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 'Ill 
 
 Essentials 
 
 OF 
 
 Chemical Physioiogy 
 
 HALLIBURTON 
 
 
New York 
 
 State College of Agriculture 
 
 At Cornell University 
 
 Ithaca, N. Y. 
 
 Library 
 
Cornell University Library 
 QP 514.H1881919 
 
 The essentials of chemical Physiology fo 
 
 ■'3"l"924 003 172 842 
 
Cornell University 
 Library 
 
 The original of tliis book is in 
 tlie Cornell University Library. 
 
 There are no known copyright restrictions in 
 the United States on the use of the text. 
 
 http://www.archive.org/details/cu31924003172842 
 
CHEMICAL PHYSIOLOGY 
 
BJBLIOGRAPHICAL NOTE 
 
 First Edition, September 1893 ; Second Edition, 
 Ap-il lSg6; Third Edition, March 1899; Fourth 
 Edition, November 1901 ; Fifth Edition, October 
 1904 ; Sixth Edition, April 1907 ; Seventh 
 Edition, October 1909 ; Eighth Edition, February 
 1914 ; Ninth Edition, September 1916 ; Tenth 
 Edition, September 191 9. 
 
THE ESSENTIALS 
 
 OF 
 
 CHEMICAL PHYSIOLOGY 
 
 FOR THE USE OF STUDENTS 
 
 BY 
 W. D. HALLIBURTON, M.D., LL.D., F.^.S. 
 
 FELLOW OF THE ROYAL COLLEGE OF PHYSICIANS 
 
 PROFESSOR OF PHYSIOLOGY IN KING's COLLEGE, LONDON 
 
 AUTHOR OF "TEXT-BOOK OF CHEMIC^AL PHYSIOLOGY AND PATHOLOGY" 
 
 TENTH EDITION 
 
 With Coloueed Plate 
 
 LONGMANS, GREEN, AND CO. 
 39 PATERNOSTER ROW, LONDON 
 
 FOURTH AVENUE & SOtii STEEET, NEW YOEK 
 
 BOMBAY, CALCUTTA, AND MADEAS 
 
 1919 
 
 All rights reserved 
 
PREFACE 
 
 TO 
 
 THE TENTH EDITION 
 
 I HAVE again to thank my colleague, Dr 0. Kosenheim, for valu- 
 able assistance in the preparation of a new edition. The great 
 difficulty experienced in revision has been to add new material 
 without increasing the size of the book. But we have accom- 
 plished the task by means of judicious , excision of matter no 
 longer necessary. In sending out this little book in "up-to- 
 date " form, I trust that its usefulness to students may continue. 
 
 W. D. HALLIBURTON. 
 
 King's College, 
 
 London, 1919. 
 
CONTENTS 
 
 PAOB 
 
 Introduction ...... 1 
 
 ELEMENTARY COURSE 
 
 LESSON 
 
 I. The Elements contained in Physiological. Compounds 5 
 
 II. Some Typical Organic Compounds . . . 11 
 
 III. The Carbohydrates . 20 
 
 IV. The Fats and Lipoids ... 33 
 V. The Proteins . 41 
 
 VI. Foods ..... .68 
 
 VII. The Digestive Juices — Saliva and Gastric Digestion . 84 
 
 VIII. The Digestive Juices (continued) — Pancreatic Digestion 
 
 and Bile . ... 107 
 
 IX. The Blood and Respiration . . 133 
 
 X. Urine . . .... 179 
 
 XI. Urine (continued) . . . 196 
 
 XII. Pathological Urine . . . 207 
 Scheme for Detecting Substances of Physiological Importance 216 
 
 ADVANCED COURSE 
 
 Introduction ... . . 220 
 
 LESSON 
 
 XIII. Carbohydrates . ' . . . 221 
 
 XIV. Carbohydrates : Action of Diastase upon Starch . 225 
 XV. Crystallisation of Proteins . . . 227 
 
 XVI. Milk ... . 228 
 
 XVII. The Proteoses 229 
 
 XVIII. . Digestion , . 230 
 
vui ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 MSSON P4»S 
 
 XIX. The Blood . . .237 
 
 XX. HEMOGLOBIN AND ITS DERIVATIVES .... 241 
 
 XXI. Muscle and Nervous Tissues . . . . 245 
 
 XXII. The Urine — Total Nitrogen, Urea, and Ammonia . . 251 
 
 XXIII. The Urine (continued)— Uric Acid and Creatinine . . 256 
 
 XXIV. The Urine {continued) — The Inorganic Salts 258 
 
 XXV. The Urine {continued) — The Pigments . . 264 
 
 APPENDIX 
 
 Hemacytometers . 268 
 
 HemoglobinomeTbrs , , . . 271 
 
 Polarisation op Light . 274 
 
 Polarimetbrs . . . 277 
 
 Relation between Circular Polarisation and Chemical 
 
 Constitution ..... . . 279 
 
 The Mercurial Air-pump (Barcroft's Modification) . . . 280 
 
 Barcroft's Differential Apparatus for Analysis of Blood Gases . 281 
 
 Haldane's Apparatus for Gas Analysis . . . 284 
 
 Solutions — Diffusion — Dialysis — Osmosis . . 286 
 
 Colloids and Colloidal Solutions . . 294 
 
 Surface Tension . . . . 297 
 
 The Reaction of Fluids . . 298 
 
 Microchemical Methods of Analysis . 299 
 
 INDEX . . . 301 
 
LIST OF ILLUSTRATIONS 
 
 FIG. 
 
 
 PAGS 
 
 1. Lactose Crystals 
 
 . Frey 
 
 28 
 
 2. Section op Pea showing Starch Grains ' 
 
 After Sachs 
 
 29 
 
 3. Cholesterol Oetstals 
 
 Frey 
 
 39 
 
 4. Leucine Crystals 
 
 Kuhne 
 
 45 
 
 5. Tyrosine Crystals 
 
 Frey 
 
 47 
 
 6. DiALYSBR 
 
 
 55 
 
 7. DiAORAM OP A Cell . Sharpey Schafer 
 
 63 
 
 8. Milk . 
 
 Yeo 
 
 73 
 
 9. Colostrum Corpuscles 
 
 Heidenhain 
 
 73 
 
 10. Schizomycetes . 
 
 After Zopf 
 
 87 
 
 11. Alveoli op Serous Gland . 
 
 Langley 
 
 96 
 
 12. Mucous Cells. 
 
 Langley 
 
 96 
 
 13. Submaxillary Gland 
 
 Heidenhain 
 
 96 
 
 14. Fundus Gland 
 
 Klein 
 
 99 
 
 15. Pyloric Gland 
 
 . Ehstein 
 
 99 
 
 16. Fundus Gland 
 
 Langley 
 
 100 
 
 17. Alveolus op Pancreas 
 
 Kilhne and Lea 
 
 110 
 
 18. H^;matoidin Crystals 
 
 Frey 
 
 121 
 
 19. 
 20. 
 
 i\. 
 
 23. 
 
 24. 
 ii. 
 26. 
 
 Villus of Eat Killed during Fat- absorption Sharpey Schafer 130 
 Mucous Membrane op Frog's Intestine during Fat- 
 
 absorption 
 Fibrin Filaments and Blood Platelets . 
 Action op Reagents on Blood Corpuscles 
 Oxyhemoglobin Crystals 
 Hemin Crystals 
 Diagram op Spectroscope 
 Arrangement op Prisms in Direct- vision Spectroscope Gscheidlen 150 
 b '" 
 
 Sharpey Schafer 131 
 Sharpey Schafer 136 
 Shcurpey Schafer 144 
 Quain's Anatomy 146 
 Preyer 147 
 150 
 
ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 FIS. 
 
 27. 
 28. 
 29. 
 30. 
 31. 
 32. 
 33. 
 34. 
 35. 
 36. 
 37. 
 38. 
 39. 
 40. 
 41. 
 42. 
 43. 
 44. 
 45. 
 46. 
 47. 
 48. 
 49. 
 50. 
 
 51. 
 
 52. 
 53. 
 54. 
 55. 
 56. 
 57. 
 
 Stand ]?ou Direct-vision Spectroscope 
 
 Absorption Spectra . 
 
 Absorption Spectra 
 
 Krogh's Tonometer . 
 
 Blood-oas Apparatus 
 
 Babcroft's Tonometer 
 
 Dissociation Curve op Hemoglobin 
 
 Dissociation Curve op Blood 
 
 Apparatus for obtaining Alveolar Air 
 
 DupRtfs Urea Apparatus 
 
 Urea Nitrate and Oxalate 
 
 Triple Phosphate Crystals 
 
 Uric Acid Crystals 
 
 Mono-Sodium Urate . 
 
 Mono- Ammonium Urate 
 
 Envelope Crystals of Calcium Oxalate 
 
 Cystine Crystals 
 
 Triple Phosphate Crystals . 
 
 Calcium Phosphate Crystals 
 
 Albuminometbr of Esbaoh . 
 
 OsAZONE Crystals 
 
 Van Slyke's Apparatus 
 
 . 151 
 
 . Rollett 151 
 
 . 152 
 
 . 1Q3 
 
 164 
 
 . 166 
 
 Ba/rcroft 168 
 
 Barcroft 169 
 
 171 
 
 Gamgee 180 
 
 Frey 185 
 
 Frey 1 95 
 
 . Frey 199 
 
 Frey 204 
 
 . Frey 204 
 
 Frey 204 
 
 . Frey 204 
 
 . 205 
 
 205 
 
 . 207 
 
 . Coloured plate to face 222 
 
 . 233 
 
 . 242 
 
 Absorption Spectra op Hemoglobin, etc. 
 Photographic Spectrum of Hemoglobin and Oxyhemo- 
 globin .... . Gamgee 243 
 Photographic Spectrum of Oxyhemoglobin and Mbthemo- 
 
 GLOBiN . ... Gamgee 243 
 
 Absorption Spectra of Myqiiematin . . 247 
 
 Kjeldahl's Method : Distilling Apparatus 252 
 
 Folin's Apparatus for Estimating Ammonia . . 253 
 
 Absorption Spectra op Urinary Pigments . After Hopkins 266 
 
 Gowers's Hemacytometbr . . 268 
 
 Thoma-Zeiss Hemacytometer . . . 2()9 
 
LIST OF ILLUSTRATIONS 
 
 FIG. 
 
 58. Pipette of Thoma-Zeiss Hemacytometer . 
 
 59. Oliver's Hemacytometer 
 
 60. GowERs's Hemoglobinometer 
 
 61. Von Fleischl's Hemometer. 
 
 62. GuVBR's HiEMOQLOBINOMETBR 
 
 63. Model to Illustrate Polarised Light 
 
 64. Model to Illustrate Polarised Light . 
 
 65. Model to Illustrate Polarised Light . 
 
 66. Diagram to Explain Polarisation of Light 
 
 67. Laurent's Polammeter 
 
 68. Diagram of Asymmetric Carbon Atoms . 
 
 69. Diagram of Barcroft's Pump 
 
 70. Barcroft's Differential Apparatus 
 
 71. Haldane's Air Analysis Apparatus 
 
 72. Diagram to Illustrate Osmosis 
 
 PAG£ 
 
 269 
 270 
 271 
 272 
 27.3 
 275 
 275 
 276 
 277 
 278 
 280 
 280 
 281 
 285 
 289 
 
ESSENTIALS OF 
 CHEMICAL PHYSIOLOGY 
 
 INTRODUCTION 
 
 Chemical Physiology is a branch of physiological science which 
 deals with the chemical composition of the body and the part played 
 by the various' substances found there in carrying out the phenomena 
 of life. It thus differs from Physiological Chemistry, which is a branch 
 of organic chemistry, and treats of the chemical composition and 
 reactions of physiological substances. These two subjects are closely 
 interwoven, and this book really deals with both, although special 
 prominence will be given to their physiological aspect. > 
 
 The substances found in the body are numerous, and in most cases 
 complex ; the majority of the foods from which the body is built up 
 are equally elaborate, for animals do not possess to such an extent as 
 plants do the power of building up complex from simple materials. 
 
 The elements found in the body are carbon, hydrogen, nitrogen, 
 oxygen, sulphur, phosphorus, .fluorine, chlorine, iodine, silicon, sodium, 
 potassium, calcium, magnesium, lithium, iron, and occasionally 
 manganese, copper, and lead. 
 
 Of these very few occur fn the free state. Oxygen and nitrogen (to 
 a small extent) are found dissolved in the blood-plasma; hydrogen 
 is formed by putrefaction in the alimentary canal. With some few 
 exceptions such as these, the elements enumerated above are found 
 combined with one another to form compounds. 
 
 The compound? found in the body are divided into — 
 
 (1) Mineral or inorganic compounds, such as water and salts. 
 
 (2) Organic compounds, or compounds of carbon. 
 
 The time-honoured classification of the organic principles into 
 Proteins 
 Carbohydrates 
 Fats 
 is a little amplified in the following table ; — 
 1 
 
2 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 i. Nitrogenous : 
 
 {a) Proteins, e.g. albumin, myosin; gelatin. 
 
 (6) Nitrogenous lipoids, e.g. the fat-like substance known as 
 lecithin. 
 
 (c) Products of protein cleavage, e.g. amino-acids, urea, ammonia, 
 ii. Non-nitrogenous : 
 
 (a) Fats, e.g. butter, fats of adipose tissue. 
 
 (6) Carbohydrates, e.g. sugar, starch. 
 
 (c) Non-nitrogenous lipoids, e.g. cholesterol. 
 
 (d) Simpler organic substances, mainly products of the break- 
 
 down of th(?se previously enumerated, e.g. glycerol, fatty 
 acids, lactic acid. 
 Living material or protoplasm is the substance of which the body 
 cells are built. It is in a continual state of unstable chemical 
 equilibrium, building itself up on the one hand, breaking down on the 
 other; the term used for the sum-total of these intramolecular 
 rearrangements is metabolism. 
 
 The chemical substances in the protoplasm which are the most 
 important from this point of view are the complex nitrogenous com- 
 pounds called Proteins and the group of substances known as the 
 Lipoids. So far as is at present known, proteins and lipoids are never 
 absent from living substance, and up to the present time they have 
 not been synthesised by laboratory processes. 
 
 The chemical structure of protoplasm can only be investigated after 
 the protoplasm has been killed. The substances it yields are : (1 ) Water ; 
 protoplasm is semi-fluid, and at least three quarters of its weight, often 
 more, are due to water. (2) Proteins. These are the most constant 
 and abundant of the solids. A protein or albuminous substance con- 
 tains the elements carbon, hydrogen, nitrogen, oxygen, with sulphur 
 and phosphorus in small quantities only. In nuclein, a protein-like 
 substance obtained from the nuclei of cells, phosphorus is more 
 abundant. The protein obtained in greatest abundance from the 
 cell-protoplasm is nueleo-protein : that is, a compound of protein with 
 varying amounts of nuclein. White of egg is a familiar instance of an 
 albuminous substance or protein, and the fact (which is also familiar) 
 that this sets into a solid on boiling will serve as a reminder that the 
 greater number of the proteins found in nature have a similar tend- 
 ency to coagulate under the influence of heat and other agencies. 
 (3) Lipoids are substances which resemble fats in their solubilities ; 
 they play an important part in metabolism ; as instances of these we 
 may mention lecithin, a fat-like substance containing phosphorus 
 
INTRODUCTION 3 
 
 and nitrogen, and cholesterol, a monohydric alcohol. (4) Inorganic 
 salts, especially phosphates and chlorides of calcium, sodium, and 
 potassium. 
 
 The fats and carbohydrates are not essential constituents of proto- 
 plasm, but they are found within many cells (cell-contents) and arc 
 mainly utilised for combustion, whereby heat and other forms of 
 energy are libei'ated. ' 
 
ELEMENTARY COURSE 
 
 LESSON I 
 THE ELEMENTS CONTAINED IN PHYSIOLOGICAL COMPOUNDS 
 
 1. Take a fragment of meat about the size of a pea and place it 
 in a porcelain crucible over a Bunsen flame. Note that it chars, showing 
 the presence of carbon, and that it gives off the unpleasant odour of 
 burning flesh, which is due to the fact that it contains the nitrogenous 
 substances called proteins. In course of time the organic material 
 is completely burnt up, and a small amount of white ash or inorganic 
 material is left behind. 
 
 2. Repeat the experiment with a pure organic substance such as 
 sugar. Note that no ash is left. Charring, as before, indicates the 
 presence of carbon, but there is no characteristic smell of burning 
 nitrogenous substances (absence of nitrogen). 
 
 3. The chief test for carbon depends on the fact that when this 
 element is oxidised it gives rise to carbon dioxide : the test for hydrogen 
 depends on the fact that when this element is oxidised it gives rise 
 to water. If all the carbon dioxide and water formed by oxidation 
 from a weighed amount of any organic substance under examination are 
 collected and estimated, the amount of carbon and hydrogen respec- 
 tively which it contains can be easily calculated. The following 
 exercises, however, deal only with the qualitative detection ot these 
 elements. 
 
 4. TESTS FOR CARBON.— The following tests can be carried 
 out with sugar. 
 
 (A) When burnt in the air it chars and subsequently the carbon 
 entirely disappears, passing off in combination with oxygen as' carbon 
 dioxide (carbonic acid gas). 
 
 (b) Mix some of the powdered sugar in a dry mortar with about 
 ten times the quantity of cupric oxide (which has been freed from 
 water by previous heating); place the mixture in a dry test-tube 
 provided with a rubber cork perforated by a bent glass tube which dips 
 into either lime water or baryta water. Heat the test-tube over a 
 
 e 
 
6 ESSiENtiALS OP CHEMICAL PHYSIOLOGY 
 
 Bunsen flame, and as the carbon of the sugar becomes oxidised carbon 
 dioxide comes off and causes a white precipitate of calcium or barium 
 carbonate, as the case may be. 
 
 5. TEST FOK HYDROOEN.— In the experiment just described 
 (4 b) note that drops of water due to oxidation of hydrogen condense 
 in the upper colder part of the test-tube. 
 
 6. TESTS FOR NITROGEN.— The greater number of tests for 
 this element are due to the circumstance that, on the breaking up 
 of organic substances which contain it, it is given off as ammonia. 
 If the ammonia is all collected and estimated, the amount of nitrogen 
 can be easily calculated. Kjeldahl's method for carrying out this 
 quantitative analysis is described in Lesson XXII. The following 
 exercises, however, are qualitative only. 
 
 (a) The characteristic odour of burning flesh, horn, hair, feathers, 
 etc., has been already noted, and, though only a rough test, is very 
 trustworthy. 
 
 (b) Take a little dried albumin and mix it thoroughly in a mortar 
 with about twenty times the amount of soda-lime and heat in a 
 test-tube over a Bunsen flame. Ammonia comes off in the vapours 
 produced, and may be recognised by (i) its odour ; (ii) it turns 
 moistened red litmus paper (held over the mouth of the tube) blue ; 
 (iii) it gives off white fumes with a glass rod (held over the mouth 
 of the tube) which has been dipped in hydrochloric acid. 
 
 (c) Mix some dried albumin with about ten times its weight of a 
 mixture of equal parts of magnesium powder and anhydrous sodium 
 carbonate. A small quantity of the mixture is then carefully heated 
 in a dry test-tube and finally heated more strongly for about half a 
 minute to red heat. Bip the tube whilst still glowing into a beaker 
 containing about 10 c.c. of distilled water ; the tube will break and 
 its contents mix with the water. Filter and label the filtrate A ; 
 divide it into two parts ; to one part add one or two drops of cold 
 saturated solution of ferrous sulphate and a drop of ferric chloride 
 solution. Warm the mixture, then cool and acidify with hydrochloric 
 acid. The fluid becomes bluish-green, and gradually a precipitate of 
 Prussian blue separates out. This test is due to the fact that some 
 of the nitrogen is fixed as sodium cyanide, and this gives the Prussian 
 blue reaction with the reagents added. 
 
 7. TESTS FOR SULPHUR.— (a) In the foregoing test (6 c) the 
 sulphur of the albumin combines with the sodium to form sodium 
 sulphide. This may be detected by taking the other part of the 
 flltrate A and adding freshly prepared solution of sodium nitro- 
 prusside ; a reddish-violet colour forms. 
 
ELEMENTS CONTAINED IN PHYSIOLOGICAL COMPOUNDS 7 
 
 (b) TEST FOR LOOSELY COMBINED SULPHUR.— Add 2 drops 
 of a neutral lead acetate solution to a few c.c. of caustic soda solution. 
 The precipitate of lead hydroxide which is first formed soon dissolves. 
 Heat a small portion of the alhumin with this alkaline solution. The 
 mixture turns black in consecLuence of the formation of lead sulphide, 
 part of the sulphur present in albumin having been split off from it 
 by the caustic soda as sodium sulphide. 
 
 (c) Take some dried albumin and fuse with a mixture of potash 
 and potassium nitrate. Cool ; dissolve in water and filter. The 
 filtrate will give the following test for sulphates : — Acidulate with 
 hydrochloric acid and add barium chloride ; a white precipitate of 
 barium sulphate is produced. 
 
 8. TEST FOR PHOSPHORUS. —The test just described (7 c) may 
 be repeated with some substance (such as caseinogen, nucleo-protein, 
 or lecithin) which contains phosphorus in organic combination ; or 
 the organic matter may be more conveniently destroyed by Neumann's 
 method, which consists in heating it with a mixture of sulphuric 
 and nitric acids. The resulting fluid in each case gives the following 
 test for phosphoric acid :— Mix it with half its volume of nitric acid ; 
 add ammonium molybdate in excess and boil ; a yellow crystalline 
 precipitate of ammonium phospho-molybdate falls. 
 
ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 SYMBOLS AND ATOMIC WEIGHTS OF THE 
 PEINCIPAL ELEMENTS 
 
 Aluminium . 
 
 Al 
 
 27-1 
 
 Magnesium 
 
 . Mg 24-32 
 
 A.ntimony 
 
 Sb 
 
 120-2 
 
 Manganese 
 
 . Mn 55-0 
 
 Arsenic 
 
 As 
 
 75-0 
 
 Mercury 
 
 . Hg 200 '0 
 
 Barium . 
 
 Ba 
 
 137-37 
 
 Nickel . 
 
 . Ni 58-7 
 
 Bismuth 
 
 Bi 
 
 208-0 
 
 Nitrogen 
 
 . N 14-01 
 
 Boron . 
 
 B 
 
 110 
 
 Osmium . 
 
 . Os 1910 
 
 Bromine 
 
 Br 
 
 79-92 
 
 Oxygen . 
 
 . 16-0 
 
 Cadmium 
 
 Cd 
 
 112-4 
 
 Phosphorus 
 
 . P ' 31-0 
 
 Calcium 
 
 Ca 
 
 40-1 
 
 Platinum 
 
 . Pt 195-0 
 
 Carbon . 
 
 c 
 
 12-0 
 
 Potassium 
 
 . K 39-10 
 
 Chlorine 
 
 CI 
 
 35-46 
 
 Silver 
 
 . Ag 107-88 
 
 Copper . 
 
 Cu 
 
 63-57 
 
 Silicon 
 
 . Si 28-3 
 
 Fluorine 
 
 F 
 
 19-0 
 
 Sodium . 
 
 - . Na 23-00 
 
 Gold . 
 
 Au 
 
 197-2 
 
 Strontium 
 
 . Sr 87-6 
 
 Hydrogen 
 
 H 
 
 1-008 
 
 Sulphur . 
 
 . S 32-07 
 
 Iodine . 
 
 I 
 
 126-92 
 
 Tin 
 
 . Sn 119 
 
 Iron 
 
 Fe 
 
 55-85 
 
 Tungsten 
 
 . W 184-0 
 
 Lead . 
 
 Pb 
 
 207-1 
 
 Zinc . ■ 
 
 .' . Zn 65-37 
 
 The above ate 
 
 mic weights are 
 
 ;akeii on the bas 
 
 is that 0=16. 
 
ELEMENTS CONTAINED IN PHYSIOLOGICAL COMPOUNDS 9 
 
 The practical exercises of the foregoing lesson show, in the first 
 instance, how the substances with which we have to deal fall under 
 the two main categories of organic and inorganic. In some of the 
 tissues of the body, such as bone and tooth, the inorganic or mineral 
 material is in excess, but in the softer portions of the organism the 
 organic compounds are in great preponderance. 
 
 Organic chemistry is sometimes defined as the chemistry of the 
 carbon compounds ; carbon is in all cases present, and is usually the 
 most abundant element. 
 
 The most important of the nitrogenous substances are the 
 proteins, as already explained in the introductory chapter, and the 
 detection and estimation of nitrogen are thus exercises of the highest 
 interest. 
 
 All the proteins contain a small amount of sulphur ; keratin, or 
 horny material, contains more than most of them do. 
 
 Phosphorus is another element of considerable importance, being 
 present in nuclein and nucleo-proteins, and also in certain complex 
 lipoids, of which lecithin may be taken as a type. Iodine occurs 
 united to protein material in the colloid substance of the thyroid gland ; 
 iron in the pigment of the blood called haemoglobin ; sodium, calcium, 
 potassium, and other metals in the inorganic substances of the body. 
 It would, however, lead us too far into the regions of pure chemistry 
 to undertake exercises for the detection of, these and other elements 
 which might be mentioned, and have been already commented upon. 
 The teacher of physiological chemistry is bound to assume that the 
 students who come before him have already passed through a course 
 of ordinary chemistry. 
 
 The main interest of the exercises selected as types lies in their 
 physiological application. As a rule an element is detected by breaking 
 up or oxidising the more or less complex molecule in which it occurs 
 into substances of simpler nature, and then performing tests for these 
 simpler products. Thus carbon is identified by the formation of carbon 
 dioxide, nitrogen by the formation of ammonia, and so forth. 
 
 A great many reactions which can be performed in the test-tube 
 imitate those which are performed in the body. Reactions in vitro 
 and in vivo, to use the technical phrases, often, though not always, 
 run parallel. Life, from one point of view, is a process of combustion 
 or oxidation ; the fuel is supplied by the food ; this is incorporated by 
 the tissues and is then burnt up by the oxygen brought to it by the 
 blood-stream, giving rise to animal heat and other manifestations of 
 energy; and finally the simple products of oxidation or chemical 
 
10 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 breakdown are carried to the organs of excretion (lung, skin, kidney, 
 etc.), where they are discharged from the body. 
 
 A candle consists principally of carbon and hydrogen ; when it is 
 burnt, the products are carbonic acid gas and water ; the former may 
 be detected by means of lime water, the latter, by holding a dry beaker 
 upside down for a few moments over the burning candle, when the 
 moisture will condense on the cold glass. 
 
 The body is more complex than a candle, but so far as its carbon 
 and hydrogen are concerned the final products of combustion are the 
 same. The carbon dioxide is discharged by the expired air, as may be 
 proved by blowing it into lime water. The water finds an outlet by 
 several channels, lungs, skin, and kidneys. The presence of nitrogen 
 in the body is perhaps the most striking chemical distinction between 
 it and a candle, and here again the process of metabolism runs a course 
 analogous in some degree to our experiments in vitro ; for the most 
 important and abundant substance which contains the waste nitrogen 
 is the simple material ammonia ; but ammonia is only discharged as 
 such to a very small extent in health. It unites with carbon and 
 oxygen to form the substance called urea (OON.^H^), which Jinds its way 
 out of the body via the urine. The urine also contains the sulphates, 
 due to the oxidation of the sulphur of the proteins, and the phosphates 
 due to the similar oxidation of the phosphorus of such substances as 
 lecithin and nuclein. Some of the salts of the urine, however, in 
 particular the chlorides, come directly from the food. This we shall 
 discuss at the proper place when we come to the study of the urine. 
 
LESSON II 
 
 SOME TYPICAL ORGANIC COMPOUNDS 
 
 1. ALCOHOL {ethyl alcohol).— The following reactions are to be 
 carried out with 96 per-cent. alcohol or with the distillate obtained 
 from an alcoholic liquid. The use of a distilling apparatus should 
 be demonstrated. 
 
 (a) Add 10 drops of alcohol to a small quantity of sodium acetate 
 crystals in a test tube. Allow about 20 drops of concentrated 
 sulphuric acid to fall on the mixture and warm gently. Acetic ester 
 (ethyl acetate) is formed, and is recognised by its characteristic 
 smell : — 
 
 C2H5OH + CH3COOH = OH3.COOO2H5 + H2O. 
 
 (b) Mix a few drops of alcohol with an equal amount of benzoyl 
 chloride and add excess of strong potash. On continuous shaking 
 the irritant smell of benzoyl chloride disappears and is replaced by 
 the fruit-like odour of benzoic ester -. — 
 
 C2H5OH + CeHsOOCl = CeHj.COOCaHs + HCl. 
 
 (c) Warm 3 drops of alcohol with two drops of concentrated sulphuric 
 acid. After cooling notice the smell of ether (ethyl ether). 
 
 (d) Warm 10 drops of alcohol with 5 drops of concentrated sulphuric 
 acid. After cooling neutralise with potash or soda. Potassium or 
 sodium ethyl sulphate is thus obtained; add a few drops of sodium 
 sulphide. On warming, ethyl merca-ptan is formed and may be 
 recognised by its garlic-like smell : — 
 
 02H5.SO^Na + NaSH = C2H5.SH + NagSOi- 
 
 (e) Add 1 drop of alcohol to about 5 c.c. water. Make alkaline 
 with strong potash and add iodine solution until the fluid remains 
 yellow. On wanning a yellow crystalline precipitate of iodoform (CHI3) 
 forms, and its characteristic smell is noticed (Lieben's reaction) : — 
 
 C2H5.OH + 6NaOH + 4I2 = CHI3 + H.COONa + 5NaI + SB.^0. 
 
 2. ALDEHYDE (acetaldehyde).—'Wa,im. a few drops of alcohol with 
 a solution of potassium bichromate and dilute sulphuric acid. The 
 
 a 
 
12 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 solution turns green, owing to reduction. Aldehyde is formed and 
 recognised by its penetrating and irritant smell: — 
 
 CH3.CH0.OH + = CH3.CHO + HjO. 
 
 Perform the following reactions with the solution of aldehyde 
 supplied. 
 
 (a) Acetaldehyde solution reduces Fehling's solution on boiling, 
 and a yellowish-red precipitate of cuprous oxide is formed : — 
 CH3CHO + 2CuO + NaOH = OHg.COONa + CUjO + HjO. 
 
 (b) Add a few drops of ammonia to silver nitrate until the white 
 precipitate just redissolves, and add a few drops of dilute aldehyde 
 solution. Then add a little potash, place the tube in a cold-water 
 bath and heat to boiling. A mirror of metallic silver is formed: — 
 
 Ag^O + CH3.CHO = Agj + CH3.COOH. 
 
 (c) On wanning an aldehyde solution with caustic alkalis a brown 
 resinous substance (aldehyde resin) is formed; this is insoluble in 
 water, but e^ily soluble in alcohol or ether. 
 
 (d) Add a small amount of an aldehyde solution to a solution of 
 phenylhydrazine hydrochloride and sodium acetate. An oily hydrazone 
 is formed :— 
 
 CeH-.NH.NH2 -t- CH3.CHO = OgHj.NH.N : CH.CH3 + H^O. 
 
 (3) FORMIC ACID.— (a) Sodium formate dissolved in a little water, 
 is acidified with dilute sulphuric acid and warmed. Formic acid 
 comes off and is recognised by its smell. 
 
 (b) Warm a cobcentrated solution of sodium formate with concen- 
 trated sulphuric acid. An odourless gas is developed which bums 
 with a blue flame {Carbon monoxide) : — 
 
 H.COOH = 00-1- HjO. 
 
 (c) Add a few drops of alcohol and a few drops of concentrated 
 sulphuric acid to some dry sodium formate In a test-tube and warm 
 the mixture. Notice the smell of formic ester. 
 
 (d) Add silver nitrate to a dilute solution of sodium formate and 
 warm. A deposit of black metallic silver is formed : — 
 
 2H.C00Ag = Agj -1- H.COOH + CO^. 
 
 (e) Heat a solution of mercuric chloride with a solution of sodium 
 formate. A white precipitate of mereurous chloride is formed : — 
 
 2H.C00Na + 2HgCl2 = HgjClj + 00^ -f- CO -i- 2NaCl + H.p. 
 
 (f) A solution of formate gives a red solution on the addition of 
 ferric chloride. On boiling a hrovmish-red precipitate is formed (a basic 
 iron formate) which dissolves on the addition of hydrochloric acid. 
 
SOME TYPICAL ORGANIC COMPOUNDS 13 
 
 4. ACETIC ACID is prepared from alcohol by oxidising it with 
 potassium permanganate and sulphuric acid, and then distilling it 
 over. It is recognised by- 
 
 (a) The characteristic taste and smell of vinegar. 
 
 (b) Neutralise a few drops of glacial acetic acid with potash or soda 
 and add ferric chloride ; the red colour of ferric acetate appears ; on 
 boiling a brownish-red precipitate of basic ferric acetate is formed. 
 
 5. OXALIC ACID.— (a) Heat a few crystals of oxalic acid on 
 platinum or nickel foil. It volatilises without charring or separation 
 of carbon. 
 
 (b) Dissolve a few crystals of oxalic acid in a few c.c. of concentrated 
 sulphuric acid and warm. Carbon monoxide and carbon dioxide are 
 evolved :— 
 
 COOH.COOH = COj + CO + H^O. 
 
 (c) Acidify a solution of oxalic acid with dilute sulphuric acid 
 and add potassium permanganate. The colour of the permanganate 
 solution is discharged :— 
 
 COOH.COOH + = 2CO2 + H2O. 
 
 (d) Calcium chloride (or barium chloride) gives a white precipitate 
 with oxalic acid solution. These oxalates are not soluble in acetic 
 acid, but are easily soluble in hydrochloric acid. 
 
 6. PHENOL {carbolic acid).—{&) Add a few drops of dilute ferric 
 chloride to a phenol solution : a purple colour is formed, which is 
 discharged by hydrochloric acid :— 
 
 SCgHsOH + FeClg = (CgH50)gFe + 3HC1. 
 
 I (b) On adding bromine water to a dilute solution of phenol, a 
 yellowish-white fliocculent precipitate of tribromophenol is produced 
 even in very dilute solutions :— 
 
 CjHgOH -I- 3Br2 = CgHgBrj.OH + 3HBr. 
 
 (c) Boil a dilute phenol solution with nitric acid. A bright 
 yellow solution results (picric acid), which turns into a brownish- 
 yellow on adding alkali : — 
 
 CjiHjOH -f- 3HNO3 = C(iH^(N02)gOH + SB^O. 
 
 (d) Millon's reagent gives a deep red colour on warming with even 
 very dilute phenol solutions. 
 
14 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 It is necessary to study first some of the simpler organic compounds 
 in order that we may understand the nature of the more elaborate 
 substances which are found in the body. 
 
 Hydrocarbons. — These are compounds of hydrogen and carbon, 
 and form the basis of classification in organic chemistry. The simplest 
 hydrocarbon known is methane, or marsh gas ; it has the formula 
 CH^ : if one of its hydrogen atoms is replaced by hydroxyl OH, we 
 obtain the simplest alcohol ; thus : — 
 
 H H 
 
 I . I 
 
 CH3 CH2.OH 
 
 [methane] [methyl alcohol] 
 
 The next alcohol in the series is formed in a similar way from the next 
 hydrocarbon CgHg (ethane) ; thus : — 
 
 I I 
 
 CHg CHj.OH 
 
 [ethane] [ethyl alcohol] 
 
 0,nd so on. 
 
 Alcoliols. — We may take ordinary or ethyl alcohol as a type of that 
 important class of organic substances known as the alcohols. These 
 are substances, the knowledge of which may be regarded as the starting 
 point of many other organic compounds. Ethylic alcohol has the 
 formula CgHgO ; one of its hydrogen atoms is replaceable by metals 
 such as sodium or potassium, and we may therefore ' write the formula 
 CjHgOH, the last hydrogen atom being the one which is replaceable by 
 other monad elements. This atom is united to oxygen to form the 
 atomic group called hydroxyl (OH). The hydroxyl can be replaced by 
 chlorine by treating alcohol with phosphorus pentachloride, and a 
 substance with the formula CjHjCl is obtained. The atomic group 
 C^Hj which is unchanged and united to OH in alcohol, and to CI in 
 ethyl chloride, is called ethyl. 
 
 There are other alcohols in which the place of ethyl is taken by 
 other organic radicals, or alkyls. Thus if the radical called methyl 
 (CH3) is united to hydroxyl we obtain methyl alcohol, CH3.OH. 
 These two alcohols, methylic and ethylic, form the first two members 
 of a group of alcohols, which are termed monohydric ; all of these 
 contain only one hydroxyl group, and the following is a list of the first 
 six members of this group with their formulae : — 
 
SOME TYPICAL ORGANIC COMPOUNDS 15 
 
 CH3.0H 
 
 methyl alcohol 
 
 C2H5.0H 
 
 ethyl 
 
 C,H..OH 
 
 propyl „ , 
 
 c,h;,.oh 
 
 butyl 
 
 C,H„.OH 
 
 amyl „ 
 
 0,H,3.0H 
 
 hexyl ,, 
 
 Each dififers from the preceding by CHg. 
 
 Aldehydes and Ketones. — In the alcohols there are possibilities for 
 isomerism to occur ; by this one means that although two (or more) 
 substances may have the same empirical formula, the arrangement of 
 the atoms or atomic groups within the molecule is different. A 
 prim.ary alcohol is one in which the hydroxyl (OH), and two hydrogen 
 atoms are attached to the same carbon atom ; it therefore contains 
 the group CHj-OH. Thus the formula for common alcohol (primary 
 ethyl alcohol) may be written : — 
 
 OH3 
 
 I 
 CHo.OH 
 
 and the formula for the next alcohol of the series (primary propyl 
 
 alcohol) is : — 
 
 CH3 
 
 I 
 CH, 
 
 I 
 CHg.OH 
 
 If a primary alcohol is oxidised, two atoms of hydrogen are removed, 
 and the oxidation product so formed is called an aldehyde (the name is 
 derived from "alcohol dehydrogenatum ") ; thus methyl alcohol yields 
 Jormaldehyde, ethyl alcohol yields acetic aldehyde, and propyl alcohol 
 yields propionic aldehyde, as shown in the following equations : — 
 
 H.CH2.OH + O = H.CHO + H3O 
 
 [methyl alcohol] [formaldehyde] 
 
 CH3.CH2OH + O = CHj.CHO + HjO 
 
 [ethyl alcohol] [acetic aldehyde] 
 
 CH3.CH2.CH2OH + O = CHg.CHj.CHO + HjO 
 
 [propyl alcohol] [propionic aldehyde] 
 
 The typical group of the aldehydes (OHO) is not stable, but is easily 
 oxidisable to form the group COOH, which is called carboxyl. The 
 readiness with which aldehydes are oxidisable renders them powerful re- 
 ducing agents, and tests for their detection mainly depend upon this fact. 
 A secondary alcohol is one in which the OH group and one hydrogen 
 
16 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 atom are attached to the same carbon atom — thus secondary propyl 
 alcohol (the first case where isomerism is possible) has the formula 
 
 CH3.CHOH.CH3. 
 The typical group is therefore the divalent radical > CHOH, and when 
 this is oxidised, the first oxidation product is called a ketone, thus : — 
 
 CH3.CHOH.CH3 + O = CHg.CO.CH3 + H2O 
 
 [secondary propyl alcohol] [propyl-ketone] 
 
 It therefore contains the group > CO. Propyl-ketone is usually called 
 acetone, the tests for which we shall have to consider in our study of 
 diabetic urine. 
 
 In the next alcohol {butyl alcohol), four isomers are possible, one of 
 which is a tertiary alcohol, i.e. it contains the trivalent radical = C - OH, 
 which breaks down on oxidation into smaller molecules. 
 
 The Fatty Acids. — These form a series of acids derived from the 
 monohydric alcohols by oxidation. Thus to take ordinary ethyl alcohol 
 CHg.CHjOH, the first stage in oxidation is the removal of two atoms 
 of hydrogen to form an aldehyde, CHg.CHO, as we have just seen ; on 
 further oxidation an atom of oxygen is added to form the acid called 
 acetic acid, CH3.COOH. A similar acid can be obtained from the 
 other alcohols of the series, thus : — 
 
 From methyl alcohol CHg.On, formic, acid H.COOH is obtained 
 „ ethyl „ C2H5.OH, acetic „ CH3.COOH 
 ,, propyl ,, 03Hy.0H, propionic ,, CgHj.COOH 
 „ butyl „ 0,Hg.OH, butyric „ O3H-.COOH 
 „ amyl „ C^Hji.OH, valeric „ C^H^j.COOH 
 „ hexyl „ CgHjg.OH, caproic „ CjHjj.COOH 
 
 and so on. 
 
 Or in general terms : — 
 
 From the alcohol with formula C„H2„+i OH the acid with formula 
 C„_jH2„_jC00H is obtained. The above series of acids constitutes that 
 known as the fatty acid series. Just as their parent alcohols contain 
 one OH group, so do the acids contain one carboxyl group (COOH) ; 
 they are therefore termed monocarhoxylic acids. 
 
 In addition to the monohydric alcohols, there are other series of 
 alcohols which diiFer from these in containing more than one OH group. 
 Those which, like glycol [C2H^.(OH2)], contain two OH grcjups are called 
 dihydric ; those which, like glycerol [C3H5(OH)g]), contain three OH 
 groups are called trihydric ; there are also tetrahydric, pentahydric, 
 hexahydric, etc., series of alcoholsi, containing respectively four, five 
 six, etc., hydroxyl groups ; and the hexahydric alcohols are particularly 
 
SOME TYPICAL ORGANIC COMPOUNDS 17 
 
 interesting to the physiologist as, they are the parent substances of the 
 chief carbohydrates. 
 
 The aldehydes or ketones obtained as the first stages in the oxida- 
 tion of these alcohols are correspondingly complex ; and by still further 
 oxidation organic acids are produced. Thus oxalic acid is an instance 
 of an acid obtained by the oxidation of a dihydric alcohol ; it is there- 
 fore a di-carboxylic acid, as it contains two carboxyl (COOH) groups, 
 just as the alcohol (glycol) from which it is derived contains two 
 hydroxyl groups. 
 
 We may give the formulae for glycol and its derivatives as an 
 example of a dihydric alcohol, but it will not be necessary at this point 
 to go into further details of other more complex alcohols : — 
 
 CH2.OH CHO COOH OHO CHO COOH 
 
 CH3.OH CH2.OH CH.,.OH CHO . COOH COOH 
 
 [glycol] [glycoUic [glyoollio [glyoxal] [glyoxylic [oxalic 
 
 aldehyde] acid] acid] acid] 
 
 Ethers are obtained by abstracting water from two molecules of an 
 alcohol, and may be regarded as the anhydrides of alcohols. Ethyl 
 ether, usually called ether simply, is obtained by heating alcohol with 
 concentrated sulphuric acid, and the reaction occurs in the two stages 
 represented by the following equations : — 
 
 C^H-.OH + HjSO, = C2H5.SO3.OH + H2O 
 
 [ethyl alcohol] "* [ethyl sulphuric acid] 
 
 OjHj.OH + CgH^.SOg.OH = C^Hj.O.OaH^ -f- H^SO^ 
 
 [ethyl alcohol] [ethyl aulphnrio acid] [ether] 
 
 It will be noticed that the sulphuric acid is recovered unchanged at 
 the end of the reaction, and therefore theoretically a small amount 
 of sulphuric acid will transform an unlimited amount of alcohol into 
 ether. We shall find later in our study of the action of enzymes that 
 these 'important agents in the chemical' transformations in the body 
 are characterised by *the same property. They probably act in a 
 manner analogous to sulphuric acid in etherification, participating 
 in intermediate reactions, but are present unchanged in the terminal 
 reaction. 
 
 Esters. — The formation of esters or compounds with acids is a 
 reaction typical of all alcohols, and is analogous to the formation of 
 salts which occurs when a metallic hydroxide reacts with an acid. 
 Thus if sodium hydroxide and nitrous acid react together we get 
 sodium nitrite and water, as shown in the following equation : — 
 
 HNO., + NaOH - NaNO^ + H,0. 
 
18 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 The similar interaction of ethyl alcohol and nitrous acid, results in the 
 formation of an ester (ethyl nitrite) and water : — 
 
 HNOj + C^Hs.OH = C2H5.NO2 + H,0 
 
 [ethyl alcohol] [ethyl nitrite] 
 
 The next equation represents the reaction between alcohol and an 
 organic acid (acetic) : — 
 
 CH3.COOH + C2H5.OH = CH3.CO.O.C2H5 -I- H^O 
 
 [acetic acid] [ethyl alcohol] [ethyl acetate or 
 
 acetic ester] 
 
 Amino-acids. — These are nitrogenous derivatives of the fatty acids, 
 and are the building stones from which the proteins are constructed ; 
 conversely they are the products obtained from proteins when these 
 complex substances are broken up. We shall consider them more fully 
 in our study of the proteins, and so we may here be content with a 
 typical example. 
 
 Acetic acid is (CH3.COOH. 
 
 If one of the hydrogen atoms in the CH3 group is replaced by the 
 amino group (NH^) we obtain CH^.NHj.COOH, which is amino-acetic 
 acid or glycine. 
 
 Aromatic Compounds. — These are derivatives of the hydrocarbon 
 called benzene, CgHg. The organic substances we have considered up 
 to this point are usually spoken of as belonging to the fatty or aliphatic 
 series ; in these the carbon atoms are united together in an open chain. 
 The aromatic compounds on the other hand are characterised by the 
 carbon atoms being united together by alternate single and double 
 bonds into a ring, and the formula for benzene, the simplest member of 
 the group, may therefore be written graphically in the following way : — 
 
 H 
 
 H— C C— H 
 
 I II, 
 H— C C— H 
 
 %/ 
 C 
 
 I 
 H 
 
 By substituting different groups (side-chains) for one or more of the 
 hydrogen atoms, all other aromatic compounds are obtained. 
 
 The benzene nucleus itself is extremely stable ; such processes as 
 oxidation and reduction can be applied to it without destroying it. 
 
SOME TYPICAL ORGANIC COMPOUNDS 19 
 
 The production of nitro-compounds by the action of nitric acid is 
 characteristic of derivatives of benzene, whereas such treatment will 
 usually oxidise and decompose aliphatic substances. A few aromatic 
 compounds are found in the animal organism, for instance, hippuric 
 acid in the urine ; some, such as tyrosine, are found among the 
 decomposition products of proteins ; hence a knowledge of these 
 substances is necessary for the student of physiology and medicine. 
 
 Phenol or carbolic acid is included among the substances tested 
 for in the accompanying practical lesson to remind the 'student of the 
 existence of aromatic compounds ; it is a hydroxyl derivative of 
 benzene, and its formula may be written CgHj.OH, or graphically : — 
 
 OH 
 
 I 
 
 
 ■^\ 
 H— C— H 
 
 H— C C— H 
 
 C 
 
 I 
 H 
 
 that is, one of the hydrogen atoms is replaced by hydroxyl (OH). It 
 is, however, usual to write it as- shown below : — 
 
 OH 
 
 \/ 
 
 the unchanged portion of the benzene nucleus being depicted by a 
 simple hexagon. 
 
 We shall come across more complicated derivatives of benzene in 
 the further study of our subject; and we shall, moreover, meet with 
 other ringed compounds (heterocyclic) in which nitrogen occurs within 
 the ring ; such substances as pyridine and pyrrol and their derivatives 
 are included in this category. They are important as the mother 
 substances of many alkaloids. 
 
LESSON III 
 THE CARBOHYDRATES 
 
 Glucose, cajie sugar, dextrin, starch, and glycogen are given round 
 as typical and important carbohydrates. 
 
 1. GLUCOSE. — Perform the following tests with a solution of 
 glucose : — 
 
 (a) Trmnmer's Test. — Put a few drops of copper sulphate solution 
 into a test-tube and add a few c.c. of strong caustic potash. On 
 adding the caustic potash a precipitate is formed, which, on addition 
 of a glucose solution, rapidly redissolves, forming a blue solution. On 
 boiling this a yellow or red precipitate (cuprous hydrate or oxide) forms. 
 
 (b) FeMiiig's Test. — Fehling's solution is a mixture of copper 
 suliihate, caustic soda, and potassium sodium tartrate (Bochelle salt) 
 of a certain strength. It is used for estimating glucose quantitatively 
 (see Lesson All). It may be used as a qualitative test also. Boil 
 some Fehling's solution ; if it remains clear it is in good condition ; 
 add to it an equal volume of solution of glucose and boil again. 
 Seduction, resulting in the formation of cuprous hydrate or oxide, 
 takes place as in Trommer's ^test. This test is more certain than 
 Trommer's, and is preferable to it. Uric acid and creatinine also 
 reduce Fehling's solution, and the sodium hydroxide has a destructive 
 action on sugar. These two disadvantages are absent in 
 
 (c; Beiiedicfs Test. — In this test sodium carbonate replaces NaOH, 
 and sodium citrate is substituted for Bochelle salt (see footnote, p. 210/. 
 Add a few drops of glucose solution to 5 c.c. of Benedict's (qualitative) 
 reagent, and boil vigorously for a few minutes. The solution becomes 
 filled with a red, yellow, or greenish fine precipitate, the colour de- 
 pending on the concentration of the glucose solution. 
 
 d) Nylander's Test. — Mix 5 c.c. of glucose solution with 1 of 
 Nylander's reagent (20 gr. of bismuth subnitrate and 50 gr. of 
 Bochelle salt dissolved in 1 litre of 8 per-cent. sodium hydroxide). 
 Boil for three minutes and allow to cooL A black precipitate of 
 metallic bismuth separates out. 
 
 N.B.— Sugars such as glucose, fructose, maltose, and lactose, 
 which give the preceding tests, are called reducing sugars. 
 
 20 
 
THE CARBOHYDRATES 21 
 
 (e) Moore's Test.— Add to the glucose solution about half its 
 volume of 20 per-cent. potash and heat. The solution becomes 
 yellowish-brown. Acidify with sulphuric acid (25 per cent.) and 
 the odour of caramel becomes apparent. 
 
 (f) Fermentation Test. — Add a fragment of dried yeast to the 
 glucose solution in a test-tube ; fill the test-tube up with mercury, 
 and invert it over mercury in a trough. Place it in an incubator at 
 body temperature for twenty-four hours. The sugar is broken up 
 into alcohol and carbon dioxide ; the latter gas collects in the upper 
 part of the test-tube. The alcohol may be detected by Lieben's 
 reaction (1 e, p. 11). 
 
 (g) Molisch's Reaction, — Add a few drops of an alcoholic solution 
 of thymol or a-naphthol to a solution of sugar, and allow a few drops 
 of concentrated sulphuric acid to run to the bottom of the test-tube. 
 A red (with thymol) or purple (with a-naphthol) forms at the surface 
 of contact. 
 
 This test is given by all the sugars, and in fact by all carbohydrates 
 with more or less intensity ; it is also given by those proteins which 
 contain a carbohydrate radical. 
 
 2. SUCROSE or CANE SUGAR.— (a) The solution of cane sugar 
 when mixed with copper sulphate and caustic potash gives a blue 
 solution. But on boiling no reduction occurs. 
 
 (b) Take some of the cane-sugar solution and boil it with a few 
 drops of 25 per-cent. sulphuric acid. This converts it into equal parts 
 of glucose and fructose. Neutralise with potash or soda. It then 
 gives Trommer's or FeMing's test in the typical way. 
 
 (c) Boil some of the cane-sugar solution with an equal volume of 
 concentrated hydrochloric acid. A deep red solution is formed. 
 Glucose, lactose, and maltose do not give this test. 
 
 (d) Cane sugar gives Molisch's reaction. 
 
 3. STARCH.— (a) Examine starch grains with the microscope. In 
 size and other minor particulars the starch grains differ according to 
 their source. Potato starch is readily obtained by mounting a scraping 
 from the surface of a freshly cut potato; these are specially large; 
 those from rice are smaller. Note the concentric markings on the 
 starch grains. If a drop of iodine solution is run in under the cover- 
 slip the grains are stained blue. 
 
 (b) Starch is not soluble in cold water. Mix a little starch with 
 cold water and pour boiling water into the paste. Continue to boil 
 until an opalescent solutioh is formed ; this, if strong, gelatinises on 
 cooling. 
 
 (c) Add iodine solution. An intense blue colour is produced, which 
 disappears on heating, and if not heated too long reappears on cooling. 
 
 N.B.— Prolonged heating drives oflf the iodine, and consequently no 
 blue colour returns after cooling. 
 
22 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 (d) Conversion into dextrin and glucose. To some starch solution 
 in a flask add a few drops of 25 per-cent. sulphuric acid, and boil for 
 fifteen minutes. Take some of the liquid, which is now clear, neutralise 
 with soda, and show the presence of dextrin and glucose. 
 
 4. DEXTRIN.— (a) Add iodine solution to solution of dextrin : a 
 reddish-brown colour is produce'd. The colour disappears on heating 
 and reappears on cooling. Many commercial dextrins give at first a 
 blue colour which changes through a purple-red to a red-brown on the 
 addition of more iodine. 
 
 (b) Saturate a dextrin solution by grinding it in a mortar with 
 finely powdered ammonium sulphate; filter. The erythro-dextrin is 
 precipitated, but only incompletely ; therefore the filtrate gives a red- 
 brown colour with a drop of iodine solution. 
 
 (c) Commercial dextrin usually gives a slight reduction with 
 Fehling's solution owing to sugar as an impurity. 
 
 5. GLYCOGEN. — Solution of glycogen is given round : (a) it is 
 opalescent like that of starch. 
 
 (b) With iodine solution it gives a brown colour very like that 
 given by dextrin. The colour disappears on heating and reappears 
 on cooling. 
 
 (c) By boiling with 25 per-cent. sulphuric acid it is converted into 
 glucose. Neutralise and test ynth Fehling's solution. 
 
 (d) Saturate the solution with ammonium sulphate as in 4 (b), and 
 filter. The glycogen is completely precipitated, and therefore the 
 filtrate gives no coloration with iodine. This easily distinguishes it 
 from dextrin. 
 
THE CARBOHYDRATES 23 
 
 The carbohydrates are found chiefly in vegetable tissues, and many 
 of them form important foods. Some carbohydrates are, however, 
 found in or formed by the animal organism. The most important of 
 these are glycogen, or animal starch; glucose; and lactose, or milk sugar. 
 
 The carbohydrates may be conveniently defined as compounds of 
 carbon, hydrogen, and oxygen, the two last-named elements being in 
 the proportion in which they occur in water. But this definition is 
 only a rough one, and if pushed too far would include many substances, 
 such as acetic acid, lactic acid, and inositol, which are not carbo- 
 hydrates. Research has shown that the chemical constitution of the 
 simplest carbohydrates is that of an aldehyde, or a ketone, and that 
 the more complex carbohydrates are condensation products of the 
 simple ones. 
 
 The meaning of the terms " aldehyde " and " ketone " has been 
 explained in the preceding lesson, but there we drew most of our 
 examples from simple aldehydes and ketones derived by oxidation from 
 monohydric alcohols. In the case of the sugars, we have to start from 
 more complex alcohols, namely, those which are called hexahydric, on 
 account of their containing six OH groups, The majority of the 
 known sugars are aldehydes (aldoses). Sugars which are ketoiies are 
 called ketoses, but only one of these, namely, fructose, is of physiological 
 interest. This constitution of the sugars explains why it is that they 
 are reducing agents. 
 
 Three hexahydric alcohols, all with the same empirical formula 
 C(jHg(OH)j, may be mentioned ; they are isomerides, and their names 
 are sorbitol, mannitol, and duloitol. By careful oxidation their alde- 
 hydes and ketones can be obtained ; these are the simple sugars ; thus, 
 glucose is the aldehyde of sorbitol ; mannose is the aldehyde of man- 
 nitol; fructose is the ketone of mannitol; and galactose is the aldehyde 
 of dulcitol. Thes6 sugars all have the empirical formula CgHjgOg. 
 
 CH„OH CH„OH CH,OH 
 
 J I I " 
 
 H— C— OH H— C— OH H— 0— OH 
 
 I- I i 
 
 H— C— OH H— C— OH OH— 0-H 
 
 I • I I 
 
 OH— C— H OH-C— H OH— C— H 
 
 I I I 
 
 H— C— OH C— O H— C— OH 
 
 I I I 
 
 C— HO CH^OH C— HO 
 
 [glucose] [fructosel [galactose] 
 
24 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 They furnish an excellent example of what is called stereochemical 
 isomerism ; that is, the position of . the atoms or groups of atoms in 
 space within the sugar molecule varies. The constitutional formulae 
 of three important simple sugars are shown on p. 23. The six carbon 
 atoms in each case form an open chain, but the way in which the 
 hydrogen and hydroxyl atoms are linked to them differs. 
 
 The aldehyde constitution of glucose and of galactose is at once 
 evident from these formulse, the typical aldehyde group (CHO or more 
 accurately 0=C — H) being at the end of the chain. The ketone 
 constitution of fructose is also shown by the typical ketone group 
 (CO) not at the end of the chain. 
 
 , By further oxidation, the sugars yield various acids. If we take 
 these sugars as typical specimens, we see that their general formula is 
 
 C„H2„0,„ 
 
 and as a general rule n = m; that is, the number of oxygen and 
 carbon atoms is equal. This number in the case of the sugars already 
 mentioned is six. Hence they are called hexoses. 
 
 Sugars are known to chemists, in which this number is 3, 4, 5, 7, etc., and 
 these are called trioses, tetroses, pentoses, heptoses, etc. The majority of 
 these have no physiological interest. It should, however, be mentioned that 
 a pentose has been obtained from certain nucleic acids presently to be 
 described (see p. 64), which are contained in animal organs (pancreas, liver, 
 etc.), and in plants (for instance, yeast). 'If the pentoses which are found 
 in various plants are given to an animal, they are excreted in great measuie 
 unchanged in the urine. 
 
