem., 10. 
 
 3 Ber. d. deutsch. chem. Gesellsch., 11. 
 
 'Kramm, Centralbl. f. d. med. Wissensch., 1897; Schmidt, cited from Chem. 
 Centralbl., 1012, 2. 
 
 5 Zeitschr. f. physiol. Chem., 4. 
 
 8 Arnold, Zeitschr. f. physiol. Chem., 49 and 83; Holobut, ibid., 56. 
 
ESTIMATION OF CREATININE. 697 
 
 acid the violet or purple-red color passes into'blue, which soon becomes pale 
 and finally a pale yellow color. It differs from the creatinine in color and 
 the absorption band as well as in that the creatinine reaction requires more 
 sodium oitroprusside. 
 
 The best method for preparing creatinine is the following, suggested 
 by Folin. 1 The creatinine is first precipitated as the double picrate 
 of creatinine and potassium by means of picric acid according to Jaffa's 
 method, and then this precipitate, while still moist, is decomposed by 
 KHCO3 and water. The solution, which contains the creatinine besides 
 potassium carbonate and small amounts of impurities, is neutralized 
 with sulphuric acid and the sulphate precipitated by alcohol. The 
 creatinine is now converted into the double zinc-chloride salt and this 
 last treated with moist lead hydroxide. After the removal of the lead, 
 the solution contains a mixture of creatinine and creatine, which last is 
 completely transformed into creatinine by heating for forty-eight hours 
 with normal sulphuric acid. After exact neutralization with barium- 
 hydroxide solution it is concentrated to the point of crystallization. 
 
 According to recent work of Folin and Blanck the creatinine-zinc chloride 
 can be dissolved in warm 10 per cent sulphuric acid when creatinine-zinc alum 
 (C\H 7 NsO) 2 SOiZnSOi, 8H 2 is obtained and from this the creatinine can be 
 obtained by decomposing with barium acetate and removing the zinc by H 2 S. 
 Creatine can, according to Folin and Denis, 2 be transformed into creatinine by 
 heating in an autoclave for 3 hours under a pressure of 4-5 kg. per qcm. 
 
 The quantitative estimation of creatinine used to be performed accord- 
 ing to Neubauer's method for the preparation of creatinine, or more 
 simply by Salkowski's 3 modification of this method. As this method 
 is now seldom used we refer the reader to other hand-books. 
 
 Folin 4 has suggested a colorimetric method for determining creatinine 
 which is based upon Jaffe's picric-acid reaction and is as follows: 10 
 cc. of the urine are treated in a graduated flask of 500 cc. capacity 
 with 15 cc. of a 1.2 per cent solution of picric acid and 5 cc. of a 10 
 per cent NaOH solution. After shaking and allowing to stand for five 
 minutes it is diluted with water to 500 cc. and mixed. This solution 
 is now compared in a Duboscq colorimeter "with a 1/2 normal potassium- 
 dicromate solution. The latter solution has in a layer 8 mm. thick 
 exactly the same intensity of color as a layer 8.1 mm. thick of a solution 
 of 10 milligrams creatinine after the addition of 15 cc. picric-acid solu- 
 tion and 5 cc. NaOH solution and dilution to 500 cc. The calculations 
 are simple. For example, in case the urine tested in a layer 7.2 mm. 
 thick has the same color as the dichromate solution in a layer 8 mm. 
 thick, then the quantity of creatinine in 10 cc. of the urine will be 
 
 8 1 
 = ^2X10, or 11.25 milligrams. This method has been tried by many 
 
 authorities and found to be trustworthy. 
 
 1 Zeitschr. f. physiol. Chem., 41. 
 
 2 Folin and Blanck, Journ. of biol. Chem., 8, with Denis, ibid., 8. 
 
 3 Zeitschr. f. physiol. Chem., 10 and 14. 
 * Ibid., 41. 
 
698 URINE. 
 
 The same method is used in the determination of creatine, which 
 for this purpose is first converted into creatinine by warming with dilute 
 mineral acid. The quantity of creatine is the difference obtained between 
 the values for creatinine before and after treatment with acid. More 
 detailed directions can be found in the cited works of Folin, v. Hoogen- 
 htjyze and Verploegh, Gottlieb and Stangassinger. 
 
 In regard to other methods, see the works of Kolisch and Gregor. 1 
 
 Xanthocreatinine, C 5 Hi N 4 O. This body, which was first prepared from 
 meat extract by Gautier, has been found, by Monari, in dog's urine after the 
 injection of creatinine into the abdominal cavity, and in human urine after several 
 hours of exhaustive inarching. According to Colasanti it occurs to a relatively 
 greater extent in lion's urine. Stadthagen 2 considers the xanthocreatinine 
 isolated from human urine after strenuous muscular activity as impure creatinine. 
 
 Xanthocreatinine forms thin sulphur-yellow plates, similar to cholesterin, 
 which have a bitter taste. It dissolves in cold water and in alcohol, and gives 
 a crystalline compound with hydrochloric acid and a double compound with 
 gold and platinum chloride. It gives a compound with zinc chloride, which 
 crystallizes in fine needles. Xanthocreatinine has a poisonous action. 
 
 Methylguanidine occurs, according to Achelis, Kutscher and Lohmann, 
 to a slight extent as a regular constituent of the urine of man, horse and dog. 
 It has been found in urines associated with dimethylguanidine by Engeland. 3 
 
 HN— CO 
 
 I I 
 Uric Acid, Ur, C5H4N4O3; 2, 6, 8-trioxypurine, OC C— NH V , has 
 
 I II >CO 
 
 HN— C— NH' 
 been prepared synthetically by Horbaczewski by fusing urea anu 
 glycocoll, or by heating trichlorlactic-acid amide with an excess of urea. 
 Behrend and Roosen prepared it from isodialuric acid and urea; it 
 is also readily produced from isouric acid on boiling with hydrochloric 
 acid (E. Fischer and Tullner) and finally E. Fischer and Ach 4 have 
 prepared uric acid from pseudouric acid by heating with oxalic acid to 
 145° C. 
 
 On strongly heating uric acid it decomposes with the formation of 
 urea, hydrocyanic acid, cyanuric acid, and ammonia. On heating with 
 concentrated hydrochloric acid in sealed tubes to 170° C. it splits into 
 glycocoll, carbon dioxide, and ammonia. By the action of oxidizing 
 agents splitting and oxidation take place, and either monoureides or 
 diureides are produced. By oxidation with lead peroxide, carbon dioxide, 
 
 1 Kolisch, Centralbl. f. innere Med., 1805; Gregor, Zeitschr. f. physiol. Chem., 31. 
 
 1 Gautier, Bull, de l'acad. demed. (2), 15, and Bull, de la soc. chim. (2), 48; Monari, 
 Maly's Jahresber., 17; Colasanti, Arch. ital. d. Biologie, 15, Fasc. 3; Stadthagen, 
 Zeitschr. f. klin. Med., 15. 
 
 1 Achelis, Centralbl. f. Physiol., 20, 455, and Zeitschr. f. physiol. Chem., 50; Kut- 
 Bcher and Lohmann, ibid., 49; Engeland, ibid., 57. 
 
 4 Horbaczewski, Monatshefte f. Chem., 6 and 8; Behrend and Roosen, Ber. d. d. 
 chem. Gesellsch., 21 ; Fischer and Tullner, ibid., 35; Fischer and Ach, ibid., 28. 
 
URIC ACID. G99 
 
 oxalic acid, urea, and allantoin, which last is glyoxyldiureide, arc pro- 
 duced (see below). By oxidation with nitric acid in the cold, urea and 
 a monoureide, the mesoxalyl urea, or alloxan, are obtained, ('.-,114X403+ 
 + H2O = C' 4 H2N204+(NH2)2C0. On warming with nitric acid, alloxan 
 yields carbon dioxide and oxalyl urea, or parabanic acid, C3H2N2O3. 
 By the addition of water the parabanic acid passes into oxaluric acid, 
 C3H4N2O4, traces of which are found in the urine and which easily splits 
 into oxalic acid and urea. In alkaline solution uric acid may, by taking 
 up water and oxygen, be transformed into a new acid, uroxanic acid, 
 CsEfeNiOe, which may then be changed into oxonic acid, C4H5N3O4. 1 
 On the oxidation of uric acid by hydrogen peroxide in alkaline solution 
 Sc iiittenhelm and Wiener 2 have obtained urea with carbonyl diurea 
 as intermediary product. Uric acid may, as F. and L. Sestini as well 
 as Gerard have shown, undergo bacterial fermentation with the forma- 
 tion of urea. According to Ulpiani and Cingolani, 3 uric acid is quan- 
 titatively split into urea and carbon dioxide, according to the equation 
 
 C5H^V>:,+2H 2 + 30 = 3C0 2 +2CO(NH2) 2 . 
 
 Uric acid occurs most abundantly in the urine of birds and of scaly 
 amphibians, in which animals the greater part of the nitrogen of the urine 
 appears in this form. Uric acid frequently occurs in the urine of carniv- 
 orous mammalia, but is sometimes absent; in urine of herbivora it is 
 habitually present, though only as traces; in human urine it occurs in 
 greater but still small and variable amounts. Traces of uric acid are 
 also found in several organs and tissues, as in the spleen, lungs, heart, 
 pancreas, liver (especially in birds), and in the brain. It always occurs 
 in the blood of birds. Traces have been found in human blood under 
 normal conditions. Under pathological conditions it occurs to an 
 increased extent in the blood, as in pneumonia and nephritis, but espe- 
 cially in leucaemia and sometimes also in arthritis. Uric acid also occurs 
 in large quantities in " chalk-stones," certain urinary calculi, and in 
 guano. It has also been detected in the urine of insects and certain 
 snails, as also in the wings (which it colors white) of certain butterflies 
 (Hopkins 4 ). 
 
 The amount of uric acid eliminated with human urine is subject to 
 considerable individual variation, but amounts on an average to 0.7 
 
 1 See Sundwik, Zeitschr. f. physiol. Chem., 20 and 41; also Behrend, Annal. d. 
 Chem. u. Pharm., 333. 
 
 2 Zeitschr. f . physiol. Chem., 62. 
 
 3 See Chem. Centralbl., 1903, where the other investigators are cited, and Centralbl. 
 f. Physiol., 19. 
 
 4 Philos. Trans. Roy. Soc, 186, B, 661. 
 
700 URINE. 
 
 gram per day on a mixed diet. The ratio of uric acid to urea varies con- 
 siderably with a mixed diet, but is on an average 1 : 50-1 : 70. In new- 
 born infants and in the first days of life the elimination of uric acid is 
 relatively increased, and the relation between uric acid and urea has been 
 found to be 1:6.42-17.1. 
 
 We used to ascribe an increasing action upon the elimination of uric 
 acid to protein food, but the investigations of Hirschfeld, Rosen- 
 feld and Orgler, Siven, Burian, and Shur, 1 and many others have 
 positively proven that a diet rich in protein does not itself increase the 
 elimination of uric acid, but only according to the amount of nucleins 
 or purine bodies contained therein. The common assumption that the 
 elimination of uric acid is smaller with a vegetable diet than with an ani- 
 mal diet, when the quantity may be 2 grams or more per twenty-four 
 hours, is explained by this. 2 
 
 Still a purine-free diet is not without some influence upon the elimina- 
 tion of uric acid, as the quantity of uric acid eliminated with a purine- 
 free diet is considerably greater than in starvation and can be increased 
 by protein feeding. The action of the food-protein is here probably an 
 indirect one, consisting in that the proteins raise the work of the digestive 
 glands and the metabolism of their cells and thereby also raise the endo- 
 genous uric acid formation (see below) somewhat. 3 Work and rest do 
 not seem to have any special influence upon the uric acid elimination, 
 although according to the confirmed statement of Siven and Leathes 4 
 the elimination in the night is less than in the morning hours. 
 
 The reports in regard to the influence of other circumstances, as well 
 as of different substances, on the elimination of uric acid are diverse. 
 This is in part due to the fact that the earlier investigators used an 
 inaccurate method (Heintz), and also that the extent of uric-acid elimina- 
 tion is dependent in the first place upon the individuality. Thus the 
 investigators are not in accord in regard to the action of drinking- 
 
 1 See the extensive review of the literature in Wiener, " Die Harnsaure," in Ergeb- 
 nisse der Physiologie, 1, Abt. 1, 1902. 
 
 2 J. Ranke, Beobachtungen und Versuche uber die Ausscheidung der Harnsaure, 
 etc. (Miinchen, 1858); Mares, Centralbl. f. d. med. Wissensch., 1888; Horbaczewski, 
 
 Wien. Sitzungsber., 100, Abt. 3, 1891. In regard to the action of various diets the 
 reader is referred to the above-cited authors, and especially to A. Hermann, Arch. f. 
 klin. Med., 43, and Camerer, Zeitschr. f. Biologie, 33, and Folin, Amer. Journ. of Physiol., 
 13. 
 
 3 See Hirschstein Arch. f. exp. Path. u. Pharm., 57; Smetanka, Pfliiger's Arch., 
 ]'.'>*> und 140 ; Mares, ibid., 134 and 140. Contrary views, Brugsch and Schitten- 
 helrn, Zeitschr. f. exp. Path. u. Therp., 4, and Siven, Pfliiger's Arch., 146. 
 
 4 Siven, Skand. Arch. f. Physiol., 11; Leathes, Journ. of Physiol, 35; see also Ken- 
 naway, Journ. of Physiol., 38. 
 
FORMATION OF URIC ACID. 701 
 
 water l and of alkalies. 2 Certain medicines, such as quinine and atro- 
 pine, diminish, while others, such as pilocarpine and, as it seems, salicylic 
 acid, 3 increase the elimination of uric acid. 
 
 There .is much diversity of opinion regarding the elimination of uric 
 acid in disease, 4 although it is known that it is increased after an abun- 
 dant destruction of nucleated cells as in pneumonia, after the crisis, 
 and in leucaemia. In the latter in most cases not only is the elimina- 
 tion to the urea increased absolutely, but also relatively ; and the relation 
 between uric acid and urea (total nitrogen calculated as urea) may in 
 lineal leucaemia even be 1 : 9, while under normal conditions, accord- 
 ing to different investigators, it is 1:50 to 70 to 100. As to the behavior 
 of uric acid in gout, authorities are by no means agreed. That the 
 blood contains uric acid in gout has been repeatedly shown, and 
 it is also found in this disease with a purine-free diet (Brugsch and 
 Schittenhelm). According to these investigators a diminished enzy- 
 motic decomposition of uric acid occurs in the body in gout and this 
 causes the occurrence of uric acid in the blood and its accumulation in 
 certain tissues. Strong arguments against this view have been presented 
 by others such as Wells and Corper, Miller and Jones. 5 
 
 Formation of Uric Acid in the Organism. Since Horbaczewski 
 first showed that uric acid could be produced by oxidation from the 
 nuclein-rich spleen-pulp or nucleins outside of the body, he also showed 
 that nucleins when introduced into the animal body caused an increase 
 in the elimination of uric acid. These observations have been confirmed, 
 and at the same time developed by the work of a great number of investi- 
 gators, and we are sure that uric acid can be produced from purine 
 bases either outside or inside the animal body, and also that food rich 
 in nucleins (especially the thymus gland) increases the elimination of 
 uric acid. It is nevertheless true that a few investigators after intro- 
 ducing pure purine bases into the organism could not observe any essential 
 rise in the uric acid or its transformation products; still we have a large 
 number of recent investigations which positively show that nucleic acids, 
 as well as purine bases, when introduced into the animal body are trans- 
 formed in abundant quantities into uric acid in tho body. 6 At present 
 
 1 See Schondorff. Pfliiger's Arch., 46, which contains the pertinent literature. 
 
 2 See Clar, Centralbl. f. d. ined. Wissensch., 1888; Haig, Journ. of Physiol., 8; and 
 A. Hermann, Arch. f. klin. Med., 43. 
 
 3 See Bohland, cited from Maly's Jahresber., 26; Schreiber and Zaudy, ibid., 30. 
 * In regard to the extensive literature on the elimination of uric acid in disease 
 
 we must refer to special works on internal diseases. 
 
 6 Brutisch and Schittenhelm, Zeitschr. f. exp. Path. u. Therp., 4; Wells and Corper, 
 Journ. of biol. Chem., 6; Miller and Jones, Zeitschr. f. physiol. Chem., 61. 
 
 6 As it is not within the scope of this book to enter into a discussion of the numer- 
 ous researches on this subject, we will refer to Wiener, " Die Harnsiiure," Ergebnisse 
 
702 URINE. 
 
 we consider the formation of uric acid from the purine bases of the nuclein 
 substances as a positively proven fact. 
 
 According to the original view of Horbaczewski the nucleins do 
 not directly (by their purine bases) cause an increased elimination of uric 
 acid, but indirectly by causing a leucocytosis with a consequent destruc- 
 tion of leucocytes. This view has been justly discarded on account, 
 of the above-mentioned conditions; still on the other hand it cannot 
 be denied that the formation of uric acid is alsto in certain regards related 
 to the formation or the destruction of leucocytes and to the metabolism 
 in the cells as a whole. 1 
 
 The uric acid, in so far as it is produced from nuclein bases, is in part 
 derived from the nucleins of the destroyed cells of the body and in part 
 from the nucleins or free purine bases introduced with the food. It 
 is therefore possible to admit, with Burian and Schur, 2 of a double origin 
 for the uric acid as well as the urinary purines (all purine bodies of the 
 urine, including the uric acid), namely, an endogenous and an exogenous 
 origin. Burtan and Schur attempted to determine the quantity of 
 endogenous urinary purines by feeding with sufficient food, but as free 
 as possible from purine bodies, and they found that this quantity was 
 constant for every individual, while it was variable for different persons. 
 The observations of many other investigators have led to similar con- 
 clusions, and we are now unanimous in our opinion that the uric acid 
 originating from the nucleins is partly endogenous and partly exogenous, 
 and that the amount of endogenous uric acid is only very slightly dependent 
 upon the protein content of the food. 
 
 The formation of uric acid from the nucleins or the purine bases seems 
 at least in great part to be of an enzymotic kind. After it was shown that 
 certain organs, such as the liver and spleen, had the power of converting 
 oxypurines into uric acid in the presence of oxygen (Horbaczewski, 
 Spitzer and Wiener 3 ), recently Schittenhelm, Burian, Jones and 
 co-workers 4 , by more careful investigations have shown that enzymes 
 
 der Physiol., 1, Abt. 1, 1002. See also Schittenhelm, Zeitschr. f. physiol. Chem., 62, 
 with Frank, ibid., 03, with Seisser, Zeitschr. f. exp. Path. u. Ther., 7; Abderhaklen, 
 London and Schittenhelm, Zeitschr. f. physiol. Chem., 61; Mendel and Lyman., Journ. 
 of bioL Chern., 8. 
 
 1 See Plimmer, Dick and Lieb, Journ. of Physiol., 39; Mares Pfliiger's Arch., 134, 
 and Smotanka, ibid., 138. 
 
 2 Pfliiger's Arch., 80, 87, and 94. 
 1 See footnote, 6, page 701. 
 
 •Schittenhelm, Zeitschr. f. physiol. Chem., 42, 43, 45, 46, 57, 63, 66, with Schmid, 
 ibid., 50, and Zeitschr. f. exp. Path. u. Therap., 4; Burian, Zeitschr. f. physiol. 
 Chem., 43; Jones and Partridge, ibid., 42; Jones with Winternitz, ibid., 44 and 60; 
 Jones, ibid., 45, 05, with Austrian, ibid., 48, with Miller, ibid., 61; Jones, Journ. of 
 biol. Chem., '.»; Wells, ibid., 7; Mendel and Mitchell, Amer. Journ. of Physiol., 20. 
 
FORMATION OF URIC ACID. 703 
 
 of different kinds act together. By means of the two deamidizing enzymes 
 adenase and gttanase, the adenine and guanine are transformed into 
 hypoxanthine and xanthine respectively, and from the latter by means 
 of an oxidizing enzyme, called xanthine oxidase by Burian, the uric 
 acid is formed. In the formation of uric acid from the nucleoproteins 
 we must admit of a gradual decomposition of these by the aid of different 
 enzymes, proteases, nucleases and deamidases. The deamidases seem 
 to be present in most organs, and we have numerous investigations upon 
 their distribution, especially those of Jones and Schittenhelm and 
 his collaborators. 1 The distribution is not the same in all animals and 
 the reports regarding it are unfortunately conflicting (Schittenhelm, 
 Jones and Miller). We must exercise the greatest caution in drawing 
 conclusions as to the occurrence of these enzymes, and from experiments 
 made with the extracts of organs, because it seems as if also other 
 unknown factors must be considered in the formation of uric acid. 
 Thus Jones has with Rohde 2 shown that in rats the organs do not con- 
 tain any xanthine oxidase, and that nevertheless the urine of this animal 
 contains uric acid. On the other hand deamidases occur in the organs 
 of monkeys (and xanthine oxidase in the liver) but the urine does not 
 contain any uric acid and only traces of allantoin (Wells). 3 The pos- 
 sibility of a uric acid formation in man and mammalia in another way 
 from the enzymotic destruction of the purines cannot, for several reasons, 
 be denied. 
 
 In birds the conditions are different, v. Mach 4 has shown that in 
 the bird family a part of the uric acid may be formed from the purine 
 bodies. The chief quantity of uric acid, however, is undoubtedly formed 
 in birds by synthesis. 
 
 The formation of uric acid in birds is increased by the administra- 
 tion of ammonium salts (v. Schroder), and urea acts in a similar 
 manner (Meyer and Jaff£). Minkowski observed, in geese with 
 extirpated livers, a very significant decrease in the elimination of uric 
 acid, while the elimination of ammonia was increased to a corresponding 
 degree. This indicates a participation of ammonia in the formation 
 of uric acid in the organism of birds; and as Minkowski has also found, 
 after the extirpation of the liver, that considerable amounts of lactic 
 acid occur in the urine, it is probable that the uric acid in birds is pro- 
 duced in the liver by synthesis, perhaps from lactic acid and ammonia ; 
 
 1 See footnote 4, page 703. 
 
 1 Jones, Zeitschr. f. physiol. Chem., 65, with Alice Rohde, Journ. of biol. Chem., 7; 
 see also Voegtlin and Jones, Zeitschr. f. physiol. Chem., 66. 
 3 Journ. of biol. Chem., 7. 
 * Arch. f. exp. Path. u. Pharm., 24. 
 
704 URINE. 
 
 although, as Salaskin and Zaleski and Lang have shown, after the 
 extirpation of the liver, and increase in the formation of lactic acid pri- 
 marily occurs, and this causes an increase in the elimination of ammonia 
 (neutralization ammonia). The direct proof for the uric-acid formation 
 from ammonia and lactic acid in the liver of birds has been given by 
 Kowalewsky and Salaskin 1 by means of blood-transfusion experiments 
 on geese with extirpated livers. They observed a relatively abundant 
 formation of uric acid after the addition of ammonium lactate and a 
 still greater formation after arginine. They not only consider ammonium 
 lactate but also amino-acids as substances from which the uric acid can 
 be produced in the liver by synthesis. That these, for example, leucine, 
 glycocoll, and aspartic acid, increase the elimination of uric acid in 
 birds was first shown by v. Knieriem. 2 
 
 The possibility of a formation of uric acid from lactic acid has been 
 shown in another manner by Wiener, 3 namely, by feeding birds with 
 urea and lactic acid and different non-nitrogenous substances, oxy-, 
 keto-, and dibasic acids of the aliphatic series. The dibasic acids, with a 
 chain of 3 carbon atoms or their ureides, showed themselves most active 
 as uric-acid formers, and Wiener is therefore of the opinion that the 
 active substances must first be converted into dibasic acids. By the 
 attachment of a urea residue the corresponding ureide is produced, 
 according to Wiener, and from this the uric acid is derived by the attach- 
 ment of a second urea residue. 
 
 Among the substances tested, only tartronic acid and its ureide, dialuric acid, 
 have shown themselves active in the experiments with the isolated organs, and 
 Wiener therefore also considers that the other acids must be first converted into 
 tartronic acid by oxidation or reduction. From lactic acid, CH 3 .CH(OH).COOH, 
 we first obtain tartronic acid, COOH.CH(OH).COOH, which by the attachment 
 
 /NH— CO\ 
 of a urea residue forms dialuric acid, CO\ /CHOH, and from this, by 
 
 \NH— CCK 
 the attachment of a second urea residue, uric acid is formed. 
 
 Recently Izar 4 has shown on perfusing blood containing urea and 
 dialuric acid through the liver of a dog and at the same time saturating 
 the blood with carbon dioxide, that an abundant formation of uric acid 
 occurred, and that a combined action between an enzyme occurring 
 
 1 v. Schroder, Zeitschr. f. physiol. Chem., 2; Meyer and Jaffa, Ber. d. f. Chem. 
 Gesellsch., 10; Minkowski, Arch. f. exp. Path. u. Pharm., 21 and 31; Salaskin and 
 Zaleski, Zeitschr. f. physiol. Chem., 29; Lang, ibid., 32; Kowalewsky and Salaskin, 
 ibid., 33. 
 
 2 Zeitschr. f. Biologic, 13. 
 
 ' Hofmeistcr's Beitrage, 2. See also Arch. f. exp. Path. u. Pharm., 42, and Ergeb- 
 nissc fi. Physiol., 1, Abt. 1, 1902. 
 
 4 Zeitschr. f. physiol. Chem., 73, see also ibid., 65. 
 
FORMATION OF URIC ACID. 705 
 
 in the blood and an alcohol-soluble co-enzyme occurring in the liver 
 and spleen took place. He has besides this also given further proof of 
 the formation of uric acid in the bird-liver from urea and ammonium 
 carbonate. 
 
 We cannot give any positive answer as to the question whether uric 
 acid is formed by synthesis in man and other mammalia. Wiener 
 has reported experiments which seem to indicate a synthetic uric-acid 
 formation in the isolated mammalian liver, and he has also obtained 
 an increase in the uric-acid elimination, although only a slight one, after 
 feeding lactic acid and dialuric acid to man. In opposition to these 
 experiments Pfeiffer l could find no increase in the elimination of 
 uric acid after feeding malonamide and tartronamide to monkeys as 
 well as tartronic acid and pseudouric acid to monkeys or human beings, 
 and he finds that a synthesis of uric acid in mammalia and man is very 
 doubtful. According to Burian 2 we have for the present no proof 
 of a synthetical formation of uric acid in the mammalian liver; in 
 view of the above-mentioned experiments of Izar, we cannot deny the 
 possibility of a synthetical formation of uric acid also in mammalia and 
 man even if we do not know to what extent this occurs. 
 
 The liver seems to be the organ in birds where the synthetical forma- 
 tion of uric acid occurs, and the fact that it was possible for Minkowski 3 
 to arrest the uric-acid formation by the extirpation of the liver, apparently 
 shows that the liver is the only organ taking part in this synthesis. If a 
 synthesis of uric acid also occurs in man and other mammalia, we must 
 consider the liver as at least one of the organs taking part in the work, 
 as shown by Wiener's and Izar's investigations. The liver is considered 
 as the most important organ in the oxidative formation of uric acid from 
 nucleins and purine bases. That this organ, at least in the dog, is not 
 the only or at least not the most important follows from the investiga- 
 tions of Abderhalden, London and Schittenhelm 4 on dogs with Eck 
 fistula. They found that, on excluding the liver in this manner, that the 
 transformation of the nucleic acid fed, the deamidation of the purine 
 bases and the oxidation of these into uric acid and allantoin was 
 undisturbed. In the dog also other organs must be considered in 
 this connection. It is not known how other animals behave in this 
 regard. 
 
 Uric acid when introduced into the mammalian organism is, as first 
 shown by Wohler and Frerichs, in the dog, and later substantiated 
 
 1 Hofmeister's Beit rage, 10. 
 
 2 Zeitschr. f. physiol. Chem., 43. 
 »1. c. 
 
 4 Zeitschr. f. physiol. Chem., 61. 
 
706 URINE. 
 
 by several experimenters, 1 in great part destroyed and more or less com- 
 pletely changed into urea. As shown by Wohler and Frerichs for the 
 dog and by later investigators 2 also for cats, rabbits and other animals, 
 that allantoin is the most essential or indeed the chief decomposition 
 product is now considered as positively proven. In man, on the con- 
 trary, the conditions are different. According to Wiechowski 3 probably 
 also a formation of allantoin from uric acid takes place in man, but it 
 is only of such an extent as to be without consideration, while in the dog 
 for example about 96 per cent of the purine base nitrogen may appear 
 as allantoin in the urine. According to the investigations of Frank and 
 Schittenhelm 4 the uric acid in man is in part transformed into urea. 
 This different behavior of uric acid in the metabolism of man and 
 animals depends, as numerous investigations 5 have shown, upon the 
 occurrence of a urocolytic enzyme in the liver and also other organs of 
 animals, which transforms the uric acid into allantoin with the taking up 
 oxygen and splitting off of carbon dioxide. This enzyme, which has been 
 called uricolase and also uricase and whose occurrence in the organs of 
 different animals varies, is absent in the organs of man. The results 
 obtained in regard to the enzymotic transformation of uric acid by 
 experiments with organ extracts must be judged with the greatest care. 
 Thus according to the statements of Wiechowski, Battelli and Stern 
 and Schittenhelm, 6 in dogs, the liver is the only organ which in a test- 
 tube shows a positive uricolysis; still in dogs, with excluded livers 
 (Eck fistula) such an abundant formation of allantoin from uric acid 
 occurs so that only 10-20 per cent of the uric acid escapes this trans- 
 formation. 7 
 
 1 Wohler and Frerichs, Annal. d. Chem. u. Pharm., 65. See also Wiener, Ergeb- 
 nisse der Physiologie, 1, Abt. 1. 
 
 2 Salkowski, Zeitschr. f. physiol. Chem., 35, and Ber. d. d. Chem. Gesellsch., 9; 
 Mendel and Brown, Amer. Jour, of Physiol., 3; Mendel and White, ibid., 12; Wie- 
 chowski, Arch. f. exp. Path. u. Pharm., 60, and Bioch. Zeitschr., 19 and 25, with Wiener, 
 Hofmeister's Beitrage, 9; Schittenhelm, Zeitschr. f. physiol. Chem., 62, with Seisser, 
 Zeitschr. f. exp. Path. v. Ther., 7; Abderhalden, London and Schittenhelm, Zeitschr. 
 f. physiol. Chem., 61. 
 
 3 Bioch. Zeitschr., 25. 
 
 4 Zeitschr. f. physiol. Chem., 63. 
 
 5 Chassevant and Richet, Comp. rend. soc. biolog., 49; Ascoli, Pfliiger's Arch., 72; 
 Jacoby, Virchow's Arch., 157; Wiener, Arch. f. exp. Path. u. Pharm., 42, and Centralbl. 
 f. Physiol., 18; Schittenhelm, Zeitschr. f. physiol. Chem., 43, 45, and 63; Burian, 
 ibid., 43; Almagia, Hofmeister's Beitrage, 7; Pfeiffer, ibid., 7; Wiechowski and Wiener, 
 ibid., 9; Galeotti, Bioch. Zeitschr., 20; Battelli and Stern, ibid., 19; Scaffidi, ibid., 
 18; Miller and Jones, Zeitschr. f. physiol. Chem., 61; Wells, Journ. of biol. Chem., 7, 
 with Corper, ibid., 6. 
 
 6 See Schittenhelm, Zeitschr. f. physiol. Chem., 63, 256. 
 
 7 Abderhalden, London and Schittenhelm, 1. c. 
 
PROPERTIES AND REACTIONS OF URIC ACID. 707 
 
 Ascoli, Izar, Bezzola and Preti * have studied the remarkable ability of 
 the liver of destroying uric acid in the blood by transfusing the arterial blood 
 through this organ and qd transfusing the blood, saturated with C0 2 , they have 
 regenerated the uric arid. It is not known what becomes of the uric acid in 
 these cases and from what substance the regeneration occurs. Preti has shown 
 that in the regeneration a combined action of an enzyme in the blood with a 
 co-enzyme of the liver, takes place. 
 
 From this power of the various organs of destroying uric acid it 
 follows that the quantity of uric acid eliminated is not a sure indication 
 of the amount of the acid formed. We must, therefore, admit that a 
 part of the uric acid formed in the body is destroyed in a manner similar 
 to that introduced from without. Burian and Schur 2 have indeed 
 suggested a factor, the so-called " integral factor," with which the quan- 
 tity of uric acid eliminated in the twenty-four hours must be multiplied 
 in order to find the quantity of uric acid formed during this time. Such 
 calculations are necessarily very uncertain and are for the present not 
 admissible. 
 
 Properties and Reactions of Uric Acid. Pure uric acid is a white, 
 odorless, and tasteless powder consisting of very small rhombic prisms 
 or plates. Impure uric acid is easilv obtained as somewhat larger, 
 colored crystals. 
 
 In rapid crystallization, small, thin, four-sided, apparently colorless, 
 rhombic prisms are formed, which can be seen only by the aid of the 
 microscope, and these sometimes appear as spools because of the round- 
 ing of their obtuse angles. The plates are sometimes six-sided, irregularly 
 developed; in other cases they are rectangular with partly straight and 
 partly jagged sides; and in other cases they show still more irregular 
 forms, the so-called dumb-Veils, etc. In slow crystallization, as when 
 the urine deposits a sediment or when treated with acid, large, invariably 
 colored crystals separate. Examined with the microscope these crystals 
 always appear yellow or yellowish brown in color. The most common 
 type is the whetstone shape, formed by the rounding off of the obtuse 
 angles of the rhombic plate. The whetstones are generally connected, 
 two or more crossing each other. Besides these forms, rosettes of pris- 
 matic crystals, irregular crosses, brown-colored rough masses of broken- 
 up crystals and prisms occur, as well as other forms. 
 
 Uric acid is insoluble in alcohol and ether; it is rather easily soluble 
 in boiling glycerin, but very insoluble in cold water, in 39480 parts at 
 18° C. (His and Paul), and in 15505 parts at 37° (Gudzent). At 
 this temperature, according to His and Paul, P. 5 per cent of the uric 
 acid is dissociated in the saturated solution. Because of the reduction 
 
 1 See Zeitschr. f . physiol. Chem., 58, 62, 64 and 63. 
 
 2 Pfliiger's Arch., 87. 
 
708 URINE. 
 
 in the dissociation on the addition of strong acids, uric acid is soluble 
 with difficulty in the presence of mineral acids. It is soluble in a warm 
 solution of sodium diphosphate, and in the presence of an excess of uric 
 acid, monophosphate and acid urate are produced. It is ordinarily- 
 assumed that sodium diphosphate forms a solvent for the uric acid in 
 the urine, while according to Gudzent this is not dissolved by the mono- 
 phosphate. Rudel l believes that urea is an important solvent, but 
 this view has not been confirmed by the observations of His and Paul. 
 Uric acid is not only dissolved by alkalies and alkali carbonates, but also 
 by several organic bases, such as ethylamine and propylamine, piperidine 
 and piperazine. Uric acid can form supersaturated solutions with alkalies 
 and these, according to Schade and Boden 2 contain colloidal uric acid 
 and they may gelatinize on cooling as well as under other conditions. 
 Uric acid dissolves, without decomposing, in concentrated sulphuric 
 acid. It is completely precipitated from the urine by picric acid (Jaffe 3 ). 
 Uric acid gives a chocolate-brown precipitate with phosphotungstic 
 acid in the presence of hydrochloric acid. 4 
 
 Uric acid is dibasic and consequently forms two series of salts, neu- 
 tral and acid. Of the alkali urates the lithium salts are the most soluble 
 and the acid ammonium salt is the most insoluble. The acid alkali 
 urates are very insoluble and separate as a sediment {sedimentum later- 
 itium) from concentrated urine on cooling. According to Gudzent 
 1 liter of water at 18° C. dissolves (as primary salts) 1.5313 grams 
 potassium, 0.8328 gram sodium, and 0.4141 gram ammonium urate, 
 and at 37° C. 2.7002, 1.5043 and 0.7413 grams of the respective urates. 5 
 The salts of the alkaline earths are soluble with great difficulty. The 
 above solubilities apply only, in Gudzent's 6 experience, to the freshly 
 prepared solution, as the solubility to a certain limit gradually dimin- 
 ishes, due to intramolecular transposition (change of the uric acid from 
 the lactam-form into the lactim-form) . 
 
 Besides the mono- and diurates also " quadriurates " have been described 
 and these occur in the excrement of snakes and birds and in the sedimentum 
 
 1 His, Jr., and Paul, Zeitschr. f. physiol. Chem., 31; Smale, Centralbl. f. physiol., 
 D; Rudel, Arch. f. exp. Path. u. Pharra., 30; Gudzent, Zeitschr. f. physiol. Chem., 
 60 and 63. 
 
 2 Zeitschr. f. physiol. Chem., 83. 
 
 3 IUd., 10. 
 
 4 In regard to the combinations of formaldehyde and uric acid, see Nicolaier, 
 Deutsch. Arch. f. klin. Med., 89 (1906). 
 
 6 Determinations of the solubility of the monourates in serum have been made by 
 Gudzent, Zeitschr. f. physiol. Chem., 63. See also Bechhold and Ziegler, Bioch. Zeitschr., 
 20. 
 
 8 Zeitschr. f. physiol. Chem., 56 and 60. 
 
PROPERTIES OF URIC ACID. 709 
 
 lateritium. Whether these quadriurates, which have recently been studied 
 by Ringer, Kohler and Schmutzer, 1 are chemical combinations of 2 molecules 
 uric acid and 1 atom of K or Xa or are mixtures, so-called solid solutions of uric 
 acid in monourates, is still a disputed question. 
 
 If a little uric acid in substance is treated on a porcelain dish with 
 a few drops of nitric acid, the uric acid dissolves on wanning, with a 
 strong development of gas, and after thoroughly drying on the water- 
 bath a beautiful red residue is obtained, which turns a purple-red (ammo- 
 nium purpurate or murexide) on the addition of a little ammonia. If 
 instead of the ammonia we add a little caustic soda (after cooling), the 
 color becomes deeper blue or bluish violet. This color disappears quickly 
 on warming, differing from certain purine bodies. This reaction is called 
 the murexide test. 
 
 A solution of phosphotungstic acid, prepared according to certain 
 directions, gives with a solution of uric acid, when treated with an excess 
 of sodium carbonate, a beautiful blue solution. This extremely delicate 
 reaction (1:500,000) was suggested by Folin and Denis. 2 
 
 Uric acid does not reduce an alkaline solution of bismuth, while, on 
 the contrary, it reduces an alkaline cupric-hydroxide solution. In the 
 presence of only a little copper salt we obtain a white precipitate consist- 
 ing of cuprous urate. In the presence of more copper salt red cuprous 
 oxide separates. The compound of uric acid with cuprous oxide is formed 
 w r hen copper salts are reduced by glucose or a bisulphite in alkaline 
 solution in the presence of a sufficient amount of urate. 
 
 If a solution of uric acid in water containing alkali carbonate is treated 
 with magnesium mixture and then a silver-nitrate solution added, a 
 gelatinous precipitate of silver-magnesium urate is formed. If a drop 
 of uric acid dissolved in sodium carbonate is placed on a piece of filter- 
 paper which has been previously treated with silver-nitrate solution, 
 a reduction of silver oxide occurs, producing a brownish-black or, in the 
 presence of only 0.002 milligram of uric acid, a yellow spot (Schiff's 
 test) . 
 
 If a weak alkaline solution of uric acid in water is treated with a soluble zinc 
 salt, a white precipitate is produced, which on the filter in the presence of alkali 
 is oxidized by the air, and becomes sky-blue in color, especially in sunlight. 
 Potassium persulphate causes a blue coloration immediately' (Ganassini's 
 reaction 3 ). 
 
 The precipitation of free uric acid from its alkali salts by means of acids can 
 be prevented to some extent by the presence of thymic acid or nucleic acid (Goto). 
 According to Seo we are here dealing with combinations of 1 molecule nucleic 
 
 1 Ringer, ibid., 67 (literature) and 75; Kohler, ibid., 70 and 72; Ringer and Schmut- 
 zer, ibid., 82. 
 
 s Journ. of biol. Chem., 12. 
 
 * Cited f. Bioch. Centralbl., 8, 250. 
 
710 URINE. 
 
 acid and 2 molecules uric acid, which protects the uric acid within the body against 
 destruction or transformation into allantoin. This view is incorrect, accord- 
 ing to Schittenhelm and Seisser. 1 According to them no constant combina- 
 tion between nucleic acid and uric acid exists, and in rabbits the nucleic acid does 
 not protect the uric acid from transformation to allantoin. 
 
 Preparation of Uric Acid from Urine. Filtered normal urine is treated 
 with 20-30 cc. of 25-per cent hydrochloric acid for each liter of urine. 
 After forty-eight hours collect the crystals and purify them by redis- 
 Bolving in dilute alkali, decolorizing with animal charcoal and repre- 
 cipitating with hydrochloric acid. Large quantities of uric acid are 
 easily obtained from the excrement of serpents by boiling it with dilute 
 caustic potash (5-per cent) until no more ammonia is developed. A 
 current of carbon dioxide is passed through the filtrate until it barely 
 has an alkaline reaction; dissolve the separated and washed acid potas- 
 sium urate in caustic potash, and precipitate the uric acid in the filtrate 
 by addition of an excess of hydrochloric acid. 
 
 Quantitative Estimation of Uric Acid in the Urine. As the older 
 method suggested by Heintz, even after recent modifications, gives 
 inaccurate results, it will not be considered here. 
 
 Salkowski and Ludwig's 2 method consists in precipitating the uric 
 acid, by silver nitrate, from the urine previously treated with magnesium 
 mixture, and weighing the uric acid obtained from the silver precip- 
 itate. Uric acid determinations by this method are often performed 
 according to the suggestion of E. Ludwig, which requires the follow- 
 ing solutions: 
 
 1. An ammoniacal silver-nitrate solution, which contains in 1 liter 26 
 grams of silver nitrate and a quantity of ammonia sufficient to redissolve com- 
 pletely the precipitate produced by the first addition of ammonia. 2. Magne- 
 sia mixture. Dissolve 100 grams of crystallized magnesium chloride in water, 
 add ammonia until the liquid smells strongly of it, and enough ammonium 
 chloride to dissolve the precipitate; then dilute the solution to 1 liter. 3. Sodium 
 sulphide solution. Dissolve 10 grams of caustic soda which is free from nitric 
 acid and nitrous acid in 1 liter of water. One half of this solution is completely 
 saturated with sulphuretted hydrogen and then mixed with the other half. 
 
 The concentration of the three solutions is so arranged that 10 cc. 
 of each is sufficient for 100 cc. of the urine. 
 
 100-200 cc, according to concentration, of the filtered urine, freed 
 from protein (by boiling after the addition of a few drops of acetic acid), 
 are poured into a beaker. In another vessel mix 10-20 cc. of the silver 
 solution with 10-20 cc. of the magnesia mixture and add ammonia, 
 and when necessary also some ammonium chloride, until the mixture 
 is clear. This solution is added to the urine while stirring, and the mix- 
 ture allowed to stand quietly for half an hour. The precipitate isi .col- 
 lected on a filter, washed with ammoniacal water, and then returned to 
 
 1 Goto, Zeitschr. f. physiol. Chem., 30; Seo, Arch. f. exp. Path. u. Pharm., 58; 
 Schittenhelm and Seisser, Zeitschr. f. exp. Path. u. Ther., 7. 
 
 2 Salkowski, Virchow's Arch., 52; Pfliiger's Arch., 5; Salkowski, Laboratory 
 Manual of Physiol, and Path. Chem., translated by Orndorff, 1904; Ludwig, Wien.. 
 med. Jahrbuch, 1884, and Zeitschr. f. anal Chem., 24. 
 
ESTIMATION OF UKIC ACID. 711 
 
 i; e same beaker by the aid of a glass rod and a wash-bottle, withoul 
 destroying the filter. Now heat i<> boiling 10-20 ce. of the alkali-sulphide 
 
 solution, which has previously been diluted with an equal volume of 
 water, and allow this solution to flow through the above filter into the 
 beaker containing the silver precipitate; wash with boiling water, and 
 
 warm the contents of the beaker on a water-bath for a time, stirring 
 
 constantly. After cooling, filter into a porcelain dish, wash the filter 
 with boiling; water, auidify the filtrate with hydrochloric acid, evaporate 
 it to about 15 cc, add a few drops more of hydrochloric acid, and allow 
 it to stand for twenty-four hours. The uric acid which has crystallized 
 is collected on a small weighed filter, washed with water, alcohol, ether, 
 and carbon disulphide, dried at 100-110° C, and weighed. For each 
 10 cc. of aqueous filtrate we must add 0.00048 gram uric acid to the 
 quantity found directly. Instead of the weighed filter-paper a glass 
 tube filled with glass wool as described in other handbooks may be sub- 
 stituted (Ludwig). Too intense or too long continued heating with 
 the alkali sulphide must be prevented, otherwise a part of the uric acid 
 may be decomposed. 
 
 Salkowski deviates from this procedure by first precipitating the 
 urine with a magnesium mixture (50 cc. to 200 cc. urine), filling up to 
 300 cc, and filtering. Of the filtrate, 200 cc. are precipitated by 10-15 
 cc. of a 3-per cent silver-nitrate solution. The silver precipitate is shaken 
 with 200-300 cc. of water acidified with a few drops of hydrochloric 
 acid, decomposed by sulphuretted hydrogen, heated to boiling, the 
 silver-sulphide precipitate boiled with fresh water, filtered, the filtrate 
 concentrated to a few cubic centimeters, treated with 5-8 drops of hydro- 
 chloric acid, and allowed to stand until the next day. According to 
 Salkowski and Kashiwabara 1 the precipitation with zinc salts can also 
 be used in the estimation of uric acid. 
 
 Hopkins 7 methcd is based on the fact that the uric acid is com- 
 pletely precipitated from the urine as ammonium urate on saturating 
 with ammonium chloride. The uric acid can either be weighed after 
 being set free by hydrochloric acid or it can be determined in several 
 ways — by titration with potassium permanganate or by the Kjeldahl 
 method. Several modifications of this method have been worked out 
 by Folin, Folin and Schaffer, Worner, and Jolles. 2 Of these methods 
 we shall describe only that suggested by Folin-Schaffer. 
 
 Folin-Schaffer Method. Treat 300 cc. urine with 75 cc. of a solu- 
 tion containing 500 grams of ammonium sulphate, 5 grams of uranium 
 acetate, and 60 cc. of 10 per cent acetic acid in a liter, and filter after 
 five minutes. This removes an unknown constituent of the urine (a 
 protein substance) which would otherwise contaminate the uric acid. 
 Take 125 cc. of the filtrate (corresponding to 100 cc. of the urine) and 
 add 5 cc. of concentrated ammonia. After twenty-four hours the pre- 
 cipitate is filtered off and washed free from chlorine on the filter by means 
 of an ammonium-sulphate solution. The precipitate is washed off the 
 
 1 Zeitschr. f. physiol. Cheni., 4. 
 
 2 Hopkins, Journ. of Path, and Bact., 1893, and Proceed. Roy. Soc, 52; Folin, 
 Zeitschr. f. physiol. Chem., 24; Folin and Schaffer, ibid., 32; Worner, ibid., 29; Jolles, 
 ibid., 29; and Wien. med. Wochenschr., 1903. 
 
712 URINE. 
 
 filter by water (total 100 cc.) into a flask, treated with 15 cc. of con- 
 centrated sulphuric acid, and titrated at 60-63° C. with N/20 potassium- 
 permanganate solution. Each cubic centimeter of this solution cor- 
 responds to 3.75 milligrams uric acid. Because of the solubility of the 
 ammonium urate a correction of 3 milligrams must be added for every 
 100 cc. of the urine. 
 
 In regard to the numerous other methods for estimating uric acid, 
 we must refer to special works on the subject, and especially to Huppert- 
 Xeubauer. Folin with Macullum Jr. and with Denis l have sug- 
 gested a colorimetric method for estimating uric acid, making use of 
 phosphotungstic acid. 
 
 Purine Bases (Alloxuric Bases) . The purine bases found in human 
 urine are xanthine, (guanine), hypoxanthine, adenine, paraxanthine, 
 heteroxanthine, episarkine, epiguanine, 1-methylxanthinc. The occur- 
 rence of guanine and carnine (Pouchet) is, according to Kruger and Salo- 
 mon, 2 not positively shown. The quantity of these bodies in the urine 
 is extremely small and varies in different individuals. Flatow and Reit- 
 zenstein 3 found 15.6-45.1 milligrams in the urine voided during twenty- 
 four hours. The quantity of alloxuric bases in the urine is regularly 
 increased after feeding with nucleins or food rich in nucleins, and after an 
 abundant destruction of leucocytes. The quantity is especially increased 
 in leucaemia. We have a number of observations on the elimination of 
 these bodies in different diseases, but they are hardly trustworthy on 
 account of the inaccuracy of the methods used in the determinations. 
 It must also be remarked that the three purine bases, heteroxanthine, 
 paraxanthine, and 1-methylxanthine, which form the chief mass of the 
 purine bases of the urine, are derived, according to numerous investiga- 
 tions 4 from the theobromine, caffeine, and theophylline which occur 
 in the food. With the purine bases we must also differentiate between 
 those of endogenous and those of exogenous origin, 5 and the same factors 
 apply as for the uric acid, viz., the endogenous purine formation represents 
 a value which is somewhat variable for different individuals and relatively 
 
 1 Journ. of biol. Chem., 13 and 14. 
 
 2 Zeitschr. f. physiol. Chem., 24; Pouchet, "Contributions a la connaissance des 
 matieres extractives de l'urine." Th6se, Paris, 1880. Cited from Huppert-Neubauer, 
 333 and 335. 
 
 3 Deutsch. med. Wochenschr., 1897. 
 
 «Albaneee, Ber. d. d. chem. Gesellsch., 32; Arch. f. exp. Path. u. Pharm., 35; 
 Bondzynski and Gottlieb, ibid., 36, and Ber. d. deutsch. chem. Gesellsch., 28; E. 
 Fischer, ibid., 30, 2405; Kruger and Salomon, Zeitschr. f. physiol. Chem., 26; Kruger 
 and Schmidt, Ber. d. d. chem. Gesellsch., 32, and Arch. f. exp. Path. u. Pharm., 45; 
 Kotake, Zeitschr. f. physiol. Chem.. 57. 
 
 4 See Burian and Schur, footnote 2, page 702, and Kaufmann and Mohr, Deutsch. 
 Arch. f. klin. Med., 74. 
 
PI KINK BASES. 713 
 
 constanl for the same individual. According to Siven, 1 with purine-free 
 
 diet the elimination of purines is lowest at night and highest in the morn- 
 ing hours. Rest and work do not show any positive difference. As 
 the four true nuclein bases have been treated in Chapter II, it only 
 remains to describe the special urinary purine bodies. 
 
 HX— CO 
 
 I I 
 Heteroxanthine, CeHjNjOi, 7-monomethylxanthine, OC C.N.CHj, was first 
 
 I I' \ch 
 
 detected in the urine by Salomon. It is identical with the monomethylxan- 
 thine which passes into the urine after feeding with theobromine or caffeine. 
 Salomon and Xeuburg 2 found heteroxanthine in the urine of a dog fed entirely 
 upon meat, and this was probably formed by a methylation in the body. 
 
 Heteroxanthine crystallizes in shining needles and dissolves with difficulty 
 in cold water (1592 parts at 18° C). It is readily soluble in ammonia and alkalies. 
 The crystalline sodium salt is insoluble in strong caustic alkali -(33-per cent) and 
 dissolves with difficulty in water. The chloride crystallizes beautifully, is rela- 
 tively insoluble, and is readily decomposed into the free base and hydrochloric 
 acid by water. Heteroxanthine is precipitated by copper sulphate and bisul- 
 phite, mercuric chloride, basic lead acetate and ammonia, and by silver nitrate. 
 The silver compound dissolves rather easily in dilute, warm nitric acid; it crystal- 
 lizes in small rhombic plates or prisms, often grown together, forming charac- 
 teristic crosses. Heteroxanthine does not give the xanthine reaction, but does 
 give Wiedel's reaction, according to Fischer (see Chapter II). 
 
 CH 3 .N— CO 
 
 I I 
 1-Methylxanthine, CoHsNA, OC C.XH , was first isolated from the 
 
 I II \ rH 
 HN— C.N/' 
 urine and studied by Kruger, and then by Kruger and Salomon. 3 It is diffi- 
 cultly soluble in cold water, but readily soluble in ammonia and caustic soda, 
 and does not give an insoluble sodium compound. It is readily soluble in dilute 
 acids, and it crystallizes from its acetic-acid solution in thin, generally hexagonal 
 plates. The chloride is decomposed into the base and hydrochloric acid by 
 water. 1-methylxanthine gives crystalline double salts with platinum and gold. 
 It is not precipitated by basic lead acetate, nor when pure by basic lead acetate 
 and ammonia. With ammonia and silver nitrate it gives a gelatinous precipitate. 
 The silver-nitrate compound crystallized from nitric acid forms rosettes of united 
 needles. With the xanthine test with nitric acid it gives an orange coloration 
 on the addition of caustic soda. It gives Weidel's reaction (according to Fischer) 
 
 beautifully. 
 
 CH 3 .X— CO 
 
 Paraxanthine, C7H8N4O2, 1.7-dimethylxanthine, OC C.XCH 3 , urotheo- 
 
 HX-C.X/' CH 
 bromine (Thudichum), was first isolated from the urine by Thudichum and 
 
 1 Skand. Arch. f. Physiol., 18. 
 
 2 Salkowski's Festschrift, Berlin, 1904. 
 
 • Kruger, Arch. f. (Anat. u.) Physiol., 1894; Kruger and Salomon, Zeitschr. f. 
 physiol. Chem., 24. 
 
714 URINE. 
 
 Salomon. 1 It crystallizes beautifully in six-sided plates or in needles. The 
 sodium compound crystallizes in rectangular plates or prisms and, like the hetero- 
 xanthine-sodium compound, is insoluble in 33-per cent caustic-soda solution. 
 The sodium compound separates in a crystalline state on neutralizing its solution 
 in water. The chloride is readily soluble and is not decomposed by water. The 
 chloroplatinate crystallizes very beautifully. Mercuric chloride precipitates it 
 only when added in excess and after a long time. The silver-nitrate compound 
 separates as white silky crystals from hot nitric acid on cooling. It gives Weidel's 
 reaction, but not the xanthine test, with nitric acid and alkali. 
 
 Episarkine is the name given by Balke to a purine body occurring in human 
 urine. The same body has been observed by Salomon 2 in pigs' and dogs' urine, 
 as well as in urine in leucaemia. Balke gives C 4 H 6 N 3 as the probable formula 
 for episarkine. It is nearly insoluble in cold water, dissolves with difficulty in 
 hot water, but may be obtained therefrom as long fine needles. Episarkine does 
 not give the xanthine - reaction with nitric acid, or Weidel's reaction. With 
 hydrochloric acid and potassium chlorate it gives a white residue which turns 
 violet with ammonia. It does not form any insoluble sodium comrjound. The 
 silver compound is difficultly soluble in nitric acid. 
 
 HN— CO 
 
 II' 
 Epiguanine, CeHrNsO, 7-methylguanine, H 2 N.C C.N.CH 3 , was first pre- 
 
 II II \ CH 
 
 pared from the urine by Kruger. 3 It is crystalline and difficultly soluble in 
 hot water or ammonia. It crystallizes from a hot 33-per cent caustic-soda solu- 
 tion on cooling in broad shining crystals and dissolves readily in hydrochloric or 
 sulphuric acid. It gives a characteristic chloroplatinate crystallizing in six-sided 
 prisms. It is precipitated neither by basic lead acetate nor by basic lead ace- 
 tate and ammonia. Silver nitrate and ammonia give a gelatinous precipitate. 
 It responds to the xanthine test with nitric acid and alkali. It acts like episarkine 
 with Weidel's test according to Fischer. 
 
 In preparing alloxuric bases from the urine, the fluid is supersaturated with 
 ammonia and precipitated by a silver-nitrate solution. The precipitate is then 
 decomposed with sulphuretted hydrogen. The boiling-hot filtrate is evaporated 
 to dryness and the dried residue treated with 3-per cent sulphuric acid. The 
 purine bases are dissolved, while the uric acid remains undissolved. This filtrate 
 is saturated with ammonia and precipitated by silver-nitrate solution. If instead 
 of precipitating with silver solution we desire to precipitate, according to Kruger 
 and Wulff, with copper suboxide, the urine may be heated to boiling, and imme- 
 diately are added, successively, 100 cc. of a 50-per cent sodium-bisulphite solu- 
 tion and 100 cc. of a 12-per cent copper-sulphate solution for every liter of urine. 
 The thoroughly washed precipitate is decomposed with hydrochloric acid and 
 sulphuretted hydrogen. The uric acid remains in great part on the filter. Further 
 details in regard to the treatment of the solution of the hydrochloric-acid com- 
 pounds may be found in Kruger and Salomon. 4 
 
 1 Thudichum, " Grundziige d. anal. med. klin. Chemie " (Berlin, 1886); Salomon, 
 Arch. f. (Anat. u.) Physiol., 1882, and Ber. d. deutsch. chem. Gesellsch., 16 and 18. 
 
 2 Balke, " Zur Kenntniss der Xanthinkorper " (Inaug.-Diss., Leipzig, 1893); Salo- 
 mon, Zeitschr. f. physiol. Chem., 18. 
 
 3 Arch. f. (Anat. u.) Physiol., 1894; Kruger and Salomon, Zeitschr. f. physiol. 
 Chem., 24 and 26. 
 
 4 Zeitschr. f. physiol. Chem., 26, and also Hoppe-Seyler-Thierf elder's Handbuch, 
 8. Aufl., 188. 
 
ESTIMATION OF PURINE BASES. 715 
 
 Quantitative Estimation of Purine Bases according to Salkowski. 1 
 400-600 cc. of the urine free from protein are first precipitated by mag- 
 nesia mixture, and then by a 3-per cent silver-nitrate solution as described 
 on page 710. The thoroughly washed silver precipitate is decomposed 
 by sulphuretted hydrogen after being suspended in 600-800 cc. of water 
 with the addition of a few drops of hydrochloric acid. It is heated to 
 boiling and filtered hot, and finally evaporated to dryness on the water- 
 bath. The residue is extracted with 20-30 cc. of hot 3-per cent sul- 
 phuric acid and allowed to stand twenty-four hours; the uric acid is 
 filtered off, washed, the filtrate made ammoniacal, and the purine bodies 
 again precipitated by silver nitrate, the precipitate collected on a small 
 chlorine-free filter, washed thoroughly, dried, carefully incinerated, 
 the ash dissolved in nitric acid, and titrated with ammonium sulpho- 
 cyanide according to Volhard's method. The ammonium-sulphocyanide 
 solution should contain 1.2-1.4 grams per liter, and its strength should 
 be determined by a silver-nitrate solution: 1 part silver corresponds 
 to 0.277 gram nitrogen x>f purine bases, or to 0.7381 gram purine bases. 
 By this method the uric-acid and purine bases can be simultaneously 
 determined in the same portion of urine. 2 
 
 Malfatti 3 determines the nitrogen of the purine bases in the hydrochloric- 
 acid filtrate from the separated uric acid. This filtrate is evaporated with mag- 
 nesia until all the ammonia has been expelled and the residue used for the Kjel- 
 dahl determination. 
 
 The nitrogen of the purine bases is also determined as the difference between 
 the uric-acid nitrogen and the total nitrogen of the purine bodies of the silver 
 precipitate (Camerer, Arxstein 4 ). Certain objections have been raised 
 against this method but they can be overcome by using the modified method 
 as suggested by Kexxaway. 5 
 
 According to the method of Kruger and Schmid 6 the uric acid and the 
 purine bases are precipitated as a cuprous compound by copper-sulphate solu- 
 tion and sodium bisulphite. The precipitate is decomposed in sufficient water 
 by sodium sulphide, and the uric acid precipitated from the concentrated filtrate 
 with hydrochloric acid, and the purine bases again precipitated from this nitrate 
 as cuprous or silver compounds. Finally, the nitrogen in the uric-acid part and 
 the part containing the mixture of purine bases is estimated. 
 
 Oxaluric Acid, C 3 HA204 = (COX 2 H 3 ).CO.COOH. This acid, whose relation 
 to uric acid and urea has been spoken of above, does not always occur in the 
 urine, and then only in traces as the ammonium salt. This salt is not directly 
 precipitated by CaCla and XH 3 , but on boiling it is decomposed into urea and 
 oxalate. In preparing oxaluric acid from urine the latter is filtered through 
 animal charcoal. The oxalurate retained by the charcoal may be obtained by 
 boiling; with alcohol. 
 
 1 Pfluger's Arch., 69. 
 
 1 In regard to the details we refer the reader to the original paper. 
 
 3 Centralbl. f. innere Med., 1897. 
 
 4 Camerer, Zeitschr. f. Biologie, 26 and 28; Arnstein, Zeitschr. f. physiol. Chem., 23. 
 8 Journ. of Physiol., 39. 
 
 6 Zeitschr. f. physiol. Chem., 45, and Hoppe-Sevler-Thierf elder's Handbuch, 8. 
 Aufl., 590. 
 
716 URINE. 
 
 COOTT 
 Oxalic Acid, C2H2O4, or • , occurs under physiological conditions 
 
 COOH 
 
 in very small amounts in the urine, about 0.02 gram in twenty-four hours 
 (Furbringer x ). According to the generally accepted view it exists in 
 the urine as calcium oxalate, which is kept in solution by the acid phos- 
 phates present. Calcium oxalate is a frequent constituent of uninary 
 sediments, and also occurs in certain urinary calculi. 
 
 The origin of the oxalic acid in the urine is not well known. Oxalic 
 acid when administered is eliminated unchanged, at least in part, by 
 the urine; 2 and as many vegetables and fruits, such as cabbage, spinach, 
 asparagus, sorrel, apples, grapes, etc., contain oxalic acid, it is possible 
 that a part of the oxalic acid of the urine originates directly from the 
 food. That oxalic acid may be formed in the animal body as a metabolic 
 product from proteins or fats follows from the observations of Mills 
 and Luthje and others, who found that in dogs on an exclusively meat 
 and fat diet, as also in starvation, oxalic acid was eliminated by the urine. 
 The oxalic acid which is eliminated in increased quantity with a diminished 
 oxygen supply and an increased protein catabolism, as found by Reale 
 and Boeri, and also by Terray, is supposed to be derived partly from 
 the greater destruction of proteins. Pure protein does not, accord- 
 ing to Salkowski and Wegrzynowski 3 increase the quantity of oxalic 
 acid eliminated; on the contrary, after meat feeding the amount of this 
 acid is increased, due in part to the meat containing oxalic acid (Sal- 
 kowski). Gelatin and gelatin-yielding tissues seem to increase the 
 excretion of oxalic acid, and the same is also true for fats or at least 
 glycerin (Wegrzynowski). After feeding nucleins no constant increase 
 in the elimination of oxalic acid has been observed. The statements 
 as to the action of carbohydrates are contradictory. The production of 
 oxalic acid due to an incomplete combustion of the carbohydrates has also 
 been suggested, and the work of Hildebrandt and P. Mayer seems to 
 indicate this under abnormal conditions. According to Darin, 4 in rabbits 
 an increased elimination of oxalic acid occurs after the introduction of 
 glycollic or glyoxylic acids, and the oxalic acid seems in many cases to 
 be an intermediary product of metabolism, which is further burnt. We 
 cannot exclude the possibility of the formation of oxalic acid in the 
 oxidation of uric acid in the animal body, yet we have no positive proof 
 
 1 Deutsch. Arch. f. klin. Med., 18. See also Dunlop, Journ. Path, and Bacterid., 3. 
 
 2 In regard to the behavior of oxalic acid in the animal body, see page 773. 
 
 3 Reale and Boeri, Wien. med. Wochenschr., 1895; Terray, Pfliiger's Arch., 65; 
 Salkowski, Berl. klin. Wochenschr., 1900; Wegrzynowski. Zeitschr. f. physiol. Chem., 
 83 which contains the literature. 
 
 4 Journ. of biol. Chem., 3, 57. 
 
ALLANTOIN. 717 
 
 of such a formation. 1 An endogenous as well as an exogenous origin 
 of oxalic acid has also been suggested. 
 
 Oxalic acid is best detected and quantitatively determined according 
 to the method suggested by Salkowski: Shaking out the oxalic acid from 
 the acidified urine by means of ether. Detailed account of this can 
 be found in Wegrzynowski. 2 
 
 .NH.CH.HN.CO.NH2, 
 Allantoin (Glyoxyldiureide), C4H6N4O3, OC\ 
 
 x NH.CO 
 
 occurs, it is claimed by earlier writers, in the urine of children within the 
 first eight days after birth, and in very small amounts also in the urine 
 of adults (Gtjsserow, Ziegler and Hermann). It is found in rather 
 abundant quantities in the urine of pregnant women (Gusserow;. 
 According to Wiechowski the urine of adults, if it contains any allan- 
 toin at all, has only traces, and he could not detect any in the urine of 
 nurslings or in the amniotic fluid, which does not agree with previous 
 reports. Allantoin has also been found in the urine of suckling calves 
 (Wohler), in urine of oxen (Salkowski), and sometimes in the urine of 
 other animals (Meissner). Wiechowski has .found it in relatively 
 large quantities in the urine of the dog, cat, rabbit and monkey, and he 
 considers that allantoin is a terminal metabolic product in these ani- 
 mals. It is also found, as first shown by Valquelin and Lassaigne, 3 
 in the allantoic fluid of the cow (hence the name). That allantoin is 
 formed from the uric acid in mammalia is almost certain, and the inves- 
 tigations on which this is based have already been given in discussing the 
 decomposition of uric acid. 4 The allantoin thus originates from the 
 purine bodies, and consequently in clogs and other animals the excretion 
 of allantoin is considerably increased, according to Minkowski, Cohn, 
 Salkowski, and Mendel and Brown. 5 after feeding thymus or pan- 
 creas. A strong allantoin excretion is also found in dogs after poisoning 
 with hydrazine (Borissow), hydroxylamine, semicarbazide, and amino- 
 guanidine (Pohl), and this increase in the excretion cf allantoin is 
 
 1 See Wiener, Ergebn. d. Physiol., 1; Tomaszewski, Zeitschr. f. exp. Path. u. Ther. f 
 7; Pohl, ibid., 8; Jastrowitz, Bioch. Zeitschr., 28. 
 
 2 Zeitschr. f. physiol. Chem., 83. 
 
 3 Ziegler and Hermann, see Gusserow, Arch. f. Gynakol.. 3 — both cited from Huppert- 
 Neibauer, Ham-Analyse, 10. Aufl., 377; Wohler, Anna! d. Chem. u. Pharm., 70; 
 Salkowski, Zeitschr. f. physiol. Chem., 42; Meissner. Zeitschr. f. rat. Me. I. (3), 31; 
 Lassaigne, Annal. de Chim. et Phys., 17; Wiechowski, Hofmeister's Beitrage, 11, and 
 Arch. f. exp. Path. u. Pharm., 60, and Bioch. Zeitschr., l',> and 25. 
 
 4 See footnote 2, page 706. 
 
 5 Minkowski, Arch. f. exp. Path. u. Pharm., 41. and Centralbl. f. innere Med., 1S98; 
 Cohn, Zeitschr. f. physiol. Chem., 25; Salkowski, Centralbl. f. d. med. Wissensch., 
 1898; Mendel and Brown, Amer. Journ. of Physiol., 3. 
 
718 URINE. 
 
 connected with the nuclein metabolism. Pohl 1 has found, in dogs on 
 poisoning with hydrazine, that the liver contained allantoin and that other 
 organs contained traces, while it does not exist in the organs of normal 
 dogs, and he has also detected the formation of allantoin in the autolysis 
 of the intestinal mucosa, liver, thymus, spleen and pancreas. It is very 
 probable that in these cases we are dealing with a destruction of cells 
 and an enzymotic uric acid formation with a subsequent uricolysis 
 with the formation of allantoin. Certain food-stuffs such as milk, wheat 
 bread, peas and beans contain, according to Ackroyd, small amounts of 
 allantoin, which are introduced into the body. Nothing is known about 
 how these traces of allantoin behave in the body. According to Po- 
 duschka and Minkowski, 2 allantoin introduced into dogs appears almost 
 entirely in the urine, while in man only a small portion of the ingested 
 substance is eliminated in the urine and seems in the last case to be chiefly 
 burned. 
 
 Allantoin is a colorless substance often crystallizing in prisms, dif- 
 ficultly soluble in cold water, easily soluble in boiling water, and also in 
 warm alcohol, but not soluble in cold alcohol or ether. A watery alla- 
 toin solution gives no precipitate with silver nitrate alone, but by the 
 careful addition of ammonia a white flocculent precipitate is formed, 
 C4HsAgN403, which is soluble in an excess of ammonia and which con- 
 sists after a certain time of very small, transparent microscopic globules. 
 The dry precipitate contains 40.75 per cent silver. A watery allantoin 
 solution is precipitated by mercuric nitrate. On continued boiling 
 allantoin reduces Fehling's solution. It gives Schiff's furfurol reac- 
 tion less rapidly and less intensely than urea. Allantoin does not give 
 the murexide test. 
 
 Allantoin is most easily prepared by the oxidation of uric acid with 
 lead peroxide or potassium permanganate. In preparing allantoin from 
 urine we must proceed differently according to whether we are using the 
 urine of animals comparatively rich in allantoin or whether we are using 
 human urine, which is very poor in allantoin. The same applies to the 
 quantitative estimation of allantoin. As the methods in both cases are 
 complicated and require certain percautions we cannot here enter into a 
 detailed description of them, and we refer to the works of Loewi and 
 Wiechowski 3 and to the complete handbooks for details. The pre- 
 
 1 Borissow, Zeitschr. f. physiol. Chem., 19; Pohl. Arch. f. exp. Path. u. Pharm., 
 46; Poduschka, ibid., 44. According to Underhill and Kleiner, Journ. of biol. Chem., 
 4, hydrazine has no other action on the excretion of allantoin than that caused by 
 the refusal to take food brought about by the poison. 
 
 2 Ackroyd, Bioch. Journ., 5; Poduschka, Arch. f. exp. Path. u. Pharm., 44; Min- 
 kowski, ibid., 41. 
 
 3 Loewi, ibid., 44; Wiechowski, Hofmeister's Beitriige, 11, and Arch, f. exp. Path, 
 u. Pharm., (50; and Bioch. Zeitschr., 19 and 25. 
 
HIPPUKIC ACID. 719 
 
 cipitation of allantoin from the urine can be accomplished by mercuric 
 nitrate and by mercuric acetate solutions, in the presence of sodium 
 acetate. 
 
 Glyoxylic Acid, C;H 4 04, /v^tt , is produced on boiling allantoin as well as 
 
 uric acid with alkalies, and also on the oxidation of many substances, among 
 which we can mention creatine and creatinine. It is also of interest that allantoin 
 can be prepared synthetically from glyoxylic acid and urea and that glyoxylic 
 acid yields oxalic acid when introduced into the body. The reports in regard 
 to its occurrence in the urine conflict, 1 as it is readily destroyed in the body, and 
 its passage into the urine is very improbable, or at least only seldom occurs. 
 
 Hippuric Acid (Benzoyl amino acetic acid), 
 
 C9H9XO3 = (C 6 H 5 CO)HN • CH2COOH. 
 
 This acid decomposes into benzoic acid and glycocoll on boiling with 
 mineral acids or alkalies, and also in the putrefaction of the urine. The 
 reverse of this occurs if these two components are heated in a sealed 
 tube, according to the following equation: C6H5COOH + XH2.CH2.COOH 
 = C6H 5 .CO.XH.CHo.COOH+H 2 0. This acid may be synthetically 
 prepared from benzamide and monochloracetic acid, CeH5.CO.NHa 
 +CH 2 Cl.COOH = C 6 H5.CO.NH.CH 2 .COOH-r-HCl, and in variou: other 
 ways, but most simply from glycocoll and benzoyl chloride in the presence 
 of alkali. 
 
 Hippuric acid occurs in large amounts in the urine of herbivora, 
 but only in small quantities in that of carnivora. The quantity of hip- 
 puric acid eliminated in human urine on a mixed diet is usually less than 
 1 gram per day; as an average it is 0.7 gram. After eating freely of vege- 
 tables and fruit, especially such fruit as plums, the quantity may be 
 more than 2 grams. Hippuric acid is also found in the perspiration, the 
 blood, the suprarenal capsule of oxen, and in ichthyosis scales. Noth- 
 ing is positively known in regard to the quantity of hippuric acid in the 
 urine in disease. 
 
 The Formation of Hippuric Acid in the Organism. Benzoic acid and 
 also the substituted benzoic acids are converted into hippuric acid and 
 substituted hippuric acids within the body. Moreover, those bodies 
 are transformed into hippuric acid which by oxidation (toluene, cinnamic 
 acid, hydrocinnamic acid) or by reduction (quinic acid) are converted 
 into benzoic acid. The question of the origin of hippuric acid is there- 
 fore connected with the question of the origin of benzoic acid; the for- 
 mation of the second component, glycocoll, from the protein substances 
 in the body is unquestionable. 
 
 1 The literature on the occurrence and detection of glyoxylic acid in the urine can 
 be found in Granstrom, Hofmeister"s Beitrage, 11. 
 
720 UKINE. 
 
 Hippuric acid is found in the urine of starving dogs (Salkowski), 
 also in dog's urine after a diet consisting entirely of meat (Meissner 
 and Shepard, Salkowski, and others 1 ). It is evident that the benzoic 
 acid originates in these cases from the proteins, and it is generally admitted 
 that it is produced by the putrefaction of proteins in the intestine. 
 Among the products of the putrefaction of protein outside of the body 
 Salkowski found phenylpropionic acid, CeH5.CH2.CH2.COOH, which 
 is oxidized in the organism to benzoic acid and eliminated as hippuric 
 acid after combining with glycocoll. Phenylpropionic acid seems to be 
 formed from the phenylalanine. The supposition that the phenylpro- 
 pionic acid is produced from tyrosine by putrefaction of the intestine 
 has not been substantiated by the researches of Baumann, Schotten, 
 and Baas. 2 The importance of putrefaction in the intestine in pro- 
 ducing hippuric acid is evident from the fact that after thoroughly dis- 
 infecting the intestine of dogs with calomel the hippuric acid disappears, 
 from the urine (Baumann 3 ). 
 
 The large quantity of hippuric acid present in the urine of herbivora 
 is partly explained by the specially active processes of putrefaction going 
 on in the intestine of these animals. According to Vasiliu 4 this can 
 hardly be correct, because, as he has found, by feeding sheep with casein, 
 this would require a too intense putrefaction of the protein (indeed 40 
 per cent of it). This author's explanation lies in part that in the her- 
 bivora only a small part of the phenylalanine is burnt, and is used to a 
 greater extent in the formation of hippuric acid than hi man and car- 
 nivora, and in part by the fact that the food of herbivora contains larger 
 quantities of a non-nitrogenous mother-substance of the benzoic acid. 
 There is hardly any doubt that the hippuric acid in human urine after 
 a mixed diet, and especially after a diet of vegetables and fruits, orig- 
 inates in part from the aromatic substances, e. g., quinic acid. 
 
 The view proposed by Weiss and others that a parallelism exists between 
 the excretion of hippuric acid and uric acid in that an increase in the first is 
 followed by a diminution in the second, and that, for example, quinic acid pro- 
 duces a diminution in the excretion of uric acid corresponding to the increased 
 formation of hippuric acid (Weiss, Lewin), cannot be considered as sufficiently 
 proven (Hupfer). 5 
 
 1 Salkowski, Ber. d. deutsch. chem. Gesellsch., 11; Meissner and Shepard, 
 Untersuch. iiber das Entstehen der Hippursiiure im thierschen Organismus. Hanover, 
 1886. 
 
 2 E. and H. Salkowski, Ber. d. deutsch. Chem. Gesellsch., 12; Baumann, Zeitschr. 
 f. physiol. Chem., 7; Schotten, ibid., 8; Baas, ibid., 11. 
 
 3 Ibid., 10, 131. 
 
 4 Vasiliu, Mitt. d. landwirt. Inst. Breslau, Bd. 4, 1907. 
 
 6 Weiss, Zeitschr. f. physiol. Chem., 25, 27, 38; Lewin, Zeitschr. f. klin. Med., 42; 
 
FORMATION OF HIPPURIC ACID. 721 
 
 As the thorough investigations of Wiechowski teach, the synthesis 
 of hippuric acid does not stand in any direct relation to the extent of 
 protein metabolism; it varies, on the contrary, with the duration of 
 circulation of benzoic acid and the quantity of glycocoll present in 
 the body. The amount of the latter in intermediary metabolism is so 
 great that in rabbits, on the administration of benzoic acid, more than 
 one-half of the total urine nitrogen may exist as glycocoll. Magnus- 
 Levy l found in rabbits and sheep up to 27.8 per cent of the total nitro- 
 gen as hippuric-acid nitrogen, and both investigators have found so much 
 hippuric-acid nitrogen that it could not be accounted for by the glycocoll 
 preformed from the proteins, which amounts to about 4-5 per cent of 
 the total nitrogen of the protein of the food and body. 
 
 In carnivora (dog) and man the conditions are different, according 
 to Brugsch and R. Hirsch, Feigin and Brugsch, as in these cases there 
 is no more glycocoll available for hippuric acid formation than is split 
 off from the proteins en hydrolysis. According to the investigations of 
 Lewinski 2 this does not seem to be correct, at least not for man. After 
 abundant introduction of benzoic acid in man about 34 per cent of the 
 total nitrogen may be excreted as hippuric acid and in a recent investiga- 
 tion he was able to obtain 50.5 grams pure crystalline hippuric acid 
 from the 24-hour urine of a man after feeding sodium benzoate. 
 
 The abundant production of hippuric acid in herbivora induced Abder- 
 halden, Gigon and Strauss to investigate the comparative supply of 
 certain amino-acids in carnivora and herbivora, and they found in cats, 
 rabbits and hens that the percentage quantity of glycocoll split off from 
 the entire organism (with the exception of the intestinal contents and 
 fat and feathers) by hydrolysis was the same, namely 2.33 to 3.34 per 
 cent of the proteins. In order to account for the large quantity of 
 glycocoll which can be eliminated as hippuric acid, we must admit of a 
 formation of glycocoll. That this occurs in animals fed with benzoic 
 acid has been recently proved by Abderhalden and Hirsch by very 
 conclusive experiments. It can be assumed that the benzoic acid com- 
 bines with higher amino-acids and that the hippuric acid is formed from 
 this combination. The investigations of Magnus-Levy to prove this 
 assumption, where he used benzoylated higher amino-acids, have not 
 
 Hupfer, Zeitschr. f. physiol. Chem., 37. See also Wiener, " Die Harnsiiure," Ergeb- 
 nisse der Physiol., 1, Abt. 1. 
 
 1 Wiechowski, Hofmeister's Beitrage, 7 (literature); A. Mangus-Levy, Munch, 
 med. Wochenschr., 1905; Ringer, Journ. of biol. Chem., 10; Epstein and Bookman, 
 ibid., 10. 
 
 2 Brugsch and Hirsch, Zeitschr. f. exp. Path. u. Therap.. 3; Brunch. Maly's 
 Jahresber., 37, 621, and Bioch. Centralbl., 8, 336; Feigin, Maly's Jahresber., 86, 
 631; Lewinski, Arch. f. exp. Path. u. Pharm., 58 and 61. 
 
•722 URINE. 
 
 given support to this assumption; Epstein and Bookman 1 found never- 
 theless in experiments with rabbits after feeding with benzoyl-leucine 
 that a great elimination of hippuric acid occurred which they consider 
 as a formation of glycocoll from this leucine. Free leucine on the con- 
 trary does not increase the hippuric acid elimination. 
 
 The kidneys may be considered in dogs as special organs for the syn- 
 thesis of hippuric acid (Schmiedeberg and Bunge 2 ). In other animals 
 as in rabbits, the formation of hippuric acid seems to take place in other 
 organs, such as the liver and muscles. The synthesis of hippuric acid is 
 therefore not exclusively limited to any special organ, though perhaps 
 in some species of animals it may be more abundant in one organ than in 
 another. 
 
 Properties and Reactions of Hippuric Acid. This acid crystallizes in 
 semi-transparent, long, four-sided, milk-white, rhombic prisms or columns, 
 or in needles by rapid crystallization. They dissolve in 600 parts cold 
 water, but more easily in hot water. They are easily soluble in alcohol, 
 but with difficulty in ether. The acid dissolves more easily (about 12 
 ; times) in acetic ether than in. ethyl ether. Petroleum-ether does not 
 dissolve hippuric acid. 
 
 On heating hippuric acid it first melts at 187.5° C. to an oily liquid 
 which crystallizes on cooling. On continued heating it decomposes, 
 producing a red mass and a sublimate of benzoic acid, with the genera- 
 tion, first, of a peculiar pleasant odor of hay and then an odor of hydro- 
 cyanic acid. Hippuric acid is easily differentiated from benzoic acid 
 by this behavior, also by its crystalline form and its insolubility in 
 petroleum ether. Hippuric acid and benzoic acid both give Lucre's 
 reaction, namely, they generate an intense odor of nitrobenzene when 
 evaporated to dryness with nitric acid and when the residue is heated 
 with sand in a glass tube. Hippuric acid in most cases forms crystal- 
 lizable salts, with bases. The combinations with alkalies and alkaline 
 earths are soluble in water and alcohol. The silver, copper, and lead 
 salts are soluble with difficulty in water; the ferric salt is insoluble. 
 
 Hippuric acid is best prepared from the fresh urine of a horse or cow. 
 The urine is boiled a few minutes with an excess of milk of lime. The 
 liquid is filtered while hot, concentrated and then cooled, and the hippuric 
 acid precipitated by the addition of an excess of hydrochloric acid. The 
 crystals are pressed, dissolved in milk of lime by boiling, and treated as 
 above; the hippuric acid is precipitated again from the concentrated 
 
 1 Abderhalden, Gigon and Strauss, Zeitschr. f. physiol. Chem., 51; Abderhalden 
 and Hirseh, ibid., 78; Mangus-Levy, Bioch. Zeitschr., 6; Epstein and Bookman, 
 Journ. of biol. Chem., 13. 
 
 2 Arch. f. exp. Path. u. Pharm., 6; alpo A. Hoffmann, ibid., 7, and Kochs, Pfluger's: 
 Arch., 20; Bashford and Cramer, Zeitschr. f. physiol. Chem., 35. 
 
PHENACETURIC ACID. BENZOIC ACID. 723 
 
 filtrate by hydrochloric acid. The crystals arc purified by recrystalliza- 
 tion and decolorized, when necessary, by animal charcoal. 
 
 The quantitative estimation of hippuric acid in the urine may be 
 performed by the following method (Binge and Schmiedeberg): 
 The urine is first made faintly alkaline with soda, evaporated nearly to 
 dryness, and the residue thoroughly extracted with strong alcohol. 
 After the evaporation of the alcohol the residue is dissolved in water, 
 the solution acidified with sulphuric acid, and completely extracted by 
 agitating (at least five times) with fresh portions of acetic ether. The 
 acetic ether is then repeatedly washed with water, which is removed by 
 means of a separatory funnel, then evaporated at a medium temperature 
 and the dry residue treated repeatedly with petroleum-ether, which 
 dissolves the benzoic acid, oxyacids, fats, and phenols, while the hippuric 
 acid remains undissolved. This residue is ncrw dissolved in a little warm 
 water and evaporated at 50-60° C. to crystallization. The crystals are 
 collected on a small weighed filter. According to Henriques and 
 Sorensen the acidified urine can be directly shaken out with acetic ether, 
 the residue after evaporation of the acetic ether boiled with hydrochloric 
 acid in order to split the hippuric acid into benzoic acid and glycocoll 
 and the quantity of nitrogen in the latter determined by a formol titra- 
 tion. Other methods have recently been suggested by Folin and 
 Flanders, by Steenbock and by Hryntschak. 1 
 
 Phenaceturic Acid, Ci HnNO3=C 6 H 5 .CH..CO.XH.CH 2 .COOH. This acid, 
 which is produced in the animal body by a combination of glycocoll with the phenyl- 
 acetic acid, CeH5.CH2.COOH, formed in the putrefaction of the proteins, has 
 been prepared from horse's urine by Salkowski, 2 but it probably also occurs in 
 human urine. According to Vasiliu 3 it is just as important a constituent of the 
 urine of herbivora as hippuric acid is. 
 
 Benzoic Acid, CyHeOo or C 6 H 5 .COOH, is found in rabbit's urine and sometimes, 
 though in small amounts, in dog's urine (Weyl and v. Anrep). According to 
 Jaarsveld and Stokvis and to Kronecker it is also found in human urine in 
 diseases of the kidneys. The occurrence of benzoic acid in the urine seems to 
 be due to a fermentative decomposition of hippuric acid. Such a decomposi- 
 tion may very easily occur in an alkaline urine or in one containing proteid(\'AN 
 de Velde and Stokvis). In certain animals — pigs and dogs — the kidneys, 
 according to Schmiedeberg and Minkowski, 4 contain a special enzyme, Schmiede- 
 berg's histozym, winch splits the hippuric acid with the separation of benzoic 
 acid. 
 
 Ethereal Sulphuric Acids. In the putrefaction of proteins in the 
 intestine, phenols — whose mother-substance is considered to be tyrosine — 
 and also indol and skatol are produced. These phenols directly, and the 
 two last-named bodies after they have been oxidized respectively into 
 
 1 Bunge and Schmiedeberg, . Arch. f. exp. Path. u. Pharm., 6; Henriques and 
 Sorensen, Zeitschr. f. physiol. Chem., 64; Folin and Flanders, Journ. of biol. Chem., 11; 
 Steenbock, ibid., 11; Hryntschak, Bioch. Zeitschr., 43. 
 
 2 Zeitschr. f. physiol. Chem., 9. 
 
 s Mitteil. d. landw. Inst., Breslau, 4. 
 
 4 Weyl and v. Anrep, Zeitschr. f. physiol. Chem., 4; Jaarsveld and Stokvis, Arch, 
 f. exp. Path. u. Pharm., 10; Kronecker, ibid., 16; Van de Velde and Stokvis, ibid., 
 17; Schmiedeberg, ibid., 14, 379; Minkowski, ibid., 17. 
 
724 UEINE. 
 
 indoxyl and skatoxyl, pass into the urine as ethereal sulphuric acids 
 after uniting with sulphuric acid. The most important of these ethereal 
 acids are phenol- and cresol-sulphuric acids — which were formerly also 
 called phenol-forming substances — indoxyl- and skatoxyl-sulphuric acids. 
 To this group also belong pyrocatechin-sulphuric acid, which occurs 
 only in very small amounts in human urine, and hydroquinone-sulphuric 
 acid, which appears in the urine after poisoning with phenol, and under 
 physiological conditions perhaps other ethereal acids occur which have 
 not been isolated. The ethereal sulphuric acids of the urine were dis- 
 covered and specially studied by Baumann. 1 The quantity of these 
 acids in human urine is small, while horse's urine contains larger quan- 
 tities. According to the determinations of v. d. Velden the quantity 
 of ethereal sulphuric acid in human urine in twenty-four hours varies 
 between 0.094 and 0.620 gram. C. Tollens found an average of 0.18 
 gram. The relation of the sulphate-sulphuric acid A to the conjugated 
 sulphuric acid B, in health, is on an average 10:1. It undergoes such 
 great variations, as found by Baumann and Herter, 2 and after them 
 by many other investigators, that it is hardly possible to consider the 
 average figures as normal. After taking phenol and certain other 
 aromatic substances, as well as when putrefaction within the organism 
 is general, the elimination of ethereal sulphuric acid is greatly increased. 
 On the contrary, it is diminished when the putrefaction in the intestine 
 is reduced or prevented. For this reason it may be greatly diminished 
 by carbohydrates and exclusive milk diet. 3 The intestinal putrefaction 
 and the elimination of ethereal sulphuric acid have also been diminished 
 in some cases by certain therapeutic agents which have an antiseptic 
 action; still the investigators do not agree in their reports. 4 
 
 Great importance has been given to the relation between the total 
 sulphuric acid and the conjugated sulphuric acid, or between the con- 
 jugated sulphuric acid and the sulphate-sulphuric acid, in the study 
 of the intensity of the putrefaction in the intestine under different con- 
 ditions. Several investigators, F. Muller, Salkowski, and v. Noorden, 5 - 
 
 1 Pfluger's Arch., 12 and 13. 
 
 2 v. d. Velden, Virchow's Arch., 70; Tollens, Zeitschr. f. physiol. Chem., 67; Herter, 
 Zeitschr. f. physiol. Chem., 1. 
 
 3 See Hirschler, Zeitschr. f. physiol. Chem., 10; Biernacki, Deutsch. Arch. f. klin. 
 Med., 49; Rovighi, Zeitschr. f. physiol. Chem., 16; Winternitz, ibid., and Schmitz, 
 ibid., 17 and 19. 
 
 * See Baumann and Morax, Zeitschr. f. physiol. Chem., 10; Steiff, Zeitschr. f. 
 klin. Med., 16; Rovighi, 1. c; Stern, Zeitschr. f. Hyg., 12; and Bartoschewitsch, 
 Zeitschr. f. physiol. Chem., 17; Mosse, ibid., 23. 
 
 ' Muller, Zeitschr. f. klin. Med., 12; v. Noorden, ibid., 17; Salkowski, Zeitschr. 
 f. physiol. Chem., 12. 
 
PHENOL- AND CKESOL-SULPHURIC ACIDS. 725 
 
 consider correct ly that this relation is only of secondary value, and that 
 it is more correct to consider the absolute value. It must be remarked 
 that the absolute values for the conjugated sulphuric acid also undergo 
 great variation, so that it is at present impossible to give the upper or 
 lower limit for the normal value. 
 
 Phenol- and p-Cresol-sulphuric Acids, C6H5.O.SO2.OH and 
 ().S0 2 .OH 
 CeH4<^ These acids are found as alkali salts in human urine, 
 
 VH 3 
 in which also orthocresol has been detected. The quantity of cresol- 
 sulphuric acid is considerably greater than of phenol-sulphuric acid. 
 In the quantitative estimation the phenols are set free from the two 
 ethereal acids and determined together as tribromphenol. The quan- 
 tity of phenols which are separated from the ethereal-sulphuric acids 
 of the urine amounts to 17-51 milligrams in the twenty-four hours 
 (Munk). In nine case investigated by Siegfried and Zimmermann 1 
 they found in the urine of healthy students in 1500 cc. urine an average 
 of 44.6 milligrams phenols, of which 26 milligrams was cresol and 18.6 
 milligrams was phenol. After the ingestion of carbolic acid, which is 
 in great part converted by synthesis within the organism into phenol- 
 sulphuric acid, also into pyrocatechin- and hydroquinon-sulphuric acid 2 
 or when the amount of sulphuric acid is not sufficient to combine with 
 the phenol, it forms phenol-glucuronic acid, 3 the quantity of phenols 
 and ethereal-sulphuric acids in the urine is considerably increased at the 
 expanse of the sulphate-sulphuric acid. The same is also true of other 
 phenols. The cresol is in great part changed into phenol in dogs, accord- 
 ing to Siegfried and Zimmermann. 4 
 
 An increased elimination of phenol-sulphuric acids occurs in active 
 putrefaction in the intestine with stoppage of the contents of the intes- 
 tine, as in ileus, diffused peritonitis with atony of the intestine, or tuber- 
 culous enteritis, but not in simple obstruction. The elimination is also 
 increased by the absorption of the products of putrefaction from 
 purulent wounds or abscesses. An increased elimination of phenol has 
 been observed in a few other cases of diseased conditions of the body. 5 
 
 1 Munk, Pfluger's Arch., 12; Siegfried and Zimmermann, Bioch. Zeitschi\, 34. 
 
 2 See Baumann, Pfluger's Arch., 12 and 13, and Baurnann and Preusse, Zeitschr. 
 f. physiol. Chem., 3, 156. 
 
 3 Schmiedeberg, Arch. f. exp. Path. u. Pharm., 11; C. Tollens, Zeitschr. f. physiol. 
 Chem., 67. 
 
 4 Bioch. Zeitschr., 46. 
 
 6 See G. Hoppe-Seyler, Zeitschr. f. physiol. Chem., 12 (this contains also all refer- 
 ences to the literature on this subject); Fedeli, Moleschott's Untersuch., 15. 
 
726 URINE. 
 
 The alkali salts of phenol- and cresol-sulphuric acids crystallize in 
 white plates, similar to mother-of-pearl, which are rather freely soluble 
 in water. They are soluble in boiling alcohol, but only slightly soluble 
 in cold alcohol. On boiling with dilute mineral acids they are decom- 
 posed into sulphuric acid and the corresponding phenol. 
 
 Phenol-sulphuric acids have been synthetically prepared by Baumann 
 from potassium pyrosulphate and potassium phenolate or p-cresolate. 
 For the method of their preparation from urine, which is rather compli- 
 cated, and also for the known phenol reactions, the reader is referred 
 to other text-books. The quantitative estimation of the phenols from 
 these etheral sulphuric acids is now ordinarily done by the following 
 methods: 
 
 Kossler and Penny's method with Neuberg's x modification. The 
 liquid containing phenol is treated with N/10 caustic soda until strongly 
 alkaline, warmed on the water-bath in a flask with a glass stopper, and 
 then treated with an excess of N/10 iodine solution, the quantity being 
 exactly measured. Sodium iodide is first formed and then sodium 
 hypoiodite, which latter forms tri-iodophenol with the phenol accord- 
 ing to the following equation: 
 
 C 6 N 5 OH+3NaIO = C 6 H 2 I 3 .OH+3NaOH. 
 
 On cooling, acidify with sulphuric acid and determine the excess of iodine 
 by titration with N/10 sodium thiosulphate solution. This process is 
 also available for the estimation of paracresol. Each cubic centimeter 
 of the iodine solution used is equivalent to 1.5670 milligrams of phenol 
 or 1.8018 milligrams of cresol. As the determination does not give any 
 idea as to the variable proportions of the two phenols, the quantity of 
 iodine used must be calculated as one or the other of the two phenols. 
 Before such a determination is carried out, the concentrated urine is 
 first distilled after acidification with sulphuric acid and the distillate 
 purified by precipitation with lead, and distilled again (Neuberg). 
 Mooser has raised objections against the use of sulphuric acid and rec- 
 ommends instead the use of phosphoric acid. In regard to the dispute 
 which has arisen between Neuberg and Mooser 2 as well as to the details 
 of Neuberg's method we must refer to the original publications and to 
 larger handbooks. 
 
 For the separate estimation of phenol and p-cresol in the urine a 
 special method has been suggested by Siegfried and Zimmermann. 3 
 The principle of the method consists in the two following estimations: 
 1. The quantity of bromine necessary to convert the phenol and cresol 
 into tribromphenol and tribromcresol is determined. 2. The quantity 
 of bromine necessary to convert the phenol into tribromphenol and the 
 
 1 Kossler and Penny, Zeitschr. f. physiol. Chem., 17; Neuberg, ibid., 27. 
 
 2 Mooser, Zeitschr. f. physiol. Chem., 63, with Liechti, ibid., 73; Neuberg and 
 Hildesheimer, Bioch. Zeitschr., 28; Marie Hensel, Zeitschr. f. physiol. Chem., 78. 
 
 3 Siegfried and Zimmermann, Bioch. Zeitschr., 29, 34 and 38; see also Ditz and Bar- 
 dach, ibid., 37 and 42. 
 
PYR0CATE0HIN-8ULPHURIC ACID. 727 
 
 cresol into dibromcresol under exactly observed conditions is deter- 
 mined and from the quantities of bromine by weight (Bi and B 2 ) 
 quantities by weight of phenol and cresol can be calculated. In regard to 
 the procedure as well as to the necessary solutions we refer to the original 
 publication. 
 
 The methods for the separate determination of the conjugated sul- 
 phuric acid and the sulphate-sulphuric acid will be spoken of later in 
 connection with the determination of the sulphuric acid of the urine. 
 
 Pyrocatechin-sulphuric Acid. This acid was first found in horse's urine in 
 rather large quantities by Baumann. It occurs in human urine only in the very 
 smallest amounts, and perhaps not constantly, but it is present abundantly in 
 the urine after taking phenol, pyrocatechin, or protocatechuic acid. 
 
 With an exclusively meat diet this acid does not occur in the urine, and it 
 therefore must originate from vegetable food. It probably originates from the 
 protocatechuic acid, which, according to Preusse, passes in part into the urine 
 as pyrocatechin-sulphuric acid. This acid may also perhaps be formed by the 
 oxidation of phenol within the organism (Baumann and Preusse 1 ). 
 
 Pyrocatechin, or o-Dioxybenzene, CeBuCOH)., was first observed in the urine 
 of a child (Ebstein and J. Muller). The reducing body alcapton, first found 
 by Bodeker - in human urine and which was considered for a long time as iden- 
 tical with pyrocatechin, is in most cases probably homogentisic acid (see below). 
 
 Pyrocatechin crystallizes in prisms which are soluble in alcohol, ether, and 
 water. It melts at 102-104° C, and sublimes in shining plates. The watery 
 solution becomes green, brown, and finally black in the presence of alkali 
 and the oxygen of the air. If very dilute ferric chloride is treated with tartaric 
 acid and then made alkaline with ammonia, and this added to a watery solution of 
 pyrocatechin, we obtain a violet or cherry-red liquid which becomes green on 
 adding excess of acetic acid. Pyroeateehin is precipitated by lead acetate. It 
 reduces an ammoniacal silver solution at the ordinary temperature, and with 
 heat reduces alkaline copper-oxide solutions but does not reduce bismuth oxide. 
 
 A urine containing pyrocatechin, if exposed to the air, especially when alkaline, 
 quickly becomes dark and reduces alkaline copper solutions when heated. In 
 detecting pyrocatechin in the urine it ie concentrated when necessary, filtered, 
 boiled with the addition of sulphuric acid to remove the phenols, and repeatedly 
 shaken, after cooling, with ether. The ether is distilled from the several ethereal 
 extracts, the residue neutralized with barium carbonate and shaken again with 
 ether. The pyrocatechin which remains after evaporating the ether may be 
 purified by recrystallization from benzene. 
 
 Hydroquinone, or p-Dioxybenzene, C 6 H 4 (OH) 2 , often occurs in the urine after 
 the use of phenol (Baumann and Preusse). The dark color which certain urines, 
 so-called " carbolic urines," assume in the air is due to decomposition products. 
 Hydroquinone does not occur as a normal constituent of urine, but only after 
 the administration of hydroquinone; and according to Lewin, 3 it may be found 
 in the urine of rabbits as an ethereal-sulphuric acid, being a decomposition product 
 of arbutin. 
 
 Hydroquinone forms rhombic crystals which are readily soluble in water, 
 alcohol, and ether. It melts at 169° C. Like pyrocatechin, it easily reduces 
 metallic oxides. It acts like pyrocatechin with alkalies, but is not precipitated 
 by lead acetate. It is oxidized into quinone by ferric chloride and other oxidiz- 
 
 1 Baumann and Herter, Zeitschr. f. physiol. Chem., 1; Preusse, ibid., 2; Baumann, 
 ibiii., 3. 
 
 - Ebstein and Muller, Virchow's Arch., 62; Bodeker, Zeitschr. f. rat. Med. (3), 7. 
 3 Lewin, Virchow's Arch., 92; Bass, Zeitschr. f. exp. Path. u. Ther., 10. 
 
7'28 URINE. 
 
 ing agents, and quinone can be detected by its peculiar odor. Hydroquinone- 
 sulphuric acid is detected in the urine by the same methods as pyrocatechin sul- 
 phuric acid. 
 
 C.O.SO2.OH 
 Indoxyl-sulphuric Acid, C 8 H 7 NS04, C 6 H 4 \ 7CH , also called 
 
 NH 
 
 urine indican, formerly called uroxanthine (Heller), occurs as an 
 alkali-salt in the urine. This acid is the mother-substance of a great 
 part of the indigo of the urine. The quantity of indigo which can be 
 separated from the urine is considered as a measure of the quantity of 
 indoxyl-sulphuric acid (and indoxyl-glucuronic acid) contained in the 
 urine. This amount, according to Jaffe, for man is 5-20 milligrams 
 per twenty-four hours, and 0.9-37.6 milligrams according to Maillard. 1 
 Horse's urine contains about twenty-five times as much indigo-forming 
 substance as human urine. 
 
 Indoxyl-sulphuric acid is derived, as previously mentioned (page 515), 
 from indol, which is first oxidized in the body into indoxyl and is then 
 conjugated with sulphuric acid. After subcutaneous injection of indol 
 the elimination of indican is considerably increased (Jaffe, Baumann 
 and Brieger, and others). It is also increased by the introduction 
 in the animal organism of orthonitrophenolpropiolic acid (G. Hoppe- 
 Seyler 2 ) . Indol is formed by the putrefaction of proteins. The 
 putrefaction of secretions rich in protein in the intestine also explains the 
 occurrence of indican in the urine during starvation. Gelatin, on the 
 contrary, does not increase the elimination of indican. 
 
 An abnormally increased elimination of indican occurs in those 
 diseases where the small intestines are obstructed, causing an increased 
 putrefaction and thus producing an abundance of indol. Such an increased 
 elimination of indican occurs on tying the small intestine of a dog, but 
 not the large intestine (Jaffe), an observation which has been recently 
 confirmed by Eldnger and Prutz. 3 They removed an intestine loop 
 in dogs and replaced it in a reversed position, the distal end of the loop 
 being attached to the proximal end of the intestine, and in this manner, 
 by the inverted peristalsis so obtained, they effected a disturbance in 
 the movement of the intestinal contents. It was shown that this obstruc- 
 tion in the small intestine caused an increased elimination of indican, 
 while an obstruction in the large intestine showed no such action. 
 
 1 Jaffe, Pfliiger's Arch., 3; Maillard, Journ. de Physiol, et de Pathol., 12. 
 
 2 Jaffe, Centralbl. f. d. med. Wissensch., 1872; Baumann and Brieger, Zeitschr. f. 
 physiol. Chem., 3; G. Hoppe-Seyler, ibid., 7 and 8. See also Porcher and Hervieux, 
 Journ. de Phyisol., 7. 
 
 * Jaffe, Virchow's Arch., 70; Ellinger and Prutz, Zeitschr. f . physiol. Chem., 38. 
 
INDOXYL-SULPHURIC ACID. 729 
 
 The putrefaction of proteins in other organs and tissues besides the 
 intestine may also cause an increase in the indican of the urine. Cer- 
 tain investigators, Blumenthal, Rosenfeld, and Lewin, claim to 
 have shown that an increased excretion of indican can also be brought 
 about without putrefaction by an increased destruction of tissue in starva- 
 tion and also after phlorhizin poisoning; but these statements are vehe- 
 mently opposed by other investigators, such as P. Mayer, Scholz, and 
 Ellinger, and are improbable. The indol, it seems, is not formed from 
 the tryptophane (indolaminopropionic acid) as intermediary step in the 
 demolition of the proteins in the animal body, but rather from the 
 putrefaction of the tryptophane in the intestine. Gentzen * has also 
 shown that tryptophane introduced subcutaneously or per os into the 
 body does not lead to an indicanuria, but only when it is exposed to bac- 
 terial decomposition in the large intestine. The reports as to the elimina- 
 tion of indican after oxalic-acid poisoning are conflicting. After poison- 
 ing with oxalic acid Harnack and v. Leyen found an increased indican 
 elimination, and Moraczewski believes he has proven a certain par- 
 allelism between the quantity of indican and the quantity of oxalic acid 
 in diabetes. Scholz, 2 on the contrary, obtained no increase in the 
 excretion of indican after oxalic-acid poisoning. 
 
 The excretion of indican is, as above stated, increased by the introduction of 
 indol, but also by indoxyl or indoxyl-c£rboxylic acid. Indol-carboxylic acid, 
 on the contrary, does not yield indican, but, according to Porcher and Her- 
 vieux, another ehromogen. Benedicenti has also shown that indigo blue or 
 analogous blue or green pigments are produced only from those derivatives 
 
 CH CH 
 
 of indol which, like n-methyl indol C 6 H 4 \ /CH, a-naphtindol, GoH 6 \ /CH or 
 
 N.CH, NH 
 
 CH, 
 
 n-methylindolin, C 6 H 4 \' y>CH 2 ,do not have the hydrogen atoms of the two methine 
 
 X.CH 3 
 groups substituted by alkyl. From those derivatives in which one or two hydro- 
 gen atoms are substituted by alkyl, such as skatol, a-methyl indol, dimethyl indol, 
 C.CH 3 CH 
 
 C 6 H 4 <J^C.CH 3 , and bz. 3, p. 2-dimethyl indol, CH 3 .GH 3 <^\c.CH3, red pig- 
 NH XH 
 
 1 Blumenthal, Arch. f. (Anat. u.) Physiol., 1901, Suppl, and 1902, with Rosenfeld, 
 Charite annalen, 27, and Hofmeister's Beitrage, 5; Lewin, Hofmeister's Beitrage, 1; 
 Mayer, Arch. f. (Anat. u.) Physiol., 1902, Zeitschr. f. klin. Med., 47, and Zeitschr. f. 
 physiol. Chem., 29, 32; Scholz, ibid., 38; Ellinger, ibid., 39; Gentzen, " Ueber die Vor- 
 stufen des Indols bei der Eiweissfaulnis im Thierkorper," Inaug.-Dissert.. Konigsberg, 
 1904. 
 
 2 Harnack, Zeitschr. f. physiol. Chemie, 29; Scholz, 1. c, Moraczewski, CentralbL 
 f. innere Med., 1903. 
 
730 URINE. 
 
 ments are produced, a behavior which Porcher and Hervieux » have observed in 
 several alkyl-substituted indols. 
 
 An increased elimination of indican has been observed in many 
 diseases, 2 and in these cases the quantity of phenol eliminated is also 
 generally increased. A urine rich in phenol is not always rich in indican. 
 
 The potassium salt of indoxyl-sulphuric acid, which was prepared 
 pure by Baumann and Brieger from the urine of dog fedj on indol, 
 has subsequently been prepared synthetically by Baumann and Thesen, 3 
 by fusing phenyl-glycine-orthocarboxylic acid with alkali and then from 
 this producing the indoxylsulphate by means of potassium pyrosulphate. 
 It crystallizes in colorless, shining plates or leaves which are easily soluble 
 in water, but less readily in alcohol. It is split by mineral acids into 
 sulphuric acid and indoxyl. The latter without access of air passes into 
 a red compound, indoxyl red, but in the presence of oxidizing reagents 
 is converted into indigo blue: 2C 8 H 7 NO+20 = Ci6HioN 2 02+2H20. The 
 detection of indican is based on this last fact. 
 
 For the rather complicated preparation of indoxyl-sulphuric acid as 
 potassium salt from urine the reader is referred to other text-books. 
 For the detection of indican in urine in ordinary cases the following 
 method of Jaffe-Obermayer, which also serves as an approximate test 
 for the quantity of indican, is sufficient. 
 
 Jaffe-Obermayer's Indican Test. Jaffe uses chloride of lime as 
 the oxidizing agent, while Obermayer employs ferric chloride. Other 
 oxidizing agents have been suggested, such as potassium permanganate, 
 potassium dichromate, alkali chlorate, and hydrogen peroxide (the 
 latter suggested by Porcher and Hervieux 4 ) . With Obermayer's 
 reagent the test is performed as follows: 
 
 The acid urine (if alkaline it must be acidified with acetic acid) (Ellin- 
 ger) is precipitated with basic lead acetate, 1 cc. for every 10 cc. of 
 the urine. 20 cc. of the filtrate are treated in a test-tube with an equal 
 volume of pure concentrated hydrochloric acid (specific gravity 1.19) 
 which contains 2-4 grams ferric chloride to the liter, and 2-3 cc. chloro- 
 form are added and the mixture immediately thoroughly shaken. The 
 chloroform is thereby colored more or less blue, depending upon the 
 amount of indican. Besides indigo blue we may also have indigo red 
 produced, whose formation has been explained in various ways. The 
 
 1 The work of Porcher and Hervieux can be found in Compt. Rend., 145, Corapt. 
 rend. soc. biol., 62, and Bull. soc. chim. (4), 1; Benedicenti, Zeitschr. f. physiol. Chem., 
 53 and Arch. f. exp. Path. u. Pharm., 1908, Suppl. (Schmiedeberg's Festschr.). 
 
 2 See Jaffe, Pfliiger's Arch., 3; Senator, Centralbl. f. d. med. Wissensch., 1877; 
 G. Hoppe-Seyler, Zeitschr. f. physiol. Chem., 12 (contains older literature; also Berl., 
 klin. Wochenschr., 1892. 
 
 3 Baumann with Brieger, Zeitschr. f. physiol. Chem., 3; with Thesen, ibid., 23. 
 
 * JafTc, Pfliiger's Arch., 3; Obermeyer, Wien. klin. Wochenschr., 1890; Salkowski, 
 Zeitschr. f. physiol. Chem., 57; Porcher and Hervieux, Zeitschr. f. physiol. Chem., 39. 
 
IN I) [CAN TESTS. 731 
 
 quantity of indigo red becomes greater the more slowly the oxidation 
 takes place, and especially when the decomposition takes place in the 
 warmth (see the works of Rosin, Bouma, Wang, Maillard, Ellinger 
 and Hervieux 1 ). 
 
 According to Ellingeb the source of the indigo-red formation may be the 
 isatin that is produced by the superoxidation of the indoxyl by the action of 
 the reagent, and this isatin forms indigo red with the indoxyl in the hydrochloric- 
 acid solution. Maillard, on the contrary, is of the view that the blue substance 
 which is taken up by the chloroform from the urine mixed with hydrochloric 
 acid is not indigotin (indigo-blue), but another substance, called by him hemi- 
 indigotin, which in alkaline solution polymerizes immediately into indigotin, 
 while in acid reaction it is converted into indirubin (indigo red). 
 
 The chloroform solution of indigo obtained in the indican test may be 
 used in the quantitative colorimetric determination by comparison with 
 a solution of indigo in chloroform of known strength (Krauss and Adrian). 
 Wang and others convert the indigo into indigo-sulphonic acid by con- 
 centrated sulphuric acid and titrate with potassium permanganate. 
 There is still doubt as to the surest and most trustworthy method for 
 the determination of indican, and especially as to the question how the 
 indigo residue is to be washed (see Wang, Bouma, Ellinger, and Sal- 
 kowski 2 ) , and for this reason we shall refer only to the works cited above. 
 
 Because of the difficulty arising from the production of indirubin 
 in addition to indigotin, Bouma has recommended the conversion of 
 all the indoxyl into indirubin by boiling the urine with hydrochloric 
 acid containing isatin. The indirubin can be taken up by chloroform 
 and determined by titration with potassium permanganate and sul- 
 phuric acid after purification of the chloroform residue. Oerum 3 has 
 also worked out a colorimetric method of estimation based upon Bouma 's 
 method. 
 
 Indol seems also to pass into the urine as a glucuronic acid, inlnyl- 
 glucuronic acid (Schmiedeberg). Such an acid has been found in the 
 urine of animals after the administration of the sodium-salt of o-nitro- 
 phenylpropiolic acid (G. Hoppe-Seyler). Porcher and Hervieux 4 
 have obtained indoxyl sulphuric acid in dogs and asses under similar 
 conditions. 
 
 Free indigo, and in fact indirubin as well as indigotin, occur in rare cases in 
 the undecomposed urine. Grober and Wang have recently observed such 
 According to Steensma 5 traces of free indol occur always in the urine. 
 
 1 Rosin, Yirchow's Arch., 123; Bouma, Zeitschr. f. physiol. Chem., 27, 30, 32, 
 39; Wang, ibid., 25, 27, 28; Ellinger, ibid., 38 and 41; -Maillard, Bull. soc. chim., 
 Paris (3), 29, and Compt. Rend., 136; also L'indoxyle urinaire et les couleurs qui en 
 derivent, Paris, 1903, and Zeitschr. f. physiol. Chem., 41; Hervieux, see Bioch. 
 Centralbl., 8, 54. 
 
 2 Krauss, Zeitschr. f. physiol. Chem., 18; Adrian, ibid., 19; Wang, ibid., 25; Sal- 
 kowski, (bid., 42. 
 
 5 Bouma, Zeitschr. f. physiol. Chem., 32; Oerum, ibid., 45. 
 
 * Schmiedeberg, Arch. f. exp. Path. u. Pharm., 14; G. Hoppe-Seyler, Zeitschr. f. 
 physiol. Chem., 7 and 8; Porcher and Hervieux, Journ. de Physiol., 7. 
 
 5 Grober, Munch, med. Wochenschr., 1904; Wang, Salkowski's Festschrift, 1904; 
 Steensma, Mary's Jahresb., 40, 314. 
 
732 URINE. 
 
 C.CH 3 
 Skatoxyl-sulphuric Acid, C9H9NSO4, C 6 H 4 <^Sc.O.S02.0H, has not 
 
 NH 
 
 been positively prepared as a constituent of normal urine, but Otto 
 has once prepared its alkali salt from diabetic urine. Perhaps skatoxyl 
 occurs in normal urine as a conjugated glucuronate (Mayer and Neu- 
 berg 1 ), and it is believed that the urine contains a skatol-chromogen 
 from which red and reddish-violet coloring-matters are obtained by 
 decomposition with strong acids and an oxidizing agent. 
 
 Skatoxyl-sulphuric acid originates, if it exists in the urine, from 
 skatol, which is formed by putrefaction in the intestine, and which is 
 then conjugated with sulphuric acid after oxidation into skatoxyl. That 
 skatol introduced into the bodj- passes partly as an ethereal-sulphuric 
 acid into the urine has been shown by Brieger. Indol and skatol act 
 differently, at least in dogs, indol producing a considerable amount 
 of ethereal-sulphuric acid, while skatol gives only a small quantity (Mes- 
 ter 2 ). Reports on this subject are at variance. 
 
 The conditions for the formation of indol and skatol by the putrefaction of 
 proteins in the intestine are decidedly different, according to Herter, as skatol 
 is produced by other putrefaction bacteria than indol. For example, bacillus coli 
 communis produces indol, but only traces of skatol, while skatol is formed by 
 certain anaerobic putrefactive bacteria. An important intermediary step in the 
 formation of skatol is the indol acetic acid (skatol carboxylic acid, according to 
 Salkowski) and this can also pass into the urine and is the chromogen of the 
 urorosein, according to Herter. 3 
 
 The potassium salt of skatoxyl-sulphuric acid is crystalline; it dis- 
 solves in water, but with difficulty in alcohol. A watery solution becomes 
 deep violet with ferric chloride. The solution becomes red with con- 
 centrated hydrochloric acid with the separation of a red precipitate. 
 This precipitate (skatol red) is, after washing with water, insoluble in 
 ether but soluble in amyl alcohol. On distillation with zinc-dust the 
 red pigment gives a strong odor of skatol. 
 
 Urines containing skatoxyl are colored dark red to violet by Jaffe's 
 indican test even on the addition of hydrochloric acid alone; with nitric 
 acid they are colored cherry red, and red on warming with ferric chloride 
 and hydrochloric acid. A red coloration of the urine can also be brought 
 about by the appearance of indigo red (indirubin) and a confusion of 
 this pigment can also take place. Rosin 4 is of the opinion that no 
 
 l Otto, Pflliger'e Arch., 33; Mayer and Neuberg, Zeitschr. f. physiol. Chem., 29. 
 
 2 Brieger, Ber. d. deutsch. chem. Gesellsch., 12, and Zeitschr. f. physiol. Chem., 
 4, 414; Mester, ibid., 12. 
 
 3 Journ. of biol. Chem., 4. 
 
 * Rosin, Virchow's Arch., 123. 
 
INDOL ACETIC ACID. 733 
 
 skatol chromogen exists in human urine, and that the observations made 
 heretofore were due to a confusion of skatol red with indigo red or uroro- 
 sein. It cannot be disputed that derivatives of skatol sometimes occur in 
 human urine, and to prevent confusion with indigo red it must be borne 
 in mind that indigo red is soluble in chloroform as well as in ether, while 
 skatol red is insoluble in these solvents. On the contrary skatol red 
 is soluble in amyl alcohol, and this solution shows absorption bands close 
 to the line D between it and E, corresponding to X = 577-550 (Porcher 
 and Hervieux 1 ). 
 
 In regard to a confusion of skatol red for urorosein it must also be 
 remarked that urorosein may also be a skatol red. The chromogen of 
 urorosein, as Herter has shown in a case, is identical with indol acetic 
 acid, which passes into skatol on splitting off carbon dioxide. According 
 to Herter 2 urorosein is not identical with skatol red, although the 
 investigations of Staal, Grosser, Porcher and Hervieux 3 indicate 
 that they are identical, and the last two investigators consider them 
 identical, because they both have the same spectrum and the same 
 chemical behavior. 
 
 C.CH 2 COOH 
 
 Indol Acetic Acid (skatol-carboxylic acid), Ci H 9 NO 2 , C 6 H 4 \ /CH 
 
 NH 
 
 This acid, whose occurrence in the urine was first shown by Salkowski, is found 
 in the urine in special putrefactive processes in the intestine (Herter) and in 
 various diseases, especially in cachectic conditions. This is of course dependent 
 upon the fact whether indol acetic acid is the actual chromogen of urorosein, and 
 also whether the experience obtained as to the occurrence of urorosein can be 
 applied to the indol acetic acid. According to Wechselmann 4 it occurs (more 
 correctly as urorosein) as traces in normal urine, abundantly.in horse urine, and 
 in especially large quantities in cow urine. When introduced into the animal 
 body it appears unchanged in the urine. 
 
 This acid crystallizes in leaves which melt at 165°, and on strongly heating 
 it yields skatol with the splitting off of carbon dioxide. The solution, acidified 
 with hydrochloric acid, when treated with a little ferric chloride solution, becomes 
 cherry red on boiling. With some acid and a little nitrite as well as with hydro- 
 chloric acid and chloride of lime the solution becomes red, then cloudy, and a 
 red pigment precipitates. This pigment is soluble in amyl alcohol and gives the 
 above-mentioned absorption bands between D and E. This red pigment is 
 urorosein. 
 
 Urorosein is the name given by Nencki 5 to a red pigment which occurs in 
 the urine under the conditions mentioned under indol acetic acid. This pig- 
 
 1 Zeitschr. f. physiol. Chem., 45. 
 
 2 Journ. of biol. Chem., 4. 
 
 3 Staal, Zeitschr. f. physiol. Chem., 46; Grosser, ibid., 44; Porcher and Hervieux, 
 ibid., 45; Compt. Rend., 138, and Journ. ile Physiol., 7. 
 
 4 Salkowski, Zeitschr. f. physiol. Chem., 9; Wechselmann, cited in Bioch. Centralbl., 
 5, 784. 
 
 5 Nencki and Sieber, Journ. f. prakt. Chem., (N. F.), 26. 
 
734 URINE. 
 
 ment is not preformed in the urine, but is produced from its chromogen (indol 
 acetic acid) when the urine is treated with ivydrochloric acid alone. The urine 
 becomes red. Urorosein differs from indirubin essentially by the same properties 
 as skatol, with which, according to some, it is identical (see above). 
 
 Nephrorosein is a pigment described by V. Arnold » which is closely related to 
 urorosein and which, like this, is produced from a chromogen when the urine is 
 treated with nitric acid or with concentrated hydrochloric acid and a little sodium 
 nitrite solution. Xephrorosein is soluble in amyl alcohol and gives a spectrum 
 with a band between b and F, reaching from b to a little beyond the middle between 
 b and F. It is changed by the action of light and finally gives a band between 
 D and E, near E. The new pigment thus obtained is called ^-urorosein to dif- 
 ferentiate it from the ordinary urorosein, a-urorosein. The nephrorosein has not 
 been observed in normal urines but only in certain pathological cases. 
 
 The pigment obtained by de Jager by precipitating the urine with HC1 and 
 formol seems to be related to urorosein and nephrorosein. According to Ellinger 
 and Flamand "- urorosein belongs probably, like skatol-red, to the group of tri- 
 indyl methane pigments prepared by them from /3-indol aldehyde by boiling in 
 pcid solution. Probably the leucobase HCCCYHeN^, which gives the red pigment, 
 HO.C : (CsH 6 X) 3 is produced by condensation. 
 
 Aromatic Oxy acids. In the putrefaction of proteins in the intes- 
 tine, paraoxyphenyl-o.cetic acid and paraoxyphenyl-propionic acid are 
 formed from tyrosine as an intermediate step, and these in great part 
 pass unchanged into the urine. The quantity of these acids is usually 
 very small. They are increased under the same conditions as the phenols, 
 especially in acute phosphorus poisoning, in which the increase is con- 
 siderable. A small portion of these oxyacids is also combined with 
 sulphuric acid. 
 
 Besides these two oxyacids which regularly occur in human urine 
 we sometimes have other oxyacids in urines. To these belong homo- 
 gentisic acid in alcaptonuria, oxyhydroparacomnaric acid, found by Blender- 
 mann in the urine on feeding rabbits with tyrosine, gallic acid, which, 
 according to Baumann, 3 sometimes appears in horse's urine, and kynu- 
 renic acid (oxyquinolincarboxylic acid), which up to the present time 
 has been found only in dog's urine. Although all these acids do not 
 belong to the physiological constituents of the urine, still they will be 
 treated in connection with these. 
 
 Paraoxyphenylacetic Acid, CsHeOj, C 6 H 4 \ , and p-Oxyphenyl- 
 
 /OK 
 
 /OF 
 
 propionic Acid (Hvdroparacoumaric Acid), C9H10O3, CeH 4 \ , are 
 
 ^ \CH2.CH2COOH 
 crystalline and are both soluble in water and in ether. The one melts at 148° C. 
 ami the other al 125° C. Both give a beautiful red coloration on being warmed 
 with Mil. 1 gent. 
 
 1 Zeitschr. f. physiol. Chem., 61 and 71. 
 
 2 de Jager, Zeitschr. f. physiol. Chem., 61; Ellinger and Flamand, ibid., 62. 
 
 3 Blendennann, Zeitschr. f. physiol. Chem., 6, 267; Baumann, ibid., 6, 193. 
 
HOMOGENTISIC ACID. 735 
 
 To detect the presence of these oxyacids proceed in the following way (Bau- 
 mann): Warm the urine for a while on the water-bath with hydrochloric acid 
 in order to drive off the volatile phenols. After cooling shake three times with 
 ether, and then shake the ethereal extracts with dilute soda Bollltion, which dis- 
 solves the oxyacids, while the residue of the phenols which are soluble in ether 
 remains. The alkaline solution of the oxyacids is now faintly acidified with sul- 
 phuric acid, shaken again with ether, the ether removed and allowed to evaporate 
 the residue dissolved in a little water, and the solution tested with Millon's 
 reagent. The two oxyacids are best differentiated by their different melting- 
 points. The reader is referred to other works for the method of isolating and 
 separating these two oxyacids. 
 
 Homogentisic Acid (Dioxyphenylacetic Acid), CsHs'^ = 
 /OH(l) 
 CeH3^-OH(4) . This acid, which was discovered by Marshall 1 
 
 \CH 2 COOH(5) 
 and calle 1 by him glycosuric acid, was isolated in larger quantities by 
 Wolkow and Baumann in a case of alcaptonuria and carefully studied 
 by them. They called it homogentisic acid because it is a homologue 
 of gentisic acid, and they showed that the peculiar properties of so-called 
 alcaptonuric urine in this case were due to this acid. This acid has later 
 been found in many cases of alcaptonuria. Glycosuric rcid, isolated from 
 alcaptonuric urine by Geyger, 2 seems to be identical with homogentisic 
 acid. 
 
 The quantity of acid eliminated, which varies in most cases between 
 3 and 7 grams per twenty-four hours, and which is higher — 14-16 grams — 
 in exceptional cases, is increased by food rich in protein. On the inges- 
 tion of tyrosine by persons with alcaptonuria, Wolkow and Baumann 
 and Embden observed a greater quantity of homogentisic acid in the 
 urine and this has been substantiated by other observers. Since Lang- 
 stein and E. Meyer showed in a case of alcaptonuria that the quantity 
 of tyrosine in the protein, even when calculated to a maximum, was 
 not sufficient to account for the quantity of homogentisic acid, and that 
 therefore we must admit of another source (the phenylalanine) for the 
 alcapton, Falta and Langstein 3 have given a direct proof that homo- 
 gentisic acid can also be formed from phenylalanine. Abderhalden, 
 Bloch and Rona 4 have shown that in alcaptonurics the excretion of 
 homogentisic acid is increased by the introduction of tyrosine or phenyl- 
 
 1 The Medical News, Philadelphia, January 8, 1887. 
 
 2 Wolkow and Baumann, Zeitschr. f. physiol. Chem., 15; Geyger, cited from Emb- 
 den, 1. c, 18. The literature can be found in Fromherz, Ueber Alkaptonurie, Inaug.- 
 Dis. Freiburg, 1908. 
 
 3 Langstein and Meyer, Deutsch. Arch. f. klin. Med., 78; Falta and Langstein, 
 Zeitschr. f. physiol. Chem., 37; Falta, Der Eiweiss-Stoffwechsel bei der Alkaptonurie, 
 Habilitationsschrift, Naumburg, a. S., 1904. 
 
 4 Zeitschr. f. physiol. Chem., 52. 
 
736 URINE. 
 
 alanine in the form of polypeptides, from dipeptides as well as tripeptides. 
 The p-tyrosine and phenylalanine are quantitatively converted into homo- 
 gentisic acid, in alcaptonuria (Falta). The m- and o-tyrosine, on the 
 contrary, are not converted, according to Blum, 1 into homogentisic acid 
 in alcaptonurics, and the dibromtyrosine yields just as little homogentisic 
 acid as the bromine or iodine derivatives of protein bodies (Falta). 
 According to the investigations of Langstein and Meyer, and especially 
 of Falta, different proteins yield varying quantities of homogentisic 
 acid in alcaptonuria, and accordingly larger amounts in proportion as 
 the protein is rich in tyrosine and phenylalanine. 
 
 On this account the quotient H ( = homogentisic acid):N (nitrogen) 
 is variable on the introduction of different proteins. For example, with 
 casein H : N is on an average much higher than with white of egg. In 
 most of the cases of alcaptonuria examined the H : N was equal to 
 40-50: 100, and with the same alcaptonuric, when no essential change 
 in the diet occurs, the quotient is relatively constant. 
 
 Wolkow and Baumann explain the formation of homogentisic acid 
 from tyrosine by an abnormal fermentation in the upper parts of the 
 intestine, but this view has now been generally rejected. The observa- 
 tions of Aberhalden, Bloch and Rona 2 that glycyl-Z-tyrosine on 
 subcutaneous injection causes an increased formation of homogentisic 
 acid, disproves this theory, and indicates a formation of homogentisic 
 acid in the tissues. This acid is also burnt in the healthy organism if 
 not too large quantities of the acid are introduced at one time, and it is 
 the general view that alcaptonuria is an anomaly in the protein metabolism. 
 
 In order to understand this anomaly and the origin of the homogentisic 
 acid we must call attention to the fact that the investigations of O. 
 Neubauer and Falta, Langstein and others 3 show that only such 
 aromatic acids are converted, in the body, into homogentisic acid, which 
 have a three-membered side-chain which is substituted by NH 2 , OH 
 or in the a-position to the carboxyl group and not in the ^-position, 
 p-tyrosine, phenylalanine, phenyl-a-lactic acid and phenyl-pyroracemic 
 acid are such acids. It can be admitted with Falta that the phenyl- 
 alanine in the body by deamidation is converted into phenyl-a-lactic 
 acid, CeH5.CH2.CHOH.COOH, from which by taking up two hydroxyl 
 groups, dioxyphenyl-a-lactic acid (uroleucic acid), (OH)2CeH3.CH2. 
 CHOH.COOH, is formed, and then from this by oxidation dioxyphenyl- 
 acetic acid (homogentisic acid), (OH) 2 C 6 H3.CH 2 .COOH, is produced. 
 Tyrosine is also supposed to undergo an analogous transformation 
 
 x Arch. f. exp. Path. u. Pharm., 59. 
 2 Zeitschr. f. physiol. Chem., 52. 
 * Ibid., 42; Frornherz, 1. c. 
 
HOMOGENTISIC ACID. 737 
 
 whereby a removal of the OH group in the para position must be 
 admitted. 
 
 According to Neubauer, 1 on the contrary, the tyrosine, as well as 
 the other amino-acids, is first transformed into the corresponding keto- 
 acid, p-oxyphenyl pyroracemic acid, OH.CeH4.CH2.CO.COOH, which 
 is then oxidized into the corresponding chinol and transformed into 
 hydroquinone pyroracemic acid, (OHjoCeHs.Cr^.CO.COOH. The homo- 
 gentisic acid is derived from this latter by the splitting off of carbon 
 dioxide by oxidative means. Phenylalanine is either changed into 
 phenyl pyroracemic acid or into p-oxyphenyl pyroracemic acid with 
 tyrosine as intermediary body and then changed as above stated. 
 
 According to the accepted hypothesis the demolition of tyrosine 
 and phenylalanine takes place into homogentisic acid, and the anomaly 
 in the metabolism of alcaptonurics consists in that in these the demoli- 
 tion stops at this point and that the ability to rupture the benzene ring 
 is absent, in the organism, in alcaptonuria. 
 
 ,The difficulties in accepting the assumption of a transformation of tyrosine 
 into homogentisic acid due to the different positions of the hydroxy! groups in 
 
 the side chain of the two bodies, as shown by the formulae HO<f yOH (homo- 
 
 CH-COOH 
 OH 
 
 gentisic acid) and ( y (tyrosine) do not exist now, since we have 
 
 CH,CHNH,COOH 
 
 learnt of other analogous processes. For exa mple, the oxidation, by Kumagai and 
 
 "Wolffensteix, 2 of paracresol H 3 C\ /OH with potassium persulphate in 
 
 OH 
 acid solution. In this manner the expected 3.4 dioxytoluene H 3 C< 
 
 was not obtained, but instead homohydroquinone HO<f /OH, and hence a 
 
 CH, 
 
 transference of the alkyl group must have occurred. 
 
 Abderhalden 3 has also shown in healthy human beings that tyrosine 
 may cause an elimination of homogentisic acid, as he positively detected 
 a small quantity of homogentisic acid in the urine of a man who had 
 taken 50 grams /-tyrosine per os (of which 44 grams were absorbed). 
 In the urine of another man he could not detect either homogentisic 
 
 1 Cited from Centralbl. f. Physiol., 23, 76. 
 2 Ber. (1. d. Chem. Gesellsch., 41. 
 ' Zeitschr. f. physiol. Chem., 77. 
 
738 URINE. 
 
 acid or any other intermediary product of the cleavage of tyrosine in 
 the urine after taking 150 grams /-tyrosine (of which 141 grams were 
 absorbed) . 
 
 Dakin l has recently opposed the above-mentioned view that in the 
 cleavage of tyrosine and phenylalanine, homogentisic acid is always 
 produced, and that the condition of alcaptonuria consists in an inability 
 of the body to burn this intermediary product of metabolism. He has 
 found that tyrosin-methyl ether, which cannot form any quinone-like 
 intermediary product, can in cats be just as completely burnt as tyrosine, 
 and the same is true for p-methylphenylalanine and for p-methoxy- 
 phenylalanine, which cannot form any quinone derivatives. Still 
 these substances can be completely burnt by alcaptonurics and accord- 
 ing to Dakin the body in alcaptonuria has still the ability to completely 
 burn the aromatic nucleus of tyrosine and phenylalanine when the 
 transformation into homogentisic acid is prevented by a proper sub- 
 stitution in the para-groups. Dakin therefore considers alcaptonuria 
 as a condition in which partly the formation of an abnormal metabolic 
 product — the homogentisic acid— takes place and where partly the ability 
 of the body to burn this product is diminished. 
 
 Garrod, 2 who has observed several cases of alcaptonuria, has also 
 tabulated a large number of cases of alcaptonuria which he finds in the 
 literature, and he shows that the anomaly of the protein metabolism 
 occurs oftener in males than in females, and also that blood relationship 
 of the parents (first cousins) predisposes to alcaptonuria. 
 
 On fusing homogentisic acid with alkali it yields gentisic acid (hydro- 
 quinone-carboxylic acid) and hydroquinone. When introduced into the 
 intestine of the dog a part is converted into toluhydroquinone, which 
 is eliminated in the form of an ethereal sulphuric acid. Homogentisic 
 acid has also been prepared synthetically by Baumann and Frankel, 
 starting with gentisic aldehyde, and by Neubauer and Flatow 3 from 
 o-oxyphenylglyoxylic acid with hydroquinone glyoxylic acid and hydro- 
 quinone glycollic acid as intermediary bodies. 
 
 Homogentisic acid crystallizes with 1 mol. of water in large, trans- 
 parent prismatic crystals, which become non-transparent at the tem- 
 perature of the room with the loss of water of crystallization. They 
 melt at 146.5-147° C, and are soluble in water, alcohol, and ether, but 
 nearly insoluble in chloroform and benzene. Homogentisic acid is 
 
 1 Journ. of Biol. Ohem., 8 and 9, with Wakeman, ibid., 9. 
 
 2 Med. chirurti. Transact., 1899 (where all cases up to that time are tabulated); 
 also The Lancet, 1901 and 1902; Garrod and Hele, Journ. of Physiol., 33. 
 
 3 Baumann and Friinkel, Zeitsohr. f. physiol. Chem., 20; Neubauer and Flatow., 
 ibid., 52. 
 
BOMOGENTISIC ACID. 739 
 
 optically inactive and non-fermentable. I^ts watery solution has the 
 properties of so-called alcaptonuric urine. It becomes greenish brown 
 
 from the surface downward on the addition of very little caustic soda 
 or ammonia with access of oxygen, and on shaking it quickly becomes 
 dark la-own or black. 
 
 If alcaptonuric mine or a homogentisic acid solution is treated with 10-40 
 per cent ordinary ammonia, a beautiful, intensive reddish-violet coloration is 
 produced on access of air according to C. Moknkk, 1 and two beautiful pigments, 
 a- and p-alcaptochrome, are formed. The first, o-alcaptochrome, is crystalline and 
 has a beautiful violel color In alkaline solution and is without fluorescence. The 
 (3-alcaptochrome is not crystalline and its alkaline solution has a more reddish 
 color with strong fluorescence in the yellowish-red. 
 
 Homogentisic acid reduces alkaline copper solutions with even slight 
 heat, and ammoniacal silver solutions immediately in the cold. It 
 does not reduce alkaline bismuth solutions. It gives a lemon-colored 
 precipitate with Millon's reagent, which becomes light brick-red on 
 warming. Ferric chloride gives to the solution a blue color which soon 
 disappears. On boiling with concentrated ferric-chloride solution an 
 odor of quinone develops. With benzoyl chloride and caustic soda in 
 the presence of ammonia we obtain the amide of dibenzoylhomogentisic 
 acid, which melts at 204° C, and which can be used in the isolation of 
 the acid from the urine, and also for its detection (Orton and Garrod). 
 Among the salts of this acid must be mentioned the lead salt containing 
 water of crystallization and 34.79 per cent Pb. This salt melts at 
 214-215° C. 
 
 In order to prepare the acid, heat the urine to boiling, add 5 grams 
 of lead acetate for every 100 cc, filter as soon as the lead acetate has 
 dissolved, and allow the nitrate to stand in a cool place for twenty-four 
 hours until it crystallizes (Garrod). The dried, powdered lead salt 
 is suspended in ether and decomposed by H2S. After the spontaneous 
 evaporation of the ether the acid is obtained in almost colorless crystals 
 (Orton and Garrod 2 ). 
 
 In regard to the quantitative estimation we proceed according to the sug- 
 gestion of Baumann by titrating the acid with a N/10 silver solution. For details 
 of this method the reader is referred to the works of Baumann, C. Th. Morner, 
 Mittelbach, Garrod and Hurtley. Deniges 3 has suggested another method. 
 
 Uroleucic acid, C 9 Hi O 6 , is, according to Htppert, probablv a dioxvphenvl- 
 lactic acid, C 6 H :i (OH) 2 .CH,.CH(OH).C()OH. This acid was first prepared by 
 Kirk from the urine of children with alcaptonuria, which also contained homo- 
 gentisic acid. Langstein and Meyer 4 have also found a small amount of this 
 acid in a case of alcaptonuria studied by them. It melts at 130-133° C. Other- 
 
 1 Zeitschr. f. physiol. Chem., 69. 
 
 2 Orton and Garrod, Journ. of Physiol., 27; Garrod, ihid., 23. 
 
 3 Mittelbach, Deutsch. Arch. f. klin. Med., 71 (which contains the work of Baumann 
 and Morner); Garrod and Hurtley, Journ. of Physiol., 33; Deniges, Chem. Centralbl., 
 1897, 1, 338. 
 
 4 Huppert, Zeitschr. f. physiol. Chem., 23; Kirk, Brit. Med. Journ., 1886 and 1888; 
 Langstein and Meyer, 1. c. 
 
740 URINE. 
 
 wise, in regard to its behavior with alkalies, with access of air, and also with 
 alkaline copper solutions and ammoniacal silver solutions, and also Millon's 
 reagent, it is similar to homogentisic acid. 
 
 Neubaueb and Flatow, who have prepared dioxyphenyl-a-lactic acid syn- 
 thetically, find that this acid has entirely different properties from the so-called 
 uroleucic acid. Garrod and Hurtley ' have also shown that an impure homo- 
 gentisic acid with a low melting-point is easily obtained, and they suggest the 
 possibility that the earlier reports in regard to uroleucic acid are due to an error. 
 
 CH COH 
 
 HC C C.COOH 
 Kynurenicacid(y-ox}'-/3-quinohncarboxvhcacid),CioH 7 N03, 
 
 HC C CH 
 
 CH N 
 
 has only been found thus far in dog's urine, but not always; its quantity is increased 
 by meat feeding. It does not occur in the urine of cats. Ellinger 2 has been 
 able to show positively that tryptophane is the mother-substance of this acid. 
 By the introduction of tryptophane in the organism he has shown the formation 
 of a kynurenic acid not only in dogs but also in rabbits. 
 
 The acid is crystalline, does not dissolve in cold water, rather well in hot alcohol, 
 and yields a barium salt which crystallizes in triangular, colorless plates. On 
 heating it melts and decomposes into C0 2 and kynurin. On evaporation to dry- 
 ness on the water-bath with hydrochloric acid and potassium chlorate a reddish 
 residue is obtained which on adding ammonia becomes first brownish green and 
 then emerald green (Jaffe's reaction 3 ). 
 
 Urinary Pigments and Chromogens. The yellow color of normal 
 urine depends perhaps upon several pigments, but in greatest part upon 
 urochrome. Besides this the urine seems to contain a very small quantity 
 of hoematoporphyrin as a regular constituent. Uroerythrin is also of 
 frequent occurrence in normal urine, but not always. Finally, the 
 excreted urine when exposed to the action of light regularly contains a 
 yellow pigment, urobilin, which is derived from a chromogen, urobilinogen, 
 by the action of light (Saillet) and air (Jaffe, Disque 4 ) and others. 
 
 Besides this chromogen, urine contains various other bodies from which color- 
 ing matters may be produced by the action of chemical agents. Humin sub- 
 stances (perhaps in part from the carbohydrates of the urine) may be formed 
 by the action of acids (v. Udranszky) without regard to the fact that such sub- 
 stances may sometimes originate from the reagents used, as from impure amyl 
 
 1 Journ. of Physiol., 30. 
 
 *Ellinger, Ber. d. d. chem. Gesellsch., 37, 1804, and Zeitschr. f. physiol. Chem., 
 43. The older literature on kynurenic acid may be found in Josephsohn, Beitrage zur 
 Kenntnis der Kynurensaure ausscheidung beim Hunde, Inaug. -Dissert., Konigsberg, 
 1898. 
 
 ' Zeitschr. f. physiol. Chem., 7. In regard to kynurenic acid, see also Huppert- 
 Neubauer, 10. Aufl., and Mendel and Jackson, Amer. Journ. of Physiol., 2; Mendel 
 and Schneider, ibid., 5; Camps, Zeitschr. f. physiol. Chem., 33. 
 
 4 Jaffp, Centralbl. f. d. med. Wissensch., 1896 and 1869, and Virchow's Arch., 47; 
 Disque, Zeitschr. f. physiol. Chem., 2; Saillet, Revue de medecine, 17, 1897. 
 
DROCHROME. 741 
 
 alcohol (v. Udraxszky ')• To these humin bodies developed by the action of 
 acid in norma] urine when exposed bo tin- air must be added the urophain of Heller, 
 the various uromelanins and other bodies described by different investigators 
 (Pl6bz, ThUDICHUM, BchunCK, DombbOWBKI *). Indigo blue (uroglaucin of 
 Heller, vrocyanin, cyanurin, and other coloring matters of earlier investigators 3 ) 
 is split off from the indoxyl-sulphuric acid or indoxyl-glucuronic acid. Red 
 coloring matter may be formed from the conjugated indoxyl and skatoxyl acids, 
 and urohodin (Heller), urorutrin (Plosz), urohcemabin (Harley), and perhaps 
 also uroroscin (Nencki and Sieber 4 ) probably have such an origin. 
 
 We cannot discuss more in detail the different coloring matters obtained 
 as decomposition products from normal urine. Haematoporphyrin has 
 already been referred to in a previous chapter (V) and will best be 
 described in connection with the pathological pigments. It only remains 
 to describe urochrome, urobilin, and uroerythrin. 
 
 Urochrome is the name given by Garrod to the yellow pigment of 
 the urine. Thudichum 5 had previously given the same name to a less 
 pure pigment isolated by himself. The accounts as to the composi- 
 tion and properties of urochrome differ so considerably that it is ques- 
 tionable whether anybody has ever had this pigment in a pure form. 
 Urochrome is free from iron, but contains nitrogen. Dombrowski 
 found 11.15 per cent nitrogen, Hohlweg found 9.89 per cent nitrogen, 
 and Klemperer found only 4.2 per cent nitrogen. According to 
 Dombrowski urochrome contains about 5 per cent sulphur, while 
 other investigators like Hohlweg, Salomonsen, and Mancini found 
 that it was free from' sulphur. 6 According to Garrod it stands in 
 close relation to urobilin and is transformed into urobilin by the action 
 of " active " acetaldehyde, while Riva 7 claims to have obtained a body 
 similar to urochrome by the oxidation of urobilin by permanganate. 
 This relation of the two pigments is denied by Dombrowski. On the 
 contrary it is the unanimous opinion that urochrome under certain con- 
 ditions may yield the pyrrol reaction. Certain investigators such as 
 Bondzynski and Dombrowski consider urochrome as a member of the 
 oxyproteic acid group (see further on), a view which does not seem to 
 
 1 v. Udranszky, Zeitschr. f. physiol. Chem., 11, 12, and 13. 
 
 * P16sz, Zeitschr. f. physiol. Chem., 8; Thudichum, Brit. Med. Journ., 201, and 
 Journ. f. prakt. Chem., 104; Schunck, cited from Huppert-Neubauer, 10. Aufl., 509; 
 
 Dombrowski, Zeitschr. f. physiol. Chem., 62. 
 s See Huppert-Neubauer, 161. 
 
 * In regard to this and other red pigments, see Huppert-Neubauer, 593 and 597; 
 Nencki and Sieber, Journ. f. prakt. Chem. (2). 26. 
 
 6 Garrod, Proc. Roy. Soc, 55; Thudichum, 1 c. 
 
 •Dombrowski, Zeitschr. f. physiol. Chem., 54 and 62; Hohlweg, Bioch. Zeitschr., 
 13; Salomonsen. ibid., 13; Mancini, ibid., 13; Klemperer, Berl. klin. Wochenschr., 
 40. 
 
 T Garrod, Journ. of Physiol., 21 and 29; Riva, cited from Huppert-Neubauer, 524. 
 
742 URINE. 
 
 have sufficient basis and in fact is denied by others such as Weisz. 1 The 
 above disputed statements as to the presence or absence of sulphur in 
 urochrome as well as the nitrogen content of urochrome make it very- 
 probable that the preparation of pure urochrome has not thus far been 
 accomplished. 
 
 Urochrome, as obtained thus far, is amorphous, brown, readily solu- 
 ble in water and ordinary alcohol, but less soluble in absolute alcohol. 
 It dissolves but slightly in acetic ether, amyl alcohol, and acetone, while 
 it is insoluble in ether, chloroform, and benzene. Urochrome is pre- 
 cipitated by copper acetate, lead acetate, silver nitrate, mercuric acetate, 
 phosphotungstic and phosphomolybdic acids. On saturating the urine 
 with ammonium sulphate a great part of the urochrome remains in solu- 
 tion. It does not show any absorption-bands and does not fluoresce 
 after the addition of ammonia and zinc chloride. Urochrome is very 
 readily decomposed, by the action of acids, with the formation of brown 
 substances. 
 
 Urochrome can be prepared according to a rather complicated method which 
 is based upon the fact that the substance remains in great part in solution on 
 saturating the urine with ammonium sulphate. If the proper quantity of alcohol 
 is added to the filtrate, a clear, yellow alcoholic layer forms on the salt solution, 
 which contains the urochrome and which can be used for the further preparation 
 of the latter (Garrod, 0. Bocchi 2 ). Klemperer, on the contrary, removes the 
 pigment from the urine by means of animal charcoal, washes it with water to 
 remove the indican and other bodies, and then extracts with alcohol and uses 
 this alcoholic extract for the further purification according to Garrod. Hohlweg, 
 Salomosen and Mancini also remove the pigment from the urine, which has 
 previously been precipitated by calcium or barium salts, by means of animal 
 charcoal. Dombrowski uses an entirely different method which is based upon 
 the precipitation of the urochrome by copper acetate. In regard to the details 
 of these different methods we refer to the original works. 
 
 Dombrowski, Browinski and Dombrowski 3 have worked out a 
 quantitative method for estimating urochrome, but its value is dependent 
 upon a further investigation as to the purity and composition of the 
 urochrome obtained by them. On this account the results found by these 
 investigators will not be given. The urochrome can be quantitatively 
 estimated, according to Klemperer, by a colorimetric method, using 
 a solution of true yellow G. If 0.1 gram of this dye is dissolved in 1 liter 
 of water and 5 cc. of this solution diluted to 50 cc. with water, then 
 this solution has the same color and shade as a 0.1 per cent urochrome 
 solution. The urine must be diluted with water until it has the same 
 depth of color. The comparison is performed in vessels with parallel 
 walls. The value of this method cannot be judged at the present time. 
 
 1 Dombrowski, 1. c; Bondzynski, Chem. Centralbl., 1910, Bd. II; Weisz, Bioch. 
 Zeitschr., 30. 
 
 2 Garrod, 1. c; Bocchi, Hofmeister's Beitrage, 11. 
 
 ' Dombrowski, Zeitschr. f. physiol. Chem., 54, with Browinski, Bull. Acad, d.. 
 d. scien. Cracovie, 1908; Klemperer, 1. c. 
 
UROBILIN. UROBILINOIDS. 743 
 
 Urobilin is the pigment first isolated from the urine by Jaff^:, 1 and 
 which is characterized by its strong fluorescence and by its absorption- 
 spectrum. Various investigators have prepared, from the urine, by dif- 
 ferent methods, pigments which differed slightly from each other but 
 behaved essentially like Jaffa's urobilin. Thus different urobilins have 
 been suggested, such as normal, febrile, physiological, and pathological 
 urobilins. 2 The possibility of the occurrence of different urobilins in 
 the urine cannot be denied; but as urobilin is a readily changeable body 
 and difficult to purify from other urinary pigments, the question as to the 
 occurrence of different urobilins must still be considered open. 
 
 In the perfectly fresh urine of healthy human beings no urobilin 
 occurs, as first suggested by Saillet, 3 but only the chromogen, urobilino- 
 gen, from which the urobilin is readily formed by the action of light 
 or by weak oxidizing agents. Pathological urines contain on the contrary 
 preformed urobilin. 
 
 Urobilinoids, i.e., bodies which are similar to urobilin in that they fluoresce 
 and show the same absorption spectrum have been prepared from bile-pig- 
 ments (by Maly and Stokvis) and from hsematin or harnatoporphyrin (by 
 Hoppe-Seyler, Le Nobel, Nencki and Sieber, MacMunn 4 ) by reduction 
 as well as by oxidation. According to H. Fischer and Meyer-Betz 5 also non- 
 stable pyrrols, which contain a non-substituted hydrogen atom in a ring carbon 
 atom, pass readily in the animal body into substances which give the character- 
 istic urobilin reactions. These reactions are also given by bodies of different 
 constitution, but which probably contain the same ehromophore groups, and it is 
 these conditions which cause the above-mentioned uncertainty as to the occur- 
 rence of different urobilins. 
 
 That urobilin is identical with the hydrobilirubin of Maly (see page 
 428) has been considered for a long time. In opposition to this view 
 we find that both bodies, not to mention other small differences, have an 
 essentially different composition. While the hydrobilirubin contains 
 9.22 per cent nitrogen, according to Maly, the urobilin contains only 
 4.09 per cent nitrogen, according to Hopkins and Garrod, and 5.93 per 
 cent nitrogen, according to Fromholdt. In the urobilin of the feces, 
 stercobilin, which is identical with urobilin, Hopkins and Garrod found 
 
 1 Centralbl. f. d. med. \\ 'issensch., 1868 and 1869, and Virchow's Arch., 47. 
 
 2 See MacMunn, Proc. Roy. Soc, 31 and 35; Ber. d. deutsch. chem. Gesellsch., 14, 
 and'journ. of Physiol., 6 and 10; Bogomoloff, Maly's Jahresber., 22; Eichholz, Journ. 
 of Physiol., 14; Ad. Jolles, Pfluger's Arch., 61. 
 
 3 Revue de medecine, 1 7, 1897. 
 
 4 Maly, Ann. d. Chem. u. Pharm., 163; Disque, Zeitschr. f. physiol. Chem., 2; 
 Stokvis, Centralbl. f. d. med. Wissensch., 1873, 211 and 449; Hoppe-Seyler, Ber. d. 
 deutsch. chem. Gesellsch., 7; Le Nobel, Pfluger's Arch., 40; Nencki and Sieber, 
 Monatshefte f. Chem., 9, and Arch. f. exp. Path. u. Pharm., 24; MacMunn, Proc. Roy. 
 Soc, 31. 
 
 6 H. Fischer and Meyer-Betz, Zeitschr. f. physiol. Chem., 75. 
 
744 URINE. 
 
 4 per cent nitrogen. Still H. Fischer 1 has now found that the stercobilin 
 has a lower nitrogen content because of a contamination with cholesterin 
 or bile acids, and it is possible that also the low nitrogen content of the 
 urine-urobilin may be caused by contamination with non-nitrogenous 
 substances. 
 
 These possibilities have, it is sure, have not been tested; but the 
 unequal nitrogen content of the two pigments does not positively exclude 
 the identity of urobilin and hydrobilirubin. Fischer and Myer-Betz 
 have in fact shown that the hemibilirubin, which forms about one-half 
 of the mixture called hydrobilirubin (see page 429) is identical with the 
 urobilinogen from human urine. 
 
 The possibility of the formation of urobilinogen and of urobilin from 
 bile pigments is assured and many physiological as well as clinical observa- 
 tions 2 support the view that this transformation of the bile pigments 
 occurs by means of putrefactive processes in the intestine. Of these 
 observations we must mention the regular appearance in the intestinal 
 tract of stercobilin, undoubtedly derived from the bile-pigmets; the 
 absence of urobilin in the urine of new-born infants, as well as on the com- 
 plete exclusion of bile from the intestine, and also the increased elimina- 
 tion of urobilin with strong intestinal putrefaction. On the other hand 
 there are investigators who, basing their opinion on clinical observations, 
 deny the enterogenous origin of urobilin and claim that the urobilin is 
 derived from a transformation of the bilirubin elsewhere than in the 
 intestine, by an oxidation of the bile-pigment or by a transformation of 
 the blood-pigments. 3 
 
 Urobilin or urobilinogen dees not occur in the urine of all animals, 
 and according to Fromholdt it is absent in the urine of rabbits. The 
 correctness of this statement is denied by Gautier and Rosso. In nor- 
 mal human blood they seem to be absent, while according to Biffi 4 
 urobilin and urobilinogen occur sometimes in disease and in cadaver blood. 
 
 1 Hopkins and Garrod, Journ. of Physiol., 22; Fromholdt, Zeitschr. f. exp. Path., 
 u. Ther., 7;*H. Fischer, Zeitschr. f. physiol. Chem., 73. 
 
 2 See Fr. Muller, Schles. Gesellsch. f. vaterl. Kultur, 1892; D. Gerhardt, " Ueber 
 Hydrobilirubin und seine Bezieh. zum Ikterus " (Inaug.-Diss., Berlin, 1889); Beck, 
 Wien. klin. Wochenschr., 1895; Harley, Brit. Med. Journ., 1896; Fischler, Zeitschr. 
 f. physiol. Chem., 48. 
 
 3 In regard to the various theories as to the formation of -urobilin, see Harley, 
 Brit. Med. Journ., 1896; A. Katz, Wien. med. Wochenschr., 1891, Nos. 28-32; Grimm, 
 Virchow's Arch., 132; Zoja, Conferenze cliniche italiane, Ser. la, 1; Hildebrandt, 
 Zeitschr. f. klin. Med., 59; Biffi, Boll. d. scienc. med. di Bologna (8), anno 78, 7; Troisier, 
 Compt. rend. soc. biol., 66 and Tsuschija, Zeitschr. f. exp. Path. u. Ther., 7; Fromholdt 
 and Nersessoff, ibid., 11. 
 
 4 Fromholdt, Zeitschr. f. physiol. Chem., 53; CI. Gautier and Russo, Compt. rend, 
 soc. biol., 64; Biffi, Folia hsematol., 4, and 1. c. Boll., 78. 
 
UROBILIN. 745 
 
 Tne quantity of urobilin in the urine under physiological conditions 
 varies widely. Saillet found 30-130 milligrams and G. Hoppe-Seyler 
 80- 140 milligrams in one day's urine. 
 
 There are numerous observations on the elimination of urobilin or 
 urobilinogen in disease, especially by Jaffe, Disque, Gerhardt, G. 
 Hoppe-Seyler, 1 and others. The quantity is increased in hemorrhage 
 and in disease where the blood-corpuscles are destroyed, as is the case 
 after the action of certain blood-poisons, such as antifebrin and anti- 
 pyrine. It is also increased in fevers, cardiac diseases, lead colic, atrophic 
 cirrhosis of the liver, and is especially abundant in so-called urobilin 
 icterus. 
 
 The properties of urobilin may vary, depending upon the method 
 of preparation and the character of the urine used; therefore only the 
 most important properties will be given. Urobilin is amorphous, brown, 
 reddish brown, red, or reddish yellow, depending upon the method 
 of preparation. It dissolves readily in alcohol, amyl alcohol, and 
 chloroform, but less readily in ether or acetic ether. It is less soluble 
 in water, but the solubility is augmented by the presence of neutral 
 salts. It may be completely precipitated from the urine by saturating 
 with ammonium sulphate, especially after the addition of sulphuric acid 
 (Mehu 2 ). It is soluble in alkalies, and is precipitated from the alkaline 
 solution by the addition of acid. It is partly dissolved by chloroform 
 from an acid (watery-alcoholic) solution; alkali solutions remove the 
 urobilin from the chloroform. The neutral or faintly alkaline solutions 
 are precipitated by certain metallic salts (zinc and lead), but not by others, 
 such as mercuric sulphate. Urobilin is precipitated from the urine by 
 phosphotungstic acid. It does not give Gmelin's test for bile-pigments. 
 It gives, on the contrary, a reaction which may be mistaken for the biuret 
 test, by the action of copper sulphate and alkali. 3 
 
 Neutral alcoholic urobilin solutions are, in strong concentration, 
 brownish yellow, in great dilution yellow or rose-colored. They have a 
 strong green fluorescence. The acid alcoholic solutions are brown, 
 reddish yellow, or rose-red, according to concentration. They are not 
 fluorescent, but show a faint absorption-band, X, between b and F, which 
 borders on F. The absorption maximum lies according to Lewin and 
 Stenger 4 at 7 = 494-497. The alkaline solutions are brownish yellow, 
 
 1 In regard to the literature on this subject we refer the reader to D. Gerhardt, 
 "Ueber Hydrobilirubin und seine Beziehungen zum Ikterus " (Berlin, 1889), and 
 also G. Hoppe-Seyler, Virchow's Arch., 124. 
 
 2 Journ. de Pharm. et Chim., 1878, cited from Maly's Jahresber., 8. 
 
 3 See Salkowski, Berlin, klin. Wochenschr., 1897, and Stokvis, Zeitschr. f. Biologie, 
 34. 
 
 4 Pfluger's Arch., 144. 
 
746 URINE. 
 
 yellow, or (the ammoniacal) yellowish green, according to concentration. 
 They show a dark band 7, which is moved somewhat toward the red 
 end of the spectrum and lies between E and F. The absorption max- 
 imum lies at X = 506-510. If some zinc-chloride solution is added to 
 an ammoniacal solution of the pigment it becomes red and shows a 
 beautiful green fluorescence and gives the same absorption bands. If 
 a sufficiently concentrated solution of urobilin alkali is carefully acidified 
 with sulphuric acid it becomes cloudy and shows a second band exactly 
 at E, and connected with 7 by a shadow (Garrod and Hopkins, Saillet x ). 
 Urobilinogen is colorless or only faintly colored, but is very quickly 
 changed in the air and by the actionof light and is transformed into urobilin. 
 The urobilinogen, which is identical with hemibilirubin, can be obtained 
 as colorless prisms by solution in hot acetic-ether and treating this with 
 ligroin, and evaporating. Urobilinogen is soluble in ether, acetic ether, 
 amyl alcohol and in chloroform, and can in part be removed from the 
 urine after adding sodium bicarbonate and shaking with chloroform 
 (Fischer and Meyer-Betz). It can also be obtained directly from the 
 urine or from the acidified urine by shaking with chloroform or ether, 
 although it is less pure. In a chloroform solution of urobilin and urobilino- 
 gen, according to Grimbert 2 , only urobilin and not urobilinogen is 
 taken up by a sodium diphosphate solution, which is not colored red by 
 phenolphthalein. Like urobilin, it is precipitated from the urine on 
 saturating with ammonium sulphate. When free from urobilin it does 
 not give auy absorption bands and no fluorescence with ammonia and 
 zinc salt. For the detection and identification of urobilinogen we make 
 use of Ehrlich's reagent (p-dimethylamino-benzaldehyde). This 
 reagent consists of dissolving 2 grams p-dimethylaminobenzaldehyde 
 in 50 cc. concentrated, fuming hydrochloric acid and diluting to 100 
 cc. with water. To 10 cc. of the urine add 1 cc. of the reagent and 
 thoroughly shake. According to the amount of urobilinogen, the solution 
 becomes pink colored or intensely red, and in the spectrum we find a 
 band between D and E. The red color can be taken up by amyl alcohol. 
 Urobilin does not give this reaction, which is common to certain haematin 
 and pyrrol derivatives. 
 
 The preparation of urobilin from the urine can be done according to the 
 original method of Jaffe, or according to the method suggested by Mehu, 
 which has been modified somewhat by Garrod and Hopkins 3 (precipita- 
 tion with ammonium sulphate) or according to Charnas* suggestion. 
 
 According to Charnas 4 the preparation is best from urobilinogen, 
 
 1 Garrod and Hopkins, Journ. of Physiol., 20; Saillet, 1. c. 
 
 I -'her and Meyer-Betz, 1. c; Grimbert, Compt. rend. soc. biol., 70. 
 » Jaffe\ 1. c; Mehu, 1. c; Garrod and Hopkins, Journ. of Physiol., 20. 
 * Charnas, Bioch. Zeitschr., 20. 
 
DETECTION AND ESTIMATION OF UROBILIN. 747 
 
 and if the urine contains urobilin, it is first allowed to undergo alkaline 
 fermentation, when the urobilin is converted into urobilinogen. The 
 urine is acidified with tartaric acid and extracted with ether. The 
 foreign pigments are precipitated from the ethereal solution by petroleum 
 ether; the ether solution is washed with water, evaporated, and the residue 
 allowed to stand with water for several hours at 38° C, when the urobilino- 
 gen is transformed into urobilin. The urobilin can now be precipitated 
 with ammonium sulphate, and the dried precipitate extracted with absolute 
 alcohol. This urobilin has about three times as much extinction ability 
 as Maly's urobilin (hydrobilirubin). Other methods of preparation 
 have been suggested. 
 
 The urobilinogen is prepared by shaking the urine, directly after 
 adding sodium bicarbonate, with chloroform (Fischer and Meyer- 
 Betz). In regard to details we must refer to the original publication. 
 
 The detection of urobilin can sometimes be done directly on the urine. 
 Otherwise the urine is shaken with ether, amyl alcohol or chloroform and 
 these solutions tested. According to Schlesinger l the urine can also be 
 precipitated by an equal volume of a saturated solution of zinc acetate in 
 alcohol and the filtrate directly tested for the fluorescence and absorption. 
 Grimbert 2 has suggested a method for the separate testing for urobilin and 
 urobilinogen by using the chloroform, after shaking the urine therewith. 
 For the detection of urobilin we always make use of the color of the acid 
 or alkaline solutions, the absorption spectrum and the beautiful fluorescence 
 of the ammoniacal solution containing zinc chloride. For the detection 
 of urobilinogen we make use of Erhlich's reagent, and the property of 
 the colorless solution of being changed into urobilin in the air and light. 
 
 In the quantitative estimation of urobilin we proceed as follows, 
 according to G. Hoppe-Seyler: 3 100 cc. of the urine are acidified with 
 sulphuric acid and saturated with ammonium sulphate. The precipitate 
 is collected on a filter after some time, washed with a saturated solu- 
 tion of ammonium sulphate, and repeatedly extracted with equal parts 
 of alcohol and chloroform after pressing. The filtered solution is treated 
 with water in a separatory funnel until the chloroform separates well 
 and becomes clear. The chloroform solution is evaporated on the water- 
 bath in a weighed beaker, the residue dried at 100° C., and then extracted 
 with ether. The ethereal extract is filtered, the residue on the filter dis- 
 solved in alcohol, and transferred to the beaker and evaporated, then 
 dried and weighed. According to this method G. Hoppe-Seyler found 
 0.08-0.14 gram of urobilin in one day's urine of a healthy person, or an 
 average of 0.123 gram. 
 
 The urobilin can also be determined according to the method sug- 
 gested by Charnas for its preparation and urobilin can also be deter- 
 mined spectroscopically by the method suggested by Saillet. 4 Further 
 details will be found in the original publications and in larger handbooks. 
 
 The quantitative estimation of urobilinogen can be accomplished 
 spectroscopically by means of Ehrmch's reagent, as suggested by Charnas. 
 
 1 Deutsch. rued. Wochenschr., 1903. 
 
 * Compt. rend. soc. biol., 70. 
 
 1 Virchow's Arch., 124. 
 
 4 Charnas, 1. c; Saillet, 1. c; see also Tsuschija, Zeitschr. f. exp. Path. u. Ther., 7. 
 
74.8 URINE. 
 
 Uroerythrin is the pigment which often gives the beautiful red color to 
 the urinary sediments (sedi?nentum lateritium) . It also frequently occurs 
 although only in very small quantities, dissolved in normal urines. The 
 quantity is increased after great muscular activity, after profuse perspira- 
 tion, immoderate eating, or partaking of alcoholic drinks, as well as after 
 digestive disturbances, fevers, circulatory disturbances of the liver, and 
 in many other pathological conditions. 
 
 Uroerythrin, which has been especially studied by Zoja, Riva, and 
 Garrod, 1 has a pink color, is amorphous, and is very quickly destroyed 
 by light, especially when in solution. The best solvent is amyl alcohol; 
 acetic ether is not so good, and alcohol, chloroform, and water are even 
 less valuable. The very dilute solutions show a pink color; but on greater 
 concentration they become reddish orange or bright red. They do not 
 fluoresce either directly or after the addition of an ammoniacal solution 
 of zinc chloride; but they have a strong absorption, beginning in the 
 middle between D and E and extending to about F, and consisting of two 
 bands which are connected by a shadow between E and b. Concentrated 
 sulphuric acid colors a uroerythrin solution a beautiful carmine red; 
 hydrochloric acid gives a pink color. Alkalies make its solution grass 
 green, and often a play of colors from pink to purple and blue is observed. 
 Porcher and Hervieux 2 claim that uroerythrin is a skatol pigment. 
 
 In preparing uroerythrin according to Garrod, the sediment is dissolved 
 in water at a gentle heat and saturated with ammonium chloride, which pre- 
 cipitates the pigment with the ammonium urate. This is purified by repeated 
 solution in water and precipitation with ammonium chloride until all the urobilin 
 is removed. The precipitate is finally extracted on the filter in the dark with 
 warm water, filtered, then diluted with water, any haematoporpr^rin remaining 
 being removed by shaking with chloroform; the precipitate is then faintly acidi- 
 fied with acetic acid and shaken with chloroform, which takes up the uroerythrin. 
 The chloroform is evaporated in the dark at a gentle heat. 
 
 Volatile fatty acids, such as formic acid, acetic acid, and perhaps also butyric 
 acid, occur under normal conditions in human urine (v. Jaksch), also in that of 
 dogs and herbivora (Schotten). The acids poorest in carbon, such as formic 
 acid and acetic acid, are more stable in the body than those richer in carbon, 
 and therefore the relatively greater part of these pass unchanged into the urine 
 (Schotten). Normal human urine contains besides these bodies others which 
 yield acetic acid when oxidized by potassium dichromate and sulphuric acid 
 (v. Jaksch). The quantity of volatile fatty acids in normal urine calculated as 
 acetic acid is, according to v. Jaksch, 0.008-0.009 gram per twenty-four hours; 
 according to v. Rokitansky, 0.054 gram; and according to Magnus-Levy 
 0.060 gram. The quantity is increased by exclusively farinaceous food (Roki- 
 tansky), in fever and in certain diseases, while in others it is diminished (v. 
 Jaksch, Rosen feld). Large amounts of volatile fatty acids are produced in the 
 alkaline fermentation of the urine, and the quantity is 6-15 times as large as in 
 
 1 Zoja, Arch. ital. di clinica med., 1893, and Centralbl. f. d. med. Wissensch., 1892; 
 Riva, Gaz. med. di Torino, Anno 43, cited from Maly's Jahresber., 24; Garrod, Journ. 
 of Physiol., 17 and 21. 
 
 2 Journ. de Physiol., 7. 
 
CARBOHYDRATES AND REDUCING SUBSTANCES. 749 
 
 normal urine (Salkowski). 1 Non-volatile fatty acids have been detected as 
 normal constituents of urine by K. Morneu and Hyhuinette. 2 
 
 Parallactic Acid. It is claimed that this acid occurs in the urine of healthy 
 persons after very fatiguing marches (Colasanti and Moscatelli). It is found 
 in larger amounts in the urine in acute phosphorus-poisoning or acute yellow 
 atrophy of the liver (Schultzen and Riess), in pregnancy (Undekhill), and 
 especially abundant in eclampsia (Zweifel and others). According to the 
 investigations of Hoppe-Seyler, Araki, and v. Terray, lactic acid passes into 
 the urine as soon as the supply of oxj^gen is decreased in any way, and this probably 
 explains the occurrence of lactic acid in the urine after epileptic attacks (Inouye 
 and Saiki). Minkowski 3 has shown that lactic acid occurs in the urine in large 
 quantities on the extirpation of the liver of birds. 
 
 Glycerophosphoric acid occurs as traces in the urine, 4 and it is probably a 
 decomposition product of lecithin. The occurrence of succinic acid in normal 
 urine is a subject of discussion. 
 
 Carbohydrates and Reducing Substances in the Urine. The occurrence 
 of glucose, as traces, in normal urine is highly probable, as the investiga- 
 tions of Brucke, Abeles, and v. UdrAnszky show. The last investigator 
 has also shown the habitual occurrence of carbohydrates in the urine, 
 and their presence has been positively proven by the investigations of 
 Baumann and Wedenski, and especially by Baisch. Besides glucose 
 normal urine contains, according to Baisch, another not well-studied 
 variety of sugar, according to Lemaire, probably isomaltose, and besides 
 this a dextrin-like carbohydrate (animal gum), as shown by Landwehr, 
 Wedenski, and Baisch. The quantity of carbohydrates eliminated under 
 normal conditions in the twenty-four hours' urine and determined by 
 the benzoylation method, which is perhaps not sufficiently trustworthy, 
 varies considerably between 1.5 and 5.09 grams. 5 
 
 The precipitate obtained from concentrated urine by the aid of alcohol and 
 whose nitrogen (colloidal nitrogen according to Salkowski) in normal urine 
 amounts to 2.34-4.08 per cent of the total nitrogen, and in pathological urines to 
 8-9 per cent, and in a case of acute yellow atrophy of the liver to 21.8 per cent 
 contains, Salkowski 6 claims, a nitrogenous carbohydrate which has strong 
 
 1 v. Jaksch, Zeitschr. f. physiol. Chem., 10; Schotten, ibid., 7; Rokitansky, Wien. 
 med. Jahrbuch, 1887; Salkowski, Zeitschr. f. physiol. Chem., 13; Magnus-Levy, 
 Salkowski's Festschrift, 1004; Rosenfeld, Deutsch, med. Wochenschr., 29. 
 
 *Skand. Arch. f. Physiol., 7. 
 
 3 Colasanti and Moscatelli, Moleschott's Untersuch., 14; Schultzen and Reiss, 
 Chem. Centralbl., 1869; Underhill, Journ. of biol. Chem., 2; Zweifel, Arch. f. Gynakol., 
 76; Araki, Zeitschr. f. physiol. Chem., 15, 16, 17, 19. See also Irisawa, ibid., 17; v. 
 Terray, PAu^er's Arch., 65; Schutz, Zeitschr. f. physiol. Chem., 19; Inouye and 
 Saiki, ibid., 37; Minkowski, Arch. f. exp. Path. u. Pharm., 21 and 31. 
 
 4 See Pasqualis, Maly's Jahresber., 24. 
 
 5 Lemaire, Zeitschr. f. physiol. Chem., 21; Baisch, ibid., 18, 19, and 20. In these 
 as well as in Treupel, ibid., 16, the works of other investigators are cited. See also 
 v. Alfthan, Deutsch. med. Wochenschr., 26. 
 
 6 Berlin, klin. Wochenschr., 1905. In regard to urinary colloids see also Lichtwitz, 
 Zeitschr. f. physiol. Chem., 61 and 72, with Rosenbach, ibid.. 61. 
 
750 • URINE. 
 
 reducing action upon alkaline copper solutions after cleavage with hydrochloric 
 acid. 
 
 Besides traces of sugar and the reducing substances previously men- 
 tioned, uric acid and creatinine, the urine contains still other bodies of this 
 character. These latter are partiy conjugated compounds of glucuronic 
 acid, C6N10O7, which is closely allied to sugar. The reducing power of ( 
 normal urine corresponds, according to various investigators, to 1.5-5.96 
 p. m. glucose. That portion of the reduction belonging to glucose 
 alone is equal to 0.1-0.6 p. m. Laveson * believes that of the total 
 reduction 17.8 per cent is due to sugar, 26.3 per cent to creatinine, 7.8 
 per cent to uric acid, and the remainder, nearly 50 per cent, is caused 
 by chiefly unknown bodies. 
 
 Conjugated glucuronates occur, as indicated by Fluckiger and first 
 positively shown by Mayer and Neuberg, in an exact manner, in very 
 small amounts in normal urine. They occur chiefly as phenol- and only 
 very small amounts of indoxyl- or skatoxylglucuronates. The quantity 
 of glucuronic acid obtained from the conjugated glucuronates is estimated 
 as 0.01 p. m. by Mayer and Neuberg, and by C. Tollens and Fr. Stern, 2 
 on the contrary it was found to be 2.5 p. m. or 0.37 gram per day. Besides 
 these conjugated glucuronates perhaps the urine sometimes contains the 
 urea glucuronic acid, the ureidoglucuronic acid prepared synthetically 
 by Neuberg and Niemann. 3 
 
 Very large amounts of these conjugated glucuronates occur in the 
 urine, on the other hand, after partaking of various therapeutic agents 
 aDd other substances, such as chloral hydrate, camphor, naphthol, borneol, 
 turpentine, morphine, and many other substances. The elimination 
 of glucuronic acid may be markedly increased in severe disturbances of 
 the respiration, severe dyspnoea, in diabetes mellitus, and by the direct 
 introduction of large amounts of glucose. According to P. Mayer, 
 in the oxidation of glucose a part of it forms glucuronic acid, hence it is 
 to be expected that the glucuronic acid can in part be derived from the 
 glucose. As a conjugation of the glucuronic acid with other bodies, 
 such as aromatic atomic complexes, prevents the combustion of this 
 acid in ,the animal body, it ought to follow that after the introduction 
 of such an atomic complex in the body during a glycosuria a correspond- 
 ing reduction of the glucose elimination would take place with the 
 increased excretion of conjugated glucuronates. In order to prove 
 this possibility, O. Loew'i 4 fed dogs with camphor during phlorhizin 
 
 1 Fluckiger, Zeitschr. f. physiol. Chem., 9; Laveson, Bioch. Zeitschr., 4. 
 
 2 Fluckiger, 1. c; Mayer and Neuberg, Zeitschr. f. physiol. Chem., 29; Tollens and 
 Stern, Zeitschr. f. physiol. Chem., 67. 
 
 ' Zeitschr. f. physiol. Chem., 44. 
 * Arch. f. exp. Path. u. Pharm., 47. 
 
CONJUGATED GLUCURONIC ACIDS. 751 
 
 diabetes and found that the above expectation was not realised. Although 
 large quantities of cainpho-gluciironic acid were excreted, the sugar 
 excretion was only slightly diminished and not in proportion to the 
 quantity of conjugated glucuronatc excreted. These negative results 
 are contradicted by the positive- results obtained by Paul Mayer. 1 
 Rabbits normally convert almost all the camphor introduced into con- 
 jugated glucuronic acid. Mayer claims that if we allow a rabbit to 
 starve several days, the animal becomes so poor in the mother-substance 
 (glycogen) yielding the glucuronic acid that the introduction of camphor 
 only brings about an elimination of small quantities of glucuronic acid. 
 By the simultaneous administration of camphor and glucose while 
 starvation is going on, the elimination of glucuronic acid rises again to 
 the same height as it was before the starvation period. This shows that 
 the sugar had conjugated itself with the camphor as glucuronic acid. 
 Hildebrandt 2 has also made experiments showing that glucuronic acid 
 can very likely be formed from sugar. The observations of Mayer are 
 not substantiated by the recent investigations of Fenyvessy, 3 and the 
 observers do not agree on this question. 
 
 The conjugated glucuronic acids are formed, based upon the investi- 
 gations of Sundwik, Fischer and Piloty, 4 by a combination taking 
 place first between the conjugator and the glucose by means of the alde- 
 hyde group, and then the end alcohol group, CHoOH, is oxidized to COOH. 
 The conjugated glucuronic acids, at least in most cases, seem to be con- 
 structed after the glucoside type, a view which has received further 
 support by the synthesis of phenolglucuronic acid and euxanthonglu- 
 curonic acids by Neuberg and Neimann. 5 Based upon their cleavage 
 (as far as they have been investigated) by kephir lactase and emulsin, 
 but not by yeast lactase (Neuberg and Wohlgemuth 6 ), the conjugated 
 glucuronic acids must belong to the /3-series of glucosides. We also 
 know of certain conjugated glucuronates that are constructed upon the 
 ester type, namely, the dimethylaminobenzoicglucuronate, discovered 
 by Jaff:e and also the benzoicglucuronic acid, after feeding benzoic 
 acid (Magnus-Levy 1 7 
 
 According to the body with which they are conjugated the glucuronates 
 vary in behavior. On taking up water they split into glucuronic acid 
 
 1 Zeitschr. f. klin. Med.. 47. 
 
 2 Arch. f. exp. Path. u. Pharm., 44. 
 8 See Maly's Jahresber., 34. 
 
 4 E. Sundwik, Akademische Abhandlung Helsingfors, 18S6; see also Maly's Jahr- 
 esber., 16, 76; Fischer and Piloty, Ber. d. d. chem. Gesellsch., 24. 
 B Zeitschr. f. physiol. Chem., 44. 
 
 6 See Neuberg, Ergebnisse der Physiologic, Bd. 3, Abt. 1, 444. 
 
 7 Jaffe, Zeitschr. f. physiol. Chem., 43; Magnus-Levy, Bioch. Zeitschr., 6. 
 
752 URINE. 
 
 and the conjugated group and this is brought about by boiling with 
 a dilute mineral acid. They are precipitated by basic lead acetate or 
 by basic lead acetate and ammonia. Most of the conjugated glucuronic 
 acids do not have a direct reducing action but are reducing after hydrolysis. 
 Certain of them, and to this group belong especially those acids of the 
 ester type, reduce copper oxide and certain other metallic oxides in alkaline 
 solution directly, and hence cause errors in the investigations of the 
 urine for sugar. The conjugated acids of the glucoside type rotate the 
 plane of polarized light to the left, while the glucuronic acid itself is dextro- 
 rotatory. The conjugated acids of the ester type, which as a rule are 
 less stable, rotate the ray of polarized light to the right. As the detection 
 of conjugated glucuronic acids is connected with the tests for sugar in 
 the urine, we will treat of this in connection with these tests. 
 
 Organic combinations containing sulphur of unknown kind, which may 
 in small part consist of sulphocyanides, 0.04 (Gscheidlen) to 0.11 p. m.- 
 (I. Munk), 1 cystine or bodies related to it, taurine derivatives, chon- 
 droitin-sulphuric acid and protein bodies, but in greater part are made up 
 of antoxyproteic acid, oxyproteic acid, alloxyproteic acid, and uroferric 
 acid, are found in human as well as in animal urines. The sulphur of 
 these mostly unknown combinations has been called " neutral," to dif- 
 ferentiate it from the " acid " sulphur of the sulphate and ethereal-sul- 
 phuric acid (Salkowski 2 ) . The neutral sulphur in normal urine is 
 13-24 per cent of the total sulphur. 3 In anaemia, cachetic conditions, 
 pulmonary tuberculosis and especially in carcinoma the quantity is 
 greatly increased (Weiss). In general it can be said that the quantity 
 is increased by an increased catabolism of protein and therefore an increase 
 in the neutral sulphur has been found in starvation (Fr. Muller), with 
 insufficient oxygen supply (Reale and Boeri, Harnack and Kleine) 
 and after chloroform narcosis (Kast and Mester). After the introduc- 
 tion of free sulphur the quantity of neutral sulphur is increased, accord- 
 ing to Presch and Yvon and to Maillard.* The quantity of neutral 
 sulphur varies, according to Benedict, within rather narrow limits 
 and especially, according to Folin, it is dependent to a less degree than 
 the sulphate excretion upon the extent of the protein metabolism. The 
 relation between the neutral and acid sulphur depends in the first place 
 
 1 Gscheidlen, Pfliiger's Arch., 14; Munk, Virchow's Arch., 69. 
 
 2 Ibid., 58, and Zeitschr. f. physiol. Chem., 9. 
 
 'Salkowski, 1. c; Stadthagen, Virchow's Arch., 100; Lepine, Compt. Rend., 91 
 and 97; Harnack and Kleine, Zeitschr. f. Biologie, 37; Mor. Weiss, Bioch. Zeitschr., 27. 
 4 Weiss, 1. c; Fr. Muller, Berl. klin. Wochenschr., 1887; Reale and Boeri, Maly's 
 Jahresber., 24; Harnack and Kleine, 1. c; Kast and Mester, Zeitschr. f. klin. Med., 
 18; Presch., Virchow's Arch., 119; Yvon, Arch, de Physiol. (5), 10; Maillard, Compt. 
 Rend., 152. 
 
ORGANIC SULPHUR COMBINATIONS. 753 
 
 upon the extent of the sulphuric-acid excretion. According to Harnack 
 and Kleine, 1 the relation of the oxidized sulphur to the total sulphur 
 changes always in the same way as the relation of the nitrogen of the 
 urea to the total nitrogen. The more unoxidized sulphur is eliminated 
 the more abundant are the nitrogen compounds, not urea, in the urine — 
 a statement which coincides with recent observations showing that the 
 neutral sulphur originates chiefly from the different proteic acids, and 
 the uroferric acid. 
 
 According to Lepine, a part of the neutral sulphur is more readily oxidized 
 (directly with chlorine or bromine) into sulphuric acid than the other, which is 
 only converted into sulphuric acid after fusing with potash and saltpeter. The 
 investigations of W. Smith 2 show that it is probable that the difficultly oxidizable 
 part of the neutral sulphur occurs as sulpho-acids. An increased elimination of 
 neutral sulphur has been observed in various diseases, such as pneumonia, cystin- 
 uria, and especially where the flow of bile into the intestine is prevented. 
 
 The total quantity of sulphur in the urine is determined by fusing the solid 
 urinary residue with saltpeter and caustic alkali or sodium peroxide, or by oxida- 
 tion with nitric acid. 3 The quantity of neutral sulphur is determined as the 
 difference between the total sulphur and the sulphur of the sulphate and ethereal- 
 sulphuric acids. The readily oxidizable part of the neutral sulphur is determined 
 by oxidation with bromine or potassium chlorate and hydrochloric acid (Lepine, 
 Jerome 4 ). 
 
 Sulphuretted hydrogen occurs in the urine only under abnormal conditions 
 or as a decomposition product. This compound may be produced from the 
 neutral sulphur of the organic substances of the urine by the action of certain 
 bacteria (Fr. Muller, Salkowski 5 ). Other investigators have given hypo- 
 sulphites as the source of the sulphuretted hydrogen. The occurrence of hypo- 
 sulphites in normal human urine, which is asserted by Heffter, is disputed by 
 Salkowski and Presch. 6 Hyposulphites occur constantly in cat's urine and, 
 as a rule, also in clog's urine. 
 
 Antoxyproteic acid is a nitrogenous acid containing sulphur which 
 Bondzynski, Dombrowski, and Panek 7 have isolated from human 
 urine. The composition of the acid was: C 43.21, H 4.91, N 24.4, S 0.61, 
 and O 26.23 per cent. A part of the sulphur can be split off by alkali. 
 This acid is soluble in water, is dextrorotatory, and is only precipitated 
 from concentrated solution by phosphotungstic acid. It does not give 
 the protein color reactions, but gives Ehrlich's diazo reaction (see below). 
 
 1 Benedict, Zeitschr. f. klin. Med., 36; Harnack and Kleine, 1. c; Folin, Amer. 
 Journ. of Physiol., 13. 
 
 2 Lepine, 1. c; Smith, Zeitschr. f. physiol. Chem., 17. 
 
 3 See Abderhalden and Funk, Zeitschr. f. physiol. Chem., 58 and 59, which also 
 cites other methods. See S. R. Benedict, Journ. of biol. Chem., 6 and 8; Denis, ibid., 
 8; Gill and Grindley, Journ. Amer. Chem. Soc, 31; Folin, ibid., 31. 
 
 4 Jerome, Pfluger's Arch., 60. 
 
 5 Fr. Muller, Berlin, klin. Wochenschr., 1887; Salkowski, ibid., 1888. 
 
 6 Heffter, Pfluger's Arch., 38; Salkowski, ibid., 39; Presch, Virchow'a Arch., 119. 
 
 7 Zeitschr. f. physiol. Chem., 46. 
 
754 URINE. 
 
 The salts with the alkalies, barium, calcium, and silver are soluble in 
 water, and of these salts that with barium and, to a still higher degree, 
 the silver salt are soluble with difficulty in alcohol. The free acid and 
 its salts are precipitated by mercuric nitrate and acetate, and by this 
 last reagent even from solutions strongly acidified with acetic acid. Basic 
 lead acetate does not precipitate the pure acid. 
 
 Oxyproteic acid is the name given by Bondzynski and Gottlieb * 
 to a nitrogenous acid containing sulphur, and which they prepared from 
 human urine, which has recently been further studied by Bondzynski, 
 Dombrowski and Panek. This acid contained C 39.62, H 5.64, N 
 18.08, S 1.12, and 35.54 per cent, and also contains sulphur which could 
 be split off. On cleavage it yields no tyrosine, nor does it give Ehrlich's 
 diazo reaction, the xanthoproteic nor the biuret reaction. It gives a 
 faint indication of a Millon reaction and is not precipitated by phos- 
 photungstic acid, hence it leads to an error in the Pfluger-Bohland 
 method for estimating urea. The acid soluble in water is precipitated 
 by mercuric nitrate and acetate in neutral solutions, but is not precipitated 
 by basic lead acetate. The salts of this acid are readily soluble in water 
 and more soluble in alcohol than the corresponding salts of antoxy- 
 proteic acid. 
 
 The acid which is found in large quantities, especially in the urine of 
 dogs poisoned with phosphorus (Bondzynski and Gottlieb), is considered 
 like the preceding acid as an intermediary oxidation product of the pro- 
 teins, and oxyproteic acid seems to represent a higher state of oxidation 
 or a demolition of the proteins than the antoxyproteic acid. 
 
 The acid called uroproteic acid by Cloetta is probably a mixture of several 
 bodies, according to the recent investigations of Bondzynski, Dombrowski, and 
 Panek. The same applies also to the barium oxyproteate prepared by Pregl 2 
 from the urine. 
 
 Alloxyproteic acid is a third acid related to the above, which was 
 first isolated by Bondzynski and Panek 3 from the urine and then care- 
 fully studied with Dombrowski. The composition is: C 41.33, H 
 5.70, N 13.55, S 2.19, and 37.23 per cent, based upon new investigations. 
 The free acid is soluble in water. It gives neither the biuret reaction 
 nor Ehrlich's reaction, and is not precipitated by phosphotungstic 
 acid. Differing from the other acids, it is precipitated by basic lead 
 acetate, and its salts are only slightly soluble in alcohol. According to 
 
 'Centralbl. f. d. med. Wissensch., 1897, No. 33. 
 
 2 Cloetta, Arch. f. pxp. Path. u. Pharm., 40; Pregl, Pfliiger's Arch., 75. 
 
 * Ber. d. d. chem. Gesellsch., 35. 
 
OXYPROTEIC ACIDS. 755 
 
 Liebermann x this acid is not a unit substance, and contains a part of 
 its sulphur as ethereal sulphuric acid, and it also contains uroferric acid. 
 
 Tho urochrome, which has been specially studied by Dombrowski, 2 is con- 
 eidered by him and Bondzynski us belonging to the oxyproteic acid group. I 
 contains about 5 per cent sulphur, is precipitated by copper acetate and yields 
 melanin-like substances on its decomposition. No positive proofs arc at hand in 
 regard to the purity of this urochrome and the reports as to its composition, 
 which are very contradictory, do not exclude the possibility that this is a mixture 
 of a yellow pigment with another substance (see page 741). 
 
 Browinski and Dombrowski 3 have carried out investigations on the nitrogen 
 tit ratable with forniol on the oxyproteic acids before and after acid hydrolysis. 
 They found that the antoxyproteic acid and the oxyproteic acid did not contain 
 any nitrogen split off as NH 3 by MgO before hydrolysis, while the alloxyproteic 
 acid as well as the urochrome yielded about 3 per cent of the total nitrogen in this 
 form. After acid hydrolysis all gave about the same quantity of ammonia. The 
 two first-mentioned acids, especially oxyproteic acid, were before hydrolysis 
 considerably richer in amino groups, tit ratable with formol, than the others. 
 This indicates that these two acids are produced from the proteins by a deeper 
 cleavage than is the alloxyproteic acid. The large amount of free amino groups, 
 which occur especially in the oxyproteic acid and which amount to 38.8 per cent 
 of the total nitrogen, is nevertheless remarkable. 
 
 The preparation of the three above-mentioned acids is based in part 
 upon the fact that alloxyproteic acid alone is precipitated by basic lead 
 acetate and that the two other acids can be precipitated from the fil- 
 trate by mercuric acetate, the antoxyproteic acid in acetic acid solution 
 and the oxyproteic acid in neutral solution. The preparation is never- 
 theless very tedious and complicated and therefore we must refer to the 
 original works for details. 
 
 Uroferric acid is an acid isolated by Thiele 4 from the urine, according to 
 Siegfried's method for preparing pure peptone. It also contains sulphur, 3.46 
 per cent, and has the formula CssHseXsSOig. The acid forms a white powder 
 which is readily soluble in water, saturated ammonium-sulphate solution, and 
 methyl alcohol. It is soluble with difficulty in absolute alcohol, insoluble in benzene, 
 chloroform, ether, and acetic ether. About one-half of the sulphur can be split 
 off as sulphuric acid on boiling with hydrochloric acid. The acid gives neither 
 the biuret test nor Millon's or Adamkiewicz's reactions. It is precipitated by 
 mercuric nitrate and sulphate, and also by phosphotungstic acid. This acid is 
 hexabasic, and its specific rotation at 18° C. (a)o = —32.5°. On cleavage it 
 yields melanine substances, sulphuric acid, aspartic acid, but no hexone bases. 
 The existence of this acid is disputed by Bondzynski, Dombrowski and Panek. 
 The investigations of Ginsberg also contradict the occurrence of such an acid, 
 because no sulphuric acid could be split off from the mixture of the oxyproteic 
 acids by hydrolysis. 
 
 Methods for the quantitative estimation of the total oxyproteic 
 acids have been suggested by Ginsberg and by Gawinski. 5 Accord- 
 
 1 Zeitschr. f. physiol. Chem., 52. 
 
 2 Ibid., 46 and 62. 
 
 3 Ibid., 77. 
 
 4 Zeitschr. f. physiol. Chem., 37. 
 
 5 Ginsberg, Hofmeister's Beitrage, 10; Gawinski, Zeitschr. f. physiol. Chem., 58. 
 
756 URINE. 
 
 ing to their determinations, in man with a mixed diet, the nitrogen of the 
 oxyproteic acids represented 3-6.8 per cent of the total nitrogen, and with 
 a milk diet it sinks to about one-half of this (Gawinski). In dogs it 
 amounts to 2 per cent of the total nitrogen (Ginsberg). In disease it 
 may rise, and in typhoid cases it may rise to 14.69 per cent of the total 
 nitrogen (Gawinski). In phosphorus poisoning this nitrogen fraction 
 is also markedly increased according to several observations. The 
 oxyproteic acids are considered, as above remarked, as intermediary 
 products of the protein metabolism, and Gawinski holds that the 
 elimination of their nitrogen runs parallel with the elimination of neutral 
 sulphur, so that this latter may serve as an approximate measure of the 
 elimination of these acids. 
 
 Abderhalden and Pregl » have shown that human urine normally contains 
 compounds which stand, perhaps, in close relation to the polypeptides, and 
 which on hydrolysis with acids yield at least a part of the moities existing in the 
 protein molecule. In the case investigated they obtained abundant glycocoll, 
 also leucine, alanine, glutamic acid, phenylalanine, and probably also aspartic 
 acid. The relation between these polypeptide-like bodies and the above-men- 
 tioned proteic acids and uroferric acid has not been investigated. 
 
 Henriques and Sorensen 2 have given further proof for the occurrence of 
 nitrogen in peptide combinations in the urine. They have shown by formol 
 titration that in normal urine amino-acid nitrogen occurs. It must be remarked 
 that they consider as amino-acid nitrogen not only the nitrogen occurring as amino- 
 acids but also the urine nitrogen directly titratable with formol, therefore also the 
 titratable amino-nitrogen in the oxyproteic acids, polypeptides or more com- 
 plicated protein derivatives. They have further shown that after boiling with 
 acid that the quantity of titratable nitrogen increases, and this increase which 
 in man may be 8.9-28.3 per cent of the amino-acid nitrogen, they consider as pep- 
 tide-like nitrogen. We have abundant literature 3 on the ways and means of 
 carrying out the formol titration in urines, considering the presence of ammonia. 
 
 Amino-acids may, when they are introduced into the body in large amounts, 
 also pass in part into the urine. This has been shown for r-alanine by R. Hirsch 
 in the dog, and by Plaut and Reese in dog and man, and for r-leucine by 
 Abderhalden and Samuely 4 in rabbits and by others using different amino-acids. 
 Embden and Reese, Forssner, Abderhalden and Schittenhelm, Samuely, 
 Embden and Marx 6 were able, by means of the naphthalene sulpho- 
 chloride method to detect glycocoll in normal human urine, and this glycocoll 
 must occur in the urine in a combination which is readily split by alkali. Although 
 
 1 Zeitschr. f. physiol. Chem., 46. 
 
 2 Henriques, Zeitschr. f. physiol. Chem., 60; Henriques and Sorensen, ibid., 63 
 and 64. 
 
 3 See Henriques and S6rensen, 1. c; Malfatti, Zeitschr. f. physiol. Chem., 61 and 
 66; de Jager, ibid., 62 and 65; Fry and Gigon, Bioch. Zeitschr., 22. 
 
 4 R. Hirsch, Zeitschr. f. exp. Path. u. Therap., 1; Plaut and Reese, Hofmeister's 
 Beitrage, 7; Abderhalden and Samuely, Zeitschr. f. physiol. Chem., 47. 
 
 5 Forssner, Zeitschr. f. physiol. Chem., 47; Abderhalden and Schittenhelm, ibid., 
 47; Samuely, ibid., 47; Embden and Reese, Hofmeister's Beitrage, 7, with Marx, 
 ihi'l.. 11, which also cites the rather conflicting deductions of Neuberg and Wolgemuth 
 and of Hirschstein. 
 
ORGANIC PHOSPHORUS COMBINATIONS. 757 
 
 there have been numerous investigations, no amino-acids besides glycocoll could 
 be detected in normal human urine, while, on the contrary, in pathological con- 
 ditions other amino-acids have been found several times. The amino-acid frac- 
 tion of the urine seems to be increased in starvation and in high altitudes ( Loewt ')• 
 The conclusions of various investigators 2 in regard to the behavior of amino- 
 acids in diseases such as gout, disagree. 
 
 Non-dialyzable substances, the so-called adialyzable bodies, or bodies that 
 dialyze with difficulty, also occur in the urine. They consist in part of chon- 
 droit in-sulphuric acid whose daily amount, according to Pons, is 0.08-0.09 gram, 
 and also of nucleic acid, mucoids, the colloidal nitrogenous bodies (see Balkowski, 
 page 749) and unknown bodies. Sasaki found 0.218-0.68 gram of such bodies 
 per liter of normal urine, and Ebbecke found 1.44 grams in men. In pregnant 
 women Savare found somewhat higher results (0.G gram per liter) than in non- 
 pregnant women (0.4 gram). The quantity is increased in fevers, in pneumonia 
 (Ebbecke), in nephritis, and especially in eclampsia, where Savare • indeed in 
 one ease found 13.84 grams per liter. The adialyzable bodies occurring in eclampsia 
 are toxic. 
 
 Organic combinations containing phosphorus such as glycerophosphoric acid, 
 phosphocarnic acid (Rockwood), etc., which yield phosphoric acid on fusing with 
 saltpeter and caustic alkali, are also found in urine (Lepine and Eymonnet, 
 Oertel). With a total elimination of about 2.0 grams total P«0 5 , Oertel found 
 on an average about 0.05 gram P 2 5 as phosphorus in organic combination. Accord- 
 ing to Kondo the quantity of organic phosphorus is increased by taking phos- 
 phatides and nucleins but not to the same extent as the quantity of phosphoric 
 acid. According to Sy.m.mers 4 the organic combined phosphorus ma}- in many 
 pathological conditions be 25-50 per cent of the total phosphoric acid. In lym- 
 phatic leucaemia, and especially in degenerative diseases of the nervous system, 
 the quantity may increase. 
 
 Enzymes of various kinds have been isolated from the urine. Among these 
 may be mentioned pepsin, diastolic enzyme and lipase. 6 
 
 Mucin. The nubecula consists, as shown by K. Morxer, 6 of a mucoid which 
 contains 12.74 per cent N and 2.3 per cent S. This mucoid, which apparently 
 originates in the urinary passages, may pass to a slight extent into solution in 
 the urine. In regard to the nature of the mucins and nucleoalbumins otherwise 
 occurring in the urine we refer the reader to the pathological constituents of the 
 urine. 
 
 Ptomaines and leucomaines, or poisonous substances of an unknown kind, 
 which are often described as alkaloidal substances, occur in normal urine, as shown 
 by earlier investigations (Pouchet, Bouchard, Aducco and others 7 ) and also 
 by recent researches of Kutscher, Lohmann and Engeland. The trimethyl- 
 
 1 Deutsch. med. Wochenschr., 1905; see also Signorelli, Bioch. Zeitschr., 39. 
 
 2 See Jastrowitz, Arch. f. exp. Path. u. Pharm., 59; Walker Hall, Bioch. Journ., 
 1; Brugsch and Sehittenhelm, Zeitschr. f. exp. Path. u. Therap., 4. 
 
 3 Pons, Hofmeister's Beitrage, 9; Sasaki, ibid., 9; Savare, ibid., 9 and 11; Ebbecke, 
 Bioch. Zeitschr., 13. 
 
 4 Rockwood, Arch. f. (Anat. u.) Physiol., 1895; Oertel, Zeitschr. f. physiol. Chem., 
 26, which cites the other works. See also Keller, Zeitschr. f. physiol. Chem., 29; 
 Mandel and Oertel, N. Y. Univ. Bull. Med. Sciences, 1; and Maly's Jahresber., 31; 
 Symmers, Journ. of Path, and Bact, 10. 
 
 5 In regard to the literature on enzymes in the urine, see Huppert-Xeubauer, 599. 
 In regard to trypsin in the urine see Johansson, Zeitschr. f. physiol. Chem., 85. 
 
 6 Skand. Arch, f . Physiol, 6. 
 
 7 A complete bibliography on the ptomaines and leucomaines of the urine is found 
 in Huppert-Xeubauer, 403. 
 
758 URINE. 
 
 amine, which originates from the phosphatides, and was first detected by de Filippi 
 and later by K. Baler, belong to the leucomanir.es and also the bases found by 
 Kutscher and by Kutscher and Lohmann, namely, methyl guanidine (also 
 found by Achelis), dimcthylguanidine, novain (previously found by Dombrowski), 
 reductonoraitt, C7H17NO2, gynesin, C19H23N3O3 (from female urine) mingin, Ci 3 Hi 8 N 2 02, 
 vitiatin (Chapter X) and methylpyridine chloride, which is not a leucomaine, but 
 is probably derived from smoking tobacco or from drinking coffee. The imidazole 
 derivatives histidine and imidazolamino-acetic acid found by Kutscher and 
 Engeland also belong to this group and the urohypertensin and urohypotensin 
 of Abelous and Bardier. 1 
 
 Under pathological conditions the quantity of leucomaines and other bodies 
 may be increased (Bouchard, Lepine and Geurin, Villiers, Griffiths, Albu, 
 and others). Within the last few years the poisonous properties of urine have 
 been the subject of more thorough investigation, especialby by Bouchard. He 
 found that the night urine is less poisonous than the day urine, and that the 
 poisonous constituents of the da}' and night urine have not the same action. 
 In order to be able to compare the toxic power of the urine under different con- 
 ditions, Bouchard determines the urotoxic coefficient, which is the weight 
 of rabbit in kilos that is killed by the quantity of urine excreted in twenty-four 
 hours by 1 kilo of the person experimented upon. 2 
 
 Many substances have been observed in animal urine which are not found in 
 human urine. To these belong the above-described kynurenic acid, urocanic acid, 
 which according to Hunter is imidazolacrylic acid, also found in dog's urine; 
 damaluric acid and damolic acid (according to Schotten, 3 probably a mixture 
 of benzoic acid with volatile fatty acids), obtained by the distillation of cow's 
 urine; and lastly lithuric acid, found in the urinary concrements of certain 
 animals. 
 
 HI. INORGANIC CONSTITUENTS OF URINE. 
 
 Chlorides. The chlorine occurring in the urine is undoubtedly com- 
 bined with the bases contained in this excretion; the chief part is in 
 combination with sodium. In accordance with this, the quantity of 
 chlorine in the urine is generally expressed as NaCl. 
 
 The question as to whether a part of the chlorine contained in the 
 urine exists as organic combinations, as considered by Berlioz and 
 Lepinois, is still disputed, although recently Baumgarten 4 has supported 
 this view. 
 
 The quantity of chlorine combinations in the urine is subject to con- 
 siderable variation. In general the amount from a healthy adult on a 
 
 1 de Filippi, Zeitschr. f. physiol. Chem., 49; Bauer, Hofmeister's Beitrage, 11; 
 Kutscher, Zeitschr. f. physiol. Chem., 51, with Lohmann, ibid., 48 and 49; Achelis, 
 ibid., 50; England, ibid., 57, and Munch, med. Wochenschr., 55; Abelous and Bardier, 
 Maly's Jahresb., 39 and 40. 
 
 2 See footnote 7, page 757. 
 
 3 Hunter, Journ. of biol. Chem., 11; Schotten, Zeitschr. f. physiol. Chem., 7. 
 
 * Berlioz and Lepinois, see Chem. Centralbl., 1894, 1, and 1895, 1; also Petit and 
 Terrat, ibid., 1894, 2, and Vitali, ibid., 1897, 2; Ville and Moitessier, Maly's Jahres- 
 ber., 31; Meillere, ibid.; Bruno, ibid., 452; Baumgarten, Zeitschr. f. exp. Path. u. 
 Therap., 5. 
 
CHLORIDES. 759 
 
 mixed diet is 10-1.") grams of NaCl per twenty-four hours. The quantity 
 of common salt in the urine depends chiefly upon the amount of sail in the 
 food, with which the elimination of chlorine increases and decreases. 
 The free drinking of water also increases the elimination of chlorine, 
 which is greater during activity than during rest (at night). Certain 
 organic chlorine combinations, such as chloroform, may increase the 
 elimination of inorganic chlorides by the urine (Zeller, Kast 1 ). 
 
 In diarrhoea, in quick formation of large transudates and exudates, 
 also in specially marked cases of acute febrile diseases at the time of the 
 crisis, the elimination of NaCl is materially decreased. The excretion 
 of chlorine may vary considerably in disease, but still the NaCl taken with 
 the food has here, as in physiological conditions, a great influence on the 
 NaCl excretion. 2 
 
 The quantitative estimation of chlorine in the urine is most simply per- 
 formed by titration with silver-nitrate solution. The urine must not 
 contain either proteid (which if present must be removed by coagulation) 
 or iodine or bromine compounds. 
 
 In the presence of bromides or iodides evaporate a measured quantity of the 
 urine to dryness, fuse the residue with saltpeter and soda, dissolve the fused 
 mass in water, and remove the iodine or bromine by the addition of dilute sul- 
 phuric acid and some nitrite, and thoroughly shake with carbon disulphide. 
 The liquid thus obtained may now be titrated with silver nitrate according to 
 Volhard's method. The quantity of bromide or iodide is calculated as the 
 difference between the quantity of silver-nitrate solution used for the titration 
 of the solution of the fused mass and the quantity used for the coresponding 
 volume of the original urine. 
 
 The otherwise excellent titration method of Mphr, according to which 
 we titrate with silver nitrate in neutral liquids, using neutral potassium 
 chromate as an indicator, cannot be used directly on the urine in careful 
 work. Organic urinary constituents are also precipitated by the silver 
 salt, and the results are therefore somewhat high for the chlorine. If 
 this method is to be employed, the organic urinary constituents must be 
 destroyed, by incineration after the addition of saltpeter free from 
 chlorine. 
 
 According to Bang and Larsson 3 the disturbing substances which react 
 with AgX0 3 can be removed by shaking with blood-charcoal. The value of this 
 surest ion is essentially diminished, because every blood-charcoal cannot be used, 
 and therefore a special testing of the blood-charcoal must be done. 
 
 teller. Zehschr. f. physiol. Chem., 8; Kast, ibid., 11; Vitali, Chem. Centralbl., 
 1899. 2. 
 
 2 On the elimination of chlorine in disease, see Albu and Neuberg, Physiol, u. Pathol. 
 des MineralsotTwe chsels, Berlin, 1906. 
 
 3 Bioch. Zeitschr., 49. 
 
760 URINE. 
 
 The silver-nitrate solution may be a N/10 one. It is often made of 
 such a strength that each cubic centimeter corresponds to 0.006 gram 
 CI or 0.01 gram NaCl. This last-mentioned solution contains 29.075 
 grams of AgXC>3 in 1 liter. 
 
 Freund and Toepfer, as well as Bodtker, 1 have suggested modifica- 
 tions of Mohr's method. 
 
 Volhard's Method. Instead of the preceding determination, Vol- 
 hard's method, which can be performed directly on the urine, may be 
 employed. The principle is as follows: All the chlorine from the urine 
 acidified with nitric acid is precipitated by an excess of silver nitrate, 
 filtered, and in a measured part of the filtrate the quantity of silver added 
 in excess is determined by means of a sulphocyanide solution. This 
 excess of silver is completely precipitated by the sulphocyanide, and a 
 solution of some ferric salt, which, as is well known, gives a blood-red 
 reaction with the smallest quantity of sulphocyanide, is used as an indicator. 
 
 We require the following solutions for this titration: 1. A silver- 
 nitrate solution which contains 29.075 grams of AgNOs per liter, and of 
 which each cubic centimeter corresponds to 0.01 gram NaCl or 0.00607 
 gram CI. 2. A saturated solution at the ordinary temperature of chlorine- 
 free iron alum or ferric sulphate. 3. Chlorine-free nitric acid of a specific 
 gravity of 1.2. 4. A potassium-sulphocyanide solution which contains 
 8.3 grams KCNS per liter, and of which 2 cc. corresponds to 1 cc. of the 
 silver-nitrate solution. 
 
 About 9 grams of potassium sulphocyanide are dissolved in water and diluted 
 to 1 liter. The quantity of KCNS contained in this solution is determined by the 
 silver-nitrate solution in the following way: Measure exactly 10 cc. of the silver 
 solution and treat it with 5 cc. of nitric acid and 1-2 cc. of the ferric-salt solu- 
 tion and dilute with water to about 100 cc. Now the sulphocyanide solution 
 is added from a burette, constantly stirring until a permanent faint-red colora- 
 tion of the liquid takes place. The quantity of sulphocyanide found in the solu- 
 tion by this means indicates how much it must be diluted to be of the proper 
 strength. Titrate once mere with 10 cc. of AgNOs solution and correct the sul- 
 phocyanide solution by the careful addition of water until 20 cc. exactly cor- 
 responds to 10 cc. of the silver solution. 
 
 The determination of the chlorine in the urine is performed by this 
 method in the following way: Exactly 10 cc. of the urine are placed in a 
 flask which has a mark corresponding to 100 cc. and which is provided 
 with a stopper; 5 cc. of nitric acid are added; dilute with about 50 cc. 
 of water and then allow exactly 20 cc. of the silver-nitrate solution to 
 flow in. Close the flask with the stopper and shake well, remove the stop- 
 per and wash it with distilled water into the flask, and fill the flask to the 
 100-cc. mark with distilled water. Close again with the stopper, care- 
 fully mix by shaking, and filter through a dry filter. Measure off 50 
 cc. of the filtrate by means of a dry pipette, add 3 cc. of ferric-salt solu- 
 tion, and allow the sulphocyanide solution to flow in until the liquid 
 above the precipitate has a permanent red color. The calculation is 
 very simple. For example, if 4.6 cc. of the sulphocyanide solution 
 
 x Freund and Toepfer, see Maly's Jahresber., 22; Bodtker, Zeitschr. f. physiol. 
 Chem., 20. 
 
PHOSPHATES. 761 
 
 was necessary to produce the final reaction, then for 100 cc. of the filtrate 
 (=10 cc. urine) 9.2 cc. of this solution arc necessary. 9.2 cc. of the 
 Bulphocyanide solution corresponds to 4.6 cc. of the silver solution, 
 and since 20—4.6 = 15.4 cc. of the Bilver solution was necessary to com- 
 pletely precipitate the chlorine in 10 cc. of the urine, then 10 cc. con- 
 tains 0.154 gram of NaCl. The quantity of sodium chloride in the urine 
 is therefore 1.54 per cent, or 15.4 p. m. If we always use 10 cc. for the 
 determination, and always 20 cc. of AgNCb solution, and dilute with 
 water to 100 cc, the quantity of Na( '1 in 1000 parts of the urine is found 
 by subtracting from 20 the number of cubic centimeters of sulphocyanide 
 (R) required with 50 cc. of the filtrate. The quantity of NaCl p. m. 
 therefore under these circumstances = 20 — R, and the percentage of 
 
 NaCl = ^. 
 
 If it is necessary to destroy the organic urinary constituents before titration, 
 this can be best performed, according to Dehn, 1 by evaporating the urine (10 cc.) 
 to dryness on the water-bath after the addition of a small amount of sodium per- 
 oxide, then faintly acidifying with nitric acid and then titrating according to 
 Volhard. Incineration is unnecessary. 
 
 For the approximate estimation of chlorine in the urine Ekehorn has made 
 use of Volhard's titration method by using for the determination a glass tube 
 closed at one end and divided into half cubic centimeters and called the chlorom- 
 eter. The reagents necessary are: (a) a mixture of 20 cc. silver-nitrate solution 
 (according to Volhard), 5 cc. nitric acid and water to 100 cc; (b) 40 cc. sul- 
 phocyanide solution and 60 cc. of a ferric alum, chlorine free and saturated at 
 the ordinary temperature. The silver-nitrate solution, of which each cubic 
 centimeter corresponds to 0.002 gm. NaCl, is equivalent to the iron sulphocyanide 
 solution. First 2 cc. of the urine are placed in the graduated tube and then 0.5 
 cc. sulphocyanide solution, and the silver-nitrate solution gradually added (shak- 
 ing the tube closed with a rubber stopper) until the coloration of the sulphocyanide 
 just disappears. 0.5 cc. is subtracted from the silver solution for the 0.5 cc. of 
 the sulphocyanide; the tube is so graduated that the quantity of XaCl in the 
 urine in parts per thousand is read off directly on the tube. The difference 
 between these results and those obtained by Volhard's titration method amounts 
 only, according to C. Th. Mobner, 2 to 0.25 to at most 0.5 p. m. 
 
 The approximate estimation of chlorine in the urine (which must be free from 
 protein) is made by strongly acidifying with nitric acid and then adding to it, 
 drop by drop, a concentrated silver-nitrate solution (1: 8). In a normal quantity 
 of chlorides the drop sinks to the bottom as a rather compact cheesy lump. In 
 diminished quantity of chlorides the precipitate is less compact and coherent, 
 and in the presence of very little chlorine a fine white precipitate or only a 
 cloudiness or opalescence is obtained. 
 
 Phosphates. Phosphoric acid occurs in acid urines partly as dihydro- 
 gen, MH2PO4, and partly as monohydrogen, M2HPO4, phosphates, 
 both of which may be found in acid urines at the same time. The 
 proportion of these may vary eonsiderabby ; acid urines contain chiefly 
 dihydrogen phosphate and in many cases the urine seems to contain only 
 dihydrogen phosphate and sometimes indeed only a small quantity of phos- 
 
 1 Zeitschr. f. physiol. Chem., 44. 
 
 2 Ekehorn, Hygiea, Stockholm, 1906; Morner, Upsala Lakaref. Forh. (N. F.), 11. 
 
762 UKINE. 
 
 phoric acid. The total quantity of phosphoric acid varies and depends on 
 the character and the quantity of food. The average quantity of P2O5 
 is in round numbers 2.5 grams, with a variation of 1-5 grams per day. 
 A small part of the phosphoric acid of the urine originates from the 
 burning of organic compounds, such as nuclein, and phosphatides 
 within the organism; on exclusive feeding with substances rich in nuclein 
 or pseudonuclein the quantity of phosphates is essentially increased; 
 still it is undecided to what extent the excretion of phosphoric acid is 
 a measure of the absorption and decomposition of these bodies. 1 The 
 greater part originates from the phosphates of the food, and the quan- 
 tity of phosphoric acid eliminated is greater when the food is rich in 
 alkali phosphates in proportion to the quantity of lime and magnesium 
 phosphates. If the food contains much lime and magnesia, large quan- 
 tities of earthy phosphates are eliminated by the excrement; and even 
 though the food contains considerable amounts of phosphoric acid in these 
 cases, the quantity excreted by the urine is small. This is especially 
 true of herbivora, in which the kidneys are the chief organs for the 
 excretion of alkali phosphates. In man, according to Ehrstrom, the 
 content of lime in the food seems to play no important role, as in his exper- 
 iments about one-half of the phosphoric acid taken as CaHP04 was 
 absorbed, still the extent of phosphoric-acid excretion through the urine 
 depends in man not only upon the total quantity of phosphoric acid in 
 the food, but also upon the relative amounts of the alkaline earths and the 
 alkali salts of the food. In carnivora, in which phosphate injected sub- 
 cutaneously is eliminated by the intestine (Bergmann), the urine is 
 habitually poor in phosphates. 2 
 
 As the extent of the elimination of phosphoric acid is mostly dependent 
 upon the character of the food and the absorption of the phosphates 
 in the intestine, it is apparent that the relation between the nitrogen and 
 phosphoric-acid excretion cannot run parallel. This is in fact so, and, 
 according to Ehrstrom, the organism has the power of accumulating 
 large quantities of phosphorus for a relatively long time independent of 
 the condition of the nitrogen balance. With a certain regular food the 
 relation between nitrogen and phosphoric acid in the urine can be kept 
 almost constant. Thus on feeding with an exclusive meat diet, as 
 observed by Voit 3 in dogs, when the nitrogen and phosphoric acid 
 
 1 See A. Gumlich, Zeitschr. f. physiol. Chem., 18; Roos, ibid., 21; Weintraud, 
 Arch. f. (Anat. u.) Physiol., 1895; Milroy and Malcolm, Journ. of Physiol., 23; Roh- 
 mann and Steinitz, Pfluger's Arch., 72; Loewi, Arch. f. exp. Path. u. Pharm., 44 and 45. 
 
 2 Ehrstrom, Skand. Arch. f. Physiol., 14; Bergmann, Arch. f. exp. Path. u. Pharm., 
 
 47. 
 
 3 Physiologie des allgemeinen Stoffwechsels und der Ernahrung in L. Hermann's 
 Handbuch, (J, Thill. 1, 7'.). 
 
PHOSPHATES. 763 
 
 (P2O5) of the food exactly reappeared in the urine and feces, the rela- 
 tion was 8.1:1. In starvation, as shown by the compilation of R. Tiger- 
 stedt, 1 the phosphorized constituents of the body are destroyed to a 
 much greater extent than when food very poor in phosphorus is given. 
 In starvation this relation is changed, namely, relatively more phosphoric 
 acid is eliminated, which seems to indicate that besides flesh and related 
 tissues another tissue rich in phosphorus is largely destroyed. The 
 starvation experiments show that this is the bone-tissue. According to 
 Preysz, Olsavszky, Klug, I. Munk and Maillard 2 the elimination 
 of phosphoric acid is considerably increased by intense muscular work. 
 As the phosphoric acid is in part derived from the nucleins, it would 
 be expected that in those diseases in which the excretion of purine 
 bodies was increased the phosphoric acid would also be augmented. 
 This is not the case, and indeed we have observed cases with an increased 
 elimination of purine bodies with a diminution in the phosphoric-acid 
 excretion. Cases of leucaemia have been observed in which the phos- 
 phoric-acid excretion was reduced, although there was a pronounced 
 increase in the number of leucocytes. In these cases there may be a 
 subsequent excretion or a retention of phosphoric acid. This last condition 
 also occurs in inflammatory and renal diseases. The earthy phosphates 
 of the urine sometimes have the tendency of precipitating either spon- 
 taneously or after warming, and this has been called phosphaturia. We 
 are here dealing with a diminished acidity and, it seems, with a dimin- 
 ished excretion of phosphoric acid and an increased elimination of lime, 
 or at least an essentially different relation between the phosphoric acid 
 and the alkaline earths of the urine, as compared with the normal (Panek 
 Iwanoff, Soetber and Krieger 3 ). 
 
 Quantitative Estimation of the Total Phosphoric Acid in the Urine. 
 This estimation is most simply performed by titrating with a solution 
 of uranium acetate. The principle of the titration is as follows: A 
 warm solution of phosphates containing free acetic acid gives a whitish- 
 yellow precipitate of uranium phosphate with a solution of a uranium 
 salt. This precipitate is insoluble in acetic acid, but dissolves in mineral 
 acids, and on this account there is always added, in titrating, a certain 
 quantity of sodium-acetate solution. Potassium ferrocyanide is used 
 as the indicator, which does not act on the uranium-phosphate precipitate, 
 but gives a reddish-brown precipitate or coloration in the presence of the 
 
 1 Skand. Arch. f. Physiol., 16. 
 
 2 Preysz, see Maly's Jahresber., 21; Olsavszky and Klug, Pfluger's Arch., 54; 
 Munk, Arch. f. (Anat. u.) Physiol., 1895; Maillard, Journ. de Phvsiol. et de Path. 
 10 and 11. 
 
 3 Panek, see Maly's Jahresber., 30, 112; Iwanoff, Biochem. Centralbl., 1, 710; 
 Soetber^and Krieger, Deutsch. Arch. f. klin. Me.!., "2; Cainpaui, Biochem. Centralbl., 
 3, 616; Tobler, Arch. f. exp. Path. u. Phann., 52. 
 
764 URINE. 
 
 smallest amount of soluble uranium salt. The solutions necessary for 
 the titration are: 1. A solution of a uranium salt of which each cubic 
 centimeter corresponds to 0.005 gram P2O5 and which contains 20.3 
 grams of uranium oxide per liter. 20 cc. of this solution corresponds 
 to 0.100 gram P2O5. 2. A solution of sodium acetate. 3. A freshly 
 prepared solution of potassium ferrocyanide. 
 
 The uranium solution is prepared from uranium nitrate or acetate. Dissolve 
 about 35 grams uranium acetate in water, add some acetic acid to facilitate solu- 
 tion, and dilute to 1 liter. The strength of this solution is determined by titrat- 
 ing withasolution of sodium phosphate of known strength (10.085 grams crystallized 
 salt in 1 liter, which corresponds to 0.010 gram P 2 6 in 50 cc). Proceed in the 
 same way as in the titration of the urine (see below), and correct the solution by 
 diluting with water, and titrate again until 20 cc. of the uranium solution cor- 
 responds exactly to 50 cc. of the above phosphate solution. 
 
 The sodium-acetate solution should contain 10 grams sodium acetate and 
 10 grams cone, acetic acid in 100 cc. For each titration 5 cc. of this solution 
 is used with 50 cc. of the urine. 
 
 In performing the titration, mix 50 cc. of filtered urine in a beaker 
 with 5 cc. of the sodium acetate, cover the beaker with a watch-glass, 
 and warm over the water-bath. Then allow the uranium solution to 
 flow in from a burette, and when the precipitate does not seem to increase, 
 place a drop of the mixture on a porcelain plate with a drop of the potas- 
 sium-ferrocyanide solution. If the amount of uranium solution added 
 has not been sufficient, the color will remain pale yellow and more 
 uranium solution must be added; but as soon as the slightest excess of 
 uranium solution has been used the color becomes a faint reddish brown. 
 When this point has been obtained, warm the solution again and add 
 another drop. If the color remains of the same intensity, the titration 
 is ended; but if the color varies, add more uranium solution, drop by 
 drop, until a permanent coloration is obtained after warming, and now 
 repeat the test with another 50 cc. of the urine. The calculation is so 
 simple that it is unnecessary to give an example. 
 
 In the above manner one determines the total quantity of phosphoric 
 acid in the urine. If we wish to know the phosphoric acid combined 
 with alkaline earths and with alkalies, we first determine the total phos- 
 phoric acid in a portion of the urine and then remove the earthy phos- 
 phates in another portion by ammonia. The precipitate is collected on 
 a filter, washed, transferred into a beaker with water, treated with acetic 
 acid, and dissolved by warming. This solution is now diluted to 
 50 cc. with water, and 5 cc. sodium-acetate solution added, then 
 titrated with uranium solution. The difference between the two deter- 
 minations gives the quantity of phosphoric acid combined with the 
 alkalies. The results obtained are not quite accurate, as a partial trans- 
 formation of the monophosphates of the alkaline earths and also calcium 
 diphosphate into triphosphates of the alkaline earths and ammonium 
 phosphate takes place on precipitating with ammonia, and the method 
 gives too high results for the phosphoric acid combined with alkalies and 
 remaining in solution. 
 
 Sulphates. The sulphuric acid of the urine originates only to a very 
 small extent from the sulphates of the food. A disproportionately 
 
SULPHATES. 765 
 
 greater part is formed by the burning within the body of the proteins 
 which contain sulphur, and it is chiefly this formation of sulphuric acid 
 from the proteins which gives rise to the previously mentioned excess 
 of acids over the bases in the urine. The quantity of sulphuric acid 
 eliminated by the urine amounts to about 2.5 grams H2SO4 per day. 
 As the sulphuric acid chiefly originates from the proteins, it follows that 
 the elimination of sulphuric acid and the elimination of nitrogen runs 
 almost parallel, and the relation Nit^SO* is about 5:1. A complete 
 parallelism can hardly be expected, as in the first place a part of the sul- 
 phur is always eliminated as neutral sulphur, and secondly because the 
 small proportion of sulphur in different protein bodies undergoes greater 
 variation as compared with the large proportion of nitrogen contained 
 therein. In general the elimination of nitrogen and sulphuric acid under 
 normal and under diseased conditions seems to run parallel. Sulphuric 
 acid occurs in the urine partly preformed (sulphate-sulphuric acid) and 
 partly as ethereal-sulphuric acid. The first is designated as A- and the 
 other as ^-sulphuric acid. 
 
 The quantity of total sulphuric acid is determined in the following 
 way, but at the same time the precautions described in other works 
 must be observed. 100 cc. of filtered urine is treated with 5 cc. of con- 
 centrated hydrochloric acid and boiled for fifteen minutes. While 
 boiling precipitate with 2 cc. of a saturated BaCb solution, and warm for 
 a little while until the barium sulphate has completely settled. The 
 precipitate must then be washed with water and also with alcohol and 
 ether (to remove resinous substances), and then treated according to 
 the usual method. 
 
 The separate determination of the sulphate-sulphuric acid and the 
 ethereal-sulphuric acid may be accomplished, according to Baumann's 
 method, by first precipitating the sulphate-sulphuric acid by BaCb from 
 the urine acidified with acetic acid, then decomposing the ethereal- 
 sulphuric acid by boiling after the addition of hydrochloric acid, and 
 finally determining the sulphuric acid set free as barium sulphate. A 
 still better method is the following, suggested by Salkowski l : 
 
 200 cc. of urine are precipitated by an equal volume of a barium solu- 
 tion, which consists of 2 vols, barium hydrate and 1 vol. barium chloride 
 solution, both saturated at the ordinary temperature. Filter through 
 a dry filter, measure off 100 cc. of the filtrate which contains only the 
 ethereal-sulphuric acid, treat with 10 cc. of hydrochloric acid of a specific 
 gravity 1.12, boil for fifteen minutes, and then warm on the water-bath 
 until the precipitate has completely settled and the supernatant liquid 
 is entirely clear. Filter and wash with warm water and with alcohol 
 and ether, and proceed according to the generally prescribed method. 
 The difference between the ethereal-sulphuric acid found and the total 
 quantity of sulphuric acid as determined in a special portion of urine 
 is taken to be the quantity of sulphate-sulphuric acid. 
 
 1 Baumann, Zeitschr. £. physiol. Chem., 1; Salkowski, Virchow's Arch., 79. 
 
766 URINE. 
 
 Folin a has suggested a method for estimating the sulphate-sul- 
 phuric acid as well as the ethereal-sulphuric acid, and also the totaL 
 sulphur, which is somewhat different from the ordinary methods. 
 
 Nitrates occur in small quantities in human urine (Schonbein), and they 
 probably originate from the drinking-water and the food. According to Weyl 
 and Citron, 2 the quantity of nitrates is smallest with a meat diet and greatest 
 with vegetable food. The average amount is about 42.5 milligrams per liter. 
 
 Potassium and Sodium. The quantity of these bodies eliminated 
 by the urine by a healthy adult on a mixed diet is, according to Salkow- 
 ski, 3 3-4 grams K2O and 5-8 grams Na20, with an average of about 2-3 
 grams K2O and 4-6 grams Xa20. The proportion of K to Na is ordinarily 
 3:5. The quantity depends above all upon the food. In starvation 
 the urine may become richer in potassium than in sodium, which results 
 from the lack of common salt and the destruction of tissue rich in potas- 
 sium. The quantity of potassium may be relatively increased during 
 fever, while after the crisis the reverse is the case. 
 
 The quantitative estimation of these bodies is made by the gravi- 
 metric methods as described in works on quantitative analysis. In 
 the determination of the total alkalies new methods have been devised 
 by Pribram and Gregor, and for the potassium alone a method by 
 Autenrieth and Bernheim. 4 
 
 Ammonia. Some ammonia is habitually found in human urine and 
 in that of carnivora. The quantity in human urine on a mixed diet is 
 an average of 0.7 gram, according to Neubauer. Maillard 5 found 
 higher values for soldiers, namely 1.11 grams. The ammonia nitrogen 
 relative to the total nitrogen is, on a mixed diet, 3.6-5.8 per cent. 
 
 As above stated (page 685), on the formation of urea from ammonia, 
 this quantity may represent the small amount of ammonia which is 
 excluded from the synthesis to urea by being combined with acids formed 
 in excess by combustion and not united with the fixed alkalies. This 
 view is confirmed by the observation that the elimination of ammonia was 
 smaller on a vegetable diet and larger on a rich meat diet than on a mixed 
 diet. After abundant meat feeding Bouchez found, for example, 1.35- 
 1.67 gram NH? in twenty-four hours. The relationship of the ammonia 
 elimination to the acid formation in the animal body corresponds also 
 to the unquestioned relation between the hydrochloric acid content of the 
 
 1 Journ. of Biol. Chem., 1, and Amer. Journ. of Physiol., 13. 
 
 5 Schonbein, Journ. f. prakt. Chem., 92; Weyl, Virchow's Arch., 96, with Citron, 
 iUd., 101. 
 
 ' Ibid., 53. 
 
 4 Pribram and Gregor, Zeitschr. f. analyt. Chem., 38; Autenreith and Bernheim, 
 Zeitschr. f. phyaol. f'hem., 37. 
 
 1 Journ. de Physiol, et de Path., 10. 
 
AMMONIA. 767 
 
 gastric juice and the ammonia elimination. Thus Hchittenhelm found 
 that with a rise in the hydrochloric acid content the percentage of ammonia 
 in the urine was raised and also the reverse. A. Loeb and v Jammfltoft l 
 have also observed a fall in the ammonia elimination a few hours ufter a 
 meal, although no satisfactory explanation of this behavior has been given. 
 That ammonia plays the role of a neutralization medium for the acids 
 produced in the body or introduced therein has been shown by various 
 observations. 
 
 In man and certain animals the elimination of ammonia is increased 
 by the introduction of mineral acids; and, as shown by Jolin, 2 organic 
 acids, such as benzoic acid, which are not destroyed in the body act in a 
 similar manner. The ammonia set free in the protein destruction is in 
 part used in the neutralization of the acids introduced, and in this way 
 a destructive removal of fixed alkalies is prevented. ■■■■■ ~^ 
 
 Acids formed in the destruction of proteins in the body act on the 
 elimination of ammonia like those introduced from without. For this 
 reason the quantity of ammonia in human urine is increased under such 
 conditions and in such diseases where an increased formation of acid 
 takes place, because of an increased metabolism of proteins. This is the 
 case with a lack of oxygen in fevers and diabetes. In the last-mentioned 
 disease, organic acids — /3-oxybutyric acid and acetoacetic acid — are pro- 
 duced, which pass into the urine combined with ammonia. 3 
 
 The liver forms urea from the ammonia supplied to it by the blood 
 and it would therefore be expected that in certain diseases of the liver 
 or with insufficient liver function that a diminished urea formation and an 
 increased ammonia elimination should take place. This condition has 
 already been mentioned above (page 685), and as there remarked we 
 must consider whether the abnormal production of acid with increased 
 elimination of neutralization ammonia is primary or whether it is a 
 diminished synthetic activity of the liver. 
 
 In close relation to what has been said stands the question whether all 
 of the ammonia occurring in the urine under normal conditions is to be 
 considered as neutralization ammonia. If this were so then probably 
 by introducing large amounts of alkali it would be possible to cause the 
 disappearance of ammonia from the urine. In Stadelmann and Beck- 
 
 : Bouchez, Journ. de Physiol, et de Path., 14; Schittenhelm, Deutsch. Archiv. f. 
 klin. Med., 77; Adam Loeb, Zeitschr. f. klin. Med., 56, and Zeitschr. f. Biol., 55; Gam- 
 meltoft, Zeitschr. f. physiol. Chem., 75. 
 
 2 Jolin, Skand. Arch. f. Physiol., 1. In regard to the behavior of ammonium salts 
 in the animal body, see Rumpf and Kleine, Zeitschr. f. Biologic, 54; Kowalewski and 
 Markewicz, Bioch. Zeitschr., 4, and the works cited on pages 682, 683. 
 
 s On the elimination of ammonia in disease, see the works of Rumpf, Virchow's 
 Arch., 143; Hallervorden, ibid. 
 
768 URINE. 
 
 mann's experiments this was not possible, still in recent experiments of 
 Janney ! it was possible, by introducing large quantities of sodium citrate, 
 which was burned in the body into carbonate, to reduce the ammonia 
 elimination to very insignificant quantities. 
 
 The detection and quantitative estimation of ammonia used to be performed 
 according to the method suggested by Schlosing. The principle of this method 
 is that the ammonia from a measured amount of urine is set free by lime-water 
 in a closed vessel and absorbed by a measured amount of N/10 sulphuric acid. 
 After the absorption of the ammonia the quantity is determined by titrating the 
 remaining free sulphuric acid with a N/10 caustic-alkali solution. This method 
 gives low results, and in exact work we must proceed as suggested by Bohland. 2 
 
 The recent methods for estimating the ammonia are all based upon 
 the distillation of the ammonia, after the addition of lime, magnesia, 
 or alkali carbonate, at low temperatures either by the aid of vacuum 
 (Nencki and Zaleski, Wurster, Kruger, Reich and Schittenhelm 
 and Schaffer) or by the aid of a current of air (Folin) and then collect- 
 ing it in a standard acid. 
 
 According to the methods suggested by Kruger, Reich and Schitten- 
 helm 3 25 cc. of the urine are placed in a distillation-flask with about 
 10 grams of NaCl and 1 gram of Na 2 CC>3, and this distilled at 43° C. 
 and a pressure of 30-40 millimeters Hg with the aid of an air-pump. 
 Alcohol is added to prevent foaming. The ammonia is absorbed in 
 N/10 acid contained in a Peligot tube surrounded by ice-water, and 
 when the distillation is finished the acid is retitrated, making use of 
 rosolic acid as indicator. In regard to details, see the original publica- 
 tions. Instead of alkali carbonate a one-half normal solution of barium 
 hvdrate in methyl alcohol can be used. According to Folin's 4 method, 
 25-50 cc. of the urine are treated in a wash-bottle with 1-2 grams soda and 
 8-10 grams sodium chloride and some petroleum, in order to prevent 
 frothing, and then a current of air is passed through and this passed 
 through a second wash-bottle containing N/10 acid. It has also been 
 suggested (Ronchese, Malfatti and others) to determine the ammonia 
 by the formol titration. This method is based upon the fact that an 
 ammonium salt yields hexamethylentetramine and free acid with for- 
 maldehyde according to the equation 4NH4Cl-f-6HCOH==C6Hi2N4 
 + 6H 2 0+4HC1. This acid is determined by titration after the addition 
 of formol. Folin 5 also recently suggested a method for the quantitative 
 colorimetric estimation of ammonia by the use of Nessler's reagent. 
 
 Calcium and Magnesium occur in the urine chiefly as phosphates. 
 The quantity of earthy phosphates eliminated daily is somewhat more 
 
 1 N. Janney, Zeitschr. f. physiol. Chem., 76, which also contains the literature. 
 
 2 Pfliiger's Arch., 43, 32. 
 
 3 Zeitschr. f. physiol. Chem., 39; Schaffer, Amer. Journ. of Physiol., 8, which contains 
 the literature. Henriques and S6rensen, Zeitschr. f. physiol. Chem., 64. 
 
 • Folin, Zeitschr. f. physiol. Chem., 37, and Journ. of biol. Chem., 8; Steel, ibid., 8. 
 
 6 Ronchese, see Maly's Jahresber., 38, 321; Malfatti, Zeitschr. f. anal. Chem., 47; 
 il. BjOrn-Anderseii and M. Lauritzen, Zeitschr. f. physiol. Chem., 64; L. de Jager, 
 ibid., 62; Folin and Maccallum, Journ. of biol. Chem., 11. 
 
CALCIUM AND MAGNESIUM. 7GD 
 
 than 1 gram, and of this amount § la magnesium and ^ calcium phos- 
 phate. This statement, as found by Renwall and Gross, is not correct, 
 or at least is not true in general, as they found more calcium than mag- 
 nesium in the urine. Long and Gephart j obtained similar results. 
 In acid urines the mono- as well as the dihydrogen earthy phosphates 
 are found, and the solubility of the first, among which the calcium salt 
 CaHP04 is especially insoluble, is particularly augmented by the presence 
 in the urine of dihydrogen alkali phosphates and sodium chloride (Ott 2 ) . 
 The quantity of alkaline earths in the urine depends on the composi- 
 tion of the food. The lime-salts absorbed are in great part excreted 
 again into the intestine, and the quantity of lime-salts in the urine is 
 therefore no measure of their absorption. The introduction of readily 
 soluble lime-salts or the addition of hydrochloric acid to the food may 
 therefore cause an increase in the quantity of lime in the urine, while 
 the reverse takes place on adding alkali phosphate to the food. Accord- 
 ing to Granstrom starvation in rabbits or the introduction of food which 
 yields an acid ash and causes an acid urine produces the same effect as 
 the introduction of acid. The observation of de Jager 3 is significant, 
 namely, he found that the partaking of CaS04 and to a higher degree 
 of MgSCU causes an increase in the urine ammonia and of acid. Noth- 
 ing is known with certainty in regard to the constant and regular change 
 in the elimination of calcium and magnesium salts in disease, and in these 
 conditions the excretion is chiefly dependent upon the diet and the forma- 
 tion and introduction of acid. 4 
 
 The quantity of calcium and magnesium is determined according to 
 the ordinary well-known methods. 
 
 Iron occurs in the urine only in small quantities, and it does not exist as a 
 salt, but as an organic combination of a colloidal nature. The earlier reports in 
 regard to the iron present seem to show that the quantity ranges from 1 to 11 
 milligrams per liter of urine. Hoffmann, Neumann and Mayer found lower 
 results — an average of 1.09 and 0.983 milligrams and according to recent determi- 
 nations of Wolter and Reich 8 the quantity is about 1 milligram. The quantity 
 of silicic acid is ordinarily stated to amount to about 0.3 p. m. H. Schulz 6 found 
 
 1 Renwall, Skand. Arch. f. Physiol., 16; Gross, Biochem. Centralbl., 4, 189; Long 
 and Gephart, Journ. Amer. Chem. Soc, 34. 
 
 2 Zeitschr. f. physiol. Chem., 10. 
 
 s Granstrom, Zeitschr. f. physiol. Chem., 58; de Jager, Bioch. Zeitschr., 38. 
 
 4 See page 758, Albu and Neuberg, 1. c, and E. Zak, Ueber Harn bei Lungenent- 
 ziindung, Wien. klin. Wochenschr., 21. 
 
 6 Kunkei, cited from Maly's Jahresber., 11; Giacosa, ibid., 16; Kobert, Arbeiten 
 des Pharm. Inst, zu Dorpat, 7; Magnier, Ber. d. deutsch. chem. Gesellsch., 7; Gott- 
 lieb, Arch. f. exp. Path. u. Pharm., 26; Jolles, Zeitschr. f. anal. Chem., 36; Hoff- 
 mann, Zeitschr. f. anal. Chem., 40; Neumann and Mayer, Zeitschr. f. physiol. 
 Chem., 37; Wolter, Bioch. Zeitschr., 24; Reich, ibid., 36. 
 
 6 Pfluger's Arch,, 144. 
 
770 URINE. 
 
 0.1046 to 0.2594 grains per day on a mixed diet. Traces of hydrogen peroxide 
 also occur in the urine. 
 
 The gases of the urine are carbon dioxide, nitrogen, and traces of 
 oxygen. The quantity of nitrogen is not quite 1 vol. per cent. The 
 carbon dioxide varies considerably. In acid urines it is hardly one-half 
 as great as in neutral or alkaline urines. 
 
 IV. THE QUANTITY AND QUANTITATIVE COMPOSITION OF URINE. 
 
 The quantity and composition of urine are liable to great variation. 
 The circumstances which under physiological conditions exercise a great 
 influence are the following: the blood-pressure, and the rapidity of the 
 blood-current in the glomeruli. The quantity of urinary constituents, 
 especially water in the blood; and, lastly, the condition of the secretory 
 glandular elements. Above all, the quantity and concentration of the 
 urine depend on the quantity of water which is introduced into the blood 
 or which leaves the body in other ways. The excretion of urine is increased 
 by drinking freely or by reducing the quantity of water otherwise removed; 
 and it is decreased by a diminished ingestion of water or by a greater loss 
 of water in other ways. Ordinarily in man just as much water is elimi- 
 nated by the kidneys as by the skin, lungs, and intestine together. At 
 lower temperatures and in moist air, since under these conditions the 
 elimination of water by the skin is diminished, the excretion of urine 
 may be considerably increased. Diminished introduction of water or 
 increased elimination of water by other means — as in violent diarrhoea or 
 vomiting, or in profuse perspiration — greatly diminishes the amount of 
 urine excreted. For example, the urine may sink as low as 500-400 cc. 
 per day in intense summer heat, while after copious draughts of water 
 the elimination of 3000 cc. of urine has been observed during the same 
 time. The quantity of urine voided in the course of twenty-four hours 
 varies considerably from day to day, the average being ordinarly cal- 
 culated as 1500 cc. for healthy adult men and 1200 cc. for women. The 
 minimum elimination occurs during the early morning between 2 and 4 
 o'clock; the maximum, in the first hours after waking and from 1-2 
 hours after a meal. 
 
 The quantity of solids excreted per day is nearly constant, even though the 
 quantity of urine may vary, and it is quite constant when the manner of living 
 is regular. Therefore the percentage of solids in the urine is naturally in inverse 
 proportion to the quantity of urine. The average amount of solids per twenty- 
 four hours is calculated as 60 grams. The quantity may be calculated with approx- 
 imate accuracy from the specific gravity if the second and third decimals of this 
 factor be multiplied by Haser's coefficient, 2.33. The product gives the amount 
 of solids in 1000 cc. of urine, and if the quantity of urine eliminated in twenty- 
 four hours be measured, the quantity of solids in twenty-four hours may be 
 easily calculated. For example, 1050 cc. of urine of a specific gravity 1.021 was 
 
QUANTITATIVE COMPOSITION. 771 
 
 eliminated in twenty-four hours; therefore the quantity of solids excreted was 
 21 X 2.33 =48.9 and iqqq = 51.35 grams. Long > has made a new determina- 
 tion of the coefficient for the specific gravity taken at 25° C. and finds that it is 
 equal to 2.<'>, which almost corresponds to Haser's coefficient at 15° C. 
 
 Those hodics which, under physiological conditions, affect the density 
 of the urine are common salt and urea. The specific gravity of the first 
 is 2.15 and the last only 1.32, so it is easy to understand, when the relative 
 proportion of these two bodies essentially deviates from the normal, 
 why the above calculation from the specific gravity is not exact. The 
 same is true when a urine poor in normal constituents contains large 
 amounts of foreign bodies, such as albumin or sugar. 
 
 As above stated, the percentage of solids in the urine generally decreases 
 with a greater elimination, and a very considerable excretion of urine 
 (polyuria) has therefore, as a rule, a lower specific gravity. An important 
 exception to this rule is observed in urine containing sugar (diabetes 
 mellitus), in which there is a copious excretion with a very high specific 
 gravity due to the sugar. In cases where very little urine is excreted 
 (oliguria), e.g., during profuse perspiration, in diarrhoea, and in fevers, 
 the specific gravity of the urine is as a rule very high; the percentage of 
 solids is also high and the urine has a dark color. Sometimes, as for 
 example, in certain cases of albuminuria, the urine may have a lew specific 
 gravity notwithstanding the oliguria, and be poor in solids and light in 
 color. 
 
 In certain cases it is interesting to know the relation between the 
 earlwn and the nitrogen, or the quotient C/N. This factor may vary 
 between 0.6 and 1 ; as a rule, it amounts on an average to 0.87, but changes 
 according to the nature of the food and is higher after a diet rich in carbo- 
 hydrates than after food rich in fat (Pregl, Tangl, Langstein and 
 Steinitz). According to Magnus-Alsleben it rises after body exer- 
 tion, but in healthy individuals the variation is independent of the 
 kind of food. In the urine analyses of Bouchez 2 a variation between 
 0.62 and 0.90 was observed which showed no regular relation to the food. 
 On account of the great variations which the composition of the urine 
 shows it is difficult and of little value to give a tabular review of the 
 composition of the urine. The following table contains only approximate 
 values and it must not be overlooked that the results are not given for 
 1000 parts of urine, but only approximate figures for the quantities 
 
 1 Journ. Amer. Chem. Soc, 25. 
 
 2 Pregl, Pfliiger's Arch., 75, which contains the earlier literature. Tangl, Arch. f. 
 (Anat. u.) Physiol., 1899, Suppl.; Langstein and Steinitz, Centralbl. f. Physiol., 19; 
 Magnus-Alsleben, Zeitschr. f. klin. Med., 68, Bouchez, footnote 1, page 767. 
 
772 URINE. 
 
 of the most important constituents which are eliminated during the 
 course of twenty-four hours in a volume of 1500 cc. of urine. These 
 figures apply only to a diet which corresponds to Voit's standard figures, 
 namely 118 grams protein, 56 grams fat, and 500 grams carbohydrate 
 per day, and to a man of average weight. 
 
 Daily quantity of solids = 55-70 grams. 
 
 Organic constituents 35-45 grams. Inorganic constituents 20-25 grams. 
 
 Urea 25-35.0 grams. Sodium chloride (NaCl) . 10-15.0 grams. 
 
 Uric acid 0.7 " Sulphuric acid (H 2 S0 4 ). . 2.5 " 
 
 Creatinine 1.5 " Phosphoric acid (P 2 5 ) . . 2.5 " 
 
 Hippuric acid 0.7 " Potash (K 2 0) 3.3 " 
 
 Ammonia (NH 3 ) 0.7 " 
 
 Magnesia (MgO) \ n fi 
 
 Lime (CaO) /■•••■ u,a 
 
 Urine contains on an average 40 p. m. solids. The quantity of urea is 
 about 20 p. m., and common salt about 10 p. m. 
 
 The physico-chemical methods are being used in urinary analysis even to a 
 greater extent than in the analysis of other animal fluids. A great number 
 of eryoscopie determinations, but fewer conduct ivitj r determinations, have been 
 made. A constant relation between the values found by physico-chemical methods 
 and the analytical methods has been sought, for example, between the freezing- 
 point depression and the specific gravity or the common salt content and others; or 
 have been made to find certain constants in the composition of the urine based 
 upon the results of various methods, and in this way to obtain an explanation 
 as to the mechanism of the excretion of urine in order to apply them for diag- 
 nostic purposes. The results obtained are, as is to be expected, so variable and 
 dependent upon so many conditions which cannot be controlled that definite 
 conclusions must be drawn with the greatest caution. In regard to the value and 
 usefulness of the various constants and relations which are based upon theoretical 
 considerations, opinions are unfortunately still too divergent and as the plan and 
 scope of this book do not allow of more detailed description of these facts we 
 must refer to larger works on the urine and diseases of the kidneys. 
 
 V. CASUAL URINARY CONSTITUENTS. 
 
 The casual appearance, in the urine, of medicinal agents or of urinary 
 constituents resulting from the introduction of foreign substances into 
 the organism is of practical importance, because such compounds may 
 interfere in certain urinary investigations; they also afford a good means 
 of determining whether certain substances have been introduced into the 
 organism or not. From this point of view a few of these bodies will be 
 spoken of in a following section (on the pathological urinary constituents) . 
 The presence of these foreign bodies, in the urine, is of special interest 
 in those cases in which they serve to elucidate the chemical transformations 
 which certain substances undergo within the organism. As inorganic 
 substances generally leave the body unchanged, 1 they are of very little 
 
 1 In regard to the behavior of certain of these bodies, see Heffter, Die Ausscheidung 
 korperfremden Substanzen im Ham, Ergebnisse d. Physiol., 2, Abt. 1. 
 
CASUAL URINARY CONSTITUENTS. 773 
 
 interest from this standpoint; but the changes which certain organic 
 substances undergo when introduced into the animal body may be studied 
 by the transformation products as found in the urine. 
 
 The bodies belonging to the fatty series undergo, though not without 
 exceptions, a combustion leading toward the final products of metab- 
 olism; still, often a greater or smaller part of the bodies in question 
 escape oxidation and appear unchanged in the urine. A part of the acids 
 belonging to this series, which are otherwise decomposed into water and 
 carbonates, and render the urine neutral or alkaline, may act in this manner. 
 The volatile fatty acids poor in carbon are less easily oxidized than those 
 rich in carbon, and they therefore pass unchanged into the urine in 
 large amounts. This is especially true of formic and acetic acids 
 (Schotten, Gr6hant and Quinquaud r ). In birds, according to Gaglio 
 and Giunti, oxalic acid is not oxidized. Opinions on the behavior of 
 oxalic acid in mammalia and man, are conflicting; the investigations 
 of Salkowski and especially of Hildebrandt and Dakin 2 show 
 that oxalic acid, when introduced in medium amounts, is in part 
 oxidized in the animal body. Racemic acid, d-l tartaric acid, passes 
 (in dogs) in part into the urine, and this unburned part is optically inactive 
 according to Neuberg and Saneyoshi. The statement of Biron 3 
 that Z-tartaric acid is more readily burned than e?-tartaric acid is accord- 
 ingly incorrect, and the cW-tartaric acid therefore does not belong to 
 those substances which are asymmetrically attacked in the animal 
 body. Malic acid and citric acid belong to those acids which are in great 
 part burned in the body. 4 
 
 The destruction of normal fatty acids with several membered chains 
 takes place, our belief being based upon the work of Knoop and Dakin 5 
 especially, in an oxidation in the 0-position, i.e., in the group which is in 
 the /3-position to the carboxyl group at the end. The conversion into an 
 
 1 Schotten, Zeitschr. f. physiol. Chem., 7; Grehant and Quinquaud, Compt. Rend., 
 104. 
 
 2 Gaglio, Arch. f. exp. Path. u. Pharm., 22; Giunti, Chem. Centralbl., 1897, 2; 
 Marfori, Maly's Jahresber., 20 and 27; Pohl, Arch. f. exp. Path. u. Pharm., 37; Sal- 
 kowski, Berl. klin. Wochenschr., 1900; Pierallini, Virchow's Arch., 160; Stradomsky, 
 i&td.,163; Klemperer and Tritschler, Zeitschr. f. klin. Med., 44; Hildebrandt, Zeitschr. 
 f. physiol. Chem., 35; Dakin, Journ. of biol. Chem., 3. 
 
 3 Biron, Zeitachr. f. physiol. Chem., 25; Neuberg and Saneyoshi, Bioch. Zeitschr., 
 36. O 
 
 4 Pohl, Arch. f. exp. Path. u. Pharm., 37, which also contains reports on the inter- 
 mediary products formed in the oxidation of the fatty bodies; K. Ohta, Bioch. 
 Zeitschr., 44. 
 
 5 F. Knoop, Hofmeister's Beitrage, 6 and Habilit.-Schrift, Freiburg, 1904; Dakin, 
 Journ. of biol. Chem., 4, 5, 6 and 9. 
 
774 URINE. 
 
 acid having two carbon atoms less takes place according to this assump- 
 tion according to the formula: 
 
 R.CH 2 .CH 2 .COOH-*R.CH(OH).CH2.COOHR.CO.CH2.COO±^H-> 
 
 R.COOH. 
 
 The animal body has therefore the ability to transform oxyacids (alcohol 
 acids) into keto-acids by oxidation as well as the reverse, the conversion 
 of keto-acids into oxyacids, and this behavior, which is indicated by the 
 above formula, makes it difficult to state which products are primary 
 and which are secondary. As example of such a reversible process we 
 will mention the following; the 0-oxybutyric acid CH 3 .CH(OH).CH 2 COOH 
 is transformed by oxidation into the keto-acid, acetoacetic acid, 
 CH3.CO.CH2COOH, and this latter by reduction is changed into /3- 
 oxybutyric acid. Both processes may take place, as Friedmann and 
 Maase, Dakin and Wakeman * have shown, in the liver, and as these 
 two so-called acetone bodies have great importance in diabetes, they 
 may serve also as an example of the first stages of a ^-oxidation (of 
 n. butyric acid). 
 
 Most of the investigations on the demolition of fatty acids have been 
 carried out by Knoop, Dakin, Friedmann and others upon substituted, 
 especially phenyl-substituted fatty acids, and in speaking of the behavior 
 of the cyclic compounds we will discuss the behavior of these. 
 
 The amino-acids are, when large amounts are introduced into the 
 animal body, eliminated unchanged, and even under physiological con- 
 ditions traces of the amino-acids formed in the animal body can pass 
 into the excretions — glycocoll in the urine and serine in the perspiration. 
 Otherwise they are as a rule decomposed and a deamidation takes place, 
 the ammonia split off serving for material for the formation of urea. 
 The two components of a racemic a-amino-acid behave differently in that 
 the alien component is burned with greater difficulty and less completely 
 than the component occurring in the body protein, which is burned 
 more readily and more completely. 
 
 In the demolition of the a-amino-acids, fatty acids, poorer in carbon, 
 are formed; the detailed manner of this demolition has been explained 
 in various ways. 
 
 According to a long-accepted view it was believed that a hydrolytic 
 splitting off of ammonia with the formation of the corresponding oxyacid 
 (alcohol acid) took place, according to the formula R.CHNH2.COOH-f- 
 H 2 = R.CH(OH).COOH+NH 3 , and then a further demolition to 
 
 1 Friedmann and Maase, Bioch. Zeitschr., 27; Dakin and Wakeman, Journ. of biol. 
 Chem., 8. 
 
CASUAL URINARY CONSTITUENTS. 775 
 
 R.COOH. The appearance of lactic acid in the urine of rabbits after 
 feeding alanine is an example of such deamidation. 1 The possibility 
 is not excluded that in the first place the keto-acid, pyruvic acid, 
 CH3.CO.COOH, is formed from the alanine and then the lactic acid, 
 CH3.CHOH.COOH, formed from this as a secondary reduction product. 
 
 In agreement with the views of Xeubauer 2 it is now rather 
 generally conceded that the hydrolytic deamidation is not as important 
 as the oxidative deamidation, with the formation of keto-acids 
 R.CH(NH 2 ).COOH+0 = R.CO.COOH+NH 3 , although this is not the 
 only possibility. The proofs for the correctness of this view have been 
 obtained essentially by experiments with aromatic amino-acids and 
 will be given as examples of such deamidation. 
 
 Dakin and Dundley 3 have shown that all a-amino-acids investi- 
 gated by them can be decomposed under special conditions so that they 
 to a certain degree yield ammonia and an a-keto-aldehyde. 
 
 R.CH.NH 2 .COOH->R.CO.CHO+NH 3 . 
 
 Thus, with alanine, and as the reaction to all appearances is reversible, 
 they consider the relationship between alanine and lactic acid is as follows - 
 
 CH 3 .CH.NH2.COOE^CH3.CO.CHO^CH 3 CHOH.COOH. 
 
 They also found it probable, that the a-keto-aldehydes represent the 
 first step in the demolition of the a-amino-acids whereby the regular 
 demolition of these acids takes place over the a-keto-acids and not over 
 oxyacids, which explains also the formation of sugar from certain amino- 
 acids (over methylglyoxal as intermediary step). 
 
 The deamidation after previous oxidation with the formation of 
 keto-acids has awakened special interest because recently in perfusion 
 experiments on dog-livers the reverse process, namely a synthesis of amino- 
 acids from keto-acids (in part also from oxyacids) and ammonia has been 
 performed (Knoop, Embden and Schmitz, Kondo 4 ). Among such 
 syntheses we can here call attention to the synthesis in the dog-liver 
 of alanine, phenylalanine and tyrosine from pyruvic acid (also lactic acid), 
 phenylpyruvic acid and p-oxyphenyl pyruvic acid, or of a-amino-n-butyric 
 acid from a-keto-butyrie acid (all as ammonium salts). 
 
 1 See Langstein and Neuberg, Arch. f. (Anat. u.) Physiol., 1903. Suppl. Bd. 
 1 Deutsch. Arch. f. klin. Med., 95, and Habilit. Schrift., Leipzig, 1908. See also 
 further on in regard to the literature on the demolition of the aromatic amino-acids. 
 
 3 Journ. of biol. Chem., 14. 
 
 4 Knoop, Zeitschr. f. physiol. Chem., 67 and 71; Embden and Schmitz, Bioch. 
 Zeitschr., 29 and 38; Kondo, ibid., 38. 
 
77G URINE. 
 
 The residue of the amino-acids remaining after deamidation can 
 naturally, according to the rule governing the fatty acids, be burned 
 and in certain cases this combustion takes place with the formation of 
 acetone bodies (which see). The fatty acid residue can also be used, be- 
 sides in the synthesis of amino-acids, also in the synthesis of other 
 substances, and in Chapter VII the formation of carbohydrates from 
 amino-acids has been mentioned. 
 
 Among the amino-acids the cystine, or better the cysteine, 
 
 CH 2 . (SH) .CH (NH 2 ) .COOH, 
 
 show a special behavior. On oxidation in the SH group and splitting 
 cff of CO2 (see page 149) it is transformed into a new amino-acid, taurine 
 (H2N)CH2.CH2(S020H). Taurine,. which when conjugated with cholic acid 
 forms taurocholic acid, occurring in the bile and which is habitually decom- 
 posed in the intestine or other parts of the animal body, can when intro- 
 duced as such into the human body, at least in part, be eliminated in 
 the urine as such or as tauro-carbamic acid, H2N.CO.NH.C2H4.SO2OH 
 (Salkowski l ). Otherwise as end-products of the demolition of cystine 
 and taurine an increased elimination of urinary sulphur, sulphuric acid and 
 thiosulphate, have been observed (Blum, Abderhalden and Samuely 2 ). 
 The sulphydryl group of cysteine also serves in the formation of sulpho- 
 cyanide, which is formed from the nitriles, introduced into the animal 
 body, by the HCN (Lang). The loosely combined sulphur of the pro- 
 teins, according to the observations of Pascheles, in alkaline reaction and 
 body temperature, can be readily transformed, with the cyan alkali into 
 sulphocyanide alkali. The alkali sulphocyanides when ingested are 
 almost quantitatively eliminated in the urine, according to Pollak. 3 
 
 By substituting one of the hydrogen atoms in the NH2 group of 
 normal a-amino-acids by an alkyl radical (methyl) the combustion of the 
 acids of the series C2 and C4 is considerably retarded and almost entirely 
 prevented in the members of the C5 and C6 series (Friedmann). 4 Sar- 
 cosine (methyl glycocoll), (CH3)NH.CH2.COOH, is not readily burnt, and 
 therefore passes in great part unchanged into the urine, but perhaps also 
 passes in small part into the corresponding uramino-acid, methylhydan- 
 toic acid, NH2.CO.N(CH 3 ).CH 2 .COOH (Schultzen 5 ), is an example 
 
 1 Ber. d. d. Chem. Gesellsch., 6, and Virchow's Arch., 58. 
 
 ' Bhjm, Hofmeister's Beitrage, 5; Abderhalden and Samuely, Zeitschr. f. physiol. 
 Chem., 46. 
 
 ' Lang, Arch. f. exp. Path. u. Pharm., 34; Pascheles, ibid.; Pollak, Hofmeister's 
 Beitrage, 2. 
 
 4 Hofmeister's Beitrage, 11. 
 
 6 Ber. d. d. Chem. Gesellsch., 5. See also Baumann and v. Mering, ibid., 8, and 
 E. Salkowski, Zeitschr. f. physiol. Chem., 4. 
 
CONJUGATION WITH SUL1T1UKIC AND GLUCURONIC ACIDS. 777 
 
 of this kind. Substitution of both hydrogen atoms of th<' ami no-group 
 by methyl groups seems to make the demolition of the amino-acids still 
 more difficult (Friedmann). Ordinary betaine (trimethyl glycocoll) 
 passes, according to Kohlhauch, 1 in part unburned into the urine in 
 camivora as well as herbivora. 
 
 The combustion of the aliphatic bodies can be retarded or prevented 
 also by substitutions of other kinds and by combining with other sub- 
 stances. 
 
 By substitution with halogens, bodies otherwise readily oxidizable are 
 converted into difficultly oxidizable ones. While the aldehydes are readily 
 and completely burnt like the primary and secondary alcohols of the 
 fatty series, the halogen-substituted aldehydes and alcohols, are, on the 
 contrary, difficultly oxidizable. The halogen-substitution products of 
 methane (chloroform, iodoform, and bromoform) are at least in part 
 destroyed, and the corresponding alkali compounds of the halogen pass 
 into the urine. 2 
 
 By conjugation with sulphuric acid, the alcohols which are otherwise 
 readily oxidizable may be protected against combustion, and conse- 
 quently the alkali salt of ethyl-sulphuric acid is not burnt in the body 
 (Salkowski 3 ). 
 
 Conjugation with other substances can prevent the combustion of 
 the aliphatic bodies as shown in the conjugation of glycocoll with benzoic 
 acid into hippuric acid. A conjugation can also be a mutual protection 
 against the combustion of two bodies as in the case of glucuronic acid 
 and certain substances. 
 
 Conjugation with glucuronic acid occurs, according to the investiga- 
 tions of Sundvik and especially of O. Neubauer, in many substituted 
 as well as non-substitued alcohols, aldehydes, and ketones. Chloral 
 hydrate, CCl3CH(OH)2, passes, after it has been converted into tri- 
 chlorethyl-alcohol by a reduction, into a levogyrate reducing acid, uro- 
 chloralic acid or trichlorethylglucuronic acid, CCI3.CH2.CGH9O7 (Musculus 
 and v. Mering). Of the primary alcohols investigated by Neubauer* 
 (upon rabbits and dogs) methyl alcohol gave no conjugated glucuronic 
 acid, and ethyl alcohol only a small amount. Isobutyl alcohol and active 
 
 1 Zeitschr. f. Biol., 57. 
 
 2 See Harnack and Griindler, Berlin, klin. Wochenschr., 1883; Zeller, Zeitschr. f. 
 physiol. Chem., 8; Kast, ibid., 11; Binz, Arch. f. exp. Path. u. Pharm., 28; Zeehuisen, 
 Maly's Jahresber., 23. 
 
 3 Pfliiger's Arch., 4. 
 
 4 Sundvik, Maly's Jahresber., 16; Musculus and v. Mering, Ber. d. deutsch. chem. 
 Gesellsch., 8; also v. Mering, ibid., 15; Zeitschr. f. physiol. Chem., 6; Kulz, Pfliiger's 
 Arch., 28 and 33; O. Neubauer, Arch. f. exp. Path. u. Pharm., 46; Saneyoshi, Bioch. 
 Zeitschr., 36. 
 
778 UEINE. 
 
 amyl alcohol yielded relatively large quantities. Secondary alcohols 
 produced conjugated glucuronic acids, and indeed to a greater .extent than 
 the primary alcohols, especially in rabbits. The ketones are reduced in 
 part into secondary alcohols and are partly excreted as the conjugated 
 acid. This could be shown for acetone with rabbits but not with dogs. 
 
 The homo- and heterocyclic compounds pass, as far as is known, 
 into the urine as such, or after a previous partial oxidation or synthesis 
 with other bodies, and appear as so-called aromatic compounds. This 
 applies at least to foreign substances that are introduced into the body. 
 
 The fact that benzene may be oxidized outside of the body into carbon 
 dioxide, oxalic acid, and volatile fatty acids has been known for a long 
 time; and as in these cases a rupture of the benzene ring must take place, 
 so also, it must be admitted, when aromatic substances undergo a com- 
 bustion in the animal body, a splitting of the benzene nucleus with the 
 formation of fatty bodies must be the result. If this does not occur, then 
 the benzene nucleus is eliminated with the urine as an aromatic compound 
 of one kind or another. The manner in which this benzene ring is opened 
 is not known. Still Jaff£ l has detected muconic acid in the urine of 
 dogs and rabbits which had been fed for a long time with benzene, and 
 suggest one way in which the benzene can be split in the animal body. 
 
 CH CH 
 
 •\ y\ 
 
 HC CH HC COOH 
 
 — * . That the demolition of the benzene nucleus 
 
 HC CH HC COOH 
 
 CH CH 
 
 occurs in certain cases, as in tyrosine and phenylalanine according to 
 the present view, over homogentisic acid, has already been mentioned. 
 The most striking example of a complete combustion of the benzene 
 nucleus is given by tyrosine, which as previously mentioned (page 737) 
 can be absorbed even in large quantities and decomposed without the 
 observer being able to detect any of the cleavage products of it in the urine. 
 Other examples of readily and at least in greatest part combustible aroma- 
 tic substances are phenyl-a-lactic acid, p-oxyphenylpyruvic acid and a-amino 
 cinnamic acid. According to Juvalta and Porcher phthalic acid is 
 also burnt in the animal body. The last investigator found that the three 
 phthalic acids have varying effects, as the o-acid is almost completely 
 burnt in dogs, while about 75 per cent of the m- and p-acids are excreted 
 unconsumed. This corresponds with the rule found by R. Cohn, 2 that 
 among the di-derivatives of benzene the ortho-compounds are more 
 
 1 Zeitschr. f. physiol. Chem., 62. 
 1 Ibid., 17. 
 
OXIDATION IN THE NUCLEUS AND SIDE CHAIN. 779 
 
 readily destroyed than the corresponding meta- and para-compounds. 
 The claims of Juvalta and Porcher are unfortunately disputed by 
 Pribram and Pohl. 1 
 
 An oxidation in the side chain of aromatic compounds is often found, 
 and may also occur in the nucleus itself. As an example, benzene is first 
 oxidized to oxybenzene (Schultzen and Naunyn), and this is then 
 further in part oxidized into dioxybenzenes (Baumann and Preusse). 
 Naphthalene appears to be converted into oxy naphthalene, and probably 
 a part also into dioxynaphthalene (Lesnik and M. Nencki). The hydro- 
 carbon with an amino- or imino-group may also be oxidized by a sub- 
 stitution of hydroxy! for hydrogen, especially when the formation of a 
 derivative in the para-position is possible (Klingenberg). For example, 
 aniline, C6H5.NH2, passes into paraminophenol, which latter passes into 
 the urine as its ethereal-sulphuric acid, H2N.C6H4.O.SO2.OH (F. Muller). 
 Acetanilid is in part converted into acetyl paraminophenol (Jaffe and 
 Hilbert, K. Morner), and carbazol into oxycarbazol (Klingenberg). 2 
 
 An oxidation of the side chain may occur by the hydrogen atoms being 
 replaced by hydroxy), or may also take place with the formation of carboxyl; 
 thus, for example, toluene, C6H5.CH3 (Schultzen and Naunyn), ethyl- 
 benzene, CGH5.C2H5, and propylbenzene, C6H5.C3H7 (Nencki and Giacosa) 3 
 besides many other bodies, are oxidized into benzoic acid. Cymene is 
 oxidized to cumic acid, xylene to toluic acid, methylpyridine to pyridine- 
 carboxylic acid in the same way. 
 
 If several side chains are present in the benzene nucleus, then only one 
 is always oxidized into carboxyl. Thus xylene, CeH^CHV^, is oxidized 
 into toluic acid, CeH^CH^OOOH (Schultzen and Naunyn); mesitylene, 
 C 6 H 3 (CH3)3, into mesitylenic acid, C 6 H 3 (CH 3 )2.COOH (L. Nencki); 
 cymene, (CH 3 )2CH.C 6 H4.CH 3 , into cumic acid, (CH 3 )CH.C 6 H 4 .COOH 
 (M. Nencki and Ziegler 4 ). 
 
 If the side-chain has several members, then the behavior is dif- 
 ferent and in these cases the demolition of aromatic amino-acids and 
 fatty acids is especially to be considered. 
 
 1 Juvalta, Zeitschr. f. physiol. Chem., 13; Pribram, Arch. f. exp. Path. u. Pharm., 
 51; Porcher, Bioch. Zeitschr., 14; Pohl, ibid., 16. 
 
 2 Schultzen and Naunyn, Arch. f. (Anat. u.) Physiol., 1867; Baumann and Preusse, 
 Zeitschr. f. physiol. Chem., 3, 156. See also Nencki and Giacosa, ibid., 4; Lesnik and 
 Nencki, Arch. f. exp. Path. u. Pharm., 24; F. Muller, Deutsch. med. Wochenschr., 
 1887; Jaffe and Hilbert, Zeitschr. f. physiol. Chem., 12; Morner, ibid., 13; Klingen- 
 berg, " Studien liber die Oxydation aromatischer Substanzen," etc., Inaug.-Diss., 
 Rostock, 1891. 
 
 3 Zeitschr. f. physiol. Chem., 4. 
 
 4 Nencki, Arch. f. exp. Path. u. Pharm., 1; Nencki and Ziegler, Ber. d. d. Chem. 
 Gesellsch.. 5; see also O. Jacobsen, ibid., 12. 
 
780 UKINE. 
 
 The aromatic amino-acids are, like the amino-acids in general, decom- 
 posed to fatty acids and have one carbon atom less. For example phenyl- 
 amino-acetic acid is in part converted into benzoic acid (O. Neubauer) ; 
 o- and ra-tyrosine yield o- and m-oxyphenylacetic acid respectively 
 (Blum, Flatow); p-chlorphenylalanine passes according to Friedmann 
 and Maase into p-chlorphenylacetic acid, and phenyl-a-aminobutyric 
 acid is changed, as Knoop x showed, into phenylpropionic acid. As 
 intermediary steps in this demolition we have, as in the other amino-acids, 
 part the hydrolytic cleavage of NH2 groups and part the demolition by 
 way of the corresponding keto-acid. 
 
 As an example of a demolition of the first kind we have for a long 
 time considered the finding by Schotten, of mandelic acid 
 
 C 6 H 5 .CH(OH).COOH 
 
 in the urine after feeding phenylaminoacetic acid, C6H5.CH(NH2).COOH. 
 According to O. Neubauer 2 the process is nevertheless of another kind, 
 namely, mandelic acid is produced secondarily by reduction from the 
 intermediarily formed keto-acid, phenylglyoxylic acid, C6H5.CO.COOH. 
 As example of a hydrolytic deamidation we will use the production, as 
 first observed by Blendermann, of p-oxyphenyl-lactic acid, 
 
 HO.C 6 H 4 .CH 2 .CH(OH).COOH 
 
 from tyrosine (in rabbits). This acid has also been found in the urine 
 by Schultzen and Riess in acute yellow atrophy of the liver, and by 
 Baumann in phosphorus poisoning, although the earlier investigators 
 incorrectly considered the acid as oxymandelic acid. That this acid, 
 which was considered as oxymandelic acid, is Z-p-oxyphenyl-lactic acid 
 has been proved by Ellinger and Kotake and Fromherz. 3 
 
 As shown especially by the investigations of O. Neubauer the demoli- 
 tion of the aromatic amino-acids passes ordinarily by way of the cor- 
 responding keto-acid. As stated above (page 737) in regard to the forma- 
 tion of homogentisic acid, the demolition of tyrosine, according to Neu- 
 bauer, passes over the p-oxyphenylpyruvic acid, HO.CeH4.CH2.CO.COOH. 
 According to him phenylamino-acetic acid also yields phenylglyoxylic acid; 
 
 1 Neubauer, Deutsch. Arch. f. klin. Med., 95; L. Blum, Arch.f. exp. Path. u. Pharm., 
 59; Flatow, Zeitschr. f. physiol. Chem., 64; F. Knoop, ibid., 67; Friedmann and 
 Maase, Bioch. 1, Zeitschr., 27. 
 
 2 Schotten, Zeitschr. f. physiol. Chem., 8; O. Neubauer, 1. c. 
 
 3 Blendemann, Zeitschr. f. physiol. Chem., 6; Schultzen and Riess, Chem. Ctotralbl., 
 1869; Baumann, Zeitschr. f. physiol. Chem., 6; Ellinger and Kotake, ibid., 65; From- 
 herz, ibid., 70. 
 
DEMOLITION OF AROMATIC FATTY ACIDS. 781 
 
 the m-tyrosine passes according to Flatow l in part as ra-oxyphenyl 
 pyruvic acid in the urine. The keto-acids give also the same end products 
 as the corresponding amino-acids. Thus o-tyrosine, like o-oxyphenyl- 
 pyruvic acid, yields o-oxyphenylacetic acid (Flatow) as end product; 
 the i>-chlnrph< ni/lalanine and the p-chlorphcnylpijrui>ic acid pass into the 
 p-chlorphenylacetic acid, which is not the case with the oxyacid, the p-chlor- 
 phenyl-lactic acid (Friedmann and Maase 2 ) . This last-mentioned case is 
 an example of the more ready combustibility of the keto-acids as com- 
 pared to the oxyacids. Another such example is the p-oxyphenyl pyruvic 
 acid, which is in great part burned, while the p-oxyphenyl-lactic acid is 
 hardly burned at all (Kotake, Suwa). A correspondingly different 
 behavior is shown by these two acids in perfusion experiments with 
 the excised liver of the dog. The oxyphenylpyruvic acid, like tyrosine, 
 shows itself to be an acetone former while oxyphenyl-lactic acid, on 
 the contrary, does not (Neubauer and Gross, E. Schmitz 3 ). The 
 ready combustibility of the keto-acids indicate that these acids and 
 not the oxyacids are the important intermediary cleavage products. 
 
 In regard to the demolition of aromatic fatty acids, Knoop 4 has found 
 that the acids with even carbon chains, such as phenyl butyric acid and 
 phenyl caproic acid, are converted into phenylacetic acid, which con- 
 jugates with glycocoll to form phenaceturic acid, while the acids with 
 uneven carbon ' chains, like phenylpropionic and phenylvaleric acid, 
 yield benzoic acid, which then is eliminated as hippuric acid. This 
 behavior is in close agreement with the generally accepted oxidation of 
 fatty at the /3-group, for which Dakin has also given important support. 
 Thus Dakin found after feeding phenylpropionic acid to cats, that 
 phenyl-&-cx])propionic add, benzoylacetic acid and acetophenone, the latter 
 passing into benzoic acid or hippuric acid, w T ere formed, which pre- 
 supposes an oxidation in the /3-position. According to the investigations 
 of Dakin and Friedmann 5 the conditions are still very complicated. 
 Certain of the processes are reversible, oxidations as well as reductions 
 occur, and a-/3-unsaturated acids may also be formed as intermediary 
 products. Dakin as well as Friedmann have obtained cinnamic acid 
 as intermediary product in the demolition of phenylpropionic acid, and 
 
 1 Neubauer, Deutsch. Arch. f. klin. Med., 95; Flatow, Zeitschr.f. physio! . Chem., 6-4. 
 1 Flatow, 1. c; Friedmann and Masse, Bioch. Zeitschr., 27. 
 
 5 Kotake, Zeitschr. f. physiol. Chem., 69; Suwa, ibid., 72; Neubauer and Gross, 
 ibid., 67; Schmitz. Bioch. Zeitschr., 28. 
 
 4 Hofmeister's Beitrage, 6, and Habilit.-Schrift, Freiburg, 1904. 
 
 6 Dakin, Journ. of biol. Chem., 4, 5, 6, 8, and 9; Friedmann, 6ee Med. Klinik,. 
 No. 28, 1911, and Bioch. Zeitschr., 35. 
 
782 
 
 URINE. 
 
 this is probably formed from the phenyl-/3-oxypropionic acid by the with- 
 drawal of water: 
 
 C 6 H 5 .CH (OH) .CH2.COOH - H 2 = C 6 H 5 .CH :CH.COOH. 
 
 Friedmann has also (in part with Sasaki) 1 studied the decomposition, of 
 fur fur propionic acid and found that pyromucic acid with furfuracrylic 
 acid as intermediary step, was formed: 
 
 C4H 3 O.CH2CH2COOH-»C4H 3 O.CH:CH.COOH^C4H30.COOH. 
 
 The above-mentioned investigators are therefore of the opinion that the 
 demolition takes place in part over the a-/3-unsaturated acids and in part 
 over the /3-keto-acids or /3-alcohol acids. 
 
 According to the investigations of Dakin and Friedmann and to the schematic 
 illustration which they give, we can consider the demolition of phenylpropionic 
 acid as follows: 
 
 C«H=.CH.,.CH,COOH (Phenylpropionic acid) 
 
 (Cinnamicacid) C,H,..CH: CH.COOH 
 
 O.H B .CO. CH .COOH (Benzoylaceticacid) 
 
 C 6 H 5 .CO .CH 3 
 
 ( Acetophenone) 
 
 C 6 H 5 .CH (OH). CH 2 .COOH 
 
 • (Phenyl-£-oxypropionic acid) 
 
 C 6 H 5 .COOH 
 
 (Benzoic add) 
 
 ■C 6 H 6 .COOH 
 
 (Benzoic acid) 
 
 C H 5 .CO. NH. CH 2 .COOH 
 
 (Hippuric acid) 
 
 Reductions may also occur and besides the examples of the reduction 
 of keto-acids to alcohol-acids, we will mention as further examples the 
 conversion, as observed by E. Meyer, 2 of nitrobenzene, C6H5NO2, or of. 
 nitrophenol, HO.CfjH4.XO2 into aminophenol, HO.C0H4.NH2, and also the 
 behavior of ?n-nitro-benzaldehyde in the animal body as mentioned below. 
 
 Syntheses of aromatic substances with other atomic groups occur 
 frequently. To these syntheses belongs, in the first place, the conjugation 
 of benzoic acid with glycocoll to form hippuric acid, the discovery of which 
 is generally ascribed to Wohler, but according to Heffter 3 more cor- 
 
 1 Sasaki, Bioch. Zeitschr., 25; Friedmann, ibid., 35. 
 * Zeitschr. f. physiol. Chem., 40. 
 
 ' Die Ausscheidung korperfremder Substanzen im Harn, Ergebnisse der Physiol., 
 4, 252. 
 
SYNTHESES OF AROMATIC SUBSTANCES. 783 
 
 rectly to Keller and Ure. All the numerous aromatic substances which 
 are converted into benzoic acid in the body are voided partly as hippuric 
 acid. This statement is not true for all species of animals. According 
 to the observations of Jaffe, 1 benzoic acid does not pass into hippuric 
 acid in birds, but after conjugation with ornithin, into the correspond- 
 ing acid, ornithuric acid, (a-5-dibenzoyldiamino valeric acid). Not only 
 are the oxybcnzoic acids and the substituted benzoic acids conjugated with 
 glycocoll, forming corresponding hippuric acids, but also the above- 
 mentioned acids, toluic, mesitylenic, cumic, and phenylacetic acids. These 
 acids are voided as toluric, mcsitylcnuric, cuminuric, and phenaceturic 
 acids. 
 
 It must be remarked in regard to the oxybcnzoic acids that a conjugation with 
 glycocoll has been shown only with salicylic and p-oxybenzoic acid (Bertagnim, 
 and others), while Baumann and Herter 2 find it only very probable for m- 
 oxyhenzoic acid. According to Baldoni, 3 in dogs, the salicylic acid docs not pass 
 into salicyluric acid, and he indeed found two acids which he calls ursalicylic 
 acid, CuHuOs and uramin-salicylic acid, CieHieNOs. The oxybcnzoic acids are 
 also in part eliminated as conjugated sulphuric acids, which is especially true for 
 ?/;-oxybenzoic acid. The three aminobenzoic acids, according to the experiments 
 of Hildebrandt, on rabbits, appeared at least in part unchanged in the urine. 
 Salkowski found, as was later confirmed by R. Cohn, 4 that in rabbits ///-amino- 
 benzoic acid passes in part into vmviiiiobenzoic acid, H 2 N.CO.HX.C 6 H.i.COOH. 
 It is also in part eliminated as aminohippuric acid. 
 
 The behavior of the halogen-substituted compounds of toluene varies in 
 different animals according to Hildebrandt's experiments. In dogs they are 
 converted into the corresponding substituted hippuric acid. In rabbits o-brom- 
 toluene is completely changed to hippuric acid, the m- and p-bromtoluene only 
 partly. The three chlortoluenes are converted in rabbits into the corresponding 
 benzoic acid and are eliminated as such and not as hippuric acid. 
 
 The substituted aldehydes are of special interest as substances which 
 may undergo conjugation with glycocoll. According to the investiga- 
 tions of R. Cohn 5 on this subject, o-nitrobcnzaldeh]/de when introduced 
 into a rabbit is only in a very small part converted into nitrobenzoic 
 acid, and the chief mass, about 90 per cent, is destroyed in the body. 
 According to Sieber and Smirnow t 6 m-nitrobenzaldchyde passes in dogs 
 into w-nitrohippuric acid, and according to Cohn into urea-m-nitro- 
 hippurate, but in rabbits a different action results. In this case not only 
 does an oxidation of the aldehyde into benzoic acid take place, but the 
 
 1 Ber. d. d. chem. Gesellsch., 10 and 11. 
 
 2 Zeitschr. f. physiol. Chem., 1, where Bertagnini's work is also cited. See also- 
 Dautzenberg, Maly's Jahresber., 11, 231. 
 
 3 Arch. f. exp. Path. u. Pharm., 1908, Suppl. Bd. (Schmiedeberg's Festschrift). 
 
 4 Salkowski, Zeitschr. f. physiol. Chem., 7; Cohn, ibid., 17; Hildebrandt, Hof- 
 meister's Beit rage, 3. 
 
 6 Zeitschr. f. physiol. Chem., 17. 
 6 Monatshefte f. Chem., 8. 
 
784 URINE. 
 
 nitro-group is also reduced to an amino-group, and finally acetic acid 
 attaches itself to this with the expulsion of water, so that the final product 
 is m-acetylaminobenzoic acid, (CH 3 .CO).NH.C6H 4 COOH. The p-nitro- 
 benzaldehyde acts in rabbits in part like the m-aldehyde and passes in 
 part into p-acetylaminobenzoic acid. Another part is converted into 
 p-nitrobenzoic acid, and the urine contains a chemical combination of 
 equal parts of these two acids. According to Sieber and Smirnow p- 
 nitrobenzaldehyde yields only urea p-nitrohippurate in dogs. The above- 
 mentioned pyridine-carboxylic acid, formed from methylpyridine 
 (a-picoline) passes into the urine after conjugation with glycocoll as 
 a-pyridinuric acid. 1 
 
 To those substances which undergo a conjugation with glycocoll 
 belongs also furfur ol (the aldehyde of pyromucic acid, C4H3O.CHO), 
 which, when introduced into rabbits and dogs, as shown first by Jaffe 
 and Cohn, 2 and then further shown by Sasaki and Friedmann, is elimi- 
 nated in two ways from the body. The furfurol can, by a similar synthesis 
 to Perkin's reaction, be transformed into the unsaturated Sicidfurfuracrylic 
 acid C 4 H 3 O.CH:CH.COOH, and also into pyromucic acid C4H3O.COOH. 
 These two acids pass, after conjugation with glycocoll, into the urine as 
 furfuracryluric acid and pyromucuric acid. In birds the pyromucic acid 
 is conjugated with ornithine and is eliminated as pyromucinornithuric acid. 
 
 It has not been proved how thiophene, C4H4S, behaves in the animal 
 body. Of methylthiophene (thiotolene), C4H3S.CH3, a very small part is 
 oxidized to thiophenic acid, C4H3S.COOH (Levy). This acid, as shown 
 by Jaffe and Levy, 3 is conjugated with glycocoll in the body (rabbits) 
 and eliminated as thiophenuric acid. 
 
 Another very important synthesis of aromatic substances is that of 
 the ethereal-sulphuric acids. Phenols, and in particular the hydroxylated 
 aromatic hydrocarbons and their derivatives are voided as ethereal-sul- 
 phuric acids, according to Baumann, Herter and others. 4 
 
 A conjugation of aromatic acids with sulphuric acid occurs less often. 
 The two previously mentioned aromatic acids, p-oxyphenylacetic and 
 p-oxyphenylpropionic acid, are in part eliminated in this form. Gentisic 
 acid (hydroquinone-carboxylic acid) also increases, according to Lik- 
 hatscheff, 5 the quantity of ethereal-sulphuric acid in the urine, while 
 
 1 In regard to the extensive literature on glycocoll conjugations we refer the reader 
 to O. Kuhling, Ueber Stoffwechselprodukte aromatischer Korper. Inaug.-Diss., 
 Berlin, 1887. 
 
 2 Ber. d.d. Chem. Gesellsch., 20 and 21; Sasaki and Friedmann, footnote 1, page 782. 
 
 3 Levy, Ueber das Verhalten einiger Thiophenderivate, etc., Inaug.-Diss., Konigs- 
 berg, 1889; Jaffe and Levy, Ber. d. d. chem. Gesellsch., 21. 
 
 * In regard to the literature, see O. Kuhling, 1. c. 
 6 Zeitschr. f. physiol. Chem., 21. 
 
CONJUGATION OF AROMATIC SUBSTANCES. 785 
 
 Rost asserts, contrary to earlier claims, that the same occurs with gallic 
 acid (trioxybenzoic acid) and tannic acid. 1 
 
 Although Nencki and Rekowski 2 have shown that acetophenone (phen- 
 ylmethylketone), CeH5.CO.CH3, is oxidized to benzoic acid and eliminated 
 as hippuric acid, the aromatic oxyketones with hydroxyl groups, such as 
 resacetophenone, 2, 4 dioxacetophenone (HO)2.CcH3.CO.CH3, pass into 
 the urine as ethereal-sulphuric acids and in part after conjugation with 
 glucuronic acid. Euxanthon, which is also an aromatic ketone, namely 
 
 CO 
 dioxyxanthon, HO.CoH3< >CeH3.0H, passes into the urine as eux- 
 
 x cr 
 
 anthic acid after conjugation with glucuronic acid. 
 
 A conjugation of other aromatic substances with glucuronic acid, 
 which last is protected from combustion, occurs rather often. The phenols, 
 as above stated (page 725), pass in part as conjugated glucuronic acids 
 into the urine. The same is true for the homologues of the phenols, for 
 certain substituted phenols, and for many aromatic substances, also 
 hydrocarbons after previous oxidation and hydration. Thus Hilde- 
 brandt and Fromm and Clemens 3 have shown that the ter penes and cam- 
 phors, by oxidation or hydration, or in certain cases by both, are converted 
 into hydroxyl derivatives when the body in question is not previously 
 hydroxylized, and that these hydroxyl derivatives are eliminated as con- 
 jugated glucuronic acids. Conjugated glucuronic acids are detected in 
 the urine after the introduction of various substances into the organsim, 
 e.g., therapeutic agents, such as ter penes, borneol, menthol, camphor (cam- 
 phoglucuronic acid was first observed by Schmiedeberg), naphthalene, 
 oil of turpentine, oxyquinolines, antipyrine, and many other bodies. 4 
 Orthonitrotoluene in dogs passes first into o-nitrobenzyl alcohol and then 
 
 1 In regard to the behavior of gallic and tannic acids in the animal body, see C. 
 Morner, Zeitschr. f. physiol. Chem., 16, which also contains the earlier literature; also 
 Harnack, ibid., 24, and Rost, Arch. f. exp. Path. u. Pharm., 38, and Sitzungsber. d. 
 Gesellsch. zur Beford. d. ges. Naturwiss. zu Marburg, 1898. 
 