 The hexoses are of great physiological importance. The principal 
 ones are glucose, fructose, and galactose, These are called mono- 
 saccha/rides. 
 
 Another important group of sugars is that of the disaccha/rides ; 
 these are formed by the combination of two molecules of mono- 
 saccharides with the loss of a molecule of water, thus : — 
 
 c„H,,o„ + c„Hj,o, = c,,iL,,o,, + up. 
 
 The principal members of the disaccharide group are sucrose, 
 lactose, and maltose. 
 
 If more than two molecules of the monosaccharide group undergo, 
 a corresponding condensation, we get what are called polysaccharides. 
 
THE CARBOHYDRATES 
 
 2S 
 
 The polysaccharides are starch, glycogen, various dextrins, cellu- 
 lose, etc. We may therefore arrange the important carbohydrates of 
 the hexose family in a tabular form as follows : — 
 
 1. Monosaccharides, 
 
 + Glucose. 
 - Fructose. 
 + Galactose. 
 
 2. Disaccharides, 
 
 CigH^jO]!. 
 
 H. Polysaccharides, 
 
 (c,H,A)". : 
 
 + Sucrose. 
 + Lactose. 
 + Maltose. 
 
 + Starch. 
 + Glycogen. 
 + Dextrin. 
 Cellulose. 
 
 The signs + and - in the above list indicate that the substances 
 to which they are prefixed are dextro- and Isevo-rotatory respectively 
 as regards polarised light.i The formulse given above are merely 
 empirical : the quantity n in the staroh group is variable and usually 
 large. The following are the chief facts in relation to each of the 
 principal carbohydrates. 
 
 MONOSACCHARIDES 
 
 Glucose. — This carbohydrate (which is also known as dextrose and 
 grape sugar) is found in fruits, honey, and in minute quantities in the 
 blood (0'12 per' cent.) and numerous tissues, organs, and fluids of the 
 body. It is the form of sugar found in larger quantities in the blood 
 and urine in the disease known as diabetes. 
 
 Glucose is soluble in hot and cold water and in alcohol. It is 
 crystalline, but not so sweet as cane sugar. When heated with strong 
 potash certain complex acids are formed which have a yellow or brown 
 colour. This constitutes Moore's test for sugar. In alkaline solutions 
 glucose reduces salts of silver, bismuth, mercury, and copper. The 
 reduction of cupric hydrate to cuprous hydrate or oxide constitutes 
 Trammer's test, which has been already described at the head of the 
 lesson. On boiling glucose with an alkaline solution of picric acid, a 
 dark red opaque solution due to the reduction of the picric to picramic 
 acid is produced. Another important property of glucose is that 
 
 ' For a description of polarised light and polarimeters see Appendix. This 
 and the other matter in the Appendix are placed there for convenience, not 
 because they are unimportant. Students are therefore urged to refer to and 
 carefully study these subjects, 
 
26 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 under the influence of yeast it is converted into alcohol and carbonic 
 acid (C,Hj,0, = 20,HeO + 200^). 
 
 Glucose may be estimated by the fermentation test, by the polari- 
 meter (its specific rotation isi[a]„= + 52-5°), and by the use of Fehling's 
 or similar solutions. The last . method is the most important : it rests 
 on the same principles as Trommer's test, and we shall study it and 
 other methods of estimating sugar in connection' with diabetic urine 
 (see Lesson XII). 
 
 Fructose. — This sugar is also known as Isevulose on account of 
 its action on polarised light. When sucrose is heated with dilute 
 mineral acids it undergoes a process known as inversion — i.e. it takes 
 up water and is converted into equal parts of glucose and fructose. 
 The previously dextro-rotatory solution of cane sugar then becomes 
 IsBVO-rotatory, the Isevo-rotatory power of the fructose ([a]i, = — 92°) 
 being greater than the dextro-rotatory power of the glucose formed. 
 Hence the term inversion. The same hydrolytic change is produced 
 by certain enzymes, such as the invertase of the intestinal juice, and 
 of yeast. 
 
 Pure fructose can be crystallised with difficulty. It gives the same 
 general reactions as glucose. Small quantities of this sugar have 
 occasionally been found in blood, urine, and muscle. 
 
 Galactose is formed by the action of dilute mineral acids or inverting 
 enzymes on lactose or milk sugar. It resembles glucose in being dextro- 
 rotatory ([«]„ = + 83°), in reducing cupric hydrate in Trommer's test, 
 and in being directly fermentable with yeast. When oxidised by means 
 of nitric acid it, however, yields an acid called mucic acid (OgHj^Og), 
 which is only sparingly soluble in water. Glucose when treated 
 in this way yields an isomeric acid — i.e. an acid with the same 
 empirical formula, called saccharic acid, which is readily soluble in 
 water. 
 
 Inositol or Inosite was discovered by Scherer in 1850 as a constituent 
 of muscle, and for a long time was known as a muscle sugar. It occurs 
 also in small quantities in other animal organs (liver, kidney, etc.), and in 
 plants it is a fairly constant constituent of roots and leaves, especially 
 growing leaves. 
 
 It has the same molecular formula as the simple sugars (GeHiaOe), 
 but it is only faintly sweet and gives none of the chemical reactions of these 
 substances. Maquenne ascertained that it has the following constitutional 
 formula ;■— 
 
THE CARBOHYDRATES 27 
 
 HOH 
 
 i 
 
 HOH— C 0— HOH 
 
 I I 
 HOH— C C— HOH 
 
 \y 
 
 
 
 I 
 HOH 
 
 A mere glance at this formula will show that it is very different from those 
 of the sugars given on p. 23. The six carbon atoms, instead of forming an 
 open chain, are linked into a ring, as in the benzene derivatives. It is 
 in fact a reduced hexa-hydroxy-benzene. It probably represents a transition 
 stage between the carbohydrates and the benzene compounds. By a closing- 
 up of the open chain of the carbohydrate molecule its formation from the 
 latter is theoretically possible. On the other hand, the opening of the 
 inositol ring would give rise .to an open chain, and it has in fact been 
 found that lactic acid is formed' from inositol by the action of certain 
 bacteria. 
 
 PISACCHABIDES 
 
 Sucrose. — This sugar (commonly known as cane sugar) is generally 
 distributed throughout the vegetable kingdom in the juices of plants 
 and fruits, especially the sugar cane, beetroot, mallow, and sugar maple. 
 It is a substance of great importance as a food. After abundant 
 ingestion of sucrose traces may appear in the urine, but the greater 
 part undergoes inversion in the alimentary canal. 
 
 Pure sucrose is crystalline and dextro-rotatory ([a],,^ +67°). It 
 holds cupric hydrate in solution in an alkaline liquid — that is, with 
 Trommer's test it gives a blue solution. But no reduction occurs on 
 boiling. After inversion it is strongly reducing. 
 
 Inversion may be brought about readily by boiling with dilute 
 mineral acids, or by means of an inverting enzyme, such as that 
 occurring in the succus entericus or intestinal juice. It then takes up 
 water and is split into equal parts of glucose and fructose :— 
 
 ^12^220]! + HgO = CgHjaOg + CgHijOj 
 
 [sucrose] [glucose] [fructose] 
 
 With yeast, sucrose is first inverted by means of a special enzyme 
 (invertase) produced by the yeast cells, and then there is an alcoholic 
 fermentation of the monosaccharides so formed, which is accomplished 
 by another enzyme called zymase. 
 
28 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 Lactose, or milk sugar, occurs in milk. It sometimes also pccurs 
 in the urine of women in the early days of lactation or after weaning. 
 
 It crystallises in rhombic prisms (see fig. 1). It is much less 
 soluble in water than cane sugar or dextrose, and has only a slightly 
 sweet taste. It is insoluble in alcohol and ether ; aqueous solutions 
 are dextro-rotatory ([«]„= +52 '5°). 
 
 Solutions of lactose give Trommer's test, but when the reducing 
 power is tested quantitatively by Fehling's solution it is found to be a 
 less powerful reducing agent than glucose. If it required seven parts 
 of a solution of glucose to reduce a given quantity of Fehling's solution, 
 it would require ten parts of a solution of lactose of the same strength 
 to reduce the same quantity of Fehling's solution. 
 
 Lactose, like cane sugar, can be hydro- 
 lysed by the same agencies as those already 
 enumerated in connection with cane sugar. 
 The monosaccharides formed are glucose and 
 galactose : — 
 
 [lactose] [g:luco8e] [galactose] 
 
 With yeast it is first inverted, and then 
 alcohol is formed. This, however, occurs 
 slowly. 
 
 The lactic-acid fermentation which occurs when milk turns sour is 
 brought about by enzymes secreted by micro-organisms which are some- 
 what similar to yeast cells. This may also occur as the result of the action 
 of putrefactive bacteria in the alimentary canal. The two stages of the 
 lactic acid fermentation are represented by the following equations : — 
 
 (1) C,,H,Ai + H,6 = 4C3H«03 
 
 [lactose] [lactic acid] 
 
 Fia. 1. — Lactose crj'stals. 
 
 (2) ^CgHgOg = 2C^H80„ -I- 400^ + 
 
 [lactic acid] [butyric acid] 
 
 4H, 
 
 Maltose is the chief end product of the action of malt diastase on 
 starch, and is also formed as an intermediate product in the action of 
 dilute sulphuric acid on the same substance. It is also the chief sugar 
 formed from Starch by the diastatio enzymes contained in the saliva 
 (ptyalin) and pancreatic juice (amylase). It can be obtained in the form 
 of acicular crystals ; it is strongly dextro-rotatory ([a]„= -f- 140°). It 
 gives Trommer's test ; but its reducing power, as measured by Fehling's 
 solution, is one-third less than that of glucose. 
 
 By prolonged boiling with water, or, more readily, by boiling with 
 
tttte CARBOHYDRATES 
 
 29 
 
 a dilute mineral acid, or by means of an inverting enzyme, such as 
 occurs in the intestinal juice, it is converted into glucose. 
 
 ^'12^22011 + HgO = 
 
 [maltofje] 
 
 :C«H,A + C,H,,0, 
 
 [glucose] 
 
 [glucose] 
 
 The three important physiological sugars (glucose, lactose, and 
 maltose) may be distinguished from one another by their relative 
 reducing action on Fehling's solution (I'O : 0'71 : 0-63), by their rota- 
 tory power, or by the phenyl-hydrazine test described in Lesson XIII. 
 
 polysacchaei'des 
 
 Starch is widely diffused through the vegetable kingdom. It occurs 
 in nature in the form of microscopic grains, varying in size and appear- 
 ance, according to their source. Each 
 consists of a central spot (hilum) round 
 which more or less concentric envelopes 
 of starch proper or granulose alternate 
 with layers of cellulose. Cellulose has 
 very little digestive value, but starch is 
 a most important food. 
 
 Starch is insoluble in cold water : it 
 forms an opalescent solution in boiling 
 water, which if concentrated gelatinises on 
 cooling. Its most characteristic reaction 
 is the blue colour it gives with iodine 
 solution. 
 
 On heating starch with dilute mineral 
 
 [G. 2. — Section of _pea showing starch 
 and aleurone grains embedded in the 
 protoplajjm of the cells ; a, aleurone 
 grains ; si, starch grains ; i, inter- 
 cellular spaces. (Yeo, after Sachs.) 
 
 acids glucose is formed. By the action 
 
 of diastatic enzymes, maltose is the chief end product. In both cases 
 
 dextrin is an intermediate stage in the process. 
 
 Before the formation of dextrin the starch solution loses its opal- 
 escence, a substance called soluble starch being formed. This, like 
 native starch, gives a blue colour with iodine solution. Although 
 the molecular weight of starch is unknown, the formula for soluble 
 starch is probably {C^Tii(iO^)2^f,. The molecules of the dextrins are 
 smaller. Equations which represent the formation of sugars and dextrins 
 from starch are very complex, and are at present hypothetical. 
 
 Dextrin is the name given to the intermediate products in the 
 hydrolysis of starch, and two chief varieties are distinguished — erythro- 
 dextrin, which gives a reddish-brown colour with iodine solution ; and 
 achroo-dextfrin, which does not. 
 
35 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 It is readily soluble in water, but insoluble in alcohol and' ether. 
 Ifc is an amorphous yellowish powder. It does not ferment with yeast. 
 It is dextro-rotatory. By hydrolysis it is converted into glucose. 
 
 Glycogen, or animal starch, is found in liver, muscle, colourless 
 blood corpuscles, and other tissues. 
 
 Glycogen is a white tasteless powder, soluble in water, but it forms, 
 like starch, an opalescent solution. It is insoluble in alcohol and ether. 
 It is dextro-rotatory. With Trommer's test it gives a blue solution, 
 but no reduction occurs on boiling. 
 
 With iodine solution it gives a reddish or port-wine -colour, very 
 similar to that given by erythro-dextrin. Dextrin may be distin- 
 guished from glycogen by (1) the fact that it gives a clear, not an 
 opalescent, solution with water; and (2) it is not precipitated by basic 
 lead acetate as glycogen is. It is, however, precipitated by basic lead 
 acetate and ammonia. (3) Glycogen is precipitated by 55 per cent, 
 of alcohol ; the dextrins require 85 per cent, or more. (4) Glycogen is 
 completely precipitated from solution by saturation with ammonium 
 sulphate ; erythro-dextrin is only partially precipitable by this means. 
 
 Cellulose. — This is the colourless material of which the cell-walls 
 and woody fibres of plants are composed. By treatment with strong 
 mineral acids, it is, like starch, converted into glucose, but with much 
 greater difficulty. The various digestive enzymes have little or no 
 action on cellulose ; hence the necessity of boiling starch before it is 
 taken as food. Boiling bursts the cellulose envelopes of the starch 
 grains, and so allows the digestive juices to get at the starch proper. 
 Cellulose is found in a few animals, as in the test or outer investment 
 of the Tunicates. 
 
 Salting out of the Colloid Carbohydrates. — By saturating solutions of the 
 colloid carbohydrates (starch, soluble starch, glycogen, and erythro-dextrin 
 partially) with such neutral salts as magnesium sulphate or ammonium sulphate 
 the carbohydrate is thrown out of solution in the form of a white precipi- 
 tate. The remaining carbohydrates (sugars and some of the smaller mole- 
 ouled dextrins such as achroo-dextrin) are not precipitated by this means. 
 We shall find in connection with the proteins that this method, known as 
 " salting out," is one largely employed there for precipitating and distinguish- 
 ing between classes of proteins. The student is therefore warned that a 
 precipitate obtained under such circumstances will not necessarily indicate 
 the presence of protein. 
 
 Glucosides. — Glucose is an aldehyde, and therefore has the power of com- 
 bining with other compounds such as alcohols, organic acids, and phenols ; a 
 hydrogen atom of the compound unites with the oxygen of the H . C : O 
 group of the sugar, and the rest of the molecule with the caibon of the 
 
TM£ CARBOHYDRATES 31 
 
 same group ; the addition compound so formed then loses water and a sub- 
 stance known as a glucoside is left. This may be illustrated by the simple 
 glucoside which can be made synthetically by warming together (in the pres- 
 ence of anhydrous hydrochloric acid) methylic alcohol and glucose. The_ 
 reaction is shown in the following equation : — 
 
 H H H 
 
 H • C • OH H • C • OH H • C • OH 
 
 H-C-OH H-C-OH , H-C-OH 
 
 H • C • OH + CH3 • OH = H . C • OH = HaO + H • C 1 
 
 H-C-OH H-C-OH H-C-OH ^ 
 
 H-C-OH H-C-OH H ■ C'- OH I 
 
 H-C-O H-C-OH H- C- 1 
 
 bCHa 6CH3 
 
 [glucosei] [methyl alcohol] [addition [water] [methylic glucoside] 
 compound] 
 
 One notes that whereas glucose contains four asymmetric carbon atoms 
 {i.e. carbon atoms united to four different atoms or atomic groups), printed in 
 thick type, the addition compound and the glucoside each contain five. It is 
 possible to obtain two methyl glucosides, a and §, one with the dextro-con- 
 figuration of the carbon atom placed lowest in the formula, the other with the 
 laevo-configuration. In one of these the lowest group of all is OCH3, as shown 
 in the above formula ; in the other it is CH3O. 
 
 Numerous glucosides are found in nature ; thus amygdalin in bitter almonds 
 is a compound of glucose with mandelic nitrile ( = benzaldehy de + hydrocyanic 
 acid) ; salicin is a compound of glucose and salicylic alcohol ; the indican of 
 plants is a compound of glucose and indoxyl, and there are many others. 
 
 Mutarotation and Tautomerism. — The optical activity of glucose when 
 freshly dissolved is about twice as great as when the solution has stood some 
 time. If the glucose is crystallised out from this solution, and again dissolved, 
 the fresh solution has again a high rotatory power, and this sinks once more 
 on standing. It is evident that a change occurs in its constitution when it 
 is left in solution, and this change is reversed on crystallisation. Tlie • 
 explanation advanced to account for this mutarotation is that in solution 
 the sugar forms an addition compound with water (a hypothetical aldehydrol), 
 and then loses water to form a ring anhydride isomeric with the original 
 glucose and analogous to a gliicoside.' The formulae will be : — 
 
 H 
 H-C-OH 
 H-C-OH 
 
 H-C 
 
 H - C - OH 
 H-C-OH I 
 
 H-C ' 
 
 OH 
 [aldehydrol] [anhydride formula of glucose or glucoside of water] 
 
 ' This formula for glucose is a general one and does not show the typical 
 stereo-chemical arrangement depicted in the formula on p. 23. 
 
 
 H 
 
 
 H 
 
 •C- 
 
 OH 
 
 H 
 
 •C- 
 
 OH 
 
 H 
 
 • c- 
 
 OH 
 
 H 
 
 • c- 
 
 OH 
 
 H 
 
 -c- 
 
 OH 
 
 H 
 
 -c. 
 
 OH 
 
 OH 
 
S2 ESStNTlALS OP CHEMICAL PHYSIOLOGY 
 
 In the anhydride formula there are seen to be five asymmetric carbon 
 atoms, so that there will again be the possibility of an a and a ^ form. In 
 a solution of glucose which has been allowed to stand, the three isomerides, 
 the original aldehyde, and the two glucosides, are all present in equilibrium 
 with one another, and the mixture has a lower rotatory power than the 
 original glucose. These mutually transformable isomerides are termed 
 tautomeric forms of glucose. Tautomerism of this kind is a frequent 
 occurrence in organic chemistry. 
 
 Our knowledge of glycosides has thrown light on the constitution of the 
 disaccharides ; thus in maltose we have one glucose molecule forming a 
 glucoside with another glucose molecule which retains its characteristic 
 aldehyde (H . C : O) group unaltered. In lactose the place of the first 
 glucose molecule is taken by galactose ; it may thus be considered as a 
 glucose-galactoside. In sucrose, on the other hand, the carboxyl groups of 
 its two constituent hexoses (glucose and fructose) are involved in the union 
 of the two molecules, which are therefore both in the glucosidic condition-, 
 as shown in the following formula : — 
 
 GH2OH.CHOH.CH.CHOH.CHOH.CH 
 
 __0 
 
 glucose residue 
 
 o 
 
 CH^OH.C.CHOH.CHOH.CH.CH^OH 
 
 I o 1 
 
 fructose residue 
 
 This indicates that there is in sucrose no leal ^ugar group (either the 
 aldehyde group H . C : O or the ketone group : : ()) at all, and this accounts 
 for the fact that sucrose does not give the typical sugar reactions (reduction with 
 Fehling's solution, and the formation of an osazone with phenyl hydrazine). 
 
 Further information regarding the carbohydrates is given in Lesson XIII. 
 
LESSON IV 
 THE FATS AND LIPOIDS 
 
 Lard and olive oil are given round as examples of fats. 
 
 1. They are insoluble in water. 
 
 2. They dissolve readily in ether. On pouring some of the ethereal 
 solution on to a piece of blotting paper, a greasy stain is left after 
 the ether has evaporated. 
 
 3. FAT SPLITTING BY LIPASE.— Boil a few c.c. of fresh milk in 
 order to destroy any lactic acid organisms which may be present ; cool 
 under the tap, and add a few drops of glycerol extract of pancreas ^ 
 containing lijmse, a few drops of phenolphthalein solution and dilute 
 potash until a faint pink colour persists. Divide this into two portions 
 labelled A and B. Boil A to destroy the enzyme, and keep both tubes 
 at a temperature of about 36° 0. The pink colour in B Will gradually 
 disappear, showing that fatty acids have been set free from the fat by 
 the action of the fat-splitting enzyme, lipase ; A undergoes no change. 
 
 4.. SAPONIFICATION BY ALKALI.— By boiling with potash, fat 
 yields a solution of soap. On adding some sulphuric acid to this the 
 fatty acid collects in a layer on the surface of the fluid. This experi- 
 ment may conveniently be performed in the following way ; — Melt some 
 lard in an evaporating basin and pour it into a solution of potash in 
 alcohoP contained in a small flask and heated carefully on a water-hath 
 nearly to boiling point. Continue to boil and saponiflcation is soon 
 completed. The completion of the process is recognised by dropping 
 some of the solution into a test-tube containing about 10 c.c. of water; 
 the solution of soap will be clear, and no oil globules should separate 
 out. If there is any separation of oil globules continue the boiling. 
 
 Then drop the soap solution into some 25 per cent, sulphuric acid 
 contained in a small beaker ; the fatty acids soon separate out and 
 float on the surface. On cooling under the tap they solidify. 
 
 5. REACTION OF FATTY ACIDS.— Wash the fatty acid obtained 
 
 ^ This is easily prepared by mixing finely minced pig's pancreas with two 
 volumes of glycerol md straining the mijfture through muslin. 
 
 ^ 30 grammes of potash are dissolved in 20 c.c. of water, and 200 o.o. of 90 
 per-oent. alcohol are added. 
 
 3 33 
 
34 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 in experiment 4 repeatedly with water, until the wash water is no 
 longer acid, and divide it into three portions. Dissolve one portion 
 in ether; this solution reacts acid to phenolphthalein ; to show this, 
 place a few drops of phenolphthalein in 5 c.c. of alcohol containing a 
 drop of 20 per-cent. potash. If this red solution is dropped into the 
 solution of fatty acid, the colour is discharged. Place the second 
 portion of fatty acid in some half-saturated solution of sodium carbonate 
 and warm ; a solution of sodium soap is obtained and carbon dioxide 
 comes off. Note with the third portion that it produces a greasy stain 
 on paper. 
 
 6. KEACTIONS OF SOAPS.— A solution of soap may be prepared 
 by heating some stearic acid with a few c.c. of water and adding 
 caustic potash drop by drop until a clear solution results, (a) Add to 
 this solution some sulphuric . or hydrochloric acid ; the fatty acid 
 separates out as described under 4. 
 
 (b) Add to the solution powdered sodium chloride and shake ; the 
 soap is salted out and is rendered insoluble. This property of soap is 
 used in soap manufacture. 
 
 (c) Add to the solution some calcium chloride. A precipitate of 
 insoluble calcium soap is formed, and the solution loses its property 
 of frothing on shaking. 
 
 7. OSMIC ACID TEST.— Fat, if it contains olein or oleic acid, is 
 blackened by osmic acid. Try this with both the lard and the olive oil. 
 
 8. TEST FOR GLYCEROL.— The most important reaction for 
 glycerol, the other constituent of a fat, is the acrolein test, which is 
 performed in the following way : — Place some lard in a dry test-tube, 
 add some crystals of acid potassium sulphate and heat. Acrolein is 
 given off, which is recognised by its characteristic unpleasant odour, 
 and by the fact that it blackens a piece of filter paper previously 
 moistened with ammoniacal silver nitrate solution. 
 
 9. EMULSIFICATION.— (a) Take two test-tubes and label them A 
 and B. Place water in A and soap solution in B. To each add a few 
 drops of olive oil and shake. In B an emulsion is formed, but not in A. 
 
 (b) Shake a few drops of rancid oil (or olive oil containing a small 
 amount of oleic acid), with a dilute solution of potash ; an emulsion 
 is formed because the potash and free fatty acid unite to form a 
 soap. Divide this into two parts, and to one of them add a little 
 gum solution or egg albumin; the emulsion is much more permanent 
 in this specimen. These experiments illustrate the favourable action 
 of soap and of a suspending medium such as mucilage upon the 
 formation of an emulsion. 
 
THE FATS AND LIPOIDS 35 
 
 Fat is found in small quantities in many animal tissues. It is, 
 however, found in large quantities in three situations, viz. bone marrow, 
 adipose tissue, and milk. The consideration of the fat in milk is post- 
 poned to Lesson VI. 
 
 The contents of the fat cells of adipose tissue are fluid during life, 
 the normal temperature of the body (37° C, or 99° F.) being con- 
 siderably above the melting-point (25° C.) of the mixture of the fats 
 found there. These fats are three in number, and are called palmitin, 
 stearin, and olein. They differ from one another in chemical com- 
 position and in certain physical characters, such as melting-point and 
 solubilities. Olein solidifies at - 5° C, palmitin at 45° C, and stearin 
 at 53-65° C. Thus, it is olein which holds the other two dissolved at 
 the body temperature. Fats are all soluble in hot alcohol, ether, and 
 chloroform, but insoluble in water. 
 
 Chemical Constitution of the Fats. — ^The fats are compounds of 
 fatty acids with glycerol, and may be termed glycerides or glyceric 
 ethers. 
 
 The fatty acids, as we have already seen (p. 16), form a series of 
 acids derived from the monohydric primary alcohols by oxidation. 
 Formic acid is the first in the series, acetic acid comes next, and so on. 
 The sixteenth term of the series is called Palmitic acid, and has the 
 formula CjrjHgj.COOH. The eighteenth is called Stearic acid, and has 
 the formula C17H35.COOH. 
 
 Oleic acid is not a member of this series of fatty acids, but belongs 
 to a somewhat similar series of acids known as the acrylic series, of 
 which the general formula is C„_iH2„_3COOH. It is the eighteenth 
 term of the series, and its formula is Cj.^Hgg.CC)OH. 
 
 The first member of the group of alcohols from which this acrylic series 
 of acids is obtained is called alltjl alnohol (CH2 : CH.CH2OH) ; the aldehyde 
 of this is acrolein (CHs : CH.CHO), and the formula for the acid (acrylic 
 acid) is CH3 : CH.COOH. It will be noticed that two of the carbon atoms 
 are united by two valencies, and these substances are therefore unsaturated ; 
 they are unstable and are prone to undergo, by uniting with another 
 element, a conversion into compounds in which the carbon atoms are united 
 by one bond only. This accounts for their reducing action, and it is owing 
 to this construction that the colour reactions with osmic acid and Sudan III. 
 (red coloration) are due. Fat which contains any member of the aci'jlio 
 series such as oleic acid blackens osmic acid, by reducing it to a lower 
 (black) oxide. The fats palmitin and stearin do not give this reaction. 
 
 The formulae for the fatty acids may also be written in a slightly 
 different way, as shown on the next page ;— 
 
36 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 Acetic acid (CHgCOjOH 
 
 Palmitic acid (Ci5H3iCO)OH 
 
 Stearic acid .... . (Ci7H3500)OH 
 
 Oleic acid (CiyHggCoioH 
 
 Each consists of a complex group placed in the above formulae within 
 brackets, united to hydroxyl. The group within brackets is called 
 the fatty, acid radical, and the fatty acid radicals of the four just 
 mentioned acids have received the following names : — 
 
 Acetyl CHgCO is the radical of acetic acid • 
 Palmityl CjjHjjCO ,, „ palmitic acid 
 
 Stearyl CjyHjjCO „ „ stearic acid 
 
 Oleyl Cj^HiggCO „ „ oleic acid 
 
 Glycerol (popularly known as glycerin) is a trihydric alcohol, 
 03115(011)3 — i.e. three atoms of hydroxyl united to &, radial glyceryl 
 (C3H5). The hydrogen in the hydroxyl atoms is replaceable by other 
 organic radicals. As an example take the radical of acetic acid called 
 acetyl (CH3.CO). The following formulae represent the derivatives that 
 can be obtained by replacing one, two, or all three hydroxyl hydrogen 
 atoms in this way : — 
 
 OH 
 C,H5-!0H 
 
 [oh 
 
 [glycerol] 
 
 OH I fOH 
 
 C3HJ OH C3H5] O.CH3.CO 
 
 O.CH3.CO (0.OH3.OO 
 
 O.CH3.CO 
 
 C3H5]O.CH3.CO 
 
 O.CH3.CO 
 
 [triacetin] 
 
 [monoacetln] 1 [diacetin] 
 
 Triacetin is a type of a neutral fat ; stearin, palmitin, and olein 
 ought more properly to be called tristearin, tripalmitin, and triolein 
 respectively. Each consists of glycerol in which the three atoms of 
 hydrogen in the hydroxyls are replaced by radicals of the fatty acid. 
 This is represented in the following formulae : — 
 
 Palmitin C3Hg(OCj5Hgj.CO)3 
 
 Stearin 03H5(OCj^H35.CO)g 
 
 Olein CgH,(OCj.H33.CO)3 
 If we substitute the letter R for the fatty acid radical, the general 
 formula for a neutral fat may be written : — 
 
 CH.,.OR 
 
 I 
 CH.OR 
 
 I 
 CH3.OR 
 
 Decomposition Products of the Fats.^The fats split up into the 
 substances out of which they are built up. 
 
tkE FAtS AND LIPOIDS 37 
 
 Under the influence of superheated steam, mineral acids, and other 
 catalysts employed in commercial processes, a fat combines with 
 water and splits into glycerol and the fatty acid. In the body fat- 
 splitting is accomplished by an organic catalyst or enzyme known as 
 lipase. The following equation represents what occurs in a fat, taking 
 tripalmitin as an example : — 
 
 C3H,(O.Cj-H3,CO)3 + 3H,0 = C3H,(OH)3 + SC^gHg^COOH 
 
 [palmitin— a fat] [glycerol] [palmitic acid— a fatty acid] 
 
 In the process of saponification, much the same sort of reaction 
 
 occurs, the final products being glycerol and a compound of the base 
 
 with the fatty acid, which is called a soap. Suppose, for instance, that 
 
 potassium hydrate is used ; we get — 
 
 C3H,(O.Cj,H3,CO)3 + 3KH0 = 03H,(OH)3 + 3Ci,H3jCOOK 
 
 [palmitin — a fat] [glycerol] [potassium palmitate— a soap] 
 
 Emulsification. — Another change that fats undergo in the body is 
 very different from saponification. It is a physical rather than a 
 chemical change ; the fat is broken up into very small globules, such 
 as are seen in natural emulsion — milk. The conditions under which 
 emulsions are formed are described in the practical exercises at the hekd 
 of this lesson. 
 
 THE LIPOIDS 
 
 The name lipoid was originally applied by Overton to a heterogeneous 
 group of substances found in the protoplasm of all cells, especially in their 
 outer layer or cell-membrane, which, like the fats, are /soluble in such 
 reagents as ether and alcohol. These substances, though present in smaller 
 amount than proteins, appear to be essential constituents of protoplasm, and 
 the labile character of their molecules is a property many of them share 
 with the proteins. The lipoids are found mixed' with fat in the ether- 
 alcohol extract of tissues and organs, and they are specially abundant in 
 nervous tissues, wheie we shall again have to refer to them (Lesson XXI). 
 
 They may be classified in the following way : — 
 
 (1) Those which, like the fats, are free from both nitrogen and 
 phosphoius. The most important member of this group is cholesterol. 
 
 (2) Those which are free from phosphorus but contain nitrogen. These 
 yield the reducing sugar called galactose when broken up, and were teimed 
 cerebro-galactosides by Thudichum. They may be simply called galacto- 
 sides. 
 
 . (3) Those which contain both phosphorus and nitrogen. These are 
 called the phosphatides, and are grouped according to the proportion of 
 nitrogen and phosphorus in their rfiolecules, as follows : — 
 
 (a) Mono-amino-mono-phosphatides, N : P = l : 1. e.gi. lecithin and 
 
 kephalin. 
 (6) Diamino-mono-phosphatides, N : P = 2 : 1. e.g. sphingo-myelin. 
 
36 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 (c) Mono-amino-diphosphatides, N : P = l : 2. One of these, named 
 
 cuorin, has been separated out from the heart by Erlandsen, 
 and a similar substance is found in egg-yolk. 
 
 (d) Diamino-diphosphatides, N : P = 2 : 2. One of these was 
 
 separated from brain by Thudichum, but has not since been 
 examined. 
 
 (e) Triamino-mono-phosphatides, N : P = 3 : 1. One of these is 
 
 present in egg-yolk. 
 
 This classification is obviously capable of extension as new phosphatides 
 are discovered. 
 
 We may now take some of the most important of these substances and 
 describe them with greater detail. 
 
 Cholesterol or cholesterin is found in small quantities in all forms 
 of protoplasm. It is a specially abundant constituent of nervous tissues, 
 particularly in the white substance of Schwann. It is found in small 
 quantities in the bile, but it may occur there in excess and form the 
 concretions known as gall stones. It can be readily extracted from the 
 brain by the use of cold acetone. In the brain it occurs in the free state. 
 
 It is a monohydric unsaturated alcohol with the empirical formula 
 C.^H45.0H. Eecent research has shown it to belong to the terpene seiies 
 which had hitherto only been found as excretory products of plant life. 
 
 Windaus finds that it contains five reduced benzene riifgs linked together 
 and a double linkage at the end of an open chain. 
 
 Cholesterol is now believed to be not merely a waste product of 
 metabolism, but to exert an important protective influence on the body cells 
 against the entrance of certain poisons called toxins. One of the poisons 
 contained in cobra venom dissolves red blood corpuscles ; the presence of 
 cholesterol in the envelope of the blood corpuscles to some extent hinders 
 this action, and it has been stated that the administration of cholesterol 
 increases the resistance of the animal. It is certainly the case that with 
 artificial blood corpuscles, membranous bags containing haemoglobin, the 
 impregnation of the membrane with cholesterol, prevents the solvent action 
 of toxins. 
 
 In order that cholesterol and its derivatives may act in this way, it 
 is necessary that the double linkage and the hydroxyl group should be 
 intact. The latter would not be the case in an ester. 
 
 From alcohol or ether containing wUter it crystallises in the form of 
 rhombic tables, which contain one molecule of water of crystallisation : 
 these are easily recognised under the microscope (fig. 3). It gives a 
 numbei' of colour tests which we shall study under Bile (Lesson VIII). 
 
 A substance called iso-cholesterol is found in the fatty secretion of the skin 
 (sebum) ; it is largely contained in the preparation called lanoline, made from 
 sheep's wool fat. It differs from cholesterol in being dextro-rotatory instead 
 of Isevo-rotatory in solution, and in some of its colour reactions. 
 
 Cholesterols isomeric with animal cholesterol are also found in many 
 plants ; these are termed phyto-cholesterols, or phytosterols for short. 
 
 Cholesterol compounds exhibit the physical phenomenon recently studied 
 by Lehmann, namely, the formation of liquid crystals ; this is also shown by 
 
THE FATS AND LIPOIDS 
 
 39 
 
 several other lipoids. Vircliow in 1855 described what he termed "myelin 
 forms " ; if brain-substance is mixed with water, where the water touches 
 the brain material, threads ai'e observable shooting out and twisting into 
 fantastic shapes ; these are termed " myelin forms," although the word 
 " myelin " has no definite chemical meaning. It has now been shown that 
 these " myelin forms " are distorted liquid crystals due to the presence of 
 cholesterol and other lipoids. The fat globules seen in the adrenal cortex, 
 dui-ing cell proliferation in cancer, and in the liver and other organs during 
 fatty degeneration, are not wholly composed of fat, foi' the polarisation micro- 
 scope shows them to be anisotropic or doubly refracting, and further investiga- 
 tion has shown them to be lipoids in the fluid crystalline condition. Pure 
 cholesterol and pure cholesterol esters do not exhibit the phenomenon ; but 
 mixtures of cholesterol and fatty acids do. 
 
 The Cerebro-Gala'ctosides. — The substance known as piotagon can be 
 separated out from the brain by means of warm alcohol ; on cooling the 
 extract, protagon is deposited as a white pre- 
 cipitate. This, however, also contains cholesterol, 
 which can be dissolved out by ether. Another 
 method of preparing protagon is to take biuin 
 and extract the cholesterol first with cold acetone ; 
 then hot acetone is employed to extract the 
 protagon. Protagon is a substance originally 
 desciibed by Couerbe, under the name cerebrote, 
 but named protagon by Liebreioh, who regarded 
 it as a definite coinpound, and the mother sub- 
 stance of all the other phosphorised and non- 
 phosphorised constituents of the brain. It has 
 now been definitely proved in confirmation of 
 what Thudichuni stated in 1874, that protagon 
 
 is not a definite chemical unit, but a mixture of phosphorised and non- 
 phosphoiised substances in such proportions that it usually contains about 
 1 per cent, of phosphorus. By treatment with appiopriate solvents and 
 recrystallisation, protagon can be separated into its constituents ; those 
 which aie free from phosphorus and sulphur and comprise about 70 per 
 cent, of the original protagon are the galactosides. Although these have 
 received many names, the known galactosides are only two in number, 
 namely, phrenodn and herasin. The former is a crystalline product and 
 is dextro-rotatory, the latter is of somewhat waxy consistency and is Isevo- 
 rotatory. Phrenosin (also called cerebrin or oerebron) yields on decom- 
 position three substances : — 
 
 (1) A reducing sugar, galactose. 
 
 (2) A base termed sphingosine (C17H35NO2), which represents an un- 
 saturated mono-amino-dihydroxy alcohol. 
 
 (3) A fatty acid called phrenosinic (or neuro-stearic acid), which has the 
 constitution of o-hydroxypentacosanic acid if^^^^i^z). 
 
 Kerasin also yields galactose and sphingosine, but the fatty acid is 
 different ; it is lignoceric acid, C2JH48O2. 
 
 Tlie Phosphatides. — The best known of these is lecithin. This is a 
 
 t'lG. 3.— Cholesterol crystals. 
 
40 ESSENTIALS Of* chemical physiology 
 
 very labile,substance, but it yields on decomposition four materials ; namely, 
 glycerol and phosphoric acid united together as glycero-phosphoric acid, 
 two fatty acid radicals, of which one is usually oleyl, and an ammonium- 
 like base termed choline (CsHuNOa). The fatty acid radicals are united to 
 glycerol as in an ordinary fat, the place of the third fatty acid radical being 
 taken by the radical of phosphoric acid, which in its turn is united in an 
 ester-like manner to the choline. The formula of choline is 
 
 CrLgv /CHo — OHoOH 
 
 chAn/ 
 
 OH3/ \0H 
 
 Kephalin resembles lecithin in being a mono-amino-mono-phosphatide. 
 It differs from lecithin in being insoluble in alcohol. On decomposition it 
 yields glycero-phosphoric acid, certain fatty acids which are less saturated 
 than oleic acid, and probably belong to the ILnoleic series. Instead of 
 choline it yields amino-ethyl alcohol (hydroxylethylamine). Kephalin is the 
 most abundant phosphatide in nerve-fibres, and has also been found in 
 «gg-yolk. . 
 
 Sphingomyelin is the phosphatide obtained from the mixture called pro- 
 tagon. It is the best known of the diamino-mono-phosphatides. If protagon 
 is dissolved in hot pyridine, and the solution allowed to cool, sphingomyelin 
 is precijpitated in an impure form as sphsero-crystals, which in suspension 
 rotate the plane of polarised light to the left. Choline, sphingosine and fatty 
 acids have been found among its cleavage products. It, however, differs 
 from lecithin by containing no glycerol. 
 
 Jecorin is a substance first separated from the liver by Drechsel and 
 since found in other organs. It appears to be a mixture or possibly a 
 compound of kephalin or a diamino-mono-phosphatide with sugar. 
 
 The Luteins or Lipochromes. — The yellow or orange-red pigments 
 which usually accompany fats in the animal organism, were called luteins 
 by Thudichum, who was the first to recognise, by spectral analysis, their 
 identity with the yellow flower pigments which are now called caiotinoids. 
 The two best-known representatives are carotin (CjoHj^), which is an un- 
 saturated hydrocarbon, and xanthophyll (C45H55O2), an oxide of carotin. 
 Their isolation from animal tissues presents great difficulties, owing to the 
 small amounts present (only 045 gr. carotin were obtained from 10,000 cows' 
 ovaries) and owing to their solubility in the usual solvents for fat, from 
 which they cannot be easily separated. They are, however, in distinction 
 from fats, not affected by saponification. In carbon disulphide solution they 
 possess typical absorption bands in the blue end of the spectrum. The lutein 
 of the ovaries, of milk and butter, consists of carotin. The lutein 
 of egg-yolk is isomeric with xanthophyll. Serum lutein (cow) consists 
 mainly of carotin, which seems to be present in a water-soluble combination 
 with serum albumin. These pigments can apparently not be synthesised by 
 the animal, but are taken up from the food, the colour of the egg, milk, or 
 butter depending greatly upon the amount of the pigments available in the 
 vegetable food given. 
 
LESSON V 
 
 THE PEOTEINS 
 
 1. TESTS FOE PROTEINS.— The following tests are to be tried 
 with a mixture of one part of white of egg to ten of water. (Egg- 
 white contains a mixture of albumin and globulin.) 
 
 (a) Heat Coagulation. — Faintly acidulate with a few drops of 
 2 percent, acetic acid and boil. The protein is rendered insoluble 
 (coagulated protein). 
 
 (b) Filter some of the egg-white solution, and acidify with a 
 few drops of 20 percent, acetic acid; a drop of potassium ferro- 
 cyanide then gives a white precipitate. 
 
 (c) Precipitation with Nitric Acid. — The addition of strong nitric 
 acid to the original solution also produces a white precipitate. 
 
 (d) Xanthoproteic Reaction. — On boiling the white precipitate 
 produced by nitric acid it turns yellow ; after cooling add ammonia ; 
 
 ,the yellow becomes orange. ^ 
 
 (e) MUlon's Test. — Millon's reagent ^ gives a white precipitate, 
 which turns brick-red on boiling. 
 
 (f) Rose's or Piotrowski's Test. — Add one drop of a 1 per-cent. 
 solution of cupric sulphate to the original solution and then caustic 
 potash, and a violet solution is obtained. 
 
 Repeat experiment (f) with a solution of commercial peptone, 
 and note that a rose-red solution is obtained. This is called the 
 biuret reaction. 
 
 (g) Rosenheim's Formaldehyde Reaction. — Add to the solution of 
 commercial peptone a very dilute solution of formaldehyde (1 : 2500), 
 and then about one-third of the volume of strong sulphuric acid 
 containing (as most commercial specimens of the acid do) a trace 
 of an oxidising agent such as ferric chloride or nitrous acid. A 
 purple ring develops at the surface of contact. This reaction is 
 the foundation of the original Adamkiewicz reaction. 
 
 (h) Adamki^icz Reaction ; here glacial acetic acid was used instead 
 
 ' Consult p. 56 for an explanation of this and the following colour react ions. 
 
 ^ Mercury is dissolved in its own weight of nitric acid. The solution so 
 obtained is then diluted with twice its volume of water. The decanted clear 
 liquid is Millon's reagent ; it is a mixture of the two nitrates of mercury con- 
 taining excess of nitric acid. 
 
 41 
 
42 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 of the formaldehyde. Most commercial specimens of glacial acetic 
 acid contain hydrogen peroxide as an impurity ; the oxidising action 
 of this on the acetic acid leads to the formation of traces of glyoxylic 
 acid and formaldehyde ; the necessary factors for the occurrence of 
 the formaldehyde reaction are thus present. Glyoxylic acid giveB the 
 reaction by virtue of its decomposition into formaldehyde, which, 
 when present in minute traces, also reacts with pure sulphuric acid. 
 
 The same Reactions (g and h) are given by the solution of egg-white, 
 but not so markedly. 
 
 2. ACTION OF NEUTRAL SALTS.— (a) Saturate the solution of 
 egg-white with magnesium sulphate by adding crystals of the salt and 
 grinding it up thoroughly in a mortar. A white precipitate of egg- 
 globulin is produced. Filter. The filtrate contains egg-albumin. The 
 precipitate of the globulin is very small. 
 
 (b) Half saturate the solution of egg-white with ammonium sulphate. 
 This may be done by adding to the solution an equal volume of a 
 saturated solution of ammonium sulphate. The precipitate produced 
 consists of the globulin ; the albumin remains in solution. 
 
 (c) Completely saturate another portion with ammonium sulphatb 
 by adding crystals of the salt and grinding in a mortar — a precipitate 
 is produced of both the globulin and albumin. Filter. The filtrate 
 contains no protein. The protein precipitate may be readily seen in 
 the crystal-magma by suspending the contents of the filter paper in 
 saturated ammonium sulphate solution. 
 
 (d) Repeat the last experiment (c) with a solution of commercial 
 peptone. A precipitate is produced of the proteoses it contains. 
 Filter. The filtrate contains the true peptone. This gives the biuret 
 reaction (see above), but large excess of strong potash must be added 
 on account of the presence of ammonium sulphate. Ammonium 
 mlphate added to saturation precipitates all proteins except peptone 
 
 3. ACTION OF ACIDS AND ALKALIS ON ALBUMIN.— Take 
 three test-tubes and label them A, B, and C. In each place an equal 
 amount of diluted egg-white. 
 
 To A add a few drops of 01 per-cent. solution of caustic potash. 
 
 To B add the same amount of O'l percent, solution of caustic 
 potash. 
 
 To C add a rather larger amount of 01 per-cent. sulphuric acid. 
 
 Put all three into the warm bath ^ at about the temperature of 
 the body (36-40° C). 
 
 After five minutes remove test-tube A, and boil. * The protein is 
 
 1 A convenient form of warm bath suitable for class purposes may be made by 
 placing an ordinary tin pot half full of water over a bent piece of iron which adts 
 as a warm stage. The stage is kept warm by a small gas flame. 
 
THE PROTEINS 43 
 
 no longer coagulated by heat, having been converted into alhali-meta- 
 protein. After cooling, colour with litmus solution and neutralise 
 with O'l per-cent. acid. At the neutral point a precipitate is formed 
 which is soluble in excess of either acid or alkali. 
 
 Next remove B. This also now contains alkali-metapfotein. Add 
 to it a few drops of sodium phosphate, colour with litmus, and neutralise 
 as before. Note that the alkali-metaprotein now reauires more acid 
 for its precipitation than in A, the acid which is first added converting 
 the sodium phosphate into acid sodium phosphate. This exercise shows 
 that the presence of inorganic salts which react with acids may modify 
 the reactions of alkali-metaprotein. 
 
 Now remove C from the bath. Boil it. Again there is no coagula- 
 tion, the proteins having been converted into acid-metaprotein. After 
 cooling, colour with litmus and neutralise with 0"1 per cent, alkali. 
 At the neutral point a precipitate is formed, soluble in excess of acid 
 or alkali. (Acid-metaprotein is formed more slowly than alkali-meta- 
 protein, so it is best to leave this experiment to the last.) 
 
 Other acids, such as acetic or oxalic, may be employed instead of 
 sulphuric acid for making acid-metaprotein. The following exercise 
 with oxalic acid gives good results : — To half a test-tubeful of diluted 
 egg-white add 5 to 10 drops of a saturated solution of oxalic acid. 
 Keep the mixture at a temperature of 40-50° 0. for a few minutes. 
 Then gradually heat the solution to boiling point ; no coagulum results. 
 
 4. GELATIN. — Take some gelatin and dissolve it in hot water. 
 On cooling, the solution sets into a jelly (gelatinisation). 
 
 Take a dilute solution of the gelatin, and try aU the protein tests 
 with it enumerated on p. 41. Carefully note down your results. 
 
 5. KERATIN. — Suspend some horn shavings or hair in water, and 
 try all the colour tests for protein with this. Bepeat also with this 
 the sulphur test (Lesson I, Exercise 7 b, p. 7). 
 
 6. MUCIN. — Add a few drops of acetic acid to some saliva. A 
 stringy precipitate of mucin is formed. 
 
 7. MUCOID. — A tendon has been soaked for a few days in lime 
 water. The fibres are not dissolved, but they are loosened from one 
 another owing to the solution of the interstitial or ground substance 
 by the lime water. Take some of the lime water extract and add acetic 
 acid. A precipitate of mucoid is obtained. The fibres themselves 
 consist of collagen, which yields gelatin on boiling. Vitreous humour 
 or the Whartonian jelly of the umbilical cord is much richer in ground 
 substance than tendon, and, if treated in the same way, a much larger 
 yield of mucoid is obtained. 
 
44 ESSENTIALS Op CHEMICAL PHYSIOLOGV 
 
 The Proteins are the most important substances that occur in 
 animal and vegetable organisms, and protein metabolism is the most 
 characteristic sign of life. 
 
 They are highly complex compounds of carbon, hydrogen, oxygen, 
 nitrogen, and sulphur occurring in a solid viscous condition, or in 
 solution in nearly all parts of the body. The different members of the 
 group present great similarities, for instance, in the large size of their 
 molecules, and in giving certain colour tests ; there are, on the other 
 hand, considerable differences between the various proteins. 
 
 The proteins in the food i^rm the source of the proteins in the body 
 tissues, but the latter are usually different in composition from the 
 former. The food proteins are in the process of digestion broken up 
 into simple substances, usually called cleavage products, and it is from 
 these that the body cells reconstruct the proteins peculiar to themselves. 
 As a result of katabolic processes in the body the proteins are finally 
 again broken down, carbonic acid, water, sulphuric, acid (combined as 
 sulphates), urea, and creatinine being the principal final products which 
 are discharged in the urine and other excretions. The intermediate 
 substances between the proteins and such final katabolites as urea will 
 be discussed under Urine. 
 
 The following figures will show how difierent the proteins are even 
 in elementary composition : Hoppe-Seyler many years ago gave the 
 variations in percentage composition as follows : — 
 
 
 c 
 
 H 
 
 N 
 
 s 
 
 
 
 From 
 
 51-5 
 
 6-9 
 
 15-2 
 
 0-3 
 
 20-9 
 
 To 
 
 54-5 
 
 7-3 
 
 17-0 
 
 2-0 
 
 23-5 
 
 and recent research has since shown that the variations are even 
 greater than those given by Hoppe-Seyler. 
 
 Differences are also seen when the cleavage products are separated 
 and estimated. These differ both in kind and in amount, but nearly 
 all of them are substances which are termed am.ino-acids. We know 
 now that the proteins are linkages of a greater or lesser number 
 of these amino-acids, and there is hope that in the future this know- 
 ledge will lead to an actual synthesis of the protein molecule, and 
 with that will come an accurate knowledge of its constitution. 
 
 When the protein molecule is broken down in the laboratory by 
 processes similar to those brought about by the digestive enzymes 
 which occur in the alimentary canal, the essential change is due to 
 what is called hydrolysis : that is, the molecule unites with water and 
 tihen breaks up into smaller molecules. The first cleavage products, 
 
THE PROTEINS 
 
 45 
 
 which are called proteoses, retain many of the characters of the original 
 protein ; and the same is true, though to a less degree, of the peptones, 
 which come next in order of formation. The peptones in their turn 
 are decomposed into short linkages of amino-acids which are called 
 polypeptides, and finally the individual amino-acids are obtained 
 separated from each other. 
 
 What we have already learnt about the fatty acids will help us in 
 understanding what is meant by an amino-acid. 
 
 If we take acetic acid, which is 6ne of the simplest of the fatty 
 acids, we see that its formula is 
 
 CH3.COOH. 
 
 If one of the three hydrogen 
 atoms in the CHg group is replaced 
 by NHj, we get a substance which 
 has the formula 
 
 CH2.NH2.COOH. 
 
 The combination NH2 which has 
 stepped in is called the amino- 
 group, and the new substance now 
 formed is called amino-acetic acid> 
 it is also termed glycine or glycocoll. 
 
 We may take another example from another fatty acid. Propionic 
 acid is CgHj.COOH ; if we replace an atom of hydrogen by the amino- 
 group as before, we obtain CjH^.NH^.OOOH, which is amino-propionic 
 acid or alanine. 
 
 If instead of propionic we take hydroxy-propionic acid, its amino- 
 derivative (amino-hydroxy-propionic acid) is termed serine. 
 
 •A fourth amino-acid is similarly obtained by the introduction 
 
 Amino-valeric acid 
 
 Fia. 4.— Leucine crystals. 
 
 C,Hc,.COOH. 
 
 of the NHj into valeric acid 
 O^Hj.NH^.COOH is called Valine. 
 
 Going to the next fatty acid in the series, caproic acid CgHjj.COOH, 
 we obtain from it in an exactly similar way, CjHjq.NHj.COOH, which 
 is amino-caproic acid or leucine. Impure leucine crystallises in 
 spheroidal clumps of crystals, as shown in fig. 4. With pure leucine 
 the needle-like crystals are obtained separately. 
 
 According to the way 'in which the amino-group is linked, a large number 
 of isomeric amino-caproic acids, all with the same empirical formula, are 
 theoretically possible. Many of these have been prepared synthetically, and 
 it has been shown that the amino-caproic acid called leucine, formed by 
 hydrolysis from most proteins, is the Isevo-rotatory variety, and 'should be 
 
46 
 
 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 more accurately named a-amino-isobutyl-acetic acid. Normal a-amino-caproic 
 acid is, however, stated to be obtained from the proteins of brain, and was 
 called, on account of its sweet taste, glycoleucine by its discoverer 
 (Thudichum). Abdeuhalden calls it norleucine. The graphic formulae of the 
 two substances are : — 
 
 Normal 
 
 a-amino-caproic 
 
 acid 
 
 OH., 
 
 CH„ 
 
 CH. 
 
 CH., 
 
 Iso-butyl 
 
 a-amino- 
 
 acetic acid 
 
 HoCCHq 
 
 V 
 CH 
 
 I 
 CH, 
 
 CH.NH., 
 
 COOH 
 
 \ 
 
 I 
 CH.NH., 
 
 COOH 
 
 [Glycoleucine.] 
 
 [Leucine.] 
 
 All the five amino-acidf mentioned (glycine, alanine, serine, valine, 
 and leucine) are found among the final cleavage products of most proteins. 
 
 A second group of amino-acids is obtained from fatty acids, which 
 
 contain two carboxyl (COOH) groups in their molecules. The most 
 
 important of the amino-derivatives obtained from these dicarboxylic 
 
 3,cids are : — 
 
 Amino-succinamic acid (asparagine). 
 
 Amino-succinic acid (aspartic acid), 
 
 Amino-glutaric acid (glutamic acid). 
 
 The third group of amino-acids is a very important one ; these are 
 termed the aromatic amino-acids ; that is, amino-acids united to the 
 benzene ring, and of these we will mention two, namely, phenyl-alanine 
 and tyrosine. We shall also have to consider a nearly related substance 
 called tryptophane. 
 
 Phenyl-alanine is alanine or amino-propionic acid in which an atom 
 of hydrogen is replaced by phenyl (C^H,). 
 
 Propionic acid has the formula CjHj.COOH. 
 
 Alanine (amino-propionic acid) is C,H^NH.i.COOH. 
 
 Phenyl-alanine is CuH^.CjHg.NH^.COOH. " 
 
 The formula of phenyl-alanine may also be written in another way. 
 
 If an H in the benzene ring (see p. 18) is replaced by the side 
 chain CH^CH.NH^.COOH, we obtain the formula of phenyl-alanine : — 
 
 CH,.CHNH,COOH 
 
THE PROTEINS 
 
 47 
 
 the remainder of the benzene ring, which is unaltered, being repre- 
 sented as usual by a simple hexagon. 
 
 Tyrosine is a little more complicated; it is p-oxyphenyl-alanine ; 
 that is, instead of phenyl (C^Hj) in the formula of phenyl-alanine, we 
 have now oxjrphenyl (CijH^.OH) ; this gives us 
 
 (C„H^.OH)C2H3.NH2.COOH 
 as the formula for tyrosine written one way, or 
 HO 
 
 CH2.CHNH2.COOH 
 
 when written in the other way. 
 Tyrosine crystallises in collec- 
 tions of very fine needles (see 
 fig. 5). 
 
 Tryptophane is more com- 
 plex still ; it is indole amino- 
 propionic acid : that is, amino- 
 propionic acid united to another 
 ringed derivative called indole. 
 Tryptophane is the portion of 
 the protein molecule which is the 
 parent substance of two evil- 
 smelling products of protein de- 
 composition called indole and scatole or methyl indole. Indole (CgHjN) 
 is a combination of the benzene and pyrrol rings, as shown below : — 
 
 Fig. 5. — Tyrosine crystals. 
 
 CH 
 
 HC 
 
 HC 
 
 CH 
 
 Tryptophane is the radical in the protein molecule which is 
 responsible for the colour test called the Adamkiewicz reaction. 
 
 In all the preceding cases, there is only one replacement of an 
 atom of hydrogen by NHg ; hence they may be grouped together as 
 mono-amino-acids. 
 
48 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 Passing to the next stage in complexity, we come to another group 
 of amino-acids which are called diamino-a^ids ; that is, fatty acids in 
 which two hydrogen atoms are replaced by NH.^ groups. Of these we 
 may particularly mention lysine, ornithine, arginine, and histidine. 
 
 Lysine is diamino-caproic acid. Caproic acid is CjHjj.COOH. 
 Mono-amino-caproic acid or leucine, we have already learnt, is 
 C.H^(,.NH.,.iDOOH. Lysine or diamino-caproic acid is C,Hc,.(NH2)2- 
 COOH. 
 
 Ornithine is diamino-valeric acid, and the following formulae will 
 show its relationship to its parent fatty acid : — 
 C^HgCOOH is valeric acid. 
 C^Hj(NH2)2COO'H is diamino-valeric acid or ornithine. 
 