 2 Arch. d. scienc. biol. de St. Petersbourg, 3, and Ber. d. deutsch. chem. 
 Gesellsch., 27. 
 
 3 Hildebrandt, Arch. f. exp. Path. u. Pharm., 45, 46; Zeitschr. f. physiol. Chem., 
 36; with Fromm, ibid., 33; and with Clemens, ibid., 37; Fromm and Clemens, ibid., 
 34. Extensive investigations on the behavior of alicylic compounds with the glucuronic 
 acid conjugation in the organism have been carried out by Hiimalainen, Skand. Arch, 
 f. Physiol., 27. 
 
 4 See O. Kiihling, 1. c, which gives the literature up to 1887; also E. Kiilz, Zeitschr. 
 f. Biologie, 27; the works of Hildebrandt, Fromm and Clemens, see footnote 3; 
 Brahm, Zeitschr. f. physiol. Chem., 28; Fenyvessy, ibid., 30; Bonanni, Hofmeister's 
 Beitrage, 1; Lawrow, Ber. d. d. chem. Gesellsch., 33. 
 
786 URINE. 
 
 Into a conjugated glucuronic acid, uronitrotoluolic acid (JaffIs 1 ). The 
 glucuronic acid split off from this conjugated acid is levogyrate and hence 
 is not identical, but only isomeric, with the ordinary glucuronic acid. 
 Diindhylaminobcnzaldehyde, according to Jaff£, is converted in part 
 into dimethylaminobenzoglucuronic acid in rabbits. The same conjugated 
 glucuronic acid is also produced, according to Hildebrandt, 2 from p- 
 dimcthijltoluidine, which is first changed into p-dimethylaminobenzoic 
 acid. Indol and skatol seem, as above stated (page 731), to be eliminated 
 in the urine partly as conjugated glucuronic acids. The mercapturic 
 acids, which will be mentioned below, also belong to those substances 
 which are conjugated with glucuronic acid, and after this conjugation 
 appear in the urine. 
 
 A conjugation of carbamic acid, NH2COOH, with amino-acids to form 
 wamino-acids, R.CH.NH.(CONH 2 )COOH, or their anhydrides, the 
 hydantoins, have also been observed in several cases, as after feeding 
 sarcosin, amino-benzoic acid, phenylalanine, taurine, tyrosine. It must 
 be remarked that according to Lippich and Dakin, 3 the uramino-acids 
 can be easily produced as transformation products from the urea in the 
 concentration of the urine by the aid of heat. 
 
 Syntheses with a simultaneous acetylation are of great interest. 
 Such a synthesis is the formation of the mercapturic acids. These acids, 
 which are produced in the body of dogs after the introduction of brom- 
 and chlorbenzene (Baumann and Preusse, Jaff£, Friedmann 4 ) are 
 acetylated derivatives of the protein cystine, and the acetylated brom- 
 phenylcysteine is CH2.S(C 6 H 4 Br).CH.NH(COCH3).COOH. Another 
 example of a synthesis with acetylation is the phenylaminoacetic acid, 
 which, as Neubauer and Warburg 5 have shown, in perfusion exper- 
 iments with dog's livers, gives among other products also acetylated 
 phenylaminoacetic acid, C6H5.CHNH(COCH3).COOH. 
 
 The synthesis of amino-acids, with simultaneous acetylation, as 
 recently observed by Knoop, are of specially great interest. After the 
 introduction of 7-phenyl-a-keto butyric acid into the body of a dog, 
 the formation of the corresponding acetylated amino-acid, 
 
 C 6 H5.CH 2 .CH 2 .CHNH(COCH 3 ).COOH 
 
 1 Zeitsehr. f. physiol. Chem., 2. 
 
 2 JafiY', Zeitsehr. f. physiol. Chem., 43; Hildebrandt, Hofmeister's Beitrage, 7. 
 
 3 Lippich, Ber. d. d. chem. Gesellsch., 41; Dakin, Journ. of biol. Chem., 8; Weiland, 
 Bioch. Zeitsehr., 38. 
 
 4 Baumann and Preusse, Zeitsehr. f. physiol. Chem., 5; Jaff6, Ber. d. deutsch. 
 chem. Gesellsch., 12; Friedmann, Hofmeister's Beitrage, 4. 
 
 6 Neubauer and 0. Warburg, Zeitsehr. f. physiol. Chem., 70. 
 
METHYLATION. 787 
 
 was observed. In perfusion experiments with dog livers, Embden and 
 Schmitz l have shown the formation of phenylalanine, tyrosine and other 
 amino-aeids, as has already been mentioned (pages 530 and 775) by 
 synthesis from the ammonium salts of the corresponding keto-arids and 
 also in part from the oxy-acids. 
 
 Mdhylation also often occurs, and as an example we will mention that 
 His has shown that 'pyridine, C5H5N, is transformed in dogs into methyl- 
 pyridine, and then passes into the urine as methyl pyridylammonium 
 hydroxide. Pyridine behaves similarly in hens (Hoshiai), pigs and goats, 
 (Totani and Hoshiai) while according to Abderhalden 2 and collabora- 
 tors, in rabbits it passes unchanged into the urine. Further examples 
 of methylation, although not aromatic substances, are the conversion 
 of guanidine acetic acid into creatine (Jaffe) and the observation of 
 Takeda 3 of the appearance of aminobutyrobetaine in the urine of dogs 
 with phosphorus poisoning. 
 
 Several alkaloids, such as quinine, morphine, and strychnine, may pass into 
 the urine. After the ingestion of turpentine, balsam of copaiba, and resins, these 
 may appear in the urine as resin acids. Different kinds of coloring-matters, such 
 as alizarin, crysophanic acid, after rhubarb or senna, and the coloring-matter of 
 the blueberry, etc., may also pass into the urine. After rhubarb, senna, or santonin 
 the urine assumes a yellow or greenish-yellow color, which is transformed into 
 a beautiful red by the addition of alkali. Phenol produces, as above mentioned, 
 a dark-brown or dark-green color which depends mainly on the decomposition 
 products of hydroquinone and humin substances. After naphthalene the urine 
 has a dark color, and several other medicinal agents produce a special coloration. 
 Thus after antipyrine it becomes yellow or blood-red. After balsam of copaiba 
 the urine becomes, when strongly acidified with hydrochloric acid, gradually 
 rose- and purple-red. After naphthalene or naphthol the urine gives with con- 
 centrated sulphuric acid (1 cc. of concentrated acid and a few drops of urine) 
 a beautiful emerald-green color, which is probably due to naphthol-glucuronic 
 acid. Odoriferous bodies also pass into the urine. After asparagus the urine 
 acquires a digusting odor which is probably due to methylmercaptan. After 
 turpentine the urine may have a peculiar odor similar to that of violets. 
 
 VI. PATHOLOGICAL CONSTITUENTS OF URINE. 
 
 Proteid. The appearance of slight traces of proteid in normal urine 
 has been observed by many investigators, such as Posner Pl6sz, v. 
 Noorden, Leube, and others. According to K. Morner 4 proteid 
 regularly occurs as a normal urinary constituent to the extent of 22-78 
 
 1 Knoop, Zeitschr. f. physiol. Chem., 67; Embden and Schmitz, Bioch. Zeitschr., 29 
 and 38. 
 
 8 His, Arch. f. exp. Path. u. Pharm., 22; Cohn, Zeitschr. f. physiol. Chem., 18; 
 Hoshiai, ibid., 62; with Totani, ibid., 68; Abderhalden and collaborators, ibil.. .V.» 
 and 62. 
 
 5 Jaff<5, Zeitschr. f. physiol. Chem., 48; Takeda, Pfluger's Arch., 133. 
 
 * Skand. Arch. f. Physiol., 6 (literature). 
 
788 URINE. 
 
 milligrams per liter. Frequently traces of a substance similar to a 
 nucleoalbumin, which is easily mistaken for mucin, and whose nature 
 will be treated of later, appears in the urine. In diseased conditions 
 proteid occurs in the urine in a variety of cases. The albuminous bodies 
 which most often occur are serglobulin and seralbumin. Proteoses (or 
 peptones) are also sometimes present. The quantity of proteid in the 
 urine is in most cases less than 5 p.m., rarely 10 p. m., and only very rarely 
 does it amount to 50 p. m. or over. Cases are known, however, where it 
 was even more than 80 p. m. 
 
 Among the many reactions proposed for the detection of proteid in 
 urine, the following are to be recommended: 
 
 The Heat Test. Filter the urine and test its reaction. An acid 
 urine may, as a rule, be boiled without further treatment, and only in 
 especially acid urines is it necessary to first treat with a little alkali. 
 An alkaline urine is made neutral or faintly acid before heating. If the 
 urine is poor in salts, add 1/10 vol. of a saturated common-salt solution 
 before boiling; then heat to the boiling-point, and if no precipitation, 
 cloudiness, or opalescence appears, the urine in question contains no 
 coagulable proteid, but it may contain proteoses or peptones. If a pre- 
 cipitate is produced on boiling, this may consist of proteid, or of earthy 
 phosphates, 1 or of both. The monohydrogen calcium phosphate decom- 
 poses on boiling, and the normal phosphate may separate out. The 
 proper amount of acid is now added to the urine, so as to prevent any 
 mistake caused by the presence of earthy phosphates, and to give a better 
 and more flocculent precipitate of the proteid. If acetic acid is used 
 for this, then add 1-3 drops of a 25 per cent acid to each 10 cc. of the 
 urine and boil after the addition of each drop. On using nitric acid, 
 add 1-2 drops of the 25 per cent acid to each cubic centimeter of the 
 boiling-hot urine. 
 
 On using acetic acid, when the quantity of proteid is very small, 
 and especially when the urine was originally alkaline, the proteid may 
 sometimes remain in solution on the addition of the above quantity of 
 acid. If, on the contrary, less acid is added, the precipitate of calcium 
 phosphate, which forms in amphoteric or faintly acid urines, is liable 
 not to dissolve completely, and this may cause it to be mistaken for a 
 proteid precipitate. If nitric acid is used for the heat test, the fact must 
 not be overlooked that after the addition of only a little acid a combina- 
 tion between it and the proteid is formed which is soluble on boiling and 
 which is only precipitated by an excess of the acid. On this account the 
 large quantity of nitric acid, as suggested above, must be added, but in 
 this case a small part of the proteid is liable to be dissolved by the excess 
 of the nitric acid. When the acid is added after boiling, which is absolutely 
 necessary, the liability of a mistake is not so great. It is on these grounds 
 that the heat test, although it gives very good results in the hands of 
 experts, is not recommended to physicians as a positive test for proteid. 
 
 1 In regard to the cause of the phosphate precipitation on boiling the urine, see 
 Malfatti, Hofrneister's Beitriige, 8. 
 
PROTEIDS IN THE URINE. 789 
 
 A confounding with mucin, when this body occurs in the urine, is 
 easily prevented in the heat test with acetic acid by acidifying another 
 portion with acetic acid at the ordinary temperature. Mucin and 
 nucleoalbumin substances similar to mucin arc hereby precipitated. If 
 in the performance of the heat and nitric-acid test, a precipitate first 
 appears on cooling or is strikingly increased, then this -hows the presence 
 of proteoses in the urine, either alone or mixed with coagulable proteid. 
 In this case a further investigation is necessary (see below). In a urine 
 rich in urates a precipitate consisting of uric acid separates on cooling. 
 This precipitate is colored and granular, and is hardly to be mistaken 
 for a proteose or proteid precipitate. 
 
 Heller's test is performed as follows (see page 99): The urine is 
 very carefully floated on the surface of nitric acid in a test-tube, or the 
 urine is placed in a test-tube and then the acid is slowly added by means 
 of a funnel, drawn out to a point, and extending to the bottom. In the 
 presence of albumin a white disk, or as we ordinarily say a white ring or 
 at least a sharply defined cloudiness, appears at the point of contact of 
 the two fluids. With this test a red or reddish-violet transparent ring 
 is always obtained with normal urine; it depends upon the indigo color- 
 ing-matters and can hardly be mistaken for the white or whitish proteid 
 ring. In a urine rich in urates, another complication may occur, due 
 to the formation of a ring produced by the precipitation of uric acid. 
 The uric-acid ring does not lie, like the proteid ring, between the two 
 liquids, but somewhat higher. For this reason two simultaneous rings 
 may exist in urines which are rich in urates and do not contain very much 
 proteid. The disturbance caused by uric acid is easily prevented by 
 diluting the urine with 1-2 vols, of water before performing the test. 
 The uric acid now remains in solution, and the delicacy of Heller's 
 test is so great that after dilution only in the presence of insignificant 
 traces of proteid does this test give negative results. In a urine very 
 rich in urea a ring-like separation of urea nitrate may also appear. This 
 ring consists of shining crystals, and it does not appear in urine previously 
 diluted. A confusion with resinous acids, which also give a whitish 
 ring with this test, is easily prevented, since these acids are soluble on the 
 addition of ether. Stir, add ether, and carefully shake the contents of 
 the test-tube. If the cloudiness is due to resinous acids, the urine gradually 
 becomes clear, and on evaporating the ether a sticky residue of resinous 
 acids is obtained. A liquid which contains true mucin does not give 
 a precipitate with this test, but it gives a more or less strongly opalescent 
 ring, which disappears on stirring. The liquid does not contain any 
 precipitate after stirring, but is somewhat opalescent. If a faint, not 
 wholly typical reaction is obtained with Heller's test after some time 
 with undiluted urine, while the diluted urine gives a pronounced reaction, 
 the presence is shown of the substance which used to be called mucin 
 or nucloealbumin. In this case proceed as described below for the detec- 
 tion of nucleoalbumin. 
 
 If the above-mentioned possible errors and the means by which they 
 may be prevented are borne in mind, there is hardly another test for 
 proteid in the urine which is at the same time so easily performed, so 
 delicate, and so positive as Heller's. With this test even 0.002 per 
 cent of albumin may be detected without difficulty. »>till the student 
 must not be satisfied with this test alone, but should apply at least a 
 
790 URINE. 
 
 second one, such as the heat test. In performing this test the (primary) 
 proteoses are also precipitated. 
 
 The reaction with meta phosphoric acid is very convenient and easily 
 performed. It is not quite so delicate and positive as Heller's test. 
 The proteoses are also precipitated by this reagent. 
 
 Reaction with Acetic Acid and Potassium Ferrocyanide. Treat the 
 urine first with acetic acid until it contains about 2 per cent, and then 
 add drop by drop a potassium-ferrocyanide solution (1:20), carefully 
 avoiding an excess. This test is very good, and in the hands of experts 
 it is even more delicate than Heller's. In the presence of a very small 
 quantity of proteid it requires more practice and dexterity than Hel- 
 ler's, as the relative quantities of reagent, proteid, and acetic acid influence 
 the result of the test. The quantity of salts in the urine likewise seems 
 to have an influence. This reagent also precipitates proteoses. 
 
 Spiegler's Test. Spiegler recommends a solution of 8 parts mercuric 
 chloride, 4 parts tartaric acid, 20 parts glycerin, and 200 parts water as a very 
 delicate reagent for proteid in the urine. A test-tube is half filled with this 
 reagent and the urine is allowed to flow upon its surface drop by drop from a 
 pipette along the wall of the test-tube. In the presence of proteid a white ring is 
 obtained at the point of contact between the two liquids. The delicacy of this 
 test is 1 :350,000. Jolles * does not consider this reagent suited for urines very 
 poor in chlorine, and for this reason he has changed it as follows: 10 grams mer- 
 curic chloride, 20 grams succinic acid, 10 grams NaCl, and 500 cc. water. 
 
 Reaction with sulphosalicylic acid. Treat the urine either with a 20 per cent 
 watery solution of sulphosalicylic acid or a few crystals of the acid. This reagent 
 does not precipitate the uric acid or the resin acids. (Roch's 2 test.) 
 
 As every normal urine contains traces of proteid, it is apparent that 
 very delicate reagents are to be used only with the greatest caution. For 
 ordinary cases Heller's test is sufficiently delicate. If no reaction is 
 obtained with this test within 2\ to 3 minutes, the .urine tested contains 
 less than 0.003 per cent of proteid, and is to be considered free from pro- 
 teid in the ordinary sense. 
 
 The use of precipitating reagents presumes that the urine to be investi- 
 gated is perfectly clear, especially in the presence of only very little 
 proteid. The urine must first be filtered. This is not easily done with 
 urine containing bacteria, but a clear urine may be obtained, as suggested 
 by A. Jolles, by shaking the urine with infusorial earth. Although 
 a little proteid is retained in this procedure and lost, it does not seem to 
 be of any importance (Grutzner, Schweissinger 3 ). 
 
 The different color reactions cannot be directly used, esspecially in deep-colored 
 urines which contain only little proteid. The common salt of the urine has a 
 disturbing action on Millon's reagent. To prove mora positively the presence 
 
 1 Spiegler, Wien. klin. Wochenschr., 1892, and Centralbl. f. d. klin. Med., 1893; 
 Jolles, Zeitschr. f. physiol. Chem., 21. 
 
 2 Pharmaceut. Centralbl., 1889, and Zeitschr. f. anal. Chem., 29. 
 
 * Jolles, Zeitschr. f. anai. Chem., 29; Grutzner, Chem. Centralbl., 1901, 1; 
 Schweissinger, ibid. 
 
PROTEIDS IN THE URINE. 791 
 
 of protein, the precipitate 1 obtained in the boiling test may be filtered, washed, 
 and then tested with MlLLON's reaction. The precipitate may also be dissolved 
 in dilute alkali and the biurel teal applied to the solution. The presence of pro- 
 teoses or peptones in the urine is directly tested for by this last-mentioned test. 
 
 In testing the urine for proteid one should never be satisfied with one 
 reaction alone, but must apply the heat test and Heller's, or the pctas- 
 sium-ferrocyanide test. In using the heat test alone the proteoses may 
 be easily overlooked, but these are detected, on the contrary, by Heller's 
 or the potassium-ferrocyanide test. If only one of these tests is employed, 
 no sufficient intimation of the kind of proteid present can be obtained, 
 whether it consists of proteoses or coagulable proteid. 
 
 For practical purposes several dry reagents for proteid have been recommended. 
 Besides the metaphosphoric acid may be mentioned Stutz's or Furbringer's 
 gelatin capsules, which contain mercuric chloride, sodium chloride, and citric 
 acid; and Geissler's albumin-test papers, which consist of strips of filter-paper 
 some of which have been dipped in a solution of citric acid, and some into a 
 solution of mercuric-cliloride and potassium-iodide solution, and then dried. 
 
 If the presence of proteid has been positively proved in the urine by 
 the above tests, it then remains necessary to determine its character. 
 
 The Detection of Globulin and Albumin. In detecting serglobulin 
 the urine is exactly neutralized, filtered, and treated with magnesium 
 sulphate in substance until it is completely saturated at the ordinary 
 temperature, or with an equal volume of a saturated neutral solution of 
 ammonium sulphate. In both cases a white, flocculent precipitate is 
 formed in the presence of globulin. In using ammonium sulphate with 
 a urine rich in urates, a precipitate consisting of ammonium urate may 
 separate. This precipitate does not appear immediately, but only after 
 a certain time, and it must not be mistaken for the globulin precipitate. 
 In detecting seralbumin heat the filtrate from the globulin precipitate 
 to boiling-point, or add about 1 per cent acetic acid to it at the ordinary 
 temperature. 
 
 For the detection and also for the quantitative estimation of the various 
 globulins (fibringlobulin, euglobulin, and pseudoglobulin) Oswald 1 has pro- 
 posed the fractional precipitation with ammonium sulphate. 
 
 Proteoses and peptones have been repeatedly found in the urine in 
 different diseases. Reliable reports are at hand on the occurrence of 
 proteoses in the urine. The statements in regard to the occurrence of 
 peptones date from a time when the conception of proteoses and pep- 
 tones was different from that of the present day, and in part they are 
 based upon investigations using untrustworthy methods. According 
 to Ito 2 true peptones are sometimes found in the urine in cases of pneu- 
 
 1 Munch, med. Wochenschr., 1904. See also Zak and Necker, Deutsch. Arch. f. 
 klin. Med., 88. 
 
 2 In regard to the literature on proteoses and peptones in urine, see Huppert- 
 Neubauer, Ham- Analyse, 10. Aufl., 466 to 492; also A. Stoffregen, Ueber das Vorkom- 
 men von Pepton im Harn, Sputum, und Eiter (Inaug.-Diss., Dorpat, 1891); E. Hirsch- 
 feldt, Ein Beitrage «ur Frage der Peptonurie (Inaug.-Diss., Dorpat, 1892); and espe- 
 
792 URINE. 
 
 monia; what has been designated as urine peptones seems to have been 
 chiefly deuteroproteoses. 
 
 In detecting the proteoses, the proteid-free urine, or urine boiled with addi- 
 tion of acetic acid, is saturated with ammonium sulphate, which precipitates the pro- 
 teoses. Several errors are here possible. The urobilin,which may give a reaction 
 similar to the biuret reaction, is also precipitated and may lead to mistakes (Sal- 
 kowski, Stokvis l ). The following modification by Bang and Devoto's 2 method 
 can be used to advantage: The urine is heated to boiling with ammonium sul- 
 phate (8 parts to 10 parts urine) and boiled for a few seconds. The hot liquid 
 is centrifuged for \ to 1 minute and separated from the sediment. The urobilin 
 is removed from this by extraction with alcohol. The residue is suspended in 
 a little water, heated to boiling, filtered, whereby the coagulable proteid is retained 
 on the filter, and any urobilin still present in the filtrate is shaken out with chloro- 
 form. The watery solution, after removal of the chloroform, is used for the biuret 
 test. For clinical purposes this method is very serviceable. 
 
 According to Salkowski the urine treated with 10-per cent hydrochloric 
 acid is precipitated with phosphotungstic acid, then warmed, the liquid decanted 
 from the resin-like precipitate, this washed with water, and then dissolved in a 
 little water with the aid of some caustic soda, warmed again until the blue color 
 disappears, cooled, and finally tested with copper sulphate. This method has 
 been somewhat modified by v. Aldor and Cerny. 3 In regard to other more 
 complicated methods we refer to Huppert-Neubauer. 
 
 Morawitz and Dietschy 4 first remove the proteid from the urine made 
 faintly acid with acid potassium phosphate by the addition of double the volume 
 of 96-per cent alcohol and warming on the water-bath for several hours. \ From 
 the concentrated filtrate acidified with a little sulphuric acid the proteoses can 
 be precipitated by saturating with zinc sulphate. After the removal of the urobilin 
 by alcohol and extracting with water, the biuret test may be applied. 
 
 If the proteoses have been precipitated from a larger portion of urine by 
 ammonium sulphate, this precipitate is tested for the presence of different pro- 
 teoses for the reasons given in Chapter II. The following serves as a preliminary 
 determination of the character of the proteoses present in the urine. If the urine 
 contains only deuteroproteose it does not become cloudy on boiling, does not give 
 Heller's test, does not become cloudy on saturating with NaCl.in neutral reaction, 
 but does become turbid on adding acetic acid saturated with this salt. In the 
 presence of only protoproteose the urine gives Heller's test, is precipitated 
 even in neutral solution on saturating with NaCl, but does not coagulate on boil- 
 ing. The presence of heteroproteose is shown by the urine behaving like the 
 above with NaCl and nitric acid, but shows a difference on heating. It gradually 
 becomes cloudy on warming and separates at about 60° C. a sticky precipitate 
 which attaches itself to the sides of the vessel and which dissolves at boiling tem- 
 perature on acidifying the urine ; the precipitate reappears on cooling. 
 
 In close relation to the proteoses stands the so-called Bence-Jones 
 proteid, which occurs in the urine in rare cases in diseases with changes 
 
 daily Stadelmann, Untersuchungen liber die Peptonurie, Wiesbaden, 1894; Ehrstrom, 
 Bidrag till kannedomen om Albumosurien, Helsingfors, 1900; Ito, Deutsch. Arch, 
 f. klin. Med., 71. 
 
 1 Salkowski, Berlin, klin. Wochenschr., 1897; Stokvis, Zeitschr. f. Biologie, 34. 
 
 1 Devoto, Zeitschr. f. physiol. Chem., 15; Bang, Deutsch. med. Wochenschr., 1898. 
 
 3 Salkowski, Centralbl. f. d. med. Wissensch., 1894; v. Aldor, Berl. klin. Wochenschr., 
 36; Cerny, Zeitschr. f. analyt. Chem., 40. 
 
 * Arch. f. exp. Path. u. Pharrn., 54. 
 
QUANTITATIVE ESTIMATION OF PROTEID IN URINE. 793 
 
 in the spinal marrow. It gives a precipitate on heating to 40-60° C, 
 which on further heating to boiling dissolves again more or less completely, 
 depending upon the reaction and upon the amount of salt present. In 
 salt-free solution the precipitate is not dissolved, on heating to boiling, 
 at least not always. It does not separate on dialysis, but can be pre- 
 cipitated from the urine by double the volume of a saturated ammonium- 
 sulphate solution or by alcohol. It has also been obtained as crystals 
 (Grutterink and de Graaff, Magnus-Levy 2 ). This body shows a 
 varying behavior in the different cases in which it has been found and its 
 nature has not been explained. From the investigations of the above- 
 mentioned and other experimenters (Moitessier, Abderhalden and 
 Rostoski) we can draw the conclusion that this proteid is similar to the 
 proteoses in several reactions, but that nevertheless it stands close to the 
 genuine protein bodies. It also yields primary as well as secondary 
 proteoses on peptic digestion (Grutterink and de Graaff), and yields 
 the same hydrolytic cleavage products as the other proteins (Abderhalden 
 and Rostoski). 
 
 Quantitative Estimation of Proteid in Urine. Of all the methods pro- 
 posed thus far, the coagulation method (boiling with the addition of 
 acetic acid) when performed with sufficient care gives the best results. 
 The average error need never amount to more than 0.01 per cent, and it 
 is generallv smaller. With this method it is best to first find how much 
 acetic acid must be added to a small portion of the urine, which has been 
 previously heated on the water-bath, to completely separate the pro- 
 teid so that the filtrate will not respond to Heller's test. Then coagulate 
 20-50-100 cc. of the urine. Pour the urine into a beaker and heat on 
 the water-bath, add the required quantity of acetic acid slowly, stirring 
 constantly, and heat at the same time, Filter while warm, wash first 
 with water, then with alcohol and ether, dry and weigh, incinerate and 
 weigh again. In exact determinations the filtrate must not give Hel- 
 ler's test. 
 
 The separate estimation of globulins and albumins is done by carefully 
 neutralizing the urine and precipitating with MgS0 4 added to saturation (Hammar- 
 stex), or simply by adding an equal volume of a saturated neutral solution of 
 ammonium sulphate (HoVmeister and Pohl 2 ). The precipitate consisting of 
 globulin is thoroughly washed with a saturated magnesium-sulphate or half- 
 saturated ammonium-sulphate solution, dried continuously at 110° C, boiled 
 with water, extracted with alcohol and ether, then dried, weighed, incinerated, 
 and weighed again. The quantity of albumin is calculated as the difference 
 between the quantity of globulin and the total proteids. 
 
 Approximate Estimation of Proteid in Urine. Of the methods suggested for 
 this purpose none has been more extensively employed than Esbach's. 
 
 'Magnus-Levy, Zeitschr. f. physiol. Chem., 30 (literature); Grutterink and de 
 Graaff, ibid., 34 and 36; Moitessier, Compt. rend. soc. biolog., 57; Ville and Derrien, 
 ibid., 62; Abderhalden and Rostoski, Zeitschr. f. physiol Chem., 46; see also Hopkins 
 and Savory, Journ. of Physiol., 42. 
 
 2 Hammarsten, Pfliiger's Arch., 17; Hofmeister and Pohl, Arch. f. exp. Path. u. 
 Pharm., 20. 
 
794 URINE. 
 
 Esbach's l Method. The acidified urine (with acetic acid) is poured into a 
 specially graduated tube to a certain mark, and then the reagent (a 2-per cent 
 citric-acid and 1 per cent picric-acid solution in water) is added to a second mark, 
 the tube closed with a rubber stopper and carefully shaken, avoiding the pro- 
 duction of froth. The tube is allowed to stand twenty-four hours, and then the 
 height of the precipitate on the graduation is read off. The reading gives directly 
 the quantity of proteid in 1000 parts of the urine. Urines rich in proteid must 
 first be diluted with water. The results obtained by this method, are, however, 
 dependent upon the temperature; and a difference in temperature of 5° to 6.5° 
 C. may cause an error of 0.2-0.3 per cent deficiency or excess in urines containing 
 a medium quantity of proteid (Christensen and Mygge). The method sug- 
 gested by Tsuschija 2 seems to be more reliable, and consists in precipitating the 
 proteid by an alcoholic solution of phosphotungstic acid containing hydrochloric 
 acid. 
 
 Other methods for the approximate estimation of proteid are the optical 
 methods of Christensen and Myg'ge, and of Walbum, 3 of Roberts and Stollni- 
 kow as modified by Brandberg, with Heller's test, which has been simplified 
 for practical purposes by Mittelbach. The density methods of Lang, Huppert 
 and Zahor are also very good. In regard to these and other methods we refer to 
 Huppert-Xeubauer's Harn-Analyse, 10. Aufl. 
 
 There is at present no trustworthy method for the quantitative estimation 
 of proteoses and peptone in the urine. 
 
 Nucleoalbumin and Mucin. According to K. Morner traces of urinary 
 mucoids may pass into solution in the urine; otherwise normal urine con- 
 tains no mucin. There is no doubt that there may be cases where true 
 mucin appears in the urine; in most cases mucin has probably been mis- 
 taken for so-called nucleoalbumin. The occurrence, under some circum- 
 stances, of nucleoalbumin in the urine is not to be denied, as such sub- 
 stances occur in the renal and urinary passages; still in most cases this 
 nucleoalbumin, as shown by K. Morner, 4 is of an entirely different kind. 
 
 All urine, according to Morner, contains a little proteid and in 
 addition substances which precipitate proteid. If the urine freed from 
 salts by dialysis is shaken with chloroform after the addition of 1-2 p. m. 
 acetic acid, a precipitate is obtained which acts like a nucleoalbumin. 
 If the acid filtrate is treated with seralbumin, a new and similar precipitate 
 is obtained, due to the presence of a residue of the substance which pre- 
 cipitates proteids. The most important of these proteid-precipitating 
 substances is chondroitin-sulphuric acid and nucleic acid, although the 
 latter appears to a much smaller extent. Taurocholic acid may in a few 
 instances, especially in icteric urines, be precipitated. The substances 
 isolated by different investigators from urine by the addition of acetic 
 acid and called " dissolved mucin " or " nucleoalbumin " are considered 
 
 1 In regard to the literature on this method and the numerous experiments to 
 determine its value, see Huppert-Neubauer, 10 Aufl., 853 and Neuberg, Der Ham, 
 g. 765. 
 
 2 Christensen, Virchow's Arch., 115; Tsuschija, Centralbl. f. Med., 1908. 
 ' Deutsch. med. Wochenschrift, 1908. 
 
 « Skand. Arch, f . Physiol., 6. 
 
DETECTION OF NUCLEOALBUMINS. 795 
 
 by Morner to be a combination of proteid chiefly with chondroitin- 
 sulphuric acid, and to a less extent with nucleic acid, and also perhaps 
 with tauroeholic acid. 
 
 As normal urine habitually contains an excess of substances capable 
 of precipitating proteids, it is apparent that an increased elimination of 
 so-called nucleoalbumin may be caused simply by an augmented excretion 
 of proteid. This happens to a still greater extent in cases where the 
 proteid as well as the proteid-precipitating substance is eliminated to an 
 increased extent. 
 
 Detection of so-called Nucleoalbumins. When a urine becomes cloudy 
 or precipitates on the addition of acetic acid, and when it gives a more 
 typical reaction with Heller's test after the dilution of the urine than 
 before, one is justified in making tests for mucin and nucleoalbumin. 
 As the salts of the urine interfere considerably with the precipitation 
 of these substances by acetic acid, they must first be removed by dialysis. 
 As large a quantity of urine as possible is dialyzed (with the addition of 
 chloroform) until the salts are removed. The acetic acid is added until 
 it contains 2 p. m., and the mixture allowed to stand. The precipitate 
 is dissolved in water by the aid of the smallest possible quantity of alkali 
 and precipitated again. In testing for chrondroitin-sulphuric acid a 
 part is warmed on the water-bath with about 5 per cent hydrochloric 
 acid. If positive results are obtained on testing for sulphuric acid and 
 reducing substance, then chondroproteid was present. If a reducing 
 substance can be detected but no sulphuric acid, then mucin is probably 
 there. If it does not contain any sulphuric acid or reducing substance, 
 a part of the precipitate is exposed to pepsin digestion and another part 
 used for the determination of any organic phosphorus. If positive results 
 are obtained from these tests, then nucleoalbumin and nucleoproteid 
 must be differentiated by special tests for nuclein bases. No positive 
 conclusion can be drawn except by using very large quantities of urine. 
 The filtrate from the nucleoalbumin can be used for the ordinary proteid 
 tests. 
 
 Nucleohistone. In a case of pseudoleucacmia A. Jolles found a phos- 
 phorized protein substance which he considers as identical with nucleohistone. 
 Histone is claimed to have been found in some cases by Krehl and AIatthes, 
 and by Kolisch and Buuian. 1 
 
 The nitrogen contained in the substances precipitated by alcohol, called the 
 "colloidal nitrogen " by Salkowski and whose quantity is doubled in carcinoma 
 as compared to the normal, consists in great part of oxyproteic acids. Accord- 
 ing to Salkowski and Kojo 2 this can be precipitated by basic lead acetate and 
 the nitrogen determined therein. 
 
 Blood and Blood-coloring Matters. The urine may contain blood from 
 hemorrhage in the kidneys or other parts of the urinary passages (ilema- 
 
 1 Jolles, Ber. d. deutsch. chem. Gesellsch., 30; Krehl and Matthes, Deutsch. Arch, 
 f. klin. Med., 54; Kolisch and Burian, Zeitschr. f. klin. Med., 29. 
 
 2 Salkowski, Berl. klin. Wochenschr., 1905 and 1910; Kojo, Zeitschr. f. physiol. 
 Chem., 73. 
 
796 URINE. 
 
 turia). In these cases, when the quantity of blood is not very small, 
 the urine is more or less cloudy and colored reddish, yellowish red, dirty 
 red, brownish red, or dark brown. In recent hemorrhages in which the 
 blood has not decomposed the color is nearer blood-red. Blood-corpuscles 
 may be found in the sediment, sometimes also blood-casts and smaller 
 or larger blood-clots. 
 
 In certain cases the urine contains no blood-corpuscles, but only dis- 
 solved blood-coloring matters, haemoglobin, or, and indeed quite often, 
 methsemoglobin (ilemoglobinuria) . The blood-pigments appear in the 
 urine under different conditions, as in dissolution of blood in poisoning 
 with arseniuretted hydrogen, chlorates, etc., after serious burns, after 
 transfusion of blood, and also in the periodic appearance of haemoglo- 
 binuria with fever. In hemoglobinuria the urine may also have an abun- 
 dant grayish-brown sediment rich in proteid which contains the remains 
 of the stromata of the red blood-corpuscles. In animals, hsemoglobinuria 
 may be produced by many causes which force free haemoglobin into the 
 plasma. 
 
 To detect blood in the urine, we make use of the microscope, the spec- 
 troscope, the guaiac test, and Heller's or Heller-Teichmann's test. 
 
 Microscopic Investigation. The blood-corpuscles may remain undis- 
 solved for a long time in acid urine; in alkaline urine, on the contrary, 
 they are easily changed and dissolved. They often appear entirely 
 unchanged in the sediment; in some cases they are distended and in 
 others unequally pointed or jagged like a thorn-apple. In hemorrhage of 
 the kidneys a cylindrical clot is sometimes found in the sediment which is 
 covered with numerous red blood-corpuscles, forming casts of the urinary 
 passages. These formations are called blood-casts. 
 
 The spectroscopic investigation is naturally of very great value; and if 
 it be necessary to determine not only the presence but also the kind of 
 coloring-matter, this method is indispensable. In regard to the optical 
 behavior of the various blood-pigments we must refer to Chapter V. 
 
 Guaiac Test. Mix in a test-tube equal volumes of tincture of 
 guaiac and old turpentine which has become strongly ozonized by the 
 action of air under the influence of light. To this mixture, which must 
 not have the slightest blue color, add the urine to be tested. In the 
 presence of blood or blood-pigments, first a bluish-green and then a beau- 
 tiful blue ring appears where the two liquids meet. On shaking the mixture 
 it becomes more or less blue. Normal urine or one containing proteid 
 does not give this reaction. According to Liebermann l this reaction 
 is brought about by the blood pigments acting as catalyst upon the 
 organic peroxides existing in the turpentine, accelerating the decomposi- 
 tion of these and the active oxygen taken up by the guaiaconic acid 
 which is oxidized to guaiac blue (guaiaconic acid ozonide). Urine con- 
 
 1 Pfluger's Arch., 104. 
 
BLOOD PIGMENTS. HiEMATOPORPHYRIN. 797 
 
 I 
 
 taining pus, even when no blood is present, gives a blue color with these 
 reagents; but in this case the tincture of guaiac alone, without tur- 
 pentine, is colored blue by the urine (Vitali '). This is at least true 
 for a tincture that has been exposed for some time to the action of air 
 and sunlight. The blue color produced by pus differs from that pro- 
 duced by blood-coloring matters by disappearing on heating the urine 
 to boiling. A urine alkaline by decomposition must first be made faintly 
 acid before performing the reaction. The turpentine should be kept 
 exposed to sunlight, while the tincture of guaiac must be kept in a 
 dark glass bottle. These reagents to be of use must be controlled by a 
 liquid containing blood. With positive results, however, this test is 
 not absolutely decisive, because other bodies may give a similar reaction, 
 but when properly performed it is so extremely delicate that when it 
 gives negative results any other test for blood is superfluous. 2 
 
 As the delicacy of the above-mentioned tests is sufficient for ordinary 
 purposes it is not necessary to give the new blood-tests suggested recently. 
 
 Heller-Teichmann's Test. If a neutral or faintly acid urine containing 
 blood is heated to boiling, one always obtains a mottled precipitate consist- 
 ing of proteid and haematin. If caustic soda is added to the boiling-hot test, 
 the liquid becomes clear and turns green when examined in thin layers (due to 
 haematin alkali), and a red precipitate, appearing green by reflected light, re-forms, 
 consisting of earthy phosphates and hsematin. This reaction is called Heller's 
 blood-test. If this precipitate is after a time collected on a small filter, it may be 
 used for the haemin test (see page 293). If the precipitate contains only a little 
 blood-coloring matter with a larger quantity of earthy phosphates, then wash 
 it with dilute acetic acid, which dissolves the earthy phosphates, and use the 
 residue for the preparation of Teichmann's haemin crystals. If, on the contrary, 
 the amount of phosphates is very small, then first add a little MgCb solution 
 to the urine, heat to boiling, and add simultaneously with the caustic potash 
 some sodium-phosphate solution. In the presence of only very small quantities 
 of blood, first make the urine very faintly alkaline with ammonia, add tannic 
 acid, acidify with acetic acid, and use this precipitate in the preparation of the 
 haemin crystals (Sturve 3 ). 
 
 0. and R. Adler 4 have recommended leucomalachite green or benzidine in 
 the presence of peroxide and acetic acid as especially sensitive reagents for blood. 
 
 Haematoporphyrin. Since the occurrence of haematoporphyrin in the 
 urine in various diseases has been made very probable by several investi- 
 gators, such as Neusser, Stokvis, MacMunn, Le Nobel, Copeman, and 
 others, 5 Salkow'ski has positively shown the presence of this pigment 
 in the urine after sulfonal intoxication. It was first isolated in a pure 
 
 1 See Maly's Jahresber., 18. 
 
 2 For more details in regard to the preparation of the reagents and the performance 
 of the reaction see O. Schumm., Zeitschr. f. physiol. Chem., 50. 
 
 3 Zeitschr. f. anal. Chem., 11. 
 
 4 Zeitschr. f. physiol. Chem., 41. 
 
 s A very complete index of the literature on haematoporphyrin in the urine may be 
 found in R. Zoja, Su qualche pigmento di alcune urine, etc., in Arch. Ital. di. clin. 
 Med., 1893. 
 
798 URINE. 
 
 crystalline state by Hammarsten * from the urine of insane women after 
 sulfonal intoxication. According to Garrod and Saillet 2 traces of 
 hsematoporphyrin (Saillet's urospectrin) regularly occur in normal 
 urines. It is also found in the urine during different diseases. It was 
 found in great abundance in a case of typhoid fever (Arnold 3 ) but 
 otherwise it generally occurs only in small amounts. It has been found in 
 considerable quantities in the urine after the lengthy use of sulfonal. 
 
 Urine containing hsematoporphyrin is sometimes only slightly colored, 
 while in other cases, as for example, after the use of sulfonal, it is more 
 or less deep red. In these last-mentioned cases the color depends, in 
 greatest part, not upon the hsematoporphyrin, but upon other red or 
 reddish-brown pigments which have not been sufficiently studied. 
 
 In the detection of small quantities of hsematoporphyrin proceed as 
 suggested by Garrod. Precipitate the urine with a 10-per cent caustic- 
 soda solution (20 cc. for every 100 cc. of urine). The phosphate pre- 
 cipitate containing the pigment is dissolved in alcohol-hydrochloric acid 
 (15-20 cc.) and the solution investigated with the spectroscope. In more 
 exact investigations make the solution alkaline with ammonia, add enough 
 acetic acid to dissolve the phosphate precipitate, shake with chloroform, 
 which takes up the pigment, and test this solution with the spectroscope. 
 
 In the presence of larger quantities of hsematoporphyrin the urine 
 is first precipitated, according to Salkowski, with an alkaline barium- 
 chloride solution (a mixture of equal volumes of barium-hydroxide solu- 
 tion, saturated in the cold, and a 10-per cent barium-chloride solution), 
 or, according to Hammarsten, 4 with a barium-acetate solution. The 
 washed precipitate, which contains the hsematoporphyrin, is allowed 
 to stand some time at the temperature of the room, with alcohol contain- 
 ing hydrochloric or sulphuric acid, and then filtered. The nitrate shows 
 the characteristic spectrum of hsematoporphyrin in acid solution and gives 
 the spectrum of alkaline hsematoporphyrin after saturation with ammonia. 
 If the alcoholic solution is mixed with chloroform and a large quantity 
 of water added and carefully shaken, sometimes a lower layer of chloro- 
 form is obtained which contains very pure hsematoporphyrin, while the 
 upper layer of alcohol and water contains the other pigments besides 
 some hsematoporphyrin. 
 
 Other methods which have no advantage over this one of Garrod have been 
 suggested by Riva and Zoja as well as Saillet. 5 
 
 Baumstark 6 found in a case of leprosy two characteristic coloring-matters 
 in the urine, " urorubrohsematin " and " urofuscohsematin," which, as their 
 
 1 Salkowski, Zeitschr. f. physiol. Chem., 15; Hammarsten, Skand. Arch. f. Physiol., 3. 
 
 2 Garrod, Journ. of Physiol., 13 (contains review of literature) and 17; Saillet,. 
 Revue de Mi'decine, 16. 
 
 3 Zeitschr. f. physiol. Chem., 82. 
 
 * Salkowski, 1. c; Hammarsten, 1. c. 
 
 6 Riva and Zoja, Maly's Jahresber., 24; Saillet, 1. c. See also Nebelthau, Zeitschr. 
 f. physiol. Chem., 27. 
 6 Pfliiger's Arch., 9. 
 
PUS. BILE ACIDS. 799 
 
 names indicate, seem to stand in close relation to the blood-coloring matters. 
 UroTubrohccmatin, CosHniNgFeiOj*, contains iron and shows in acid solution an 
 absorption-hand in front of D and a broader one back of D. In alkaline solution 
 it shows four bands— behind D, at E, beyond F, and behind G. It is not soluble 
 either in water, alcohol, ether, or chloroform. It gives a beautiful brownish-red 
 non-dichroic liquid with alkalies. Urofuscohoematin, CssHiobNsC^, which is free 
 from iron, shows no characteristic spectrum; it dissolves in alkalies, producing 
 a brown color. It remains to be proven whether these two pigments are related 
 to (impure) lueinatoporphyrin. 
 
 Melanin. In the presence of melanotic cancers dark pigments are some- 
 times eliminated with the urine. K. Morner has isolated two pigments from 
 such a urine, of which one was soluble in warm 50-75 per cent acetic acid, while 
 the other, on the contrary, was insoluble. The one seemed to be phymatorhusin 
 (see Chapter XV). Usually the urine does not contain any melanin, but a 
 chromogen of melanin, a meianogen. In such cases the urine gives Eislet's 
 reaction, becoming dark-colored with oxidizing agents, such as concentrated 
 nitric acid, potassium bichromate, and sulphuric acid, as well as with free sulphuric 
 acid. They also give Thormahlen's reaction namely a beautiful blue coloration 
 with sodium nitroprusside and then acetic acid. Urine containing melanin or 
 meianogen is colored black by a ferric-chloride solution (v. Jaksch *)■ 
 
 In a case of melanotic sarcoma H. Eppinger 2 has isolated from the urine 
 a crystalline meianogen of the composition C 9 Hi 2 N 2 S04, and which was insoluble 
 in ether. It gave the ordinary meianogen reactions and, according to him is 
 probably an amidated ethereal sulphuric acid of methylpyrrolidinoxycarboxylic 
 acid, which is derived from tryptophane. 
 
 Pus occurs in the urine in various inflammatory affections, especially 
 in catarrh of the bladder and in inflammation of the pelvis of the 
 kidneys, or of the urethra. 
 
 Pus is best detected by means of the miscroscope. The pus-cells are 
 rather easily destroyed in alkaline urines. In detecting pus we make 
 use of Donne's pus test, which is performed in the following way: Pour 
 off the urine from the sediment as carefully as possible, place a small 
 piece of caustic alkali on the sediment, and stir. If the pus-cells have 
 not been previously changed, the sediment is converted by this means 
 into a slimy tough mass. 
 
 The pus-corpuscles swell up in alkaline urines, and dissolve, or at least 
 are so changed that they cannot be recognized under the microscope. 
 The urine in these cases is more or less slimy or fibrous, and the proteid 
 can be precipitated in large flakes by acetic acid, so that it might possibly 
 be mistaken for mucin. The closer investigation of the precipitate 
 produced by acetic acid, and especially the appearance or non-appearance 
 of a reducing substance after boiling it with a mineral acid, demonstrates 
 the nature of the precipitated substance. Urine containing pus always 
 contains proteid. 
 
 Bile-acids. The reports in regard to the occurrence of bile-acids in the 
 urine under physiological conditions do not agree. According to Dragen- 
 dorff and Hone traces of bile-acids occur in the urine; according to Mao 
 
 ^hormahlen, Virchow's Arch., 108; v. Jaksch, Zeitschr. f. physiol. Chem., 13. 
 1 Bioch. Zeitschr., 28. 
 
800 URINE. 
 
 kay and v. Udranszky and K. Morner 1 they do not. Pathologically 
 they are present in the urine in hepatogenic icterus, although not invar- 
 iably. 
 
 Detection of Bile-acids in the Urine. Pettenkofer's test gives the most 
 decisive reaction ; but as it gives similar color reactions with other bodies, it must 
 be supplemented by the spectroscopic investigation. The direct test for bile- 
 acids is easily performed after the addition of traces of bile to a normal urine. 
 But the direct detection in a colored icteric urine is more difficult and gives very 
 misleading results; the bile-acid must therefore always be isolated from the urine. 
 This may be done by the following method of Hoppe-Seyler, which is slightly 
 modified in non-essential points. 
 
 Hoppe-Seyler's Method. Concentrate the urine and extract the residue 
 with strong alcohol. The filtrate is freed from alcohol by evaporation and then 
 precipitated by basic lead acetate and ammonia. The washed precipitate is 
 treated with boiling alcohol, filtered hot, the filtrate treated with a few drops 
 of soda solution, and evaporated to dryness. The dry residue is extracted with 
 absolute alcohol, filtered, and an excess of ether added. The amorphous or, 
 after a longer time, crystalline, precipitate consisting of the alkali salts of the 
 biliary acids is used in performing Pettenkofer's test. 
 
 Bile-pigments occur in the urine in different forms of icterus. A 
 urine containing bile-pigments is always abnormally colored — yellow, 
 yellowish brown, deep brown, greenish yellow, greenish brown, or nearly 
 pure green. On shaking it froths, and the bubbles are yellow or yellowish 
 green in color. As a rule icteric urine is somewhat cloudy, and the sedi- 
 ment is frequently, especially when it contains epithelium-cells, rather 
 strongly colored by the bile-pigments. 
 
 Detection of Bile-coloring Matters in Urine. Many tests have been 
 proposed for the detection of these substances. Ordinarily we obtain 
 the best results with the following three tests: 
 
 • Gmelin's test may be applied directly to the urine; but it is better to 
 use Rosenbach's modification. Filter the urine through a very small 
 filter, which becomes deeply colored from the retained epithelium-cells 
 and bodies of that nature. After the liquid has entirely passed through 
 apply to the inside of the filter a drop of nitric acid which contains only 
 very little nitrous acid. A pale-yellow spot will be formed which is sur- 
 rounded by colored rings which appear yellowish red, violet, blue, and 
 green from within outward. This modification is very delicate, and it 
 is hardly possible to mistake indican and other coloring-matters for the 
 bile-pigments. Several other modifications of Gmelin's direct test, e.g., 
 with concentrated sulphuric acid and nitrate, etc., have been proposed, 
 but they are neither simpler nor more delicate than Rosenbach's modifica- 
 tion. 
 
 Huppert's Reaction. In a dark-colored urine or one rich in indican 
 good results are not always obtained with Gmelin's test. In such cases, 
 as also in urines containing blood-coloring matters at the same time, 
 the urine is treated with lime-water, or first with some CaCb solution, 
 
 'Cited from Huppert-Neubauer, Harn-Analyse, 10. Aufl., 229. 
 
BILE PIGMENTS. 801 
 
 and then with a solution of sodium or ammonium carbonate. The pre- 
 cipitate which contains the bile-coloring matter is filtered, washed, dis- 
 solved in alcohol which contains 5 cc. of concentrated hydrochloric acid 
 in 100 cc. (I. Munk), and heated to boiling, when the solution becomes 
 green or bluish green. According to Xakayama i this reaction is more 
 delicate on using a mixture of ferric chloride, acid, and alcohol. 
 
 Hammarsten's Reaction. For ordinary cases it is sufficient to add 
 a few drops of urine to about 2-3 cc. of the reagent (see page 432), when 
 the mixture immediately after shaking turns a beautiful green or bluish 
 green, which color remains for several days. In the presence of only 
 very small quantities of bile-pigments, especially when blood or other 
 pigments are simultaneously present, pour about 10 cc. of the acid or 
 nearly neutral (not alkaline) urine into the tube of a small centrifugal 
 machine and add BaCb solution and centrifuge for about one minute. 
 The liquid is decanted and the sediment stirred with about 1 cc. of the 
 reagent and centrifuged again. A beautiful green solution is obtained 
 which may be changed, by the addition of increased quantities of the acid 
 mixture, to blue, violet, red, and reddish yellow. The green color may 
 be obtained in the presence of 1 part bile-pigment in 500,000-1,000,000 
 parts urine. In the presence of large amounts of other pigments calcium 
 chloride is better suited than barium chloride. 
 
 Bouma 2 has suggested the use of alcohol containing ferric chloride 
 and hydrochloric acid instead of the above-mentioned acid mixture. He 
 has also w r orked out a colorimetric method of quantitative estimation 
 of bilirubin in urine by means of this reagent. 
 
 As above indicated, we have a great many other tests besides these 
 given above. A very complete summary of these tests and the literature 
 thereof can be found in the work of Obermayer and Popper. 
 
 For ordinary purposes the above-mentioned tests are sufficiently 
 delicate, and according to Hammarsten it is not advisable, as also in the 
 case of the detection of proteid, sugar, etc., to increase the delicacy of 
 a test so that it shows the presence of the traces of the questionable 
 substance in normal urine. If in certain cases a greater delicacy is 
 required than is obtained with the above tests, then we must recommend 
 the flotation test of Obermeyer and Popper 3 with iodine and salt. 
 
 Medicinal coloring-matters produced from santonin, rhubarb, senna, etc., 
 may give an abnormal color to the urine and may be mistaken for bile-pigments, 
 or, in alkaline urines, perhaps for blood-eoloring matters. If hydrochloric acid 
 is added to such a urine, it becomes yellow or pale yellow, while on the addition 
 of an excess of alkali it takes on a more or less beautiful red color. 
 
 1 Munk, Arch. f. (Anat. u.) Physiol., 1898; Nakayama, Zeitschr. f. physiol. Chem., 
 36. 
 
 * Deutsch. med. Wochenschr., 1902 and 1904 
 
 * Wien. med. Wochenschr., 21. 
 
802 URINE. 
 
 Sugar in Urine. 
 
 The occurrence of traces of glucose in the urine of perfectly healthy 
 persons has been, as above stated (page 749), quite positively proven. If 
 sugar appears in the urine in constant and especially in large quantities, 
 it must be considered as an abnormal constituent. In a previous chapter 
 several of the principal causes of glycosuria in man and animals were men- 
 tioned, and the reader is referred to Chapters VII and VIII for the essen- 
 tial facts in regard to the appearance of sugar in the urine. 
 
 In man the appearance of glucose in the urine has been observed 
 under various pathological conditions, such as lesions of the brain and 
 especially of the medulla oblongata, abnormal circulation in the abdomen, 
 diseases of the heart, lungs and liver, cholera, and many other diseases. 
 The continued presence of sugar in human urine, sometimes in very con- 
 siderable quantities, occurs in diabetes mellitus. In this disease there 
 may be elimination of 1 kilogram or even more of glucose per day. 
 In the beginning of the disease, when the quantity of sugar is still very 
 small, the urine often does not appear abnormal. In the more developed, 
 typical cases the quantity of urine voided increases considerably, to 
 3-6-10 liters per day. The percentage of the physiological constituents 
 is as a rule very low, while their absolute daily quantity is increased. 
 The urine is pale, but of a high specific gravity, 1.030-1.040 or even higher. 
 The high specific gravity depends upon the quantity of sugar present, 
 which varies in different cases, but may reach 10 per cent. The urine is 
 therefore characterized in typical cases of diabetes by the very large 
 quantity voided, by the pale color and high specific gravity, and by its 
 containing sugar. 
 
 That the urine after the introduction into the system of certain medici- 
 nal agents or poisonous bodies contains reducing substances, conjugated 
 glucuronic acids, which may be mistaken for sugar, has already been men- 
 tioned. 
 
 Glucose in urine. The properties and reactions of this sugar have been 
 considered in a previous chapter, and it remains but to mention the methods 
 for the detection and quantitative determination of glucose in the urine. 
 
 The detection of sugar in the urine is ordinarily, in the presence of not 
 too small quantities, a very simple task. The presence of only very small 
 quantities may make its detection sometimes very difficult and laborious. 
 A urine containing proteid must first have the proteid removed by coagu- 
 lation with acetic acid and heat before it can be tested for sugar. ■ 
 
 The tests which are most frequently employed and are especially 
 recommended are as follows: 
 
 Trommer's Test. In a typical diabetic urine or one rich in sugar this 
 test succeeds well, and it may be performed in the manner suggested on 
 
SUGAR IN URINE. 803 
 
 page 214. This test may lead to very great mistakes in urine poor in 
 sugar, especially when they have at the same time normal or increased 
 amounts of physiological constituents, and therefore it cannot be recom- 
 mended to physicians or to persons inexperienced in such work. Normal 
 urine contains reducing substances, such as uric acid, creatinine, and others, 
 and therefore a reduction takes place in all urines on using this test. A 
 separation of copper suboxide does not generally occur, but still if one 
 varies the proportion of the alkali to the copper sulphate and boils, there 
 takes place an actual separation of suboxide in normal urines, or a peculiar 
 yellowish red liquid due to finely divided cuprous hydroxide. This occurs 
 especially on the addition of much alkali or too much copper sulphate, 
 and by careless manipulation the inexperienced worker may therefore 
 sometimes obtain apparently positive results in a normal urine. On the 
 other hand, as the urine contains substances such as creatinine and 
 ammonia (from the urea), which in the presence of only a little sugar 
 may keep the copper suboxide in solution, the investigator may easily 
 overlook small quantities of sugar that may be present. 
 
 The delicacy of Trommer's test can be increased by the suggestion made by 
 Worm-Muller. 1 As "by this rather complicated and tedious method small 
 amounts of sugar cannot be detected in certain urines, and also as special urines 
 from healthy persons readily give inconclusive results, and finally as Schondorff 
 has shown in numerous cases that the physiological sugar content of the urine 
 responds to this test in perfectly healthy persons because of its extreme delicacy, 
 it does not seem advisable in Hammarsten's opinion to recommend this test to 
 the physician. Bang and Bohmansson 2 have recently also shown its unre- 
 liability. 
 
 Almen's bismuth test, which has been incorrectly called Nylander's 
 test, is performed with the alkaline-bismuth solution prepared as described 
 on page 214. For each test 10 cc. of urine are taken and treated with 
 1 cc. of the bismuth solution and boiled for a few minutes. In the 
 presence of sugar the urine becomes dark yellow or yellowish brown; 
 then it grows darker, cloudy, dark brown, or nearly black, and non- 
 transparent. After a longer or shorter time a black deposit appears, 
 the supernatant liquid gradually clears, but still remains colored. In 
 the presence of only very little sugar the test does not become black or 
 dark brown, but simply deeper colored, and not until after some time 
 is there seen on the upper layer of the phosphate precipitate a dark or 
 black layer (of bismuth?). In the presence of much sugar a larger 
 amount of the reagent may be used without disadvantage. In a urine 
 poor in sugar only 1 cc. of the reagent for every 10 cc. of the urine must 
 be employed. 
 
 1 In regard to this test see Pfluger, Pfluger's Arch., 105 and 106; Hammarsten, 
 ibid., 116, and Zeitschr. f. physiol. Chem., 50. 
 
 2 Schondorff, Pfluger's Arch., 121; Bohmansson, Bioch. Zeitschr., 19. 
 
804 URINE. 
 
 Small amounts of proteid may retard this reaction and reduce the 
 delicacy of the test. Large quantities of proteid may, however, give 
 rise to an error by forming bismuth sulphide, and therefore it must 
 always be first removed. The assertion of Bechhold that mercury 
 compounds in the urine disturb the test has not been substantiated by 
 Zeidlitz on properly performing the test, and recently Rehfuss and 
 Hawk x came to the same conclusion. Those sources of error which 
 in Trommer's test are caused by the presence of uric acid and creatinine 
 are removed by using this test. The bismuth test is, moreover, readily 
 performed, and on this account is to be recommended to the physician. 
 
 The bumping and ejection of the fluid can be readily prevented by heating 
 over a very small flame after the test has been brought to a boil, and by gently 
 shaking the contents of the not too narrow test-tube. The recommendation 
 of heating for a longer time in the water-bath, fifteen minutes or more, is to be 
 discarded, as the delicacy of the test is thereby so much increased that it gives 
 a reaction with a physiological sugar content of 0.02 per cent. 
 
 When the amount of sugar in the urine is not less than 0.1 per cent 
 a positive reaction is obtained if the test is boiled for 2-3 minutes and 
 then allowed to stand quietly for 5 minutes. The phosphate precipitate 
 is then black or nearly black. In detecting smaller quantities of sugar 
 — 0.05 per cent, the test as a rule must be boiled longer — about 5 minutes. 
 
 The value of this test lies in the fact that it positively detects small 
 quantities of sugar — 0.1 per cent or somewhat less, and that when the 
 urine gives negative results we can consider it free from sugar in a clinical 
 sense. Like Trommer's test it is a reduction test, and shows also certain 
 other reducing bodies besides the sugar. These bodies are certain con- 
 jugated glucuronic acids which may appear in the urine. After the use 
 of certain therapeutic agents, such as rhubarb, senna, antipyrine, salol, 
 turpentine and others, the bismuth test gives .positive results. From 
 this it follows that we should never be satisfied with this test alone, espe- 
 cially when the reduction is not very great. 
 
 According to Bohmansson and Bang this test is perfectly reliable 
 if about 20 cc. of the urine is treated with 5 cc. of 25 per cent HC1 and 
 2 grams blood-charcoal (a teaspoonful) added and shaken every once in 
 a while during five minutes and then filtered. The filtrate on neutraliza- 
 tion with caustic soda is used for the Almen test. The disturbing reduc 
 ing substances are removed by the animal charcoal, but the sugar is not. 
 
 According to Andersen 2 this procedure cannot be used in the quan- 
 
 1 Bechhold, Zeitschr. f. physiol. Chem., 46; Zeidlitz, Upsala Lakaref. Forh. (N. F.), 
 11 (Hammarsten Festschr.); Rehfuss and Hawk, Journ. of biol. Chem., 7. 
 
 2 Bohmansson and Bang, Bioch. Zeitschr., 19, and Zeitschr. f. physiol. Chem., 
 63; Andersen, Bioch. Zeitschr., 37. 
 
SUGAR IN URINE. 805 
 
 titative estimation of sugar as a part of the sugar is retained by the 
 use of hydrochloric acid and blood-charcoal. According to Andersen 
 the pigments and the disturbing substances can be removed by per- 
 cipitation with mercuric nitrate. It can be more simply done by treating 
 40 cc. of the urine with 10 cc. acetic acid of 50 per cent strength and 4 
 grams blood-charcoal, shaking as above described and filtering. In the 
 presence of acetic acid no sugar is taken up by the charcoal and as this 
 simple method can be used for the quantitative estimation it can therefore 
 be used in the qualitative tests for sugar. 
 
 Fermentation Test. On using this test the process must vary accord- 
 ing as the bismuth test shows small or large quantities of sugar. If a 
 rather strong reduction is obtained, the urine may be treated with yeast 
 and the presence of sugar determined by the generation of carbon 
 dioxide. In this case the acid urine, or that faintly acidified with a little 
 tartaric acid is treated with compressed yeast, or yeast which has pre- 
 viously been washed by decantation with water. Pour this urine 
 to which the yeast has been added into a Schrotter's gas burette or a 
 Lohnstein's saccharimeter (see below). As the fermentation proceeds, 
 the carbon dioxide collects in the upper part of the tube, while a correspond- 
 ing quantity of liquid is expelled below. As a control in this case two 
 similar tests must be made, one with normal urine and yeast to learn 
 the quantity of gas usually developed, and the other with a sugar solu- 
 tion and yeast to determine the activity of the yeast. According to 
 Victorow l the fermentation is complete after six hours at a tem- 
 perature of 34-36° C. 
 
 If, on the contrary, only a faint reduction with the bismuth test 
 is found, no positive conclusion can be drawn from the absence of any 
 carbon dioxide or the appearance of a very insignificant quantity. The 
 urine absorbs considerable amounts of carbon dioxide, and in the presence 
 of only small amounts of sugar the fermentation test as above performed 
 may lead to negative or inaccurate results. In this case proceed in the 
 following way: Treat the acid urine, or urine which has been faintly 
 acidified with tartaric acid, with yeast whose activity has been tested 
 by a special test on a sugar solution, and allow it to stand six to twelve 
 hours at about 34-36° C. Then test again with the bismuth test, and 
 if the reaction now gives negative results, then sugar was previously 
 present. But if the reaction continues to give positive results, then it 
 shows, if the yeast is active, the presence of other reducing, unfer- 
 mentable substances. 
 
 In performing the fermentation test care should be taken that the urine 
 be acid before as well as after fermentation. If the reaction becomes 
 
 1 Pfliiger's Arch., 118. 
 
806 URINE. 
 
 alkaline during fermentation (alkaline fermentation), then the test 
 must be discarded. The vessel must be perfectly clean and strongly 
 heated before use. To make sure the urine may be boiled before fer- 
 mentation. 1 
 
 If a good polariscope is at hand it must not be forgotten to control 
 the results of the fermentation by determining the rotation before and 
 after fermentation. The phenylhydrazine test also, in many otherwise 
 doubtful cases, gives good service in testing urines for sugar. 
 
 Phenylhydrazine Test. Can be performed in the following manner: 
 20-25 cc. urine in a test-tube or in a beaker covered with a watch-glass 
 are treated with 1 gram phenylhydrazine hydrochloride and 2 grams 
 sodium acetate, and after solution of the salts it is warmed on the water- 
 bath for three-quarters of an hour. In the presence of sugar even dur- 
 ing the warming, a precipitate occurs, or in the presence of only a little 
 sugar, at least after the gradual cooling, a yellow, crystalline precipitate 
 forms. If the precipitate is very slight, it can be collected to advantage 
 by means of a centrifuge and investigated by aid of the microscope. 
 One finds at least a few phenylglucosazone crystals in the sediment 
 while the appearance of smaller or larger yellow platelets or strongly 
 refractive, brown globules is not indicative of sugar. In the presence 
 of large amounts of sugar in the urine a large quantity of the yellow 
 needles of phenylglucosazone or a mass of them are obtained. 
 
 This reaction is very reliable, and by it the presence of 0.03 per cent 
 sugar can be detected (Rosenfeld, Geyer 2 ). In doubtful cases it is 
 necessary to investigate the nature of the precipitate. For this purpose 
 dissolve a large quantity of the crystals in hot alcohol, treat the filtrate 
 with water, and boil off the alcohol. Still better, the precipitate is 
 dissolved, according to Neuberg, in some pyridine, and again precipi- 
 tated as crystals by the addition of benzene, ligroin, or ether. If the 
 characteristic yellow crystalline needles, whose melting-point (204- 
 205° C.) may also be determined, are now obtained, then this test is 
 decisive for the presence of sugar. It must not be forgotten that fructose 
 gives the same osazone as glucose, and that a further investigation is 
 necessary in certain cases, and also that the impure crystals of phen- 
 ylducosazone have a much lower melting-point than the pure ones. 
 
 The following modification by A. Neumann is simple, practical, and at the 
 same time sufficiently delicate. 5 cc. of the urine are treated with 2 cc. of acetic 
 acid (30-per cent) saturated with sodium acetate, 2 drops of pure phenylhydrazine 
 
 1 On the performance of the fermentation test and certain sources of error, see 
 Salkow.sk i, Berlin, klin. Wochenschr., 1905 (Ewald-Festnummer), and Pfliiger, Pfliiger's 
 Arch., 105 and 111. 
 
 2 Rosenfeld, Deutsch. med. Wochenschr., 1888; Geyer, cited from Roos, Zeitschr.. 
 f. physiol. Chem., 15. 
 
SUGAR IN URINE. 807 
 
 added, and the mixture boiled in a test-tube until it measures 3 cc. After quickly 
 cooling warm again and then allow it to cool slowly. After 5-10 minutes beautifully 
 formed crystals are obtained even in the presence of only 0.02 per cent sugar. 
 According to the experience of ElAMMABSTEN this modification, even in the presence 
 of 0.1 per cent sugar in concentrated urines, does not always give a positive reac- 
 tion. Salkowski ' has suggested an even more simple method. 
 
 The value of the phem ihydrazine test lias been considerably debated, 
 and the objection has been made that glucuronic acids also give a similar 
 precipitate. A confounding with glucuronic acid is, according to Hirschl, 
 not to be apprehended when the test is heated in the water-bath for a 
 long time (one hour). Kistermann found this precaution insufficient, 
 and Roos states that the phenylhydrazine test always gives a positive 
 result with human urine, which coincides with E. Holmgren's 2 and 
 Hammersten's experience. This test only show's a non-physiological 
 quantity of sugar when a rather abundant crystallization is obtained from 
 a small quantity of urine (about 5-10 cc.) Too great a delicacy of this 
 test is not to be recommended. 
 
 Rubxer's test is performed as follows: The urine is precipitated with an excess 
 of a concentrated lead-acetate solution and the nitrate carefully treated with 
 enough ammonia to produce a flocculent precipitate. It is then heated to boiling, 
 when the precipitate becomes flesh-colored or pink in the presence of sugar. 
 
 Polarization. This test is of great value, especially as in many cases 
 it quickly differentiates between glucose and other reducing, sometimes 
 levogyrate, substances, such as the conjugated glucuronic acids. In 
 the presence of only very little sugar the value of this test depends on 
 the delicacy of the instrument and the dexterity of the observer. As a 
 urine which show r s no rotation or is actually faintly levorotatory, may 
 contain 0.2 per cent glucose or perhaps even more, this test must be 
 combined with the fermentation test if we are seeking very small amounts 
 of sugar. The sugar in these cases can be detected only by the use of a 
 very accurate and delicate instrument. This method is in many cases 
 not serviceable for the physician. If the urine is to be clarified and 
 partly decolorized by precipitation with lead acetate, it must be done in 
 acid solution with acetic acid. 3 
 
 In the isolation of sugar and carbohydrates from the urine the benzoic-acid 
 esters may be prepared according to Baumann's method. The urine is made 
 alkaline with caustic soda to precipitate the earthy phosphates, the filtrate treated 
 with 10 cc. of benzoyl chloride and 120 cc. of 10 per cent caustic soda solution 
 for even' 100 cc. of the filtrate (Reixbold 4 ), and shaken until the odor of benzoyl 
 
 1 Neumann, Arch. f. (Anat. u.) Physiol., 1899, Suppl. See also Margulies, Berlin. 
 klin. Woehenschr., 1900; Salkowski, Arbeiter aus dem pathol. Inst., Berlin, 1906. 
 
 * Hirschl, Zeitschr. f. physiol. Chein., 14; Kistermann, Deutsch. Arch. f. klin. 
 Med., 50; Roos, 1. c; Holmgren, Maly's Jahresber., 27. 
 
 1 See Grossmann, Bioch. Zeitschr., 1. 
 
 4 Pfluger's Arch., 91. 
 
808 URINE. 
 
 chloride has disappeared. After standing sufficiently long the ester is collected, 
 finely divided, and saponified with an alcoholic solution of sodium ethylate in the 
 cold according to Baisch's method, 1 and the various carbohydrates separated 
 according to his suggestion. 
 
 If small quantities of sugar are to be isolated from the urine, precipitate the 
 urine first with sugar of lead, filter, precipitate the filtrate with ammoniacal 
 basic lead acetate, wash this precipitate with water, decompose it with H 2 S when 
 suspended in water and use the filtrate for the special tests. Schondorff 2 
 has suggested a method for the detection and estimation of very small amounts 
 of sugar based upon the work of Patein and Dufau. This method depends 
 upon precipitating the nitrogenous substances with mercuric nitrate. 
 
 To the physician, who naturally wants simple and quick methods, 
 the bismuth test is especially to be recommended. If this test gives neg- 
 ative results, the urine is to be considered as free from sugar in a clinical 
 sense. If it gives positive results, the presence of sugar must be con- 
 trolled by other tests, especially by the fermentation test. 
 
 Other tests for sugar, as, for example, the reaction with orthonitrophenyl- 
 propiolic acid, picric acid, diazobenzene-sulphonic acid, are superfluous. The 
 reaction with a-naphthol, which is a reaction for carbohydrates in general, for 
 glucuronic acid and mucin, may, because of its extreme delicacy, give rise to 
 mistakes, and is therefore not to be recommenced to physicians. Normal urines 
 give this test, and if the strongly diluted urine gives the reaction the presence 
 of great quantities of carbohydrates may be suspected. In these cases more 
 positive results are obtained by using other tests. This test requires great clean- 
 liness, and it has the inconvenience that sufficiently pure sulphuric acid is not 
 always readily procurable. Several investigators, such as v. Udransky, Luther, 
 Roos and Treupel, 3 have investigated this test in regard to its applicability 
 as an approximate test for carbohydrates in the urine. 
 
 Quantitative Determination of Sugar in the Urine. The quantity of 
 sugar can be determined by titration, by fermentation of the sugar, by 
 -polarization, and also in other ways. 
 
 The titration methods are based upon the property of the sugar to 
 reduce metallic oxides in alkaline solutions. As the titration liquids 
 (cupric oxide solution in the Fehling-Soxhlet, Pavy, Bang, Bertrand 
 methods and mercuric oxide in Knapp's method) are also reduced by 
 other urinary constituents, these reduction methods always give too high 
 results. When large quantities of sugar are present, as in typical 
 diabetic urine, which generally contains a lower percentage of normal 
 reducing constituents, this is indeed of little account; but when small 
 quantities of sugar are present in an otherwise normal urine, the mistake 
 may, on the contrary, be important, as the reducing power of normal 
 urine may correspond to 5 p. m. glucose (see page 749). In such cases 
 the titration procedure must be employed in connection with the fer- 
 mentation method, which will be described later. 
 
 1 Zeitschr. f. physiol. Chem., 19. 
 
 J Pfluger's Anh., 121, which cites the work of Patein and Dufau. 
 
 1 See Roos and Treupel, Zeitschr. f. physiol. Chem., 15 and 16. 
 
ESTIMATION OF SUGAR IN URINE. 809 
 
 Of the titration methods with copper solutions the method suggested 
 by Bang is the simplest, and at the same time seems to be more reliable 
 than any of the others. For this reason we will describe only this method 
 and refer to the original works and to Hoppe-Seyler-Thierfelder, 
 Handbuch der Chem. Analyses, 1 ( .)()'.), for description of the titration of 
 Fehling's solution according to Soxhlet l and to the titration accord- 
 ing to Pavy and Kumagawa-Suto. 2 
 
 Bang's First Method. 3 The principle of this method is that when urine 
 is boiled with an excess of a solution of potassium carbonate, potassium 
 thiocyanate and copper sulphate, copper thiocyanide is formed, and this 
 remains in solution as a colorless compound. The excess of cupric 
 oxide remaining is determined by titration with a solution of hydroxyl- 
 amine until the blue color disappears. The quantity of sugar is calculated 
 from the quantity of hydroxylamine used. 
 
 The following solutions are necessary: (a) A copper salt solution 
 containing 25 grams cupric sulphate in 2 liters, and (6) a solution con- 
 taining 0.55 grams hydroxylamine sulphate in 2 liters. 
 
 The copper solution is prepared in the following manner: Dissolve 100 grams 
 potassium bicarbonate in 1300 ec. water in a 2-liter graduated flask, and if nec- 
 essary warm to 50-60° C. After complete solution of the bicarbonate, add 
 400 grains potassium thiocyanate and 500 grams potassium carbonate. To 
 this solution, which must have the temperature of the room, add very slowly 
 150 cc. of a copper sulphate solution, which contains 166.67 grams copper sul- 
 phate (CuS04+5H^O) per liter, then add water up to 2 liters. This solution 
 unfortunately does not keep indefinitely, still, according to Andersen, it can be 
 kept in the dark up to 3 months and its strength controlled by titration with the 
 hydroxylamine solution. The hydroxylamine solution is prepared by dissolving 
 200 grams potassium thiocyanate in about 1500 cc. water in a 2-liter graduated 
 flask and adding a solution of 6.55 grams hydroxylamine sulphate in water; then 
 add water to the 2-liter mark. This solution, on the contrary keeps, but it must 
 be kept in a dark-colored bottle. Equal volumes of each of these two solutions 
 should exactly correspond to each other, and this can be determined by titrating 
 at ordinary temperature 50 cc. of the copper solution (plus 10 cc. water) with the 
 hydroxylamine solution. 
 
 The presence of proteid does not interfere with the reaction, and it 
 is not necessary to remove the proteid. The urine for titration should 
 not contain more than 0.6 per cent sugar. If the amount is lower, then 
 10 cc. of urine is used directly; if it is higher, then the urine is corre- 
 spondingly diluted and of this diluted urine we also make use of 10 cc. 
 in the titration. The quantities of sugar given in the table below vary 
 between 0.9 and 60 milligrams in 10 cc. 
 
 Performance of the Determination. 10 cc. of the sugar fluid are placed 
 in a glass flask and treated with 50 cc. of the copper solution. This is 
 heated on a wire-gauze to boiling, boiled for three minutes, cooled 
 quickly with water to the temperature of the room and then the hydrox- 
 
 1 Journ. f. prakt. Chem., (N. F.), 21. 
 
 2 Pavy, The Physiology of the Carbohydrates, London, 1894; Kumagawa and Suto, 
 Salkowski's Festschr., 1904; Sahli, Deutsch. med. Wochenschr., 1905. 
 
 J Bang, Bioch. Zeitschr., 2, 11, 32, and 38. See also Funk, Zeitschr. f. physiol. 
 Chem., 56 and 69; Jessen-Hansen, Bioch. Zeitschr., 10 and Andersen, ibid., 15 and 26. 
 
810 
 
 URINE. 
 
 ylamine solution allowed to flow in from a burette until the blue color 
 disappears and the solution is colorless, or, in urine poor in sugar, is yellow. 
 The sugar in milligrams is directly obtained from the amount of hydroxyl- 
 amine solution used by referring to the following reduction table : l 
 
 Hydroxyl- 
 
 amine 
 
 solution 
 
 used. 
 
 Milligrams 
 sugar. 
 
 Hydroxyl- 
 
 amine 
 
 solution 
 
 used. 
 
 Milligrams 
 sugar. 
 
 Hydroxyl- 
 
 amine 
 
 solution 
 
 used. 
 
 Milligrams 
 sugar. 
 
 Hydroxyl- 
 
 amine. 
 
 solution 
 
 used. 
 
 Milligrams 
 sugar. 
 
 0.75 
 
 60.0 
 
 13.00 
 
 39.0 
 
 25.50 
 
 23.5 
 
 38.00 
 
 10.4 
 
 1.00 
 
 59.4 
 
 13.50 
 
 38.3 
 
 26.00 
 
 22.9 
 
 38.50 
 
 9.9 
 
 1.50 
 
 58.4 
 
 14.00 
 
 37.7 
 
 26.50 
 
 22.3 
 
 39.00 
 
 9.4 
 
 2.00 
 
 57.3 
 
 14.50 
 
 37.1 
 
 27.00 
 
 21.8 
 
 39.50 
 
 9.0 
 
 2.50 
 
 56.2 
 
 15.00 
 
 36.4 
 
 27.50 
 
 21.2 
 
 40.00 
 
 8.5 
 
 3.00 
 
 55.0 
 
 15.50 
 
 35.8 
 
 28.00 
 
 20.7 
 
 40.50 
 
 8.1 
 
 3.50 
 
 54.3 
 
 16.00 
 
 35.1 
 
 28.50 
 
 20.1 
 
 41.00 
 
 7.6 
 
 4.00 
 
 53.4 
 
 16.50 
 
 34.5 
 
 29.00 
 
 19.6 
 
 41.50 
 
 7.2 
 
 4.50 
 
 52.6 
 
 17.00 
 
 33.9 
 
 29.50 
 
 19.1 
 
 42.00 
 
 6.7 
 
 5.00 
 
 51.6 
 
 17.50 
 
 33.3 
 
 30.00 
 
 18.6 
 
 42.50 
 
 6.3 
 
 5.50 
 
 50.7 
 
 18.00 
 
 32.6 
 
 30.50 
 
 18.0 
 
 43.00 
 
 5.8 
 
 6.00 
 
 49.8 
 
 18.50 
 
 32.0 
 
 31.00 
 
 17.5 
 
 43.50 
 
 5.4 
 
 6.50 
 
 48.9 
 
 19.00 
 
 31.4 
 
 31.50 
 
 17.0 
 
 44.00 
 
 4.9 
 
 7.00 
 
 48.0 
 
 19.50 
 
 30.8 
 
 32.00 
 
 16.5 
 
 44.50 
 
 4.5 
 
 7.50 
 
 47.2 
 
 20.00 
 
 30.2 
 
 32.50 
 
 15.9 
 
 45.00 
 
 4.1 
 
 8.00 
 
 46.3 
 
 20.50 
 
 29.6 
 
 33.00 
 
 15.4 
 
 45.50 
 
 3.7 
 
 8.50 
 
 45.5 
 
 21.00 
 
 29.0 
 
 33.50 
 
 14.9 
 
 46.00 
 
 3.3 
 
 9.00 
 
 44.7 
 
 21.50 
 
 28.3 
 
 34.00 
 
 14.4 
 
 46.50 
 
 2.9 
 
 9.50 
 
 44.0 
 
 22.00 
 
 27.7 
 
 34.50 
 
 13.9 
 
 47.00 
 
 2.5 
 
 10.00 
 
 43.3 
 
 22.50 
 
 27.1 
 
 35.00 
 
 13.4 
 
 47.50 
 
 2.1 
 
 10.50 
 
 42.5 
 
 23.00 
 
 26.5 
 
 35.50 
 
 12.9 
 
 48.00 
 
 1.7 
 
 11.00 
 
 41.8 
 
 23.50 
 
 25.8 
 
 36.00 
 
 12.4 
 
 48.50 
 
 1.3 
 
 11.50 
 
 41.1 
 
 24.00 
 
 25.2 
 
 36.50 
 
 11.9 
 
 49.00 
 
 0.9 
 
 12.00 
 
 40.4 
 
 24.50 
 
 24.6 
 
 37.00 
 
 11.4 
 
 
 
 12.50 
 
 39.7 
 
 25.00 
 
 24.1 
 
 37.50 
 
 10.9 
 
 
 
 For every tV cc. hydroxylamine solution used more than given in the table 
 between 49.00-15.00, subtract 0.1 milligram from the corresponding sugar value 
 and 0.2 milligram between 15.00-1.0. 
 
 The yellow color of the urine may be somewhat disturbing for the 
 end reaction so that with little experience an error of 0.5 cc. hydroxylamine 
 solution ( = about 0.5 milligram sugar) may occur. In order to decolorize 
 the urine we can precipitate, according to Andersen, with mercuric 
 nitrate, when the greatest part of the disturbing reducing substances are 
 removed, and then the excess of mercury removed by caustic soda and 
 shaking with zinc. Still simpler is the suggestion mentioned on page 
 805 with blood-charcoal after acidification with acetic acid. 
 
 Bang 2 decolorizes by the addition of 2 cc. alcohol of 95-97 per cent 
 and a teaspoonful of blood-charcoal to 18 cc. urine, shaking and filtering 
 immediately. By this means 50 per cent of the other reducing substances 
 
 1 This table is given with the permission of the publisher, Julius Springer, Berlin, 
 where it can be obtained at a low cost. 
 
 2 Andersen, Bioch. Zeitschr., 15 and 37; Bang, ibid., 38. 
 
ESTIMATION OF SUGAR IN URINE. 811 
 
 are removed. If an acidified urine is used for the titration then the urine 
 is added to the copper solution and not the reverse. 
 
 Bang's Second Method. As the reagents necessary for the preceding titration 
 are expensive, and as the copper solution only keeps for three months, and the 
 preparation of the solutions requires great exactitude and is somewhat difficult, 
 and as the method gives somewhat higher results than other reduction methods 
 due to the high alkali and salt content of the solutions, Bang « has recently 
 modified his original method. Instead of potassium thiocyanate he uses potassium 
 chloride, which can also keep the cuprous oxide in solution as a colorless com- 
 pound. Also the non-reduced cupric oxide remaining, as in the early method, is not 
 determined, but the cuprous oxide formed in the reduction with the sugar is directly 
 determined by titration. This is done by means of a N/100 (or N/10 or X/25) 
 iodine solution, which in the alkaline liquid acts oxidizinidy with the formation 
 of cupric oxide, according to the formula: CuCl-f-I+K 2 C03 =CuC0 3 -(-KC*l-(-KI. 
 Starch solution is used as indicator. As the potassium chloride can only hold 
 small amounts of cuprous oxide in solution, and as the end-reaction with the blue 
 iodine-starch cannot be determined with ease in the presence of large amounts 
 of cupric oxide in solution, but can easily be clone with the faintly blue coloration 
 due to cupric oxide, by this method a maximum of 10 milligrams sugar can only be 
 determined. On this account a urine rich in sugar must be diluted considerably 
 before titration. It must also be remarked that the iodine does not only react 
 with the cuprous oxide but also with other urinary constituents, and the importance 
 of this method on titration with rich urines, poor in sugar, has not been sufficiently 
 investigated. This method has given good results with pure sugar solutions and 
 with blood; but as its use for the determination of sugar in the urine has not 
 been sufficiently tested, we have only given the chief points of the method. 
 
 Bertrand's 2 Titration Method is more complicated than Bang's method and 
 does not seem to have any special advantages over this latter, at least in regard 
 to the determination of sugar in the urine. A part of the cuprous oxide here also 
 remains in solution and like the titration, according to Fehling, the cuprous 
 oxide sometimes settles only with difficulty. As this method seems to be used 
 extensively we will give the principles of the method. 
 
 The method consists in boiling the sugar solution (sugar urine) with an excess 
 of Fehling's solution. The cuprous oxide, freed from copper salt by decantation 
 and washing (under special precautions), is dissolved by ferric sulphate in sulphuric 
 acid, and the ferrous sulphate produced is determined by titration with potassium 
 permanganate, standardized by oxalic acid. The equations of the reactions are 
 as follows: 
 
 1. Cu 2 0+Fe 2 (S0 4 ) 3 +H 2 S0 4 = H 2 0-r-2CuS0 4 +2FeS0 4 
 
 2. 10FeSO 4 +2KMnO 4 +8H 2 SO 4 =8H 2 0+5Fe 2 (S0 4 )3+2MnS0 4 +K 2 S0 4 . 
 
 2 Cu are equivalent to 2 Fe, and as these are equivalent to 1 mol. oxalic acid, 
 then from the amount of oxalic acid (ammonium oxalate) used in the standardiza- 
 tion of the potassium permanganate solution the quantity of copper separated as 
 cuprous oxide can be readily calculated. The corresponding quantity of sugar 
 may be found in a special table. 
 
 For exact determinations of sugar the method as suggested by Allihn 
 and modified by Pfluger 3 is the best suited. 
 
 1 Bioch. Zeitschr., 49. 
 
 1 Bulletin de la Soc. chim., (3), 35, (1906). 
 
 1 Pfluger's Arch., 66. 
 
812 URINE. 
 
 The titration according to Knapp depends on the fact that mercuric 
 cyanide in alkaline solution is reduced to metallic mercury by glucose. The 
 titration liquid should contain 10 grams of chemically pure dry mercuric cyanide 
 and 100 cc. of caustic-soda solution of a specific gravity of 1.145 per liter. When 
 the titration is performed as described below (according to Worm-Muller and 
 Otto), 20 cc. of this solution should correspond to exactly 0.05 gram of glucose. 
 If the process is carried out in other ways, the value of the solution is different. 
 
 In this titration also, the quantity of sugar in the urine should be between 
 \ and 1 per cent, and the extent of dilution necessary be determined by a pre- 
 liminary test. To determine the end-reaction as described below, the test for the 
 excess of mercury is made with sulphuretted hydrogen. 
 
 In performing the titration allow 20 cc. of Knapp's solution to flow into a 
 flask and dilute with SO cc. of water, or when the urine contains less than 0.5 
 per cent of sugar use only 40-60 cc. After this heat to boiling and allow the diluted 
 urine to flow gradually into the hot solution, at first 2 cc, then 1 cc, then 0.5 cc, 
 then 0.2 cc, and lastly 0.1 cc. After each addition let it boil \ minute. When 
 the end-reaction is approaching, the liquid begins to clarify and the mercury 
 separates with the phosphates. The end-reaction is determined by taking a 
 drop of the upper layer of the liquid into a capillary tube and then blowing it out 
 on pure white filter-paper. The moist spot is first held over a bottle containing 
 fuming hydrochloric acid and then over strong sulphuretted hydrogen. The 
 presence of a minimum quantity of mercury salt in the liquid is shown by the 
 spot becoming yellowish, which is best seen when it is compared with a second 
 spot that has not been exposed to the gas. The end-reaction is still clearer when 
 a small part of the liquid is filtered, acidified with acetic acid, and tested with 
 sulphuretted hydrogen (Otto) 1 . As the added quantity of urine contains 0.050 
 gram sugar the calculation of the percentage content in sugar, bearing in mind 
 the extent of dilution, is very simple. 
 
 This titration, unlike the previous one, may be performed equally well by 
 daylight and by artificial light. It is applicable even when the quantity of sugar 
 in the urine is very small and that of the other urinary constituents is normal. It 
 is more easily performed, and the titration liquids may be kept without decom- 
 posing for a long time (Worm-Muller and his pupils 2 ) . There is diversity of 
 opinion, nevertheless, among investigators on the value of this titration method. 
 
 Estimation of the Quantity of Sugar by Fermentation. This 
 may be done in various ways: the simplest method, and one at the same 
 time sufficiently exact for ordinary cases, is that of Roberts. This 
 consists in determining the specific gravity of the urine before and after 
 fermentation. In the fermentation of sugar, carbon dioxide and alcohol 
 are formed as chief products, and the specific gravity is lowered, partly 
 on account of the disappearance of the sugar and partly on account of 
 the production of alcohol. Roberts found that a decrease of 0.001 in 
 the specific gravity corresponded to 0.23 per cent sugar, and this has been 
 substantiated since by several other investigators (Worm-Muller and 
 others). If the urine, for example, has a specific gravity of 1.030 before 
 fermentation and 1.008 after, then the quantity of sugar contained therein 
 was 22X0.23 = 5.06 per cent. 
 
 In performing this test the specific gravity must be taken at the same 
 temperature before and after the fermentation. The urine must be 
 faintly acid, and when necessary it should be acidified with a little hydro- 
 
 1 Journal f. prakt. Chem., 26. 
 
 2 Pfluger'e Arch., 16 and 23. 
 
ESTIMATION OF SUGAR IN URINE. 813 
 
 chloric acid or sulphuric acid. The activity of the yeast must, when, 
 necessary, be controlled by a special test. Place 200 cc. of the urine 
 in a 400 cc. flask, add a piece of compressed yeast the size of a pea, and 
 subdivide the yeast through the liquid by Bhaking; close the flask with 
 a stopper provided with a finely- drawn-out glass tube, and allow the test 
 to stand at the temperature of the room or, still better, at 30-35° C. 
 After twenty-four hours the fermentation is ordinarily ended, but this 
 must be verified by the bismuth test. After complete fermentation filter 
 through a dry filter, bring the filtrate to the proper temperature, and 
 determine the specific gravity. 
 
 If the specific gravity be determined with a good pycnometer sup- 
 plied with a thermometer and an expansion-tube, this method, when the 
 quantity of sugar is not less than 0.4-0.5 per cent, gives, according to 
 Worm-Muller, very exact results, but this has been disputed by Budde. 1 
 For the physician the method in this form is not serviceable. Even when 
 the specific gravity is determined by a delicate urinometer which can 
 give the density to the fourth decimal, exact results are not obtained, 
 because of the ordinary errors of the method (Budde); but the errors 
 are usually smaller than those which occur in titrations made by unskilled 
 hands. 
 
 When the quantity of sugar is less than 1.5 per cent, these methods 
 cannot be used. Such small amounts cannot, as already mentioned, 
 be determined by titration directly, because of the reducing power of nor- 
 mal urine. In such cases, it is better to first determine the reducing power 
 of the urine by titration according to Bang or Knapp, then ferment the 
 urine with the addition of yeast and titrate again. The difference 
 found between the two titrations calculated as sugar gives the true 
 quantity of the latter. 
 
 The determination of the sugar by fermentation can be so performed 
 that the loss in weight due to the CO2 can be estimated, or the volume 
 of the gas measured. For this last purpose Lohnstein 2 has constructed 
 a special fermentation saccharometer, and his " precision saccharometer " 
 is to be recommended. Based upon Lohnstein 's instrument, Wag- 
 ner 3 has constructed a "fermentation saccharo-manometer," which 
 has certain advantages over Lohnstein 's apparatus. 
 
 Estimations of Sugar by Polarization. In this method the 
 urine must be clear, not too deeply colored, and, above all, must not 
 contain any other optically active substances besides glucose. The 
 urine may contain several levorotatory substances such as proteids, 
 0-oxybutyric acid, conjugated glucuronic acids, the so-called Leo's sugar 
 and less often cystine, all of which are unfermentable. The proteid is 
 removed by coagulation, and the others are detected by the polariscope 
 after complete fermentation. The fermentable fructose is detected in a 
 special manner (see below), and the dextrorotatory milk-sugar differs 
 from glucose in its not fermenting readily. By using a delicate instru- 
 
 1 Roberts, The Lancet, 1862; Worm-Muller, Pfluger's Arch., 33 and 37; Budde, 
 ibid., 40, and Zeitschr. f. physiol. Chem., 13. See Lohnstein, Pfluger's Arch., 62. 
 
 2 Berlin, klin. Wochenschr., 35, and Allg. med. Central-Ztg., 1899; Goldman, Chem. 
 Centralbl., 1907, 1, 1149. 
 
 ' Munch, med. Wochensch., 1905. 
 
$14 URINE. 
 
 ment and with sufficient practice very exact results can be obtained by 
 this method. The value of this procedure consists in the rapidity with 
 which the determination can be made. In using instruments specially 
 constructed for clinical purposes the accuracy is less than with the less 
 expensive fermentation test. Under such circumstances, and as the 
 estimation by means of polarization can be performed with exactitude 
 only by specially trained chemists, it is hardly worth while to give this 
 method in detail, and the reader is referred to handbooks for hints in the 
 use of the apparatus. 
 
 Hasselbach and Lindhard 1 have recently suggested a method for the 
 quantitative estimation of sugar which is based on the decolorization of an alkaline 
 safranin solution in the presence of sugar. 
 
 Fructose (levulose). Levogyrate urines containing sugar have been 
 noted by several investigators, although the nature of the sugar was not 
 well known to the earlier observers. In recent years several positively 
 authentic cases of levulosuria have been described, and also cases of 
 diabetes have been found where fructose exists in the urine besides glucose. 
 Reports on this subject do not agree, however. 2 
 
 Fructose may be detected as follows: The urine is levorotatory, and 
 the levorotatory substance ferments with yeast. The urine gives the 
 ordinary reduction tests and the ordinary phenylglucosazone. With 
 methylphenylhydrazine it gives the characteristic fructose methyl- 
 phenylosazone, and it also gives Seliwanoff's reaction on heating 
 after the addition of an equal volume of hydrochloric acid and a little 
 resorcin. With this test it must be remarked that too lengthy or too 
 strong heating must not be applied, since other carbohydrates may also 
 give the reaction (see page 218 and the works of Rosin and Umber 3 ). 
 In the presence of fructose a red coloration appears. After cooling it 
 can be neutralized with soda and shaken out with amyl alcohol, (Rosin) 
 or with acetic ether (Borchardt). The amyl alcohol removes a red 
 pigment which gives a band in the spectrum between E and b and on 
 stronger concentration also a band in the blue at F. The acetic ether 
 in the presence of fructose becomes yellow, and this is more characteristic 
 according to Borchardt than Rosin's method, which has certain 
 fallacies. The simultaneous presence of nitrites and indican disturbs 
 the test, and in this case first remove the nitrous acid by boiling the 
 urine, acidified with acetic acid or hydrochloric acid for one minute. In 
 order to remove other disturbing pigments, Malfatti suggests the 
 oxidation of the urine with a little hydrochloric acid and potassium 
 permanganate. Jolles 4 has suggested a method for detecting fructose 
 besides glucose by means of a diphenylamine solution. 
 
 1 Bioch. Zeitschr., 27. 
 
 *See Borchardt, Zeitschr. f. physiol. Chem., 55 and 60; W. Voit, ibid., 58 and 61; 
 Adler, Pfluger's Arch., 139. 
 
 a Umber, Salkowski's Festschrift, Berlin, 1904; Rosin, ibid., and Zeitschr. f. 
 physiol. Chem., 38. 
 
 4 Rosin, 1. c; Borchardt, 1. c; Malfatti, Zeitschr. f. physiol. Chem., 58; Jolles 
 and Mauthner, Chem. Centralbl., 1910, 1, 483. 
 
LACTOSE IN URINE. 815- 
 
 Maltose sometimes occurs in the urine according to Lepine and Boulud, 
 Geelmuyden, 1 who also held this view, now states that maltose does not occur 
 in the urine. 
 
 Laiose is a substance named by Huppert and found by Leo - in diabetic urines 
 in certain cases, and which he considers as a sugar. It is levogyrate, amorphous, 
 and does not taste sweet, but rather sharp and salty. Laiose lias a reducing 
 action on metallic oxides, does not ferment, and gives a non-crystalline, yellowish- 
 brown oil with phenylhydrazine. There is no positive proof as yet that this 
 substance is a sugar. 
 
 Lactose. The appearance of lactose in the urine of pregnant women 
 was first shown by the observations of De Sinety and F. Hofmeister, 
 and this has been substantiated by other investigators. After the inges- 
 tion of large quantities of milk-sugar some lactose may be found in the 
 urine (see Chapter VIII on absorption). Langstein and Steinitz have 
 observed the passage of lactose and also of galactose 3 into the urine of 
 nurslings with disease of the stomach. The passage of lactose into the 
 urine is called lactosuria. 
 
 The positive detection of this sugar in the urine is difficult, because 
 it is, like glucose, dextrogyrate, and also gives the usual reduction tests. 
 If urine contains a dextrogyrate, non-fermentable sugar which reduces 
 bismuth solutions, then it is very probable that it contains lactose. It 
 must be remarked that the fermentation test for lactose is, according to 
 the experience of Lusk and Voit, 4 best performed by using pure cultivated 
 yeast (saccharomyces apiculatus). This yeast only ferments the glucose 
 while it does not decompose the milk-sugar. Voit claims that if Rub- 
 ner's test is performed without heating to boiling, but only to 80° C, 
 the color becomes yellow or brown in the presence of lactose, instead 
 of red. The most positive means for the detection of this sugar is to 
 isolate the sugar from the urine. This may be done by the method 
 suggested by F. Hofmeister. 5 
 
 R. Bauer 6 detects galactose as well as lactose in the urine by oxidation with 
 concentrated nitric acid, producing mucic acid. 
 
 Cammidge's reaction, which is recommended in the diagnosis of acute diseases 
 of the pancreas, consists in that certain urines do not give the phenylhydrazine 
 reaction directly, but only after boiling with an acid. The reason of this is not 
 known and the reaction is partly due to cane-sugar, in part to pentoses or gluco- 
 ronic acid and in part to mixtures of bodies. 
 
 1 Lepine and Boulud, Compt. Rend., 132; Geelmuyden, Zeitschr. f. klin, Med., 70. 
 
 2 Virchow's Arch., 107. 
 
 3 Hofmeister, Zeitschr. f. physiol. Chem., 1, which also contains the pertinent 
 literature. See also Lemaire, ibid., 21; Langstein and Steinitz, Hofmeister's Beitrage, 7. 
 
 4 Carl Voit, Ueber Die Glycogenbiklung nach Aufnahme verschiedener Zuckeraten, 
 Zeitschr. f. Biologic, 28. 
 
 5 Hofmeister, Zeitschr. f. physiol. Chem., 1, which also contains the pertinent 
 literature. 
 
 6 Zeitschr. f. Physiol. Chem., 51. 
 
816 URINE. 
 
 Pentoses. Salkowski and Jastrowitz first found in the urine of 
 persons addicted to the morphine habit a variety of sugar which was a 
 pentose and yielded an osazone which melted at 159° C. Since this 
 several other cases of pentosuria have been observed, and according to 
 Kulz and Vogel and others small amounts of pentose also occur in the 
 urine of diabetics, as also in the urine of dogs with pancreatic or phlorhizin 
 diabetes. 1 
 
 The pentose isolated by Neubeeg from the urine in chronic pentosuria 
 was cW-arabinose. Luzzatto and Klercker studied cases of pento- 
 suria and found Z-arabinose. In alimentary pentosuria the £-arabinose 
 of the plant food may be found in the urine. The appearance of pentoses 
 in the urine after eating fruits and fruit-juices has been repeatedly 
 observed by Blumenthal and also by v. Jaksch. According to Comi- 
 notti 2 pentoses habitually occur in human urine on a mixed diet. 
 
 A urine containing pentose reduces bismuth as well as copper solu- 
 tions, although the reduction is not so rapid, but appears gradually. 
 If only pentose is present, the urine does not ferment, but in the presence 
 of glucose small amounts of pentose may also undergo fermentation. 
 The preparation of the osazone serves in the detection of pentoses and 
 when obtained from the urine it melts at 156-160° C. The phloroglucin 
 or orcin tests can also be employed (see page 209). Of these the last is 
 most preferable, especially as it excludes a confusion with the conjugated 
 glucuronic acids. 
 
 The orcin test can be performed as follows: 5 cc. of the urine are mixed 
 with an equal volume of HC1 sp.gr. 1.19, a small amount of orcin added and 
 the whole heated to boiling. As soon as a greenish cloudiness appears 
 cool the mixture off and shake carefully with amyl alcohol. The amyl- 
 alcohol solution is used in the spectroscopic examination. The pre- 
 cipitation of a bluish-green pigment is in itself significant. 
 
 Bial a uses as reagent 30 per cent hydrochloric acid, which contains 1 gram 
 of orcin and 25 drops of a ferric-chloride solution (62.9 per cent of the crystalline 
 salt) in 500 cc. of the acid. 4.5 cc. of the reagent are heated to boiling and then a 
 few drops ("not more than 1 cc.) of the urine are added to the hot but not boiling 
 liquid. In the presence of pentose the liquid turns a beautiful green. The use- 
 fulness of Bial's reagent is questioned by several experimenters. The delicacy 
 is too great and the possibility of confounding with other carbohydrates is not 
 excluded. In regard to the numerous modifications of this test and to Jolles' 
 reaction we refer to page 209. The same for the quantitative estimation of pen- 
 
 1 In regard to the literature, see footnote 1, page 20S. See also Blumenthal, 
 "Die Pentosurie," Deutsche Klinik, 1902. 
 
 2 Blumenthal, Deutsche Klinik, 1902; v. Jaksch, Centralbl. f. innere Medizin, 
 1906; Cominotti, Bioch. Zeitschr., 22. 
 
 ' Deutsch. med. Wochenschr., 1903; see also footnote 4, page 209. 
 
CONJUGATED GLUCURONIC ACIDS. 817 
 
 toses. Jolles 1 considers the preparation of the osazonc as a specially conclusive 
 test and the distillation of the osazone with hydrochloric acid and testing the 
 distillate with Bial's reagent. 
 
 Rosenberger 2 believes he has detected a heptose in the urine in a case of 
 diabetes. According to him and to Geelmuyden 3 probably different varieties 
 of sugar, which are not well known, can possibly occur in urine of diabetics. 
 
 Conjugated Glucuronic Acids. Certain conjugated glucuronic acids 
 such as menthol- and turpentine-glucuronic acid may spontaneously 
 decompose in the urine, and in this case they may readily lead to a con- 
 fusion with pentoses. The urine should always be fresh as possible 
 for these examinations. 
 
 A confusion of the glucuronic acids, which have a reducing power on 
 copper or bismuth solutions, with glucose and fructose, can be pre- 
 vented by the fermentation test. They may also be distinguished from 
 glucose by their optical behavior, as the conjugated glucuronic acids 
 are levogyrate. On boiling with an acid, dextrorotatory glucuronic acid 
 is produced and the levorotation is changed to dextrorotation. 
 
 The conjugated glucuronic acids, like the pentoses, give the phloro- 
 glucin-hydrochloric-acid test. On the contrary they do not give the 
 orcin test directly, but only after cleavage with the setting free of glucuronic 
 acid. On using Bial's reagent no mistaking for pentoses occurs, although 
 this statement requires further substantiation. The pentoses may also 
 be isolated and identified by their osazones. Certain readily decomposable 
 glucuronic acids can here give phenylhydrazine compounds. In order to 
 detect glucuronic acid in the osazone precipitate, we can, as suggested 
 by Xeuberg and Saneyoshi 4 take a knife point (about 8 milligrams) 
 of the precipitate, dissolve in 4 cc. strong hydrochloric acid, dilute with 
 4 cc. water, heat to boiling, add at least 0.1 gram naphthoresorcin, warm 
 for \ minute, allow to slowly cool to 50° and shake with benzene. In the 
 presence of glucuronic acid the benzene solution is violet with an absorp- 
 tion in the yellowish-green . 
 
 The occurrence of conjugated glucuronic acids in the urine is shown 
 when the urine does not give the orcin-hydrochloric-acid reaction directly, 
 but only after boiling with the acid. The naphthoresorcin reaction, 
 as suggested by Tollens, can also be used. To 5 cc. urine add 0.5 cc. 
 of a 1 per cent alcoholic solution of naphthoresorcin and 5 cc. hydro- 
 chloric acid (sp.gr. 1.19), boil for one minute, allow to stand four minutes, 
 
 1 Jolles, Bioch. Zeitschr., 2, Centralbl. f. inn. Med., 1907 and 1912, and Zeitschr. 
 f. anal. Chem., 46. 
 
 2 Zeitschr. f. physiol. Chem., 49. 
 
 3 Rosenberger, Centralbl. f. inn. Med., 28; Geelmuyden, Zeitschr. f. klin. Med., 
 58, 63, and 70. 
 
 4 Bioch. Zeitschr., 36. 
 
818 URINE. 
 
 cool and shake with ether. In the presence of glucuronic acid the ether 
 becomes violet or blue, and shows the absorption bands, given on page 
 223. According to Neuberg this test, which is not specific for glucuronic 
 acid, is best performed with the naphthoresorcin in substance. This 
 test is more conclusive, if, as suggested by Neuberg and Schewket, 1 
 the residue from an ethereal extract of the acidified urine is used. 
 
 The surest method is that suggested by Mayer and Neuberg, which 
 consists in precipitating the urine with basic lead acetate, decomposing 
 the precipitate with H2S, boiling with dilute sulphuric acid in order to 
 split the conjugated acid, and then after neutralizing with soda, prepar- 
 ing the characteristic bromphenylhydrazine compound of glucuronic 
 acid (see page 223) with ^-bromphenylhydrazine hydrochloride and 
 sodium acetate. Hervieux 2 has slightly modified this method. In 
 regard to the quantitative estimation of glucuronic acid we must refer 
 to the work of C. Tollens. 3 
 
 Inosite seems to be a normal urinary constituent, although it occurs 
 only in very small quantities (Hoppe-Seyler, Starkenstein 4 ) . In 
 diabetes insipidus, as well as after excessive drinking of water, it occurs 
 in large quantities in the urine because of a more abundant washing- 
 out of the tissues. 
 
 For the detection of inosite we make use of the method given on page 581, 
 with the modifications suggested by Meillere and Starkenstein. 
 
 Acetone Bodies (acetone, acetoacetic acid, /3-oxybutyric acid). These 
 bodies, whose occurrence in the urine and formation in the organism have 
 been the subject of numerous investigations, occur in the urine espe- 
 cially in diabetes mellitus, but also in many other diseases. 5 According 
 to v. Jaksch and others, acetone is a normal urinary constituent, though 
 it may occur only in very small amounts (0.01 gram in twenty-four hours). 
 
 In regard to the origin of these bodies it was formerly considered that 
 they were produced by an increased destruction of protein. One of the 
 various reasons for this was the increase in the elimination of acetone 
 
 1 B. Tollens, Ber. d. d. chem. Gesellsch., 41, 1788, and C. Tollens, Zeitschr. f. physiol. 
 Chem., 56; Neuberg, Bioch. Zeitschr., 24; Neuberg and Schewket, ibid., 44; see also 
 Mandel and Neuberg, ibid., 13. 
 
 2 Mayer and Neuberg, Zeitschr. f. physiol. Chem., 29; Hervieux, Compt. rend, 
 eoc. biol., 63. 
 
 3 Zeitschr. f. physiol. Chem., 61. 
 
 4 Starkenstein, Zeitschr. f. exp. Path. u. Therap., 5, which contains the literature. 
 
 '■> In regard to the extensive literature on acetone bodies the reader is referred to- 
 Huppcrt-N'eubauer, Ham-Analyse, 10. Aufl., and v. Noorden's Lehrb. d. Pathol, des 
 Stoffwechsels. Berlin, 1906, and for recent work, Magnus-Levy, Die Azetonkorper,. 
 Ergbn. d. inn. Med. u. Kinderheilk., I. 
 
ACETONE BODIES. 819 
 
 and acctoacetic acid during inanition (v. Jaksch, Fr. Muller : ). This 
 also stands in accord with the observations that a considerable increase 
 in the quantity of acetone and acctoacetic acid eliminated is observed 
 in such diseases as fevers, diabetes, digestive disturbances, mental dis- 
 eases with abstinence and cachexia, where the body protein is largely 
 destroyed. The formation of acetone bodies from protein is also indi- 
 cated by the fact that acetone has been obtained as an oxidation prod- 
 uct from gelatin and protein (Blumenthal and Neuberg, Orgler 2 ). 
 The investigations of Embden and collaborators are more conclusive. 
 After Embden and Kalberlah showed that the liver is an organ where 
 acetone is formed, Embden, Salomon and Schmidt 3 showed by exper- 
 iments on extirpated livers, that butyric acid, oxybutyric acid, leucine, 
 tyrosine and in fact those aromatic bodies which, like tyrosine, phenyl- 
 alanine, phenyl-a-lactic acid and homogentisic acid contain a combustible 
 benzene nucleus, are transformed, in the liver, into acetone. Research, 
 which has been continued further by Embden and his collaborators 
 and substantiated by others, such as Baer, and Blum, Borchardt 
 and Lange, Neubauer and Gross, Schmitz and Fr. Sachs 4 has shown 
 that there can be no doubt that certain amino-acids, especially leucine, 
 are strong acetone formers, and consequently that acetone can be formed 
 from protein. Protamines and histones can also increase the acetone 
 elimination (Borchardt) or, as we say, may have a " ketoplastic " 
 action, and it is therefore possible that acetone can be formed from 
 arginine with a-amino-valerianic acid as intermediary step (Borchardt 
 and Lange). 
 
 As we cannot deny the possibility of a formation of acetone from pro- 
 teins, on the other hand we have observations which are inconsistent with 
 the origin of the acetone bodies entirely from the proteins. Thus no par- 
 allelism exists between the acetone bodies and the nitrogen excretion 
 in diabetics, and the fact, that in man no certain relation exists between 
 the acetone elimination and the nitrogen and sulphur excretion, seems to 
 show that the acetone bodies are not entirely derived from the proteins. 
 In man the excretion of acetone does not increase with the rise in the 
 
 1 v. Jaksch, Ueber Acetonurie und Diaceturie. Berlin, 1885; Fr. Muller, Bericht 
 iiber die Ergebnisse des an Cetti ausgefiihrten Hungerversuches. Berlin, klin. Wochen- 
 echr., 1887. 
 
 2 Blumenthal and Neuberg, Deutsch. med. Wochenschr., 1901; Orgler, Hofmeis- 
 ter's Beit rage, 1. 
 
 1 Hofmeister's Beitriige, 8. 
 
 4 Embden, ibid., 11, with Marx, Engel, Lattes and Michaud, ibid., 11; Baer and 
 Blum, Arch. f. exp. Path. u. Pharm., 55, 56, and 62; Borchardt, ibid, 53, with Lange, 
 Hofmeister"s Beitriige, 8; Neubauer and Gross, Zeitschr. f. physiol. Chem., 67; Schmitz, 
 Bioch. Zeitschr., 28; Fr. Sachs, ibid., 27. 
 
820 URINE. 
 
 quantity of protein, and an increase in the latter above the average causes 
 a diminution in the elimination of acetone (Rosenfeld, Hirschfeld, 
 Fr. Voit 1 ). 
 
 The carbohydrates cannot be considered as material for the forma- 
 tion of acetone bodies. It is generally admitted that in man the 
 exclusion of carbohydrates from the food or the diminution in their 
 amount or their assimilation may lead to more or less increased elimina- 
 tion of acetone bodies. This behavior may occur in diabetes as well as 
 in starvation and in the above-mentioned diseased conditions. The 
 increased elimination of acetone with food lacking carbohydrates also 
 occurs in healthy persons with a fatty diet but with a sufficient supply of 
 calories in other ways (alimentary acetonuria). With an abundant 
 supply of carbohydrates the elimination of acetone bodies may be greatly 
 diminished or even stopped entirely. The carbohydrates therefore 
 act " antiketoplastic," and a similar retarding action can be produced 
 by certain other substances, such as glycerin (Hirschfeld), lactic acid 
 and glutaric acid (Baer and Blum) alanine and asparagin (Forssner, 
 Borchardt and Lange 2 ). Certain bodies like glycerine, lactic acid, 
 alanine, asparagin, which cause a sugar formation or increased elimina- 
 tion of sugar, act in the same way. 
 
 It must not be overlooked that the conditions are different in man 
 and in other carnivora (Geelmuyden, Fr. Voit). In dogs the elimina- 
 tion of acetone bodies is not increased in starvation, but is reduced; it 
 is augmented with increased quantities of meat, runs parallel with the 
 nitrogen excretion, and is not diminished by carbohydrates (Fr. Voit 3 ) . 
 In spite of this divergent behavior an unmistakable relation also exists 
 in the dog between the elimination of acetone bodies and the carbo- 
 hydrate metabolism, because in phlorhizin diabetes the acidosis occurs 
 only after the glycogen has been consumed (Marum 4 ). 
 
 As the carbohydrates cannot be acetone-formers, then a second 
 source only remains, namely, the fats. As proof of this there are certain 
 cases of diabetes with strong elimination of acetone bodies (/3-oxybutyric 
 acid) where the quantity of protein transformed was too small to account 
 for the acetone bodies (Magnus-Levy). The free elimination of acetone 
 bodies in starvation may also depend upon the fact that a great part of 
 
 1 Hirschfeld, Zeitschr. f. klin. Med., 28; Geelmuyden, see Maly's Jahresber., 26, 
 and Zeitschr. f. physiol. Chem., 23 and 26; Rosenfeld, Centralbl. f. innere Med., 16; 
 Voit, Deutsch. Arch. f. klin. Med., 66. 
 
 2 Borchardt and Lange, 1. c; Hofmeister's Beitrage, 9; which also cites other 
 works; Baer and Blum, ibid., 10; Forssner, Skand. Arch. f. Physiol., 25. 
 
 1 See footnote 1. 
 
 4 Hofmeister's Beitrage, 10. 
 
ACETONE BODIES. 821 
 
 the body fat is consumed, and in several cases a certain relation has 
 been found between the fat consumed and the acetone bodies eliminated. 
 Certain investigators (Geelmuyden, Schwarz, Waldvogel) have 
 also observed an increase in the acetonuria on partaking of fatty food, 
 and Forssner l has indeed found a certain parallelism lei ween the 
 acetone elimination and the fat taken up. For the present the fats are 
 considered as the most important source of the acetone bodies. 
 
 The three acetone bodies occurring in the urine, as above stated, are 
 acetone, acetoacetic acid and /3-oxybutyric acid, and this last is considered 
 as the mother-substance of the other two. If /3-oxybutyric acid, 
 CH3.CHOH.CH2.COOH, is introduced into the animal body, it is burnt 
 if the quantity is not too great, while if in excess it passes into the urine 
 as acetoacetic acid, CH3.CO.CH2COOH. This acid can also be burnt, 
 but if large quantities are introduced it appears in part in the urine and 
 readily splits into acetone, CH3.CO.CH3, and CO2. Acetone is in part 
 burnt in the animal body, but a part is eliminated by the kidneys and 
 especially by the lungs. We can imagine that the /3-oxybutyric acid is 
 a physiological metabolic product which normally is completely changed 
 into acetoacetic acid and acetone, and in diabetes and especially with 
 lack of carbohydrates is formed to an increased extent, or its combustion 
 made more difficult, so that in the first place acetone and acetoacetic 
 acid pass into the urine and in severe cases also /3-oxybutyric acid (acidosis). 
 In this connection it must be borne in mind that, because of the previously- 
 mentioned (page 774) reversibility of the process, the direction may also 
 be reversed, that is acetoacetic acid can also be changed into /3-oxybutyric 
 acid in the animal body and this has been proven by perfusion of livers 
 (Friedmann and Maase) as well as in animals (Dakin) and in diabetics 
 (O. Neubauer) 2 . 
 
 Since leucine in perfusion experiments with livers yields acetoacetic acid 
 (Embden and Engel) and also, as Baer and Blum 3 found, that leucine and iso- 
 valeric acid increased the /3-oxybutyric acid elimination in diabetics, it has been 
 accepted that a formation of /3-oxybutyric acid takes place from the leucine 
 with isovaleric acid as an intermediary product : 
 
 (leucine CH 3 ) 2 CH.CH 2 .CH(XH 2 ).Co6h->(CH 3 ) 2 CH.CH2.COOH, isovaleric acid). 
 Valine (a-amino-valeric acid (CH 3 )2CH.CH(NH 2 ).COOH) is, on the contrary, 
 not an acetone former. 
 
 1 Magnus-Levy, Arch. f. exp. Path. u. Pharm., 42; Geelmuyden, 1. 0., and Norsk, 
 Magasin for Laegevidenskaben, 1900; see also Zeitschr. f. physiol. Chem., 41; Schwarz, 
 Deutsch. Arch. f. klin. Med., 1903; Waldvogel, Centralbl. f. inn. Med., 20; Forssner, 
 jskand. Arch. f. Physiol., 22 and 23. 
 
 2 Friedmann and Maase, Bioch. Zeitschr., 27; Dakin, Journ. of biol. Chem., 8; 
 Neubauer, Maly's Jahresb., 40, 849. 
 
 3 Arch. f. exp. Path. u. Pharm., 55 and 56; Embden and Engel, Hofme.ster's 
 Beitrage, 11. 
 
822 URINE. 
 
 In regard to the formation of acetone bodies from fat it must be 
 remarked that glycerin has an antiketoplastic action, and that the 
 fatty acids can only be considered. As to the behavior of these in the 
 formation of acetone, Embden and Marx 1 have shown that only those 
 normal fatty acids which contain an even number of carbon atoms are 
 acetone formers, while those with an uneven number of carbon atoms 
 are without action in this regard. This is true at least for the acids from 
 n-decanoic acid to n-butyric acid, which latter is a strong acetone former. 
 As in diabetics a greater number of oxybutyric acid molecules can be 
 eliminated than corresponds to the number of fatty acid molecules decom- 
 posed, it seems as if more than one molecule of /3-oxybutyric acid is pro- 
 duced from one molecule of fatty acid. We cannot therefore admit 
 of a simple demolition of the fatty acids to butyric acid (by consecutive 
 oxidation attacks in the /3-position) , but rather a destruction of the fatty 
 acid molecules into several parts, and these take part in the formation of 
 /3-oxybutyric acid. 
 
 A synthetical formation of /3-oxybutyric acid has been accomplished by Geel- 
 muyden and others, but especially by Magnus-Levy, starting' with acetaldehyde, 
 according to the hypothesis of Spiro. It is also interesting that Friedmann 2 
 has shown by perfusion experiments with livers that aldehyde ammonia, and to 
 a greater extent aldol, are acetone formers. It must therefore be admitted that 
 first a condensation of the aldehyde to aldol takes place, CH 3 .COH-f CH 3 .COH 
 = CH 3 .CH(OH).CH 2 .COH, and that /3-oxybutryic acid, CH 3 .CH(OH).CH 2 .COOH, 
 is formed from this by oxidation. 
 
 According to the above-mentioned perfusion experiments it must 
 be admitted that the liver is important in the formation of acetone 
 bodies, and Embden and Lattes have found that the ability of the liver 
 of the dog with pancreas diabetes or phloridzin diabetes to produce 
 acetone is much greater than the liver of the normal animal. On the 
 other hand, as shown by Embden and Michaud, 3 in dogs and oxen the 
 liver also has a strong destructive action upon acetoacetic acid. A 
 similar action is also found in the kidneys, muscles and spleen of dogs 
 and pigs. The destructive action of fresh organs is much stronger upon 
 acetoacetic acid than upon acetone. They could not find any special 
 cleavage products, and the above-mentioned, so-called demolition may 
 therefore perhaps in part be a reformation of /3-oxybutyric acid from the 
 acetoacetic acid. 
 
 Acetone, C.3HoO, dimethylketone, CH3.CO.CH3, is a thin, water- 
 clear liquid, boiling at 56.3° and possessing a pleasant odor of fruit, 
 
 1 Hofmeister's Beitriige, 11. 
 
 2 Geelmuyden, Zeitschr. f. physiol. Chem., 23 and 26; Magnus-Levy, Arch. f. exp. 
 Path. u. Pharm., 42; Friedmann, Hofmeister's Beitriige, 11. 
 
 3 Embden and Lattes, Hofmeister's Beitriige, 11; Embden and Michaud, ibid., 11. 
 
ACETONE. 823 
 
 which in diabetes gives a pomaceous or fruit odor to the urine as well as 
 the expired air. It is Lighter than water, with which it mixes in all 
 proportions, also with alcohol and ether. The most important reactions 
 for acetone are the following: 
 
 Lieben's Iodoform Test. When a watery solution of acetone is treated 
 with alkali and then with some iodo-potassium-iodide solution and gently 
 warmed, a yellow precipitate of iodoform is produced, which is known 
 by its odor and by the appearance of the crystals (six-sided plates or stars) 
 under the microscope. This reaction is very delicate, but it is not char- 
 acteristic of acetone. Gunning's modification of the iodoform test con- 
 sists in using an alcoholic solution of iodine and ammonia instead of the 
 iodine dissolved in potassium iodide and alkali hydroxide. In this case, 
 besides iodoform, a black precipitate of nitrogen iodide is formed, but 
 this gradually disappears on standing, leaving the iodoform visible. This 
 modification has the advantage that it does not give any iodoform with 
 alcohol or aldehyde. On the other hand, it is not quite so delicate, 
 but still it detects 0.01 milligram of acetone in 1 cc. 
 
 Frommer's l Test. This reagent is a 10 per cent alcoholic solution 
 of salicylaldehyde. Add 1-2 cc. of this solution to 10 cc. of the solution 
 (urine) and add to this mixture 1 gram KOH in substance, when a 
 carmine-red color will be observed. If necessary warm to about 70° C. 
 This reaction is just as delicate as the above. 
 
 Reynold's Mercuric-oxide Test is based on the power of acetone to 
 dissolve freshly precipitated HgO. A mercuric-chloride solution is pre- 
 cipitated by alcoholic caustic potash. To this add the liquid to be 
 tested, shake well, and filter. In the presence of acetone the filtrate 
 contains mercury, which may be detected by ammonium sulphide. 
 This test has about the same delicacy as Gunning's test. Aldehydes 
 also dissolve appreciable quantities of mercuric oxide. 
 
 Legal's Sodium Nitroprusside Test. If an acetone solution is treated 
 with a few drops of a freshly prepared sodium-nit roprusside solution 
 and then with caustic-potash or soda solution, the liquid is colored ruby- 
 red. Creatinine gives the same color; but if the mixture is saturated 
 with acetic acid, the color becomes carmine or purplish red in the presence 
 of acetone, but yellow and then gradually green and blue in the presence 
 of creatinine. With this test paracresol responds with a reddish-yellow 
 color, which becomes light pink when acidified with acetic acid and can- 
 not be mistaken for acetone. Rothera 2 has suggested a modification 
 which is more delicate by using ammonium salts and ammonia. 
 
 Penzoldt's Indigo Test depends on the fact that orthonitrobenzaldehyde 
 in alkaline solution with acetone yields indigo. A warm saturated and 
 
 1 Berl. klin. Wochenschr., 1905. 2 Journ. of Physiol., 37. 
 
824 URINE. 
 
 then cooled solution of the aldehyde is treated with the liquid to be tested 
 for acetone and next with caustic soda. In the presence of acetone the 
 liquid first becomes yellow, then green, and lastly indigo separates; and 
 this may be dissolved with a blue color by shaking with chloroform; 
 1.6 milligrams acetone can be detected by this test. 
 
 Acetoacetic Acid, C4HGO3, acetylacetic acid, diacetic acid, CH3.CO. 
 CH2.COOH, is a colorless, strongly acid liquid which mixes with water, 
 alcohol, and ether in all proportions. On heating to boiling with water, 
 and especially with acids, it decomposes into carbon dioxide and ace- 
 tone, and therefore gives the above-mentioned reactions for acetone. 
 It differs from acetone in that it gives a violet-red or brownish-red 
 color with a dilute ferric-chloride solution. For the detection of this 
 acid we make use of the following reactions which may be applied directly 
 to the urine: 
 
 Gerhardt's Reaction. Treat 10-15 cc. of the urine with ferric- 
 chloride solution until it fails to give a precipitate filter, and add some 
 more ferric chloride. In the presence of acetoacetic acid a wine-red 
 color is obtained. The color becomes paler at the room temperature 
 within twenty-four hours, but more quickly on boiling (differing from 
 salicylic acid, phenol, sulphocyanides) . A portion of the urine slightly 
 acidified and boiled does not give this reaction on cooling, on account 
 of the decomposition of the acetoacetic acid. 
 
 Arnold and Lipliawsky's Reaction. 6 cc. of a solution contain- 
 ing 1 gram of p-aminoacetophenone and 2 cc. of concentrated hydro- 
 chloric acid in 100 cc. of water are mixed with 3 cc. of a 1 per cent potas- 
 sium-nitrite solution and then treated with an equal volume of urine. 
 A few drops of concentrated ammonia are now added and violently 
 shaken. A brick-red coloration is obtained. Then take 10 drops to 
 2 cc. of this mixture (according to the quantity of acetoacetic acid in 
 the urine), add 15-20 cc. HO of sp.gr. 1.19, 3 cc. of chloroform, and 
 2-4 drops of ferric-chloride solution and mix without shaking. In the 
 presence of acetoacetic acid the chloroform is colored violet or blue 
 (otherwise only yellowish or faintly red). This reaction is more delicate 
 than the preceding test and reacts with 0.04 p.m. acetoacetic acid. Large 
 amounts of acetone (but not the quantity occurring in urines) give this 
 reaction according to Allard. 1 
 
 Bondi and Schwarz's 2 Reaction. 5 cc. of the urine is treated drop by drop 
 with iodine-potassium iodide solution until the color is orange-red. Then warm 
 gently and when the orange-red color has disappeared add the iodine solution 
 again until the color remains permanent on warming. Then boil, when the irritat- 
 ing vapors of iodo-acetone will attack the eyes. Acetone does not give this reaction. 
 
 1 Arnold, Wicn. klin. Wochenschr., 1899, and Centralbl. f. innere Med., 1900; 
 Lipliawsky, Deutsch. med. Wochenschr., 1901; Allard, Berl. klin. Wochenschr., 1901. 
 " Wxn. klin. Wochenschr., 1906. 
 
ACETOACETIC ACID. (9-OXYBUTYRIC ACID. 825 
 
 Detection of Acetone and Acetoacetic Acid in the Urine. Before test- 
 ing for acetone test for acetoacetic acid; as this acid gradually decom- 
 poses on allowing the urine to stand, the specimen must be as fresh as 
 possible. In the presence of acetoacetic acid the urine gives the above- 
 mentioned tests. In testing for acetone in the presence of acetoacetic 
 acid make the urine slightly alkaline and shake in a separatory funnel 
 with ether free from alcohol and acetone. Remove the ether and shake 
 it with water, which takes up the acetone, and test for acetone in the 
 watery solution. 
 
 In the absence of acetoacetic acid the acetone may be tested for 
 directly in the urine; this may be done by Frommer's test or Legal's 
 test. These tests, which are only approximate, are of value only when 
 the urine contains a considerable amount of acetone. 
 
 For a more accurate test \vc distill at least 250 cc. of the urine faintly acidified 
 with sulphuric acid, care being taken to have a good condensation. Most of the 
 acetone is contained in the first 10-20 cc. of the distillate. A better result may 
 be obtained by distilling a large quantity of urine until about tV has been dis- 
 tilled off, acidify the distillate with hydrochloric acid, redistill and repeat this 
 several times, collecting the first portion of each distillation. The final distillate 
 is used for the above reactions. 1 Salkowski and Borchardt have called attention 
 to the fact that in the distillation of an acidified urine containing sugar for the 
 detection or estimation of acetone, a substance giving iodoform can be formed 
 from the sugar if the distillation is carried too far. According to Borchardt 2 
 the urine must therefore first lie diluted with water, or the concentration prevented 
 by the addition of water dropwise during distillation. 
 
 The quantitative estimation of acetone (also that formed from the 
 acetoacetic acid) is done by distilling the urine after the addition of acetic 
 acid or a little sulphuric acid. The quantity of acetone in the distillate 
 can be determined, according to the Huppert-Messinger method, by 
 converting it into iodoform by means of potassium iodide and then titrat- 
 ing the quantity of iodine used in the formation of the iodoform. The 
 precipitation of the acetone as p-nitrophenylhydrazone-acetone by means 
 of p-nitrophenylhydrazine in acetic acid solution can also be used for 
 determining the acetone in the distillate (v. Ekenstein and Blanksma 
 and Moller). In regard to these methods we refer to. 3 Embden and 
 Schliep and Folin 4 have suggested methods for determining the quan- 
 tity of acetone and acetoacetic acid separately. In regard to these 
 estimations we must refer to the work of Embden and Schmitz. 5 
 
 0-Oxybutyric Acid, C , 4H 8 U 3 , = CH ? .CH(OH).CH 2 .COOH, ordinarily 
 forms an odorless syrup, but may also be obtained as crystals. It is readily 
 soluble in water, alcohol, and ether. It is levorotatory ; (a) D =— 24.12° 
 
 1 See also Salkowski, Pfluger's Arch., 56. 
 * Hofmeister's Beitnige, 8. 
 
 3 Hoppe-Seyler, Thierfelder, 8. Aufl., 617 and 618. 
 
 4 Embden and Schliep, Centralbl. f. d. ges. Phys. u. Path. d. Stoffwechsel, 1907; 
 Folin, Journ. of biol. Chem., 3. In regard to the quantitative estimation of acetone 
 see also 0. Sammet, Zeitschr. f. physiol. Chem., 83. 
 
 8 Abderhalden's Handbuch der biochemischen Arbeitsmethoden, Bd. 3. 
 
826 URINE. 
 
 for solutions of 1-11 per cent and has a disturbing action upon the deter- 
 mination of sugar by means of the polariscope. It is not precipitated 
 by basic lead acetate or by ammoniacal lead acetate, neither does it 
 ferment. On boiling with water, especially in the presence of a mineral 
 acid, this acid decomposes into a-crotonic acid, which melts at 71-72° 
 C, and water, CH : ,.CH(OH).CH2.COOH = H 2 0-r-CH3.CH:CH.COOH. 
 It yields acetone on oxidation with a chromic-acid mixture. 
 
 Detection of 0-Oxybutyric Acid in the Urine. If a urine is still levo- 
 gyrate after fermentation with yeast, the presence of oxybutyric acid 
 is probable. A further test may be made, according to Rulz, by evaporat- 
 ing the fermented urine to a syrup and, after the addition of an equal 
 volume of concentrated sulphuric acid, distilling directly without cool- 
 ing, a-crotonic acid is produced, which distills over, and, after collect- 
 ing in a test-tube, crystals which melt at +72° C. separate on cooling. 
 If no crystals are obtained, shake the distillate repeatedly with ether and 
 let this spontaneously evaporate. The crystals which separate out 
 can be purified according to Embden and Schmitz by redissolving in ether, 
 evaporating the chief part of the ether and precipitating with petroleum- 
 ether, which removes the volatile fatty acids and benzoic acid. 
 
 The quantitative estimation is done by complete extraction of the 
 P-oxy butyric acid by ether and determining the specific rotation. The 
 extraction can be done according to Magnus-Levy l or according to 
 Bergell. 2 Other methods of estimating /3-oxybutyric acid have been 
 suggested by Darmstadter, Boekelman and Bouma. 3 In regard to 
 the quantitative estimation we refer to Embden and Schmitz. 4 
 
 Ehrlich's 5 Urine Test. Mix 250 cc. of a solution which contains 50 cc. of 
 HC1 and 1 gram of sulphanilic acid in one liter, with 5 cc. of a \ per cent solution 
 of sodium nitrite (which produces very little of the active body, sulphodiazo- 
 benzene). In performing this test treat the urine with an equal volume of this 
 mixture and then supersaturate with ammonia. Normal urine will become 
 yellow or orange after the addition of ammonia (aromatic oxyacids may after a cer- 
 tain time give red azo bodies which color the upper layer of the phosphate sediment). 
 In pathological urines there sometimes occurs (and this is the characteristic 
 diazo reaction) a primary yellow coloration, with very marked secondary red 
 coloration on the addition of ammonia, and the froth is also tinged with red. 
 The upper layer of the sediment becomes greenish. The body which gives this 
 reaction is unknown, but it especially occurs in the urine of typhoid patients 
 (Ehrlich). Opinions differ in regard to the significance of this reaction. If 
 the urine is made alkaline with sodium carbonate instead of ammonia and treated 
 
 1 See Hoppe-Seyler, Thierfelder's Handbuch, 8. Aufl., G19, and Geelmuyden, Ham- 
 marsten's Festschr., 1906. 
 
 5 Zeitschr. f. physiol. Chem., 33. 
 
 a Darmstadter, ibid., 37; Boekelman and Bouma, see Maly's Jahresber, 31. 
 
 1 Bee footnote •",, page 825. 
 
 'Ehrlich, Zeitschr. f. klin. Med., 5. See also Clemens, Deutsch. Arch. f. klin. 
 Med., (53 (literature). Kutscher and Engeland, footnote 1, page 758. 
 
CYSTINE. 827 
 
 with a freshly prepared solution of diazobenzene sulphonic acid made alkaline 
 with sodium carbonate, normal urine also rives an orange or Bordeaux-red 
 coloration. The known normal urinary constituents which give the diazo reac- 
 tion are the aromatic oxyacidfl, antoxyproteic acid and the imidazole derivative 
 found by Engeland (see page 758). 
 
 Ehrlich's reaction with p-dime1 hylaminobenzaldehyde has already been 
 discussed in connection with urobilinogen. 
 
 Hosenbach's urine test, which consists in adding nitric acid drop by drop 
 to the boiling-hot urine and obtaining a claret-red coloration and a bluish-red 
 foam on shaking, depends upon the formation of indigo substances, especially 
 indigo-red. 1 
 
 Fat in the Urine. The elimination of a urine which in appearance and rich- 
 ness in fat resembles chyle is called chyluria. It habitually contains a proteid 
 and often fibrin. Chyluria occurs mostly in the inhabitants of the tropics. 
 Lipuria, or the elimination of fat with the urine, may appear in apparently healthy 
 persons, sometimes with and sometimes without albuminuria, in pregnancy, and 
 also in certain diseases, as in diabetes, poisoning with phosphorus, and fatty 
 degeneration of the kidneys. 
 
 Fat is usually detected by the microscope. It may also be dissolved with 
 ether, and may invariably be detected by evaporating the urine to dryness and 
 extracting the residue with ether. 
 
 Cholesterin is also sometimes found in the urine in chyluria and in a few other 
 cases. 
 
 Amino-acids. Leucine and tyrosine have been repeatedly found in urine by 
 the older methods, especially in acute yellow atrophy of the liver, in acute phos- 
 phorus poisoning, and in severe cases of typhoid and smallpox. Since (3-naphtha- 
 lene sulphochloride has been used in the detection of amino-acids these bodies have 
 not only been repeatedly found in normal urine (glycocoll, see page 756), but also 
 in pathological urines. 2 
 
 Cystine. Baumann and Goldmann claim that a substance similar 
 to cystine occurs in very small amounts in normal urine. This substance 
 occurs in large quantities in the urine of dogs after poisoning with phos- 
 phorus. Cystine itself is only found with positiveness, and even then 
 very rarely, in urinary calculi and in pathological urines, from which 
 it may separate as a sediment. Cystinuria occurs oftener in men than 
 in women. Baumann and v. Udranszky found in urine in cystinuria 
 the two diamines, cadaverine (pentamethylendiamine) and putrescine 
 (tetramethylendiamine), which are produced in the putrefaction of 
 proteins. Cases of cystinuria may occur with or without the occurrence 
 of diamines in the urine, and only rarely are the diamines found in the 
 urine as well as in the feces, which perhaps depends upon the fact, as 
 found by Cammidge and Garrod 3 in one case, that the diamines occur 
 
 1 See Rosin, Virchow's Arch., 123. 
 
 2 Ignatowski, Zeitschr. f. physiol. Chem., 42; Abderhalden and Schittenhelm, 
 ibid., 45; Abderhalden and Barker, ibid., 42. See also footnote 5, page 756, and 2, 757. 
 
 3 Baumann, Zeitschr. f. physiol. Chem., 8. In regard to the literature on cystinuria 
 6ee Brenzinger, ibid., 16; Baumann and Goldmann, ibid., 12; Baumann and v. 
 Udranszky, ibid., 13; Stadthagen and Brieger, Berlin, klin. Wochenschr., 1889; 
 Cammidge and Garrod, Journ. of Path, and Bacteriol., 1900 (literature on diamines 
 in the urine and feces); Loewy and Neuberg, Bioeh. Zeitschr., 2; Wolf and Schaffer, 
 Journ. of biol. Chem., 4; Williams and Wolf, ibid., 6. 
 
828 UKINE. 
 
 only from time to time in the feces. Cystinuria is generally admitted as 
 rather an anomaly in the protein metabolism where the cystine for 
 unknown reasons is not destroyed as ordinarily. It is remarkable 
 that the cystine of the food-proteins is eliminated by the urine, while in 
 cystinurics, at least sometimes, such cystine introduced is quantitatively 
 transformed. 1 Certain observations, such as the appearance of lysine 
 in the urine of cystinurics (Ackermann and Kutscher 2 ), make it probable 
 that the demolition of other amino-acids is diminished in cystinuria. 
 The properties and reactions of cystine have been given on pages 148 
 and 149. 
 
 Cystine is easily prepared from cystine calculi by dissolving them 
 in alkali carbonate, precipitating the solution with acetic acid, and redis- 
 solving the precipitate in ammonia. The cystine crystallizes on the 
 spontaneous evaporation of the ammonia. The cystine dissolved in 
 the urine is detected, in the absence of proteid and sulphuretted hydrogen, 
 by boiling with alkali and testing with a lead salt or sodium nitroprusside. 
 To isolate cystine from the urine, acidify the urine strongly with acetic 
 acid. The precipitate containing cystine is collected after twenty-four 
 hours and digested with hydrochloric acid, which dissolves the cystine 
 and calcium oxalate, leaving the uric acid undissolved. Filter, supersat- 
 urate the filtrate with ammonium carbonate, and treat the precipitate 
 with ammonia, which dissolves the cystine and leaves the calcium oxalate. 
 Filter again and precipitate with acetic acid. The precipitated cystine 
 is identified by the microscope and the above-mentioned reactions. 
 Cystine as a sediment is identified by the microscope. It must be purified 
 by dissolving in ammonia and precipitating with acetic acid; it is then 
 further tested. Traces of dissolved cystine may be detected by the 
 production of benzoyl-cystine, according to Baumann and Goldmann. 
 For the detection and estimation of cystine we can proceed to advantage 
 in the following manner, suggested by Gaskell. 3 The urine freed from 
 oxalates and phosphates by means of ammonia and calcium chloride is 
 treated with an equal volume of acetone and with acetic acid. The 
 crystals which precipitate are dissolved in ammonia and then purified 
 by reprecipitation with acetone. 
 
 VH. URINARY SEDIMENTS AND CALCULI. 
 
 Urinary sediment is the more or less abundant deposit which is found 
 in the urine after standing. This deposit may consist partly of organized 
 and partly- of non-organized constituents. The first, consisting of cells 
 of various kinds, yeast-fungi, bacteria, spermatozoa, casts, etc., must 
 be investigated by means of the microscope, and the following only 
 applies to the non-organized deposits. 
 
 1 See Wolf and Schaffer, Journ. of biol. Chem., 4; and Hele, Journ. of Physiol.. 39. 
 
 2 Zeitschr. f. Biol., 57. 
 
 8 Journ. of Physiol., 36. 
 
URINARY SEDIMENTS. 829 
 
 As previously mentioned (page 674), the urine of healthy individuals 
 may sometimes, even on voiding, be cloudy on account of the phosphates 
 present, or become so after a little while because of the separation of 
 urates. As a rule, urine just voided is clear, and after cooling shows 
 only a faint cloud (nubecula) which consists of urine mucoid, a few epithe- 
 lium-cells, mucous corpuscles, and urate particles. If an acid urine is 
 allowed to stand, it will gradually change; it becomes darker and deposits 
 a sediment consisting of uric acid or urates, and sometimes also calcium- 
 oxalate crystals, in which yeast-fungi and bacteria are often to be seen. 
 This change, which the earlier investigators called " acid fermenta- 
 tion of the urine," is generally considered as an exchange of the dihy- 
 drogen alkali phosphates with the urates of the urine. Monohydrogen 
 phosphates besides acid urates, quadriurates (page 708) or free uric acid 
 or a mixture of both, according to conditions, 1 are thus formed. 
 
 Sooner or later, sometimes only after several weeks, the reaction 
 of the original acid urine changes and becomes neutral or alkaline. The 
 urine has now passed into the " alkaline fermentation," which con- 
 sists in the decomposition of the urea into carbon dioxide and ammonia 
 by means of lower organisms, micrococcus ureae, bacterium ureae, and 
 other bacteria. Museums 2 has isolated an enzyme from the micro- 
 coccus ureae which decomposes urea, which is soluble in water and is called 
 urease. During the alkaline fermentation volatile fatty acids, especially 
 acetic acid, may be produced, chiefly by the fermentation of the car- 
 bohydrates of the urine (Salkowski 3 ). A fermentation by which 
 nitric acid is reduced to nitrous acid, and another where sulphuretted 
 hydrogen is produced, may sometimes occur. 
 