 Arginine is a somewhat more complex substance, which icontains 
 the ornithine radical. It belongs to the same group of substances as 
 creatine, another important cleavage product of the protein molecule. 
 Creatine is methyl-guanidine acetic acid, and has the formula 
 
 HN 
 
 ^C — N(CH3)CH2.COOH 
 
 On boiling it with baryta water, it takes up water (HjO) and splits at 
 the dotted line into urea (CO(NH,),) and sarcosine, as shown below. 
 
 H,N 
 
 V = 
 
 NH.CH3.CH2.COOH 
 
 [urea] [sarcosine or niethyl-glycine] 
 
 Arginine splits in a similar way, urea being split off on the left, 
 and ornithine instead of sarcosine on the right. Arginine is, therje- 
 fore, a compound of ornithine with a urea group. 
 
 Histidine, though not strictly speaking a diamino-acid, is a diazine 
 derivative (imidazole-amino-propionic acid) and so may be included in 
 the same group. 
 
 These substances we have hitherto described as acids, but they 
 may also play the part of bases, for the introduction of a second basic 
 group into the fatty acid molecules confers upon them basic properties. 
 The three substances : — 
 
 Lysine . . CgHj^N^O, 
 
 Arginine . O^Hj^N^Og 
 
 Histidine . C^HgNgO, 
 
THE PROTEINS 49 
 
 are in fact often called the hexone bases, because each of them contains 
 6 atoms of carbon, as the above empirical formulse show. 
 
 Cystine is a complex diamino-acid in which sulphur is present, and 
 in which the greater part of the sulphur of the protein molecule is 
 contained. The sulphur test described in Lesson I, Exercise 7 6, p. 7, 
 is due to the presence of the cystine group in the protein molecule, and 
 it is particularly well shown by such proteins as keratin, which are rich 
 in cystine. 
 
 Chemically it is di- (thio amino propionic acid), and its formula is 
 
 C xio — S — S — CH., 
 
 I I ' 
 
 CH.NH, CH.NH., 
 
 I I ■ 
 
 COOH COOH 
 
 We may summarise what we have learnt up to this point by 
 enumerating, the principal members of these various groups of amino- 
 acids : — 
 
 (1) The mono-amino-acids : 
 
 (a) Of the mono-carboxylic group : glycine, alanine, serine, 
 valine, and leucine. 
 
 (6) Of the dioa/rhoxylic group : asparagine, aspartic acid, and 
 glutamic acid. 
 
 (c) Of the ringed group : phenyl-alanine, tyrosine, and trypto- 
 phane. 
 
 (2) The diamino-acids : lysine, ornithine, arginine, and cystine. 
 We have still to mention : — 
 
 (3) Pyrrolidine Derivatives. — These are derivatives of a ring which 
 reminds us of the benzene ring, but nitrogen is included in the ring- 
 formation ; the most important are pyrrolidine-carboxylic acid, or 
 proline, and its hydroxy derivative oxyproline. The formula for proline, 
 which is a very constant product of protein cleavage, is 
 
 H,C— CH, 
 
 "I I 
 H„C CH.COOH 
 
 NH 
 
 (4) Pyrimidine Bases. — These are derivatives of the pyrimidine 
 ring, another heterocyclic nucleus. 
 
 The pyrimidine bases obtainable from the cleavage of certain 
 " 4 
 
50 
 
 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 proteins (nucleo-proteins) are cytosine, thymine, and uracil. Their 
 formulae are as follows : — 
 
 HN— CO 
 
 I I 
 00 CH 
 
 I II 
 HN— CH 
 
 [uracil or dioxypyrimidine] 
 
 HN— CO 
 
 I I 
 OC C.CH, 
 
 I II 
 HN— CH 
 
 [thymine or methyl uracil] 
 
 HN— C.NHg 
 
 I I 
 OC CH 
 
 I II 
 HN— CH 
 
 [cytosine or amino oxypyrimidine] 
 
 (5) Purine Bases. — These, like the preceding, are obtained from the 
 nucleic acid complex of nucleo-proteins, and will be described on p. 64. 
 
 (6) Ammonia. 
 
 Our list now represents the principal groups of chemical nuclei 
 united together in the protein molecule, and its length makes one 
 realise the complicated nature of that molecule and the difficulties 
 which beset its investigation. We may put the problem another way. 
 In the simple sugars, with sfx atoms of carbon, there are as many as 
 twenty-four different ways in which the atomic groups may be linked 
 up ; the formulae on p. 23 give only three of these which represent the 
 structure of glucose, fructose, and galactose ; but the majority of the 
 remainder have also been prepared by the chemists. The molecule of 
 albumin has at least 700 carbon atoms, so the possible combinations 
 and permutations must be reckoned by many thousands. 
 
 Many workers are steadily analysing the various known proteins, 
 taking them to pieces and identifying and estimating the fragments. 
 The following brief table gives the results obtained with some of the 
 cleavage products of a few proteins. The numbers given are per- 
 centages : — 
 
 
 e 
 
 
 "3 
 
 Id 
 
 
 S 
 
 is 
 
 i 
 
 3 
 E 
 
 E 
 o 
 
 
 J2 
 
 < 
 
 B 
 
 u 
 
 a; 
 
 1 
 
 bo 
 
 o 
 
 3 
 s 
 
 a j= 
 1° 
 
 
 
 IS 
 .S8 
 
 P- 
 
 e 
 s 
 
 a 
 
 is 
 
 ■S 2 
 
 3 
 
 
 
 
 
 
 
 
 H — 
 
 
 
 Glycine 
 
 
 
 
 
 3-6 
 
 0-.5 
 
 lli-5 
 
 4-7 
 
 1-2 
 
 
 
 02 
 
 Leucine 
 
 20 
 
 61 
 
 18-7 
 
 10-5 
 
 21 
 
 7-1 
 
 15-5 
 
 18-6 
 
 5-6 
 
 Glutamic acid 
 
 7-7 
 
 8-0 
 
 8-5 
 
 110 
 
 0-9 
 
 3-7 
 
 17-2 
 
 18-3 
 
 37-3 
 
 Tyrosine 
 
 2-1 
 
 1-1 
 
 2-5 
 
 4-5 
 
 
 
 3-2 
 
 2-1 
 
 3-5 
 
 1-2 
 
 Arginine 
 
 
 
 
 4-8 
 
 7-6 
 
 
 11-7 
 
 1-2 
 
 3-2 
 
 Tryptophane 
 
 + 
 
 + 
 
 + 
 
 1-5 
 
 
 
 
 + 
 
 
 
 + 
 
 Cystine 
 
 2-5 
 
 0-3 
 
 0-7 
 
 0-06 
 
 «{ 
 
 More 
 than 10 
 
 0-2 
 i 
 
 
 0-4 
 
THE PROtElNS 51 
 
 Such numbers, of course, are not to be committed to memory, 
 especially as the methods of analysis cannot in all cases be considered 
 satisfactory, but they are sufficient to convey to the reader the 
 differences between the proteins. There are several blanks left, on 
 account of no estimations having yet been made. Where the sign + 
 occurs, the substance in question has been proved to be present, but 
 not yet determined quantitatively. Among the more striking points 
 brought out are : — • 
 
 1. The absence of glycine from albumins. 
 
 2. The high percentage of glycine in gelatin. 
 
 3. The absence of tyrosine and tryptophane in gelatin. 
 
 4. The high percentage of the sulphur-containing substance (cystine) 
 in keratin. 
 
 5. The high percentage of glutamic acid in vegetable proteins. 
 
 We have next to consider the way in which the amino-acids are 
 linked together into groups ; and the culmination of this branch of 
 research will be the discovery of the way in which such groups are 
 linked together to form the protein molecule. 
 
 The groups are termed polypeptides; many of these have been 
 made synthetically in the laboratory, and so the synthesis of the 
 protein molecule is foreshadowed. 
 
 We may take as our examples of the polypeptides some of the 
 simplest, and may write the formulae of a few amino-acids as follows': — 
 
 NHj.CH^.COOH Glycine 
 
 NHg.CjH^.COOH Alanine 
 
 NHj.CjHip.COOH Leucine 
 or in general terms 
 
 HNH.R.COOH. 
 
 Two amino-acids are linked together as shown in the following 
 formula : — 
 
 HNH.R.CO \ OH.H 
 
 NH.R.COOH. 
 
 What happens is that the hydroxyl (OH) of the carboxyl (COOH) 
 group of one acid unites with one atom of the hydrogen of the next 
 amino (HNH) group, and water is thus formed as shown within the 
 dotted lines : this is eliminated and the rest of the chain closes 
 up. In this way we get a dipeptide. The names glycyl, alanyl, 
 leucyl, etc., are given to the NHj.R.CO group which replaces the 
 hydrogen of the next NH., group. Thus glycyl-glycine, glycyl-leucine, 
 leucyl-alanine, alanyl-leucine, and numerous other combinations are 
 
5'2 ESSENTIALS OP CHEMICAL PHYSIOLOGY 
 
 obtained. If the same operation is repeated we obtain tripeptides 
 (leucyl-glycyl-alanine, alanyl-leucyl-tyrosine, etc.) ; then come the 
 tetrapeptides and so on. In the end, by coupUng the chains sufficiently 
 often, and in appropriate order, Fischer has already obtained substances 
 which give some of the reactions of peptone. 
 
 Hausmann's Method. — The ideal aim of the chemist would be to 
 separate the complex mixture of cleavage products quantitatively in 
 such a way as to account for the whole of the carbon, nitrogen, sulphur, 
 etc., in the original protein. This idea has not yet been attained on 
 account of the secondary reactions taking place during hydrolysis, such 
 as formation of brown and black pigments, splitting off of carbonic 
 acid, etc. Even with the best methods at his disposal, Fischer and his 
 colleagues succeeded in separating at the utmost 50 to 70 per cent, of 
 the amino-acids present in the cleavage products, and the chief loss 
 appears to be in the mono-amino-acids. A new method recently 
 introduced by Dakin has, however, given better results. 
 
 Under these circumstances it is of the greatest value to be able to 
 obtain, by a short and trustworthy procedure, at least an approximate 
 knowledge of the nitrogen distribution in the protein molecule, even if 
 this does not allow us to determine quantitatively the individual cleavage 
 products. Such a method has been worked out, mainly in Hofmeister's 
 laboratory, by Hausmann, and has been subsequently used by Osborne 
 and others. 
 
 Hausmann's method is shortly as follows : — The total nitrogen of 
 the protein is estimated by KJeldahl's method. A weighed amount 
 of the substance is then hydrolysed by means of hydrochloric acid. 
 After complete hydrolysis the cleavage products are separated into 
 three classes and the nitrogen estimated in each as — 
 
 1. Amide-N or ammonia nitrogen. This comprises the nitrogen 
 of that part of the protein molecule which is easily split off as 
 ammonia, and is determined by distilling off the ammonia after adding 
 magnesia. 
 
 2. Diamino-N. The fluid, free from ammonia, is precipitated by 
 phosphotungstic acid, and the nitrogen present in the precipitate 
 determined. This represents the nitrogen of the diamLno-acids (histi- 
 dine, arginine, etc.). 
 
 3. Mono-amino-N. This is the nitrogen contained in the residual 
 fluid after removal of the amide and diamino-N. 
 
 This method has furnished most valuable information when applied 
 to different animal and vegetable proteins, as is shown in the following 
 table from the analyses of Osborne : — 
 
THE PROTEINS 
 
 
 Total N. ■ 
 
 Amide-N. 
 
 Diamino-N. 
 
 Mono- 
 amino-N. 
 
 Zein (maize) .... 
 
 16-l.S 
 
 2-97 
 
 0-49 
 
 12-51 
 
 Hordein (barley) 
 
 17-21 
 
 4-01 
 
 0-77 
 
 12-04 
 
 Gliadln (wheat) 
 
 17-66 
 
 4-20 
 
 0-98 
 
 12-41 
 
 Glntenin (wheat) 
 
 17-49 
 
 3 -.30 
 
 2-05 
 
 11-95 
 
 Leuoosin (wheat) 
 
 16-93 
 
 1-16 
 
 3-50 
 
 11-83 
 
 Edestin (hemp) 
 
 18-64 
 
 1-88 
 
 5-91 
 
 10-78 
 
 Caseinogen (milk) . 
 
 15-62 
 
 1-61 
 
 3-49 
 
 10-31 
 
 These figures show interesting differences between otherwise similar 
 proteins. New characteristics are given for some protein groups, e.g. the 
 alcohol-soluble vegetable proteins, which possess a high amide-N and 
 low diamino-N. In Osborne's analyses (not given) of various edestins, 
 great differences of the diamino-N were revealed. The method has also 
 proved useful for the differentiation of proteoses, and interesting deduc- 
 tions as to the food value of various proteins have been drawn from its 
 results. As 80 to 90 per cent, of the carbon of proteins (according to 
 Kossel) is present in combination with nitrogen, the method is likely to 
 give important clues as to the constitution of different proteins. 
 
 Van Slyke's Method. — A further differentiation of the units in 
 the protein molecule has been made possible by a method Van Slyke has 
 introduced. The details of the method are given in Lesson XVIII ; it 
 • is an application of the well-known reaction of nitrous acid on sub- 
 stances containing an amino-group. Since nitrous acid liberates nitrogen 
 only from the amino-group, it is possible, by estimating the total 
 nitrogen, to determine by difference the non-amino-nitrogen in a protein 
 (that is, the part of the nitrogen which is in heterocyclic combination in 
 proline, oxyproline, tryptophane, and histidine). By making use of these 
 facts, and applying them to Hausmann's metljod. Van Slyke has been 
 successful in determining from 98 to 100 per cent, of the nitrogenous 
 products of a complete protein hydrolysis, and the whole operation can 
 be carried out ^vith 2 or .3 grammes of the protein material. After 
 complete hydrolysis of the protein, the ammonia nitrogen is estimated 
 by vacuum distillation after adding magnesia. Arginine, histidine, 
 lysine, and cystine are precipitated, as in Hausmann's method, by 
 phosphotungstic acid ; this precipitate is dissolved, and the total nitrogen 
 and the ajnino-nitrogen in it are estimated. The difference gives the 
 non-amino-nitrogen in the histidine (which contains two-thirds non- 
 amino-nitrogen) and arginine (which contains three-quarters non-amino- 
 nitrogen). The remaining nitrogen of this fraction is contained in the 
 
54 
 
 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 bases lysine and cystine ; these contain only amino-nitrogen ; cystine is 
 , determined separately by a sulphur estimation, and lysine by difference. 
 Of the other pair of amino-acids, arginine is estimated by boiling with 
 potash, which splits off half its nitrogen as ammonia, and histidine by 
 difference. The mono-amino-acids contained in the phosphotungstic 
 filtrate are separated into two fractions ; (1) the acids which contain 
 only amino-nitrogen, and (2) those which also contain secondary 
 nitrogen in the pyrrolidine ring (proline and oxyproline), or in the 
 indole ring (tryptophane). 
 
 During the distillation of the ammonia (amide nitrogen), a black 
 colouring matter is formed ; the nitrogen in this is estimated and is 
 designated humin nitrogen. The following table gives the amounts in 
 percentages of the total nitrogen, of the nitrogen in the various fractions 
 of certain proteins : — 
 
 
 g 
 
 'A 
 
 |zi 
 
 ,^ 
 
 3) 
 
 ^ 
 
 o^ 
 
 .^' 
 
 ^ 
 
 
 o 
 
 a 
 
 1 
 
 ' a 
 
 
 » 
 
 c 6 
 
 S 6 
 
 
 
 "a 
 S 
 
 B 
 a 
 
 a 
 
 ■a 
 
 ■s 
 
 ^ 
 
 
 OS 
 
 Wheat gliadin . 
 
 25-52 
 
 0-86 
 
 1-25 
 
 5-71 
 
 6-20 
 
 0-75 
 
 51 -98 
 
 8-50 
 
 99-771 
 
 Edestin . 
 
 9-99 
 
 1-98 
 
 1-49 
 
 27 05 
 
 5-75 
 
 3-86 
 
 47-55 
 
 1-70 
 
 99-37; 
 
 Keratin (from 
 
 
 
 
 
 
 
 
 1 i 
 
 hair) 
 
 1005 
 
 7-42 
 
 6-60 
 
 15-33 
 
 3-48 
 
 5-37 
 
 47-50 
 
 3-10 
 
 98-851 
 
 Gelatin . 
 
 2-25 
 
 0-07 
 
 0-00 
 
 14-70 
 
 4-48 
 
 6-32 
 
 56-30 
 
 14-90 
 
 99-02 
 
 Fibrin 
 
 8-32 
 
 3-17 
 
 0-99 
 
 13-86 
 
 4-83 
 
 11-51 
 
 54-30 
 
 2-70 
 
 99-58 
 
 Hasmoglobin . 
 
 5-24 
 
 3-60 
 
 000 
 
 7-70 
 
 12-70 
 
 10-90 
 
 57-00 
 
 2-90 100-04 
 
 It will be seen that practically 100 per cent, of the total nitrogen is 
 accounted for. A further striking result is the high non-amino-nitrogen 
 percentage in gelatin, indicating that larger quantities of proline are 
 contained in it than older analyses had revealed. The large amount of 
 lysine in haemoglobin is also unexpected. The method has a great 
 future before it. 
 
 TESTS FOR PROTEINS 
 
 Solubilities. — The proteins are insoluble in alcohol and ether. 
 Some are soluble in water,i others insoluble. Many of the latter 
 are soluble in weak saline solutions. Some are insoluble, others 
 soluble in concentrated saline solutions. 
 
 ' The proteins are not truly soluble in water ; they are in a state of colloidal 
 solution, a condition intermediate between true solution and suspension. Many of 
 t^beir properties are due to this fact. (See Appendix. ) 
 
THE PROTEINS 
 
 55 
 
 All proteins are soluble with the aid of heat in concentrated 
 mineral acids and alkalis. Such treatment, however, decomposes as 
 well as dissolves the protein. Proteins are also soluble in gastric and 
 pancreatic juices ; but here, again, they undergo a change, as we have 
 already seen. 
 
 Heat Coagulation. — Most native proteins, such as white of egg, are 
 rendered insoluble when their solutions are heated. The temperature 
 of heat coagulation differs in different proteins ; thus myosinogen and 
 fibrinogen coagulate at 56° C, serum albumin and serum globulin at 
 about 75° C. 
 
 The proteins which are coagulated by heat come mainly under two 
 classes ; the albumins and Jhe gjobulins. These differ in solubility ; 
 the albumins are soluble in distilled water, the 
 true globulins require salts to hold them in 
 solution. 
 
 IndifEiisibility. — The proteins (peptones ex- 
 cepted) belong to the class of substances called 
 colloids by Thomas Graham ; that is, they pass 
 with difficulty, or not at all, through animal 
 membranes. In the construction of dialysers, 
 vegetable parchment is largely used. 
 
 Proteins may thus be separated from 
 diffusible (erystalloid) substances such as salts, 
 but the process is a tedious one. If some 
 serum or white of egg is placed in a dialyser 
 (fig. 6) and distilled water outside, the greater 
 amount of the salts passes into the water 
 through the membrane and is replaced by 
 water ; the two proteins albumin and globulin 
 remain inside ; the globulin is, however, precipitated, as the salts 
 which"^reviously kept if in solution are removed. 
 
 The terms " diffusion," " dialysis," and " osmosis " should be distinguished 
 from one another. 
 
 If water is carefully poured on the surface of a solution of any substance, 
 this substance gradually spreads through the water, and the oompoaition of 
 the mixture becomes uniform in time. The time occupied is short for sub- 
 stances like sodium chloride, and long for substances like albumin. The 
 phenomenon is called diffusion. If the solutions are separated by a mem- 
 brane the term diali/sis is employed. The word osmosis is properly restricted 
 to the passage of water through membranes, and can be best studied when 
 semi-permeable membranes are employed. See more fully article Osmosis 
 in Appendix. 
 
 Fig. 6. — In this form of dialyser 
 the substance to be dialysed 
 is placed within the piece 
 of tubing suspended in the 
 larger vessel of water. The 
 tubing is made of (i^arch- 
 ment paper. 
 
56 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 Crystallisation. — Hfemoglobin, the red pigment of the blood, is a 
 protein substance and is crystallisable (for further details, see The 
 Blood, Lesson IX). Like other proteins it has an enormously large 
 molecule ; though crystalline, it is not, however, crystalloid in 
 Graham's sense of that term. Blood pigment, however, is not the 
 only crystallisable protein. Long ago crystals of protein (globulin 
 or vitellin) were observed in the aleurone grains of many seeds, 
 and in the somewhat similar granules occurring in the egg-yolk 
 of some fishes and amphibians. By appropriate methods these 
 have been separated and recrystallised. Further, egg-albumin itself 
 has been crystallised. If a solution of white of egg is diluted with 
 an equal volume of saturated solution of ammonium sulphate, the 
 globulin present is precipitated and is removed by filtration. The 
 filtrate is now allowed to remain some days at the temperature 
 of the air, and as it becomes more concentrated from evaporation, 
 minute spheroidal globules, and finally minute needles of egg-albumin, 
 either aggregated or separate, make their appearance (Hofmeister). 
 Crystallisation is more rapid if a little acetic or sulphuric acid is 
 added (Hopkins). Serum albumin (from some animals) has also been 
 similarly crystallised (Giirber). 
 
 Action on Polarised Light. — All proteins are Isevo-rotatory, the 
 amount of rotation varying with individual proteins. Several of 
 the compound proteins, e.g. hsemoglobin, and nucleo-proteins are 
 dextro-rotatory, though their protein components are Isevo-rotatory 
 (Gamgee). 
 
 Colour Beactions. — The principal colour reactions have been already 
 described in the heading of this lesson. 
 
 ('1 ) The xanthoproteic reaction depends on the conversion of the 
 aromatic group of the protein molecule into nitro-derivatives. 
 
 (2) Millon's reaction is due to the presence of the tyrosine group, 
 and is given by all benzene derivatives which contain a hydroxy! group 
 (OH) reJDlacing hydrogen. 
 
 (3) The formaldehyde reaction (and the Adamkiewicz reaction) is 
 due to the presence of the tryptophane radical (indole amino-propionic 
 acid). 
 
 The presence, absence, or intensity of these colour tests in various 
 proteins depends respectively on the presence, absence, or amount of 
 the groups to which they are due. 
 
 (4) In the copper sulphate test the proteoses and peptones behave 
 differently from the native proteins ; the latter give a violet and the 
 former a rose-red colour, which is called the biuret reaction, because the 
 
THE PROTEINS 57 
 
 same tint is also given by the substance called biuret, i The name does 
 not imply that biuret is present in protein, but both biuret and protein 
 give the reaction because they possess the same atomic groups, namely, 
 two CONHg groups linked either to a carbon atom, or to a nitrogen 
 atom, or directly to one another (Schilf). The native proteins give 
 a violet colour because the red tint of the copper compound with the 
 biuret group is mixed with another copper compound which has a 
 blue colour. 
 
 Precipitants of Proteins. — Proteins are precipitated by a large 
 number of reagents ; the peptones and proteoses are exceptions in many 
 cases, and will be considered separately afterwards (see Lesson VII). 
 
 Solutions of the proteins are precipitated by — 
 
 1. Strong acids, such as nitric acid. 
 
 2. Picric acid. 
 
 3. Acetic acid and potassium ferrocyanide. 
 
 4. Acetic acid and excess of neutral salts such as sodium sulphate. 
 
 5. Salts of the heavy metals, such as copper sulphate, mercuric 
 chloride, lead acetate, silver nitrate, etc. ^ 
 
 6. Tannin. 
 
 7. Alcohol. 
 
 8. Saturation with certain neutral salts, such as ammonium sulphate. 
 It is necessary that the words coagulation and precipitation should, 
 
 in connection with the proteins, be carefvilly distinguished. The term 
 coagulation is used when an insoluble protein (coagulated protein) is 
 formed from a soluble one. This may occur — 
 
 1. When the protein is heated — heat coagulation. 
 
 2. Under the influence of an enzyme ; for instance, when a curd 
 is formed in milk by rennet or a clot in shed blood by the fibrin 
 ferment — enzyme coagulation. 
 
 There are, however, certain precipitants of proteins in which the 
 precipitate formed is readily soluble in suitable reagents, such as saline 
 solutions, and the protein continues to show its typical reactions. This 
 precipitation is not coagulation. Such a precipitate is produced by 
 saturation with ammonium sulphate. Certain proteins, called globulins, 
 are more readily precipitated by such means than others. Thus, 
 serum . globulin is precipitated by half -saturation with ammonium 
 sulphate. Full saturation with ammonium sulphate precipitates all 
 proteins but peptone. The globulins are precipitated by certain salts 
 
 ' Biuret is obtained by- heating solid- urea; ammonia is given off and leaves 
 biuret thus ; — 
 
 2CON2H4 = C2O2N3H5 + NHj 
 [urea] [biuret] [ammonia] 
 
58 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 such as sodium chloride and magnesium sulphate, which do not 
 precipitate the albumins. The precipitation of proteins by salts in 
 this way is conveniently termed " salting out." 
 
 The precipitate produced by alcohol is peculiar in that after a time 
 it becomes a coagulum. Protein freshly precipitated by alcohol is 
 readily soluble in water or Saline media ; but after it has been allowed 
 to stand some time under alcohol it becomes more and more insoluble. 
 Albumins and globulins are most readily rendered insoluble by this 
 method ; proteoses and peptones are never rendered insoluble by the 
 action of alcohol. This fact is of value in the separation of these proteins 
 from others. 
 
 CLASSIFICATION OF PROTEINS 
 
 The knowledge of the chemistry of the proteins which is slowly 
 progressing will no doubt in time enable us to give a classification of 
 these substances on a strictly chemical basis. The following classifica- 
 tion must be regarded as a provisional one, which, while it retains the 
 old familiar names as far as possible, yet attempts also to incorporate 
 some of the Mw ideas. The classes of animal proteins, then, beginning 
 with the simplest, are as follow ; — 
 
 1. Protamines. 6. Phospho-proteins. 
 
 2. Histones. 7. Conjugated proteins. 
 
 3. Albumins. (a) Gluco-proteins. 
 
 4. Globulins. (6) Nuclea-proteins. 
 
 5. Sclero-proteins. (c) Chromo-proteins. 
 We shall take these classes one by one. 
 
 1. The Protamines 
 
 These substances are obtainable from the heads of the spermatozoa 
 of certain fishes, where they occur in combination with nuclein. 
 Kossel's view that they are the simplest proteins in nature has met 
 with general acceptance, and they give such typical protein reactions 
 as the copper sulphate test (Rose's or Piotrowski's reaction). On hydro- 
 lytic decomposition they first yield substances of smaller molecular 
 weight analogous to the peptones which are called protones, and then 
 they split up into amiuo-acids. The number of resulting amino-acids is 
 small as compared with other proteins ; hence the hypothesis that they 
 are simple proteins is confirmed. Notable among their decomposition 
 products are the diamino-acids or hexone bases, especially arginine. 
 The protamines difier in their composition according to their source, 
 and yield these products in difierent proportion^. 
 
THE PROTEINS 
 
 59 
 
 Salmine (from the salmon roe) and cliipeine (from the herring roe) 
 appear to be identical, and have the empirical formula Cg^Hj^NijOg : 
 its principal decomposition product is arginine, but small amounts of 
 valine, serine, and proline are also found. Sturine (from the sturgeon) 
 yields the same products with lysine and histidine in addition. With 
 one exception, the protamines yield no aromatic amino-acids ; the 
 exception is cydopterine (from Cyclopterus lumpus) ; this substance is 
 thus an important chemical link between the other protamines and 
 the more complex members of the protein family. 
 
 2. The Histones 
 
 These are substances which have been separated from blood cor- 
 puscles ; globin, the protein constituent of haemoglobin, is a well- 
 marked instance. They yield a larger number of amino compounds 
 than do the protamines, but diamino-acids are still relatively abundant. 
 They are coagulable by heat, soluble in dilute acids, and precipitable 
 from such splutions by ammonia. The precipitability by ammonia is 
 a property possessed by no olher protein group. ' 
 
 3. The Albumins 
 
 These are typical proteins, and yield the majority of the cleavage 
 products enumerated on pp. 45 to 50. 
 
 They enter into colloidal solution in water, in dilute saline solutions, 
 and in saturated solutions of sodium chloride and magnesium sulphate. 
 They are, however, precipitated by saturating their solutions with 
 ammonium sulphate. Their solutions are coagulated by heat usually 
 at 70-73° C. Serum albumin, egg-albumin, and lact-albumin are 
 instances. 
 
 4. The Globulins 
 
 The globulins give the same general tests as the albumins : they 
 are coagulated by heat, but differ from the albumins mainly in their 
 solubilities. This difference in solubility may be stated in tabular form 
 as follows : — 
 
 Reagent. 
 
 Albumin. 
 
 Globulin. 
 
 Water .... . . 
 
 Dilute saline solution .... 
 
 Saturated solution of magnesium sulphate 
 
 or sodium chloride .... 
 Half-saturated solution of ammonium 
 
 sulphate 
 
 Saturated solution of ammonium sulphate 
 
 soluble 
 soluble 
 
 soluble 
 
 soluble 
 insoluble 
 
 insoluble 
 soluble 
 
 insoluble 
 
 insoluble 
 insoluble 
 
60 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 In general terms globulins are more readily salted out than albumins ; 
 they may therefore be precipitated and thus separated from the albumins 
 by saturation with such salts as sodium chloride, or, better, magnesium 
 sulphate, or by half saturation with ammonium sulphate. 
 
 The typical globulins are also insoluble in water, and so may be 
 precipitated by removing the salt which keeps them in solution. This 
 may be accomplished by dialysis (see p. 55). 
 
 Their temperature of heat coagulation varies considerably. The 
 following are the commoner globulins : — fibrinogen and serum globulin 
 in blood ; egg-globulin in white of egg, para-myosinogen in muscle, and 
 crystallin in the crystalline lens. We must also include under the same 
 heading certain proteins which are the result of enzyme coagulation on 
 globulins such as fibrin (sep Blood) and myosin (see Muscle). 
 
 The most striking and real distinction between globulins and 
 albumins is that the latter on hydrolysis yield no glycine, whereas 
 the globulins do. 
 
 5. The Sclero-proteins 
 
 These substances form a heterogeneous group of substances, which 
 were formerly termed albuminoids. The prefix sclera- indicates the 
 skeletal origin and often insoluble nature of the members of the group. 
 The principal proteins under this heading are the following : — 
 
 1. CoUasen, the substance of which the white fibres of connective 
 tissue are composed. Some observers regard it as the anhydride of 
 gelatin. 
 
 2. Ossein. — This is the same substance derived from bone. 
 
 In round numbers the solid matter in bone contains two-thirds inorganic 
 or earthy matter, and one-third organic or animal matter. The inorganic 
 constituents are calcium phosphate (84 per cent, of the ash), calcium carbonate 
 (13 per cent.), and smaller quantities of calcium chloride, calcium fluoride, 
 and magnesium phosphate. The organic constituents are ossein (this is the 
 most abundant), elastin from the membranes lining the Haversian canals, 
 lacunae, and oanaliculi,and other proteins and nuclein from the bone corpuscles. 
 There is also a small quantity of fat even after removal of all the marrow. 
 Dentine is like bone chemically, but the proportion of earthy matter is rather 
 greater. Enamel is the hardest tissue in the body ; the mineral matter is 
 like that found in bone and dentine ; but the organic matter is so .small 
 in quantity as to be practically non-existent (Tomes). Enamel is epiblastic, 
 not mesoblastic like bone and dentine. 
 
 3. Gelatin. — This substance is produced by boiling collagen with 
 water. It possesses the peculiar property of setting into a jelly when 
 
 . a solution made with hot water cools. On digestion it is like ordinary 
 
THE PROTEINS 61 
 
 proteins converted into peptone-like substances and is readily absorbed. 
 Though it will replace in diet a certain quantity of such proteins and 
 thus acts as a " protein sparing " food, it cannot altogether take their 
 place as a food. Animals whose sole nitrogenous food is gelatin waste 
 rapidly. The reason for this is that gelatin contains neither the 
 tyrosine nor the tryptophane radicals, and so it gives neither Millon's 
 nor the Adamkiewicz reaction. Animals which receive in their food 
 gelatin to which tyrosine and tryptophane are added thrive better. 
 
 4. Chondrin. — This is the name given to the mixture of gelatin and 
 mucoid which is obtained by boiling cartilage. 
 
 5. Elastin. — This is the substance of which the yellow or elastic 
 fibres of connective tissues are composed. It is a very insoluble material. 
 The sarcolemma of muscular fibres and certain basement membranes 
 are composed of a similar substance. 
 
 6. Keratin, or horny material, is the substance found in the surface 
 layers of the epidermis, in hairs, nails, hoofs, and horns. It is very 
 insoluble, and chiefly differs from most other proteins in its high 
 percentage of the sulphur-containing amino-acid called cystine. A, 
 similar substance, called neurokeratin, is found in neuroglia and nerve 
 fibres. In this connection it is interesting to note that the epidermis 
 and the nervous system are both formed from the same layer of the 
 embryo — the epiblast. 
 
 6. The Phosplio-proteins 
 
 Vitelliti, (from egg-yoke), caseinogen, the principal protein of milk, 
 and casein, the result of the action of rennet on caseinogen (see Milk), 
 are the principal members of this group. Among their decomposition 
 products is a considerable quantity of phosphoric acid. They have 
 been frequently confused with the nucleo-proteins which we shall be 
 studying immediately ; but they do not yield the products (purine and 
 other bases) which are characteristic of nucleo-compounds. The 
 phosphorus is contained within the protein molecules, and not in another 
 molecular group united to the proteinj as in the case of the nucleo- 
 proteins. The phospho-proteins are of special value in the nutrition 
 of young and embryonic animals. Many other proteins, such as the 
 globulin of blood serum, contain traces of phosphorus. 
 
 7. The Conjugated Proteins 
 
 These very complex substances are compounds in which the protein 
 molecule is united to other organic materials, which are, as a rule, also 
 of complex nature. This second constituent of the compound is usually 
 
62 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 termed a jn-osthetic groitp. They may be divided into the following 
 sub-classes. 
 
 i. Chromo-proteins. — Thefee are compounds of proteins with a 
 pigment, which usually contains iron. They are exemplified by haemo- 
 globin and its allies, 'which will be fully considered under Blood. 
 
 ii. Gluco-proteins. — These are compounds of protein with a carbo- 
 hydrate group. This class includes the mucins and the mucoids. 
 
 The mucins are widely distributed and may occur in epithelial cells, 
 or be shed out by these cells (mucus, mucous glands, goblet cells). The 
 mucins obtained from different sources vary in composition and re- 
 actions, but they all agree in the following points : — 
 
 (a) Physi(^l character. "Viscid and tenacious. 
 
 \b) They are soluble in dilute alkalis, such as lime water, and are 
 precipitable from solution by acetic acid. 
 
 The mucoids generally resemble the mucins, but differ from them in 
 minor details. The term is applied to the mucin-like substances which 
 form the chief constituent of the ground substance of connective tissues 
 (tendo-mucoid, chondro-mucoid, etc.). Another, ovo-mucoid, is found 
 in white of egg, and others (pseudo-mucin and para-mucin) are occasion- 
 ally found in dropsical effusions, and in the fluid of ovarian cysts. 
 
 The differences between the mucins and mucoids are possibly due to the 
 nature of the protein part of the molecule, and also to the nature of the 
 conjugated sulphuric acids which they contain. Those from cartilage, tendon, 
 and aorta furnish chondroitin sulphuric acid which on fui-ther hydrolysis 
 yields the amino-sugar chondrosamine. The mucoids from cornea, vitreous 
 humor, gastric mucosa, serum mucoid, and ovo-mucoid on the other hand 
 contain niucoitin sulphuric acid, which yields (f-glucosamine (chitosamine) 
 another amino-sugar (CeHjiOsNHij) on hydrolysis. 
 
 Pavy and others have shown that a small quantity of the same 
 carbohydrate derivative can be split off from various other proteins 
 which we have already placed among the albumins and globulins. It 
 is, however, probable that this must not be considered a prosthetic 
 group, but is more intimately united within the protein molecule. 
 
 iii. Nucleo-protems. — These are compounds ^ of protein with a com- 
 plex organic acid called nucleic acid which contains phosphorus. They 
 are found both in the nuclei and cell-protoplasm of cells. In physical 
 characters they often simulate mucin. 
 
 Nuclein is the name given to the chief constituent of cell-nuclei. 
 It is identical with the chromatin of histologists (see fig. 7). 
 
 On decomposition it yields an organic acid called nucleic acid, 
 
 ' Walter Jones in his recent monograph is doubtful if they are really definite 
 compounds. 
 
THE. PROTEINS 63 
 
 together with a variable but usually small amount of protein. It 
 contains a high percentage (10-11) of phosphorus. The nuclein obtained 
 from the nuclei or heads of the spermatozoa consists of nucleic acid 
 without any protein admixture. In fishes' spermatozoa, however, there 
 is an exception to this rule, for there it is, as we have already seen, 
 united to protamine. 
 
 The nudeo-proteins- of' cell protoplasm are compounds of nucleic 
 acid with a much larger quantity of protein, so that they usually 
 contain only 1 per cent, or less of phosphorus. Some also contain 
 iron, and it is probable that the normal supply of iron to the body is 
 contained in the nucleo-proteins or hcematogens (Bunge) of plant or 
 animal cells. 
 
 Nucleo-proteins may be prepared from xellular structures such as 
 thymus, testis, kidney, etc., by two principal methods : — 
 
 1. Wooldridge's Method. — The organ is minced and soaked in water 
 
 Fig. 7.— l5iagram of a cell : p, protoplasm composed of apong^ioplasm 
 and hyaloplasm ; n, nucleus with intranuclear network of chromatin 
 or nuclein ; and n\ nucleolus (Schafer). 
 
 for twenty-four hours. Dilute acetic acid added to the aqueous extract 
 precipitates the nucleo-protein. 
 
 2. Sodium chloride Method. — The minced organ is ground up in a 
 mortar wjth solid sodium' chloride ; the resulting viscous mass is 
 poured into excess of water, and the nucleo-protein rises in strings to 
 the top of the water. 
 
 The solvent usually employed for a nucleo-protein, by whichever 
 method it is prepared, is a 1 per-cent. solution of sodium carbonate. 
 The relationship of nucleo-proteins to the coagulation of the blood is 
 described under that heading. 
 
 Nucleic acid yields, among its decomposition products, phosphoric 
 acid, a carbohydrate, various bases of the purine group, and bases also 
 of the pyrimidine group. The following diagrammatic way of repre- 
 senting the decomposition of nucleo-protein will assist the student in 
 remembering the relationships of these substances ; — 
 
64 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 NUCLEO-PKOTEIN 
 
 subjected to gastric digestion yields 
 
 Protein — converted into pep- Nuclein, which remains as an 
 tone, which goes into solution. insoluble residue. If this is 
 
 dissolved in alkali, and hydro- 
 chloric acid added, it yields 
 
 Protein — converted into acid A precipitate consisting of nucleic 
 meta-protein in solution. acid. If this is heated in a 
 
 sealed tube with hydrochloric 
 acid, it yields the following; 
 substances. 
 
 Phosphoric acid. Carbohydrate. Purine bases. Pyrimidine 
 
 bases. 
 
 Recent research on the nucleic acids obtained from various 
 
 mammalian organs indicates that they fall into two main classes : — 
 
 (1) Nucleic Acid Proper. — This yields on decomposition — 
 
 (a) Phosphoric acid. 
 
 (6) A carbohydrate of the hexose group which has not yet been 
 
 identified. In nucleic acid from vegetable cells {e.g. 
 
 from yeast) the sugar which takes its place is a pentose 
 
 (c?-ribose). (Levene). 
 
 (c) Two members of the purine group in the same proportion, 
 
 namely, adenine and guanine. 
 
 {d) Two pyrimidine bases, namely, cytosine and thymine. 
 
 In yeast nucleic acid, uracil takes the place of thymine 
 
 (see p. 50). 
 
 The purine bases are specially interesting because of their close 
 
 relationship to uric acid (which see, p. 200). They are all derivatives 
 
 of a ringed complex, named purine by Fischer, and their relationship 
 
 to each other is best seen by their formulae : — 
 
 /Purine CjH^N^ 
 
 I Hypoxanthine (monoxy-purine) CjH^N^O 
 
 . I Xanthine (dioxy-purine) C.H.N .0., 
 
 Purine bases<, . j ■ / • • \ o tt xt VtVt 
 
 \ Adenine (amino-purine) CjHgNj.NH^ 
 
 I Guanine (amino-oxy-purine) CjHgN^O.NH^ 
 
 \Uric acid (trioxy-purine) C^H^N^Og 
 
THE PROTEINS 65 
 
 The two bases obtained from nucleic acid are the two which contain 
 the NH2 group. If xanthine and hypoxanthine are obtained, they 
 are the secondary effects of oxidative and de-aminising enzymes. 
 
 (2) Guanylic Acid. — This is a simpler form of nucleic acid found 
 in certain organs (pancreas, liver, etc.), mixed with the nucleic acid 
 proper. It yields on decomposition only three substances, namely : — 
 
 (a) Phosphoric acid. 
 
 (b) A carbohydrate of the pentose group. 
 
 (c) Guanine. 
 
 From his work on the nucleic acid of yeast, Levene finds that it is 
 composed of complexes 'consisting of phosphoric acid, carbohydrate (ribose)^ 
 and a base. These are termed nucleotides. Guanylic acid, described above, 
 is a mono-nucleotide, but the majority of nucleic acids are poly-nucleotides. 
 When these are broken down by chemical reagents, the first change is the 
 removal of the phosphoric acid, leaving intact the combinations of base and 
 carbohydrate ; these latter combinations are called nucleosides ; thus — 
 
 Adenine + ribose = adenosine. 
 Guanine + ribose = guanosine. 
 
 These nucleosides may be further split into base and ribose ; or they may be 
 de-aminised (i.e. the amino-group is removed) and nucleosides obtained in 
 which hypoxanthine and xanthine are united with the ribose, and these in 
 their turn may be split into base and ribose. 
 
 The same cleavages are accomplished in the body by the action of tissue- 
 enzymes contained in varying degrees in the different organs and tissues. 
 As these enzymes are specific, the number which may come into successive 
 play in the decompositions which occur in the body is very numerous. These 
 enzymes are spoken of under the general term nucleases. 
 
 Protein-hydrolysis 
 
 When protein material is subjected to hydrolysis, as it is when 
 heated with mineral acid, or superheated steam, or when acted upon 
 by such enzymes as trypsin in the alimentary canal, it is finally resolved 
 into the numerous amino-acids of which it is built. But before this 
 ultimate stage is reached, it is split into substances of progressively 
 diminishing molecular size, which still retain many of the protein 
 characters. These may be classified in order of formation as follows ; — ■ 
 
 1. Meta-proteins. 
 
 2. Proteoses. 
 
 3. Peptones. 
 
 4. Polypeptides. 
 
 5. Amino-acids, 
 
66 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 The polypeptides are linkages of two or more amino-acids as already 
 explained. Although most of the polypeptides at present known are 
 products of laboratory synthesis, some have been definitely separated 
 from the digestion of proteins, and so they must appear in our classifi- 
 cation. The proteoses, peptones, and some of the more complex poly- 
 peptides give the biuret reaction; the peptones, which are probably 
 complex polypeptides, cannot be salted out of solution like the proteoses ; 
 their molecules are smaller than those of the proteoses. We shall study 
 them more fully under Digestion. 
 
 It is, however, convenient to add here a brief description of the 
 meta-proteins, since some of the practical exercises at the head of this, 
 lesson deal with them. 
 
 They are obtained as the first stage of hydrolysis by enzymes, and 
 also by the action of dilute acids or alkalis on either albumins or 
 globulins. The general properties of the acid 'and alkali meta-proteins 
 which are thereby formed are as follows : — they are insoluble in pure 
 water, but are soluble in either acid or alkali and are precipitated 
 by neutralisation unless certain disturbing influences such as sodium 
 phosphate are present. They are precipitated, as globulins are, by 
 saturation with such neutral salts as sodium chloride or magnesium 
 sulphate. They are not coagulated by heat if in solution. In alkali- 
 meta-protein some of the sulphur in the original protein is removed. 
 
 A variety of meta-protein (probably a compound containing a large 
 quantity of alkali) may be formed by adding strong potash to undiluted 
 white of egg. The resulting- jelly is called Lieberlcuhn's jelly. A similar 
 jelly is obtainable by adding strong acetic acid to undiluted egg-white. 
 
 The word " albuminate " is used for compounds of protein with mineral 
 substances. Thus if a solution of copper sulphate is added to a solution of 
 albumin a precipitate of copper albuminate is formed. Similarly, by the 
 addition of other salts of the heavy metals, other metallic albuminates are 
 obtainable. The halogens (chlorine, bromine, iodine) also form albuminates 
 in this sense, and may be used for the precipitation of proteins. 
 
 It should be noted, in conclusion, that the foregoing classification of pro- 
 teins is mainly applicable to those of animal origin. The vegetable proteins 
 may roughly be arranged under the same main headings, although it is 
 dovibtfulif a real and complete analogy exists in all cases. The cleavage 
 products of the vegetable proteins are in the main the same as those of the 
 animal proteins, but the quantity of each yielded is usually different. Many 
 vegetable proteins, for instance, give a very much higher yield of glutamic 
 acid than do those of animal origin. 
 
 There are also certain vegetable proteins, such as gliadin from the gluten 
 of wheat, liordein from barley, and zein from maize, which stand apart from 
 all other members of the group in being soluble in alcohol. 
 
1. Aiuunims, suuii as leuuosm iii vvueat. 
 
 2. Globulins, such as edestin of hemp and other seeds ; most of these are 
 readily crystallisable. 
 
 3. Glutelins. These are insoluble in water and saline solutions, and are 
 soluble only in dilute alkali. They are probably not very strongly marked 
 jff from the globulins, since it has been shown that the solubility of globulins 
 in dilute saline solutions is also due to a trace of alkali. The best example 
 af this third class is the glutenin of wheat gluten. 
 
 4. Gliadins ; the proteins soluble in alcohol just alluded to. They are 
 aharacterised also by the absence of lysine among their cleavage products, 
 md usually yield a very high percentage of glutamic acid on decomposition. 
 The gluten of wheat flour, which is formed when water is added to it, has 
 been shown to consist of two proteins — one (gliadin) soluble in alcohol, the 
 other (glutenin) soluble in alkali. It is to the former that the gluten of 
 dough owes its cohesiveness ; and grains such as rice, which contain no 
 gliadin, cannot in consequence be employed for making bread. 
 
LESSON VI 
 rooDs 
 
 A. MILK.— 1. Examine a drop of milk with the microscope. 
 
 2. Note the specific gravity of fresh milk with the lactometer ; com- 
 pare this with the specific gravity of milk from which the cream has 
 been removed (skimmed milk). The specific gravity of skimmed milk 
 is higher owing to the removal of the lightest constituent — the cream. 
 
 3. The reaction of fresh milk is neutral or slightly alkaline to litmus. 
 
 4. Warm some milk in a test-tube to the temperature of the body, 
 and add a few drops of rennet. After standing a curd is formed ftom 
 the conversion of caseinogen, the chief protein in milk, into casein. The 
 casein entangles the fat globules. The liquid residue is termed whc'if 
 No curdling is produced if the rennet solution is previously boiled, 
 because heat destroys rennet as it does all enzymes. 
 
 5. Take some milk to which 0-2 per cent, of potassium oxalate has 
 been added ; warm to 40°. C. and add rennet. No curdling takes place 
 because the oxalate has precipitated the calcium salts which are 
 necessary in the coagulation process. 
 
 Take a second specimen of oxalated milk and add a few drops of 
 2 per-cent. solution of calcium chloride, and then rennet ; curdling or 
 coagulation takes place if the mixture is kept warm in the usual way. 
 
 6. To another portion of warm milk diluted with water add a few 
 drops of 20 per-cent. acetic acid. A lumpy precipitate of caseinogen 
 entangling the fat is formed. 
 
 7. Filter off this precipitate, and in the filtrate test for lactose or 
 milk-mgar by Fehling's solution (see Lesson III) ; for lact-albumin by 
 boiling, or by Millon's reagent (see Lesson V) ; and for earthy (that 
 is, calcium and magnesium) phosphates by ammonia, which precipitates 
 these phosphates. Phosphates may also be detected in the following 
 way : add nitric acid, boil and filter ; warm the filtrate with ammonium 
 molybdate ; a yellow crystalline precipitate of ammonium phospho- 
 molybdate is formed. 
 
 8. Fat (butter) may be extracted from the precipitate obtained in 6 
 by shaking it with ether ; on evaporation of the ethereal extract the 
 fat is left behind, forming a greasy stain on paper. The presence of fat 
 
 68 
 
t-OobS 69 
 
 may also be demonstrated by the black colour produced by the addition 
 of osmic acid to the milk. 
 
 9. Shake up a little milk with twice its volume of ether; the 
 opacity of the milk remains nearly as great as before. Repeat this, 
 but first add to the milk a few drops of caustic potash before adding 
 the ether and then shake. The milk which lies beneath the ethereal 
 solution of fat becomes translucent. As a matter of fact ether dissolves 
 the fat without the addition of alkali, and the opacity of milk is there- 
 fore not due to the fat globules alone, but largely to their protein 
 envelopes. The clearing which takes place when ether and alkali are 
 added is due to an action of the reagents on the caseinogen. 
 
 1 0. Caseinogen, like globulin, is precipitated by saturating milk with 
 sodium chloride or magnesium sulphate, and by half saturation with 
 ammonium sulphate, but differs from the globulins in not being 
 coagulated by heat. The precipitate produced by saturation with 
 salt floats to the surface with the entangled fat, and the clear salted 
 whey is seen below after an hour or two. 
 
 B. FLOUR. — Mix some wheat flour with a lit|;le water into a stiff 
 dough. Wrap this up in a piece of muslin and knead it under a tap 
 or in a dish of water. The starch grains come through the holes 
 in the muslin (identify by iodine test), and an elastic sticky mass 
 remains behind. This is a protein called gluten. Suspend a fragment 
 of gluten in water ; add nitric acid and boil ; it turns yellow ; cool and 
 add ammonia ; it turns orange (xanthoproteic reaction). Boil another 
 fragment with Millon's reagent ; it turns a brick-red colour. 
 
 C. BREAD contains the same constituents as flour, except that 
 some of the starch has been converted into dextrin and glucose 
 during baking (most flours, however, contain a small quantity of 
 sugar). Extract breadcrust with cold water, and test the extract 
 for dextrin (iodine test) and for glucose (Trommer's or Fehling's 
 test). If hot water is used, starch also passes into solution. 
 
 D. MEAT. — This is our main source of protein food. Cut up some 
 lean meat into fine shreds and grind these up with salt solution. 
 Filter and test for proteins. 
 
76 ESSENTIALS OF CHEMICAL PHYSIOLOGV 
 
 THE PEINCIPAL FOOD-STUFFS 
 
 We can now proceed to apply the knowledge we have obtained of 
 the proteins, carbohydrates, and fats to the investigation of some 
 important foods. The chief chemical substances in food are : — 
 
 1. Proteins \ 
 
 2. Carbohydrates V organic. 
 
 3. Fats ) 
 
 4. Water 
 
 V inorganic. 
 
 5. Salts ' * 
 
 }'■ 
 
 In milk and in eggs, which form the exclusive foods, of young 
 animals, all varieties of these principles are present in suitable propor- 
 tions. Hence they are spoken of as perfect foods. Eggs, though a 
 perfect food for the developing bird, contain too little carbohydrate 
 for a mammal. In most vegetable foods carbohydrates are in excess, 
 while in animal foods, such as m^t, the proteins are predominant. 
 In a suitable diet these should be mixed in proper proportions, which 
 vary for herbivorous and carnivorous animals. We must, however, 
 limit ourselves to the omnivorous animal, man. 
 
 A healthy and suitable diet must possess the following characters : — 
 
 1. It must contain the proper amount and proportion of the various 
 chemical substances. 
 
 2. It must be adapted to the climate, to the age of the individual, 
 and to the amount of work done by him. 
 
 3. The food must contain, not only the necessary amount of 
 chemical substances, but these must be present in a digestible form. 
 As an instance of this, many vegetables (peas, beans, lentils) contain 
 even more protein than beef and mutton, but are not so nutritious, as 
 they are less digestible, much passing off in the faeces unused. 
 
 The nutritive value of a diet depends mainly on the amount of 
 carbon and nitrogen it contains in a really digestible form. A man 
 doing a moderate amount of work, and taking an ordinary diet, will 
 eliminate, chiefly from the lungs in the form of carbonic acid, from 
 250 to 280 grammes of carbon per diem. During the same time he will 
 eliminate, chiefly in the form of urea in the urine, about 1 5 to 1 8 grammes 
 of nitrogen. These substances are derived from the food, and from 
 the metabolism of the tissues ; various forms of energy, work and 
 heat being the chief, are simultaneously liberated. During muscular 
 exercise the output of carbon greatly increases ; the increased excretion 
 of nitrogen is insignificant. Taking, then, the state of moderate 
 exercise, it is necessary that the waste of the tissues should be replaced 
 
FOODS 
 
 71 
 
 by fresh material in the form of food ; and the proportion of carbon to 
 nitrogen should be the same as in the excretions: 250 to 15, or 16 '6 
 to 1. The proportion of carbon to nitrogen in protein is, however, 53 
 to 15, or 3 "5 to 1. The extra supply of carbon comes from non-nitro- 
 genous foods — viz. fat and carbohydrate. 
 Voit gives the following daily diet : — 
 
 Protein 120 grammes. 
 
 Fat 100 
 
 Carbohydrate 333 „ 
 Ranke's diet closely resembles Voit's ; it is — 
 
 Protein 100 grammes. 
 
 Fat 100 
 
 Carbohydrate 250 „ 
 In preparing diet tables, such adequate diets as those just given 
 should be borne in mind. The following peace-time dietary (from 
 G. N. Stewart) will be seen to be rather more liberal, but may be taken 
 as fairly typical of what is usually consumed by an adult man in the 
 twenty-four hours, doing an ordinary amount of work. 
 
 Food-stuflf. 
 
 
 
 Grammes of 
 
 Quantity. 
 
 
 
 
 
 
 
 
 
 Nitro- 
 gen. 
 
 Car- 
 bon. 
 
 Pro- 
 teins. 
 
 Fats. 
 
 Carbo- 
 hydrates. 
 
 Salts. 
 
 
 1 
 English 
 
 
 
 
 
 
 
 Metric System. ! Weisfhts. 
 
 
 
 
 
 
 
 Lean meat 
 
 250 grammes i 9 oz. 
 
 8 
 
 33 
 
 55 
 
 8-5 
 
 
 
 4 
 
 Bread . 
 
 500 „ ' 18 „ 
 
 6 
 
 112 
 
 40 
 
 7-5 
 
 245 
 
 6-5 
 
 Milk . 
 
 500 ,, J-pint 
 
 3 
 
 35 
 
 20 
 
 20 
 
 25 
 
 3-5 
 
 Butter . 
 
 30 „ 1 oz. 
 
 
 
 20 
 
 
 
 27 
 
 
 
 0-5 
 
 Fat with meat 
 
 30 „ 1 „ 
 
 
 
 22 
 
 
 
 30 
 
 
 
 
 
 Potatoes 
 
 450 „ 16 „ 
 
 1-5 
 
 •47 
 
 10 
 
 
 
 95 
 
 4-5 
 
 Oatmeal 
 
 A 
 
 75 „ 3 „ 
 
 1-7 
 
 30 
 
 10 
 
 4 
 
 48 
 
 2 
 
 
 
 20-2 
 
 299 
 
 135 
 
 97 
 
 413 
 
 21 
 
 For a certain time (as during rationing due to war conditions) a 
 man will maintain his health on a scantier diet. In the necessity for 
 such precautions the workers must be given their proper supply of 
 energy-producing food (fat and carbohydrate). Long before the war 
 Chittenden urged that the normal diet should contain only about 
 half the customary quantity of protein, and our recent experience 
 has shown that a Chittenden diet can be kept up for a long time. 
 The body certainly does not assimilate the larger amount usually taken, 
 
72 teSSENTlALS OF CttEMICAL PHVSlCLOGy 
 
 for the greater part of the nitrogenous constituents is converted into 
 amino-acids, which are rapidly transformed by the liver into urea and 
 cast out of the body, leaving the non-nitrogenous remainder to be 
 utilised in the same way as fats and carbohydrates are, for the produc- 
 tion of heat and energy. Chittenden's views will, however, bring home 
 to many people that temperance is necessary in food as well as in drink. 
 The majority of well-to-do people certainly eat an excess of meat, and so 
 throw an unnecessary strain upon their digestive and excretory organs. 
 One should hesitate, however, in accepting Chittenden's conclusions to 
 the full, for it is doubtful if the minimum is also the optim,um diet. 
 It may be that there is a real need for an excess of protein beyond the 
 apparent minimum. In diamond mining a large quantity of earth 
 must be crushed to obtain the precious stones. It may be that among 
 the many cleavage products of protein the majority may be compared 
 to this waste earth, and we get rid of them as quickly as possible in the 
 excretions, but some few (such as tyrosine and tryptophane) are 
 unusually precious for protein synthesis or metabolic processes in the 
 body, and that, in order to get an adequate supply of these, a 
 comparatively large amount of protein must be ingested. 
 
 Recent research has shown that there is something else in an 
 adequate diet which is necessary, especially during the time of growth. 
 These unknown constituents (vitamines) will be discussed in the 
 concluding section of this chapter. 
 
 MILK 
 
 Milk is often spoken of as a "perfect food," and it is so for infants. 
 For those who are older it is so voluminous that unpleasantly large 
 quantities of it would have to be taken in the course of the day to 
 ensure the proper supply of nitrogen and carbon. Moreover, for adults 
 it is relatively too rich in protein and fat. It also contains too little 
 iron (Bunge) ; hence children weaned late become anaemic. 
 
 The microscope reveals that it consists of two parts : a clear fluid and 
 a number of minute particles that float in it. These consist of minute 
 fat globules, varying in diameter from 0'0015 to O'OOS millimetre. 
 
 The milk secreted during the first few days of lactation is called 
 colostrum. It contains very little caseinogen, but large quantities of 
 globulin instead. Microscopically, cells from the acini of the mammary 
 gland are seen, which contain fat globules in their interior ; they are 
 called colostrum corpuscles. 
 
 Beaction and Specific Gravity. — The reaction of fresh cow's milk and 
 
POODS 
 
 73 
 
 of human milk is amphoteric towards litmus. This is due to the presence 
 of both acid and alkaline salts ; the latter are usually in excess. Milk 
 readily turns acid or sour as the result of fermentative change, part 
 of its lactose being transformed into lactic acid (see p. 28). The 
 specific gravity of ,milk- is usually ascertained with the hydrometer. 
 That of normal cow's milk vaiies from 1028 to 1034. When the milk 
 is skimmed the specific gravity rises, owing, to the removal of the 
 light constituent, the fat, to 1033 to 1038. In all cases the specific 
 
 
 *P o O OftSs 
 
 Fig. 8. — Microscopic appearance of millc in the 
 early stage of lactation, showing colostrum 
 corpuscles (a) in addition to fat globules. ( Yeo. ) 
 
 Fig. d.—a, b, colostrum 
 corpuscles with line and 
 coarse fat globules re- 
 spectively ; c, d, e, pale 
 cells devoid of fat. 
 (Heidenhain.) 
 
 gravity of water, with which other substances are compared, is taken 
 as 1000. 
 
 Composition. — Bunge gives the following table, contrasting the 
 milk of woman and cow : — 
 
 
 Woman. 
 
 Cow. 
 
 Proteins (chiefly caseinogen) 
 
 Butter (fat). 
 
 Lactose 
 
 Salts . . ... 
 
 Per cent. 
 ■ 1-7 
 
 3-4 
 
 6-2 
 
 0-2 
 
 Per cent. 
 3-5 
 3-7 
 4-9 
 0-7 
 
 Hence, in feeding infants on cow's milk, it will be necessary to dilute 
 it, and add sugar and a little cream to make it approximately equal to 
 natural human milk. 
 
 The Proteins of Milk. — The principal protein in milk is called 
 caseinogen : this is the one which is coagulated by rennet to form casein. 
 
74 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 Cheese consists of casein with the entangled fat. The other proteins in 
 milk, present in small amounts, are lact-albumin, lacto-glohulvn, and 
 traces of a protein which is soluble in alcohol. Lacto-globulin closely 
 resembles serum-globulin, but lact-albumin differs from serum-albumin 
 in percentage composition, specific rotation, coagulation temperature, 
 etc. It has been obtained in the crystalline condition. 
 
 The Coagulation of Milk. — Rennet is the agent usually employed 
 for this purpose ; it is an enzyme secreted by the stomach, especially 
 by sucking animals, and is generally obtained from the calf. 
 
 The curd consists of the casein and entangled fat : the liquid 
 residue called whey contains the sugar, salts, and the other proteins of 
 the milk. 
 
 Caseinogen itself may be precipitated by acids such as acetic 
 acid, or by saturation with neutral salts. This, however, is not 
 coagulation, but precipitation. The precipitate may be collected 
 and dissolved in lime water ; the addition of rennet then produces 
 coagulation in this solution, provided that a sufficient amount of 
 calcium salts is present. 
 
 In milt also, rennet produces coagulation, provided that a sufficient 
 amount of calcium salts is present. If the calcium salts are precipi- 
 tated by the addition of potassium oxalate, rennet causes no formation 
 of casein. The process of curdling in milk is a double one ; the 
 first action due to rennet is to produce a change in caseinogen ; the 
 second action is that of the calcium salt, which precipitates the altered 
 caseinogen as casein. In blood also calcium salts are necessary for 
 coagulation ; but there they act in a diflierent way, namely, in the 
 production of fibrin-ferment (see Coagulation op Blood, p. 136). . 
 
 Caseinogen is not coagulable by heat. We have already classed it 
 with vitellin as a phospho-protein (see p. 61). 
 
 Caseinogen, as was originally pointed out by Hammarsten, is a protein 
 with acid properties : it is quite insoluble in water, but it forms soluble salts 
 with such metallic bases as potassium, sodium, and calcium. The caseinogen 
 as it exists in milk is combined with calcium as calcium caseinogenate. 
 When acetic acid is added to milk, we therefore get calcium acetate, and a 
 precipitate of free caseinogen. On " dissolving " this caseinogen in an alkali 
 such as soda or potash, we have the formation of sodium caseinogenate or 
 potassium caseinogenate, as the case may be. The precipitate obtained in 
 milk by the addition of alcohol, or by " salting out," is not free caseinogen, 
 but calcium caseinogenate. When we add potassium oxalate to milk, we get 
 the reaction represented in the following equation : — Calcium caseinogenate -I- 
 potassium oxalate = calcium oxalate -I- potassium caseinogenate. When we add 
 calcium chloride to oxalated milk, the following equation represents what 
 
FOODS 75 
 
 occurs : — Potassium caseinogenate + calcium cliloride*= calcium caseinogenate 
 + potassium chloride. 
 
 Calcium caseinogenate forms an opalescent or colloidal solution in water 
 and reacts with the I'ennet enzyme. The oaseinogenates of magnesium, 
 barium, and strontium have similar characters. The caseinogenates of 
 potassium, sodium, and ammonium difter from the above by forming a nearly 
 clear solution in watei', and they do not react with the rennet enzyme (W. A. 
 Osborne). 
 
 The Fats of Milk. — The chemical composition of the fat of milk 
 (butter) is very like that of adipose tissue. It consists chiefly of 
 palmitin, stearin,' and olein. There are, however, smaller quantities of 
 fats derived from fatty acids lower in the series, especially butyrin and 
 caproin. The old statement that each fat globule is surrounded by a 
 film of protein is, according to Ramsden's work, correct. Milk also con- 
 tains small quantities of lipoids, namely, phosphatides .and cholesterol ; 
 and a yellow fatty pigment or lipochrome (see p. 40). 
 
 Milk Sugar or Lactose. — This is a disaccharide {C-^2^22^u)- ^^^ 
 properties have already been described in Lesson III, p. 28. 
 
 Souring of Milk. — When milk is allowed to stand, the chief change 
 Vhich jt is apt to undergo is a conversion of a part of its lactose into 
 lactic' acid. ' This is due to the action of micro-organisms, and would 
 not occur if the milk were contained in cold sterilised vessels. Equa- 
 tions showing the change produced are given on p" 28. When souring 
 occurs, the acid which is formed precipitates a portion of the caseinogen. 
 This must not be confounded with the formation of casein from casein- 
 ogen which is produced by rennet. There are, however, some bacterial 
 growths which like rennet produce true coagulation. 
 
 Alcoholic Fermentation in Milk. — When yeast is added to milk, 
 the sugar does not readily undergo the alcoholic fermentation. Other 
 somewhat similar fungoid growths are, however, able to produce the 
 change, as in the preparation of koumiss ; the milk sugar is first inverted, 
 that is, glucose and galactose are formed from it (see p. 28), and it is 
 from these sugars that alcohol and carbonic acid originate. 
 
 The Salts of Milk. — The principal salt is, calcium phosphate; a 
 small quantity of magnesium phosphate is also present. The other 
 salts are chiefly chlorides of sodium and potassium. 
 
 Differences between different Milks. — It is an undoubted fact that 
 the milk provided by nature for the growing offspring is different in the 
 various classes of the animal kingdom. The quantitative variations are 
 often enormous, and it has been shown that the milk best adapted for 
 the nutrition of the young animal is that which comes from its mother, 
 
76 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 or, at least, from an ahimal of the same species. The practical applica- 
 tion of this rule comes home most to us when dealing with the feeding 
 of children, and it is universally acknowledged that, after all, cows' milk 
 is but a poor substitute for huma^ milk. Cows' milk is, of course, 
 diluted, and sugar and cream added, so as to make it quantitatively 
 like mother's milk, but even then the question arises whether the 
 essential difference between the two kinds of milk is not deeper than 
 one of mere quantity; and, in particular, the pendulum of scientific 
 opinion has swung backwards and forwards in relation to the question 
 whether the principal protein, called caseinogen in both, is really 
 identical in the two cases. The caseinogen of human milk curdles in 
 small flocculi in the stomach, so contrasting with the heavy curd which 
 cows' milk forms; and even although the curdling of cows' milk be 
 made to occur in smaller fragments by mixing the milk with barley 
 water or lime water, its digestion proceeds with comparative slowness 
 in the child's alimentary canal. These are practical points well known 
 to every clinical observer, and in the past they have been attributed, 
 not so much to fundamental differences in the caseinogen itself, as to 
 accidental concomitant factors ; the excess of citric acid in human 
 milk, for instance, or its paucity in calcium salts, having been held 
 responsible for the differences observed in the physical condition Of the 
 curd and in its digestibility. 
 
 This question is far from settled even to-day, but there are some 
 data now available that point to a qualitative difference between 
 caseinogens. Some of these depend on the application of the " biological 
 test " carried out on the line of immunity experiments, which has been 
 so signally successful in the distinction between the blood-proteins of 
 different species of animals (see Lesson IX). The differences, however, 
 which lead to the formation of specific precipitins are so slight, that 
 ordinary chemical methods of analysis are, at present, unable to 
 reveal them. But, in the case of milk, there are differences which the 
 chemist can detect. One cannot lay much stress on mere percentage 
 composition, although differences have been noted in that, because we 
 have no guarantee that the proteins investigated were separated from 
 all impurities ; there are also differences in the percentage of amino- 
 acids obtained after hydrolysis, but the present methods of estimating 
 these with accuracy leave much to be desired. A deeper chemical 
 distinction noted is contained in the recent work of Bienenfeld, who 
 finds that human caseinogen contains a carbohydrate complex which is 
 absent from that of the cow. 
 
 A few years ago it was stated that human caseinogen will not 
 
FOODS 77 
 
 curdle with rennet ; but this has been shown to be a mistake. The 
 conditions of rennet curdling are somewhat different in the two kinds 
 of milk we are considering, but provided the reaction in the stomach 
 is acid, human milk is curdled by rennet when acted on by gastric 
 juice. 
 
 EGGS 
 
 In this country the eggs of hens and ducks are those particularly 
 selected as foods. The shell is made of calcareous matter, especially 
 calcium carbonate. The white is composed of a richly albuminous 
 fluid enclosed in a network of firmer and more fibrous material. The 
 amount of solids is 13'3 per cent. : of this 12-2 is protein in nature. 
 The proteins are albumin, with smaller quantities of egg-globulin and 
 ovo-mucoid (p. 62). The remainder is made up of sugar (0"5 per cent.), 
 traces of fats, lecithin (and other phosphatides) and cholesterol, and 
 0'6 per cent, of inorganic salts. The yolk is rich in food materials for 
 the development of the future embryo. In it there are two varieties 
 of yolk-spherules, one kind yellow and opaque (due to admixture with 
 fat and a yellow lipocbrome, see p. 40), and the other smaller, transparent 
 and almost colourless : these are protein in nature, consisting of the 
 phosphoprotein called vitellin (p. 61). Lecithin, kephalin, cholesterol, 
 small quantities of sugar, and inorganic salts are also present. ' 
 
 The nutritive value of eggs is high, as they are so readily digestible ; 
 but the more an egg is cooked the more insoluble do its protein 
 constituents become. 
 
 MEAT 
 
 This is composed of the muscular and connective (including adipose) 
 tissues of certain animals. The flesh of some animals is not eaten ; 
 in some (Jases this is a matter of fashion ; some flesh like that of the 
 carnivora, is stated to have an unpleasant taste ; and in other cases 
 {e.g. the horse) it is more lucrative to use the animal as a beast of 
 burden. 
 
 Meat is the most concentrated and most easily assimilable, of 
 nitrogenous foods. It is our chief source of nitrogen. Its chief solid 
 constituent is protein, and the principal protein is myosin. In addition 
 to the extractives and salts contained in muscle, there is always a certain 
 percentage of fat, even though all visible adipose tissue is dissected off. 
 The fat-cells are placed between the muscular fibres, and the amount 
 of fat so situated varies in different animals. It is particularly 
 abundant in pork ; hence the indigestibility of this form of flesh ; the fat 
 
78 
 
 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 prevents the gastric juice from obtaining ready access to the muscular 
 fibres. 
 
 The following table gives the chief substances in some of the 
 principal meats used as food : — 
 
 Constituents. 
 
 Ox. 
 
 Calf. 
 
 Pig- 
 
 Horse. 
 
 Fowl. 
 
 Pike. 
 
 Water ... 
 
 76-7 
 
 75-6 
 
 72-6 
 
 74-3 
 
 70-8 
 
 79-3 
 
 Solids .... 
 
 23-3 
 
 24-4 
 
 27-4 
 
 25-7 
 
 29-2 
 
 20-7 
 
 Proteins, including gelatin . 
 
 20-0 
 
 19-4 
 
 19-9 
 
 21-6 
 
 22-7 
 
 18-3 
 
 Fat . . 
 
 1-6 
 
 2-9 
 
 6-2 
 
 2-5 
 
 4-1 
 
 0'7 
 
 Carbohydrate 
 
 0-6 
 
 0-8 
 
 0-6 
 
 0-6 
 
 1-3 
 
 0-9 
 
 Salts 
 
 1-2 
 
 1-3 
 
 1-1 
 
 1-0 
 
 11 
 
 0-s 
 
 The large percentage of water in meat should be particularly noted ; 
 if a man wished to take his daily quantity of 100 grammes of protein 
 entirely in the form of meat, it would be necessary for him to consume 
 about 500 grammes (i.e. a little more than 1 lb.) of meatier diem. 
 
 FLOUK 
 
 The best wheat flour is made from the interior of wheat grains, 
 and contains the greater proportion of the starch of the grain and 
 most of the protein. .Whole flour is made from the whole grain minus 
 the husk, and thus contains, not only the white interior, but also the 
 " germ " or embryo plant, and the harder and browner outer portion 
 of the grain. This outer region contains a somewhat larger proportion 
 of the proteins of the grain. Whole flour contains 1 to 2 per cent, 
 more protein than the best white flour, but it has the disadvantage of 
 being less readily digested (see also Vitamines, p. 82). Brown flour 
 contains a certain amount of bran in addition; it is still less digestible, 
 but is useful as a mild laxative, the insoluble cellulose mechanically 
 irritating the intestinal canal as it passes along. 
 
 The best flour contains very little sugar. The presence of sugar 
 indicates that germination has commenced in the grains. In the 
 manufacture of malt from barley this is purposely allowed to go on. 
 
 When mixed with water, wheat flour forms a sticky adhesive mass 
 called dough. This is, due to the formation of gluten, and the forms 
 of grain poor in gluten cannot be made into dough (oats, rice, etc.). 
 Gluten does not exist in the flour as such, but is formed on the addition 
 of water from the pre-existing soluble proteins in the flour. It is a 
 mixture of the two proteins gliadin and glutenin (see p. 67). 
 
FOODS 
 
 79- 
 
 The following table contrasts the composition of some of the more 
 important vegetable foods : — 
 
 Constituents. 
 
 Wheat. 
 
 Barley. 
 
 Oats. 
 
 Rice. 
 
 Lentils. 
 
 Peas. 
 
 Potatoes. 
 
 Water . 
 
 13-6 
 
 13 8 
 
 12-4 
 
 131 
 
 12-5 
 
 14-8 
 
 76-0 
 
 Protein . 
 
 12-4 
 
 111 
 
 10-4 
 
 7-9 
 
 24-8 
 
 23-7 
 
 2 
 
 Fat 
 
 1-4 
 
 2-2 
 
 5-2 
 
 0-9 
 
 1-9 
 
 1-6 
 
 0-2 
 
 Starch . 
 
 67-9 
 
 64-9 
 
 57-8 
 
 76-5 
 
 54-8 
 
 49-3 
 
 20-9 
 
 Cellulose 
 
 2-5 
 
 5-3 
 
 11-2 
 
 0-6 
 
 36 
 
 7-5 
 
 0-7 
 
 Mineral salts . 
 
 1-8 
 
 2-7 
 
 3 
 
 1-0 
 
 2-4 
 
 31 
 
 1-0 
 
 We see from this table — 
 
 1. The great quantity of starch always present. 
 
 2. The small quantity of fat ; that bread is generally eaten with 
 butter is a popular recognition of this fact. 
 
 Protein, except in potatoes, is pretty abundant, and especially so 
 in the pulses (lentils, peas, etc.). The protein in the pulses is not 
 gluten, but consists mainly of globulins. 
 
 In the mineral matters of vegetables, salts of potassium and 
 magnesium are, as a rule, more abundant than those of sodium and 
 calcium. 
 
 BREAD 
 
 Bread is made by cooking the dough of wheat flour mixed with 
 yeast, salt, and flavouring materials. An enzyme in the flour acts at 
 the commencement of the process when the temperature is kept a 
 little over that of the body, and forms dextrin and sugar from the 
 starch, and then the alcoholic fermentation, due to the action of the 
 yeast, begins. The bubbles of carbonic acid, burrowing passages 
 through the bread, make it light and spongy. This enables the 
 digestive juices subsequently to soak into it readily and affect all parts 
 of it. During baking the gas and alcohol are expelled from the bread, 
 the yeast is killed, and a crust forms from the drying of the outer 
 portions of the loaf. 
 
 White bread contains, in a hundred parts, 7 to 10 of protein, 55 of 
 carbohydrate, 1 of fat, 2 of salts, and the rest water. 
 
 COOKING OF FOOD 
 
 The cooking of foods is a development of civiUsation, and much 
 relating to this subject is a matter of education and taste rather than 
 of physiological necessity. Cooking, however, serves many useful 
 ends : — 
 
'80 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 1. It destroys all parasites and danger of infection. This relates 
 not only to bacterial growths, but also to larger parasites, such as 
 tapeworms and triohinse. 
 
 2. In the case of vegetable foods it breaks up the starch grains, 
 bursting the cellulose and allowing the digestive juices to come into 
 contact with the starch proper. 
 
 3. In the case of animal foods it converts the insoluble collagen 
 of the universally distributed connective tissues into the soluble 
 gelatin. The loosening of the fibres is assisted by the formation of 
 steam' between them. By thus loosening the binding material, the 
 more important elements of -the food, such as muscular fibres, are 
 rendered accessible to the gastric and other juices. Meat before it is 
 cooked is generally kept a certain length of time to allow rigor mortis 
 to pass oflf. 
 
 Of the two chief methods of cooking, roasting and boiling, the 
 former is the more economical, as by its means the meat is first sur- 
 rounded with a coat of coagulated protein on its exterior, which keeps 
 in the juices to a great extent, letting little else escape but the dripping 
 (fat). Whereas in boiling, unless bouillon and bouilli are used, there is 
 considerable waste. Cooking, especially boiling, renders the proteins 
 more insoluble than they are in the raw state, but this is counter- 
 balanced by the other advantages that cooking possesses. 
 
 Beef Tea. — In making beef tea and similar extracts of meat it 
 is necessary that the meat should be placed in cold water, and this 
 is gradually and carefully warmed. In cooking a joint it is usual to 
 put the meat into boUing water at once, so that the outer part is 
 coagulated, and the loss of material minimised. 
 
 An extremely important point in this connection is that beof tea 
 and similar meat extracts should not be regarded as important foods. 
 They are valuable as pleasant stimulating drinks for invalids, but they 
 contain very little of the nutritive material of the meat, their chief 
 constituents, next to water, being the salts and extractives (creatine, 
 hypoxanthine, lactic acid, etc.) of flesh. 
 
 Many invalids restricted to a liquid diet get tired of milk, and 
 imagine that they get sufficient nutriment by taking beef tea instead. 
 It is very important that this erroneous idea should be corrected. One 
 of the greatest difficulties that a physician has to deal with in these 
 cases is the distaste which many adults evince for milk. It is essential 
 that this should be obviated as far as possible by preparing the milk in 
 difierent ways to avoid monotony. Some can take koumiss ; but a less 
 expensive variation may be introduced in the shape of junkets, which, 
 
FOODS Si 
 
 although well known in the West of England, are comparatively un- 
 known in other parts. The preparation of a junket consists of adding 
 to warm milk in a bowl or dish a small quantity of rennet (Clark's 
 essence is very good for this purpose) and flavouring material according 
 to taste. The mixture is then put aside, and in a short time the milk 
 sets into a jelly (coagulation of casein), which may then be served with 
 or without cream. 
 
 Soup contains the extractives of meat, a small proportion of the 
 proteins, and the principal part of the gelatin. The gelatin is usually 
 increased by adding bones and fibrous tissue to the stock. It is the 
 presence of this substance which causes the soup when cold to gelatinise. 
 
 ADJUNCTS TO FOOD 
 
 Among these must be placed alcohol, the value of which within 
 moderate limits is not as a food, but as a stimulant; condiments 
 (mustard, pepper, ginger, curry powder, etc.), which are stomachic 
 stimulants, the abuse of which is followed by dyspeptic troubles ; and 
 tea, coffee, cocoa, and similar drinks. These are stimulants chiefly to 
 the nervous system : tea, coffee, mate (Paraguay), guarana (Brazil), 
 cola nut (Central Africa), bush tea (South Africa), and a few other 
 plants used in various countries all owe their chief property to an 
 alkaloid called theine or caffeine (CjHjpN^Og) ; cocoa to the closely 
 related alkaloid, theobromine (CjHgN^Oj) ; coca to cocaine. These 
 alkaloids are all poisonous, and used in excess, even in the form of 
 infusions of tea and coffee, produce over-excitement, loss of digestive 
 power, and other disorders well known to physicians. Coffee differs 
 from tea in being rich in aromatic matters ; tea contains a bitter 
 principle, tannin. To avoid the injurious solution of too much tannin, 
 tea should only be allowed to infuse (draw) for a few minutes. Cocoa 
 is not only a stimulant, but contains more food-stufis ; it contains 
 about 50 per cent, of fat and 12 per cent, of protein. In cocoa, as manu- 
 factured for the market, the amount of fat is reduced to 30 per cent., and 
 the amount of protein rises proportionately to about 20 pet cent. 
 
 Green vegetables are taken as a palatable adjunct to other foods 
 rather than for their nutritive properties. Their potassium salts are, 
 however, abundant. Cabbage, turnips, and asparagus contain 80 to 
 92 water, 1 to 2 protein, 2 to* 4 carbohydrates, and 1 to 1-5 cellulose 
 per Cent. The small amount of nutriment in most green foods accounts 
 for the large meals made by and the vast capacity of the alimentary 
 canal of herbivorous animals. (See also Vitamines, next page.) 
 6 
 
82 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 VITAMINES 
 
 If an animal is fed upon a mixtjire of pure protein, fat, and carbo- 
 hydrate, with a due admixture of salts and water, it does not thrive, 
 but shows evidence of malnutrition, although the quantities given may 
 be theoretically correct. If a growing animal is fed on such a diet it 
 ceases to grow. But if, as Hopkins showed originally, a small amount of 
 a natural food, such as milk, is mixed with the artificial diet just referred 
 to, the animals thrive and grow normally. There is something extra, 
 something which is at present unknown, which is absolutely essential, 
 and quite small amounts of it are usually sufficient. 
 
 If the unknown constituents are absent from a mean's diet, he under- 
 goes just the same sort of malady, and illnesses so produced, such as 
 scurvy, rickets, and Beri-beri, are termed " Deficiency diseases.'' 
 
 A great deal of work is going on now in relation to these diseases ; 
 we may take Beri-beri as an example. This was prevalent among the 
 natives of Japan when their staple article of diet was polished rice, 
 that is, rice grains deprived of their external layer. This disease is 
 characterised by general malnutrition and neuritis or inflammatiQn 
 of the nerves followed by nerve-degeneration and paralysis. It can 
 also be produced in birds by feeding them on polished rice; and in 
 both man and bird can be rapidly cured by adding the polishings of 
 the rice grains. The germ or embryo contained in the outer layer 
 of the grain contains the extra something, and it is to this substance 
 that the term vitamine of accessory food substance has been applied. 
 At present its chemical composition is unknown. 
 
 Vitamine is not confined to rice grains, but is found in many other 
 vegetable and animal foods. The value of whole meal bread, for 
 ex'ample, does not depend on the small extra amount of protein it 
 contains, but here also upon a vitamine. The amount of vitamine 
 varies considerably. Thus, in pigeons fed upon polished rice, as much 
 as 20 grammes of meat daily must be added to prevent the occurrence 
 of Beri-beri ; whereas 3 grammes of egg-yblk are sufficient, and half a 
 gramme of yeast is enough.' 
 
 The best known of these vitamines or accessory substances are : — 
 Fat soluble A : this is contained in most animal fats, and is specially 
 abundant in butter and cod-liver oil. It is absent from vegetable 
 fats, but present in the green parts of vegetables. Animals cannot 
 make it for themselves, so lactating mothers must receive it in their 
 food. 
 
 Water soluble B : The Beri-beri condition is the specific result of 
 
FOODS 83 
 
 the absence of this. Both A and B are essential for growth in 
 young animals. 
 
 Water soluble C : Contained in juices of fruits (especially oranges 
 and lemons) and vegetables (especially turnips). Absence of this in 
 the food leads to scurvy. 
 
 MABOABINE 
 
 This has now become a staple article of diet, and the old prejudice 
 against this butter substitute has largely disappeared, partly because of the 
 stress of war, but mainly because margarine makers have learnt how to make 
 it palatable. The best margarine is called oleo-margarine, and is made with 
 beef fat as its chief basis. It is a valuable food, and contains the fat-soluble 
 accessory substance. The present-day margarines are mainly made from 
 vegetable oils (palm-kernel oil, cotton-seed oil, etc.), which undergo a process 
 known as hardening or hydrogenation to render them solid at ordinary 
 temperatures. This consists in passing hydrogen gas through the oil at 250° C. 
 in the presence of a metallic catalyst. The process can be arrested at any stage 
 when the desired amount of hardening is obtained. Such margarines are 
 destitute of the fat-soluble accessory, and though their calorific value is of 
 the usual magnitude, they are of inferior value, especially for growing infants 
 and children. Even if the fat which is hydrogenated contains fat-soluble A 
 to start with {e.g. whale oil), the high temperature destroys it. 
 
LESSON VII 
 THE DIGESTIVE JUICES 
 
 SALIVA 
 
 1. The reaction of saliva is alkaline to litmus paper. 
 
 2. To a little saliva in a test-tube add acetic acid. Mucin is 
 precipitated in stringy flakes. 
 
 3. Filter off the mucin thus precipitated ; the filter will also catch 
 any cells which may be present. Apply the xanthoproteic or MiUon's 
 test to the filtrate ; the presence of a small amount of protein is shown. 
 
 4. The presence of potassium thiocyanate (KCNS) in saliva may be 
 shown by the red colour given by a drop of ferric chloride solution ; 
 this colour is discharged by the addition of a drop of mercuric chloride 
 solution. The presence and amount of potassium thiocyanate in saliva 
 
 •are, however, very inconstant. 
 
 5. Put some 05 per-cent. solution of starch into three test-tubes, 
 A, B, and C. Add some saliva to B and C ; faintly acidulate C with 
 0'2 per-cent. hydrochloric acid; then place the three tubes in the 
 water-bath at 40° 0. After five or six minutes remove the test-tubes 
 and examine their contents. The starch to which no saliva was added 
 (tube A) will be unaltered, and will give a deep blue colour on the 
 addition of a drop of iodine solution. The same is true for the acidu- 
 lated specimen (tube C). ThO' contents of test-tube B will give a red- 
 brown colour with iodine owing to the presence of erythro-dextrin, and 
 will also contain a reducing sugar (maltose), as can be shown by boiling 
 with Fehling's solution. 
 
 6. THE ACHROMIC POINT.— By the action of ptyalin, the starch- 
 splitting enzyme of saliva, starch is converted into (1) soluble starch, 
 (2) erythro-dextrin, which gives a red-brown colour with iodine; (3) 
 achroo-dextrin, which gives no colour with iodine ; and (4), finally, 
 maltose. In accurate work with amylolytic enzymes, it is usual to 
 determine the rate of their action by determining the exact point when 
 iodine ceases to give a coloration; this corresponds to the moment 
 when all the erythro-dextrin has disappeared, having been converted 
 into achroo-dextrin and maltose ; this is known as the achromic point. 
 This may be determ,ined with saliva in the following way : Rinse out 
 the mouth thoroughly with warm distilled water; then collect some 
 saliva, dilute it with five times its volume of distilled water and filter. 
 
 Place a number of drops of iodine solution on a white testing slab. 
 
 Then mix 5 c.c. of a 0'5 per-cent. solution of starch with an equal 
 quantity of the diluted filtered saliva, and place the mixture in the 
 water-bath at 40° C. Every half minute or so transfer a drop of the 
 digesting mixture with a glass rod to a drop of iodine solution. At 
 first, as long as starch is preseilt, the colour struck will be blue ; then 
 as erythro-dextrin appears the colour will be violet, owing to the 
 mixture of the blue (due to starch) and of the red (due to erythro- 
 dextrin) ; a little later the colour struck will be the red-brown due to 
 
 84 
 
GASTRIC DIGESTION 85 
 
 the presence of erythro-dextrin ; with successive drops after this the 
 colour will get fainter and fainter, until finally the achromia point is 
 reached. Note the time this has taken from the moment when the 
 starch and saliva mixture was placed in the warm bath. Compare 
 this time with that obtained by other members of the class, and a 
 relative measure of the activity of the saliva of different people will 
 in this way be obtained. 
 
 GASTEIO DIGESTION 
 
 1. Half fill four test-tubes — 
 
 A with water; B with 0'2 per-cent. hydrochloric acidi; C 
 with 0-2 per-cent. hydrochloric acid; D with solution of 
 white of egg (1 to 10 of water). 
 
 2. To A add a few drops of glycerol extract of stomach- (this 
 contains pepsin) and a piece of solid protein like fibrin. 
 
 To B also add pepsin solution and a piece of fibrin. 
 To C add only a piece of fibrin. 
 
 To D add a few drops of pepsin solution and fill up the tube with 
 0''2 per-cent. hydrochloric acid. 
 
 3. Put the tubes into the water-bath at 40° C, and observe them 
 carefully. 
 
 In A the fibrin remains unaltered. 
 In B it becomes swollen, and gradually dissolves. 
 In C it becomes swollen, but does not dissolve. 
 These experiments show that neither pepsin nor hydrochloric acid 
 alone digest protein, but that both must be present for this purpose. 
 After half an hour examine the solution in test-tube B. 
 
 (a) Colour some of the liquid with litmus and neutralise with 
 dilute alkali. Acid-meta-protein is precipitated. 
 
 (b) Take another test-tube, and put into it a drop of 1 per-cent. 
 solution of copper sulphate ; empty it out so that the merest trace of 
 copper sulphate remains adherent to the wall of the tube ; then add 
 the solution from test-tube B and a few drops of strong caustic potash. 
 A pink colour (biuret reaction) is produced. This should be carefully 
 compared with the violet tint given by unaltered albumin. 
 
 (c) To a third portion of the fiuid in test-tube B add a drop of 
 nitric acid ; proteoses or propeptones are precipitated. This precipitate 
 dissolves on heating and reappears on cooling. 
 
 Bepeat these three tests with the digested white of egg in test- 
 tube D. 
 
 4. Examine an artificial gastric digestion which has been kept a 
 week. Note the absence of putrefactive odour ; in this it contrasts 
 very forcibly with an artificial pancreatic digestion under similar 
 conditions. 
 
 ' Made by adtling 6 c.c. of commercial concentrated hydrochloric acid to 
 994 c.c. of water. 
 
 '^ Benger's liquor pepticus may be used instead of the glycerol extract gf 
 stomach. 
 
86 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 ENZYMES 
 
 The word fermentation was first applied to the change of sugar 
 into alcohol and carbonic acid by means of yeast. The evolution of 
 carbonic acid causes frothing and bubbling ; hence the term " fennen-' 
 tation." The agent yeast which produces this used to be called the 
 ferment. Microscopic investigation shows that yeast is composed of 
 minute rapidly growing unicellular organisms belonging to the fungus 
 group of plants. 
 
 The souring of milk, the transformation of urea into ammonium 
 carbonate in decomposing urine, and the formation of vinegar (acetic 
 acid) from alcohol are produced by the growth of very similar 
 organisms. The complex series of changes known as putrefaction, 
 which are accompanied by the formation of malodorous gases, and 
 which are produced by the growth of various forms of rapidly multi- 
 plying bacteria, also come into the same category. 
 
 That the change or fermentation is produced by these organisms is 
 shown by the fact that it occurs only when the organisms are present, 
 and stops when they are removed or killed by a high temperature or 
 by certain substances (carbolic acid, mercuric chloride, etc.) called 
 antiseptics. 
 
 The " germ theory " of disease explains the infectious diseases by 
 considering that the change in the system is of the nature of fermen- 
 tation, and, like the others we have mentioned, produced by microbes ; 
 the transference of the bacteria or their spores from one person to 
 another constitutes infection. The poisons produced by the growing 
 bacteria appear to be either alkaloidal (ptomaines) or protein in nature. 
 The existence of poisonous proteins is a very remarkable thing, as no 
 profound chemical differences have yet been shown to exist between 
 them and those which are not poisonous, but which are useful as foods. 
 Snake venom is an instance of a very virulent poison of protein nature. " 
 The micrdfecopic appearances of some of the micro-organisms con- 
 cerned are shown in the accompanying illustration (fig. 10). 
 
 All these micro-organisms require moisture in which to act. They 
 act best at a temperature of about 40° C. Their activity is stopped, 
 but the organisms are not destroyed by cold ; even after being sub- 
 jected to the intense cold of liquid air, they resume activity when once 
 more raised to a suitable temperature. The organisms are, however, 
 like other living cells, killed by too great heat. Some micro-organisms 
 act without _/ree oxygen ; these are called anaerobic, in contradistinction 
 to those that require free oxygen and which are therefore called aerobic. 
 
EN2YMES 
 
 87 
 
 Another well-known fact concerning micro-organisms is that the 
 substances they produce in time put a stop to their activity ; thus in 
 the case of yeast, the aleohol produced, and in the case of putrefactive 
 bacteria acting on proteins, the phenol, cresol, etc., produced, first stop 
 the growth of and ultimately kill the organisms in question. 
 
 For a long time it was uncertain how micro-organisms were able to 
 
 Fig. 10. — Typical fotms of Schizortiycetes (after Zopf) : a, micrococcus-; h, macrococcus ; c, bac- 
 terium ; d, ' bacillus ; e, Clostridium ; /, monas Okenii ; ff, leptothrix ; h, i, vibrio ; k, 
 spirillum ; I, spirulina ; m, spiromonas ; n, spirochsete ; o, cladothrix. 
 
 effect these chemical transformations. It is now, however, definitely 
 proved that they do so by producing agents of a chemical nature which 
 were formerly called soluble ferments, but are now usually spoken of 
 as enzymes. This was first demonstrated in connection with the 
 invertase of yeast cells, and with the enzyme secreted by the micro- 
 coccus urese which converts urea into ammonium carbonate in putrefying 
 urine. For a long time, however, efforts to obtain from yeast cells an 
 enzyme capable of bringing about the alcoholic fermentation vere 
 
88 
 
 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 unsuccessful. This is because the enzyme does not leave the yeast 
 cells, but acts intracellularly. Buchner finally, by crushing the yeast 
 cells, succeeded in obtaining from them the long-sought enzyme, and 
 he termed it zymase ; and since then other enzymes have been obtained 
 from other micro-organisms by similar means. 
 
 Enzymes are also formed by the cells of the higher organisms, both 
 in animal and vegetable life; and in animals those which are pro- 
 duced by the cells of the digestive glands are those which have been 
 longest known. Familiar instances of these are ptyalin, the starch- 
 splitting enzyme of saliva, and pepsin, the protein-splitting enzyme 
 of gastric juice. The substance upon which the enzyme acts is spoken 
 of as the substrate. 
 
 We may therefore place these essential facts concerning enzyme 
 action in the following tabular way, restricting ourselv&s for the 
 present to those we have already mentioned. 
 
 The Living Cell. 
 
 The Enzyme 
 produced. 
 
 The Substrate. 
 
 The Products of Action. 
 
 The yeast cell. 
 The salivary cell. 
 The gastric cell. 
 
 Zymase. 
 Ptyalin. 
 Pepsin. 
 
 Glucose. 
 
 Starch. 
 
 Protein. 
 
 Alcohol and carbon 
 dioxide. 
 
 Dextrins and malt- 
 ose. 
 
 Proteoses and pep- 
 tones. 
 
 The enzymes concerned in digestion, the study of which we are 
 now commencing, fall under the following five principal headings : — 
 
 1 . Amylolytic or amyloclastic — those which convert polysaccharides 
 (starch, glycogen) into sugar with intermediate dextrins. Examples : 
 the diastase of vegetable seeds, the ptyalin of saliva, the amylase of 
 pancreatic juice. 
 
 2. Inverting — those which convert disaccharides into mono- 
 saccharides. Examples : — 
 
 (a) Invertase of yeast cells ; invertase of intestinal juice ; these 
 convert sucrose into equal parts of glucose and fructose. 
 
 (6) Maltase of intestinal juice ; this converts maltose into glucose. 
 
 (c) Lactase of intestinal juice ; this converts lactose into equal 
 parts of glucose and galactose. 
 
 3. Lipolytic or lipoclastic — those which split fats into fatty acids 
 and glycerol. An example, lipase, is found in pancreatic juice. 
 
 4. Proteolytic or proteoclastic-^those which split proteins into 
 
ENZYMES o9 
 
 proteoses, peptones, pol3^eptides, and finally amino-acids. Examples : 
 the pepsin of gastric, and the trypsin of pancreatic jaice. 
 
 5. Peptolytic or peptoclastic — those which split proteoses and 
 peptones into polypeptides and amino-acids. The erepsin of intestinal 
 juice is an example of these. 
 
 The enzymes in the foregoing lists are hydrolytic ; that is, water is 
 added to the substrate, which then splits into simpler molecules. This 
 is seen in the following examples : — 
 
 (a) Conversion of cellulose into carbonic acid and methane by the 
 enzyme secreted by putrefactive organisms : — 
 
 (CoHijOj),, + nB.fi = 3nC0^ + 3nCB.^ 
 
 [celliUose] [water] [carbonic acid] [methane] 
 
 (b) Inversion of sucrose by invertase : — 
 
 C12H22O11 + H2O = CoHj^Og + CeHjjOu 
 
 [sucrose] [water] [glucose] [fructose] 
 
 In addition to these there are many others which are concerned in 
 other processes than those of digestion. Of these we may mention : — 
 
 6. Coagulative enzymes — those which convert soluble, into in- 
 soluble proteins ; the best example of this class is thrombin or fibrin- 
 ferment, which comes into play in blood-coagulation, converting the 
 soluble protein in blood-plasma called fibrinogen into fibrin. The 
 similar conversion of myosinogen in muscle into myosin during rigor 
 mortis is also possibly due to the activity of an enzyme. Rennet or 
 rennin found in the gastric juice converts the soluble caseinogenate of 
 milk into casein, and may therefore be included with these enzymes ; 
 it comes into action during the digestion of milk in the stomach. 
 
 7. Intracellular or Autolytic Enzymes. — These comeinto play during 
 cell life, and are important in the metabolic or intracellular chemical 
 changes which occur in protoplasm ; they also may be subdivided into 
 proteolytic, peptolytic, lipolytic, etc., according to the substrate upon 
 which they act. After death their activity ' continues, and so they 
 produce self-digestion or autolysis of the cells in which they are 
 situated, if the tissue or organ is kept at an appropriate temperature 
 and under aseptic conditions. 
 
 8. Oxidases, which are oxygen carriers and produce oxidation : 
 they are mainly found as intracellular enzymes, and are important in 
 tissue respiration. 
 
 9. Reductases. — These are the counterpart of the oxidases and 
 produce reduction in the tissues. 
 
 10. Deaminases. — These remove the amino-group from amino- 
 compounds, 
 
90 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 These ten groups do not by any means exhaust the list ; there is 
 in the living organism no group of agents as important as enzymes in 
 bringing about the chemical changes necessary for its continued 
 metabolism and well-being. We shall come across many examples of 
 enzymic action in our subsequent studies, for instance in the formation 
 of urea, of uric acid, etc., etc. 
 
 There are, however, certain considerations of a more general nature 
 which we may enumerate here. The first of these is : — 
 
 The Chemical Nature of Enzymes. — This is a subject which is very 
 difficult to investigate ; the enzymes are substances which to a great 
 extent elude the grasp of the chemist. No one has ever yet been 
 successful in obtaining any enzyme in a state of absolute purity ; but 
 we are perhaps safe in saying that if they are not protein in character, 
 they are substances closely allied to the proteins. 
 
 Zymogens. — These are the parent substances or precursors of the 
 enzymes. The granules seen in many secreting cells consist very 
 largely of zymogen, which in the act of secretion is converted into 
 the active enzyme. Thus pepsin is formed from pepsinogen, trypsin 
 from trypsinogen, thrombin from thrombogen or prothrombin, and so 
 forth. 
 
 Activation of Enzymes. Co-enzymes. — Many enzymes contained in 
 secretions are in a condition ready for action. The activity of 
 other enzymes only occurs after they have been rendered energetic 
 by the presence or action of other substances termed activating 
 agents or co-enzymes. The modus operandi of the co-enzyme appears 
 to vary in different cases ; the co-enzyme may be itself an enzyme, 
 or it may be a more or less simple organic substance, or even an 
 inorganic material. Pepsin, for instance, will only act in an 
 acid medium, and its most favourable ally is hydrochloric acid ; a 
 compound of the two substances, pepsin-hydrochloric acid, appears to 
 be the effective agent in the proteolysis which occurs in the stomach. 
 Trypsin is not present as such in the fresh pancreatic juice ; what is 
 present is trypsinogen ; this is converted into the active enzyme 
 trypsin when it meets the entero-kinase of the succus entericus ; and 
 entero-kinase is itself an enzyme, an enzyme of enzymes, as Pavloff 
 terms it. Thrombo-kinase is regarded by some as the activating agent 
 for thrombin or fibrin ferment, though here, as also -in rennet action, 
 the presence of calcium is essential too. Bile salts act as coadjutors in 
 the action of pancreatic lipase, phosphatfes and other phosphorus-contain- 
 ing substances in the action of zymase, and so forth. 
 
 The Specificity of Enzyme Action. — An enzyme which acts uponstarcb 
 
ENZYMES 91 
 
 will not act upon protein ; and one which acts upon protein will not act 
 upon starch or fat. In some cases the action of an enzyme is extraordin- 
 arily limited ; thus there are three separate enzymes to hydrolyse the 
 three principal disaccharides, sucrose, lactose, and maltose, neither of 
 which will act upon either of the other two sugars in the list. Arginase 
 splits arginine into ornithine and urea, but will act upon no other 
 substance. Some of the peptolytic enzymes obtained in extracts of 
 tissues and organs will act upon certain polypeptides, resolving them 
 into their constituent amino-acids, whereas others act in a similar way 
 on other groups of polypeptides. The "lock and key" simile first 
 introduced by Emil Fischer will aid us in understanding this specificity 
 of action. Each lock must have its special key : so the configuration 
 of an enzyme must be related in some way to the configuration of 
 the substrate to enable it to enter and unlock its parts from one 
 another. 
 
 The Inexhaustibility of Enzymes. — A small amount of enzyme will 
 act on an unlimited amount of substrate, provided sufficient time is 
 given, and provided also the products of action are removed. " A little 
 leaven leaveneth the whole lump." This is perhaps analogous to the 
 part played by sulphuric acid in the etherification of alcohol (seep. 17). 
 The enzyme appears to take a share in intermediate reactions, and 
 there is some evidence that in certain stages it combines with the 
 substrate ; but subsequently when the substrate breaks up into simpler 
 materials, the enzyme is liberated unchanged, and so ready to similarly 
 act on a fresh amount of substrate in which the same series of events is 
 repeated. 
 
 Catalytic Action of Enzymes. — The analogy of enzymic action is, in 
 fact, so close to that of inorganic catalysts, that the view at present 
 current regarding it is that the action is a catalytic one. This is to say, 
 the presence of the enzyme induces a chemical reaction to occur rapidly 
 which in its absence also occurs, but so slowly that any action at all is 
 difficult to discover. To use the technical phrase, its action is to increase 
 the velocity of chemical reactions. It is, for instance, quite conceivable 
 that, if starch and water were mixed together, the starch would in time 
 take up the water and split into its constituent molecules of sugar. 
 But an action of this kind would be so slow, occupying perchance 
 many years, that for practical purposes it does not take place at all. If 
 an inorganic catalyst is added, such as sulphuric acid, and the tempera- 
 ture raised to boiling point, the action takes place in a few minutes ; 
 if an organic catalyst, such as the enzyme ptyalin, is added, the velocity 
 of the change is equally great or even greater, but what is of more 
 
92 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 importance for the well-being of the animal, a moderate temperature, 
 namely that of the body, ainply suffices. The organic catalysts or 
 enzymes are, however, colloidal in nature (possibly protein), and this 
 explains the points in which they differ from the inorganic catalysts, 
 for instance in their destructibility by high temperatures. 
 
 Reaction Velocity. — Most of the reactions in inorganic chemistry take 
 place between electrolytes, that is substances which are conductors of the 
 electric current. They conduct electricity when they are in solution 
 because they are largely broken up into their constituent ions ; thus 
 sodium chloride is broken up into ions of sodium and ions of chlorine ; 
 the sodium ions are called kations because they become charged with 
 positive electricity and move towards the kathode or negative pole ; 
 the chlorine ions are called anions because they are charged with 
 negative electricity and move towards the anode or positive pole. 
 Substances which are not ionised in solution are called non-electrolytes 
 and do not conduct electricity. 
 
 The reactions of inorganic salts, bases, and acids are really reactions 
 between ions, and ionic reactions occur at such enormous velocity as 
 to be almost instantaneous. Ionic reactions take place between the 
 inorganic constituents of living cells, and such reactions occurring as 
 they do in a colloidal medium are somewhat slowed down, but even so 
 are completed in an immeasurably short time. The most important 
 substances (fats, carbohydrates, proteins) in living tissues are, however, 
 not electrolytes, and reactions between them are spoken of as molehilar 
 reactions, and occur so slowly that it is possible to ascertain the rate at 
 which they take place. Reaction velocity is defined as the quantity of 
 the substance transformed, measured in gramme-molecules per litre, 
 which disappears in the unit of time (one minute). When starch is 
 transformed into sugar, or protein into amino-acids, there is only one 
 substance transformed, and such reactions, which compose the majority 
 of the reactions in living cells, are called unimolecular reactions, or 
 reactions of the first order. When, for instance, starch is changed into 
 sugar by the action of an acid, it is the starch alone which is altered ; 
 the acidity undergoes no diminution. Similarly when the change is 
 brought about by an enzyme, the starch only is changed ; the enzyme 
 is still presenj; in its original quantity. Reaction velocity is thus of 
 special importance in a study of the changes produced by enzymes, and 
 these are the most frequent of all changes in living structures. 
 
 Since the quantity of the substance acted upon is continually 
 diminishing, the velocity of the reaction cannot remain the same 
 throughout, but must diminish in a certain ratio. Suppose 20 parts 
 
ENZYMES 93 
 
 out of 100 are transformed in the first minute, there will be only 80 
 parts remaining at the commencement of the second minute : — 
 
 100 - — = 80. 
 5 
 
 Similarly at the commencement of the third minute we have only 64 
 left, 16 having disappeared : — 
 
 80-— = 64. 
 5 
 
 In the fourth minute, 12-8 disappears and 51-2 is left : — 
 
 64--i = 51-2; 
 5 
 
 and so on. 
 
 In order to express this in general terms, we may label the original 
 
 concentration 100 by the symbol C„, and for 80, 64, 51-2, etc., use the 
 
 terms Cj, Cg, C3, etc. . . . C,. The constant figure in the above example 
 
 is J or 0'2. This may be represented by h. The equations then run : — 
 
 Further 
 or. 
 
 Further 
 Finally 
 
 If this is plotted out in the form of a curve, we obtain the curve known 
 as a logarithmic curve. 
 
 In other cases the law is a different one, and we find that the 
 reaction velocity is not directly proportional to the quantity of reacting 
 substance, but to the square of this quantity. In all such cases, two 
 substances are simultaneously changed in their concentration. Such a 
 process takes place in the decomposition of esters (compounds of organic 
 acids with alcohols), under the influence of an alkali ; here not only is 
 the amount of ester becoming less, but the alkali is also used up in 
 the formation of salts of the organic acid. Such reactions are called 
 himolecular reactions, or reactions of the second order. Certain reactions 
 in living cells are of this order, but reactions of higher orders still are 
 not as yet known in living cells. 
 
 l^he Optimum Tem/peratwte of Enzym,e Action. — As the temperature 
 rises the velocity of the action increases until a temperature is reached 
 at which the activity of the enzyme is greatest. Most enzymes act best 
 at 40° C, but there are exceptions to this rule; malt diastase, for 
 instabce, acts best at 60° C. Beyond the optimum temperature a 
 
 Co 
 
 -GJc = C,., 
 
 , or 
 
 c„(i- 
 
 -k) 
 
 =c. 
 
 0, 
 
 i\~k)-C 
 
 Wi- 
 
 ■k)y. 
 
 k= 
 
 C,; 
 
 
 Co(l- 
 
 -kf 
 
 = c,. 
 
 
 
 
 Co(l- 
 
 -kf 
 
 = €3. 
 
 
 
 
 Co(l- 
 
 -k)' 
 
 -c. 
 
 
 
94 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 further rise inhibits activity, until a point is reached when the enzyme 
 is destroyed. The susceptibiUty of enzymes to the influence of a high 
 temperature is a striking piece of evidence in favour oi the* view that 
 these agents are protein-like in nature. The fatal temperature is as a 
 rule in the neighbourhood of 50° C. 
 
 The effect of a rise of temperature is thus complicated by being of 
 a twofold nature. In the first place, and between certain limits, a rule 
 known as the law of Arrhenius is followed : that is a rise of 10° C. 
 doubles or more than doubles the velocity of the action of the enzyme, 
 as it does other chemical reactions. But as the temperature rises, 
 the velocity of the disintegration of the enzyme increases also. The 
 optimum temperature will therefore be one at which the accelerating 
 effect is strong enough to finish the reaction quickly, and the retarding 
 effect due to destruction of the enzyme is not so great as to paralyse 
 the accelerating effect. 
 
 Reversibility of Enzyme Action. — We have just seen that the 
 majority of en^ymic actions are unimolecular, and that the law followed 
 is the simple logarithmic law. 
 
 But in these reactions we usually meet with the peculiarity that 
 it is not completed when the reaction ceases. A certain quantity 
 of the substrate never disappears. Thus a small amount of sucrose 
 remains unchanged whether the hydrolysis is brought about by the 
 action of an acid or of an enzyme. This phenomenon is due to the fact 
 that two reactions are always taking place in opposite directions. 
 Simultaneously with the splitting up, the synthetical reaction begins, 
 and synthesis or building up increases in proportion as the splitting of 
 the compound advances. The velocity of the splitting process decreases 
 at the same rate as the velocity of the synthetic process increases. At 
 a certain point, both have the same velocity, and therefore no further 
 change occurs in the mixture when this condition of equilibrium is 
 reached. This rule is expressed by writing the chemical equation 
 connected by a double arrow instead of the sign of equation. Two 
 examples follow : — ' 
 
 CjHj . OH + CHg . COOHllrCaHj . COO . CHg + H^O 
 
 [Ethyl alcohol] [Acetic acid] [Ethyl acetate] [Water] 
 
 C„Hj,0, + C,H,,0,^C,,H,,Oii + H,0 
 
 [Glucose] [Fructose] [Sucrose] [Water] 
 
 This phenomenon is termed "reversibility," and was first demon- 
 strated by Croft Hill in his experiments with sucrose and invertase. 
 
 In intracellular action this is a factor of importance, for the same 
 enzyme can in the presence of different proportions of the substrate and 
 
SALIVA 95 
 
 its cleavage products both tie (in anabolism) and untie (in katabolism) 
 the knot. 
 
 It should further be noted that hydrolytic actions are isothermic ; 
 that is, the total energy of the products is equal to that of the 
 substance broken up. 
 
 The simple logarithmic law of enzyme action has been demonstrated 
 for the majority of enzymes (invertase, trypsin, erepsin, lipase, etc.). 
 The effect in a given time is directly proportional to the quantity 
 of enzyme present. But pepsin is an exception to this rule as was 
 first pointed out by Schiitz in 1885. He found that peptic activity 
 is proportional to the square root of the amount of pepsin present. 
 Thus if a certain quantity of pepsin produced an amount of 
 digestive action which we call a, in order to produce a digestive 
 action equal to 2a in the same time, it would be necessary to 
 employ four times the amount of pepsin ; and in order to produce a 
 digestive action equal to 3a, it would be necessary to use nine times 
 the amount of pepsin. This rule (Schiitz's law) has been often con- 
 firmed, and a few years ago Arrhenius explained it on mathematical 
 lines. 
 
 Anti-enzymes. — Many chemical substances, such as strong acids and 
 alkalis, alcohol, formaldehyde, iodine, potassium cyanide, and salts of 
 the heavy metals, hinder enzyme activity. But the term anti-enzyme 
 is generally limited to substances produced in the metabolism of living 
 oi-gauisms. Excess of these organic anti-enzymes can be readily pro- 
 duced by injecting an enzyme into the blood-stream of an animal. 
 This stimulates the production of an anti-enzyme, so that whpn the 
 blood-serum is mixed with the original enzyme, its power is inhibited. 
 Anti-enzymes are specific, that is, they inhibit the enzyme which was 
 injected into the blood, and no other. 
 
 THE SALIVA 
 
 r 
 
 The secretion of saliva is a reflex action ; the taste or smell of food 
 excites the nerve-endings of the afferent nerves (glossopharyngeal and 
 olfactory) ; the efferent or secretory nerves are contained in the chorda 
 tympani (a branch of the seventh cranial nerve), which supplies the 
 submaxillary and Sublingual glands, and in a branch of the glosso- 
 pharyngeal, which supplies the parotid. The sympathetic branches 
 which supply the blood vessels with constrictor nerves contain in some 
 animals secretory fibres also. 
 
 The parotid gland is called a serous or albuminous gland ; before 
 
96 
 
 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 Fig. 11.— Alveoli of serous gland ; A, loaded before secretion ; B, after a short period of active 
 secretion ; C, after a prolonged period. • (Lang-ley.) 
 
 Pig. 12. — Mucous cells from a fresh submaxillary gland of dog: a, loaded with mucinogen 
 granules before secretion ; b, after secretion ; the granules are fewer, especially at the 
 attached border of the cell ; a' and b' represent cells in a loaded and discharged condition 
 respectively which have been irrigated with water or dilute acid. The mucous granules are 
 swollen into a transparent mass of mucin traversed by a network of protoplasmic cell- 
 substance. (Foster, after Langley.) 
 
 Fl6. IS.^Section of part of the human submaxillary gland. (Heidenhain.) .To the ria-ht is 
 group of mucous alveoli, to the left a group of serous alveoli. 
 
Saliva 97 
 
 secretion the cells of the acini are swollen out with granules ; after 
 secretion has occurred the cells shrink, owing to the granules having 
 been shed out to contribute to the secretion (see fig. 11) being con- 
 verted into ptyalin. 
 
 The submaxillary and sublingual glands are called mucous glands : 
 their accretion contains mucin. Mucin is absent from parotid 
 saliva. The granules in the cells are larger than those of the parotid 
 gland : they are composed of mucinogen, the precursor of mucin 
 (see fig. 12). 
 
 Jn a section of a mucous gland prepared in the ordinary way the 
 mucinogen granules are swollen out, and give a highly refracting 
 appearance to the mucous acini (see fig. 13). 
 
 COMPOSITION OF SALIVA 
 
 On microscopic examination of mixed saliva a few epithelial 
 scales from the mouth and salivary corpuscles from the tonsils 
 are seen. The liquid is transparent, slightly opalescent, of slimy 
 consistencyt and may contain lumps of nearly pure mucin. On 
 standing it becomes cloudy owing to the precipitation of calcium 
 carbonate, the carbonic acid which held it in solution as bicarbonate 
 escaping. 
 
 Of the three forms of saliva which contribute to the mixture found 
 in the mouth, the sublingual is richest in solids (2-75 per cent.). The 
 submaxillary saliva comes next (2-1 to 2-5 per cent.). When artificially 
 obtained by stimulation of nerves in the dog the saliva obtained by 
 stimulation of the sympathetic is richer in solids than that obtained 
 by stimulation of the chorda tympani. The parotid saliva is poorest in 
 total solids (0-3 to 0-5 per cent.), and contains no mucin. Mixed saliva 
 contains in man an average of about 0'5 per cent, of solids : it is alka- 
 line in reaction, due to the salts in it ; and has a specific gravity of 
 1002 to 1006. 
 
 The solid constituents dissolved in saliva may be classified thus ;— 
 
 I a. Mucin : this may be precipitated by acetic acid. 
 . I 6. Ptyalin ; an amylolytic enzyme, 
 rgamc ^ Protein : of the nature of a globulin. 
 \d. Potassium thiocyanate. 
 
 e. Sodium ehloride : the most abundant salt. 
 
 f. Other salts : sodium carbonate ; calcium phosphate 
 ' and carbonate; magnesium phosphate; potassium 
 
 chloride. 
 
98 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 THE ACTION OF SALIVA 
 
 The action of saliva is twofold, physical and chemical. , 
 
 The physical use of saliva consists in moistening the mucous mem- 
 brane of the mouth, assisting the solution of soluble substances in the 
 food, and in virtue of its mucin lubricating the bolus of food to facilitate 
 swallowing. 
 