 When the alkaline fermentation has advanced only so far as to render 
 the reaction neutral, there often occur in the sediment fragments of uric- 
 acid crystals, sometimes covered with prismatic crystals of alkali urate; 
 dark-colored spheres of ammonium urate, crystals of calcium oxalate, 
 and sometimes crystallized calcium phosphate are also found. Crystals 
 of ammonium-magnesium phosphate (triple phosphate) and spherical 
 ammonium urate are specially characteristic of alkaline fermentation. 
 The urine in alkaline fermentation becomes paler and is often covered 
 with a fine membrane which contains amorphous calcium phosphate 
 and glistening crystals of triple phosphate and numerous micro-organisms. 
 
 1 See Huppert-Xeubauer, 10. Aufl., and A. Ritter, Zeitschr. f. Biologie, 35. 
 
 5 Museulus, Pfliiger's Arch., 12. 
 
 1 Salkowski, Zeitschr. f. Physiol. Chem., 13. 
 
830 URINE. 
 
 Non-Organized Sediments. 
 
 Uric Acid. This acid occurs in acid urines as colored crystals which 
 are identified partly by their form and partly by their property of giving 
 the murexid test. On warming the urine they are not dissolved. On 
 the addition of caustic alkali to the sediment the crystals dissolve, and 
 when a drop of this solution is placed on a microscope-slide and treated 
 with a drop of hydrochloric acid, small crystals of uric acid are obtained 
 which can be easily seen under the microscope. 
 
 Acid Urates. These occur only in the sediment of acid or neutral 
 urines. They are amorphous, clay-yellow, brick-red, rose-colored, or 
 brownish-red. They differ from other sediments in that they dissolve 
 on warming the urine. They give the murexid test, and small micro- 
 scopic crystals of uric acid separate on the addition of hydrochloric 
 acid. Crystalline alkali urates occur very rarely in the urine, and as a 
 rule only in such as have become neutral but not alkaline, by alkaline 
 fermentation. The crystals are somewhat similar to those of neutral 
 calcium phosphate; they are not dissolved by acetic acid, however, 
 but give a cloudiness therewith due to small crystals of uric acid. 
 
 Ammonium urate may indeed occur as a sediment in a neutral urine 
 which at first was strongly acid and has become neutralized by the alkaline 
 fermentation, but it is only characteristic of ammoniacal urines. This 
 sediment consists of yellow or brownish rounded spheres which are often 
 covered with thorny-shaped prisms and, because of this, are rather 
 large and resemble the thorn-apple. It reacts to the murexid test. It 
 is dissolved by alkalies with the development of ammonia, and crystals 
 of uric acid separate on the addition of hydrochloric acid to this solution. 
 
 Calcium oxalate occurs in the sediment generally as small, shining, 
 strongly refractive quadratic octahedra, which on microscopical examina- 
 tion remind one of a letter-envelope. The crystals can only be mistaken 
 for small, not fully developed crystals of ammonium-magnesium phos- 
 phate. They differ from these by their insolubility in acetic acid. The 
 oxalate may also occur as flat, oval, or nearly circular disks with central 
 cavities which from the side appear like an hour-glass. Calcium oxalate 
 may occur as a sediment in an acid as well as in a neutral or alkaline 
 urine. The quantity of calcium oxalate separated from the urine as 
 sediment depends not only upon the amount of this salt present, but 
 also upon the acidity of the urine. The solvent for the oxalate in the 
 urine seems to be the diacid alkali phosphate, and the greater the quan- 
 tity of this salt in the urine the greater the quantity of oxalate in solu- 
 tion. When, as previously mentioned (page 829), the simple-acid phos- 
 phate is formed from the diacid phosphate, on allowing the urine to 
 stand, a corresponded part of the oxalate may be separated as sediment. 
 
URINARY SEDIMENTS. 831 
 
 Calcium carbonate occurs in considerable quantities as sediment in 
 the urine of herbivora. It occurs in but small quantities as a sediment 
 in human urine, and in fact only in alkaline urines. It either has the 
 same appearance as amorphous calcium oxalate or it occurs as some- 
 what larger spheres with concentric bands. It dissolves in acetic acid 
 with the generation of gas, which differentiates it from calcium oxalate. 
 It is not yellow or brown like ammonium urate, and does not give the 
 murexid test. 
 
 Calcium sulphate occurs very rarely as a sediment in strongly acid urine. It 
 appears as long, thin, colorless needles, or generally as plates grouped together. 
 
 Calcium Phosphate. The calcium triphosphate, Ca3(P04) 2 , which 
 occurs only in alkaline urines, is always amorphous and occurs partly 
 as a colorless, very fine powder, and partly as a membrane consisting of 
 very fine granules. It differs from the amorphous urates in that it is 
 colorless, dissolves in acetic acid, but remains undissolved on warming 
 the urine. Calcium Diphosphate, CaHP04+2H20, occurs in neutral 
 or only in very faintly acid urine. 1 It is found sometimes as a thin 
 film covering the urine and sometimes as a sediment. In crystallizing, 
 the crystals may be single, or they may cross one another, or they may 
 be arranged in groups of colorless, wedge-shaped crystals whose wide 
 end is sharply defined. These crystals differ from crystalline alkali 
 urates in that they dissolve without a residue in dilute acids and do not 
 give the murexid test. 
 
 Ammonium-magnesium phosphate, triple phosphate, may separate 
 from an amphoteric urine in the presence of a sufficient quantity of ammo- 
 nium salts, but it is generally characteristic of a urine which is ammo- 
 niacal through alkaline fermentation. The crystals are so large that they 
 may be seen with the unaided eye as colorless glistening particles in the 
 sediment, on the walls of the vessel, and in the film on the surface of the 
 urine. This salt forms large prismatic crystals of the rhombic system 
 (coffin-shaped) which are easily soluble in acetic acid. Amorphous 
 magnesium triphosphate, Mg3(P04)2, occurs with calcium triphosphate 
 in urines rendered alkaline by a fixed alkali. Crystalline magnesium 
 phosphate, Mg3(P04)2+22H20, has been observed in a few cases in 
 human urine (also in horse's urine) as strongly refractive, long rhombic 
 plates. 
 
 As more rare sediments we fine* cystine, tyrosine, hippuric acid, xanthine, hama- 
 toidine. In alkaline urine blue crystals of indigo may also occur, due to a decom- 
 position of indoxyl-glucuronic acid. 
 
 1 In regard to the conditions for the appearance of these sediments in urines see 
 C. Th. Morner, Zeitschr. f. physiol. Chem., 58. 
 
832 UEINE. 
 
 Urinary Calculi. 
 
 Besides certain pathological constituents of the urine, all those urinary- 
 constituents which occur as sediments take part in the formation of 
 urinary calculi. Ebstein 1 considers the essential difference between an 
 amorphous and crystalline sediment in the urine on the one side and urinary 
 sand or large calculi on the other to be the occurrence of an organic 
 frame in the latter. As the sediments which appear in normal acid urine 
 and in a urine alkaline through fermentation are diverse, so also are the 
 urinary calculi which appear under corresponding conditions. 
 
 .If the formation of the calculus and its further development take place 
 in an un decomposed urine, it is called a primary formation. If, on the 
 contrary, the urine has undergone alkaline fermentation and the ammonia 
 formed thereby has given rise to a calculus formation by precipitating 
 ammonium urate, triple phosphate, and earthy phosphates, then it is 
 called a secondary formation. Such a formation takes place, for 
 instance, when a foreign body in the bladder produces catarrh accom- 
 panied by alkaline fermentation. 
 
 We discriminate between the nucleus or nuclei — if such can be seen — 
 and the different layers of the calculus. The nucleus may be essentially 
 different in 'different cases, for quite frequently it consists of a foreign 
 body introduced in the bladder. The calculus may have more than 
 one nucleus. In a tabulation made by Ultzmann of 545 cases of vesic- 
 ular calculi, the nucleus in 80.9 per cent of the cases consisted of uric 
 acid (and urates); in 5.6 per cent, of calcium oxalate; in 8.6 per cent, 
 of earthy phosphates; in 1.4 per cent, of cystein; and in 3.5 per cent, 
 of some foreign body. 
 
 During the growth of a calculus it often happens that, for some reason 
 or other, the original calculus-forming substance is covered with another 
 layer of a different substance. A new layer of the original substance may 
 deposit on the outside of this, and this process may be repeated. In 
 this way a calculus consisting originally of a simple stone may be con- 
 verted into a so-called compound stcne with several layers of different 
 substances. Such calculi are always formed when a primary is changed 
 into a secondary formation. By the continued action of an alkaline 
 urine containing pus, the primary constituents of a primary calculus 
 may be partly dissolved and be replaced by phosphates. Metamor- 
 phosed urinary calculi are formed in this way. 
 
 Die Natur unci Behandlung der Harnsteine. Wiesbaden, 1884. 
 
URINARY CALCULI. 833 
 
 Uric-acid calculi are very abundant. They are variable in size and 
 form. The size of the bladder-stone varies from that of a pea or bean to 
 that of a goose-egg. Uric acid stones are always colored; generally 
 they are grayish-yellow, yellowish-brown, or pale red-brown. The upper 
 surface is sometimes entirely even or smooth, sometimes rough or uneven. 
 Next to the oxalate calculus the uric-acid calculus is the hardest. The 
 fractured surface shows regular concentric, unequally colored layers 
 which may often be removed as shells. These calculi are formed pri- 
 marily. Layers of uric acid sometimes alternate with other layers of 
 primary formation, most frequently with layers of calcium oxalate. 
 The simple uric-acid calculus leaves very little residue when burnt on 
 a platinum foil. It gives the murexid test, but there is no material 
 development of ammonia when acted on by caustic soda. 
 
 Ammonium urate calculi occur as primary calculi in new-born or nurs- 
 ing infants, rarely in grown persons. They often occur as a secondary 
 formation. The primary stones are small, with a pale yellow or dark- 
 yellowish surface. When moist they are almost like dough; in the 
 dry state they are earthy, easily crumbling into pale powder. They 
 give the murexid test and develop much ammonia with caustic soda. 
 
 Calcium-oxalate calculi are, next to uric-acid calculi, the most abundant. 
 They are either smooth and small (hemp-seed calculi) or larger, of the 
 size of a hen's egg, with rough, uneven surface, or their surface is cov- 
 ered with prongs (mulberry calculi). These calculi produce bleeding 
 easily, and therefore they often have a dark-brown surface due to decom- 
 posed blood-coloring matters. Among the calculi occurring in man 
 these are the hardest. They dissolve in hydrochloric acid without 
 developing gas, but are not soluble in acetic acid. After gently heating 
 the powder, it dissolves in acetic acid with frothing. With more intense 
 heat it becomes alkaline, due to the production of quicklime. 
 
 Phosphate Calculi. These, which consist mainly of a mixture of the 
 normal phosphate of the alkaline earths with triple phosphate, may be 
 very large. They are as a rule of secondary formation and contain 
 besides these phosphates also some ammonium urate and calcium oxalate. 
 These calculi ordinarily consist of a mixture of three constituents — 
 earthy phosphate, triple phosphate, and ammonium urate — surrounding 
 a foreign body as a nucleus. Their color is variable — white, dingy white, 
 pale yellow, sometimes violet or lilac-colored (from indigo red). The 
 surface is always rough. Calculi consisting of triple phosphate alone 
 are seldom found. They are ordinarily small, with granular or radiated 
 crystalline fracture. Stones of mono-acid calcium phosphate are also 
 seldom obtained. They are white and have beautiful crystalline texture. 
 The phosphatic calculi do not burn up, the powder dissolves in acid 
 without effervescence, and the solution gives the reactions for phos- 
 
834 URINE. 
 
 phoric acid and the alkaline earths. The triple-phosphate calculi generate 
 ammonia on the addition of an alkali. 
 
 Calcium-carbonate ^calculi occur chiefly in herbivora. They are seldom found 
 in man. They have mostly chalky properties, and are ordinarily white. They 
 are completely or in great part dissolved by acids with effervescence. 
 
 Cystine calculi occur but seldom. They are of primary formation, of various 
 sizes, sometimes as large as a hen's egg. They have a smooth or rough surface, 
 are white or pale yellow, and have a crystalline fracture. They are not very 
 hard and are consumed almost entirely on the platinum foil, burning with a bluish 
 flame. They give the above-mentioned reactions for cystine. 
 
 Xanthine calculi are very rarely found. They are also of primary formation. 
 They vary from the size of a pea to that of a hen's egg. They are whitish, yel- 
 lowish-brown or cinnamon-brown in color, of medium hardness, with amorphous 
 fracture, and on rubbing appear like wax. They burn up completely when heated 
 on a platinum foil. They give the xanthine reaction with nitric acid and alkali, 
 but this must not be mistaken for the murexid test. 
 
 Urostealith calculi have been observed only a few times. In the moist state 
 they are soft and elastic at the temperature of the body, but in the dry state 
 they are brittle, with an amorphous fracture and waxy appearance. They burn 
 with a luminous flame when heated on platinum foil and generate an odor similar to 
 resin or shellac. Such a calculus, investigated by Krukenberg, 1 consisted of 
 paraffin derived from a paraffin bougie used as a sound on the patient. Perhaps 
 the urostealith calculi observed in other cases had a similar origin, although the 
 substances of which they consisted have not been closely studied. Horbaczew- 
 ski has recently analyzed a case of urostealith which, to all appearances, was 
 formed in the bladder. This calculus contained 25 p. m. water, 8 p. m. inorganic 
 bodies, 117 p. m. bodies insoluble in ether, and 850 p. m. organic bodies soluble 
 in ether, among which were 515 p. m. free fatty acids, 335 p. m. fat, and traces of 
 cholesterin. The fatty acids consisted of a mixture of stearic, palmitic, and 
 probably myristic acids. - 
 
 Horbaczewski 2 has also analyzed a bladder stone which contained 958.7 
 p. m. cholesterin. 
 
 Fibrin calculi sometimes occur. They consist of more or less changed fibrin- 
 coagulum. On burning they develop an odor of burnt horn. 
 
 The chemical investigation of urinary calculi is of great practical impor- 
 tance. To make such an examination actually instructive it is necessary 
 to investigate, separately, the different layers which constitute the cal- 
 culus. For this purpose saw the calculus, previously wrapped in paper, 
 with a fine saw so that the nucleus becomes accessible. Then peel off the 
 different layers, or, if the stone is to be kept, scrape off enough of the 
 powder from each layer for examination. This powder is then tested by 
 heating on the platinum foil. It must not be forgotten that a calculus 
 is never entirely burnt up, and also that it is never so free from organic 
 matter that on heating it does not carbonize. Do not, therefore, lay too 
 great stress on a very insignificant unburnt residue or on a very small 
 
 l Chem. Untersuch. z. wissensch. Med., 2. Cited from Maly's Jahresber., 19, 422. 
 2 Zeitschr. f. physiol. Chem., 18. 
 
URINARY CALCULI. 835 
 
 amount of organic matter, but consider the calculus in the former case 
 as completely burnt and in the latter as unaffected. 
 
 When tlif powder is in great part burnt up, but a significant quantity 
 of unburnt residue remains, then the powder in question contains as a 
 rule urat<- mixed with inorganic bodies. In such cases remove the urate 
 with boiling water and then test the filtrate for uric acid and the sus- 
 pected bases. The residue is then tested according to the following 
 scfu me of Heller, which is well adapted to the investigation of urinary 
 calculi. In regard to the more detailed examination the reader is 
 referred to special works on the subject. 
 
836 
 
 URINE. 
 
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CHAPTER XV 
 THE SKIN AND ITS SECRETIONS 
 
 In the structure of the skin of man and vertebrates many different 
 kinds of substances occur which have already been considered, such as 
 the constituents of the epidermal formation, the connective and fatty 
 tissues, the nerves, muscles, etc. Among these the different horn struc- 
 tures, the hair, nails, etc., whose chief constituent, keratin, has been 
 spoken of in another chapter (Chapter II), are of special interest. 
 
 The cells of the horny structure show, in proportion to their age, a 
 different resistance to chemical reagents, especially fixed alkalies. The 
 younger the horn-cell the less resistance it has to the action of alkalies; 
 with advancing age the resistance becomes greater, and the cell-mem- 
 branes of many horn-formations are nearly insoluble in caustic alkalies. 
 Keratin (or the keratins) occurs in the horn structure mixed with other 
 bodies, from which it is isolated with difficulty. These are detected by 
 microchemical investigations, and according to Unna l three different 
 substances can be detected in the horn substance, designated by him A-, 
 B- and C-keratin. 
 
 The il-keratin, which forms the envelope of the horn and hair cells and the 
 outer layer of the hair, is the purest keratin. It is not dissolved by fuming nitric 
 acid at the ordinary temperature and does not give the xanthoproteic reaction, 
 and its keratin nature is doubtful. The 5-keratin, which occurs as the contents 
 of the nail cells, gives the xanthoproteic reaction like the C-keratin occurring in 
 hair, but differs from the C-keratin by being soluble in fuming nitric acid. 
 
 Besides these substances, which have been called keratins, the horn 
 structure also contains other proteins which are soluble in pepsin- 
 hydrochloric acid. Among these we find residue of nuclei and the 
 so-called trichohyalin in the hair, which is a substance of unknown 
 constitution and characterized by great insolubility. From these 
 statements it is evident that we are here dealing with a mixture of dif- 
 ferent substances and for this reason it is unnecessary to give the older 
 elementary analyses of the various epidermoidal structures. 
 
 1 Monatsch. f. prakt. Dermat., 44. 
 
 837 
 
838 THE SKIN AND ITS SECRETIONS. 
 
 The quantity of sulphur and of mineral bodies is of certain interest. 
 The sulphur and cystine content of these structures can be found on 
 pages 113, 114 and in this connection it must be mentioned that, accord- 
 ing to the investigations of Rutherford and Hawk, 1 the sulphur content 
 of human hair is higher in men than in women, at least for the Caucasian 
 race, and also that red hair has the highest sulphur content irrespective 
 of race or gender. Hair on incineration leaves considerable ash, which 
 in human hair varies between 2.6 and 16 p. m., and in animal hair 
 is still greater, even up to 71 p. m. in the hair of the deer. The ash 
 consists of large amounts of alkali and calcium sulphate, and its sulphur 
 probably originates from the organic substance, which make the state- 
 ments as to the composition of the ash of hair of little value. Calcium 
 occurs in larger amounts, especially phosphate as well as carbonate, and is 
 most abundant in white hair. The amount of iron oxide in 1000 grams 
 of the ash of human hair varies between 42.2 grams in blond and 108.7 
 grams in brown hair, and silicic acid between 66.1 grams in black and 
 424.6 grams in red hair (Baudrimont). The nails are rich in calcium 
 phosphate, and the feathers rich in silicic acid, especially the feathers 
 of grain-eating birds. According to v. Gorup-Besanez 2 the quantity 
 of silicic acid in grain-eating birds was 400 p. m., and in meat, berries 
 and insect-eating birds the amount was only 270 p. m. of the total ash. 
 Drechsel claims that at least a part of the silicic acid exists in the 
 feathers in organic combination as an ester while according to Cerny 3 it 
 exists only as an accidental contamination. 
 
 According to Gautier and Bertrand 4 arsenic also occurs in the 
 epidermal formations. Gautier says that arsenic is of importance in 
 the formation and growth of the formations, and on the other hand the 
 hair, nails, and epidermis-cells are of great importance in the excretion 
 of arsenic. 
 
 The ability of the skin to take up chlorides as observed by Wahlgren 
 aDd by Padtberg, 5 is remarkable. According to them the skin is an 
 important chloride depot, which stores up chlorides when supplied in 
 excess and gives them up when necessary. 
 
 The skin of invertebrates has been the subject, in a few cases, of 
 chemical investigation, and in these animals various substances have 
 been found, of which a few, though little studied, are worth discussing. 
 Among them tunicin, which is found especially in the mantle of the 
 
 1 Journ. of Biol. Chem., 3. 
 
 1 Lehr. d. physiol. Chem., 4. Aufl., 660, 661; Baudrimont, ibid. 
 
 * Drechsel, Centralbl. f. Physiol., 11, 361; Cerny, Zeitschr. (. physiol. Chem., 62. 
 
 ♦Gautier, Compt. Rend., 129, 130, 131; Bertrand, ibid., 134. 
 
 ' Wahlgren, Arch. f. exp. Path. u. Pharm., 61; Padtberg, ibid., 63. 
 
TUNICIN. CHITIN. 839 
 
 tunicata, and the widely diffused chitin, found in the cuticle-formation of 
 invertebrates, arc of interest. 
 
 Tunicin. Cellulose seems, from the investigations of Amukonn, to occur 
 rather extensively in the animal kingdom in the aithropoda and the mollusks. 
 It has been known for a long time as the mantle of the tunicata, and this animal 
 cellulose was called tunicin by Berthelot. According to the investigations 
 of Winterstein there does not seem to exist any marked difference between 
 tunicin and ordinary vegetable cellulose. On boiling with dilute acid, tunicin 
 yields glucose, as shown first by Franchimont and later confirmed by Win- 
 terstein. By the action of acetic acid anhydride and sulphuric acid, upon 
 tunicate-cellulose, Abderhalden and Zemplen 1 obtained octoacetyl-cellobiose, 
 which also indicates the relationship with the plant cellulose. 
 
 Chitin is not found in vertebrates. In invertebrates chitin is alleged 
 to occur in several classes of animals; it occurs chiefly in cephalopods 
 (sepia scales) and especially in the arthropods, in which it forms the 
 chief organic constituent of the shells, etc. It has been found in the 
 plant kingdom as in fungi (Gilson, Winterstein 2 ). The question 
 whether there are two or more chitins or whether there is only one 
 is still disputed (Krawkow, Zander, Wester 3 ). No formula can be 
 given for the same reasons (Sundwik, Araki, Brach 4 ). 
 
 Chitin is decomposed on boiling with mineral acids and yields, as 
 shown by Ledderhose, glucosamine and acetic acid. Hoppe-Seyler 
 and Araki found, on heating with alkali and a little water to 180°, that 
 chitin was split into a new substance, chitosan, and acetic acid, and that 
 this chitosan contained acetyl groups as well as glucosamine. Frankel 
 and Kelly as well as Offer 5 have obtained acetylglucosamine, 
 (C6Hi2N05)COCH3 and acetyldiglucosamine (Ci2H23N 2 Cq)COCH3 as 
 cleavage products of chitin, and they consider chitin as a polymeric 
 monacetyldiglucosamine. 
 
 The chitosan which v. Furth and Russo 6 have obtained as a crys- 
 talline hydrochloric acid combination and which E. Loewy has obtained 
 as a crystalline sulphate is, according to the latter, a polymeric monacetyl- 
 diglucosamine with at least two monacetyldiglucosamine groups. Accord- 
 
 1 Ambronn, Maly's Jahresber., 20; Berthelot, Annal. de Chim. et Phys., 56, Compt. 
 Rend., 47; Winterstein, Zeitschr. f. physiol. Chem., 18; Franchimont, Ber. d. deutsch. 
 chem. Gesellsch., 12; Abderhalden and Zemplen, Zeitschr. f. physiol. Chem., 72. 
 
 2 Gilson, Compt. Rend., 120; Winterstein, Ber. d. d. chem. Gesellsch., 27 and 28. 
 'Krawkow, Zeitschr. f. Biol., 29; Zander, Pfliiger's Arch., 66; Wester, Chem. 
 
 Centralbl., 1909, II. 
 
 4 Sundwik, Zeitschr. f. physiol. Chem., 5; Araki, ibid., 20; Brach, Bioch. Zeitschr., 
 38. 
 
 5 Ledderhose, Zeitschr. f. physiol. Chem., 2 and 4; Araki, 1. c, Frankel and Kelly, 
 Monatshefte f. Chem., 23; Offer, Bioch. Zeitschr., 7. 
 
 fr v. Furth and Russo, Hofmeister's Beitrage, 8; Loewy, Bioch. Zeitschr., 23; Brach, 
 1. c. 
 
840 THE SKIN AND ITS SECRETIONS. 
 
 ing to v. Furth and Russo on cleavage it yields 25 per cent acetic acid 
 and 60 per cent glucosamine. The formula is (C28H5oN40i9)z according 
 to v. FtiRTH and collaborators and splits according to the equation: 
 (C 2 8H 5 oN40i9)4+5:cH20 = 43(C 6 Hi3N05)+2x(CH 3 COOH). According 
 to Brach, who admits of at least four glucosamine groups in chitosan, 
 the formula for chitin is (C32H 5 4N402i)x and contains 4 acetyl for every 
 4 nitrogen atoms. The transformation into chitosan consists in a 
 rupture of one-half of the acetic acid groups in the chitin. 
 
 In a dry state chitin forms a white, brittle mass retaining the form 
 of the original tissue. It is insoluble in boiling water, alcohol, ether, 
 acetic acid, dilute mineral acids, and dilute alkalies. It is soluble in 
 concentrated acids. It is dissolved without decomposing in cold con- 
 centrated hydrochloric acid, but is decomposed by boiling hydrochloric 
 acid. According to Krawkow the various chitins behave differently 
 with iodine or with sulphuric acid and iodine, in that some are colored 
 reddish brown, blue, or violet, while others are not colored at all. Accord- 
 ing to Wester chitin free from chitosan is not colored by iodine. 
 
 Chitin may be easily prepared from the wings of insects or from the shells 
 of the lobster or the crab, the last-mentioned having first been extracted by an 
 acid so as to remove the lime salts. The wings or shells are boiled with caustic 
 alkali until they are white, afterward washed with water, then with dilute acid 
 and water. The pigments remaining can be destroyed by permanganate. The 
 excess of this last can be removed by a dilute solution of bisulphite, washed with 
 water and then extracted with alcohol and ether. 
 
 Hyalin is the chief organic constituent of the walls of hydatid cysts. From a 
 chemical point of view it stands close to chitin, or between it and protein. In 
 old and more transparent sacs it is tolerably free from mineral bodies, but in 
 younger sacs it contains a great quantity (16 per cent) of lime salts (carbonate, 
 phosphate, and sulphate). 
 
 According to Lucke l its composition is'. 
 
 C 
 
 From old cysts 45 3 
 
 From young cysts. 44 . 1 
 
 It differs from keratin on the one hand and from proteins on the other by the 
 absence of sulphur, also by its yielding, when boiled with dilute sulphuric acid, 
 a variety of sugar in large quantities (50 per cent), which is reducing, fermentable, 
 and dextrogyrate. It differs from chitin by the property of being gradually 
 dissolved by caustic potash or soda, or by dilute acids; also by its solubility on 
 heating with water to 150° C. 
 
 The coloring matters of the skin and horn-formations are of different 
 kinds, but have not been extensively studied. Those occurring in the 
 stratum Malpighii of the skin, especially of the negro, and the black 
 or brown pigment occurring in the hair, belong to the group of those 
 substances which have received the name melanins. 
 
 1 Virchow'e Arch., 19. 
 
 H 
 
 N 
 
 O 
 
 6.5 
 
 5.2 
 
 43.0 
 
 6.7 
 
 4.5 
 
 44.7 
 
MELANINS. 841 
 
 Melanins. This group includes several different varieties of amorphous 
 black or brown pigments which are insoluble in water, alcohol, ether. 
 chloroform, and dilute acids, and which occur in the skin, hair, chorioidia, 
 in sepia, in certain pathological formations, and in the blood and urine 
 in disease. From the true native melanins we must differentiate the 
 humus-like products obtained on boiling proteins with mineral acids 
 and which have been called melanoidins or melanoidic acid (Schmiedeberg) 
 and whose relation to the true melanins is still unknown. 
 
 The melanoidins are readily soluble in dilute alkali while the melanins 
 show a different behavior in this regard. Of the melanins a few such as 
 Schmiedeberg's sarcomelanin, and that from the melanotic sarcomata 
 of horses, the hippomelanin (Nencki, Sieber, and Berdez), which are 
 soluble with difficulty in alkalis, while others, such as the coloring matter 
 of certain pathological swellings in man, the phymatorhusin (Nencki and 
 Berdez) are readily soluble in alkalies. The melanins, as above stated, 
 are in general insoluble in dilute mineral acids; from black sheep- 
 wool Gortner l has isolated a melanin which was soluble in acetic acid 
 and in dilute mineral acids (see below). 
 
 Among the melanins there are a few, for example the choroid pig- 
 ment, which are free from sulphur (Landolt and others); others, on the 
 contrary, as sarcomelanin and the pigment of the hair (Sieber) are rather 
 rich in sulphur (2-4 per cent), while the phymatorhusin found in cer- 
 tain swellings and in the urine (Nencki and Berdez, K. Morner) is 
 very rich in sulphur (8-10 per cent). Whether any of these pigments, 
 especially the phymatorhusin, contains any iron or not is an important 
 though disputed point, for it leads to the question whether these pigments 
 are formed from the blood-coloring matters. 
 
 According to Nencki and Berdez the pigment, phymatorhusin, isolated by 
 them from a melanotic sarcoma did not contain any iron, and according to them 
 is not a derivative of haemoglobin. K. Morner and later also Brandl and L. 
 Pfeiffer found, on the contrary, that this pigment did contain iron, and they 
 consider it as a derivative of the blood-pigments. The sarcomelanin (from a sar- 
 comatous liver) analyzed by Schmiedeberg contained 2.7 per cent iron which 
 was partly in organic combination and could not be completely removed by 
 dilute hydrochloric acid. The sarcomelanic acid prepared by Schmiedeberg 
 by the action of alkali on this melanin contained 1.07 per cent iron. The sar- 
 comelanin investigated by Zdarek and v. Zeynek also contained 0.4 per cent iron. 
 Recently Wolff 2 prepared two pigments from a melanotic liver, of which one 
 was no doubt modified. The other, which was soluble in a soda solution, con- 
 
 1 Gortner, Journ. of biol. Chem., 8, and Bioch. Bulletin, 1, 1911. 
 
 i Zdarek and v. Zeynek, Zeitschr. f. physiol. Chem., 36; Wolff, Hofmeister's Beitrage, 
 5. The literature on the melanins may be found in Schmiedeberg, " Elementarformeln 
 einiger Eiweisskorper, etc." Arch. f. exp. Path. u. Pharm., 39; also in Robert, Wiener 
 Klinik, 27 (1901), and Spiegler, Hofmeister's Betrage, 4, and especially v. Furth, 
 Centralbl. f. allg. Path. u. Path. Anat., ,15, 1907, 617. 
 
842 THE SKIN AND ITS SECRETIONS. 
 
 tained 2.51 per cent sulphur and 2.63 per cent iron, which was in great part 
 split off by 20 per cent hydrochloric acid. From another liver he, on the con- 
 trary, obtained melanin free from iron but with 1.67 per cent sulphur. From 
 this melanin he obtained, by treatment with bromine, a hydro-aromatic body 
 which was related to xyliton (a condensation product of acetone). A similar 
 product could not be obtained from the pigment of the hair (Spiegler) nor from 
 hippomelanin (v. Furth and Jerusalem 1 ). 
 
 The difficulties which attend the isolation and purification of the 
 melanins have not been overcome in certain cases, while in others it is 
 questionable whether the final product obtained has not another com- 
 position from the original coloring matter, owing to the energetic chemical 
 processes resorted to in its purification. The elementary composition 
 shows widely varying results in the different melanins, namely, 48-60 
 per cent carbon, and 8-14 per cent nitrogen. Under these circumstances, 
 and as no doubt we have a large number of melanins having different 
 composition, it seems that a tabulation of the analyses of the different 
 preparations can only be of secondary importance. 
 
 Gortner differentiates between two different groups of melanins. 
 The one, to which the melanin isolated by him from sheeps-wool belongs, 
 is soluble in very dilute acid, has a protein nature and is called melano- 
 protein. By the action of strong alkali the nitrogen and hydrogen con- 
 tent is much reduced and the quantity of carbon increased. The melanin 
 is now insoluble in dilute acids, like the second group of melanins. The 
 melanoprotein on hydrolysis with hydrochloric acid yields besides amino- 
 acids, a black pigment, rich in carbon and insoluble in acids. The 
 melanin isolated by Piettre 2 form sarcomatous horse tumors, on alkali 
 hydrolysis, yielded amino-acids and a melanin much richer in carbon 
 and poorer in nitrogen, a melainin. The sepia melanin and also the 
 artificially prepared melanin by means of tyrosinase, had a similar behavior. 
 The melanin is, therefore, according to Piettre, composed of a protein 
 group and a pigment residue, which is insoluble in acids. 
 
 So little is known about the structural products of the melanins or 
 melanoids that it is impossible to give the origin of these bodies. As 
 undoubtedly there are several distinct melanins, their origin must also be 
 distinct. The ferruginous melanins should be considered as originating 
 from the blood-pigments until further research proves otherwise. Others, 
 on the contrary, cannot have this origin; for example, the pigments 
 of the hair and choroid, which are free from iron and which do not yield 
 hamopyrrol according to Spiegler. Several melanins — and this is also 
 
 1 Wolff, Hofmeister's Beitrage, 5; Speigler, ibid., 10; v. Furth and Jerusalem, 
 ibid., 10. 
 
 2 Gortner, 1. c. and Bull. Soc. Chim. de France (4) 11; Piettre, Compt. Rend., 153. 
 and Congres, internat., de Path. Comparee, Paris, 1912. 
 
MELANINS. 843 
 
 true of the melanoids produced from proteins on cleavage with acids 
 (Samuely ! ) — yield indol or skatol and a pyrrol substance on fusion 
 with alkali, while hippomelanin, according to v. Furth and Jebubalem, 
 gives a fecal odor on this treatment, but does not yield any indol or skatol. 
 More characteristic than the two last mentioned bodies is a phenol-like 
 substance, which occurs to a slight extent, and gives a bluish-black 
 color with ferric chloride (v. Furth). 
 
 The cyclic complexes of the proteins are rightly considered as the 
 mother-substance of the melanins (Samuely and v. Furth and others), 
 and this view has received support by the behavior of tyrosine with 
 oxidases. It has been found that by the action of. a plant oxidase, 
 Bertrand's tyrosinase, 2 upon tyrosine, colored products and then 
 melanin-like substances are formed, v. Furth with Schneider and 
 Pribram, Gessard, Neuberg, Dewitz and others 3 have shown that 
 similar-acting tyrosinases also occur in the animal kingdom, in insects 
 and sepia, in melanotic tumors and in pigmented skin, and v. Furth 
 and Jerusalem have prepared an artificial melanin from tyrosine which 
 shows great similarity to hippomelanin. Finally Neuberg and Jager 4 
 have also prepared extracts from melanotic growths which formed a dark- 
 brown pigment from adrenalin. As indicated above, we tend more and 
 more to accept the view that the melanins are derived from the cyclic 
 components of the proteins. 
 
 In addition to the coloring matters of the human skin it is in place here to 
 treat of the pigments found in the skin or epidermal formation of animals. 
 
 The beautiful color of the feathers of many birds depends in certain cases on 
 purely physical causes (interference-phenomena), but in other cases on coloring 
 matters of various kinds. Such a coloring matter is the amorphous reddish- 
 violet turacin, which contains 7 per cent copper and whose spectrum is very similar 
 to that of oxyhemoglobin. It must be remarked that according to Laidlaw 5 
 turacin or at least a pigment with the same properties can be obtained on boiling 
 lurmatoporphyrin in dilute ammonia with ammoniacal copper solution. Kruken- 
 berg 6 found a large number of coloring matters in bird's feathers, namely zooery- 
 thnn, zoofulrin turacoverdin, zoorubin psittacofulvin, and others which cannot be 
 enumerated here. 
 
 Tetronerythrin, so named by Wurm, is a red amorphous pigment which is 
 soluble in alcohol and ether, and which occurs in the red warty spots over the eyes 
 of the heathcock and the grouse, and which is very widely spread among the 
 invertebrates (Halliburton, De Merejkowski MacMunn). Besides tetronery- 
 
 1 Hofmeister's Beitrage, 2. 
 
 2 Compt. Rend., 122. 
 
 3 The literature can be found in v. Furth and Jerusalem, Hofmeister's Beitrage, 
 10. 
 
 4 Neuberg, Virchow's Arch., 192; Jager, ibid., 198. 
 6 Journ. of Physiol., 31. 
 
 s Vergleichende physiol. Studien, Abth. 5, and (2. Reihe) Abth. 1, 151, Abth. 2, 1, 
 and Abth. 3, 128. 
 
844 THE SKIN AND ITS SECRETIONS. 
 
 thrin MacMunn found in the shells of crabs and lobsters a blue coloring matter, 
 cyanocrystallin, which turns red with acids and by boiling water. Hcematoporphyrin, 
 according to MacMunn, also occurs in the integuments of certain of the lower 
 animals. The blue pigment occurring in the fins of the fish, crenilabrus pavo, 
 is according to v. Zeynek * a chromoprotein. 
 
 In certain butterflies (the pieridinse) the white pigment of the wings consists, 
 as shown by Hopkins, 2 of uric acid, and the yellow pigment of a uric-acid deriva- 
 tive, lepidotic acid, which yields a purple substance, lepidoporphyrin, on warming 
 with dilute sulphuric acid. The yellow and red pigment of the Vanessa are, 
 according to Linden, 3 of an entirely different kind. In this case we are dealing 
 with a compound between protein and a pigment which is allied to bilirubin or 
 urobilin, i.e., a compound similar to haemoglobin. 
 
 In addition to the coloring matters thus far mentioned a few others found in 
 certain animals (though not in the skin) will be spoken of. 
 
 Carminic Acid, or the red pigment of the cochineal, gives on oxidation, accord- 
 ing to Liebermann and Voswinckel, 4 cochenillic acid, CioH 8 7 , and coccinic acid. 
 C 6 H 8 0t, the first being the tri-carboxylic acid, and the other the di-carboxylic 
 acid, of m-cresol. The beautiful purple solution of ammonium carminate has two 
 absorption-bands between D and E which are similar to those of oxyhemoglobin. 
 These bands lie nearer to E and closer together and are less sharply defined. Pur- 
 ple is the evaporated residue from the purple-violet secretion, caused by the action 
 of the sunlight, upon the so-called " purple gland " of the mantle of certain species 
 of murex and purpura. According to Friedlander 5 the pigment is a bromine 
 derivative of indigo and indeed di-bromindigo. 
 
 Among the remaining coloring matters found in invertebrates may be men- 
 tioned blue stentorin, actiniochrom, bonellin, poly pery thrin, pentacrinin, antedonin, 
 crustaceorubin, janthinin, and chlorophyll. 
 
 Sebum when freshly secreted is an oily semi-fluid mass which solidifies 
 on the upper surface of the skin, forming a greasy coating. Rohmann 
 and Linser hold that sebum is a mixture of the secretion of the sebaceous 
 glands and of the constituents of the epidermis. Hoppe-Seyler found, 
 in the sebum, a body similar to casein besides albumin and fat, while 
 Rohmann and Linser claim that true fat occurs only to a very slight 
 extent. On saponification the sebum gives an oil, dermohin, which 
 combines readily with iodine, and another body, dermocerin, which 
 melts at 64-65° and which occurs to a considerable extent in dermoid 
 cysts, and which is perhaps identical with the constituent of cysts, 
 called cetyl alcohol by v. Zeynek. According to Ameseder this der- 
 mocerin is not a pure substance, and the cetyl alcohol, obtained from 
 the fat of dermoid cysts is an eicosyl alcohol, C20H42O, corresponding to 
 arachinic acid. Cholesterin is found in especially large quantities in 
 
 1 Wurm, cited from Maly's Jahresber., 1; Halliburton, Journ. of Physiol., 6; Merej- 
 kowski, Compt. Rend., 93; MacMunn, Proc. Roy. Soc, 1883, and Journ. of Physiol., 7; 
 v. Zeynek, Zeitschr. f. physiol. Chem., 34 and 36, and Wien. Sitz.-Ber. 121, 1912. 
 
 ' Phil. Trans., 186. 
 
 ' Pfluger's Arch., 98. 
 
 4 Ber. d. deutsch. chem. Gesellsch., 30. 
 
 * Ibid., 42. 
 
SEBUM. CERUMEN. 845 
 
 the verniz caseosa. Ruppel l found on an average in the vernix caseosa 
 348.52 p. m. water and 138.72 p. m. ether extractives, and also mentions 
 the presence of isocholesterin. These claims are disputed by Unna. 2 
 In his experience isocholesterin does not occur in the vernix fat nor in 
 the sebum of man, although all kinds of sebum contain cholesterin. 
 
 According to Unna and Golodetz 3 the fat secretion (of the skin), 
 as the fat of the ball of the foot, and sebum are rich in oxycholesterin, 
 while the cell fats of the outer skin does not contain any oxycholesterin. 
 The nails, which are rather rich in oxycholesterin, are an exception. 
 
 On account of the opinion generally held that the wax of the plant 
 epidermis serves as protection for the inner parts of the fruit and plant, 
 Liebreich 4 has suggested that these combinations of fatty acids with 
 monatomic alcohols are the cause of the waxes having a greater resistance 
 as compared with the glycerin fats. He also considers that the choles- 
 terin fats play the role of a protective fat in the animal kingdom, and he 
 has been able to detect cholesterin fat in human skin and hair, in vernix 
 caseosa, whalebone, tortoise-shell, cow's horn, the feathers and beaks 
 of several birds, the spines of the hedgehog and porcupine, the hoofs of 
 horses, etc. He draws the following conclusion from this, namely, 
 that the cholesterin fats always appear in combination with the keratinous 
 substance, and that the cholesterin fat, like the wax of plants, serves 
 as protection for the skin-surface of animals. Of the sebum fats inves- 
 tigated by Unna all contained, with the exception of the epidermis fat, 
 besides cholesterin, greater or smaller amounts of cholesterin ester. The 
 epidermis fat, on the contrary, was almost free from esters and consisted 
 chiefly of free cholesterin. 
 
 In the fatty protective substance secreted by the Psylla altii, Sundvik 5 
 found psylla-alcohol, CsnHesO, which exists there as an ester in combination with 
 peyllic acid, C32H65COOH. This alcohol has also been found in the wax of the 
 humble-bee. 
 
 Cerumen is a mixture of the secretion of the sebaceous and sweat 
 glands of the cartilaginous part of the outer passages of the ear. It 
 chiefly contains soaps and fat, fatty acids, cholesterin and protein, and. 
 besides these a red substance easily soluble in alcohol and with a bitter- 
 sweet taste. 6 
 
 1 Hoppe-Seyler, Physiol. Chem., 760; Linser with Rohmann, Centralbl. f. Physiol., 
 19, 317; see also reference in ibid., 18, from Deutsch. Arch. f. klin. Med., 1904; Riippel,. 
 Zeitschr. f. physiol. Chem., 21; Ameseder, ibid., 52; Zumbusch, ibid., 59. 
 
 2 Monatsch. f. prakt. Dermat., 45. 
 
 3 Bioch. Zeitschr., 20. 
 
 4 Virchow's Arch., 121. 
 
 5 Zeitschr. f. physiol. Chem., 17, 25, 32, 53, 54 and 72. 
 • See Lamois and Martz, Maly's Jahresber., 27, 40. 
 
S46 THE SKIN AND ITS SECRETIONS. 
 
 The preputial secretion, smegma prceputii, contains chiefly fat, also 
 cholesterin and ammonium soaps, which probably are produced from 
 decomposed urine. The hippuric acid, benzoic acid, and calcium oxalate 
 found in the smegma of the horse probably have the same origin. 
 
 We may also consider as a preputial secretion the castoreum, which is secreted 
 by two peculiar glandular sacs, in the prepuce of the beaver. The castoreum is a 
 mixture of proteins, fats, resins, traces of phenol (volatile oil), and a non-nitrog- 
 enous body, castorin, crystallizing from alcohol in four-sided needles, insoluble 
 in cold water, but somewhat soluble in boiling water, and whose composition is 
 little known. 
 
 In the secretion from the anal glands of the skunk, butyl mercaptan and alkyl 
 sulphides have been found (Aldrich, E. Beckmann l ). 
 
 Wool-fat, or the so-called fat-sweat of sheep, is a mixture of the secretion of 
 the sudoriparous and sebaceous glands. There is found in the watery extract a 
 large quantity of potassium which is combined with organic acid, volatile and non- 
 volatile fatty acids, benzoic acid, phenol-sulphuric acid, lactic acid, malic acid, 
 succinic acid, and others. The fat contains, among other bodies, abundant quan- 
 tities of ethers of fatty acids with cholesterin and isocholesterin. Darmstadter 
 and Lifschutz have found other alcohols in wool-fat besides myristic acid, also 
 two oxyfatty acids, lanoceric acid, CjoHeoCj, and lanopalmitic acid, C16H32O3. 
 Isocholesterin, oxycholesterin and carnaubyl alcohol, C24H49OH, are besides the 
 two last-mentioned acids, substances that are characteristic of wool-fat. Accord- 
 ing to Rohmann 2 wool-fat contains a body lanocerin, which is the internal anhy- 
 dride of the above-mentioned lanoceric acid. 
 
 The secretion of the coccygeal glands of ducks and geese contains a body similar 
 to casein, besides albumin, nuclein, lecithin, and fat, but no sugar (De Jonge). 
 The chief constituent is octadecyl alcohol, Ci 8 H s8 0, which represents 40-45 per 
 cent of the ethereal extract (Rohmann). The fatty acids are oleic acid, small 
 amounts of caprylic acid, palmitic acid, and stearic acid, and optical isomers of 
 lauric and myristic acid. The fatty acids are in great part combined with the 
 octadecylic alcohol, and this is probably formed by the reduction of stearic acid or 
 oelic acid. The secretion also contains a substance related to lanocerin which 
 Rohmann calls pennacerin. Poisonous bodies have been found in the secretion 
 of the skin of the salamander and the toad, namely, samandarin (Zaleski, Faust) 
 and bufidin (Jornara and Casali), bufotalin and the disputed bodies bufonin 
 and bvjotenin (Faust, Bertrand and Phisalix 3 ). The active constituents in 
 the poison of the rattle-snake and cobra, the crotalotoxin and the ophiotoxin have 
 been isolated and studied by Faust. 4 They are free from nitrogen and have a 
 similar composition, namely, C34H54O21 and C34H62O20 and are classified in the 
 pharmacological group of sapotoxins by Faust. Thalassin is the crystalline body 
 discovered by Richet 5 which is the poisonous constituent of the feelers of the 
 sea nettle. 
 
 1 Aldrich, Journ. of Exp. Med., 1; Beckmann, Maly's Jahresber., 26, 566. 
 
 2 Darmstader and Lifschutz, Ber. d. d. Chem., Gesellsch., 29 and 31; Rohmann, 
 Hofmeister's Beitrage, 5, and Centralbl. f. Physiol., 19, 317. See also Unna, 1. c, 45; 
 and Lifschutz and Unna, ibid., p. 234. 
 
 a De Jonge, Zeitschr. f. physiol. Chem., 3; Rohmann, 1. c; Zaleski, Hoppe-Seyler's 
 Med.-chem. (Jntersuch., p. 85; Faust, Arch. f. exp. Path. u. Pharm., 41; Jornara and 
 Casali, Maly's Jahresbr., 3; Faust, Arch. f. exp. Path. u. Pharm., 47 and 49; Bertrand, 
 Compt. Rend., 135; Bertrand and Phisalix, ibid. 
 
 4 Arch. f. exp. Path. u. Pharm., 56 and 64. 
 
 6 Pfluger's Arch., 108. 
 
PERSPIRATION. 847 
 
 The Perspiration. Of the bodies secreted by the skin, whose quantity 
 amounts to about ^j of the weight of the body, a disproportionately 
 large part consists of water. Next to the kidneys, the skin, in man, is 
 the most important means for the elimination of water. As the glands 
 of the skin and the kidneys stand near to each other in regard to their 
 functions, they may to a certain extent act vicariously. 
 
 The circumstances which influence the secretion of perspiration are numerous, 
 and the quantity of sweat secreted must consequently vary considerably. The 
 secretion differs in different parts of the skin, and it has been stated that the per- 
 spiration of the cheek, that of the palm of the hand, and that under the arm stand 
 to each other as 100:90:45. From the unecjual secretion on different parts of the 
 body it follows that no results as to the quantity of secretion for the entire surface 
 of the body can be calculated from the quantity secreted by a small part of the 
 skin in a given time. In determining the total quantity a stronger secretion is as 
 a rule produced, and as the glands can with difficulty work for a long time with 
 the same energy, it is hardly correct to estimate the quantity of secretion per day 
 from a strong secretion during only a short time. 
 
 The perspiration obtained for 'investigation is never quite pure, but 
 contains cast-off epidermis-cells, also cells and fat-globules from the 
 sebaceous glands. Filtered perspiration is a clear, colorless fluid with 
 a salty taste and of different odors from different parts of the body. The 
 physiological reaction is acid, according to most reports. Under certain 
 conditions an alkaline sweat may be secreted (Trumpy and Luchsinger, 
 Heuss). An alkaline reaction may also depend on a decomposition 
 with the formation of ammonia. According to a few investigators the 
 physiological reaction is alkaline, and an acid reaction depends upon 
 an admixture of fatty acids from the sebum. Camerer found that 
 the reaction of human perspiration in certain cases was acid and in 
 others alkaline. Moriggia found that the sweat from herbivora was 
 ordinarily alkaline, while that from carnivora was generally acid. 
 Smith j showed that horse's sweat is strongly alkaline. 
 
 Kittsteiner, 2 who has found that human perspiration is nearly always 
 acid, has also found that the perspiration from the vola manus, when 
 not contaminated with sebum, is acid in reaction and that an acid reac- 
 tion is not necessarily dependent upon an admixture with sebum. 
 
 The specific gravity of human perspiration varies between 1.001 
 and 1.010. It contains 977.4-995.6 p. m., average about 982 p. m. 
 water. The solids are 4.4-22.6 p. m. The molecular concentration 
 also varies widely and the freezing-point depression depends essentially 
 
 1 Trumpy and Luchsinger, Pfli'iger's Arch., 18; Heuss, Maly's Jahresbcr., 22; 
 Camerer, Zeitschr. f. Biologie, 41; Moriggia, Moleschott's Untersuch. zur Xaturlehre, 
 11; Smith, Journ. of Physiol., 11. In regard to the older literature on perspiration, 
 Bee Hermann's Handbuch, 5, Thl. 1, 421 and 543. 
 
 2 Arch. f. Hyg., 73 and 78. 
 
848 THE SKIN AND ITS SECRETIONS. 
 
 upon the content of NaCl. Ardin-Delteil found A = —0.08-0.46°, 
 average— 0.327°. Brieger and Disselhorst found with perspiration 
 containing 2.9, 7.07 and 13.5 p. m. NaCl that the A was equal to-0.322°, 
 — 0.608° and —1.002°, respectively. Tarugi and Tomasinelli * found A 
 to be 0.52° as an average. Kittsteiner 2 found that perspiration had an 
 average specific gravity of 1.0046 and the average quantities of nitrogen 
 and sulphur were 0.5 and 0.08 p. m. respectively. The NaCl content 
 increased with the rapidity of secretion while the nitrogen content 
 . diminished. The organic bodies are neutral fats, cholesterin, volatile fatty 
 acids, traces of protein (according to Leclerc and Smith always in 
 horses, and according to Gaube regularly in man, while Leube 3 claims 
 only occasionally after hot baths, in Bright's disease, and after the use 
 of pilocarpin), creatinine (Capranica), aromatic oxyacids, ethereal-sulphuric 
 acids of phenol and skatoxijl (Kast 4 ), sometimes also of indoxyl, serine 
 (page 145) and lastly urea. The quantity of urea has been determined by 
 Argutinsky. In two steam-bath experiments, in which in the course of 
 £ and | hour respectively he obtained 225 and 330 cc. of perspiration, he 
 found 1.61 and 1.24 p. m. urea. Of the total nitrogen of the perspiration 
 in these two experiments 68.5 per cent and 74.9 per cent respectively 
 belong to the urea. From Argutinsky's experiments, and also from 
 those of Cramer, 5 it follows that of the total nitrogen a portion, not to 
 be disregarded, is eliminated by the perspiration. This portion was 
 indeed 12 per cent, in an experiment of Cramer, at high temperature 
 and powerful muscular activity, and Zuntz and his collaborators find 
 indeed more than 13 per cent in high altitudes. Cramer also found 
 ammonia in the perspiration. In uraemia and in anuria in cholera, 
 urea may be secreted in such quantities, by the sweat-glands, that crystals 
 deposit upon the skin. The mineral bodies consist chiefly of sodium 
 chloride with some potassium chloride, alkali sulphate and phosphate. 
 The relative quantities of these in perspiration differ materially from 
 the amount in the urine (Favre, Kast 6 ). The relation, according to 
 Kast, is as follows: 
 
 Chlorine 
 
 In perspiration 
 
 In urine 
 
 Phosphate 
 0.001.5 
 0.1320 
 
 Sulphate: 
 0.009 
 0.397 
 
 1 Ardin-Delteil, Maly's Jahresber., 30; Brieger and Disselhorst, Deutsch. med. 
 Wochenschr., 29; Tarugi and Tomasinelli, cited in Physiol. Centralbl., 22, 748. 
 
 2 I.e. 
 
 3 Leclerc, Compt. Rend., 107; Gaube, Maly's Jahresber., 22; Leube, Virchow's 
 Arch., 48 and 50, and Arch. f. klin. Med., 7. 
 
 * Capranica, Maly's Jahresber., 12; Kast, Zeitschr. f. physiol. Chem., 11. 
 5 Argutinsky, Pfluger's Arch., 46; Cramer, Arch. f. Hygiene, 10. 
 •Compt. Rend., 35, and Arch, gener. de Med. (5), 2; Kast, 1. c. 
 
EXCHANGE OF GAS THROUGH THE SKIN. 849 
 
 Kast found that the proportion of ethereal-sulphuric acid to the 
 sulphate-sulphuric acid in perspiration was 1:12. After the administra- 
 tion of aromatic substances the ethereal-sulphuric acid does not increase 
 to the same extent in the perspiration as in the urine (see Chapter XIV). 
 The quantity of mineral substances was on an average 7 p. m. 
 
 Sugar may pass into the perspiration in diabetes, but the passage of the bile- 
 coloring matters has not been positively shown in this secretion. Benzoic acid, 
 succinic acid, tartaric acid, iodine, arsenic, mercuric chloride and quinine pass 
 into the perspiration. Uric acid has also been found in the perspiration in gout 
 and cystine in cystinuria. 
 
 Chromnidrosia is the name given to the secretion of colored perspiration. 
 Sometimes perspiration has been observed to be colored blue by indigo (Bizio), 
 by pyocyanin, or by ferro-phosphate (Collmann l ). True blood-sweat, in which 
 blood-corpuscles exude from the opening of the glands, has also been observed. 
 
 The exchange of gas through the skin is of great importance for non- 
 scaly amphibians; in mammalia, birds and human beings it is of little 
 importance compared with the exchange of gas by the lungs. The 
 absorption of oxygen by the skin, which was first shown by Regnault 
 and Reiset, is small, and according to Zuelzer amounts under the 
 most favorable circumstances to T fo- of the oxygen absorbed by the 
 lungs. The quantity of carbon dioxide eliminated by the skin increases 
 with the rise of temperature (Aubert, Rohrig, Fubini and Ronchi, 
 Barratt and according to Willebrand beginning at 33°) 2 . It especially 
 increases with hyperemia of the skin and in particular after muscular 
 activity. It is also greater in light than in darkness. It is greater dur- 
 ing digestion than when fasting, and greater after a vegetable than after 
 an animal diet (Fubini and Ronchi). The quantity calculated by differ- 
 ent investigators for the entire skin surface in twenty-four hours varies 
 between 2.23 and 32.8 grams. According to Schierbeck and Wille- 
 brand 3 the average quantity is 7.5-9 grams, and it is ordinarily given as 
 about 1.5 per cent of the quantity eliminated by the lungs. In a horse, 
 Zuntz, with Lehmann and Hagemann, 4, found for twenty-four hours 
 an elimination of carbon dioxide by the skin and intestine which amounted 
 to nearly 3 per cent of the total respiration. Less than four-fifths of 
 this carbon dioxide came from the skin respiration. The same investi- 
 gators found that the skin respiration equals 2\ per cent of the simulta- 
 neous lung respiration. 
 
 1 Bizio, Wien. Sitzungsber., 39; Collmann, cited from v. Gorup-Besanez's Lehrbuch, 
 4. Aufl., 555. 
 
 2 Zuelzer, Zeitschr. f. klin. Med., 53; Aubert, Pfluger's Arch., 6; Rohrig, Deutsch. 
 Klin., 1872, 209; Fubini and Ronchi, Moleschott's Untersuch. z. Naturlehre, 12; 
 Barratt, Journ. of Physiol., 21; Willebrand, Skand. Arch. f. Physiol., 13. 
 
 1 See Hoppe-Seyler, Physiol. Chem., 580; Schierbeck, Arch. f. (Anat. u.) Physiol., 
 1892; Willebrand, 1. c. 
 
 4 Arch. f. (Anat. u.) Physiol., 1894, and Maly's Jahresber., 24. 
 
CHAPTER XVI. 
 RESPIRATION AND OXIDATION. 
 
 During life a constant exchange of gases takes place between the 
 animal body and the surrounding medium. Oxygen is inspired and 
 carbon dioxide expired. This exchange of gases, which is called respira- 
 tion, is brought about in man and vertebrates by the nutritive fluids, 
 blood and lymph, which circulate in the body and which are in constant 
 communication with the outer medium on one side and the tissue-elements 
 on the other. Such an exchange of gaseous constituents may take place 
 wherever the anatomical conditions offer no obstacle, and in man it may 
 go on in the intestinal tract, through the skin, and in the lungs. As 
 compared with the exchange of gas in the lungs, the exchange already 
 mentioned, which occurs in the intestine and through the skin, is very 
 insignificant. For this reason we will discuss in this chapter only the 
 exchange of gas between the blood and the air of the lungs on one side 
 and the blood and lymph and the tissues on the other. The first is often 
 designated as external respiration, and the other, internal respiration. 
 Besides this we will discuss the oxidation processes caused by the internal 
 respiration. 
 
 I. THE GASES OF THE BLOOD. 
 
 Since the pioneer investigations of Magnus and Lothar Meyer, the 
 gases of the blood have formed the subject of repeated careful investiga- 
 tions by prominent experimenters, among whom must be mentioned first 
 C.LuDwiGand his pupils, and E. Pfluger and his school; and C. Bohr. 
 By these investigations not only has science been enriched by a mass of 
 facts, but also the methods themselves have been made more perfect 
 and accurate. In regard to these methods, as also in regard to the laws 
 of the absorption of gases by liquids, dissociation, and related questions, 
 the reader is referred to text-books on physiology, on physics, and on 
 gasometric analysis. 
 
 The gases occurring in blood under physiological conditions are 
 oxygen, carbon dioxide and nitrogen, and traces of argon, and perhaps 
 also carbon monoxide. Traces of hydrogen and marsh-gas also some- 
 times occur. The nitrogen is found only in very small quantities, on an 
 average 1.2 vols, per cent. The quantity is here, as in all following 
 
 850 
 
GASES OF THE BLOOD. 851 
 
 experiments, calculated for 0° C. and 760 mm. mercury pressure. The 
 nitrogen seems to be simply absorbed by the blood, at least in great 
 part. It appears, like argon, to play no direct part in the processes of 
 life, and its quantity varies but slightly in the blood of different blood- 
 vessels. 
 
 The oxygen and carbon dioxide behave otherwise, as their quantities 
 have significant variations, not only in the blood from different blood- 
 vessels, but also because many factors, such as a difference in the rapidity 
 of circulation and the ventilation of the lungs, a different temperature, 
 alkalinity of the blood, rest and activity cause a change. In regard to the 
 gases they contain, the greatest difference is observable between the blood 
 of the arteries and that cf the veins. 
 
 The quantity of oxygen in the arterial blood (of dogs) is on an average 
 22 vols, per cent (Pfluger, Bohr and Henriques). In human blood 
 Setschenow found about the same quantity, namely, 21. G vols, per 
 cent. Leowy in another manner has determined the quantity of oxygen 
 which the blood can take up by first shaking human venous blood with 
 air and then calculating from this the quantity of oxygen in human 
 arterial blood. He calculates the average amount as 18 vols, per cent. 
 Lower figures have been found for the blood of herbivora (such as horse, 
 sheep, rabbits) and birds (hen and ducks) namely, 14-10.7 per cent (Zuntz 
 and Hagemann, Sczelkow, Walter, Jolyet). Venous blood in dif- 
 ferent vascular regions has variable quantities of oxygen. By sum- 
 marizing a great number of analyses by different experimenters, Zuntz 
 has calculated that the venous blood of the right side of the heart con- 
 tains on an average 7.15 per cent less oxygen than the arterial blood. 
 
 The quantity of carbon dioxide in the arterial blood (of dogs) is about 
 40 vols, per cent (Ludwig, Setschenow, Pfluger, P. Bert, Bohr 
 and Henriques and others), or a little above. In herbivora and the 
 above-mentioned birds the quantity of carbon dioxide in the arterial 
 blood is higher than in the carnivorous dog. Setschenow found 40.3 
 vols, per cent in human arterial blood. The quantity of carbon dioxide 
 in venous blood varies still more (Ludwig, Pfluger, and their pupils, 
 P. Bert, Mathieu and Urbain, and others). According to the calcula- 
 tions of Zuntz, the venous blood of the right side of the heart contains 
 about 8.2 per cent more carbon dioxide than the arterial. The average 
 amount may be put down as 50 vols, per cent. Holmgren found in 
 blood after asphyxiation even 69.21 vols, per cent carbon dioxide. 1 
 
 1 All the figures given above may be found in Zuntz 's " Die Gase des Blutes " in 
 Hermann's Handbuch d. Physiol., 4, Thl. 2, 33-43, which also contains detailed state- 
 ments and the pertinent literature, and Bohr in Xagel's Handbuch der Physiologie dcs 
 Menschen, Bd. 1, Hefte 1, 1905, and in Loewy, Handb. d. Bioch. of C. Oppenheimer, 
 Bd. 4. 
 
852 RESPIRATION AND OXIDATION. 
 
 Oxygen is dissolved only in a small extent by the plasma, whose 
 absorbability for oxygen is 97.5 per cent of that of water, according to 
 Bohr. The greater part or nearly all of the oxygen is loosely combined 
 with the haemoglobin. The quantity of the oxygen which is contained in 
 the blood of the dog corresponds closely to the quantity which, from the 
 activity of the haemoglobin, we should expect to combine with oxygen 
 and from the quantity of haemoglobin contained therein. It is difficult 
 to ascertain how far the circulating arterial blood is saturated with oxygen, 
 as immediately after bleeding a loss of oxygen always takes place. Still 
 it seems to be unquestionable that it is not quite completely saturated 
 with oxygen, in life. The laws which regulate the binding of the oxygen 
 in the blood will be found in the discussion of the gas exchanged between 
 the blood and the air of the lungs. 
 
 The carbon dioxide of the blood occurs in part, and indeed, accord- 
 ing to the investigations of Alex. Schmidt, 1 Zuntz, 2 and L. Fredericq, 3 
 to the extent of at least one-third in the blood-corpuscles, also in part, 
 and in fact the greatest part, in the plasma or serum. Bohr 4 claims 
 that about 30 mm. may be considered as the average pressure of the 
 carbon dioxide in the organism, and with such a pressure the quantity of 
 physically dissolved CO2 in 100 cc. of the blood amounts to 2.01 cc. As 
 the blood with this tension takes up about 40 vols, per cent CO2, there- 
 fore about 5 per cent of the total carbon dioxide is simply dissolved. 
 Under the assumption that the blood corpuscles make up about one- 
 third of the volume of the blood, of the physically dissolved CO2, 0.59 
 cc. exists with the corpuscles and 1.42 cc. with the plasma. 
 
 As the blood corpuscles in 100 cc. blood as above stated take up at 
 the above pressure about 14 cc. CO2, only a small part of its CO2 is physi- 
 cally dissolved. The chief mass of the CO2 is loosely combined and the 
 constituent of these cells which unites with the CO2 seems to be the 
 alkali combined with phosphoric acid, oxyhemoglobin or haemoglobin, 
 and globulin on one side and the haemoglobin itself on the other. That 
 in the red blood-corpuscles alkali phosphate occurs in such quantities 
 that it may be of importance in the combination with carbon dioxide 
 is not to be doubted ; and it must be allowed that from the diphosphate, 
 by a greater partial pressure of the carbon dioxide, monophosphate and 
 alkali carbonate are formed, while by a lower partial pressure of the 
 carbon dioxide, the mass action of the phosphoric acid again comes into 
 play, so that, with the carbon dioxide becoming free, a reformation of 
 
 1 Ber. d. k. sachs. Gesellsch. d. Wissensoh. math.-phys. Klasse, 1867. 
 •Centralbl. f. d. med. Wissensch., 1867, 529. 
 
 3 Recherches sur la constitution du Plasma sanguin, 1878, 50, 51. 
 
 4 In regard to the work of Bohr we will refer here and in future to Nagel's Handbuch 
 der Physiologje des Menschen, Bd. 1. 
 
CARBON DIOXIDE IN THE BLOOD. 853 
 
 alkali diphosphate takes place. It is generally admitted that the blood- 
 coloring matters, especially the oxyhemoglobin, which can expel carbon 
 dioxide from sodium carbonate in vacuo, acts like an acid, and as the 
 globulins also act similarly (see below), these bodies may also occur in 
 the blood-corpuscles as an alkali combination. The alkali of the blood- 
 corpuscles must, therefore, according to the law of mass action, be divided 
 between the carbon dioxide, phosphoric acid, and the other constituents 
 of the blood-corpuscles which possess acidic properties, and among these 
 especially the blood pigments, because the globulin can hardly be of 
 importance on account of its small quantity. By greater mass action 
 or greater partial pressure of the carbon dioxide, bicarbonate must be 
 formed at the expense of the diphosphates and the other alkali combina- 
 tions, while at a diminished partial pressure of the same gas, with the 
 escape of carbon dioxide, the alkali diphosphate and the other alkali 
 combinations must be reformed at the cost of the bicarbonate. 
 
 Haemoglobin must nevertheless, as the investigations of Setschenow l 
 and Zuntz, and especially those of Bohr and Torup, 2 have shown, be 
 able to hold the carbon dioxide loosely combined even in the absence 
 of alkali. Bohr has also found that the dissociation curve of the car- 
 bon dioxide haemoglobin corresponds essentially to the curve of the 
 absorption of carbon dioxide, on which ground he and Torup consider 
 the haemoglobin itself as of importance in the binding of the carbon 
 dioxide of the blood, and not its alkali combinations. According to 
 Bohr the haemoglobin takes up the two gases, oxygen and carbon dioxide, 
 simultaneously by the oxygen uniting with the pigment nucleus and the 
 carbon dioxide with the protein component. But as according to the 
 researches of Zuntz 3 the combination of haemoglobin with the alkali is 
 first split to any great extent with a carbon dioxide tension of more than 
 70 mm., it must be admitted that with the ordinary CO2 pressure in 
 the organism, the combination of the carbon dioxide in the blood cor- 
 puscles does not essentially take place through the agency of the alkali 
 but chieriy by means of the haemoglobin. 
 
 The chief part of the carbon dioxide of the blood is found in the 
 blood-plasma or the blood-serum, which follows from the fact that the 
 serum is richer in carbon dioxide than the corresponding blood itself. 
 By experiments with the air-pump on blood-serum it has been found 
 that the chief part of the carbon dioxide contained in the serum is given 
 off in a vacuum, while a smaller part can be removed only after the 
 
 1 Centralbl. f. d. med. Wissench., 1877. See also Zuntz in Hermann's Handbuch, 
 76. 
 
 1 Zuntz, 1. c, 76; Bohr, Maly's Jahresber., 17; Torup, ibid. 
 1 Centralbl. f. d. med. Wissensch., 1867. 
 
854 RESPIRATION AND OXIDATION. 
 
 addition of an acid. The red blood-corpuscles also act as an acid, and 
 therefore in blood all the carbon dioxide is expelled in vacuo. Hence 
 a part of the carbon dioxide is in firm chemical combination in the serum. 
 
 Absorption experiments with blood-serum have shown us further that 
 the carbon dioxide which can be pumped out is in greater part loosely 
 chemically combined, and from this loose combination of the carbon 
 dioxide it necessarily follows that the serum must also contain simply 
 absorbed carbon dioxide. For the form of binding of the carbon dioxide 
 contained in the serum or the plasma, there are the three following pos- 
 sibilities: 1. A part of the carbon dioxide is simply absorbed; 2. Another 
 part is in loose chemical combination; 3. A third part is in firm chemical 
 combination. 
 
 The quantity of physically dissolved carbon dioxide in the serum 
 cannot be higher than about 2 vols, per cent, as the quantity of carbon 
 dioxide in the plasma corresponding to 100 cc. of blood is given above 
 as 1.42 cc. 
 
 The quantity of carbon dioxide in the blood-serum which is combined 
 as a firm chemical union depends upon the quantity of simple alkali 
 carbonate in the serum. This amount is not known, and it cannot be 
 determined either by the alkalinity found by titration, nor can it be cal- 
 culated from the excess of alkali found in the ash, because the alkali is not 
 only combined with carbon dioxide, but also with other bodies, especially 
 with protein. The quantity of carbon dioxide in firm chemical combi- 
 nation cannot be ascertained after pumping out in vacuo without the 
 addition of acid, because to all appearances certain active constituents 
 of the serum, acting like acids, expel carbon dioxide from the simple 
 carbonate. The quantity of carbon dioxide not expelled from dog- 
 serum by vacuum alone without the addition of acid amounts to 4.9 
 to 9.3 vols, per cent, according to the determinations of Pfluger. 1 
 
 From the occurrence of simple alkali carbonates in the blood-serum 
 it naturally follows that a part of the loosely combined carbon dioxide 
 of the serum which can be pumped out must exist as bicarbonate. The 
 occurrence of this combination in the blood-serum has also been directly 
 shown. In experiments with the pump, as well as in absorption experi- 
 ments, the serum behaves in other ways differently from a solution of bicar- 
 bonate, or carbonate of a corresponding concentration; and the action 
 of the loosely combined carbon dioxide in the serum can be explained 
 only by the occurrence of bicarbonate in the serum. By means of a 
 vacuum, the serum always allows much more than one-half of the carbon 
 dioxide to be expelled and it follows from this that in the pumping out 
 
 1 E. Pflvip;er's Ueber die Kohlensiiure des Blutes, Bonn, 1864, 11. Cited from 
 Zuntz in Hermann's Handbuch, 65. 
 
CARBON DIOXIDE IN THE BLOOD. 855 
 
 not only may a dissociation of the bicarbonate take place, but also a 
 conversion of the double sodium carbonate into a simple salt. As we 
 know of no other carbon-dioxide combination, besides the bicarbonate, 
 in the serum, from which the carbon dioxide can be set free by simple 
 dissociation in vacuo, it must be assumed that the serum contains other 
 weak acids, in addition to the carbon dioxide, which contend with it for 
 the alkalies, and which expel the carbon dioxide from simple carbonates 
 in vacuo. The carbon dioxide which is expelled by means of the pump, 
 and which, without regard to the quantity merely absorbed, is generally 
 designated as " carbon dioxide in loose chemical combination," is thus 
 only obtained in part in dissociable loose combinations; in part it origi- 
 nates from the simple carbonates, from which it is expelled, in vacuo, by 
 other weak acids. 
 
 These weak acids are thought to be in part phosphoric acid and in 
 part globulins. The importance of the alkali phosphates in the car- 
 bon dioxide combination has been shown by the investigations of Fernet; 
 but the quantity of these salts in the serum is, at least in certain kinds 
 of blood, for example, in ox-serum, so small that it can hardly be of 
 importance. In regard to the globulins, Setschenow is of the opinion 
 that they do not act as acids themselves, but form a combination with 
 carbon dioxide, producing carboglobulinic acid, which unites with the 
 alkali. According to Sertoli, 1 whose views have found a supporter 
 in Torup, the globulins themselves are the acids which are combined 
 with the alkali of the blood-serum. In both cases the globulins would 
 form, directly or indirectly, that chief constituent of the plasma or of 
 the blood-serum which, according to the law of mass action, contends 
 with the carbon dioxide for the alkalies. By a greater partial pressure 
 of the carbon dioxide the latter deprives the globulin alkali of a part of 
 its alkali, and bicarbonate is formed; by low partial pressure carbon 
 dioxide is set free and it is abstracted from the bicarbonate by the 
 globulin alkali. It must also be added that the above-mentioned car- 
 boglobulinic acid can perhaps be considered as a dissociable combination 
 of carbonic acid and protein. 
 
 The assumption that the proteins of the blood are bodies active in 
 combining with the carbon dioxide has received some support from the 
 investigations of Siegfried 2 on the combination of carbon dioxide with 
 amphoteric amino bodies. Siegfried has found that amino-acids com- 
 bine with carbon dioxide, thereby being converted into carbamino- 
 
 1 Hoppe-Seyler, Med. chem. Untersuch., 350. 
 
 2 Zeitschr. f. physiol. Chem., 44 and 46. 
 
856 RESPIRATION AND OXIDATION. 
 
 H 
 
 I 
 acids— glycocoll for example, into carbamino acetic acid, CH2 — N — COOH, 
 
 COOH 
 
 and that the carbon dioxide can be readily split off from these compounds. 
 The peptones and serum proteins in the presence of calcium hydroxide 
 may also act in the same manner as amino-acids. Protein carbamino- 
 acids are formed, and the possibility of such a binding of carbon dioxide 
 must also be considered. 
 
 In the foregoing it has been assumed that the alkali is the most essen- 
 tial and important constituent of the blood-serum, as well as of the blood 
 in general, in uniting with the carbon dioxide. The fact that the quan- 
 tity of carbon dioxide in the blood greatly diminishes with a decrease 
 in the quantity of alkali strengthens this assumption. Such a condi- 
 tion is found, for example, after poisoning with mineral acids. Thus 
 Walter found only 2-3 vols, per cent carbon dioxide in the blood of 
 rabbits into whose stomachs hydrochloric acid had been introduced. In 
 the comatose state of diabetes mellitus the alkali of the blood seems to 
 be in great part saturated with acid combinations, /3-oxybutyric acid 
 (Stadelmann, Minkowski), and Minkowski x found only 3.3 vols, 
 per cent carbon dioxide in the blood in diabetic coma. 
 
 Gases of the Lymph and Secretions. 
 
 The gases of the lymph are the same as in the blood-serum, and the 
 lymph stands close to the blood-serum in regard to the quantity of the 
 various gases, as well as to the kind of carbon-dioxide combination. The 
 investigations of Daenhardt and Hensen 2 on the gases of human 
 lymph are at hand, but it still remains a question whether the lymph 
 investigated was quite normal. The gases of normal dog-lymph were 
 first investigated by Hammarsten. 3 This gas contained traces of 
 oxygen and consisted of 37.4-53.1 per cent CO2 and 1.6 per cent N at 0° 
 C. and 760 mm. Hg pressure. About cne-half of the carbon dioxide was 
 in firm chemical combination. The quantity was greater than in the 
 serum from arterial blood, but smaller than from venous blood. 
 
 The remarkable observation of Buchner, that the lymph collected 
 after asphyxiation is poorer in carbon dioxide than that of the breathing 
 
 1 Walter, Arch. f. exp. Path. u. Pharm., 7; Stadelmann, ibid., 17; Minkowski, 
 Mittheil a. d. med. Klinik in Konigsberg, 1888. 
 
 2 Virchow's Arch., 37. 
 
 1 Ber. d. k. sachs. Gesellsch. d. Wissensch., math.-phys. Klasse, 23. 
 
GASES OF THE LYMPH AND .SECRETIONS. 857 
 
 animal, is explained by Zuntz ' by the formation of acid in the tissues, 
 ami especially in the lymphatic glands, immediately after death, and 
 this acid in pari decomposes the alkali carbonates < f the Lymph. 
 
 The secretions, with the exception of the saliva, in which Pfluger 
 and Kulz found respectively 0.6 per cent and 1 per cent oxygen, are 
 almost free from oxygen. The quantity of nitrogen is the same as in blood, 
 and the chief mass of the gases consists of carbon dioxide. The quantity 
 of this gas is chiefly dependent upon the reaction, i.e., upon the quan- 
 tity of alkali. This follows from the analyses of Pfluger. He found 
 19 per cent carbon dioxide removable by the air-pump and 54 per cent 
 firmly combined carbon dioxide in a strongly alkaline bile, but, on the 
 contrary, G.6 per cent carbon dioxide removable by the air-pump and 
 0.8 per cent firmly combined carbon dioxide in a neutral bile. Alkaline 
 saliva is also very rich in carbon dioxide. As average for two analyses 
 made by Pfluger of the submaxillary saliva of a clog we have 27.5 per 
 cent carbon dioxide removable by the air-pump and 47.4 per cent chem- 
 ically combined carbon dioxide, making a total of 74.9 per cent. Kulz 2 
 found a maximum of 65.78 per cent carbon dioxide for the parotid saliva, 
 of which 3.31 per cent was removable by the air-pump and 62.7 per 
 cent was firmly combined. From these and other reports as to the 
 quantity of carbon dioxide removable by the air-pump and chemically 
 combined in the alkaline secretions it follows that bodies occur in them, 
 although not in appreciable quantities, which are analogous to the pro- 
 tein bodies of the blood-serum and which act like weak acids. 
 
 The acid or at any rate non-alkaline secretions, urine and milk, con- 
 tain, on the contrary, considerably less carbon dioxide, which is almost 
 all removable by the air-pump, and a part seems to be loosely combined 
 with the sodium phosphate. The figures found by Pfluger for the 
 total quantity of carbon dioxide in milk and urine are 10 and 18.1-19.7 
 per cent respectively. 
 
 Ewald 3 made investigations on the quantity of gas in pathological 
 transudates. He found only traces, or at least only very insignificant 
 quantities of oxygen in these fluids. The quantity of nitrogen was about 
 the same as in blood; that of carbon dioxide was greater than in the 
 lymph (of dogs), and in certain cases even greater than in the blood after 
 asphyxiation (dog's blood). The tension of the carbon dioxide was 
 greater than in venous blood. In exudates the quantity of carbon 
 dioxide, especially that firmly combined, increases with the age of the 
 
 1 Buchner, Arbeiten aus der physiol. Anstalt zu Leipzig, 1876; Zuntz, 1. c, 85. 
 
 2 Pfluger, Pfltiger'a Arch., 1 and 2; Kitlz, Zcitschr. f. Biologie, 23. It seems as if 
 Kiilz's results were not calculated at 760 millimeters Hg. but rather at 1 meter. 
 
 3 C. A. Ewald, Arch. f. (Anat. u.) Physiol., 1873 and 1876. 
 
858 KESPIEATION AND OXIDATION. 
 
 fluid, while, on the contrary, the total quantity of carbon dioxide, and 
 especially the quantity firmly combined, decreases with the quantity 
 of pus-corpuscles. 
 
 H. THE EXCHANGE OF GAS BETWEEN THE BLOOD, ON THE ONE HAND, 
 AND PULMONARY AH* AND THE TISSUES, ON THE OTHER. 
 
 In Chapter I (page 42) it was stated that we are to-day of the 
 opinion, derived especially from the researches of Pfluger and his 
 pupils, that the oxidations of the animal body do not take place in the 
 fluids and juices, but are connected with the form-elements and tissues. 
 It is nevertheless true that oxidations take place in the blood itself, al- 
 though, only to a slight extent; but these oxidations depend, it seems, 
 upon the form-elements of the blood, hence it does not contradict the 
 above statement that the oxidations exclusively occur in the cells and 
 chiefly in the tissues. 
 
 The gaseous exchange in the tissues, which has been designated 
 internal respiration, consists chiefly in that the oxygen passes from the 
 blood in the capillaries to the tissues, while the great bulk of the carbon 
 dioxide of the tissues originates therein and passes into the blood of the 
 capillaries. The exchange of gas in the lungs, which is called external 
 respiration, consists, as is seen by a comparison of the inspired and 
 expired air, in the blood taking oxygen from the air in the lungs and giving 
 off carbon dioxide. This does not exclude the fact that in the lungs, as in 
 every other tissue, an internal respiration takes place, namely, a com- 
 bustion with a consumption of oxygen and formation of carbon dioxide. 
 According to Bohr and Henriques x the lungs take a variable but 
 sometimes a very important part in the total metabolism. This part, 
 which on an average is 33 per cent, but may even rise above 60 per 
 cent of the total metabolism, depends, these experimenters say, upon 
 the fact that the intermediary metabolic products formed in the tissues 
 are burnt in the lungs. It is also in part represented by the specific 
 work of the lungs. 
 
 What kind of processes take part in this double exchange of gas? 
 Is the gaseous exchange simply the result of an unequal tension of the 
 blood on one side and the air in the lungs or tissues on the other? Do 
 the gases pass from a place of higher pressure to one of a lower, according 
 to the laws of diffusion, or are other forces and processes active? 
 
 These questions are closely related to that of the tension of the 
 oxygen and carbon dioxide in the blood and in the air of the lungs and 
 tissues. 
 
 1 Centralbl. f. Physiol., 6, and Maly's Jahresber., 27. 
 
GAS EXCHANGE. OXYGEN TENSION. 859 
 
 Oxygen occurs in the blood in a disproportionately large part as 
 oxyhemoglobin, and the law of the dissociation of oxyhemoglobin is 
 of fundamental importance in the study of the tension of the oxygen 
 in the blood. 
 
 Attempts have been made to prove this law by investigations on 
 pure solutions of haemoglobin, and Hufner * has made very careful and 
 important determinations on such solutions. Recent investigations 
 of Bohr 2 and his pupils, as well as of Loewy and Zuntz, 3 have shown 
 that the conditions in the blood are different from a pure hemoglobin 
 solution, which, in part, may be due to a change in the hemoglobin 
 brought about in its preparation. A hemoglobin solution in which 
 alcohol is used in preparing it, combines more firmly with oxygen than 
 the blood, and the dissociation tension of the oxygen is greater in blood 
 than in such a hemoglobin solution. 
 
 The oxygen tension may be variable, as Loewy 4 has shown, with 
 different individuals, and it is not the same in the blood of different 
 animals with the same oxygen pressure; for example, it is less in cat's 
 blood than in the dog, horse and human blood. The temperature is also 
 of great importance, as the dissociation tension increases with a rise in 
 temperature, and with the same pressure the blood combines with less 
 oxygen at a high temperature than at a low temperature. The influence 
 of the concentration of the hemoglobin manifests itself in that in dilute 
 solutions the oxygen is more firmly combined (Hufner, Loewy and Zuntz, 
 Bohr) and that consequently blood made laky with water has a lower 
 dissociation tension and a firmer binding of the oxygen than undiluted 
 blood. 
 
 Of especial interest is the finding of Bohr, Hasselbalch and Krogh 5 
 that the CO2 present also influences the oxygen taken up, in that as the 
 carbon dioxide tension (also within physiological limits) increases the 
 oxygen taken up diminishes. The laws of oxygen absorption must 
 be determined by determinations upon blood itself, at the same time 
 observing the temperature and the carbon dioxide tension. A series of 
 determinations made by Krogh 6 upon horse's blood at 38°, and a con- 
 stant carbon dioxide tension, is given below. In calculating the results 
 in column 4 the quantity of oxygen chemically combined at 150 mm. 
 oxygen pressure is equal to 100. 
 
 1 Arch. f. (Anat, u.) Physiol., 1890 and 1894. 
 
 2 See Nagel's Handbuch, and Krogh, Skand. Arch. f. Physiol., 16. 
 »Arch. f. (Anat. u.) Physiol., 1904. 
 
 « Ibid. 
 
 6 Centralbl. f. Physiol., 17, and Skand. Arch. f. Physiol., 16. 
 
 6 Skand. Arch. f. Physiol., 16. 
 
860 RESPIRATION AND OXIDATION. 
 
 In 100 cc. blood Oxygen taken up 
 
 Oxygen Chemically ,9 x yp n , ? er cen ,^ Dissolved in 
 
 tension in mm. combined dissolved chemically 1QQ laama 
 
 v^uatuu iii ii.»u. oxygen in plasma combined i^ac^xa 
 
 10 6.0 0.020 30.0 0.030 
 
 20 12.9 0.041 64.7 0.061 
 
 30 16.3 0.061 81.6 0.091 
 
 40 18.1 0.081 90.4 0.121 
 
 50 19.1 0.101 95.4 0.152 
 
 60 19.5 0.121 97.6 0.182 
 
 70 19.8 0.141 98.8 0.212 
 
 80 19.9 0.162 99.5 0.243 
 
 90 19.95 0.182 99.8 0.273 
 
 150 20.00 0.303 100.0 0.455 
 
 From the above table we see that even, with an oxygen tension which 
 amounts to only one-half of the oxygen pressure in the air, the haemoglobin 
 is saturated in greatest part with oxygen. The dissociation is hence at 
 70-80 mm. pressure only slightly more than with a pressure of 150 mm. 
 and indeed even with as low a pressure as 40-30 mm., still 90-80 per cent 
 of the entire quantity of oxygen taken up chemically at 150 mm. is com- 
 bined with the haemoglobin. 
 
 Frcm these and other observations it follows that the oxygen partial 
 pressure may sink to one-half of that existing in the atmospheric air 
 without markedly influencing the oxygen content of the blood. This 
 also coincides with the experience of Frankel and Geppert x on the 
 action of low air pressures upon the oxygen content of the blood of dogs. 
 With an air pressure of 410 mm. Hg, they found that the oxygen content 
 of arterial blood was normal. With an air pressure of 378-3G5 mm. 
 it was slightly diminished, and only on reducing the pressure to 300 
 mm. was a noticeable decrease observed. A. Loewy 2 found that 
 the lowest oxygen pressure of the alveolar air wherein the exchange 
 of material can go on normally both qualitatively and quantitatively, 
 is equal to 30 mm. Hg. 
 
 In regard to the above-mentioned action of low air pressure it must 
 be remarked that the alveolar oxygen tension is regulated by changes 
 in the respiration, so that with great diminution in the quantity of oxygen 
 of the inspired air, the alveolar air contains the same quantity of oxygen 
 as with a higher oxygen partial pressure of the inspired air (Loewy). 
 For example, Loewy found the same quantity of oxygen, namely, 6.1 
 per cent, in the alveolar air with 16 and with 10.5 per cent oxygen in 
 the inspired air, because the respiration in the latter case was 8.5 liters 
 per minute against only 4.9 liters in the first case. 
 
 It may be concluded from the large quantity of oxygen or oxyhaemo- 
 
 1 Ueber die Wirkungen der verdiinnten Luft auf den Organismus, Berlin, 1883. 
 1 A. Loewy, Untersuch. iiber die Respiration und Zirculation, etc., Berlin, 1895; 
 also Centralbl. f. Physiol., 13, 449, and Arch. f. (Anat. u.) Physiol., 1900. 
 
OXYGEN TENSION IN BLOOD. 861 
 
 globin in the arterial blood that the tension of the oxygen in the arterial 
 blood must be relatively higher. This is substantiated by the earlier 
 observations of Bert and Hufner, as well as by the determination.-, 
 of Herter, Fredericq and others, 1 using aerotonometric methods, 
 which will be mentioned below in connection with the carbon dioxide 
 tension. Herter found the oxygen tension in the arterial blood of 
 dogs to be equal, on an average, to a pressure of 78.7 mm. Hg and Fre- 
 dericq, by a better method, found that it was equal to 91-99 mm. Hg. 
 
 The oxygen tension of the venous blood of dogs has been found by 
 aerotonometric means to be equal to 20.6-27.7 mm. (Strassburg, 
 Falloise), and by means of the lung-catheter (see below) equal to 25.5-27 
 mm. (Wolfberg, Nussbaum). For human venous blood Loewy and v. 
 Schrotter 2 found an average of 37.68 mm. Concerning the question 
 as to the mechanism of taking up oxygen in the lungs these figures are of 
 less interest than the oxygen tension in the arterial blood, that is, that 
 which has left the lungs, whose tension is estimated as 90 to about 100 
 mm. Hg as given above. 
 
 These results do not coincide with the investigations of Bohr, 3 who 
 in many cases obtained essentially higher figures for the oxygen tension 
 in arterial blood. 
 
 He experimented on dogs, allowing the blood, whose coagulation had been 
 prevented by the injection of peptone solution or infusion of the leech, to flow 
 from one bisected carotid to the other, or from the femoral artery to the femoral 
 vein, through an apparatus called by him an haemat aerometer. The apparatus, 
 which is a modification of Ludwig's rheometer (stromuhr), allowed, according 
 to Bohr, of a complete interchange between the gases of the blood circulating 
 through the apparatus and a quantitj r of gas whose composition was known at 
 the beginning of the experiment and inclosed in the apparatus. The mixture 
 of gases was analyzed after an equalization of the gases by diffusion. In this 
 way the tension of the oxygen and carbon dioxide in the circulating arterial blood 
 was determined. During the experiment the composition of the inspired and 
 expired air was also determined, the number of inspirations noted, and the extern 
 of respiratory exchange of gas measured. To be able to make a comparison 
 between the gas tension in the blood and in an expired air whose composition was 
 closer to the unknown composition of the alveolar air than the ordinary expired 
 air, the composition of the expired air at the moment it passed the bifurcation of 
 the trachea was ascertained by special calculation. The tension of the gases ; ; 
 this " bifurcated air " could be compared with the tension of the gases of the blood 
 and in such a way that the comparison took place simultaneously. Recentlv 
 
 1 Pert, La pression barometrique, Paris, 1878; Herter, Zeitschr. f. phvsiol. Chem. 
 3; Hufner, 1. c; Fredericq, Centralbl. f. Physiol., 7, and Travaux der laborat. de 
 l'inst. de phvsiol. de Liege, 5, 1896. 
 
 - StrasBburg, Pfluger's Arch., 6; Falloisi:, Bull. Acad. Roy. Belg., 1902. Wolfberg, 
 Pfliiger's Arch., 4 and 6; Xussbauni, ibid., 1; Loewy and v. Schrotter, cited by Loewy 
 in Gppenheimer'a Elandb., 4, 76. 
 
 * Skand. Arch. f. Physiol., 2, and Nagel's Handbuch der Physiologic 
 
862 RESPIRATION AND OXIDATION. 
 
 Krogh • constructed an apparatus, called by him microtonometer, to be used 
 for the same purpose. 
 
 Bohr found remarkably high results for the oxygen tension in arterial 
 blood in this series of experiments. They varied between 101 and 144 
 mm. Hg pressure. In eight out of nine experiments on the breathing 
 of atmospheric air, and in four out of five experiments on breathing air 
 containing carbon dioxide, the oxygen tension in the arterial blood was 
 higher than the " bifurcated air." The greatest difference, where the 
 oxygen tension was higher in the blood than in the air of the lungs, was 
 38 mm. Hg. 
 
 Hufner and Fredericq 2 have made the objection to Bohr's experi- 
 ments and views that a perfect equilibrium had probably not been 
 attained between the air in the apparatus and the gases of the blood. 
 Fredericq, by new experiments, presents strong objections to the 
 acceptance of Bohr's findings, while on the other hand Bohr not only 
 defends his experiments, but also finds errors in the experiments of his 
 opponents, while Haldane and Smith's 3 experiments, making use of 
 an entirely different principle, tend to corroborate the high results attained 
 by Bohr. 
 
 Haldane's method is as follows : The individual experimented upon is allowed 
 to inspire air containing an exactly known but small quantity of carbon monoxide 
 (0.045-0.06 per cent), until no further absorption of carbon monoxide takes place 
 and the percentage saturation of the haemoglobin in the arterial blood with carbon 
 monoxide has become constant, as shown by a special titration method. This 
 percentage saturation is dependent upon the relation between the tension of the 
 oxygen in the blood and the tension of the carbon monoxide, as known from the 
 composition of the inspired air. When this last and the percentage saturation 
 with carbon monoxide and oxygen are known the oxygen tension in the blood 
 can be easily calculated. 
 
 According to this method Haldane and Smith found still higher 
 figures than Bohr for the oxygen tension in the blood, and they calculated 
 the average tension of the oxygen in human arterial blood to be equal 
 to 293 mm. Hg. 
 
 Based upon the experiments of A. and M. Krogh, which will be dis- 
 cussed below (page 864) A. Krogh 4 has presented objections to the 
 experiments of Haldane and Smith. 
 
 Let us now compare the figures for the oxygen tension of the arterial 
 blood as found by various investigators with the tension of the oxygen 
 in the air of the lungs. 
 
 1 Skand. Arch. f. Physiol., 20. 
 
 2 Hufner, Arch. f. (Anat. u.) Physiol., 1890; Fredericq, Centralbl. f. Physiol., 7, 
 and Traveaux du laboratorie de l'institute de physiologie de Liege, 5, 1896. 
 
 3 Haldane, Journ. of Physiol., 18; Haldane and Smith, ibid., 20. 
 ♦Skand. Arch. f. Physiol., 23, 217, 253. 
 
THE ALVEOLAR AIR. 8G3 
 
 Numerous investigations as to the composition of the inspired atmos- 
 pheric air as well as the expired air are at hand, and it can be said that 
 these two kinds of air at 0° C. and a pressure of 700 mm. Hg have the 
 following average composition in volume per cent. 
 
 Oxvizen Nitrogen Carbon 
 
 vxygen (aQ( , argon j Dioxide 
 
 Atmospheric air 20.90 79.02 0.03 
 
 Expired air 16.03 79.59 4.38 
 
 The partial pressure of the oxygen of the atmospheric air corresponds 
 at a normal barometric pressure of 760 mm. to a pressure of 150 mm. 
 Hg. The loss of oxygen which the inspired air suffers in respiration 
 amounts to about 4.93 per cent, while the expired air contains about 
 one hundred times as much carbon dioxide as the inspired air. 
 
 The expired air is therefore a mixture of alveolar air with the residue 
 of inspired air remaining in the air-passages; hence in the study of 
 the gaseous exchange in the lungs the alveolar air must first be con- 
 sidered. There exists no direct determination of the composition of the 
 alveolar air in man, but only approximate calculations. From the 
 average results found by Vierordt in normal respiration for the carbon 
 dioxide in the expired air, 4.63 per cent, Zuntz l has calculated the 
 probable quantity of carbon dioxide in the alveolar air as equal to 5.44 
 per cent. If we start from this value, with the assumption that the 
 quantity of nitrogen in the alveolar air does not essentially differ from 
 the expired air, and admit that the quantity of oxygen in the alveolar 
 air is 6 per cent less than the inspired air, it will be seen that the alveolar 
 air contains 15 per cent oxygen. As the total pressure of the air of the 
 lungs after deducting the aqueous tension of about 50 mm. can be cal- 
 culated as about 710 mm. the partial pressure of the oxgyen in man 
 can be put at about 106 mm. and that of the carbon dioxide as about 
 45 mm. 
 
 Based upon several respiration experiments upon different persons, 
 Loewy has been able to calculate the composition of the alveolar air 
 of human beings almost at the atmospheric pressure, from the com- 
 position of the expired air and the depth of inspiration and expiration, 
 taking into consideration the air in the upper air-passages. He obtained 
 results which varied between 101 and 105 mm. Hg for the oxygen tension 
 and between 32-42 mm. for the carbon dioxide tension. 
 
 The alveolar oxygen tension in dogs can be calculated from the car- 
 bon dioxide content of the alveolar air and is also found to be above 
 100 mm. Hg. 
 
 If the oxygen partial pressure in the alveoli is put at about 105 
 
 1 See Zuntz, 1. c, Hermann's Handbuch, 105 and 106. 
 
S64 RESPIRATION AND OXIDATION. 
 
 i 
 mm. Hg and we compare this with the highest results obtained for the 
 oxygen tension of the arterial blood as determined by tonometric means, 
 we find that the taking up of oxygen in the lungs can be simply explained 
 according to physical laws as a diffusion process. The conditions are 
 quite different if we start with the high-tension results of Bohr, 101-144 
 mm. Hg, or the still higher results of Haldane and Smith. The oxygen 
 tension in the blood is, in many cases, according to these latter authors, 
 always higher than the tension in the lungs, as average for various races 
 of animals. In these cases the passage of oxygen from the lungs to the 
 blood cannot be explained simply by a diffusion. We must therefore 
 with Bohr, accept a special activity cf the lungs, and according to, 
 him a secretory activity of the lungs also exists besides diffusion. In his 
 most recent work Bohr x presents the view that the specific action of the 
 lungs essentially consists in maintaining a necessary difference in pressure 
 for the diffusion. Nevertheless besides this a secretory process is nec- 
 essary, especially for the taking up of oxygen. Based upon newer 
 measurements Douglas and Haldane 2 also advocate the view that the 
 taking up of oxygen can be brought about by diffusion alone, but that 
 with the existing lack of oxygen in the tissues an active secretion of 
 oxygen takes place in the lungs. 
 
 By means of a tonometer described by A. Krogh, he and M. Krogh 
 have compared the oxygen tension in the arterial blood with that in the 
 alveolar air. In these experiments the tension in the blood was always 
 found lower than in the alveolar air. From this A. Krogh 3 concludes 
 that the exchange of gas in the lungs is chiefly brought about by diffusion. 
 Fredericq 4 has recently arrived at the same view by his experiments 
 on the respiratory exchange of gas in aquatic animals. 
 
 As reports on the taking up of oxygen are conflicting so also are those 
 on the giving up of carbon dioxide. 
 
 The tension of the carbon dioxide in the blood has been determined 
 in different ways by Pfluger and his pupils Wolffberg, Strassbtjrg, 
 and Nussbaum. 5 
 
 According to the aero tonometric method the blood is allowed to flow directly 
 from the artery or vein through a glass tube which contains a gas mixture of a 
 known composition. If the tension of the carbon dioxide in the blood is greater 
 than the gas mixture, then the blood gives up carbon dioxide, while in the reverse 
 case it takes up carbon dioxide from the gas mixture. The analysis of the gas 
 
 »Centralbl. f. Physiol. 23, 274; Skand. Arch. f. Physiol., 22, 221 (1909). 
 *Skand. Arch. f. Physiol., 25, 169 (1911); Proc. Roy. Soc, 1911; see also Journ. 
 of Physiol., 44, 305 (1912). 
 
 'Skand. Arch. f. Physiol., 20, 203; 23, 179, 200, 213, (1910). 
 
 fAfdh. intern, de Physiol., 10, 391 (1911). 
 
 * Wolffbonr, Pfltigef's Arch., fi; Strassburg, ibid.; Nussbaum, ibid., 7. 
 
CARBON DIOXIDE TENSION. 865 
 
 mixture after passing the blood through it will also decide if the tension of the 
 Carbon dioxide in the blood is greater or less than in the gas mixture; and by a 
 sufficiently great number of determinations, especially when the quantity of car- 
 bon dioxide of the gas mixture corresponds as closely as possible, in the beginning, 
 to the probable tension of this gas in the blood, we may learn the tension of the 
 carbon dioxide in the blood. As above mentioned the oxygen tension can be 
 determined by the same method. 
 
 According to this method the carbon-dioxide tension of the arterial 
 blood is on an average 2.8 per cent of an atmosphere, 1 corresponding to 
 a pressure of 20 mm. mercury (Strassburg). In the blood from the 
 pulmonary aveoli NlJSSBAUM found a carbon-dioxide tension of 3.81 
 per cent of an atmosphere, corresponding to a pressure of 27 mm. mer- 
 cury. Strassburg, who experimented in non-tracheotomized dogs 
 in which the ventilation of the lungs was less active and therefore the 
 carbon dioxide was removed from the blood with less readiness, found 
 in the venous blood of the heart, a carbon-dioxide tension of 5.4 per 
 cent of an atmosphere, corresponding to a partial pressure of 38.3 mm. 
 mercury. 
 
 Another method, which was first used by Pfluger and his pupils 
 Wolffberg and Nussbaum, depends upon excluding a part of the lungs 
 be means of the lung catheter 
 
 The principle of this method is as follows: By the introduction of a catheter, 
 of a special construction, into a branch of a bronchus the corresponding lobe of 
 the lung may be hermetically sealed, while in the other lobes of the same lung, 
 and in the other lung, the ventilation remains unchanged, so that no accumulation 
 of carbon dioxide takes place in the blood. When the cutting off lasts so long that 
 a complete equalization between the gases of the blood and the retained air of 
 the lungs is assumed, a sample of this air of the lungs is removed by means of 
 the catheter and analyzed. 
 
 When a complete exchange between the gases of the inclosed part of 
 the lungs and the gases of the circulating venous blood has taken place, 
 the tension of the gases in this part of the lungs can be considered as a 
 measure for the gas tension in the venous blood, if we admit that the 
 gas exchange is due only to physical forces. In their experiments 
 Wolffberg and Nussbaum found only 3.6 per cent CO2 in the air taken 
 out with the catheter. Nussbaum also determined the carbon-dioxide 
 tension in the blood from the right heart in a case simultaneous with 
 the catheterization of the lungs. He found almost identical results, 
 namely, a carbon-dioxide tensioD of 3.84 per cent and 3.81 per cent 
 of an atmosphere, which also shows that complete equalization between 
 the gases of the blood and lungs in the inclosed parts of the lungs had taken 
 
 1 Here and in the following discussion we mean by atmospheric pressure the pressure 
 in the lungs after subtracting the aqueous vapor tension (about 50 mm.), namely, 
 760 — 50 = 710 mm. mercury pressure. 
 
866 RESPIRATION AND OXIDATION. 
 
 place. The method of catheterizing the lungs is, as shown by Loewy 
 and v. Schrotter, 1 also applicable to man, and they found that the 
 carbon dioxide tension of human venous blood was equal to 6 per cent 
 of the atmospheric pressure in the lungs = 42.6 mm. Hg, while according 
 to Loewy's calculations the carbon dioxide tension in the respired lung 
 aveoli varied between 31.8 and 41.8 mm. Hg with an average of 37.3 
 mm. Hg for eleven cases. 
 
 According to these investigations the giving up of carbon dioxide 
 may also be explained by physical laws; but Bohr, in his experiments 
 above mentioned (page 861), has arrived at other results in regard to 
 the carbon-dioxide tension. In eleven experiments with inhalation of 
 atmospheric air the carbon-dioxide tension in the arterial blood varied 
 from to 38 mm. Hg, and in five experiments with inhalation of air con- 
 taining carbon dioxide, from 0.9 to 57.8 mm. Hg. A comparison of the 
 carbon-dioxide tension in the blood with the bifurcated air gave in several 
 cases a greater carbon-dioxide pressure in the air of the lungs than in 
 the blood, and as maximum this difference amounted to 17.2 mm. in 
 favor of the air of the lungs in the experiments with inhalation of atmos- 
 pheric air. As the aveolar air is richer in carbon dioxide than the bifur- 
 cated air this experiment unquestionably proves, according to Bohr, 
 that the carbon dioxide has migrated against the high pressure. 
 
 In opposition to these investigations, Fredericq, 2 in his above-men- 
 tioned experiments, obtained the same figures for the carbon-dioxide 
 tension in arterial peptone blood as Pfluger and his pupils found for 
 normal blood. Weisgerber, 3 in Fredericq's laboratory, has made 
 experiments with animals which respired air rich in carbon dioxide, and 
 these experiments confirm Pfluger's theory of respiration. Recently 
 Falloise has made determinations of the carbon-dioxide tension of 
 venous blood by means of Fredericq's aerotonometer. The carbon- 
 dioxide tension was found to equal 6 per cent of an atmosphere, hence 
 somewhat higher than the results found by Pfluger's pupils. To these 
 investigations Bohr has presented strong objections; he has demon- 
 strated the principles for the construction of the tonometer, and claims that 
 the earlier experiments with the tonometer are not conclusive, as a 
 complete equilibrium of the gas tension was not attained. 
 
 A certain importance has been ascribed to oxygen in regard to the 
 elimination of carbon dioxide in the lungs, in that it has an expelling 
 action on the carbon dioxide from its combinations in the blood. This 
 theory, first advanced by Holmgren, has recently found an advocate 
 
 1 1. c, footnote 2, page 861. 
 
 2 See footnote 1, page 861. 
 
 •Centralbl. f. Physiol., 10, 482; Falloise, see Maly's Jahresber., 32. 
 
INTERNAL RESPIRATION. 867 
 
 in Werigo. Still Zuntz has presented weighty objections to Werigo's 
 experiments, and Bohr 1 has later also shown that we have no positive 
 basis for the above assumption. 
 
 The conditions as to the elimination of carbon dioxide in the lungs 
 are not quite clear. On the one hand we have advocates of the view 
 that the gas exchange takes place simply according to physical laws 
 and is chiefly considered as a diffusion process. According to the former 
 views of Bohr, 2 which he has supported by recent experiments, a diffusion 
 does take place, but the lung is a gland which has the power of secreting 
 gases, and the gas exchange in the lungs is essentially a secretory proc- 
 ess. In his most recent work Bohr, 3 after a thorough criticism of the 
 methods used in the measurement of the lung diffusion and based upon a 
 new calculation of the extent of diffusion, has come to the conclusion 
 that the specific activity of the lungs consists in that a difference in 
 pressure necessary for the diffusion is brought about. 
 
 That a true secretion of gases occurs in animals follows from the composition 
 and behavior of the gases in the swimming-bladder of fishes. These gases con- 
 sist of oxygen and nitrogen with only small quantities of carbon dioxide. In 
 fishes which do not live at any great depth the quantity of oxygen is ordinarily 
 as high as in the atmosphere, while fishes which live at great depths may, accord- 
 ing to Biot and others, contain considerably more oxygen and even above 80 per 
 cent. AIoreau has also found that after emptying the swimming-bladder by 
 means of a trocar, new air collected after a time, and this air was richer in oxygen 
 than the atmospheric air, and contained even 85 per cent oxygen. Bohr, "who 
 has proven and confirmed these statements, also found that this accumulation 
 is under the influence of the nervous system, because on the section of certain 
 branches of the pneumogastric nerve it is discontinued. It is beyond dispute that 
 there is here a secretion and not a diffusion of oxygen. Recently Jaeger * has 
 given a further explanation as to the secretory activity of the swimming-bladder. 
 
 From what has been said above (page 858) in regard to the internal 
 respiration, one can conclude that it consists chiefly that in the capil- 
 laries the oxygen passes from the blood into the tissues, while the car- 
 bon dioxide passes from the tissues into the blood. 
 
 The assertion of Estor and Saint Pierre that the quantity of oxygen 
 in the blood of the arteries decreases with the remoteness from the heart 
 has been shown to be incorrect by Pfluger, 5 and the oxygen tension in 
 blood on entering the capillaries must be higher. The oxygen tension 
 
 1 Holmgren, Wien. Sitzungsber., 48; Werigo, Pfliiger's Arch., 51 and 52; Zuntz, 
 ibid., 52; Bohr, see Nagel's Handbuch der Physiologic 
 
 2 Centralbl. f. Physiol., 21, 367. 
 
 3 Ibid., 23, 374; Skand. Arch. f. Physiol., 22, 221 (1909). 
 
 4 Biot, see Hermann's Handbuch d. Physiol., 4, Thl. 2, 151; Moreau, Compt. 
 Rend., 57; Bohr, Journ. of Physiol., 15. See also Hiifner, Arch. f. (Anat. u.) Physiol. 
 1892; Jaeger, Pfluger 's Arch., 94. 
 
 5 Estor and Saint Pierre with Pfluger in Pfliiger's Arch., 1. 
 
868 RESPIRATION AND OXIDATION. 
 
 of the plasma is of importance in the giving up of oxygen to the tissues, 
 as the blood corpuscles contain a supply of oxygen only sufficient to 
 replace that removed from the plasma by the tissue. This quantity 
 of oxygen, which is dissolved in the plasma and at the disposal of the 
 tissues, is dependent upon the oxygen tension in the blood and only 
 indirectly dependent upon the total quantity of oxygen in the blood. 
 As this tissue is almost or entirely free from oxygen, a considerable dif- 
 ference in regard to the oxygen pressure must exist between the blood 
 and the tissues. The possibility that this difference in pressure is suf- 
 ficient to supply the tissues with the necessary quantity of oxygen is 
 hardly to be doubted. 
 
 The animal body, it seems, also has the command over means of regu- 
 lating and varying the oxygen tension, and such a means is the carbon 
 dioxide produced in the tissue which, according to Bohr, Hasselbalch, 
 and Krogh, 1 raises the oxygen tension. This is of special importance 
 when the tension of the oxygen in the blood of the capillaries is very low; 
 then the ability of the carbon dioxide to raise the dissociation tension 
 of the ox3'hsemoglobin comes into play, especially with low oxygen tension." 
 Another regulating moment is, Bohr claims, the specific oxygen capacity 
 of the blood, which means the relation of the maximum oxygen combina- 
 tion to the quantity of iron of the blood or the haemoglobin solution. 
 
 In regard to the carbon-dioxide tension in the tissue it must be 
 assumed a priori that it is higher than in the blood. This is found to 
 be true. Strassburg 2 found in the urine of dogs and in the bile a car- 
 bon-dioxide tension of 9 per cent and 7 per cent of an atmosphere, 
 respectively. The same experimenter has, further, injected atmospheric 
 air into a ligatured portion of the intestine of a living dog and analyzed 
 the air taken out after some time. He found a carbon-dioxide tension 
 of 7.7 per cent of an atmosphere. The carbon-dioxide tension in the 
 tissues is considerably greater than in the venous blood, and there is 
 no opposition to the view that the carbon dioxide simply diffuses from 
 the tissues into the blood according to the law of diffusion. 
 
 Several methods have been suggested for the study of the quantitative 
 relation of the respiratory exchange of gas. The reader must be referred 
 to other text-books for details as to these methods, and we will here 
 mention only the chief features of the most important methods. It must 
 also be remarked, in regard to these methods, that those of Regnault 
 and Reiset and of Pettenkofer, determine the total gas exchange, 
 and indeed for a long time, while the other three methods determine the 
 respiratory gas exchange alone, and this only for a short time. 
 
 1 Centralbl. f. Physiol., 17, and Skand. Arch. f. Physiol., 16. 
 
 2 Pfiuger's Arch., 6. 
 
METHODS FOR DETERMINING RESPIRATORY EXCHANGE. £69 
 
 Regnault and Reiset's Method. According to this method the animal 
 •or person experimented upon is allowed to respire in an inclosed Bpace. The 
 carbon dioxide is removed from the air, as it forms, by Strong caustic alkali, from 
 which the quantity may be determined, while the oxygen is replaced continually 
 in exactly measured quantities. This method, which also makes possible a direct 
 determination of the oxygen used as well as the carbon dioxide produced, has 
 since been modified by other investigators, such as Pfluger and his pupils, Seegen 
 and Nkwak, and Hoppe-Seyler, Rosenthal and Oppenheimer and especially 
 by Atwater and Benedict. 1 
 
 Pettenkofer's Method. According to this method the individual to be 
 experimented upon breathes in a room through which a current of atmospheric 
 air is passed. The quantity of air passed through is carefully measured. As it 
 is impossible to analyze all the air made to pass through the chamber, a small 
 fraction of this air is diverted into a branch line during the entire experiment, 
 carefully measured, and the quantity of carbon dioxide and water determined. 
 From the composition of this air the quantity of water and carbon dioxide con- 
 tained in the large quantity of air made to pass through the chamber can be 
 calculated. The consumption of oxygen cannot be directly determined in this 
 method, but may be calculated indirectly by difference, which is a defect in this 
 method. The large respiration apparatus of Sonden and Tigerstedt as well 
 as of Atwater and Rosa 2 are based upon this principle. 
 
 Speck's Method. 3 For briefer experiments on man Speck used the follow- 
 ing: He breathes through a mouthpiece with two valves, closing the nose with a 
 clamp, into two spirometer-receivers, where the gas-volume can be read off very 
 accurately. The air from one of the spirometers is inhaled through one valve 
 and the expired air passes through the other into the other spirometer. By means 
 of a rubber tube connected with the expiration-tube an accurately measured part 
 of the expired air may be passed into an absorption-tube and analyzed. 
 
 Zuntz and Geppert's Method.* This method, which has been improved by 
 Zuntz and his pupils from time to time, consists in the following: The individual 
 being experimented upon inspires pure atmospheric air through a very wide feed- 
 pipe leading from the open air, the inspired and the expired air being separated by 
 two valves (human subjects breathe with closed nose by means of a soft-rubber 
 mouthpiece, animals through an air-tight tracheal canula). The volume of the 
 expired air is measured by a gas-meter and an aliquot part of this air collected and 
 the quantity of carbon dioxide and oxygen determined. As the composition of 
 the atmospheric air can be considered as constant within a certain limit, the 
 production of carbon dioxide as well as the consumption of oxygen may be readily 
 calculated (see the works of Zuntz and his pupils). 
 
 Hanriot and Richet's Method 6 is characterized by its "simplicity. These 
 investigators allow the total air to pass through three gasometers, one after the 
 other. The first measures the inspired air, whose composition is known. The 
 second gasometer measures the expired air, and the third the quantity of the 
 
 x See Zuntz in Hermann's Handbuch, 4, Thl. 2, and Hoppe-Seyler, Zeitschr. f. 
 physiol. Chem., 19; Rosenthal, Arch. f. (Anat. u.) Physiol., 1902; Zuntz and Oppen- 
 heimer, Arch.' f. (Anat. u.) Physiol., 1905, and Bioch. Zeitschr., 14; Atwater and 
 Benedict, Bull. Dept. of Agriculture, Washington, 69, 109, and 136. See also Krogh, 
 Wien. Sitz. Ber., 115, III., and Skand. Arch. f. Physiol., 18. 
 
 2 Pettenkofer's method; see Zuntz, 1. c; Sond6n and Tigerstedt, Skand. Arch, 
 f. Physiol., 6; Atwater and Rosa, Bull, of Dept. of Agriculture, 63. Washington. 
 
 1 Speck, Physiologie des menschlichen Atmens. Leipzig, 1892. 
 
 4 Pfluger's Arch., 42. See also Magnus-Levy in Pfluger's Arch., 55, 10, in which 
 the work of Zuntz and his pupils is cited. 
 
 5 Compt. Rend., 104. 
 
870 RESPIRATION AND OXIDATION. 
 
 expired air after the carbon dioxide has been removed by a suitable apparatus. 
 The quantity of carbon dioxide produced and the oxygen consumed can be_readily 
 calculated from these data. 
 
 Appendix 
 
 THE LUNGS AND THEIR EXPECTORATIONS 
 
 Besides 'proteins and the albuminoids of the connective-substance 
 group, lecithin, taurine (especially in ox-lungs), uric acid, and inosite 
 have been found in the lungs. Poulet 1 claims to have found a special 
 acid in the lung-tissue, which he has called -pulmotartaric acid. Glyco- 
 gen occurs abundantly in the embryonic lung, but is absent in the adult 
 organ. The proteolytic enzymes also belong to the physiological con- 
 stituents of the lungs. They are active in the autolysis of the 
 lungs (Jacoby) as well as in the solution of pneumonic infiltrations (Fr. 
 Muller) ? 
 
 The lungs have a strong reducing property, which Bohr explains by 
 the extensive oxidation processes in the lungs. According to N. Sieber 
 they also have the ability to decompose neutral fats, while Riehl 3 
 says they do not have the ability to invert milk sugar. 
 
 The black or dark-brown pigment in the lungs of human beings and domestic 
 animals consists chiefly of carbon, which originates from the soot in the air. The 
 pigment may in part also consist of melanin. Besides carbon, other bodies, such 
 as iron oxide, silicic acid, and clay, may be deposited in the lungs, being inhaled 
 as dust. 
 
 Among the bodies found in the lungs under pathological conditions 
 must be specially mentioned, proteoses (and peptones?) in pneumonia 
 and suppuration, glycogen, a slightly dextrorotatory carbohydrate 
 differing from glycogen, found by Potjchet in consumptives, and finally 
 also cellulose, which, according to Freund, 4 occurs in the lungs, blood, 
 and pus of persons with tuberculosis. 
 
 C. \V. Schmidt found in 1000 grams of miners 1 bodies from the normal 
 human lung the following: NaCl 130, K 2 13, Na 2 195, CaO 19, MgO 
 19, Fe 2 3 32, P 2 5 485, S0 3 8, and sand 134 grams. According to 
 Oidtmann, 5 the lungs of a 14-day old child contained 796.05 p. m. water, 
 198.19 p. m. organic bodies, and 5.76 p. m. inorganic bodies. 
 
 1 Cited from Maly's Jahresber., 18, 248. 
 
 2 Jacoby, Zeitschr. f. physiol. Chem., 33; Muller, Verhandl. d. Kongress. f. inn. 
 Medizin, 1002. 
 
 1 X. Sieber, Zeitschr. f. physiol. Chem., 55; Riehl, Zeitschr. f. Biol., 48. 
 4 Pouchet, Compt. Rend., 96; Freund, cited from Maly's Jahresber., 16, 471. 
 'Schmidt, cited from v. Gorup-Besanez, Lehrbuch, 4. Aufi., 727; Oidtmann, 
 ibid., 732. 
 
PHYSIOLOGICAL OXIDATION PROCESSES. 871 
 
 The sputum is a mixture of the mucous secretion of the respiratory- 
 passages, of saliva and buccal mucus. Because of this its composition 
 is variable, especially under pathological conditions when various prod- 
 ucts mix with it. The chemical constituents are, besides the mineral 
 substances, chiefly mucin with a little proteid and nuclein substance. 
 Inilcr pathological conditions proteoses and peptones (?), which are 
 probably produced by bacterial action cr by autolysis (Wanner, Simon 1 ), 
 volatile fatty acids, glycogen, Charcot's crystals, and also crystals of 
 cholesterin, hsematoidin, tyrosine, fat and fatty acids, triple phosphates, 
 etc., have been found. 
 
 The form constituents are, under physiological circumstances, epithe- 
 lium-cells of various kinds, leucocytes, sometimes also red blood-cor- 
 puscles and various kinds of fungi. In pathological conditions elastic 
 fibers, spiral formations consisting of a mucin-like substance, fibrin 
 ceagulum, pus, pathogenic microbes of various kinds and the above- 
 mentioned crystals occur. 
 
 The lung concretions contain chiefly calcium and phosphoric acid as inorganic 
 constituents. Silicic acid is, in Zickgraf's opinion; an essential and constant 
 constituent, but according to Gerhartz and Strigel 2 is not always constant. 
 
 in. HOW ARE THE PHYSIOLOGICAL OXIDATION PROCESSES BROUGHT 
 
 ABOUT? 
 
 After the oxygen passes from the blood to the tissues a very extensive 
 oxidation is there carried out, which in conjunction with cleavage processes 
 yields finally the products carbon dioxide, water, urea and other bodies. 
 Little is known as to the manner in which the organism carries out such 
 complete oxidations. Attempts have been made for a long time to 
 explain the mechanism of the oxidation processes. Thus Schonbein 3 
 believed in the presence in the organism of oxygen in a peculiar form, 
 suited for the oxidation. Hoppe-Seyler 4 connects the oxidation with a 
 simultaneous reduction; reducible or readily oxidizable substances first rup- 
 ture the oxygen molecule ( = 02) into atoms and take one up; the other 
 at the moment it is set free is especially able to oxidize. M. Traube 5 
 believes that in the case that a readily oxidizable (auto-oxidizable) substance 
 is present, the oxidation is produced by means of the entire oxygen 
 molecule, and indeed in the manner that water is transformed at the same 
 time to hydrogen peroxide, for example A-r-2H20+Oo = A(OH)2 + H202. 
 
 1 Wanner, Deutsch. Arch. f. klin. Med., 75; Simon, Arch. f. exp. Path. u. Pharm., 49. 
 
 * Gerhartz and Strigel, Beitr. z. klin. d. Tuberkulose, 10, which also cites Zickgraf. 
 'Baseler, Verh., Bd. 1, 339 (1853); Sitzungsber. Bayer. Akad. Wiss., 1863, BA. 
 
 1, 274. 
 
 4 Zeitschr f. physiol. Chem., 2, 1 (1878). 
 
 * Ber. d. d. chem. Gesellsch., Bd. 15 to 26 (1882 to 1893). 
 
872 RESPIRATION AND OXIDATION. 
 
 With these views as basis and at the same time although independently 
 of each other Engler 1 and Bach 2 have developed a theory which for 
 the present is the one generally accepted. According to this theory, 
 peroxides of the hydrogen peroxide type are always formed as primary 
 oxidation products. The peroxides are either formed by a direct attach- 
 ment of oxygen with readily oxidizable substances or in consequence of a 
 simultaneous oxidation with other substances — in the last way, for 
 example, the formation of H2O2 in the oxidation of indigo-white to indigo 
 according to the formula: 
 
 Indigo^ +02 = Indigo + H2O2 
 
 Indigo-white 
 
 Only certain substances have the ability either directly or indirectly of 
 forming peroxides. Certain protein-like substances occurring especially 
 in the plants which have this ability, have been called oxygenases by 
 Bach and Chodat. 3 Most of the substances which are oxidized within 
 the organism lose their ability to be directly oxidized. The oxidation 
 of such substances can, according to Bach, 4 be accomplished in that the 
 oxygen is transported to the substance to be oxidized from the peroxide 
 simultaneously present by means of special enzymes, the peroxidases. 
 These latter were first prepared from pumpkins and from horse-radish 
 roots. In the absence of peroxides or oxygenases the peroxidases are 
 without action. Chodat and Bach 5 have also found that certain 
 preparations, which have previously been called oxidases, can be decom- 
 posed into oxygenases and peroxidases. According to Bach's theory the 
 formation of peroxides is a constant process going on in the organism, 
 to which the organism accommodates itself, in that the cells by means of 
 the peroxidases can make use of the peroxides for the oxidation processes. 
 Besides this the organism also forms other enzymes, the so-called 
 catalases, which have the ability of decomposing the peroxides with the 
 formation of molecular oxygen (O2) and in this way making a possible 
 excess of peroxides harmless. 6 In reference to the behavior of the perox- 
 
 1 Verh. naturw. Verein, Karlsruhe, 20, XI (1896), Bd. 13, 72; see also Zeitschr. 
 f. physiol. Chem., 59, 327 (1909). 
 
 2 Compt. Rend., 124, 951 (1897); see also Bioch. Centralbl., 1, 417, and 457 (1903); 
 9, 1 (1909). 
 
 a Ber. d. d. chem. Gesellsch., 36, 600 (1903). 
 
 *Ibid., 36, 600 (1903). 
 
 5 Ibid., 36, 606 (1903). 
 
 « Bioch. Centralbl., 1, 460. 
 
PHYSIOLOGICAL OXIDATION PROCESSES. 873 
 
 idases to heat the views are contradictory. Czyhlauz and v. Fiuni 
 found that the peroxidases from animal tissues were remarkably resistant 
 to high temperatures, while Batelli and Stern x find that animal peroxi- 
 dases arc destroyed even at 66° C. 
 
 According to Bach's theory on the one hand, substances are nec- 
 essary for the oxidation, which take up oxygen with the formation of 
 peroxides (oxygenases) and on the other hand, substances which are 
 able to transport the oxygen from the peroxides to the oxidizable bodies 
 (peroxidases). In certain oxidations, for example in the phenols, the 
 peroxidases can be replaced by certain metallic combinations. 2 The 
 iron, of the blood pigments, acts in this way in the guaiacum reaction 
 (see Chapter XIV). The oil of turpentine here represents the peroxide 
 and can be replaced by hydrogen peroxide. The oxidizable substance, 
 which becomes blue in the reaction, is the guaiaconic acid in the guaiac 
 gum. 3 
 
 Irrespective of whether the division of the oxidation enzymes into 
 oxygenases and peroxidases can be carried out in all cases, there are 
 various oxidation processes, whose occurrence by a combination of oxy- 
 genase (or peroxide) with peroxidase (or metallic salt) can be explained only 
 with difficulty. According to Bertrand's 4 view the action of plant 
 oxidation enzymes is connected with their manganese content. Never- 
 theless Bach 5 has been able to prepare enzymes from plants which were 
 entirely free from iron as well as manganese salts. Starting from Ber- 
 trand's view, Trillat 6 has prepared solutions of manganese salts, alkali 
 and colloidal substances, which acted like oxidizing enzymes. Dony- 
 Henault 7 has prepared artificial "oxidases" from a faintly alkaline 
 solution of gum treated with a solution of manganese salt. According to 
 Euler and Bolin 8 the salts of certain organic acids have the ability of 
 setting the oxidation power of manganese salts free. Similar observations 
 have been made by Wolff. 9 In the oxidation of auto-oxidizable substances 
 the presence of extremely small amounts of iron salts may be of advantage, 
 
 1 Czyhlarz and v. Ftirth, Hofmeister's Beitrage, 10, 358 (1907); Batelli and Stern, 
 Bioch. Zeitschr., 13, 44 (1908). 
 
 2 Ber. d. d. chem. Gesellsch., 43, 366 (1910). 
 
 3 C. E. Carlson, Zeitschr. f. physiol. Chem., 48, 69 (1906). P. Richter, Arch. 
 d. Pharm., 244, 90 (1906). 
 
 <Compt. Rend., 124, 1032, 1355 (1897). 
 
 6 Ber. d. d. ehem. Gesellsch., 43, 364 (1910). 
 8 Compt. Rend., 138, 274 (1904). 
 
 7 Bull. acad. roy. de Belgique, 1908, 105. 
 
 8 Zeitschr. f. physiol. Chem., 57, 80 (1908). 
 
 9 Ann. inst. Past., 24 (1910). 
 
87-4 RESPIRATION AND OXIDATION. 
 
 for example with the lecithins (Thunberg, Warberg and Meyerhof l 
 as well as in the oxidation of certain thio-compounds. 2 
 
 Batelli and Stern 3 have made careful investigations as to the 
 occurrence of peroxidases in the animal organism. In order to eliminate 
 the action of catalases which are present in the tissues and which, 
 as shown by earlier investigators, decompose the hydrogen peroxide, 
 these experimenters used ethyl hydrogen peroxide, C2H5.O.O.H, on 
 which the catalases do not act. With ethyl hydrogen peroxide and 
 hydroidic acid nearly all animal tissues gave the peroxidase reaction, 
 wherein free iodine was formed. Scheunert, Grimmer and Andryewski 4 
 make use of the following solution as a reagent for peroxidases: 100 cc. 
 fresh tincture of guaiacum and 0.1 to 0.2 cc. 3 per cent H2O2 solution. 
 Blood does not give any blue coloration with this reagent, but in the pres- 
 ence of large quantities of H2O2 or other superoxide solutions (ethylhy- 
 drogen peroxide, oil of turpentine) it does give a blue coloration. With 
 this active tincture of guaiacum these experimenters were able to detect 
 peroxidases in the salivary glands, as well as the mucous membrane 
 of the stomach and intestine of certain varieties of animals. The liver 
 was always free from peroxidases. On the other hand, Batelli and Stern 5 
 also tested the ability of various tissues of acting upon formic acid in the 
 presence of H2O2 with the evolution of carbon dioxide. In later works 
 these experimenters claim that in all animal tissues there exists a substance 
 of an unknown nature, the pnein, which has the ability of bringing about 
 the respiration in all animal tissues. Pnein, which is soluble in water, 
 dializable and resistant to temperature, increases the so-called chief respira- 
 tion, which is connected with the life of the cells and which stops more 
 or less rapidly after the death of the animal. The so-called accessory 
 respiration continues quite a long time, after death, and this can continue 
 in the absence of cell elements and is of an enzymotic character. Thun- 
 berg 6 , who has constructed an apparatus for measuring the respiratory 
 exchange of gas in small organs and organisms (microrespirometer) finds 
 that the salts of certain organic acids (succinic acid, citric acid, malic 
 acid, fumaric acid) accelerate more or less the gas exchange in surviv- 
 ing frog's muscles. In their last communication Batelli and Stern 7 
 differentiate between two kinds of oxidation catalysts: the oxidases 
 and the oxidones. The first to which, among others the tyrosinase, alcohol- 
 
 1 Skand. Arch. f. Physiol., 24, 90 (1911); Zeitschr. f. physiol. Chem., 85, 412 (1913). 
 
 2 Thunberg, Lunds Univ. Arsskr., N. F., 2, Bd. 9 (1913). 
 « Bioch. Zeitschr., 13, 44 (1908). 
 
 * Ibid., 53, 300 (1913). 
 
 *Ibid., 21, 487 (1910); 30, 172 (1910); 33, 315 (1911). 
 
 ■ Skand. Arch. f. Physiol., 17, 23, 24, 25 (1911). 
 
 7 Bioch. Zeitschr., 46, 317, 343 (1912); Compt. rend., soc. biol., 74, 212 (1913). 
 
PHYSIOLOCAL OXIDATION PROCESSES. 875 
 
 oxidase, xanthin oxidase (see below) belong, are soluble in water, re- 
 sistant to alcohol and acetone and can be heated to 55-60° C. The 
 oxodones, which for example oxidize succinic acid to malic acid and 
 act upon p-phenyldiamine, cannot be extracted from the tissues by water; 
 they are injured or destroyed by alcohol, acetone or by being heated 
 to 55-60° C. 
 
 Warburg ! has carried on extensive experiments on the influence of 
 foreign bodies upon the respiration in the cells and has conformed the 
 results to Overton's theory of lipoid membrane. 
 
 No deep oxidation processes have been produced under the influence 
 of the mentioned oxidizing substances outside of the organism. The 
 various divergent views on the nature of the oxidizing substances strik- 
 ingly indicates how little exact knowledge we have of this subject. 
 Perhaps the oxidation within the body takes place step by step, and it 
 seems possible that the consecutive stages of the reaction can be brought 
 about by different agents. A positive division of the so-called oxidizing 
 enzymes cannot be made at the present time. For in many cases it 
 is undecided whether we are dealing with enzymotic processes or with 
 non-enzymotic catalytic action of metallic salts, especially as the 
 reports on the heat-resistance of the active substances are contra- 
 dictory. In the enzymotic oxidations we are in many cases in doubt 
 whether the oxygen is transported directly to the oxidizing substance or 
 whether the oxidation is brought about by the system peroxide plus 
 peroxidase. When the oxidation cannot be shown as due to the just- 
 mentioned system, then the active enzyme is called simply oxidase. 
 
 In consideration of the substances upon which the oxidation enzymes act, 
 we can divide them for the present into the following groups, according to 
 Oppexheimer: 2 
 
 1. Alcoholases, which transform alcohols into acids, for example the acetic- 
 acid-forming enzyme of certain varieties of bacteria. 
 
 2. Aldehydases, which oxidize aldehydes into acids, for example, salicylase. 
 
 3. Purine-oxidases, which oxidize hypoxanthine and xanthine into uric acid and 
 which act upon uric acid with the formation of allantoin. This last reaction is 
 produced by the action of the so-called uriease. 
 
 4. Phenol-oxidases, which oxidize various phenols and related bodies with the 
 formation of pigments. The guaiac reaction is of this kind. 
 
 5. Tyrosinases, which oxidize tyrosine and closely related bodies into dark 
 pigments. 
 
 The system peroxide -f peroxidase has been shown only in connection with 
 enzymes of group 4. 
 
 Besides the above-mentioned bodies, upon which the different classes of oxidiz- 
 ing enzymes act more specifically, we can mention the following as oxidase reagents. 
 
 x Zeitschr. f. physiol. Chem., 67. 69, 70, (1910); 71, 76 (1911). 
 
 1 Die Fermente und ihre Wirkungen, 3. Aufl., Spec. Teil, 351 (1909). 
 
876 RESPIRATION AND OXIDATION. 
 
 1. Iodides in acid solution in the presence of H 2 2 . According to Bach and 
 Chodat > this reaction is completely parallel with the guaiac reaction. 
 
 2. Formic acid with H 2 2 (see above). 
 
 3. Amines (especially p-phenyldiamine) which forms colored products on 
 t aking up oxygen. 2 
 
 4. Leucobases or mixtures of their formers, which by oxidation are converted 
 into pigments. A solution of a mixture of a-naphthol and p-phenylen-diamine 
 made alkaline with soda gives indophenol on taking up oxygen (Rohmann and 
 Spitzer 3 ). 
 
 5. Certain benzene derivatives which on oxidation and loss of water are 
 transformed into diphenol derivatives, for example vanillin into dehydrodivanillin. 4 
 
 For quantitative estimation of the extent of oxidation Bach and Chodat 5 
 use the transformation of pyrogallol into purpurogallin, which latter can be weighed. 
 Bach 6 determines the amount of iodine set free in the reaction between hydrogen 
 peroxide and hydriodic acid and Batelli and Stern 7 determine the quantity of 
 C0 2 formed in the oxidation of formic acid. 
 
 There is no doubt that reductions occur to a great extent in the animal 
 body and often go hand in hand with oxidations. The question as to 
 the extent in which special reduction enzymes are concerned, is still 
 undecided. As the oxidations are explained by the action of special 
 enzymes, so also we can admit of special reduction enzymes, so-called 
 reductases or hydrogenases. To this group belongs the so-called " philo- 
 thion" (De Rey-Pailhade), which in the presence of sulphur and 
 water develops sulphuretted hydrogen, while others on the contrary do 
 not accept this view, and deny the enzyme nature of philothion. 8 The 
 investigations of Nasse and Rosing 9 on the oxidation of protein in the 
 presence of sulphur contradict the enzymotic nature of this formation 
 of sulphuretted hydrogen, and the recent investigations of Heffter 10 
 have shown that certain reductions occurring in the tissues are not pro- 
 duced by enzymes. He has also shown that those reductions, which are 
 not influenced by HCN, like the reduction of pigments (methylene blue),. 
 
 1 Ber. d. d. chem. Gesellsch., 35, 2466 (1902). 
 
 2 Bioch. Zeitschr., 46, 317 (1912). 
 
 » Ber. d. d. chem. Gesellsch., 28, 567 (1894). 
 
 * In regard to this and other reagents, see Zeitschr. f. physiol. Chem., 59, 359 
 (Engler and Herzog). 
 
 5 Ber. d. d. chem. Gesellsch., 37, 1342 (1904). 
 *IUd., 37, 3785 (1904). 
 
 7 Bioch. Zeitschr., 31, 443; 33, 282 (1911). 
 
 8 De Rey-Pailhade, Recherches exp6r. sur le Philothion, etc., Paris (G. Masson), 
 1891, and Nouvelles recherches sur le Philothion, Paris (Masson), 1892; Bull. soc. 
 chim. (4), 1; Pozzi-Escot, Bull. soc. chim. (3), 27, and Chem. Centralbl., 1904, 1, 
 1645; Chodat and Bach, Ber. d. d. chem. Gesellsch., 36; Abelous and Ribaut, Compt. 
 Rend., 137, and Bull. soc. chim., (3), 31. 
 
 »E. Rosing, Unters. liber die Oxydation von Eiweiss in Gegenwart von Schwefel, 
 I norg. -Dissert, Rostock, 1891. 
 
 10 Med.-naturw. Arch., 1, 81-104; Marburg, cited in Chem. Centralbl., 2, 1907,, 
 822; Thunberg, Ergebn. d. Physiol., 11. 
 
REDUCTION PROCESSES. 877 
 
 of sulphur to H2S and others, can be brought about by the labile H of the 
 Bulphydryl-compounds. In this manner for example the cysteine 
 (see Chapter II) arts and quickly reacts with sulphur with the formation 
 of H2S and similarly acting substances have been detected by IIkffter 
 in various organs and organ extracts. We have here a group of reductions 
 which are not of an enzyme nature. 
 
 From the investigations of Abelous and Aloy l it follows that 
 plants as well as animal organs have the ability at the same time of oxidiz- 
 ing salicylaldehyde and of reducing nitrates to nitrites. On the other 
 hand ScHARDiNGEit, Trommsdorff and Bach 2 have shown that fresh 
 cow's milk, which alone is without action upon methylene blue and on 
 nitrates, reduces these bodies in the presence of aldehydes into leucobases 
 or nitrites. Boiled milk does not have this power and the action is 
 explained by the presence of a reductase, the so-called Schardinger 
 enzyme. The optimum of action lies at about 70° C. Bach found the same 
 action in various animal organs. The process may to be just as well 
 considered as an oxidation under the influence of an aldehydase whereby 
 the methylene blue or the nitrate gives up the oxygen for the oxidation 
 of the aldehyde. On the other hand Strassner 3 ascribes the reduction 
 of the methylene blue to the above-mentioned reducing action of the 
 sulphydryl groups. 
 
 1 Compt. Rend., 138, 382 (1904); see also Pozzi-Escot, ibid., 13S, 511. 
 
 2 Schardinger, Zeitschr. f. Unters. d. Nahrune;s- und Genussmittel, 5, 22 (1902); 
 Trommsdorff, Centralbl. f. Bakter., 49, 291 (1909); Bach, Ber. d. d. chem. Gesellsch., 
 42. 4463 (1909); Bioch. Zeitschr., 31, 443; 33, 2S2; 38, 154 (1911). 
 
 » Ibid., 29, 295 (1910). 
 
CHAPTER XVII. 
 
 METABOLISM WITH VARIOUS FOODS, AND THEIR NECESSITY 
 
 TO MAN. 
 
 I. GENERAL DISCUSSION AND METHODS USED IN THE STUDY OF MATTER 
 AND FORCE METABOLISM. 
 
 The conversion of chemical energy into heat and mechanical work 
 which characterizes animal life, leads to the formation of relatively 
 simple compounds — carbon dioxide, urea, etc. — which leave the organism, 
 and which, moreover, being very poor in energy, are for this reason of 
 little or no value to the body. It is therefore absolutely necessary for 
 the continuance of life and the normal course of the functions of the body 
 that the organism and its different tissues should be supplied with new 
 material to replace that which has been exhausted. This is accom- 
 plished by means of food. Those bodies are designated as food which 
 have no injurious action upon the organism and which serve as a source 
 of energy and can replace those constituents of the body that have been 
 consumed in metabolism or that can prevent or diminish the consumption 
 of such constituents. 
 
 Among the numerous dissimilar substances which man and animals 
 take with the food all cannot be equally necessary or have the same value. 
 Some perhaps are unnecessary, while others may be indispensable. We 
 have learned by direct observation and a wide experience that besides the 
 oxygen, which is necessary for oxidation, the essential foods for animals 
 in general, and for man especially, are water, mineral bodies, proteins, 
 carbohydrates, and fats. 
 
 It is also apparent that the various groups of food-stuffs necessary for 
 the tissues and organs must be of varying importance; thus, for instance, 
 water and the mineral bodies have another value than the organic foods, 
 and these again must differ in importance among themselves. The 
 knowledge of the action of various nutritive bodies on the exchange of 
 material from a qualitative as well as a quantitative point of view must 
 be of fundamental importance in determining the value of different 
 nutritive substances relative to the demands of the body for food under 
 various conditions, and also in deciding many other questions — for instance, 
 the proper nutrition for an individual in health and in disease. 
 
 878 
 
EXCRETA OF THE ORGANISM. 879 
 
 Such knowledge can be attained only by a series of systematic and 
 thorough observations, in which the quantity of nutritive material, rela- 
 tive to the weight of the body, taken and absorbed in a given time is 
 compared with the quantity of final metabolic products which leave the 
 organism at the same time. Researches of this kind have been made by 
 investigators, but above nil should be mentioned those made by BlBCHOFF 
 and Voir, by Pettenkofer and Voit, and by Voit and his pupils, by 
 Rubner, Zintz and by Atwater. 
 
 It is absolutely necessary in researches on the exchange of material 
 to be able to collect, analyze, and quantitatively estimate the excreta 
 of the organism, so that they may be compared with the quantity and 
 composition of the nutritive bodies ingested. In the first place, one must 
 know what the habitual excreta of the body are and in what way these 
 bodies leave the organism. One must also have trustworthy methods 
 for their quantitative estimation. 
 
 The organism may, under physiological conditions, be exposed to 
 accidental or periodic losses of valuable material — such losses as occur 
 only in certain individuals, or in the same individual only at a certain 
 period; for instance, the secretion of milk, the production of eggs, the 
 ejection of semen or menstrual blood. It is therefore apparent that these 
 losses can be the subject of investigation and estimation only in special 
 cases. 
 
 The regular and constant excreta of the organism are of the very 
 greatest importance in the study of metabolism. To these belong, in 
 the first place, the true final metabolic products — carbon dioxide, urea 
 (uric acid, hippuric acid, creatinine, and other urinary constituents), 
 and a part of the water. The remainder of the water, the mineral bodies, 
 and those secretions or tissue constituents — mucus, digestive fluids, sebum, 
 perspiration, and epidermal formations — which are either poured into 
 the intestinal tract, or secreted from the surface of the body, or broken 
 off and thereby lost to the body, also belong to the constant excreta. 
 
 The remains of food, sometimes indigestible, sometimes digestible but not 
 acted upon, which are contained in the feces, and which vary considerably in 
 quantity and composition with the nature of the food, also belong to the excreta 
 of the organism. Even though these remains, which are never absorbed and 
 therefore are never constituents of the animal fluids or tissues, cannot be con- 
 sidered as excreta of the body in a strict sense, still their quantitative estimation 
 is absolutely necessary in certain experiments on the exchange of material. 
 
 The determination of the constant loss is in some cases accompanied with the 
 greatest difficulties. The loss from the detached epidermis, from the secretion 
 of the sebaceous glands, etc., cannot be determined with exactness without dif- 
 ficulty, and therefore— as they do not occasion any appreciable loss because of 
 their small quantity — they need not he considered in quantitative experiments 
 on metabolism. This also applies to the constituents of the mucus, bile, pancreatic 
 and intestinal juices, etc., occurring in the contents of the intestine, and which, 
 leaving the body with the feces, cannot be separated from the other contents of 
 
880 METABOLISM. 
 
 the intestine and therefore cannot be quantitatively determined separately. The 
 uncertainty which, because of the intimated difficulties, attaches itself to the 
 results of the experiment, is very small as compared to the variation which is caused 
 by different individualities, different modes of living, different foods, etc. Only 
 approximate values can therefore be given for the constant excreta of the human 
 body. 
 