 The chemical action of saliva is due to its enzyme ptyalin. 
 
 The starch is first split, into erythro-dextrin, which gives a red colour 
 with iodine, achroo-dextrin, which gives no colour with iodine, _ and 
 maltose. Brown and Morris give the following hypothetical equation : — 
 
 [starch] [water) 
 
 = 4nCi,H,-0„ + (C,H 0,)„ + {G,n,,0,)„ 
 
 [maltose] [achroo-dextrin] [erythro-dextrin] 
 
 The erythro-dextrin is then converted into achroo-dextrin, and 
 finally into maltose. 
 
 • Ptyalin acts in a similar way, but more slowly on glycogen : ,it has 
 no action on cellulose; hence it is inoperative on uncooked starch 
 grains, for in these the cellulose layers are intact. 
 
 Ptyalin acts best at about the temperature of the body (35-40°), 
 and in a neutral medium ; a small amount of alkali makes but little 
 difference ; a very small amount of acid stops its activity. The con- 
 version of starch into sugar by saliva in the stomach continues for a 
 variable time, for the swallowed masses which fall into the fundus of 
 the stomach are not subjected to peristalsis and admixture with gastric 
 juice until a later stage in the digestion ; but the admixture of the 
 contents of the fundus with the rest of the gastric contents wUl occur 
 more qiackly if the person moves about. The hydrochloric acid 
 which is' poured out by the gastric glands first neutralises the saliva 
 and combines with the proteins in the food ; but immediately free 
 hydrochloric acid appears the ptyalin is destroyed, so that it does 
 not resume work even when the semi-digested food once more becomes 
 alkaline in the duodenum. 
 
 THE SECRETION OF GASTRIC JUICE 
 
 The juice secreted by the glands in the mucous membrane of the 
 stomach varies in composition in the different regions, but the mixed 
 juice is a solution of a proteolytic enzyme (pepsin) in a saline solution, 
 which also contains a little free hydrochloric acid. 
 
 The gastric juice can be obtained during the life of an animal by 
 
GASTRIC JUICE 
 
 99 
 
 Fia. IB.— A pyloric gland from a 
 section of the dog's stomach 
 (Elbstein) : m, mouth ; n, neck ; 
 tr, B. deep portion of tubule cut 
 transversely. 
 
 Fta. 14.— A fundus gland from the dog's stomach (Klein) : d, duct or mouth of the gland ; b, base 
 of one of its tubules ; on the right the base of a tubule is more highly magnified ; c, central 
 cell ; p, parietal cell. 
 
100 
 
 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 means of a gastric fistula. Gastric fistulse have also been made in 
 human beings, either by accidental injury or by surgical operations. 
 The most celebrated case is that of Alexis St Martin, a young Canadian 
 who received a musket wound in the abdomen in 1822. Observations 
 made on him by Dr Beaumont formed the starting point for our correct 
 knowledge of the physiology of the stomach and its secretion. 
 
 We can make artificial gastric juice by 
 mixing weak hydrochloric acid (0'2 to 0-4 
 • per cent.) with a glycerol or aqueous extract 
 of the stomach of a recently killed animal. 
 This acts like the normal juice. 
 
 Three kinds of glands are distinguished 
 in the stomach, which diflfer from each other 
 in their position, in the character of their, 
 epithelium, and in their secretion. The car- 
 diac glands are simple tubular glands quite 
 close to the cardiac orifice. Thefundus glands 
 are those situated in the remainder of the 
 cardiac half and fundus of the stomach : their 
 ducts are short, their tubules long in pro- 
 portion. The latter are fiUed with polyhedral 
 cells, only a small lumen being left; they 
 are more closely granular than the corre- 
 sponding cells in the pyloric glands. They 
 are called principal or central cells. Between 
 them and the basement membrane of the 
 tubule are other cells which stain readily 
 with aniline dyes. They are called parietal 
 or oxyntic {i.e. acid-forming) cells. The 
 pyloric glands, in the pyloric canal, have 
 long ducts and short tubules lined with 
 cubical cells. There are no parietal cells 
 (see figs. 14 and 15). 
 
 The central cells of the fundus glands and 
 to a less degree the cells of the pyloric glands 
 are loaded with granules. During secretion they discharge their granules, 
 those which remain being chiefly situated near the lumen, leaving in each 
 cell a clear outer zone (see fig. 16). These are the cells which secrete 
 the pepsin. Like secreting cells generally, they select certain materials 
 from the lymph that bathes them : these materials are worked up by 
 the protoplasmic activity of the cells into the secretion, which is then 
 
 Oamic acid prepara- 
 tion (Langley); c, columnar 
 epithelium of the surface ; n, 
 neck of the gland, with cen- 
 tral and parietal cells ; /, base 
 occupied only by principal or 
 central cells, which exhibit the 
 granules accumulated towards 
 the lumen of the gland. 
 
GASTRIC JUICE 101 
 
 discharged into the lumen of the gland. The most important substance 
 in a digestive secretion is the enzyme. In the case of a gastric juice 
 this is pepsin. We can trace an intermediate step in this process by 
 the presence of the granules. The granules are not, however, composed 
 of pepsin, but of a mother-substance, which is readily converted into 
 pepsin. We have seen a similar enzyme precursor in the salivary cells 
 (p. 97), and shall find others in the pancreas, and the term zymogen is 
 applied to these enzyme precursors. The zymogen in the gasiric cells 
 is called pepsinogen. The rennin that causes the curdling of milk is 
 formed by the same cells. 
 
 The parietal cells are also called oxyntic cells, because they secrete 
 the hydrochloric acid of the juice. Heidenhain succeeded in making 
 in one dog a cul-de-sac of the fundus, in another of the pyloric region 
 of the stomach ; the former secreted a juice containing both acid and 
 pepsin ; the latter, pq,rietal cells being absent, secreted a viscid alkaline 
 juice containing pepsin. The formation of a free acid from the alkaline 
 blood and lymph is an important but puzzling problem. There is no 
 doubt that it is formed from the chlorides of the blood and lymph, and 
 of the chemical theories advanced as to how this is done, Maly's is the 
 most satisfactory. He considers that the acid originates by the inter- 
 action of sodium chloride and sodium dihydrogen phosphate, as is 
 shown in the following equation : — • 
 
 NaHjPO^ + NaCU 
 
 Na^HPO.-fHCl 
 
 [sodium di- [sodium 
 hydrogen chloride] 
 phosphate] 
 
 [disodium [hydro- 
 hydrogen chloric 
 phosphate] acid] 
 
 The sodium dihydrogen phosphate in the above equation is probably 
 derived from the interaction of the' disodium hydrogen phosphate and 
 the carbonic acid of the blood, thus : — 
 
 NajHPO^ -I- CO2 -I- HgO = NaHCOg -1- NaH^PO^. 
 
 Other theories have tried to explain the formation of such a strong 
 acid as hydrochloric by the law of " mass action.'' We know that by 
 the action of large quantities of carbonic acid on salts of the mineral 
 acids the latter may be liberated in small quantities. We know, 
 further, that small quantities of acid ions may be continually formed 
 in the organism by ionisation. But in every case we can only make 
 use of these explanations if we assume that the small quantities of 
 acid are carried away as soon as they are formed, and thus give room 
 for the formation of fresh acid. ' Even then it is impossible to explain 
 the whole process. A specific action of the cells is no doubt exerted, 
 for these reactions can hardly be considered to OQCur in the blood 
 
102 
 
 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 generally, but rather in the oxyntic cells, which possess the necessary 
 selective powers in reference to the constituents of the blood, and the 
 hydrochloric acid, as soon as it is formed, passes into the secretion of 
 the gland in consequence of its high power of diffusion. 
 
 COMPOSITION OF GASTEIC JUICE 
 
 The following table gives the percentage composition of the gastric 
 juice, of man and dog : — 
 
 Constituents. 
 
 Human. 
 
 Dog. 
 
 Water 
 
 Organic substances (chiefly pepsin) 
 
 CaCIa ' . 
 NaCl 
 
 Ka 
 NH4C1 
 
 Ca^lPOJa . 
 
 Mg,PO,. ... 
 
 99-44 
 0-32 
 0-20 
 0-006 
 0-14 
 0-05 
 
 L 0-01 
 
 97-30 
 1-71 
 0-50 
 0-06 
 0-25 
 0-11 
 0-05 
 017 
 0-02 
 0-008 
 
 In the foregoing table one also sees the great preponderance of 
 chlorides over other salts : apportioning the total chlorine to the 
 various metals present, that which remains over must be combined 
 with hydrogen to form the free hydrochloric acid of the juice. The 
 freshly secreted juice contains about 0-5 per cent, of the acid (as 
 shown in the analysis of dog's gastric juice in the table). When the 
 juice remains in the stomach this is in part neutralised by the food 
 and saliva, and also by pancreatic juice which enters the stomach from 
 the duodenum, so that the ultimate percentage is only 0-2. 
 
 Pepsin stands apart from nearly all other enzymes by requiring 
 an acid medium in order that it may act. A compound of the two 
 substances caUed pepsiri^hydrocMoric acid is the really active agent. 
 Other acids may take the place of hydrochloric acid, but none act 
 so well. Lactic acid is often found in gastric juice ; this, however, is 
 derived by fermentative processes from the food. 
 
 Pavloff has shown that in dogs the secretory fibres for the gastric 
 glands are contained in the vagus nerves. 
 
 By an ingenious surgical operation he succeeded in separating off 
 from the stomach a diverticulum which pours its secretion through an 
 opening in the abdominal wall. This small stomach was found to act 
 in every way like the main stomach of the animal. The pure juice 
 
GASTRIC JUICE 103 
 
 so obtained is clear and colourless ; it has a specific gravity of 1003 
 to 1006. ,It is feebly dextro-rotatory, and gives some of the protein 
 reactions. It contains from 0-4 to 0'6 per cent, of hydrochloric acid. 
 It is strongly proteolytic, and inverts cane sugar. When cooled to 
 0° C. it deposits a fine precipitate of pepsin j this settles in layers, 
 and the layers, first deposited contain most of the acid, which is 
 loosely combined with and carried down by the pepsin. Pepsin is 
 also precipitable by saturation with ammonium sulphate (Ktihne). 
 
 The juice is most abundant in the early periods of digestion, but 
 it continues to be secreted in declining quantity as long as any food 
 remains to be dealt with. When there is no food given there is no 
 Juice. But sham feeding with meat will cause it to flow. 
 
 The larger the proportion of pnotein in the diet, the more abundant 
 and active is the juice secreted, provided the animal is hungry ; the 
 psychical element is important. 
 
 THE ACTIONS OF GASTRIC JUICE 
 
 Gastric juice has the following five actions : — 
 
 1. It is antiseptic, owing to the hydrochloric acid present ; putre- 
 factive processes do not normally occur in the stomach, and the micro- 
 organisms which produce such processes, many of which are swallowed 
 with the food, are in great measure destroyed, and thus the body is 
 protected from them. 
 
 2. It inverts sucrose into glucose and fructose. This also is 
 due to the acid of the juice, and is frequently assisted by inverting 
 enzymes contained in the vegetable food swallowed. The juice has 
 no action on starch. 
 
 3. It contains lipase, or a fat-splitting ferment. The protein 
 envelopes of the fat cells are first dissolved by the pepsin-hydrochloric 
 acid, and the solid fats are melted. They are , then split in small 
 measure into their constituents, glycerol and fatty acids. This action 
 is mainly produced by a regurgitation of the contents of the duodenum 
 mixed with pancreatic juice ; but even after the pylorus has been 
 ligatured and regurgitation prevented, the gastric juice itself produces 
 a small amount of fat-splitting, and therefore contains lipase. It is a 
 remarkable fact that the administration of fat in the food increases the 
 regurgitation from the duodenum. 
 
 4. It curdles milk. This is due to the action of the rennet enzyme 
 or rennin. The conditions of this action we have already discussed 
 under Milk (see p. 74) ; but it may here be added that Pavloff has 
 ai^vanced the view- that rennin is not a distinct and separate enzyme, 
 
104 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 but that milk-curdling is only one of the activities of pepsin. This 
 hypothesis has been accepted by numerous physiologists ; but, on the 
 other hand, there is a number of equally eminent observers who still 
 maintain that pepsin and rennin are two separate enzymes. Whichever 
 view is correct, the curd of casein formed from the caseinogen is 
 subsequently digested as other proteins are. 
 
 5. It is proteolytic ; this is the most important action of all. The 
 proteins of the food are converted by the pepsin-hydrochloric acid into 
 peptones. It has been stated that the prolonged action of the juice 
 leads to the further splitting of the peptones into amino-acids, but 
 accurate work has shown that pepsin-hydrochloric acid does not split 
 any of the known polypeptides into their ultimate cleavage products. 
 
 This action is a process of hydrolysis ; and peptones may be formed 
 by other hydrolysing agencies, such as superheated steam or heating 
 with dilute mineral acids. The first stage in the process of hydrolysis 
 is that of acid meta^protein ; the next step is the formation of proteoses. 
 The word "proteose" includes the albumoses (from albumin), globuloses 
 (from globulin), vitelloses (from vitellin), etc. Similar substances are 
 also formed from gelatin (gelatoses) and elastin (elastoses). Then 
 peptone (a mixture of polypeptides) is produced. The products of 
 digestion of protein may be arranged according to the order in which 
 they are formed, as follows : — 
 
 1. Acid meta-protein. 
 
 !(a) Proto-proteose "| The primary proteoses, i.e. 
 (6) Hetero-proteose }■ those which are formed 
 J first, 
 (c) Deutero- or secondary 
 proteose. 
 
 3. Peptone (Polypeptides). 
 
 1. Acid Meta-protein. — The general properties of the meta-proteins, 
 the first degradation products in the cleavage of the proteins which 
 occurs during digestion, are described on p. 66. We shall find later 
 that, in pancreatic digestion, an alkalimeta-protein is formed instead 
 of the acid modification. The theory has been put forward that a 
 protein is capable of playing the part of a base in virtue of its NH, 
 groups, and also of an acid in virtue of its COOH groups. 
 
 2. Proteoses. — They are not coagulated by heat ; they are pre- 
 cipitated but not cqagulated by alcohol : like peptone they give the 
 pink biuret reaction. They are precipitated by nitric acid, the pre- 
 cipitate heing soluble on heating, and reappea/ring when the liquid cools. 
 
GASTRIC JUICE 
 
 105 
 
 This last is a distinctive property of proteoses. They are slightly 
 diffusible. 
 
 The primary proteoses are precipitated by saturation with magnesium 
 sulphate or sodium chloride ; deutero-proteose is not ; it is, however, 
 precipitated by saturation with ammonium sulphate. Proto- and 
 deutero-proteose are soluble in water ; hetero-proteose is not ; it requires 
 salt to hold it in solution. ' 
 
 3. Peptones. — They are soluble in water, are not coagulated by 
 heat, and are not precipitated by nitric acid, copper sulphate, ammo- 
 nium sulphate, and a number of other precipitants of proteins. They 
 are precipitated but not coagulated by alcohol. They are also precipi- 
 tated by tannin, picric acid, potassio-mercuric iodide, phospho-molybdic 
 acid, and phospho-tungstic acid. 
 
 They give the biuret reaction (rose-red solution with a trace of 
 copper sulphate and caustic potash or soda). • 
 
 Peptone is readily diffusible through animal membranes. 
 
 The annexed table will give us at a glance the chief characters of 
 peptones and proteoses in contrast with those of the native proteins, 
 albumins, and globulins. 
 
 Variety 
 
 of 
 Protein. 
 
 Action 
 
 of 
 Heat. 
 
 Action 
 
 of 
 Alcohol. 
 
 Action 
 
 of 
 
 Nitric Acid. 
 
 Action of 
 Ammonium 
 Sulphate. 
 
 Action of 
 
 Copper 
 
 Sulphate 
 
 and Caustic 
 
 Potash. 
 
 f 
 
 Diffusi- 
 bility. 
 
 Albumin. 
 Globulin. 
 Proteoses. 
 
 Peptones. 
 
 Coagulated. 
 
 Ditto. 
 
 Not 
 coagulated. 
 
 Not 
 coagulated. 
 
 Precipitated, 
 then coagu- 
 lated. 
 
 Ditto. 
 
 Precipitated, 
 but not co- 
 agulated. 
 
 Precipitated, 
 but not co- 
 agulated. 
 
 Precipitated 
 in the cold ; 
 not readily 
 soluble on 
 heating. 
 
 Ditto. 
 
 Precipitated 
 in the cold ; 
 readily sol- 
 nble on 
 heating; the 
 precipitate 
 reappears 
 on cooling. 1 
 
 Not precipi- 
 tated. 
 
 Precipitated 
 by complete 
 saturation. 
 
 Precipitated 
 by half satu- 
 ration; alto 
 precipitated 
 by MgS04. 
 
 Precipitated 
 by satura- 
 tion. 
 
 Not precipi- 
 tated. 
 
 Violet 
 colour. 
 
 Ditto. 
 
 Rose-red 
 colour 
 (biuret 
 
 reaction). 
 
 Rose-red 
 
 colour 
 ' (biuret 
 reaction). 
 
 Nil. 
 Ditto. 
 Slight. 
 
 i 
 .Great. 
 
 1 In th? case of de«tero-a,lbumos^ this reaction only occurs in the presence of excess gf sftlt. 
 
106 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 It will be noted that proteoses and peptones are classified mainly 
 on physical differences such as solubility and "salting out." It is, 
 however, convenient to retain the various names for the present until 
 more is known of their true chemical nature. They are doubtless 
 mixtures of coniplex polypeptides, and the peptide chains become 
 shorter as digestive cleavage progresses. 
 
 The question has been often raised why the stomach does not digest itself 
 during life. The mere fact that the tissues are alkaline and pepsin requires 
 an acid medium in which to act is not an explanation, but only opens up a 
 fresh difficulty as to why the pancreatic juice which is alkaline does not digest 
 the intestinal wall. To say that it is the vital properties of the tissues that 
 enable them to resist digestion only shelves the difficulty and gives no real 
 explanation of the mechanism of defence. Recent studies on the important 
 question of immunity (see Lesson IX) have furnished us with the key to 
 the problem ; just as poisons introduced from without stimulate the cells to 
 produce antitoxins, so harmful substances produced within the body are pro- 
 vided with anti-substances capable of neutralising their eifects ; for this 
 reason the blood does not normally clot within the blood-vessels, and Wein- 
 land has shown that the gastric epithelium forms an antipepsin, the intestinal 
 epithelium an antitrypsin, and so on. The bodies of parasitic worms that 
 live in the intestine are particularly rich in these anti-bodies. 
 
LESSON VIII 
 
 THE DIGESTIVE JUICES {continued) 
 
 PANCREATIC DIGESTION 
 
 1. A 1 per-cent. solution of sodium carbonate, to which a little 
 glycerol extract of pancreas ^ has been added, forms a good artificial 
 pancreatic fluid. 
 
 2. Half fill three test-tubes with this solution. 
 
 A. To this add half its bulk of diluted egg-white (1 in 10). 
 
 B. To this add a piece of fibrin. 
 
 C. Boil this ; cool ; then add fibrin. 
 
 3. Put all into the water-bath at 40° 0. After half an hour, test A 
 and B for'alkali-metaprotein by neutralisation, for proteoses by nitric 
 acid, and for proteoses and peptone by the biuret reaction. 
 
 4. Note in B that the fibrin does not swell up and dissolve, as in 
 gastric digestion, but that it is eaten away from the edges to the 
 interior. 
 
 5. In C no digestion occurs, as enzymes are destroyed by boiling. 
 
 6. Take a solution of starch, equal quantities in three test-tubes. 
 
 D. To this add a few drops of extract of pancreas (without 
 
 the sodium carbonate). 
 
 E. To this add a few drops of bile. 
 
 F. To this add both bile and pancreatic extract. 
 
 7. Put these into the water-bath, and test small portions of each 
 every half-minute by the iodine reaction. It disappears first in F ; 
 then in D ; while E undergoes no change. Test D and F for malbose 
 by Fehling's solution. 
 
 This shows the favourable influence bile exerts on pancreatic 
 digestion. It is, however, more marked still in the case of fats. 
 (See 9, below.) 
 
 8. Shake up a few drops of olive oil with artificial pancreatic juice 
 
 ^ Benger's liquor pancreatioua diluted with two or three times its volume of 
 1 per-cent. sodium carbonate may be used instead. 
 
 107 
 
108 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 (extract of pancreas and sodium carbonate). A milky fluid (emulsion) 
 is formed, from which the oil does not readily separate on standing. 
 
 9. Boil 10 c.c. of fresh milk ; cool ; colour with an indicator (for 
 instance, a few drops of an alcoholic solution of phenolphthalein), and 
 divide it into two parts of 5 c.c. each. To one add a few drops of 
 glycerol extract of pancreas ; to the other, the same amount of 
 pancreatic extract and a few drops of bile. Put each into the warm 
 bath. In each the pink colour disappears as fatty acids are liberated 
 by lipase ; this occurs more rapidly in the specimen containing bile. 
 
 The foregoing experiments illustrate the action that pancreatic 
 juice has on all three classes of organic food. 
 
 BILE 
 
 1. Ox bile is given round. Observe its colour, taste, smell, and 
 reaction to litmus paper. 
 
 2. Acidulate a little bile with 20 per-cent. acetic acid. A stringy 
 precipitate of a mucinoid substance is obtained. Filter this off and 
 boil the filtrate ; no protein coagulable by heat is present. 
 
 3. Add a few drops of bile to (a) acid-metaprotein prepared as 
 described in Lesson V, and to (b) solution of proteoses to which half 
 its volume of 0-2 per-cent. hydrochloric acid has been added. A 
 precipitate occurs ia each case. Bile salts precipitate the unpeptonised 
 protein which leaves the stomach. 
 
 4. PETTBNKOFEE'S TEST FOR BILE SALTS.— To a thin film of 
 bile in a capsule add a drop of solution of cane sugar and a drop of 
 concentrated sulphuric acid. A purple colour is produced. This occurs 
 more quickly on the application of heat. The test may also be per 
 formed as follows : — Shake up some bile and cane sugar solution in a 
 test-tube until a froth is formed. Pour concentrated sulphuric acid 
 gently down the side of the tube : it produces a purple colour in 
 the froth. 
 
 5. GMELIN'S TEST FOB BILE PIGMENTS.— On to a Uttle filming 
 nitric acid (i.e. nitric acid containing nitrons acid in solution) in a test- 
 tube pour gently a little bile. Notice the succession of colours — green, 
 blue, red, and yellow — at the junction of the two liguids. This test 
 may also be performed on a flat porcelain dish ; place a drop of fuming 
 nitric acid in the middle of a thin film of bile : it becomes surrounded 
 by rings of the above-mentioned colours. Huppert's test for bile 
 pigments is specially applicable for their detection in urine, and so is 
 postponed to Lesson XII. 
 
 6. HAT'S TEST FOB BILE SALTS.— Take two beakers or test tubes 
 foil of water ; to one add a few drops of bile, or solution of bile salts. 
 
BILE 109 
 
 Sprinkle a little flowers of sulphur on the surface of each. It remains 
 floating- on the pure water ; but where bile is present the surface 
 tension of the water is reduced, and the sulphur consequently rapidly 
 sinks. This test is very sensitive, and may be employed for the 
 detection of bile salts in urine. 
 
 7. CHOLESTEROL.— (a) Examine crystals of this substance with 
 the microscope. Heat these on a slide with a drop of sulphuric acid 
 and water (5:1); the edges of the crystals turn red. 
 
 (b) SalkowsM's Beadion, — Dissolve some cholesterol in chloroform 
 in a dry test-tube, and gently shake with an equal amount of concen- 
 trated sulphuric acid ; the solution turns red, and the subjacent acid 
 acquires a green fluorescence. The chloroformic solution of cholesterol 
 so reddened is rendered colourless by pouring it into a wet test-tube, 
 and the colour is restored by the addition of sulphuric acid. 
 
 (c) Liebermann-Burchard Beaction.—Tvro or three drops of acetic 
 anhydride are added to a chloroformic solution of cholesterol and 
 then sulphuric acid drop by drop. A rose-red colour first develops; 
 this becomes blue and finally bluish-green. 
 
 (d) Preparation of Cholesterol from Bram. — Ox or sheep's brain is 
 minced, and in order to remove the water is misred with three times 
 its weight of plaster of Paris. After some hours the mixture sets 
 into a hard mass, which can easily be broken up. Some of this 
 powdered material is given round. Add to the quantity supplied 
 suf&cient acetone to cover it. Allow it to stand for ten minutes, 
 shaking frequently. Filter the acetone solution through a dry filter 
 into a beaker, and allow the acetone to evaporate spontaneously. 
 Cholesterol crystallises out ; dissolve this in hot alcohol ; place a drop 
 on a glass slide and examine the typical crystals with the microscope. 
 
110 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 THE PANCREAS 
 
 The Pancreas is a compound tubulo-racemose gland ; between the 
 secreting acini are situated little masses of epithelial cells without ducts 
 called " islets of Langerhans." Examination of the secreting cells in 
 different stages of activity reveals changes comparable to those already 
 described in the case of salivary and gastric cells. Granules indicating 
 the presence of zymogens crowd the cells before secretion : these are 
 discharged during secretion, so that in an animal whose pancreas has 
 been powerfully stimulated to secrete, the granules are seen only at 
 the free border of the cells (see fig. 17). 
 
 Fig. 17. — Part of an alveolus of the rabbit's pancreas : A, before discharge ; B, after. 
 (From Foster, after Kuhne and Lea.) 
 
 As in the case of gastric juice, experiments on the pancreatic 
 secretion are usually performed with an artificial juice, made by 
 mixing a weak alkaline solution (1 per cent, sodium carbonate) with an 
 extract of pancreas. The pancreas should be kept some time before 
 the extract is made, so as to ensure that the transformation of 
 trypsinogen into tr3^sin has taken place. 
 
 Quantitative analysis of human pancreatic juice gives the following 
 results : — 
 
 Water . 97-6 per cent. 
 
 Organic solids . . 1-8 „ 
 
 Inorganic salts . . 0'6 „ 
 
 Dog's pancreatic juice is considerably richer in solids. 
 
 The organic substances in pancreatic juice are : — 
 
 (a) Enzymes. These are the most important both quantitatively 
 and functionally. They are four in number : — 
 
 i. Trypsin, a proteolytic enzyme. In the fresh juice, however, this 
 is present in the form of trypsinogen. 
 
 ii. Amylase, an amylolytic enzyme. 
 
PANCREATIC DIGESTION 111 
 
 iii. Lipase, a fat-splitting enzyme. 
 
 iv. A milk-curdling enzyme. 
 
 - (6) A small amount of protein matter, coagulable by heat. 
 
 (c) Traces of leucine, tyrosine, xanthine, and soaps. 
 
 The inorganic substances in pancreatic juice are : — 
 
 Sodium chloride, which is the most abundant, and smaller quantities 
 of potassium chloride, and phosphates of sodium, calcium, and 
 magnesium. The alkalinity of the juice is due to phosphates and 
 carbonates, especially of sodium. 
 
 THE SECRETION OF PANCREATIC JUICE 
 
 One of the most effective ways of producing a flow of the juice is to 
 introduce acid into the duodenum, and no doubt the acid of the gastric 
 juice is the normal stimulus for the pancreatic flow. This flow still 
 occurs when all the nerves supplying the duodenum and pancreas are 
 cut, and it was held by Popielski and by Wertheimer and Le Page 
 that it must be due to a local reflex, the centres being situated in the 
 scattered ganglia of the pancreas and of the solar plexus. Starling 
 and Bayliss, however, pointed out that it cannot be a nervous reflex, 
 since it occurs after extirpation of the solar plexus, and destruction of 
 all nerves passing to an isolated loop of intestine. Moreovef , atropine 
 does not paralyse the secretory action. It must therefore be due to 
 direct excitation of the pancreatic cells by a substance or substances 
 conveyed to the gland from the bowel by the blood-stream. 
 
 The exciting substance is not acid ; injection of 04 per cent, of 
 hydrochloric acid into the blood-stream has no influence on the 
 pancreas. The substance in question must be produced in the intestinal 
 mucous membrane under the influence of the acid. This conclusion 
 was confirmed by experiment. If the mucous membrane of the 
 duodenum or jejunum is exposed to the action of 0'4 per cent, hydro- 
 chloric acid, a substance is produced which, when injected into the 
 blood-stream in minimal doses, produces a copious secretion of pan- 
 creatic juice. The substance is termed secretin. It is associated with 
 another substance which lowers arterial blood-pressure. The two 
 substances are not identical, since acid extracts of the lower end of the 
 ileum produce a lowering of blood-pressure, but have no excitatory 
 influence on the pancreas. 
 
 Secretin is split off from a precursor, prosecretin, which is present 
 in relatively large amounts in the duodenal mucous membrane, and 
 gradually diminishes in amount throughout the intestine until it 
 entirely disappears in the ileum. Pro-secretin can be dissolved out of 
 
112 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 the mucous membrane by normal saline solution. It has no influence 
 on the pancreatic secretion. Secretin can be split oif from it by boiling 
 or by treatment with acid. 
 
 What secretin is chemically, we do not yet know. It is soluble in 
 alcohol and ether. It is not a protein, but probably is an organic 
 substance of low molecular weight. It is, moreover, the same sub- 
 stance in all animals, and not specific to diiferent kinds of animals. 
 
 The question arises whether there are any secretory nerves for 
 the pancreas. Pavloff thought he had discovered them in the vagus ; 
 but as he did not exclude in his experiments the passage of the 
 acid chyme from the stomach into the duodenum, it is probable that 
 the pancreatic secretion he obtained was due to that circumstance and 
 the consequent formation of secretin. Nerves of this nature have been 
 proved to exist by Anrep, but their action is a feeble one. 
 
 Injection of secretin also stimulates the flow of bile and of succus 
 entericus. 
 
 Secretin is an instance of the chemical messengers or hormones 
 (Starling) of the body. Evidence is accumulating to show that hor- 
 mones are extremely important. It has already, for instance, been 
 shown that one called gastrin is formed as the result of salivary diges- 
 tion, and stimulates the flow of gastric juice. Another is formed in 
 the corpus luteum of the ovary, which, passing into the mother's 
 circulation, stimulates the mammse to enlarge and secrete milk. 
 
 The pancreatic juice does not act alone on the food in the intestines. 
 There are, in addition, the bile, the succus entericus and bacterial action 
 to be considered. 
 
 The succus entericus or intestinal juice has no action on native 
 proteins. It is stated to possess a slight lipolytic action, and it 
 appears to have to some extent the power of converting starch into 
 sugar ; its best known action on carbohydrates, however, is due to an 
 enzyme it contains called invertase, which converts sucrose into glucose 
 and fructose (see p. 26). The term " inversion " may be extended to 
 include the similar hydrolysis of other disaccharides, although there 
 may be no formation of laevo-rotatory substances. There are two other 
 inverting enzymes in the succus entericus, one of which acts on maltose, 
 and the other on lactose. 
 
 A few years ago, however, Pavlo£f showed that succus entericus has 
 a still more important action, which is to activate the proteolytic 
 power of the pancreatic juice. Fresh pancreatic juice has very little 
 power on proteins, for what it contains is not trypsin, but its precursor, 
 trypsinogen. 
 
PANCREATIC DIGESTION 113 
 
 If fresh pancreatic and intestinal juices are mixed together, the 
 result is a very powerful proteolytic mixture, though neither Juice by 
 itself has any proteolytic activity. The substance is the intestinal 
 juice that activates trypsinogen or, in other words, liberates trypsin 
 from trypsinogen, and has been called by Pavloff an enzyme of enzymes, 
 or enter o-kinase. 
 
 Dixon and Hamill's recent work has made still clearer the mechanism 
 of pancreatic secretion. There are in the pancreas three precursors 
 of enzymes, namely, protrypsinogen, proamylase, and prolipase. 
 Secretin combines chemically, or at any rate acts chemically, on all 
 three ; it liberates amylase and lipase from their precursors, and these 
 two active enzymes pass into the pancreatic juice. It liberates tryp- 
 sinogen from protrypsinogen, and trypsinogen passes into the juice; 
 finally trjfpsinogen is converted into the active enzyme trypsin by the 
 entero-kinase of the succus entericus. Trypsinogen appeal's to be a 
 complex consisting of trypsin united to a protein moiety, and as long 
 as the enzyme is combined in this way, it is inactive ; entero-kinase is a 
 proteolytic enzyme which digests the protein moiety, and thus liberates 
 the trypsin (J. Mellanby and Wooley). The view has been advanced by 
 Herzen that the spleen sends a hormone to the pancreas which assists 
 in the elaboration of trypsinogen, but the evidence in favour of this 
 hypothesis is not generally regarded as convincing. 
 
 Intestinal juice contains another enzyme called erepsin by its dis- 
 coverer, Otto Cohnheim ; this is a peptolytic enzyme, and- breaks up 
 proteoses and peptones into their final cleavage products, the amino- 
 acids, and so assists the action of trypsin. The only native protein 
 which it digests is caseinogen. 
 
 ACTION OF PANCREATIC JUICE 
 
 The action of pancreatic juice, which when activated is the most 
 poweriul and important of all the digestive juices, may be described 
 under the headings of its four enzytaes. 
 
 1. Action of Trypsin. — Trypsin acts like pepsin, but with certain 
 differences, which are as follows : — 
 
 (a) It acts in an alkaline, pepsin in an acid medium. 
 
 (6) It acts more rapidly than pepsin ; deutero-proteoses can be 
 detected as intermediate products in the formation of peptone; the 
 primary proteoses have not been detected. 
 
 (c) Alkali-metaprotein is formed in place of the acid-metaprotein 
 of gastric digestion. 
 
114 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 (d) It acts more powerfully on certain proteins (such as elastin) 
 which are difficult of digestion in gastric juice. It does not, 'however, 
 digest collagen. , 
 
 (e) Acting on solid proteins such as fibrin, it eats them away from 
 the surface to the interior ; thei'e is no preliminary swelling as in 
 gastric digestion. 
 
 (f) Trypsin acts further than pepsin, and rapidly ' splits up the 
 proteose and peptone which have left the stomach into simpler sub- 
 stances, the polypeptides. The polypeptides in their turn are resolved 
 into their constituent amino-acids, such as leucine, tyrosine, alanine, 
 aspartic acid, glutamic acid, arginine, tryptophane, and many others. 
 The constitution and properties of these cleavage products are 
 described on pp. 45 to 50. In addition to these there is a certain 
 amount of ammonia. The red colour which a tryptic digest strikes 
 with chlorine or bromine water is due to the presence of tryptophane 
 (indole-amino-propionic acid). 
 
 When once the peptone stage is passed, the products of further 
 cleavage no longer give the biuret reaction ; hence they are frequently 
 termed abiuretie. 
 
 A variable fraction of the protein molecule is broken off with 
 comparative ease, so that certain free amino-acids appear in the 
 mixture, at a time when the remainder are still linked together as 
 polypeptides. But ultimately the whole molecule is resolved into 
 amino-acids, either entirely separated, or in very short polypeptide 
 linkages. 
 
 It will thus be seen that there are two important differences between 
 pepsin and trypsin ; one is a difference of degree, trypsin being by far 
 the more powerful and rapid catalyst ; the second is a difference of 
 kind, pepsin not being able to cleave polypeptides into amino-acids in 
 the way trypsin can. The preliminary action of pepsin, however, is 
 beneficial, for trypsin cleavage occurs more readily after pepsin has 
 acted on a protein. 
 
 2. Action of Amylase. — The conversion of starch into maltose 
 is the most rapid of all the actions of the pancreatic juice. Its power 
 in this direction is much greater than that of saliva, and it will act 
 even on unboiled starch. The absence of this enzyme in the pancreatic 
 juice of infants is an indication that milk, and not starch, is their 
 natural diet. 
 
 3. Action on Fats. — These are split by pancreatic lipase into 
 glycerol and fatty acids. The fatty acids unite with the alkaline 
 bases present to form soaps [saponification, see p. 37). If a glycerol 
 
BACTERIAL ACTION 115 
 
 extract of pancreas is filtered, the filtrate has no lipoclastic action ; the 
 material deposited on the filter is also inactive, but on mixing it with 
 the inactive filtrate once more, a strongly lipoclastic material is 
 obtained. In this way lipase is separable into two fractions : the 
 material on the filter is inactive lipase; the material in the filtrate 
 is its co-enzyme; the latter is jiot destroyed by boiling. Bile salts 
 also activate the inactive lipase, and this explains the fact that bile 
 favours fat-splitting. 
 
 Pancreatic juice also assists in the emulsification of fats; this it 
 is able to do because it is alkaline, and it is capable of liberating fatty 
 acids which form soaps with the alkali present ; the soap forms a film 
 on the outer surface of each globule, and this prevents them running 
 together. Emulsions are much more permanent in the presence of 
 colloids, such as gum or protein. The presence of protein in the 
 pancreatic juice renders it therefore specially suitable for the purpose 
 of emulsification. ' 
 
 4. Milk-curdling Enzyme. — The addition of pancreatic extracts or 
 pancreatic juice' to milk causes clotting ; but this action (which differs in 
 some particulars from the clotting caused by gastric rennet) can hardly 
 ever be called into play, as the milk upon which the juice has to act 
 has been already curdled by the rennin of the stomach. This action is, 
 as in the case of pepsin, possibly a function of trypsin. 
 
 BACTERIAL ACTION 
 
 The gastric juice is an antiseptic ; the pancreatic juice is not. An 
 alkaline fluid like pancreatic juice is just the most suitable medium 
 for bacteria to flourish in. In an artificial digestion the fluid soon 
 becomes putrid, unless special precautions to exclude or kill bacteria 
 are taken. It is often difficult to say where pancreatic action ends 
 and bacterial action begins, as many of the bacteria that grow in the 
 intestinal contents (having reached that situation in .spite of the gastric 
 juice) produce enzymes which act in the same way as the pancreatic 
 juice. Some form sugar from starch, others peptone, and amino- 
 acids from proteins, while others, again, break up fats. There are, 
 however, certain actions that are entirely due to these putrefactive 
 l)rganisms. 
 
 i. On carbohydrates. The most frequent fermentation they set 
 up is the lactic acid fermentation ; this may go further and result in 
 the formation of carbonic acid, hydrogen, and butyric acid (see p. 28). 
 Cellulose is broken up into carbonic acid and methane, This i§ the 
 
116 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 chief cause of the gases in the intestine, the amount of which is 
 increased by vegetable food. 
 
 ii. On fats. In addition to acting like lipase, they produce lower 
 acids (valeric, butyric, etc.). The formation of acid products from fats 
 and carbohydrates gives to the intestinal contents an acid reaction. 
 Recent researches show that the contents of the intestine become acid 
 much higher up than was formerly supposed. These organic acids do 
 not, however, hinder pancreatic digestion. 
 
 ., iii. On proteins. Fatty acids and amino-acids are produced ; but 
 the enzymes of these putrefactive organisms have a specially powerful 
 action in liberating substances having an evil odour, such as indole 
 (CgHyN), skatole (CgHgN), and phenol (C^B-fi). The indole and 
 skatole originate from the tryptophane radical of proteins. There are 
 also gaseous products in some cases. 
 
 Ammonia-producing organisms flourish best in the lower regions of 
 the small intestine ; the ammonia neutralises the organic acids produced 
 higher up, and in the large intestine the contents are consequently 
 alkaline. 
 
 Bases from Amino-acids. — A very characteristic action of putrefactive 
 bacteria is exerted on the amino-acids ; this change consists in the 
 splitting off of carbonic acid from their carboxyl (COOH) group, and 
 the production of amines according to the following equation : — 
 
 R.NH2 R.NHj 
 
 I = I +C0,. 
 
 QOOH H 
 
 R in the above equation represents the radical which varies in the 
 different members of the amino-acid group (CH^ in glycine, CjH^ in 
 alanine, CjHjq in leucine, etc.). 
 
 It has been known for some time that the well-known putrefactive 
 bases putrescine and cadaverine are formed in this way from ornithine 
 and lysine respectively : — 
 
 NH2.CH2.CH2.CH2.CH.NH2.COOH = NH2.CH2.CH2.CH2.CH2.NH2 -1- CO2 
 
 [ornithine or diamino-valeric acid] [putrescine or tetraniethylene-diamine] 
 
 NH2.cH2.CH2.CH2.CH2CH.NH2.cooH 
 
 [lysine or diamino-eaproic acid] = NH2.CH2.CH3.CH2.CH2.CH2.NH2. + COg. 
 
 [cadaverine or pentamethylene-diaminc] 
 
 Recently, however, it has been recognised that this " decarboxyla- 
 tion" of amino-acids is a general reaction of certain putrefactive 
 organisms, and that a whole series of bases is obtainable, which are of 
 considerable physiological interest. For example it was found that 
 
Bacterial ActioN 
 
 11? 
 
 during the putrefaction of meat and placenta, leucine and tyrosine 
 are converted into the bases iso-amylamine and oxyphenylethylamine 
 respectively : — 
 
 [leucine or amino-caproie acid] 
 
 CH.CH2.CH.NH2.(X)OH=^^^^CH.CH2.CH2.NH2+C02 
 
 [iso-amylamine] 
 
 OH.C6H,.CH2.CH.NH2COOH=OH.C6H4.CH2.CH2.NH2+C02. 
 
 [oxyphenylethylamine] 
 
 [tyrosine or oxyphenylalanine] 
 
 These two bases exert a pressor action on arterial blood-pressure 
 similar to that of adrenaline, and it is most probable that the pressor 
 bases contained in normal urine during a liberal meat diet owe their 
 origin to bacterial action in the intestine. The base oxyphenylethyl- 
 amine has also been found in extracts of ergot — the fungus which 
 develops in the ovaries of certain grasses {e.g. rye) ; the enzymes of this 
 fungus are able, like bacterial enzymes, to " decarboxylise " not only 
 tyrosine, but also histidine and arginine, the bases formed being imi- 
 dazolethylamine and agmatine respectively, as shown below : — 
 
 HO— NH 
 
 >CH 
 
 > 
 
 (NH)C/ 
 
 V 
 
 0— N 
 
 I 
 CHs 
 
 I 
 CH.NH, 
 
 I 
 COOH 
 
 [histidine] 
 
 HC— NH 
 
 II I >CH 
 
 C— N 
 I 
 
 I 
 CH2.NH2 
 
 [ imidazolethylamine] 
 
 (NH)C<^' 
 
 NH2 
 
 ■NH.CH2 
 
 I 
 CH2 
 
 I 
 CHa 
 
 I 
 CH.NH2 
 
 I 
 
 COOH 
 
 [arginine] 
 
 NH2 
 
 ^NH.CH, 
 
 I 
 CH2 
 
 I 
 OH2 
 
 CH2.NH2 
 [agmatine] 
 
 Imidazolethylamine has also been obtained from histidine owing to 
 the action of putrefactive bacteria in the intestine. Very small doses 
 lower blood-pressure enormously. The base agmatine was first dis- 
 covered in herring roe. There is no doubt that the pharmacological 
 action of ergot is in part due to these bases. 
 
 Quite analogous to them is the formation of amino-butyric acid from 
 
118 
 
 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 glutamic acid, and of ^-alanine from aspartic acid during putre- 
 faction : — 
 
 COOH 
 
 1 
 
 COOH 
 
 1 
 
 
 COOH 
 
 1 
 
 COOH 
 
 1 
 
 CH^ 
 
 CHa 
 
 
 CH2 
 
 CH, 
 
 1 
 
 1 
 
 H-COa 
 
 1 
 
 = 1 
 
 CHa 
 
 1 
 
 CHs 
 
 1 
 
 
 CHNH. 
 
 1 
 
 CH2.NH 
 
 [p-alanine] 
 
 CH.NHa' 
 
 1 
 
 CH2.NH2 
 
 [amino- 
 
 
 COOH 
 
 [aspartic acid] 
 
 
 COOH 
 
 butyric acid] 
 
 
 
 
 ■ [glutamic acid] 
 
 
 
 
 
 +C02 
 
 EXTIRPATION OF THE PANCREAS 
 
 Complete removal of the pancreas in animals and diseases of the 
 pancreas in man produce a condition of diabetes, in addition to the 
 loss of pancreatic action in the intestines. Grafting the pancreas 
 from another animal into the abdomen of the animal from which 
 the ■ pancreas has been previously removed relieves the diabetic 
 condition. 
 
 How the pancreas acts otherwise than in producing the pancreatic 
 juice is not precisely known. It must, however, have other functions 
 related to the general metabolic phenomena of the body, which are 
 disturbed by removal or disease of the gland. This is an illustration of 
 a universal truth — viz. that each part of the body does not merely do 
 its own special work, but is concerned in the great cycle of changes 
 which is called general metabolism. Interference with any organ 
 upsets, not only its specific function, but causes disturbances ■ through 
 the body generally. The interdependence of the circulatory and 
 respiratory systems is a well-known instance. Removal of the thyroid 
 gland upsets the whole body, producing widespread changes known 
 as myxcedema. Removal of the testes produces, not only a loss of the 
 spermatic secretion, but changes the whole growth and appearance 
 of the animal. This is accounted for by the hypothesis that such 
 glands produce an internal secretion, which leaves the gland via the 
 lymph or venous blood, and is then distributed to minister to parts 
 elsewhere. Removal of such glands as the thyroid or suprarenal 
 produces disease or death because this internal secretion can no longer 
 be formed. In the case of the pancreas, the external secretion of the 
 pancreas (that is, pancreatic juice) is formed by the cells lining the 
 acini, and the internal secretion, stoppage of which in some way leads 
 to diabetes, has been attributed to the islets of Langerhans. 
 
DIABETES 119 
 
 Olycosuria (Sugar in the urine) occurs in many conditions. It may be a 
 temporary condition, as in alimentary glycosuria, which is due to excess of 
 carbohydrate food, or a comparatively inactive liver which is incapable of 
 dealing with the usual carbohydrate supply. It may be produced by injury 
 to the floor of the fourth ventricle (Claude •Bernard's celebrated puncture ex- 
 periment), but only when the liver has withiri it a store of glycogen. The 
 injury to the bulbar centre influences thus the nervous mechanism which 
 regulates the glycogenic function of the liver. In diabetes mellitus, the body 
 is unable to utilise sugar by burning it, and so liberating heat and energy ; 
 sugar therefore accumulates in the blood and overflows into the urine. In 
 some cases rigid abstention from carbohydrate food makes little or no differ- 
 ence, and the sugar must come from the protein constituents of protoplasm, 
 alanine being one of the most important of the intermediate products. When 
 the pancreatic functions are in abeyance, the diabetic state is due to an im- 
 paired capacity to oxidise sugar down to its ultimate products, carbonic acid 
 and water. The destruction of sugar by the tissues is termed glycolysis, and 
 one view advanced is that the internal secretion of the pancreas a^ctivates the 
 glycolytic enzyme ; therefore when the internal secretion of the pancreas is 
 absent, the activating impulse is absent also. There are many difficulties, 
 however, in accepting this view. Adrenaline, the secretion of the suprarenal 
 body, produces an increased discharge of sugar from the liver, but under 
 normal conditions this is inhibited by an antagonistic hormone secreted by 
 the pancreas. The most satisfactory view of pancreatic diabetes is that it is 
 due to the absence of this antagonistic hormone. 
 
 Many drugs and poisons produce glycosuria, but the most potent of them 
 is phloridzin ; this substance causes diabetes in animals which have no 
 glycogen in their tissues, and phloridzin-diabetes is analogous to those severe 
 forms of diabetes mellitus in man in which the sugar must come from protein 
 katabolism. Curiously enough, in phloridzin-diabetes the blood shows no 
 excess of sugar, but this is probably because the drug renders the kidney 
 so permeable fo sugar that the outflow into the urine occurs at such a rapid 
 rate that the percentage in the blood is kept at a low figure. 
 
 Acidosis. — This condition is seen in diabetes ; poisonous acids in the blood 
 produce a state of coma, or deep unconsciousness, which may finally cause 
 death. Tor a diabetic patient is not only unable to burn and So utilise carbo- 
 hydrate, but he fails in a similar way in his utilisation of fat. Butyric acid 
 and p-hydroxybutyric acid are probably normal intermediate products in fat 
 katabolism, but a healthy man on a normal diet is able still further to oxidise 
 them into carbonic acid and water. < But on an abnormal diet, for instance, 
 when carbohydrate food is absent, fat-cleavage largely stops short at the 
 hydroxy butyric acid stage ; consequently this and possibly other related 
 fatty acids accumulate and cause acidosis ; this condition is increased the 
 more fat is given in the food, and the acidosis of diabetes is similarly in- 
 creased by fatty food. These poisonous acids were once believed to originate 
 from proteins ; if that were so there ought to be an increase of other protein 
 katabolites in the urine, which there is not. The acids decrease the alkalinity 
 and carbonic acid of the blood, and the ammonia of the urine .is increased ; 
 this indicates an attempt of the body to neutralise the acids. 
 
120 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 The hydroxybutyric acid does not pass entirely unchanged into the urine. 
 /3-Hydroxybutyric acid is CH3.CHOH.CH2.COOH. By oxidation, the two 
 hydrogen atoms in thick type are removed to form water, and this leaves 
 CH3.CO.CH2.COOH, which is aceto-acetic acid : when the COO in thick 
 type is removed we get acetone (CH3.CO.CH3), which gives the breath and 
 urine of such patients an apple-like smell. 
 
 In these changes the liver plays an important part by means of certain 
 enzymes which Dakin has proved to 'exist. One enzyme, called P-hyd/roxy- 
 buti/rase, is an oxidase ; it oxidises the /3-hydroxybutyric into aceto-acetic 
 acid, and its action is increased by the addition of blood or oxyhsemoglobin, 
 which furnishes the necessary oxygen. It probably is active in health as well 
 as in disease, the aceto-acetic acid being finally burnt into carbonic acid and 
 water. The other enzyme which forms acetone is not an oxidative one, and 
 acetone formation probably never occurs in the healthy state. 
 
 The question of diabetes is an important one, and the foregoing paragraphs 
 have treated it only in outline ; the student should consult a general text- 
 book on Physiology or Pathology for a full consideration of the subject. 
 
 THE BILE 
 
 The liver is an organ which has many functions ; one of these, the 
 glycogenic function, is referred to in the preceding section ; it also plays 
 a part in the metabolism of proteins (see formation of urea and uric 
 acid) and of fats. In this place, however, we are specially concerned 
 with its secretion, namely, the Bile. 
 
 Bile is the secretion of the liver which is poured into the duodenum : 
 it has been collected in living animals by means of a biliary fistula ; the 
 same operation has occasionally been performed in human beings. At 
 death the gall bladder yields a good supply of bile which is more con- 
 centrated than that obtained from a fistula. 
 
 Though the chief blood supply of the liver is by a vein (the portal 
 vein), the amount of blood in the liver varies with its needs being 
 increased during the periods of digestion. This is due to the fact 
 that in the area from which the portal vein collects blood — stomach, 
 intestine, spleen, and pancreas — the arterioles are all dilated, and the 
 capillaries are thus gorged with blood. Further, the active peristalsis 
 of the intestine and the pumping action of the spleen are additional 
 factors in driving more blood onwards to the liver. 
 
 The bile is secreted from the portal blood at a much lower pressure 
 than one finds in glands, such as the salivary glands, the blood supply 
 of which is arterial. Herring and Simpson have found that the pres- 
 sure in the bile duct averages 30 mm. of mercury, which is considerably 
 above that in the portal vein. 
 
THE BILE 121 
 
 The increase in the flow of bile which occurs after the arrival of 
 the semi-digested food (chyme) in the intestine has been explained by 
 the circumstance that secretin is a stimulant of the liver as well as of 
 the pancreas. The action of secretin on bile is not, however, a pro- 
 nounced one. The most efficient cholagogue known consists of the 
 bile salts themselves ; these, after entering the intestine, are reabsorbed 
 and return to the liver, and once more stimulate that organ to activity. 
 
 The chemical processes by which the constituents of the bile are 
 forined are obscure. We, however, know that the biliary pigment is 
 produced by the decomposition of haemoglobin. Bilirubin is, in fact, 
 identical with the iron-free derivative of haemoglobin called hsematoidin, 
 which is found in the form of crystals in the old blood clots such as 
 occur in the brain after cerebral htemor- 
 rhage((see fig. 18). Moreover, bilirubin / * ^f 
 
 yields hsemopyrrol, a substance also ^ ^B *>^ r-^A 
 
 obtained from blood-pigment. ff ^ ^2» fc <^ ^ ' j^P 
 
 An injection of haemoglobin into the f * 9 ^% ^' ^ ^ 
 portal vein, or of substances such as _ ' g |j||i ^^ 
 water which liberate hsemoglobin from ^ ^ - »' 7"^' 
 
 the red blood corpuscles, produces an .' mtMkv^' 
 
 increase of bile pigment. If the spleen ^911^^^^ ■* ' 
 takes any part in the elaboration of bile 
 
 , .,1 , 1 ,. , I'^IG- 18- — Hgematoidin crystals. 
 
 pigment, it does not proceed so tar as to 
 
 liberate haemoglobin from the corpuscles. No free hsemoglobin is 
 
 discoverable in the blood-plasma in the splenic vein. 
 
 The amount of bile secreted is differently estimated by different 
 observers ; the amount secreted daily in man appears to vary from 
 500 c.c. to 1 litre (1000 c.c). 
 
 THE CONSTITUENTS OF BILE 
 
 The constituents of the bile are the bile salts proper (taurocholate 
 and glycocholate of soda), the bile pigments (bilirubin, biliverdin), a 
 mucinoid substance, small quantities of fats, soaps, cholesterol, lecithin, 
 urea and mineral salts, of which sodium chloride and the phosphates 
 of iron, calcium, and magnesium are the most important. 
 
 Bile is a yellowish, reddish-brown or green fluid, according to the 
 relative preponderance of its two chief pigments. It has a musk-like 
 odour, a bitter-sweet taste, and a neutral or faintly alkaline reaction. 
 
 The specific gravity of human bile from the gall-bladder is 1026 to 
 1032; that from a fistula, 1010 to 1011. The greater concentration 
 
122 
 
 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 of gall-bladder bile is partly but not wholly explained by the addition 
 to it from the walls of that cavity of the mucinoid material. 
 
 The amount of solids in gall-bladder bile varies from 9 to 14 per 
 cent., in fistula bile from 1-5 to 3 per cent. The following, table shows 
 that this low percentage of solids is almost entirely due to want of bile 
 salts. ' This can be accounted for in the way first suggested by Schiff — 
 that there is normally a bile circulation going on in the body ; a large 
 quantity of the bile salts which paSs into the intestines is first split up, 
 then reabsorbed and again secreted. Such a circulation would obviously 
 be impossible in cases where all the bile is discharged to the exterior. 
 
 The following table gives some important analyses of human 
 bile :— 
 
 Constituents. 
 
 Fistula Bile 
 
 (healthy woman. 
 
 Copeman and 
 
 Winston). 
 
 Fistula Bile (case 
 of cancer. Yeo 
 and Herroun). 
 
 Normal Bite 
 (Frerichs). 
 
 Sodinm glycoeholate . 
 Sodium taurocholate . 
 Fats and lipoids . .« 
 Mucinoid material 
 Pigment 
 Inorganic salts 
 
 1 0-6280 1 
 
 0-0990 
 0-1725 
 0-0725 
 0-4510 
 
 0-165 
 0-055 
 0-038 
 
 1 0-148 
 
 0-878 
 
 V 9-14 
 1-18 
 2-98 
 0-78 
 
 Total solids . 
 
 Water (by difference) . 
 
 1-4230 
 98-5770 
 
 1-284 
 98-716 
 
 14-08 
 85-92 
 
 
 '100-0000 
 
 100-000 
 
 100-00 
 
 Bile Mucin. — -There has been considerable diversity of opinion as 
 to whether the bile mucin is really mucin. The most recent work in 
 Hammarsten's laboratory shows that difierences occur in different 
 animals. Thus in the ox there is very little true mucin, but a great 
 amount of nucleo-protein ; in human bile, on the other hand, there is 
 very little if any nucleo-protein ; the mucinoid material present there 
 is really mucin. (On the general characters of Mucin and Nucleo- 
 PHOTEiNS, see pp. 62 to 65.) 
 
 The Bile Salts. — The bile contains the sodium salts of complex 
 acids called the bile acids. The acids most frequently found are 
 glycocholic and taurocholic acids. The former is more abundant 
 in the bile of man and herbivora ; the latter in carnivora such as the 
 
THE BILE 123 
 
 dog. The most important difference between the two acids is that 
 taurocholic acid contains sulphur, and glycocholic acid does not. 
 
 Grlycocholic acid (CjgH^gNOu) is hydrolysed by the action of dilute 
 acids and alkalis, and also in the intestine, and split into glycine or 
 amino-acetic acid and cholalic acid : — 
 
 Cj^H^gNO^ + H,0 = CH2.NH„.C00H + a,H,„0 
 
 [gfycochoUo acid] [glycine] ' [cholalic acid] 
 
 The glycocholate of soda has the formula Cg^H^.^NaNOi;. 
 Taurocholic acid (Co^H^jNOyS) similarly splits into taurine or 
 amino-ethylrsulphonic acid and cholalic acid : — 
 
 [taurocholic acidl [taurine] [cholalic acid] 
 
 The taurocholate of soda has the formula CggH^NaNO^S. 
 
 Glycocholic and taurocholic acids have lately been prepared syu- 
 thfetically from cholalic acid and glycine, and taurine respectively. 
 
 The colour reaction called Pettenkofer's reaction, described in the 
 practical exercises at the head of this lesson, is due to the presence 
 of cholalic acid. The sulphuric acid acting on sugar forms a small 
 quantity of furfuraldehyde, in addition to other products. It is the 
 furfuraldehyde which gives the purple colour with cholalic acid. 
 