 The following figures represent the quantity of excreta for twenty- 
 iour hours from a grown man, weighing 60-70 kilos, on a mixed diet. 
 The figures are compiled from the results of different investigators: 
 
 Grams. 
 
 Water 2500-3500 
 
 Salts (with the urine) 20-30 
 
 Carbon dioxide 750-900 
 
 Urea 20-40 
 
 Other nitrogenous urinary constituents 2-5 
 
 Solids in the excrement 20-50 
 
 These total excreta are approximately divided among the various 
 excretions in the following way; but still it must not be forgotten that 
 this division may vary to a great extent under different external circum- 
 stances: By respiration about 32 per cent, by the evaporation from the 
 skin 17 per cent, with the urine 46-47 per cent, and with the excrement 
 5-9 per cent. The elimination by the skin and lungs, which is sometimes 
 differentiated by the name " per spir alio insensiblis " from the visible 
 elimination by the kidneys and intestine, is on an average about 50 per 
 cent of the total elimination. This proportion, quoted only relatively, 
 is subject to considerable variation, because of the great difference in 
 the loss of water through the skin and kidneys under varying circum- 
 stances. 
 
 The nitrogenous constituents of the excretions consist cniefly of urea, 
 or uric acid in certain animals, and the other nitrogenous urinary con- 
 stituents. A disproportionately large part of the nitrogen leaves the body 
 with the urine, and, as the nitrogenous constituents of this excretion are 
 final products of the metabolism of proteins in the organism, the quantity 
 of proteins catabolized in the body may be easily calculated by multiply- 
 ing- the quantity of nitrogen in the urine by the coefficient 6.25 ( :L 1 Q 6 (l = 6.25), 
 if it is admitted that the proteins contain in round numbers 16 per cent 
 of nitrogen. 
 
 Still another question is whether the nitrogen leaves the body only 
 with the urine or by other channels. The latter is habitually the case. 
 The discharges from the intestine always contain some nitrogen, which 
 consists in part of non-absorbed remnants of the food, but in chief part 
 and sometimes entirely of constituents of the epithelium and the secre- 
 tions. Under these circumstances it is apparent that one cannot give 
 any exact figures which are valid for all cases for that part of the nitrogen 
 of the excrement which originates in the digestive tract and in the digestive 
 
NITROGEN ELIMINATION. NITROGEN DEFICIT. 881 
 
 fluids. It may not vary in different individuals only, but also in the same 
 individual after more or less active secretion and absorption. In the 
 attempts made to determine this part of the nitrogen of the excrement 
 it has been found that in man, on non-nitrogenous or nearly nitrogen- 
 free food, it amounts in round numbers to somewhat less than 1 gram 
 per twenty-four hours (Rieder, Rubner). Even with such food the 
 .absolute quantity of nitrogen eliminated by the feces increases with 
 the quantity of food because of the accelerated digestion (Tsuboi 1 ), 
 and is greater than in starvation. Muller 2 found in his observations 
 on the faster C'etti that only 0.2 gram nitrogen was derived from the 
 intestinal canal. 
 
 The quantity of nitrogen which leaves the body under normal circum- 
 stances by means of the hair and nails, with the scaling off of the skin, 
 and with the perspiration cannot be accurately determined. It is 
 nevertheless so small that it may be ignored. Only in profuse sweating 
 need the elimination by this channel be taken into consideration. 
 
 The view was formerly held that in man and carnivora an elimination 
 of gaseous nitrogen took place through the skin and lungs, and because of 
 this, on comparing the nitrogen of the food with that of the urine and 
 feces, a nitrogen deficit occurred in the visible elimination. 
 
 This question has been the subject of much discussion and of numerous 
 investigations, the most recent by Krogh and Oppenheimer. 3 These 
 researches have shown that the above assumption is unfounded, and 
 moreover several authorities, especially Pettenkofer and Voit, and 
 Gruber, 4 have shown by experiments on man and animals that with 
 the proper quantity and quality of food the body can be brought into 
 nitrogenous equilibrium, in which the quantity of nitrogen voided with 
 the urine and feces is equal or nearly equal to the quantity contained in 
 the food. Undoubtedly we must admit, with Voit, that a deficit of nitro- 
 gen does not exist, or it is so insignificant that in experiments upon 
 metabolism it need not be considered. Ordinarily, in investigations on 
 the catabolism of proteins in the body, it is only necessary to consider the 
 nitrogen of the urine and feces, but it must be remarked that the nitrogen 
 of the urine is a measure of the extent of the catabolism of the proteins 
 
 1 Rieder, Zeitschr. f. Biologie, 20; Rubner, ibid., 15; Tsuboi, ibid., 35. 
 
 2 Berlin, klin. Wochenschr., 1887. 
 
 3 See Regnault and Reiset, Annal. d. chem. et phys. (3), 26, and Annal. d. Chem. 
 U. Pharm., 73; Seegen and Nowak, Wien. Sitzungsber., 71, and Pfliiger's Arch., 25; 
 Pettenkofer and Voit, Zeitschr. f. Biologie, 16; Leo, Pfliiger's Arch., 26; Krogh, 
 Skand. Arch. f. Physiol., 18, and Wien. Sitz. Ber., 115, III; Oppenheimer, Bioch. 
 Zeitschr., 4. 
 
 4 Pettenkofer and Voit, in Hernnan's Handbuch, 6, Thl. 1; Gruber, Zeitschr. f. 
 Biologie, 16 and 19. 
 
882 METABOLISM. 
 
 in the body, while the nitrogen of the feces (after deducting about 1 gram 
 on a mixed diet) is a measure of the non-absorbed part of the nitrogen of 
 the food. The nitrogen of the food, as well as of the excreta, is generally- 
 determined by Kjeldahl's method. 
 
 In the oxidation of the proteins in the organism, their sulphur is oxidized 
 into sulphuric acid, and on this depends the fact that the elimination of 
 sulphuric acid by the urine, which in man is but to a small extent derived 
 from the sulphates of the food, nearly makes equal variations with the 
 elimination of nitrogen by the urine. If the amount of nitrogen and sul- 
 phur in the proteins is considered as 16 per cent and 1 per cent respectively, 
 then the proportion between the nitrogen of the proteins and the sulphuric 
 acid, H2SO4, produced by their combustion is in the ratio 5.2 : 1, or about 
 the same as in the urine (see page 765). The determination of the quantity 
 of sulphuric acid eliminated in the urine gives us an important means of 
 controlling the extent of the transformation of proteins, and such a con- 
 trol is especially important in cases in which it is expected to study the 
 action of certain nitrogenous non-albuminous bodies on the metabolism 
 of proteins, or to decide the question whether a true protein combustion 
 and not only a washing out of the nitrogenous products of metabolism 
 from the tissues is taking place. A determination of the nitrogen alone 
 is naturally not sufficient in such cases. A perfectly positive measure 
 of the protein catabolism cannot be made from the sulphuric acid of the 
 urine, as the various protein substances have a rather variable sulphur 
 content, and on the other hand also a variable quantity of the sulphur 
 in the urine exists as so-called neutral sulphur. 
 
 In metabolism experiments the total sulphur of the urine as well as the 
 feces must be determined, and it may also be of importance to determine 
 the relation between the sulphuric-acid sulphur and the neutral sulphur 
 of the urine. The elimination of the sulphur originating from the proteins 
 does not, according to v. Wendt, Hamalainen and Helme and Ch. 
 Wolff x always run parallel with the protein nitrogen, and for the 
 white of egg the maxima of the elimination curves may indeed be 
 separated during a period of twenty-four hours (Wolff). The sulphur 
 is more quickly eliminated than the nitrogen, and this behavior of sul- 
 phur gives in certain cases a more positive picture of the temporal 
 catabolism of protein than the nitrogen. This is of importance, as the 
 elimination of the nitrogen corresponding to a certain amount of protein 
 requires several days for completion. Falta has also observed that the 
 chief amount of nitrogen in man on taking different proteins is secreted 
 with varying rapidity, and the same is true, according to Hamalainen 
 
 1 Wendt, Skand. Arch. f. Physiol., 17; Hamalainen and Helme, ibid., 19; Falta, 
 Deutsch. Arch. f. klin. Med., 86; Ch. G. Wolff, Bioch. Zeitschr., 40. 
 
CALCULATION OF METABOLISM. 883 
 
 and Helme, for the elimination of sulphur, as in their experiments the 
 sulphur elimination from white of egg required about six days and from 
 casein only two days. These conditions must be considered in metab- 
 olism experiments. 
 
 Besides lecithins and other phosphatides the body takes with its food 
 pseudonucleins as well as true nucleins, and these are absorbed more or 
 less completely from the intestinal tract and then assimilated. On the 
 other hand, the phosphorized protein substances, lecithins and phos- 
 phatides, are also decomposed within the body, and their phosphorus is 
 chiefly eliminated as phosphoric acid and also in part as organic phos- 
 phorus (see page 757). For these reasons the phosphorus is of great 
 importance in certain investigations on metabolism. 
 
 It is found, on comparing the nitrogen of the food with that of the 
 urine and feces, that there is an excess of the first ; this means that the 
 body has increased its stock of nitrogenous substances — proteins. If, 
 on the contrary, the urine and feces contain more nitrogen than the food 
 taken at the same time, this denotes that the body is giving up part of its 
 nitrogen — that is, part of its own proteins has been decomposed. 
 
 We can, from the quantity of nitrogen, as above stated, calculate the corre- 
 sponding quantity of proteins by multiplying by 6.25. x Usually, according to 
 Voit's proposition, the nitrogen of the urine is not calculated as decomposed 
 proteins, but as decomposed muscle-substance or flesh. Lean meat contains on 
 an average about 3.4 per cent nitrogen; hence each gram of nitrogen of the urine 
 corresponds in round numbers to about 30 grams of flesh. The assumption that 
 lean meat contains 3.4 per cent nitrogen is arbitrary, and the relation of N : C 
 in the proteins of dried meat, which is of great importance in certain experiments 
 on metabolism, is given differently by various experimenters, namely, 1 : 3.22- 
 1 : 3.68. Argutinsky found in beef, after complete removal of fat and subtrac- 
 tion of glycogen, that the relation was 1 : 3.24 (see Chapter X). 
 
 The carbon leaves the body chiefly as carbon dioxide, which is elimi- 
 nated by the lungs and skin. The remainder of the carbon is excreted in 
 the urine and feces in the form of organic compounds, in which the quan- 
 tity of carbon can be determined by elementary analysis. It was for- 
 merly considered sufficient to calculate the quantity of carbon in the urine 
 from the quantity of nitrogen according to the relation N:C = 1:0.67 to 
 0.72. This does not seem to be trustworthy, as this relation varies and 
 depends, according to Tangl, Pfluger, Langstein, and Steinitz, 2 upon 
 the kind of food. Tangl has shown that the richer the food is in car- 
 bohydrates the more carbon and hence the more heat of combustion per 
 
 1 In calculating the protein catabolism from the nitrogen of the urine it must not 
 be forgotten that the food often contains nitrogenous extractives whose nitrogen cannot 
 be calculated as protein and for which a special correction must be made, if necessary. 
 
 2 Tangl, Arch. f. (Anat. u.) Physiol., 1899, Suppl. Bd.; Pfluger in Pfliiger's Arch., 
 79; Langstein and Steinitz, Centralbl. f. Physiol., 19. 
 
884 METABOLISM. 
 
 gram of nitrogen does the urine contain. He found the following for 1 
 gram of nitrogen in the urine: With diet rich in fat 0.747 gram C and 9.22 
 calories; for carbohydrate-rich diet he found 0.936 gram C and 11.67 
 calories. The quantity of carbon in the feces can be calculated from the 
 
 C 
 
 quantity of nitrogen in the feces by using the quotient — = 9.2 (average 
 
 with mixed diet, according to Atwater and Benedict. 1 ) 
 
 The extent of the gas exchange can be determined by any of the 
 methods given on page 869. By multiplying the quantity of carbon dioxide 
 found by 0.273 one obtains the quantity of carbon eliminated as CO2. 
 If the total quantity of carbon eliminated in various ways is compared 
 with the carbon contained in the food, some idea can be obtained as to 
 the transformation of the carbon compounds. If the quantity of carbon 
 in the food is greater than in the excreta, then the excess is deposited; 
 while if the reverse be the case, it shows a corresponding loss of body 
 substance. 
 
 The nature of the substances here deposited or lost, whether they consist 
 of proteins, fats, or carbohydrates, is learned from the total quantity of the 
 nitrogen of the excretions. The corresponding quantity of proteins may be cal- 
 culated from the quantity of nitrogen, and, as the average quantity of carbon 
 in the proteins is known, the quantity of carbon which corresponds to the decom- 
 posed proteins may be easily ascertained. If the quantity of carbon thus found 
 is smaller than the quantity of the total carbon in the excreta, it is then obvious 
 that some other nitrogen-free substance has been consumed besides the proteins. 
 If the quantity of carbon in the proteins is considered in round numbers as 52.5 
 per cent, then the relation between carbon (52.5) and nitrogen (16) is 3.28, or in 
 round numbers 3.3 : 1. If the total quantity of nitrogen eliminated is multiplied 
 by 3.3, the excess of carbon in the eliminations over the product found represents 
 the carbon of the decomposed non-nitrogenous compounds. For instance, in 
 the case of a person experimented upon, 10 grams of nitrogen and 200 grams of 
 carbon were eliminated in the course of twenty-four hours; then these 62.5 grams of 
 protein correspond to 33 grams of carbon, and the difference, 200 — (3.3X10) =167, 
 represents the quantity of carbon in the decomposed non-nitrogenous compounds. 
 If we start from the simplest case, starvation, where the body lives at the expense 
 of its own substance, then, since the quantity of carbohydrates as compared with 
 the fats of the body is extremely small, in such cases in order to avoid mistakes 
 the assumption must be made that the person experimented upon has used only 
 fat and proteins. As animal fat contains on an average 76.5 per cent carbon, 
 the quantity of transformed fat may be calculated by multiplying the carbon by 
 
 ~-v = 1.3. In the case of the above example, the person experimented upon 
 
 would have used 62.5 grams of proteins and 167X1.3=217 grams of fat, of his 
 own body, in the course of the twenty-four hours. 
 
 Starting from the nitrogen balance, it can be calculated in the same way 
 whether an excess of carbon in the food as compared with the quantity of carbon 
 in the excreta is retained by the body as proteins or fat or as both. On the other 
 hand, with an excess of carbon in the excreta one can determine how much of the 
 loss of the substance of the body is due to a consumption of the proteins on the 
 one side and of non-nitrogenous bodies on the other side. How to especially 
 
 1 Bull, of Dept. of Agric, U. S., Washington, No. 136. 
 
CALORIC VALUES OF FOOD-STUFFS. 885 
 
 calculate the part taken by the fats and carbohydrate will be shown in connection 
 with the calculation of the energy metabolism. 
 
 The quantity of water and mineral bodies voided with the urine and 
 feces can easily be determined. The quantity of w r ater eliminated by 
 the skin and lungs may be directly estimated by means of the large 
 respiration apparatus. 
 
 The organic constituents of the body as well as the foodstuffs intro- 
 duced, represent a sum of chemical' energy which the body can use 
 for force. The exchange of material is also an exchange of force, 
 and the first stands in such close relation to the second that the study 
 of one cannot be separated from the other. The energy theory of 
 metabolism has exercised an extraordinarily fruitful influence upon 
 the entire study of metabolism and nutrition, and this is due in great 
 measure to the work of Rubner. 
 
 This energy of the various foods may be represented by the amount 
 of heat which is set free in their combustion. This quantity of heat is 
 expressed as calories, and a small calorie is the quantity of heat necessary 
 to warm 1 gram of water from 0° to 1° C. A large calorie is the quantity 
 of heat necessary to warm 1 kilo of water 1° C. Here and in the follow- 
 ing pages large calories are to be understood. There are numerous 
 investigations by different experimenters, such as Frankland, Dan- 
 ilewski, Rubner, Berthelot, Stohmann, Benedict and Osborne, 
 and others, on the calorific value of different foodstuffs. The following 
 results, which represent the calorific value of a few nutritive bodies on 
 complete combustion outside of the body to the highest oxidation prod- 
 ucts, are taken from Stohmann's 1 work. 
 
 Calories. 
 
 Casein 5 . 86 
 
 Ovalbumin 5 . 74 
 
 Conglut in 5 . 48 
 
 Protein (average) 5.71 
 
 Animal tissue-fat 9 . 50 
 
 Butter-fat 9 . 23 
 
 Cane-sugar 3 . 96 
 
 Milk-sugar 3 . 95 
 
 Glucose 3 . 74 
 
 Starch 4 . 19 
 
 Fats and carbohydrates are completely burnt in the body, and one can 
 therefore consider their combustion equivalent as a measure of the living 
 force developed by them within the organism. We generally designate 
 9.3 and 4.1 calories for each gram of substance as the average for the 
 physiological calorific value of fats and carbohydrates respectively. 
 
 1 See Rubner, Zeitschr. f. Biologic, 21, which also cites the works of Frankland 
 and Danilewski; see also Berthelot, Compt. Rend., 102, 104, and 110; Stohmann, 
 Zritschr. f. Biologie, 31; Benedict and Osborne (vegetable proteins), Journ. of biol. 
 Chem., 3. 
 
886 METABOLISM. 
 
 The proteins act differently from the fats and carbohydrates. They 
 are only incompletely burnt, and they yield certain decomposition prod- 
 ucts, which, leaving the body with the excreta, still represent a certain 
 quantity of energy which is lost to the body. The heat of combustion 
 of the proteins is smaller within the organism than outside of it, and they 
 must therefore be specially determined. For this purpose Rubner 1 
 fed a dog on washed meat, and he subtracted from the heat of combustion 
 of the food the heat of combustion of the urine and feces, which cor- 
 responded to the food taken plus the quantity of heat necessary for the 
 swelling up of the proteins and the solution of the urea. Rubner has 
 also tried to determine the heat of combustion of the proteins (muscle- 
 proteins) decomposed in the body of rabbits in starvation. According 
 to these investigations, the physiological heat of combustion in calories 
 for each gram of substance is as follows: 
 
 1 gram of the dry substance Calories. 
 
 Protein from meat 4.4 
 
 Muscle 4.0 
 
 Protein in starvation 3.8 
 
 Fat (average for various fats) 9.3 
 
 Carbohydrates (calculated average) 4.1 
 
 The physiological combustion value of the various foods belonging to 
 the same group is not quite the same. It is, for instance, 3.97 calories 
 for a vegetable protein, conglutin, and 4.42 calories for an animal protein 
 body, syntonin. According to Rubner the normal heat value per 1 
 gram of animal protein may be considered as 4.23 calories, and of vegetable 
 protein as 3.96 calories. When a person on a mixed diet takes about 
 60 per cent of the proteins from animal foods and about 40 per cent from 
 vegetable foods, the value of 1 gram of the protein of the food is equivalent 
 to about 4.1 calories. The physiological value of each of the three 
 chief groups of organic foods, by their decomposition in the body, is in 
 round numbers as follows: 
 
 Calories. 
 
 1 gram protein 4.1 
 
 1 gram fat 9.3 
 
 1 gram carbohydrate 4.1 
 
 1 gram alcohol 7.1 
 
 These figures are generally used in the calculation of the energy con- 
 tent of various foodstuffs and diets. 
 
 The extent of gas exchange and the so-called respiratory quotient 
 is, besides the extent of nitrogen elimination, of the greatest importance 
 in the calculation of the extent of energy metabolism and the division 
 of the energy between the protein, fat and carbohydrate. 
 
 < >n comparing the inspired and expired air we learn, on measuring 
 them when dry and at the same temperature and pressure, that the volume 
 
 1 Zeitschr. f . Biologie, 21. 
 
RESPIRATORY QUOTIENT. Ss7 
 
 of the expired air is less than that of the inspired air. This depends 
 upon the fact that not all of the oxygen appears again in the expired 
 air as carbon dioxide, because it is not only used in the oxidation of car- 
 bon, hut also in part in the formation of water, sulphuric acid, and other 
 bodies. The volume of expired carbon dioxide is regularly less than the 
 
 CO 
 volume of the inspired oxygen, and the relation -rr 11 , which is called the 
 
 respiratory quotient, is generally less than 1. 
 
 The magnitude of the respiratory quotient is dependent upon the kind 
 of substances destroyed in the body. In the combustion of pure carbon 
 one volume of oxygen j'ields one volume of carbon dioxide, and the 
 quotient is therefore equal to 1. The same is true in the burning of 
 carbohydrates, and in the exclusive decomposition of carbohydrates in 
 the animal body the respiratory quotient must be approximately 1. In 
 the exclusive metabolism of proteins it is close to 0.80, and with the decom- 
 position of fat it is 0.7. In starvation, as the animal draws on its own 
 flesh and fat, the respiratory quotient must be a close approach to the 
 latter figure. The respiratory quotient, which is calculated with exclusive 
 combustion of carbohydrate, fat and protein, as respectively, 1, 0.707 and 
 0.809 and with alcohol is 0.667, also gives important information as to 
 the quality of material decomposed in the body, especially with the 
 supposition that the carbon dioxide elimination is not influenced by some 
 special condition such as a change in the respirator}' mechanism. Another 
 supposition is that no incomplete oxidation step in combustion is elimi- 
 nated. 
 
 The respiratory quotient can also be strongly influenced by inter- 
 mediary processes in the animal bod}', as by the formation of glycogen 
 from protein, or from fat or by the formation of fat from carbohydrates. 
 In the first case the quotient may be lower than 0.7 and in the last case 
 it can be higher than 1. 
 
 Knowledge as to the extent of oxygen consumption is of special 
 importance in the calculation of the energy metabolism from the extent 
 of gas exchange, and one can under some circumstances approximately 
 calculate the energy exchange from the calorific value of the oxygen 
 alone — with regard to the respiratory quotient (Zuntz and co-workers). 
 The calorific value of oxygen must be different for each of the three men- 
 tioned foodstuffs, as they require different quantities of oxygen for their 
 combustion. For fat and carbohydrate this calorific value can be readily 
 calculated, as these bodies are completely burnt into carbon dioxide and 
 water. One gram of starch uses 828.8 cc. oxygen in its combustion 
 and produces 828.8 cc. carbon dioxide, and 4183 calories of heat are 
 developed. For one liter ( = 1.43 gram) oxygen, 5047 calories are pro- 
 duced, therefore for every liter ( = 1.966 gram) carbon dioxide formed, 
 
888 METABOLISM. 
 
 the same number, 5047 calories, are produced. In an analogous manner 
 the average calorific value of fat for 1 liter of oxygen, 4686 calories, and 
 for 1 liter carbon dioxide, 6629 calories, can be calculated. 
 
 These figures, which represent the physiological combustion values 
 per 1 gram of food-stuffs, derived from the carbon dioxide output or the 
 oxygen in-take in (grams or) liters which are represented by the quotients 
 
 ' or „' , have been called the calorific coefficients. 
 L.CO2 L.O2 
 
 With proteins, because of the unequal composition of the different 
 proteins, the results are uncertain and variable, and the calculation is 
 much more complicated. As example we will give the following calcula- 
 tion of Zuntz J for the fat-free dry substance of meat. 
 
 This substance consisted in 100 parts 
 
 52.38g.C.; 7.27g.H.; 22.68g.O.; 16.65g.N.; 1.02g.S. 
 Of which were found in the urine. 9.406 2.663 14.099 16.28 1.02 
 
 Of which were found in the feces . 1.471 0.212 0.889 0.37 
 
 Retained 41.50C; 4.40H; 7.690; 0.0N; 0.0S. 
 
 From this residue, with the taking up of 96.63 liters of oxygen, besides 39.6 
 grams water, 77.39 liters carbon dioxide were formed and the respiratory quotient 
 is therefore 0.801. Now 100 grams of such dry meat substance on complete 
 combustion yields 563.09 calories, and if we subtract the calorific value of the 
 corresponding urine (=113.70 calories) and feces ( = 17.76 calories), the sum, 
 131.46 calories, then 431.63 calories were set free in the body. For every gram 
 
 of nitrogen eliminated in the urine (16.28 gram) there is produced - „ ~q =26.51. 
 
 calories; the corresponding quantity of oxygen is ' n =5.91 liter O and the 
 
 77.39 
 corresponding quantity of C0 2 produced is '„» =4.75 liters C0 2 . The calorific 
 
 cyo r -I 
 
 value for 1 liter of oxygen consumed is therefore _ Q . = 4.485 calories, and for 
 
 1 liter of carbon dioxide produced , 7C . = 5.579 calories. 
 
 For milk protein Zuntz has calculated for 1 gram urea nitrogen 5.8 liters 
 oxygen, 4.6 liters carbon dioxide and 27 calories. The calorific value can be cal- 
 culated from this for 1 liter 0=4.66 and for 1 liter C0 2 = 5.87 calories. If we 
 
 Cal. 
 take the average of these calculations we obtain the calorific coefficients T rt ' =4.57 
 
 L/.U2 
 
 Cal. 
 and > r<n =5.73 for protein. 
 
 For the three foodstuffs we have the following calorific values: 
 
 Per 1 liter Relative Per 1 liter Relative 
 
 Oxygen. value. Carbon dioxide. value. 
 
 Protein 4.57 100 5.73 113.4 
 
 Fat 4.69 102.6 6.63 131.3 
 
 Carbohydrate 5 . 05 110. 5 5 .05 100 . 
 
 1 Zuntz, Loewy, Miiller and Caspari, Hohenklima und Bergwanderungen, Berlin, 
 1906, pages 102, 103. 
 
CALCULATION OF THE CALORIC VALUE. 889 
 
 The figures for the oxygen vary less than those for the carbon dioxide, 
 and this is a reason why the oxygen values are better suited than the 
 CO2 values for calculating the energy production from the extent of gas 
 exchange. Other investigators have obtained results which correspond 
 more or less with the above values for the heat value of oxygen, and E. 
 Voit and Kummaciiek, 1 who have made calculations in another way, 
 have obtained still smaller differences for the relative oxygen value. 
 
 From what was said above we can calculate the extent of protein 
 metabolism, the corresponding development of energy and the correspond- 
 ing absorption of oxygen and carbon dioxide formation, from the quantity 
 of nitrogen in the urine. If we subtract the oxygen and carbon dioxide 
 values from the total, directly determined gas exchange, the result repre- 
 sents the fats and carbohydrates used. According to Zuntz from this 
 residue we can calculate the heat value of the oxygen used as well as the 
 division of the decomposition of the fat and carbohydrate by consider- 
 ing the respiratory quotient. For this purpose Zuntz and Schumburg 
 have constructed a table, an abstract of which we give below, taken 
 from the work of Magnus-Levy. 2 
 
 Division in per cent. 
 
 Carbohydrate. Fat. 
 
 100 
 
 83 17 
 
 66 34 
 
 49 51 
 
 32 68 
 
 15 85 
 
 100 
 
 As the calorific oxygen values in the combustion of protein, fat 
 and carbohydrate show no great differences among themselves, in those 
 cases where, as in starvation, the part taken by the proteins in the total 
 metabolism is relatively small, one can calculate the total energy exchange, 
 without any striking error, from the respiratory quotient and the oxygen 
 used. This is especially important in experiments of short duration 
 where the protein metabolism cannot be directly determined, but is 
 calculated from the nitrogen elimination occurring during a longer time. 
 The method of Zuntz and Geppert, mentioned on page 869, has shown 
 itself especially useful in the study of the material and force exchange 
 in these experiments of short duration, while the respiration apparatus 
 constructed on Pettenkofer's or the Regnault-Reiset principle are 
 only useful in experiments over a longer period. 
 
 Kaufmann ' incloses the individual to be experimented upon in a capacious 
 sheet-iron room, which serves both as a respiration-chamber and a calorimeter, 
 
 1 Voit, Zeitschr. f. Biol., 44; Kummacher, ibid. 
 
 ■ A. Magnus-Levy in v. Noorden's Handb. d. Pathol, des Stoffwechsels, Bd. 1. (1906). 
 
 1 Arch. d. Physiologie (5), 8. 
 
 R. Q. 
 
 Calories value 
 
 
 per 1 liter O. 
 
 1.000 
 
 5.047 
 
 0.950 
 
 4.986 
 
 0.900 
 
 4.924 
 
 0.S50 
 
 4.803 
 
 0.800 
 
 4.801 
 
 0.750 
 
 4.740 
 
 0.707 
 
 4.086 
 
890 METABOLISM. 
 
 and which permits the estimation of the nitrogen of the urine and the carbon 
 dioxide expired, as well as the inspired oxygen and the quantity of heat produced. 
 If we start from the theoretically calculated formulae for the various possible 
 transformations of the proteins, fats, and carbohydrates in the body, it is clear 
 that other values must be obtained for the heat, carbon dioxide, oxygen, and 
 nitrogen of the urine, when one, for example, admits of a complete combustion 
 of proteins to urea, carbon dioxide, and water, or of a partial splitting off of fat. 
 Another relation between heat, carbon dioxide, and oxygen is also to be expected 
 when the fat is completely burnt or when it is decomposed into sugar, carbon 
 dioxide, and water. In this way, by a comparison of the values found in special 
 cases with the figures calculated for the various transformations, Kaufmann 
 attempts to explain the various decomposition processes in the body under dif- 
 ferent nutritive conditions. 
 
 The organic foodstuffs serve in part to replace the necessary losses 
 of the organs and in part as sources of energy. Under all circumstances 
 a restitution of the protein-like constituents of the organs is necessary. 
 This replacement is, according to Rubner, represented by the so-called 
 wear-and-tear quota (see below) which amounts to about 4-6 per cent of 
 the total energy transformed and which can be supplied by proteins only. 
 For the supply of the remaining exchange, which according to Rubner 
 serves as source of energy, all three groups of organic foodstuffs can be 
 used, and investigations carried out by Rubner have taught that these 
 foodstuffs can act as sources of energy in the animal body in a proportion 
 which corresponds with the respective figures of their heat value. This 
 is apparent from the following table. In this is found the weight of 
 the various foods equal to 100 grams of fat, a part determined from 
 experiments on animals and a part calculated from figures of the heat 
 values: 
 
 From Experiments From the Difference, 
 
 on Animals. Heat Value. per cent. 
 
 Syntonin 225 213 +5.6 
 
 Muscle-flesh (dried) .... 243 235 +4.3 
 
 Starch 232 229 +1.3 
 
 Cane-sugar 234 235 -0 
 
 Glucose 256 255 -0 
 
 From the given isodynamic value of the various foods it follows that 
 these substances replace one another in the body almost in exact ratio 
 to the energy contained in them. Thus in round numbers 227 grams of 
 protein and carbohydrate are equal to or isodynamic with 100 grams of 
 fat in regard to source of energy, because each yields 930 calories on com- 
 bustion in the oody. 
 
 By means of recent very important calorimetric investigations, Rub- 
 ner ! has shown that the heat produced in an animal in several series of 
 experiments extending over forty-five days corresponded to within 0.47 
 per cent of the physiological heat of combustion calculated from the decom- 
 
 Zeitschr. f. Biologie, 30. 
 
ISODYNAMICS OF FOOD-STUFFS. 891 
 
 posed body and foods. Atwater and his collaborators x have made some 
 very thorough investigations on this subject on men. In their experi- 
 ments they made use of a large respiration calorimeter, which not only 
 exactly determined the excreta, but also made a calorimetric determina- 
 tion of the heat given out by the person experimented upon, i.e., the work 
 performed. From the results of these experiments they found an almost 
 absolutely complete agreement between the calories found directly and 
 those calculated. 
 
 This isodynamic law is of fundamental value in the study of metabo- 
 lism and nutrition. The quantity of energy in the transformed foods 
 or the constituents of the body may be used as a measure for the total 
 consumption of energy, and the knowledge of the quantity of energy 
 in the foods must also be the basis for the calculation of dietaries for 
 human beings under various conditions. 
 
 The isodynamic theory has been accepted by a large number of inves- 
 tigators, but not by all. Certain of them, especially the French, accept 
 an isoglucosic instead of the isodynamic. According to this theory the 
 organism for its physiological functions can use glucose only, and as a 
 formation of glucose is possible from proteins as well as fats, those quan- 
 tities of food-stuffs are to be considered as equivalent which yield an 
 equal amount of glucose. 
 
 The heat value of a foodstuff can be directly determined in a calorim- 
 eter, but may also be calculated from its composition. If one subtracts 
 from the gross heat value of the food obtained in one way or another 
 the combustion heat of the feces and urine with the same diet, there is 
 obtained the net calorific value of the diet. This value, calculated in 
 percentage of the total energy content of the food, is called the physio- 
 logical availability by Rubner. 2 In order to elucidate this we will give 
 a few of Rubner's values. The loss in calories, as well as the physio- 
 logical availability, is calculated in percentages of the total energy 
 content of the food. 
 
 p 00( j r Loss in per cent. Total loss Availability 
 
 In Urine. In the Feces, in per cent, in per cent. 
 
 Cow's milk 5.13 5.07 10.20 89.8 
 
 Mixed diet (rich in fat) 3.87 5.73 9.60 90.4 
 
 Mixed diet (poor in fat) 4.70 6.00 10.70 89.3 
 
 Potatoes 2.00 5.60 7.60 92.4 
 
 Graham bread 2.40 15.50 17.90 82.1 
 
 Rve bread 2.20 24.30 26.50 73.5 
 
 Meat 16.30 6.90 23.20 76.8 
 
 In order to simplify the calculation of the energy exchange there exist other 
 standard factors, besides the above-mentioned standard figures for the physiological 
 
 1 Bull, of Dept. of Agric, Washington, 44, 63, 69, and 109, and Ergebnisse der 
 Physiologie, 3. 
 
 2 Zeitschr. f. Biologie, 42. 
 
892 METABOLISM. 
 
 calorific value of the organic foodstuffs, also for the carbon of the carbon dioxide, 
 and for the oxygen. Thus for 1 gram of meat (dry substance) free from fat 
 and extractives we have the calculated value of 5.44-5.77 calories. Kohler x 
 found 5.678 calories for 1 gram of ash and fat-free dried-meat substance of the 
 ox and 5.599 calories for horse meat. According to Frentzel and Schreuer 2 
 45.4 calories is calculated for 1 gram of nitrogen in fat and ash-free dried-meat 
 feces (dog), while 6.97 to 7.45 calories is calculated for 1 gram of nitrogen in meat- 
 
 urine. The calorific urine quotient — ^ — seems still, as above given, not to be 
 
 constant for human beings, but is dependent upon the variety of food. 
 
 H. METABOLISM IN STARVATION AND WITH INSUFFICIENT NUTRITION. 
 
 In starvation the decomposition in the body continues uninterruptedly, 
 though with decreased intensity; but, as it takes place at the expense of 
 the substance of the body, it can continue for a limited time only. When 
 an animal has lost a certain fraction of the mass of the body, death is the 
 result. This fraction varies with the condition of the body at the begin- 
 ning of the starvation period. Fat animals succumb when the weight of 
 the body has sunk to one-half of the original weight. Otherwise, accord- 
 ing to Chossat, 3 animals die as a rule when the weight of the body has 
 sunk to two-fifths of the original weight. The period when death occurs 
 from starvation not only varies with the varied nutritive condition at the 
 beginning, of starvation, but also with the more or less active exchange 
 of material. This is more active in small and young animals than in large 
 and older ones, but different classes of animals show an unequal activity. 
 Children succumb in starvation in 3-5 days after having lost one-fourth 
 of their body mass. Grown persons may, as observed upon Succi, 4 and 
 other professional fasters, starve for twenty days or more without lasting 
 injury; and there are reports of cases of starvation extending over a 
 period of even more than forty to fifty days. Dogs may starve, accord- 
 ing to several observers, 50-60 days. Hawk 5 and co-workers have 
 recorded a case where a dog was starved for 117 days and lost about 
 63 per cent of its original weight. Snakes and frogs can starve for one- 
 half a year or even a whole year. 
 
 In starvation the weight of the body decreases. The loss of weight is 
 greatest in the first few days, and then decreases rather uniformly. In 
 small animals the absolute loss of weight per day is naturally less than 
 in larger animals. The relative loss of weight — that is, the loss of weight 
 
 1 Zeitschr. f. physiol. Chem., 31. 
 
 2 The works of Frentzel and Schreuer may be found in Arch. f. (Anat. u.) Physiol., 
 1901, 1002, and L903. 
 
 3 Cited from \'<>it in Hermann's Handbuch, 6, Thl. 1, 100. 
 * See Luciani, Das Hungern. Hamburg u. Leipzig, 1890. 
 
 6 P. B. Hawk, P. E. Howe, and H. A. Mattil, Journ. of biol. Chem., 11. 
 
METABOLISM IN STARVATION. 893 
 
 of the unit of the weight of the body, namely, 1 kilo — is, on the contrary, 
 greater in small animals than in larger ones. The reason for this is that 
 the smaller animals have a greater surface of body in proportion to their 
 mass than larger animals, and the greater loss of heat caused thereby 
 must be replaced by a more active consumption of material. 
 
 It follows from the decrease in the weight of the body that the absolute 
 extent of metabolism must diminish in starvation. If, on the contrary, 
 the extent of metabolism is referred to the unit of weight of the body, 
 namely, 1 kilo, it appears that this quantity remains almost un- 
 changed during starvation. The investigations of Zuntz, Lehmann, 
 and others, 1 on the professional faster Cetti, showed on the third and 
 sixth days of starvation an average consumption of 4.65 cc. oxygen per 
 kilo in one minute, and on the ninth to eleventh day an average of 4.73 
 cc. The calories, as a measure of the metabolism, fell on the first to 
 fifth day of starvation from 1850 to 1G00 calories, or from 32.4 to 30 per 
 kilo, and it remained nearly unchanged, if referred to the unit of body 
 weight. 2 In man the average daily energy consumption in starvation 
 amounts to about 30-32 calories per kilo. 
 
 The extent of the metabolism of proteins, or the elimination of nitrogen 
 by the urine, which is a measure of the same, diminishes as the weight 
 of the body diminishes. This decrease is not regular or the same during 
 the entire period of starvation, and the extent depends, as the experi- 
 ments made upon carnivora have shown, upon several circumstances. 
 During the first few days of starvation the excretion of nitrogen is greatest, 
 and the richer the body is in protein, due to the food previously taken, 
 the greater is the protein catabolism or the nitrogen elimination, accord- 
 ing to Voit. The nitrogen elimination diminishes the more rapidly — 
 that is, the curve of the decrease is more sudden — the richer in proteins 
 the food was which was taken before starvation. This condition is 
 apparent from the following table of data of three different starvation 
 experiments made by Voit 3 on the same dog. This dog received 2500 
 grams of meat daily before the first series of experiments, 1500 grams of 
 meat daily before the second series, and a mixed diet relatively poor in 
 nitrogen before the third series. 
 
 Day of Starvation. grams of Urea Eliminated in Twenty-four Hgurs.^ 
 
 First 60.1 ' 26.5 ' 13.8 
 
 Second 24.9 18.6 11.5 
 
 Third 19.1 15.7 10.2 
 
 Fourth 17.3 14.9 12.2 
 
 Fifth 12.3 14.8 12.1 
 
 Sixth 13.3 12.8 12.6 
 
 Seventh 12.5 12.9 11.3 
 
 Eighth 10.1 12.1 10.7 
 
 1 Berlin, klin. Wochenschr., 1887. 
 
 2 See also Tigerstedt and collaborators in Skand. Arch. f. Physiol., 7. 
 1 See Hermann's Handbuch, 6, Thl. 1, 89. 
 
894 METABOLISM. 
 
 In man and also in animals sometimes a rise in the nitrogen excretion 
 is observed about the second or third starvation day, which is then fol- 
 lowed by a regular diminution. This rise is explained by Prausnitz, 
 Tigerstedt, Landergren, 1 as follows: At the commencement of star- 
 vation the protein metabolism is reduced by the glycogen still present 
 in the body. After the consumption of the glycogen, which takes place 
 in great part during the first days of starvation, the destruction of pro- 
 teins increases as the glycogen action decreases, and then decreases again 
 when the body has become poorer in available proteins. 
 
 Other conditions, such as varying quantities of fat in the body, have 
 an influence on the rapidity with which the nitrogen is eliminated during 
 the first days of starvation. After the first few days of starvation the 
 elimination of nitrogen is more uniform. It may diminish gradually 
 and regularly until the death of the animals or there may be a rise in the 
 last days, a so-called premortal increase. Whether the one or the other 
 occurs depends upon the relation between the protein and fat content 
 of the body. 
 
 Like the destruction of proteins during starvation, the decomposi- 
 tion of fat proceeds uninterruptedly, and the greatest part of the' calories 
 needed during starvation are supplied by the fats. According to Rubner 
 and Voit the protein catabolism varies only slightly in starving animals 
 at rest and at an average temperature, and forms a constant portion 
 of the total exchange of energy; of the total calories in dogs 10-16 per 
 cent comes from the protein decomposition and 84-90 per cent from the 
 fats. This is at least true for starving animals which had a sufficiently 
 great original fat content. If on account of starvation the animal has 
 become relatively poorer in fat and the fat content of the body has fallen 
 below a certain limit, then in order to supply the calories necessary, a 
 larger quantity of protein is destroyed and the premortal increase now 
 occurs (E. Voit). The reason for this premortal rise in protein catabol- 
 ism is still not completely understood (Schulz and collaborators 2 ). 
 
 Since the fat has a diminishing influence on the destruction of pro- 
 teins corresponding to what was said above, the elimination of nitrogen 
 in starvation is less in fat than in lean individuals. For instance, only 
 9 grams of urea were voided in twenty-four hours during the later stages 
 of starvation by a well-nourished and fat person suffering from disease 
 of the brain, while I. Munk found that 20-29 grams urea were voided 
 daily by Cetti, 3 who had been poorly nourished. 
 
 'Prausnitz, Zeitsohr. f. Biologie, 29; Tigerstedt and collaborators, 1. c; Landergren, 
 Undersokningar ofver menniskans agghviteomsattning, Inaug.-Diss. Stockholm,. 
 1902. 
 
 2 Voit, Zeitschr. f. Biologie, 41; 167 and 502. See also Kaufmann, ibid., and N_ 
 Schulz, ibid., and Pflu^-r's Arch., 76, with Mangold, Stubel and Hempel, ibid., 114. 
 
 » Berl. klin. Wochenschr., J887. 
 
METABOLISM IN STARVATION. 895 
 
 The investigations on the exchange of gas in starvation have shown, 
 as previously mentioned, that its absolute extent is diminished, hut 
 that when the consumption of oxygen and elimination of carbon dioxide 
 are calculated on the unit weight of the body, 1 kilo, this quantity 
 quickly sinks to a minimum and then remains unchanged, or, on the 
 continuation of the starvation, may actually rise. It is a well-known 
 fact that the body temperature of starving animals remains almost con- 
 stant, without showing any appreciable decrease, during the greater 
 part of the starvation period. The temperature of the animal first sinks 
 a few days before death, which occurs at about 33-30° C. 
 
 From what has been said about the respiratory quotient it follows 
 that in starvation it is about the same as with fat and meat exclusively 
 as food, i.e., approximately 0.7. This is often the case, but it may occa- 
 sionally be lower, 0.65-0.50, as observed in the cases of Cetti and Succi. 
 This can be explained by an elimination of acetone bodies by the urine; 
 a part can be accounted for perhaps by a formation and deposition of 
 glycogen from protein. 
 
 Water passes uninterruptedly from the body in starvation even when 
 none is taken. If the quantity of water in the tissues rich in proteins 
 is considered as 70-80 per cent, and the quantity of proteins in them 
 20 per cent, then for each gram of protein destroyed about 4 grams of 
 water are set free. This liberation of water from the tissues is generally 
 sufficient to supply the loss of water, and starvation is ordinarily not 
 accompanied with thirst. 
 
 The loss of water calculated on the percentage of the total organism must 
 naturally be essentially dependent upon the previous amount of fatty tissue in the 
 body. In certain cases the starving animal body has indeed been found richer 
 in water; but if we bear these conditions in mind, then, it seems, according to 
 Bohtlingk, 1 that, from experiments upon white mice, the animal body is poorer 
 in water during inanition. The body loses more water than is set free by the 
 destruction of the tissues. 
 
 The mineral substances leave the body uninterruptedly in starvation 
 until death, and the influence of the destruction of tissues is plainly 
 perceptible by their elimination. Because of the destruction of tissues 
 rich in potassium the proportion between potassium and sodium in 
 the urine changes in starvation, so that, contrary to the normal condi- 
 tions, the potassium is eliminated in proportionately greater quantities. 
 
 Contrary to the above Bohtlingk with starving white mice, and Katsuyama ! 
 with starving rabbits found a greater excretion of sodium than potassium. 
 
 1 Arch, des sciences biol. de St. Petersbourg, 5. 
 
 * Bohtlingk, 1. c; Katsuyama, Zeitschr. f. physiol. Chem., 26. 
 
896 
 
 METABOLISM. 
 
 Munk observed, in Cetti's case, an increase in the elimination of 
 phosphoric acid in relation to the iV-elimination, which indicates an 
 increased decomposition of bone-substance, and this explanation is 
 supported by the fact that a simultaneous increase in the elimination of 
 lime and magnesia occurs. Recently Wellmann * showed that in rabbits, 
 the increase in the elimination of phosphorus, calcium and magnesium 
 in starvation corresponds to the loss in the bones of these constituents. 
 
 The question as to the participation of the different organs in the loss 
 of weight of the body during starvation is of special interest. In elucida- 
 tion of this point we give the following results of Chossat's experiments 
 on pigeons, and those of Voit 2 on a male cat. The results are percentages 
 of weight lost from the original weight of the organ. 
 
 Pigeon (Chossat). 
 
 Adipose tissue 93 per cent. 
 
 Spleen 71 
 
 Pancreas 64 
 
 Liver 52 
 
 Heart 45 
 
 Intestine 42 
 
 Muscles 42 
 
 Testicles — 
 
 Skin 33 
 
 Kidneys 32 
 
 Lungs 22 
 
 Bones 17 
 
 Nervous system 2 
 
 Male Cat (Voit). 
 97 per cent. 
 67 
 17 
 54 
 
 3 
 18 
 31 
 40 
 21 
 26 
 18 
 
 4 
 
 3 
 
 The total quantity of blood, as well as the quantity of solids contained 
 therein, decreases, as Panum and others 3 have shown, in the same pro- 
 portion as the weight of the body. Concerning the loss of water by 
 different organs authorities disagree, Lukjanow 4 claiming that the 
 various organs differ from each other in this respect. 
 
 The above-tabulated results cannot serve as a measure of the metabol- 
 ism in the various organs during starvation. For instance, the nervous 
 system shows only a small loss of weight as compared with the other 
 organs, but from this it must not be concluded that the exchange of 
 material in this system of organs is least active. The conditions may be 
 quite different; for one organ may derive its nutriment during starva- 
 tion from some other organ and exist at its expense. A positive con- 
 clusion cannot be drawn in regard to the activity of the metabolism in 
 an organ from the loss of weight of that organ in starvation. Death 
 by starvation is not the result of the death of all the organs of the body, 
 
 1 Munk, Berl. klin. Wbchenschr., 1887; Wellmann, Pfliiger's Arch., 121. 
 
 2 Cited from Voit in Hermann's Handbwh, fi. Part I, 96 and 97. 
 
 5 Panum, Virchow's Arch., 29; London, Arch. d. scienc. biol. de St. P£tersbourg, 4.. 
 • Zeitechr. f. physiol. Chem., 13. 
 
METABOLISM IN STARVATION. 897 
 
 but it depends more likely upon the disturbance in the nutrition of a few 
 less vitally important organs (E. Voit 1 ). 
 
 In calculating or determining the loss of weight of the organs in 
 starvation the original fat content of the organs must be considered. 
 With the consideration of the fat content of the organs, determined or 
 estimated in a special way before the starvation period and at the end, 
 E. Voit 2 found the following loss of weight in the supposed fat-free 
 organs in starvation, namely, muscles 41 per cent, viscera 42 per cent, 
 skin 28 per cent, and skeleton 5 per cent. 
 
 The quantity of urine nitrogen sinks in starvation corresponding to 
 the protein catabolism, but to a varying degree in different individuals. 
 The lowest value observed thus far in man was 2.82 grams per diem as 
 found by E. and 0. Freund on the twenty-first day in the faster Succi. 
 Calculated on 1 kilogram of body weight, the urine nitrogen, as is to be 
 expected, shows striking differences in different persons; in Cetti and 
 Succi it was 0.150-0.200 gram on the fifth to tenth day of starvation. 
 The division of the nitrogen in the urine in starvation is unl'.ke that in 
 the normal condition. The relative amount cf urea diminishes, as 
 shown by E. and (). Freund, Brugsch and Cathcart, 3 so that instead 
 of being about 85 per cent of the total nitrogen under normal conditions 
 it can sink to 54 per cent (Brugsch). At the same time because of the 
 abundant formation of acetone bodies (starvation acidosis) the quantity 
 of ammonia increases considerably (Brugsch, Cathcart). A relative 
 increase in the neutral sulphur of the urine also takes place (Benedict, 
 Cathcart 4 ). Creatine also occurs in starvation urine and according 
 to Hawk 5 and co-workers the elimination of creatine is much greater than 
 the creatinine a few days before the premortal nitrogen elimination. 
 
 One must differentiate between the real starvation metabolism and the 
 metabolism in the inanition condition, the basal requirement (Magnus- 
 Levy) or the maintenance value (Loewy 6 ). With this we understand the 
 metabolism in uniform, medium temperature, with absolute bodily rest 
 and inactivity of the intestinal canal. As a measure of this we deter- 
 mine the gas exchange in a person lying down with as perfect com- 
 plete muscular rest as possible, or sleeping in the early morning and 
 at least twelve hours after a light meal not rich in carbohydrates. This 
 
 1 Zeitschr. f. Biologie, 41. 
 
 2 Ibid., 46. 
 
 J E. and O. Freund, Wien. klin. Rundschau, 1901, Nos. 5 and G; Brugsch, Zeitschr. 
 f. exp. Path. u. Therap., 1 and 3; Cathcart, Bioch. Zeitschr., 6. 
 
 4 Zeitschr. f. klin. Med., 36; Cathcart, 1. c. 
 
 5 Journ. of biol. Chem., 11. 
 
 6 Magnus-Levy in v. Noorden's Handbuch, and Loewy in Oppenheimer's Handbuch 
 d. Biochemie, Bd. 4. 
 
898 METABOLISM. 
 
 basal requirement is the measure of the energy necessary for the per- 
 formance of all the functions necessary to maintain life during rest; and 
 all work above this minimum activity is called productive increase by 
 Magnus-Levy. The basal requirement is almost constant for the same 
 individual and serves as the starting point in the study of the action of 
 different influences such as work, food, diseased conditions, etc., upon 
 metabolism. The extent of this basal requirement, as determined by 
 the gas exchange according to the Zuntz-Geppert method, and by 
 Johansson l and collaborators amounts in men of 60-70 kilos body 
 weight to about 220-250 cc. oxygen and 160-200 cc. carbon dioxide per 
 minute, which equals 20-24 grams carbon dioxide per hour. Johansson 
 found in forced complete muscular rest 20.7 grams CO2 per hour and 24.8 
 grams CO2 in the ordinary resting. Gigon 2 found about 23.4 grams 
 CO2 and 21 grams oxygen for the basal requirement. According to Mag- 
 nus-Levy the total daily metabolism can be calculated for the basal 
 requirement as 1625 calories, or including the rise due to the partaking 
 of food as 1800 calories. According to Gigon the basal requirement 
 consists of 15.22 per cent protein, 15-35.2 per cent carbohydrates and 
 44.5-70 per cent fat. 
 
 The food may be quantitatively insufficient, and the final result of 
 this is absolute inanition. The food may also be qualitatively insufficient 
 or, as we say, inadequate. This occurs when any of the necessary 
 nutritive bodies are absent in the food, while the others occur in sufficient 
 or perhaps even in excessive amounts. 
 
 Lack of Water in the Food. The quantity of water in the organism is 
 greatest during fcetal life and then decreases with increasing age. Nat- 
 urally, the quantity differs essentially in different organs. The enamel, 
 with only 2 p. m. water, is the tissue poorest in water, while the teeth 
 contain about 100 p. m. and the fatty tissue 60-120 p. m. water. The 
 bones, with 140-440 p. m., and the cartilage with 540-740 p. m. are 
 somewhat richer in water, while the muscles, blood and glands, with 750 
 to more than 800 p. m., are still richer. The quantity of water is even 
 greater in the animal fluids (see preceding chapter), and the adult body 
 contains in all about 630 p. m. water. 3 It .follows from what has been 
 given in Chapter I in regard to the great importance of water for living 
 processes, that if the loss of water is not replaced by fresh supply, the 
 organism must succumb sooner or later. Death occurs indeed sooner 
 from lack of water than from complete inanition (Landauer, Nothwang). 
 
 1 The literature can be found in the works of Magnus-Levy and Loewy. 
 1 Johansson, Skand. Arch. f. Physiol., 7, 8, 21, and Nord. Med. Arch. Festband, 
 1897; see also Magnus-Levy; Gigon, Pfluger's Arch., 140. 
 * See Voit, in Hermann's Handbuch, 6, part 1, 345. 
 
LACK OF MINERAL SUBSTANCES. 899 
 
 If water is withdrawn for a certain time, as Landauer and espe- 
 cially Straub have shown, it has an accelerating influence upon the 
 decomposition of protein. This increased destruction has, according to 
 Landauer, the purpose of replacing a part of the water removed, by the 
 production of water by means of the increased metabolism. The depriva- 
 tion of water for a short time may, according to Spiegler, 1 especially in 
 man, cause a diminution in the protein metabolism by means of a reduced 
 protein absorption. 
 
 Lack of Mineral Substances in the Food. In the previous chapters 
 attention has repeatedly been called to the importance of the mineral 
 bodies and also to the occurrence of certain mineral substances in certain 
 amounts in the various organs. The mineral content of the tissues and 
 fluids is not very great as a rule. With the exception of the skeleton, 
 which contains as average about 220 p. m. mineral bodies (Volkmann 2 ), 
 the animal fluids or tissues are poor in inorganic constituents, and the 
 quantity of these amounts as a rule, only to about 10 p. m. Of the 
 total quantity of mineral substances in the organism, the greatest part 
 occurs in the skeleton, 830 p. m., and the next greatest in the muscles, 
 about 100 p. m. (Volkmann). 
 
 The mineral bodies seem to be partly dissolved in the fluids and partly 
 combined with organic substances, but nothing definite can be given as 
 to the kind of combination, or whether they occur in stoichiometric 
 proportions, or whether they are simply adsorption combinations. In 
 accordance with this the organism persistently retains, with food poor 
 in salts, a part of the mineral substances, also such as are soluble, as the 
 chlorides. On the burning of the organic substances the mineral bodies 
 combined therewith are set free and may be eliminated. It is also 
 admitted that they in part combine with the new products of the com- 
 bustion, and in part with organic nutritive bodies poor in salts or nearly 
 salt-free, which are absorbed from the intestinal canal and are thus retained 
 (Voit, Forster 3 ). 
 
 If this statement is correct, it is possible that a constant supply of 
 mineral substances with the food is not absolutely necessary, and that the 
 amount of inorganic bodies which must be administered is insignificant. 
 The question whether this is so or not has not, especially in man, been 
 sufficiently investigated; but generally we consider the need of mineral 
 
 1 Landauer, Maly's Jahresber., 24; Nothwang, Arch. f. Hyg., 1892; Straub, Zeitschr. 
 f. Biol., 37 and 38; Spiegler, ibid., 41. 
 
 2 See Hermann's Handbuch., 6, pt. 1, 353. 
 
 3 Forster, Zeitschr. f. Biologie, 9. See also Voit, in Hermann's Handbuch, 6, 
 Part 1, 354. In regard to the occurrence and the behavior of the various mineral 
 constituents of the animal body see the work of Albu and Neuberg, Physiologie und 
 Pathologie des Mineralstoffwechsel, Berlin, 1906. 
 
900 METABOLISM. 
 
 substances by man as very small. It may, however, be assumed that 
 man usually takes with his food a considerable excess of mineral sub- 
 stances. 
 
 Experiments to determine the results of an insufficient supply of 
 mineral substances with the food in animals have been made by several 
 investigators, especially Fcrster. He observed, on experimenting with 
 dogs and pigeons with food as poor as possible in mineral substances, 
 that a very suggestive disturbance of the functions of the organs, par- 
 ticularly the muscles and the nervous system, appeared, and that death 
 resulted in a short time, earlier in fact than in complete starvation. On 
 observations made upon himself, Taylor l found on partaking less than 
 0.1 gram salts per diem that the chief disturbance occurred in the mus- 
 cular system. 
 
 Bunge in opposition to these observations of Forster's has suggested 
 that the early death of these cases was not caused by the lack of mineral 
 salts, but more likely by the lack of bases necessary to neutralize the sul- 
 phuric acid formed in the combustion of the proteins in the organism; 
 these bases must then be taken from the tissues. In accordance with 
 this view, Bunge and Lunin 2 also found, in experimenting with mice, 
 that animals which received nearly ash-free food with the addition of 
 sodium carbonate were kept alive twice as long as those which had the 
 same food without the sodium carbonate. Special experiments also 
 show that the carbonate cannot be replaced by an equivalent amount of 
 sodium chloride, and that to all appearances it acts by combining with 
 the acids formed in the body. The addition of alkali carbonate to the 
 otherwise nearly ash-free food may indeed delay death, but cannot pre- 
 vent it, and even in the presence of the necessary amount of. bases death 
 results from lack of mineral substances in the food. 
 
 With an insufficient supply of chlorides with the food the elimination 
 of chlorine by the urine decreases constantly, and at last it may stop 
 entirely, while the tissues still persistently retain the chlorides. It has 
 already been stated (Chapter VIII) how chloride starvation influences 
 other functions, especially the secretion of gastric juice. If there be a 
 lack of sodium as compared with potassium, or if there be an excess of 
 potassium compounds in any other form than KC1, the potassium com- 
 binations are replaced in the organism by NaCl, so that new potassium 
 and sodium compounds are produced which are voided with the urine. 
 The organism becomes poorer in NaCl, which therefore must be taken 
 in greater amounts from the outside (Bunge). This occurs continuously 
 
 1 University of California Publications, Pathol., 1. 
 
 2 Bunge, Lehrbuch der physiol. Chem., 4. Aufl., 97; Lunin, Zeitschr. f. physiol. 
 Chem., 5. 
 
ALKALI CARBONATES. PHOSPHATES AND EARTHS. ( J01 
 
 in herbivora, and in man with vegetable food rich in potash. For human 
 beings, and especially for the poorer classes of people who live chiefly 
 on potatoes and foods rich in potash, common salt is not only a condi- 
 ment, but a necessary addition to the food (Bunge l ). On the behavior 
 of chlorides, especially sodium chloride, in the animal body as well as the 
 elimination or the retention of NaCl in diseases, we have an abundance 
 of investigations, which may be found in Albu and Neuberg's 2 work, 
 previously cited. 
 
 Lack of Alkali Carbonates or Bases in the Food. The chemical processes 
 in the organism are dependent upon the presence in the tissues and tissue- 
 fluids of a certain reaction, and this reaction, which is habitually alkaline 
 toward litmus and neutral toward phenolphthalein, is chiefly due to the 
 presence of alkali carbonates and carbon dioxide and in a lesser degree to 
 alkali phosphates. The alkali carbonates are also cf great importance, 
 not only as a solvent for certain protein bodies and as constituents of 
 certain secretions, such as the pancreatic and intestinal juices, but they 
 are also a means of transportation of the carbon dioxide in the blood. 
 It is therefore easy to understand that a decrease below a certain point 
 in the quantity of alkali carbonate must endanger life. Such a decrease 
 not only occurs with lack of bases in the food which brings about various 
 disturbances and death by a relatively great production of acids through 
 the burning of the proteins, but it also occurs when an animal is given 
 dilute mineral acids for a period. The importance of ammonia as a 
 means of neutralizing the acids produced or introduced into the body 
 as well as the unequal resistance of man and other animals toward this 
 action of acids has already been discussed in Chapter XIV. 
 
 Lack of Phosphates and Earths. With the exception of the value of 
 the alkaline earths as carbonates and more especially as phosphates in 
 the physical composition of certain structures, such as the bones and 
 teeth, their physiological importance is almost unknown. The importance 
 of calcium for certain enzymotic processes and of calcium ions for the 
 functions of the muscles, and especially for cell life, gives an indication of 
 the necessity of the alkaline earths to the animal organism. Little is 
 known of the need of these earth in adults, and no average results can 
 be given. According to Kochmann and Petzsch 3 we cannot conceive 
 of a certain calcium minimum (in dogs) as the Ca needs vary with dif- 
 ferent foods. With a Ca equilibrium we can cause an increased elimi- 
 nation of calcium by increasing the quantity of protein, of fat, or of 
 carbohydrate in the food and this probably depends upon a giving up 
 
 1 Zeitschr. f . Biologie, 9. 
 
 * See footnote 3, page 899. 
 
 * Kochmann, Bioch. Zeitschr., 31, with Petzsch, ibid., 32. 
 
902 METABOLISM. 
 
 of calcium phosphate by the skeleton. It is impossible to give 
 positive figures for the need of phosphates or phosphoric acid, whose 
 value is recognized not only in the construction of the bones, but also' 
 in the functions of the muscles, the nervous system, the glands, the 
 organs of generation, etc. The extent of this need is most difficult to 
 determine, as the body shows a strong tendency, when increased amounts 
 of phosphorus are introduced, to retain more than is necessary. The 
 need of phosphates, which, according to Ehrstrom, 1 corresponds in adults 
 to a minimum of 1 to 2 grams phosphorus, is relatively smaller in adults 
 than in young, developing animals, and in these latter the question of 
 the result of an insufficient supply of earthy phosphates and alkaline 
 earths upon the bone tissue is of special interest. For details we refer 
 to Chapter IX and to the cited work of Albu-Neuberg. 
 
 Another important question is, How far do the phosphates take part 
 in the construction of the phosphorized constituents of the body or to 
 what extent are they necessary? The experiments of Rohmann and his 
 pupils 2 with phosphorized (casein, vitellin) and non-phosphorized pro- 
 teins (edestin) and phosphates show that with the introduction of casein 
 and vitellin a deposition of nitrogen and phosphorus takes place, while 
 with non-phosphorized protein and phosphates this does not seem to 
 occur. The body apparently does not have the power of building up 
 the phosphorized cell constituents necessary for cell life from non-phos- 
 phorized proteins and phosphates. On the contrary, according to the 
 observations of several investigators, the lecithins seem to possess this 
 power. As known from the investigations of Meischer, the develop- 
 ment of the generative organs of the salmon, which are very rich in nuclein 
 substances and phosphatides, from the muscles which are relatively poor 
 in organic-combined phosphorus, seem to indicate a synthesis of phos- 
 phorized organic substance from the phosphates. The investigations 
 of Hart, McCollum and Fuller, 3 who found that pigs with food 
 poor in phosphorus develop just as well with inorganic phosphates as, 
 with organic phosphorus compounds, also indicate such a formation. 
 The recent investigations of McCollum 4 on rats show that these animals 
 can take up the entire need of phosphorus for the skeleton as well as for 
 the reformation of nucleins and phosphatides in the form of inorganic 
 
 1 Skand. Arch. f. Physiol., 14. 
 
 2 The literature on feeding experiments with phosphorized and non-phosphorized 
 food can be found in McCollum, Amer. Journ. of Physiol., 25. 
 
 1 Hart, McCollum and Fuller, Amer. Journ., of Physiol., 23. See also Lipschiitz, 
 Pfliiger's Arch., 143. The literature on the phosphorus metabolism can also be 
 found in Albu and Neuberg, Physiologie und Pathologie des Mineralstoffwechsel,. 
 Berlin, 1906. 
 
 4 Amer. Journ. of Physiol., 25. 
 
LACK OF IRON. 903 
 
 phosphorus. Also the investigations of v. Wendt and Holsti l show 
 that a synthesis of organic phosphorized substances from phosphates is 
 very probable. The feeding experiments of Osborne and collabora- 
 tors, which we will soon discuss, and which extend over a long period 
 where the animals were fed with proteins, fat, carbohydrates and min- 
 eral substances free from phosphorus, give especially strong proof of 
 the ability of the animal to construct phosphatides and nucleins from only 
 inorganic phosphorus. 
 
 Lack of Iron. As iron is an integral constituent of haemoglobin, 
 absolutely necessary for the supply of oxygen, it is an indispensable 
 constituent of food. Iron is a never-failing constituent of the nucleins 
 and nucleoproteins, and herein lies another reason for the necessity 
 of the introduction of iron. Iron is also of great importance in the 
 action of certain enzymes, the oxidases. In iron starvation, iron is 
 continually eliminated, even though in diminished amounts; and with 
 an insufficient supply of iron with the food the formation of haemoglobin 
 decreases. The formation of haemoglobin is not only enhanced by the 
 supply of organic iron, but also, according to the general view, by inor- 
 ganic iron preparations. The various divergent reports of this question 
 have already been given in a previous chapter (on the blood). 
 
 In the absence of protein bodies in the food. the organism must nourish 
 itself by its own protein substances, and with such nutrition it must sooner 
 or later succumb. By the exclusive administration of fat and carbohy- 
 drates the consumption of proteins in these cases is very considerably 
 reduced. For a long time we believed in the view suggested by C. and E. 
 Voit 2 that with a nitrogen- free diet the protein metabolism could never 
 be reduced to as small a value as in starvation, but now, due to the investi- 
 gations Of HlRSCHFELD, IvUMAGAWA, KLEMPERER, SlVEN, LaNDERGREN 
 
 and recently those of Thomas, 3 we learn that the protein metabolism 
 with such a diet can be smaller than in complete starvation. With exclusive 
 feeding of sugar, according to Thomas, the nitrogen elimination can be 
 reduced in a few days to the wear and tear quota, and he has observed 
 an elimination of only 30 milligrams nitrogen per day and per kilo of 
 body weight. 
 
 The absence of fats and carbohydrates in the food affects carnivora and 
 herbivora somewhat differently. It is not known whether carnivora 
 
 1 v. Wendt, Skand. Arch. f. Physiol., 17; Holsti; ibid., 23. See also Gregersen, 
 Zeitschr. f. physiol. Chem., 71. 
 
 2 Zeitschr. f. Biologie, 32. 
 
 ' Hirschfeld, Yirchow's Arch., 114; Kumagawa, ibid., 116; Klemperer, Zeitschr. 
 f. klin. Med., 16; Siven, Skand. Arch. f. Physiol., 10 and 11; Landergren, 1. c, 11; 
 footnote 1, page 894, also Maly's Jahresber., 32; Karl Thomas, Arch. f. (Anat. u.) 
 Physiol., 1909 and 1910, Suppl. Bd. 
 
904 METABOLISM. 
 
 can be kept alive for any length of time by food entirely free from fat 
 and carbohydrates. 1 But it has been positively demonstrated that they 
 can be kept alive a long time by feeding exclusively with meat freed as 
 much as possible from visible fat (Pfluger 2 ) . Human beings and 
 herbivora, on the contrary, cannot live for any length of time on such 
 food. On the one hand they lose the property of digesting and assimilating 
 the necessarily large amounts of meat, and on the other a distaste for 
 large quantities of meat or proteins soon appears. The elimination of 
 acetone bodies with an exclusion of carbohydrates from the food of man 
 is of interest (see Chapter XIV) . 
 
 A question of greater importance is whether it is possible to maintain 
 life in an animal for anj r length of time with a mixture of simple organic 
 and inorganic foodstuffs. The earlier experiments carried out by many 
 investigators to decide this question have not yielded satisfactory results, 
 and Rohmann 3 was first able, by feeding a mixture of several proteins 
 with fat, starch, glucose and salts, to keep mice alive for a long time, and 
 was also able to raise young mice by artificial feeding of the mother and 
 then the small animals. Rohmann concludes from his experiments 
 that for the continuous maintenance or for development of the animal 
 a mixture of different proteins is necessery, but more recently he 4 has 
 found that this can be accomplished by a single protein, and the results 
 of his experiment coincide well in this regard with the investigations of 
 Osborne and Mendel (and E. Ferry 5 ). 
 
 In experiments with white mice these investigators have found that 
 on feeding with a mixture of only one protein with cane-sugar, starch, 
 fat, agar-agar and mineral substances, adult mice could be kept for 169- 
 259 days without changing their body weight. The reason why the 
 adult mice could not be maintained for a still longer time and why young 
 mice did not grow was that certain substances of unknown kind were 
 lacking. Such substances occur in milk, and by adding to the food, milk 
 from which the proteins have been removed, although the food contained 
 only one protein, the animals can be kept alive for a longer time— 500-600 
 days, and the normal growth accomplished as well. These proteins were, 
 especially, casein, lactalbumin, ovalbumin, hemp-seed edestin, wheat 
 glutenin and excelsin, while on the contrary they were not able to pro- 
 
 1 See Horbaczewski, Maly's Jahresber., 31, 715. 
 
 2 Pfliiger's Arch., 50. 
 
 * F. Rohmann, Klin, therap. Wochenschr., No. 40, 1902, and Allg. med. Centralbl. 
 Zeitung, 1908, No. 9. 
 
 * Rohmann, Bioch. Zeitschr., 39. 
 
 * Th. B. Osborne and L. B. Mendell, Science, 34. The Carnegie Institution, Wash- 
 ington, Parts 1 and 2, 1911; with Edna Ferry, Journ. of biol. Chem., 12 and 13, and 
 Zeitschr. f. physiol. Chem., 80. 
 
FEEDING WITH FOOD-STUFFS AND LIPOIDS. S05 
 
 duce a sufficient growth with pea-legumin, zein, gliadin and hordein when 
 added to the other foodstuffs and protein-free milk. These experiments 
 showed that animals fed with gliadin as the only protein had the normal 
 ability to produce offspring and had the ability to produce milk necessary 
 for their food. 
 
 In another series of experiments it was shown that the protein-free 
 milk could be replaced by a proper mixture of salts and that the organic 
 constituents of such milk were not necessary. On feeding with fat, car- 
 bohydrates, casein and such a salt mixture they were able to attain 
 normal growth in a series of experiments of more than 80 or 100 days. 
 Growth was produced in the animals also in the absence of substances 
 soluble in ether (lipoids). This is remarkable, as according to the 
 observations and experiments of Stepp, lipoids are necessary for the 
 normal nutrition. 
 
 According to Stepp l a food which is adequate but not quite genuine 
 for mice can be made genuine by the addition thereto of certain substances 
 soluble in alcohol-ether from milk, egg-yolk, brain, etc. These substances, 
 which are neither fat nor cholesterin, and which he calls lipoids, are partly 
 heat-labile and correspondingly lose their action by continuously boiling 
 with alcohol or by a lengthy boiling of the natural food-stuffs with alcohol 
 or water. A proper food for mice can be so changed by continuous 
 boiling with alcohol so that all animals fed with it die, while the changes 
 in the food brought about in this way can be counteracted by the lipoids 
 obtained under conditions where the lengthy action of heat is prevented. 
 Mice, which die with an otherwise sufficient food but free from lipoids 
 may be kept alive by the addition of the undestroyed lipoids to the 
 same food. 
 
 Recently it has been suggested that beside the foodstuffs in the ordinary 
 sense, other constituents of our food exist which are of the very greatest 
 importance for life. The investigations of Funk as well as those of 
 Suzuki, Shimamura and Odake on the constituents of rice-bran give a 
 specially striking proof of this. According to C. Funk 2 rice-bran contains 
 a substance called vitamine, C17H20X2O7, which belongs to the pyrimidine 
 group and which also occurs in yeast, milk residue and beef-brains. This 
 substance, which is absent in polished rice, causes the disease Beri-Beri 
 in man and polyneuritis in birds. Suzuki, Shimamura and Odake have 
 also isolated from rice-bran a substance which they call oryzanine, which 
 is soluble in alcohol and necessary for animal life. With mixtures of 
 protein, carbohydrates, fat and salts without oryzanine these investiga- 
 tors could not keep hens, pigeons and mice alive and dogs could not be 
 
 1 Bioch. Zeitschr., 22, and Zeitschr. f. Biol., 57 and 59. 
 
 2 C. Funk, Journ. of Physiol., 43 and 45; Suzuki, Shimamura and Odake, Biocb. 
 Zeitschr., 43. 
 
906 METABOLISM. 
 
 kept alive with boiled meat and polished rice. They emaciate quickly 
 and rapidly recover again if they receive oryzanine. 
 
 It follows from the above that there exists a certain unexplainable 
 contradiction between the important observations of Stepp and those 
 of the other investigators on the one side and the very interesting, prolonged 
 experiments of Osborne and Mendel with pure foodstuffs on the 
 other side. 
 
 ffl. METABOLISM WITH VARIOUS FOODS. 
 
 For carnivora, as above stated, meat as poor as possible in fat may 
 be a complete and sufficient food. As the proteins moreover take a special 
 place among the organic nutritive bodies by the quantity of nitrogen they 
 contain, it is proper that we first describe the metabolism with an exclu- 
 sively meat diet. 
 
 Metabolism with food rich in proteins, i.e., feeding only with meat as 
 poor in fat as possible. 
 
 By an increased supply of proteins the catabolism and the elimination 
 of nitrogen is increased, and this in proportion to the supply of proteins. 
 
 If a certain quantity of meat has daily been given to carnivora as 
 food and the quantity is suddenly increased, an augmented catabolism 
 of proteins, or an increase in the quantity of nitrogen eliminated, is the 
 result. If the animal is daily fed for a certain time with larger quantities, 
 of the same meat, a part of the proteins accumulates in the body, but 
 this part decreases from day to day, while there is a corresponding daily 
 increase in the elimination of nitrogen. In this way a nitrogenous 
 equilibrium is established; that is, the total quantity of nitrogen eliminated 
 is equal to the quantity of nitrogen in the absorbed proteins or meat. 
 If, on the 'contrary, an animal in nitrogenous equilibrium, having been 
 fed on large quantities of meat, is suddenly given a small quantity 
 of meat per day, it uses up its own body proteins, the amount de- 
 creasing from day to day. The elimination of nitrogen and the catab- 
 olism of proteins decrease constantly, and the animal may in this case, 
 also pass into nitrogenous equilibrium, or almost into this condition 
 These relations are illustrated by the following table (Voit): 1 
 
 Grams of Meat in the Food per Day. 
 
 1 
 
 Before the Teat. 
 
 500 
 
 During the Test. 
 
 1500 
 
 
 1500 
 
 1000 
 
 
 Grams of Flesh Metabolized in Body per Day. 
 
 
 1 
 
 1222 
 1163 
 
 2 3 4 5 
 1310 1390 1410 1440 
 lOSC. 1088 1080 1027 
 
 G 7 
 1450 1500 
 
 1 Hermann's Handbueh, 6, Part I, 110. 
 
METABOLISM WITH FOOD RICH IN PROTEINS. 907 
 
 In the first case (1) the metabolism of meat before the beginning of 
 the actual experiment on feeding with 500 grams of meat was 447 grams, 
 and it increased considerably on the first day of the experiment, after 
 feeding with 1500 grams of meat. In the second case (2), in which the 
 animal was previously in nitrogenous equilibrium with 1500 grams of 
 meat, the metabolism of flesh on the first day of the experiment, with 
 only 1000 grams meat, decreased considerably, and on the fifth day an 
 almost nitrogenous equilibrium was obtained. During this time the 
 animal gave up daily some of its own proteins. Between that point below 
 which the animal loses from its own weight and the maximum, which 
 seems to be dependent upon the digestive and assimilative capacity of 
 the intestinal canal, a carnivore may be kept in nitrogenous equilibrium 
 with varying quantities of proteins in the food. 
 
 The supply of proteins, as well as the protein condition of the body, 
 affects the extent of the protein metabolism. A body which has become 
 rich in proteins by a previous abundant meat diet must, to prevent a loss 
 of proteins, take up more protein with the food than a body poor in pro- 
 teins. 
 
 In regard to the rapidity with which the protein catabolism takes 
 place Falta x found in man but not, or at least not to the same extent, 
 in dogs, that quite great differences exist between the different proteins. 
 Thus on feeding pure proteins the chief amount of the nitrogen is more 
 quickly eliminated after feeding casein than after genuine ovalbumin. 
 This latter is more easily demolished after a previous modification by 
 coagulation than in the native state, which indicates that an unequal 
 resistance of the different proteins toward the digestive juices plays a 
 part. Hamalainen and Helme 2 have also obtained similar results. 
 Even on feeding with easily decomposable proteins it always takes several 
 days before the total nitrogen corresponding thereto is eliminated, which 
 depends, according to Falta, upon a progressive demolition of the pro- 
 tein. From the unequal rate at which the different proteins are decom- 
 posed it follows that in the passage from a diet poor in protein to one 
 rich in protein the time within which nitrogenous equilibrium occurs 
 depends chiefly upon the kind of protein contained in the food. 
 
 Pettenkofer and Voit have made investigations on the ?netabolism 
 of fat with an exclusively protein diet. These investigations have shown 
 that by increasing the quantity of proteins in the food the daily metab- 
 olism of fat decreases, and they have drawn the conclusion from these 
 experiments, that there may even take place a formation of fat under 
 these circumstances. The objections presented by Pfluger to these 
 
 1 Deutsch. Arch. f. klin. Med., 86. 
 1 Skand. Arch. f. Physiol., 19. 
 
908 METABOLISM. 
 
 experiments, as well as the proofs of the formation of fat from proteins, 
 are also given in Chapter IX. 
 
 According to Pfluger's doctrine, the protein can influence the formation of 
 fat only in an indirect way, namely, in that it is consumed instead of the non- 
 nitrogenous bodies and hence the fat and fat-forming carbohydrates are 
 spared. If sufficient protein is introduced with the food to satisfy the total nu- 
 tritive requirements, then the decomposition of fat stops; and if non-nitrogenous 
 food is taken at the same time, this is not consumed, but is stored up in the animal 
 body, the fats as such, and the carbohydrates at least in great part as fat. 
 
 Pfluger defines the " nutritive requirement " as the smallest quantity of 
 lean meat which produces nitrogenous equilibrium without causing any decom- 
 position of fat or carbohydrates. At rest and at an average temperature it is 
 found in dogs to be 2.073 to 2.099 grams of nitrogen 1 (in meat fed) per kilo of 
 flesh weight (not body weight, as the fat, which often forms a considerable fraction 
 of the weight of the body, cannot as it were be used as dead measure). Even 
 when the supply of protein is in excess of the nutritive requirements, Pfluger 
 found that the protein metabolism increases with an increased supply until 
 the limit of digestive power is reached, which limit is about 2600 grams of meat 
 with a dog weighing 30 kilos. In these experiments of Pfluger's not all of the 
 excess of protein introduced was completely decomposed, but a part was retained 
 by the body. Pfluger therefore defends the proposition " that a supply of 
 proteins only, without fat or carbohydrate does not exclude a protein fattening." 
 
 From what has been said on protein metabolism in starvation and 
 with exclusive protein food, it follows that the protein catabolism in the 
 animal body never stops, that the extent is dependent in the first place 
 upon the extent of protein supply, and that the animal body has the prop- 
 erty, within wide limits, of accommodating the protein catabolism to the 
 protein supply. 
 
 These and certain other peculiarities of protein catabolism have led 
 Voit to the view that not all proteins in the body are decomposed with 
 the same ease. Voit differentiates between the proteins fixed in the 
 tissue-elements, so-called organized proteins, tissue-proteins, from those 
 proteins which circulate with the fluids in the body and its tissues and 
 which are taken up by the living cells of the tissues, from the interstitial 
 fluids washing them, and destroyed. These circulating proteins or supply 
 proteins are, he claims, more easily and quickly destroyed than the tissue- 
 proteins. When, therefore, in a fasting animal which has been previously 
 fed with meat, an abundant and quickly decreasing decomposition of 
 proteins takes place, while in the further course of starvation this protein 
 catabolism becomes less in quantity and more uniform, this depends upon 
 the fact that the supply of circulating proteins is destroyed chiefly in the 
 first days of starvation and the tissue-proteins in the last days. 
 
 The tissue-elements constitute an apparatus of a relatively stable 
 nature, which has the power of taking proteins from the fluids washing 
 the tissues and appropriating them, while their own proteins, the tissue- 
 
 1 See Schondorff, Pfluger's Arch., 71. 
 
METABOLISM WITH FOOD RICH IX PROTEINS. 909 
 
 proteins, are ordinarily catabolized to only a small extent, about 1 per 
 cent daily (Voit). By an increased supply of proteins the activity of 
 the cells and their ability to decompose nutritive proteins is also increased 
 to a certain degree. When nitrogenous equilibrium is obtained after 
 an increased supply of proteins, it indicates that the decomposing power 
 of the cells for proteins has increased so that the same quantity of proteins 
 is metabolized as is supplied to the body. If the protein metabolism is 
 decreased by the simultaneous administration of other non-nitrogenous 
 foods (see below), a part of the circulating proteins may have time to 
 become fixed and organized by the tissues, and in this way the mass of 
 the flesh of the body increases. During starvation or with a lack of pro- 
 teins in the food the reverse takes place, for a part of the tissue protein- 
 is converted into circulating proteins which are metabolized, and in this 
 case the flesh of the body decreases. 
 
 Voit's theory has been criticised by several investigators and espe- 
 cially by Pfluger. Pfluger's belief, based on an investigation made 
 by one of his pupils, Schondorff, 1 is that the extent of protein destruc- 
 tion is not dependent upon the quantity of circulating proteins, but 
 upon the nutritive condition of the cells for the time being — a view 
 which does not widely differ from Voit if the author does not misunder- 
 stand Pfluger. Voit 2 has, as is known, stated that the conditions f < >r 
 the destruction of substances in the body exist in the cells, and also that 
 the circulating protein is first catabolized after having beon taken up 
 by the cells from the fluids washing them. Besides this, certain inves- 
 tigations conclusively show that the extent of protein catabolism is depend- 
 ent upon the concentration of the decomposable proteins at the place 
 where the decomposition is taking place. Thus in confirmation with 
 the earlier investigations of v. Gebhardt and Krummacher, Thomas, 
 v. Hoesslin and Lesser 3 have recently shown that on feeding with 
 a certain quantity of protein, less protein was catabolized when the pro- 
 tein was supplied piecemeal, i.e., in several small portions during the day 
 instead of at one time. That the peculiarity of the nitrogen elimination 
 in starvation and after sufficient protein supply depends essentially 
 upon the concentration of the decomposable proteins (or more correctly the 
 decomposable nitrogenous substances) is no doubt also generally admitted. 4 
 
 1 Pfluger, Pfluger's Arch., 54; Schondorff, ibid., 54. 
 
 2 Zeitschr. f. Biologie, 11. 
 
 3 K. Thomas. Arch. f. (Anat. u.) Physiol., 1909; H. v. Hoesslin and E. J. Lesser, 
 Zci schr. f. physiol. Chem., 73, when also the works of v. Gebhardt and Kummacher are 
 cited. 
 
 4 See also E. Voit and A. Korkunoff, Zeitschr. f. Biol., 32, and O. Frank and R. 
 Trommsdorff, ibid., 43. 
 
910 METABOLISM. 
 
 Recent investigations, especially those of Folin, 1 which show that the 
 amount of certain nitrogenous urinary constituents, such as creatinine, 
 uric acid and the combinations containing neutral sulphur, are almost 
 independent of the quantity of protein taken as food, while the quantity 
 of urea is determined by the protein partaken of, tend to substantiate 
 Voit's view that we must differentiate between the real cell protein 
 and the food protein. This has also led Folin to differentiate between 
 endogenous and exogenous protein metabolism. The chief point in 
 Voit's theory that all the proteins in the body do not behave alike 
 and that the organized proteins which have been fixed in the cells and 
 have been introduced into the cell structure are less readily catabolized 
 than the proteins occurring in the nutritive fluids or temporarily taken 
 up from these, must also be considered as not disputed. Rtjbner 2 
 differentiates also between the deposited protein (growth protein, and 
 deposited by the activity of the cells melioration protein) in the body on 
 the one hand and the protein temporarily incorporated with the body 
 (supply protein and catabolized in passing to a protein-poor diet, transitory 
 protein) on the other hand. 
 
 This question is intimately connected with another, namely, whether the 
 food proteins taken up by the cells are metabolized as such or whether they are 
 first organized, i.e., are converted into specific cell protein. The observations 
 of Panum, Falck, Asher and Haas and others 3 on dogs have shown that the 
 nitrogen elimination increases almost immediately after a meal and in the fifth or 
 sixth hour according to these experimenters, when according to Schmidt-Mulheim * 
 about 59 per cent of the eaten protein is absorbed, do not indicate that a trans- 
 formation of the food protein into organized protein occurs before it is catab- 
 olized. The recent investigations upon the deep cleavage of proteins in digestion 
 and the generally accepted protein syntheses from ammo-acids have made this 
 question lose its special interest. 
 
 On account of the above-stated action of the concentration of the 
 catabolizable nitrogenous material upon the protein decomposition or 
 nitrogen elimination, it is not possible to replace the quantity of protein 
 catabolized in starvation by the exclusive feeding of protein administered 
 at one time and in quantities corresponding to the food proteins. This 
 always requires large amounts of protein. Even on the fractional intro- 
 duction of natural protein v. Hoesslin and Lesser were unable to pro- 
 duce a nitrogen equilibrium with quantities of protein equal to the starva- 
 
 1 Amer. Journ. of Physiol., 13. 
 
 ; Anh. f. (Ariat. u.) Physiol., 1911. 
 
 *Panum, Nord. Med. Arkiv., 6; Falck, see Hermann's Handbuch, 6, Part I, 107; 
 Asher and Hums, Bioch. Zeitsr-hr., 12. For further information in regard to the curve 
 of nitrogen elimination in man, see Tschenloff, Korrespond. Blatt Schweiz. Aerzte, 
 1896; Rosemann, Pflliger's Arch., 65, and Veraguth, Journ. of Physiol., 21; Schlosse, 
 Maly's Jahresber., 31. 
 
 4 Arch. f. (Anat. u.) Physiol., 1879. 
 
NUTRITIVE VALUE OF GELATIN. 911 
 
 tion protein; the elimination of nitrogen was always somewhat increased. 
 
 On the fractional introduction <>f protein, Thomas 1 was nevertheless able 
 in dogs to produce nitrogenous equilibrium without essentially raising 
 the protein metabolism (in comparison with the starvation value). In 
 experiments upon himself he was not able to produce this. 
 
 It has been stated above that other foods may decrease the catab- 
 olism of proteins. Gelatin is such a food. Gelatin and the gelatin-formers 
 do not seem to be converted into protein in the body, and this last cannot 
 be entirely replaced by gelatin in the food. For example, if a dog is fed 
 on gelatin and fat, its body sustains a loss of proteins even when the 
 quantity of gelatin is great enough so that the animal with an amount of 
 fat and meat containing just the same quantity of nitrogen as the gelatin 
 in question, remains in nitrogenous equilibrium. On the other hand, 
 gelatin, as Voit, Panum and Oerum 2 have shown, has great value as 
 a means of sparing the proteins, and it may decrease the catabolism of 
 proteins to a still greater extent than fats and carbohydrates. This is 
 apparent from the following summary of Voit's experiments upon a dog: 
 
 Food per Day. Flesh. 
 
 Meat. Gelatin. Fat. Sugar. Catabolized. On the Body. 
 
 400 200 450 -50 
 
 400 250 439 -39 
 
 400 200 356 +44 
 
 I. Munk 3 has later arrived at similar results by means of more deci- 
 sive experiments, and the recent investigations of Krummacher and 
 Kirchmann 4 show the extent of the sparing action of gelatin upon pro- 
 teins. The extent of protein destruction during gelatin feeding was com- 
 pared with the extent of protein catabolism in starvation, and it was 
 found that 35-37.5 per cent of the quantity of protein decomposed in 
 starvation could be spared by gelatin. The physiological availability 
 of gelatin was found by Krummacher to be equal to 3.88 calories for 1 
 gram, which corresponds to about 72.4 per cent of the energy-content of 
 the gelatin. 
 
 The value of gelatin has been found by Murlin 5 to be dependent to 
 a high degree upon the protein condition of the body, on the calorific 
 value of the food and the quantity of carbohydrates in the latter. If 
 in a man weighing 70 kilos, 51 calories per kilo were partaken, the quan- 
 tity of nitrogen eliminated w T as 10 per cent more than the starvation 
 
 1 v. Hoesslin and Lesser, 1. c; Thomas, Arch. f. (Anat. u.) Physiol., Suppl. Bd.,1910. 
 - Voit, 1. c, 123; Panum and Oerum, Nord. Med. Arkiv., 11. 
 
 3 Pfluger's Arch., 58. 
 
 4 Krummacher, Zeitschr. f. Biologie, 42; Kirchmann, ibid., 40. 
 * Amer. Journ. of Physiol., 19. 
 
912 METABOLISM. 
 
 value, and when two-thirds of the total calories partaken of were sup- 
 plied by carbohydrates, 63 per cent of the total nitrogen could be replaced 
 by gelatin nitrogen. 
 
 The reason why gelatin cannot entirely replace protein has been sought 
 for in the fact that gelatin does not contain all the amino-acids of the 
 proteins (such as tyrosine and tryptophane), or does not contain a suf- 
 ncient amount of the various amino-acids. The correctness of this 
 explanation was first shown by Kaufmann by an experiment on himself, 
 where he showed that gelatin after addition of tyrosine, tryptophane 
 and cystine could be made equivalent to protein. The conclusive proof 
 was given later by Abderhalden 1 when he showed that completely 
 decomposed gelatin on the addition of a mixture of amino-acids, among 
 them also tyrosine and tryptophane, could be made equivalent to proteins. 
 
 As it has been possible to replace the proteins in the food by their 
 cleavage products or mixtures of amino-acids, 2 it is easily understandable 
 that also proteoses or peptones can completely or partly replace the 
 protein. Their ability in this regard is essentially dependent upon their 
 constitution, i.e., their content of the different amino-acids. As the 
 proteoses and peptones are produced by cleavage and as therefore in 
 one proteose we hae certain atomic comp lcxes and in others again 
 these may be absent or only exist to a slight extent, it is conceivable that 
 different investigators 3 have obtained contradictory results because of 
 the use of different proteoses and peptones. 
 
 We have a number of investigations on the action of amides upon 
 metabolism, which are mostly connected by the use of asparagin. These 
 investigations have in part led to conflicting results; but they indicate 
 that carnivora and herbivora act differently, that the results are depen- 
 dent upon the rapidity with which the asparagin is absorbed and also 
 upon the bacterial action in the intestine, and that in herbivora a protein- 
 sparing action can be brought about by asparagin. 4 If, as is generally 
 
 1 Martin Kaufmann, Pfiuger's Arch., 109; Abderhalden, Zeitschr. f. physiol. Chem., 
 77. 
 
 2 See Abderhalden and collaborators, Chapter VIII; also Abderhalden, Zeitschr. 
 f. physiol. Chem., 77, and especially 83. 
 
 3 In regard to the literature on the nutritive value of the proteoses and peptones 
 see Maly, Pfliiger's Arch., 9; P16sz and Gyergyay, ibid., 10; Adamkiewicz, 'Die Natur 
 und der Niihrwerth des Peptones" (Berlin, 1877); Pollitzer, Pfiuger's Arch., 37, 301; 
 Zuntz, ilrid., 37, 313; Munk, Centralbl. f. d. med. Wissensch., 1889, 20, and Deutsch. 
 med. Wochenschr., 1889; Ellinger, Zeitschr. f. Biologie, 33 (literature). Blum, Zeitschr. 
 f. physiol. Chem., 30; Henriques and Hansen, Zeitschr. f. physiol. Chem., 48. 
 
 4 Weiske, Zeitschr. f. Biologie, 15 and 17, and Centralbl. f. d. med. Wissensch., 1890, 
 945; Munk, Virchow's Arch., 94 and 98; Politis, Zeitschr. f. Biologie, 28. See also 
 Mauthner, ibid., 28; Gabriel, ibid., 29; and Voit, ibid., 29, 125; Kellner, Maly's Jahres- 
 
METABOLISM WITH A MIXED DIET. 913 
 
 admitted, the amino-acids can serve in the building up of the proteins, 
 then there is no use denying that their amides can also be used by the 
 animal body. 
 
 Recently Grafe, Abderhalden l and their collaborators have carried 
 on investigations on the value of ammonia and of urea as protein sparers 
 and protein formers. These investigations have shown that ammonia 
 or urea under special conditions of experimentation may cause a nitrogen 
 retention, but we are not justified in believing that a synthesis of protein 
 from ammonia takes place. 
 
 Metabolism on a Diet Consisting of Protein, with Fat or Carbohydrates. 
 As the various foodstuffs can replace each other as sources of energy in 
 the food it follows that the non-nitrogenous foodstuffs can be used 
 instead of the proteins and can reduce the catabolism of these. Thus 
 the fat cannot completely arrest or prevent the catabolism of proteins, 
 but it can decrease it and so spare the proteins. This is apparent from 
 the following table by Voit. 2 A is the average for three days, and B 
 for six days. 
 
 Food. Flesh. 
 
 A 
 
 B 
 
 Meat. 
 
 1500 
 
 1500 
 
 Fat. 
 
 
 150 
 
 Metabolized. 
 
 1512 
 
 1474 
 
 On the Body. 
 
 -12 
 +26 
 
 According to Voit the adipose tissue of the body acts like the food- 
 fat, and the protein-sparing effect of the former may be added to that of 
 the latter, so that a body rich in fat may not only remain in nitrogenous 
 equilibrium, but may even add to the store of body proteins, while in a 
 lean body with the same food containing the same amount of proteins 
 and fat there would be a loss of proteins. In a body rich in fat a greater 
 quantity of proteins is protected from metabolism by a certain quantity 
 of fat than in a lean body. 
 
 Like the fats the carbohydrates have a sparing action on the proteins. 
 By the addition of carbohydrates to the food, carnivora not only remain 
 in nitrogenous equilibrium, but the same quantity of meat which in 
 itself is insufficient and which without carbohydrates would cause a loss 
 
 ber., 27, and Zeitschr. f. Biologie, 39; Pfliiger's Arch., 113; Kellner and Kohler, Chem. 
 Centralbl., 1, 1906; Voltz, Pfliiger's Arch., 107, 117, with Yakuwa, ibid., 121; v. 
 Strusiewicz, Zeitschr. f. Biol., 47; Rosenfeld and Lehmann, Pfliiger's Arch., 112; 
 Lehmann, ibid., 115; M. Miiller, ibid., 117; Henriques and Hansen, Zeitschr. f. physiol. 
 Chem., 54. 
 
 1 Grafe, Zeitschr. f. physiol. Chem., 78, 82, 84, with Schlapfer, ibid., 77, with 
 Turban, ibid., 83; Voltz, ibid., 74; Abderhalden with Hirsch or Laupe, ibid., 80, 
 82-84; Peschek, Bioch. Zeitschr. 45. 
 
 2 Voit, in Hermann's Handb., 6, 130. 
 
914 METABOLISM. 
 
 of weight in the body may with the addition of carbohydrates produce 
 a deposit of proteins. This is apparent from the following table: 1 
 
 Food. Flesh. 
 
 Meat. 
 
 Fat. 
 
 Sugar. 
 
 Starch. 
 
 Metabolized. 
 
 On the Body 
 
 500 
 
 250 
 
 
 
 558 
 
 - 58 
 
 500 
 
 
 300 
 
 
 466 
 
 + 34 
 
 500 
 
 
 200 
 
 
 505 
 
 - 5 
 
 800 
 
 
 
 250 
 
 745 
 
 + 55 
 
 800 
 
 200 
 
 
 
 773 
 
 + 27 
 
 2000 
 
 
 
 200^300 
 
 1792 
 
 +208 
 
 2000 
 
 250 
 
 
 
 1883 
 
 + 117 
 
 The sparing of protein by carbohydrates is greater, as shown by the 
 table, than by fats. According to Voit the first is on an average 9 per 
 cent and the other 7 per cent of the administration protein without a 
 previous addition of non-nitrogenous bodies. Increasing quantities of 
 carbohydrates in the food decrease the protein metabolism more regularly 
 and constantly than increasing quantities of fat. Atwater and Bene- 
 dict 2 also found that the carbohydrates had a somewhat greater sparing 
 action upon proteins than fats. 
 
 Because of this great protein-sparing action of carbohydrates the her- 
 bivora, which as a rule partake of considerable quantities of carbohydrates, 
 assimilate proteins readily (Voit). 
 
 The greater protein-sparing action of carbohydrates as compared with 
 that of the fats occurs, as shown by Landergren, 3 to a still higher degree 
 with food poor in nitrogen or in nitrogen starvation, in which cases the 
 carbohydrates have double the protein-sparing action as compared with 
 an isodynamic quantity of fat. This different behavior of the fats 
 and the carbohydrates is also shown in the experiments of Rubner and 
 Thomas 4 that on the exclusive feeding of sugar the nitrogen elimination 
 is reduced to the wear and tear quota while on the exclusive feeding of 
 fats the nitrogen requirement was about two to three times as great as 
 the wear and tear quota. 
 
 The protein-sparing action of the carbohydrates and fats has generally 
 been studied through the one-sided feeding with one or the other of these 
 two groups of foodstuffs. The question may be raised whether the differ- 
 ence observed between the fats and carbohydrates could not also be 
 brought about by the simultaneous supply of carbohydrates and fat in 
 varying proportions. Tallquist 5 made a series of experiments on this 
 
 1 Voit, in Hermann's Handb., 6, page 143. 
 
 2 See Ergebnisse der Physiologie, 3. 
 
 3 1. c, Inaug.-Diss., and Skand. Arch. f. Physiol., 14. Wimmer, Zeitschr. f. Biol., 
 57, has given further proofs of the strong protein-sparing action of carbohydrates in 
 nitrogen starvation. 
 
 * See Thomas, Arch. f. (Anat. u.) Physiol. Suppl. Bd., 1910. 
 
 6 Finska Lakaresallskapets Handl., 1901. See also Arch. f. Hygiene, 41. 
 
LIMIT OF PROTEIN REQUIREMENT. 915 
 
 subject. In one of the periods 16.27 grams X, 14 grama fat, and 166 
 grains carbohydrate were given; in a second, Hi. (is grams X, 1 10 grama 
 fat, and 250 grams carbohydrate, containing almost the .same number 
 of calorics, namely, 2807 and 2873. In both cases an almoal complete 
 nitrogenous equilibrium was reached and the carbohydrate did not 
 spare more protein than the fat. It is therefore possible that the fat has 
 about the same protein-sparing action as an isodynamic amount of car- 
 bohydrate when the quantity of carbohydrates does not sink below a 
 certain minimum, which is not known for the present. 
 
 This condition as well as the extent of protein-sparing action of the 
 carbohydrates stands, according to Landergren, in close relation to 
 the formation of sugar in the body. The animal bod}- always needs 
 sugar, and a lack of carbohydrates in the food leads to a part of the pro- 
 teins being used in the sugar formation. This part can be spared by 
 carbohydrates but not by fats, from which, according to Landergren, 
 the carbohydrates cannot be formed. In this also lies the probable 
 reason why the fats, on being fed exclusively but not with a sufficient 
 supply of carbohydrates, have a much lower protein-sparing action than 
 the carbohydrates. The fats cannot prevent the protein catabolism 
 necessary for the formation of sugar on a diet lacking in carbohydrates. 
 The law as to the increased protein catabolism with increased pro- 
 tein supply also applies to food consisting of protein with fat and car- 
 bohydrates. In these cases the body tries to adapt its protein catabolism 
 to the supply; and when the daily calorie-supply is completely covered 
 by the food, the organism can, within wide limits, be in nitrogenous 
 equilibrium with different quantities of protein. 
 
 The upper limit to the possible protein catabolism per kilo and per 
 day has been determined only for herbivora. For human beings it is 
 not known, and its determination is from a practical standpoint of second- 
 ary importance. What is more important is to ascertain the lower limit, 
 and on this subject we have several older experiments upon man as well 
 as upon dogs by Hirschfeld, Kumagawa, Klemperer, Munk, Rosen- 
 heim, 1 and others. It follows from these experiments that the lower 
 limit of protein requirement for human beings, for a week or less, is about 
 30-40 grams or 0.4-0.6 gram per kilo with a body of average weight, 
 v. Noorden 2 considers 0.6 gram protein (absorbed protein) per kilo 
 and per day as the lower limit (threshold of protein requirement). The 
 above-mentioned figures are valid only for short series of experiments; 
 still there exists the observation of E. Voit and Constantinidi 3 on 
 
 1 See footnote 3, page 903; also Munk, Arch. f. (Anat. u.) Physiol., 1S91 and 1896 
 Rosenheim, ibid., 1891; Pfluger's Arch., 54. 
 
 2 Grundriss einer Methodik der Stoffwechseluntersuchungen. Berlin, 1892. 
 * Zeitschr. f. Biologie, 2a. 
 
916 METABOLISM. 
 
 the diet of a vegetarian when the protein condition was kept almost 
 but not completely normal for a long time with about 0.6 gram of pro- 
 tein per kilo. Caspari x has also made observations upon a vegetarian 
 for a period of 14 days with an average of 0.1 gram nitrogen (recalculated 
 as equal to 0.62 gram protein) per kilo, where a nearly complete nitrog- 
 enous equilibrium was observed as the average result. 
 
 According to Voit's normal figures, which will be spoken of below, 
 for the nutritive need of man, an average workingman of about 70 kilos 
 weight, requires on a mixed diet about 40 calories per kilo (true calories 
 or net calories). In the above experiments with food very poor in pro- 
 tein the demand for calories was considerably greater; as, for instance, 
 in certain cases it was 51 (Kumagawa) or even 78.5 calories (Klemperer). 
 It therefore seems as if the above very low supply of protein was pos- 
 sible only with great waste of non-nitrogenous food; but in opposition 
 to this it must be recalled that in Voit and Constantinidi's experiments 
 upon the vegetarian, who for years was accustomed to a food poor in 
 protein and rich in carbohydrate, the calories amounted to only 43.7 
 per kilo. In the case studied by Caspari a supply of 41 calories per 
 kilo was entirely sufficient. 
 
 Siven has shown by experiments upon himself that the adult human 
 organism, at least for a short time, can be maintained in nitrogenous 
 equilibrium with a specially low supply of nitrogen without increasing 
 the calories in the food above the normal. With a supply of 41-43 
 calories per kilo he remained in nitrogenous equilibrium for four days 
 with a supply of nitrogen of 0.08 gram per kilo of body weight. Of the 
 nitrogen taken, a part was of a non-protein nature and the quantity of 
 true protein nitrogen was only 0.045 gram, corresponding to about 0.3 
 gram of protein per kilo of body weight. That this low limit, which 
 by the way holds only for a short time, has no general validity follows 
 from other observations. Thus Caspari 2 also, in an experiment on him- 
 self, could not attain complete nitrogenous equilibrium on a much greater 
 nitrogen supply. The protein minimum also seems to vary in different 
 individuals. 
 
 The protein minimum can also be different for other reasons. It 
 varies, as mentioned by Rubner, not only with the kind of foodstuffs, 
 but also with the nutritive condition of the body. The needs of the 
 cells for protein varies with the nutritive condition of the body. Where 
 the protein is eagerly demanded, less supply of protein suffices, and where 
 the demand is low more protein must be offered (Rubner). The more the 
 
 1 Physiologische Studien iiber Vegetarismus, Bonn, 1905. 
 
 2 Sivf'n, Skand. Arch. f. Physiol., 10 and 11; Caspari, Arch. f. (Anat. u.) Physiol., 
 1901. 
 
WEAR AND TEAR QUOTA. 917 
 
 body has become reduced the lower is the protein minimum, according to 
 RUBNBB. 1 
 
 As mentioned in the early part of this chapter, the body always suffers 
 a certain loss of nitrogen through the falling out of the hair and other 
 epidermis formations, by the secretions, etc.; but to this also belongs the 
 constant loss of nitrogenous substance which every cell sustains because 
 of its activity. This unpreventable loss of nitrogen has been included 
 by Rubner under the name " wear and tear " quota, and this quota, 
 which corresponds to the nitrogen elimination with a perfectly nitrogen- 
 free diet, and hence is a protein minimum, may rise to 4 to 6 per cent of 
 the total calorific needs. The energy supply of the food is under these 
 conditions entirely assumed by the non-nitrogenous foodstuffs, and when 
 this quota is replaced by protein the body is in a condition of lowest 
 nitrogenous equilibrium. 
 
 All proteins do not have the same value in replacing the protein 
 minimum. Michaud 2 determined the protein minimum in dogs by 
 feeding entirely with nitrogen-free food, and he found that this min- 
 imum can be covered by the corresponding quantity of protein specific 
 of the animal, but not by the same quantity of an alien protein, like 
 gliadin and edestin. v. Hoesslin and Lesser have found on the con- 
 trary in experiments with dogs that proteins specific to the animal were 
 only unessentially superior to the proteins of horse flesh, and E. Voit 
 and Listerer found for the three kinds of protein, beef-muscle, aleuronat 
 and casein, that the relation was 100 : 106 : 121. Thomas 3 has carried 
 out experiments on man with different foods and has found that the 
 nitrogen of various kinds of proteins has an unequal value in replacing 
 the wear and tear quota. By the expression " biological equivalence " 
 of the nitrogenous foodstuffs he denotes the number of parts of body 
 nitrogen which can be replaced by 100 parts of the food-nitrogen and 
 he found the following equivalence: for beef =104.7, milk = 99.7, casein = 
 70.14, wheat flour = 39.6, potatoes = 78.9, peas = 55.7, and corn = 29.5. 
 Also in consideration of the different content of nitrogenous extractives 
 in the food these figures therefore show that different proteins have essen- 
 tially different values for the replacement of the nitrogen minimum. 
 
 The purposes of the protein as foodstuff are, according to Rubner, as 
 follows: (1) To compensate for the wear and tear quota; (2) betterment 
 of the condition of the cells; and (3) dynamogenic purpose. In the 
 accomplishment of this third purpose the protein splits into a nitrogenous 
 
 1 Rubner, Theorie d. Ernahrung nach Vollendung des Wachstums, Arch. f. Hyg., 
 66, 1-80, and Ernahrungsorgange behn Wachstum des Ivindes, ibid., 66, 81-126. 
 
 2 Zeitschr. f. physiol. Chem., 59. 
 
 1 v. Hoesslin and Lesser, 1. c, E. Voit and Listerer, Zeitschr. f. Biol., 53; Thomas, 
 Arch. f. (Anat. u.) Physiol., 1909. 
 
918 
 
 METABOLISM. 
 
 and a non-nitrogenous part. The potential energy set free immediately 
 as heat in the combustion of the nitrogenous part, which is quantitatively 
 used within the region of the chemical heat regulation but is otherwise 
 lost, has been called the specific dynamic action by Rubner. 1 The 
 remainder of the energy which is represented by the non-nitrogenous 
 part of the proteins, serves, like all other foodstuffs, in satisfying the 
 energy requirement of the cells. According to Rubner only non-nitrog- 
 enous groups (of the proteins, fats and carbohydrates) come almost 
 entirely, if not completely, in consideration for purposes of energy. 
 
 In close relation to the second purpose, the betterment of the condi- 
 tion of the cells, stands the question as to the conditions favoring the deposi- 
 tion of flesh in the body, which is closely associated with the question as 
 to the conditions of fattening the body. In this connection it must be 
 remembered in the first place that all fattening presupposes an overfeed- 
 ing, i.e., a supply of foodstuffs which is greater than that catabolized in 
 the same time. 
 
 In carnivora a flesh deposition may take place on the exclusive feeding 
 with meat. This is not generally large in proportion to the quantity of 
 protein catabolized. In man and herbivora, who cannot cover their 
 calorific needs by protein alone, this is not possible, and the question as 
 to the conditions of fattening with a mixed diet is of importance. 
 
 These conditions have also been studied in carnivora, and here, as 
 Voit has shown, the relation between protein and fat (and carbo- 
 hydrates) is of great importance. If much fat is given in proportion 
 to the protein of the food, as with average quantities of meat with con- 
 siderable addition of fat, then nitrogenous equilibrium is but slowly 
 attained and the daily deposit of flesh, though not large, is quite constant, 
 and may become greater in the course of time. If, on the contrary, 
 much meat besides proportionately little fat is given, then the deposit 
 of protein with increased catabolism is smaller day by day, and nitrog- 
 enous equilibrium is attained in a few days. In spite of the somewhat 
 larger deposit per diem, the total flesh deposit is not considerable in 
 these cases. The following experiment of Voit may serve as example: 
 
 Number of 
 Days of Ex- 
 perimentation. 
 
 Food. 
 
 Total 
 
 Deposit of 
 
 Fleah. 
 
 Daily 
 
 Deposit of 
 
 Flesh. 
 
 
 Meat, Grams. 
 
 Fat, Grams. 
 
 Equilibrium. 
 
 32 
 
 7 
 
 .500 
 
 1800 
 
 250 
 250 
 
 1792 
 
 8.54 
 
 56 
 122 
 
 Not attained 
 Attained 
 
 The greatest absolute deposition of flesh in the body was obtained in 
 these cases with only 500 grams of meat and 250 grams of fat, and even 
 
 1 Rubner, 1. c, and Gesetze des Energieverbrauch.es, 70. 
 
PROTEIN FATTENING. 919 
 
 after 32 days nitrogenous equilibrium had not occurred. On feeding 
 with 1800 grams of meat and 250 grams of fat nitrogen us equilibrium 
 was established ;ifter seven days; and though the deposition of flesh 
 per day was greater, still the absolute deposit was not one-half as great 
 as in the former case. 
 
 The possibility of a protein fattening in man and animals (dogs, sheep) 
 is shown by the series of experiments of Krug, Bornstein, Schreuer, 
 Henneberg, Pfeiffer and Kalb and others 1 and there is no doubt 
 that such a fattening is possible. That we are here not dealing with an 
 increase in the number of cells, but rather an enlargement of the volume 
 of the same is the generally accepted view. Theories as to the value and 
 nature of this protein-fattening are still divergent, as we must differentiate 
 between flesh accumulation or actual organ formation and protein 
 accumulation or deposition of dead protein, and opinions vary in 
 regard to the question how far the one or the other of these occur. 
 By determining the relation between P2O5 and N in muscles, kidneys 
 and liver in dogs and hens in starvation and in fattening, Grund 2 has 
 tested this possibility experimentally. If we are dealing with the deposi- 
 tion of dead protein then the relationship of the P2O5 to the N would 
 change in favor of the nitrogen; Grund found only a very slight change 
 of this kind, which was not conclusive, and according to him the various 
 organs have correspondingly a certain tendency of maintaining the rela- 
 tion between phosphorus and nitrogen unchanged in starvation as well 
 as in fattening. 
 
 It is difficult to produce a permanent flesh deposit in adult man by 
 overfeeding alone. It is to a much greater degree a function of the specific 
 growth energy of the cells and the cell-work than the excess of food. 
 Therefore there is observed, according to v. Noorden, abundant flesh 
 deposition (1) in each growing body; (2) in those no longer growing, but 
 whose body is accustomed to increased work; (3) whenever, by previous 
 insufficient food or by disease, the flesh condition of the body has been 
 diminished and therefore requires abundant food to replace it. The 
 deposition of flesh is in this case an expression of the regenerative energy 
 of the cells. 3 > *< T-/.I> ' 
 
 The experiences of graziers show that in food-animals a flesh deposit 
 does not occur, or at least is only inconsiderable, on overfeeding. The 
 
 1 Krug, Cited by v. Noorden, Lehrb. der Path, des Stoffwechsel, 1. Aufl., p. 120; 
 Bornstein, Berl. klin. Woehenschr., 1898, and Pfluger's Arch., 83 and 106; Bornstein 
 and Schreuer, Pfliiger's Arch., 110; Henneberg and Pfeiffer, see Maly's Jahresb., 
 20; Pfeiffer and Kalb, ibid., 22. 
 
 2 G. Grund, Zeitschr. f. Biol., 54. 
 
 J See also Svenson, Zeitschr. f. klin. Med., 43. 
 
920 METABOLISM. 
 
 individuality and the race of the animal are of importance for flesh deposi- 
 tion. 
 
 The conditions in young, growing individuals differ from those in 
 adults. In the first the protein is necessary for the building up of the 
 growing tissue and in them an abundant true flesh deposition takes place. 
 For this protein fattening the amount of supply does not take first place, 
 but rather the energy of development. 
 
 As above stated (Chapter IX) , in regard to the formation of fat in the 
 animal body, the most essential condition for a fat deposition is an over- 
 feeding with non-nitrogenous foods. The extent of fat deposition is 
 determined by the excess of calories administered over those actually 
 needed. But as protein and fat are expensive nutritive bodies as com- 
 pared with carbohydrates, the supply of greater quantities of carbo- 
 hydrates is important for fat deposition. The body decomposes less 
 substances at rest than during activity. Bodily rest, besides a proper 
 combination of the three chief groups of organic foods, is therefore 
 also an essential requisite for an abundant fat deposit. 
 
 E. Grafe and D. Graham 1 report an experiment on a dog in which they were 
 able to keep the body weight nearly constant for about two months by excessive 
 food with about 210 per cent of the minimum need of calories and with a diet 
 very rich in non-nitrogenous food-stuffs. No fattening occurred in this case; 
 the calories produced were considerably increased and the author considers this 
 case as an accommodation to the food and a luxus-consumption of non-nitrogenous 
 food-stuffs. 
 
 Action of Certain Other Bodies on Metabolism. Water. If a quan- 
 tity in excess of that which is necessary, is introduced into the organism, 
 the excess is quickly and principally eliminated with the urine. This 
 increased elimination of urine causes in fasting animals (Voit, Forster), 
 but not to any appreciable degree in animals taking food (Seegen, Sal- 
 kowski and Munk, Mayer, Dubelir 2 ), an increased elimination of 
 nitrogen. The reason for this increased nitrogen excretion is to be found 
 in the fact that the drinking of much water causes a complete washing 
 out of the urea from the tissues. Another view, which is defended by 
 Voit, is that because of the more active current of fluids, after taking 
 large quantities of water, an increased metabolism of proteins takes place. 
 Voit considers this explanation the correct one, although he does not 
 deny that by the liberal administration of water a more complete washing 
 out of the urea from the tissues takes place. Opinions on this subject 
 are not yet in accord, and recently Heilner has advocated Voit's 
 
 1 Zeitschr. f. physiol. Chem., 73. 
 
 2 Voit, Untersuch. uber den Einfluss des Kochsalzes, etc. (Munchen, 1860); Forster, 
 cited from Voit in Plennann's Handbuch, 6, 153; Seegen, Wien. Sitzungsber., 63; 
 Salkowski and Munk, Virchow's Arch., 71; Mayer, Zeitschr. f. klin. Med., 2; Dubelir, 
 Zeitschr. f. Biologie, 28. 
 
ACTION OF SALTS AND ALCOHOL UPON METABOLISM. 921 
 
 view. The recent investigations of Abderhalden 1 show a washing 
 out of the retained nitrogen by the partaking of water. 
 
 We have the thorough investigations of Hawk 2 and his co-workers 
 on the action of drinking of water upon the digestion and absorption of 
 foods as well as upon the putrefaction processes in the intestine and the 
 elimination of allantoin and purine bodies in the urine. 
 
 When the body has lost a certain amount of water, then the abstinence 
 from water (in animals) is accompanied by a rise in the protein metabo- 
 lism (Landauer, Straib 3 ). In regard to the action of water on the 
 formation of fat and its metabolism, the theory that the free drinking 
 of water is favorable for the deposition of fat seems to be generally 
 admitted, while the drinking of only very little water acts against its 
 formation. For the present we have no conclusive proofs of the correct- 
 ness of this view. 
 
 Salts. In regard to the action of salts — for example sodium chloride 
 and the neutral salts — which partly depends upon the use of large and 
 varying amounts of salt in the experiments, the authors disagree. Inves- 
 tigations of Stratjb and Rost 4 show that the action of salts stands in 
 close relation to their power of abstracting water. Small amounts of 
 salt which do not produce diuresis have no action on metabolism. On 
 the contrary, larger amounts, which bring about a diuresis, which is 
 not compensated by the ingestion of water, produce a rise in the pro- 
 tein metabolism. If the diuresis is compensated by drinking water, 
 then the protein metabolism is not increased by salts, but is diminished 
 to a slight degree. An increased nitrogen excretion caused by taking 
 salts can be increased by the ingestion of water, thus increasing the 
 diuresis, and the action of salts seems to bear a close relation to the 
 demand and supply of water. 
 
 Alcohol. The question as to how far the alcohol absorbed in the 
 intestinal canal is burnt in the bod}-, or whether it leaves the body 
 unchanged by various channels, has been the subject of much discussion. 
 To all appearances the greatest part of the alcohol introduced (95 per 
 cent or more) is burnt in the body (Stubbotin, Thudichum, Bodlander, 
 Benedicenti 5 ). As the alcohol has a high calorific value (1 gram = 7.1 
 
 1 See R. Neumann, Arch. f. Hygiene, 36; Heilner, Zeitschr. f. Biologie, 47 and 49; 
 Hawk, University of Pennsylvania Med. Bull., xviii; Abderhalden, Zeitschr. f. physiol. 
 Chem., 59. 
 
 2 See Journ. of biol. Chem., 10 and 11, Arch, of internat. Med., 1911,Journ. of 
 Amer. Chem. Soc., 33 and 34. 
 
 3 Landauer, Maly's Jahresber., 24; Straub, Zeitschr. f. Biologie, 37. 
 
 4 W. Straub, Zeitschr. f. Biologie, 37 and 38; Rost, Arbeiten aus d. Kaiserliche 
 Gesundheitsamte, 18 (literature). See also Griiber, Maly's Jahresber., 30, 612. 
 
 s Arch. f. (Anat. u.) Physiol., 1896. which contains the literature. 
 
922 METABOLISM. 
 
 calories), then the question arises whether it acts sparingly on other 
 bodies, and whether it is to be considered as a nutritive substance. The 
 earlier investigations made to decide these questions have led to no 
 decisive result. The thorough investigations of Atwater and Benedict, 
 Zuntz and Geppert, Bjerre, Clopatt, Neumann, Offer, Rosemann, 1 
 and others, seem to show positively that, in man, alcohol can diminish the 
 consumption not only of fat and carbohydrates, but also the proteins, 
 although at first, due to its poisonous properties, it may increase the pro- 
 tein metabolism for a short time. The nutritive value of alcohol can 
 be of special importance in certain cases only, as large amounts of alcohol 
 taken at one time, or the continued use of smaller quantities, has an injur- 
 ious action on the organism. Alcohol may therefore be regarded as a 
 foodstuff only in exceptional cases, and in other respects must be con- 
 sidered as an article of luxury. 
 
 Coffee and tea have no action on the exchange of material which can 
 be positively proven, and their importance lies chiefly in their action 
 upon the nervous system. It is impossible to enter into the effect of 
 various therapeutic agents upon metabolism. 
 
 IV. THE DEPENDENCE OF METABOLISM ON OTHER CONDITIONS. 
 
 The so-called basal requirement which was previously mentioned, 
 i.e., the extent of metabolism with absolute rest of body and inactivity 
 of the intestinal tract, serves best as a starting-point for the study of 
 metabolism under various external circumstances. The metabolism 
 going on under these conditions leads in the first place to the production 
 of heat, and it is only to a subordinate degree dependent upon the work 
 of the circulatory and respiratory apparatus and the activity of the glands. 
 According to a calculation by Zuntz, 2 only 10-20 per cent of the total 
 calories of the basal requirement belongs to the circulation and respira- 
 tion work. 
 
 The magnitude of the basal requirement depends in the first place 
 upon the heat production necessary to cover the loss of heat, and this 
 heat production is in turn dependent upon the relation between the weight 
 and the surface of the body. 
 
 Weight of Body and Age. The greater the mass of the body the greater 
 the absolute consumption of material; while, on the contrary, other 
 
 1 In regard to the literature on this subject, see the works of O. Neumann, Arch, 
 f. Hygiene, 36 and 41, and Rosemann, Pfliiger's Arch., 86 and 94. A summary of 
 the entire literature upon alcohol can be found in Abderhalden, " Bibliographie der 
 gesamten wissenschaftlichen Literatur iiber den Alcohol und den Alcoholismus," 
 Berlin and Wien, 1904. See also Rosemann in Oppenheimer's Handb. d. Bioch., Bd. 
 4,1. 
 
 2 Cited from v. Xoorden's Handbuch, 1. Aufl., page 97. 
 
DEPENDENCE OF WEIGHT OF BODY AND AGE. 923 
 
 things being equal, a small individual of the same species of animal metab- 
 olizes absolutely less, but relatively more as compared with the unit of 
 the weight of the body. With increasing bodily weight the total metab- 
 olism per kilo of animal diminishes, which is true first for individuals of 
 the same species of animals, but also seems to have a certain correctness 
 on the comparison of different species of animals. It must be remarked 
 that the relation between flesh and fat in the body exerts an important 
 influence. The extent of the metabolism is dependent upon the quantity 
 of active cells, and a very fat individual therefore decomposes less sub- 
 stance per kilo than a lean person of the same weight. According to 
 Rubner l the importance of the size of the flesh or cell-mass in the body 
 is overestimated. In his investigations on two boys, one of whom was 
 corpulent and the other normally developed, and on comparing the food- 
 need with that found by Camerer for boys of the same weight, Rubner 
 came to theVesult that the exchange of force in the corpulent boy almost 
 completely corresponded with that in the non-corpulent boy of the same 
 weight. By approximately estimating the quantity of fat in the body 
 Rubner was also able, from the protein condition, to compare the cal- 
 culated exchange of energy with that actually found. The exchange 
 per kilo amounted to 52 calories in the lean and 43.6 calories in the fat 
 boy, while, if the protein condition was a measure, one would expect 
 an exchange of calories of only 35 calories for the fat person. We can- 
 not therefore admit of a diminished activity of the cell-mass in the fat boy, 
 but rather an increased activity. According to Rubner it is not the 
 flesh - mass (protein mass) alone, but its variable functional changes, 
 which determine the extent of decomposition. In women, who generally 
 have less body weight and a greater quantity of fat than men, the metab- 
 olism in general is smaller, and the latter is ordinarily about four-fifths 
 that of men. 
 
 The essential reason why small animals catabolize relatively more 
 substance than large ones, when calculated per kilo body weight, is that 
 the bodies of smaller animals have greater surface in proportion to their 
 mass. On this account the loss of heat is greater, which causes increased 
 heat production, i.e., a more active metabolism. This is also the reason 
 why young individuals of the same kind show a relatively greater metab- 
 olism than older ones. If the heat production and carbon-dioxide elim- 
 ination is calculated on the unit of surface of the body, we find, on the 
 contrary, as the experiments of Rubner, Richet, 2 and others show, 
 that they vary only slightly from a certain average in individuals of 
 different weights. 
 
 1 Beitrage zur Ernahrung im Knabenalter, etc. Berlin, 1902. 
 
 * Rubner, Zeitschr. f. Biologie, 19 and 21; Richet, Arch, de Physiol., 5 (2). 
 
924 METABOLISM. 
 
 According to Rubner's rule as to the influence of the surface, which 
 has been recently formulated by E. Voit, the need of energy in homceo- 
 thermic animals is influenced by the development of their surface when 
 their body is given rest, medium surrounding temperature, and relatively 
 equal protein condition. This rule applies not only to adult human beings, 
 but also to children and growing individuals (Rubner, Oppenheimer, 
 Schlossmann and Murschhauser) . The surface is the essential factor 
 in determining the extent of exchange of energy. In order to show 
 this we will give here, from a work of Rubner, 1 the figures representing 
 the quantity of heat in calories for 1 square meter of surface for twenty- 
 four hours: 
 
 Adult, medium diet, rest 1189 calories. 
 
 Adult, medium die x , work 1399 ' ' 
 
 Suckling 1221 " 
 
 Child with medium diet 1447 ' ' 
 
 Aged men and women 1099 ' ' 
 
 Women 1004 ' ' 
 
 The variation in the calorific values 2 found by many investigators, 
 which is sometimes not very small, suggests the fact that the surface 
 rule is not alone decisive for the exchange of material in resting animals. 
 Still it is generally considered that it is of the greatest importance in 
 metabolism. 
 
 The more active metabolism in young individuals is apparent when 
 we measure the gaseous exchange as well as the excretion of nitrogen. 
 As example of the elimination of urea in children the following results of 
 Camerer 3 are of value : 
 
 Age. 
 
 Weight of Body 
 
 Urea in Grams. 
 
 
 in Kilos. 
 
 Per Day. 
 
 Per Kilo. 
 
 1 \ years 
 
 10.80 
 
 12.10 
 
 1.35 
 
 3 " 
 
 13.30 
 
 11.10 
 
 0.90 
 
 5 " 
 
 16.20 
 
 12.37 
 
 0.76 
 
 7 " 
 
 18.80 
 
 14.05 
 
 17.27 
 
 0.75 
 
 9 " 
 
 25.10 
 
 0.69 
 
 12J " 
 
 32.60 
 
 17.79 
 
 0.54 
 
 15 " 
 
 35.70 
 
 17.78 
 
 0.50 
 
 In adults weighing about 70 kilos, from 30 to 35 grams of urea per day 
 are eliminated, or 0.5 gram per kilo. At about fifteen years of age the 
 destruction of proteins per kilo is about the same as in adults. The 
 relatively greater metabolism of proteins in young individuals is explained 
 partly by the fact that the metabolism of material in general is more 
 active in young animals, and partly by the fact that young animals are, 
 as a rule, poorer in fat than those full grown. 
 
 1 Rubner, Ernahrung im Knabenalter, page 45; E. Voit, Zeitschr. f. Biologie, 41; 
 Oppenheimer, ibid., 42; Schlossmann and Murschhauser, Bioch. Zeitschr., 18 and 26. 
 1 See Magnus-Levy, Pfluger's Arch., 55; Slowtzoff (u. Zuntz), ibid., 95. 
 » Zeitschr. f . Biologie, 16 and 20. 
 
SEX. REST AND WORK. 925 
 
 That young individuals show a more active metabolism than adults, 
 follows, as above stated, principally from the relatively greater body 
 surface in the first as compared to the latter. According to Tigerstedt 
 and Sonden, the greater metabolism in young animals depends neverthe- 
 less, also in part, on the fact that in these individuals the decomposition 
 in itself is more active than in older ones. The period of growth has a 
 considerable influence on the extent of metabolism (in man), and indeed 
 the metabolism, even when calculated on the unit of surface of body, is 
 greater in youth than in old age. This view is strongly disputed by 
 Rubner. He does not deny that differences exist between young and 
 adult individuals which may be considered as a deviation from the above 
 rule; still these differences may, he claims, be dependent upon the work 
 performed, the food, and the nutritive condition. Magnus-Levy and 
 Falk l have reported observations which support the conclusions of 
 Sonden and Tigerstedt. 
 
 Nurslings have a behavior different from older children, as with them 
 during the first months of life, and especially the first three days, the 
 metabolism, calculated on the unit of surface, is strikingly low, and 
 lower than with adults. After about two weeks it attains about the same 
 height as adults (Scherer, Forster 2 ). 
 
 In old age the metabolism is very much reduced; and even when calcu- 
 lated upon the square meter of surface of body it is lower than in an 
 individual of medium age. 
 
 The question as to what extent sex specially influences metabolism 
 remains to be investigated. Tigerstedt and Sonden found that in the 
 young the carbon-dioxide elimination, per kilo of body weight, as well as 
 per square meter of body surface, was considerably greater in males 
 than in females of the same age and the same weight of body. This 
 difference between the sexes seems to disappear gradually, and at old 
 age it is entirely absent. The investigations of Magnus-Levy and 
 Falk oppose these observations. They investigated by means of the 
 Zuntz-Geppert method, not only children, but also adults and old 
 persons of both sexes, but could not observe any positive influence of 
 the sex upon metabolism. 3 
 
 1 Tigerstedt and Sonden, Skand. Arch. f. Physiol. 6; Rubner, 1. c; and Arch. f. 
 Hygiene, 66; Magnus-Levy, Arch. f. (Anat. u.) Physiol., 1899, Suppl. 
 
 1 Cited by A. Loewy in Oppenheimer's Handb., Bd. 4, 189. 
 
 * Tigerstedt and Sonden, Skand. Arch. f. Physiol., 6; Magnus-Levy and Falk, 
 Arch. f. (Anat. u.) Physiol., 1899, Suppl. In regard to metabolism and its relation 
 to the phases of sexual life and especially under the influence of menstruation and 
 pregnancy, see the investigations of A. Ver Eecke (Bull. acad. roy. de m£d. de Bel- 
 gique, 1897 and 1901, and Maly's Jahresber., 30 and 31). See also Magnus-Levy 
 in- v. Noorden's Handb. d. Pathol, d. Stoffwechsels. 
 
926 METABOLISM. 
 
 As the metabolism may be kept at its lowest point by absolute rest 
 of body and inactivity of the intestinal tract, it is manifest that work 
 and the ingestion of focd have an important bearing on the extent of 
 metabolism. 
 
 Rest and Work. During work a greater quantity of chemical energy 
 is converted into kinetic energy, i.e., the metabolism is increased more or 
 less on account of work. 
 
 As explained in a previous chapter (X), work, according to the gen- 
 erally accepted view, has no material influence on the excretion of nitro- 
 gen. It is nevertheless true that several investigators have observed,, 
 in certain cases, an increased elimination of nitrogen; this increase does 
 not seem to be directly related to the work, but to be caused by secondary 
 circumstances. These observations have been explained in other ways. 
 For instance, work may, when it is connected with violent movements 
 of the body, easily cause dyspnoea, and this last, as Frankel j has shown, 
 may occasion an increase in the elimination of nitrogen, since diminution 
 of the oxygen supply increases the protein metabolism. In other series 
 of experiments the quantity of carbohydrates and fats in the food was 
 not sufficient; the supply of fat in the body was decreased thereby, and 
 the destruction of proteins was correspondingly increased. Other condi- 
 tions, such as the external temperature and the weather, 2 thirst, and 
 drinking of water, can also influence the excretion of nitrogen. The 
 prevailing sentiment is that muscular activity has hardly any influence 
 on the metabolism of proteins. 
 
 On the contrary, work has a very considerable influence on the elimi- 
 nation of carbon dioxide and the consumption of oxygen. This action, 
 which was first observed by Lavoisier, has later been confirmed by 
 many investigators. Pettenkofer and Voit 3 have made investigations 
 tions on a full-grown man as to the metabolism of the nitrogenous as well 
 as of the non-nitrogenous bodies during rest and work, partly while 
 fasting and partly on a mixed diet. The experiments were made on a 
 full-grown man weighing 70 kilos. The results are contained in the 
 following table: 
 
 Fasting . . 
 Mixed diet . 
 
 1 Virchow's Arch., 67 and 71. In regard to disputed views see C. Voit, Zeitschr. 
 f. Biol., 49, and Frankel, ibid., 50. 
 
 1 See Zuntz and Hchumburg, Aroh. f. (Anat. u.) Physiol., 1895. 
 1 Zeitschr. f . Biologie, 2. 
 
 
 - 
 
 Ci 
 
 jnsumpt 
 
 ion of 
 
 
 
 Proteins. 
 
 
 Fat. 
 
 Carbohydrates. CO2 Eliminated. 
 
 O Consumed. 
 
 Rest . . 
 
 . 79 
 
 
 209 
 
 716 
 
 761 
 
 Work . 
 
 ...75 
 
 
 380 
 
 1187 
 
 1071 
 
 Rest. . 
 
 ...137 
 
 
 72 
 
 352 912 
 
 831 
 
 Work . 
 
 . . . 137 
 
 
 173 
 
 352 1209 
 
 980 
 
WORK AND GAS EXCHANGE. 927 
 
 In these cases work did not seem to have any influence on the destruc- 
 tion of proteins, while the gas exchange was considerably increased. 
 
 Zuntz and his pupils l have made important investigations on the 
 extent of the exchange of gas as a measure of metabolism during work 
 and caused by work. These investigations not only show the impor- 
 tant influence of muscular work on the catabolism of material, but they 
 also indicate, in a very instructive way, the relation between the extent 
 of metabolism of material and its utilization for work of various kinds. 
 We can refer only to those which are of special physiological interest. 
 
 The action of muscular work on the gas exchange does not alone appear 
 with hard work. From the researches of Speck and others we learn that 
 even very small, apparently quite unessential movements may increase 
 the production of carbon dioxide to such an extent that by not observing 
 these, as in numerous older experiments, very considerable errors may 
 creep in. Johansson 2 has also made experiments upon himself, and 
 finds that on the production of as complete a muscular inactivity as 
 possible the ordinary amount of carbon dioxide (31.2 grams per hour 
 at rest in the ordinary sense) may be reduced nearly one-third, or to an 
 average of 22 grams per hour. 
 
 The quantity of carbon dioxide eliminated during a working period 
 is uniformly greater than the quantity of oxygen taken up at the same 
 time, and hence a raising of the respiratory quotient was usually con- 
 sidered as caused by work. This rise does not seem to be based upon the 
 cl.aracter of the chemical processes going on during work, as we have a 
 series of experiments made by Zuntz and his collaborators, Lehmann, Kat- 
 zenstein and Hagemann, 3 in which the respiratory quotient remained 
 almost wholly unchanged in spite of work. According to Loewy 4 the 
 combustion processes in the animal body go on in the same way in work 
 as in rest, and a raising cf the respiratory quotient (irrespective of the 
 transient change in the respiratory mechanism) takes place only with 
 insufficient supply of oxygen to the muscles, as in continuous fatiguing 
 work or excessive muscular activity for a brief period, also with local 
 lack of oxygen caused by excessive work of certain groups of muscles. 
 This varying condition of the respiratory quotient has been explained by 
 
 1 See the works of Zuntz and Lehmann, Maly'a Jahresber., 19; Katzenstein, Pfliiger's 
 Arch., 49; Loewy, ibid.; Zuntz, ibid., 68; Zuntz and Slowtzoff, ibid., 95; and especially 
 the large work " Untersuch. iiber den Stoffwechsel des Pferdes bei Ruhe und Arbeit," 
 Zuntz and Hagemann, Berlin, 1898; Hohenklima und Bergwanderungen by Zuntz, 
 Loewy, Miiller and Caspari, which also contains a bibliography. 
 
 ^ord. Med. Arkiv. Festband, 1897; also Maly's Jahresber., 27; Speck, "Physiol, 
 des menschl. Atmens," Leipzig, 1892. 
 
 3 See footnote 1 . 
 
 4 Pfliiger's Arch., 49. 
 
928 METABOLISM. 
 
 Katzenstein by the statement that during work two kinds of chemical 
 processes act side by side. The one depends upon the work which is 
 connected with the production of carbon dioxide, also in the absence 
 of free oxygen, while the other brings about the regeneration which takes 
 place by the taking up of oxygen. When these two chief kinds of chemical 
 processes make the same progress the respiratory quotient remains 
 unchanged during work; if by hard work the decomposition is increased 
 as compared with the regeneration, then a raising of the respiratory 
 quotient takes place. If, on the contrary, moderate work is continued 
 and performed in a way so that irregularities and occasional changes 
 in the circulation and respiration are excluded or are without importance, 
 then the respiratory quotient may correspondingly remain the same 
 during work as in rest. Its extent is thus determined in the first place 
 by the nutritive material at its disposal (Zuntz and his pupils). 
 
 The theory of Loewy and Zuntz, that the raising of the respiratory quotient 
 during work is to be explained by an insufficient supply of oxygen, is opposed 
 by Laulanie. 1 He has observed the reverse, namely, a diminution in the 
 respiratory quotient during continuous excessive work, and this is not reconcilable 
 with the above statements. He considers that sugar is the source of muscular 
 energy, and that the rise in the respiratory quotient is due to an increased combus- 
 tion of sugar. Its diminution, he explains, is caused by a re-formation of sugar 
 from fat which takes place at the same time and is accompanied by an increased 
 consumption of oxygen. 
 
 In sleep metabolism decreases as compared with that during waking 
 hours, and the most essential reason for this is the muscular inactivity 
 during sleep. The investigations of Rubner upon a dog, and of Johans- 
 son 2 upon human beings, teach us that if the muscular work is elim- 
 inated the metabolism during waking hours is not greater than in sleep. 
 
 The action of light also stands in close connection with the question 
 of the action of muscular work. It seems positively proven that metabo- 
 lism is increased under the influence of light. Most investigators, such as 
 Speck, Loeb, and Ewald, 3 consider that this increase is due to the move- 
 ments caused by the light or an increased muscle tonus, and in man an 
 increase in metabolism under the influence of light with complete rest 
 has not been observed. Divergent results have been obtained in animals, 
 and our knowledge of the truth is not yet complete. 4 
 
 Menial activity docs not seem to have any influence on metabolism 
 according to the means at hand for studying this influence. 
 
 1 Arch, de Physiol. (5), 8, 572. 
 
 1 Rubner, Ludwig-Festechr., 1887; Loewy, Berl. klin. Wochenschr., 1891, 434;, 
 Johansson, Skand. Arch. f. Physiol., 8. 
 
 1 Speck, 1. c; Loeb, Pfluger's Arch., 42; Ewald, Journ. of Physiol., 13. 
 4 See larger handbooks for the literature on this question. 
 
HEAT REGULATION' IN ANIMALS. 929 
 
 The Action of the External Temperature also stands in close relation 
 to muscular work, namely to the question as to whether the chemical 
 heat regulation is independent of the muscular activity. The heat 
 regulation, as is well known, is of two kinds, namely the chemical heat 
 regulation, which consists in a change in the metabolism and which man- 
 ifests itself as an increased heat production due to the increased metabo- 
 lism at low temperatures, and the physical heat regulation, which occurs 
 generally at higher temperatures and is caused by changes in the con- 
 ditions in the heat elimination of the thermal equilibrium. 
 
 In regard to the chemical heat regulation, which will only be discussed 
 here, we must differentiate between cold-blooded and warm-blooded 
 animals. In the first the metabolism rises with an increase in the surround- 
 ing temperature, while in the second group the conditions are different. 
 The experiments of Speck, Loewy and Johansson j on human beings 
 have shown that the lowering of the external temperature is without 
 influence upon the extent of metabolism (measured by the gas exchange) 
 only as long as all natural and non-voluntary movements of the muscles 
 are excluded; otherwise the metabolism is raised. A chemical heat regu- 
 lation, i.e., a rise in metabolism without noticeable movements of the 
 muscles, is not accepted in man, or at least it has not been proven. The 
 heat regulation, in man, at lower temperatures seems to be brought about 
 by the natural or reflex production of muscle action, nor has a chemical 
 heat regulation in the reverse sense, namely, a fall in the catabolism by 
 raising the external temperature, been shown in man. The investigations 
 of Eykman 2 upon inhabitants of the tropics also show the same result, 
 namely, that in human beings no appreciable chemical heat regulation 
 occurs. 
 
 In animals the conditions are different so far as that a chemical heat 
 regulation in the true sense has been positively shown. The investiga- 
 tions of Rubner 3 on various animals have shown that the reduction 
 of the external temperature with these, causes a considerable chemical 
 heat regulation by increasing the metabolism without any chill or shiver 
 movements. On sufficient cooling the temperature of the body may fall 
 irrespective of the increased metabolism, and at a certain limit of body 
 temperature the exchange of material becomes still lower with decreasing 
 temperature. According to Rubner many animals can bear a temperature 
 of 0° C. for days in absolute rest. If the natural muscular activity is 
 eliminated by poisoning with curare or by section of the spinal cord, then, 
 as shown by Pfluger 4 and his pupils, the warm-blooded animal behaves 
 
 1 Speck, 1. c; Loewy, Pfliiger's Arch., 46; Johansson, Skand. Arch. f. Physiol., 7. 
 
 2 Virchow's Arch., 133, and Pfluger's Arch., 64. 
 
 ' Arch. f. Hyg., 37, and Handbuch d. Hyg., Bd. 1, Leipzig, 1911. 
 4 See footnote 2, page 591. 
 
930 METABOLISM. 
 
 like a cold-blooded animal, and the metabolism decreases parallel with 
 the body temperature. In normal animals, on the contrary, the body 
 temperature can be kept constant, on lowering the external temperature, 
 by an increased metabolism; but also in such animals because of a rise 
 in the external temperature a rise in the metabolism above a certain 
 limit can also take place. 
 
 A very interesting and important question is the action of high altitude 
 upon the oxidation processes, the economy of temperature, the protein 
 exchange and the general metabolism. The results of the laborious 
 and important investigations on this subject may, be found in the large 
 work of N. Zuntz, A. Loewy, F. Muller and W. Caspari. 1 
 
 That the ingestion of food raises the metabolism has been known for 
 a rather long time, and this has been studied by Zuntz, v. Mering, Mag- 
 nus-Levy, Voit, Rubner, Johansson and collaborators, also by Heilner 
 and by Gigon. 2 It follows from these investigations that this rise in 
 metabolism, which in man, on sufficient supply of food, amounts to a rise 
 of 10-15 per cent of the basal requirement and with abundant supply of 
 food may be still larger (35 per cent in the researches of Johansson, 
 Tigerstedt and collaborators), has a double cause, namely, partly a 
 digestion work (Zuntz) and partly a chemical decomposition (specific 
 dynamic action of Rubner) which takes place at the same time. 
 
 The sum of all the work which is necessary for the chemical trans- 
 formation of the foods, as well as for the mechanical division and trans- 
 portation of the food in the intestinal canal, is called the digestion work by 
 Zuntz. That such work exists has been shown by Zuntz and v. Mering 
 by comparative tests of the different action upon metabolism by 
 foods introduced per os and intravenously, and recently Cohnheim 3 
 has shown that in sham feeding an increased catabolism of non- 
 nitrogenous body constituents took place. The influence of digestion 
 work in Zuntz's sense is especially apparent in herbivora, in which this 
 work, according to Zuntz and collaborators, may amount to the consump- 
 tion of more than 50 per cent of the total energy content of the raw 
 fodder. 
 
 1 Hohenklima und Bergwanderungen in ihrer Wirkung auf den Menschen, Berlin, 
 1906. 
 
 2 Zuntz and v. Mering, Pfluger's Arch., 15; Zuntz, Naturw. Rundschau, 21 (1906), 
 with Hagemann, 1. c, with Magnus-Levy, Pfluger's Arch., 49; Magnus-Levy, ibid., 
 55, and v. Noorden's Handbuch; Voit, Hermann's Handbuch, 6; Rubner, Zietschr. 
 f. Biol., 19 and 21; and Arch. f. Hyg., 66; Johansson, Skand. Arch. f. Physiol., 21, 
 with Koraen, ibid., 13; Heilner, Zeitschr. f. Biol., 48 and 50; Gigon, Pfluger's Arch., 
 140. 
 
 'Arch. f. Hyg., 57. 
 
SPECIFIC DYNAMIC ACTION. 931 
 
 On partaking of largo amounts of food, especially proteins, by car- 
 nivora, the digestion work in the above sense is not sufficient to account 
 for the increase in metabolism, and in these cases, besides this, we must 
 accept an increase in the chemical transformation process in the animal 
 body brought on by the foodstuffs in an unknown manner (specific 
 dynamic action of foodstuffs, according to Rubner). The only real 
 difference in opinion between the various experimenters consists, so far 
 as Hammarsten can see, in that according to the Zuntz school, normally 
 on supplying sufficient food it is the digestion work in the above sense 
 which chiefly causes the rise in metabolism after taking food, while 
 according to the views of Voit-Rubner, with which Heilner agrees, 
 it is on the contrary the specific dynamic action. 
 
 That the proteins or their cleavage products, without regard to the 
 digestion work, cause a rise in the metabolism seems to be generally 
 accepted. This rise, according to Gigon, is not proportional to the 
 protein supply, as on supplying quantities of protein represented by 
 1:2:4:3 the oxygen absorption was in the proportion 1:3:6:9 and the 
 carbon dioxide elimination was in the proportion 1:4:8:12. On the 
 introduction of glucose Gigon found, as first shown by Johansson, 1 
 that the introduction of carbohydrate caused a proportional rise in the 
 carbon dioxide elimination to a maximal limit of 150 grams. The 
 conditions on supplying fat are harder to judge, but Gigon found no rise 
 in metabolism on introducing oil. 
 
 The rise in the gas exchange occurring after feeding protein and sugar 
 is added, according to Gigon, entirely to the basal metabolism. A 
 substitution in the basal metabolism of the catabolized body constituents 
 by the food taken does not take place according to Gigon and, as example, 
 the protein is not replaced from catabolism by the sugar introduced. 
 The isodynamic law does not apply to the metabolism occurring the 
 first few hours after supplying food, as shown by Johansson and Hell- 
 gren, and Gigon 2 believes that the foodstuffs first pass into the various 
 depots of the body to be later used for purposes of energy. Proteins 
 serve only to a slight degree to replace the catabolized body protein; 
 the remainder is stored up in part as glycogen and in part as fat. The 
 fat is deposited as such and the carbohydrates are deposited as glycogen 
 and fat. 
 
 As the three foodstuffs influence the metabolism in very different ways we 
 can, according to Gigon, speak of a specific action of the foodstuffs. This 
 action, according to him, is more of a material than of a dynamic kind, and the 
 expression, specific dynamic action, may lead to an erroneous conception. 
 
 l Skand. Arch. f. Physiol., 21. 
 
 2 Johansson with Hellgren, Hammarsten's Festschrift, 190C; Gigon, Skand. Arch. f. 
 Physiol., 21. 
 
932 METABOLISM. 
 
 V. THE NECESSITY OF FOOD BY MAN UNDER VARIOUS CONDITIONS. 
 
 Various attempts have been made to determine the daily quantity of 
 organic food needed by man. Certain investigators have calculated 
 from the total consumption of food by a large number of similarly fed 
 individuals — soldiers, sailors, laborers, etc. — the average quantity of 
 foodstuffs required per head. Others have calculated the daily demand 
 for food from the quantity of carbon and nitrogen in the excreta, or cal- 
 culated it from the exchange of force of the persons experimented upon. 
 Others, again, have calculated the quantity of nutritive material in a 
 diet by which an equilibrium was maintained in the individual for one or 
 several days between the consumption and the elimination of carbon 
 and nitrogen. Lastly, still others have quantitatively determined, dur- 
 ing a period of several days, the organic foodstuffs daily consumed by 
 persons of various occupations who chose their own food, by which they 
 were well nourished and rendered fully capable of work. 
 
 Among these methods a few are not quite free from objection, and 
 others have not as yet been tried on a sufficiently large scale. Neverthe- 
 less the experiments collected thus far serve, partly because of their 
 number and partly because the methods correct and control one another, 
 as a good starting-point in determining the diet of various classes and 
 similar questions. 
 
 If the quantity of foodstuffs taken daily be converted into calories 
 produced during physiological combustion, we then obtain some idea of 
 the sum of the chemical energy which under varying conditions is intro- 
 duced into the body. It must not be forgotten that the food is never 
 completely absorbed, and that undigested or unabsorbed residues are 
 always expelled from the body with the feces. The gross results of calo- 
 ries calculated from the food taken must therefore, according to Rubner, 
 be diminished by at least 8 per cent. This figure is true at least when 
 the human being partakes of a mixed diet of about 60 per cent of the 
 proteins as animal, and about 40 per cent of the proteins as vegetable 
 foodstuffs. With more one-sided vegetable food, especially when this 
 is rich in undigestible cellulose, a much larger quantity must be sub- 
 tracted. 
 
 The following summary contains a few examples of the quantity of 
 food which is consumed by individuals of various classes of people under 
 different conditions. In the last column we also find the quantity of 
 living force which corresponds to the quantity of food in question, calcu- 
 lated as calories, with the above-stated correction. The calories are 
 therefore net results, while the figures for the nutritive bodies are gross 
 results. 
 
FOOD REQUIREMENT IN MAN. 933 
 
 Proteins. Fat. ^J^tea" Calories. Authority. 
 
 Soldier during peace 119 40 529 2784 Playfair.'- 
 
 Soldier light service 117 35 447 2424 Hildesheim. 
 
 Soldier in field 146 40 504 2852 Hildesheim. 
 
 Laborer 130 40 550 2903 Moleschott. 
 
 Laborer at rest 137 72 352 2458 Pettenkofer and Voit. 
 
 Cabinetmaker (40 years). 131 68 494 2835 Forster.-" 
 
 Young physician 127 89 302 2002 Forster. 
 
 Young phvsi.ian 134 102 292 2476 Forster. 
 
 Laborer (36 years) 133 95 422 2902 Forster. 
 
 English smith 176 71 666 3780 Playfair. 
 
 English pugilist 288 88 93 2189 Playfair. 
 
 Bavarian wood-chopper. . 135 208 876 5589 Liebig. 
 
 Laborer in Silesia 80 16 552 2518 Meinert. 3 
 
 Seamstress in London ... 54 29 292 1688 Playfair. 
 
 Swedish laborer 134 79 485 3019 Hultgren and Landergren. 4 
 
 Japanese student 83 14 622 2779 Eijkma.v 5 
 
 Japanese shopman 55 6 394 1744 Tawara. 5 
 
 We have a very large number of complete investigations upon the 
 diet of people of different vocations in America, but they are too exten- 
 sive to enter into, hence we must refer to the original publications of 
 Atwater. 6 
 
 It is evident that persons of essentially different weight of body 
 who live under unequal external conditions must need essentially dif- 
 ferent food. It is also to be expected (and this is confirmed by the table) 
 that not only the absolute quantity of food consumed by various persons, 
 but also the relative proportion of the various organic nutritive substances, 
 shows considerable variation. Results for the daily need of human 
 beings in general cannot be given. For certain classes, such as soldiers, 
 laborers, etc., results may be given which are valuable for the calculation 
 of the daily rations. 
 
 Based on extensive investigations and a very wide experience, Voit 
 has proposed the following average quantities for the daily diet of adults: 
 
 Proteins. Fat. Carbohydrates. Calories. 
 
 For men 118 grams 56 grams 500 grams 2810 
 
 But it should be remarked that these data relate to a man weighing 
 70 to 75 kilos and who was engaged daily for ten hours in not too fatiguing 
 labor. 
 
 The quantity of food required by a woman engaged in moderate work 
 
 1 In regard to the earlier researches cited in this table, we refer the reader to Voit, 
 in Hermann's Handbuch, 6, 519. 
 
 2 Ibid., and Zeitschr. f. Biologie, 9. 
 
 3 Armee- und Volksernahrung, Berlin, 1880. 
 
 4 Untersuching uber die Ernahrung schwedischer Arbeiter bei frei gewahlter Kost, 
 Stockholm, 1891. Maly's Jahresber., 21. 
 
 5 Cited from Kellner and Mori in Zeitschr. f . Biologie, 25. 
 
 6 Report of the Storrs Agric. Expt. Station, Conn., 1891-1895, and 1896, and U. S. 
 Depart, of Agriculture, Bull. 53, 1898. 
 
934 METABOLISM. 
 
 is about four-fifths that of a laboring man, and we may consider the 
 following as a daily diet with moderate work : 
 
 Proteins. Fat. Carbohydrates. Calories. 
 
 For women 94 grams 45 grams 400 grams 2240 
 
 The proportion of fat to carbohydrates is here as 1:8-9. Such a 
 proportion often occurs in the food of the poorer classes who chiefly live 
 upon the cheap and voluminous vegetable food, while this ratio in the 
 food of wealthier persons is 1 :3-4. It would be desirable if in the above 
 rations the fat were increased at the expense of the carbohydrates, but 
 unfortunately on account of the high price of fat such a modification 
 cannot always be made. 
 
 In examining the above figures for the daily rations it must not 
 be forgotten that those for the various foodstuffs are gross results. 
 They consequently represent the quantity of those which must be taken 
 in, and not those which are really absorbed. The figures for the calories 
 are, on the contrary, net results. 
 
 The various foods are, as is well known, not equally digested and 
 absorbed, and in general the vegetable foods are less completely consumed 
 than animal foods. This is especially true of the proteins. When, 
 therefore, Voit, as above stated, calculates the daily quantity of pro- 
 teins needed by a laborer as 118 grams, he starts with the supposition 
 that the diet is a mixed animal and vegetable one, and also that of the 
 above 118 grams about 105 grams are absorbed. The results obtained 
 by Pfluger and his pupils Bohland and Bleibtreu x on the extent of 
 the metabolism of proteins in man with an optional and sufficient diet 
 correspond well with the above figures, when the unequal weight of body 
 of the various persons experimented upon is sufficiently considered. 
 
 As a rule, the more exclusively a vegetable food is employed, the 
 smaller is the quantity of proteins in it. The strictly vegetable diet 
 of certain people, as that of the Japanese and of the so-called vegeta- 
 rians, is therefore a proof that, if the quantity of food be sufficient, a 
 person may exist on considerably smaller quantities of proteins than 
 Voit suggests. It follows from the investigations of Hirschfeld, Kuma- 
 gawa and Klemperer, Siven, and others (see pages 903, 915) that an 
 almost complete or indeed a complete nitrogenous equilibrium may be 
 attained by the sufficient administration of non-nitrogeneous nutritive 
 bodies with relatively very small quantities of proteins. 
 
 If we bear in mind that the food of people of different countries 
 varies greatly, and that the individual also takes essentially different 
 nourishment according to the external conditions of living and the influence 
 of climate, it is not remarkable that a person accustomed to a mixed 
 
 1 Bohland, Pfluger's Arch., 36; Bleibtreu, ibid., 38. 
 
PROTEIN REQUIREMENT. 935 
 
 diet can exist for some time on a strictly vegetable diet deficient in pro- 
 teins. No one doubts the ability of man to adapt himself to a heteroge- 
 neously composed diet when this is not too difficult of digestion and is 
 sufficient in quantity; nor can we deny that it is possible for a man 
 to exist for a long time with smaller amounts of protein than Voir 
 suggests, namely 118 grams. Thus 0. Neumann ' experimented on him- 
 self during 746 days in three series of experiments, and his diet consisted 
 of 74.2 grams protein, 117 grams fat, and 213 grams carbohydrates = 23G7 
 gross calories, with a weight of 70 kilos and with ordinary laboratory 
 work. These figures cannot be compared with those obtained by Voit's 
 worker, weighing 70 kilos, whose work was harder than a tailor's and 
 easier than a blacksmith's; for example, the work of a mason, carpenter, 
 or cabinet-maker. The very extensive investigations recently performed 
 by Chittenden 2 on the determination of the extent of protein necessary 
 are of great interest. These investigations, upon a total of twenty- 
 six persons, extended over a period of five to twenty months, and con- 
 sisted of careful observations upon the manner of living, food taken, 
 nitrogen elimination, and the ability of performing work. The individuals 
 were divided into three groups. The first consisted of five professional 
 men (four assistants and one professor). The second group was composed 
 of thirteen soldiers (of the sanitary corps of the United States army) who 
 besides their daily work were given gymnastic exercises for six months. 
 The third group consisted of eight athletic students who were trained in 
 different kinds of sport. 
 
 In all the persons experimented upon the original nitrogen content 
 of the food, which corresponded to Voit's value or were somewhat higher, 
 was gradually reduced more or less. The total calories supplied were 
 not increased above the original value, but rather diminished to a reason- 
 able extent. The bodily as well as the mental ability was repeatedly 
 tested. As it is not possible to enter into the details of the investiga- 
 tion the following will be sufficient to show the results. With a diet 
 corresponding to Voit's values the amount of urine nitrogen per day is 
 16 grams, corresponding to a total protein catabolism in the body of 100 
 grams, or 1.43 grams per kilo. The corresponding results for the above 
 three groups may be found in the following table, where for comparison 
 Hammarsten also includes the figures for Voit's diet: 
 
 Urine Nitrogen. Catabolized Protein. Protein per Kilo. 
 
 Mm. Max. Min. Max. Min. Max. 
 
 Group 1 5.69 8.99 35.6 56.19 0.61 0.86 
 
 Group 2 7.03 8.39 43.9 52.44 0.74 0.87 
 
 Group 3 7.47 11.06 46.7 69.10 0.75 0.92 
 
 Voit's figures 16 100 1.43 
 
 1 Arch. f. Hygiene, 45. 
 
 * R. H. Chittenden, Physiological Economy in Nutrition, New York, 1904. 
 
936 METABOLISM. 
 
 The chief results from these investigations are that on partaking of 
 amounts of protein much smaller than Voit's figures, without changing 
 the original supply of calories and indeed diminishing the same, the 
 persons experimented upon remained not only in nitrogenous equilibrium, 
 but in perfect health, and were not only able to perform ordinary work, 
 but were indeed regularly able to perform much greater work. 
 
 From these investigations, which extended over a long period and 
 were carried on with special care in exactitude, it cannot be denied that 
 man can for a long time exist with much smaller quantities of protein 
 than Voit's figures call for, which is also derived from the experience of 
 vegetarians, and from people living almost entirely upon vegetable food. 
 On the other hand it must not be forgotten that Voit's figures represent 
 average results not theoretically necessary, but which have been shown 
 to be the actual diet developed from habit, custom, conditions of life and 
 climate, with sufficient nourishment and free selection for centuries in 
 Middle and North Europe. A rational change in this food requirement 
 based upon scientific facts is just as difficult to determine as it is to carry 
 out practically. Certain standard figures for the general needs of nutri- 
 tion cannot be established because the conditions in various countries 
 are different and must necessarily be so. The numerous compilations 
 (of Atwater and others l ) on the diet of different families in America 
 have given the figures 97-113 grams protein for a man, and the very 
 careful investigations of Hultgren and Landergren have also shown 
 that the laborer in Sweden with moderate work and an average body 
 weight of 70.3 kilos, with optional diet, partakes 134 grams protein, 79 
 grams fat, and 522 grams carbohydrates. The quantity of protein 
 is here greater than is necessary, according to Voit. On the other hand 
 Lapicque 2 found 67 grams protein for Abyssinians and 81 grams for 
 Malaysians (per body weight of 70 kilos), materially lower figures. 
 
 If we compare the figures on page 933 with the average figures pro- 
 posed by Voit for the daily diet of a laborer, it would seem at the first 
 glance as if the food consumed in certain cases was considerably in excess 
 of the need, while in other cases, as, for instance, that of a seamstress 
 in London, it was entirely insufficient. A positive conclusion cannot, 
 therefore, be drawn if we do not know the weight of the body, as well 
 as the labor performed by the person, and also the conditions of living. 
 
 1 Atwater, Report of the Storrs Agric. Expt. Station, Conn., 1891-1895 and 1896; 
 also Nutrition investigations at the University of Tennessee, 1896 and 1897; U.S 
 Dept. of Agriculture, Bull. 53, 1898. See also Atwater and Bryant, ibid., Bull. 75. 
 Jaffa, ibid., 84; Grindley, Sammis, and others, ibid., 91. 
 
 2 Hultgren and Landergren, 1. c; Lapicque, Arch, de Physiol. (5), 6. 
 
WORK AND FOOD REQUIREMENT. 937 
 
 It is certainly true that the amount of nutriment required by the body 
 is not directly proportional to the body weight, for a small body consume! 
 relatively more substance than a larger one, and varying quantities of 
 fat may also cause a difference; but a large body, which must maintain 
 a greater quantity, consumes an absolutely greater amount of substance 
 than a small one, and in estimating the nutritive need one must also 
 always consider the weight of the body. According to Voit, the diet 
 for a laborer with 70 kilos body weight requires 40 calories for each kilo. 
 Ekholm 1 calculates, basing it upon his experiments, that for a man 
 weighing 70 kilos, busied with reading and writing, the net calories are 
 24 .",0 and the gross calories 2700, or 35 and 38.6 calories per kilo. In the 
 ordinary sense for a resting man, the general food requirement is calculated 
 in round numbers as 30 calories for every kilo. The minimum figure 
 for metabolism during sleep and in as complete rest as possible has been 
 found by Sonden, Tigerstedt and Johansson 2 to be 24-25 calories. 
 
 As several times stated above, the demands of the body for nourish- 
 ment vary with different conditions of the body. Among these condi- 
 tions two are especially important, namely, work and rest. 
 
 In a previous chapter, in which muscular labor was spoken of, it was 
 seen that all foodstuffs have almost the same power of serving as a source 
 of muscular work, and that the muscles, it seems, select that foodstuff 
 which is supplied to them in the greatest quantity. As a natural sequence 
 it is to be expected that muscular activity requires indeed an increased 
 supply of foodstuffs, but no essential change in their relation as compared 
 to rest. 
 
 Still this does not seem to hold true in daily experience. It is a well- 
 known fact that hard-working individuals — men and animals — require 
 a greater quantity of proteins in the food than less active ones. This 
 contradiction, is however, only apparent, and it depends, as Voit has 
 shown, upon the fact that individuals used to violent work are more 
 muscular. For this reason a person performing severe muscular labor 
 requires food containing a larger proportion of proteins than an individual 
 whose occupation demands less violent exertion. Another fact is that 
 the diet rich in proteins is often concentrated and less bulky, and also 
 that in many cases of training, a diet yielding as little fat as possible is 
 selected. 
 
 If we compare the results for the needs of food in work and rest which 
 are obtained under conditions which can be readily controlled, it is found 
 that the above statements are in general confirmed. As example of this 
 
 1 Skand. Arch, f . Physiol., 11. 
 
 * Sonden and Tigerstedt, Skand. Arch. f. Physiol., 6; Johansson, ibid., 7; Tigerstedt, 
 Nord. Med. Arkiv. Festband., 1897. 
 
938 METABOLISM. 
 
 the following tables give the rations of soldiers in peace and in the field 
 and the average figures from the detailed data of various countries : 1 
 
 A. Peace Ration. B. War Ration. 
 
 Fat. 
 
 Carbohydrates. 
 
 Calories. 
 
 40 
 
 551 
 
 2900 
 
 59 
 
 557 
 
 3250 
 
 Fat. 
 
 Carbohydrates. 
 
 Calories. 
 
 80 
 
 500 
 
 3013 
 
 100 
 
 500 
 
 3218 
 
 Proteins. Fat. Carbohydrates. Proteins. Fat. Carbohydrates. 
 
 Minimum. . . 108 22 504 126 38 484 
 
 Maximum. . . 165 97 731 197 95 688 
 
 Mean 130 40 551 146 59 557 
 
 The following figures for the daily ration are obtained from the above 
 averages : 
 
 Proteins. 
 
 In peace 130 
 
 In war 146 
 
 If we calculate the fat in its equivalent quantity of starch, then the 
 relation of the proteins to the non-nitrogenous foods is : 
 
 In peace 1 : 4 . 97 
 
 In war 1 : 4 . 79 
 
 The relation in both cases is nearly the same. Similar results are 
 obtained when we start with Voit's figures for a soldier in manceuver A 
 (hard work) and B (strenuous work) in war. 
 
 Proteins. 
 
 A 135 
 
 B 145 
 
 The relation here, when the fat is recalculated as starch, in both cases is 
 the same, or equal to 1 : 5. 
 
 If we calculate that portion of the total calories supplied which falls 
 to each group of the foodstuffs, it is found that 16-19 per cent comes 
 from the protein in rest as well as with medium and strenuous work. 
 For the fat and the carbohydrates the variations are greater; the chief 
 quantity of calories comes from the carbohydrates. Of the total calories 
 16-30 per cent comes from the fat and 50-60 per cent from the carbo- 
 hydrates. 
 
 The importance of the food-demand for working individuals is shown 
 by the figures given on page 933 for a wood-chopper in Bavaria. A 
 need of more than 4000 calories occurs but seldom, and with very hard 
 work the demand may rise even to 7000 calories (Atwater and Bryant, 
 Jaffa 2 ). 
 
 1 Germany, Austria, Switzerland, France, Italy, Russia, and the United States. 
 It is not known by the author whether these figures have been changed in the last 
 few years in the various countries, and hence whether they must be modified or not. 
 
 * flee footnote 1, page 936. 
 
WORK AND FOOD REQUIREMENT. 939 
 
 As more work requires an increase in the absolute quantity of food, 
 so the quantity of food must he diminished when little work is performed. 
 The question as to how far this can be done is of importance in regard 
 to the diet in prisons and poorhouses. We give below the following as 
 example of such diets: 
 
 Proteins. Fat. Carbohydrates. Calories. 
 
 Prisoner (not working).. 87 22 305 1667 Schuster. 1 
 
 Prisoner (not working).. 85 30 300 1709 Voit. 
 
 Man in poorhouse 92 45 332 1985 Forster. 2 
 
 Woman in poorhouse. . . 80 49 266 1724 Forster. 
 
 The figures given by Voit are, he says, the lowest reported for a non- 
 working prionser. He considers the following as the lowest diet for old 
 non-working people: 
 
 Proteins. Fat. Carbohydrates. Calories. 
 
 Men 90 40 350 2200 
 
 Women 80 35 300 1723 
 
 In calculating the daily diet it is in most cases sufficient to ascertain 
 how much of the various foodstuffs must be administered to the body 
 in order to keep it in the proper condition to perform the work required 
 of it. In other cases it may be a question of improving the nutritive 
 condition of the body by properly selected food; and there are also cases 
 in which it is desired to diminish the mass or weight of the body by an 
 insufficient nutrition. This is especially the case in obesity, and all the 
 dietaries proposed for this purpose are chiefly starvation cures, which 
 is readily apparent if we study such dietaries. 
 
 1 See Voit, Unters. der Kost, Munchen, 1877, page 142. See also Hirschfeld, 
 Maly's Jahresber., 30. 
 * Ibid., page 186. 
 
940 
 
 FOOD TABLES. 
 
 TABLE I.— FOODS. 1 
 
 1. Animal Foodstuffs. 
 
 1000 Parts contain 
 
 a o 
 
 0) > 
 
 m -^ 
 
 IS 
 
 O V 
 
 Relation of 
 1:2:3. 
 
 a. Meat without Bones. 
 
 Fat beef 2 
 
 Beef (average fat J ) 
 
 Beef 2 
 
 Corned beef (average fat) 
 
 Veal 
 
 Horse, salted and smoked 
 
 Smoked ham 
 
 Pork, salted and smoked 3 
 
 Meat from hare 
 
 " " chicken 
 
 " " partridge 
 
 " " wild duck 
 
 b. Meat with Bones. 
 
 Fat beef 2 
 
 Beef (average fat 1 ) 
 
 Beef, slightly corned 
 
 Beef, thoroughly corned 
 
 Mutton, very fat 
 
 ' ' average fat 
 
 Pork, fresh, fat 
 
 ' ' corned, fat 
 
 Smoked ham 
 
 c. Fishes. 
 
 River eel, fresh, entire 
 
 Salmon, " 
 
 Anchovy, 
 
 Flounder, " " 
 
 River perch, fresh, entire 
 
 Torsk " " 
 
 Pike, " " 
 
 Herring, salted, entire 
 
 Anchovy, " " 
 
 Salmon (side), salted 
 
 Kabeljau (salted haddock) 
 
 Codfish (dried ling) 
 
 " (dried torsk) 
 
 Fish-rneal from variety of Gadtjs 
 
 183 
 
 166 
 
 196 
 
 98 
 
 190 
 
 120 
 
 218 
 
 115 
 
 190 
 
 80 
 
 318 
 
 65 
 
 255 
 
 365 
 
 100 
 
 660 
 
 233 
 
 11 
 
 195 
 
 93 
 
 253 
 
 14 
 
 246 
 
 31 
 
 156 
 
 141 
 
 167 
 
 83 
 
 175 
 
 93 
 
 190 
 
 100 
 
 135 
 
 332 
 
 160 
 
 160 
 
 100 
 
 460 
 
 120 
 
 540 
 
 200 
 
 300 
 
 89 
 
 220 
 
 121 
 
 67 
 
 128 
 
 39 
 
 145 
 
 14 
 
 100 
 
 2 
 
 86 
 
 1 
 
 82 
 
 1 
 
 140 
 
 140 
 
 116 
 
 43 
 
 200 
 
 108 
 
 246 
 
 4 
 
 532 
 
 5 
 
 665 
 
 10 
 
 736 
 
 7 
 
 11 
 
 18 
 
 18 
 
 117 
 
 13 
 
 125 
 
 100 
 
 40 
 
 12 
 
 11 
 
 14 
 
 12 
 
 9 
 
 15 
 
 85 
 
 100 
 
 8 
 10 
 
 5 
 60 
 70 
 
 6 
 10 
 11 
 
 11 
 
 6 
 100 
 107 
 132 
 178 
 106 
 59 
 87 
 
 640 
 688 
 672 
 550 
 717 
 492 
 280 
 130 
 744 
 701 
 719 
 711 
 
 544 
 585 
 480 
 430 
 437 
 520 
 365 
 200 
 340 
 
 352 
 469 
 489 
 580 
 440 
 455 
 461 
 280 
 334 
 460 
 472 
 257 
 116 
 170 
 
 150 
 
 150 
 
 167 
 
 180 
 
 88 
 
 150 
 
 70 
 
 80 
 
 90 
 
 333 
 333 
 333 
 250 
 450 
 450 
 450 
 340 
 400 
 100 
 100 
 100 
 150 
 
 100 
 100 
 100 
 100 
 100 
 100 
 100 
 100 
 100 
 100 
 100 
 100 
 
 100 
 100 
 100 
 100 
 100 
 100 
 100 
 100 
 100 
 
 100 
 100 
 100 
 100 
 100 
 100 
 100 
 100 
 100 
 100 
 100 
 100 
 100 
 100 
 
 90 
 
 50 
 
 63 
 
 53 
 
 42 
 
 20 
 
 143 
 
 660 
 
 5 
 
 48 
 
 6 
 
 13 
 
 90 
 
 49 
 
 53 
 
 53 
 
 246 
 
 100 
 
 460 
 
 450 
 
 150 
 
 246 
 
 56 
 
 31 
 
 9 
 
 2 
 
 1 
 
 1 
 
 100 
 
 37 
 
 54 
 
 1 
 
 1 
 
 1 
 
 1 
 
 ' The re'sults in the following tables are chiefly compiled from the summary of Alm^n and of 
 KoNi'i We here designate as " waste " that part of the foods which is lost in the preparation 
 or that which is not used by the body; for instance, bones, skin, egg-shells, and the cellulose 
 vegetable foods. . 
 
 » Meat such as is ordinarily sold in the markets in bweden. 
 
 • Pork, chiefly from the breast and belly, such as occurs in the rations of Swedish soldiers. 
 
ANIMAL FOODS. 
 TABLE I.— FOODS— (Continued). 
 
 941 
 
 1. Animal Foodstuffs. 
 
 1000 Parts contain 
 
 Relation of 
 11:2: 3. 
 
 1 
 
 
 100 
 
 89 
 
 10!) 
 
 2S 
 
 100 
 
 50 
 
 100 
 
 65 
 
 100 
 
 17 
 
 100 
 
 113 
 
 1U 
 
 1 
 
 d. Inner Organs (Fresh). 
 
 Brain 
 
 Beef-liver 
 
 Beef-heart 
 
 Heart and lungs of mutton 
 
 Veal-kidney 
 
 Ox tongue (fresh) 
 
 Blood from various animals 
 
 (average results) 
 
 e. Other Animal Foods. 
 Variety of pork-sausage (Mett- 
 
 wurst) 
 
 Same for frying 
 
 Butter 
 
 Lard 
 
 Meat extract 
 
 Cow's milk (full) 
 
 " " (skimmed) 
 
 Buttermilk 
 
 Cream 
 
 Cheese (fat) 
 
 " (poor) 
 
 Whey cheese (poor) 
 
 Hen's egg, entire 
 
 " " without shell 
 
 Yolk of egg 
 
 White of egg 
 
 2. Vegetable Foodstuffs. 
 
 Wheat (grains) 
 
 Wheat-flour (fine) 
 
 ' ' (very fine) , 
 
 Wheat-bran 
 
 Wheat-bread (fresh) 
 
 Macaroni 
 
 Rye (grains) , 
 
 Rye-flour 
 
 Rye-bread (dry) 
 
 (fresh, coarse).. . . 
 
 (fresh, fine) 
 
 Barley (grains) 
 
 Scotch barley 
 
 Oat (grains) 
 
 ' ' (peeled) 
 
 Corn 
 
 Rice (peeled for boiling) 
 
 French beans 
 
 Peas (yellow or green, dry) . . 
 Flour from peas 
 
 116 
 196 
 184 
 163 
 221 
 150 
 
 182 
 
 190 
 
 220 
 
 7 
 
 3 
 
 304 
 
 35 
 
 35 
 
 41 
 
 37 
 
 230 
 
 334 
 
 89 
 
 106 
 
 122 
 
 160 
 
 103 
 
 123 
 
 110 
 
 92 
 
 150 
 
 88 
 
 90 
 
 115 
 
 115 
 
 114 
 
 77 
 
 80 
 
 111 
 
 110 
 
 117 
 
 140 
 
 101 
 
 70 
 
 232 
 
 220 
 
 270 
 
 103 
 56 
 92 
 
 106 
 38 
 
 170 
 
 150 
 160 
 850 
 990 
 
 35 
 
 7 
 
 9 
 
 257 
 
 270 
 
 66 
 
 70 
 
 93 
 
 107 
 
 307 
 
 17 
 10 
 11 
 
 39 
 10 
 
 3 
 17 
 15 
 20 
 10 
 14 
 21 
 10 
 60 
 60 
 58 
 
 7 
 21 
 15 
 15 
 
 11 
 
 50 
 
 50 
 
 38 
 
 35 
 
 40 
 
 50 
 
 456 
 
 4 
 
 5 
 
 676 
 740 
 768 
 439 
 550 
 768 
 688 
 720 
 725 
 480 
 514 
 654 
 720 
 563 
 660 
 656 
 770 
 537 
 530 
 520 
 
 50 
 55 
 15 
 
 175 
 
 7 
 
 7 
 
 7 
 
 6 
 
 60 
 
 50 
 
 56 
 
 8 
 
 10 
 
 13 
 
 18 
 
 8 
 
 3 
 
 50 
 
 17 
 
 8 
 
 18 
 
 20 
 
 15 
 
 16 
 
 11 
 
 26 
 
 7 
 
 30 
 
 20 
 
 17 
 
 2 
 
 36 
 
 25 
 
 25 
 
 770 
 720 
 714 
 721 
 728 
 670 
 
 807 
 
 610 
 565 
 119 
 7 
 217 
 873 
 901 
 905 
 665 
 400 
 500 
 329 
 654 
 756 
 520 
 875 
 
 140 
 120 
 120 
 130 
 330 
 131 
 140 
 110 
 110 
 400 
 370 
 140 
 146 
 130 
 100 
 140 
 146 
 137 
 150 
 125 
 
 135 
 
 26 
 12 
 
 6 
 192 
 
 5 
 
 22 
 20 
 16 
 
 17 
 11 
 48 
 
 7 
 
 100 
 
 20 
 
 28 
 
 5 
 37 
 60 
 45 
 
 100 
 100 
 
 100 
 100 
 
 100 
 100 
 100 
 100 
 100 
 100 
 100 
 100 
 100 
 100 
 100 
 
 100 
 100 
 100 
 100 
 100 
 100 
 100 
 100 
 100 
 100 
 100 
 100 
 100 
 100 
 100 
 100 
 100 
 100 
 100 
 100 
 
 79 
 
 73 
 
 12 1C0 
 
 33000 
 
 100 
 20 
 22 
 695 
 117 
 19 
 79 
 
 192 
 
 7 
 
 
 
 
 
 100 
 
 
 
 143 
 
 143 
 
 93 
 
 95 
 
 17 
 
 15 
 
 512 
 
 4 
 
 4 
 
 
 
 7 
 
 549 
 654 
 835 
 292 
 625 
 853 
 600 
 626 
 634 
 623 
 634 
 589 
 654 
 481 
 471 
 662 
 1100 
 231 
 240 
 192 
 
942 
 
 FOOD TABLES. 
 TABLE I.— FOODS— (Continued). 
 
 
 1000 Parts contain 
 
 Relation of 
 1:2:3. 
 
 
 1 
 
 2 
 
 3 
 
 4 
 
 5 
 
 6 
 
 1 
 
 ■ 2 
 
 :3 
 
 2. Vegetable Foodstuffs. 
 
 ■a m 
 
 
 
 
 
 
 
 
 
 
 CD "o 
 
 "O cS 
 
 °r1 
 
 A~ W 
 
 
 o H 
 
 id 
 
 ■Si 
 
 
 
 
 
 
 Potatoes 
 
 20 
 14 
 
 2 
 2 
 
 200 
 74 
 
 10 
 
 7 
 
 760 
 893 
 
 8 
 10 
 
 100 
 100 
 
 10 
 14 
 
 1030 
 
 Turnips 
 
 529 
 
 Carrot (yellow) 
 
 10 
 
 2 
 
 90 
 
 10 
 
 873 
 
 15 
 
 100 
 
 20 
 
 900 
 
 Cauliflower 
 
 25 
 19 
 27 
 31 
 
 4 
 2 
 1 
 5 
 
 50 
 49 
 66 
 33 
 
 8 
 12 
 
 6 
 19 
 
 904 
 900 
 
 888 
 908 
 
 9 
 18 
 12 
 
 8 
 
 100 
 100 
 100 
 100 
 
 16 
 
 11 
 
 4 
 
 16 
 
 200 
 
 Cabbage 
 
 258 
 
 Beans 
 
 244 
 
 Spinach 
 
 106 
 
 Lettuce 
 
 14 
 10 
 
 3 
 1 
 
 22 
 23 
 
 10 
 
 4 
 
 944 
 956 
 
 7 
 6 
 
 100 
 100 
 
 21 
 10 
 
 157 
 
 Cucumbers 
 
 230 
 
 Radishes 
 
 12 
 32 
 
 1 
 
 4 
 
 38 
 60 
 
 7 
 9 
 
 934 
 
 877 
 
 8 
 18 
 
 100 
 100 
 
 8 
 12 
 
 317 
 
 Edible mushrooms (average).. . 
 
 188 
 
 Same dried in the air (average). 
 
 219 
 
 25 
 
 412 
 
 61 
 
 160 
 
 123 
 
 100 
 
 12 
 
 188 
 
 Apples and pears 
 
 4 
 
 
 130 
 
 3 
 
 832 
 
 31 
 
 100 
 
 
 3250 
 
 Various berries (average) 
 
 5 
 
 
 90 
 
 6 
 
 849 
 
 50 
 
 100 
 
 
 1800 
 
 Almonds 
 
 242 
 
 537 
 
 72 
 
 29 
 
 54 
 
 66 
 
 100 
 
 222 
 
 30 
 
 Cocoa 
 
 140 
 
 480 
 
 180 
 
 50 
 
 55 
 
 95 
 
 100 
 
 343 
 
 129 
 
 TABLE II.— MALT LIQUORS 
 
 1000 Parts by Weight contain 
 
 Porter 
 
 Beer (Swedish) 
 
 ' ■ (Swedish export). . 
 
 Draught-beer 
 
 Lager-beer 
 
 Bock-beer 
 
 Weiss-beer 
 
 Swedish " Svagdricka " 
 
 
 0> 
 
 
 
 
 
 o?5 
 2 * 
 ■£.2 
 
 o 
 A 
 
 o 
 
 o 
 
 o 
 
 S3 
 
 u 
 
 X 
 
 W 
 
 13 
 O 
 
 871 
 
 2 
 
 54 
 
 76 
 
 7 
 
 887 
 
 
 28 
 
 — 
 
 15 
 
 885 
 
 
 32 
 
 — 
 
 7 
 
 911 
 
 2 
 
 35 
 
 55 
 
 8 
 
 903 
 
 2 
 
 40 
 
 58 
 
 4 
 
 881 
 
 2 
 
 47 
 
 72 
 
 6 
 
 916 
 
 3 
 
 25 
 
 59 
 
 5 
 
 945 
 
 — 
 
 22 
 
 — 
 
 7 
 
 13 
 
 65 
 73 
 
 10 
 
 7 
 
 13 
 
 31 
 
 47 
 
 
 d 
 
 
 a 
 
 
 
 
 
 
 <u 
 
 T3 
 
 
 
 
 
 
 < 
 
 O 
 
 3.0 
 
 — 
 
 2.0 
 
 2 
 
 1.5 
 
 2 
 
 1.7 
 
 — 
 
 4.0 
 
 — 
 
 — 
 
 — 
 
 23 — — 
 
WINES AND OTHER ALCOHOLIC LIQUORS. 
 
 943 
 
 TABLE III.— WINES AND OTHER ALCOHOLIC LIQUORS 
 
 1000 Parts by Weight contain 
 
 Bordeaux wine 
 
 White wine (Rheingau) 
 
 Champagne 
 
 Rhine wine (sparkling). 
 
 Tokay 
 
 Sherry 
 
 Port wine 
 
 Madeira 
 
 Marsala 
 
 Swedish punch 
 
 Brandy 
 
 French cognac 
 
 Liqueurs 
 
 1 
 
 
 
 
 
 
 
 
 It 
 
 o 
 JO 
 
 o 
 a 
 
 ti 
 
 85 
 
 111 
 
 a a £ 
 
 c 
 a 
 
 3 
 
 
 ■ o . 
 
 g CO 
 
 03 
 
 
 G 
 
 M 
 
 w 
 
 Ml 
 
 ~ — — 
 < 
 
 >> 
 O 
 
 A 
 
 ■ 
 < 
 
 as : — 
 
 o 
 
 883 
 
 94 
 
 23 
 
 6 
 
 5.9 
 
 
 
 2.0 
 
 
 863 
 
 115 
 
 23 
 
 4 
 
 5.0 
 
 — 
 
 2.0 
 
 
 776 
 
 90 
 
 134 
 
 115 
 
 6.0 
 
 1.0 
 
 1 
 
 } 60-70 
 
 801 
 
 94 
 
 105 
 
 87 
 
 6.0 
 
 1.0 
 
 2.0 
 
 SOS 
 
 120 
 
 72 
 
 51 
 
 7.0 
 
 9.0 
 
 3.0 
 
 
 795 
 
 170 
 
 35 
 
 15 
 
 5.0 
 
 6.0 
 
 5.0 
 
 
 774 
 
 164 
 
 62 
 
 40 
 
 4.0 
 
 2.0 
 
 3.0 
 
 
 791 
 
 156 
 
 53 
 
 33 
 
 5.0 
 
 3.0 
 
 3.0 
 
 
 790 
 
 164 
 
 46 
 
 35 
 
 5.0 
 
 4.0 
 
 4.0 
 
 
 479 
 
 263 
 460 
 
 — 
 
 332 
 
 — 
 
 — 
 
 — 
 
 
 — 
 
 550 
 
 
 
 
 
 
 
 
 442-590 
 
 — 
 
 260-475 
 
 — 
 
 — 
 
 — 
 
 
S44 INDEX TO SPECTRUM PLATE. 
 
 SPECTRUM PLATE 
 
 1. Absorption spectrum of a solution of oxyhemoglobin. 
 
 2. Absorption spectrum of a solution of haemoglobin, obtained by the action of an 
 
 ammoniacal ferro-tartrate solution on an oxyhemoglobin solution. 
 -3. Absorption spectrum of a faintly alkaline solution of melhcemoglobin. 
 A. Absorption spectrum of a solution of hoemalin in ether containing oxalic acid. 
 
 5. Absorption spectrum of an alkaline solution of hcematin. 
 
 6. Absorption spectrum of an alkaline solution of hcemochromoaen, obtained by the 
 
 action of an ammoniacal ferro-tartrate solution on an alkaline-haematin solution 
 
 7. Absorption spectrum of an acid solution of hamatoporphyrin. 
 
 8. Absorption spectrum of an ammoniacal solution of urobilin after the addition of a 
 
 zinc-chloride solution. 
 
INDEX OF AUTHORS 
 
 Abderhalden, E., enzymes, 50, 53, 54, 62, 
 65, 525, protein hydrolysis, 84, 106, 
 107, 109, 114, 121-125, 132, 133, 142, 
 146, 154, 262, 288, 507, 605, 634, 662; 
 polypeptides, 86-90, 132, 133, 509; 
 protein reaction, 101; ichthylepidin, 
 121; proteoses. 127; cystine and cys- 
 tinuria, 148, 776; tryptophane, 157, 
 158; histidine, 160, 161; diamino- 
 trioxydodecanoic acid, 165; glyco- 
 proteid, 167; proteoses in the blood, 
 263, 264; blood serum, 266, 269, 270; 
 blood corpuscles, 275, 277, 304; blood 
 analyses, 328, 334, 338; iron per- 
 parations. 340; blood and high altitudes, 
 341; adrenalin, 376, 380; melanin, 380; 
 cholesterin, 445, 448; gastric contents, 
 481; digestion, 484, 513; duodenal 
 secretion, 489, 490; absorption and 
 synthesis of proteins, 529, 530; fat, 
 559; milk, 646, 659, 662, 667; urea 
 formation, 682; nuclein metabolism, 
 702, 705; amino-acid storage, 721, 722; 
 alcaptonuria, 735-737; urinary sul- 
 phur, 752; polypetides in the urine, 
 763; amino acids in the urine, 756, 827: 
 pyridine, 787; Bence-Jones proteid, 
 792, 793; tunicin, 839; nutritive value 
 of gelatin, 912; ammonia as protein 
 sparer, 912; water and metabolism 
 920; alcohol, 921; nin - hydrin, 101; 
 caprine, 144; tyrosine reagent, 154 
 
 Abel, J., 379, 621, 683 
 
 Abeles, M., 266, 749 
 
 Abelmann, M., 532, 539, 540 
 
 Abelous, J., 757, 877 
 
 Abelsdorf, G., 616 
 
 Ach, L., 698 
 
 Achalme, 307 
 
 Achard, Ch., 265 
 
 Acheles, \V., 698, 757 
 
 Ackermann, D., putrefaction bases, 47; 
 histidine, 82, 159, 160; proline, 154; 
 aporrhegmen, 166; blood corpuscles, 
 274, 275; meat bases, 578; lysine in 
 the urine, 828 
 
 Ackrovd, H., 717 
 
 Adam, H., 311 
 
 Adamkiewicz, A., 100, 912 
 
 Adamson, 17 
 
 v. Anrep, R., 287, 723 
 
 Adler, O., proteoses, 209; fructose, 217, 
 
 814; ochronosis, 550; blood, 797 
 Adler, R., proteoses, 134; pentoses, 209; 
 
 fructose, 217; blood, 797 
 Adrian, C, 730 
 Adriance, J., 662, 665 
 Adriance, V., 662, 665 
 Aducco, V., 675, 757 
 Agulhon, 50 
 Albanese, M., 711 
 Albert, R., 41 
 
 Albertoni, P., 415, 502, 533 
 Albrecht, E., 273 
 Albro, A., 506 
 Albu, A., gastric juice, 466; urine poison, 
 
 758; mineral metabolism, 758, 769, 
 
 899, 901, 902 
 v. Alder, L., 792 
 Aldrich, J. B., 846 
 Alexander, F., 133, 652 
 v. Alfthan, K., 749 
 Allard, E., 825 
 Allaria, G. B., 659 
 Allen, S., 471 
 Alleis, R. A., 157, 380 
 Allihn, F., 656, 811 
 Allison, F. G., 689 
 Almagia, M. f 410, 583, 706 
 Almen, A., xanthine, 188; sugar test, 
 
 214; meat, 599, 600; food-stuffs, 940 
 Aloy, J., 877 
 Alsberg, C., 104, 184 
 Altmann, R., 179 
 Amberg, S., 71, 642, 643 
 Ambronn, H., 839 
 Amerman, G., 470 
 Ameseder, Fr., 239, 627, 844 
 Amiradzibi, S., 575 
 Amthor, K., 237, 559 
 Andersen, A. C., 154, 804, 809, 810 
 Anderson, R J., 580 
 Anderson, N., 332 
 Andersson, J., 376 
 Andryewskij, P., 874 
 Anselm, R., 435 
 Ansiaux, G., 97, 253 
 Anthon, 213 
 Appleyard, J. R., 28 
 Araki, T., blood pigments, 285; nucleic 
 
 945 
 
946 
 
 INDEX OF AUTHORS 
 
 acids, 508; lactic acid, 582-585, 748, 
 
 749- chitin, 839 
 Ardin-Delteil, P., 848 
 Argiris, A., 112, 114, 606 
 Argutinsky, P., 600, 848, 883 
 Arinkin, M., 43 
 Armstrong, E. T., enzyme, 55, 56, 58, 
 
 59, 65; glucoside, 200; osone, 203 
 Armstrong, H. E., 59, 65 
 Arnheim, J., 408 
 Arnold, J., 377 
 Arnold, V., protein reaction, 101; cystein, 
 
 149; urine reaction, 696; nephrosein, 
 
 734; hffimatoporphyrin, 797; aceto- 
 
 acetic acid, 824, 825 
 Arnold, W., 2S8 
 Arnschink, L., 537 
 Arnstein, R., 715 
 Aron, H., 281, 552, 554, 555 
 Arrhenius, S., dissociation theory, 4; 
 
 catalysis, 33, 34; enzymes, 53; Schutz's 
 
 rule, 57; toxin-antitoxin combination, 
 
 67, 68 
 Arronet, H., 328 
 Ateaga, T. F., 400 
 Arthus, M., blood coagulation, 250, 251, 
 
 313, 314, 316-318, 320; fibrinolysis, 
 
 255; serum lipase, 265, glycolysis, 332; 
 
 casein, ,648, 650 
 Artmann, P., 23 
 Asahina, Y., 297 
 Ascher, E., 145 
 Ascherson, 646 
 Ascoli, A., antolysis, 43; nucleic acids, 
 
 172, 185; uracil 194; glutinase, 505; 
 
 protein absorption, 526; placenta, 642; 
 
 urea formation, 682; uric acid, 706 
 Asher, L., blood sugar, 264; lymph, 364, 
 
 349-352; spleen, 372; thyroids, 374; 
 
 liver, 381, absorption, .528; nitrogen 
 
 elimination, 910 
 Aso, K., 97 
 Astaschewsky, 593 
 Athanasiu, J., 384, 561 
 Atwater, W. O., metabolism, 595, 597, 
 
 879, 891, 914; respiration apparatus, 
 
 868,869; urine quotient C:N, 884; 
 
 alcohol, 921; diets, 932, 936, 939 
 Aubert, H., 849 
 Auche, A., 267, 431 
 Austin, A. E., 392 
 Austrian, C. R., 702 
 Autenrieth, W., 766 
 Aynand, M., 308, 314, 315 
 Ayres, W. C, 615 
 Akerman, J., 477 
 
 Baas, H., 501, 720 
 
 Babcock, 653 
 
 Babkin, B., 107, 499, 682 
 
 Bach, A., oxygenases, peroxides and 
 peroxidases, 871, 873; catalases, 873; j 
 oxidation processes, 875; philothion, ' 
 877; reduction processes, 877 I 
 
 Bacmeister, 439 
 
 Baer, J., 43; cystin, 148; thiolactic acid, 
 151; lactic acid, 583; ammonia elimina- 
 tion, 675, 676; acetone bodies, 818,. 
 819, 822 
 
 v. Baeyer, A., 37, 38, 212 
 
 Baginsky, A., 438, 555 
 
 Baglioni, S., 333 
 
 Bailie, A., 387 
 
 Bainbridge, F. A., 53, 351 
 
 Baisch, C, 749, 808 
 
 Baker, J. L., 226 
 
 Balch, A., 414 
 
 Baldi, D., 385 
 
 Baldoni, A., 509, 783 
 
 Balean, H., 301 
 
 Balke, P., purine bases, 188, 193, 714; 
 phosphocarmic acid, 578 
 
 Bang, B., 671 
 
 Bang, 1., histone, 108; guanylic acid, 
 183, 185; nucleohistone, 307; blood 
 sugar, 329; blood, 329; estimation of 
 sugar, 329, 808-811; lymph glands 
 and thymus, 365-368; glycogen, 402; 
 saliva, 457; rennin enzyme, 473; chlor- 
 ine estimation in the urine, 759; pro- 
 teoses in the urine, 792; sugar tests, 803, 
 804; sugar formation, 399 
 
 Barbara, A. G., 356, 349, 415 
 
 Barbieri, J., 152, 448 
 
 Barbieri, N. A., 615, 630 
 
 Barcroft, J., 277-279 
 
 Bardach, Fr., 726 
 
 Bardach, K., 684 i 
 
 Bardier, E., 757 
 
 Barendrecht, H. P., 65 
 
 Barker, B., 307 < 
 
 Barker, L. F., 827 
 
 Barral, 332 
 
 Barratt, W., 849 
 
 Barszczewski, C, 210 
 
 Bartholomans, E., 297, 295, 429 
 
 Bartoschewitsch, S. F., 724 
 
 Basch, K., 667-669 
 
 Baserin, O., 443 
 
 Bashford, E., 722 
 
 Baskoff, 385, 386 j 
 
 Bass, 727 
 
 Basso w, 461 
 
 Bastianelli, G., 492 
 
 Batelli, F., fermentation, 407; uricolysis, 
 706; peroxidases, 873, 874; oxidation 
 processes, 874, 875 
 
 Baudrimont, 838 
 
 Bauer, Fr., 182 
 
 Bauer, H., 646 
 
 Bauer, J., 525, 561 
 
 Bauer, K., 757 
 
 Bauer, M., 160 
 
 Bauer, R., 113, 815 
 
 Baum, Fr., 159 
 
 Baumann, E., diamines, 47; cystine, and 
 cystinuria, 149, 827, 828; thiolactic 
 acid, 149, 150; carbohydrates, 215; 
 
INDEX OF AUTHORS 
 
 947 
 
 iodothyrin, 376; deamidation, 410; 
 intestinal putrefaction, 514, 720: ethereal 
 sulphuric acids, 515, 724-729, 784; 
 hippuric acid, 720; oxyacids, 734, 
 homogentisic acid, 735, 73ii, 73s, 7:;'."; 
 carbohydrates in the urine, 749, SOS; 
 urinary sulphuric acids, 764; sarcosine, 
 77t>; behavior of aromatic bodies, 77s, 
 780, 783: mercapturic acids, 786 
 
 Baumann. L., 157, 288, 484, 513 
 
 Baumgarten, A., 360 
 
 Baumgarten, O., 403, 758 
 
 Baumstark, v., brain constituents, 605, 
 606, 612, 613; urinary pigments, 799 
 
 Baumstark, Rob., 512, 524 
 
 Haver. H., 136 
 
 Bayer, R.. 372 
 
 Bavliss. W. M., enzvme, 54, enterokinase, 
 492, 496; secretin. 492, 498; intestinal 
 enzymes, 492, 493, 503; trypsinogen 
 and trypsin, 496, 497, 503, 506; casein 
 digestion, 652 
 
 Beattv. W. A., 89 
 
 Beaumont, W., 461, 481 
 
 Bebeschin, K., 673 
 
 Beccari, L., 435 
 
 Bechamp, A., 633, 655, 661 
 
 Bechhold, H., colloids, 17, 18, 30, 32, 51; 
 uric acid, 70S; sugar determination, 804 
 
 Becht, F. C, 349 
 
 Beck. A., 744 
 
 Beck. C, 595 
 
 Beckmann. E., 4 
 
 Beckmann, Ernst. 846 
 
 Beckmann. \Y., 768 
 
 Becquerel, A., 33S, 665 
 
 Beger, C, 667 
 
 Behrend, R.. 698, 699 
 
 v. Behring, E., 66 
 
 Beier, Karl, 441 
 
 Beitler, C, 152 
 
 Bellamv. H.. 496 
 
 Bellori, E.. 653 
 
 van Bemmelen, J. M., 29, 31 
 
 Bence, J., 311 
 
 Bence Jones, H., 792 
 
 Bendix, E., 208, 209, 394 
 
 Benedicenti, A., 126, 729, 921 
 
 Benedict, F. G., metabolism in work, 
 595, 597; respiration, 868; calorimetry, 
 885; sparing of proteins, 914; alcohol, 
 921 
 
 Benedict, H., 595. 752, 897 
 
 Benedict, S. R., 689, 693, 752 
 
 Benedikt, R., 236 
 
 Benedikt. St.. 214 
 
 Benrath, A.. 477 
 
 Berard. E.. 97. 633 
 
 Berdez. J., 841 
 
 Berenstein, M., 521 
 
 Bergell. P., carbohydrates in proteins, 
 84, 262; lecithin, 245; polvpeptides, 
 508: placenta. 642; casein, 662; 
 oxybutyric acid, 826 
 
 Berber, W. M., 456 
 Bergh, E., 116 
 
 Ber^holz, R., 50!) 
 
 Bergin, T. J., 519 
 
 v. Bergmann, G., 266, 267, 442 
 
 Bergmann, P., 376, 466, 542-543 
 
 Bergmann, VVolfg, 761 
 
 Berlioz, A., 758 
 
 Berlinerblau, M., 334 
 
 Bernard, Claude, blood sugar, 332; gly- 
 colysis, 332; glycogen, 389; 390, 398, 
 sugar puncture, 402; diabetes, 405; 
 pancreas, 495, 501, 502, fat absorption, 
 538; muscle glycogen, 592 
 
 Bernert, R., 83, 233, 358 
 
 Bernheim, A, 357, 359 
 
 Bernheim, R., 766 
 
 Bernstein, J., 602 
 
 Bernstein, N. O., 495 
 
 Bert, P., mammary glands, 643, 669; 
 gases of blood, 851, 861 
 
 Bertagnini, C., 783 
 
 Bertarelli, E., 64 
 
 Berthelot, M. P. E., division law, 27; 
 fat cleavage, 501; tunicin, 839, calor- 
 imetry, 885 
 
 Bertin-Sans H., 285, 287 
 
 Bertrand, G., arsenic, 72, 373, 838; 
 xylonic acid, 210; sugar determina- 
 tion, 808, 811; tyrosinase, 842; reptile 
 poison, 846; oxidases, 873 
 
 Bertz, F., 558 
 
 Berzelius, J. J., catalytic reactions, 33; 
 saliva, 458 -i 
 
 Besbokaia, M., 496 
 
 Best, Fr., 481 
 
 Bezzola, C., 706 
 
 Bial, M., pentoses. 208, 209, 816; glucur- 
 onic acids, 221; diastase, 265, 346, 
 398; glvcogen, 396 
 
 Bialobrzeski. M., 292 
 
 Bialocour, F., 485 
 
 v. Bibra, E., 389 
 
 Bickel, A., 464, 465 
 
 Bidder, F., buccal mucus, 453, 554; 
 saliva, 458; gastric juice, 465; pan- 
 creatic juice, 499; bile, 518; fat 
 absorption, 538 
 
 Biedert, Ph., 660 
 
 Biehler, A., 124 
 
 Biel, J., 658, 662 
 
 Bielfeld, P., 387 
 
 Bienenfeld, B., 660 
 
 Bienstock, B., 519, 520 
 
 Biernacki, E., blood, 309, 326; pepsin, 
 467; trypsin, 503; intestinal putrefac- 
 tion, 517, 519, 724 
 
 Bierry, H., cataphoresis, 50; filtration. 51 ; 
 enzvmes, 53, 71, 231; pancreatic juice, 
 500 
 
 Biffi, U., 267, 652, 744 
 
 Billard, G., 45 
 
 Billitzer, J., 26, 27 
 
 Biltz, W., glvcogen, 19, 20; colloids, 22, 
 
948 
 
 INDEX OF AUTHORS 
 
 23; adsorption, 28, 29, 69; dextrin 
 B, 230 
 Binet, P., 541 
 
 Bing, H. J., 245, 331, 385, 398 
 Bingel, A., 266 
 Bing, C, 777 
 Biondi, C, 42 
 Biot, J. B., 867 
 Birchard, Fr., 134, 144 
 Bisearo, G., 653 
 Bischoff, Th., 879 
 Bizio, G., 849 
 Bizio, J., 390 
 Bizzozero, J., 307, 314 
 Bjerre, P., 921 
 Bjorn-Andersen, H., 769 
 Blachstein, A., 582 
 Blanck, F. C, 696 
 Blankenhorn, E., 606 
 Blanksma, J., amino-sugar, 167; 219, 
 oxymethylfurfurol, 211, 215, 217; ace- 
 tone, 825 
 Bleibtreu, L., 326, 690, 934 
 Bleibtreu, M., 326, 563, 636 
 Bleile, A. M., 332 
 Blendermann, H., 410, 734, 780 
 Bliss, C. L., 126 
 Blix, M. G., 326 
 Bloch, Br., 735, 736 
 Blondlot, N., 518 
 Blood, A., 52 
 
 Blum, F., haloginized protein, 82; Mil- 
 Ions reaction, 99; adrenalin glycosuria, 
 380 
 Blum, L., autolysis, 45; protein nitrogen, 
 77; protogon, 126; alcoptonuria, 735, 
 cystin, 776; tvrosin cleavage, 779, ace- 
 tone bodies, 818, 819, 822; food value 
 of albumoses, 912 
 Blumenthal, F., gelatin, 83; indol and 
 skatol, 159; pentoses, 208, 815, 816; 
 nucleoproteins, 383; glycogen, 394; 
 assimilation limit, 534; urine indican, 
 728; acetone, 818 
 Boas, J., 488 
 Bocarius, N., 621 
 Bocchi, O., 741 
 Bock, C., 398, 400 
 Bock, J., 285 
 Bode, A., 658 
 Boden, E., 707 
 Bodliinder, G., 921 
 Bodon, K., 356 
 Bodong, A., 318, 323 
 Boedeker, G\, 727 
 Bddtker, E., 680, 759 
 Boehm, P., 381 
 Boehm, I!.. 448, 581, 590,591 
 
 Moaner, H., 154 
 
 Boehtlingk, R., 895, 896 
 
 Hofikfjlmann. W. A., 826 
 Bor-ri, C,., 71 11, 752 
 Boettger, 214 
 Bogdanow, E., 5X6, 596 
 
 Bogdanow-Beresowski, 455 
 
 Bogen, H., 464 
 
 Bogomoloff, Th., 743 
 
 Bohland, K., urea nitrogen, 680; urea, 
 690; uric acid, 701; ammonia, 768; 
 protein need, 934 
 
 Bohm, V., 402 
 
 Bohmansson, G., 803, 804 
 
 Bohr, Chr., blood pigments, 276, 278, 
 279, 282, 287; egg hatching, 637; gases 
 of blood, 850-853; metabolism in 
 lungs, 858, 869; oxygen tension, 859, 
 861-863, 867; carbon dioxide tension, 
 865, 866; specific action of lungs, 864, 
 867; air-bladder, 867; oxygen capacity, 
 868 
 
 Du Bois-Reymond, E., 592, 602 
 
 Du Bois, Reymond, R., 312 
 
 Bokorny, T.', 212 
 
 Bolafno, C, 630 
 
 Boland, G. W., 365 
 
 Boldyreff, W., gastric digestion, 481, 511; 
 intestinal juice, 490-493 
 
 Boljarski, N., 694 
 
 Bolin, J., 873 
 
 Boll, F., 615 
 
 Bonamartini, G., 96 
 
 Bonanni, A., 437, 438, 785 
 
 Bondi, J., 642, 643 
 
 Bondi, S., lipoproteids, 87; sericin, 122, 
 123; bile acids, 419, 421, 423; acetoace- 
 tic acid, 825 
 
 Bondzinski, St., oxyproteic acid, 83; 
 koprosterin, 448; ovalbumin, 633; 
 urine purines, 711; urochrom, 741; 
 oxyproteic acids in urine, 753, 754 
 
 Bonnema, A., 646 
 
 Bookman, S., 721, 722 
 
 Boos, P., 454 
 
 Borchardt, L., elastin albumose, 263, 
 527; sugar formation, 398; fructose 
 uria, 814; acetone, 818, 819, 825 
 
 Bordet, J., antienzymes, 64; sensibiliza- 
 tors, 69; blood coagulation, 314, 318, 
 320 
 
 Borissow, P., 462, 717 
 
 Borkel, C., 136 
 
 Bornstein, K., 595, 918 
 
 Boruttau, H., 581 
 
 Bosshard, E., 149 
 
 Bostock, G., 676 
 
 Bottazzi, Ph., freezing point, lowering of 
 blood, 12; blood corpuscles, 304; gly- 
 cogen, 384, 390, 391; heart muscle, 569; 
 smooth muscle, 602; placenta, 640 
 Bouchard, Ch., 393, 757,. 758 
 Bouchet, A., 680 
 Bouchez, 767, 772 
 Boudet, 264 
 
 Boulud, glucuronic acids, 221, 331; pen- 
 toses, 264; sugar in blood, 264, 329- 
 332; glycolysis,333; maltose in urine, 814 
 Bouma, .1., indican, 730; bile pigments, 
 801, oxybutyric acid, 826 
 
INDEX OF AUTHORS 
 
 949 
 
 Bourcet, P., 269, 336 
 
 Bourquelot, E., 395 
 
 Bouveault, L., 144 
 
 Brach, H., 839 
 
 Bradley, H. C, 500 
 
 Brahm, C, 559, 785 
 
 Brahm, B., 179, 182, 183, 211 
 
 Brand, J., 414, 437, 438 
 
 Brandberg, J., 793 
 
 Brandenburg, K., 310 
 
 Brandl, J., Ml 
 
 Brasch, VV., 144, 534 
 
 Brat, H., 209 
 
 Brauer, L., 140 
 
 Braun, K., 04 
 
 Braunstein, A., 408 
 
 Brautlecht, C, 78 
 
 Bredig, G., colloidal metals 14; surface 
 tension, 26; catalysis, 33-37; assym- 
 etric synthesis, 59 
 
 Brienl, F., 103 
 
 Brenzinger, K., 827 
 
 Bretschneider, A., 329, 330 
 
 Brewster, J. F., 155 
 
 Brieger, L., putrefaction products, 47; 
 neurine, 246; intestinal putrefaction, 
 514; skatol, 521; neuridine, 606, 612, 
 628; urine indican, 727, 729; skatoxyl- 
 sulphuric acid, 732 \ cystinuria, 827; 
 perspiration, 848 
 
 Briggs, C. E., 402 
 
 Brig], P., 179, 183 
 
 Brion, A., 774 
 
 Brodie. T. G.,'256, 400, 509 
 
 Brodlev, H. C, 72 
 
 Brook, F. \Y , 82 
 
 Brooks, CI., 399 
 
 Browinski, J., 266, 741, 754 
 
 Brown. A. J., 55, 56, 65 
 
 Brown, E. W ., 705, 717 
 
 Brown, H. T., inverting enzymes, 55; 
 starch hydrolysis, 226, 229, 456, sacha- 
 rase, 492 
 
 Brown. R., 19 
 
 Brown, T. Graham, 594 
 
 Browne, C. A. (jr.), 647 
 
 Brubacher, H., 554, 557 
 
 v. Brucke. E., blood coagulation, 316; 
 glycogen, 392; pepsin 467-469, fat 
 emulsion, 511; protein absorption, 
 525; carbohydrates in urine, 749 
 
 Brugsch, Th., bile pigments, 443; pan- 
 creas, 532; uric acid, 700, 701; hip- 
 puric acid, 721; amino-acids in urine, 
 757; urine in hunger, 897 
 
 Bruhns, G., 193 
 
 Brunner, E., 36 
 
 Brunner, Th., 665 
 
 Bruno, G., 501, 502, 506, 510, 758 
 
 Brunton-Blaikie, 572 
 
 de Bruyn, Lobrv, 19, 201, 220 
 
 Bryant, A. P., 936, 938 
 
 Buchanan, A., 256 
 
 Buchner, E., alcohol fermentation, 41, 
 
 205; lactic ackl fermentation, 207; 
 sugar test, 213 
 
 Buchner, H„ 41, 856 
 
 Buchtala, H., 113-115, 147, 426 
 
 Budde, V., Sl_> 
 
 Billow, K., <).-», 227 
 
 Biinz, R., 612, 613 
 
 Burger, L., 545 
 
 Burker, K., 314, 315 
 
 Bufalini, 339 
 
 Bugarszky, St., 271, 326 
 
 Buglia, G., 510, 572, 602, 603 
 
 Bull, H., 237 
 
 v. Bunge, G., serum, 270; blood cor- 
 puscles, 304; blood coagulation, 313; 
 blood analysis, 328; iron preparations, 
 340; blood and high altitude, 341; 
 iron in liver, 387; gastric juice, 485; 
 cartilage, 550; hamatogen, 629, 637; 
 milk, 657, 664,666-668; hippuric acid, 
 722, 723; mineral requirement, 900, 
 901 
 
 Bunsen, R., 690 
 
 Buntzen, J., 339 
 
 Buraczewski, J., 83 
 
 Burchard, H., 447 
 
 Burckhardt, A. E., 270 
 
 Burian, R., purine bases and their 
 enzymes, 193, 195, 371, 373, 572, 594, 
 702, 703, 713; uric acid formation, 700, 
 792, 703, 705; uric acid cleavage, 706; 
 histone in urine, 795 
 
 Burow, R., 371, 663 
 
 Busch, P. W., 349, 350 
 
 Butlerow, A., 211, 212 
 
 Butterfield, E., 277, 303 
 
 Bywaters, H. W., 260, 263 
 
 Cade, A., 464 
 
 Cahn, A., 476, 615 
 
 Camerer, W., milk, 656, 657, 662, 664- 
 666; urine nitrogen, 680; uric acid elim- 
 ination, 700; purine bases, 715; meta- 
 bolism, 922, 924 
 
 Camerer, W., jr., 847 
 
 Cameron, A. T., 78, 109 
 
 Cam is, M., 279, 592 
 
 Cammidge, P. J., 815, 827 
 
 Campani, A., 763 
 
 Campbell, G., 628, 629, 633, 634 
 
 Campbell, J. F„ 432-434 
 
 Camps, R., 739 
 
 Camus, L., pancreatic juice, 496; secretin, 
 498; vesiculase, 622 
 
 Cannon, W. B., 479-484, 524 
 
 Cappelli, J., 304, 602 
 
 Cappezzuoli, C, 369, 557 
 
 Capranica, St.. 190. 848 
 
 Carbone, D., 613 
 
 Carlier, E. \\\, 313, 347 
 
 Carlinfanti, E., 235, 236 
 
 Carlini, C, 381 
 
 Carlson, A. J., 348, 349, 454 
 
 Carlson, C. E., 873 
 
950 
 
 INDEX OF AUTHORS 
 
 Carnot, Ad., 552, 558 
 
 Carvallo, J., 485, 48(5 
 
 Casali, A., 847 
 
 Caspari, W., high altitude 341, 888; 
 
 protein metabolism, 595, 916, milk fat; 
 
 669; vegetable diet, 915-916 
 Castoro, N., 161 
 Catheart, E. P., autolysis, 44; glycogen, 
 
 396; stomach, 479; gastric digestion, 
 
 4S1; protein absorption, 527; creatine 
 
 and creatinine, 594, 693; milk sugar, 
 
 670; starvation urine, 897 
 Cavazzani, E., cerebrospinal fluid, 361; 
 
 glycogen cleavage, 399; absorption, 
 
 535, phosphocarnic acid, 578; muscular 
 
 work, 592; semen, 620 
 Cernj, C, 838 
 Cerny, T., 583, 792 
 Chabbas, J., 617 
 Chabrie, C, 557 
 Chandelon, Th., 592 
 Chaniewski, 563 
 Charnas, D., 746, 747 
 Chassevant, A., 706 
 Chauveau, A., sugar formation, 412; 
 
 fat formation, 562; muscular work; 
 
 592, 597 
 Cherry, Th., 68 
 Chigin, P., 462 
 Chistoni, A., 314, 315 
 Chittenden, R. H., keratin, 119; elastin, 
 
 116, 117; gelatin, 118, 120; albumoses 
 
 and peptones, 128-131, 137; saliva, 
 
 455-458; pepsin, 470, 471; trypsin, 506; 
 
 tendon mucoid, 545, 551; myosin, 
 
 567, 568; neurokeratin, 605, 613, 614; 
 
 protein requirement, 934 
 Chodat, R., oxidases, peroxides and 
 
 peroxidases, 871; oxidation processes, 
 
 875; ," philothion," 877 
 Chossat, Th., 892, 896 
 Christenn, G., 662 
 Christensen, A., 793 
 Ciamician, G., 159 
 Cingolani, M., 699 
 Citron, H., 766 
 Clapp, S. H., 89, 107 
 Clar, C, 701 
 Clarke, T. W., 287 
 Claus, R., 408 
 
 Clausmann, P., 693, 552, 558 
 Clemens, Paul, 785. 827 
 Clemm, C. G., 665 
 Clerk, B., 265 
 Cleve, P. T., 422, 423 
 Cloetta, M., 290, 292, 293, 753 
 Cloez, 441 
 Clopatt, A., 921 
 Closson, O. E., 400 
 Cobliner. 332 
 Cohn, Felix, 485 
 Cohn, Max, 500 
 Cohn, Michael, 455 
 Cohn, R., leucinimid, 145; carbohydrate 
 
 formation, 410, 411; fate of aromatic 
 substances in the animal organism, 778, 
 783, 784; furfurol, 784, pyridin, 787 
 
 Cohn, Th., 621, 717 
 
 Cohnheim, J., 456 
 
 Cohnheim, O., lipoid action, 10; proteins, 
 76, 95; blood and high altitudes, 341, 
 340; glycolysis; 407, 408; gastric 
 juice, 465; peptic digestion, 481, 484; 
 absorption, 484, 528, 542-543; erepsin, 
 491, 493; pancreatic juice, 497; con- 
 nective tissue, 512; peristalsis, 524; 
 digestion work, 930; accommodation 
 of digestive enzymes, 53 
 
 Cohnstein, J., 336 
 
 Cohnstein, W., 309, 352 
 
 Colasanti, G, 591; 593, 698, 748, 749 
 
 Cole, S. W., protein reaction, 100; trypto- 
 phane, 155-158 
 
 Collmann, 849 
 
 Comaille, A., 653, 668 
 
 Comesatti, G., 380 
 
 Comiotti, L., 816 
 
 Connstein, W., 265 
 
 Conradi, H., 45, 324, 520 
 
 Constantinidi, A., 535, 915, 916 
 
 Constantino, A., 572 
 
 Contejean, Ch , phlorhizin diabetes, 
 400; gastric juice, 465, 476; pyloric 
 secretion, 477 
 
 Copemann, M., 797 
 
 Coranda, G., 682 
 
 Cordua, H., 301 
 
 Corin, G., protein coagulation, 97; fibrino- 
 gen, 253; proteins of egg-white, 633 
 
 Coronedi, G., 560 
 
 Corper, H. J., 372, 449-450, 701, 706 
 
 Corvisart, L., 502 
 
 Costantino, A., 586, 602, 603, 748 
 
 Le Count, E. R., 114 
 
 Courant, G., milk, 644, 649, 650, 659 
 
 Cousin, H., 242, 248 
 
 Couvreur, E., 393 
 
 Cramer. C. D., 253 
 
 Crameri E., 122, 848 
 
 Cramer, Tr., 531 
 
 Cramer, W., absorption, 525, 528, 530; 
 protagon, 606-608; placenta, 640; 
 creatine, 692; hippuric acid, 722; 
 blood coagulation, 320 
 
 Cremer, M., glycogen, 58; 332, 390, 
 393, 395; pentoses, 208; glycolysis, 
 332; phlorizin-diabetes, 400; sugar 
 formation, 412; fat formation, 562 
 
 Cristea, G., 336 
 
 Crittenden, A. L., 454 
 
 Croftan, A., 334 
 
 Croft-Hill, A., 58, 225 
 
 Croner, W., 488. 536 
 
 Cronheim, W.. 488 
 
 Croockewitt, J. H., 122 
 
 Csdkae, J., 648, 658 
 
 v. Csonka, F., 657 
 
 Cummis, G. W., 506, 567, 568 
 
INDEX OF AUTHORS 
 
 951 
 
 Cunningham, K. II , ">10 
 Curtius, Th., 85, So, 422 
 Cutler, W. D., 168, 544, 545 
 Cybulski, N., 379 
 Czernecki, W., 266, 358 
 Czerny, Adalb, 307 
 Czernv, F., 407, 
 Czerny, V., 485, 525 
 v. Czyhlarz, E., 873 
 
 Daddi, L., 339, 669 
 
 Daenhardt, C, 856 
 
 Dakin, H. D., autolysis, 44; mandelic 
 acid ester, 62; arginase, 89, 161, 574, 
 681, 682; protamine, 109, 110; valine, 
 141; serine, 145; proline, 154; color 
 reactions, 157; lysin, 163; hexon bases, 
 164; oxalic acid, 716, 773; alcap- 
 tonuria, 737; cleavage of fatty acids 
 774, 781; uramino acids, 786; acetone 
 formers, 822; sugar formation, 412; 
 lactic acid, 583; a-amino acids, 775 
 
 Daland, J., 329 
 
 van Dam, W., 474, 650 
 
 Danilewski, A., plasteines, 58, 135; pro- 
 tein sulphur, 79; retarding substances, 
 487, 492; muscle protein, 566-569; 
 milk globules, 646 
 
 Danilewsky, B., 885 
 
 Danilewsky, W., 241 
 
 Dareste, C, 620, 628 
 
 Darmstadter, E., 826 
 
 Darmstadter, J., 846 
 
 Darmstadter, L., 448 
 
 Dastre, A., fibrinogen, 252; fibrinolysis, 
 255; glycogen, 307, 346; blood coagula- 
 tion, 314; liver, 383, 384, 388; gly- 
 cogen, 395, 397, 399; bile, 414, 434, 
 435, 511; enterokinase, 496, 497; fat 
 absorption, 538 
 
 Dantzenberg, P. J. W., 783 
 
 Danwe, F., 49 
 
 Davidoff, W., 75 
 
 Day, H., 480 
 
 Dean, A. L., 228, 529 
 
 Decaisne, E., 667 
 
 Deetjen, H., 308, 314, 315 
 
 Dehn, W. M., 760 
 
 Dekhuysen, C, 13 
 
 Delezenne, C , enzyme retardation, 65; 
 papain, 65; blood coagulation, 250, 
 313, 318, 320, 324, 325, 351; intestinal 
 juice, 490-492, enterokinase, and pan- 
 creatic juice, 493, 495-497, 509; secre- 
 tin, 498 
 
 Delfino, A., 640 
 
 Demant, B., 491 
 
 Detnoor, J., 453 
 
 Denig^s, G., tyrosin, 154; indol and 
 skatol reactions, 158, 159; inosite, 
 580; homogentisic acid, 739 
 
 Denis, P. S., 257 
 
 Denis, W., tyrosin, 154; urea in blood, 
 333, 336; hypophysis, 592; creatine, 
 
 692, 606: uric acid, 708, 711, 334; 
 urine sulphur, 752 
 
 Denk, \\\, 336 
 
 Dennemark, L., 658 
 
 Derrien, E., glucose, 215; blood pig- 
 ments, 281, 285, oxymethylfurfurol, 
 419; Bence-Jones, protein, 792 
 
 Desgrez, A., 393 
 
 Dencher. P., 532, 539, 540 
 
 Devilard, P., 360 
 
 Devoto, L., 792 
 
 Dewitz, J., 843 
 
 Diakanow, C, 243-245 
 
 Diamare, V., 494 
 
 Dick, AL, 702 
 
 Diels, O., 445, 446, 448 
 
 Diesselhorst, G., 848 
 
 Dietrich, M., 104, 652 
 
 Dietschv, R., 792 
 
 Dietz, VV., 56, 60 
 
 Dietze, Alb., 506 
 
 Dillner, H., 633 
 
 Dimitz, L., 248, 614 
 
 Disque, L., 740, 742, 744 
 
 Ditthorn, Fr., 167, 173, 219 
 
 Dittrich, P., 285 
 
 Ditz, H., 726 
 
 Doblin, A., 329, 333 
 
 Dorpinghaus, Th., carbohydrates in pro- 
 tein substances, 84;' protein hydrolysis, 
 113, 141, 142, 252 
 
 Dohm, M., 406 
 
 Dombrowsky, St., urinary pigments, 
 740, 741; oxyproteic acids in urine, 
 753, 754; urinary bases, 757 
 
 de Domenicis, A., 287 
 
 de Dominicis, N., 404 
 
 Donath, J., 360 
 
 Donne, A., 799 
 
 Donv-Henault, O., 873 
 
 Donze, G., 680 
 
 Doree, Ch., 448 
 
 Dormann, E., 298 
 
 Dorner, G., 574 
 
 Douglas, C. G., 286 
 
 Douglas, Gordon C, 864 
 
 Doyon, M., fibrinogen, 252, 253, 335; 
 serum lipase, 265; blood coagulation, 
 324, 325; glycolysis, 332; bile, 415, 416, 
 434, 439 
 
 Dragendorff, D„ 800 
 
 Drechsel, E., proteins, 76, 79, 94, 123; 
 diamino-acetic acid, 163; lysin, 163, 
 164; purin bases, 188; thyroid gland, 
 375; jecorin, 385; urea formation, 
 681, 683; carbamic acid, 683; silicic 
 acid ester, 838 
 
 Dreser, H., 13, 71 
 
 Dreyfus, G. L., 465 
 
 Droop-Richmond, H., 646 
 
 Drosdoff, W. f 335 
 
 Dubelir, D., 920 
 
 Ducceschi, V., 475, 569 
 
 Duclaux, E., 97, 647 
 
952 
 
 INDEX OF AUTHORS 
 
 Ducleau, E., 55 
 Dudley, H. W., 583, 775 
 During, Fr., 554 
 Dufau, E., 80S 
 Dufourt, 415, 416, 439, 592 
 Duggan, C. W., 97 
 Dull, G., 229 
 Dumas, J. A., 684 
 Dunham, E., 180, 239, 673 
 Dunlop, J. C, 595, 715 
 
 Ebbeke, U., 757 
 
 Ebstein, E., 114, 208, 209 
 
 Ebstein, W., 457, 727, 831 
 
 Eckhard, C, 452 
 
 Edelstein, E., 43 
 
 Edelstein, F., 657, 662 
 
 Edic, E., 246, 402 
 
 Edkins, J. S., 463, 477, 509 
 
 verEecke, A., 925 
 
 Ehrenfeld, R., proteins, 83; leucine, 143; 
 tyrosin, 153 
 
 Ehenreich, M., 49, 505 
 
 Ehrenthal, W., 521 
 
 Ehrlich, F., amino-acids, 143, 144, 153, 
 206; fermentation, 157; fusel oil, 206 
 
 Ehrlich, P., side chain theory, 67, 
 68; amboceptors, 69; dimethylamino- 
 benzaldehyde, 159, 219, 746, 827; 
 bilirubin, 431; urine test, 826 
 
 Ehrmann, R., 405 
 
 Ehrstrom, R., histone, 108; phosphate, 
 761,762,796; albumoseuria, 791 
 
 Eichholz, A., 260, 633, 743 
 
 Eichhorst, H., 525 
 
 Einhorn, M., 159 
 
 Ekbom, A., 426 
 
 Ekehorn, G., 760 
 
 van Ekenstein, A., amino-sugar, 167, 
 219; carbohydrates, 201, oxymethyl- 
 furfurol, 211, 215, 217; acetone, 825 
 
 Ekholm, K., 936 
 
 Elias, H., 609 
 
 Ellenberger, W., 479, 480, 658, 659 
 
 Ellinger, A., isoserin, 145; tryptophan, 
 155, 156; arginin, 163; blood coagula- 
 tion, 324; lymph formation, 351, 352; 
 pancreatic secretion, 500; urine in- 
 dican, 728, 730; tri-indylmethane pig- 
 ments, 734; kynurenic acid, 739; 
 oxyphenyllactic acid, 780; food value 
 of.albumoses, 912 
 
 Ellmer, A., 237 
 
 Ely, J., 455, 457 
 
 Embden, G., cystein and cystine, 79, 
 147, 149; serine, 145; glycolysis, 333, 
 408; carbohydrate formation. 410; 
 liver blood perfusion, 529, 530, 775, 
 786; lactic acid, 583, 584, 333; gly- 
 cocoll in urine, 756; acetone bodies, 819 
 822, 825, 826 
 
 Embden, H., 735 
 
 Emerson, R. L., 507 
 
 Emich, Fr., 517 
 
 Emmerling, A., 113 
 
 Emmerling, O., 58, 59, 225 
 
 Emsmann, Otto, 463 
 
 Engel, 662 
 
 Engel, H., Upases, 57, 476; lactic acid 
 
 formation, 585; acetone bodies, 818, 
 
 821 
 Engel, St., 658, 663 
 Engeland, R., agmantin, 162; aporrheg- 
 
 men, 166; carnitin, 577; methvl-guani- 
 
 din, 698; urine bases, 757, 826^ 827 
 Engelmann, G. J., 595 
 Engler, C., 871, 875 
 Eppinger, H., 375, 406, 676, 799 
 Eppinger, P., 290, 292, 293, 354 
 Epstein, A., 721, 722 
 
 Erben, Fr., oxystearic acid, 233; leuco- 
 cytes, 307, 342; blood analysis, 342; 
 
 chyle fat, 346; urine nitrogen, 681, 
 
 urein, 691 
 Erdelvi, A., 482 
 Erikson, A., 50, 63 
 Erlandsen, A., phosphatides, 242, 243, 
 
 245, 246, 586; cuorin, 248, 249; phlor- 
 
 hizindiabetes, 400 
 Erlanger, J., 541 
 Erlenmeyer, E., 142, 152 
 Erlenmeyer, E. (jr.) 145, 148, 152 
 d'Errico, G., 391 
 Esbach, G., 793 
 Escher, Heins, H., 623, 631 
 Estor, A., 867 
 Etard, A., 578 
 Etti, C., 642 
 Ettinger, J., 43 
 Eulenburg, 398 
 v. Euler, H., activators, 52; enzymes, 53,. 
 
 56, 70; phosphoric acid ester, 205; 
 
 erepsin, 493; oxidation processes, 873 
 Eves, F., 457 
 Ewald, Aug., proteins, 112, 119; haema- 
 
 toidin, 301; digestion, 508; visual 
 
 purple, 615; corpora lutea, 623 
 Ewald, C. A., 857, 928 
 Ewins, A. I., 380 
 Eykman, C., 326, 929, 932 
 Eymonnet, 757 
 
 Fabian, E., 396 
 
 Fahr, G., 587 
 
 Fajans, K., 35 
 
 Falck, 910 
 
 Falk, Edm, 642 
 
 Falk, Ernst, 924, 925 
 
 Falk, Fr., 248, 613 
 
 Falloise, A., bile, 416; gastric lipase,. 
 
 476; intestinal enzyme; 492, 493; 
 
 chloralsecretin, 499; blood gas tension, 
 
 861, 866 
 Falta, W., thyroid gland, 375; diabetes 
 
 405, 406, 409, 412; alcaptonuria, 735, 
 
 736; protein metabolism, 882, 883, 907 
 Fano, G., 251 
 Farkas, K., 638 
 
INDEX OF AUTHORS 
 
 953 
 
 Farmer, Ch., 688 
 
 Farwik, B., 599 
 
 Fasal, H., 106, 107, 158, 652 
 
 Faust, E., sepsine, 47; gelatin, 118: 
 poisons in the secretion of the skin and 
 salivary glands, 847 
 
 Favre, P. A., 848 
 
 Fawitzki, A., 685 
 
 Fedeli, C, 686 
 
 Feder, L., 682 
 
 Fehling, H., 214, 808 
 
 Fehrsen, A., 338 
 
 Fetgin, P., 721 
 
 Feinshmidt. J., 408 
 
 v. Fenyvessy, Bela, 751, 785 
 
 Fermi, CI., 255, 486, 505 
 
 Fernet, E., 855 
 
 Ferrv, E., 904 
 
 Kick, A., 596 
 
 Fiessinger, N., 307, 364 
 
 Fiiehne, \V., 440 
 
 Filhol, 670 
 
 De Filippi, F., 397, 534, 757 
 
 Fine, M. S., 692 
 
 Fingerling, G., 667 
 
 Fink, H., 295 
 
 Finkler, D., 41 
 
 Fischer, Ch., 140 
 
 Fischer, Kuril, enzymes, 58, 59; specif- 
 icity of enzymes action, 62, 65; amino- 
 acids, 85, 140-145, 148, 152-155 
 polypeptides, 86-89, 124, 132, 133 
 136; protein hydrolysis, 113, 119, 120 
 132, 133, 136, 507; ornithin, 162 
 lysin, 163; diaminotrioxydodecanoic 
 acid, 165; purin bases, 186, 188-191 
 pyrimidine bases, 194, 195; carbo- 
 hydrates, 197-202, 211, 212, 215, 216 
 glucosamine, 218; glucuronic acid, 220 
 isomaltose, 225; lactose fermentation 
 654; uric acid, 698; urine purines, 711 
 conjugated glucuronic acids, 751 
 
 Fischer, Hans, blood pigment derivatives 
 295-297, 295; bile acids, 425, 427 
 bilirubin, hydrobilirubin, urobilinogen 
 and urobilin, 429, 744, 746, 747, 429 
 koprosterin, 448; urobilinoids, 743 
 bilirubic acid, 429 
 
 Fischer, H. W., 587 
 
 Fischer, Martin, 400 
 
 Fischler, E., 686 
 
 Fischler, M., 434, 744 
 
 Fisher, H. L., 694 
 
 Fiske, P. S., 36, 38 
 
 Fitzgerald, 476 
 
 Flack, M., 374 
 
 Flacher, Fr., 379 
 
 Planum.!, CI., 152, 153, 734 
 
 Flanders, Fr., 723 
 
 Flatow, L., 738, 739, 779, 780 
 
 Flatow, R., 711 
 
 Fleckseder, R., 455 
 
 Fleig, C. 416, 498, 499 
 
 Fleischer, R., 402 
 
 Fleischl, E., 303, 441 
 
 Fleischmann, W., (>47 
 
 Fleischer, M. S., 320 
 
 Fleitmann, 79 
 
 Fletcher, W. M., 459, 585, 593 
 
 Flint, A., 448, 488 
 
 Floresco, N. f 3X4, 388, 434 
 
 Fluckiger, M., 749, 750 
 
 Foa C, 455, 64.^, 659 
 
 Folin, ()., cystine, 149; tyrosine test, 154; 
 blood alkalinity, 309; urea, 333, 335; 
 urine acidity, 677; nitrogen determina- 
 tion, 688; urea determination, 688- 
 690; urein, 691; methylurea, 691; 
 creatinine and creatine, 691-693, 696, 
 697; uric acid, 700, 708, 711, 334; 
 hippuric acid, 723; urinary sulphur, 
 752, 766; ammonia, 768, 769; acetone, 
 825; protein metabolism, 910 
 
 Fordos, M., 365 
 
 La For-e, F., 179, 180, 185 
 
 Forrest, J. R.. 553 
 
 Forschbach, J., 396 
 
 Forssner, G., 756, 819, 820 
 
 Forster, J., mineral metabolism, 72, 899; 
 transfusion, 344; water and metabol- 
 ism, 920; metabolism of sucklings, 925; 
 diets, 932, 939 
 
 Fosse, R., 681 
 
 Foster, M. L., 159 
 
 Foster, N. B., 694 
 
 Franckel, P., 465 
 
 Frankel, A., 685, 860 
 
 Frankel, Sigm., proteins, 79, 102; pep- 
 tones, 131; thiolactic acid, 151; his- 
 tidin, 160; albumin, 219; cephalin, 
 248; thyroids, 375; adrenalin, 380; 
 glycogen, 396; gastric juice, 476; 
 chondrosin, 548; brain phosphatides, 
 605, 609; brain analyses, 613, 614; 
 neottin, 630, kidney phosphatides, 
 673; homogentisic acid, 738; chitin, 
 839; protein cleavage, 925 
 
 Frankel, W., 34 
 
 Framm, F., 120, 213, 332 
 
 Franchimont, A. P., 839 
 
 Frank, E., 265, 329-332 
 
 Frank, Fr., 702, 706 
 
 Frank, O., 535-537, 591. 910 
 
 Frankland, E., 885 
 
 Franz, Fr., 251 
 
 Frauenberger, Fr., 547 
 
 Frazer, J. C. W., 3 
 
 Fredericq, L., proteincoagulation 97; 
 serumglobulin, 260; haemocyanin, 303, 
 304; blood coagulation, 313" blood 
 gases, 852, 861, 862, 864, 866 
 
 Frehn, A., 663 
 
 Fremv, E., 603, 636 
 
 Frenkel-Heiden, 360 
 
 Frenzel, J., glvcogen, 391, 393; work and 
 fat destruction, 596, 598; meat, 600; 
 calories and nitrogen, 892 
 
 Frerichs, F. Th., synovia, 362, human 
 
^54 
 
 INDEX OF AUTHORS 
 
 bile, 437; saliva, 458; uric acid de- 
 composition, 705 
 
 Freudberg, A., 309 
 
 Freund, E., serumglobulins, 259; albu- 
 moses in blood, 263; haematinof: en, 299; 
 blood coagulation, 313, 320; glycogen, 
 395; .digestion blood, 530; chlorine 
 determination, 759; lungs, 870; starva- 
 tion metabolism, 987 
 
 Freund, O., 897 
 
 Freundlich, H., 22; surface tension, 28; 
 adsorption, 29 
 
 Prey, W., 756 
 
 Frevtag, Fr., cerebrosides, 364, 607, 610, 
 protagon, 606, 607, 610 
 
 Fridericia, L. S., 597 
 
 Friedenthal, PL, 467, 525 
 
 Fried jung, J. K., 664 
 
 Friedlander, G., 525 
 
 Frienlander, P., 843 
 
 Friedmann, E., protein sulphur, 79; 
 thiolactic acid, 113, 151; albumoses, 
 134; isoleucine, 144; cystine, 148; 
 cysteinic acid and taurine, 148, 151, 
 442; adrenalin, 379; demolition of 
 fatty acids, 774, 776, 781; of aromatic 
 substances, 779-781, 736; furfurol, 
 784; acetone former, 820, 822 
 
 Friend, W. M., 274, 356 
 
 Fries, H., 593 
 
 Frohlich, 379 
 
 Fromherz, K., 735, 736, 780 
 
 Fromholdt, G., 743, 744 
 
 Fromm, E., 785 
 
 Fromme, A., 57, 476 
 
 Frommer, V., 823 
 
 Frouin, A., thyroid gland, 377; gastric 
 juice, 463, 464; intestinal juice, 490- 
 492; pancreatic juice, 496 
 
 Frugoni, C., 405 
 
 Fubini, S., 849 
 
 Fuchs, D., 114, 154 
 
 Fiirbringer, P., 715, 791 
 
 v. Furth, O., peroxyproteic acids, 83; 
 xanthoprotein, 83; nucleic acids, 184; 
 cholin, 247; iodothyrin, 376; supra- 
 renin, 379; adrenalinglucosuria, 405; 
 cholic acid, 424; secretin, 498; bile 
 and fat splitting, 502; muscles, 566- 
 572, 589, 590; camosin, 575; smooth 
 muscles, 602; chitosin, 839; melanins, 
 841-843; tyrosinase, 843, peroxydase, 
 873 
 
 Fuld, E., rennin action, 57, 474, 650; 
 fibrin formation, 256, 318, 319, 324; 
 pepsin determination, 470; womans' 
 milk, 660, 662 
 
 Fuller, J. G., 902 
 
 Funk, C., amino-aoid combination, 87, 
 88; glutamic acid, 146; urinary sul- 
 phur, 752; sugar determination, 809; 
 vitamin'', 905 
 
 v. Funke, 595 
 
 Gabriel, S., cystine, 148; bones, 552; 
 teeth, 558; ovalbumin, 633; food- 
 value of aspargin, 912 
 
 Gache, J., 496 
 
 Gaglio, G., 334, 582, 773 
 
 Galdi, F., 354 
 
 Galeotti, G., 96, 706 
 
 Galimard, J., 113 
 
 Galli, P., 267 
 
 Gallois, 580 
 
 Gamgee, A., nucleoproteins, 175; nucleic 
 acids, 182; blood pigments, 280; 
 intestinal juice, 490; protagon, 606, 
 607; pseudocerebrin, 611 
 
 Gammeltoft, S. A., 689, 690, 767 
 
 Ganassini, D., 455, 456, 709 
 
 Gansser, E., 278 
 
 Gardner, J. A., 448 
 
 Garrod, A. E., hsematoporphyrin, 295, 
 299, 797; alcaptonuria, 738; homo- 
 gen tisic acid, 738, 739; urochrome, 740, 
 741; urobilin, 743, 745; uroerythrin, 
 748; cystinuria, 827 
 
 Gaskell, J. F., 828 
 
 Gassmann, Th., 552, 557, 558 
 
 Gatin-Gruzewska, Z., glycogen, 19, 20, 
 390, 391; seralbumin, 261 
 
 Gaube, T., 848 
 
 Gaule, J., 340 
 
 Gaunt, 41 
 
 Gautier, A., ptomaines, 47; arsenic, 72, 
 269, 336, 368, 373, 838; glycogen, 392; 
 fat formation, 562; muscles, 578; 
 protein of hen egg, 633, 634; xantho- 
 creatininine, 698; fluorine, 552, 558 
 
 Gautier, CI., 252, 253, 654, 744 
 
 Gawinski, W., 756 
 
 v. Gebhardt, F., 591, 909 
 
 Geelmuvden, H. C., sugar in urine, 814, 
 816; acetone bodies, 819, 820, 822, 826 
 
 Geiger, W., 145 
 
 Geissler, 791 
 
 Generali, F., 374 
 
 Gengou, O., 64, 314, 318, 320 
 
 v. Genser, 665 
 
 Gentzen, M., 728 
 
 Geoghegan, E. G., 609, 610, 614 
 
 Gephart, F., 245, 680, 689, 769 
 
 Geppert, J., blood alkalinity, 309; respira- 
 tion, 860, 869, 890; alcohol, 921 
 
 Gerard, E., 446, 699 
 
 Gerber, N., 662 
 
 Gerhardt, C., 824 
 
 Gerhardt, D., 744, 745 
 
 Gerhartz, H., 870 
 
 Gerngross, O., 195 
 
 Gertner, W., 416 
 
 Gessard, C., 843 
 
 Gewin, J. W. A., 475 
 
 Geyer, J., 806 
 
 Geyger, A., 735 
 
 Giacosa, P., mucins, 167; blood pigments, 
 302; frog egg, 636; iron in urine, 770; 
 aromatic substances, 779 
 
INDEX OF AUTHORS 
 
 955 
 
 Giaja, J., 71, 231 
 
 Gibson, R., 103 
 
 Giertz, H., 104, 177 
 
 Gies, W. J., elastin, 116-1 IS; hexone 
 bases, 164; mucin substances, 16,N, 
 L89, 472, 644, 645, 551; lymph, 349 
 351; pancreatic juice, ■"><>(>; ligaments 
 and tendons, 545; bones, f>f)l; pro- 
 tagon, 606, 608; phrenosin, 611; 
 urein, 691 
 
 Gigon, A., polypeptides, 65; diabetes, 
 405, 409; amino-acid supply, 721, 
 722; amino-acide in urine, 756; basal- 
 requirement, 898, 931; metabolism, 
 929-931 
 
 Gilbert, 531, 563 
 
 Cull, F. W., 689, 752 
 
 Gilson, E., 242, 245, 839 
 
 Ginsberg, S., 534 
 
 Ginsberg, VV., 754, 756 
 
 Githens, Th., St., 270 
 
 Giunti, L., 773 
 
 Gizelt, A., 498 
 
 Gjaldbak, I. K., 58, 135, 165 
 
 Glaessner, K., pseudopepsin, 466, 489, 
 490; erepsin, 493; pancreatic juice, 
 500, 509 
 
 Gleiss, W., 593 
 
 Glendinning, T. A., 55 
 
 Gley, E., iodine in blood, 269; blood 
 coagulation, 324; lymphagogues, 351; 
 thyroidea, 374; pancreatic juice, 496, 
 498; heart muscle; 599, vesiculase, 
 622 
 
 Glikin, W., fat, 233; lecithin, 241, 244, 
 245, 663; liver nitrogen, 387; choles- 
 terin, 445; milk, 663 
 
 Glund, W., 87 
 
 Gmelin, L., 432, 458 
 
 Gmelin, W., 465 
 
 Gogitidse, S., 669 
 
 Goldmann, E., ovstine and cystinuria, 
 149,827,828; iodothyrin, 376 
 
 Goldmann, F., 813 
 
 Goldschmidt, C, 687 
 
 tioldschmidt, F., 133 
 
 Goldschmidt, H., 36, 456 
 
 Golodetz, L., 844 
 
 Gompel, M., 65 
 
 Gonnermann, M., 140, 508 
 
 Goodbodv, W., 515 
 
 Goodwin, R., 130 
 
 Gorodecki, 443 
 
 Gortner, R. A., 841, 842 
 
 v. Gorup-Besanez, E. F., 356, 437, 838 
 
 Gossmann, H., 495 
 
 Gosio, B., 582 
 
 Goto, M., 110, 111, 709 
 
 Gottlieb, R., urea, 333; bile, 435; creatine 
 and creatinine, 573, 692, 698; urine 
 purines, 711; oxyproteic acid, 754; 
 iron in urine. 770 
 
 Gouban, F., 366, 367 
 
 Gourhay, F., 373 
 
 • IrCraaf, C. J. H., 792, 793 
 Grabowski, J., 297 
 
 Graebe, C., 524 
 
 Graffenberger, L., 554 
 
 Grafe, E., 54, 912, 919 
 
 Graham, I)., 919 
 
 Graham, Th., 13, 14, 18, 31, 90 
 
 Granstrdm, E., 718, 769 
 
 Grawite, E., 340 
 
 Grebe, F., 396 
 
 Green, E. H., 121 
 
 Green, J. R., 255, 316 
 
 Greer, James Richard, 348, 349 
 
 Gregersen, J. P., 903 
 
 Gregor, A., 698 
 
 Gregor, G., 766 
 
 G reliant, N., 333, 335, 684, 773 
 
 Griesbach, W., 333 
 
 Griffiths, A. B., 614, 758 
 
 Grimaux, E., 85, 86, 323 
 
 Grimbert, L., 677, 746, 747 
 
 Grimm, F., 744 
 
 Grimmer, W., 482, 490, 874 
 
 Crindley, H. S., 689, 752, 936 
 
 Griswofd, W., 457 
 
 Grober, J., 467 
 
 Grober, A., 732 
 
 Groho, B., 485 
 
 Groll, S., 339 
 
 Grosjean, A., 324 
 
 Gross, A., 358, 629 
 
 Gross, O., pepsin determination, 469; 
 trypsin determination, 506; lens, 619; 
 alkaline earths in urine, 769 
 Gross, W., 463, 780, 818 
 Grossenbacher, H., 372 
 Grosser, P., 733 
 Grossmann, H., 808 
 Grouven, H., 599 
 Grube, K., 396, 400 
 Gruber, D., 229 
 Gruber, M., 8S1, 920 
 Griibler, G., 94 
 Griinbaum, D., 642 
 Griindler, J., 777 
 Griinhagen, A., 361 
 < initzner, B., 790 
 
 Gri'itzner, P., pepsin determination, 469; 
 sast'ic contents, 479, 480; Brunner'a 
 glands, 489, 490; pancreatic diastase, 
 501 
 Grund, G.. 208, 209, 389, 919 
 Grutterink, A., 792, 793 
 Gryns, G., 7 
 Gscheidlen, R., rhodan, 455, 456, 751; 
 
 lactic acid, 582; urea, 684 
 Gubler, A., 348, 349 
 Gudzent, F., 707, 708 
 Gumbel, Th., 77 
 Gunther, G., 622 
 
 Gurber, A., ion permeability, 8, 310; 
 serumalbumin, 261, 263; serum, 267; 
 bile, 440; amniotic fluid, 642 
 Guerin, G., 758 
 
956 
 
 INDEX OF AUTHORS 
 
 Guest, H. H., 106, 107 
 
 Guggenheim, M., 50, 380 
 
 Guigan, H., Mc, 399 
 
 Guillemonat, A., 371, 387 
 
 Guinochet, E., 358 
 
 Guldberg, C. M., 32 
 
 Gulewitsch, W., arginine, 161, 162; 
 thymin, 195; choline, 247; trypsin, 
 508; meat extractive, bases, 575-578 
 
 Gullbring, A., 421 
 
 Gumilewski, 491 
 
 Gumlich, E., 680, 685, 761 
 
 Gundermann, K., 82 
 
 Gunning, J. W., 823 
 
 Gusserow, A., 716, 717 
 
 Guth, F., 233 
 
 Guyenot, E., 457 
 
 Gyergyai, A., 912 
 
 de Haan, J., 306 
 
 Haas, E., 528, 910 
 
 Haberlandt, L., 265 
 
 Habermann, J., proteins, 83; amino- 
 acids, 143, 146, 153 
 
 Hamalainen, J., glucuronic acid conjuga- 
 tion, 785; sulphur elimination, 882, 
 883; "protein catabolism, 907 
 
 Handel', M., 549 
 
 Hansel, E., 487, 663 
 
 Haser, 771 
 
 Hausermann, E., 340 
 
 Hafner, A., 233 
 
 Hagemann, O., skin breathing, 849; 
 blood gases, 851; metabolism, 926, 
 927, 929 
 
 Hagen, W., 193 
 
 Hahn, M., fermentation, 41; casein 
 digestion, 651, urea formation, 683, 
 684 
 
 Haig, A., 701 
 
 Haiser, F., inosinic acid, 178-180; 182, 
 183; carnine, 577 
 
 Haldane, J., blood pigments, 285, 286, 
 302; amount of blood, 343; oxygen 
 tension, 862, 863 
 
 Hall, W., purine bases, 193, 520, 572; 
 iron absorption, 340; amino-acids in 
 
 urine, 757 
 
 Hallauer, B., 440 
 
 Halle, W. L., 380 
 
 v. Hallervorden, E., 682, 685, 767 
 
 Halliburton, W. D., protein coagula- 
 tion, 97; dextrins, 234, choline; 246; 
 fibrin ferment, 256; eerum albumin, 
 261; blood serum, 270; stroma of 
 blood corpuscles, 274; tetronerythrin, 
 304, 843; leucocytes, 306; blood 
 coagulation, 323; pericardial fluid, 
 356; cerebro-spinal fluid, 360; liver, 
 382; glycogen, 391; pancreatic renin, 
 509; myxcedema, 545; bone marrow, 
 553; muscles, 566-570; brain proteins, 
 604, 605; diseases of the nervous sys- 
 tem, 614; kidneys, 673 
 
 Halpern, H, 372 
 
 Halsey, J. T., 152 
 
 Ham, C. E., 301 
 
 Hamburger, C., 265, 456 
 
 Hamburger, E. W., 435 
 
 Hamburger, H. J., blood corpuscles, 6-8, 
 273, 304, 305, 311; blood alkalinity, 
 269; blood serum, 271; phagocytoses, 
 306; blood alkalinity, 309, 310; lymph 
 formation, 350, 351; ascitic fluid, 359; 
 intestinal juice, 490, 491; enterokinase, 
 496; absorption, 542-543 
 
 Hammarsten, O., rennin action, 54, 63, 
 473, 474, 475, 650; nucleoaJbumin, 
 105; mucin substances, 168-169; 
 helicoproteid, 173; nucleoproteins, 
 208, 383, 494, 495 ; fibrinogen, 
 fibrin and blood coagulation, 253, 
 254, 257, 263, 316; fibrin globulin, 
 258; globulins, 259-261, 263, serum- 
 albumin, 262; blood plasma and serum, 
 267-270, hsematoporphyrin, 299, 797, 
 798; gases of lymph, 346, 856; transu- 
 dates, 354, 356, 357, 359; synovia, 362; 
 human bile, 414, 437, 438; bile of 
 various animals, 417, 427, 439; bile acids 
 417, 420-427, 438; bile pigments, 267, 
 432, 434, 801; phosphatides, 435, 
 439; saliva, 457; pepsin, 466, 467; 
 trypsin, 503; pseudomucin, 625, 626; 
 perch eggs, 630, 636; casein, 649; 
 lactoprotein, 653; area in bile, 679; 
 protein in urine, 793; sugar determina- 
 tion in urine, 806 
 
 Hammerbacker, F., 458 
 
 Hammerl, H, 521 
 
 Hammerschlag, A., 308, 309 
 
 Hammerschlag, G., 637 
 
 Handovsky, H, 18 
 
 Henriot, M., lipases, 59, 265; djiabetes, 
 404; respiration quotient, 563; respira- 
 tion, 869 
 
 Hansen, C., lecithin, 242; protein syn- 
 thesis, 529; fatty tissue, 559; fat of 
 yolk, 630; fat of milk, 669; food value 
 of albumoses, 912; asparagin, 912 
 
 Hansen, W., 233 
 
 Hanssen, O., amyloid, 171, 172, 173, 
 547 
 
 Harden, A., co-enzyme, 52, 205; phos- 
 phoric acid ester, 60, 205; dioxyacetone, 
 205; glycolysis, 407, 583 
 
 v. Hardt-Stremayr, 77 
 
 Hardy, W. B., colloids, 15, 20, 21, 25, 26, 
 30; ovalbumin, 634 
 
 Hari, P., 54 
 
 Harkink, I., 594, 693 
 
 Harley, V., sugar of blood, 398; intestinal 
 putrefaction, 515; pancreas, 532, 539, 
 540; large intestines, 541, urinary 
 pigments, 740, 744 
 
 Harms, H, 552 
 
 Harnack, E., ash-free protein, [95; iodo- 
 spongin, 122; blood pigments, 286, 
 
INDEX OF AUTHORS 
 
 957 
 
 287; hydramnion, 642; oxalic acid 
 poisoning, 728, 729; sulphur in urine, 
 752; demolition of halogen substi- 
 tuted methane, 777; galic and tannic 
 acid, 785 
 
 Harries, C, S3 
 
 Harris, J. F., 77, 179, 185 
 
 Hart, A. 8., 116, 117 
 
 Hart, E., protein nitrogen, 77; albumoses, 
 134; histidine, 160; hexone bases, 164; 
 
 Hart, E. B.. 902 
 
 Hartley, P., 384, 385 
 
 Hartogh, 412 
 
 Hartung, C, 636 
 
 Hartwell, J. A., 130 
 
 Hasebroek, K., lecithin, 245, 515; peri- 
 cardial fluid, 356; digestion products, 
 472 
 
 Haskins, H. D., 680, 683, 691 
 
 Haslam, H. C, salting out of proteins, 
 95, 250; albumoses, 139 
 
 Hasselbalch, K. A., reaction of blood, 75, 
 310; methsemoglobin, 283, 284; egg- 
 hatching, 637; sugar determination, 
 814; oxygen intake, 859; oxygen 
 tension, 867 
 
 Hatcher, R. A., 397 
 
 Hauff, 665 
 
 Hausmann, W., nitrogen in proteins, 77, 
 119; haematoporphyrin, 295; kopros- 
 terin, 448; cholesterin, 449-450 
 
 Hawk, P. B., keratin, 112; bones, 551; 
 Eck's fistula, 683; sugar determina- 
 tion, 804; hair, 838; metabolism, 892, 
 897, 920 
 
 Hay, M., 358 
 
 Haycroft, J. B., protein coagulation, 97; 
 blood coagulation, 251, 313; diabetes, 
 403; biliverdin, 433 
 
 Havem, G., 307, 342 
 
 Heckel, F., 141 
 
 Hedenius, 116, 434 
 
 Hedin, S. G., blood corpuscles, 7, 8; 
 autolysis, 42-44; adsorption, 49, 50; 
 enzymes, 54-57; retardation of enzyme 
 action, 63-65; rennin-antirennin bind- 
 ing, 68; elastin, 116; histidine, 159, 
 160; arginine, 161; lysine, 163; blood, 
 326; haematocrit, 326; lienases, 371; 
 rennin zymogen. 473; rennin enzyme, 
 474; muscle protease, 571 
 
 Hedon, E., pancreas diabetes, 406; 
 absorption of sugar, 533; of fat, 539, 
 540 
 
 Heffter, A., liver, 385; muscle, 565, 589; 
 lactic acid, 593; hyposulphite in urine, 
 753; foreign substances in urine, 773; 
 hippuric acid synthesis, 783; reduc- 
 tions, 877 
 
 Heger, P., 381 
 
 Heidenhain, M., 101 
 
 Heidenhain, R., lvmphagogues and lymph, 
 11, 345, 349-351; transudates, 353; 
 bile, 414, 415; saliva, 453, 459; stom- 
 
 ach, 461, 476, 477; pyloric secretion, 
 477; pancreas and its secretion, 494, 
 495, 497, 503; absorption, 528, 534; 
 smooth muscles, 602 
 
 Heilner, E., protein assimilation, 526; 
 metabolism, 920, 929; specific dynamic 
 action, 930 
 
 Heinemann, H. N., 597 
 
 Heinsheimer, Fr., 476 
 
 Heintz, W., 235, 582 
 
 Heiss, E., 555 
 
 Heitzmann, C., 555 
 
 Heckma, E., intestinal juice, 490-493, 
 enterokinase, 496, 497 
 
 Hele, T. Sh., 738, 828 
 
 Heller, Fl., protein test, 99, 789; uroxan- 
 thin, 727; urinary pigments, 740; 
 blood test, 796; urinary calculi, 834- 
 835 
 
 Hellgren, W., 931 
 
 Hellwig, 602 
 
 Helm, 461 
 
 Helme, W., 882, 883, 907 
 
 Hetper, J., 292 
 
 Hemmeter, J. C., 463, 464, 519 
 
 Hempel, E., 894 
 
 Henderson, L. J., 309, 675, 677 
 
 Henderson, Y., 77, 402, 529 
 
 Henkel, Th., 647 
 
 Henneberg, W., 510, 918 
 
 Henninger, A., 137 
 
 Henogque, A., 303 
 
 Henri, V., cataphoresis, 50, 51; enzymes, 
 56; white of egg, 65; saccharase, 65; 
 diastase, 71 
 
 Henriques, R., 239 
 
 Henriques, v., plastein, 58, 135; formol 
 titration, 165; lecithin, 242; lecithin 
 sugar, 331, 332; protein synthesis, 
 529; fat tissue, 559; fat of yolk, 630; 
 fat of milk, 669; urea determination, 
 689, 690; hippuric acid, 723; urine nitro- 
 gen, 756; ammonia, 768: gases of blood, 
 851; metabolism in lungs, 85S; food 
 value, albumoses, 912; asparagin, 912 
 
 Hensel, Marie, 726 
 
 Hensen, V., 349, 856 
 
 Henze, M., gorgonin and iodogorgonic 
 acid, 123, 146; protein of octopoilcs, 
 175; hsemocyanin, 303, 304; bloo 1 
 corpuscles in ascitic fluid, 304; liver, 
 388; spongosterin, 448; muscles, 603 
 
 Heptner, F. K., 438 
 
 l'Heritier, 665 
 
 Herlaut, I... 185 
 
 Herlitzka, A., 455 
 
 Hermann, E., 72 
 
 Hermann, L., blood in starvation, 339; 
 formation of feces, 521; muscular 
 work, 592; allantoin, 716, 717 
 
 Heron, J., 229, 456, 492 
 
 Herrmann, A., 507, 700, 701 
 
 Hermann, Edm., 336 
 
 Herry, A., 325 
 
958 
 
 INDEX OF AUTHORS 
 
 Herter, C. A., indol and skatol, 159, 732; 
 
 urorosein, 732, 733 
 Herter, E., saliva, 458; ethereal sulphuric 
 
 acids, 724, 726, 784; oxybenzoic acids, 
 
 783; oxvgen tension, 861 
 Herth, R.,* 137 
 Hervierux, Ch., indol and indican, 266, 
 
 728-732; skatol red and urorosein, 
 
 733; uroerythrin, 748; glucuronic acids, 
 
 817 
 v. Herwerden, M., 651 
 Herzen, A., spleen and digestion, 372; 
 
 gastric juice secretion, 462; pancreatic 
 
 juice, 496, 498 
 Herzfeld, A., 203, 224 
 Herzfeld, E., 267, 433 
 Herzog, R. O., enzymes, 41; histidin, 
 
 160; lactic acid, 582, 616; oxydase 
 
 reactions, 875 
 Hess, K., 297 
 Hess, L., 43 
 Heubner, O., 25S, 569 
 Heuss, E., 847 
 Hewlett, A. W., 541 
 Hewlett, R. T., 97 
 Hewson, W., 6, 313 
 Heyl, F. W\, 185 
 Hevmann, F., 626 
 Heynsius, A., 128, 432-434 
 Hiestand, O., 245 
 Hilbert, P., 779 
 Hildebrandt, H., oxalic acid, 716, 773; 
 
 amino-benzoic acids, 783; toluols, 783; 
 
 glucuronic acid conjugation, 785, 786 
 Hildebrandt, P., antiemulsin, 64; mam- 
 mary glands, 644, 668; glucuronic 
 
 acids, conjugation, 750 
 Hildebrandt, W., 267, 744 
 Hildesheim, 932 
 Hildesheimer, A., 726 
 ililger, 171 
 Hilger, A., 579 
 Hill, A. V., 277, 278 
 ililler, E.. 554 
 Hirsch, Rahel., glycolysis, 408; hippuric 
 
 acid, 721; amino-acids in urine, 756 
 Hirsch, Paul, 722, 912 
 Hirschberg, A., 307 
 Hirschfeld, E., 485 
 
 Hirschfeld, F., work and nitrogen elimina- 
 tion, 595; uric acid, 700; acetone 
 
 bodies, 819; protein catabolism, 903, 
 
 915, 934; daily ration, 939 
 Herschfeld, H., 791 
 Hirsfhl, J. A., 806 
 Hirschler. A., 517. 518, 519, 724 
 Hirechstein, L., 700, 756 
 His, W., 550 
 
 Hifl, \Y. (jr), 193. 707, 787 
 Hlasiwetz, H.. 146 
 Hochhaus, H., 340 
 Hober, R., osmotic pressure, 13. 15; 
 
 precipitation properties of ions, 25; 
 
 alkalinity of blood, 310, 311; absorp- 
 
 tion, 542; permeability, 588; urine 
 acidity, 677 
 
 Hone, J., 800 
 
 Hoernes, Ph., 78 
 
 Horth, F., 582 
 
 v. Hoesslin, H., 909-911, 917 
 
 Hoyrup, M., 161 
 
 Hofbauer, L., 456 
 
 van't Hoff, J., osmotic pressure, 2, 3; 
 catalysis, 33, 35; glucosides, 59 
 
 Hoffman, Ch., 152 
 
 Hoffmann, A., 722 
 
 Hoffmann, F. A., transudates, 354, 357, 
 361, 362; sugar in blood, 398; glucos- 
 curia, 400 
 
 Hoffmann, J., 675 
 
 Hoffmann, P., 770 
 
 Hofmann, tyrosin test, 153 
 
 Hofmann, Fr., 559-561 
 
 v. Hofmann, Karl, 621 
 
 Hofmann, K. B., 116, 124 
 
 Hofmeister, F., gelatin, 31; cell enzymes, 
 44; protein nitrogen, 77, 82; amino- 
 acids, binding of 85; removal of pro- 
 teins, 102; collagen and gelatin, 118, 
 120; albumoses and peptones, 133, 136, 
 139; grouping of proteins, 159; serum- 
 globulins, 259, 261; pus, 364; actions 
 of stomach, 479, 480; protein absorp-^ 
 tion, 527, 528; assimilation limit, 533; 
 blood serum and earthy phosphates, 
 557; ovalbumin and protein crystalliza- 
 tion, 633, 634; urea formation, 684; 
 creatinine, 694; protein in urine, 793; 
 lactose in urine, 815 
 
 Hofmeister, V., 4S0, 510 
 
 Hohlweg, H., 267, 740, 741 
 
 Holde, D., 233 
 
 Hollinger, A., 328 
 
 Holmgren, E. S., 452, 806 
 
 Holmgren, Fr., 852, 866 
 
 Holmgren, I., 489, 566, 569 
 
 Holobut, Th., 696 
 
 v. Hoist, G., 169, 354, 362 
 
 Holsti, O., 903 
 
 Honore, Ch., 526 
 
 v. Hoogenhuyze, C. J., creatine and 
 creatinine, 573, 594, 691-694 
 
 Hooker, D., 351 
 
 Hooper, C. W., 442 
 
 Hopkins, F. G., halogen proteins, 82; 
 protein reaction, 100; tryptophan, 155- 
 158; protein crystallization, 263, 633, 
 634; lactic acid formation, 533; uric 
 acid, 699, 710, 711, 843; urobilin, 743, 
 746; Hence-Jones protein, 792; butter- 
 flies, S4M 
 
 Hoppo-Seyler, F., oxydation, 41; ovovi- 
 tellin, 105; collagen, 118; proteins,. 
 167; aucleins, 175; xanthin, 189; 
 lecithin, 243-245; blood plasma, 269; 
 blood corpuscles, 274, 304, blood pig- 
 ments, 275-278, 281, 283, 286, 287- 
 290, 293-302; urobilinoids, 295, 743; 
 
INDEX OF AUTHORri 
 
 959 
 
 glycogen, 307, 390; blood analysis, I 
 328; chyle, 347; pericardial fluid, 366; 
 pus, 303-365; strumacvstica, 374; 
 bile, 435, 430, 440; excretin, 523; 
 cartilage, 549, 550; bones and teeth, 
 553, 558; lactic acids, 583, 585, 503, 
 748; retina, 015; ovovitellin, 028; 
 milk, 047, 04N, 666; bile acids in urine, 
 800; inositc, S17; chitin, 839; sebum, 
 844; skin breathing, M0; respiration 
 apparatus, 80S; oxydation, 871 
 
 Hoppe-Seyler, G., blood pigment deter- 
 mination, 302; phenol elimination, 
 725; indoxvl, 728-732, urobilin, 744, 
 745, 747 
 
 Hop wood, A., 87 
 
 Horbaczewski, J., keratin, 112; elastin, 
 116, 117; purine bases, 188, 189; 
 uric acid, 373, 098, 700-702; urostealith, 
 834, S35; metabolism, 904 
 
 Hornborg, A. J., 464, 465 
 
 Honre, R. M., 251 
 
 Horodynski, W., 334, 336 
 
 Horowitz, L. M., 520 
 
 Horton, E., 59 
 
 Hoshiai. Z., 787 
 
 Hougardv, A., 261 
 
 Howe, P. E., 892 
 
 Howel, W. H., 257, 266, 319, 322 
 
 Hrvntsehak, Th., 723 
 
 Huber, A., 251, 255 
 
 Hudson, C. S., 55, 57, 199 
 
 v. Hiibl, 236, 238 
 
 Huebner, R., 159 
 
 Hufher, G., leucin, 142; blood pigments, 
 277-279, 283-286, 289; spectrophoto- 
 metry, 302, 303; bile, 420; gases 
 of bird egg, 636; urea determination, 
 690; oxygen tension, 859, S61, 862; 
 air bladder, 867 
 
 Hurthle, K., 264 
 
 Huaounenq, L., biliverdin, 434: ha?mato- 
 gen, 629; clupervin, 636; ash of milk, 
 and of child, 666 
 
 Huiskamp, VV., fibrinogen and fibrin for- 
 mation, 253, 254, 256, 258. 320; nucleo- 
 protein in blood, 258; thymus histone. 
 366. 367 
 
 Hulett. G. A., 29 
 
 Hultgren, E. O., utilization of nutrtives, 
 531,535,542, 543; diets, 9L 2, 936 
 
 Hummelberger. F., 126 
 
 Humnicki, V., 448 
 
 Hundeshagen, F., 122, 242 
 
 Hunter, A., 100. Ill, 134, 758 
 
 Hupfer. Fr., 721 
 
 Huppert. H., Schutze's rule. 57; diges- 
 tion products, 133: elycopen, 307, 391; 
 bile pigment reactions, 432; pepsin de- 
 termination. 4(10: Mesh, 601; urea, 686; 
 uroleucinic acid, 739; urine albumin, 
 794; laiose, 814; acetone, 825 
 
 Hurtley, W. H., 287, 739 
 
 Hurwitz, S. H.. 253 
 
 Husson, 071 
 Hutchinson, Hob., 553 
 Hybbinette, 8., 748 
 
 Ibrahim, J., 497 
 
 Ide, 578 
 
 Ignatowski, A., 827 
 
 Inagaki, ('., scrum albumin, 262; serum- 
 globulin, 270; blood corpuscles, 341; 
 protein absorption, 528; muscle rigor, 
 590 
 
 Inoko, Y., 277 
 
 Inouye, K., 179, 574, 748, 749 
 
 Inove, Z., 470 
 
 Irisawa, T., 334, 749 
 
 Ishihara, H., 423 
 
 Ito, M., 791 
 
 Iversen, A., 621, 623 
 
 Iwanoff, 763 
 
 Iwanoff, L., 182, 206, 508 
 
 Izer, G., autolysis, 43; uric acid, 704-706 
 
 Jaarswelde, G. J., 723 
 
 Jablonsky, J., 499 
 
 Jackson, H. C., 739 
 
 Jacobs, W. A., serine, 145; nucleic acid, 
 178-185; ribose, 211, glucothionic 
 acid, 369; sphingosin, 611; cerebronic 
 acid, 611 
 
 Jacobsen, A., 331 
 
 Jacobsen, C. A., 673 
 
 Jacobsen, O., 437, 438, 779 
 
 Jacoby, M., autolysis, 42, 45, 869; phos- 
 phorous poisoning, blood, 253, 255, 
 256; pepsin determination, 470; tryp- 
 sin determination, 506; fertilization, 
 640; uric acid demolition, 706 
 
 Jacubowitsch, 458 
 
 Jaeckle, H.. 233, 235, 559 
 
 Jaderholm. A., 285, 289 
 
 Jarger. A., 867. 843 
 
 Jaha, M. E., 936, 939 
 
 JaO'c. M., ornithine and ornithuric acid, 
 162, 7S3; bile pigments. 432, 433; 
 urobilin, 521; creatine and creatinine, 
 574, 575, 693, 695; phenylsemiear- 
 bazide, 687: urethane, 692; uric 
 acid, 703, 704, 708; indican in urine, 
 727-730; kyenuric acid, 739; urobilin, 
 740. 741, 744, 740; conjugated glu- 
 curonic acids, 751, 786; behavior of 
 organic substances, 778, 779, 783, 786; 
 furfurol, 7S4; thiophene, 784; guani- 
 dine acetic acid, 787 
 
 de Jager, L., 675, 677, 734, 756, 769 
 
 Jahnson-Blohm, G., 03 
 
 Jakowsky, M., 513 
 
 v. Jaksch, R.. blood alkalinitv, 309; 
 urea, 333; brain, 005; volatile fatty 
 acids, 748; melanin, 799; pentoseuria, 
 816; acetone, 818 
 
 Jalowetz, E., 226 
 
 Jamieson, G. S., 123 
 
 Janney, N., 768 
 
960 
 
 INDEX OF AUTHORS 
 
 Jansen, B. C. P., 491, 581 
 
 Jappelli, G., 453 
 
 Jaquet, A., 277 
 
 Jastrowitz, H., 469, 716, 757 
 
 Jastrowitz, M., 208, 815 
 
 Jelinek, J., 407, 583 
 
 Jensen, P., 581, 5S7 
 
 Jerome, W., Smith, 753 
 
 Jerusalem, E., nucleic acids, 184; lactic 
 acid,48S; melanins, 841-843 
 
 Jesner, S., 617 
 
 Jess, A., 619 
 
 Jessen-Hansen, H., 165, 809 
 
 Joachim, Jul., 259, 353, 357, 359 
 
 Jochmann, G., 307 
 
 Jodlbauer, A., 50, 552 
 
 Johansson, F., 757 
 
 Johansson, J., E., serumalbumin, 262; 
 tissue and gas metabolism, 597, 898, 
 927-931, 936; digestion work and 
 specific dynamic action, 930 
 
 Johnson, St", 691, 694 
 
 Johnson, T. B., protein . sulphur, 79; 
 thiopolypeptides, 87; nucleic acid, 185; 
 cvtosine, 194 
 
 John, S., 376 
 
 Jolles, A., pentoses, 209, 210, 816; bile 
 pigments, 431; milk, 664; urine 
 acidity, 677; urinometer, 679; uric 
 acid, 711; urobilin, 743; albumin in 
 urine, 787; nucleohistone, 795; fructose 
 determination, 814 
 
 Joly, 670 
 
 Jolvet, 851 
 
 Jones, D. B., 84, 106, 107 
 
 Jones, W., autolysis, 44; nucleopro- 
 teids, 175; nucleic acids, 182, 183; 
 thymine, 195; purin substances and 
 their enzymes, 368, 371, 373, 701-703, 
 706 
 
 Jonescu, D., 65 
 
 de Jonge, D., 846, 847 
 
 Jornara, D., 847_ 
 
 Josephsohn, A., 739 
 
 Junger, E., 427 
 
 Jureensen, E., 20, 97 
 
 Jungfleisch, E., 27, 585 
 
 Junkersdorf, P., 413 
 
 Juschtschenko, A., 373 
 
 Just, L, 339 
 
 Justus, J., 71 
 
 Juvalta, X., 778 
 
 Kaas, K., 78, 634 
 3\ahn, R., 402 
 Kalb, <",., 918 
 Kalbcrlah, It.. 585,818 
 Kalmus, E., 2X<) 
 Kanitz. A.. 506 
 Kapfberger, G., 53 
 Kareff, N., 253 
 Kashiwabara, M., 710 
 Kast, A., intestinal putrefaction, 519; 
 urinary sulphur, 752; chlorine excre- 
 
 tion, 758; halogen substituted methane, 
 777; perspiration, 848 
 
 Kastle, J. H., 56, 59 
 
 Kato, Kan, 636 
 
 Katsuyama, K., 161, 334, 582, 896 
 
 Katz, A., 744 
 
 Katz, J., 598, 599 
 
 Katzenstein, A., 926, 927 
 
 Kauder, G., 261 
 
 Kaufmann, M., glycogen, 397; sugar of 
 blood, 398; sugar formation, 412; 
 fat formation, 562; urea, 572; sugar 
 utilization, 592; lactose, 670; urea 
 formation, 684; metabolism experi- 
 ments, 890 
 
 Kauffmann, M. (Frankfurt), 247, 360, 713 
 
 Kaufmann, Martin, 894, 912 
 
 Kaup, J., 595 
 
 Kausch, W., 395 
 
 Kautzsch, K.j polypeptides, 87; glu- 
 tamic acid, 147; proline, 154; adrenalin 
 379; digestion, 484, 513 
 
 Kaznelson, H., 464 
 
 Keller, A., 757 
 
 Keller, Fr., 525 
 
 Keller, W., 783 
 
 Kellner, ()., utilization of nutriments, 
 531; protein catabolism in work, 595: 
 asparagin, nutrition value, 912; diets, 
 932 
 
 Kelly, A., 839 
 
 Kempe, M., 157, 158 
 
 Kennawav, E. L., 83, 109, 700, 715 
 
 Kendall, A. J., 520 
 
 Kermauner, F., 521 
 
 Kerner, G., 694 
 
 Kiermaver, 211 
 
 Kiesel, A., 247 
 
 Kikkoji, T., 640 
 
 Kiliani, H., 197 
 
 Kirchmann, J., 911 
 
 Kirk, R., 739 
 
 Kirschbaum, P., 613 
 
 Kistermann, C., 806 
 
 Kitagawa, F., 608, 611 
 
 Kittsteiner, C., 847 
 
 Kjeldahl, J., methods for nitrogen deter- 
 mination, 687, 688 
 
 Klages, A., 427 
 
 Klatte, F., 205 
 
 v. Klaveren, H. K. L., 288 
 
 Klecki, K., 521 
 
 Klein, \\ ., 675 
 
 Kleine, F., 752. 767 
 
 Kleiner, 1. S., 717 
 
 Klemensiewicz, R., 476, 477 
 
 Klemperer, G., urochrom, 740, 741; 
 oxalic acid, 773; protein metabolism,. 
 903, 915, 934 
 
 af Klercker, O., 691, 692, 694, 815 
 
 Klingemann, F., 670 
 
 Klinuemann, W., 484 
 
 Klingenberg, K., 779 
 
INDEX OF AUTHORS 
 
 961 
 
 K'ug, F., tryptophan, 155; pepsin, 466, 
 472; trypsin, ">02, 503; phosphoric 
 acid excretion, 762 
 
 v. Knafil-I.cn/, E.„ 124, .390 
 
 Knapp, K . sugar test, 214; sugar deter- 
 mination, sos. 81 1 
 
 v. Knieriem, W., cellulose, 510; urea 
 
 formation, 682j uric acid formation, 704 
 
 Knopfclmaclicr, \\ '., 233, 559 
 
 Knoop, 1'., liistidin, 159, 160; methyl- 
 imidasol, 201; demolition of fatty 
 acids, 774, 781; synthesis of amino 
 acids, 775, 786; demolition of aromatic 
 substances, 779, 781 
 
 Knop, W., 690 
 
 Knorr, L., 297 
 
 Kobert, H. W., 276 
 
 Kobert, R., cyanmethemoglobin, 285; 
 iron in urine, 770; melanins, 841 
 
 Kobrak, E., 662 
 
 Koch, \\\, lecithins, 241-245; cephalin, 
 248; brain analyses, 605, 607, 609, 
 612-614; milk, 663 
 
 Kocher, Th., 3(5 
 
 Kochs, \\\, 722 
 
 Kochmann, M., 902 
 
 Kobner, 440, 441 
 
 Koefoed, E., 647 
 
 Kohler, A., 600, 892, 912 
 
 Koelichen, K., 35 
 
 Koelker, A. H., 455 
 
 Konig, J., 599, 657-659 
 
 Koppe, H., blood corpuscles, 7, 8, 273, 
 305, 326; hydrochloric acid of stomach, 
 477 
 
 y. Korosy, K., digestion, 484, 513; 
 parenternally introduced protein, 525; 
 digestion blood, 527, 530 
 
 Koster, H., 650 
 
 Koettgen, E., 616 
 
 Kohler, R., 708 
 
 Kohlrauch, A., 776 
 
 Kojo, K., 795 
 
 Kolisch, R., 698, 795 
 
 Kondo, K., indol and skatol, 159; lactic 
 acid, 333, 585; eholesterin, 385; chon- 
 droitin sulphuric acid, 548, 549; urinary 
 phosphorous, 757 
 
 Koraen, G., 598, 929 
 
 Kordnvi, A., 13, 311, 351 
 
 Korkunoff, A., 910 
 
 Korn, A.. 469 
 
 Korndofer, G., 572 
 
 Korowin, 500 
 
 Kossel, A., protein nitrogen, 77, 78; 
 nitration of protamines, 83; arginase, 
 89, 161, 574. 681, 682; histones, 107- 
 109; protamines, 109-111, 137: pro- 
 tein hydrolysis, 122, 141, 145, 154; 
 histidin, 159, 1 1 '»( ) : hexone bases, 161, 
 164; arginine, 161, 162; agmatine, 162; 
 ornithin, 163; lysin, 163; nucleopro- 
 teins, 174; nucleic acids, 179, 185; 
 purin bases. 186, 187, 190-193, 367, 
 
 386, 495; pyrimidine bases, 194, 195; 
 primary and secondary cell constituents, 
 240; hemoglobin, 277; nucleohistone, 
 307, 366; blood plates, 308; pus, 304; 
 protagon, 606, 007, 610; cerebrosides, 
 607, 610; ichthulin, 630 
 
 Kossler, A., 725 
 
 Kostin, S., 2S7 
 
 Kostvtschew, S., 184, Is.", 
 
 Kotake, Y., 170. 393, 394, 711, 780 
 
 Kowalewski, K., 132, 185, 704, 767 
 
 Kowarski, A., 572 
 
 Kraft, F., 17 
 
 Kramm, W., 695 
 
 Kranenburg, W. R. H., 476 
 
 Kraske, B., 333 
 
 Krasnosselsky, T., 369 
 
 Kratter, Jul., 560 
 
 Kraus, Fr., 384, 410, 411, 583 
 
 Krause, R. A., 692 
 
 Krauss, E., 730 
 
 Krauze, L., 83 
 
 Krawkow, X. P., amyloid, 171, 172; 
 chitin, S39. S40 
 
 Kreglinger, G., 341 
 
 Krehl, L., 795 
 
 Kreis, H., 233 
 
 Kresteff, S., 477 
 
 Kreuzhage, C, 595 
 
 Krieger, H., 261, 263, 763 
 
 Krimberg, R., 575-578 
 
 Kristeller, L., 692 
 
 Krober, E., 209 
 
 Kronig, B., 71 
 
 Krogh, A., oxygen in blood, 859, 862, 864, 
 867; microtonometer, 861, 864; car- 
 bon dioxide tension, 864; metabolism 
 experiments, 868, 881 
 
 Krogh, M., 690, 862, 864, 866 
 
 Kronecker, F., 723 
 
 Krshyschkowsky, K., 463 
 
 Kruger, A., 79, 120, 230 
 
 Kruger, Fr., modification of proteins, 97; 
 haemoglobin, 280; leucocytes, 305; 
 blood, 335, 336; spleen, 371; liver, 
 388; sulphocyanides in saliva, 455; 
 milk, 654 
 
 Kruger, M., purin bases, 186, 188; in 
 feces, 520; in urine, 711-715; ammonia, 
 768 
 
 Kruger, Th. R., 136, 578, 598 
 
 Krug, B., 918 
 
 Krukenberg, F. C. W., keratinalbumoses 
 113; skeletins, 121; cornein. 122 
 cornicrystallin, 123; hyalogens, 170 
 171; lipochrome, 267; hsemoerythrin 
 303. 304; muscle extractives, 572, 577 
 bird egg, 630; uroostealith, 834-5 
 bird feathers, 843 
 
 Krumbholz, C. J., 536 
 
 Krummacher, ()., sugar formation, 412; 
 work, 595; calorific power, 889; protein 
 metabolism, 909; nutritive value of 
 gelatin, 911 
 
962 
 
 INDEX OF AUTHORS 
 
 Krzemecki, A., 82 
 
 Kudo, T., 476, 479, 506 
 
 Kiibel, F., 457 
 
 Kiihling, ()., 784, 785 
 
 Kuhne, W., enzymes, 40; neurokeratin, 
 112, 605, 614; gelatin, 119; albumoses 
 and peptones, 128, 129, 132, 135-139 
 paraglobulin, 258; luematoidin, 301 
 glycogen, 390; gastric digestion, 472 
 485; pancreas and its enzymes, 500 
 502, 503, 508, 509; fat emulsion, 536 
 muscles, 566-568, 589; smooth mucles 
 602; pigments of the eye, 615, 616 
 corpora lutea, 623 
 
 Kiilz, C, 355 
 
 Kiilz, E., cystine, 147; pentoses, 208, 815; 
 isomaltose, 225; glycogen, 390-394, 
 397, 592; diabetes, 400, 403; saliva, 
 454, 456; gastric juice, 476, 477; 
 pancreatic diastase, 500; gases of milk, 
 664; conjugated glucuronic acids, 777, 
 785; oxybutyric acid, 826 
 
 Kiilz, R., 283, 857 
 
 Kueny, L., 215 
 
 Kiister, F. W., 28 
 
 Kuster, \V., blood pigments, 283, 289- 
 298, 430; bile pigments, 425, 428-431, 
 433, 434, 429 
 
 Kiittner, S., 652 
 
 Kuhn, 617 
 
 Kuliabko, A., 494, 691 
 
 Kullberg, S., 205 
 
 Kumagai, T., 737 
 
 Kumagawa, M., fat determination, 238; 
 fat formation, 562; sugar determination, 
 809; protein metabolism, 903, 915, 934 
 
 Kunkel, A. J., arsenic, 72; carbon 
 monoxide blood test, 287; iron prep- 
 arations, 340; bile, 435, 443; iron in 
 urine, 770 
 
 Kuprianow, J., 582 
 
 Kurajeff, D., coagulose, 58, 135; halogen 
 protein, 83; protamine, 109; trypto- 
 phan, 155 
 
 Kurbatoff, D., 237 
 
 Kurpjuweit, O., 520 
 
 Kusmine, K., 351 
 
 Kusumoto, Ch., 448, 449 
 
 Kutscher, Fr., protein nitrogen, 77 
 gelatin, 83; proteolysis, 106, 107, 122 
 146; histone, 107, 108; protamine, 109 
 digestion products, 132; histidine, 159 
 160; arginine, 161, 162; agmatin, 162 
 lysine, 163, 164; hexone bases, 164 
 aporrhegmen, 166; cytosin, 194 
 thymin, 368; erepsin, 493; guanidine 
 507; choline, 507; intestinal digestion 
 513; absorption, 527, 528; bases of 
 meat extract, 575, 576; methylguani- 
 dine, 60S; urin:iry bases, 757, 826, 828 
 
 Kuwsehinski, P., 499 
 • Kyes, P., 241 
 
 Laache, S., 342 
 
 Ladenburg, A., 621 
 Laidlaw, P. P., 301, 380, 843 
 Laitinen, T., 309 
 Lambling, E., 680 
 Lampe, A., 912 
 Lampel, H., 78, 126 
 Lanceraux, 406 
 Landau, A., 361 
 Landauer, A., 518, 899, 920 
 
 Landergren, E., utilization of nutriments, 
 531, 535; metabolism, 894, 903, 914; 
 diets, 932, 935 
 
 Landois, L., 275, 343 
 
 Landolt, H., 841 
 
 Landsteiner, K., 69 
 
 Landwehr, H., 749 
 
 Lane-Clavpton, J. E., 43 
 
 Lang, G.; 484, 793 
 
 Lang, J., 151, 349 
 
 Lang, S., 703, 704, 776 
 
 de Lange, C, 242, 664 
 
 Lange, F., 818, 819 
 
 Langer, L., 235 
 
 Langgaard, A., 660 
 
 Langhaus, A., 230 
 
 Langhaus, Th., 301 
 
 Langbeld, K., SI, 426 
 
 Langley, J. N., 457, 459, 477 
 
 Langstein, L., carbohydrates in proteins, 
 84, 168, 262; digestion products, 132; 
 skatosin, 159; fibrin and leucocytes, 
 253; blood globulin, 260; rest nitro- 
 gen in blood, 267; serumprotein, 270; 
 deamidation, 410, 411, 775; lactic acid, 
 583; ovoglobulin 633; ovalbumin, 633, 
 634; ovomucoid, 634; casein, 662; 
 alcaptonuria, 735, 736, 739; C:N 
 quotient, 772, 884; lactose in urine, 815 
 
 Lankester, E. R., 303, 304 
 
 Lannois, E., 533, 846 
 
 Lapicque, L., 371, 387, 388, 936 
 
 Lappe, J., 492 
 
 Laptschinsky, M., 619 
 
 Laqueur, E., autolysis, 43; lipase, 476; 
 casein and rennin coagulation, 648-651 
 
 Laqueur, J., 340 
 
 Larin, A. M., 469 
 
 Larsson, K. O., 759 
 
 Lassaigne, J. L., 717 
 
 Lassar, O., 309 
 
 Lassar-Cohn, 422-425, 427, 435 
 
 Latarjet, A., 464 
 
 Latschenberger, J., bile pigments, 441, 
 442; iron in bile and liver, 443; pro- 
 tein absorption, 525 
 
 Latschinoff, P., 422-426 
 
 Lattes, L., 822 
 
 Laulanio, F., 563, 597, 927 
 
 Lauritzen, M., 769 
 
 de Laval, G., 656 
 
 Laves, E., 660 
 
 Laves, M., 397, 403, 404 
 
 Laveson, H., 479 
 
 Lavoisier, M., 926 
 
INDEX OF AUTHORS 
 
 963 
 
 Lawes, 563 
 
 Lawrow, D., roaguk>6e, 58, 135; histone, 
 
 ln7, 1(IS; digestion products, 132) his- 
 
 tidine, 160; blood pigments, 288; 
 conjugated glucuronic acids, 7s.", 
 
 Laxa, <>.. 649, 651 
 
 Lazarus-Barlow, W. 8., 351 
 
 Lea, Sh., 65 
 
 Leathes, J. B., lymph, 13; autolysis, 44; 
 liver fat, 384, 385; protein absorption, 
 627j ovarial fluid, 626; uric acid, 700 
 
 Leavenworth, Ch., 78, 84, L63 
 
 Lebedeff, A.,»384, 660, 501, 009 
 
 v. Lebedew, A., 205 
 
 Leclerc, A., 848 
 
 Leconte, P., 403 
 
 Ledderhose, G., 218, 839 
 
 Ledoux, A., 324 
 
 Leers, O., 285 
 
 van Leersum, E. C, 209, 221 
 
 Lefevre, K. I'., 222 
 
 Lefmann, G., 092 
 
 Legal, E., 158, 823 
 
 Lenmann, C, fat formation, 503; meta- 
 bolism in hunger, 893; in work, 920, 
 927; asparagin, food value, 912 
 
 Lehmann, C. G., 458, 632 
 
 Lehmann, Fr., 849 
 
 Lehmann. K. B., 288, 560 
 
 Lehndorff. H., 300 
 
 Leichtenstern, O., 338, 339 
 
 Leick, 559 
 
 Lemaire, F., 749, 815 
 
 Lenk, E., 423, 589, 590 
 
 Leo, H., liver fat, 384, 501; diabetes, 
 404; acidity in gastric juice, 4S9; 
 laiose, 814; nitrogen deficit, 881 
 
 Lepage, L., 498, 499 
 
 Lepine, R., glucuronic acids, 221, 331; 
 pentoses, 264; sugar in blood, 264, 329, 
 330; glycolysis, 332, 333, 34G, 407, 
 408; glycogen, 393. phlorhizin-diabetes, 
 400; absorption, 533; urinary sulphur, 
 752, 753; urinary phosphorous, 757; 
 urinary poisons, 758; urine maltose, 814 
 
 Leponois, E., 758 
 
 Lerch, 019 
 
 Lesem, W., 606, 607 
 
 Lesnik, M., 778, 779 
 
 Lesser, E. J., 909-911, 917 
 
 Lesser, K. A., 350 
 
 Letsche, E., blood serum, 264, 266; 
 methaemofdobin, 283; bile acids, 419, 
 420, 423 
 
 Leube, W., 787, 848 
 
 Leuchs, H., serine, 145; arabinose, 201; 
 glucosamine, 218; oxyprolin, 155 
 
 Levene, P. A., autolysis, 44, 620; poly- 
 peptides, 89 ; nucleoalbumin, 104; 
 albumin hvdrolysis, 106, 107, 119, 134, 
 141, 143, 144, 146; plasteins, 135; 
 tendon-mucin, 168, 544; nucleic acids, 
 178-185, 643; guanine, 190; pyrimi- 
 dine bases, 194, 195, 507, ribose; 211; 
 
 ghicothionic acids, 307, 369, 387, 547, 
 043; spleen, 369; liver, 383; phlor- 
 izin diabetes, 400; glycolysis, 408, 
 333; trypsin, 502, 503; brain protein, 
 604; cerebroside, 609, 611; Bphingosin, 
 611; cerebronic acid, 011; ichthulin, 
 630; urea, 082, 089 ; creatin and 
 creatinine, 692; lactic acid, 583 
 
 Levi-Malvano, M., 235, 230 
 
 Levison, L., 470 
 
 Levites, S., 78, 449-450, 476, 514 
 
 Levy, A. G., 309 
 
 Levy, H., 784 
 
 Levy, Ludw., 571 
 
 Levy, M., 557 
 
 Lewandowskv, M., 525 
 
 Lewin, Karl, 721, 728 
 
 Lewin, L., blood pigments, 281, 285, 286, 
 288, 289; hydroquinone, 727; urobilin, 
 745 
 
 Lewinsky, J., 269, 270, 721 
 
 Lewis, D. H., 53 
 
 Lewis, Th., 602 
 
 Lewy, B., 621 
 
 v. d. Leyen, E., 728 
 
 Lichtwitz, L., 66, 749 
 
 Liddle, L. M., 107 
 
 Lieb, Ch., 702 
 
 Lieben, A., 301, 306, 823 
 
 Liebermann, C., 447, 636, 843 
 
 Liebermann, H., 754 
 
 Liebermann, L., protein reaction, 100; 
 lecithalbumins, 105; nucleins, 175, 
 176; hen's egg, 628, 630, 632, 637, 
 638; kidneys, 673; Guaiac test, 796 
 
 Liebermeister, G., 258 
 
 v. Liebig, J., mineral substances, 7:; 
 fat formation, 563; work and meta- 
 bolism, 594, 596; urea, 686; diets, 932 
 
 Lieblein, V., 361, 684 
 
 Liebrecht, A., 82 
 
 Liebreich. O.. 606, 844 
 
 Liechti, P., 726 
 
 Liepmann, W., 642 
 
 van Lier, E. H. B., 545 
 
 van Lier, G. A., 8 
 
 Lifschutz, J., oleic acid, 237; cholesterin, 
 447, 449; isocholesterin, 446; wool fat, 
 846 
 
 Likhatscheff, A., 785 
 
 Lilienfeld, L., nucleo histone, 108, 307; 
 fibrin-ferment and blood coagulation, 
 256, 314, 316; blood plates, 308; 
 thymus, 366, 368 
 
 Lillie, R. S., colloids, 16, 17, 31; salt 
 action, 73 
 
 v. Limbeck, R., 309, 310 
 
 Limpricht, H., 572 
 
 Lindberger, W., 506, 517 
 
 Lindemann, L., 208 
 
 v. Linder, M., 843 
 
 Linden, 8. E., 23, 26 
 
 Lindhard, J., 814 
 
 Lindvall, V., 112 
 
964 
 
 INDEX OF AUTHORS 
 
 Ling, A. R., 226 
 
 Linn, K., 445, 446 
 
 Linnert, K., 612, 613 
 
 Linossier, G., 675 
 
 Linser, P., 844 
 
 Lintner, C. J., 229 
 
 Lipliawskv, A., 824, 825 
 
 Lipp, A., 152 
 
 Lippich, Fr., metallic albuminates, 96; 
 
 leucine, 143; excrements, 521; urein, 
 
 691 ; uramino acids, 786 
 v. Lippmann, E. O., 153 
 Lipschiitz, A., 902 
 Lister, J., 313 
 Ljubarsky, E., 239 
 Ljungdahl, M., 402 
 Lloyd- J ones, E., 308 
 Lochhead, A. C, 606 
 Lochhead, J., 640 
 Loche, F. S., 72, 592 
 Lockemann, G., 307 
 Locquin, R., 144 
 Loeb, A., 415, 767, 333 
 Loeb, J., muscles, 9; Overton's theory, 10; 
 
 enzymes, 70; antagonistic salt action, 
 
 72, 73; artificial fertilization, 639, 640; 
 metabolism, 928; fundulustrials, 70, 
 
 73, 74 
 
 Loeb, L., 256, 319 
 
 Loeb, W., 37, 212, 597 
 
 Loebisch, Wilh., 643, 668 
 
 Loebisch, W. F., 434, 544 
 
 Lohlein, W., 469, 505, 506 
 
 Loning, H., 611 
 
 Lonnberg, J., 549, 673 
 
 Lonnqvist, B., 462 
 
 Loeper, M., 265 
 
 Lorcher, G., 474 
 
 Loeschcke, K., 392 
 
 Lotsch, E., 484 
 
 Loevenhart, A. S., enzymes, 56, 59, 71, 
 501, 502, 563 
 
 Loew, O., protein, 78, 82, 97, 126; sugar 
 synthesis, 212 
 
 Lowe, S., 10 
 
 Lowenthal, S., 43 
 
 Loewenthal, W., 511 
 
 Loewi, O., phlorhizin-diabetes, 400; sugar 
 formation, 412; protein synthesis, 529; 
 
 , urea formation, 682; allantoin, 718; 
 
 ' conjugated glucuronic acids, 750; phos- 
 phorous metabolism, 761 
 
 Loewit, M., 314 
 
 Loewy, 4.., diamines, 163; blood alkalin- 
 ity, 310; high altitudes, 341, 929; 
 liver nitrogen, 387; work and meta- 
 I bolism, 595; acid action, 676; amino- 
 acids in urine, 757; cystinuria, 827; 
 gases of blood, 851, 852, 859, 861, 865; 
 
 I alveolar air, 860, 863, 865; metabol- 
 ism, 888, 925-929; maintenance value, 
 s',17, SOS 
 
 Loewy, E., 839 
 
 Lohmann, A., lysine, 164; choline, 246, 
 
 247, 379, 507; methylguanidine, 698; 
 urinary bases, 757 
 
 Lohnstein, Th., 679, 804, 813 
 
 Lohrisch, H., 510 
 
 Lombardi, M., 96 
 
 Lombroso, U., 532, 535, 536, 539, 540 
 
 London, E. S., enzymes, 53; nucleic 
 acids, 472; gastric lipase, 476; diges- 
 tion, 482-485, 513, 514; pancreatic 
 juice, 498; Eck's fistula, 529, 702; 
 small intestines, 541; creatinine, 694; 
 nuclein metabolism, 702, 705; starva- 
 tion blood, 896 
 
 Long, J. H., lecithin, 244, 245; casein, 
 648, 649; urine nitrogen, 680; urinary 
 coefficient, 771; elimination of alkali 
 earths, 769 
 
 Longcope, W. T., 43 
 
 L6pez-Su£rez, J., 476 
 
 Lorrain-Smith, J., 343 
 
 Losev, G., 29 
 
 Lossen, F., 83 
 
 Lossen, J., 325 
 
 Lottermoser, A., 19, 23 
 
 Luchsinger, B., 394, 847 
 
 Luciani, L., 339, 892 
 
 Luckhardt, A. B., 348 
 
 Ludwig, C., bile, 441; gastric digestion, 
 485; pancreatic juice, 495; absorption 
 of proteins, 526, 527; of sugar, 534; 
 gases of blood, 850, 851 
 
 Ludwig, E., 239, 627, 709, 710 
 
 Lucke, A., hyalin, 171, 840; pus, 365; 
 benzoic acid reaction, 722 
 
 Liidecke, T., 243 
 
 Liidecke, K., 247 
 
 Luthje, H., sugar formation, 408, 410, 
 412; nitrogen retention, 530; oxalic 
 acid, 715 
 
 Lukjanow, S., 415, 896 
 
 Lukomnik, I., 136 
 
 Lummert, W., 384, 563 
 
 Lundsgaard, Chr., 75, 310 
 
 Lunin, N., 900 
 
 Lusk, Gr., phlorhizin-diabetes, 399, 400, 
 407, 409, 412, 413; lactose in intestines, 
 532; in urine, 815 
 
 Lussana, F., 398 
 
 Luther, E., 808 
 
 Luzzatto, A., 815 
 
 Lyman, I. F., 702 
 
 Lyon, E. P., 73 
 
 Lyttkens, H., 265, 329, 332 
 
 Maas, O., 126 
 
 Maase, C., 774, 779, 780, 822 
 
 Macadam, J., 595 
 
 Macallum, A. B., 271, 340, 586, 769 
 
 Maccallum, A., (jr.), 711 
 
 MacCallum, J. B., 490 
 
 McClerulen, J. F., 630 
 
 McClendon, 73 
 
 MacCollmn, E. V., 902, 903 
 
 McCrudden, F. H., 557 
 
INDEX OF AUTHORS 
 
 «J65 
 
 Macfadven, A., 41, 512 
 
 v. Mach, W.j 703 
 
 Markay, J. C. EL, MM) 
 
 Markic. W. ( '.. 573, 57 1 
 
 MacLean, H.. 243, 247 
 
 Maclean, H., 407, 583 
 
 Macleod, J., glycoecuria, 402; bone mar- 
 row, 553; phosphocarnic acid, 578, 
 593; carbamic acid, 683, 691 
 
 MacMunn, Ch., A., ha-matoporphyrin, 
 295, 797, 843; echinochrom, 301; 
 cholohu'inatin. 435; myohaematin, 
 571; urobilinoid, 743; tetronerythrin, 
 843 
 
 Madsen, Th., 50, 57, 449-450 
 
 Mamianini, G., 159 
 
 Magne, H., 670 
 
 Magnier. 770 
 
 Magnus-Alsleben, E., 772 
 
 Magnus, G., S50 
 
 Magnus, R., 52, 524 
 
 Magnus Lew, A., spleen, 372; thyroid 
 gaud, 374', 376; liver, 382, 388, 389; 
 diabetes, 413; salivary glands, 452; 
 pancreas, 495 ; analyses of muscles, 599, 
 600; of brain tissue, 614; kidneys, 673; 
 hippuric acid, 721, 722; fatty acids 
 in urine, 748; benzoicglucuronic acid, 
 751; Bence-Jones' protein, 792; ace- 
 tone bodies, 818, 820, 821, 826; respira- 
 tion, 869; metabolism, 8S9, 923-925, 
 929, 897, 898 
 
 Maignon, ¥., 581 
 
 Maillard, L. C, creatinine, 693; indoxyl 
 sulphuric acid, 727, 730; urinary sul- 
 phur, 752; urinary phosphorous, 762; 
 ammonia, 766 
 
 Maillard, M. L., 71 
 
 Majert, W., 621 
 
 Makris, C, 660 
 
 Malcolm, J., 761 
 
 Malengreau, F., 242, 366, 367 
 
 Malenuck, W. D., 109, 110 
 
 Malfatti, H., urine, purin bases, 715, 
 amino-acids, 756; ammonia, 768, 769; 
 phosphorous elimination, 788; fruc- 
 tose determination, 814 
 
 Mall, F., 121 
 
 Mallevre, A., 510 
 
 Maly, R., oxyproteic acids, 82, 83; 
 peptones, 137; bile pigments, 429, 
 431-433, 743; saliva, 453; hydro- 
 chloric acid secretion, 477; putrefac- 
 tion, 517; luteins, 631 
 
 Manasse, P., 385 
 
 Manche, M., 592 
 
 Manchot, \Y., 279, 286, 298 
 
 Mancini, St., 208, 267, 740, 741 
 
 Mandel, J. A., glutamic acid. 147; nucleic 
 acids, 179, 643; adenine-hexose com- 
 pound, 180; guanylic acid, 183; naph- 
 thoresorcin reaction, 223; glucothionic 
 acids, 307, 369, 387, 643, 673; spleen, 
 369; liver, 383; mammary glands, 668; 
 
 reno-sulphuric acid, 673; urinary phos 
 phorouB, 757 
 
 Mandel, H., 107 
 
 Mandelstaiiiin. B., 415 
 
 Mangold. E., 390, 894 
 
 Manicardi, ( '.. 578 
 
 Mann. 8., 612, 613 
 
 Manning, T. D., 191 
 
 Mansfeld, G., 265, 360, 377 
 
 Maquenne, L, starch, 227, 229, 230 
 
 cellose, 231: protein assimilation, 525 
 
 sugar absorption, 534; iuosite, 579. 
 
 sarcosin, 776; digestion work, 929! 
 