 The Bile Pigments. — The two chief bile pigments are bilirubin 
 and biliverdin. Bile which contains chiefly the former (such as dog's 
 bile) is of a golden or orange-yellow colour, while the bile of many 
 herbivora, which contains chiefly biliverdin, is either green or bluish 
 green. Human bile is generally described as containing chiefly bili- 
 rubin, but there have been some cases described in which biliverdin 
 was in excess. The bile pigments show no absorption bands with the 
 spectroscope. 
 
 Bilirubin has the formula CggH^gN^Ou : it is thus an iron-free 
 derivative of haemoglobin. The iron is apparently stored up in the 
 liver cells, perhaps for future use in the manufacture of new 
 haemoglobin. 
 
 The bile contains only a trace of iron. 
 
 Biliverdin has the formula (Cjf|Hjg!N.20^)„ {i.e. more oxygen than bili- 
 rubin) : it may occur as such in bile ; it may be formed by simply 
 exposing red bile to the oxidising action of the atmosphere ; or it may 
 be formed, as in Gmelin's test, by the more vigorous oxidation produced 
 by fuming nitric acid. 
 
 Gmelin's test consists of a play of colours — green, blue, red, and 
 finally yellow — produced by the oxidising action of fuming nitric acid 
 
IM ESSEN tlALS OF CHEMICAL PHVSIOLOGV 
 
 (that is, nitric acid containing nitrous acid in solution). The end or 
 yellow product is called choletelin, CjgHguN^Ojj. Occasionally in various 
 animals these products of intermediate oxidation are found in the bile ; 
 thus bilicyanin (the blue pigment) and bilipurpurin (the purple one) 
 may be present. 
 
 Hydrobilirubin. — If a solution of bilirubin or biliverdin in dilute 
 alkali is treated with sodium amalgam or allowed to putrefy, a 
 brownish pigment is formed called hydrobilirubin, CgjH^N^Oy. With 
 the spectroscope it shows a dark absorption band between h and F, and 
 a fainter band in the region of the D line. 
 
 Urobilin. — Hydrobilirubin is interesting because a similar substance 
 is formed from the bile pigment by reduction processes in the intestine ; 
 this is stercohUin, the pigment of the fseces. Some of this is absorbed 
 and ultimately leaves the body in the urine as one of its pigments 
 called urobilin. A small quantity of urobilin is sometimes found 
 preformed in the bile. The identity of urobilin and stercobilin has 
 been frequently disputed, but the work of Garrod and Hopkins has 
 confirmed the old statement that they are the same substance with 
 different names. Urobilin has a well-marked absorption band in the 
 region of the F line, and when partially precipitated from an alkaline 
 solution by acidification, it also shows an absorption band in the region 
 of the E line. Hydrobilirubin differs from urobilin in containing much 
 more nitrogen in its molecule (9'2 instead of 4'1 per cent.), and is 
 probably a product of less complete reduction than urobilin. (See 
 further. Lesson XXV.) Urobilin is also formed by the oxidation of 
 hsemopyrrol (see Haemoglobin, p. 148). 
 
 Cholesterol. — In bile this substance is normally present in small 
 quantities only, but it may occur in excess, and so form the concretions 
 known as gallstones, which are generally more or less tinged with 
 bilirubin. Its chemical characters and physiological importance are 
 discussed on pp. 38, 39 ; its colour reactions are given in to-day's 
 practical exercises (p. 109). 
 
 THE USES OF BILE 
 
 Bile is doubtless, to a certain extent, excretory. In some animals 
 it has a slight action on fats and starch, but it appears to be rather a 
 coadjutor to the pancreatic juice (especially in the digestion of fat) than 
 bo have any independent digestive activity. Its auxiliary, action in 
 the digestion of fat and starch has been shown in our practical 
 exercises (pp. 107-108). It has a similar slight assisting power in the 
 digestion of proteins. 
 
THE BILE 125 
 
 Bile is said to be a natural antiseptic, lessening the putrefactive 
 processes in the intestine. This is very doubtful. Though the bile 
 salts are weak antiseptics, the bile itself is readily putrescible, and 
 the power it has of diminishing putrefaction in the intestine is due 
 chiefly to the fact that by increasing absorption it lessens the amount 
 of putrescible matter in the bowel. 
 
 When the bile meets the chyme the turbidity of the latter is 
 increased, owing to the precipitation of unpeptonised protein. This 
 is an action due to the bile salts, and it has been surmised that this 
 conversion of the chyme into a more viscid mass is to hinder somewhat 
 its progress through the intestine : it clings to the intestinal wall, thus 
 allowing absorption to take place. The neutralisation of the acid 
 gastric juice by the bile also allows the alkalinity of the pancreatic 
 juice to have full play. Bile is a solvent of ^fatty acids, and assists the 
 absorption of fat. 
 
 THE FATE OF THE BILIARY CONSTITUENTS 
 
 We have seen that fistula bile is poor in solids as compared with 
 normal bile, and that this is explained on the supposition that the 
 normal bile circulation is not occurring — the liver cannot excrete 
 what it does not receive back from the intestine. Schiff was the first 
 to show that if the bile is led back into the duodenum, or even if the 
 animal is fed on bile, the percentage of solids in the bile excreted is 
 at once raised. It is on these experiments that the theory of a bile 
 circulation is mainly founded. The bile circulation relates, however, 
 chiefly, if not entirely, to the bile salts : they are found but sparingly 
 in the faeces ; tliey are only represented to slight extent in the urine ; 
 hence it is calculated that seven-eighths of them are reabsorbed from 
 the intestine. Small quantities of cholalic acid, taurine, and glycine 
 are found in the fseces ; the greater part of these products of the 
 decomposition of the bile salts is taken by the portal vein to the liver, 
 where they are once more synthesised into the bile salts. Some of the 
 taurine is absorbed and excreted as tauro-carbamic acid (CgH^NHCO. 
 NHjHSOg) in the urine. Some of the absorbed glycine may be excreted 
 as urea. The pigment is changed into stercobilin, a substance like 
 hydrobilirubin. Some of the stercobilin is absorbed, and leaves the 
 body as the urinary pigment, urobilin. The cholesterol in the ffeces 
 was formerly supposed to be a bile residue ; but in some animals, 
 especially those which feed on gra^ss, the source of the frecal cholesterol 
 is the vegetable cholesterol (phytosterol) of the food. In some cases 
 it is reduced to form a derivative- termed coprosterol. 
 
126 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 The faeces are alkaline in reaction, and on an ordinary mixed diet 
 contain comparatively little food residues, and a small quantity is 
 excreted even during starvation. Voit and Hermann showed inde- 
 pendently that an intestinal loop which had been emptied and separated 
 from the rest of the bowel contained, a few days later, material identical 
 with faeces, and consiisting of intestinal juice, desquamated epithelium 
 cells, and bacteria. The increase in the amount of fseces which occurs 
 when food is taken, even when the food is free from cellulose, is due 
 to the mechanical and chemical stimulation which leads to an increase 
 in the succus entericus, and in the shedding of epithelial cells. The 
 fseces contain about 1 per cent, of nitrogen, but this is chiefly contained 
 in the bodies of bacteria, and the disintegrated epithelial cells. Addi- 
 tion of protein to the diet makes practically no diiference to the 
 nitrogen in the faeces under normal conditions. 
 
 The addition of cellulose to the diet increases the bulk of the 
 fseces, partly because most of the cellulose is unchanged, partly 
 because it stimulates the mucous membrane to secrete more 
 succus entericus, and finally because the larger food residue favours 
 the development of bacteria. On an average, from one-third to one-fifth 
 (varying with the diet) of the weight of dried fseces consists of bacteria. 
 The average weight of dried bacteria excreted daily is 8 grammes ; 
 this contains OS gramme of nitrogen, or about half the nitrogen of 
 the fKces. Strasburger estimated that about 128,000,000,000,000 
 bacteria are evacuated in the fseces of a man every day. The vast 
 majority of these are dead. 
 
 When cellulose is absent from the diet, the faeces contain from 
 65 to 75 percent, of water; the dry residue contains about 7 per cent, 
 of nitrogen, and the non-nitrogenous material consists of about equal 
 quantities of ash and substances soluble in ether, with small quantities 
 of stercobilin and other bile residues. The ash contains mainly calcium 
 phosphate, with small amount* of iron and magnesium. The ethereal 
 extract contains cholesterol, lecithin, fatty acids, soaps, and a very 
 small amount of neutral fat. The proteins are chiefly mucin and 
 nucleo-protein, and are derived not from the food, but from the 
 intestinal wall, or are contained in the bacteria ; no doubt a large 
 part of the ethereal extract is also supplied by the bacteria. 
 
 Cellulose is thus the only important constituent of the food which 
 is unafiected by the digestive juices, although a variable amount, 
 which is largest in herbivorous animals, undergoes bacterial decom- 
 position. The presence of cellulose also interferes with the absorption 
 of proteins, for the digestive juices have difliculty in penetrating the 
 
ABSORPTION ■ 127 
 
 cellulose membranes of vegetable cells. Thus "Voit found that 42 per 
 cent, of the nitrogen in the food were lost in the faeces of a vegetarian. 
 This is due solely to the cellulose and not to any difference in the 
 digestibility of animal and vegetable proteins, for if vegetable food is 
 finely subdivided, and then thoroughly cooked and softened, this loss 
 is lessened, and if vegetable protein is entirely freed from cellulose 
 it is as thoroughly absorbed as animal protein. Fifteen per cent, of 
 the dry substance of green vegetables and brown bread, 20 per cent, 
 of carrots and turnips, and a still larger amount of beans are lost in 
 the fsecal residue. 
 
 .The intestinal contents travel more rapidly when vegetables are 
 present, for the indigestible cellulose stimulates peristalsis and there- 
 fore a large quantity of water escapes absorption in the colon. Thus 
 on an ordinary mixed diet 35 grammes of dry substance and 100 
 grammes of water are daily excreted in the faeces, whereas on a 
 vegetable diet the quantities are 75 and 260 grammes respectively. 
 
 MECONIUM 
 
 Meconium is the name given to the greenish-black contents of the 
 intestine of new-born children. It is chiefly concentrated bile, with 
 debris from the intestinal wall. The pigment is a mixture of bilirubin 
 and biliverdin ; it is not stercobilin. 
 
 ABSORPTION 
 
 Food is digested in order that it may be absorbed. It is absorbed 
 in order that it may be assimilated — that is, become an integral part 
 of the living material of the body. 
 
 Having now considered the action of digestive juices, we can study 
 the absorption which follows. In the mouth and oesophagus the 
 thickness of the epithelium and the quick passage of the food through 
 these parts reduce absorption to a minimum. Absorption takes place 
 to a small extent in the stomach ; the small intestine, with its folds 
 and villi to increase its surface, is, however, the great place for absorp- 
 tion ; and, although the villi are absent from the large intestine, 
 absorption (mainly of water) occurs there also, but to a less extent. 
 
 Foods such as water and soluble salts like sodium chloride are 
 absorbed unchanged. The organic foods, however, are considerably 
 changed, colloid materials such as starch and protein being converted 
 respectively into the dilfusible materials sugar and aminoacids. 
 
 There ar? two channels of absorption, the blood-vessels (portal 
 capillaries) and the lymphatic, vessels or lacteals. 
 
128 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 Absorption, however, is no mere physical process of diffusion and 
 filtration. We must also take into account the fact that the cells 
 through which the absorbed substances pass are living, and in virtue 
 of their vital activity not only select materials for absorption, but also 
 may change those substances while in contact with them. These cells 
 are of two kinds : (1) the columnar epithelium that covers the surface; 
 and (2) the lymph cells in the lymphoid tissue beneath. It is now 
 generally accepted that of the two the former, the columnar epithelium, 
 is the more important. When these cells are removed, or rendered 
 inactive by sodium fluoride, absorption practically ceases, though the 
 opportunities for simple filtration or diffusion would by such means- be 
 increased. 
 
 Absorption of Carbohydrates. — Though the sugar formed from 
 starch by ptyalin and amylase is maltose, that found in the blood 
 is glucose. Under normal circumstances little if any is absorbed by 
 the lacteals. The glucose is formed from the maltose by the succus 
 entericus. Sucrose and lactose are also converted into mono- 
 saccharides before absorption ; but if they are injected into the blood- 
 stream direct, they are unaltered by the liver and finally leave the 
 body by the urine. 
 
 The carbohydrate food which enters the blood as glucose is taken 
 to the liver, and there stored up in the form of glycogen — a reserve 
 store of carbohydrate material for the future needs of the body. 
 Glycogen, however, is found in animals which take no carbohydrate 
 food. It must then be formed by the protoplasmic activity of the 
 liver cells from their protein constituents. The carbohydrate store 
 leaves the liver in the blood of the hepatic vein as glucose once more. 
 
 The above is a brief statement of the glycogenic function of the 
 liver as taught by Claude Bernard, and accepted by the majority of 
 physiologists. It was strongly contested by Pavy, who held that the 
 glycogen formed in the liv^r from the sugar of the portal blood is never 
 during life reconverted into sugar, but is used in the formation of other 
 substances, especially fat ; and it certainly is the case that carbohydrate 
 food is fattening. The main fate of glycogen is, however, to be con- 
 verted into glucose for distribution to the tissues. 
 
 Absorption of Proteins. — It has been stated that under abnormal 
 conditions a certain amount of soluble protein is absorbed unchanged. 
 Patients fed per rectum are supposed to derive nourishment from 
 protein food; proteoclastic enzymes are not present in this part of 
 the intestine, and no cogent proof of protein absorption in this way 
 has ever been adduced, 
 
ABSORPTION 129 
 
 Under normal conditions, however, the food proteins are broken 
 up into substances with smaller molecules, and the ready diffusibility 
 of peptones led most physiologists to consider that protein was usually 
 absorbed as peptone, or as -proteose and peptone. But proteose 
 and peptone are absent from the blood and lymph in all circum- 
 stances, even from the portal blood during the most active digestion. 
 It is fortunate that this is so, for proteose and peptone when intro- 
 duced into the blood produce poisonous effects ; the coagulability of 
 the blood is lessened, blood pressure falls, secretion ceases, and in 
 the dog 0-3 gramme of commercial peptone per kilogramme of body- 
 weight is often sufficient to produce death. 
 
 This absence of "peptone" (using the word to include the proteoses) 
 did not, however, absolutely negative the idea that "peptone" is the 
 form in which proteins are absorbed, and the difficulty was met by 
 supposing that during absorption the products of proteolysis were 
 reconverted into native proteins (albumins and globulins). This 
 synthesis was further considered to be accomplished by the epithelial 
 cells that line the intestine. 
 
 This view has now had its day, and the change of opinion that 
 has relegated it to the past is due (1) to our increased knowledge of 
 the power of trypsin and erepsin ; (2) to a more careful examination 
 of the intestinal contents, and of the blood during absorption. We 
 now know that in the intestine the proteins are, by the two enzymes 
 trypsin and erepsin, broken down beyond the peptone stage into 
 their final cleavage products, the amino-acids, and that these pass 
 into the blood as such, for the amount of non-protein nitrogen in 
 that iluid is increased during absorption. If an animal is fed on the 
 cleavage products obtained from a pancreatic digest, nitrogenous 
 equilibrium is still maintained. These amino-acids are partly utilised 
 by the cells of the body to repair their waste, but partly and to a still 
 greater extent converted by the liver into the waste substance tirea, 
 which is finally excreted by the kidneys. The view that the absorptive 
 epithelium of the alimentary tract has any special power in building up 
 proteins from these simple cleavage products has been abandoned. 
 
 We thus see that the cells of the body possess the power of rebuilding 
 the proteins peculiar to themselves from the fragments of the molecules 
 of the food proteins. This accounts for the fact that the animal tissues 
 retain their chemical individuality in spite of the great variations in the 
 composition of the diet the animal takes.. 
 
 If a man wishes to build a new house, and to employ for the purpose 
 the bricks previously used in the building of another house, he takes the 
 9 
 
130 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 old house to pieces and uses the bricks and stones most appropriate for 
 liis purpose, rearranges them in such a way that the new house has its 
 own special architectural features, and discards as waste the bricks 
 and stones which are not suitable. This idea underlies the custom 
 of speaking of the cleavage products of protein as "building stones." 
 Each tissue has special architectural features in its protein molecules, 
 and these molecules are reconstructed by using the building stones that 
 previously had been used in the building of other protein molecules, 
 either in another animal or in vegetable structures. The building 
 
 op str 
 
 'Via. 19.— Section of tht \'illus of a rat killed flnrinf,^ fat-al).sor|ition (K. S. Schafer) : 
 ejt, epithelium ; t>ti; striated border ; c, 1,\ inph cells ; c', l;i'mph cells in the ' 
 epithelium ; I, central lacteal containiji{j disinte^^rating lymph cells. 
 
 stones which are in excess or are unsuitable are simply got rid of as 
 waste substance. 
 
 A large number of them are never actually built into protoplasm, 
 but are carried to the liver when the amino-group is removed ; this is 
 termed decuniuation ; the nitrogenous portion is then converted into 
 urea, and the non-nitrogenous portion of the protein molecule is then 
 available for calorific processes (see Urea formation). One can now 
 definitely state which are the ones that on p. 72 we compared to 
 diamonds, because they are unusually precious for the synthe.sis of 
 protein by tissue cells ; they are principally phenyl-alanine and its near 
 relations tyrosine and tryptophane, for if they are injected into the 
 
ABSORPTION 
 
 131 
 
 blood-stream they do not give rise to any increase in the urea formed ; 
 moreover, proteins destitute of these amino acids are of inferior nutritive 
 value. Lysine and histidine are also in the same category. 
 
 I Absorption of Fats. — The fats undergo in the intestine two changes : 
 one a physical change (emulsification), the other a chemical change 
 (saponification). The lymphatic vessels are the great channels for 
 fat absorption, and their name, lacteals, is derived from the milk-like 
 appearance of their contents (chyle) during the absorption of fat. 
 
 The way in which the minute fat globules pass from the intestine 
 into the lacteals has been studied by killing animals at varying periods 
 after a meal of fat and making osmic acid microscopic preparations 
 of the villi. Figs. 19 and 20 illustrate the appearances observed. 
 
 The columnar epithelium cells become first filled with fatty globules 
 of varying size, which are generally larger near the free border. The 
 globules pass down the cells, the larger 
 ones breaking up into smaller ones 
 during the journey ; they are then 
 transferred to the amoeboid cells of 
 the lymphoid tissue beneath ; these 
 ultimately penetrate into the central 
 lacteal, where they either disintegrate 
 or discharge their cargo into the lymph- 
 streatn. The globules are by this time 
 divided into immeasurably small ones, 
 the molecular basis of chyle. The 
 chyle enters the blood-stream by the 
 thoracic duct, and after an abundant fatty meal the blood-plasma is 
 quite milky ; the fat droplets are so small that they circulate without 
 hindrance through the capillaries. The fat in the blood after a 
 meal is eventually stored up in the connective tissue cells of adipose 
 tissue. It must, however, be borne in mind that the fat of the body 
 is not exclusively derived from the fat of the food, but it may 
 originate also from carbohydrates and in the opinion of .most 
 physiologists, from protein as well. 
 
 As the fat globules were never seen penetrating the striated border 
 of the epithelial cells, there was a difficulty in understanding how they 
 reached the interior of these cells ; the cells will not take up other 
 particles, and it is certain that they do not in the higher animals 
 protrude pseudopodia from their borders (this, however, does occur 
 in the endoderm of some of the lower invertebrates). 
 
 Recent research has solved this difficulty. In the first place, 
 
 Fig. 20. — Mucous membrane of frog's in- 
 testine during fat-absorption (E. S. 
 Schafer) : ep, epitiielium ; str, striated 
 border ; 0, lymph cells ; I, lacteal. 
 
132 ESSENTIALS OF CHEMICAL PHYSIOLOGV 
 
 particles may be present in jihe epithelium and lymphoid cells while 
 no fat is being absorbed. These particles are protoplasmic in nature, 
 as they stain with reagents that stain protoplasmic granules ; but as 
 they also stain darkly with osmic acid, they are apt to be mistaken 
 for fat. There is, however, no doubt that the particles found d ' g 
 fat-absorption are composed of fat. There is also no doubt tha le 
 epithelial cells have the power of again forming fat out of the y 
 acids and glycerol into which it has been broken up in the intestine.' 
 These substances, being soluble, pass readily into the epithelium cells, 
 and these cells perform the 83mthetic act of building them into fat 
 once more ; the fat so formed appears in the form of small globules, 
 surrounding or becoming mixed with the protoplasmic granules that 
 are ordinarily present. A remarkable fact which Munk made out 
 is that after feeding an animal on fatty acids the chyle contains fat. 
 The necessary glycerol must have been formed by protoplasmic activity 
 during absorption. Preliminary emulsification, though advantageous 
 for the action of lipase, is not essential. 
 
 Bile aids the digestion of fat by co-operating with the pancreatic 
 lipase as explained on p. 124. It is also a solvent of fatty acids, 
 and it probably assists fat-absorption by reducing the surface tension 
 of the intestinal contents ; membranes moistened with bile allow fatty 
 materials to pass through them more readily than would otherwise be 
 the case. In cases of disease in which bile is absent from the intestines 
 a large proportion of the fat in the food passes into the faeces. 
 
 In conclusion it must be mentioned that the lymphocyte? are greatly 
 increased in the blood during absorption, and some physiologists hold 
 the view that these come from the intestinal mucous membrane, and 
 share in the work of transporting absorbed materials. 
 
LESSON IX 
 THE BLOOD AND RESPIRATION 
 
 BLOOD PLASMA 
 
 1. The coagulation of the blood has been prevented in specimen A 
 by the addition of neutral salt (an equal volume of saturated sodium- 
 sulphate solution, or a quarter of its volume of saturated magnesium- 
 sulphate solution). The corpuscles have settled, and the supernatant 
 salted plasma has been siphoned off. 
 
 2. The coagulation of the blood in specimen B has been pre- 
 vented by the addition of an equal volume of a 04 percent, solution 
 of potassium oxalate in normal saline solution. 
 
 3. Put a small quantity of A into three test-tubes and dilute each 
 with about ten times its volume of liquid : 
 
 A 1. With distilled water. 
 
 A 2. With solution of fibrin-ferment containing a little calcium 
 chloride.^ 
 
 A 3. With the same. 
 
 4. Put A 1 and A 2 into the water-bath at 40° 0. ; leave A 3 at the 
 temperature of the air. A 1 coagulates slowly or not at all; A 2 
 coagulates rapidly : A 3 coagulates less rapidly than A 2. 
 
 5. Add to some of B a few drops of dilute (2 per-cent.) calcium-chloride 
 solution : it coagulates, and more quickly, if the temperature is 40° C. 
 
 BLOOD SEBUM 
 
 Blood serum is the fluid residue of the blood after the separation of 
 the clot; it is blood plasma minus the fibrin which it yields. The 
 general appearance of fibrin obtained by whipping fresh blood will 
 already be familiar to the student, as he has used it in experiments 
 on digestion. 
 
 Serum has a yellowish tinge due to serum lutein, but as generally 
 obtained it is often contaminated with a small amount of oxyhaemo- 
 globin, and so looks reddish. It contains proteins (giving the general 
 tests already studied in Lesson V), extractives, and salts in solution. 
 The proteins are serum albumin and serum globulin. Fibrin-ferment 
 
 ' An easy way of preparing an impure but eflSoient solution of fibrin-ferment is 
 to take 5 o. e. of blood serum and dilute it with a litre of distilled water. A partial 
 precipitation of globulin takes place, and carries down the ferment with it. After 
 a few hours pour off the supernatant flaid and dissolve the precipitate iif half a litre 
 of tap water to which a few drops of 2 percent, solution of calcium chloride have 
 been added. The solution can be then given round to the class as fibrin-ferment 
 
 133 
 
134 ESSENTIALS OF CHEMICAL PHYSIOLOCV 
 
 or thrombin is also present, and in the following experiments is 
 precipitated with serum globulin. 
 
 SEPARATION OF THE SERUM PROTEINS.— (a) Dilute serum 
 with fifteen times its volume of water. It becomes cloudy owing to 
 the partial precipitation of serum globulin. Add a few drops of 2 
 per-cent. acetic acid; the precipitate becomes more abundant, but 
 dissolves in excess of the acid. 
 
 (b) Pass a stream of carbonic acid through serum diluted with 
 twenty times its bulk of water. A partial precipitation of serum 
 globulin occurs. 
 
 (c) Saturate some serum with magnesium sulphate by adding 
 crystals of the salt and grinding in a mortar. A precipitate of 
 serum globulin is produced. 
 
 (d) Half saturate the serum with ammonium sulphate by adding 
 to it an equal volume of a saturated solution of the salt. Serum 
 globulin is precipitated. 
 
 (e) Completely saturate the serum with ammonium sulphate by 
 adding ccystals of the salt and grinding in a mortar; a precipitate 
 is produced of both the globulin and the albumin. Filter through a 
 dry filter paper; the filtrate contains no protein. 
 
 (f) Put some serum in a dialyser with distilled water in the outer 
 vessel. In a day or two, especially if the water in the outer vessel is 
 changed frequently, the salts dialyse out into the water ; the proteins 
 remain inside the dialyser ; of these the serum albumin and a fraction 
 of the serum globulin (called pseudo-globulin) remain in solution, but 
 the other fraction of the serum globulin (called euglobulin) is pre- 
 cipitated, as it requires salt to hold it in solution. 
 
 (g) Whether serum albumin is a single protein or a mixture of 
 several proteins is a matter of dispute. The only method by which 
 it can be at present fractionated is the somewhat uncertain one of 
 heat coagulation. If the globulin is removed by saturation with 
 magnesium sulphate, the filtrate, which contains serum albumin, when 
 heated after faint acidulation with 2 per-cent. acetic acid, deposits a 
 flocculent precipitate at about 73° C. Filter this ofT, and on heating the 
 filtrate a second coagulum is obtained at about 77° C, and similarly a 
 third and very small fraction separates out at 86° C. 
 
 HAEMOGLOBIN 
 
 1. THE SPECTROSCOPE.-^'Direct the spectroscope to the window 
 and carefully focus Fraunhofer's lines. Note especially D in the yellow, 
 and E, the next well-marked line, in the green. 
 
 Direct the spectroscope to a luminous gas flame; these lines are 
 absent. Place a little sodium chloride in the flame. Notice the bright 
 yellow line in the position of the D line. 
 
 2. SPECTROSCOPIC EXAMINATION OF BLOOD.-Take a series 
 of six test-tubes of about equal size. Fill the first with diluted 
 defibrinated ox-blQod (1 part pf bipod to 30 of water) ; then fill the 
 
HAEMOGLOBIN 135 
 
 second, tube with the same mixture diluted with an equal bulk of water 
 (1 in 60) ; half fill the third tube with this and fill up the tube with 
 an equal bulk of water (1 in 120), and so on. The sixth tube will contain 
 1 part of blood to about 1000 of water and will be nearly colourless. 
 
 3. Into another series of six test-tubes pour some of the contents 
 of each of the first series and add one drop (from a dropping bottle) of 
 a 10 per-cent. sodium hydrosulphite solution. Note the change of tint 
 from red to purple. Another reducing agent called Stokes's reagent 
 may be employed in this experiment instead. It must always be 
 freshly prepared ; it is a solution of ferrous sulphate to which a little 
 tartaric acid has been added, and then ammonia till the reaction is 
 alkaline. The ammonia should not be added until just before it is used. 
 
 4. Examine the tubes with the spectroscope and map out on a 
 chart the typical absorption bands of oxyhsemoglobin in the first 
 series, and of reduced heemoglobin in the second series. Notice 
 that in the more dilute specimens of reduced haemoglobin the bands 
 are no longer seen, whereas those of oxyhsemoglobin in specimens 
 similarly diluted are still visible. 
 
 5. Take a tube which shows the single band of reduced haemoglobin 
 and shake it with the air ; the bright red colour returns to it and it 
 shows spectroscopically the two bands of oxyhsemoglobin for a short 
 time. Continue watching the two bands, and note that they fade and 
 are replaced by a single band as reduction again occurs. 
 
 6. BLOOD CRYSTALS.— Mix a drop of defibrinated rat's blood on 
 a slide with a drop of water, or mount it in a drop of Canada balsam. 
 Exaimine the crystals of oxyhaemoglobin as they form. Five to ten 
 minutes usually elapses before the crystals are seen. 
 
 7. Smear a little blood, obtained "by pricking the finger, on a slide, 
 and allow it to dry ; cover, and run glacial acetic acid under the 
 cover slip, and boil ; when cool repeat this with fresh acid and then 
 examine microscopically for the dark brown crystals of hsemin. 
 
 8. CHEMICAL TESTS FOR BJjOOB.—GtMiacum. Test.— Take 
 some tincture of guaiacum, and add a small quantity of blood to it ; 
 add to the mixture a little hydrogen peroxide (or most specimens of 
 commercial turpentine will do as well) and a blue colour is developed. 
 This test is due to the iron-containing radical in haemoglobin, and is 
 given even after the blood has been previously boiled : repeat the test 
 using some boiled blood. 
 
 9. Adler's Test. — Take half a test-tube of diluted blood (so dilute 
 as to be practically colourless) ; add a few drops of benzidine dissolved 
 in glacial acetic acid, and a few drops of hydrogen peroxide : a blue 
 colour develops immediately. Spectroscopically, this blue solution 
 shows an absorption band in the yellow. This test is far more delicate 
 than the guaiacum test, but its intensity is lessened if the blood has 
 Ijeen previously boiled. It is a very convenient test for blood in urine, 
 
136 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 COAGULATION OF BLOOD 
 
 Microscopic investigation of vertebrate blood shows it to consist of 
 a iiuid (the blood-plasma or liquor sanguinis) which holds in suspension 
 large numbers of corpuscles; the corpuscles are red or coloured 
 (erythrocytes) ; white or colourless (leucocytes), and the blood platelets. 
 
 After blood is shed it rapidly becomes viscous and then sets into 
 a firm red jelly. The jelly soon contracts and squeezes out a straw- 
 coloured fluid called the serum, in which the shrunken clot ultimately 
 floats. With the microscope, filaments of fibrin are seen forming a net- 
 work, throughout the fluid, many radiating from sma^l clumps of blood 
 
 .f^jfir^ 
 
 Fig. 21. — Fibrin filaments and blood platelets ; A, network of fibrin shown after washing away 
 the corpuscles from a preparation of blood that has been allowed to clot. Many of the 
 filaments radiate from small clumps of blood' platelets. B (from Osier), blood corpuscles and 
 blood platelets within a small vein. 
 
 platelets. It is the formation of fibrin which is the essential act of 
 coagulation : this entangles the corpuscles and forms the clot. Fibrin 
 is formed from the plasma, and may be obtained free from corpuscles 
 when blood-plasma is allowed to clot, the corpuscles having previously 
 been removed. It may also be obtained from blood by whipping it 
 with a bunch of twigs ; the fibrin adheres to the twigs and entangles 
 but few corpuscles. These may be removed by washing with water. 
 Serum is plasma minus the fibrin which it yields. The relation of 
 plasma, serum, and clot can be seen at a glance in the following scheme 
 of the constituents of the blood : — 
 
 / -o, ( Serum 
 
 j Plasma | j,.^^^.^ 
 
 \ Corpuscles ) 
 
 Blood , 
 
 Clot 
 
 It may be roughly stated that in 100 parts by weight" of blood 60-65 
 parts consist of plasma and 35-40 of corpuscles. 
 
 The buffy coai.is seen when blood coagulates slowly, as in horse's 
 blood. The red corpuscles sink more rapidly than the white, and 
 the upper stratum of the clot (buify coat) consists mainly of fibrin 
 and white corpuscles. 
 
 Coagulation is hastened by — 
 
 1. A temperature a little over that of the body. 
 
 2. Contact with foreign matter. 
 
COAGULATION OF BLOOD 137 
 
 3. Injury to the vessel walls. 
 
 4. Agitation. 
 
 5. Addition of calcium salts. 
 
 6. Injection of nucleo- protein produces intravascular clotting 
 (positive phase). Very minute doses, however,- produce the opposite 
 effect : namely, delay of coagulation (negative effect). 
 
 Coagulation is hindered or prevented by — 
 
 1. A low temperature. In a vessel cooled by ice, coagulation may 
 be prevented for an hour or more. 
 
 2. The addition of a large quantity of neutral salts, such as sodium 
 sulphate or magnesium sulphate. 
 
 3. Addition of a soluble oxalate, fluoride or citrate. 
 
 4. Injection of commercial peptone (which consists chiefly of 
 proteoses) into the circulation of the living animal. 
 
 5. Addition of leech extract to the blood, or injection of leech 
 ejttract into the circulation while the animal is alive. 
 
 6. Contact with the living vascular walls. 
 
 7. Contact with oil. 
 
 The cause of the coagulation of the blood may be briefly stated as 
 follows : — 
 
 When blood is within the vessels one of the constituents of the 
 plasma, a protein of the globulin class called fibrinogen, exists in a 
 soluble form. 
 
 When the blood is shed the fibrinogen molecule is altered in such 
 a way that it gives rise to the comparatively insoluble material fibrin. 
 
 The statement has been made that the fibrinogen molecule is split 
 into two parts : one part is a globulin (fibrinoglobulin), which remains 
 in solution : the other and larger part is the insoluble substance fibrin. 
 It is, however, doubtful if this really represents what occurs, for recent 
 work seems to show that the fibrinoglobulin iS not a product of 
 fibrinogen, but exists in the blood-plasma beforehand. At any rate, 
 whether this is so or not, the fact remains that fibrin is the important 
 product and the only one which need concern us. 
 
 The next question is. What causes the transformation of fibrinogen 
 into fibrin ? And the answer to that is, that the change is due to the 
 activity of a special enzyme called fibrin-ferment or thrombin. 
 
 This enzyme does not,exist in healthy blood contained in healthy 
 blood-vessels, but is formed by the disintegration of the blood platelets 
 and colourless corpuscles which occurs when the blood leaves the blood- 
 vessels or comes into contact with foreign matter. Hence the blood 
 does not coagulate during life. But, it will be said, disintegration of 
 
138 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 the blood corpuscles occurs during life ; why, then, does the blood not 
 coagulate? The reason is that although the formed elements do 
 disintegrate in the living blood, such a phenomenon takes place vety 
 slowly and gradually, so that there can never in normal circumstances 
 be any massive liberation of fibrin-ferment, and, further, that there are 
 agencies at work to neutralise the fibrin-ferment as it is formed. The 
 most noteworthy of these neutralising agencies is the presence in the 
 blood of an anti-enzyme called antithromhin, analogous to the antipepsin 
 and antitrypsin which, we have seen, are efficacious in preventing the 
 stomach and intestines from undergoing self-digestion. 
 
 Nucleo-proteins (see pp. 62-65) obtained from most of the cellular 
 organs of the body produce intravascular clotting when injected into 
 the circulation of the living animal. In certain diseased conditions 
 intravascular clotting, or thrombosis, sometimes occurs. This must be 
 due to the entrance of nucleo-protein into the circulation from dis- 
 integrated cells. Nucleo-protein and thrombin are probably not 
 identical ; but it is possible that the nucleo-protein may hold in loose 
 combination or admixture another material called thrombo-kinase, 
 which we shall be considering immediately. 
 
 Thrombin is believed to originate chiefly from the blood platelets 
 and in part from the leucocytes. Birds' blood clots very slowly, and 
 the absence of blood platelets in this variety of blood will in part 
 account for this. Lymph which contains colourless corpuscles, but 
 no platelets, also clots, so in this case the colourless corpuscles must 
 be the source of the enzyme. One should, however, be careful in 
 speaking of the disintegration of leucocytes to remember that the 
 word " disintegration '' does not mean complete breakdown, leading to 
 disappearance ; the colourless corpuscles do not appreciabl}' diminish 
 in number when the blood is shed, but what occurs in the surviving 
 leucocytes is a shedtling out of certain products, among which fibrin- 
 ferment is one. 
 
 We have now traced fibrin-formation, the essential cause of blood- 
 clotting, to the activity of thrombin ; it is next necessary to allude 
 to what has been discovered in relation to the origin of thrombin. 
 Like other enzymes, it is preceded by a mother substance or zymogen. 
 This zymogen is called prothrombin, or thrombogen, and there appear 
 to be two necessary agents concerned in the jcon version of thrombogen 
 into thrombin : one of these is the action of calcium salts, the other is 
 the presence of an activating agent, analogous to entero-kinase (see 
 pp. 90 and 113), and called thrombo-kinase. The exact rdle played by 
 ?E^ch is still a matter of speculation. 
 
COAGULATION OF BLOOD 139 
 
 The part played by calcium salts is well illustrated by the fact 
 that coagulation is prevented by the decalcification of the blood. 
 This can be accomplished by the addition of a small amount of soluble 
 oxalate or fluoride to the blood immediately it is shed. The calcium 
 of the blood - plasma is then immediately precipitated as insoluble 
 calcium oxalate or fluoride, and is thus not available for the transforma- 
 tion of thrombogen into thrombin. The addition of the oxalate or 
 fluoride must be rapidly performed, otherwise time will be given for 
 the conversion of thrombogen into thrombin, and thrombin, when 
 formed, will act upon fibrinogen whether the calcium has been removed 
 or not. In other words, calcium is only necessary for the formation 
 of fibrin-ferment and not for the action of fibrin-ferment on fibrinogen. 
 Fibrin is thus not a compound of calcium and fibrinogen. 
 
 The action of a soluble citrate is also in a certain sense a decalcifying 
 action, for although calcium citrate is a soluble salt it does not ionise in 
 solution so as to liberate the free calcium ions which are essential for 
 thrombin formation. 
 
 Oxalated blood (or oxalated plasma) will clot when calcium is once 
 more restored by the addition of a small amount of calcium chloride, 
 but such an addition to fluoride plasma will not induce clotting ; 
 thrombin must be added also. In some way sodium fluoride interferes 
 with the formation of thrombin, probably by preventing the liberation 
 of thrombo-kinase from the formed elements of the blood. 
 
 The other activating agent, thrombo-kinase, is in part liberated 
 from the formed elements of the, blood, but it is also obtained from 
 many other tissues. If a haemorrhage takes place under ordinary 
 conditions the blood as it flows from the wound passes over the 
 muscles and skin which have been cut', and rapidly clots owing to the 
 thrombo-kinase supplied by those tissues. If blood is obtained by 
 drawing it off through a perfectly clean cannula into a clean vessel 
 without allowing it to touch the tissues, it remains unclotted for a 
 long time ; in the case of birds' blood this time may extend to many 
 days ; but the addition of a small piece of tissue such as muscle, or of 
 an extract of such a tissue, produces almost immediate clotting. If a 
 solution of fibrinogen is prepared and calcium added it will not clot ; 
 if thrombin or a fluid such as serum which contains thrombin is added 
 also it will clot. It will not clot if birds' plasma obtained as above' is 
 added to it ; nor if tissue extract is added to it ; but if both are added 
 it will. In other words, the thrombogen of the birds' plasma plus the 
 thrombo-kinase of the tissue extract have the same effect as thrombin. 
 
 The next point to consider is why blood obtained after the previous 
 
140 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 injection of proteoses (or commercial peptone) into the circujation 
 does not clot. It certainly contains calcium salts, and probably both 
 thrombogen and thrombo-kinase, for it can be made to clot without 
 the addition of either ; for instance, by dilution or the passage of a 
 stream of carbon dioxide through it. There must be something in 
 peptone blood which antagonises the action of thrombin. This some- 
 thing is an excess of antithrombin. Peptone will not hinder blood 
 coagulation, or only very slightly if it is added to the blood after it 
 is shed. The antithrombin must therefore be added to the blood while 
 it is circulating in the body. We can even go further than this and 
 say what part of the body it is which is concerned in the production 
 •of antithrombin : it is the liver, for if the liver is shut off from the 
 circulation, peptone is ineffective in its action. The converse experi- 
 ment confirms this conclusion, for if a solution of peptone is artificially 
 perfused through an excised surviving liver, a substance is formed 
 which has the power of hindering or preventing the coagulation of 
 shed blood. 
 
 We are thus justified in two conclusions : — 
 
 (1) That the antithrombin which is normally present in healthy 
 blood in sufficient quantities to prevent intravascular clotting, is 
 formed in the liver. 
 
 (2) That commercial peptone in virtue of the proteoses it contains 
 stimulates this action of the liver to such an extraordinary degree, 
 that the accumulation of antithrombin in the blood becomes sufficiently 
 great to prevent the blood from clotting even after it is shed. 
 
 It should be noted, however, that this effect upon the liver varies 
 in different animals, and is most marked in the dog. Peptone blood is 
 very poor in leucocytes, but the cause of their disappearance is not 
 clear. 
 
 We shall conclude by considering only one more of the hindrances 
 to coagulation, and that by no means the least interesting. The 
 leech lives by sucking the blood of other animals ; from the leech's 
 point of view it is therefore necessary that the blood should flow 
 freely and not clot. The glands at the head end of the leech, often 
 spoken of roughly as the salivary glands, secrete something which 
 hinders the blood from coagulating, and every one who has been treated 
 by leeches knows by experience how difficult it is to prevent a leech-bite 
 from bleeding after the leech has been removed ; complete cleansing is 
 necessary to wash away the leech's secretion from the wound. Now if 
 an extract of leech's heads is made, with salt solution and filtered, that 
 fiiiid will prevent coagulation, whether it is injected into the blood- 
 
THE PLASMA AND SERUM 
 
 141 
 
 stream or added to shed blood. The substance in the extract is called 
 hirudin, and this is believed to be antithrombin itself. 
 
 We may summarise our present knowledge of the causes of coagula- 
 tion in the following tabular way : — 
 
 From the platelets, 
 and to a lesser degree 
 from the leucocytes, a 
 material is shed out 
 called — 
 
 Thrombogen. 
 
 Prom the formed 
 elements of the blood, 
 but also from the tissues 
 over which the escaping 
 blood flows, is shed out 
 an activating agent 
 called 
 Theombo-kinase. 
 
 In the blood-plasma 
 a protein substance ex- 
 ists called 
 
 Fibrinogen. 
 
 In the presence of calcium salts, thrombo- 
 kinase activates thrombogen in such a way that 
 an active enzyme is produced, which is called 
 Thrombin. 
 
 Throm.bin or fibrin-ferment acts on fibrinogen in such a way that it 
 is transformed into the inf oluble stringy material which is called 
 
 Fibrin. 
 
 The views enumerated in the preceding .paragraphs are in the main 
 those of Morawitz. Howell agrees that the lack of coagulation seen in 
 birds' blood and peptone blood is due to excess of antithrombin, but 
 holds that thrombo-kinase (or thromboplastin, as he terms it) brings 
 about clotting not by activating thrombogen but by neutralising anti- 
 thromljin. He further thinks that thrombo-plastin is a lipoid. 
 
 THE PLASMA AND SERUM 
 
 The liquid in which the corpuscles float may be obtained by employ- 
 ing one or other of the methods already described for preventing the 
 blood from coagulating. The corpuscles, being heavy, sink, and the 
 supernatant plasma can then be removed by a pipette or siphon ; the 
 separation can be eifected more thoroughly and rapidly by the use of 
 a centrifugal machine. 
 
 On counteracting the influence which has prevented the blood from 
 coagulating, the plasma then itself coagulates. The plasma obtained 
 by the use of cold, clots on warming gently ; plasma which has been 
 
142 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 decalcified by the action of soluble oxalate clots on the addition of 
 a calcium salt; plasma obtained by the use of a strong solution of 
 salt coagulates when this is diluted by the addition of water, the 
 addition of thrombin being necessary in most cases ; where coagulation 
 occurs without the addition 'of thrombin, no doubt some is present 
 from the partial disintegration of the corpuscles which has already 
 occurred. Pericardial and hydrocele fluids resemble pure plasma very 
 closely in composition. As a rule, however, they contain few or no 
 white corpuscles, and do not clot spontaneously, but after the addition 
 of thrombin or liquids such as serum which contain thrombin they 
 always yield fibrin. 
 
 Pure plasma may be obtained from horse's veins by what is known 
 as the "living test-tube" experiment. If the jugular vein is ligatured 
 in two places, so as to include a quantity of blood within it, then 
 removed from the animal and hung in a cool place, the blood will not 
 coagulate for many hours. The corpuscles settle and the supernatant 
 plasma can be removed with a pipette. 
 
 The plasma is alkaline, yellowish in tint, and its specific gravity is 
 about 1026 to 1029. 
 
 Its chief constituents may be enumerated as follows : — 
 1000 parts of plasma contain — 
 
 Water .... .. 902-90 
 
 Solids . . . 97-10 
 
 Proteins : 1, yield of fibrin 4-05 
 
 2, other proteins . 78-84 
 
 Extractives (including fat) . 5-66 
 
 Inorganic salts . . 8*55 
 
 In round numbers plasma contains 10 per cent, of solids, of which 8 are 
 
 protein in nature. 
 
 Serum contains the same three classes of* constituents — proteins, 
 extractives, and salts. The extractives and salts are the same in the 
 two liquids. The proteins diifer, as is shown in the following table : — 
 
 Proteins of Plasma. 
 Fibrinogen. 
 Serum globulin. 
 
 Proteins of Serum. 
 Serum globulin. 
 Serum albumin. ■ 
 
 Serum albumin. Thrombin. 
 
 The gases of the plasma and serum are small quantities of oxygen, 
 nitrogen, and carbonic acid. The greater part of the oxygen of the 
 blood is combined in the red corpuscles with haemoglobin ; the carbonic 
 acid is chiefly combined as carbonates (see Respiration). 
 
THE PLASMA AND SERUM 143 
 
 We may now consider one by one the various constituents of, the 
 plasma and serum. 
 
 A. Proteins. — Fibrinogen. — This is the parent substance of fibrin. 
 It is a globulin. It differs from serum globulin, and may be separated 
 from it by the fact that half saturation with sodium chloride precipi- 
 tates it. It coagulates by heat at the low temperature of 56° C. As 
 judged from the yield of fibrin, it is the least abundant of the proteins 
 of the plasma (see table on p. 1 42). 
 
 Serum Globulin and Serum Albumin. — These substances, which are 
 typical of the globulin and alljumin groups of proteins, are considered 
 in the practical exercises at the head of this lesson ; see also Lesson 
 V, p. 59. Both serum globulin and serum albumin probably consist of 
 more than one protein substance (see practical ekercises f and g in 
 to-day's lesson). 
 
 'Thrombin. — Schmidt's method of preparing it is to take serum and 
 add excess of alcohol. This precipitates all the proteins, and also 
 thrombin. After some weeks the alcohol is poured off; the serum 
 globulin and serum albumin have been by this means rendered insoluble 
 in water ; an aqueous extract is, however, found to contain thrombin, 
 which is not so easily coagulated by alcohol as the proteins are. 
 A simpler method of preparing fibrin ferment in an impure but efficient 
 form is given in the footnote on p. 133. 
 
 B. Extractives. — These are non-nitrogenous and nitrogenous. The 
 non-nitrogenous are sugar (0-12 per cent.), fats, serum lutein (see p. 40), 
 soaps, cholesterol and cholesterol esters ; and the nitrogenous are urea 
 (0"02 to 0'04 per cent.) and still smaller quantities of uric acid, creatine, 
 creatinine, xanthine, hypoxanthine, and amino-acids. 
 
 C. Salts. — The most abundant salt is sodium chloride; it consti- 
 tutes between 60 and 90 per cent, of the total mineral matter. Potassium 
 chloride is present in much smaller amount. It constitutes about 4 per 
 cent, of the total ash. The other salts are phosphates and sulphates. 
 
 Schmidt gives the following table : — 
 
 1000 parts of plasma yield — 
 
 Mineral matter 8 5 50 
 
 Chlorine . 3-640 
 
 SO3 . 0-115 
 
 P2O5 .... 0-191 
 
 Potassium . *•. 0-323 
 
 Sodium 3-341 
 
 Calcium phospliate . . 0-311 
 
 Magnesium phosphate 0-222 
 
144 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 THE WHITE BLOOD CORPUSCLES 
 
 These corpuscles are typical animal cells. Their nucleus consists 
 of nuclein ; their cell-protoplasm yields protein belonging to the nucleo- 
 protein and globulin groups. The protoplasm of these cells also 
 contain small quantities of fat, lipoids, and glycogen. 
 
 THE RED BLOOD CORPUSCLES 
 
 The red blood corpuscles are much more numerous than the white, 
 averaging in man 5,000,000 per cubic millimetre, or 400 to 500 red to 
 each white corpusclfe. The methods of enumeration of the corpuscles 
 are described in the Appendix. 
 
 They vary in size and structure in diiFerent groups of vertebrates. 
 
 In mammals they are biconcave (except in the camel tribe, where they 
 
 are biconvex) non-nucleated discs, in man averaging -^yu^ inch in 
 
 diameter ; during foetal life nucleated red 
 
 A ^ ^ iWk /^ corpuscles are, however, found. In birds, 
 
 e @ ^ *^ ^>^ reptiles, amphibians, and fishes they are 
 
 biconvex oval discs with nucleus; they 
 
 ■ '"^Jr tr/"''^ ^^^ largest in the amphibia. 
 
 I"V K.^ Water causes the corpuscles to swell up, 
 
 ''waS-^'^irbroorco^'utie! and dissolves out the red pigment (oxyh«mo- 
 iitsol^ti™?raotionofunn'j; globin), leaving a globular colourless mem- 
 on a red corpuscle. brane Or stroma. This is termed laking the 
 
 blood. Laking or hmmolysis is also produced by soaps, bile salts, and 
 other substances. Strong salt solution causes the corpuscles to shrink : 
 they become crenated or wrinkled. The action of water and salt 
 solution is explained by the existence of a membrane on the surface 
 of the corpuscles through which osmosis takes place. Physiological 
 salt solution (0-9 per-cent. sodium chloride) produces no change as 
 it has the same osmotic pressure as blood-plasma. Dilute alkalis 
 (0-2 per-cent. potash) dissolve the corpuscles. Dilute acids (1 percent, 
 acetic acid) act like water, and in nucleated corpuscles render the 
 nucleus distinct. Tannic acid causes a discharge of hsemoglobin from 
 the stroma, but this is immediately altered and precipitated. It 
 remains adherent to the stroma as a bro^n globule, consisting prob- 
 ably of hsematin. Boric acid acts similarly, but in nucleated red 
 corpuscles the pigment collects chiefly round the nucleus, which may 
 then be extruded from the corpuscles. 
 
THE RED BLOOD CORPUSCLES 145 
 
 Composition. — 1000 parts of red corpuscles 
 
 contain — 
 
 Water .... 
 
 . 688 ■ parts 
 
 Solids r^'""''. ■ ' ■ 
 (^inorganic 
 
 . 303-88 „ 
 . 8-12 „ 
 
 100 parts of dried corpuscles contain — 
 
 
 Protein 
 
 . 5 to 1 2 parts 
 
 Hsemoglobin . ... 
 
 . 86 „ 94' ., 
 
 Phosphatides calculated as lecithin 
 
 . 1-8 part 
 
 Cholesterol . ... 
 
 . 0-1 
 
 The protein present appears to be identical with the nucleo-protein 
 of white corpuscles. The mineral matter consists chiefly of chlorides 
 of potassium and sodium, and phosphates of calcium and magnesium. 
 In most animals, including man, potassium chloride is more abundant 
 than sodium chloride. 
 
 Oxygen is contained in combination with the hsemoglobin to form 
 oxyhsemoglobin. The corpuscles also contain a certain amount of 
 carbonic acid (see Respiration, at the end of this lesson). 
 
 The Pigment of the Eed Corpuscles. — The pigment is by far the 
 most abundant and important of the constituents of the red corpuscles. 
 It differs from most other proteins in containing the element iron ; it 
 is also readily crystallisable. 
 
 It exists in the blood in two conditions : in arterial blood it is com- 
 bined loosely with oxygen, is of a bright red colour, and is called oxy- 
 hsemoglobin ; the other condition is the deoxygenated or reduced 
 hsemoglobin. This is found in the blood after asphyxia. It also 
 occurs in all venous blood — that is, blood which is returning to the 
 heart after it has supplied the tissues with oxygen. Venous blood, 
 however, always contains a considerable quantity of Oxyhsemoglobin 
 also. Hsemoglobin is the oxygen-carrier of the body, and it may be 
 called a respiratory pigment. 
 
 Crystals of oxyhsemoglobin may be obtained with readiness from 
 the blood of such animals as the rat, guinea-pig, or dog ; with difficulty 
 from other animals, such as man, ape, and most of the common 
 mammals. The following methods are the best : — • 
 
 1. Mix a drop of defibrinated blood of the rat on a slide with a 
 drop of water ; put on a cover glass ; in a few minutes the corpuscles 
 are rendered colourless, and then the oxyhsemoglobin crystallises 
 out from the solution so formed. 
 
 2. Microscopical preparations may also be made by Stein's method, 
 10 
 
146 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 which consists in using Canada balsam instead of water in the above 
 experiment. ■ 
 
 3. On a larger scale the crystals may be obtained by laking the 
 blood by shaking it with one-sixteenth of its volume of ether. After 
 a period, varying from a few minutes 'to days, abundant crystals are 
 deposited. The laking of blood by ether and similar reagents is due 
 to their solvent effects on the lipoids of the cell membrane. The 
 accompanying illustrations (fig. 23) represent the form of the crystals 
 so obtained. 
 
 In nearly all animals the crystals are rhombic prisms ; but in the 
 guinea-pig they are rhombic tetrahedra (four-sided pyramids) ; in the 
 
 squirrel, hexagonal plates ; and in the 
 hamster, rhombohedra and hexagonal 
 plates. r 
 
 The crystals also contain a varying 
 amount of water of crystallisation : 
 this may explain their different crystal- 
 line forms and solubilities. The vary- 
 ing form of the crystals does not there- 
 fore prove that the oxyhsemoglobin 
 of different animals has a different 
 composition, for it has been shown 
 ^» A. " 'v ^^'^ the crystals of one form can be 
 ^H Ji. ^W transformed on recrystallisation into 
 
 those of another form and then back 
 again into the original form. The 
 Fig. 23.— Oxyhemoglobin crystals magni- crystalline form of oxyhsemoglobin is, 
 
 fied : 1, from human blood ; 2, from the 'pi 
 
 guinea-pigr; 3, squirrel; 4, hamster. however, no guarantee of the purity of 
 
 the substance, for after many recrystal- 
 lisations, although the crystalline form remains unaltered, the cleavage 
 products obtained on hydrolysis are different ; for instance, no glycine 
 is found after many recrystallisations (Abderhalden). The haemoglobin 
 molecule contains carbon, hydrogen, nitrogen, oxygen, sulphur, and 
 iron. The percentage of iron is about 0'4, but varies in different pre- 
 parations. Oxyhsemoglobin may be estimated in th,e blood (1) by the 
 amount of iron in the ash, or (2) by certain colorimetric methods 
 which are described in the Appendix. 
 
 Hsemoglobin is a conjugated protein, and on the addition of an 
 acid or alkali it is broken up into two parts, a protein called globin 
 (one of the histones, see p. 59), and a brown pigment called hcematin, 
 which contains all the iron of the original substance. 
 
 h^f4 ^ 
 
THE RED BLOOD CORPUSCLES 147 
 
 Haematin has the formula Cg^Hgg „ ggO^N^Fe. It presents different 
 spectroscopic appearances in acid and alkaline solutions. As obtained 
 from oxyhsemoglobin it should be termed oxyhcematin. It may be 
 reduced in alkaline solution by adding a reducing reagent, and the 
 well-marked absorption spectrum of reduced hcematin forms the most 
 delicate of the spectroscopic tests for blood pigment. A pyridine 
 compound of reduced hsematin has been obtained in crystalline 
 form.. 
 
 Hsemin is of great importance, as the obtaining of this substance 
 in a crystalline form is the best , ^ 
 
 chemical test for blood. Hsemin tH^j^ *!' '^ 
 
 crystals, sometimes called Teioh- ■ 'Ty/'^ '-4>*' *^ ^-4- 
 mann's crystals, are prepared for i -h^ >v ^ *^ "~ ■*> 
 
 microscopic examination by boiling ^ "is ^ ^ ^^^ 
 
 a fragment of dried blood with a *j '^s* | . X -^,^ ^ ^ 
 drop of gl&cial acetic acid on a slide; ' ■^■k.j^^ ^ -=>^'ix 
 
 on cooling, dark brown plates and j, ^ -a-a^ ^^-^ >^' 
 
 prisms belonging to the triclinic ' ^ T*- SUff^ * 
 
 system, often in star-shaped clusters _ »/ i- ■* 
 
 and with rounded angles (fig. 24), ^- % / "^=' 
 
 Fig. 24. — Hsemin crystals magnified. (Preyer.) 
 
 separate out. 
 
 In the case of an old blood-stain it is necessary to add a crystal 
 of sodium chloride. Fresh blood contains sufficient sodium chloride 
 in itself. The action of the acetic acid is to split the hsemoglobin into 
 hiematin and globin. A hydroxyl group of the hsematin is then re- 
 placed by chlorine. It is similarly easily replaceable by an atom of 
 bromine or iodine. Nencki has further shown that, when prepared in 
 this way, hsemin also contains the acetyl group. It has the empirical 
 formula OggH320^N4FeCl. 
 
 Haematoporphyrin (CgjHggOgN^) is iron-free hsematin : it may be pre- 
 pared by mixing blood with strong sulphuric acid; the iron is taken out 
 as ferrous sulphate. It has been obtained in crystals by Wildstatter. 
 This substance is also found sometimes in nature; it occurs in certain 
 invertebrate pigments, and may also be found in certain forms of 
 pathological urine. Even normal urine contains traces of it. It 
 shows well-marked spectroscopic bands, and so is not identical with 
 the iron-free derivative of hsemoglobin called hsematoidin which is 
 formed in extravasations of blood in the body (see p. 121). 
 