 930 
 Marcet, 188, 523 
 Marchetti, < ... 560 
 Marchlewaki, 1... leaf and blood pigments, 
 
 276, 277, 296, 297; hajmin, 292; 
 
 cholohu'inatin and bilipurpuriu,435 
 Marcus, E., 259 
 Marcuse, \Y., 592, 593 
 Mares, ¥., 700, 702 
 Marfori, P., 773 
 Margulies, 806 
 Maiie. P. L., 307, 364 
 Marino-Zucco, 246 
 Mark, H., 385 
 Markewicz, M., 767 
 Marquardsen, E., 519 
 Marshall, J., 735 
 Martin, C. J., fibrin ferment, 57, 256; 
 
 toxin-antitoxin combinations, 68; blood 
 
 coagulation, 323 
 Martin, S. H., 510 
 Martz, 846 
 Marum, A., 820 
 Marx, A., 756, 818, 822 
 Marxer, A., 406 
 Maschke, O., 94, 695 
 Masius. J. B., 429, 521 
 Massen, V., 683 
 Masuyama, M., 628 
 Mathews, A , arbacin, 108; protamine, 
 
 109, 623; lysine, 163; nucleic acids, 
 
 184; fibrinogen, 252, 253 
 Mathieu, E., 851 
 Matthes, M., 519, 795 
 Mattili, H. A., 892 
 Mauthner, J., 149, 445, 814, 912 
 Mawas, I., 253 
 Maximowitsrh, S., 261, 262 
 May, R., 208 
 
 Mayeda, M.. 172, 173, 462 
 Mayer, Arthur, 770 
 Mayer, A., L05 
 Mayer, E. W.. 445. 448 
 Maver, J., 920 
 Mayer, L.. 532, 540 
 Mayer. Mart., 253. 270 
 Mayer, P., isoserin, 145; cystin, 147-149; 
 
 mannoses, 201, 203; glucuronic acids, 
 
 221, 222; lecithin, 244; conjugated 
 
 glucuronic acids, 331, 749, 750, 817; 
 
 phosphatides, 385; deamidation, 410; 
 
966 
 
 INDEX OF AUTHORS 
 
 1 inosite, 579; oxalic acid, 716; mdican, 
 
 728; skatoxyl-glucuronic acid, 732 
 Mayo-Robson, A. VY., 414 
 Mays, K., 502, 503, 616 
 Mazurkiewicz \Y.. 499 
 Meara, F. S., 131 
 Medigreceanu, Fl., 182, 277, 481 
 Meek, W. I., 253 
 Mehu, C, 351, 745, 746 
 Meigs, E. B., 589, 590, 603 
 Meikere, G., inosite, 579, 580, 818; 
 
 urinary chlorine compounds, 758 
 Meinert.'C. A., 531, 932 
 Meinertz, J., 385 
 
 Meisenheimer, J., 41, 205, 207, 213 
 Meissl, E., 563 
 Meissl, Th., 642 
 Meissner, F., digestion products, 471, 
 
 472; urea formatiom, 684; allantoin, 
 
 716, 717; hippuric acid, 720 
 Meister, V., 684 
 Mellanby, E., 573, 574 
 Mellanby, J., serum proteins, 259, 322; 
 
 peptone-blood, 325; creatine and crea- 
 tinine, 692, 694 
 Mendel, Lafayette B., enzymes, 52, 53; 
 
 lymph formation, 351; saliva, 454; 
 
 trypsinogen, 496; protein absorption, 
 
 525; creatine, 693; uric acid, 702, 705; 
 
 allantoin, 717; kynurenic acid, 740; 
 
 artificial feeding, 904, 906 
 Mendes de Leon, M. A., 662 
 Menozzi, A., 449-450 
 Menschutkins, 35 
 Menzies, J. A., 285 
 de Merejkowski, C, 843 
 v. Mering, J., urochloralic acid, 221, 777; 
 
 sugar in blood, 264; blood from portal 
 
 vein, 336, 532; glycogen, 394; phlorizin 
 
 diabetes, 399, 400; pancreatic diabetes, 
 
 404; amylolysis, 456, 500 
 Merunowicz, J., 293 
 Mesermitzki, 628 
 Messinger, J., 825 
 Messner, E., 529 
 Mester, Br., 519, 732, 752 
 Mett, S., 469 
 Meyer-Betz, Fr., haematoporphyrin, 295; 
 
 bilirubin, 429, 744; urobilinoids, 743; 
 
 urobilinogen, 744, 746, 747 
 Meyer, C, 388 
 v. Meyer, E., 23 
 Meyer, E., 440 
 Meyer, Erich, 735, 739, 783 
 Meyer, G., 531 
 Meyer, G. M., amino-acids in blood, 266; 
 
 glucose, 408, 333; urea, 682, 689; 
 
 lactic acid, 583 
 Meyer, H., 221, 703, 704 
 Meyer, Hans, 267 
 de Meyer, J., 408 
 Meyer, K., 64 
 Meyer, Kurt, 32, 396 
 Meyer, Lothar, 860 
 
 Meyer, P., 425, 429 
 
 Meyer-Wedell, L., 385 
 
 Michaelis, H., 265 
 
 Michaelis, L., cataphoresis, 20; colloid 
 envelopment, 23; adsorption, 49-51, 
 97, 102; reaction of blood, 75; coagula- 
 tion of proteins by heat, 20, 97; albu- 
 moses, 134; sugar-of blood, 264, 235, 
 328, 329; butyrinases, 265; protein 
 absorption, 525; milk, 657 
 
 Michaud, L., 401, 818, 822, 917 
 
 Michel, A., 261, 262, 263 
 
 Micheli, F., 139 
 
 Micko, K, 521, 578 
 
 v. Middendorff, M., 336 
 
 Mieg, W., 631 
 
 Miescher, F., protamines, 109-111; nu- 
 clein, 175; nucleic acid, 179; pus, 364, 
 365; spermatozoa, 622, 623; salmon 
 metabolism, 902 
 
 Miethe, A., 281, 285, 286, 289 
 
 Migay, Th., 481 
 
 Miller, I. R., 701-703, 706 
 
 Millon, M. E., 99, 653 
 
 Mills, W., 715 
 
 Milroy, J. A., 290 
 
 Milroy, T. H., 175, 761 
 
 Minami, D., 501 
 
 Minkowski, O., blood alkalinity, 309; 
 ascitic fluid, 358; glycogen, 397; 
 sugar of blood, 293; phlorhizin- 
 diabetes, 399, 400; pancreatic diabetes, 
 403-406; duodenal diabetes, 405; bile 
 pigment, 442, 443; pancreas and 
 absorption, 532; fat absorption, 535, 
 539, 540; lactic acid, 582, 749; uric 
 acid, 703-705; allantoin, 717; his- 
 tozym, 723; blood in diabetes, 856 
 
 Mitchell, P. H., 702 
 
 Mitjukoff, K., 626 
 
 Mittelbach, F., 254, 739, 793 
 
 v. Mituch, A., 637 
 
 Miura, K., 264, 393, 492 
 
 Miyamota, S., 467 
 
 Moeckel, K., 265 
 
 Mollenberg, R., 338 
 
 Moeller, J., 521 
 
 Moller, S'., 825 
 
 Morner, C., Th., albumoid, 114, 546. 549, 
 617; gelatin, 118, 120, 546; ichthy- 
 lepidin, 121; gorgonin and pennatulin, 
 122; cornicrystalline, 123; proteins 
 of anthozoa, 123; tyrosin test, 154; 
 membranin, 171, 550, 617; fructose, 
 217; vitreous humor, 545, 616; car- 
 tilage tissue, 546-550; cornea, 550; 
 mucoid, 545, 550, 551; bones, 555; 
 crystalline lense, 617, 618; sugar 
 in egg-white, 632; ovomucoid, 634, 
 635; perca globulin, 636; homogentisic 
 acid, 738, chlorine determination, 
 760; gallic and tannic acid, 785; cal- 
 cium diphosphate in urine sediment, 
 831 
 
INDEX OF AUTHORS 
 
 967 
 
 Morner, K. A. H., sulphur of proteins, 
 79; cystine and cystein, 100, 113, 114, 
 147-149; thiolactic acid, 79, 113, 151; 
 albuminate, 120, 509; pyro-racemic 
 acid, 150; proteins of serum, '259, 200, 
 262; lucrum, 292, 293; blister fluid, 
 301; hydrochloric acid determination, 
 489; chondroitin sulphuric acid, 547, 
 073; muscle pigments, 571 • urinary 
 nitrogen, 080, 085; urea determina- 
 tion, 688, 689j fatty acids in urine, 
 7 IS; urinary nubecula, 757; acetanilid, 
 779; albumin in urine, 787; nucleo- 
 albumin of urine, 794; melanins, 799, 
 841 ; bile acids in urine, 800 
 
 Mohr, Fr., 488, 759 
 
 Mohr, L., diabetes and sugar formation, 
 400, 412, 413; purines in urine, 713 
 
 Mohr, P., 112 
 
 Moitessier, J., 287, 758, 792, 793 
 
 Moleschott, J., 932 
 
 Molisch, H., 215 
 
 Moll, L., 103 
 
 Monari, A., 593, 594, 098 
 
 Monod, O., 054 
 
 Moor, Ovid., 090, 091 
 
 Moore, J., 213 
 
 Moore, B., theory of Overton, 10; adsor- 
 pates, 12, 29; colloids, 10; glycoseuria, 
 402; bile and fatty acids, 511, 530; 
 intestinal contents, 519; fat synthesis, 
 535; fat emulsion, 530 
 
 Mooser, YV., 720 
 
 Moraczewski, W., excrements, 521; heart 
 muscle, 599; pseudonuclein, 051; in- 
 dican of urine, 728, 729 
 
 Morat, J., 592 
 
 Morawitz, P., fibrin and leucocytes, 147; 
 serum proteins, 270; blood coagula- 
 tion, 314, 315, 318-321, 324, 325; 
 detection of albumoses in urine, 792 
 
 Morax, V., 724 
 
 Moreau, A., 807 
 
 Moreau, J., 229, 230 
 
 Morel, A., fibrinogen, 252, 253; serum 
 lipase, 205; blood coagulation, 234; 
 glycolysis, 332; hsematogen, 029; milk, 
 054 
 
 Morel, I., 077 
 
 Morel, L., 501 
 
 Moreschi, A., 449 
 
 Morgen, A., 007 
 
 Morgenroth, J., 04, 08 
 
 Mori, Y., 531, 932 
 
 Moriggia, A., 847 
 
 Morishima, K., 382 
 
 Moritz, 479 
 
 Moritz, F., 354, 400, 401, 075 
 
 Moriya, G., 582, 606 
 
 Morkowin, N., 109 
 
 Morochowetz, L., 540 
 
 Morris, G. H., 41, 220, 229 
 
 Morse, H. N., 3 
 
 Moruzzi, G., 247 
 
 Moscatelli, R., allantoin, 355, 359; lactic 
 acid, 593, 748, 749 
 
 Moscati, <i., starch assimilation, 532; 
 glycogen, 581, 591; placenta, 040 
 
 Mosen, l;., 308 
 
 Mosse, ML, pseudochylous appearance, 
 358; sugar in blood, 398; hydrochloric 
 • acid in stomach, 470; ethereal sulphuric 
 acids, 7l' I 
 
 Mott, F. W., choline in blood, 240; in 
 cerebrospinal fluid, 300; diseases of the 
 nervous system, (>14 
 
 Mottram, \\ . H., 384, 385 
 
 Mouton, H., 65 
 
 Muhle, P., 130 
 
 Miihsam, J., 335 
 
 Miiller, A., 15, 23 
 
 Midler, Alb., 489 
 
 Miiller, Eduard, 307 
 
 Muller, Erich, 510 
 
 Midler, Ernst, 419, 421, 423 
 
 Muller, Fr., 130 
 
 Muller, Franz, 377, 378, 888, 920, 929 
 
 Muller, Friedrich, autolysis of pneu- 
 monic infiltrations, 45, 304, 809; glu- 
 cosamine from proteins, 83, 84, 108, 
 109, 170, 620; high altitudes, 341; 
 starvation (indican) 510; fat absorp- 
 tion, 538, 539; ethereal sulphuric 
 acids, 724; urobilin, 744; sulphur of 
 urine, 752, 753; aniline, 779; acetone 
 bodies, 818; feces nitrogen, 831 
 
 Muller, Joh., 580, 592, 028 
 
 Muller, Jul., 727 
 
 Muller, Max, 000, 721 
 
 Muller, Martin, 578 
 
 Muller, Paul, 448, 521 
 
 Muller, Paul, Th., 252, 253, 270, 553 
 
 Muller, W., 009, 01 
 
 Muntz, A., 669 
 
 Miinzer, E., 682, 685 
 
 Miither, A., 210 
 
 Muirhead, A., 083 
 
 Mulder, G. J., Ill 
 
 Munk, I., chyle and lymph, 340-349; 
 sulphocyanides, 455, 450, 751; in- 
 testinal contents, 519; absorption of 
 protein, 525, 520; of sugar, 534; of fat, 
 535, 537, 538; fat synthesis and fat 
 formation, 500, 503; work and meta- 
 bolism, 595; smooth muscles, 002; 
 milk, 055, 050; urea, 082; phenol 
 elimination, 725; phosphoric acid elim- 
 ination, 702; bile pigment reaction, 801 ; 
 starvation metabolism, 895, 890; 
 nutritive value of gelatin, 911; of 
 albumoses, 912; of asparagin, 912; 
 protein requirement. 915; water and 
 metabolism, 920 
 
 Murlin, J. R., 911 
 
 Murray, Fr. \V.. 500 
 
 Murschhauser. H., 923 
 
 Musculus, F., amylolysis, 229, 450, 500; 
 urochloralic acid, 777; urease, 829 
 
968 
 
 INDEX OF AUTHORS 
 
 Myers, V., 195, 692, 693 
 Mygge, J., 793 
 
 Mvlius, F., starch iodide, 228; bile acids, 
 419, 422-425 
 
 v. NagelilC, 227 
 
 Nageli, K., 5 
 
 Nageli, O., 676, 677 
 
 Nagano, J., 491, 532, 533 
 
 Nakaseko, R., 393 
 
 Nakayama, M., 182, 493, 801 
 
 van Name, W. G., 118, 121 
 
 Nasse, H., muscle experiments, 9; blood, 
 336,337; lymph, 349; spleen, 371 
 
 Nasse, O., proteins, 78, 99, glutin, 120; 
 dextrins, 230; glycolysis, 332; gly- 
 cogen, 390, 391, 590-592; saliva, 456; 
 457; musculin, 568, 570; oxidations, 877 
 
 Naunyn, B., glycogen, 394; bile pig- 
 ments and liver, 442, 443; demolition 
 of aromatic substances, 778, 779 
 
 Nawratzki, 360 
 
 Nebelthau, E., 393, 799 
 
 Necker, F., 791 
 
 Nef, J. U., 213, 214 
 
 Neilson, C. H., 37, 53, 454 
 
 Neimann, W., glucuronic acids, 221, 222, 
 750, 751 
 
 Nelson, L., 109, 110 
 
 Nencki, L., 779 
 
 Nencki, M., protein sulphur, 78; trypto- 
 phan, 155; skatol acetic acid, 155; 
 indol, 157; blood pigments, 276, 277, 
 280, 291-295; haematoporphyrin, 295; 
 diabetes, 403; gastric juice, 465-467, 
 476, 477, 485; enzymes of the stomach, 
 475;cleavage of esters, 501; intestinal 
 digestion, 511, 512; intestinal put- 
 refaction, 514; reaction in intestine, 
 519; ammonia, 528, 683, 768; urea 
 572, 682-684; carbamic acid, 683 
 nrosein, 733, 740; urobilinoids, 743, 
 demolition of aromatic substances, 
 778, 780, 785, melanins, 841 
 
 Neppi, B., 505 
 
 Nerking, J., lecithin, 241, 244; glycogen, 
 392; bone marrow, 553; milk, 663 
 
 Nernst, \Y\, permeability of membranes, 
 9; division rule, 27; diffusion, 36; 
 toxin-antotixin reaction, 69; gas chains, 
 74, 272 
 
 Nerseasoff, N., 744 
 
 Neubauer, C, creatin, 575; creatinine, 
 691; ammonia, 766 
 
 Neubauer, E., 245, 404 
 
 Neubauer, O., protein reaction, 100; 
 acids of alcaptonurics, 736, 738, 739; 
 demolition of amino-acids, 775, 779, 
 780, 786; conjugation of glucuronic 
 acid, 777; acetone formation, 780, 818, 
 
 Neuberg, C, autolysis, 43; putrefactive 
 products of proteins, 82; gelatin, 83; 
 artificial phosphoproteins, 105; iso- 
 
 leuctne, 144; isoserine, 145; oxyamino 
 succinic acid, 147; cystine, 147-149 
 proline, 154; tryptophan, 157, 158 
 diamine formation, 163; amyloid, 171 
 172; nucleic acids, 178, 179, 182, 183 
 sugar demolition, 200; mannoses, 201 
 203; pentoses, 203, 208-211, 815 
 sugar-free fermentation, 206; glucose 
 215; galactose, 216; laevulose, 217, 218 
 355; glucoseamine, 219, 626, 629 
 aminoaldehyde, 219, 220; glucuronic 
 acids, 221, 223, 749-751, 817; amino- 
 acids in blood, 267; glucothionic acid, 
 369, 673; melanin, 380; softening of 
 liver, 386, 387; glycogen, 393; deamdia- 
 tion, 410, 411; cholesterin, 445, 447; 
 chondrosin, 548; inosite, 579; lactic 
 acid, 583; lactose, 655; renosulphuric 
 acid, 673; urine, 678; heteroxan thine, 
 713; phenol determination, 725, 726; 
 skatoxyl-glucuronic acid, 732; amino- 
 acids in urine, 756; mineral meta- 
 bolism, 758, 769; tataric acid, 774; 
 phenylhydrazine test, 806; acetone 818; 
 cystinuria, 827; tyrosinase, 843; min- 
 eral metabolism, 899, 901, 902 
 
 Neumann, Alb., nucleic acids, 179, 184, 
 185; pyrimidine bases, 194, 195; orcin 
 test, 209; iron in urine, 770; phenyl- 
 hydrazine test, 806 
 
 Neumann, E., 443 
 
 Neumann, Jul., 338 
 
 Neumann, O., 921, 934 
 
 Neumann, R., 920 
 
 Neumann, Walt, 136 
 
 Neumeister, R., keratins, 112; albumoses 
 and peptones, 128-131; tryptophan, 
 155; dextrins, 230; glycogen, 391; 
 protein assimilation, 525; ovomucoid, 
 634 
 
 Neusser, E., 797 
 
 Nickles, J., 635 
 
 Nicloux, M., 56, 264 
 
 Nicolaier, A., 710 
 
 Niemann, A., 532, 539 
 
 Nierenstein, E., 469 
 
 Nilson, G., 228 
 
 Nilson, L. F., 658 
 
 Nishi, M., 404 
 
 Njegovan, VI., 245 
 
 Le Nobel, C., 295, 743, 797 
 
 Noel-Paton, D., lymph, 349; liver, 384, 
 385; glycogen, sugar formation, 399; 
 bile, 414, 438; creatine metabolism, 
 573-575; lactose. 670 
 
 Noguchi, H., 449-450 
 
 Nogueira, A., 673 
 
 Nolf, P., osmotic pressure, 13; fibrinogen 
 252, 253; fibrinolysis, 255, 256; albu- 
 moses in blood, 263: blood coagulation, 
 318, 319-322, 324, 325, 320; saliva, 
 452; absorption, 526, 527; carbamic 
 acid, 683, 691 
 
 Noll, A., 184, 614, 535 
 
INDEX OF AUTHORS 
 
 9G9 
 
 v. Norden, C, spectrophotometry, 302; 
 diabetes, 401, 404, 405, 412; liver 
 and urinary nitrogen, 685; ethereal 
 sulphuric acids, 724; albumin in urine, 
 787; acetone bodies, 818; metabolism, 
 
 915, *r > 1 * > 
 
 v. Noorden, K., 333 
 Nothwang, Fr., 899 
 Noskin, J., 375 
 
 Novi, J., 435, 459 
 Now. V., 126 
 Nowak, .!.. con, 808, 881 
 Nuremberg, A., 370 
 Niirenbenr, A., 56 
 Nussbaum, M., 860, 864, 865 
 Nuttal, C. 516 
 Nylander, E., 214 
 Nylen, S., 457.. 
 
 Obermayer, Fr., protein pieeipitation, 
 102; globulins, 103; bile pigments, 
 434, 801, 802; indican detection, 729, 
 730 
 
 Oberniuller, K., 445, 446 
 
 Odake, 8., 905, 906 
 
 Oddi, R., 511, 675 
 
 Odenius, R., 643 
 
 Oertel, H., 757 
 
 Oertmann, E., 41 
 
 Oerum, H. P., (sr.), 627, 911 
 
 Oerum, H. P. T. (jr.), 342, 437, 730 
 
 Oesterberg, E., 680, 693 
 
 Offer, Th. R., pentose-amine, 219; gly- 
 cogen, 396; chitin, 839; alcohol, 921 
 
 Offringa, J., 282 
 
 Ofner, R., 217, 218 
 
 Ogata, M., 485 
 
 Ohta, K., 774 
 
 Oidtmann, H., 366, 368, 870 
 
 Oker-Blom, M., 8, 377 
 
 Okunew, W., 135 
 
 Olinger, J., 529 
 
 Oliver, G., 379 
 
 Ollendorff, G., 203 
 
 Olsavszky, V., 762 
 
 Omeliansky, V., 510 
 
 Omi, K., 492, 526 
 
 van Oondt, 401 
 
 Opie, E. L., 307 
 
 Oppenheimer, C., serumalbumin, 261; 
 parenteral protein assimilation, 525; 
 respiration, 868; oxydation enzymes, 
 875; nitrogen elimination, 881; sur- 
 face rule, 923 
 
 Oppenheimer, Max, 583 
 
 Oppenheimer, 8., 583 
 
 Oppler, B., 329, 513 
 
 Orbdn, R., 492 
 
 Orgler, A., acetone, 83, 818; chondrosin, 
 548; uric acid, 638 
 
 Orndorff, W. R., 428, 430, 431 
 
 Orton, K., 738, 739 
 
 Osborne, Th. B., proteins, 77-79, 84, 
 104, 106-108, 163; polypeptides, 89; 
 
 nucleic acids, 179, 186; ovovitellin, 
 628, 620; protein of white of egg, 633, 
 634; calorimetry, 886; phosphorous 
 
 metabolism, 903; artificial nutrition. 
 904, '.toe, 
 
 Osborne, W. A., 581 
 
 Ostertag, 659 
 
 Ostwald, Willi., 28, 29; catalvsis, 33, 35, 
 36 
 
 Ostwald, Wo., 15, 31, 279 
 
 Ostwald, A., halogen protein, 82; thy- 
 roid glands, 373, 375, 376; di-iodotyrosin, 
 508; urinary globulin, 701 
 
 Otori, J., mucin, 169; transudates, 355; 
 guanidine, 507, 574; pseudomucin, 
 626 
 
 Ott, A., 769 
 
 Otte, P., 486 
 
 Otto, J. G., sugar of blood, 265; blood 
 pigments, 277, 283, 284. 302; blood, 
 328, 329, 335, 338, 339, 532; skatoxyl- 
 sulphuric acid, 732; sugar determina- 
 tion, 811 
 
 Overton, E., plasmolysis, 6, 8; muscle 
 experiments, 9, 588; amphibians, 9; 
 theory of permeability, 9-11 
 
 Owen, Rees, 347 
 
 Paal, C., proteins, 78; gelatin peptones, 
 
 120; alkali albuminate, 126; peptone, 
 
 130, 131, 137 
 Pacchioni, D., 381 
 Pachon, V., blood coagulation, 324; 
 
 stomach extirpation, 485, 486; trypsino- 
 
 gen, 496 
 Paderi, 332 
 Padtterg, J. H., 838 
 Pages, C, blood coagulation, 251, 316, 
 
 317; rennin action, 650; milk, 667 
 Pagniez, Ph., 314, 315 
 Paigkull, L., exudates, 353, 354, 357, 358, 
 
 bile, 417 
 Painter, H. M., 130, 457 
 Panek, K., 753, 754, 763 
 Panella, A., 266, 578, 602 
 Panormoff, A., 581, 633, 635 
 Panum, P., serumcasein, 258; starvation 
 
 blood, 338, 339, 896; transfusion, 340, 
 
 344; amount of blood, 343; nitrogen 
 
 elimination, 910, nutritive value of 
 
 gelatin, 911 
 Panzer, Th., proteins, 83; chylus, 347, 
 
 cerebrospinal fluid, 360; bile, 423; 
 
 colloid, 625, 626 
 Pappenhusen, Th., 484 
 Paraschtschuk, S., 669 
 Parastschuk, S. W., 474 
 Parcus, E., 610 
 Parke, J. L., 632, 635 
 Parker, W. H., 16, 536 
 Parmentier, E., 660 
 Parnas, J., 248, 583 
 Partridge, C. L., 371, 702 
 Paschutin, V., 350, 492 
 
970 
 
 INDEX OF AUTHORS 
 
 Pascucci, O., 273, 274 
 
 Pascheles, W., 776 
 
 Pasqualis, G., 749 
 
 Pasteur, L., 40, 41, 516 
 
 Patein, G., 808 
 
 Patten, A. J., 79, 149, 160 
 
 Patten, J. B., 457 
 
 Paul, Th., 71, 707 
 
 Pauli, \V., colloids, 17, 18, 20, 25, 30;- 
 proteins, 95, 97; gelatin, 31, 119 
 
 Pauly, H., histidin, 82, 159-161, adrenalin, 
 379 
 
 Pautz, W., 361, 492, 617 
 
 Pavy, F. W.. carbohydrate groups in 
 proteins, 84, 396; isomaltose. 264; 
 glycogen, 392, 398; sugar in blood and 
 diabetes, 400; self-digestion of stomach, 
 486; work and metabolism, 595; sugar 
 determination, 808, 809 
 
 Pawlow, J. P., secretion of enzymes, 52, 
 53; Schutze's rule, 57; bile fistula, 414; 
 saliva, 454; stomach and gastric juice, 
 461-463, 465, 468; stomach enzymes, 
 474; pyloric reflex, 481; intestinal 
 juice, 490; pancreatic juice and enzymes 
 of pancreas, 49.5-498, 499, 501; diges- 
 tion in intestines, 513; ammonia in 
 blood, 683; Eck's fistula, 684 
 
 Payer, A., 338 
 
 Pearce, R. G., 399 
 
 Peiper, G., 309 
 
 Pierce, G., 71 
 
 Peju, P., 253 
 
 Pekelharing, C. A., cataphoresis, 50, 51; 
 fibrin ferment and blood coagulation, 
 256, 257, 316, 317, 319, 320; nucleo- 
 proteins, 258, 569; stomach enzymes, 
 466-468; creatin and creatinine, 594, 
 692, 693 
 
 Penny, E., 725 
 
 Penzoldt, F., 402, 824 
 
 Pernou, M., 371 
 
 Perrin, J., 15, 20 
 
 I eschek, E., 912 
 
 I etersen, P., 599 
 
 Petit, A., 758 
 
 Petrowa, M., 416 
 
 Petry, E., 651 
 
 v. Pettenkofer, M., bile acid test, 418; 
 fat formation, 560, 561; work and 
 metabolism, 594, 596, 597; respira- 
 tion apparatus, 868, 869; metabolism 
 experiments, 879, 881, 908, 926; diets, 
 932 
 
 Pittibone, C., 690 
 
 I'd rone, A., 315 
 
 Petzseh, E., 902 
 
 Pfaff, P., 414 
 
 Pfannenstiel, J., 620 
 
 Pfaundler, M., 132, 133, 681 
 
 Pfeffer, W., 2, 3 
 
 Pfeiffer, E., 662, 665 
 
 Pfeiffer, I,., 841 
 
 Pfeiffer, Th., 918 
 
 Pfeiffer, Wilh., 705 
 
 Pfleidener, 470, 471 
 
 Pfluger,. E., oxydations, 41; life of cells, 
 45; carbonic acid of lymph, 347; gly- 
 cogen, 390, 392, 393, 394, 413, 549; 
 diabetes and sugar formation, 405, 408, 
 412, 413; duodenum and diabetes, 405; 
 gases of saliva, 453, 857; bile and fatty 
 acids, 511, 536; fat formation, 561, 
 562; muscle metabolism, 591, 595, 
 597; gases of milk, 657, 857; urinary 
 nitrogen, 680; urea determination, 690; 
 sugar tests, 803, 805; sugar determina- 
 tion, 811; gases of blood and respira- 
 tion, 850, 851, 854, 858, 866-868; 
 N:C quotient in urine, 884; protein 
 metabolism, 904, 9/)8, 909; external 
 temperature and metabolism, 929; 
 protein allowance, 934 
 
 Phisalix, C., 847 
 
 Picard, J., 616, 617 
 
 Piccard, 109 
 
 Piccolo, G., 301, 623 
 
 Pick, A., 470 
 
 Pick, E. P., albumoses and peptones, 131, 
 133-135, 139; serumglobulins, 259; 
 peptozym, 325; thyreoidea and adren- 
 alin-glycosuria, 376 
 
 Pick, F.,*399 
 
 Pickardt, M., 355, 549, 550 
 
 Pickering, J. W., 85, 86, 323 
 
 Picton, H., 22, 26 
 
 Pierallini, G., 773 
 
 Piettre, M., stroma of blood corpuscles, 
 275; blood pigment, 281, 285; hsemin, 
 292; hyoglycocholic acid, 421; melanin, 
 842 
 
 Pigeand, J., 354 
 
 Pighini, G., 613 
 
 Pilotv, O., blood and blood pigments, 
 220, 277, 290, 292-298, 295, 297; 
 bile pigments, 429, conjugated glu- 
 curonic acids, 751 
 
 Pilz, O., 137 
 
 Pilzecker, A., 440 
 
 Pimenow, P., 462 
 
 Pincussohn, L., 23 
 
 Pineles, Fr., 376 
 
 Pinkus, S. N., proteins and their crystal- 
 lization, 82, 95, 263, 633, 634 
 
 Piontkowski, L. F., 463 
 
 Piria, 153 
 
 Planer, J., 486 
 
 Plattner, E., 418 
 
 Plaut, M., 756 
 
 Playfair, 932 
 
 Pletnew, D., 493, 532 
 
 Plimmer, R. H., Aders, enzymes, 53; 
 gelatin hydrolysis, 119; nucleopro- 
 teins, 174,' 630; livetin, 629; ichthulin, 
 630; hatching of the egg, 637; casein 
 digestion, 652; uric acid, 702 
 
 P16sz, P., blood corpuscles, 274; liver 
 382, 383; urinary pigments, 740; 
 
INDEX OF AUTHORS 
 
 971 
 
 proteid in urine, 787, albumoses, 
 nutrition value, 912 
 
 Poda, H., 521 
 
 Poduschka, P., 717 
 
 Poehl, A., 517, 021 
 
 Pohl, J., dextrin, 230; globulin determina- 
 tion, 261, 793; liver, 383; acid poison- 
 ing, 676; urea, 682; allantoin, 716, 717; 
 oxalic acid, 773; demolition of fatty 
 acids, 774; phthalic acid, 778 
 
 Poleck, 631 
 
 Policard, A., 324 
 
 Polimanti, O., 561 
 
 Politis, G., 912 
 
 Pollak, H., 105 
 
 Pollak, L., 505, 776 
 
 Pollitzer, S., 912 
 
 Polwzowa, W., 482, 514 
 
 Ponfick, E., 343, 344 
 
 Ponamarevv, 489, 490 
 
 Pons, Ch., 364, 548, 757 
 
 Popel, \\\, 339 
 
 Popielska, Helene, 463 
 
 Popielski, L., enzymes, 53, 324; saliva, 
 454; pancreatic juice, 496, 498 
 
 Popowsky, N., 158 
 
 Popper, H., glvcogen, 395; bile pigments, 
 434, 801, 802; pancreatic juice, 500, 
 509 
 
 Porcher, Ch., lactose, 670; indican of 
 urine, 728-732; skatol red and uroro- 
 sein, 733; uroerythrin, 748; phthalic 
 acid, 778 
 
 Porges, O., 245, 259 
 
 Porteret, E., 393 
 
 Portier, P., 407, 532 
 
 Posner, C, 620, 621, 787 
 
 Posner, E. R., 169, 472, 606 
 
 Posselt, L., 122 
 
 Posternak, S., 578, 579 
 
 Pottevin, H., 59, 226 
 
 Pouchet, A. G., carnine, 577, 711; 
 urinarv poisons, 757; lungs, 870 
 
 Poulet, V., 869 
 
 Poulsen, W., 550 
 
 Poulsson, E.. 572 
 
 Pozerski, E., 65, 498 
 
 Pozzi-Escot, E., 877 
 
 Prausnitz, W., 400. 521, 894 
 
 Prayon, I., 693 
 
 Pregyl, Fr., keratins, 112, 114; koilin, 
 115, 124; carbon monoxy haemoglobin, 
 290; dehydrocholan, 418; bile acids, 
 423, 426; intestinal juice, 490, 491; 
 colloid, 626; ovalbumin, 634; oxypro- 
 teic acid, 753; polypeptides in urine. 
 756; €:N quotient, 772 
 
 Presch, W., 752, 753 
 
 Preti, L., 64, 71, 706 
 
 Preusse, C, phenols in urine, 725-727; 
 behavior of aromatic substances in 
 animal body, 778, 779, 786 
 
 Prevost, J. L., 541, 684 
 
 Prever, W., 288, 642 
 
 Preysz, K., 762 
 
 Pribram, E., 778 
 
 Pribram, H., 106 
 
 Pribram, R., 766 
 
 Pridgent, G., 242 
 
 Pringle, H., histopeptone ,109; prota- 
 mines, 109,110; absorption, 525, 528, 
 530; blood coagulation, 320 
 
 Pringsheim, H., 230, 510 
 
 Pristley, J. H., 37 
 
 Prochownik, L., 642, 643 
 
 Proscher, F., 431, 659, 667 
 
 Profitlich, W., 389 
 
 Prutz, W., 728 
 
 Prym, O., 372, 496 
 
 Przibram, H., 571 
 
 Pugliese, A., 319, 476 
 
 Pulvermacher, G., 212 
 
 Pupkin, Z., 309 
 
 Pyman, F. L„ 160 
 
 Quadiarello, G., 675 
 Quevenne, Th., 348, 349 
 Quincke, G., 23, 64(5 
 Quincke, H., 301, 340 
 Quinquand, Ch., urea, 333, 335; muscle 
 work, 592; fatty acids in urine, 773 
 Quinton, R., 13 
 
 Raaschou, C. A., 183 
 
 Rabinowitsch, A. G., 484 
 
 Rachford, B. K., 501, 506, 511 
 
 Radenhausen, P., 646 
 
 Radziejewski, 8.. 559, 560 
 
 Radzikowski, C, 462 
 
 Raehlmann, E., 616, 617 
 
 Raineri, G., 642 
 
 Rakoczv, A., 475 
 
 Ramsden, W., 30, 97 
 
 Ranc, A., 267 
 
 Ranke, H., 700 
 
 Ranke, J., 344 
 
 Ransom, H., 449 
 
 Raper, H. 8., 136 
 
 Rapp, R., 41 
 
 Raske, K., 140, 148 
 
 Rauchwerger, D., 445, 447 
 
 Raudnitz, R. W., 644, 649 
 
 Reach, F., 381, 466, 597 
 
 Reale, E., 716, 752 
 
 Reemlin, E. B., 513 
 
 Reese, H., 756 
 
 Regnault, H. V.. 849, 868, 881 
 
 Reh, A., 104, 366, 652 
 
 Rehfuss, M., 804 
 
 Rehn, E., 253 
 
 Reich, M., 770 
 
 Reich. O., 771 
 
 Reichel, H., 650 
 
 Reich-Herzberge, F., 508 
 
 Reid, E. W., 16 
 
 v. Reinbold, B., 283, 507, 808, 283 
 
 Reinecke, 558 
 
 Reiset, J., 849, 868, 881 
 
972 
 
 INDEX OF AUTHORS 
 
 Reiss, E., 260 
 
 Reiss, W., 619 
 
 Reitzenstein, A., 711 
 
 Rekowski, L., 786 
 
 Rennie, J., 494 
 
 RenVall, G., 769 
 
 Rettger, I., 257, 322 
 
 Rettger, L. F., 496 
 
 Reuss, A., 355, 357 
 
 Rewald, B., 179, 180, 210, 211 
 
 Reye, W., 254 , 
 
 Remolds, J. E., 823 
 
 de Rev-Pailhade, J., 877 
 
 Rhodin, N. J., 43 
 
 v. Rhorer, L., 677 
 
 Ribaut, H., 877 
 
 Richards, A. X., albumoids, 116, 117, 118; 
 hexone bases, 164; saliva, 455, 456 
 
 Richaud, A., 599 
 
 Richet, Ch., gastric juice, 465; fat forma- 
 tion, 458; urea, 682; uric acid, 706; 
 thalassin, 846; respiration, 869; sur- 
 face rule, 923; spleen, 372 
 
 Richter, Max, 621 
 
 Richter, P., 873 
 
 Richter, P. F., osmotic pressure, 13; 
 amino-acids in blood, 267; softening 
 of the liver, 386, 387, 685 
 
 Rieder, H., 881 
 
 Riegel, 464 
 
 Riegel, M., 647 
 
 Riegler, E., 290 
 
 Riehl, M., 869 
 
 Riess, L., 748, 749, 780 
 
 Riesser, O., 162, 611 
 
 Rinaldi, U., 572 
 
 Ringer, A. I., 721 
 
 Ringer, A. J., 412 
 
 Ringer, L., 316 
 
 Ringer, S., 72 
 
 Ringer, W. E., 50, 51, 675, 677, 708 
 
 Ringstedt, O. T., 675 
 
 Ritter, A., sugar in blood, 398; phlor- 
 hizin-diabetes, 400; fat absorption, 
 535; urinary fermentation, 829 
 
 Ritter, F., 439, 440, 445, 449 
 
 Rittausen, H., proteins, 94; leucinimide, 
 143; milk, 655 
 
 Riva, A., 740, 748, 799 
 
 Rivalta, F., 354 
 
 Roaf, H. E., lipoid theory, 10; adsorpate 
 12, 29; osmotic pressure of protein, 17, 
 17; lecithin, 246; glycoseuria, 402 
 
 Roberts, F., 279 
 
 Roberts, W., 509, 793, 812, 813 
 
 Robertson, T. B., lipoid theory, 10; 
 protein salts, 95; crystallized albumin, 
 262; globulins, 263, 270; casein, 648, 
 649 
 
 Roch, G., 790 
 
 Rockwood, C. W., 757 
 
 Rockwood, D., bile and fatty acids, 511, 
 536; intestinal contents, 519 
 
 Rockwood, E. W., 525 
 
 Rodhe, E., 100 
 
 Rodier, A., 338 
 
 Roden, H., 63 
 
 Roeder, G., 194, 195 
 
 Rohmann, F., amvlolvsis and diastase, 
 225, 265, 346, 456; glycolysis, 333; 
 blood, 335; glycogen 393, 398; in- 
 testinal juice, 491, 492; bile and 
 putrefaction, 518; absorption, 532- 
 534, 538; muscles, 565, 589; casein, 
 649; phosphorous metabolism, 761 
 902; sebum, 844; wool fat, 846 
 coccygeal glands secretion, 846, 847 
 oxydase action, 875; artificial feeding, 
 904 
 
 Rohrig, A., 591, 849 
 
 Rose, B., 650 
 
 Rose, C., 558 
 
 Rose, Heinrich, 429, 295 
 
 Rosing, E., 877 
 
 Roger, G. H., 381 
 
 Roger, H., 457, 510 
 
 Rogozinski, F., 136 
 
 Rohde, A., 703 
 
 Rokitansky, P., 748 
 
 Rona, P., cataphoresis, 20; enzymes, 56; 
 adsorption, 97, 102; thymuslhistone; 
 109; gelatin, 119; albumoses, 134; 
 sugar in blood, 264, 265; butyrinases 
 265; calcium in serum, 269; sugar 
 in blood, 328, 329; glucose, 333, 
 duodenal, secretion, 489, 490; protein 
 absorption, 525, 529; milk, 657; alcap- 
 tonuria, 735, 736 
 
 Ronchese, A., 768, 769 
 
 Ronchi, J., 849 
 
 Roos, E., 376, 761, 806, 807 
 
 Roosen, O., 698 
 
 Rorive, F., 217 
 
 Rosa, E. B., 869 
 
 Rose, W. C., 692, 693 
 
 Rosemann, R., gastric juice, 465, 479; 
 milk, 670; nitrogen elimination, 910; 
 alcohol, 921 
 
 Rosenbach, O., 749, 800, 827 
 
 Rosenbaum, A., 408 
 
 Rosenberg, Br., 401 
 
 Rosenberg, S., duodenal diabetes, 405; 
 bile, 415; pancreatic juice, 497; putre- 
 faction, 518; pancreas and absorp- 
 tion, 532, 535, 539 
 
 Rosenberger, F., 579, 816 
 
 Rosenfeld, B., 484 
 
 Rosenfeld, F., 728, 748, 912 
 
 Rosenfeld, G., fat and fat formation, 384, 
 560-562, 669; uric acid, 700; phenyl- 
 hydrazine test, 806; acetone bodies, 819 
 
 Rosenfeld, L., 136 
 
 Rosenfeld, M., 290, 293 
 
 Rosenfeld, R., 264 
 
 Rosenheim, O., tryptophane reaction, 157; 
 cerebrospinal fluid, 360; pancreatic 
 lipase, 502; muscle-work, 592; pro- 
 tagon, 606-609 
 
INDEX OF AUTHORS 
 
 973 
 
 Rosenheim, Th., 915 
 
 Rosenqvist, E., 412 
 
 Rosensbein, A., chvle and lymph, 347, 
 348, 349; absorption, 520, 534, 537 
 
 Rosenstein, \V., 403 
 
 Rosenthal, J., 868 
 
 Rosenthaler, L., 59, 66 
 
 Rosin, 11., fructose, 217, 814; indican, 
 730; skatol pigments, 733; Rosen- 
 bach's urine test, 827 
 
 Rossi, O., 360 
 
 Rost, K., 920 
 
 Rost, Ft., 265 
 
 Rostoski, ()., 792, 793 
 
 Roth, (>., 207 
 
 Roth, W., 351 
 
 Rothberger, C. J., 683 
 
 Rothera, C. H., 77, 148, 824 
 
 Rothmann, A., 573, 692 
 
 Rotmann, F., 355 
 
 Rotechy, A.. 295 
 
 Roux, E., 227 
 
 Rovida, C. L., hyaline substance, 274, 
 304 
 
 Rovighi, A., 517, 724 
 
 Rowland, S., 41-43, 371, 571 
 
 Rowntree, L. G., 183, 642, 643 
 
 Rozenblat, H., 462 
 
 Rubbrecht, R., 270 
 
 Rubner, M., protein sulphur, 79; sugar 
 test, 215, 806, 815; utilization of 
 nutiiments, 531, 534, 535, 538; fat 
 formation, 502, 563; milk, 664; meta- 
 bolism experiments, 879, S85, 890, 
 894, 922, 923, 928, 929; nitrogen 
 of excrements, 881 ; heat of combus- 
 tion, 885, 886, 891, 932: utilization 
 quota, 890, 910; protein catabolism, 
 910, 914, 916, 918; specific dynamic 
 action, 917, 930; surface rule, 923, 924 
 
 Rubow, W., 586 
 
 Rudinger, ('., 375, 406 
 
 Riidel, G., 707 
 
 Ruff, O., 200, 203 
 
 Ruge, E., 515 
 
 Rulot, H., 255, 256 
 
 Rumpf, Th., 412, 767 
 
 Runeberg, J. W., 357 
 
 Ruppel, W. G., 606, 660, 844 
 
 Russel, 653 
 
 Russo, M., 839 
 
 Russo, Ph., 744 
 
 Rutherford, A., 838 
 
 Rutherford, Th. A., 112 
 
 Ryan, L. A., 603 
 
 Rywosch, D., 273 
 
 Sabanejew, A., 137 
 
 Sabbatani, S., 316 
 
 Bachaijin, 328 
 
 Bacharow, X., 135 
 
 Sachs, Fritz, papain, 65; pentoses, 210; 
 nuclease, 182, 493, 508; hydrochloric 
 acid secretion, 477; acetone formers, 818 
 
 Sachs, H., 64 
 
 Sachsse, K , 215, 228 
 
 Sackur. <>.. 648, 649 
 
 Saaikoff, W., Us 
 
 Sagelmann, A., 482 
 
 Sahli, H., 303, 325, 809 
 
 Saiki, T., 603, 748, 749 
 
 Saillet, hsematoporphyrin, 295, 797, 798; 
 
 • urobilin and urobilinogen, 740, 743, 
 744, 740, 747 
 
 Sainsburv, 316 
 
 de Saint-Martin, L., 286 
 
 Saint Pierre. C, 867 
 
 Saito, S.. 334, 582 
 
 Salaskin, S., digestion products, 132; 
 plastein, 135; leucinimid, 14)i: blood 
 alkalinity, 309; ammonia, 334, 336, 
 528, 683; erepsin, 393; urea, 682, 684; 
 liver and acid formation, 685; uric 
 acid formation, 703, 704 
 
 Salkowski, E., autolysis, 42; denaturing 
 of proteins, 97; pseudonuclein, 104, 
 651; albumoses, 131; in urine, 792; 
 putrefaction products, 141, 514; skatol 
 carbonic acid, 155, 732, 733; indol, 158; 
 pentoses, 208, 209, 815; glucuronic 
 acid, 221; cerebrospinal fluid, 360; 
 synovin, 302; liver-proteins, 383, 384; 
 glycogen, 393; eholesteiin, 447: saliva, 
 459; trypsin, 503; putrefaction, 517; 
 corpse wax, 500; flesh, 600; dermoid 
 cyst, 027; dextrose in white of egg, 
 032; ovomucoid, 034; casein, 651, 
 652; urea, 082, 684, 690; creatinine, 
 696; uric acid, 705, 709, 710; purine 
 bases, 714; oxalic acid, 710, 773; 
 allantoin, 716, 717; hippuric acid, 720; 
 phenaceturic acid, 723; ethereal-sul- 
 phuric aoi.ls, 724; indican, 729, 730; 
 urobilin, 745; urine, fatty acids, 748, 
 829; carbohydrates, 749, 815; sulphur 
 compounds, 752, 753; adializable 
 urinary constituents, 757, 795; urinary 
 sulphur acids, 704; alkalies, 700; 
 demolition of various substances, 770, 
 777, 783; ha^matoporphyrin, 797; 
 sugar tests, 805, 806; acetone deter- 
 mination, 825; water and metabolism, 
 920 
 
 Salkowski, H., 141, 514, 720 
 
 Salomon, Georg, purin bases, 187; glyco- 
 gen, 207; lactic acid, 334; urinary purines, 
 711-713 
 
 Salomon, H., 410, 818 
 
 Salomon, W., 082 
 
 Salomone, G., 78 
 
 Salomonsen, K. E., 740, 741 
 
 Samec, M., 141 
 
 Sammet, O., 825 
 
 Sammis, J. L. 936 
 
 Samuely, F., proteolysis, 107; amino- 
 acids in urine, 756; cystine demoli- 
 tion, 776; melanoids, 843 
 
 Sandgren J., 265, 329, 332 
 
974 
 
 INDEX OF AUTHORS 
 
 Sandmeyer, W., pancreatic diabetes, 405; 
 
 absorption, 532, 539, 540 
 Sandstrom, J., 374 
 Saneyoshi, S., 774, 777, 817 
 Sasaki, K., 757 
 Sasaki, T., 146, 147, 781, 784 
 Sato, T., 369 
 Satta, G., 681 
 Sauer, K., 402 
 Sauerbeck, E., 494 
 Savare, M., 640, 641, 757 
 Savory, H., 792 
 Sawitsch, W., 496, 499 
 Sawjalow, W., 58, 135, 475 
 Saxl, P., 43, 387, 569 
 Scaffidi, V., ferratin, 384; iron in liver, 
 
 387; purine bases, in muscles, 572, 
 
 594, 603; uric acid, 706 
 Schade, H., 707 
 Schafer, E., 379 
 Schaeffer, 50, 51 
 Schaffer, F., 78 
 
 Schaffer, Ph., 692, 711, 768, 827, 828 
 Schalfejeff, M., 292, 293 
 Schardinger, F., 581, 877, 230 
 Scheele, M. H., 454 
 Scheermesser, W., 136 
 Scheibe, A., 659, 664 
 Schemiakine, A. J., 477, 478 
 Schenck, Fr., 83, 398 
 Schenck, M., 422, 423 
 Schepowalnikoff, N. P., 495 
 Scherer, Fr., 925 
 v. Scherer, J., lymph, 348; inosite, 579, 
 
 580; meta and para-albumin, 625 
 Scheuer, M., 464 
 Scheunert, A., gastric digestion, 479, 482; 
 
 duodenal secretion, 490; pancreatic 
 
 stones, 509; cellulose, 510; peroxydase, 
 
 874 
 Schewket, O., 817 
 Schierbeck, N. P., saliva, 457; gases of 
 
 stomach, 486; trypsin action. 506; 
 
 excrements, 520; skin breathing, 849 
 Scruff, A., 469 
 Schiff, H., protein, 78; biuret test, 101; 
 
 cholesterin, 448; urea, 686; uric acid, 709 
 Schiff, M., spleen, 372; liver, 381, sugar 
 
 of liver, 398; bile, 416, 541; charging 
 
 theory, 477, 496 
 Schindler, S., 368 
 Schittenhelm, A., nucleic acid, 182, 514; 
 
 blood coagulation, 318, 323; deamida- 
 
 tion enzymes, 371; purine bases, 520; 
 
 urea formation, 682; uric acid and its 
 
 formation, 373, 699-703, 705, 706, 709; 
 
 amino-acids in urine, 756, 757, 827; 
 
 ammonia, 767, 768 
 Schlapfer, V., 912 
 Schlatter, K., 485, 486 
 Sdilcsinger, A., 457 
 Sfhlcsinger, W., 747 
 Bchliep, L„ 825 
 Sclilosing, Th., 768 
 
 Schloessing, C. 22 
 
 Schlossberger, J. E., 665, 670 
 
 Schlossmann, A., 656, 658, 659, 663, 923 
 
 Schmey, M., 387, 599 
 
 Schmid, Jul., 702, 715 
 
 Schmidt, Ad., 513, 523 
 
 Schmidt, Albr., 621 
 
 Schmidt, Alex., blood coagulation, 256, 
 257, 305, 314-317, 319, 321-323; 
 fibrinoplastic substances, 258, 319, 
 320; blood corpuscles, of frogs, 375; 
 leucocytes, 305, 306; cell protein, 306, 
 307, 367; saliva, 453, 454; gases of 
 blood, 852 
 
 Schmidt, C, serum, 270; blood, 328; 
 lymph, 348; transudates, 353; saliva, 
 458; mucus of the mouth, 453, 454; 
 gastric juice, 465; pancreatic juice, 
 499, 500; bile, 518; fat absorption, 
 538; osteomolacia, 555 
 
 Schmidt, C. H. L., 82 
 
 Schmidt, C. W., 870 
 
 Schmidt, E., 572 
 
 Schmidt, Ernst., 695 
 
 Schmidt, Fr., 818 
 
 Schmidt, Hub.. 101 
 
 Schmidt, P., 711 
 
 Schmidt-Muhlheim, 251, 526, 910 
 
 Schmidt-Nielsen, Signe, 50 
 
 Schmidt-Nielsen, Sigvae, rennin, 50, 651; 
 casein, 649, 650 
 
 Schmiedeberg, O., albumin crystals, 94; 
 salmine, 110, 110; alkali albuminate, 
 126; onuphin, 171; nucleic acids, 179, 
 184, 185; nucleosin, 195; glucuronic 
 acids, 221, 725, 730, 731, 785; ferra- 
 tin, 383; chondroitin sulphuric acid, 
 547-549; urea, 682; hippuric acid, 
 722, 723; histozym, 723; melanin 
 substances, 840, 841 
 
 Schmitz, E., 775, 780, 786, 818, 826 
 
 Schmitz, H, 136 
 
 Schmitz, K., 517, 519, 724 
 
 Schmutzer, I., 708 
 
 Schneider, A., 328 
 
 Schneider, E., 455, 739 
 
 Schoffer, A., 142 
 
 Schonbein, C. F., 766, 871 
 
 Schondorff, B., urea, 333, 572, 664, 679 
 690; thyroidea, 376; glycogen, 390 
 394, 396, 581; phlorhizin action, 400 
 uric acid, 701 ; sugar in urine, 803, 808 
 protein metabolism, 908, 909 
 
 Schone, A., 208 
 
 Scholz, H, 728, 729 
 
 Schottelius, M., 516 
 
 Schotten, C., fellic acid, 427; intestinal 
 putrefaction, 720; fatty acids in urine, 
 748, 773; damaluric and damolic 
 acid, 758; behavior of aromatic sub- 
 stances in animal body, 780 
 
 Schoubenko, G., 79 
 
 Schoumow-Simanowski, E. O., 467, 477, 
 485 
 
INDEX OF AUTHORS 
 
 975 
 
 Schreiber, E., 701 
 
 Schreiner, Ph., G21 
 
 Schrener, M., flesh nitrogen, 600; calorific 
 
 value of nitrogen, 892; protein feeding, 
 
 918 
 Schrodt, M., 553 
 v. Schroder, W., urea, 12, 333, 079, 682, 
 
 684; uric acid, 334, 703, 704 
 Schroter, F., 182 
 
 v. Schrotter, H., 130, 131, 137, 861, 865 
 Schryver, S. B., 43, 427 
 Schule, 464 
 Schule, A., 456 
 Schutz, E , Schutz's rule, 57; digestion 
 
 products, 133; pepsin determination, 
 
 469; stomach movement, 479; lactic 
 
 acid in urine, 749 
 Schutz, J., pepsin determination, 469; 
 
 action, 471; hydrochloric acid, 489; 
 
 bile and fat splitting, 502, 511 
 Schtitze, A., 64 
 
 Schiitzenberger, P., 85, 86, 130 
 Scbultze, B., 563 
 Schultze, E., 680 
 Schultze, F. E., 72 
 Schultzen, O., diabetes, 403; urea, 682, 
 
 683; lactic acid in urine, 748, .749; 
 
 sarcosin, 776; behavior of aromatic 
 
 substances in the animal body, 778- 
 
 780 
 Schulz, Art., 299 
 Schulz, Fr. N., proteins, 79, 95, 98; oxy- 
 
 proteins, 83; histone, 108; galactos- 
 
 amine, 167, 173, 219; serumalbumin, 
 
 261, globin, 288; starvation blood, 339; 
 
 premortal protein metabolism, 894 
 Schulz, H., 376, 546, 770 
 Schulz, C, 457 
 Schulz, E., products of hydrolysis of 
 
 proteins, 141, 153; phenylalanine, 152; 
 
 histidine, 160; arginine, 161; lysine 
 
 163; hexone bases, 164; vernine, 178- 
 
 180; hemicelluloses, 231; phosphatides, 
 
 245; isocholesterin, 448 
 Schulze, E., 650 
 Schulze, F., 22 
 Schulze, H., 14, 21 
 Schumburg, W., 474, 889, 926 
 Schumm, O., blood, 342, 796; chyle fat, 
 
 347; sugar formation, 412; pancreatic 
 
 cvst, 500 
 Schunck, C. A., 276, 277, 296, 631 
 Schunck, E., 740 
 Schur, H., uric acid, 700, 702, 706; 
 
 urinarv purines, 702, 713 
 Schurig, 387 
 Schuster, A., 531, 938 
 Scnuurmanns-Stekhoven, 282 
 Schwalbe, E., 315, 384, 561 
 Schwann, Th.. 414, 528" 
 Schwarz, Carl, choline, 247; iodothyrin, 
 
 376; glycoseuria, 405; digestion, 483; 
 
 secretin, 498 
 Schwarz, H., 216 
 
 Schwarz, Hugo, 116, 117, 118 
 
 Schwarz, Karl, 572, 575 
 
 Schwarz, L., 126, 477, 820 
 
 Schwarz, (>., 402, 487, S25 
 
 Schwareschild, M., 502, 508 
 
 Schweisslnger, O., 790 
 
 Schwiening, 42, 43 
 
 Schwinge. \\ ., 33s 
 
 Scofield, H., 433 
 
 Scott, F. H., 174, 630, 637 
 
 Scott, L., 122 
 
 Sczelkow, 851 
 
 Sebauer, it., 555 
 
 Sebelien, J., peptones, 130; milk, 644, 
 651, 652, 655, 656, 658; casein diges- 
 tion, 651 ; carbohydrates in milk, 655 
 
 Seegen, J., sugar in blood, 264, 398, 592 
 597; sugar formation, 405; amylolysis, 
 456; respiration, 868; nitrogen deficit, 
 881 ; water and metabolism, 920 
 
 Seelig, P., 517 
 
 Seeman, J., protein substances, oxyda- 
 tion, 83; ealactoseamine, 167; erepsin, 
 493; intestinal contents, 513; absorp- 
 tion, 527, 528; creatine and creatinine, 
 573; ovomucoid, 635 
 
 Segale, M., 72 
 
 Seisser, Ph., 702, 705, 709 
 
 Seitz, W., 381, 389 
 
 Seliwanoff, Th., 217 
 
 Selmi, 47 
 
 Semmer, G., 275 
 
 Senator, H., 729 
 
 Senter, G., 56 
 
 Sera, Y., 393, 394 
 
 Sertoli, E., 855 
 
 Sestini, F. and L., 699 
 
 Setschenow, J., 851, 853, 855 
 
 Shackell, L. F., 73 
 
 Shepard, C. W., 720 
 
 Shibata, N., 384, 561 
 
 Shimamura, T., 905, 906 
 
 Shinidzu, Y., 238 
 
 Shaw-Mackenzie, J. A., 502 
 
 Siau, R. L., 264, 400 
 
 Sieber, N., protein sulphur, 79; blood 
 pigments, 280, 292, 293; ha?matopor- 
 phyrin, 295; glycolysis, 333; diabetes, 
 403; gastric juice, 361, 362, 485; 
 stomach enzymes, 475; intestinal diges- 
 tion, 512; Umikoff's reaction, 664; 
 urosein,7 33, 740; urobilinoids, 743; 
 nitrobenzaldehyde, 784; melanins, 841; 
 lungs, 869 
 
 Siedentopf, H., 19 
 
 Siegert, F., 559 
 
 Siegfried, M.; peptone substances and 
 kyrines, 121, 132, 133, 136-138, 146: 
 reticulin, 121, 544; lysine, 163; car- 
 bamino reaction, 166, 855; jecorin, 385; 
 phosphocarnic acid, 578, 585, 593, 598; 
 orylic acid, 653; milk-nucleon 653,663; 
 phenol excretion, 725, 726 
 
 v. Siewert, A., 292 
 
976 
 
 INDEX OF AUTHORS 
 
 Signorelli, E., 757 
 Sikes, A. W., 663 
 Silbermann, M., 145, 147 
 Simacek, E., 407, 583 
 Simon, Fr., 606 
 Simon, G., 656, 658 
 Simon, L., G., 510 
 Simon, O., 45, 364, 870 
 de Sinetv, L., 815 
 Sittia;, 0\, 355 
 
 Siven, V. O., uric acid, 700; urinary 
 purines, 713; protein metabolism, 903; 
 916, 934 
 Sivrc, A., 482 
 Siwertzow, D., 241, 244 
 Sjoqvist, J., enzymes, 57; peptones 137; 
 hydrochloric acid determination, 488, 
 489; intestinal concrements, 524; 
 urinary nitrogen, 680, 685; urea 
 determination, 688, 689 
 Skita, A., 145 
 v. Skramlik, E., 675 
 
 Skraup, Zd., protein nitrogen, 77, 78; 
 hydrolysis of proteins, 119. 124, 146, 
 147; alkali albuminate, 126; oxyamino- 
 acids, 165; carbohydrate, 215 
 Skworzow, W., 575, 576 
 Slanskv, P., 585 
 Slavw,'379, 380 
 Slosse, A., 333, 684, 910 
 Slowtzoff, B., pentosan, 208; liver, 389; 
 semen, 620; milk coagulation, 651; 
 metabolism, 923, 926 
 v. Slyke, D. D., deamidation, 77, 78, 88; 
 hydrolysis of albumin, 106, 107, 134, 
 141, 143, 144; plastein, 135; amino- 
 acids in blood, 266; casein, 648, 649 
 van Slyke, L. L., 648, 649 
 Small. Fr., 707 
 Smetanka, F., 700, 702 
 Smirnow, A., 784 
 Smith, F., 847, 848 
 Smith, Herbert, 457, 551 
 Smith, Lorrain, 862, 863 
 Smith, W. J., 752 
 Socin, C. A., 395 
 Socoloff, N., 437 
 Soldner, F., milk, 647-650, 655-657, 662, 
 
 664-668 
 Sorensen, S. P. L., isoelectric point, 20; 
 determination of the reaction, 74, 75; 
 coagulation of proteins by heat. 20, 
 97; glycocoll, 140; phenylalanine, 152; 
 proline, 154; arginine, 161; ornithin, 
 162, 163; formol titration, 165; hip- 
 puric acid, 723; urinary nitrogen, 756; 
 ammonia, 768 
 Soetber, F., 763 
 Solera, L., 455, 456 
 Solley, Fr., 118, 120 
 
 Sollrnann, T., chyle, 347; bile, 440; mus- 
 cles, 566, 567, 570; uterine fibroma, 627 
 Sommer, A., 384 
 Sommerfeld, 438 
 
 Sommerfeld, P., 465 
 
 Sonden, K., respiration apparatus, 869; 
 
 metabolism, 924, 925, 936 
 Sorby, H. C., 636 
 Soret, J., 281 
 Sourder, C., 502 
 Le Sourd, L., 314 
 Soudat, 665 
 Southgate, 506 
 
 Soxhlet, glucose, 216; galactose, 216; 
 maltose, 225; fat formation, 563; 
 milk, 647, 650, 656, 667. 669; sugar 
 titration, 808, 809 
 Spack, Wl., 124 
 Spanpani, G., 669 
 Spangaro, S., 250 
 Spanjer-Herford, R., 455 
 Speck, C., 869, 926, 928 
 Spiegler, A., 899 
 Spiegler, E., 790, 841, 842 
 Spiro, K., colloids, precipitation, 25, 102; 
 swelling, 31; diffusion, 32; gelatin, 119; 
 glycocoll, 140; pyrazinedicarbonic, 
 • acid, 220; serumglobulins, 259; blood 
 coagulation, 31S, 319, 324; rennin 
 action, 650; urine acidity, 675; oxy- 
 butvric acid formation, 822 
 Spiro," P., 593 
 
 Spitzer, W., glycolysis, 333, 407; liver, 
 383; uric acid formation, 702, 875 
 Spriggs, E. J., 469 
 Spring, 14 
 
 Staal, J. Ph., 676, 733 
 Stade, W., 57, 476 
 
 Stadelmann, E., tryptophan, 155; icterus, 
 301; adrenal bodies, 377; bile, 414- 
 416, 438, 441-444, 541; intestinal 
 putrefaction, 519; nitrogen excretion, 
 685; ammonia, 768; pentoseuna, 791; 
 diabetes, blood, 856 
 Stadthagen, M., diamines, 47; adenine, 
 191; xanthocreatinine, 698; urinary 
 sulphur, 752, cystinuria, 827 
 Stadler, G., 434 
 Staehlin, R., 354 
 Stanek, V., 247 
 
 Stangassinger, R., 573, 692, 698 
 Stange, M., 233 
 Starke, .1., 102 
 Starke, K., 262, 263 
 
 Starkenstein, E., 398,402, 579, 817, 818 
 Starling, E. H, colloids, 16; enzymes, 5'; 
 lymph formation, 351; hormones, 375; 
 enterokinase, 492, 496; secretin, 492, 
 498; intestinal enzymes, 492, 493; 
 pancreatic erepsin, 493, 503; trypsino- 
 gen and trvsin, 496, 497 
 Stassano, H., 496, 497 
 Stassow, B. [)., 541 
 Stauber, A., 493 
 Stavenhagen, A., 41 
 Steel, M., 768 
 Steenbock, H., 723 
 Steensma, F. A., 158, 488, 732 
 
INDEX OF AUTHORS 
 
 977 
 
 Steiff, R., 724 
 
 Steiger, E., 161 
 
 Steil, H., 599 
 
 Stein, G., 445 
 
 Steinitz, Fr., phosphorous metabolism, 
 
 701; C:N quotient, 772, 884; lactose 
 
 in urine, 815 
 Stenger, E., blood pigments, 281, 285, 
 
 286,289; urobilin, 745 
 Stepanek, J. O., 628 
 Stepp, W., 905, 906 
 Stern, E., 35 
 Stern, Fr., 750 
 Stern, H., 441, 442 
 Stern, Heinrich, 621 
 Stern, L., uricolysis, 706; peroxidase, 
 
 873, 874; oxydation processes, 874, 875 
 Stern, M., 248, 630 
 Stern, R., 440, 724 
 Steudel, H., arginine, 162; hexone bases, 
 
 164; mucin, 169; nucleic acids, 179, 
 
 181, 183, 184; pyrimidine bases, 194, 
 
 195; glucoseamine, 219 
 Stewart, C. W., 471 
 Stewart, G. X., 326, 566, 567, 570 
 Steyrer, A., 571 
 Sticker, G., 455, 458 
 Stiles, P. G., 400, 457 
 Stock, J., 297, 297 
 Stockmann, R., 595 
 Stoltzner, H., 554 
 Stoft'regen, A., 791 
 Stohmann, F., 510, 885 
 Stokes, 282, 289 
 Stoklasa, J., lecithin, 241; glycolysis, 
 
 407, 408, 509; lactic acid formation, 
 
 583; fermentation enzyme of milk, 653 
 Stokvis, B. J., bile pigments. 432, 433, 
 
 743; benzoic acid, 723; urobilin, 745, 
 
 792; hannatoporphyrin, 797 
 Stolnikow, J., 793 
 Stolte, K., 220, 396, 682 
 Stoltzner, W., 554 
 Stolz, Fr., 379 
 Stomberg, H., 256, 322 
 Stone, W., 208 
 Stookev, L. B., 136, 394 
 Stoop, F., 145, 148 
 Starch, \ ., 646, 671 
 Strada, Fr., 364 
 Stradomskv, N., 773 
 Strashesko, N. D., 462 
 Strassburg, G., gases of lymph, 347; 
 
 tension of the gases of blood, 861, 864, 
 
 868 
 Strassburger, J., 523 
 Strassuer, \Y., 877 
 Straub, W., 403, 89V), 920 
 Strauch, F. W\, 124 
 Strauss, Edw., 122 
 .Strauss, H., fructose, 217, 218, 264 
 
 blood, 264; transudates, 264, 355, 358 
 
 bile, 437; lactic acid fermentation, 485 
 
 amino acid storage, 721, 722 
 
 Strecker, A., 242,245, 422 
 Strickler, E., 658 
 Strigel, A., 870 
 Strohmer, F., 563 
 
 Strusiewicz, B., 912 
 
 Struve, H., 797 
 
 Strzyzowski, C., 293 
 
 Stubel, H., 894 
 
 Stutz, 791 
 
 Subbotin, V., 339, 921 
 
 Suckrow, Fr., 400 
 
 Sugg, E., 653 
 
 Suida, W., 445 
 
 Suleima, Th., 513 
 
 O'Sullivan, C., 54, 55, 57 
 
 Sundberg, C., 467, 468 
 
 Sunde, E., 655 
 
 Sundvik, E., purin bases, 188, 190 
 glucoseamine, 218; uric acid, 699 
 conjugated glucuronic, acids, 751, 777 
 chitin, 839; psylla alcohol, 846 
 
 Suter, F., 79, 113, 151 
 
 Suto, K., 238, 809 
 
 Suwa, A., 572, 780 
 
 Suzuki, \\\, cystine, 148; muscles, 572; 
 crab meat, 578; phytase, 579; orvza- 
 ninc, 905, 906 
 
 Svedberg, The., 14, 20 
 
 Svenson, N., 919 
 
 Swain, R. E., 159 
 
 Symmers, D., 757 
 
 Syniewski, V., 227, 229 
 
 v. Szontagh, F., 653, 658, 660, 662 
 
 Szydlowski, Z., 474 
 
 Szymonowicz, L., 379 
 
 Tachan, H., 145 
 
 Takahashi, D., 269, 329 
 
 Takaishi, M., 579 
 
 Takamine, J., 379 
 
 Takada, K., 787 
 
 Tallqvist, T. W., 914 
 
 Tammann, G., 60, 66 
 
 Tanaka, T., 371 
 
 Tangl, Fr., blood serum, 264, 271; blood 
 
 analysis, 326; sugar in blood, 398; 
 
 fat, 482; egg, development, 637, 638; 
 
 casein, 648, 658; milk, 668; C:N 
 
 quotient, 772, 884 
 Tanret, C., 200 
 v. Tappeimer, H., enzymes, 50; cellulose, 
 
 510, 515; bile acids, 541 
 v. Tarchanoff, J., 441, 632 
 Tarugi, B., 848 
 Tarulli, L., 675 
 Tawara, 932 
 Taylor, A. E., enzymes, 56; liver fat, 
 
 384; fat formation, 561; mineral 
 
 starvation, 900 
 Tebb, Chr., reticulin, 121; glycogen, o!)2; 
 
 amylolysis, 456; saecliarate, 492; mal- 
 tose, 500; protogon, 606-609; choles- 
 
 terin in brain, 613 
 Tedesko, Fr., 676 
 
178 
 
 INDEX OF AUTHORS 
 
 Teeple, J., 428, 430, 431 
 
 Teichrnann, L., 292 
 
 Tengstrom, B. St., 418 
 
 Terrat, P., 758 
 
 v. Terray, P., bile and putrefaction, 518, 
 
 520; oxalic acid, 71(5; lactic acid in 
 
 urine, 748, 749 
 Terroine, E. F., 105, 500-503 
 Terry, O. P.. 454 
 Terunchi, Y., 6S2 
 Tezner, E., 455, 456 
 Thannhauser, S. J., 429 
 Theissier, 247 
 Thesen, J., 572, 729 
 Thevenot, 247 
 Thiele, O., 754 
 Thiemich, M., 384 
 Thierf elder, H., barium, 72; galactose, 
 
 216, 610, 611; glucuronic acid, 223; 
 
 cephalin, 248; digestion and micro- 
 organism, 516; protagon, 607, 608; 
 
 cerebron and cerebroside, 608-611; 
 
 yolk phosphatides, 630; mammary 
 
 glands, 643, 669; sphingosin, 611; 
 
 cerebronic acid, 611 
 Thies, Fr., 379, 380 
 Thiroloix, J., 406 
 Thiry, L., 490, 491 
 Thorner, W., 644 
 Thomas, K., 595, 611 
 Thomas, Karl, 903 
 Thomas, P., 492 
 
 Thomas, W., 488, 909, 911, 914, 917 
 Thompson, W. H., 574, 682 
 Thorns, H., 687 
 Thormahlen, J., 799 
 Thudichum, L. W., phosphatides, 239, 
 
 240, 242, 245, 248; bilirubin, 431; 
 
 brain phosphatides, 605, 607-609; cere- 
 
 brosides, 609-611; sphingosin, 611; 
 
 lutein, 631; paraxanthine, 7l4; urinary 
 
 pigments, 740; alcohol in animal 
 
 organism, 921 
 Thunberg, T., 873, 874, 876 
 Tichomirow, N. P., 468 
 Tklemann, F., 458 
 Tiemann, H., 652, 658 
 Tigerstedt, K., 762 
 Tigerstedt, R., respiration apparatus, 
 
 869; metabolism, 893, 894, 924, 925, 
 
 936; digestion work, 930 
 Tissot, J., 592 
 Tobler, L., 341, 484, 763 
 Toepfer, G., 530, 759 
 Tollens, B., carbohydrates, 197, 208-211, 
 
 216, 217; glucuronic acids, 223, 817; 
 
 urea, 687; naphthoresorcin-reaction, 
 
 223 817 
 Tollens, C, 724, 725, 750, 817 
 Tolmatscheff, 656, 662, 665 
 Tomasinelli, <.., 848 
 Tomaszewski, Zd., 716 
 Tompeon, E., 54, 55, 57 
 Torup, S., carbon monoxyhsemoglobin, 
 
 287, 288; globulins and carbonic 
 
 acids, 853, 855 
 Totani, G., 161, 787 
 Tower, R. W., 121 
 Towles, C., 692, 693 
 Toyonaga, M., 388 
 Traube, J., 10, 11; absorption, 542; 
 
 oxydation, 871 
 Traube, M., 1, 9 
 Traxl, W., 78 
 Treupel, G., 749, 808 
 Treves, Z., 78 
 
 Trier, G., 178-180, 240, 243 
 Trifanowski, D., 437 
 Trillat, A., 873 
 Tritschler, F., 773 
 Troisier, J., 744 
 Troller, J., 464 
 Trommer, C., 214 
 Trommsdorff, R., 877, 910 
 Tnimpy, D., 847 
 Trunkel, H., 119, 120 
 Truthe, W., 230 
 Tschenloff, R., 910 
 Tschernoruzki, M., 307 
 Tscherwinsky, N., 563 
 Tschirjew. S., 344 
 Tsuboi, J., 339, 881 
 Tsuschija, J., 744, 747, 793 
 Tuczek, F., 459 
 Tullner, H., 698 
 Turk, W., 146 
 Turkel, R., 585 
 Turban, K., 912 
 Turby, H., 491 
 
 Udranszky, L., diamines, 47; bile acids, 
 419, 800; urinary pigments and humus 
 substances, 740; carbohydrates in 
 urine, 749, 808; cystine, 827 
 
 Uffelmann, J., 488 
 
 Uhlik, M., 280, 282, 283 
 
 Ulrich, Chr., 226 
 
 Ultzmann, R., 832 
 
 Umber, F., albumoses, 133; nucleins, 
 175; transudates, 354; gastric juice, 
 464, 465; proteins of pancreas, 494; 
 kevuloseuria, 814 
 
 Umikoff, N., 664 
 
 Underhill, F. P., protozym, 324; glyco- 
 seuria, 400, 402; saliva, 454; allantoin, 
 717; lactic acid in urine, 748, 749 
 
 Unna, P. G., 837, 844, 845 
 
 Ulpiani, C., 244, 699 
 
 Urano, F., 573, 587 
 
 Urbain, V., 851 
 
 Ure, A., 783 
 
 Usher, Fr., 37 
 
 Ussow, 506 
 
 Ustjanzew, W., 542 
 
 Vahlen, E., 425 
 Valenciennes, A., 603, 6S6 
 Valenti, A., 663 
 
INDEX OF AUTHORS 
 
 97 J 
 
 de Vamossv. Z., 370 
 
 Vandegrift, G. W , 545 
 
 Vandevelde, A. J. J., 053, 
 
 Vanlair, C, 129, 521 
 
 Vnsilin. H.. 720, 723 
 
 Vassale, ( ;., 374 
 
 Vaubd, W.. 82, 09 
 
 Vauquelin. L. N., 717 
 
 Vav, Fr., 3S3, 592 
 
 v. d. Velde, A.. 723 
 
 v. d. Velden, R., 724 
 
 Velichi. J., 602 
 
 Vella, L.. 490 
 
 Veraguth, O., 910 
 
 Verhaegen, A., 405 
 
 Verneuil, 401 
 
 Vernois, M., 665 
 
 Vernon, H. M., erepsin, 57, 492, 493; 
 white of egg, 05; pancreatic enzymes, 
 497, 500, 501, 503, 507, 509; muscle 
 rigor, 590 
 
 Verploegh, H., creatin and creatinine, 
 573, 091-094, 098 
 
 Verzar, Fr., 404 
 
 Viault. P., 340 
 
 Victorow, C, S04 
 
 Vierordt, K., 302, 803 
 
 Vigno, L., 520 
 
 Vignon, L., 122 
 
 Vila, A., blood corpuscles, 275, 281; 
 blood pigments, 285, 292; musculamine, 
 578 
 
 Ville. J., oxymethyl furfurol, 215; blood 
 pigments, 281, 285; bile acids, 419; 
 fat absorbtion, 539, 540; urinary 
 chlorine compounds, 758; Bence-Jones 
 protein, 792 
 
 Villiers, A., 758 
 
 Vincent, Sw., 370-002 
 
 Vines, S. H., 493, 502 
 
 Vinci, S., 314, 315 
 
 Virchow, R., 171, 301 
 
 Vitali, A.. 790 
 
 Vitali, D., 758 
 
 Vitek, 407 
 
 Voegtlin, C, 507, 092, 094, 703 
 
 Volte, \Y., 040, 912 
 
 Vogel, H., 372 
 
 Vogel, J., pentoses, 208, 815; isomaltose, 
 225; lactase, 492; amylolysis, 450, 500 
 
 Vogelius, 391 
 
 Voges, O.. 080 
 
 Vohl, H., 579 
 
 Voit. C, glycogen, 390, 393, 395, 534; 
 bile and putrefaction, 518; excrements, 
 520; absorption, 525, 537, 538; fat 
 formation, 500, 501, 003; work and 
 metabolism. 594, 597, 925; nitrogen 
 in meat, 000, 883; urea foimation, 
 t>>4; phosphoric acid excretion, 702; 
 standard numbers, 772, 915; lactose 
 detection, 815; metabolism experiments, 
 879, 881, 920, 920, 929; starvation 
 metabolism, 893, 903; water content 
 
 of body, SOS; mineral metabolism, 899; 
 protein catabolism, 903, 908, 908 910, 
 913, 914; nutritive value of gelatin 
 diets, 932-930, 938 
 
 Voit, E., glycogen, 395; bones, 554, 555; 
 fat formation, 562, 563j calorific value 
 of oxygen, 889; of nutritive substances, 
 S02; starvation metabolism, 894, 898 
 897; nitrogen excretion, 910; vege- 
 table diet, 915; protein minimum, 917; 
 surface rule, 023 
 
 Voit, Fr., galactose fermentation, 210; 
 thyroidea and metabolism, 370; glyco- 
 gen formation, 305; sugar elimina- 
 tion, 305, 534; feces formation, 521; 
 lactose, 532; curare poisoning, 591; 
 acetone bodies, 819 
 
 Voit, W., 814 
 
 Voitinovici, A., 100, 114, 121 
 
 Volhard, F., 409, 470, 500 
 
 Volhard, J., 759, 700 
 
 Volkmann, A. W., 899 
 
 Vorlander, D., 247 
 
 v. Vornveld, J. A., 341 
 
 Vossius, A., 441 
 
 Voswinckel, H., 843 
 
 Vozarik, A., 075, 077 
 
 de Vries, H., 2, 5, 
 
 Vulpian^A., 377, 441 
 
 Waage, P., 32 
 
 Wachsmann, M., 501 
 
 Waehsmuth, L., 350 
 
 Wakchli, G., 117 
 
 de Waele, H., 053 
 
 Wagner, B., 813 
 
 Wagner, H., 572, 577 
 
 Wahlgren, V.. 417, 420, 440, 838 
 
 Wait, Ch., 595 
 
 Wakemann, A. J., 380, 387, 737, 774 
 
 Walbum, L. E., 50, 793 
 
 Wald vogel, R., 820 
 
 Walker, J., 28 
 
 Wallace, G. B., 89 
 
 Walter, Fr., 082, 851, 856 
 
 Walter, G., 173, 030 
 
 v. Walther, P., 535, 537 
 
 Walton, J. H., 35 
 
 Wanach, R., 304 
 
 Wang, E., 730, 731 
 
 Wanklyn, J. A., 047 
 
 Wauner, Fr., 870 
 
 Warburg, O., leucine, 142, 143; fertiliza- 
 tion, 040; phenylamino acetic acid, 780; 
 oxvdation processes, 873, 874 
 
 Warfield, L. M., 455 
 
 Warmbold, H., 047 
 
 Warren, J., 593 
 
 Wasbuteki, M., 519 
 
 Wassiliew, W., 499 
 
 Wastenevs, H., 73 
 
 Waterman, N., 379 
 
 Wavmouth, Reid, E.. 533 
 
 Weber, 635 
 
980 
 
 INDEX OF AUTHORS 
 
 Weber, O. H., 341, 555, 340 
 
 Weber, S., 594, 692, _693 
 
 Wechselmann, Ad., 733 
 
 Wechsler, E., 83, 117 
 
 Wedenski, X., 749 
 
 Wegrzvnowski, L., 716 
 
 Wehrle, E., 404 
 
 Weidel, H., 189 
 
 Weigert, Fr., 163 
 
 Weigert, R., 265 
 
 Weil, Arth., 161, 605, 144 
 
 Weil, F. J., 423 
 
 Weiland, W., 786 
 
 Weinland, E., lactase, 53, 492, 532; 
 
 glycogen, 390, 395; sugar formation, 
 
 412; retarding substances, 466, 487, 
 
 892; fat formation, 561; 
 Weintraud, W., 403, 404, 685, 761 
 Weis, Fr., 506 
 Weisbach, 613 
 Weiser, St., 264 
 Weisberger, G., 866 
 Weiske H., cellulose 510; bones, 554, 
 
 555; asparagin, nutritive value, 912 
 Weiss, F., 78, 83, 109, 111, 162 
 Weiss, H. R., 505, 506 
 Weiss, J., 721 
 Weiss, Sigm., 395, 592 
 Weisz, Mor., 741, 752 
 Weizmann, Ch., 87 
 Wellman, O., 896 
 Wells, H. G., keratin, 114; hypophysis, 
 
 380; liver nitrogen, 387; uric acid, 
 
 701-703, 706 
 Wendel, A., 143 
 v. Wendt, G., 882, 903 
 Wenz, R., 128 
 Wenzel, F., 17S-180, 577 
 Werenskjold, F., 659 
 Werigo, B., 95, 866 
 Werneken, G., 651 
 Werner, A., 552 
 Wertheimer, E., 440, 498, 499 
 Werther, M., 459, 590, 591, 593 
 v. Westenrijk, N. f 309 
 Wester, D. H., 839, 840 
 Wetzel,'G., 122, 160, 178 
 Weyl, Th., albumin crystals, 94; car- 
 
 bonmonoxy methsemoglobin, 287; amni- 
 otic, fluid, 642. 643; creatinine, 695; 
 
 benzoic acid, 723; nitrate, 766 
 Wheeler, H., L., iodogorgonic acid, 123; 
 
 phenylalanine, 152; nucleic acid, 185; 
 
 cytosine, 194 
 Whipple, G. H., 253, 442 
 White, B., 705 
 Withnev, J. L., 406 
 Wichmann, A., 261, 652 
 Widdicombe, J. H., 492 
 Wichowski, W., urea determination, 088; 
 
 uric arid, and allantoin, 705, 706, 
 
 716-718; hippuric acid, 721 
 Wicland, H., 423 
 Wiener, H., autolysis, 43; serumglobulins, 
 
 259, 261, 335, nucleic acids, 514; uric 
 acid, 699, 700, 702, 704-706; oxalic 
 acid, 716 
 
 Wiener, K., 182 
 
 Wihelmv, 33 
 
 Wilke, K., 298 
 
 Willcock, Ed., 634 
 
 Willdenow, C., 164 
 
 v. Willebrand, E. A., 849 
 
 Willheim, R., 103 
 
 Williams, D., 510 
 
 Williams, H. B., 827 
 
 Willstatter, R., proline 154; lecithin, 263; 
 glycerinphosphoric acid, 247; chloro- 
 phyll and blood pigments, 277, 296, 
 297; cholesterin, 445, 448; carotein 
 yolk lutein, xanthophyll, 631 
 
 Wilson, R. A., 606, 608 
 
 Wimmer, M., 914 
 
 Windaus, A., histidin, 159, methylimi- 
 dazol, 201; cholesterin, 445, 448, 449 
 
 Windrath, H., 529 
 
 Winkler, 559 
 
 Winogiadow, A., 416 
 
 Winteler, L., 416 
 
 Winter, J., 660 
 
 Winterberg, A., 334, 675, 683 
 
 Winternitz, Hugo, blood pigment deter- 
 mination, 302; amounts of haemoglobin, 
 338; bile, 440; putrefaction, 517, 
 724; iodized fat, 560, 668, 669 
 
 Winternitz, M. C., 371, 702 
 
 Winterstein, E., amino-acids, 141, 153; 
 arginine, 161; lysine, 163; hexone 
 bases, 164; phosphatides, 245; phytin, 
 579; colostrum, 658; tunicin, 839; 
 chitin, 839 
 
 Wislicenus, J., 596 
 
 Wittmaack, K., 663 
 
 Woeber, A., 126 
 
 Wohler, Fr., hippuric acid synthesis, 39, 
 783; urea, 689; demolition of uric acid, 
 705, allantoin, 716, 717 
 
 Worner, E., 607, 611, 711 
 
 Wohl, A., 200 
 
 Wohlgemuth, J., autolysis, 43; enzymes, 
 53, 71; oxyaminosuberic acid, 147; 
 oxvdiamino-sebacic acid, 165; pen- 
 toses, 203, 208, 210; diastase in blood, 
 298; ferratin, 383; glycogen, 393, 394; 
 cystine demolition, 442; gastric juice, 
 476; pancreatic juice, 500; pancreatic 
 diastase, 501; pancreatic rennin, 509; 
 enzyme of egg-yolk, 628; woman's 
 milk, 660, 661; conjugaged glucuronic 
 acids, 751 ; amino acids in urine, 755 
 
 Wolf, C. G., glycogen, 397; urinary 
 nitrogen, 680; creatinine, 693; cystin- 
 uria, 827, 828; urinary sulphur, 882 
 
 Wolfenstein, R., 737 
 
 v. Wolff, E., 595 
 
 Wolff, H., 219, 358, 841 
 Wolff, .1., 873 
 
 Wolff, L. W., 19 
 
INDEX OF AUTHORS 
 
 981 
 
 Wnlffherg, 8., 395, 861, 864, 865 
 
 Wolkow, M., 735, 736 
 
 Woll, F. W., 645 
 
 Wolter. O., 770 
 
 Woltenng, H., 340, 383 
 
 Woods, II. 8.. 246 
 
 Wooldridge, L. C, stroma, of blood cor- 
 puscles. 274; tissue fibrinogen, 306, 
 307; blood coagulation, 315, 321, 323, 
 324 
 
 Worm-Muller, J., blood, 339, 340, 343, 
 344; sugar te-it, 803; sugar determina- 
 tion, 811-813 
 
 Worms, W., 635 
 
 Woronsow, W. N., 381 
 
 Wright, A., fibrin ferment and coagula- 
 tion, 256, 313, 323; blood alkalinity, 
 309; diabetes, 400 
 
 Wr6blewski, A., fermentation, 41 ; pseudo- 
 nuclein, 104; starch, 227; pepsin, 466, 
 470; enzyme action, 510; enzymes 
 of brain, (J06; milk, 660, 661 
 
 Wulff, C. 190, 714 
 
 Wurm, W. A., 843 
 
 Wurster, C, 768 
 
 Wurtz, A., 346, 625 
 
 Yagi, 8., 448 
 
 Yakuvva, G., 912 
 
 Yoshimoto, 443 
 
 Yoshimura, K., 579 
 
 Young, P. A., 435 
 
 Young, R. A., 230, 391 
 
 Young, W. J., co-enzymes, 52, 205; car- 
 
 bohydrate-phosphoiic acid ester, 60, 
 
 205; dioxyacetone, 205 
 Yvon, 752 
 
 Zangerle, M., 626 
 
 Zah6r, H., 794 
 
 Zaitschek, A., milk, 651, 653, 658-662, 
 
 668 
 Zak, E., 354, 769, 791 
 Zaleski, J., leaf and blood pigments, 277, 
 
 296; blood pigments, 291-295, 301; 
 
 ammonia, 334, 336, 528, 683, 768; 
 
 urea, 684; liver and acid formation, 
 
 685, 703, 704 
 Zaleski, St., iron of liver, 383, 388; milk, 
 
 666, 667; reaction of intestinal contents 
 
 519 
 Zaleskv, N., 552, 847 
 Zander, E., S39 
 Zanetti, C. U., ,seromucoid, 260, 263; 
 
 bile, 417; ovomucoid, 635 
 
 Zangermcistcr, \Y., 302, 642 
 
 Zaribnickv, It., '■> Is 
 
 Zaudy, 70] 
 
 Zdaiek, E., 841 
 
 v. Zebrowski, E., 453 
 
 Zerhui.-en, II., 777 
 
 Zeidlite. P. V., 804 
 
 Zegla, P., 398 
 
 Zeller, A., 75s, 777 
 
 Zemplen, G., 839 
 
 v. Zeynek, R.„ dermoid-cyst-fat, 239, 627, 
 844; blood pigments, 283, 285, 289, 
 290; liver, 602; bile, 438; sarcomelanin 
 841; chromoproteid, 843 
 
 Zickgraf, G., 83, 870 
 
 Ziegler, E., 779 
 
 Zieuler, J., 32, 708, 716, 717 
 
 Zillessen, H., 593 
 
 de Zilwa, L., 500 
 
 Zimmermann, R., 372, 725, 726 
 
 Zimnitzki, S., 517 
 
 Zink, J., 237, 559 
 
 Zinnowsky, O., 277 
 
 Zinsser, A., 476 
 
 Zisteier, J., 917 
 
 Zobel, S., 581 
 
 Zoja, L., oxyproteic acids, 83; elastin, 122; 
 ovalbumin, 633; urobilin, 744; uroery- 
 thrin, 748; ha-matoporphyrin, 797, 798 
 
 Zsigmondy, R., colloids, 15, 19, 21, 23 
 
 Zuelzer, G., lecithin, 245; diabetes, 
 405, 406; skin breathing, 849 
 
 v. Zumbusch, L., 434, 844 
 
 Zuntz, N., blood, 310, 338, 340, 341; 
 glycogen, 391; sugar in blood, 398; 
 plilorhizin diabetes, 400; digestion, 
 506, 542; protein assimilation, 525; 
 muscle fat, 586, 596; muscle metabol- 
 ism, 591, 595, 597; pig milk, 659; 
 high altitudes, 341, 848; skin breath- 
 ing, 849; gases of blood, 851-853, 859, 
 866, carbonic acid of lymph, 856; 
 alveolar air, S63; respiration, 868. 
 869; metabolism, 879, 884, 890, 893 1 
 922, 923, 926, 927, 929; calculation 
 of calorific power, 888; nutritive value 
 of albumoses, 912; alcohol, 921; diges- 
 tion work, 930 
 
 Zuntz, L., 309 
 
 Zuntz, E., digestion products, 132-134, 
 139; gastric digestion, 482,484; absorp- 
 tion, 484, 526, 532, 540; trypsinogen 
 and its activation, 496, 497; intestinaL 
 diaestion, 513; muscles ,572 
 
 Zweifel, P., 456, 500, 748, 749 
 
GENERAL INDEX 
 
 Abderhalden and Schmidt's reaction for 
 
 proteins, 101 
 Abiuret products, 132, 513, 526 
 Absorption, 524-543 
 
 chemical processes during, 
 
 524-525 
 effect of extirpation of the 
 pancreas upon, 
 531,532 
 removing portions 
 of intestine upon, 
 541, 542 
 of amino acids from intes- 
 tine, 526-528 
 of bile constituents, 540, 541 
 carbohydrates, degrees of 
 
 rapiditv of, 532-534 
 of fat, 535-540 
 of nonbiuret giving products 
 
 from intestine, 526-528 
 of mineral substances, 540 
 peptones from intestine, 526- 
 
 528 
 of proteose from intestine, 
 
 526-528 
 of undigested protein, 525, 
 
 526 
 theory of, 542, 543 
 Acetanilide, fate of in organism, 779 
 Acethaemin, 292 
 Acetone bodies, 776 
 
 in urine, 818-828 
 origin of, 818, 819 
 fate in organism, 821 
 formation from fat, 820-822 
 formation from protein, 819 
 former, 781 
 in urine, 822-824 
 quantitative estimation of, in 
 urine, 825 
 Acetophenone, fate of in organism, 785 
 Acetylation in organism, 786 
 Acetyldiglucosamine, 839 
 Acetyl equivalent of fats, 238 
 Acetylglucosamine, 784 
 Acetylene Iwemoglobin, 288 
 Acetylpara-amidophenol, 780 
 Achilles tendon, 545 
 Acholia, pigmentary, 439 
 
 Achroodextrin, 229, 458 
 Acid abietinic, 447 
 
 acetic, in intestines, 513 
 in stomach, 465 
 in urine, 748, 773 
 acetoacetic in urine, 821, 824, 825 
 
 fate in organism, 821 
 acetylaminobenzoic, 7S4 
 albuminates, 125-127 
 
 absorption of, 533 
 in peptic digestion, 472 
 alloxyproteic, in urine, 752, 754, 755 
 amides, behavior in organism, 775 
 aminoacetic. See Glycocoll. 
 aminocaproic. See Leucine, 141 
 a-diamino-/3-dithiolactic, 148 
 0-7-diaminovaleric,, 163 
 a-e-diaminocaproic, 163 
 d-a-amino-n-caproic, 144 
 aminoethylsulphonic, 150 
 a-aminoalutaric, 146, 117 
 a-aminoisobutylacetic. See Leucine, 
 a-amino-valeric, 140. See Valine, 
 aminobenzoic, behavior in organ- 
 ism, 783 
 aminoeinnamic, 778, 780 
 a-amino-/3-oxypropionic, 145. See 
 
 Serum. 
 a-amino-/3-thiolactic, 148, 150. See 
 
 Cystine, 
 a-aminopropionic, 140. See Alanine, 
 aminoglucuronic, 548 
 aminohippuric, 783 
 aminophenylacetic, behavior in or- 
 ganism, 781 
 aminosuccinic. See Aspartic acid, 
 
 146 
 antoxvproteic in urine, 752 
 arachidic, 232, 627,645,845 
 aspartic, 85, 146 
 
 quantitv in proteins, 106, 
 
 107, 115, 125 
 relation to formation of 
 
 urea, 682, 77* I 
 relation to formation of 
 uric acid, 702 
 /3-amino-a-oxypropionic. See Iso- 
 
 serine, 14fi 
 /3-imidazol-a-aminopropionic, 159 
 
 983 
 
984 
 
 GENERAL INDEX 
 
 Acid benzoic, conjugation of, in organism, 
 782 
 in urine, 723 
 glucuronic, 751 
 
 benzoyl-amino acetic. See Hippuric 
 acid, 719 
 
 bilinic, 429 
 
 bilianic, 423 
 
 bilirubinic, 430 
 
 bromphenylmercapturic, 780 
 
 butyric, fermentation, 214, 516 
 
 camphoglucuronic, 222, 751, 786 
 
 capric, 232 
 
 caproic, 232 
 
 caprylic, 232 
 
 carbamic, 267 
 
 conjugation of, 786 
 fate of in organism, 786 
 in blood, 267, 683 
 in urine, 691, 692 
 
 carbamino-acetic, 164, 856 
 
 carboglobulinic, 855 
 
 carbolic. See Phenol. 
 
 carminic, 844 
 
 carnaubic, 239, 673 
 
 carnic, 653 
 
 caseanic, 85, 165 
 
 caseinic, 85, 165 
 
 cephalic, 248 
 
 cepholinic, 248 
 
 cerebrinic, 612 
 
 cerebrinic-phosphoric, 612 
 
 cerebronic, 611 
 
 cerotic, 239 
 
 cheno-taurocolic, 422, 427 
 
 chitaminic, 219 
 
 chitaric, 219 
 
 chlorhodinic, 366 
 
 chlorphenylmercapturic, 786 
 
 cholalic, 423-425 
 
 constitution of, 423 
 
 cholanic, 425 
 
 choleic, 425 
 
 cholesterinic, 423 
 
 cholic, 423, 424 
 
 properties of, 424 
 tests for, 424 
 
 choloidanic, 423 
 
 choloidic, 427 
 
 cholylic, 423 
 
 chondroitic. See Chondroitinsul- 
 phuric acid, 547 
 
 chondroitin-sulphuric, 171, 173, 547 
 
 chrysophanic, elimination in urine, 
 787 
 
 cilianic, 423 
 
 cinnamic, behavior in organism, 
 719 
 
 citric, in milk, 647, 658, 663 
 
 coccinic, 844 
 
 cochenillic, 844 
 
 combined hydrochloric, 488 
 
 cresol-sulphuric, 724-727 
 
 erotonic, 825 
 
 Acid cumic, conjugation of in organism. 
 783 
 cuminuric, synthesis of in organism, 
 
 783 
 cyanuric, 687, 698 
 cysteinic, 149 
 damaluric, 758 
 damolic, 758 
 dehydrobillinic, 429 
 dehydrocholeic, 425 
 desaminoalbuminic, 127 
 desoxycholic, 425, 426 
 
 properties of, 426 
 diacetic. See Aceto-acetic 
 dialinic, relation to formation of 
 
 urine, 704 
 diamino acetic, 85 
 diaminotrioxydodecanoic, 85, 165 
 dimethylaminobenzoglucuronic in 
 
 urine, 751, 786 
 p-dimethylaminobenzoic, 786 
 dioxydiaminosuberic, 165 
 dioxyphenylacetic in urine, 735 
 dioxyphenyl-lactic, 738 
 dioxystearic, 236, 647 
 doeglic, 237 
 elaic, 236 
 elaidic, 236 
 ellagic, 524 
 equivalent, 238 
 erucic, 232 
 ethylidenelactic, 582 
 euxanthic, 222, 223 
 
 in urine, 785 
 euxanthonglucuronic, 222 
 excretolic, 523 
 fellic, 426 
 fermentation lactic, 582 
 
 in blood, 335 
 in coagulation of 
 milk, 585, 645. 
 in muscle, 604 
 in stomach, 487,, 
 488 
 formic, fate in organism, 748, 773 
 furfuracrylic in urine, 784 
 furfuracryluric in urine, 784 
 gadoleic, 237 
 galactonic, 217 
 gallic, fate of in organism, 785 
 
 in urine, 734 
 gentisic, 336, 737 
 
 behavior in organism, 784 
 gluconic, 198 
 
 in diabetes, 403 
 glucosaminic, 201, 219, 221 
 in milk, 643 
 glucothionic, 387, 673 
 
 in milk, 643 
 glucuronic, 221-223 
 
 conjugation with, 785 
 elimination, effect of for- 
 eign substances on, 
 750 
 
GENERAL INDEX 
 
 985 
 
 Acid glucuronic, formation of in organism, 
 751 
 in bile, 417, 433 
 in blood, 264 
 in urine, 750 
 isolation of, 222 
 preparation of, 223 
 properties of, 222 
 quantitative estimation 
 of, 223 
 glutamic, 82, 86, 106, 107, 115, 125, 
 
 147 
 glutinic, 119 
 glyceric, 145 
 glycerophosphoric, 248 
 
 in urine, 749, 757 
 glychollic, fate of in organism, 713 
 glycocholic, 417, 419, 420 
 
 absorption of, 548 
 in feces, 517 
 preparation of, 421 
 properties of, 420 
 glycocholeic, 420-421 
 
 preparation of, 421 
 properties of, 420 
 glycosuric in urine, 735 
 glycuronic, 221 
 glyoxylic, 719 
 
 as reagent, 100 
 guanidineacetic, fate of in organism. 
 
 See Glycosamine, 787 
 5-guanido-a-aminovaleric. See Arg- 
 
 inine, 161 
 guano bile, 421 
 guanvlic, 178, 181, 183, 184 
 
 of liver, 383 
 hEematinic, 296, 430 
 haematopyrrolidinic, 291 
 haemoglobin, 285 
 hippuric, 267, 719-723 
 
 formation of, 139 
 
 in organism, 
 719 
 in urine of herbivora, 720, 
 
 721 
 occurrence of, 719 720 
 preparation of, 722-723 
 properties of, 722 
 quantitative estimation of, 
 
 723 
 reactions for, 722 
 synthesis of in organism, 
 
 782 
 synthetical preparation of, 
 
 719 
 theories of formation in 
 organism, 721 
 homogentisic, 778 
 
 formation of in organ- 
 ism, 735-737 
 in urine, 727, 734-740 
 mother substances of, 
 
 735-736 
 preparation of, 739 
 
 Acid homogentisic, properties of, 738-739 
 quantitative estima- 
 tion of, 739 
 quantity eliminated, 
 
 735 
 test tor. 7:;'.» 
 homoglytisic, origin of, 736 
 hydrochloric action upon ptyalin, 455 
 secretion of 
 
 bile, 416 
 6ecretion of 
 pancrea- 
 tic juice, 
 498 
 pylorus, 
 480, 482 
 antifermentive action, 
 485 
 hydrochinon sulphuric, 724 
 hydrocinnamic, behavior in animal 
 
 body, 720 
 hydrocyanic, effect on blood pig- 
 ments, 285 i 
 pepsin diges- 
 tion, 471 
 trypsin di- 
 gestion, 506 
 hydroparacoumaric, in intestinal pu- 
 trefaction^ 15 
 in urine, 734 
 hydroquinone carboxylic, 738 
 hydroquinonesulphurir, 691, 724, 728 
 hyoglycocholic, 421, 427 
 hypogaeic, 239 
 
 imidazolaminoacetic in urine, 758 
 imidazol propionic, 82 
 indolacetic, 82 
 
 in urine, 733 
 indola:ninoproj;ionic, 82, 155 
 indol-carboxylic, 728 
 indolTpropionic, 82, 155 
 indoxyl-earboxylic, 728 
 indoxylglucuronic, 728 
 indoxyl-sulphuric, 724 
 
 formation of in 
 
 organism, 728 
 in urine, 728-731 
 inosinic, 17S, 181-183, 185 
 iodogorgonir, 123 
 isobilianic, 423 
 isocholanic, 425 
 isocholic, 427 
 
 isophonopyrrol carboxylic, 429, 430 
 isovaleric, 774 
 jecoleic, 237 
 kynurenic, 740, 758 
 
 in urine, 734 
 kyroproteic, 83 
 lactic, 145 
 
 formation of in active 
 muscles, 593 
 
 in blood, 333 
 in blood, 334 
 in cerebrospinal fluid, 360 
 
986 
 
 GENERAL INDEX 
 
 Acid lactic, in relation to diabetes, 408 
 origin of, 583, 584 
 quantitative determination in 
 
 gastric contents, 488 
 test for in gastric contents, 488 
 transformation into uric acid, 
 704 
 lactophosphocarnic, 645, 653 
 lanoceric, 846 
 lanopalmitic, 846 
 lauric, 232, 647, 846 
 lepidotic, 844 
 leucinic, 142 
 levulinic, 211, 654 
 linolenic, 232 
 linolic, 232 
 lithobilic, 524 
 lithocholic, 427 
 lithofellic, 427, 524 
 lithuric, 758 
 lysalbinic, 127 
 lysuric, 164 
 maleic, 290 
 
 malic, fate in organism, 668, 676 
 mandelic, 780 
 mannonic, 212 
 margaric, 235 
 melanoidic, 841 
 menthol-glucuronic, 817 
 mesitylenic, conjugation of in organ- 
 ism, 783 
 mesitylenuric synthesis of in organ- 
 ism, 783 
 metaphosphoric as precipitant of 
 proteids, 98, 789 
 
 in nucleins, 176 
 in pseudonucleins, 
 177 
 methylethyl-a-aminopropionic, 143. 
 
 See Isoleucine. 
 methylguanidine-acetic. See Crea- 
 tine, 573 
 methylhydantoie, 776 
 monoxystearic, 233, 236 
 mucic, 217, 394, 654 
 muconic, 778 
 myristic, 232 
 naphtholglucuronic, 787 
 neurostearic, 611 
 nitrobenzoic, 784 
 nitrohippuric, 783 
 
 nitrophenolpropiolic, fate in organ- 
 ism, 728, 731 
 test for dex- 
 trose, 216,808 
 norisosaooharic, 219, 630 
 nucleic, 622 
 
 of milk, 643 
 yeast, 178, 185 
 nucleotinic, 179 
 nuoleotinphosphoric, 179 
 oleic, 232 
 
 properties of, 236 
 tests for, 237 
 
 Acid ornithuric, 163 
 
 synthesis of in organism, 
 783 
 orotic, 654 
 orylic, 653 
 
 oxalic in urine, 716, 717 
 oxaluric, 699, 715 
 oxaminic, 83 
 oxonic, 699 
 
 oxyaminosuberic, 85, 148 
 oxyaminosuccinic, 85, 147 
 oxy-a-pyrolidinecarboxylic. See oxy- 
 
 proline. 
 oxybenzoic, fate in organism, 783 
 /3-oxybutyric, 821, 825-827 
 
 detection of in urine, 
 
 826 
 fate in organism, 821 \ 
 formation from amino 
 
 acids, 821 
 properties of, 825, 826 
 quantitative estima- 
 tion of, in urine 826 
 oxydiaminosebacic, 85, 165 
 oxydiaminosuberic, 85, 164 
 oxyhydroparacoumaric, in urine, 734 
 oxyisovaleric, 774 
 oxymandelic, 739 
 
 oxymethylpyrazine-carboxylic, 221 
 p-oxyphenylacetic, 515 
 
 in urine, 734, 784 
 p-oxyphenyl-a-aminopropionic . See 
 
 Tyrosine, 152 
 p-oxyphenylpropionic, 515, 734 
 
 in urine, 784 
 oxyphenylpyroracemic, 737 
 oxyproteic in urine, 752, 754 
 oxyprotosulphonic acid, 83 
 oxyquinoline carboxylic. See Kynu- 
 
 renic acid, 
 palmitic, 232, 235 
 
 properties of, 236 
 parabanic, 699 
 paralactic, 582 
 
 origin of, 583, 584 
 in urine, 749 
 paranucleic, 652 
 paraoxyphenyl-acetic in urine, 734, 
 
 735 
 paraoxyphenylpropionic, 82 
 
 in urine, 
 734, 735 
 pepsin-hydrochloric, 473 
 peroxyproteic ; 83 
 phenaceturic in urine, 723 
 phenol glucuronic, 222, 725, 749, 751 
 phenol-sulphuric, 724, 725-727 
 phenylacetic, 82 
 
 conjugation of in or- 
 ganism, 783 
 phenylaminoacetic, 780 
 phenyl-a-aminonropionic. See Phe- 
 nylalanine, 151 
 phenylbutyric, 781 
 
GENERAL INDEX 
 
 987 
 
 Acid phenylcaproic, 781 
 
 phenylketopropionic, 781 
 phenyllactic, 736, 778, 780 
 phenyl oxypropionic, 781 
 phenylpropiomc, 82 
 phenyl pyroracemic, 737 
 phenylvaleric, 781 
 
 in perspiration, 848 
 in urine, 724 
 phonopyrrolcarboxylic, 298 
 phosphocarnic, 267, 572, 577-579 
 
 in active muscles, 594 
 
 in urine, 757 
 
 source of muscular 
 
 energy, 598 
 as nuclein amounts 
 in blood of differ- 
 ent animals, 326 
 in urine, quantitative 
 estimation of, 763- 
 764 
 phthalic, fate in organism, 779 
 physetoleic, 239 
 plasm inic, 186 
 polypeptide phosphoric, 652 
 protalbinic, 127 
 protic, 572 
 protocatechuic, fate in organism, 
 
 726 
 pseudonucleic. See Paranucleic acid 
 psyllic, 845 
 pulmotartaric, 870 
 pyinic, 366 
 
 pyridine-carboxylic, fate of in or- 
 ganism, 784 
 pyridinuric, 784 
 pyrocatechin-sulphuric, 724 
 
 in urine, 727 
 pyrocholoidanic, 424 
 a-pyrolidine carboxylic. See Pro- 
 line, 154 
 pyromucic in urine, 784 
 pyromucinornithuric in urine, 784 
 pyromucuric, 784 
 pyroracemic, 150 
 
 effect of yeast upon, 
 206 
 pyrrolidone-carboxylic. See a-Pro- 
 
 line, 113 
 pyruvic, effect of yeast upon, 206 
 quinic, 719 
 
 racemic, fate in organism, 773 
 renosulphuric, 673 
 rhodizonic, 580 
 saccharic, 198, 222 
 
 behavior in diabetics, 403 
 relation to glycogen for- 
 mation, 394 
 salicylic, conjugation of in organism- 
 
 783 
 salmo-nucleic, 178 
 sarcolactic, 582 
 
 in brain, 606 
 
 in lymphatic glands, 366 
 
 Acid sarcolactic, in muscular work and 
 rigor, 591-595 
 in the bones, 556 
 passage into urine, 748 
 earcomelanic, 841 
 scymnol, 417 
 scymnol-sulphuric, 417 
 sebacic, 236 
 silicic, in blood, 268 
 in bones, 555 
 in connective tissue, 549 
 in feathers and hair, 839 
 in hen's eggs, 632, 637, 639 
 in urine, 769 
 skatol acetic, 82, 156 
 skatolaminoacetic, 156 
 akatol-carboxylic in urine, 733 
 skatoxylglucuronic, 222, 732, 750 
 skatoxyl-sulphuric, 724, 732, 733 
 origin of, 732 
 stearic, 232 
 
 properties of, 235 
 succinic, 83 
 
 in fermentation of milk, 645 
 in intestines, 513 
 in perspiration, 848 
 in spleen, 370 
 in thyroid, 373 
 in transudates, 355, 359, 361 
 in urine, 749, 773 
 sulphosalicylic as a reagent, 790 
 sulphuric, action on peptic digestion, 
 469. See also Ethereal sulphates 
 and Mineral substances, 
 tannic, fate of in organism, 785 
 tartaric, relation to glycogen for- 
 mation, 394 
 in organism, 773 
 in perspiration, 848 
 tartronic, 704 
 taurocarbamic, 776 
 taurocholic, 417, 421-422 
 
 preparation of, 421, 422 
 properties of, 422 
 taurocholeic, 422, 423 
 tetraoxyaminocaproic, 548 
 therapinic, 237 
 thiolactic, 150 
 thiophenuric in urine, 784 
 thymic, 179 
 
 thymonucleic, groups, 181 
 thiolactic, 85 
 toluic, conjugation of in organism, 
 
 783 
 toluric, synthesis of in organism, 783 
 triehlorethylglucuronic. See Uro- 
 
 chloralic acid, 
 triticonucleic, 178, 185 
 2-, 4-, S-trimethvlpvrrol-S-propionic, 
 
 429 
 trioxy benzoic. See Gallic. 
 turpentine glucuronic, 785, 817 
 tyrosinesulphuric, 153 
 uraminobenzoic, 783 
 
988 
 
 GENERAL INDEX 
 
 Acid uram in-salicylic, 783 
 urea glucuronic, 750 
 uric, 187, 267 
 
 amounts formed in organism, 
 
 707 
 as a pigment, 844 
 effect of food on elimination of, 
 
 700 
 endogenous origin, 702 
 exogenous origin, 702 
 fate of in organism, 705, 706 
 formation of from purines, 701, 
 702 
 in birds, 703, 704 
 of in the organism, 
 
 701-707 
 from lactic acid, 704 
 in blood, 334 
 in muscles, 572 
 in urinary sediment, 830 
 in urine, 698-712 
 occurrence of, 699, 700 
 preparation from urine, 710 
 preparation of, 698 
 properties of, 698-699, 707, 708 
 quantitative estimation of in 
 urine, 710 
 relation to urea 
 elimination, 701 
 quantity in various urine, 699, 
 
 700 
 synthetic formation of, in or- 
 ganism, 705 
 tests for, 709 
 
 various factors effecting elim- 
 ination, 700-702 
 Urocanic, 758 
 urochloralic, 777 
 uroferric in urine, 752, 755 
 uroleucic in urine, 736, 739 
 uronitrotoluolic in urine, 786 
 uroproteic in urine, 754 
 uroxanic, 699 
 ursalicylic in urine, 783 
 ursocholeic, 427 
 valeric, 80, 142 
 vanillinic, 780 
 whey, 645 
 
 xanthobilirubinic, 429 
 xanthopyrrolcarboxylic, 299 
 yeast-nucleic, 179, 185 
 Acids, amino, 92 
 
 aromatic, fate of in organ- 
 ism, 780-783 
 investigations on, 
 of demolition in 
 organism, 780- 
 783 
 as acetone formers, 819 
 as carbon dioxide binders, 
 
 866, 856 
 conjugation with, 786 
 deamidation, 682 
 fate of, in organism, 774-776 
 
 Acids, amino, formation in tryptic diges- 
 tion, 507 ; 
 of, in liver, 530 
 how absorbed, 528 
 in globin of blood pig- 
 ments, 289 
 in homogentissic acid for- 
 mation, 736-738 
 in liver tissue, 383 
 in lymphatic glands, 366 
 in muscles, 572 
 in transudates and exu- 
 dates, 355 
 in serum, 267 
 in urine, 756, 827 
 investigation on the demo- 
 lition of, in organism, 
 774-776 
 of pseudomucin, 626 
 Sorensen's, formol titration 
 
 for, 139-167 
 synthesis of, in organism, 
 
 786 
 sugar formation from, in 
 
 liver, 412 
 transformation of, into 
 urea, in the organism, 
 683,684 
 aromatic, fate of, in organism, 784 
 
 -oxy, in urine, 733, 734 
 bile, 267 
 
 detection of, 418, 419, 427, 428 
 biliary, properties of alkali salts, 
 
 418 
 caseonphosphoric, 652 
 cholic, preparations of, 426 
 conjugated glucuronic in urine, 
 
 817-818 
 desaminoproteic, 83 
 diamino, 84, 159-163 
 ethereal sulphuric, 723-733 
 
 amounts in 
 
 urine, 724 
 in urine, 765 
 quantitative es- 
 timation of, 
 726 
 synthesis in 
 liver, 381 
 excitants for bile secretion, 416 
 fatty, 232-239, 265 
 
 amounts in blood of differ- 
 ent animals, 328 
 in brain, 605 
 in blood, 334 
 in perspiration, 848 
 in pus, 365, 366 
 in urine, 748, 773, 829 
 investigation on the demol- 
 ition of, in organism, 773, 
 774 
 series, fate of, in organism, 
 773, 774 
 glucuronic, conjugated, 222 
 
GENERAL INDEX 
 
 989 
 
 Acids, glycocholie, preparation of, 421 
 
 in large intestine, from putrefac- 
 tion, 515 
 in spleen, 370 
 in thymus, 368 
 in thyroid gland, 373 
 in transudates and exudates, 355 
 kyroproteic, 83 ' 
 lactic, 582-586 
 
 detection of, 585 
 in bones, 556 
 in urine, 703, 704, 748 
 mercaptic, elimination of, 786 
 
 properties of, 585 
 melanoidic, 841 
 
 mineral, alkali-removing action of, 
 
 and action on the elimination of 
 
 ammonia, 675, 676, 762, 768, 856 
 
 893 
 
 monoamino. See Acids, Amino. 
 
 neutralization of, in the organism, 
 
 675, 676 
 nucleic, 175, 177-186 
 complex, 181 
 effect of gastric juice on, 
 
 473 
 enzymotic, cleavage of, 508 
 plant, 185-186 
 simple, 181 
 organic, behavior in animal body, 
 
 767, 772, 773, 776, 777 
 oxyamino, 79, 80, 84, 148, 219 
 oxyfatty, in animal fat, 232, 238 
 oxy, in urine, detection of, 735 
 oxypropionic, 582 
 oxyproteic in urine, quantitative 
 
 estimation of, 755, 751} 
 phthalic, fate of, in the organism, 
 
 77S 
 proteic in blood, 267 
 in serum, 267 
 in urine, 752, 754 
 thymonueleic, 178 
 thymua-onucleic, 173 
 uramino, 786 
 ureido glucuronic, 750 
 volatile fatty, in urine, 748 
 Acidosis, 820, 821 
 A. Tire, 212 
 Acrolein, 234, 237 
 a-aerose, 212 
 /3-acrose, 212 
 Actinioehrom, 844 
 Activators. 60 
 
 Adamkiewicz-Hopkin's reaction for tryp- 
 tophane, 157 
 Adamkiewicz'a reaction for protein, 100 
 Adaptation of the glands, 454, 462, 463, 
 
 494-496 
 Addison's disease, 377, 379 
 Adelomorphic cells, 489 
 Adenase, 48, 188 
 
 Adenine, 178, 181, 185, 187, 188, 192, 193, 
 712 
 
 Adenine-hexose compound, 180 
 Adenosine, 180 
 
 Adialyzable bodies in urine, 757 
 Adipoeere, 560 
 Admissibility, theories of, 9 
 Adrenalin, 378-380 
 
 bodies, 377, 378 
 
 constitution of, 378 
 
 function of, 379, 380 
 
 properties of, 379 
 
 tests for, 379 
 " Adsorpates," 12 
 Adsorption, 49, 62, 63, 69 
 
 in relation to permeability, 
 11, 12 
 J^agrophiUe, 524 
 /Erotonometric method, 864 
 Age, effect upon metabolism, 922, 924 
 Agglutenins, 47, 69 
 Agmatine, 162 
 
 Air bladder, of fishes, gases of, 867 
 Alanine, 85, 106, 107, 109, 111, 113, 125, 
 
 140, 145 
 Alanylalanine, 88 
 Alanvlalanineglycin, 86 
 Alanylglycine, 87, 508 
 Alanylleucine, 86, 508 
 Albamine, 220 
 Albumin, 49 
 
 detection of, in urine, 791 
 Albuminates, 104, 125-127 
 acid, 92, 126 
 
 alkali, 91, 92, 125, 126, 533 
 Albuminoids, 92, 112 
 Albuminose, 623 
 Albumins, 91, 92, 93, 102, 103, 106 
 
 quantitative estimation of, in 
 urine, 71)3 
 
 properties of, 102, 103 
 Albumoids, preparation of, 549 
 Albuminuria, 771, 787 
 Albuminoids, 92 
 
 Albuminous bodies, in general, 94-97 
 Albumoid. properties of, 618 
 Albumose, alkali, 127 
 Albumoses, 130 
 Alcapton in urine, 727 
 Alcaptonuria, 735-740 
 Alcohol, aminoethvl, 240 
 cetyl, 239 
 melissyl, 239 
 myricyl, 239 
 Alcoholase, 47, 48 
 Alcoholases, 875 
 Alcoholic fermentation, 41 
 Aldehydases, 875 
 Aldehydes, fate of, in organism, 775, 780, 
 
 781 
 Aldoses, 197 
 Alexines, 266 
 
 Alimentary acetonuria, 820 
 glycosuria, 533 
 Alizarin.'elimination in urine. 556, 787 
 Alkali earths. See Mineral substances. 
 
990 
 
 GENERAL INDEX 
 
 Alkaloids, fate of, in organism, 787 
 Alkyl sulphides, 846 
 Allantoin, 699, 717-719 
 
 elimination of during poison- 
 ing, 717, 718 
 formation of, in organism, 717 
 occurrence of, 717 
 preparation of, 718 
 properties of, 718 
 tests for, 718 
 Alloisoleucine, 145 
 Alloxan, 86 
 Alloxuric bases, 186, 712 
 
 preparation of, 714. See 
 Purines. 
 Alveolar air, 853 
 Ambergris, 524 
 Amboceptors, 69 
 Ambrain, 524 
 Amicrons, 19 
 Amidases, 702 
 Amidoguanine, 718 
 Amidomj'elin, 609 
 Amidulin, 227 
 
 Amino acids. See Acids, Amino. 
 Amino aldehyde, 219 
 
 butyrobetaine in urine, 787 
 cerebrinic acid, glucoside, 612 
 oxypurine. See Guanine, 
 oxypyrimidine. See Cytosine. 
 sugars, 219-221 
 Aminophenol, 779 
 Aminopurine, 191 
 Aminosuccinic-acid amide. See Aspara- 
 
 gine 
 Ammonia, amounts in urine, 766 
 
 detection of, in urine, 768 
 elimination in relation to acid 
 
 formation, 766, 767 
 importance of to organism, 767 
 in blood, 334 
 in urine, 766-768 
 in venous blood, 336 
 quantitative estimation of, in 
 
 urine, 768 
 transformation of, in the liver, 
 767 
 Ammonium magnesium phosphate, in 
 intestinal calculi, 232 
 in urinary calculi, 834 
 salts, relation to "glycogen 
 formation, 394 
 relation to urea for- 
 mation, 683, 767 
 relation to uric acid 
 formation, 702 
 urate, in urinary calculi, 834 
 in urinary sediment, 
 829 
 Amniotic fluid, 642 
 Amphicreatine, 578 
 Arnygdalase, 48 
 Amygdalin, cleavage of, 59 
 Amylase, 47, 48 
 
 Amylodextrin, 227, 229 
 
 Amyloid, 172, 231 
 
 preparation of, 174 
 
 Amylopectin, 227 
 
 Amylopsin, 501 
 
 Amylose, 227 
 
 soluble, 227 
 
 Amylum. See Starch, 226 
 
 Anaphylaxis, 70 
 
 Aniline, fate of in organism, 779 
 
 Animal oxidation, 42 
 
 gum in urine, 749 
 
 Anions, 5 
 
 Anisotropous muscle substance, 565 
 
 Antedonin, 844 
 
 Antialbumate, 472 
 
 Antialbumid, 472 
 
 Antibodies, 66, 68 
 
 Antidonin, 844 
 
 Antienzyme, 63 
 
 Antigens, 66, 68, 69 
 
 Antiketoplastic action, 820 
 
 Antimony, action on N-elimination, 679 
 
 passage into milk, 671. See. 
 Mineral substances. 
 