 Haemopyrrol, which is formed by reduction from hseinatoporphyrin, 
 has been proved to be a mixture of several pyrrol derivatives. It is 
 also similarly obtained from, the derivative of chlorophyll called 
 
148 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 phylloporphyrin, a fact which illustrates the near relationship of the 
 principal animal and vegetable pigments. 
 
 The relationships of the derivatives of blood pigment is shown in 
 the following simple scheme : — 
 
 Oxyheemoglobin minus protein = Oxyhsematin. 
 
 minus Oxygen. minus Oxygen. 
 
 I • I 
 
 Reduced hsemoglobin minus protein = Reduced hsematin. 
 Hsematin minus Iron = Hsematoporphyrin. 
 Hsematin with an OH replaced by CI = Hsemin. 
 Hsematoporphyrin on reduction yieldspyrrolderivatives(H8emopyrrol). 
 
 COMPOUNDS OF HAEMOGLOBIN WITH GASES 
 
 Hifimoglobin forms at least four compounds with gases : — 
 
 -,„.., f 1. Oxyhsemoglobin. 
 
 With oxygen . . . { •', ^ , 
 
 •' \ 2. Methsemoglobm. 
 
 With carbon monoxide . 3. Carbonic oxide haemoglobin. 
 
 With nitric oxide . . 4. Nitric oxide hiiemoglobin. 
 
 These compounds have similar crystalline forms : each consists of 
 a molecule of haemoglobin combined with one of the gas. They part 
 with the combined gas somewhat readily, and are arranged in order of 
 stability in the above list, the least stable first. 
 
 Oxyhaemoglobin is the compound that exists in arterial blood. The 
 oxygen linked to the haemoglobin, which is removed by the tissues 
 through which the blood circulates, may be called the respiratory 
 oxygen of hcBmoglohin. The processes that occur in the lungs and 
 tissues, resulting in the oxygenation and deoxygenation respectively 
 of the haemoglobin, may be imitated outside the body, using either 
 blood, or pure solutions of haemoglobin. The respiratory oxygen can 
 be removed, for example, in the Torricellian vacuum of a mercurial 
 air-pump, or by passing a neutral gas such as hydrogen through the 
 blood or by the use of reducing agents such as ammonium sulphide 
 or Stokes's reagent (an ammoniacal solution of ferrous tartrate), or, 
 best of all, sodium hydrosulphite. One gramme of htemoglobin will 
 combine with 1 '34 c.c. of oxygen. 
 
 If any of these methods for reducing oxyhaemoglobin is used, the 
 bright red (arterial) colour of oxyhaemoglobin changes to the purplish 
 (venous) tint of hsemoglobin. On once more allowing oxygen to come 
 into contact with the hsemoglobin, as by shaking the solution with the 
 air, the bright arterial colour returns. 
 
COMPOUNDS OF HEMOGLOBIN WITH GASES 149 
 
 These colour-changes may be more accurately studied with the 
 spectroscope, and the constant position of the absorption bands seen 
 constitutes an important test for blood pigment. 
 
 The Spectroscope. — When a ray of white light is passed through a 
 prism, it is refracted or bent at each surface of the prism; the whole ray 
 is, however, not equally bent, but it is split into its constituent colours, 
 which may be allowed to fall on a screen. The band of colours beginning 
 with the red, passing through orange, yellow, green, blue, and ending 
 with violet, is called a spectrum : this is seen in nature in the rainbow. 
 
 The spectrum of sunlight is interrupted by numerous dark lines 
 crossing it vertically called Fraunhofer's lines. These are pwfectly 
 constant in position, and serve as landmarks .in the spectrum. The 
 most prominent are A, B, and C, in the red ; D, in the yellow ; E, b, 
 and F, in the green ; G and H, in the violet. These lines are due to 
 certain volatile substances in the solar atmosphere. If the light from 
 burning sodium or its compounds is examined spectroscopically, it 
 will be found to give a bright yellow line, or rather two bright yellow 
 lines very close together. Potassium gives two bright red lines and one 
 violet line ; and the other elements, when incandescent, give character- 
 istic lines, but none so simple as sodium. If now the ilame of an 
 ordinary lamp be examined, it will be found to give a continuous spectrum 
 like that of sunlight in the arrangement of its colours, but unlike it in 
 the absence of dark lines ; but if the light from the lamp is made to 
 pass through sodium vapour before it reaches the spectroscope, the 
 bright yellow light will be found absent, and in its place a dark line, or 
 rather two dark lines very close together, occupy the same position 
 as the two bright lines of the sodium spectrum. The sodium vapour 
 thus absorbs the same rays as those which it itself produces at a higher 
 temperature. Thus the ~D line, as we term it, in the solar spectrum is 
 due to the presence of sodium vapour in the solar atmosphere. The 
 other dark lines are similarly accounted for by other elements. 
 
 The large form of spectroscope (fig. 25) consists of a tube A, called 
 the pollimator, with a slit at the end S, and a convex lens at the end L. 
 The latter makes the rays of light passing through the slit from the 
 source of light parallel ; they fall on the prism P, and then the spectrum 
 so formed is focused by the telescope T. 
 
 A third tube, not shown in the figure, carries a small transparent 
 scale of wave-lengths, so that the position of any point in the spectrum 
 may be given in terms of the corresponding wave-lengths. 
 
 If we now interpose between the source of light and the slit S a 
 piece of coloured glass (H in fig. 25), or a solution of a coloured substance 
 
150 
 
 ESSENTIALS OF CHEMICAL PHYStOLOGV 
 
 contained in a vessel with parallel sides, the spectrum is found to be no 
 longer continuous, but is interrupted by a number of dark shadows, or 
 absorption bands, corresponding to the light absorbed by the coloured 
 medium. Thus a solution of oxyhsemoglobin of a certain strength 
 gives two bands between D and E lines ; reduced haemoglobin gives only 
 
 Fig. 25. — Diagram of spectroscope. 
 
 one ; and other red solutions, though to the naked eye similar to 
 oxyhsemoglobin, will give characteristic bands in other positions. 
 
 A convenient form of small spectroscope is the direct vision spectro- 
 scope, in which, by an arrangement of alternating prisms of crown and 
 flint glass (see fig. 26), the spectrum is observed by the eye in the 
 same line as the tube furnished with the slit. Such small spectro- 
 
 FfG. 26. — Arrangement of prisms in direct-vision spectroscope. 
 
 scopes may for convenience be mounted on a stand provided with' a 
 gas-burner and a receptacle for, the test-tube (see fig. 27). In the 
 examination of the spectrum of small coloured objects, a combination 
 of the microscope and direct-vision spectroscope, called the micro-spectro- 
 scope, is used. 
 
 Fig. 28 illustrates a method of representing absorption spectra 
 diagrammatically. The solution was examined in a layer 1 centimetre 
 thick. The base line has on it at the proper distances the chief Fraun- 
 
COMPOUNDS OF HAEMOGLOBIN WITH GASES 
 
 151 
 
 hofer lines, and along the right-hand edges as the percentage amounts 
 of oxyhsemoglobin present in I, of reduced hsemoglobin in II. The width 
 
 Fio. 27.— Stand for direct-vision spectroscope : S, spectroscope ; T, test-tube for 
 coloured substance under investigation. 
 
 Fig. 28. — Graphic representations of the amount of absorption of light by solution (I) of oxy- 
 hemoglobin ; (II) of reduced haemoglobin, of different strengths. The shading indicates the 
 amount of absorption of the spectrum ; the figures on the right border express percentages. 
 (BoUett.) 
 
 of the shadings of each level represents the position and amount of 
 absorption corresponding to the percentages. 
 
 The characteristic spectrum of oxyhsemoglobin, as it actually 
 
152 
 
 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 appears through the spectroscope, is seen in the next figure (fig. ^9, 
 spectrum 2). There arc two distinct absorption bands, between the 
 D and E lines ; the one nearest to D (the <i. band) is narrower, darker, 
 and has better defined edges than the other (the (i band). As will be 
 seen on looking at fig. 28, a solution of oxyhsemoglobin of concentration 
 greater than O'CS per cent, and less than 0-8.5 per cent, (examined in a 
 cell of the usual thickness of 1 centimetre) gives one thick band over- 
 lapping both D and E, and a stronger solution only lets the red light 
 through between C and D. A solution which gives the two character- 
 istic bands must therefore be a very dilute one. The single band (y band) 
 
 
 
 ( 
 
 : I 
 
 ) 
 
 
 I 
 
 : 
 
 h E 
 
 G 
 
 1 
 
 <::::, 
 
 o 
 bN 
 
 
 
 ^ 
 
 
 
 
 
 
 
 
 
 
 
 
 
 2 
 3 
 
 1 
 
 
 1 
 
 
 
 
 
 lil^H 
 
 
 
 
 
 
 1 
 
 H 
 
 
 ■ 
 
 
 
 
 .^n^^ 
 
 
 
 
 
 
 
 1 
 
 4 
 
 1 
 
 
 III 
 
 
 
 1 i^^^^B 
 
 
 
 
 
 
 
 1 1 
 
 5 
 
 1 
 
 1 
 
 
 H 
 
 ■■■■■■ 
 
 Fig. 99. — 1, Solar siiectruiTi ; 2, spectrum of ox.vhasniofrlobin fO'o? per cent, solution): 3, spectnim 
 of rediK.ed hienio;,'lobin ; 4, ypectrum of CO-htenioglobiu ; 5, spectrum of methiemoglobin 
 (concentrated solution). 
 
 of haemoglobin (fig. 29, spectrum 3) is not so well defined as the a and 
 [i bands. On dilution it fades rapidly, so that in a solution of such a 
 strength that both bands of oxyh;emoglobin would be quite distinct 
 the single band of reduced haemoglobin has disappeared from view. The 
 oxyhfemoglobin bands can be distinguished in a solution which contains 
 only one part of the pigment to 10,000 of water, and even in more dilute 
 solutions which seem to be colourless the a band is still visible. 
 
 Methasmoglobin. — This may be produced artificially by adding 
 such reagents as piitassium ferricyanide or amyl nitrite to a solution of 
 oxyhsemoglobin ; it may also occur- in certain diseased conditions in the 
 urine ; it is therefore of considerable practical importance. It can be 
 
COMPOUNDS OF HEMOGLOBIN WITH GASES 153 
 
 Crystallised, and is usually stated to contain the same amount of oxygen 
 as oxyhsemoglobin, only combined differently. Buckmaster's recent 
 work, however, has shown that methsemoglobin only contains half as 
 much oxygen as oxyhsemoglobin. This oxygen is not removable by the 
 air-pump, nor by a stream of a neutral gas such as hydrogen. It can, 
 however, by reducing agents such as ammonium sulphide, be made to 
 yield haemoglobin. Methsemoglobin is of a brownish-red colour and 
 gives a characteristic absorption band in the red between the C and D 
 lines (fig. 29, spectrum 5). 
 
 The ferricyanide of potassium or sodium not only causes the 
 conversion of oxyhsemoglobin into methsemoglobin, but if the reagent 
 is added to blood which has been previously laked by the addition of 
 twice its volume of water there is an evolution of oxygen. If a small 
 amount of sodium carbonate or ammonia is added as well to prevent 
 the evolution of any carbonic acid, and the oxygen is collected and 
 measured, it is found that all the oxygen previously combined in 
 oxyhsemoglobin is discharged. This is at first sight puzzling, because, 
 as just stated, methsemoglobin contains also oxyg6n. What occurs 
 is that, after the oxygen is discharged from oxyhsemoglobin, fresh 
 oxygen takes its place from the reagents added. The oxygen atoms 
 of the methsemoglobin must be attached to a different part of the 
 hsematin group from the oxygen atoms of the oxyhsemoglobin, so that 
 the hsematin group when thus altered loses its power of combining 
 with oxygen and carbonic oxide to form compounds which are dissoci- 
 able in a vacuum. 
 
 Carbonic Oxide Haemoglobin may be readily prepared by passing 
 a stream of carbonic oxide or coal gas through blood or through a 
 solution of oxyhsemoglobin. It has a peculiar cherry-red eolour. Its 
 absorption spectrum is very like that of oxyhsemoglobin, but the two 
 bands are slightly nearer the violet end of the spectrum (fig. 29, spec- 
 trum 4). - Reducing agents, such as ammonium sulphide, do not change 
 it ; the gas is more firmly combined than the oxygen in oxyhsemoglobin. 
 CO-hsemoglobin forms crystals like those of oxyhsemoglobin : it resists 
 . putrefaction for a very long time. 
 
 Carbonic oxide is given off during the imperfect combustion of carbon 
 such as occurs in charcoal stoves ; it is a powerful poison combining 
 with the haemoglobin of the blood, and thus it interferes with normal 
 respiratory processes. The colour of the blood and its resistance to 
 reducing agents are in such cases characteristic. 
 
 Nitric Oxide Hsemoglobin. — When ammonia is added to blood, and 
 then a stream of nitric oxide is passed through it, this compound is 
 
154 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 formed. It may be obtained in crystals isomorphous with oxy- and 
 CO-hseraoglobin. It also has a similar spectrum. It is even more 
 stable than CO-hsemoglobin ; it is not only of theoretical importance 
 as completing the series, but is of some practical interest in cases of 
 poisoning by gas liberated from high explosives. 
 
 TESTS FOE BLOOD 
 
 These may be gathered from preceding descriptions. Briefly, they 
 are microscopic, spectroscopic, and chemical. The best chemical test 
 is the formation of hsemin crystals. The old test with tincture of 
 guaiacum and hydrogen peroxide, the blood causing the tincture 
 to become blue, is not very trustworthy, as it is also given by many 
 other organic substances. The test for instance is given by milk, and 
 is there due to the presence of an enzyme called a peroxidase, which is 
 destroyed by boiling. Boiled blood, however, gives the test as well as 
 fresh blood, and the reaction is due to the presence of the iron- 
 containing radical of hsemoglobin. 
 
 In medico -legal cases it is often necessary to ascertain whether a 
 red fluid or stain upon clothing is or is not blood. In any such case 
 it is advisable not to rely upon one test only, but to try every means 
 of detection at one's disposal. To discover whether it is blood or not 
 is by no means a difficult problem, but to distinguish human blood 
 from that of the common mammals is possible only by the " biological " 
 test described at the end of the next section. 
 
 IMMUNITY 
 
 The chemical defences of the body against injury and disease are 
 numerous. The property of coagulating which the blood possesses is a 
 defence against haemorrhage ; the acid of the gastric juice is a protection 
 against harmful bacteria introduced with food. Bacterial activity in 
 urine is inhibited by the acidity of that secretion. 
 
 Far more important and widespread in its effects than any of the 
 foregoing is the bactericidal {i.e. bacteria-killing) action of the blood 
 and lymph ; a study of this question has led to many interesting results, 
 especially in connection with the important problem of immunity. 
 
 It is a familiar fact that one attack of many of the infective maladies 
 protects us against anothe.r attack of the same disease. The person is 
 said to be immune, either partially or completely, against that disease. 
 Vaccination produces in a patient an attack of cowpox or vaccinia. 
 This disease is either closely related to smallpox, or may be it is small- 
 pox modified and rendered less malignant by passing through the body 
 
IMMUNITY . 155 
 
 of a calf. At any rate, an attack of vaccinia renders a person immune 
 to smallpox for a certain number of years. Vaccination is an instance 
 of what is called protective inoculation, which is now practised with 
 such great success in reference to other diseases, such as plague 
 and typhoid fever. The study of immunity has also rendered 
 possible what may be called curative inoculation, or the injection 
 of antitoxic material as a cure for diphtheria, tetanus, shake- 
 poisoning, etc. 
 
 The power the blood possesses of slaying bacteria is not limited to 
 the colourless corpuscles or phagocytes, but is also a property of the 
 fluid part of the blood, at any rate in the case of some micro-organisms. 
 The chemical characters of the substances which kill the bacteria are 
 not fully known; but they appear to be protein in nature. The 
 bactericidal powers of blood are destroyed by heating it for an hour 
 to 55° C. The substances, whatever be their source or their chemical 
 nature, are called hacterio-lysins. 
 
 Closely allied to the bactericidal power of blood, or blood serum, 
 is its globulicidal power. By this one means that the blood serum of 
 one animal has the power of dissolving the red blood corpuscles of 
 another species. If the serum of one animal is injected into the blood- 
 stream of an animal of another species, the result is a destruction of 
 its red corpuscles, which may be so excessive as to lead to the passing 
 of the liberated hsemoglobin into the urine (hsemoglobinuria). The 
 substances in the serum that possess this property are called hemolysins, 
 and though there is some doubt whether bacterio-lysins and hsemolysins 
 are absolutely identical, there is no doubt that they are closely related. 
 
 Normal blood thus possesses not only phagocytes, which eat up 
 bacteria, but also a certain amount of chemical substances which are 
 inimical to the life of our bacterial foes. But suppose a person gets 
 " run down " ; every one knows he is then more liable to " catch any- 
 thing." This coincides with a diminution in the bactericidal power of 
 his blood. But even a perfectly healthy person has not an unlimited 
 supply of bacterio-lysins, and if the bacteria are sufficiently numerous 
 he will fall a victim to the disease they produce. Here, however, 
 comes in the remarkable part of the defence. In the struggle he will 
 produce more and more bacterio-lysin, and if he gets well it means 
 that the bacteria are finally vanqjiished, and his blood remains rich 
 in the particular bacterio-lysin he has produced, and so will render 
 him immune to further attacks from that particular species of bacterium. 
 Every bacterium seems to cause the development of a specific anti- 
 substance. 
 
156 _ ESSENTIALS OF CHEMICAL PHYSIOLOGV 
 
 Immunity can more conveniently be produced gradually in animals, 
 and this applies, not only to the bacteria, but also to the toxins they 
 form. If, for instance, the bacilli which produce diphtheria are grown 
 In a suitable medium, they produce the diphtheria poison, or toxin, 
 much in the same way that yeastcells will produce alcohol when grown 
 in a solution of sugar. Diphtheria toxin is associated with a proteose, 
 as is also the case with the poison of snake venom. If a certain small 
 dose called a " lethal dose " is injected into a guinea-pig the result is 
 death. But if the guinea-pig receives a smaller dose it will recover ; 
 a few days after it will stand a rather larger dose ; and this may be 
 continued until, after many successive gradually increasing doses, it 
 will finally stand an amount equal to many lethal doses without any 
 ill effects. The gradual introduction of the toxin has called forth the 
 production of an antitoxin. If this is done in the horse instead of the 
 guinea-pig the production of antitoxin is still more marked, and the 
 serum obtained from the Ijlood of an immunised horse may be used 
 for injecting into human beings suffering from diphtheria, and it 
 rapidly cures the disease. The two actions of the blood, antitoxic 
 and anti-bacterial, are frequently associated, but may be entirely 
 distinct. 
 
 The antitoxin is also a protein probably of the nature of a globulin ; 
 at any rate it is a protein of larger molecular weight than a proteose. 
 This suggests a practical point. In the case of snake-poisoning the 
 poison gets into the blood rapidly owing to the comparative ease with 
 which it diffuses, and so it is quickly carried all over the body. In 
 treatment with the antitoxin or antivenin, speed is everything if life 
 is to be saved ; injection of this material under the skin is not much 
 good, for the diffusion into the blood is too slow. It should be injected 
 straight away into a blood-vessel. 
 
 There is no doubt that in these cases the antitoxin neutralises the 
 toxin much in the same way that an acid neutralises an alkali. If the 
 toxin and antitoxin are mixed in a test-tube, and time allowed for the 
 interaction to occur, the result is an innocuous mixture. The toxin, 
 however, is merely neutralised, not destroyed ; for if the mixture 
 in the test-tube is heated to 68° C, the antitoxin is coagulated and 
 destroyed, and the toxin remains as poisonous as ever. 
 
 The substances which on injection' provoke the appearance of antidotes 
 of this nature are either proteins, or are protein-like. They are called 
 antigens. ' 
 
 Immunity is distinguished into active and passive. Active immunity is 
 pioduced by the development of protective substances in the body ; passive 
 
IMMUNITY 157 
 
 immunity by the injection of a protective serum. Of the two the former is 
 the more permanent. 
 
 Ricin, the poisonous protein of castor-oil seeds, and ahrin, that of the 
 Jequirity bean, also produce when gradually given to animals an immunity, 
 due to the production of antiricin and antiabrin respectively. 
 
 Ehrlich's hypothesis to explain such facts is usually spoken of as the 
 side-chain theory of immunity. He considers that the toxins are capable of 
 uniting with the protoplasm of the living cells by possessing groups of atoms 
 like those by which nutritive proteins are united to cells during normal assimi- 
 lation. He terms these haptophor groups, and the groups to which these are 
 attached in the cells he terms receptor groups. The introduction of a toxin 
 stimulates an excessive production of receptors, which are finally thrown out 
 into the circulation, and the free circulating receptors constitute the antitoxin. 
 The comparison of the process to assimilation is justified by the fact that non- 
 toxic substances such as milk or egg-white introduced gradually by successive 
 doses into the blood-stream cause the formation of anti-substances capable 
 of coagulating them. 
 
 Up to this point I have .spoken only of the blood, but workers are steadily 
 bringing forward evidence to show that other cells of the body may by similar 
 measures be rendered capable of producing a corresponding protective 
 mechanism. 
 
 One further development of the theory must be mentioned. At least two 
 different substances are necessary to render a serum bactericidal or globulicidal. 
 The bacterio-lysin or haemolysin consists of these two substances. One of 
 these is called the amboceptor, the other the complement. We may illustrate 
 the use of these terms by an example. The repeated injection of the blood 
 of one animal (e.g. the goat) into the blood of another animal (e.g. a sheep) 
 after a time renders the latter animal immune to further injections, and at 
 the same time causes the production of a serum which dissolves readily the 
 red blood corpuscles of the first animal. The sheep's serum is thus hsemo- 
 lytic towards goat's blood corpuscles. This power is destroyed by heating 
 to 56° C. for half an hour, but returns when fresh serum of any animal is 
 added. The specific immunising substance formed in the sheep is called the 
 amboceptor ; the enzyme-like substance destroyed by heat is the comple- 
 ment. The latter is not specific, since it is furnished by the blood of non- 
 immunised animals, but it is nevertheless essential for haemolysis. Ehrlich 
 believes that the amboceptor has two side groups — one which unites with 
 the receptor of the red corpuscles, and one which unites with the haptophor 
 group of the complement, and thus renders possible the enzyme-like action 
 of the complement on the red corpuscles. 
 
 To put it another way : the cell-dissolving substances cannot act on their 
 objects of attack without an intermediate substance to anchor them on 
 the substance in question. This intermediary substance, known as the 
 amboceptor, is specific, and varies with the substance to be attacked (red 
 corpuscles, bacterium, toxin, etc.). The complement may be compared 
 to a person who wants to unlock a door ; to do this eff'ectively he must be 
 provided with the proper key (amboceptor). 
 
 Many antigens in small doses cause an increased sensitiveness of an 
 
158 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 animal to the foreign protein ; so that a second small dose injected a few 
 weeks later may produce death. This is called anaphylaxis. 
 
 Quite distinct from the bactericidal, globjilicidal, and antitoxic 
 properties of blood is its agglutinating action. This is another, result 
 of infection with many kinds of bacteria or their toxins. The blood 
 acquires the property of rendering immobile and clumping together 
 the specific bacteria used in the infection. The test applied to the 
 blood in cases of typhoid fever, and generally called Widal's reaction, 
 depends on this fact. The substances that produce this eifect are called 
 agglutinins. They also are probably protein-like in nature, but are 
 more resistant to heat than the lysins. Prolonged heating to over 
 60° C is necessary to destroy their activity. 
 
 We thus see that the means the body possesses of combating 
 bacterial invasion are numerous. In some cases the bacteria are 
 killed by bacterio-lysins, and in other cases they are directly attacked 
 and devoured by the phagocytes. Bacteria which are destroyed in 
 this way produce no evil results, whereas those which are not destroyed 
 are called pathogenic, or disease-producing organisms. There is stiU 
 another line of defence, for if the bacteria are not destroyed the 
 poisons or toxins they produce are in certain other cases neutralised 
 by antitoxins. 
 
 Metschnikoflf's view,' which is very widely accepted by bacterio- 
 logists, is that the most stress should be laid upon phagocytosis as 
 the principal factor in the resistance of the body to bacteria ; and the 
 discovery of opsonins by Sir A. E. Wright not only emphasises this 
 opinion, but shows how the body fluids co-operate with the phagocytes 
 in the process. The word " opsonin " is derived from a Greek word 
 which means " to prepare the feast.'' Washed bacteria from a culture 
 are distasteful to leucocytes, and would therefore, other things being 
 equal, be pathogenic if injected into an animal's body. But if the 
 bacteria have been previously soaked in serum, especially if that serum 
 has been obtained from the blood of an animal previously immunised 
 against that special bacterium, then the leucocytes devour them eagerly. 
 It was at first supposed that something had been added to the bacterium 
 to make it tasty, and that each kind of bacterium requires its own 
 special sauce or opsonin. It is, however, equally possible that the serum 
 has not added anything to the bacterium, but removed from it some- 
 thing that previously made it distasteful. At any rate the ultimate 
 eflFect is the same, and the bacterium is rendered non-pathogenic. When 
 a person is attacked by some invading organism, say the tubercle 
 bacillus, if that person's blood is naturally rich in the proper kind of 
 
IMMUNITY 159 
 
 opsonin he will not be troubled with tuberculosis ; but if the opsonic 
 power of his blood is low tte bacillus will produce the disease. The 
 modern treatment of tuberculosis aims at increasing the opsonic 
 power of the blood by improving the general condition of the patient 
 by good food and pure air, and also by the injection of the appropriate 
 opsonin into his blood. 
 
 Lastly, we come to a question which more directly appeals to the 
 physiologist than the preceding, because experiments in relation to 
 immunity have furnished us with what has hitherto been lacking, a 
 means of distinguishing human blood from the blood of other animals. 
 
 The discovery was made by Tchistovitch (1899), and his original 
 experiment was as follows : — Rabbits, dogs, goats, and guinea-pigs 
 were inoculated with eel-serum, which is toxic ; he thereby obtained 
 from these animals an antitoxic serum. But the serum was not only 
 antitoxic ; it also produced a precipitate when added to eel-serum, 
 though not when added to the serum of any other animal. In other 
 words, not only has a specific antitoxin been produced, but also a 
 specific precipitin. Numerous observers have since found that this is 
 a general rule throughout the animal kingdom, including man. If, for 
 instance, a rabbit is treated with human blood, the serum ultimately 
 obtained from the rabbit contains a specific precipitin for human blood; 
 that is to say, a prbcipitate is formed on adding such a rabbit's serum 
 to human blood, but not when added to the blood of any other animal, i 
 The great value of the test is its delicacy : it will detect the specific 
 blSod when it is greatly diluted, after it has been dried for weeks, or 
 even when it is mixed with the blood of other animals. 
 
 The lipoids contained in the membrane of cells play some part in the 
 relationship of such cells to toxins. The matter has been mainly studied in 
 relation to red corpuscles, and the toxins (such as saponin and the hsemolysin 
 of snake venom) which attack them. There is some evidence that the 
 cholesterol in the envelope of the red corpuscles is a protective agent (see.also 
 p. 38). A few years ago, Preston Kyes stated that lecithin is the amboceptor 
 which anchors the hsemolysin on to the red cells. But more recent research 
 has failed to substantiate this view, and the compounds which Kyes described 
 and called lecithides are impure mixtures of several substances. It is much 
 more probable that the real agent at work in hseraolysis is a lipolytic or fat- 
 splitting enzyme ; this splits up the lecithin of the cell- wall, liberating oleic . 
 aoid and desoleolecithin (that is, lecithin minus its oleic acid radical), and it 
 is these cleavage products which dissolve out the hfemoglobiij and so destroy 
 the corpuscles. 
 
 ^ There may be a slight reaction with the blood of allied animals ; for instance, 
 with monkey's blood in the case of man. 
 
160 
 
 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 CHEMISTKY OF RESPIRATION 
 
 The consideration of the blood, and especially of its pigment, is so 
 closely associated with respiration that a brief account of that process 
 follows conveniently here. 
 
 The air in the alveoli of the lungs, and the blood in the pulmonary 
 capillaries are only separated by the thin capillary and alveolar walls. 
 The blood parts with its excess of carbonic acid and watery vapour to 
 the alveolar air ; the blood at the same time receives from the alveolar 
 air the oxygen which renders it arterial. 
 
 The intake of oxygen is the commencement, and the output of 
 carbonic acid the end, of the series of changes known as respiration. 
 The intermediate steps take place all over the body, and constitute 
 what is known as internal or tissue respiration. The exchange of 
 gases which occurs in the lungs is sometimes called in contradistinction 
 external respiration. We have already seen that the oxyhaemogl»bin 
 is only a loose compound, and in the tissues it parts with its oxygen. 
 The oxygen does not necessarily undergo immediate union with carbon 
 to form carbonic acid, and with hydrogen to form water, but in most 
 cases, as in muscle, is held in reserve by the tissue itself. Ultimately, 
 however, these substances pass into the venous blood, and the carbonic 
 acid and a portion of the water find an outlet by the lungs. 
 
 Inspired and Expired Air. — The composition of the inspired and 
 expired air may be compared in the following table : — 
 
 
 Inspired or 
 Atmospheric Air. 
 
 Expired Air. 
 
 Oxygen . 
 
 Nitrogen .... 
 
 Carbonic acid 
 
 Watery vapour 
 
 Temperature. 
 
 20-96 vols, per cent. 
 79 
 0-04 „ 
 
 variable 
 
 J) 
 
 16-03 vols, per cent. 
 79 
 4'4 „ 
 
 saturated 
 thatof body (37°C.). 
 
 The nitrogen remains unchanged. The recently discovered gases, 
 argon, crypton, etc., are in the above table reckoned in with the nitrogen. 
 They are, however, only present in minute quantities. The chief 
 change is in the proportion of oxygen and carbonic acid. The loss of 
 oxygen is about 5, the gain in carbonic acid 4-5. If the inspired and 
 expired airs are carefully measured at the same temperature and 
 barometric pressure, the volume of expired air is thus rather less than 
 that of the inspired. The conversion of oxygen into carbonic acid would 
 
THE GASES OF THE BLOOD 161 
 
 not cause any change in the volume of the gas, for a molecule of oxygen 
 (Oj) would give rise to a molecule of carbonic acid (OO^) which would 
 occupy the same volume (Avogadro's law). It must, however, be 
 remembered that carbon is not the only element which is oxidised. 
 Fats contain a number of atoms of hydrogen which during metabolism 
 are oxidised to form water ; a certain small amount of oxygen is also 
 used in the formation of urea. Carbohydrates contain sufficient oxygen 
 in their own molecules to oxidise their hydrogen : hence the apparent 
 loss of oxygen is least when a vegetable diet (that is, one consisting 
 largely of starch and other carbohydrates) is taken, and greatest when 
 
 much fat and protein are eaten. The quotient — 2 given o ^^ called 
 
 O2 absorbed 
 
 4'5 
 the respiratory quotient. Normally it is -^ = 0'9, but this varies 
 
 considerably with diet, as just stated. 
 
 THE GASES OF THE BLOOD 
 
 Before we can understand either the chemistry of respiration or 
 its regulation, which is in part a chemical process, it is necessary that 
 s we should study the fundamental laws which regulate the retention 
 of oxygen and carbonic acid in the blood ; and as the blood presents 
 many complications, it will be best at the outset to consider the solution 
 of gases in such a simple medium as water. 
 
 Solution of Gases in Water 
 
 If water is shaken up with oxygen, a certain definite amount of 
 oxygen will become dissolved in the water. Under the same conditions 
 the same quantity of oxygen would always be dissolved, and in the 
 following argument it is assumed throughout that the temperature 
 remains constant. The amount dissolved, then, depends upon two cir- 
 cumstances, each of which can be measured. The first is the pressure 
 of the oxygen to which the water is exposed when shaken ; the second 
 is a property of the oxygen itself, namely, its solubility in water. The 
 solubilities of dififerent gases differ very much ; some (for instance, 
 oxygen) are not readily soluble in water, whilst others, such as carbonic 
 acid, are very soluble. 
 
 If a cubic centimetre of water is introduced into a large air-tight 
 bottle containing pure oxygen at the atmospheric pressure, and another 
 cubic centimetre of water is similarly placed in a bottle containing 
 pure carbonic acid at thie same pressure, the former would be found 
 to have dissolved 0-04 c.c. of oxygen, the latter 1 c.c, of carbonic acid, 
 11 
 
162 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 These figures represent the degrees to which the two gases are soluble 
 in water under similar circumstances, and are called their coefficients 
 of solubility. The coefficient of solubility of gas in a liquid is therefore 
 the amount of a gas which 1 c.c. of liquid will dissolve at 760 mm. of 
 mercury, that is, at atmospheric pressure. 
 
 The quantity of gas which a liquid will dissolve depends also upon 
 the pressure of the gas to which the liquid is exposed. Thus, in the 
 instance given above, if the oxygen had been rarefied in the bottle until 
 it only exerted a pressure of one-fifth of an atmosphere, the water would 
 have only taken up not 0'04 c.c. of oxygen, but only one-fifth of that 
 amount, O'OOS c.c. If we represent the coefficient of solubility of a 
 gas by K, and the pressure of the gas to which the liquid is exposed by 
 P', and the atmospheric pressure by P ; then the quantity (Q) of the 
 gas dissolved by 1 c.c. of the liquid may be found by the following 
 formula : — 
 
 Dalton-Henry Law. — What has been said above is as true of gases 
 which are mixed together as of pure gases. For instance, we have seen 
 that a cubic centimetre of water shaken up with oxygen at one-fifth of 
 an atmosphere (152 mm. pressure) will absorb 0'04 x'i = 0'008 c^c. ; 
 if it is shaken with nitrogen at a pressure of four-fifths of an atmosphere, 
 it will dissolve 0'02 x i = 0'016 c.c. If now a c.c. of water is shaken 
 with air (a mixture of one part of oxygen to four of nitrogen) it will 
 dissolve 0-008 c.c. of oxygen and 0'016 c.c. of nitrogen. This fact has 
 been stated as the Dalton-Henry law in the following words : — When 
 two or more gases are mixed together, they each produce the same 
 pressure as if they separately occupied the entire space and the other 
 gases were absent. The total pressure of the mixture is the sum of 
 the partial pressures of the individual gases in the mixture. 
 
 The Tension of Gases in Fluids 
 
 In the cases which have been discussed up to this point, a condition 
 of equilibrium exists between the gas dissolved in the fluid and the 
 gas in the atmosphere to which the fluid is exposed, so that as many 
 molecules of the gas leave the .surface of the fluid as enter it. The 
 gas dissolved in the fluid, therefore, exercises a pressure which is the 
 same as that of the gas in the atmosphere when equilibrium exists. 
 For the sake of convenience the word Tension is applied to the pressure 
 of the gas in the fluid. 
 
 Definition of Tension. — The tension of a gas dissolved in a fluid is 
 
THE GASES OF THE BLOOD 
 
 163 
 
 equal to the pressure of the same gas in an atmosphere with which the 
 
 gas in the fluid would be in equilibrium. Above, we have called the 
 
 pressure which the gas exerts on the liquid, P'. If we call the tension 
 
 of the gas in the liquid T, we find that when equilibrium exists P' = T. 
 
 In the case of all true solutions, therefore, we may replace P' in our 
 
 T 
 previous equation by T ; therefore Q = K x ^. We thus arrive at a 
 
 relation between two separate things, which must be most carefully 
 distinguished from one another — the quantity of the gas dissolved in 
 the liquid and its tension. 
 
 Measurement of Tension in Fluids — Aerotonometer. — Numerous 
 instruments have been invented for measuring tension. They are called 
 tonometers. Krogh's (fig. 30) is the best. 
 It consists of a T-shaped cannula (A) 
 introduced into the blood-vessel, say the 
 carotid artery. The blood fills the cavity 
 B and leaves it at C, so that a constant 
 stream of blood is kept flowing. Into 
 it a small bubble of air (D) is introduced. 
 Exchange of gases takes place between 
 the bubble and the blood, and the former 
 very soon gets into equilibrium with the 
 latter., When it has done so, the bubble 
 is withdrawn up the capillary tube E, 
 taken away, and analysed. 
 
 As an example, suppose the bubble on analysis proved to consist 
 of 4 per-cent. carbonic acid and 12 per-cent. oxygen, together with 
 nitrogen and aqueous vapour. The gas in the instrument was com- 
 pressed by the pressure of the arterial blood (say 120 mm. of mercury) 
 in addition to the atmospheric pressure of 760 mm. of mercury, and 
 therefore its -total pressure was 120 -)- 760 = 880 mm. of mercury. Four 
 per cent, of this would have been due to the carbonic acid ; 4 per cent, 
 of 880 is 35"2. Twelve per cent, would have been due to the oxygen ; 
 12 per cent, of 880 is 105'6. That is, the carbonic acid and oxygen 
 tensions would have been in round figures 35 and 106 mm. of mercury 
 respectively. 
 
 Measurement of the Quantity of a Gas in a Fluid 
 
 The most general method of determining the quantity of gas in a 
 fluid is by boiling a measured quantity of the fluid in a vacuum. The 
 gas is all given off; it may be collected and measured. In the case 
 
 Fig. 30.— Krogh's Tonometer. 
 
164 
 
 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 I. 
 
 of blood, which is the only fluid that need be considered, th'is process 
 is carried out by means of a mercurial air-pump known as the blood- 
 gas pump. One of the numerous forms of mercury pump is described 
 in the Appendix. 
 
 The total gas obtained is first measured; the carbonic acid is 
 removed by caustic potash, and the gas that remains, which consists 
 of oxygen and nitrogen, is measured ; the oxygen is then removed by 
 pyrogallic acid, and the residual gas is nitrogen. 
 Haldane's apparatus for carrying this out is de- 
 scribed in the Appendix. Another method is the 
 following : — 
 
 Chemical Method of Blood-gas Analysis. — When a 
 solution of oxyhsemoglobin is shaken with potassium 
 ferricyanide, it yields the same amount of oxygen 
 to the air as it would if boiled in a vacuum. In 
 much the same way urea when treated with sodium 
 hypobromite yields up all its nitrogen, and the 
 apparatus used for determining the oxygen in blood 
 is very similar to Dupre's urea apparatus (see 
 Lesson X). The blood (5 c.c.) is placed in the large 
 bottle (fig. 31, F) underneath a layer of dilute 
 ammonia solution. The blood is thus protected 
 from the air whilst the apparatus becomes equal in 
 temperature to the bath in which it is placed. The 
 blood is shaken with the ammonia solution, which 
 lakes it thoroughly ; the ferricyanide solution is 
 then spilt into the laked blood from the tube G 
 and the oxygen is shaken out of the solution. When 
 the oxygen has been determined the bottle is opened 
 
 Fio. 31.— Apparatus for , . ... , , . , ,, , ^ 
 
 obtaining tlie blood and tartaric acid IS placed in the small tube G ; 
 
 this is subsequently spilt into the mixture of blood, 
 ammonia, and ferricyanide ; it liberates the carbonic acid which is also 
 shaken out of the fluid. The carbonic acid does not come completely 
 out, however, and a correction has to be introduced for the quantity 
 which remains in solution. The gas (oxygen or carbonic acid, as the 
 case may be) passes over into the tube C, which was previously filled 
 up to the zero mark (H) with water, and connected to a reservoir (A) ; 
 this would drive water out of A into the open tube D, and the water 
 will therefore rise' in D; but in practice it is convenient to keep the 
 gas always at the same volume ; this may be done by raising the 
 pressure in the open limb (D) of the pressure gauge by squeezing 
 
tiiE GASES OF THE BLOOb 165 
 
 some of the water, with which the gauge is filled, out of a rubber 
 reservoir (B) which forms the base of the gauge, thus the level of the 
 water in C is maintained at the zero mark, while that in D rises 
 from H to I. The actual measurement then is the increase of pressure 
 (i.e. the height of the column of water H I) which is necessary to 
 keep the gas at the same volume after the oxygen or carbonic 
 acid has been shaken ofif as it previously occupied. From tiiis the 
 quantity coming off can be calculated. The apparatus just described 
 illustrates the principle of the method. Barcrof t's most recent improved 
 instrument is described in the Appendix. 
 
 The chemical method is not quite so accurate as the vacuum pump, 
 but it is much more convenient for the study of many problems, as 
 it requires less blood, and, owing to its simplicity, a great number of 
 observations can be made upon a single animal. It can also be used 
 for observations on human blood. 
 
 Belation between Quantity and Tension of Gases in Blood 
 
 In the preceding paragraphs the methods of measuring the tension 
 and the quantity of gas in a given sample of blood have been described. 
 It is now necessary to consider the relationship between them. 
 
 On p. 163 we have seen that for gases in solution in water, 
 
 T 
 Q = K X -, where Q is the quantity of gas dissolved, T the tension, K 
 
 the coefficient of solubility, and P the atmospheric pressure. Since 
 K and P are constant, it follows that Q varies directly in proportion to T ; 
 that is to say, if the tension is doubled, the quantity of gas dissolved 
 is also doubled ; if the tension is trebled, the quantity of gas is trebled, 
 and so on. These results might be plotted out on a curve in which the 
 quantities are placed on the ordinate, and the tensions on the abscissa. 
 Such a curve would gjve the quantity of gas dissolved at any given 
 tension, and, in the case of water, it would turn out to be a straight line. 
 
 But in the case of both the oxygen and the carbonic acid in blood, 
 the curve showing the relationship between the tension of gas and the 
 volume which can be pumped off is not a straight line. 
 
 Oxygen in Blood, — Prom every 100 _c.c. of arterial blood, about 
 20 c.c. of oxygen can be removed by the air-pump. Nearly all of 
 this oxygen is chemically combined with haemoglobin : the amount 
 in actual solution in the blood is 0-7 c.c. for every 100 c.c. of 
 blood. Haemoglobin owes its value as a respiratory pigment to two 
 principal facts : (1) It can unite with a large quantity of oxygen, and 
 therefore blood can carry about thirty times as much oxygen to the 
 
166 
 
 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 tissues as plasma would under the same circumstances. (2) The inter- 
 action between haemoglobin and oxygen is a reversible one ; the two 
 unite in the lungs, where the pressure of oxygen is high ; but when 
 oxygen is absent or at a low pressure, as in the tissues, the oxyhemo- 
 globin parts with its store of oxygen. 
 
 We will now consider the nature of this union, and the conditions 
 under which it takes place. 
 
 The reaction between haemoglobin and oxygen is a chemical one, 
 At most, 1 gramme of hasmoglobin can unite with 1-34 c.c. of 
 oxygen. This figure is not quite constant, probably on account of 
 slightly difiierent forms of globin (the protein constituent of haemo- 
 globin) united with the hsematin (the iron-containing constituent) 
 in dififerent animals. The relation between the respiratory oxygen 
 and the iron of the haemoglobin is, however, quite constant, and is 
 
 Fig. 32. — Barcroft's Tonometer, suspended horizontally 
 m warm bath in which it is rotated. 
 
 called the " specific oxygen capacity." Each gramme of iron in 
 haemoglobin unites with 400 c.c. of oxygen ; these figures are in the 
 relation of one atom of iron to two. atoms of loxygen. The reversible 
 nature of the reaction may, therefore, be expressed by the equation 
 H6 + 0.^"t"H60n. A reversible reaction means that it will go in either 
 direction according to the concentration of the substances present ; 
 thus if the concentration of oxygen in solution is increased, more of 
 the hfemoglobin will become oxyhaemoglobin ; and if it is diminished, 
 oxyhaemoglobin will break up into reduced haemoglobin and oxygen. 
 
 The quantity of gas in solution (that is, not united with haemoglobin) 
 varies in proportion to the oxygen pressure to which the haemoglobin 
 solution is exposed ; therefore the problem before us is to ascertain the 
 relative quantities of oxy- and reduced haemoglobin when a haemoglobin 
 solution is shaken up with oxygen at different pressures. 
 
 This can be done by means of Barcroft's tonometer (fig. 32). 
 Suppose we have six of these tubes, and each contains the same 
 
THE GASES OF THE BLOOD 
 
 167 
 
 amount (a few c.c.) of hsemoglobin solution, and gases of the following 
 composition : — 
 
 No. 1. Nitrogen and no oxygen. 
 
 No. 2. Nitrogen and enough oxygen to give 5 mm. oxygen pressure. 
 
 No. 3. „ „ „ 10 „ 
 
 No. 4. „ „ „ 20 „ 
 
 No. 5. „ „ „ 50 „ 
 
 No. 6. „ „ „ 100 „ 
 
 Each tonometer is rotated round and round in a bath at body tem- 
 perature until the hsemoglobin and the oxygen are in equilibrium ; this 
 will take about fifteen minutes ; the solution is then withdrawn and 
 examined in order to ascertain the relative quantities of oxy- and reduced 
 hsemoglobin in each of the six vessels. 
 
 The figures for a pure solution of hsemoglobin would be : — 
 
 
 No. 1. 
 
 No. 2. 
 
 No. 3. 
 
 No. 4. 
 
 No. 5. 
 
 No. 6. 
 
 Percentage of reduced haBmoglobin 
 Percentage of oxyhsemoglobin 
 
 100 
 
 
 63 
 37 
 
 45 
 55 
 
 28 
 72 
 
 13 
 
 87 
 
 6 
 
 94 
 
 100 
 
 100 
 
 100 
 
 ioo 
 
 100 
 
 100 
 
 The same answer may be expressed graphically ; if the pressures of 
 oxygen are plotted horizontally, and the percentages of oxy- and reduced 
 hsemoglobin in the solution are plotted vertically, we get the curve 
 shown in the accompanying diagram (fig. 33), which is called the dis- 
 sociation curve of hoimoglobin. 
 
 A solution of pure hsemoglobin is, however, not the same thing as 
 blopd, and the dissociation of oxyhsemoglobin in the latter fluid during 
 life is influenced by various conditions, especially by (1) temperature, (2) 
 the presence of salts, and (3) the presence of acids, especially carbonic 
 acid. These factors make the oxyhsemoglobin molecules break down 
 more rapidly, and form more rapidly. It is clearly necessary that the 
 two processes (the union of hsemoglobin with oxygen, and the liberation 
 of oxygen from oxyhsemoglobin) should occur at the same rate, that 
 is under one second, which is about the time occupied by any given 
 portion of blood in travelling along the capillaries. 
 
 It would be futile to have an oxygen carrier in the blood which took 
 a fraction of a second to acquire its oxygen in the lungs, and a fraction of 
 an hour to release it in the tissues. Yet a solution of pure hsemoglobin 
 
168 
 
 ESSENTIALS OE CHEMICAL PHYSIQLOGV 
 
 is just such a substance. In the actual blood, however, the three factors 
 just mentioned, the salts, especially the potassium salts of the red 
 corpuscles, the high temperature, and the presence of carbonic acid at 
 40 mm. pressure, increase the rate of dissociation of oxyhsemoglobin so 
 greatly that it equals the rate at which the union of oxygen and 
 haemoglobin occurs in the lungs. Nature has adapted the conditions of 
 
 Total Hssmoglobin 100 
 
 Percentage Percentage 
 of reduced of oxyhssmo- 
 hEBmoglobin, globin. 
 
 28 
 
 45 
 
 63 
 
 100 
 
 100^ 
 94 
 87 
 
 72 
 
 55 
 
 37 
 
 
 }^ 
 
 ■■.: 
 
 ' /, ' 
 
 ''■'■-■ 
 
 
 
 
 
 ^____ ■ ■ ^ 
 
 
 
 
 ^ 
 
 
 
 
 / 
 
 
 
 ■l 
 
 1 
 
 
 ' 
 
 
 i 
 
 
 
 
 
 5 10 20 
 
 50 
 
 100 
 
 Oxygen Pressure in mm. of Mercury. 
 
 Fig. 33. — Dissociation curve of haemoglobin at 37° G. The shaded area 
 
 represents reduced haemoglobin, the white, oxyhsemoglobin. 
 
 life so admirably that the needs of the body are served by a substance 
 hsemoglobin, which by itself is inefficient for oxygen transport. 
 
 The next figure (fig. 34) shows the dissociation curve in the actual 
 blood, and it should be carefully compared with fig. 33. The two 
 present to the eye graphically the superiority of hsemoglobin as an 
 oxygen carrier when it is present in the living blood over that which it 
 possesses in a pure solution. 
 
 In the second curve, that of the blood itself, it will be seen that at 
 an oxygen pressure of over 60 mm. of mercury (the pressure in the lung 
 alveoli is about 100) the blood will nearly saturate itself with oxygen, 
 and that at pressures below 50 the blood loses its oxygen rapidly, whilst 
 
THE GASES OF Tttfe BLOOD 
 
 169 
 
 at 10 mm. pressure it is nearly completely reduced. As the rate at 
 which oxygen can diffuse out of the capillaries into the surrounding 
 tissues depends upon the pressure it exerts in the plasma, it is important 
 that the blood should be capable of a considerable degree of reduction 
 when it is in contact with fluid containing oxygen at a pressure such as 
 one finds in the tissues (20 to 30 mm. of mercury). 
 
 Carbonic Acid in Blood. — Pure distilled water dissociates to a 
 trifling extent into H and OH ions, which of necessity are equal in 
 
 100 
 
 90 
 
 ■ ■ ■ 
 
 : ' '' ' '- ''X 
 
 
 611 
 
 80 
 
 'P 
 
 
 o 
 
 
 J3 
 
 /O 
 
 -*:s 
 
 
 ^ 
 
 
 T) 
 
 fin 
 
 
 
 
 
 fq 
 
 50 
 
 40 
 
 30 
 
 20 
 
 10 (^ 
 
 10 20 30 40 50 60 70 60 90 100 
 
 Oxygen Pressure in mm. of Mercury. 
 
 Fig. 34. — Dissociation curve of hsemoglobin in the blood. The shaded area as before 
 represents reduced haemoglobin, the white area, oxyhaemoglobin. 
 
 number, so we speak of water as neutral, not because it is neither 
 acid nor alkaline, but because it is both in equal degree. Blood, though 
 alkaline to litmus, nevertheless contains H ions, and so has a certain 
 degree of acidity. The unit of acidity is the concentration of H ions 
 in a normal solution of HCl (36-5 gr. per litre) ; compared to this 
 the H-ion concentration in blood is very small indeed, being only 
 0.000,000,032. Nevertheless, variations in this figure produce pro- 
 nounced effects, an increase leading to stimulation of the respiratory 
 centre. The principal acid to which this is due ik carbonic acid 
 (HjjCOg), and though COj is being continually thrust into the blood 
 by the tissues especially during activity the H-ion concentration in 
 
170 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 health varies but little, for certain substances in the blood spoken of 
 as " buffers " seize it, and hold it in combination. These are principally 
 sodium bicarbonate, sodium phosphate, and proteins. The first- 
 named secures most of the COj, the proteins get about a third, and 
 among the proteins Buckmaster finds haemoglobin the most efficient. 
 Whether COj haemoglobin is in the same category as other compounds 
 of this pigment with gases is uncertain ; finally, about 5 per cent, is 
 present in simple solution. The total COj is about equal to what 
 water would absorb at 760 mm. pressure, but most of this is in com- 
 bination, so small an amount being free that the blood is in equilibrium 
 with a OOj pressure of only 40 mm. Hg (5 per cent of an atmosphere). 
 Much in the same way that oxyhaemoglobin dissociates in the tissues, 
 the COj compounds dissociate in the lungs, and COj is discharged into 
 the air. 
 
 Differences between Arterial and Venous Blood. — The average 
 quantities of gases in human 'blood are as seen in the following 
 
 table : — 
 
 For 100 volumes of blood. 
 
 Arterial. Venous. 
 
 Oxygen . . . 18-5 , 12 
 
 COg . ... 50 56 
 
 Nitrogen . . 2 2 
 
 Nitrogen is simply dissolved from the air and has no physiological 
 significance. The other two gases are important, and the numbers for 
 venous blood vary a good deal ; tissue activity makes venous blood 
 still more venous. But on the average, every 100 c.c. of blood which 
 pass through the lung gain 6'5 c.c. of oxygen and lose 6 c.c. of COg. 
 We have now to study how this interchange is effected. 
 
 THE MECHANISM OF GASEOUS EXCHANGE IN THE LUNG 
 
 1. Oxygen. — The simplest explanation of the passage of oxygen 
 from the alveolar air into the blood is that the process is one of 
 diffusion. This view can be maintained if it can be proved that the 
 pressure of oxygen in the alveolar air is as great as or greater than 
 the tension of oxygen in the arterial hlood, and therefore a fortiori 
 greater than that of oxygen in the venous blood. 
 
 The conception of respiration based upon this view would be that 
 the oxygen in the air of the alveoli, though less than that in the 
 atmosphere, is greater than that in venous blood ; hence oxygen passes 
 from the alveolar air into the blood-plasma ; the oxygen immediately 
 combines with the haemoglobin, and thus leaves the plasma free to 
 
MECHANISM OF GASEOUS EXCHANGE IN THE LUNG 171 
 
 absorb more oxygen ; and this goes on until the haemoglobin is entirely, 
 or almost entirely, saturated with oxygen. The reverse change occurs, 
 in the tissues when the partial pressure of oxygen is lower than in the 
 plasma, or in the lymph r that bathes the tissue elements ; the plasma 
 parts with its oxygen to the lymph, the lymph to the tissues ; the 
 oxyhsemoglobin then undergoes dissociation to supply more oxygen to 
 the plasma and lymph, and thus in turn to the tissues once more. 
 This goes on until the oxyhsemoglobin loses about half its store of 
 oxygen. 
 
 This view is regarded as the correct one, owing to the accurate 
 determinations which can now be made, first, of the oxygen pressure in 
 
 ™'~'^' -aani, ,. FMniJTHPiFnF 
 
 SAMPLING TUBE 
 
 Fig. 35. — Apparatus for obtaining alveolar air. 
 
 alveolar air, and secondly, of the oxygen tension in blood. We will take 
 the two in the order named. 
 
 i. The Pressureof Oxygen in the Alveolar Air. — Haldane and Priestley 
 introduced a very simple method of collecting alveolar air. A piece of 
 rubber tubing is taken about 1 inch in diameter and about 4 feet long 
 (fig. §5). A mouthpiece is fitted into one end. About 2 inches from 
 the mouthpiece a small hole is made into which is inserted tlie tube of 
 a gas-receiver, or sampling-tube, as in the figure (fig. 35). The gas- 
 receiver is fitted at the upper end with a three-way tap, and the lower 
 end is also closed by a tap ; before it is used, it is filled with mercury. 
 The subject of the experiment breathes normally through the tube for 
 a time, and then, at the end of a normal inspiration, he expires quickly 
 and very deeply through the mouthpiece and instantly closes it with 
 his tongue. The lower tap of the receiver is then turned, and as the 
 mercury runs out, a sample of the air takes its place and fills the 
 receiver ; this sample is then analysed. A second experiment is then 
 done, in which the subject expires deeply at the end of a normal 
 
172 '" EssEl^tiAL^ oir chemical PHVsioiOGV 
 
 expiration, and another sample obtained. The mean result of the two 
 analyses gives the composition of alveolar air. 
 
 It is found on analysis that the normal oxygen pressure in the 
 alveoli is approximately equal to 100 mm. of mercury, and this is 
 equivalent to 13 per cent, of an atmosphere. 
 
 ii. The Tension of Oxygen in the Bloods — This is estimated by Krogh's 
 tonometer (fig. 30, p. 163), and the experiments show that the tension of 
 oxygen in the blood is lower than the alveolar oxygen pressure. If the 
 latter is artificially raised or lowered, the oxygen tension in the blood 
 rises and falls in the same way, but always remains lower than the 
 oxygen pressure in the alveoli. 
 
 Some authorities consider that in cases of definite oxygen want, such as 
 during violent muscular exercise, or on the tops of high mountains, the lining 
 epithelium of the pulmonary alveoli can, by a process of active secretion, 
 transfer oxygen from the alveolar air to the blood. In the case of exercise the 
 observations made by different workers are at variance, whilst at high altitudes 
 they are so few as to make further work desirable before physiologists generally 
 can accept the possibility of the secretion of oxygen by the lung. That secre- 
 tion is not impossible is shown by the fact that a similar secretion of oxygen - 
 is known to occur in the swim-bladder of certain fishes. The swim-bladder 
 corresponds morphologically with the lungs of a mammal, and the oxygen 
 stored in it is far in excess of anything that can be explained by mere difi'usion 
 from the sea-water. This storage of oxygen, moreover, ceases when the • 
 vagus nerves which supply the sWim-bladder are divided. 
 
 2. Carbonic Acid. — Here, again, the same two measurements are 
 necessary and are obtained in the same way. The alveolar tension of 
 this gas is always lower than that of the arterial blood ; the pressure 
 differences are less than in the case of oxygen ; this coincides with the 
 ease with which carbonic acid passes out through the membrane which 
 separates the blood from the air. 
 
 It is unnecessary to suppose that the alveolar epithelium actively 
 excretes CO^, for mere diffusion will explain the passage of that gas 
 from the blood to the alveolar air. 
 
 The following table summarises the main facts in relation to the two 
 gases, and the arrows show how they always pass from situations of 
 higher to those of lower pressure. 
 
 Pressure (Tension) of Gases 
 Lungs. Arterial Blood. Venous Blood. Tissues. 
 
 Oxygen- 100 mm. yi ,„„ [■ >-35 mm. — >-35 mm. to zero. 
 
 Carbonic"! ,„ fJust over"! , ,- ,e 
 
 Acid I *^ ™™-^-| 40 mm. K"^^ mm.^-over 45 mm. 
 