 Antipepsin. See Enzymes, 464 
 
 Antipeptone, 130 
 
 Antipyrine, fate of, in organism, 785 
 
 Antirennin, 69, 474 
 
 Antithrombin, 322 
 
 Antitoxins, 47, 66 
 
 Antitrypsins, 503 
 
 Anuria in cholera, 848 
 
 Aorta elastin, 116 
 
 Apatite in bone ash, 553 
 
 Amphopeptone, 129 
 
 Aporrhegmas, 167 
 
 Aqueous humor, 361 
 
 Arabinoses, 198, 210. Structural formu- 
 lae for, 198 
 
 d-arabinosinine, 201 
 
 Arabite, 198 
 
 Arachnoidal fluid, 355 
 
 Arbacin, 108 
 
 Arbutin, 394, 727 
 
 Arginase, 48, 91, 161 
 
 Arginine, 85, 106, 107, 108, 109, 111, 113, 
 115, 117, 119, 125, 161, 162 
 in various proteins, 165 
 cleavage into creatine, 575 
 
 Arginine-histone peptone, 139 
 
 Argon in blood, 850. See also Gases 
 
 Arnold and Lipliawsky's reaction for 
 acetoacetic acid in urine, 824 
 
 Arnold's reaction for proteins, 100 
 urine reaction, 696 
 
 Aromatic combinations, fate in organism,. 
 776-784 
 
 Arsenic poisoning, 439-441 
 
 on nitrogen elimination, 679 
 
 Arterin, 276 
 
 Ascitic fluid, 357 
 
 Asparagine, 146 
 
 Asparagus, effect on odor of urine, 764 
 
GENERAL INDEX 
 
 991 
 
 Assimilation limit, 533 
 
 Atheromatous cysts, 142 
 
 Atmidalbumin, 130 
 
 Atmidalbumose, 130 
 
 Atmidkeratin, 113 
 
 Atmidkeratose, 113 
 
 Atropine, effect on uric acid elimination, 
 
 700 
 Auto digestion, 43 
 Auto-oxidation, 
 Autolysis, 43 
 
 as a protective agent, 46 
 effect of arsenic upon, 44 
 C0 2 on, 44 
 in organic colloids, on 
 
 44 
 oxygen upon, 44 
 radium on, 44 
 reaction upon, 43 
 importance of enzymes on, 45 
 various processes in, 44 
 Autolytic processes in life, 45 
 Autotoxines, influence on putrefaction, 529 
 
 Bacteria, influence on putrefaction, 520 
 
 Bacterial action in intestinal canal, 518 
 
 Bacteriolysins, 69 
 
 Bacterium urea?, 829 
 
 Balsam of copaiba, fate of, in organism, 
 
 787 
 Bang's method of estimating sugar, 809 
 Barium, 22 
 
 Basal requirement, S97, 922 
 Basedow's disease, 375 
 Baumann's test for dextrose, 216 
 Beer vinegar bacteria, enzymes of, 10 
 Beeswax, 239 
 
 Bela v. Bitto's reaction for acetone, 823 
 Bence-Jones' protein, 792 
 Benzaldehyde, 36 
 
 Benzene homologues, fate of, in organism, 
 779 
 ring, theory for splitting of, in 
 organism, 77S 
 Benzidine blood test, 798 
 Benzoylation of carbohvdrates, 215, 216, 
 
 749, 808 
 Benzoylchloride test for dextrose, 215 
 Benzoyl cystine, 150 
 Betaine, 246, 572 
 Bezoar stone, oriental, 426, 532 
 Bifurcated air, 853 
 Bile-acids, 419-428 
 
 detection of, 418, 419 
 
 in animal fluids, 
 427, 428 
 formation of, 442 
 in blood, 267, 333 
 in urine, 799-800 
 orisrin of, 440 
 
 relation to bile pigments, 444 
 Bile, 413, 442 
 
 absorption of, from the liver, 444 
 amounts secreted, 414-445 
 
 Bile, chemical formation of, 440-444 
 
 coloring matters, detection of, in 
 blood, 435 
 
 composition of, in disca.se, 43!) 
 concretions, cholesterin stones, 445 
 
 pigment atones, 445 
 constituents, absorption of, 540, 541 
 constituents of, 417, 435 
 effect of in absorption of fats, 536- 
 
 540 
 effect on putrefaction, 518, 519 
 gases of, 857 
 Hufner's, 419 
 human, 437 
 
 composition of, 438 
 pigments, in, 438 
 properties of, 437 
 importance in absorption of fat, 511 
 mucus, 415, 437 
 passage into blood, 444 
 urine, 444 
 phosphorized constituents of, 439 
 properties of, 416, 417 
 putrefaction of, in intestine, 516 
 quantitative composition of, 436, 437 
 pigments, 427 
 
 formation of, 440-442 
 in feces, 522 
 in urine, 800, 801 
 origin of, 440, 441 
 relation to bile acids, 444 
 relation to blood pig- 
 ments, 430, 442, 443 
 relation to urinary pig- 
 ments, 743. 744 
 test for, 432, 433 
 salts, 417-419 
 
 properties of, 418 
 tests for, 418, 419 
 secretion, cholagogues, 415 
 
 effect of therapeutic agenta 
 on, 415 
 Biliary fistula, 413, 421 
 Bilicyanin, 435 
 Bilifulvin, 427 
 Bilifuscin, 434 
 Bilihumin, 435 
 Biliphaein, 427 
 Biliprasin, 434 
 Bilipurpurin, 435 
 Bilirubin, 427, 431, 516 
 
 derivatives of, 428-430 
 detection of, in blood, 435 
 Ehrlich's test for, 429 
 Hedenin's test for, 433 
 hemi-, 429 
 
 Huppert's test for, 429 
 hydro-, 428 
 of bloo;l serum. 26S 
 preparation of, 433 
 properties of, 430-432 
 quantitative estimation of, 433 
 Biliverdin, 433, 434 
 
 in excrements, 530 
 
992 
 
 GENERAL INDEX 
 
 Biliverdin in feces, 530 
 properties, 434 
 preparation of, 434 
 Biological equivalence, 917 
 
 protein reactions, 266, 533 
 Bismuth, passage into milk, 670 
 Bitter substances, effect upon secretion, 
 
 461, 482 
 Biuret, 86 
 
 base, 86 
 
 reaction for proteins, 101 
 Blister-fluid, 362 
 Blood, 308-344 
 
 acid lactic, in, 334 
 
 alkalies in, 310, 311 
 
 ammonia in, 334 
 
 analyses of blood from various 
 
 animals, 328 
 arterial, 335, 336 
 
 quantity of carbon di- 
 oxide in, 851 
 quantity of oxygen in, 851 
 at different periods of life,, 
 " buffy coat," 312 
 casts in urine, 796 
 clot, 251 
 
 coagulation, accelerating sub- 
 stances, in cell, 315, 
 316 
 calcium salts in, 316, 
 
 317 
 lime salts in, 316 
 methods of retarding, 
 
 312 
 prevention of, 251 
 retarding substances 
 
 in, 312-326 
 theories for, 317, 321- 
 323 
 changes in viscosity, 311 
 coloring matters, in urine. See 
 also Blood 
 p i g m e n ts, 
 795-799 
 relation to bile 
 pigments, 
 442, 443 
 corpuscles, 250, 272-305-308^ 
 constituents of, 274 
 determination of vol- 
 ume in blood, 326, 
 327 
 effect of water on, 6 
 aalte on, 6 
 experiments with, 6 
 ha;maKKlutination, 275 
 haemoglobin, 274, 276 
 haemolysis of, 273 
 in lymph, 346 
 isolation of, 274 
 mineral bodies in, 304 
 number of, 272 
 size of, 272 
 non-nucleated, 250 
 
 Blood-corpuscles, nucleated, 250 
 
 protection of salts upon, 
 
 6 
 quantitative constitu- 
 tion of, 304 
 quantitative determi- 
 nation of, 326, 327, 
 328 
 red, osmotic phenom- 
 ena with, 8 
 stromata, 274 
 stroma-fibrin, 275 
 stroma, 272, 273, 274 
 white, 250, 305-308 
 
 number of, 305 
 constituents of, 
 306-308 
 defibrinated, 252 
 degree of dissociation in, 271 
 determination of reaction, 271, 272 
 form elements of, 272-276 
 detection of, in urine, 796 
 determination of H ions in, 310 
 during pregnancy, 337 
 effect of alkalinity on carbon- 
 dioxide content, 856 
 electrical conductivity,cleavages in, 
 enzymes in, 53, 332 
 fat in, 334 
 fatty acids in, 334 
 from muscular veins, 337 
 from veins of glands, 337 
 gas exchange in, 858-870 
 gases of, 857 
 gas tension in, 859-868 
 hepatic vein, from the, 336 
 human, analysis of, 328 
 importance of haemoglobin in oxy- 
 gen-carbon dioxide exchange in,. 
 853 
 influence of food on, 338 
 injection of, 343, 344 
 in urine, 795-799 
 laky, 312 
 leucsemic, properties of, 342 
 
 constituents of, 342 
 leucocytes, increase in number of, 
 
 342 
 manner of binding of carbon 
 
 dioxide in, 853, 854 
 menstrual, 337 
 mineral substances in, 335 
 non-coagubility of circulating, the- 
 ories for, 319, 320 
 of various animals, analysis of,, 
 
 328 
 of the two sexes, 337 
 of woman, analysis of, 328 
 oxidation in, 858 
 pigment arterin, 276 
 pigments, 276-305 
 
 acid haematinic, 296 
 acid hemoglobin, 286 
 carbohsemoglobin, 288 
 
GENERAL INDEX 
 
 993 
 
 Blood pigment -<, carbon dioxide -haemo- 
 globin, 288 
 
 carbon monoxide-haemo- 
 globin, 286, 287 
 carbon-monoxide methae- 
 
 moglobin. 287 
 ehlorocruorm, 303 
 cryptopyrrol, 297, 299 
 cyanluemoglobin, 2N5 
 cyanmethsemoglobin. 285 
 decomposition, products 
 
 of, 288, 289 
 detection of, 286, 287 
 formation of, 286 
 globin, 289 
 
 properties of, 289 
 haematin, 277, 290-292 
 formula for,291 
 preparation of, 
 
 291 
 properties of, 
 
 291 
 spectroscopic 
 action of ,292 
 haematin, reduced, 289 
 haematinogen, 300 
 haMiiatocrystallin, 278 
 haematoglobulin, 278 
 haematoidin, 301 
 
 relation to 
 bile pig- 
 ments, 301 
 haematoporphyrin, 277 
 haematoporphyrin, spec- 
 troscopic examination 
 of, 300 
 haematoporphyrin, prep- 
 aration of, 300 
 haematoporpbvrin, pro- 
 duction of, 294-301 
 haematoporphyrin, for- 
 mula for, 294 
 haematoporphyrin, rela- 
 tion to haematin, 294 
 haematoporphyrin, rela- 
 tion to bile pigments, 
 295 
 haematoporphyrin, be- 
 havior in animal body, 
 295 
 haematoporphryin, rela- 
 tion to plant pigments, 
 296 
 haemin, 292-294 
 
 formula for, 292 
 properties of, 293 
 preparation of, 
 293 
 ha?machromogen,2S9, 290 
 haemerythrin, 303 
 haemin crystals, 292 
 
 prepara- 
 tion, of, 
 
 293 
 
 Blood pigments, hacmochrom, 276 
 haemocvanin, 303 
 haemoglobin, 274, 276 
 haemoglobin, composi- 
 tion of, 277 
 hemoglobin, molecular 
 
 weight of, 278 
 haemoglobin, gas combin- 
 ing ability, 280 
 haemoglobin, prepara- 
 tion from oxyhaemo- 
 globin, 283 
 haemoglobin, quantita- 
 tive determination of, 
 301 
 haemoglobin Hoppe-Sey- 
 ler's colonmetric meth- 
 od, 301 
 haemoglobin, reduced, 
 
 282 
 haemoglobin, spectro- 
 scopic quantitative 
 method for, 302 
 haemopyrrol, 297, 
 
 299 
 hsemorrhodin, 288 
 haemochromogen, 276 
 haemoverdin, 288 
 in urine, 795-799 
 isohaemopyrrol, 297 
 kathaemoglobin, 288 
 mesoporphyrin, 294, 
 
 300 
 methaemoglobin, 283- 
 
 285 
 methaemoglobin, proper- 
 ty of, 284, 285 
 methaemoglobin, prepa- 
 
 aration of, 285 
 nitric oxide-haemoglobin, 
 
 288 
 neutral haematin, 288 
 phlebin, 276 
 phonoporphyrin, 295 
 phvllohaemin, 296 
 phyllopyrrol, 298, 299 
 phylloporphyrin, 296 
 porphyrinogen, 295 
 oxyhaematin, 290 
 oxyhaemocyanin, 303 
 oxyhaemoglobin, prop- 
 erties of. 280, 281 
 parahaemoglobin, 281 
 parahaemoglobin, prop- 
 erties of, 281 
 photomethsmoglobin, 
 
 285 
 purple cruorin, 282 
 quantitative estimation 
 
 of. 301-304 
 sulphhaemoglobin. 287 
 sulnhur methaemoglobin, 
 
 287 
 tetronerytl'.rin, 303 
 
994 
 
 GENERAL INDEX 
 
 Blood plasma, 250, 252-264 
 
 analysis of, 269 
 plates, 250, 308 
 
 in coagulation, 314 
 poikillocytosis, 342 
 portal vein, from the, 336 
 quantitative composition of, 326- 
 335 
 estimation of urea in, 
 •691 
 quantity of, 343 
 
 bleeding of, 343 
 in organs, 344 
 reaction of, 76, 309-311 
 red corpuscles, decrease in number 
 of, 341 
 effect of hemorrhage 
 
 on, 341 
 effect of transfusion, 
 
 340 
 effect of transuda- 
 tion from, 340 
 effect of pressure on, 
 
 340 
 increase of, 340 
 increase of, theories 
 for, 341 
 refraction coefficient of serum, 311 
 rennin acting enzyme in, 474 
 serum, acids in, 267 
 
 action of, on starch, 226 
 analyses of, 269 
 constituents of, 264-269 
 properties of, 264 
 rest in nitrogen, 267 
 pigments of, 268 
 quantitative analysis of 
 
 mineral bodies, 270, 271 
 specific gravity, determina- 
 tion of, 309 
 spleenic vein, from the, 336, 337 
 " sucre virtual," 331 
 " sucre immediat," 331 
 urea in, 333, 334 
 uric acid in, 334 
 vascular regions, composition of, 
 
 335-344 
 venous, 335, 336 
 
 quantity of carbon diox- 
 ide in, 851 
 quantity of oxygen in, 851 
 Blueberry, pigments of, in urine, 787 
 Blue milk, 671 
 Blue stentorin, 844 
 Boar sperms, 622 
 
 Boas' test, for lactic acid, in gastric juice, 
 487 
 for HCL, 486, 487 
 Body, relation of weight and age of, to 
 absolute consumption of mate- 
 rial, 913 
 weight decrease during starvation, 
 885 
 Boiling-point, elevation of by colloids, 17 
 
 Bombicesterin, 449 
 
 Bondi-Schwarz's test for acetoacetic acid, 
 
 824 
 Bone, 551-558 
 
 ash, analysis of, 553 
 at different ages, 555 
 catabolism in starvation, 896 
 components of, 551 
 earth, analysis of, 552 
 effect of food upon, 556 
 marrow, 554 
 rachitic, 557 
 rachitis of, 555 
 softening of, 555 
 Bonellin, 844 
 
 Bony structure, matrix of, 551 
 Borneol, fate of, in organism, 785 
 Bottcher's spermine crystals, 621 
 Bottger-Almen's test, 215, 802 
 " Bowman's disks," 566 
 Boyle-Marriot's law, 28 
 Bradoxidizable substances, 4 
 Brain, constituents of, 604-606 
 epileptic, analysis of. 613 
 gray and white substance, com- 
 pared, 605 
 human, analysis of, 612-613 
 paralytics, analysis of, 613 
 phosphatides of, 606-609 
 quantitative composition of, 612 
 British gum, 229 
 Bromhaemin, 294 
 Bromides, in saliva, 459 
 
 relation to formation of gas- 
 tric juice, 477 
 Bromine, 22, 123 
 
 Bromoform, behavior in animal body, 775 
 Bromthymine, 195 
 Bromtoluene, behavior in animal body, 
 
 775 
 Brownian molecular motion, 20 
 Briicke's quantitative method for pep- 
 sin, 469 
 Buccal mucus, 453 
 " Buffy coat," 312 
 Bufidin, 846 
 Bufonin, 846 
 Bufotalin, 846 
 Bufotenin, 846 
 Bufotin, 846 
 Bunge and Schmeideberg, quantitative 
 
 method for hippuric acid, 723 
 Burbot, sperms of, 181 
 Bursae mucosa;, 361 
 Butalanine, 495 
 Butter, 647 
 
 Butterflies, pigments of, 844 
 Butylmercaptan, 844 
 Butyrinase in blood, 266 
 Butvrine, mono enzymotic synthesis of, 60 
 Byssus, 92, 122, 123 
 
 Cadaver alkaloids, 82 
 Cadaverine, 47, 82, 164 
 
GENERAL INDEX 
 
 995 
 
 Cadaverine in urine, 827 
 Caecum, 514 
 Caffeine, 187 
 
 Calcium carbonate in urinary sediment, 
 831 
 importance to enzymotic proc- 
 esses, 255, 256, 307, 312, 313, 
 498, 649. 650 
 in urine, 768(769 
 manner of excretion, 769 
 oxalate in urinary sediment, 830 
 phosphate in urinary sediment, 
 
 831 
 salts in blood coagulation, 308, 
 
 309 
 sulphate in urinary sediments, 
 831. See also Mineral sub- 
 stances. 
 Calculi-ammonium urate, 833 
 -calcium carbonate, 834 
 
 oxalate, 833 
 -cystin, 834 
 -fibrin. 834 
 Heller's scheme for investigating, 
 
 833 
 hemp seed, 833 
 intestinal, 531 
 -mulberry, 833 
 pancreatic, 509 
 -phosphate, 833 
 salivary, 459 
 -uric acid, 833 
 urinary, 828-829, 832-836 
 urinary, scheme for chemical anal- 
 ysis of, 836 
 urinarv, chemical investigation of, 
 
 834-836 
 -urostealith, 834 
 -xanthine, 834 
 Caliphora larvae, fat formation, 561 
 Calomel, effect upon excrement, 529 
 Calorific coefficients, 888 
 value of fat, 888 
 value of proteins. 888 
 
 milk protein, 888 
 starch, 887 
 urine quotient, 884 
 Cammidge's reaction for sugar, 815 
 Camphors, fate of, in organism, 000 
 Cane-sugar, 49, 224. See Sucrose. 
 Canirine, 578 
 d-Caprine, 144 
 
 Capronica's test for guanine, 191 
 Capsule of crystalline lens, 170, 617 
 Caramel, 214, 225 
 
 Carbamino reaction, Siegfried's, 166 
 Carbazole, fate of. in organism, 779 
 Carbohaemoglobin, 287 
 Carbohydrate (phosphatized), 244'. 
 Carbohydrates, absorption of, 532-534 
 
 as a source of muscular 
 
 energy, 598 
 benzoyl esters of, 202 
 classification of, 197 
 
 Carbohydrates, cyanhydrin synthesis for, 
 200 
 effect of gastric juice on, 
 
 473 
 ether-iike combinations, 
 
 202 
 fate of, in organism, 885 
 fermentation of, 203-207 
 hydrazones, 202-203 
 in urine. 749-752 
 in venous blood, 336 
 osazones, 202-203 
 phosphoric acid esters of, 
 
 204-205 
 relation to histidine and 
 
 purines, 202 
 See Various sugars. 
 Carbolic urines, 728 
 
 Carbon dioxide, acids, amino as binders 
 of, 855, 856 
 effect of alkalinity on 
 
 content in blood, 856 
 formation, calculation of, 
 
 889 
 haemoglobin, 287, 853 
 
 as a binder 
 of, 853 
 influencing oxygen ab- 
 sorption, 859 
 manner of binding in 
 
 blood, 853-854 
 mechanism of elimina- 
 tion, 852-853 
 physical explanation of 
 
 the giving up of, 866 
 proteins as binders of, 855 
 quantity- in arterial blood, 
 851 
 
 venous blood 
 851 
 tension, 865 
 tension in tissue, 868 
 Carbon, elimination in organism, 883-884 
 monoxide blood, test for, 286 
 
 haemochromogen, 288 
 haemoglobin, 279, 285, 
 
 288, 300 
 methaemoglobin, 286 
 poisoning, 285, 402, 
 582, 679 
 Carboxylase, 206 
 Carnaubon, 240, 673 
 Carniferrine, 578 
 Carnine, 572, 577, 712 
 Carnitine, 572, 577 
 Carnomuscarine, 578 
 Carnosine, 572, 576-577 
 Carotin, 631 
 Cartilage, 546-550 
 
 components of, 540 
 constituents of, 549-550 
 gelatin, 549 
 
 hyalin, action of trvpsin upon, 
 508 
 
996 
 
 GENERAL INDEX 
 
 Caseid, 649 
 
 Casein, 49, 84, 91, 106, 647-652 
 cleavage products, of, 106 
 coagulation of, 649 
 
 a two-faced proc- 
 ess, 650 
 theory, of, 650, 651 
 composition of, 647 
 properties of, 647, 648 
 hexone bases in, 165 
 human, preparation of, 652 
 of woman's milk, 661 
 peptic digestion of, 651, 652 
 preparation of, 652 
 solutions of, properties of, 649 
 Caseinates, 648, 649 
 Caseinokyrin, 136 
 Caseoses, 130 
 Castoreum, 846 
 Castor lipase, 234 
 Castorin, 846 
 
 Catabolism of protein, 907, 908, 909, 911 
 Catalases, 43, 872 
 
 definition of, 33 
 equilibrium constant of, 33 
 in heterogeneous systems, 36 
 mass action on, 32 
 measurement of, 32 
 of benzaldehyde, 36 
 of diaceton alcohol, 35 
 of diazoacetic ether, 34 
 of esters, 32 
 
 of hydrogen peroxide, 35 
 reaction velocity of, 32 
 velocity coefficient, 34 
 function of, 7 
 Catalysts, 35 
 Catalytic processes, compared (organic 
 
 and inorganic), 37 
 Cataphoresis, 20, 50 
 Cataract of lens, 619 
 Cations, 5 
 Cell-globulin, 274 
 
 -membrane, animal effect of gastric 
 juice on, 473 
 of plant, effect of gastric 
 juice on, 473 
 Cells, adelomorphic, 461 
 
 boundary layer of, 21 
 <cover, 46l 
 delomorphic, 461 
 lymphoid, 300 
 mineral constituents of, 22 
 pepsin, 461 
 permeability of, 34 
 rennin, 461 
 Cellobiose, 231 
 Cellose, 231 
 Cellulose, 231 
 
 fermentation of, in the intes- 
 tine, 511 
 Celluloses, 226 
 Cement, of teeth, 557 
 Cephalin, 248-249, 605 
 
 Cephalopoda, flesh of, 572, 604 
 Cerebrin, 607, 609-610 
 
 in pus-corpuscles, 364, 365 
 Cerebron, 605, 609, 611-612 
 
 cleavage products of, 611 
 preparation of, 611 
 properties of, 611 
 Cerebrosides, 605, 607, 609 
 Cerebrospinal fluid, 360-361 
 Cerolein, 239 
 Cerumen of skin, 845 
 Cetin, 238, 239 
 Cetyl alcohol, 239, 627, 845 
 Chalaza, 634 
 Charcoal bone, ability to absorb trypsin, 
 
 703 
 Charcot's crystals, 871 
 Charcot-Leyden crystals, 621 
 Chemical processes, plants and animals, 
 
 37 
 " Chemical tonus," 591 
 Chief cells. See Adelomorphic cells. 
 Chitin, 122, 839, 839-840 
 Chitosamine, 219, 626 
 Chitosan, 168, 840 
 Chitose, 221 
 Chloral hydrate, fate of, in organism, 777 
 
 secretin, 415, 500 
 Chloramine, 40 
 Chlorbenzene, behavior in animal body, 
 
 786 
 Chlorides. See Mineral substances, 
 of urine, 758-761 
 in urine, quantitative estima- 
 tion of, 759 
 quantity of, 759 
 Chlorine, in teeth, 558 
 in blood, 333 
 Chlorochrome, 384 
 Chlorocruorin, 299 
 
 Chloroform, behavior in animal body, 760, 
 775 
 influence upon elimination of 
 
 chlorine, 760 
 influence upon muscles, 591 
 influence upon protein, 96 
 Chlorometer, 762 
 Chlorophan, 617 
 Chlorophyl, 38, 844 
 Chlorosis, 340 
 Chlortoluene, behavior in animal body, 
 
 783 
 Cholagogues, 415 
 Cholecyanin, 430 
 Choleprasein, 434 
 Cholepyrrhin, 427 
 
 Cholera bacilli, behavior toward gastric 
 juice, 485 
 blood, 334 
 perspiration, 848 
 Cholestanol, a and /3, 446 
 Cholestenon, 443 
 Cholesteriline, 445 
 Cholesterin, 10, 265, 613 
 
GENERAL INDEX 
 
 997 
 
 Cholesterin, amounts in blood of different 
 animate, 328 
 
 constitution of, 445 
 
 derivatives of, 146 
 
 ester, 264, 360, 444, 445, 613. 
 
 844 
 importance of. 149 
 in ascitic fluid, 359 
 in chyle, 347 
 in lymphatic glands, 366 
 in lymph, 348 
 in pus corpuscles, 364 
 in pus serum, 363, 365 
 in spleen, 370 
 occurrence of, 446 
 of sebum of skin, 845 
 preparation of, 450 
 properties of, 446 
 tests for, 447-448 
 Cholesterone, 445 
 Choline, 240, 246-248, 607 
 
 in cerebrospinal fluid, 360 
 in thyroid, 373 
 occurrence of, 247 
 preparation of, 247 
 properties of, 247 
 Cholohaematin, 435 
 Chondrigen, 546 
 Chondrin, 121 
 Chondrin-balls, 549 
 
 Chondroalbuminoid, elementary composi- 
 tion of, 551 
 Chondroproteins, 168 ,172-174 
 Chondromucoid, 172, 546-547 
 
 elementary composition 
 
 of, 551 
 preparation of, 548 
 Chondroitin, 547 
 Chondrosin, 171, 547 
 Chorda saliva, 448 
 Choroid coat, 619 
 Chromaffine tissue, 378 
 Chromhidrosis, 849 
 Chromogens. See Urinary pigments. 
 Chromoproteins, 92, 93, 167 
 Chyle, quantitative composition of, 346- 
 
 347 
 Chyluria, 827 
 Chylous ascites, 358 
 Chyme, 478 
 Chymosin, 474 
 Ciamician and Magnanini's reaction for 
 
 indol, 158 
 Circulating proteins, 908-910 
 Cleavage, hydrolytic, 16 
 
 processes, 15 
 Clupeine, 110 
 Coagulation, intravascular, 324 
 
 method for quantitative pro- 
 tein in urine, 793 
 Coagulins of blood, 320, 321 
 Coagulose, 59 
 Coagu loses, 135 
 Coapeptides, 136 
 
 Coaproteoscs, 130 
 Cobra-poison, 310, 320 
 
 Cochineal, s J \ 
 Cocosite, 581 
 Codfish, eggs, 630 
 
 sperms, 107 
 Co-enzymes, 60 
 
 Coffee, action on metabolism, 912 
 Coilin, 92 
 Collagen, 92, 549, 551 
 
 analysis of, 118 
 in lymphatic glands, 366 
 preparation of, 118, 121 
 properties of, 119 
 Colloid, 171, 624, 625 
 
 cysts, 624 
 
 effect of charge upon, 23 
 
 effect of various ions upon, 22 
 
 envelope, 45 
 
 from uterine fibroma, 627 
 
 substances, non-permeability of, 
 9 
 
 suspension, electrolytic precipita- 
 tion, 21 
 " Colloidal nitrogen," 795 
 
 substances, preparation of. 14 ' 
 Colloids, 49 
 
 adsorption, 27-30 
 
 boiling-point, elevation of, 17 
 
 character of, 14 
 
 classification of, 15 
 
 diffusion of, 18 
 
 disperse phase, 27 
 
 dispersion means, 27 
 
 effects of the different ions, 25 
 
 electrical transportation of sus- 
 pended particles, 20 
 
 electrolyte precipitation of, 24 
 
 emulsion, 15 
 
 examples of, 14 
 
 filterability of, 17 
 
 freezing-point, depression of, 17 
 
 hydrophile, 15 
 
 in relation to surface tension and 
 adsorption, 24 
 
 irreversible, 21 
 
 migration of to poles, 21 
 
 molecular movement of, 20 
 
 optical properties of, 19 
 
 precipitation of, 21 
 
 precipitation phenomena, the- 
 ories of, 25 
 
 protective, 23 
 
 relationship to crystalloids, 14 
 
 relative size of, 18 
 
 reversible, 21 
 
 suspension, 15 
 
 suspension, precipitation of, 70 
 
 Tyndal's phenomenon, 19 
 
 ultra microscope, use of in, 19 
 Colon, effect of extirpation of, 549 
 Coloring matter. See Pigments. 
 Colostrum, 658 
 
 composition of, 658 
 
998 
 
 GENERAL INDEX 
 
 Colostrum of woman's milk, 665 
 Combustion, physiological heat of, 879 
 Complements, 69 
 Conalbumin, 633 
 Conchiolin, 122, 123 
 
 Concrements, intestinal, 523, 524. See 
 also Calculi, 
 prostatic, 623 
 Concretions of lungs, 871 
 Conglutin, 878, 879 
 
 Conjugated glucuronic acids, fate of in 
 s organism, 777 
 
 sulphuric acids, fate of in 
 organism, 777 
 Connective tissue, analysis of, 545 
 
 components of, 544 
 fibrils of, 545 
 Copper, occurrence in blood, 268, 333 
 in bile, 416, 433, 440 
 in liver, 387 
 in pigments, 299 
 Cornea, 550 
 
 Corneal mucoid, elementary composition 
 of, 551 
 tissue, 619 
 Cornein, 122, 123-124 
 Cornicrystallin, 123 
 Corpora lutea, 623 
 Corpse-wax, 560 
 Corpus callosum, 613, 614 
 Corpuscles, blood. See Blood corpuscles, 
 colloid, 624 
 colostrum, 645 
 Gluge's, 624 
 Corpuscula amvlacea, 612 
 Cover cells, 456, 476, 478 
 Crab extract, 576 
 Crangitine, 578 
 Crangonine, 578 
 Creatine, 267 
 
 detection of, 576 
 formation of, in active muscles, 
 594 
 in organism, 787 
 in ascitic fluids, 359 
 in urine, 692 
 
 mother substance of, 574 
 origin of, 574 
 preparation of, 573, 576 
 production from arginine, 575 
 properties, 575-576 
 relation of to creatinine, 692-693 
 relation to catabolism of pro- 
 tein, 574 
 Creatinine, 572, 573 r 576 
 
 elimination, effect of disease 
 upon, 694 
 effect of muscular 
 activity upon, 
 694 
 effect of starva- 
 tion upon, 694 
 Folin's folorimetric method 
 for, 697 
 
 Creatinine, formation of, in active mus- 
 cles, 594 
 mother substances of, 693 
 preparation of, 697 
 properties of, 695, 696 
 quantitative estimation of, 697 
 quantity of, in urine, 692 
 relation of, to creatine, 692- 
 
 693 
 -zinc chloride, 695 
 Crenilabrine, 110 
 Crenilabrus pavo, 844 
 Croners-Conheim's test for lactic acid in 
 
 gastric juice, 488 
 Crude fibre, digestion of, 549 
 
 silk, 121, 122 
 Cruor, 252 
 Cruorin, purple, 282 
 Crusocreatinine, 578 
 Crustaceorubin, 844 
 Crusta infiammatoria, 306 
 
 phlogistica, 306 
 Cryptopyrrol, 430 
 Crystalbumin, 618 
 Crystalfibrin, 618 
 Crystallin, alpha and beta, properties of,, 
 
 618 
 Crystalline lens, 617-619 
 Crystalloids, 13 
 Crystals, Charcot-Leyden, 621 
 Cuorin, 249 
 
 Curare, 348, 398, 588, 920 
 Cyanhaemoglobin, 284 
 Cyanhydrins, formation of, 200 
 Cyanmethaemoglobin, 284 
 Cyanocrystallin, 637, 844 
 Cyanurin, 741 
 Cyclopterine, 110 
 Cymene, fate of, in organism, 779 
 Cyprinine, 110 
 
 hexone bases in, 165 
 Cysteine, 80, 85, 148. 150, 619 
 Cystic fluid, protein bodies in, 626 
 Cystine, 80, 85, 100, 107, 113, 114, 115,. 
 116, 125, 148-150 
 in urine, 827-828 
 preparation of, 828 
 protein-. 148 
 stone-, 148 
 Cystinuria, 827, 828 
 
 Cysts, characteristic constituents of. 624 
 colloid, 624 
 dermoid, 627 
 intraligamentary, 627 
 myxoid, 624 
 papillary, 627 
 parovarial, 627 
 proliferous, 624 
 serous, 624 
 tubo-ovarial, 627 
 Cytidine, 180, 185 
 Cytin, 368 
 
 Cvtoglobin, 307, 315, 366 
 Cytosine, 178, 181, 185, 193-194 
 
GENERAL INDEX 
 
 999 
 
 Cytosine, detection of, 194 
 Cytotoxin, 69 
 Cytozym, 319 
 
 Deamidation, 411. 530, 582, 702, 774 
 Dehydrochloride nsemin, 293 
 Dehydrocholon. 418, 423 
 Deniges's test for tyrosine, 154 
 Dentin, 553 
 Dermocerin, 844 
 Derrnolein, 844 
 Desamidoprotein, 78 
 Descernet's membrane, 171, 551, 018 
 Desoxyha-matoporphyrin, 291 
 Deutercaseoses, 129 
 Deuteroelastose, 117 
 Deuteromyosinose, 130 
 Deuteroproteose, 129 
 Deuterospongenose, 122 
 Deuterovitellose, 130 
 Development, work of, 042 
 Dextrin, 229 
 
 hydrolytic cleavage products of, 
 229 
 Dextrins, 220 
 
 Dextrose, 212. See Glucose. 
 Diabetes, duodenal, 400 
 mellitus, 403 
 
 acetone bodies in, 818 
 pancreas, artificial, 405 
 
 relat ion to adrenals 
 and thyroids, 400 
 phlorhizin, 400 
 
 sugar eliminated in, origin of, 
 409-411 
 Diabetic sugar, 212 
 Diaceton alcohol, 35 
 Diamine, 24, 131, 103, 757, 928 
 Dialysis, 14 
 Diarginylalanine, 111 
 Diarginylproline, 111 
 Diarginylserine, 111 
 Diarginylvaline, 111 
 Diastase. See Enzymes, 48, 04. 
 
 action of, on starch paste, 226 
 Diazca^etic ether, 34 
 
 Diazohenzenesulphonic-acid test for dex- 
 trose, 210 
 Diazo-reaction, Ehrlich's, 755 
 
 for histidine, 101 
 Dibenzoylornithine, 161 
 Diet, average daily adult, 923 
 
 for people in different vocations, 924, 
 927 
 Diffusion, 1 
 
 of colloids, 18 
 streams, 5 
 Digestion, effect of extirpation of pan- 
 creas upon, 531-532 
 gastric, effect of fats on, 481 
 in the stomach, 478-489 
 
 time of, 481 
 movement of food in stomach 
 during, 479-480 
 
 Digestion, movements of stomach during, 
 
 1 7'. i 
 peptic, 132 
 
 Digestion-work, 930-931 
 Diglucosamine, 220 
 Diglycyl-gfycine, 85 
 Dihydrocholesterin, 443, 446 
 1 K-isobutyldiacytpiperazine, 142 
 Di-iodotyrosin, 123 
 Di-leucyl-cystine, 85 
 Di-leucy 1-glycyl-glycine, 85 
 Dimethylaminobenzaldehyde, fate of, in 
 
 organism, 786 
 Dimethylfulvene, 7 
 Dimethylguanidine in urine, 758 
 Dimethylindol, 729 
 Dimethylketone, 822 
 Dimethyltoluidine, fate of, in organism, 
 
 786 
 Dimethylxanthine (1, 7), 713 
 Dioxyacetone, 205 
 Dioxybenzene, 778 
 o-Dioxybenzene. 727 
 yj-Dioxybenzene, 728 
 Dioxymethylene creatinine, 574 
 Dioxynapththaline, 778 
 Dioxypurine, 188 
 Dioxypyrimidine. 220 
 Dipalmitylolein, 233 
 Dipentosamine, 220 
 Dipeptides, 85-89 
 Diphosphates, 239 
 Disaccharides, 223-226 
 Disperse phase, 47 
 Dispersion means, 47 
 Dissociation, degree of, 5 
 Di-stearyl lecithin. 148, 241, 242 
 Di-stearylolein, 233 
 Di-stearyl-palmatin, 233 
 Di-tetraoxybutylpyrazine, 220 
 Dithiopiperidine, 88 
 Donne's pus test, 798 
 Dotterplattchen, 93, 628, 638 
 Dulcite, 198 
 Dye stuffs, behavior of living cells 
 
 toward, 10 
 Dyslvsins, 427 
 Dysoxidizable substances, 4 
 Dyspeptone, 472 
 Dysproteoses, 129 
 
 Ear, fluids of the inner, 619 
 
 Earthy phosphates. See Phosphates. 
 
 earthy 
 Echinochrom, 299 
 Echinoooccus cysts, fluid of, 362 
 Eck'e fistula operation, 398, 537, 542, 680 
 Edestan, 108, 408 
 Edestin, 84 
 
 cleavage products of, 107 
 
 hexone bases in, 165 
 Eel-meat, 601 
 
 scrum. 250, 327 
 Egg shell, 030-641 
 
1000 
 
 GENERAL INDEX 
 
 Egg, white of the, 632-636 
 
 yolk of hen's, 628 
 Eggs, chemical energy in, 639 
 development of, 637 
 fertilization of, 639-640 
 incubation, 637-638 
 
 change in solid con- 
 tent during, 638 
 exchange of gases, 637- 
 638 
 Loeb's experiments on fertilization, 
 639 
 Ehrlich's diazo reaction, 753, 847 
 glucosamine test, 221 
 side chain theory, 67, 71 
 test for bilirubin, 429 
 urine test, 826 
 Eicosyl alcohol, 844 
 Elaidin, 236 
 
 Elastic substance, action of trypsin on, 
 508 
 tissue, analysis of, 545 
 Elastin, 92 
 
 analysis of, 116 
 effect of gastric juice on, 473 
 hexone bases in, 165 
 in lymphatic glands, 366 
 peptone, 117 
 preparation of, 117 
 properties of, 117 
 Elastoses. 130 
 
 Electrolytes, amphoteric, 90, 93 
 Elephant, bones of, 553 
 Emulsin, 48, 58, 59, 62, 64 
 Emulsoids, properties of, 15 
 Enamel of teeth, 557, 891 
 Encephalin, 607, 609, 610 
 Endocrinic glands, 374, 375 
 Endoenzymes, 52 
 Endolymph, 619 
 
 Energy content of various food stuffs, 886 
 development, calculation of, 889 
 exchange, 891 
 
 calculation of, 889 
 metabolism calculation of, 886, 
 887 
 Enterokinase, 496 
 Enzymes, 37-70 
 
 action on glucosides, 62 
 action, specificity of. 61 
 action, retardation of, 62 
 activators, 52 
 adenase, 703 
 alcoholases, 875 
 aldehydases, 875 
 amylopsin, 501 
 anti, 63-64, 266 
 ant i pepsin, 467 
 arginase, 682 
 butyrinases, 266 
 catalases, 266, 872 
 classification of, 47 
 co-,52 
 deamidizing, 703 
 
 Enzymes, deviation, 64 
 diastases, 266 
 
 effect of bile upon, 511-512 
 endo-, 52 
 esterases, 266 
 extra cellular, 52 
 fat splitting, 501-503 
 fermentation, 653 
 formation of, 52 
 gastric lipase, 476-478 
 general properties, 48 
 glutinase, 505 
 glycolytic, 332 
 glyoxylase, 584 
 guanase, 703 
 heat production of, 54 
 histozyn, 723 
 hydrogenases, 876 
 in amniotic fluid, 642 
 in ascitic fluids, 359 
 in blood, 52 
 in blood serum, 266 
 in bile, 435 
 in brain, 608 
 in fatty tissue, 558 
 in gastric juice, 466 
 in intestinal juice, 491-492 
 in leucocytes, 307 
 in liver, 386 
 in lungs, 870 
 in lymph, 346 
 in mammary glands, 643 
 in milk, 653 
 in muscle, 572 
 
 in pancreatic gland, 495, 500 
 in pancreatic juice, 496 
 in placenta. 641 
 in prostate, 621 
 in pus cells, 365 
 in pyloric secretion, 478 
 in saliva, 455 
 in spleen, 370, 371 
 in thymus, 369 
 in thyroid gland, 373 
 intracellular, 52 
 in urine, 757 
 in yolk, 628 
 lipases, 260 
 . maltase, 266, 458 
 modes of action of, 54 
 myosin ferment, 570 
 nuclease, 504 
 nucleases, 703 
 oxidases, 266 
 oxidones, 875 
 oxygenase, 872 
 pancreas press juice, action of, 
 
 62 
 pancreatic rennin, 509 
 pepsin, 466-476 
 peroxidases, 872 
 phenol-oxidases, 875 
 phytase, 579 
 polypeptide-splitting, 266 
 
GENERAL INDEX 
 
 1001 
 
 Enzymes, proteolytic, 82, 266, 703 
 pseudopepsin, 166 
 ptyalin, 156 400 
 
 purine-oxidases, 875 
 quantitative determination of, 
 
 58 
 reactivation, 52, 64 
 reductases, s7l>, 877 
 rennin, 266, 474 
 retardation by charcoal, 62 
 retarding substances, 65 
 reversibility of enzyme action, 
 
 58 
 salivary diastase, 456 
 Schutz's rule for, 58 
 secretion of, 52 
 steapsin, 501-503 
 syntheses, 38, 39 
 synthesis of hippuric acid, 39 
 trypsin, 503-509 
 tyrosinases, 875 
 urease, 829 
 uricase, 706 
 uricolase, 706 
 urocolytic, 706 
 xanthin oxidase, 703 
 Enzymotic processes, 40 
 
 fermentation processes, 41 
 hydrolytic cleavages, 40, 58 
 synthetic processes, 58 
 reactions, 55 
 
 laws of, 55, 56, 57 
 Epidermis, 112, 834, 835, 845 
 Epidermoidal structures, 837-838 
 Epiguanine, 187 
 Epiguanine in urine, 712, 714 
 Epinephrin, 378 
 Episarkine, 187 
 
 in urine, 712, 714 
 Epitoxiod, 71 
 Equilibrium constant, 33 
 
 nitrogenous, 906 
 Erepsin. See Enzymes, 48, 593-494 
 
 action of, 493 
 Ereptases, 503 
 Erythrite, relation to glycogen formation, 
 
 393 
 Erythrocytes, 275 
 Erythrodextrin, 229 
 Erythropsin. See Visual purple, 615 
 Esbach's quantitative method for pro- 
 tein in urine, 794 
 Ethal, 239 
 
 Ether, action on blood, 273 
 Ethyl alcohol, action on metabolism, 912 
 behavior in animal body, 
 775, 912 
 benzene, behavior in animal bodv, 
 
 778 
 butyrate, enzymotic synthesis of, 60 
 mercaptan, behavior in animal 
 
 bodv, 776 
 secretin, 823 
 sulphide, from protein, 79 
 
 Ethyl sulphide, behavior in animal bodv, 
 776 
 sulphuric acid, behavior in animal 
 body, 776 
 Ethylene glycol, glycogen formation, 394 
 Euglobulin, 259 
 
 Euxanthon, fate of, in organism, 785 
 Excelsin, 108, 160 
 Exchange of force, 871, 877, 879 
 Excrements, 521, 549, 873, 874 
 
 in biliary fistula-, 519 
 Excreta, regular and constant. ,S79 
 analysis of, 880 
 
 nitrogenous constituents of, 880 
 Excretin, 523 
 Exostosis, 556 
 
 Expectorations of lungs, 870-871 
 Extirpation of large intestine, effect of, 548 
 Extra cellular enzymes, 52 
 Extractive bodies of brain, 606 
 
 of kidneys, 673 
 
 of mammary glands, 
 
 643 
 of milk plasma, 647 
 non-nitrogenous, 579 
 of pancreatic gland, 495 
 substances of liver, 386 
 
 of muscles, 572-588 
 Extractives nitrogenous, of muscle, 572 
 of bone marrow, 554 
 of bodies of cystic fluids, 627 
 of testes, 620 
 of yolk, 628 
 Exudates, gases of. 857 
 Eye, fluids of, 615-619 
 
 lens of, insoluble protein of, 618 
 soluble protein of, 618 
 pigments of, 615-617 
 tissues of, 615-619 
 
 Fat-cells, membrane of, 558 
 
 action of trypsin 
 on, 508 
 -globule, 646 
 Fats, 38, 232-249 
 
 absorption of, 535-540 
 
 effect of bile upon, 
 536-540 
 acetone formers, 820, 822 
 amounts in blood of different animals, 
 
 328 
 catabolism in starvation, 894 
 chemical methods for investigating, 
 
 238 
 deposition of, 920 
 destruction of, during work, 596 
 detection of, 237 
 effect of extirpation of pancreas on 
 
 absorption of, 539. 540 
 effect of gastric juice on, 473 
 effect on glycogen content of liver, 
 
 394 
 emulsion of, 511 
 fate of, in organism, 885 
 
1002 
 
 GENERAL INDEX 
 
 Fats, "format ion from carbohydrates, 563 
 from glycogen in liver, 
 
 397 
 from protein, 560-562 
 of, in organism, 559 
 hydrolysis of, 234 
 human, 559 
 in blood, 334 
 
 serum, 265 
 in chyle, 346, 347 
 in kidney, 673 
 in liver, 384 
 in lymph, 348 
 in lymphatic glands, 366 
 in muscle, 586 
 in pus corpuscles, 364 
 in pus-serum, 363 
 in spleen, 370 
 in synovial fluid, 362 
 in thymus, 368 
 in urine, 827 
 in woman's milk, 660 
 in yolk, 630 
 
 manner of absorption of, 535-536 
 metabolism of, in starvation, 894 
 
 with an exclusive pro- 
 tein diet, 907-908 
 muscular energy, source of, 598 
 of different animals, 559 
 pancreatic splitting of, 502 
 properties of, 233 
 saponification of, 234 
 storing up of, 563 
 syntheses of, 60 
 Fatty degeneration, 385, 560 
 
 series, fate of, in organism, 773-774 
 tissue, 558-564 
 
 analysis of, 558 
 constituents of, 558 
 Feathers, mineral substances of, 838 
 
 pigments of, 843-844 
 Feces, appearance of, 521, 522 
 constituents of, 521, 879 
 pigments in, 522 
 reaction of, 522 
 Feeding experiments to show value of 
 
 different foodstuffs, 904-906 
 Fermentation, 8, 9, 203, 207 
 
 lactic acid, in stomach, 485 
 processes, 41 
 
 test in urine, 803, 804, 809, 
 810, 812 
 for sugar, 803 
 Ferments, 41 
 Ferratin, of liver, 383 
 Ferrine, 384 
 Fertilization membrane, bringing about, 
 
 640 
 Fever elimination of ammonia. 768 
 Fibrin, 100, 251, 254-261 
 action of, 257 
 cleavage products of, 106 
 coagulation, 256, 257 
 elementary composition of, 263 
 
 Fibrin, ferment, 256 
 
 globulin, 258, 259 
 Henle's, 620 
 
 in blood coagulation, 317 
 in blood during pregnancy, 337 
 in venous blood, 336 
 manner of formation, 322 
 peptic digestion of, 472 
 plastic substance, 258 
 preparation of, 254-255 
 production, manner of, 315 
 properties of, 255 
 quantitative estimation of, 255 
 Fibrinogen, 91, 252-261 
 
 amounts in blood, in poison- 
 ing, 253 
 detection of, 254 
 elementary composition, 263 
 formation, seat of, 252 
 in coagulation of blood, 317 
 in venous blood, 336 
 occurrence of, 252 
 preparation of, 254 
 properties of, 253 
 purification of, 254 
 quantitative estimation, 254 
 relation to fibrin, 257, 258 
 transformation into fibrin, 256 
 Fibrinolysis, 255, 322 
 Fibroin, 92, 122, 124 
 
 analysis of, 125 
 properties of, 124 
 Filterability of colloids, 17 
 
 preparation of the filter, 18 
 Fischer-Weidel's reaction, 189 
 Fleischl's hsemometer, 299 
 Florence's sperm reaction, 621 
 Fluorine content in teeth, 558 
 
 content in organs and tissues, 
 553 
 Folin and Dennis' test for tyrosine, 154 
 Folin's method for urea, 689 
 Folin-Schaffer's quantitative method for 
 
 uric acid, 711-712 
 Food, amount of, for an average daily 
 diet, 933, 934 
 chemical energy introduced with, 
 
 932 
 definition of, 878 
 influence on blood, 338 
 necessity under various conditions, 
 
 932-939 
 needs of, in work and rest, 937-939 
 requirements for men in various 
 
 vocations, 933 
 stuff, determination of heat value, 
 891 
 energy content of, 886 
 organic, uses for in organism, 
 
 890 
 physiological availability of, 
 891 
 Foods, energy of, 885 
 
 essential to life, 878 
 
GENERAL INDEX 
 
 1003 
 
 Foods, importance of various, 878 
 Formaldehyde, 38 
 
 formation of, from carbon- 
 ic acid, 2 
 relation to glycogen for- 
 mation, 397 
 transformation into sugar, 
 1 
 Formol titrable nitrogen in urine, 755, 756 
 titration, Sorensen's, for amino 
 acids, 166 
 Freezing-point depression, for mamma- 
 lians, 12 
 in the frog, 12 
 in the inverte- 
 brates, 12 
 in the lower 
 
 fishes, 13 
 in the higher 
 
 fishes, 13 
 in the eel, 13 
 in urine, 13 
 of colloids, 17 
 molecular lowering of, 4 
 Fructosazine, 221 
 Fructose, 217-218 
 
 in blood serum, 265 
 in urine, 814-815 
 structural formula 1 for, 197, 199 
 tests for, 217-218 
 Fruit-sugar, 217. See Levulose. 
 Fuld and Levison's method for testing 
 
 pepsin, 470 
 Furbringer's test for proteid, 790 
 Furfurol, 208-209 
 
 fate of, in organism, 784 
 Fuscin, 617 
 
 Gadushistone, 108 
 Galactosamine, 220 
 Galactose,, 654, 816 
 
 structural formula for, 199 
 tests for, 217 
 Galactose, 202 
 
 Gall bladder, secretion of, 416, 437 
 Gall-stones, 444 
 Gallois' inosite test, 578 
 Ganassini's reaction for uric acid, 707 
 Gas exchange, a measure of metabolism, 
 927-928 
 between blood and pul- 
 monary air, 858-870 
 between blood and tissues, 
 
 858-870 
 in muscle activity, 592 
 in starvation, 895 
 methods for the quantita- 
 tive determination of, 
 868-870 
 through skin, 849 
 rise of, 931 
 tension in blood, 859-868 
 
 methods of determining, 864- 
 866 
 
 Gases, in bile, 857 
 
 in birds' eggs, 636 
 in blood, 857, 850-856 
 in exudates, 867 
 in blood serum, 26'.) 
 in gastric digestion, 485-486 
 in lymph, 346, 856-858, 
 in milk, 657, 658 
 in muscles, 588 
 in saliva, 857 
 in stomach, 485-486 
 in transudates, 355 
 in urine, 770, 857 
 in woman's milk, 68 I 
 produced in putrefaction, 515-516 
 Gastric contents, indicators for determin- 
 ing acids in. 487 
 nature of acids in, 487 
 quantitative determina- 
 tion of lactic acid in, 
 488 
 test for lactic acid in, 
 488 
 digestion, absorption of cleavage 
 products in stomach, 
 484 
 degree of, 483, 484 
 effect of fats on, 481 
 gases in, 485, 486 
 time of passage through 
 the stomach of dif- 
 ferent foods, 482, 483 
 juice, 461-466 
 
 action of foreign substances 
 
 on ser-retion of, 462 
 action of saliva on secre- 
 tion of, 463 
 composition of, 465 
 constituents in, 466 
 degree of acidity in, 487 
 obtainment, free of saliva 
 
 461-482 
 origin of hydrochloric acid 
 
 in, 477 
 secretion of, 461, 462-463 
 in man, 464 
 lipase, 476-478 
 Geissler's albumin-test papers, 790 
 Gel, 15 
 Gels, 30-32 
 
 Gelatin, 31, 78, 80, 118, 119-121, 152 
 analysis of, 118 
 as a foodstuff, 912 
 forming substances of bones, pep- 
 tic digestion of, 472 
 forming substances of cartilage, 
 
 peptic digestion of, 472 
 forming substance of connecting 
 tissue, action of trypsin on, 508 
 forming substances of connective 
 tissue, peptic digestion of, 472 
 from peptic digestion, 473 
 hexone bases in, 165 
 in egg development, 639 
 
1004 
 
 GENERAL INDEX 
 
 Gelatin, in protein catabolism, 911 
 oxidation of, 83 
 pancreatic digestion of, 508 
 peptic digestion of, 473 
 peptones, 120, 121 
 preparation of, 118 
 properties of, 119 
 protein-sparer, 911, 912 
 Gelatose, proto-, 120 
 
 deutero-, 120 
 Gelatoses, 120, 473 
 Generation, organs of, 620-642 
 Generative organs, female, 623-642 
 secretions, male, 620-623 
 Gerhardt's test for acetoacetic acid, 824 
 Glands, albuminous, 541 
 
 Brunner's, secretion of, 489 
 fundus, 460, 461 
 Lieberkuhn's, secretion of, 490 
 mammary, 643 
 
 constituents of, 643 
 mixed, 451 
 mucous, 451, 460 
 
 membrane, in the intes- 
 tine, 489-493 
 membrane of stomach, 
 460 
 pancreatic, 494-495 
 pyloric, 460, 461 
 salivary, 451-460 
 
 analysis of, 451 
 Gliadin, cleavage products of, 107 
 Globan, 104, 276 
 Globulins, 78, 91, 92, 93, 106 
 
 detection of, in urine, 791 
 properties of, 104 
 quantitative estimation of, in 
 urine, 791, 793 
 Globuloses, 130 
 Glucocyanhydrin, 200 
 Glucomaines in urine, 757, 758 
 Glucoproteins, 167, 168-174 
 Gluconose, 206 
 Glucopeptose, 200 
 Glucoproteins, phosphorized, 105 
 Glucoproteose, 133 
 Glucosamine, 84, 219 
 
 from blood globulin, 260 
 from seralbumin, 262 
 preparation of, 220 
 tests for, 219 
 d-glucosamine, 201 
 Glucosan, 213 
 Glucose, 59, 212-217 
 in blood, 330 
 in urine, 749, 802-814 
 from blood globulin, 260 
 osimine formation, 201 
 preparation of, 216 
 properties of, 213 
 structural formula for, 197, 198, 
 
 199 
 tests for, 213-216 
 Glucosidee, 202 
 
 Glucosides, action of enzymes on, 62 
 Glucoside-splitting enzymes, 16, 202, 491 
 Glucosoxime, 200 
 
 Glucuronates, conjugated, properties of, 
 751, 752 
 conjugated in urine,750-752 
 Glucurone, 222 
 Gluge's corpuscles, 623 
 Gluteins, 119 
 Glutelins, 92 
 Gluten casein, hexone bases in, 165 
 
 proteins, hexone bases in, 165 
 Glutin, 92 
 Glutinase, 505 
 Glutokyrin, 138 
 Glycerides, tri-, 232 
 Glycerine, 265 
 
 relation to glycogen formation, 
 394, 397, 412 
 Glycine. See Glycocoll, 139 
 Glycocoll, 85, 106, 107, 109, 113, 115, 119, 
 124, 125, 139 
 amounts in proteins, 106, 107, 
 
 113, 115, 125 
 conjugation with, 782-785 
 formation of, in organism, 721 
 importance of, in uric acid 
 formation, 720, 721 
 Glycocyamine, 693 
 
 Glycogen, 229, 390^14, 581-582, 637 
 amount in liver, 390, 391 
 amount in muscles, 390, 391 
 consumption of, in muscles, 592 
 content increased by, 393, 394, 
 
 396, 397 
 fat formation from, 397 
 formation, a cell function, 398 
 formation from sugar, 395 
 in lymph, 346 
 in lymphatic glands, 366 
 in placenta, 641 
 origin of, 393 
 origin in muscles, 397 
 preparation of, 392 
 properties of, 391, 392 
 pseudoglycogen-formers, 395, 
 
 396 
 quantitative estimation of, 393 
 synthesis of, 58 
 synthesis of, in liver, 381 
 true glycogen formers, 395, 396 
 transformation into sugar, 398, 
 399 
 Glycolaldehyde, 38 
 Glycolysis, 265, 332, 407-409, 570 
 Glycolytic enzyme, 265 
 Glycoproteins, 92, 167, 168-174 
 
 in blood, 264 
 Glycosuria adrenalin, 402, 403 
 
 relation of pancreas 
 and adrenals, 406 
 alimentary, 401 
 diabetic, relation of pancreas 
 to, 405 
 
GENERAL INDEX 
 
 1005 
 
 Glycosuria piqure, 402 
 *alt, 401 
 
 BUgar-puncture, 402 
 Glycylalanine, 86 
 Glycylasparaginyl leucine, 86 
 Glycyiglycin, 86 
 Glycyl leucine, 86 
 
 proline anhydride, 86 
 
 tyrosine, 86 
 
 valanine anhydride, 86 
 
 Glyoxal-methyl, 202 
 
 Glyoxyldiureide. See Allantoin, 717 
 
 Gmelin's test, for bile pigments, 429 
 
 in urine, 800 
 Goitre, 376 
 Gorgonin, 123 
 Graafian follicles, 623, 624 
 Grape sugar, 212. See Glucose. 
 Guaiac test for blood, 281, 795 
 Guanadine, 162 
 Guanase, 48, 188, 187, 3S2, 368, 702. See 
 
 Enzymes. 
 Guanidobutvlamine, 162 
 Guanine, 178, 181, 183, 185, 187, 188, 190, 
 191, 712 
 Capronica's test for, 191 
 epi-, 187 
 -hexoside, 180 
 Weidel's reaction for, 191 
 Guano, 188, 699 
 Guanosine, ISO 
 Guanovulit, 637 
 Gulose, 211 
 Gums, 229, 230 
 plant, 226 
 vegetable, 230 
 Gunning's modified Lieben's test, 822 
 Gunzburg's test for HC1 in gastric juice, 
 
 487 
 Gvnesin in urine, 758 
 
 Hacmaphilia, 329 
 Hu'inase, 50 
 Ha;mataerometer, 861 
 Hiematin, 289-291 
 
 neutral, 215 
 reduced, 288 
 Haematinogen, 296 
 Haematinometer, 297 
 Haematocrit, 326 
 Haematocrystallin, 278 
 Haematogen, 629, 637 
 Hsematoidin, 871 
 Haematoblasts, 308 
 Hajmatoporphyrin, 294-296, 844 
 
 relation to bilirubin, 
 
 295, 428, 440 
 relation to chlorophyl, 
 
 276, 295 
 relation to urobilin, 
 
 295, 440, 742 
 in urine, 740, 797-798 
 Haematoscope, 303 
 
 Haematuria, 795, 796 
 Hsemerythrin, 299 
 Bsmin, 292 294, 297, 800 
 
 crystals, 292, 293, 800 
 
 Haemochrom, 275, 27'.) 
 Hsemochromogen, 276, 288, 289 
 Haemocyanin, 92 
 Haemoglobin, 92, 108 
 
 amounts in blood, 328 
 carbon dioxide binder, 853 
 exudation of, <i 
 in blood during pregnancy, 
 
 337 
 in (X) 2 — () 2 exchange in 
 
 blood, 853 
 in venous blood, 326 
 transformation into bile pig- 
 ments, 442 
 Haemoglobinuria, 796 
 Haemoglutination, 275 
 Haemolysins, 69 
 Haemolysis, 273 
 Haemometer, 303 
 
 Ha>mopyrrol, 276,' 291, 295, 428, 843 
 Haemorrhodin, 287 
 Haemoverdin, 287 
 Hair, 837 
 
 -balls, 524 
 
 human, sulphur content of, 838 
 lanugo, 642 
 Hammarsten's test for bile pigments, 430, 
 
 800 
 Haptogen-membrane, 646 
 Haptophore, 67 
 
 Hanriot and Richet's method for deter- 
 mining respiratory exchange, 869 
 Haser's coefficient, 771 
 Heat regulation, chemical, 929 
 physical, 929 
 Hedenius' bilirubin test, 433 
 Helicoproteid, 174 
 Heller's blood test, 797 
 
 scheme for investigating calculi, 
 837 
 Heller-Teichmann's test, 797 
 
 in urine, 788 
 for proteid, 98 
 Hemicelluloses, 231 
 Hemicollin, 120 
 Hemielastin, 117 
 
 in blood, 264 
 Hemipeptone, 130 
 Hemolysins, 70 
 Hemp seed calculi, 835 
 Henle's fibrin, 620 
 Henoeque's haematoscope, 299 
 Henriques and Gammeltoft's method for 
 
 urea, 690 
 Heparphosphatide, 386 
 Hepatopancreas, 494 
 Heptapeptides, 85, 87 
 Heptose in urine, 817 
 Heptoses, 197 
 Heterocaseoses, 129 
 
1006 
 
 GENERAL INDEX 
 
 Heterocyclic compounds, fate of, in 
 
 organism, 778-787 
 Heterolysis, 18 
 Heterosponginose, 122 
 Heterosyntonose hexone bases in, 165 
 Heteroxanthine, 187 
 
 in urine, 712, 713 
 Hexapeptides, 85 
 Hexobioses, 224 
 Hexone bases, 161 
 
 in various proteins, 165 
 Hexoses, 197, 211-218 
 
 syntheses of, 212 
 Hippokoprosterin, 449 
 Hippomelanin, 841 
 Hirudin, 251 
 
 Histidine, 85, 106, 107, 109, 110, 111, 115, 
 117, 119, 125, 159-161 
 diazo reaction for, 161 
 in various proteins, 165 
 Weidel's reaction for, 160 
 Histidyl-histidine, 85 
 Histone in urine, 795 
 Histone-peptone, 109 
 Histones, 78, 91, 92, 93 
 Gadus-, 108 
 hexone bases in, 165 
 Lota-, 108 
 
 properties of, 108-109 
 Histozym, 48 
 
 Hoffmann's test for tyrosine, 153 
 Holozym, 319 
 
 Homocerebrin, 607, 609, 610 
 Homocyclic compounds, fate of, in organ- 
 ism, 778-787 
 Hopkin's quantitative method for uric 
 
 acid, 711 
 Hoppe-Seyler's reaction for xanthine, 190 
 CO blood test, 286 
 colorimetric method, 297 
 test for bile acid, 179 
 Hordein, 107, 156 
 Hormone, 375 
 Hormones, 407 
 Horn structures, 837 
 
 mineral content of, 838 
 sulphur content of, 838 
 See also Keratin. 
 Hufner's bile, 419 
 Humor, aqueous, 352, 359, 617 
 Huppert-Messinger method of estimating 
 
 acetone, 825 
 Huppert-Schiiltz's method of testing 
 
 pepsin, 468 
 Huppert's test for bile pigments, 429, 
 
 430, 799 
 Hyalin, 840 
 Hyalines, 171 
 Hyaline substance, 364 
 Hyalogens, 171 
 Hyalomucoid, 617 
 Hydantoins, 786 
 Hydremia, 340. 352 
 Hydramnion, 642 
 
 Hydrazine poisoning, 718 
 
 Hydrazones, 202-203 
 
 Hydrobilirubin, 743 
 
 Hydrocele and spermatocele fluids, 359, 
 
 360 
 Hydrochinon in urine, 727 
 Hydrogel, 14 
 Hydrogen, colorimetric method, 75 
 
 determination of, 75 
 
 determination of, in fluids con- 
 taining CO2, 76 
 
 electromotive method, 75 
 
 ion content, 75 
 
 peroxide, 35 
 Hydrogenases, 876 
 Hydrolytic cleavage processes, 40 
 Hydroquinone, 727 
 Hydrosol, 14 
 
 Hydroxylamine poisoning, 718 
 Hyperacidity, 486 
 Hyperglycemia, 401, 402 
 Hyperthyreoidismus, 378 
 Hypertonic solution, 6 
 Hypnotics and glycogen formers, 394 
 Hypophysis, 380 
 Hypotonic solution, 6 
 Hypoxanthine, 178, 187, 188, 191, 192, 
 572, 712 
 detection of, 191 
 
 Ichthidin, 630, 636 
 
 Ichthin, 636 
 
 Ichthulin, 92, 174, 630, 636 
 
 Ichthylepidin, 122 
 
 Icterne, 413, 428, 431, 440, 441 
 
 urine, 799 
 Ignotine, 576 
 Ileum, extirpation of, 549 
 Imbibition, 30 
 Imidazol derivatives in urine, 757, 826 
 
 structural formula of, 186 
 Immune bodies, 66, 69 
 Immunity, 63-70 
 
 active, 70 
 passive, 70 
 
 theory of, Arrhenius', 67, 68 
 theory of Erlich, 67, 68 
 Immunization, 66 
 Indican, 517, 728 
 
 elimination of, 728 
 
 effect of putrefaction 
 on, 729 
 excretion of, 729, 730 
 quantitative method for, 731 
 tests for, 730, 731 
 Indigo-blue of urine, 741 
 Indigo-red formation, source of, 731 
 Indigotin in urine, 728, 731 
 Indirubin in urine, 731 
 Indol, 46, 82, 117, 157-159, 267, 515, 522, 
 843 
 fate of, in organism, 786 
 formation of, in organism, 728, 729 
 
GENERAL INDEX 
 
 1007 
 
 Indol, methyl-, 157 
 tests for, 158 
 Indoxyl, 724 
 Infraproteins, 93 
 Inosine, ISO, 577 
 Inosite, 579-581 
 
 in adrenal bodies, 377 
 preparation of, 580 
 properties of, 580 
 Inositogen, 577 
 
 "Integral factor," in uric acid elimina- 
 tion, 705 
 Internal friction, 18 
 
 effect of acids and alka- 
 lies upon, 19 
 effect of NaCl upon, 19 
 of hydrophile colloids, 19 
 of suspension colloids, 18 
 Intestinal fermentation, 512, 513 
 
 juice, constituents of, 491, 492 
 
 properties of, 491 
 putrefaction, 512, 513 
 Intestine, chemical processes in, 510-524 
 decomposition by microbes in, 
 
 513 
 large, putrefaction in, 515 
 small, digestion and absorption 
 of various foods in, 513-514 
 walls of, chemical processes in, 
 527-529 
 Intracellular enzymes, 52 
 Intraligamentary cysts, 627 
 Inulin, 228 
 
 Inversion of sugars, 224 
 Invert sugar, 224 
 Invertase. See Enzymes, 48 
 Invertin, 225. See Enzymes. 
 Iodine combinations, passage into milk, 
 667 
 passage into per- 
 spiration, 849 
 passage into saliva, 
 457 
 in blood, 268, 333, 337 
 in glands, 378, 379 
 in perspiration, 848 
 in proteins, 81, 125 
 Iodoform, fate in organism, 795 
 Iodospongin, 124 
 Iodothyrin, 373, 376 
 
 of thymus, 368 
 Iodothyrioglobulin, 373, 376 
 Ions, 5, 70-76 
 
 action of, on enzymes, 70 
 Iron, absorption, 338-339 
 and blood, 339, 639 
 elimination, 433, 440, 770 
 importance of. in metabolism, 903 
 in spleen, 371 
 in urine, 769 
 results in lack of, 895 
 Islands of Langerhans, 407 
 Iso-casein, 649 
 Isocholesterin, 449 
 
 Isodynamic law in metabolism, 890-892 
 Isoelectric point, 21, 26 
 Isolactose svnthesis of, 58 
 [soleucine, 86, 106. 107, 143-145 
 I s< .maltose, 223, 224, 225-226 
 
 in urine, 749 
 
 synthesis of, 58 
 
 splitting of, 58 
 Isoserine, 146 
 Isosmotic solutions, 3 
 Isotonic solution, 5 
 lsotropous muscle substance, 564 
 
 Jacoby's quantitative method for pepsin,. 
 
 470 
 Jaffe's creatinine reaction, 695 
 Jaffe-Obermayer's indican test, 729 
 Janthinin, 844 
 "Jaune indien," 222 
 Jecorin, 605 
 
 in liver, 385 
 
 in spleen, 370 
 Jejunum, effect of extirpation of. 549 
 Jerusalem's test for lactic acid in gastric 
 juice, 488 
 
 Kaolin, 16 
 Karyogen, 623 
 Kephir, 518, 654, 659 
 
 effect on putrefaction, 518 
 -lactase, 57. 58 
 Kerasin, 607, 609, 610-611, 613 
 Keratin, A, B, C of horn and hair, prop- 
 erties of, 837 
 action of gastric juice on, 473 
 Keratins, 92, 112-116 
 
 composition of, 113 
 Ketone, behavior in animal body, 776, 777 
 Ketoplastic action, 819 
 Ketoses, 197 
 Kidneys, constituents of, 672-674 
 
 formation of hippuric acid in,. 
 
 722 
 quantitative analysis of, 673 
 relation to formation of urea, 686 
 Kinase, in thrombin formation, 323 
 occurrence in intestine, 492 
 Kinases, 51 
 Kjeldahl's method for total nitrogen, 
 
 688 
 Klupeovin, 698 
 
 Knapp's method of estimating sugar, 
 812 
 reaction for dextrose, 215 
 Knop-Hupner's method for urea, 696 
 Koilin, 115 
 Koprosterin, 44s 
 
 Kossel's reaction for hypoxanthine, 192 
 Kossler and Penny's method for phenol- 
 sulphuric acids, 726 
 Kramm's creatinine reaction, 696 
 Kumyss, 654, 659 
 
1008 
 
 GENERAL INDEX 
 
 Kyrin, 137 
 
 caseino- 137 
 
 gluto-, 137 
 
 proto-, 137 
 Kyrines, 167 
 
 carbamino reaction with, 167 
 
 Lactacidase, 207 
 Lactalbumin, 91, 652-654 
 
 cleavage products of, 106 
 composition of, 652 
 properties of, 653 
 Lactase, 48, 64 
 Lactimide, 156 
 Lactocrit, 657 
 Lactoglobulin, 652 
 Lactones, 198 
 Lactoprotein, 653 
 Lactose, 223, 224, 647, 654-655 
 
 in milk, conversion into lactic 
 
 acid, 644 
 in urine, 815 
 preparation of, 655 
 properties of, 654 
 tests for, 655 
 
 theory of formation in mammary 
 gland, 643 
 Lactosuria, 811 
 Laiose in urine, 815 
 Langerhans, islands of, 494 
 Lanocerin, 844 
 Lanolin, 446 
 Lanugo hair, 642 
 Large intestine, effect of extirpation of, 
 
 548 
 Latebra, 624 
 Lead in blood, 333 
 
 in the liver, 389 
 passage into milk, 670 
 Lecithin, 105, 586 
 
 amounts in blood of different 
 
 animals, 328 
 from blood serum, 261 
 in chyle, 347 
 in lymph, 348 
 in pus corpuscles, 364, 365 
 in pus-serum, 363 
 quantitative determination of, 
 246 
 Lecithins, 10, 242-249 
 
 hydrolytic products of, 342 
 in metabolism, importance of, 
 
 902 
 preparation of, 246 
 properties of, 244 
 structure of, 242 
 Lecithoproteins, 92 
 Legal's reaction for indol, 158 
 Legumin, cleavage products of, 107 
 Lense, of oxen, analysis of, 619 
 Leo's sugar, 810 
 Lepidoporphyrin, 844 
 Lethal, 238, 239 
 Leucaemia, blood, 187, 341, 698 
 
 Leucaemia, spleen, 375 
 
 urine, 710, 764 
 Leucine, 85, 106, 107, 109, 111, 113, 115, 
 123, 124, 125, 141-143, 267 
 conversion into isoamyl alcohol, 
 
 206 
 in brain, 607 
 in lymphatic glands, 366 
 in spleen, 370 
 in thyroid gland, 373 
 in urine, 755, 827 
 Leucinimide, 143 
 Leucocytes, 305 
 
 constituents affecting coagu- 
 lation, 315 
 in venous blood, 336 
 osmotic phenomena with, 8 
 See also white blood cor- 
 puscles 
 Leucomaines, 25 
 Leuconuclein, 367 
 
 in blood, 316 
 Leucopolun, 609 
 Leucyl peptides, 85-88 
 Levulins, 228 
 Levulose, 217 
 
 in urine, 814-815 
 Lichenin, 228 
 
 Lieberkiihns solid alkali albuminate, 126 
 Lieben's iodoform test for acetone, 822 
 Liebermann-Burchart's, for cholesterin, 
 
 447 
 Liebermann's reaction for protein, 100 
 Liebig's method for urea, 688 
 Lienases, 371 
 
 Lifschiitz's reaction for cholesterin, 445 
 Ligamentum nuchas, 115, 116, 546 
 Lignin, 231 
 
 Lime salts, manner of excretion, 769 
 Lipanin, absorption of, 545 
 Lipase, 47, 48 
 
 pancreatic, 57 
 Lipases, plants, 64. See also Enzymes. 
 Lipochromes, 268 
 Lipopeptides, 88 
 Lipoids, 241 
 
 as a special limiting layer, 10 
 in brain, 605 
 
 in relation to osmosis in cells, 10 
 sulphurized, 605 
 Lipuria, 827 
 Lithium, 22, 333 
 Liver, 381-450 
 
 acetone formation in, 819, 822 
 
 amino acids, synthesis of, 382 
 
 autolysis of, 387 
 
 assimilation in, 381 
 
 bile pigments, formation of, 381 
 
 blood pigments, destruction of, 
 
 388 
 -cells, reaction of, 382 
 conjugation of glucuronic acid in, 
 
 382 
 constituents of, 382 
 
GENERAL INDEX 
 
 1(09 
 
 Liver, creatin-creatine metabolism, 694, 
 695 
 deamidation in, 382 
 destroying of uric acid in, 707 
 ethereal sulphuric acids, formation 
 
 of, 382 
 formation of amino acids in, 530 
 formation of bile in, 382 
 glycogen formation, 381, 390 
 glycogen content in, extirpation of 
 
 the adrenals, 402 
 glycolysis in, theories for, 408 
 iron in, 387, 388 
 " organ plasma " of, 382 
 pigments of, 384 
 processes going on in, 381, 382 
 protein synthesis in, 529, 530 
 storehouse for protein, 389 
 synthetical formation of uric acid 
 
 in, 705 
 urea formation, 382 
 
 of. from ammonia, 
 767 
 uric acid formation in, 702, 703 
 Livetin, 629 
 Lotah istone, 108 
 Lungs, concretions of, 871 
 constituents of, 870 
 expectorations of, 870, 871 
 gas, exchange in, 867 
 Lutein, 631 
 
 Luteins, in blood serum, 268 
 Lymph, 345-380 
 
 amount secreted, 349 
 composition of, 345-348 
 coagulation of, 346 
 formation of, 350-352 
 gases of, 856-858 
 origin of, 345 
 osmotic pressure in, 348 
 secretion of, circumstances in- 
 fluencing, 349, 350 
 Lymphagogues, 350 
 Lymphatic glands, 366 
 
 quantitative composi- 
 tion of, 366 
 Lymphocytes, 300 
 Lymphoid cells, 305 
 Lysatine, 160 
 Lysatinine. 160 
 
 Lysine, 85, 106, 107, 109, 111, 113, 115, 
 117, 119, 123, 124, 125, 163, 
 164, 267 
 in various proteins, 165 
 peptone, 138 
 Lysines, 47 
 
 Lysylglycylpeptide, 89 
 Lysyl lysine, 85 
 
 Magnesium in urine, 768, 769 
 
 triphosphate in urinarv sedi- 
 ment, 831 
 Maintenance value, 897 
 Maltase, 48, 458. See also Enzymes. 
 
 Malt dextrin, 229 
 Malt diastase, 225 
 
 action of, upon starch, 226 
 Maltose, 223, 224, 225 
 in urine, 815 
 synthesis of, 58 
 Malt sugar, 225 
 Mammary gland, theory of formation of 
 
 lactose in, 643 
 Mandelic acid ester, racemic, 62 
 
 nitrile glucoside, 59 
 Manganese, 268, 333, 442 
 Mannite, 198 
 Mannose, 198, 216 
 Manonose, 206 
 Marcitine, 47 
 Margarin, 235 
 
 Maschke's reaction for creatinine, 695 
 Mass action, 32, 74 
 Mastic, 22 
 Meconium, 523 
 
 Medicinal coloring matters in urine, 801 ' 
 Medulla, 607 
 
 Medullary fibers, composition of, 614 
 Melanin, 617, 842 
 
 in urine, 799 
 Melanins, of skin, 841 
 
 mother substances of, 843 
 Melanogen in urine, 799 
 Melanoidin nitrogen, 78 
 Melanoidins, 841 
 Melano-protein, 842 
 Melanotic cancers, 802, 842, 843 
 Membrane, semi-permeable, 26 
 Membranin, 171, 550 
 
 properties of, 617 
 Menthol, fate of, in organism, 785 
 Mercuric salts poisoning, 23 
 Mesitylene, fate of, in organism, 779 
 Mesoinosite, 582 
 Mesoporphyrin, 295 
 Mesoxalyl urea, 698 
 Metabolism, 878-939 
 
 affected by sleep, 928 
 as affected by external tem- 
 perature, 929 
 as affected by high alti- 
 tude, 930 
 as affected by ingestion of 
 
 food, 930 
 as affected by light, 928 
 as affected by mental activ- 
 ity, 928 
 basal requirement, 897, 898 
 calculation of, 889 
 conditions affecting, 922-931 
 effect of age upon, 922-925 
 effect of alcohol on, 921, 922 
 effect of coffee and tea upon, 
 
 922 
 effect of rest and work upon, 
 
 926-«)2-< 
 effect of salts on, 921 
 ' effect of sex upon, 925, 926 
 
1010 
 
 GENERAL INDEX 
 
 Metabolism, effect of weight of body 
 upon, 922-925 
 effect of water on, 920, 921 
 importance of lecithins in, 
 
 902 
 importance of phosphates 
 
 in, 902 
 in active and inactive mus- 
 cles, 591-598 
 inanition condition, 897-898 
 in starvation, 892-906 
 lack of mineral substances in 
 food, 899, 900 
 water in food, 898 
 maintenance value, 897-898 
 measured by gas exchange, 
 
 927, 923 
 with absence of carbohy- 
 drates in food, 903, 904 
 with absence of fats in food, 
 
 903-904 
 with absence of proteins 
 
 in food, 903 
 with a mixed diet, 913-920 
 of fat with an exclusive 
 
 protein diet, 907, 908 
 with foods rich in protein, 
 
 906-913 
 with insufficient supply of 
 
 chlorides, 900, 901 
 with lack of bases in foods, 
 
 901 
 with lack of earths in food, 
 
 901, 902 
 with lack of iron in foods, 
 
 903 
 with lack of phosphates in 
 
 food, 901, 902 
 with various foods, 906-922 
 Metacasein, 651 
 Metalbumin, 624, 625 
 Metanitrobenzaldehyde, fate of, in the 
 
 body, 784 
 Metaproteins, 93 
 Metazym, 319 
 Methaemoglobin of thyroid gland, 374. 
 
 See also Blood pigments. 
 Methal, 239 
 Mf-those, 212 
 
 Methylation in organism, 787 
 Mfthylenitan, 212 
 
 Mfthylethylmaleic acid anhydride, 290 
 Methylglycocoll. See Sarcosin. 
 Methylglyoxal in relation to lactic acid 
 
 fermentation, 584 
 Methylguanidine, 578, 698 
 
 in urine, 758 
 Methyl guanine (7), 714 
 Methylindol, 157 See Skatol. 
 Methylindolm, 729 
 
 Methylimidazol formation of, 201-202 
 Methyl pentoses, 199, 208 
 Methylphenylhydrazine, test for levulose, 
 218 
 
 Methyl pyridine, behavior in animal body,. 
 778, 781 
 chloride in urine, 756, 
 758 
 Methylpyridylammonium hydroxide, 787 
 Methylthiophene, 784 
 Methyluracil, 194 
 Methyluramine, 574, 694 
 Methyl-urea, 691 
 
 in urine, 712, 713 
 Methylxanthine, 187 
 Mett's quantitative method for pepsin, 
 
 469 
 Micro-organisms in intestines, 511, 514, 
 
 517, 520 
 Microrespirometer, 874 
 Microtonometer, 861 
 Milk, 643-671 
 
 albumin, quantitative determina- 
 tion of, 656 
 ash, quantitative composition of, 
 
 666 
 casein, quantitative determination 
 
 of, 656 
 coagulation, a two-faced process, 
 
 650 
 coagulation of, 645 
 cows, 644, 645 
 cows, quantitative composition of, 
 
 657 
 effect on putrefaction, 518 
 fat, quantitative determination of, 
 657 
 constituents of, 647 
 theory for origin of, 669 
 foodstuffs in. 666 
 gases of, 857 
 globules, 645-647 
 goat's, 659 
 human, 660-671 
 human, compared to cow's milk,. 
 
 660, 661, 663 
 influence of food on composition of, 
 
 667, 668 
 mare's, 659 
 lactose quantitative determination,. 
 
 of, 657 
 mineral bodies, quantitative de- 
 termination of, 656, 657, 658 
 of carnivora, 659 
 of various animals, composition of, 
 
 660 
 passage of foreign substances into, 
 
 670 
 plasma, 647 
 
 preparation, composition of, 659 
 proteins, quantitative determina- 
 tion of, 656 
 quantitative analysis of, 656, 657 
 secretion, chemistry of, 668-671 
 solids, quantitative determination! 
 
 of, 656 
 souring of, 645 
 sugar, 226. See Lactose. 
 
GENERAL INDEX 
 
 1011 
 
 Milk, sugar, theory for origin of, 670 
 
 quantitative determination 
 of, 657 
 theory for origin of casein, tills 
 
 permanent emulsion, 646 
 uterine, 641 
 
 various fermentations of, til.") 
 woman's, quantitative composition, 
 652, 653 
 quantity of mineral sub- 
 stances in, 654 
 Millon's reaction for proteins, 99 
 
 in hyurocele an I 
 spermatocele Quids, 
 . 359 
 Mineral bodies in milk, 656 
 Mineral substances, absorption of, 540 
 
 amounts in blood of 
 different animals, 
 328 
 distribution of, 72 
 effect on metabolism, 
 
 921 
 importance to life of 
 
 cells, 72 
 in bile, 436 
 in blood, 335 
 in blood serum, 26S, 
 
 269 
 in bone structure, 552 
 in brain, 615 
 in cartilage, 550 
 in cells, 72 
 in cerebrospinal fluid, 
 
 361 
 in chyle, 347 
 in connective tissue, 
 
 546 
 in cystic fluid, 627 
 in egg shell, 636 
 in feathers, 838 
 in gastric juice, 466 
 in hair, 838 
 in intestinal juice, 491 
 in kidneys, 673 
 in liver, 387-389 
 in lungs, 870 
 in lymph, 346, 348 
 in muscles, 586-588, 
 
 599 
 in nails, 838 
 in pancreatic gland, 
 
 495 
 in pancreatic juice, 
 
 500 
 in pericardial fluid, 
 
 356 
 in pus corpuscles, 365 
 in pus-serum, 363 
 in retina, 615 
 in semen, 621 
 in smooth muscles, 
 
 602 
 in spermatozoa, 622 
 
 Mineral substances, in starvation, 895, 
 896 
 in synovial fluid, 362 
 in urine, 758-770 
 in white of egg, 635 
 in woman's milk, 654 
 in volk, 632 
 lack of, in food, 899, 
 
 900 
 of spleen, 361 
 toxicity of, 73 
 Mingen in urine, 756, 75s 
 Mohr's quantitative method for chlorides, 
 
 759 
 Molisch's test for sugar, 216 
 Monaminophosphatides, 241 
 Monomethylxanthine, 712 
 Monosaccharides, 197-218 
 
 cyanhydrin formation, 
 
 200 
 oxime formation, 200 
 transformation of, 201 
 Moore's test for sugar, 214, 224 
 Morner-Sjoqvist and Folin's method for 
 
 urea, 6S9 
 Morphine, elimination in urine, 749, 787 
 
 in milk, 670 
 Moss-starch, 228 
 Mucin, 619 
 
 action of trypsin on, 508 
 " dissolved," 794 
 effect of gastric juice on, 473 
 in urine, 757, 794 
 pseudo, 171 
 substances, 168-172 
 Mucilages, 226 
 
 vegetable, 230 
 Mucinoids, 171 
 Mucins, analysis of, 169 
 
 amino acids in. 170 
 cleavage products of, 170 
 true, 168, 169 
 Mucoid, osseo-, 172 
 Mucoids, 169, 171 
 
 effect of gastric juice on, 473 
 Mucous of urine, 674 
 Mulberry calculi, 833 
 Murexide test, 707 
 Muscarine, 246 
 Muscle-pigments, 571 
 Muscle-plasma, 566 
 
 preparation and proper- 
 ties of 566, 567 
 proteins of, 569 570 
 Muscle-serum, 566 
 
 smooth, 601-603 
 
 constituents of, 602 
 extractives in, 602 
 " snow," 566 
 "-stroma," 569 
 svntonin, 569 
 Muscles,'565-603 
 
 acid rigor of. 590 
 
 active, acid reaction of, 593 
 
1012 
 
 GENERAL INDEX 
 
 Muscles, active, characteristics of, 596 
 
 amount of fat in, 600 
 
 an isotropous substance of, 555, 
 556 
 
 chemical rigor of, 590 
 
 dead, proteins of, 567 
 
 elementary analysis of, 601 
 
 extractive bodies of, 572-588 
 
 "flesh quotient,'' 601 
 
 haemoglobin, 571 
 
 heat rigor of, 590 
 
 imbibition rigor of, 590 
 
 isotropous substance of, 555, 556 
 
 metabolism in, 591-598 
 
 of different animals, ash anal- 
 yses of, 599 
 
 of different animals, analyses of, 
 599 
 
 proteins of, 566-572 
 
 quantitative composition of, 598- 
 601 
 
 reaction of, 565 
 
 rigor mortis of, 588-591 
 
 striated, 565-601 
 
 water rigor of, 590 
 Musculamine, 578 
 Muscular action, source of, 597 
 Musculin, 568, 570 
 Myelin, 608 
 " Myeline forms," 605 
 Myoalbumin, 569 
 Myocynine, 578 
 Myogen, 570, 571 
 Myogen-fibrin, 570, 571 
 
 soluble, 570 
 
 preparation of, 570, 571 
 Myoglobulin, 568, 571 
 Myohaematin, 571 
 Myoproteid, 571 
 Myosin, 84, 567, 568, 570 
 
 as related to fibrinogen, 254 
 
 coagulation of, 570 
 
 ferment, 570 
 
 fibrin, 568, 570 
 
 in blood corpuscles, 306 
 
 preparation of, 568 
 Myosinogen, 569, 570, 571 
 Myosinoses, 130 
 Myri»in, 239 
 Mytilite, 581 
 Mytolin, 569 
 Myxcedema, 374 
 Myxoid cysts, 624 
 
 Nails, 837 
 
 mineral substances of, 838 
 Naphthalene, fate of, in organism, 779, 
 
 785 
 Napthoresorcin, reaction for conjugated 
 glucuroric acid, 218, 223, 
 
 818 
 reaction for levulose, 218 
 Naphthylisocyanate compounds of amino- 
 acids. See various Amino-acids. 
 
 Narcotics, relation to glycogen formation, 
 
 394 
 Neosine, 578 
 Neossin, 171 
 Neottin, 631 
 Neozym, 320 
 
 Nephrorosein in urine, 734 
 Nerve fibres, analysis of, 614 
 Nerves, 604-615 
 Neubauer's and Rolide's reaction for 
 
 proteins, 100 
 Neuberg's test for levulose, 218 
 Neuberg-Rauchwerger's reaction for cho- 
 
 lesterin, 444 
 Neuridine, 606, 612, 628 
 Neurine, 246 
 Neurochitin, 614, 615 
 Neuroglia, 601 
 
 Neuroglobulin, a and /3, 604, 605 
 Neurokeratin, 605, 614, in various nerves, 
 
 amounts in, 614 
 Nicotine, effect upon gases of stomach, 
 
 485 
 Ninhydrin reaction, 101 
 Nitrates in urine, 766 
 Nitriles, fate in organism, 775 
 Nitrobenzaldehydes, fate of, in organism, 
 
 783, 784 
 Nitrobenzene, fate of, in organism, 782 
 Nitrogen colloidal, 795 
 
 elimination in starvation, 893, 
 894 
 through various 
 
 channels, 880, 881 
 gaseous elimination of, 881. 
 See also Gases in various tis- 
 sues and fluids. 
 Nitrogenous equilibrium, 918, 919, 934 
 Nitrotoluol, behavior in animal body, 
 
 786 
 Novaine, 577 
 
 in urine, 758 
 Nuclease, 504, 508 
 
 Nucleases, 48, 182. See also Enzymes. 
 Nucleinases, 182 
 Nuclein bases, 186 
 
 in lymphatic glands, 366 
 plates, 308 
 in pus-cells, 365 
 true, 176 
 Nucleins, 176, 177 
 
 action of trypsin on, 508 
 effect of gastric juice on, 473 
 pseudo-, 177 
 Nucleoalbumins, 91, 92, 93, 177 
 
 detection of in urine, 795 
 properties of, 104 
 in urine, 794 
 Nucleohistone, 307 
 
 in blood, 316 
 in urine, 795 
 of thymus, 367, 368 
 Nucleon, 578. 620, 666 
 Nucleo proteins, 92, 167, 174-177, 252 
 
GENERAL INDEX 
 
 1013 
 
 Nucleo proteins, action of trypsin on, 508 
 cleavage products of , 177— 
 
 195 
 of blood, 258 
 of milk, 653 
 of the spleen, 369 
 peptic digestion of, 472 
 Nucleosidases, 182 
 Nucleosides, 180, 211 
 Nucleosin, 196 
 Nucleotides, 179 
 
 mono-, 179 
 poly-, 179 
 Nucleotin, 179, 182 
 Nucleotoprotamines, 174, 623 
 Nutritive need of man, 916 
 requirement, 908 
 Nylander Almen's test, 215 
 
 sugar test, 802 
 
 Obermayer's indican test, 729 
 
 Obermuller's cholesterin reaction, 445 
 
 Oblitine, 578 
 
 Ochronose, 550 
 
 Octodecapetide, 85, 87 
 
 Oedematous fluid, and its constituents, 363 
 
 Oesophageal fistula, 459 
 
 Oil-turpentine, behavior in animal body, 
 
 749, 784 
 Olein, 236 
 
 mono and tri enzymotic synthesis 
 of, 60 
 Oligemia, 340 
 Oligocythemia, 340 
 Oliguria, 771 
 Olive oil, absorption, 544 
 
 effect upon bile secretion, 415 
 Onuphin, 171 
 Oocyan, 636 
 Oorodein, 636 
 Opalisin, 652 
 
 Opium, passage of, into milk, 670 
 Organic phosphorous compounds in urine, 
 757 
 in urine, 762 
 sulphur compounds in urine, 752, 
 765 
 Orcin-hydrochloric acid test, 209, 223 
 Orcin, test for pentoses in urine, 817 
 Oriental bezoar-stone, 524 
 Ornithine, 85, 162, 163 
 Orthonitrobenzaldehyde, fate of in the 
 
 body, 784 
 Orthonitrophenylpropiolic acid, test for 
 
 dextrose, etc. See Acid nitrophenyl- 
 
 propiolic. 
 Orthonitrotoluene, fate of, in organism, 785 
 Osamines, 204 
 Osazones, 202, 203, 215 
 Osimines, 201 
 Osmometer, 16 
 Osmosis, 5 
 Osmotic pressure, 1-13 
 
 determination of, 16 
 
 Osmotic pressure, effective, 12 
 
 effect of various sub- 
 stances upon, 17 
 electrolytes, abnormal, 
 
 due to ionization, 
 in higher animals, 13 
 in lower sea animals, 13 
 in Lymph, 13 
 in milk and bile, 13 
 non-electrolytes, 4 
 of animal fluids, 12 
 of colloids, 16-18 
 of the blood, in selachii, 
 
 13 
 in saliva, 13 
 in urine, 13 
 Osones, 203 
 Ossein, 551 
 
 Osseoalbuminoid, elementary composi- 
 tion of, 551 
 Osseomucoid, 172, 551 
 
 elementary composition of. 
 551 
 Osteomalacia, 556, 557 
 Osteosclerosis, 556 
 Otoliths, 619 
 Ovalbumin, 633-635 
 
 cleavage products of, 106 
 preparation of, 634 
 Ovaries, 623 
 
 cysts in, 624 
 Ovin, 631 
 
 Ovoglobulin, 84, 633 
 Ovokeratin, 114 
 Ovomucin, 634 
 
 Ovomucoid, 171. 633, 635, 636 
 properties, 635 
 preparation, 635 
 Ovovitellin, 84, 91 
 
 composition of, 628, 629 
 preparation of, 630 
 Ovum, 623, 628 
 Oxamide, 83 
 Oxidases, 188 
 
 artificial, 873. See also En- 
 zymes 
 Oxidation in animal tissues, theories of, 42 
 
 in the organism, 850-877 
 Oxidones, 874, 875 
 Oxydase, 47, 48 
 
 Oxygen absorption, calculation of, 889 
 activation, 4-8 
 tension in blood, 859-861 
 tension, regulation of in organism, 
 868 
 Oxygenases, 872 
 Oxyhaematin, 289-291 
 Oxyhaemocyanin, 299 
 Oxyhemoglobin, 276, 278-282 
 
 absorption bands of 281, 
 
 282 
 action of trypsin on, 508 
 effect of gastric juice on, 
 473 
 
1014 
 
 GENERAL INDEX 
 
 Oxyhemoglobin, law of dissociation of, 
 859 
 preparation of, 282 
 See also Blood pig- 
 ments. 
 
 Oxyphenylethvlamine, 82 
 
 Oxyproline, 85, A 106, 107, 125, 155 
 
 Oxyprotein, 83 
 
 ( )xypurine, 190 
 
 Oxypyrimidine, 195 
 
 Oxyquinolines, fate of, in organism, 785 
 
 Ozone, occurrence in the organism, 4 
 
 Palmitin, 235 
 Pancreas, 494-510 
 
 importance of, to absorption of 
 carbohydrates, 535 
 .Pancreatic diastase, 501 
 
 gland, constituents of, 495 
 juice, 494-501 
 
 action on fats, 502 
 action of, on starch, 226 
 amount secreted, 500 
 constituents of, 500 
 excitants for the secre- 
 tion of, 498 
 properties of, 500 
 lipase, 57. See also En- 
 zymes, 
 secretion of, 496 
 rennin. 509. See also En- 
 zymes, 
 secretion, human, 500, 501 
 Papain, 51 
 Paracasein, 650 
 Parachymosin, 474, 567 
 Paracresol, 515 
 
 formation in putrefaction, 515, 
 723, 724 
 Paraglobulin, 258 
 Parahsemoglobin, 280 
 Parahistone, 109 
 Paralbumin, 624, 625 
 Paraminophenol, 779 
 Paramucin, 626 
 Paramyosinogen, 568, 570, 571 
 Paranuclein. See Pseudonuclein 
 Paraovarial cysts, 627 
 Paraoxypropiophenone, 785 
 Parapeptone, 472 
 Parathyroids, 377 
 
 Paraxanthine in urine, 712, 713, 714 1 
 Parenchymatous organs, action of trypsin 
 
 on, 508 
 Parenteral canal, introduction of pro- 
 tein by way of, 533-511 
 Parotid, 448 
 
 saliva, 450 
 Peaglobulin, 84 
 Pectin bodies, 229, 231 
 Pemphigus chronicus, 359 
 Pennac-frin, S 10 
 Pennatulin, 123 
 Pentacrinin, 844 
 
 Pentamethylenediamine, 47. See Cada- 
 
 verin 
 Pentapeptides, 85 
 Pentosamines, 220 
 Pentosans, 207 
 
 Pentoses, 197, 198, 199, 207-211 
 in urine, 816 
 
 quantitative estimation, 209 
 test, orcin-hydrochloric, 209 
 tests for, 209 
 Pentosides, 180 
 Pentosuria, 816 
 
 Penzoldt's test for acetone, 823 
 Pepsin, 57, 64, 466-476 
 
 action on proteins, 468-473 
 characteristic property of, 468 
 digestion of proteins, products of, 
 
 472 
 digestion, effect of foreign sub- 
 stances upon rapidity of, 470 
 digestion, rapidity of, 471 
 digestion of various bodies, 472, 
 
 473 
 mother substances of, 477 
 nature of, 467 
 occurrence of, 466 
 preparation of, 468 
 properties of, 467, 468 
 quantitative methods for, 469, 470 
 relation to rennin, 475 
 test for, 469 
 
 testing; for, in gastric juice, 487 
 Pepsinogen, 477 
 
 Peptases, 503. See also Enzymes. 
 Peptic-glutin peptone, 135 
 peptones, 135 
 
 zymolysis, effect of bile upon, 511 
 Peptidases, 48 
 Peptides, 68, 85-89, 93, 126, 127, 156 
 
 behavior toward enzymes, 8, 
 266, 492, 510, 511. See Poly- 
 peptides, 
 in urine, 755 
 
 relation to alcaptonuria, 735 
 relation to urea formation, 777 
 Peptinuria, 791 
 Peptochondrin, 549 
 Peptoid, 507 
 Peptone-plasma, 251 
 Peptones, 92, 127-132 
 anti-, 132 
 as foodstuff, 912 
 carbamino reaction with, 167 
 from peptic digestion 472 
 in urine, 791 
 properties of, 131 
 quantitative estimation, 138 
 separation from proteoses, 136, 
 138 
 Percaglobulin, 637 
 Pericardial fluid, 356, 357 
 
 analysis of, 356 
 Perilymph, 619 
 Peritoneal fluid, 357 
 
GENERAL INDEX 
 
 1015 
 
 rermeability, causes for, 
 
 of blood corpuscles, 7 
 
 ( hrerton'a solubility theory, 
 
 10 
 shortcomings of, 10, 11 
 Traube's solution tenacity 
 theory, 1 1 
 Peroxidases, 872. See also Enzymes. 
 Peroxydase, 50 
 Perspiration, 847-849 
 
 circumstances influencing, 
 
 847 
 constituents of, 848 
 properties of, 847 
 Pettenkofer's method for determining 
 respiratory change, 869 
 Pit test for bile-acid, 798 
 test lor bile, 418 
 Pfaundler's method of precipitating urine, 
 
 681 
 Phaseomannite, 579 
 Phenol, 515 
 
 as precipitant of proteids, 98 
 in urine, 515, 723, 726, 785, 787 
 Phenol-oxidases, 875 
 Phenols, 117, 723 
 
 fate of, in organism, 775, 784 
 Phenylalanine, 85, 109, 114, 151, 152 
 
 amounts in proteins, 106, 
 107, 113, 115, 125 
 Phenylethylamine, 82 
 Phenylieocyanate compounds of amino 
 acids. See various 
 Acids, amino, 
 peptone, 138 
 Phenylmethvlketone, 785 
 Philothion, 876 
 Phlebm, 276 
 
 Phosphates, importance of, in metabolism, 
 902 
 in urine, 761-764 
 in urine, quantity of, 762 
 See also Mineral substances. 
 Phosphatides, 239-249, 265, 586 
 
 in adrenal bodies, 377 
 in bile, 435, 439 
 in kidney, 673 
 in liver, 385 
 in yolk, 630 
 Phosphaturia, 763 
 Phosphoglycoproteins, 174, 177 
 Phosphoproteins, 91, 92, 93 
 
 properties of, 104 
 Phosphorus compounds, action on bile, 
 437, 441 
 action on blood, 
 
 252, 254 
 action on urea 
 elimination, 
 679, 685 
 organic, in urine, 
 
 756 
 metabolism, 875, 
 876, 894 
 
 Phosphorus-containing urinary constitu- 
 ents, 756, 875 
 elimination in organism, 883 
 elimination in relation to ni- 
 trogen elimination, 762, 763 
 elimination in relation to pur- 
 ine metabolism, 7*13 
 Phosphatides, properties of, 241 
 Photometha;moglobin, 2.S4 
 Phrenin, 612 
 Phrenosin, 609, 610, 611 
 Phyllocyanin, 295 
 Phylloervthrin, 435 
 Phylloporphyrin, 276, 295 
 Phymatorhusin, 841 
 
 in urine, 799 
 Physical chemistry in biology, 1 
 Physiological oxidation processes, 871-877 
 
 salt solution, 7 
 Phytase, 579 
 Phytin, 579 
 Phytosterines, 446 
 
 Picolin, behavior of, in animal body, 784 
 Pigments, fate of, in organism, 787 
 medicinal in urine, 801 
 of bile, 427, 433, 435, 436 
 of birds' eggs, 636 
 of blood, 275, 298 
 of blood serum, 268 
 of butterflies, 844 
 of corpora lutea, 297, 623 
 of eye, 614, 616 
 of feathers. 843, 844 
 of human skin, 840-843 
 of liver, 384 
 of lobster, 638, 844 
 of lower animals, 299, 844 
 of lungs, 870 
 of muscle, 571 
 of placenta, 641 
 of pus, 366 
 of skin, 840-844 
 of stones, 441 
 of urine, 733. 734, 740, 748, 798, 
 
 799 
 of yolk, 631 
 Pilocarpine, effect upon elimination of 
 C0 2 in stomach, 485 
 effect upon elimination of 
 
 uric acid, 700 
 effect upon secretion of in- 
 testinal juice, 490 
 effect upon secretion of sali- 
 va, 457 
 Piperdine-glvcosuria, 403 
 Piquie, 402 
 
 Pina's test for tvrosine, 153 
 Placenta, 641 
 Plant cells, 5 
 
 osmotic experiments with, 5 
 gums, 226 
 Plasmolysbg power of differently con- 
 structed salts, 6 
 Plasmolysis, 5 
 
1016 
 
 GENERAL INDEX 
 
 Plaamolvsis, substances bringing about, 5 
 Plasmolytic experiments with animal 
 
 cells, 7 
 Plasmoschisis, 314 
 Plasmozym, 319 
 Plastein, 59 
 Plasteines, 135 
 1 lastin, 178 
 
 Plattner's crystallized bile, 396 
 Pleural fluid, 357 
 Pnein, 874 
 Pneumonic infil .rations, solutions of, 18, 
 
 368, 86j 
 Poikilocytosis, Oj.2 
 Polycythtc.. ia, 3*0 
 Polynucleotides, 179 
 Polypeptide-like 1 odies in urine, 756 
 Polypeptides, bd-ui, 92. See Peptides, 
 action of try] sin on, 509 
 cleavage of, by enzymes, 62 
 E. Fischer's synthetical 
 
 preparation of, 88 
 methylated b8 
 syntheses ol , 86 
 synthetically produced, 87 
 sulphur containing, 88 
 synthetic, comparison of 
 properties with natural pro- 
 teins, 90. See also Pep- 
 tides. 
 Polyperythrin, 844 
 Polysaccharides, colloid, 226-231 
 Polyuria, 771 
 
 Potassium in urine, 766. See also Min- 
 eral substances. 
 Praglobin, 307 
 
 Precipitation, Traube's membrane, 1 
 Precipitins, 66 
 Preglobulin, 315, 368 
 Preputial, secretion of skin, 846 
 Proenzymes, 51 
 Prolamine, 78 
 Prolamins, 106 
 Proline, 85, 109, 111, 154. 155 
 
 amounts in protsins, 106, 107, 
 113, 115, 125 
 Propepsin, 477 
 Propeptone, 126 
 Propylalanine, 85 
 Propyl benzene, behavior in animal body, 
 
 778 
 Propylene glycol, relation to glycogen 
 
 formation, 394 
 Prosecretin, 463, 499 
 Prostate, secretion of, 621 
 Prostatic concrements, 172, 611, 623 
 Prosthetic group, 174 
 Protagon, 605, 606-608. 609 
 
 cleavage prod rets of, 607 
 elementary composition of, 606 
 nature of, 606, 607 
 preparation of, 608 
 properties of, 608 
 Protamine nucleate, 623 
 
 Protamines, 91, 92, 93, 108 
 
 hexone bases in, 165 
 preparation of, 112 
 properties of, 109-112 
 Proteans, 93 
 Protease, 47, 48 
 
 alpha retardation of, 63 
 Proteases, 48 
 
 Protective colloids, 37, 44, 131 
 Protein, absorption, effect of cellulose on, 
 531 
 catabolism, 907, 908, 909, 911 
 
 importance of sulphur 
 
 in, 882, 883 
 in active muscle, 594 
 in starvation, 894 
 destruction of, during work, 595 
 ochrome, 155 
 requirement, 933-936 
 
 lower limit, 915J 
 sparers, .913-915 
 
 substances, diarginylalanine, 111 
 diarginylproline, 111 
 diarginylserine, 111 
 diarginylvaline, 111 
 Proteins, 38, 77-195 
 
 absorption of, 525-531 
 acid, 125 
 
 action of neutral salts on, 98 
 action of nitrous acid upon, 79 
 action of pepsin on, 468-473 
 action of trypsin on, 505, 507 
 adsorption compounds, 95 
 adsorption of, 97 
 albuminates, 104 
 albuminoids, 112 
 albuminous, 102-138 
 albumins, 106 
 albumose, alkali, 127 
 alcohol soluble, 92 
 alkali, 126 
 
 amino acids in, linking of, 90 
 amounts in blood of different 
 
 animals, 328 
 analysis, 118 
 atmidkeratin, 113 
 atmidkeratose, 113 
 Bence-Jones, 792 
 byssus, 122, 123 
 carbon dioxide binders in blood 
 
 855 
 casein, 106 
 chitin, 122 
 chondrin, 121 
 chondro-, 168, 172-174 
 chromo-, 167 
 circulating, 908-910 
 classification of, 91-93 
 cleavage products, 93, 106 
 clupeine, 110 
 coagulated, 93 
 coagulation of, 96 
 collagen, 118, 119 
 color, reaction for, 99 
 
GENERAL INDEX 
 
 1017 
 
 Proteins, composition of, 77, 94 
 compound. Ill', ld7-177 
 concliiolm, 122, 12:1 
 conjugated, ( .*2, 93 
 cornein, 122, 123, 124 
 cornicrystalline, 123 
 crystalline, 94 
 cyclopterine, 110 
 cyprinine, 1 10 
 derived, 93 
 deutero, 120 
 deuteroelastose, 117 
 effect on glycogen content of 
 
 liver, 394, 397 
 elastin, 116-118 
 fate of, in organism, 886 
 fattening, 919 
 fibrin, 106 
 fibroin, 122, 124 . 
 foodstuff purposes of, 917, 918 
 forms of nitrogen in, 77, 78 
 gelatin, 118, 119-121 
 gelatin-peptones, 120 
 gelatose, 120 
 gelatoses, 120 
 globan, 104 
 globulins, 103-104 
 gluco-, 167, 168-174 
 glucoproteins, phosphorized, 105 
 gluteins, 119 
 glyco-, 92, 167, 168-174 
 gorgonin, 123 
 helico-, 174 
 hemicollin, 120 
 hemielastin, 117 
 hordein, cleavage of, 107 
 hydrolysis of, 81 
 ichthylepidin, 122 
 in amniotic fluid, 642 
 in aqueous humor, 361 
 in ascitic fluids, 359 
 in bone, 551 
 in bone marrow, 554 
 in brain, 604 
 in cartilage, 546 
 in cerebrospinal fluid, 360 
 in chyle, 347 
 in connective tissue, 544 
 in crystalline lens, 617, 618 
 in cystic fluids, 625-627 
 in dead muscles, 567 
 in fat globules, 647 
 in horn substance, 837 
 in hydrocele and spermatocele 
 
 fluids, 359 
 in kidneys, 672 
 in liver, 382, 383 
 in lungs, 870 
 in lymph, 346, 348 
 in lymphatic glands, 366 
 in mammary glands, 643 
 in milk, 647-654 
 in milk plasma, 647 
 in muscles, 566-572 
 
 Proteins, in muscle-plasma, 569, 570 
 
 in pancreatic gland, l { '."> 
 in pericardial Quid, 356 
 
 in placenta, til 1 
 
 in prostate secretion, 621 
 
 in pus corpuscles, 364, 365 
 
 in pus-serum, 363 
 
 in retina, 615 
 
 in salivary glands, 451 
 
 in sebum of .skin, 844 
 
 in semen, 620 
 
 in smooth muscle, 602 
 
 in spermatozoa, 623 
 
 in sputum, S71 
 
 in synovial fluid, 362 
 
 in testes, 620 
 
 in thymus, 366, 367 
 
 in transudates and exudates, 
 353, 354 
 
 in urine, 787-795 
 
 in urine, detection of, 788 
 
 in urine, quantitative estima- 
 tion of, in urine, 793 
 
 in veins, 336 
 
 in venous blood, 336 
 
 in white of egg, 633-636 
 
 in yolk, 628-630 
 
 iodo. 123 
 
 keratins, 112-116 
 
 koilin, 115 
 
 lecithalbumins, 105 
 
 lecitho, 92 
 
 Lieberkuhn's solid alkali albu- 
 minate, 126 
 
 metabolism calculation of, 889 
 
 metabolism of, in starvation, 
 893, 894 
 
 metabolism with food rich in 
 proteins, 906, 913 
 
 method of synthesis, 86 
 
 membranins, 617 
 
 modified, 96 
 
 molecular weight, 98 
 
 mucins, true, analysis of, 168- 
 172 
 
 mucinoi Is, 171 
 
 mucoids, 171 
 
 native, 96 
 
 Neubauer and Rohde's test, 
 100 
 
 nuclei of aliphatic series, 85 
 
 nuclei of carbocyclic series, 85 
 
 nuclei of heterocvclic series, 85 
 
 nucleo, 92, 105, 167, 174-177 
 
 nucleo albumins, 104, 105 
 
 ovokeratin, 114 
 
 ovovitellin, 105, 106 
 
 parenterally introduced, absorp- 
 tion of, 525 
 
 pennatulin, 123 
 
 phospho, 92, 104 
 
 phosphoglvcoproteins, 174, 177 
 
 phosphorized importance of, in 
 metabolism. 902 
 
1018 
 
 GENERAL INDEX 
 
 Proteins, precipitation of, 95, 96, 98, 99 
 preparation, 118, 121 
 prolamine, 106 
 properties of, 94-96, 107, 108, 
 
 119 
 protamine, constitution of, 111 
 protamines, 109-112 
 proto-, 120 
 protoelastin, 117 
 protones, 110 
 pseudomucins. 171 
 pseudonucleins, 104 
 putrefaction of, 
 putrefactive products of, 82 
 quantitative estimation, 101 
 reaction of, 91 
 reactions, precipitation, 98 
 reticulin, 121, 122 
 salmine, 110 
 salting out of, 95 
 scombrine, 110 
 semiglutin, 120 
 seralbumin, cleavage products 
 
 of. 106 
 [serglobulins, cleavage of, 106 
 sericin, 122, 124 
 simple, 91, 92, 93, 94 
 
 cleavage products of, 
 125-195 
 skeletins, 122 
 
 constitution, 122 
 source of muscular energy, 595- 
 
 597 
 Bpongin, 122, 123 
 sturine, 110 
 sulphur content, 
 syntheses from amino acids, 
 
 529, 530 
 synthesis of, in organism, 530 
 syntheticallv formed, 59 
 tests for, 99, 100, 101 
 tissue, 908-910 
 xanthoprotein reaction, 99 
 zein, cleavage products of, 107 
 Proteose-like substances in blood serum, 
 
 264 
 Proteoses, 92, 127-132 
 
 absorption, 534, 538 
 
 as foodstuff , 912 
 
 carbamino reaction with, 167 
 
 detection of, in urine, 792 
 
 deutero, 130 
 
 dys, 130 
 
 from peptic digestion, 472 
 
 gluco-, 134 
 
 hetero 130, 134 
 
 in blood, 262, 263, 534, 535 
 
 in intravascular coagulation, 
 
 in lungs, 870 
 
 in stomach, 483. 484 
 in urine, 791 
 primary, 130 
 properties of, 131 
 
 Proteoses, proto-, 130, 134 
 
 quantitative estimation, 138 
 
 secondary, 130 
 
 separation from peptones, 136, 
 
 138 
 syn-, 134 
 Prothrombin, 255, 256, 257, 312, 314, 315, 
 317 
 in circulating plasma, 318 
 Protocaseoses, 129 
 Protoelastose, 117 
 Protogelatose, 119 
 Protogen, 126 
 Protokyrins, 138 
 Protones, 110 
 Protoplasm, 5 
 
 Protoproteose, 131, 133, 135 
 Protosyntonose hexone bases in, 165 
 Proto-toxoids, 71 
 Pseudocerebrin, 611 
 Pseudoglobulin, 259 
 Pseudohsemoglobin, 283 
 Pseudomucin, 171 
 
 beta, 625, 626 
 detection of, 626 
 hydrolytic cleavage prod- 
 ucts of, 626 
 Pseudonuclein, 629 ; 651 
 Pseudonucleins, 104, 177 
 Pseudopepsin, 464, 489 
 Pseudoxanthine, 578 
 Psittacofulvin, 843 
 Psylla-alcohol, 845 
 Ptomaines, 46, 82 
 
 in urine, 757, 758 
 Ptyalin, 48, 456-460 
 
 action of, 456, 457 
 best reaction for, 457 
 condition of, in the intestine, 510 
 effects of foreign substances upon, 
 
 457, 458 
 preparation of, 456 
 Purine bases, 186-193, 267, 715 
 detection of, 193 
 in active muscles, 594 
 in adrenal bodies, 377 
 in spleen, 370 
 preparation of, 193 
 quantitative estimation of, 
 
 715 
 transformation into uric 
 acid, 702, 703 
 bodies, in pus-corpuscles, 364 
 
 composition of, 187 
 oxidases, 875. See Enzymes, 
 skeleton, 186 
 
 structural formula of, 186 
 tri-oxy-, 2, 6, 8, 187 
 Purines, in lymphatic glands, 366 
 in thymus, 36S 
 in thyroid gland, 373 
 Pus, 363 
 
 cells, analysis of, 365 
 corpuscles, 364-366 
 
GENERAL INDEX 
 
 1019 
 
 Pus, constituents of, 364 
 in urine, 7W 
 serum, 363 
 
 analyses of, 363, 364 
 analyses of ash of, 364 
 Putrefaction, 43, Hi 
 
 factors influencing, 516-520 
 in intestines, importance of, 
 
 517 
 
 preventive substances for, 
 
 518 
 
 Putrefactive products, absorption of, 515 
 
 conjugation of, 515 
 
 elimination of, in 
 
 urine, 515 
 in large intestine, 
 515 
 Putrescine, 47, 82, 162 
 
 in urine, 827 
 Putrine, 47 
 Pyin, 363, 366 
 Pyloric secretion, 478 
 Pyocyaneus protease, 57 
 Pyocyanin, 366 
 Pyogenin, 364 
 Pyosin, 364 
 Pyoxanthose, 366 
 
 Pyrazine, formation from glycocoll, 220 
 Pyridine, fate of in organism, 787 
 
 structural formula of, 186 
 Pyrimidine, 193 
 
 bases, 177, 178, 193-195 
 Pyrin, 357 
 
 Pyrocatechin in urine, 727 
 Pyrrol derivatives, 154, 158, 276, 290, 
 
 740 
 Pyrrolidone-carboxylic acid, 113 
 
 Quercinite, 581 
 
 Quercite, 394 
 
 Quinidine, as catalyst, 60 
 
 Quinine, as catalyst, 60 
 
 effect upon spleen, 375 
 
 Cal 
 ■Quotient, T ^ r . , 888 
 
 Cal ftfi 
 
 LA' 888 
 
 — 892 
 
 C to N, 771 
 
 flesh, 602 
 
 nitrogen to homogentistic acid, 
 
 735 
 respiratorv quotient, 412, 563, 
 
 593, 879, 887, 918 
 urea to nitrogen, 777, 877 
 calorific urine, 892 
 
 ^,887 
 
 C 
 
 N 
 
 in feces, £84 
 
 Quotient, ^, in urine, 771 
 
 N 
 
 =-, in urine, 883 
 
 N 
 
 ■5-, in urine, 882 
 
 ft 
 
 calculation of, 889 
 in starvation, 895 
 respiratory, 887, 927, 928 
 
 Rachitis, 556, 557 
 
 Reaction, of a solution, determination of 
 74 
 velocity, 32 
 Reactivation enzymes, 52 
 Receptors, 67 
 
 Reducing substances in urine, 749-752 
 Reductase, 47, 48 
 Reductases, 876, 877 
 Reductions in animal body, 876 
 Reductonovaine in urine, 758 
 Regnault-Reiset's method for determining 
 
 respiratory exchange, 869 
 Reichert-Meissl's equivalent for fats, 238 
 Rennet, 649 
 Rennin, 48, 49, 57, 474-476 
 
 action of on charcoal, 62 
 
 anti, 64, 69 
 
 occurrence of, 474 
 
 pancreatic, 509 
 
 preparation of. 475 
 
 properties of, 475 
 
 relation to pepsin, 475 
 
 retardation of, 63 
 
 retarding substances for, 474 
 
 testing for in gastric juice, 487 
 
 See also Enzymes, 
 zymogen, 474 
 Resacetophenone, 785 
 Resins, fate of in organism, 787 
 Respiration, 850-877 
 
 apparatus, experiments with, 
 
 889, 890 
 external, 858 
 
 gas tension in blood, 859-868 
 importance of haemoglobin 
 in oxygen-carbon dioxide 
 exchange in blood, 853 
 internal, 858, 867 
 mechanism of carbon dioxide 
 
 elimination, 852, 853 
 processes taking part in the 
 gas exchange, 858 
 Respiratory exchange, methods for deter- 
 mining. 86S-S70 
 quotient in active muscle, 596 
 in diabetes 412-413 
 Rest carbon, 268 
 Rest nitrogen, in serum, 267 
 
 in stomach, 483, 484 
 and work, effect on metabolism, 
 926-928 
 
1020 
 
 GENERAL INDEX 
 
 Reticulin, 92, 121, 544 
 
 composition of, 121 
 in lymphatic glands, 336 
 properties of, 121 
 preparation of J 122 
 Retina, constituents of, 615 
 
 pigments of, 615-617 
 Reynold's test for acetone, 823 
 Rhamnose, 208 
 
 relation to glycogen formation, 
 394 
 Rhodophan, 617 
 Rhodopsin, 615 
 
 Ribose, structural formula for, 199, 211 
 tf-Ribose, 178 
 Ricin, 470, 505 
 
 in testing pepsin, 470 
 Rigor mortis, 601 
 
 of muscles, 588-591 
 Ringer-Locke's solution, 75 
 Ritthausen's method of determining pro- 
 teins in milk, 656 
 Roch's test for proteid, 995 
 Rosenbach's bile pigment test, 799 
 
 urine test, 827 
 Rotation, specific, 199 
 Rovida's hyaline substance, 274, 302, 367 
 Rubner's reaction for dextrose, 215 
 sugar test in urine, 809 
 
 Saccharase, 48, 49, 57. See also Enzymes, 
 action of charcoal on, 63 
 retardation of, 63 
 Saccharides, mono, 197-218 
 di, 197, 223-226 
 poly, crystalline, 197 
 poly, colloid, 197, 226 
 tri, 197-226 
 Saccharose, 223, 224, 225. See Sucrose. 
 Sachsse's reaction for dextrose, 295 
 Sahidin, 609 
 Sahli's hsemometer, 299 
 Saliva, the, 451-460 
 
 action of, in stomach, 480 
 
 on starch, 226 
 chorda, 452 
 constituents of, 453 
 diastatic power of, 71 
 gases of, 857 
 
 human, quantitative composition 
 of, 458 
 quantity secreted, 459 
 mixed buccal, constituents of, 455 
 paralytic, 452 
 parotid, 453, 454 
 physiological importance of, 459, 
 
 460 
 properties of, 453, 454, 455 
 sublingual, 453 
 submaxillary, 452 
 sympathetic, 452 
 Salivary concrements, 460 
 Salkowski's creatinine reaction, 696 
 
 reaction for chloesterin, 444 
 
 Salmine, 110 
 
 Salt action on enzymes, 70-76 
 glycosuria, 402 
 plasma, 251 
 Samandarin, 846 
 Sandmeyer's method of inducing pancreas 
 
 diabetes, 405 
 Santonin, elimination of in urine, 787, 800 
 Sapokrinin, 499 
 Saponin, 273, 446, 447 
 Sarcolemma, relation to permeability, 9 
 Sarcomelanin, 841 
 Sarcosine, fate in organism, 786 
 Sarkine. See Hypoxanthine, 190 
 Sarkosine, 776 
 Schalfejeff's, haemin, 292 
 Scherer's inosite test, 578 
 Schiff s reaction, 686 
 
 reaction for cholesterin, 445 
 Schreiner's bases, 621 
 Schutz-Borrissow's rule of ferment action,. 
 
 54, 471, 476, 503, 506 
 Schutz's rule, 58 
 Schweitzer's reagent, 231 
 Scleroproteins, 93 ^ 
 Sclerotic, 619 
 Scombrine, 110 
 Scombron, 108 
 Scyllite, 370, 581 
 Scymnol, 418 
 Sebelien's method of determining proteins 
 
 in milk, 656 
 Sebum of skin, 844 
 Secretin, 463, 492, 498, 499 
 Secretion enzymes, 52 
 
 of prostate, 621 
 Sedimentum lateritium, 708 
 Segregation, 52 
 Seliwanoff's reaction for levulose, 21S r 
 
 815 
 Semen, 620, 621 
 
 Semicarbazide, poisoning therewith, 718 
 Semiglutin, 119, 120 
 Seminose, 216, 217 
 Semi-permeable, membrane, 1 
 Senna, elimination of coloring matter of, 
 
 in urine, 787, 802, 827 
 Sensibilizators, 69 
 Sepia, 381, 841, 843 
 Sepsine. 47 
 
 Seralbumin, 84, 91, 252, 261-264 
 crystalline, 262 
 composition of, 262 
 elementary composition of,. 
 
 263 
 preparation of. 262, 263 
 properties of, 262, 263. See- 
 also Proteins, 
 quantitative estimation of, 
 263 
 Serglobulin, 84, 91, 252, 258-261 
 detection of, 261 
 elementary composition of, 
 263 
 
GENERAL INDEX 
 
 1021 
 
 Serglobulin, para-, 258 
 
 preparation, 259, 201 
 
 properties, 260 
 quantitative estimation, 261. 
 See also Proteins. 
 Sericin, 92, 122, 124 
 Serine, 106, 107, 111, 113, 115, 125, 145 
 
 is<>-, 146 
 Serolin, 265 
 Seromucoid, 261, 264 
 Serosamucin, 354 
 
 Serum, casein, 258. See Serum glo- 
 bulin. 
 normal. See Blood serum. 
 Sham feeding, 450, 464, 821 
 Shark, bile, 41(1, 433, 681 
 urea, 334, 433, 681 
 Shell, membrane of hen's egg, 636-641 
 Side-chain theory, 71 
 Siegfried and Zimmermann's method for 
 
 phenol-sulphuric acids, 726 
 Siegfried's earbamino reaction, 166 
 Silk gelatin, 123, 124 
 Sinistrin. animal, 174 
 Skatol, 46, 82, 117, 157-159, 515 
 522, 723, 732, 843 
 fate of in organism, 786 
 Skatosine, 159 
 Skatoxyl, 724 
 
 tests for, 732 
 Skeletins, 122 
 
 composition of, 122 
 Skin, 837-849 
 
 exchange of gas through, 849 
 horn formations of, 840 
 human, pigments of, 840-843 
 melanins of, 841 
 perspiration of, 847-849 
 pigments of 840-844 
 sebum of, 844 
 secretion, preputial, 846 
 secretions of, 837-849 
 
 of various animals, 846 
 Smegma praputii, 844 
 Snake poison, effect upon blood, 255, 310, 
 
 325 
 Soaps, 265 
 
 in chyle, 347 
 Sodium alcoholate as a saponification 
 agent, 234, 237 
 compounds, division among form 
 
 elements and fluids, 22, 23 
 in urine, 766. See Mineral sub- 
 stances. 
 Solanin, 273 
 Solution tenacity, 10 
 
 in relation to Osmosis Relation- 
 ship to surface tension, 1 1 
 Sorbin, 218 
 Sorbinose, 218 
 Sorbite, 198 
 Sorensen's formol titration for amino- 
 
 acids, 166 
 Specific dynamic action, 918 
 
 Speck's method for determining respira- 
 tory exchange, 860 
 Spermaceti, 238 
 
 oil, 238, 239 
 Spermatin, 623 
 Spermatocele fluids, 359, 360 
 Spermatozoa, 020, 622, 623 
 
 properties of, 622 
 
 constituents of, 622 
 
 Florence's test for, 621 
 osmotic phenomena with, 8 
 Spermine, 621, 022 
 
 crystals, Bottcher's, 621 
 Sphygmogenin, 378 
 Sphyngomyelin, 608, 609 
 Sphyngosin, 011 
 Spiegler's test for proteids, 789 
 Spinal marrow, 014 
 .Spirograph in, 171 
 Spleen, 369-373 
 
 analyses of, 372 
 constituents of, 370 
 pathological processes in, 372 
 physiological functions of, 372 
 uric acid formation in, 702, 703 
 Spongin, 92, 122, 123 
 
 iodo-, 123 
 Sponginoses, 122 
 Spongosterin, 449 
 Sputum, 871 
 
 form constituents of, 871 
 Stachyose, 226 
 Starch, 220-228 
 
 artificial, 227 
 cellulose, 227 
 granulose, hydrolysis, 228 
 gum, 229 
 soluble, 227 
 Starvation, bone, catabolism in, 896 
 catabolism of fat in, 894 
 gas exchange in, 895 
 loss in weight of different 
 
 organs in, S90 
 metabolism, 892-906 
 metabolism of fats in, 894 
 mineral substances in, 895, 
 
 896 
 nitrogen content of urine in, 
 
 897 
 nitrogen elimination in, 893, 
 
 894 
 protein in, 894 
 
 metabolism in, 893, 
 894 
 time interval to death, 892 
 urea content in, 897 
 urinary constituents in starva- 
 tion, 899 
 water in, 896 
 Steapsin, 476, 501-503 
 
 effect upon, of bile, 51 1 
 Steapsinogen, 478 
 
 activation of, by bile, 511 
 Stearine, 234, 235 
 
1022 
 
 GENERAL INDEX 
 
 -Steensma's modification of Gunzburg's 
 
 test, 487 
 Stenson's test, 590 
 Stentorin blue, 844 
 Stercobilin, 428, 429, 529, 743 
 
 in feces, 532 
 Stercorin, 448 
 Stethal, 239 
 
 Stoke's reduction fluid, 282 
 Stomach, absorption of cleavage products 
 in, 484 
 action of salivary diastase in, 480 
 contents. See Chymus. 
 digestion and absorption of 
 
 various food in, 513, 514 
 fistulas, 365, 460 
 gases in, 485, 486 ' 
 glands, 458 
 
 lactic acid fermentation in, 485 
 movement of food in, during 
 
 digestion, 479, 480 
 movement within, during diges- 
 tion, 479 
 self-digestion of, 486 
 Stone-cystine, 148, 150 
 Stroma-fibrin, 275 
 
 of blood corpuscles, 712, 
 273 
 Stromata, 273, 274 
 Strychnine, and glycogen transformation, 
 
 391, 394 
 Sturgeon, spermataozoa of, 110, 181 
 Sturine, 110 
 
 hexone bases in, 165 
 Stutz's test for proteid, 794 
 Subcutaneous oedema, fluid of, 362 
 .Sublingual glands, 448 
 saliva, 450 
 Submaxillary glands, 446 
 Submicrons, 19 
 Substrate, 47 
 "Sucre immediat," 331 
 " Sucre virtuel," 331 
 
 Sugar, amounts in blood of different 
 animals, 328 
 amounts in blood, 331 
 detection of in urine, 802-808 
 fats, in liver, 412, 413 
 gelatin, 139 
 glycogen, 393, 395 
 in aqueous humor, 361 
 in ascitic fluids, 359 
 in blood, 329, 330 
 in blood serum, 265 
 in cerebrospinal fluid, 360 
 in lymph, 346 
 
 in transudates and exudates, 355 
 in urine, 802-818 
 in venous blood, 336 
 proteins, in liver, 409-411 
 Sulphaemoglobin, 286 
 Sulphate quantitative estimation of, in 
 
 urine, 765 
 Sulphates, amounts in urine, 765 
 
 Sulphates in urine, 764-766. See also 
 Mineral substance, 
 ethereal in urine, 765 
 quantitative estimation of, 765 
 Sulphatide, 609 
 Sulphocyanides, occurrence in urine, 752, 
 
 775 
 Sulphonal intoxication, urine, 294, 797 
 Sulphur, compounds in urine, 752, 753 
 
 elimination, • importance of in 
 
 protein metabolism, 882, 883 
 in proteins, 78, 79. See also 
 
 various proteins, 
 in urine, 752, 874, 875, 890 
 methsemoglobin, 286 
 Sulphuretted hydrogen in urine, 753 
 Suprarenin, 387 
 
 Surface tension, in relation to osmosis. 10 
 Suspension colloids, precipitation of, 70 
 Suspensoids, properties of, 15 
 Sympathetic saliva, 449 
 Synovia, 360 
 
 Synovial cavities around joints, fluid in, 
 360 
 analysis of, 362 
 fluid, 362 
 Synoviamucin, 362 
 Synovin, 362 
 Synproteose, 135 
 Syntonin, hexone bases in ,165 
 Syntoxoids, 71 
 
 Taenia, 389 
 Talose, 211 
 Tartar, 460 
 Tatalbumin, 632 
 Taurine, 150, 151 
 
 fate of in organism, 786 
 Tears, 619 
 
 Teichmann's crystals, 292, 796 
 Tendon mucoid, 544 
 
 elementary composition of, 551 
 Terpenes, fate of in organism, 785 
 Testes, 620 
 Tetanolysin, 72 
 
 Tetanotoxine and gastric juice, 485 
 Tetraglyclyglycine, 85, 510 
 Tetramethylene diamine, 47. See Putras- 
 
 cine. 
 Tetrapeptides, 85, 87, 89, 510 
 Tetronerythrin, 843, 844 
 Tetroses, 197 
 Theobromine, 187 
 Theophylline, 187 
 
 Thiophene, behavior in animal body, 784 
 Thiotolene, 784 
 Thrombin, 48, 256, 315, 317, 324 
 
 action of, 256 
 
 formation of, 318 
 
 preparation of, 257 
 
 properties of, 256 
 
 pro-, 256 
 Thrombogen, 318, 321 
 
 formation of, 322 
 
GENERAL INDEX 
 
 1023 
 
 Thrombokinase. 318, 310 
 Thromb< (plastic substances, 326 
 Thrombosin, 320 
 
 -lime, 320 
 Thrombozvm, 321 
 
 formation of, 322 
 Thymine, 178, 181, 185, 195 
 Thymus, 366-309 
 
 analysis of, 369 
 constituents of, 368 
 functions of, 369 
 Thyreoglobulin, 375, 376 
 Thyre< >pint ciil, 375 
 Thyroid, gland. 373-376 
 
 constituents of, 373 
 effect of extirpation of, 374 
 Tissue, connective, analysis of, 545 
 elastic analysis of, 545 
 fatty, 558-564 
 fibrinogen, 307 
 proteins. 906-910 
 Tissues, end products of oxidation in, 871 
 gas exchange in, 858-870 
 gelatinous. 545 
 mucous. 545 
 oxidation in. 858 
 physiological oxidation in, 871 
 theories for oxidation processes 
 in. 871, 872 
 Tollen's reaction for pentoses, 209 
 
 Roriye test for levulose, 218 
 test for glucuronic acid, 223 
 Toluene, fate of in organism, 779 
 Toluylenediamine, poisoning with, 441 
 Tonus, chemical, 590 
 Tooth, cement, 557 
 dentin, 557 
 enamel, 557 
 structure, 557 
 Toxin, cysts. 69 
 Toxins, 46, 66 
 
 a method of measuring, 67 
 Toxoid, 67 
 
 proto, syn, epi, 67 
 Toxons, 67 
 Toxophore. group. 07 
 Tradescantiadiscolor, 6 
 Transfusion, fluid for mamallian heart, 72 
 
 composition of, 72, 73 
 Transudates and exudates, constituents 
 found in. 353-355 
 distinguishing feature in, 356 
 formation of. 352, 353 
 osmotic pressure in, 356 
 pathological, gases of, 857 
 reaction of, 356 
 Trichohyalin. 837 
 Triglycerides. 232 
 Triglyclyglycin. 85. 510 
 
 ethyl ester, 86 
 Trimethylamine in urine. 7">7 
 Trimethylvinylammonium hydroxide, 246. 
 
 See Neurine. 
 Triolein, 233, 236 
 
 Trioses, 197 
 
 Trioxypurine, 2, 6, 8, 187. See Uric acid. 
 Tripahnitin, 2:53, 235 
 Tripeptides, 85, 50!), 510 
 Triple phosphates, in urinary concremente 
 S3 1-833 
 in urinary sediments, 
 831 
 Trisaccharides, 200 
 Tristearin, 233, 23 1 
 Trommer's test, 214, 793, 803 
 Trypsin 49, 57, 503-509 
 
 action of, 504, 505 
 action of charcoal on, 62 
 digestion action of foreign bodies 
 
 on, 506 
 formation of, 497 
 preparation of, 504 
 properties of, 503 
 quantitative estimation of, 505 
 rapidity of, under various con- 
 ditions, 506 
 retardation of, 63 
 tests for. 505. See also Enzymes, 
 upon other bodies, 508 
 Trvpsinogen, 504 
 
 activation of, 496, 497, 498 
 activators of. 497 
 Tryptic digestion, 505 
 
 products, of 507 
 Tryptophane, 85, 111, 119, 155-157 
 
 Adamkiewicz - Hopkin's 
 
 reaction, 157 
 amounts in proteins, 106, 
 
 107 
 effect of yeast upon, 206 
 quantitative, colorimetric 
 method for, 157 
 Trvptophol, 157. 206 
 Tubo-ovaria, 628 
 Tunicin. 838, 839 
 Turacin, 843 
 Turacoverdin, 844 
 Turpentine, fate of in organism, 787 
 Tyrosinases, 875 
 Tvndall's phenomenon, 40 
 Tyrosine, 85, 109, 111, 119, 123, 124, 
 152-154. 267 
 amounts in proteins, 106, 107, 
 
 113, 115, 125 
 effect of yeast upon, 206 
 importance of in homogentisic 
 
 acid formations, 737 
 tests for, 153 
 Tyrosol, 153, 206 
 
 Uffelmann's test for free lactic acid in 
 
 gastric juice, 487 
 Umikoff's reaction, 663 
 Uracil. ITS. IS.",. 101. 195 
 
 Weidel's reaction for, 104 
 Wheeler and Johnson reaction for, 
 194 
 Uranium salts, and glycosuria, 402 
 
1024 
 
 GENERAL INDEX 
 
 Urate, ammonium, in urinary sediment, 830 
 Urates, acid, in urinary sediment, 830 
 
 of urine, 674 
 Ultra violet rays, 50 
 Umbilical cord, 54.5 
 Urea, 267, 679, 691 
 
 compounds of, 687 
 creatinine, 682 
 
 .formation, anhydride theory, 684 
 from ammonium salts, 6S3 
 from arginine, 682 
 in ascitic Quids, 359 
 in blood. 333, 334 
 in cerebrospinal fluid, 360 
 in lymph, 346 
 in muscles, 572 
 in the organism, 682 
 in transudates and exudates, 355 
 in veins, 336 
 mesoxalyl, 699 
 
 mother substances of, 682, 683 
 nitrate, 686 
 occurrence of, 679 
 other organs besides liver, 685 
 oxalate, 687 
 oxidation theory, 684 
 physiological significance of, 680 
 preparation of, 679, 687, 688 
 properties of, 686 
 
 quantitative methods for 689-691 
 quantity voided, 68 
 tests for, 686 
 Urease, 48, 829 
 Urein, 691 
 
 Ureotheobromine, 713 
 Urethane, 692 
 Uric acid stones, 832 
 Uricolvsis, 704 
 Uridine, 180, 185 
 Urinary calculi, 828, 829, 832-836 
 
 calcium carbonate, 834 
 calcium oxalate, 833 
 chemical investigation of, 
 
 834-836 
 cystine, 834 
 fibrin, 834 
 phosphate, 833 
 scheme for chemical, 
 
 analysis of, 836 
 uric acid, 833 
 urostealith, 834 
 xanthine, 834 
 pigments, 740-748 
 sediments, 674, 828, 829 
 
 acid hippuric in, 831 
 ammonium magnesium 
 
 phosphate in, 831 
 ammonium urate in. 
 
 830 
 calcium carbonate in, 
 
 831 
 calcium oxalate in, 830 
 calcium phosphate in, 
 831 
 
 Urinary sediments, calcium sulphate in, 
 831 
 cystin in, 831 
 haematoidine in, 831 
 magnesium triphos- 
 phate in, 831 
 non-organized, 830, 
 
 831 
 triple phosphates in, 
 
 831 
 tyrosine in, 831 
 urates, acid in, 830 
 uric acid in, 830 
 xanthine in, 
 Urine, 670-836 
 
 abnormal color of, due to foreign 
 
 substances, 787 
 acetoacetic acid in, 824, 825 
 acetone bodies in, 818-828 
 acetone in, 822-824 
 acid fermentation of, 829 
 alcaptonuric, test for, 739 
 alkaline due to ammonium car- 
 bonate, 676 
 fermentation of, 829 
 Almen's bismuth test for sugar, 
 
 803 
 amino-acids in, S27 
 ammonium urate calculi, 833 
 average composition of, 772 
 Bang's quantitative methods for 
 
 sugar, 809-811 
 Bertrand's quantitative method 
 
 for sugar, 811 
 Bial's test for pentoses, 816 
 bile-acids in, 799, 800 
 bile-pigments in, 800-801 
 casual constituents of, 772-787 
 cholesterin in, 827 
 cloudy, reasons for, 674 
 color of, 674 
 conjugated glucuronic acids in, 
 
 817,818 
 cystine in, 827, 828 
 degree of acidity, 675 
 detection of acetone and aceto- 
 acetic acid in, 825 
 Bence-Jones protein in, 
 
 792 
 betaoxybutyric acid in, 
 
 826 " 
 bile-acids in. 800 
 blood in, 796 
 conjugated glucuronic 
 
 acids in, 817, 818 
 fructose in, 814 
 hsematoporphvrin in, 
 
 798 
 lactose in, 815 
 pentose in, 816 
 pigments in, 800, 801 
 proteins in, 787-781 
 proteoses in 791, 792 
 sugar in, 802-808 
 
GENERAL INDEX 
 
 1025 
 
 Urine, determination of acidity, 070,677, 
 ton acidity, 677, 
 
 078 
 specific gravity 
 678, 079 
 division of the nitrogen of, 681 
 Khrlich's urine test, 826 
 end products of acids amino 
 aromatic, 780-783 
 amino-aeids, 774- 
 
 776 
 benzene ring and 
 homologues, 778, 
 779 
 fatty series, 773,774 
 heterocyclic com- 
 pounds, 778-787 
 homocyclic com- 
 pounds, 778-787 
 fat in, 827 
 
 fermentation test for sugar, 805 
 fructose in, 814, 815 
 gases of, 857 
 glucose in, 802-814 
 guaiac test for blood in, 796 
 luvmatoporphyrin in, 797, 798 
 Heller-Teichmann's test for blood 
 
 in, 797 
 heptose in, 817 
 indican, 728 
 
 inorganic constituents of, 758-770 
 inosite in, 818 
 isolation of sugar from, 807 
 Kjeldahl's method for total nitro- 
 gen, 688 
 Knapp's quantitative method for 
 
 sugar, 812 
 lactose in, 815 
 laiose in, 815 
 levulose in, 814, 815 
 maltose in, 815 
 
 medicinal coloring matter in, 801 
 melanin in. 799 
 microscopic investigation of blood 
 
 corpuscles, 796 
 nitrogen content during starvation, 
 
 897 
 Nylander's test for sugar, 803 
 odor of, 674 
 organic phvsiological constituents 
 
 of, 679-758 
 osmotic pressure of, 678 
 /3-oxybutyric acid in, 825-827 
 passage of sugar into, 534 
 pathological constituents of, 787- 
 
 828 
 pentoses in, 816 
 percentage division of nitrogenous 
 
 substances, 631 
 phenylhydrazine test for sugar, 806 
 physical properties of, 674r-679 
 physico-chemical methods in, 772 
 phvsiological constituents of, 679- 
 787 
 
 Urine, pigments in, 798, 799 
 
 polarization of, for sugar, 807 
 pus in, 799 
 
 quantitative composition of, 770- 
 277 
 determination of su- 
 gar, 808-814 
 determination of pro- 
 
 teid, 793 
 determination of total 
 
 nitrogen, 0.XK 
 estimation of acetone 
 
 in, 825 
 estimation of /3-oxy- 
 
 butyric acid, 826 
 estimation of sugar 
 by fermentation, 
 812 
 estimation of sugar 
 by polarization, 
 813-814 
 method for nitrogen 
 in very small quan- 
 tities of urine, 689 
 quantity of, 770 
 
 conditions affecting, 
 
 770 
 of solids excreted, 770, 
 771 
 reaction of, 674, 675 
 Rubner's test for sugar, 807 
 sedimentatum lateritium, 674 
 specific gravity of, 678 
 spectroscopic investigation for 
 
 blood, 796 
 sugar in, 802-818 
 taste of, 674 
 transparency of, 674 
 Trommer's test for sugar, 802 
 urea content in starvation 
 Urinometer, 678 
 Urobilin, 429, 516, 740, 743-746 
 detection of, 747 
 formation of in organism, 744 
 in blood serum, 268 
 preparation of, 746 
 properties of, 745 
 quantitative estimation of, 747 
 tests for, 745, 746 
 Urobilinogen, 740 
 
 detection of in urine, 746 
 formation of in organism, 
 
 744, 746. 747 
 in blocd serum, 268 
 preparation of, 746, 747 
 properties of, 746 
 quantitative estimation of, 
 747 
 Urobilinoids, 743 
 Urochrome, preparation of, 742 
 
 quantitative estimation of,742 
 Urochrome, 740, 741, 742 
 in urine, 755 
 Urocyanin, 741 
 
1026 
 
 GENERAL INDEX 
 
 Uroerythrin, 740, 748 
 
 tests for, 748 
 Urofuscohaematin, 798 
 Uroglaucin, 741 
 Urohaeniatin, 741 
 Urohodin, 741 
 Urohypertensin, 758 
 Urohypotensin, 758 
 Uromelanins, 741 
 Urophain, 740 
 Uropyrryl, 741 
 Urorosein, 741 
 
 in urine, 733 
 Urorubin, 741 
 Urospectrin, Saillet's, 797 
 Urostealith calculi, 834 
 Urotheobromine, 713 
 Urotoxic coefficient, 758 
 Urorubrohsematin, 798 
 Uroxanthine, 728 
 Uterine milk, 642 
 Uterus, colloid, 627 
 
 Valine, 111, 140, 141 
 
 amounts in proteins, 106, 107, 113, 
 115, 125 
 
 Vanillin, behavior in animal body, 779 
 
 Van Slyke's method for amino acids, 79 
 
 Van't Hoff's rule, 201 
 
 Velocity coefficient, 34 
 
 Vernine, 178 
 
 Vernix caseosa, 845 
 
 Vesiculase, 621, 622 
 
 Viridinine, 47 
 
 Visual purple, 615-617 
 
 preparation of, 617 
 properties of, 616 
 regeneration of, 616 
 
 Visual red, 615 
 
 Vitali's pus-blood test, 796 
 
 Vitamine, 905 
 
 Vitellin, 628 
 
 cleavage products of, 106 
 
 Vitellolutein, 632 
 
 Vitellorubin, 632 
 
 Vitelloses, 130 
 
 Vitiatine, 578 
 
 in urine, 758 
 
 Vitreous humor, 617 
 
 Voit'a normal average diet, 933 
 
 Volhard and Lohlein's quantitative 
 method for pepsin, 470 
 
 Volhard's quantitative method for 
 chlorides, 760 
 
 Walden's reversion, 89 
 Water, importance to life, 71, 72 
 
 lack of in food, 899 
 Wear and tear quota, 890, 917 
 Weidel's reaction for histidine, 160 
 xanthine, 190 
 
 Weiss' sparing theory, 396 
 
 Weyl's creatinine reaction,695 
 
 Wheeler and Johnson reaction for Uracil, 
 
 194 
 Whey, 645 
 
 acid, 645 
 
 protein, 650 
 
 sweet, 645 
 Witch's milk, 665 
 Wool-fat constituents of, 846 
 Work affecting metabolism, 926-928 
 Worm, Muller's sugar test, 803 
 
 Xanthine, 86, 178, 187, 188, 189, 190, 712 
 in ascitic fluids, 359 
 in urinary calculi, 833, 834 
 in urinary sediments, 831 
 in urine, 710 
 para, 187 
 
 Seyler's reaction for, 190 
 Weidel's reaction for, 190 
 
 Xantho creatinine, 578 
 
 in urine, 698 
 
 Xantho melanin, 84 
 
 Aanthoproteic reaction for proteins, 99, 
 173 
 
 Xanthoprotein, 84 
 
 Xanthophan, 617 
 
 Xanthosine, 180 
 
 Xylene, fate of in organism, 779 
 
 Xyliton, 842 
 
 Xyloses, structural formula for, 198, 210 
 
 Yeast, 59, 62 
 
 action of, on glucose, 226 
 maltase, 266 
 Yoghurt, 654, 659 
 Yolk, constituents of, 628 
 
 fat of, 630 
 
 membrane, 628 
 
 mineral bodies of, 632 
 
 of hen's egg, 628 
 
 pigments of, 631 
 
 phosphatides of, 630 
 
 proteins of, 628-630 
 
 spherules, 628, 636 
 
 Zein, 77, 105, 163 
 Zinc, in the bile, 433 
 
 in the liver, 389 
 
 passage into milk, 670 
 Zooerythrin, 843 
 Zoofulvin, 843 
 Zoorubin, 843 
 
 Zuntz and Geppert's method for deter- 
 mining respiratory exchange, 869 
 Zymase, 41 
 Zymogen, rennin, 474 
 Zymogens, 51, 461 
 Zymoplastic substances in blood, 315 
 
COLUMBIA UNIVERSITY LIBRARY 
 
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 as provided by the rules of the Library or by special ar- 
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