CAUSE AND REGULATION OF RESPIRATION 173 
 
 CAUSE AND REGULATION OF RESPIRATION 
 
 The activity of the muscles of respiration is controlled by a 
 specialised small district of grey matter in the floor of the fourth 
 ventricle, which is called the respiratoiy centre. The activity of this 
 centre is regulated by two main factors : (1) the action upon it by 
 afferent nerves, of which the most important are the vagus nerves from 
 the lungs, and (2) the chemical condition of the blood. 
 
 If the lungs of an animal are alternately and forcibly inflated and 
 deflated with air, or if a man voluntarily takes a number of deep breaths 
 rapidly for a minute or two, breathing then ceases for a variable time, 
 and this condition is termed apnoea. This condition is not due, as was 
 at one time supposed, to an over-oxygenation of the blood, nor is it 
 produced, as Head considered, purely as a result of the excitation of 
 the vagus nerve-endings . in the lung, for it occurs after the vagus 
 nerves are severed. Fredericq has always maintained that apnoea has 
 a chemical rather than a nervous origin ; he attributed it, however, not 
 to over-oxygenation of the blood, but to a lessening of the carbonic 
 acid which is swept out of the body by the powerful respiratory efforts. 
 Haldane and Priestley corroborated this view by their important 
 researches. 
 
 They found that, under constant atmospheric pressure, in man the 
 alveolar air contains a nearly constant percentage of carbon dioxide 
 in the same person. In different individuals this percentage varies 
 somewhat, but averages 5-1 per cent, of an atmosphere in men, and 
 4 '7 in women and children. 
 
 With varying atmospheric pressures, the percentage varies inversely 
 as the atmospheric pressure, so that the pressure or tension of the 
 carbon dioxide remains constant. The oxygen pressure, however, 
 varies widely under the same conditions. 
 
 These observations and the next to be immediately described 
 furnish the chemical key to the cause of the amount of pulmonary 
 ventilation, and play an important part in conjunction with the 
 respiratory nervous system in the regulation of breathing. For the 
 respiratory centre is not only affected by the impulses reaching it by 
 the vagi and other afferent nerves, but it is also very sensitive to any 
 rise in the tension of carbon dioxide in the blood that supplies it. The 
 changes in the tension of this gas in the arterial blood are normally 
 proportional to the changes in the carbon dioxide pressure in the alveoli, 
 and the changes in the lung alveoli are transmitted to the respiratory 
 centre by the blood. They found that a rise of 0'2 per cent, in the 
 
174 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 alveolar carbon dioxide pressure is sufficient to double the amount 
 of alveolar ventilation during rest. During work the alveolar carbon 
 dioxide pressure increases slightly, and the pulmonary ventilation is 
 consequently increased. 
 
 Changes in the oxygen pressure within wide limits have no such 
 influence ; the normal chemical stimulus to respiration is, therefore, 
 an increase of carbon dioxide, and not diminution of oxygen. If these 
 limits are exceeded, as when the oxygen pressure falls below 13 per 
 cent, of an atmosphere, the respiratory centre begins to be excited 
 for want of oxygen. Physiologists now agree that fatigue products, 
 such as sarcolactic acid, assist the carbon dioxide in stimulating the 
 respiratory centre. The important stimulus is not any particular acid, 
 but the total hydrogen-ion concentration. 
 
 In connection with the relative importance of the nervous and 
 chemical factors in breathing, F. H. Scott has shown that the principal 
 respiratory nerves (the pneumo-gastrics) regulate the rate or rhythm 
 of the respiratory movements, whilst the chemical factor specially 
 regulates the amount of pulmonary ventilation, that is, the depth of 
 the individual respiratory efforts ; for when these nerves are divided, 
 a rise in the alveolar tension of carbon dioxide (or great diminution 
 of the oxygen in the respired air) increases the depth, but not the 
 rate of breathing. 
 
 In a normal respiration the chemical and nervous factors would, 
 therefore, appear to be related somewhat as follows : — The inspiratory 
 centre makes an effort, the degree of exaltation of the centre, and, 
 therefore, the magnitude of the effort, more especially in the 
 matter of depth, is governed by the tension of carbonic acid in 
 the blood, but it is cut short by an inhibitory impulse passing 
 up the vagus, only to begin again when the effects of this inhibitory 
 impulse are removed. 
 
 TISSUE KBSPIRATION 
 
 It must be borne in mind that pulmonary respiration is but the 
 means of supplying the tissues with oxygen and removing from the 
 body the waste products of tissue activity such as carbon dioxide. The 
 amount of respiratory exchange which takes place in the tissues is 
 connected with the degree of metabolism which occurs there. 
 
 Tissue respiration consists in the passage of oxygen from the blood 
 of the capillaries to the cells of the tissues, and the passage of carbonic 
 acid in the reverse direction. This gaseous interchange is no doubt 
 brought about by a simple process of diffusion. The oxygen passes out 
 
TISSUE RESPIRATION _ 175 
 
 of the plasma of the blood through the capillary wall, and then through 
 the lymph until it reaches the cell in which it is goiiig to be used. In 
 order that a constant stream of oxygen may pass from the blood to the 
 cell, there must be a difference of oxygen pressure between the oxygen 
 dissolved in the plasma and that dissolved in the lymph, and the latter 
 must be at a greater pressure than that dissolved in the cell. The 
 amount of oxygen which passes will, other things being equal, be directly 
 proportional to these pressure differences, and as the amount varies 
 greatly at different times, it is obvious that the pressure differences 
 vary greatly also. When, for instance, a muscle is at rest, the oxygen 
 pressure in the capillaries is very near to that in the muscle fibre ; 
 when the muscle is active and using large quantities of oxygen, the 
 intra-capillary oxygen pressure is much greater than the intra-muscular 
 oxygen pressure. Such a change might be brought about by a rise 
 in the intra-capillary oxygen pressure, or a fall in the intra-muscular 
 oxygen pressure, or by both taking place simultaneously. Let us 
 therefore inquire what is known about these quantities. 
 
 The tension of oxygen in muscle has recently been calculated as 
 being at most equal to 19 mm. of mercury ; from this it may vary 
 down to zero. Within these limits the conditions for diffusion can be 
 increased by a drop in the intra-muscular oxygen pressure. 
 
 There is, in addition, a mechanism for raising the intra-capillary 
 oxygen pressure. This is the increased quantity of acid (carbonic and 
 sarcolactic acids) which is thrown into the blood as the result of 
 the metabolism in the muscles and other tissues. 
 
 In glandular structures the oxygen pressure is higher than in 
 muscle ; probably owing to the relatively more copious blood-supply of 
 glands, equilibrium is more readily established between the blood and 
 the gland cells, the oxygen pressure in the cells being almost that 
 present in venous blood. 
 
 The quantity of oxygen used by different tissues varies not only 
 with the degree of their activity, but also with the nature of the tissues. 
 On the whole it may be said that, weight for weight, glandular tissue 
 uses most oxygen ; next in order come the muscular tissues, and last of 
 all, the connective tissues. There are some important tissues, notably 
 the nervous system, about' which little is known in this connection. 
 The amount of oxygen used by an organ or tissue per gramme per 
 minute is called its coefficient of oxidation. 
 
 In order to obtain the coefficient of oxidation, it is necessary : — (1) 
 to estimate the gases in the blood going to and emerging from the 
 organ ; (2) to determine the amount of blood passing through the 
 
176 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 organ in a given time, say one minute ; and (3) at the conclusion of 
 the experiment the organ is weighed so that its gaseous exchange can 
 be calculated. 
 
 In order to measure the gaseous exchange of J-n organ over a long 
 period the organ is supplied with blood which alternately traverses the 
 organ and aSrates itself in a closed chamber. The amount of oxygen 
 in the chamber is kept constant by the addition of that gas to the air 
 of the chamber at the same rate at which the circulation acquires it. 
 The amount of oxygen so added is measured. The method has 
 recently been applied with conspicuous success to the gaseous exchange 
 of the heart. 
 
 Relation of Tissue Respiration to Functional J^ctivity. — In all 
 organs, so far as is known, increased activity is accompanied by 
 increased oxidation. 
 
 Much interest centres about the question of the order of time in 
 which these events take place. This matter has been investigated in 
 the case of skeletal muscle and the submaxillary gland, both of 
 which organs can be thrown into profound activity for a short space 
 of time ; in each case most of the oxidation follows upon the activity, 
 and not the activity upon the oxidation. The important inference is 
 drawn that the contraction or secretion, as the case may be, is not 
 caused by the oxidation in the sense that the machinery of a locomotive 
 is driven by the energy derived from the oxidation of the coal ; rather 
 is the mechanism like that of a spring which is liberated at the moment 
 of doing the work, and has to be rewound subsequently ; the process 
 of rewinding involves oxidation. In the c&,se of muscle, the heat- 
 formation which occurs in the period following activity only takes 
 place if the muscle is supplied with oxygen. The output of carbonic 
 acid, in its turn, follows upon the intake of oxygen. The order of 
 events, is, therefore, (1) increase of functional activity, (2) increase 
 of heat formation and oxygen taken in, and (3) increase of carbonic 
 acid put out. 
 
 The table on the next page shows the coeflScients of oxidation for 
 resting organs, and the extent to which they are increased in activity. 
 Intertsity of Respiration. — Most of the figures relating to gaseous 
 metabolism given in that table were obtained from the examina- 
 tion of the tissues and organs of the dog. If all the tissues were 
 examined in turn and their relative weights known, an average 
 might be struck which would give the gaseous metabolism for the body 
 taken as a whole, and this might be expressed as the amount of oxygen 
 used per minute per gramme of body-weight. An easier and more 
 
TISSUE RESPIRATION 
 
 177 
 
 Organ. 
 
 Condition of 
 Rest. 
 
 Oxygen used 
 
 per minute 
 
 per gramme 
 
 of organ. 
 
 Condition of 
 Activity. 
 
 Oxygen used 
 
 per minute 
 
 per gramme 
 
 of organ. 
 
 Voluntary 
 muscle. 
 
 Nerves cut. 
 Tone absent. 
 
 003 0.0. 
 
 Tone existing in rest. 
 Gentle contraction. 
 Active contraction. 
 
 0-006 c.c. 
 020 C.C. 
 0080 C.C. 
 
 Unstriped 
 muscle. 
 
 Resting. 
 
 0-004 c.e. 
 
 Contracting. 
 
 0-007 CO. 
 
 Heart. 
 
 Very slow and 
 feeble con- 
 tractions. 
 
 0-007 c.c. 
 
 Normal contractions. 
 Very active. 
 
 0-05 C.C. 
 08 C.C. 
 
 Submaxillary 
 gland. 
 
 Nerves cut. 
 
 0-03 C.C. 
 
 Chorda stimulation. 
 
 0-10 C.C. 
 
 Pancreas. 
 
 Not secreting. 
 
 0-03 C.C. 
 
 Secretion after injec- 
 tion of secretion. 
 
 0-10 C.C. 
 
 Kidney. 
 
 Scanty secre- 
 tion. 
 
 0-03 CO. 
 
 After injection of 
 diuretic. 
 
 0-10 C.C. 
 
 Intestines. 
 
 Not absorbing. 
 
 0-02 C.C. 
 
 Absorbing peptone. 
 
 03 C.C. 
 
 Liver. 
 
 In fasting 
 animal. 
 
 0-01 to 
 0-02 0.0. 
 
 In fed ^nimal. 
 
 0-03 to 
 0-05 C.C. 
 
 Suprarenal 
 gland. 
 
 Normal. 
 
 0-045 C.C. 
 
 
 
 practicable method would be to weigh the animal, and then from the 
 composition of the inspired and expired air and the amount of oxygen 
 taken in, and given out, calculate how much is retained and utilised. 
 In the dog, the amount is about 0-016 c.c. of oxygen per minute per 
 gramme of body-weight. This figure, however, is not the same in all 
 animals, and the size of the figure will indicate what we may term the 
 intensity of respiration. Thus in cold-blooded animals, especially fishes 
 with their small supply of oxygen, the figure is very much smaller. 
 But in warm-blooded animals great variations are seen ; the intensity 
 of respiration, for instance, is much greater in birds than in mammals. 
 12 
 
178 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 Among the mammals, the intensity of respiration varies, roughly, 
 inversely with the size of the animal ; thus, in the mouse, an animal 
 that breathes with extreme rapidity, the intensity is probably ten to 
 fifteen times greater than -in the dog, and in the elephant very much 
 less. In man the average is about half that in the dog, that is, 0-008 c.c. 
 of oxygen per gramme of body-weight per minute. 
 
 Acidosis. — Carbonic acid is the principal acid of the blood, and 
 increase in it raises . the hydrogen-ion concentration of that fluid ; 
 this stimulates the respiratory centre to increased activity ; excess of 
 the acid, however, does not in health raise the H-ion concentration 
 beyond physiological limits, because certain substances (called "buffers ") 
 in the blood (sodium bi-carbonate, protein, etc.) combine with the 
 COj. Other acids may, however, accumulate ; one example is lactic 
 acid; this occurs after excessive muscular activity, in oxygen want 
 and in some forms of renal disease, hence the breathlessness of such 
 states. In diabetes, hydroxybutyric acid leads also to acidosis (p. 119). 
 The reader is referred to larger text-books on Physiology and 
 Pathology for a fuller discussion of the subject, the importance of 
 which is now fully recognised. Hence, in disease, an important 
 determination is the power the blood has of neutralising acids, and the 
 COj combining power of the blood is often termed its alkaline reserve. 
 This can be estimated in several ways of which Van Slyke's is the 
 most often used ; his apparatus directly estimates the COg of blood- 
 plasma (J. Biol. Chem., 1917, xxx., p. 347). It depends on the 
 vacuum extraction principle, and quite small amounts of blood-plasma 
 are needed : the analyses can be performed in jthree minutes ; hence 
 the method is suitable for clinical use. Another method he has 
 introduced is to calculate the COj of the plasma from the rate of acid 
 excretion in the urine. 
 
 I have not included any practical exercises on Respiration at the head of 
 this lesson, because I am aware how variable are the resources of physio- 
 logical laboratories for this purpose. I would only suggest that as far as 
 practicable the following experiments might be performed or shown by way 
 of demonstration. 
 
 1. The use of the mercurial pump. 
 
 2. Analysis of blood gases by the chemical method. 
 
 .3. Collection of alveolar air by the Haldane-Priestley method. 
 
 4. An analysis of a mixture of gases, say in expired air, by Haldane's 
 apparatus described in the Appendix. 
 
 5. The use of the aerotonometer. 
 
 6. The estimation of the alkaline reserve by Van Slyke's method. 
 
LESSON X 
 UEINE 
 
 1. Test the reaction of urine with litmus paper. 
 
 2. Determine its specific gravity by the urinometer. 
 
 3. Test for the foUowing INORGANIC SALTS :— 
 
 (a) Chlorides. — Acidulate with nitric acid and add silver nitrate ; 
 a white precipitate of silver chloride, soluble in ammonia, is produced. 
 The object of acidulating with nitric acid is to prevent phosphates 
 and urates being precipitated by the nitrate of silver. 
 
 (b) Sulphates. — Acidulate with hydrochloric acid and add barium 
 chloride. A white precipitate of barium sulphate is produced. Hydro- 
 chloric acid is added first, to prevent precipitation of phosphates. 
 
 (c) Phosphates. — i. Add ammonia ; a white crystalline precipitate 
 of earthy (that is, calcium and magnesium) phosphates is produced. 
 This becomes more apparent on standing. The alkaliTie (that is, 
 sodium and potassium) phosphates remain in solution. 
 
 ii. Mix another portion of urine with half its volume of nitric acid ; 
 add ammonium molybdate, and boil. A yellow crystalline precipitate 
 of ammonium phospho-molybdate falls. This test is given by both 
 kinds of phosphates. 
 
 4. UEEA. — Take some urea crystals. Observe that they are 
 readily soluble in water, and that effervescence occurs when fuming 
 nitric acid (i.e. nitric acid containing nitrous acid in solution) is added 
 to the solution. The effervescence is due to the breaking up of the 
 urea. Carbonic acid and nitrogen come off. A similar bubbling, due 
 to evolution of nitrogen, occurs when an alkaline solution of sodium 
 hypobromite is added to another portion of the solution. 
 
 5. Heat some urea crystals in a dry test-tube. Biuret is formed, 
 and ammonia comes off. 
 
 2CON2Hi = CjOgNgHg + NHg 
 [urea] [biuret] [ammonia] 
 
 After cooling add a drop of copper-sulphate solution and a few drops 
 of 20 percent, potash. A rose-red colour is produced. 
 
 6. Quantitative estimation of urea. 
 
 179 
 
180 
 
 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 For this purpose Dupr^'s apparatus (fig. 36) is the most convenient. 
 It consists of a bottle united to a measuring tube by indiarubber tubing. 
 The measuring tube (an inverted burette will do very well) is placed 
 within a cylinder of water, and can be raised and lowered at will. 
 Measure 25 c.c. of an alkaline solution of sodium hypobromite (made by 
 
 mixing 2 c.c. of bromine with 23 c.c. of 
 a 40 per-cent. solution of caustic soda) 
 into the bottle. Measure 5 c.c. of urine 
 into a small tube, and lower it care- 
 fully, so that no urine spills, into the 
 bottle. Close the bottle securely with 
 a stopper perforated by a glass tube : 
 this glass tube^ is connected to the 
 measuring tube by indiarubber tubing 
 and a T-piece. The third limb of the 
 T-piece is closed by a piece of india- 
 rubber tubing and a pinch-cock, seen 
 at the top of the figure. Open the 
 pinch-cock and lower the measuring 
 tube until it touches the bottom 
 of the tall cylinder. Note the level 
 of the water iii the burette. Close 
 the pinch-cock, and raise the measur- 
 ing tube tp ascertain whether the, 
 apparatus is air-tight. Then lower it 
 again. Tilt the bottle so as to upset 
 the urine, and shake well for a minute 
 or so. During this time there is an 
 evolution of gas. Then immerse the 
 bottle in a large beaker containing 
 water of the same temperature as that 
 in the cylinder. After two or three 
 minutes raise the measuring tube until 
 the surfaces of the water inside and 
 outside it are at the same level, the 
 gas being thus under atmospheric pressure. Bead the level of the 
 meniscus as before ; the difference in the two readings gives the 
 volume of gas evolved. This gas is nitrogen. The carbonic acid 
 resulting from the decomposition of urea has been absorbed by the 
 excess of soda in the bottle. 354 c.c. of nitrogen are yielded by 
 
 1 The efficiency of the apparatus is increased by having a glass bulb blown on 
 this tube to prevent froth passing into the rest of the apparatus. This is not 
 shown in the figure. 
 
 Fig. 36. — Dupr6*s urea apparatus. 
 
URINE 181 
 
 1 gramme of urea. From this the auantity of urea in the 5 c.c. of 
 urine and the percentage of urea can be calculated. If the total 
 urea passed in the twenty-four hours is to be ascertained, the 
 twenty-four hours' urine must be carefully measured and thoroughly 
 mixed. A sample is then taken from the total for analysis; and 
 then, by a simple sum in proportiion, the total amount of urea 
 is ascertained. More accurate methods for estimating urea are 
 described in Lesson XXII. 
 
 7. CREATININE.— (a) Weyl's Test— Add a little sodium nitro- 
 prusside and caustic soda to the urine. A red colour develops, which 
 changes in a short time to yellow. If glacial acetic acid is added to 
 the yellow solution, it becomes green on boiling, and a sediment of 
 Prussian blue forms on standing. (Acetone gives a similar colour 
 reaction, but the colour changes to purple on acidifying.) 
 
 (b) Jaffa's Test. — Add picric acid and a few drops of strong potash; 
 a deep red colour results. This method may be employed quantitatively 
 (see Lesson XXIII). If the urine contains sugar, the fluid becomes so 
 dark as to be opaque. 
 
182 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 The kidney is a compound tubular gland, the tubules of which 
 differ much in the character of the epithelium that lines them in 
 various parts of their course. The obviously secreting part of the 
 kidney is the glandular epithelium that lines the convoluted portion 
 of the tubules ; there is in addition to this what is usually termed 
 the filtering apparatus: tufts of capillary blood-vessels called the 
 Malpighian glomeruli are supplied \^ith afferent vessels from the renal 
 artery ; the efferent vessels that leave these have a smaller calibre, and 
 thus there is high pressure in the Malpighian capillaries. Certain con- 
 stituents of the blood, especially water and salts, pass through the 
 thin walls of these vessels into the surrounding Bowman's capsule, 
 which forms the commencement of each renal tubule. Bowman's 
 capsule is lined by a flattened epithelium, which is reflected over the 
 capillary tuft. Though the process which occurs here is generally 
 spoken of as a filtration, some physiologists contest the correctness of 
 this view. During the passage of this dilute urine 'through the rest 
 of the renal tubule it gains the constituents, urea, urates, etc., which 
 are poured into it by the secreting cells of the convoluted tubules. 
 
 GENERAL CHARACTERS OF URINE 
 
 Quantity. — A man of average weight and height passes from 1400 
 to 1600 c.c, or about 50 oz., daily. This contains about 60 grammes 
 (1| oz.) of solids. The urine should be collected in a tall graduated 
 glass vessel capable of holding 3000 c.c, which should have a smooth- 
 edged neck accurately covered by a ground-glass plate to exclude dust 
 and avoid evaporation. A few drops of chloroform should be added as 
 an antiseptic. From the total quantity thus collected in the twenty- 
 four hours, samples should be drawn off for examination. 
 
 Colour. — This is some shade of yellow which varies considerably in 
 health with the concentration of the urine. It appears to be due to a 
 mixture of pigments : of these urobilin is the one of which we have the 
 most accurate knowledge. Urobilin has a reddish tint and is ultimately 
 derived from the blood pigment, and, like bile pigment, is an iron-free 
 derivative of haemoglobin containing the pyrrole ring. The bile pig- 
 ment (and possibly also the hsematin of the food) is in the intestines 
 converted into stercobilin ; most of the stercobilin leaves the body 
 with the freces ; but some is reabsorbed and is excreted with the urine 
 as urobilin. Urobilin is very like the artificial reduction product of 
 bilirubin called hydrobilirubin (seep. 124). Normal urine, however, 
 contains very little urobilin. The actual body present is a chromogen 
 or mother substance called urobilinogen, which by oxidation (such as 
 
GENERAL CHARACTERS OF URINE 183 
 
 occurs when the urine stands exposed to light and air) is converted 
 into the pigment proper. In certain diseased conditions the amount 
 of urobilin is considerably increased. 
 
 The most abundant urinary pigment is a yellow one called uro- 
 chrome. Some regard it as a derivative of urobilin, but it probably is 
 not related to that substance (see Lesson XXV). 
 
 Keaction. — The reaction of normal urine is acid to litmus. This 
 acidity is mainly due to acid salts, especially acid sodium phosphate. 
 In certain circumstances the urine becomes less acid and even alkaline ; 
 the most important of these are as follows : — 
 
 1. During digestion. Here there is a formation of free acid in the 
 stomach, and a corresponding liberation of bases in the blood which 
 passing into the urine diminish its acidity, or even render it alkaline. 
 This is called the alkaline tide ; the opposite condition, the acid tide, 
 occurs after a fast — for instance, before breakfast. Leathes states that 
 respiration is a more important factor than gastric secretion in producing 
 the change of reaction ; during sleep respiration is comparatively in- 
 active, hence COj accumulates, and the increase in H-ion concentration 
 is reflected in the urine. With the activity associated with daytime, 
 this effect passes off. 
 
 2. In herbivorou animals and vegetarians. The food here contains 
 excess of alkaline salts of acids such as tartaric, citric, malic, etc. These 
 acids are oxidised into carbonates, which passing into the urine give it 
 an alkaline reaction (see further on Reaction, Appendix p. 298). 
 
 Specific Gravity. — This should be taken in a sample of the twenty- 
 four hours' urine with a good urinometer. The specific gravity varies 
 inversely as the quantity of urine passed under normal conditions from 
 1015 to 1025. A specific gravity below 1010 should excite suspicion of 
 hydruria ; one over 1030 of a febrile condition, or of diabetes, a disease 
 in which it may rise to 1050. The specific gravity has, however, been 
 known to sink as low as 1002 (after large potations, urina potus), or to 
 rise as high as 1035 (after great sweating) in perfectly healthy persons. 
 Composition. — The following table gives the average amounts of the 
 urinary constituents passed by a man (taking an ordinary diet contain- 
 ing about 100 grammes of protein) in the twenty-four hours : — 
 Total quantity of urine . . 1500-00 grammes 
 
 Water . . .' 1440-00 
 
 Total solids 60-00 
 
 Urea . . 35-00 
 
 Uric acid 0-75 
 
 Hippuric acid . 1-05 
 
184 
 
 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 Creatinine 
 Sodium chloride 
 Sulphuric acid . 
 Phosphoric acid 
 Chlorine . 
 Ammonia 
 Potassium 
 Sodium . 
 Calcium . 
 Magnesium 
 
 0'91 grammes 
 16-5 
 2-01 
 3-16 
 11-00 
 0-65 
 2-50 
 5-50 
 0-26 
 0-21 
 
 The most abundant constituents of the urine are water, urea, and 
 sodium chloride. In the foregoing table the student must not be 
 misled by seeing the names of the acids and metals separated. The 
 acids and the bases are combined to form salts : urates, chlorides, etc. 
 
 UREA 
 
 The time-honoured structural formula of urea as carbamide 
 CO<^-j^tt2 must be replaced, according to the recent work of Werner, 
 
 by the cyclic formula H-N:C' 
 
 /' 
 
 I , i.e. iso- carbamide. 
 O 
 
 This formula 
 
 offers a satisfactory explanation for the behaviour of urea during 
 hydrolysis by acids, etc. It has the same empirical, but not the . same 
 structural formula, as ammonium cyanate, (NH^)C]S'0, from which 
 it was first prepared synthetically by Wohler in 1828. 
 
 When separated from the urine, it is found to be crystalline and 
 readily soluble in water, alcohol, and acetone : it has a saltish taste, 
 and is neutral to litmus paper. Pure urea melts at 132° C. 
 
 When treated with nitric acid, nitrate of urea (CON2H4.HNO3) 
 is formed ; this crystallises in lozenge-shaped tablets, or hexagons 
 (fig. 37, a). When treated with oxalic acid, prismatic crystals of urea 
 oxalate, (CONgHJj.HjC^O^, are formed (fig. 37, b). 
 
 These crystals may be readily obtained in an impure form by 
 adding excess of the respective acids to urine which has been concen- 
 trated to a third or a quarter of its balk. 
 
 Under the influence of an enzyme secreted by certain micro- • 
 organisms, such as the Micrococcus urece, which grows readily in stale 
 urine, urea takes up water, and is converted into ammonium carbonate 
 [COlSTgH^ -(- 2H2O = (NH4)2COg]. Hence the ammoniacal odour of 
 putrid urine. 
 
 Many leguminous seeds, especially the soy-bean, contain an enzyme 
 
UREA 
 
 185 
 
 (urease) which converts urea into ammonia and carbon dioxide ; this 
 may be used for estimating urea (see Lesson XXII). 
 
 By means of nitrous acid, urea is broken up into carbonic acid, water, 
 and nitrogen. This may be used as a teat for urea. Add fuming nitric 
 acid (i.e. nitric acid containing nitrous acid in solution) to a solution of 
 urea, or to urine ; an abundant evolution of gas bubbles takes place. 
 
 Hypobromite of soda decomposes urea in the following way : — 
 
 CON",H^+3NaBrO = 
 
 [urea] 
 
 : CO, + N, + 2H,0 + SNaBr 
 
 [sodium 
 bromide] 
 
 [sodium [carbonic [nitro- [water] 
 hypobromite] acid] g^en] 
 
 This reaction is important, for on it one of the readiest methods 
 for estimating urea depends. If the experiment is performed as 
 directed on p. 180, nitrogen is the only gas that comes off, the carbonic 
 acid being absorbed by excess of soda. The amount of nitrogen is a 
 measure of the amount of urea. 
 
 Fig. 37. — a, nitrate ; b, oxalate of urea. 
 
 The quantity of urea excreted is somewhat variable, the chief cause 
 of variation being the amount of protein food ingested. In a man 
 taking the usual Voit diet containing about 100 grammes of protein 
 (which will contain about 16 grammes of nitrogen) the quantity of 
 urea excreted daily averages 33 grammes (500 grains). The percentage 
 in human urine would then be 2 per cent. ; but this also varies, because 
 the concentration of the arine varies considerably in health. The 
 excretion of urea is usually at a maximum three hours after a meal, 
 especially after a meal rich in protein. 
 
 Muscular exercise has but little effect on the amount of nitrogen 
 discharged. This is strikingly different from what occurs in the case 
 of carbonic acid ; the more the muscles work, the more carbonic acid 
 do they send into venous blood, which is rapidly discharged by the 
 
186 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 expired air. Muscular energy is derived normally from the combustion 
 of non-nitrogenous material ; this is very largely carbohydrate. If 
 the muscles, however, are not supplied with the proper amount of 
 carbohydrate and fat, or if the work done is very excessive, then they 
 consume some of their more precious protein material. 
 
 Where is Urea formed? — The older authors considered that it 
 was formed in the kidneys, just as they also erroneously thought that 
 carbonic acid was formed in the lungs. Prevost and Dumas were the 
 first to show that after complete extirpation of the kidney the forma- 
 tion of urea and other waste products goes on, and these accumulate 
 in the blood and tissues. Similarly, in those cases of disease in 
 which the kidneys cease work, urea is still formed and accumulates. 
 This condition is called urcemia (or urea in the blood), and unless the 
 waste substances are discharged from the body the patient dies. 
 
 UrtBmia. — This term was originally applied on the erroneous supposition 
 that it is urea or some antecedent of urea which acts as the poison. There is no 
 doubt that the poison is not any constituent of normal urine ; if tte kidneys 
 of an animal are extirpated the animal dies in a few days, but there are no 
 ursemic convulsions. In man also, if the kidneys are healthy or approxi- 
 mately so, and suppression of urine occurs from the simultaneous blocking 
 of both renal arteries by clot, or of both ureters by stones, again uraemia does 
 not follow. On the other hand, uraemia may occur even while a patient with 
 diseased kidneys is passing a considerable amount of urine. What the poison 
 is that is responsible for the convulsions and coma is unknown. It is doubt- 
 less some abnormal katabolic product, but whether this is produced by the 
 diseased kidney cells, or in some other part of the body, is also unknown. 
 
 Where, then, is the seat of urea formation ? The facts of experiment 
 and of pathology point very strongly in support of the theory that urea 
 is formed in the liver. The principal are the following : — 
 
 1. After removal of the liver in such animals as frogs, urea formation 
 almost ceases, and ammonia is found in the urine instead. 
 
 2. In mammals, the extirpation of the liver is such a serious 
 operation that the animals die. But the liver of mammals can be very 
 largely thrown out of gear by the operation known as Eck's fistula, 
 which consists in connecting the portal vein directly to the inferior 
 vena cava. In these circumstances the liver receives blood only by 
 the hepatic artery. The amount of urea is lessened, and its place is 
 taken by ammonia. 
 
 3. When degenerative changes occur in the liver, as in cirrhosis of 
 that organ, the urea formed is much lessened, and its place is taken 
 by ammonia. In acute yellow atrophy, urea is almost absent from the 
 urine, and, again, there is considerable increase in the ammonia. In 
 
UREA 187 
 
 this disease amino-acids such as leucine and tyrosine are also found in 
 the urine ; these arise from the disintegration of the proteins of the 
 liver cells, but they may in part originate in the intestine, and, escaping 
 further decomposition in the degenerated liver, pass as such into the 
 urine. 
 
 We have to consider next the intermediate stages between protein 
 and urea. In order that the student may grasp the meaning of urea 
 formation it would be advisable for him to turn again to p. 71 and read 
 the paragraph there relating to Chittenden's views of diet, and to pp. 1 28 
 and 131, which treat of protein absorption, for the question. What is a 
 normal diet 1 is intimately bound up with the question, What is a 
 normal urine 1 If, for instance, the diet of the future is to contain 
 only half as much protein as in the past, the urine of the future will 
 naturally show a nitrogenous output of half of that which has hitherto 
 been regarded as normal. In people on such a reduced diet, Folin has 
 shown that the decrease in urinary nitrogen falls mainly on the urea, 
 but certain other nitrogenous katabolites, particularly one called 
 creatinine, remain remarkably constant in absolute amount in spite of 
 the great reduction in the protein ingested. 
 
 The laws governing the composition of urine are obviously the 
 effect of the laws that govern protein katabolism. Many years ago 
 Voit supposed that the protein ingested was utilised partly in tissue 
 formation, and partly remained in the circulating fluids as " circulating 
 protein '' ; he further considered that the breakdown of the protein in 
 the tissues was accomplished with much greater difficulty than that in 
 blood and lymph, and that the small amount of " tissue protein '' which 
 disintegrates as the result of the wear and tear of the tissues was 
 dissolved and added to the " circulating protein," in which alone the 
 formation of final katabolic products such as urea was supposed to 
 occur.. As time went on, it was shown that many facts were incom- 
 patible with this theory, and so it was largely displaced by Pfliiger's 
 view, in which it was held that the food protein must first be assimilated, 
 and become part and parcel of living cells before katabolism occurs. 
 We now know that neither of these views is correct, but that nitro- 
 genous katabolism is of two kinds : one kind varies with the food ; 
 it is therefore variable in amount, and occurs almost immediately or 
 within a few hours after the food is absorbed ; the amino-acids absorT)ed 
 from the intestine are in great measure never built into living proto- 
 plasm at all, and are simply taken to the liver, where they are 
 deaminised, and their nitrogenous part converted into urea. This 
 variety of katabolism is called exogenous. The other kind of metabolism 
 
188 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 is constant in quantity and smaller in amount, and is due to the actual 
 breakdown of protein matter in the body cells and tissues, which had ' 
 been built into them previously. This form of metabolism is called 
 endogenous or tissue katabolism, and the final product is in part urea, 
 but the waste nitrogen finds its way out of the body in other substances 
 also, of which creatinine appears to be important. This form of 
 metabolism sets a limit to the lowest level of nitrogenous requirement 
 attainable ; the protein sufficient to maintain it is indispensable. 
 Whether the amount of protein which is exogenously metabolised can 
 be entirely dispensed with is at present questionable, and those who 
 seek to replace it entirely by non-nitrogenous food are living dangerously 
 near the margin. A point of considerable importance in this connection 
 is, that the nitrogen of the protein is split off from it by hydrolysis 
 without oxidation. There is thus very little loss of potential energy, 
 the energy of the products being nearly equal to that of the original 
 protein ; it is, however, the non-nitrogenous residue which is mainly 
 available for oxidation, and thus for calorific processes. The fact that 
 muscular work does not normally increase nitrogenous metabolism 
 becomes intelligible in the light of the consideration that protein 
 kktabolism, in so far as its nitrogen is concerned, is independent of the 
 oxidations which give rise to heat, or to the energy which is converted 
 into work. The body is very economical in so far as protein is 
 concerned, and tissue or endogenous katabolism is kept at a low level. 
 
 What is the proportion between exogenous and endogenous nitrogen 
 katabolism 1 It is very difficult to give any exact estimate. We do 
 know that in ordinary diets, the former is far in excess, and probably 
 in a man excreting 1 6 grammes of nitrogen daily (that is, the amount 
 corresponding to an intake of 100 grammes of protein), only a quarter 
 of this or even less represents tissue breakdown. 
 
 The view we have advanced concerning urea formation, then, is 
 that it is mainly the result of the conversion, by the liver, of amino- 
 acids absorbed from the intestine into that substance. This view 
 receives confirmation from experiments in which certain amino-acids, 
 such as glycine, leucine, and arginine, have been injected direct into 
 the blood-stream. Tlie result is an increased formation of urea. In 
 the case of arginine the exact cheiiiical decomposition which takes place 
 is known. We have already seen that arginine is a compound of a urea 
 radical and a substance called ornithine (diamino-valeric acid, see 
 p. 48) ; the liver is able to hydrolyse arginine, and so urea is liberated. 
 This power is due to the action of a special enzyme called arginase, 
 which, although it is also found in other organs, is specially abundant 
 
UREA 189 
 
 in the liver. In addition to this the ornithine itself is further broken 
 up, and so an extra quantity of urea is formed. On the other hand, 
 there are some amino-acids (e.g. tyrosine) which on injection do not lead 
 to any increase in urea formation. 
 
 If, however, we glance at the formula of ornithine, we see that it 
 has one point in common with other amino-acids, such as glycine and 
 leucine, to take simple examples : — 
 
 Ornithine CjHj^N^Oj 
 
 Glycine C^H^NOj 
 
 Leucine CgHjgNOj 
 That is, in -all cases the carbon atoms are more numerous than the 
 nitrogen atoms. In urea, CONjH^, the reverse is the case. The 
 amino-acids must therefore be split into simpler compounds, which 
 unite with one another to form urea. Urea formation is thus in part 
 synthetic. These simpler compounds are ammonium salts. Schroder's 
 work proved that ammonium carbonate is one of the urea precursors, 
 if not the principal one. The equation which represents the reaction 
 is as follows ; — 
 
 (NHJ2CO3 = CON^H, + 2H2O 
 
 [ammonmm [urea] [water] 
 
 carbonate] 
 
 Schroder's principal experiment was this : a mixture of defibrinated 
 blood and ammonium carbonate was injected into the liver by the 
 portal vein ; the blood leaving the liver by the hepatic vein was 
 found to contain urea in abundance. This does not occur when, the 
 same experiment is performed with any other organ of the body, so 
 that Schroder's experiments also prove the great importance of the 
 liver in urea formation. Similar results were obtained by Nencki with 
 ammonium carbamate. 
 
 We must further remember that ammonia itself is one of the pro- 
 ducts of digestion of protein in the intestine, and it may possibly, to a 
 small extent, be a result of tissue katabolisra. This ammonia passes 
 into the blood, where it unites with carbonic acid to form either the 
 carbamate or carbonate of ammonium. Thus ammonia, whether formed 
 directly or by the breakdown of amino-acids, is the principal immediate 
 precursor of urea. 
 
 The following structural formulas show the relationship between 
 ammonium carbonate, ammonium carbamate, and urea : — 
 
 [ammonium carbonate] [ammonium carbamate] [urea] 
 
 The loss of one molecule of water from ammonium carbonate pro- 
 
190 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 duces ammonium carbamate ; the loss of a second molecule of water 
 produces urea. But this view, though simple, will have to be modified 
 if Werner's conception of the structure of urea is accepted (see p. 184). 
 
 AMMONIA 
 
 The urine of man and carniyora contains small quantities of 
 ammonium salts. The reason that some ammonia always slips through 
 into the urine is that a part of the ammonia-containing blood passes 
 through the kidney before reaching organs, such as the liver, which are 
 capable of synthesising urea. In man the daily amount of ammonia 
 excreted varies between 0'3 and 1'2 gramme : the average i§ 0'7 gramme. 
 The ingestion of ammonium carbonate does not increase the amount of 
 ammonia in the urine, but increases the amount of urea, into which 
 substance the ammonium carbonate is easily converted. But if a more 
 stable salt, such as ammonium chloride, is given, it appears as such in 
 the urine. 
 
 In normal circumstances the amount of ammonia depends on the 
 adjustment between the production of acid substances in metabolism 
 and the supply of bases in the food. Ammonia formation is the 
 physiological remedy for deficiency of bases. 
 
 When the production of acids is excessive (as in diabetes, or when 
 mineral acids are given by the mouth or injected into the blood-stream, 
 the result is an increase of the physiological remedy, and excess of 
 the ammonia passes over into the urine. Under normal conditions 
 ammonia is kept at a minimum, being finally converted into the less 
 toxic substance urea, which the kidneys easily excrete. The defence 
 of the organism against acids which are very toxic is an increase of 
 ammonia formation, or, to put it more correctly, less of the ammonia 
 formed is converted into urea. 
 
 Under the opposite conditions — namely, excess of alkali, either in 
 food or given as such — the ammonia disappears from the urine, all 
 being converted into urea. Hence the diminution of ammonia in the 
 urine of man on a vegetable diet, and its absence in the urine of 
 herbivorous animals. 
 
 Not only is this the case, but if ammonium chloride is given to a 
 herbivorous animal like a rabbit, the urinary ammonia is but little 
 increased. It reacts with sodium carbonate in the tissues, forming 
 ammonium carbonate (which is excreted as urea) and sodium chloride. 
 Herbivora also suffer much more from, and are more easily killed by, 
 acids than camivora, their organisation not permitting a ready supply 
 of ammonia to neutralise excess of acids. ' 
 
CREATINE AND CREATININE 191 
 
 CEEATINE AND CREATININE 
 
 Creatine is an abundant constituent of muscle ; its chemical 
 structure is very like that of arginine ; it contains a urea radical, 
 and by boiling it with baryta it splits into urea and sarcoaine (methyl- 
 glycine), as shown in the following equation : — 
 
 C^HgNgOg + H^O = CONjH, + CgH^NOj 
 
 [creatine] [water] [urea] [sarcoaine] 
 
 The same decomposition is shown graphically on p. 48. 
 
 Creatine is absent from normal urine, but it is present in the urine 
 of infants ; also during starvation, in acute fevers, in women during 
 involution of the uterus, and in certain other conditions in which 
 there is rapid loss of muscular material. 
 
 Its ;iormal fate in the body is unknown ; it may be converted into 
 urea as in the foregoing equation, but injection of creatine into the 
 blood-stream does not cause any increase in urea formation ; the creatine 
 injected is almost wholly excreted unchanged. 
 
 It also is not converted into creatinine ; although it has been 
 generally assumed that this conversion does occur. The transformation 
 of creatine into creatinine is shown in the following equation : — 
 
 C.HpNgOj - H^O = C.H-NjO 
 
 [creatine] [water] [creatinine! 
 
 Recent researches have entirely failed to substantiate the view that 
 the urinary creatinine originates from the muscular creatine. If 
 creatine (an innocuous neutral substance) were converted by the loss 
 of water in the muscles into creatinine (a strongly basic substance), 
 it would be contrary to all that is known of the chemical changes that 
 occur in the body. 
 
 Creatinine is present in the urine. Amongst all the inconstances 
 of urinary composition, it appears to be the substance most constant 
 in amount, diet and exercise having no effect on it. Folin's view, that 
 its amount is a criterion of the extent of endogenous nitrogenous 
 metabolism, has steadily gained ground, and the work of the past 
 few years has shown that the liver and not the muscles is the seat 
 of its formation. Some observers have supposed that certain tissue 
 enzymes, termed creatase and creatinase, are agents in its formation 
 and destruction ; others have failed to discover the presence of these 
 enzymes in the liver. On this and on other points there are differ- 
 ences of opinion, but without discussing the pros and cons of minor 
 details, the following view of Mellanby may be taken as a working 
 
192 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 hypothesis of the metabolic history of the substances in question. 
 Mellanby took as his starting-point the investigation of the contra- 
 dictory data relating to the proportion of creatine and creatinine in 
 muscle, and by improved methods showed that creatinine is never 
 present in muscle at all, even after prolonged muscular work. He then 
 studied in the developing bird the amount of creatine at different stages, 
 and found that it is entirely absent in the chick's musculature up to 
 the twelfth day of incubation ; after this date the liver and the muscular 
 creatine develop pari passu. After hatching, the liver still continues 
 to grow rapidly, and the creatine percentage in the muscles increases 
 also, although the development in the size of the muscles occurs very 
 slowly. This and other experiments on the injecti(m of creatine and 
 creatinine into the blood-stream finally led Mellanby to the following 
 hypothesis : Certain products of protein katabolism, the nature of 
 which is uncertain, are carried by the blood to the liver, and from 
 these the liver forms creatinine ; this is transported to the muscles 
 and there stored as creatine; when the muscles are saturated with 
 creatine, excess of creatinine is then excreted by the kidneys. The small 
 amount of creatinine excreted in diseases of the liver also supports the 
 view that this organ is responsible for creatinine formation. 
 
 These views have been and will be subjected to the usual tests of 
 criticism and renewed research ; they certainly appear to explain some 
 of our previous difficulties, though the ultimate fate of the muscular 
 creatine is still unsolved. 
 
 THE INORGANIC CONSTITUENTS OF URINE 
 
 The inorganic or mineral constituents of urine are chiefly chlorides, 
 phosphates, sulphates, and carbonates; the metals with which these 
 are in combination are sodium, potassium, ammonium, calcium, and 
 magnesium. The total amount of these salts excreted varies from 19 
 to 25 grammes daily. The most abundant is sodium chloride, which 
 averages in amount 10 to 16 grammes per diem. These substances 
 are derived from two sources — first from the food, and secondly as the 
 result of metabolic processes. The chlorides and most of the phos- 
 phates come fi'om the food ; the sulphates and some of the phosphates 
 are the result of metabolism. The sulphates are derived from the 
 changes that occur in the proteins ; the nitrogen of proteins leaves the 
 body chiefly as urea ; the sulphur of the proteins is oxidised to form 
 sulphuric acid, which passes into the urine in the form of sulphates. 
 The excretion of sulphates, moreover, runs parallel to that of urea. 
 Sulphates, like urea, are the result of exogenous protein metabolism ; 
 
THE INORGANIC CONSTITUENTS OF URINE 193 
 
 endogenous metabolism, so far as sulphur is concerned, is represented 
 in the urine chiefly by less fully oxidised compounds of sulphur. The 
 chief tests for the various salts have been given in the practical exer- 
 cises at the head of this lesson. 
 
 Chlorides. — The chief chloride is that of sodium; The ingestion of 
 sodium chloride is followed by its appearance in the urine, some on the 
 same day, some on the next day. Some is decomposed to form the 
 hydrochloric acid of the gastric juice. The salt, in passing through 
 the body, fulfils the useful office of stimulating metabolism and 
 excretion. 
 
 Sulphates. — The sulphates in the urine are principally those of 
 potassium and sodium. They are derived from the metabolism of 
 proteins in the body. Only . the smallest trace enters the body with 
 the food. Sulphates have an unpleasant bitter taste (for instance, 
 Epsom salts) ; hence we do not take food that contains them. The 
 sulphates vary in amount from 1 -5 to 3 grammes daily. 
 
 In addition to these sulphates there is a small quantity of sulphuric 
 acid, comprising about one-tenth of the total, which is combined with 
 organic radicals ; the compounds are known as ethereal sulphates, and 
 they originate mainly from putrefactive processes occurring in the in- 
 testine. The most important of these ethereal sulphates are phenyl 
 sulphate of potassium and indoxyl sulphate of potassium. The latter 
 originates from the indole formed in the intestine, and as it yields 
 indigo when treated with certain reagents it is sometimes called indican. 
 It is very important to remember that the indican of urine is not the 
 same thing as the indican of plants. Both yield indigo, but there the 
 resemblance ceases. 
 
 The equation representing the formation of potassium phenyl- 
 sulphate is as follows : — 
 
 C,H,OH + SO,<;g| = SO,<ggH^ + H,0 
 
 [phenol] [potassium [potassium [water] 
 
 hydrogen sulphate] phenyl-sulphate] 
 
 The formation of potassium indoxyl-sulphate may be represented 
 
 CH 
 as follows : — Indole, CgH^<^^CH on absorption is converted 
 
 NH 
 
 COH 
 into indoxyl : CeH4<Q:CH. 
 
 NH 
 
 13 
 
194 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 Indoxyl then interacts with potassium hydrogen sulphate as 
 follows : — 
 
 CgH.NO + SO,<g| ^ SO^g^sHflN ^ ^^q 
 
 [indoxyl] [potassiuin - [potassium [water] 
 
 hj'drogen sulphate] indoxyl-sulphate] 
 
 The formation of such sulphates is important ; the aromatic sub- 
 stances liberated by putrefactive processes in the intestine are poisonous, 
 but their conversion into ethereal sulphates renders them innocuous. 
 (For teats for indoxyl in urine see Advanced Course, Lesson XXV.) 
 
 Carbonates. — Carbonates and bicarbonates of sodium, calcium, 
 magnesium, and ammonium are present in alkaline urine only. They 
 arise from the carbonates of the food, or from vegetable acids (malic, 
 tartaric, etc., in the food). They are, therefore, found in the urine of 
 herbivora and vegetarians, whose urine is thus rendered alkaline. Urine 
 containing carbonates becomes, like saliva, cloudy on standing, the 
 precipitate consisting of calcium carbonate, and also phosphates. 
 
 Phosphates. — Two classes of phosphates occur in normal urine : — 
 
 1. Alkaline phosphates — that is, phosphates of sodium (abundant) 
 and potassium (scanty). 
 
 2. Earthy phosphates, that is, phosphates of calcium (abundant) 
 and magnesium (scanty). 
 
 The composition of the phosphates in urine is liable to variation. 
 In acid urine the acidity is due to the acid salts. These are chiefly 
 sodium dihydrogen phosphate, NaH2PO^, and calcium dihydrogen 
 phosphate, Ca(H2P04)2. 
 
 In neutral urine, in addition, disodium hydrogen phosphate 
 (NajHPO^), calcium hydrogen phosphate, CaHPO^, and magnesium 
 hydrogen phosphate, MgHPO^, are found. In alkaline urine there may 
 be instead of, or in addition to the above, the normal phosphates of 
 sodium, calcium, and magnesium [Na^PO^, Cag(P0^)2, Mg3(PO^)2]. 
 
 The earthy phosphates are precipitated by rendering the urine 
 alkaline by ammonia. In urine undergoing putrefaction, ammonia is 
 formed from the urea : this also precipitates the earthy phosphates. 
 The phosphates most frequently found in the white creamy precipitate 
 which occurs in decomposing urine are — 
 
 (1) Triple phosphate or ammonium - magnesium phosphate 
 (NH^MgPO^ + eHgO). This crystallises in "coffin-lid" crystals (see 
 iig. 38) or feathery stars. 
 
 (2) Stellar phosphate. Of calcium phosphate, which crystallises in 
 star-like clusters of prisms. 
 
 As a rule, normal urine gives no precipitate when it is boiled ; but 
 
THE INORGANIC CONSTITUENTS OF URINE 195 
 
 sometimes neutral, alkaline, and occasionally faintly acid urine give 
 a precipitate of calcium phosphate when boiled ; this precipitate is 
 amorphous, and is liable to be mistaken for albumin. It may be 
 distinguished readily from albumin, as it is soluble in a few drops of 
 acetic acid, whereas coagulated protein does not dissolve. 
 
 Fig. 38. — Ammonium-magnesium 
 or triple phosphate. 
 
 The phosphoric acid in the urine chiefly originates from the 
 phosphates of the food, but is partly a decomposition or katabolic 
 product of the phosphorised organic materials in the body, such 
 as lecithin and nuclein. The amount of PgOj in the twenty-four 
 hours' urine varies from 2 -5 to 3 '5 grammes, of which the earthy 
 phosphates contain about half (1 to 1'5 gr.). 
 
LESSON XI 
 
 UBINE {contimied) 
 
 1. UREA NITRATE. — Evaporate some urine in a dish to a 
 quarter of its bulk. Pour the concentrated urine into a watch-glass ; 
 let it cool, and add a few drops of strong, but not fuming, nitric acid. 
 Crystals of urea nitrate separate out. Examine these microscopically. 
 
 2. UREA OXALATE. — Concentrate the urine as in the last 
 exercise, and add oxalic acid. Crystals of urea oxalate separate out. 
 Examine these microscopically. 
 
 3. URIC ACID.— Examine microscopically the crystals of uric acid 
 in some urine, to which 5 per cent, of hydrochloric acid has been added 
 twenty-four hours previously. Note that they are deeply tinged with 
 pigment, and to the naked eye look like granules of cayenne pepped 
 
 When microscopically examined, the crystals are seen to be large 
 bundles, principally in the shape of barrels, with spicules projecting 
 from the ends, and whetstones. If oxalic acid is used instead of 
 hydrochloric acid in this experiment, the crystals are smaller, and 
 more closely resemble those observed in pathological urine in cases 
 of uric acid gravel (see fig. 39). 
 
 Dissolve the crystals in caustic potash and then carefully add 
 excess of hydrochloric acid. Small crystals of uric acid again form. 
 
 Mureayide Test. — Place a little uric acid, or a urate (for instance, 
 serpent's urine), in a dish ; add a little dilute nitric acid and evaporate 
 to dryness on a water-bath. A yellowish-red residue is left Add a 
 little ammonia carefully. The residue turns to violet. This is due to the 
 formation of murexide or purpurate of ammonia. On the addition of 
 potash the colour becomes bluer. 
 
 Schifs Test. — Dissolve some uric acid in sodium carbonate solu- 
 tion. Put a drop of this on blotting paper, add a drop of silver 
 nitrate, and warm gently ; the black colour of reduced silver is seen 
 on the paper. 
 
 Folin's Test. — Suspend a minute quantity of uric acid in a few 
 c.c. of water and add two or three drops of a saturated solution of 
 sodium carbonate to dissolve it. To the clear solution add 1 or 2 c.c. 
 of Folin's phosphotungstic acid reagent (see Lesson XXIII.), and 
 
 196 
 
tJRiNE id*? 
 
 enough saturated sodium carbonate solution (or a few crystals of 
 sodium carbonate) to render the mixture alkaline. A blue colour 
 develops. This test may be employed for the estimation of uric acid 
 in urine (see Lesson ZXIII.). 
 
 Beduction of Fehling's Solution.— THasolve some uric acid by 
 boiling it with sodium carbonate solution. Add Fehling's solution 
 and boil again ; a white precipitate of copper urate is formed, and 
 on boiling for some time, reduction occurs with the formation of 
 cuprous oxide. Repeat the experiment with Nylander's solution, 
 or with Benedict's qualitative reagent (p. 20) ; no reduction occurs ; 
 this demonstrates the advantage of such solutions when testing 
 diabetic urine for small quantities of glucose. 
 
 4. DEPOSIT OF URATES OR LITHATES (LATERITIOUS 
 DEPOSIT). — The specimen of urine from the hospital contains excess 
 of urates, which have become deposited on the urine becoming cool. 
 They are tinged with pigment (uroerythrin), and have a pinkish 
 colour, like brickdust; hence the term " lateritious." Examine 
 microscopically. The deposit is usually amorphous— that is, non- 
 crystalline. Sometimes crystals of calcium oxalate (envelope crystals 
 — octahedra) are seen also; these are colourless. 
 
 The deposit of urates dissolves on heating the urine. 
 
 5. DEPOSIT OF PHOSPHATES.— Another specimen of patho- 
 logical urine contains excess of phosphates, which have formed a 
 white deposit on the uiine becoming alkaline. This precipitate does 
 not dissolve on heating ; it may be increased. It is, however, soluble 
 in acetic acid. Examine microscopically for cof^-lid crystals of triple 
 phosphate (ammonium-magnesium phosphate), or crystals of stellar 
 (calcium) phosphate, and for mucus. Mucus is flocculent to the 
 naked eye, amorphous to the microscope. 
 
 N.B.— On boiling neutral, alkaline, or even faintly acid urine it 
 may become turbid from deposition of phosphates. The solubility 
 of this deposit in a few drops of acetic acid distinguishes it from 
 albumin, for which it is liable to be mistaken. 
 
 Some of the facts described in the foregoing exercises have been 
 already dwelt upon in the preceding lesson. They are, however, 
 conveniently grouped together here, as all involve the use of the 
 microscope. 
 
19B ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 URIC ACID 
 
 Uric acid (CjN^H^Og) is in mammals the medium by which only a 
 small quantity of nitrogen is excreted from the body. It is, however, 
 in birds and reptiles the principal nitrogenous constituent of their 
 urine. It is not present in the free state, but is combined with bases 
 to fbrm urates. 
 
 It may be obtained from human urine by adding 5 c.c. of hydro- 
 chloric acid to 100 c.c. of the urine, and allowing the mixture to stand 
 for twelve to twenty-four hours. The crystals which form are deeply 
 tinged with urinary pigment, and though by repeated solution in 
 caustic soda or potash, and reprecipitation by hydrochloric acid, they 
 may be obtained free from pigment, pure uric acid is more readily 
 obtained from the solid urine of a serpent or bird, which consists 
 principally of the acid ammonium urate. This is dissolved in soda, 
 and then the addition of hydrochloric acid produces as before the 
 crystallisation of uric acid from the solution. 
 
 The pure acid crystallises in colourless rectangular plates or prisms. 
 In striking contrast to urea it is a most insoluble substance; at 37° C. 
 uric acid dissolves in pure water in the proportion of 1:15000 
 (Qudzent), and at 18° in the proportion 1 : 39500 (His and Paul). The 
 forms which uric acid assumes when precipitated from human urine, 
 either by the addition of hydrochloric acid or in certain pathological 
 processes, are very various, the most frequent being the whetstone 
 shape ; there are also bundles of crystals resembling sheaves, barrels, 
 and dumb-bells (see fig. 39). 
 
 The murexide test which has just been described among the practical 
 exercises is the principal test for uric acid. The test has received the 
 name on account of the resemblance of the colour to the purple of the 
 ancients, which was obtained from certain snails of the genus Murex. 
 
 Another reaction that uric acid undergoes (though it is not applicable 
 as a test) is that on treatment with certain oxidising reagents urea and 
 oxalic acid can be obtained from it. It is, however, doubtful whether a 
 similar oxidation occurs in the normal metabolic processes of the body. 
 
 Uric acid does not contain the carboxyl group (COOH) which is 
 typical of organic acids, and in aqueous solution its reaction is neutral. 
 Nevertheless one of its hydrogen atoms is replaceable by a metallic 
 radical, and it acts, therefore, in aqueous solutions as a monobasic acid, 
 and forms prima/ry salts (also called mono-, hi-, or acid urates). In the 
 presence of strong bases it forms secondary salts (also called neutral, 
 normal, or di- urates). The secondary salts, however, only exist in the 
 
URIC ACID 199 
 
 solid condition or in the presence of strong alkali. By water they are 
 decomposed nt once into primary salt and alkali ; by carbonic acid they 
 are decomposed into primary salt and alkali carbonate. A third series 
 of salts (quadri-urates or hemi-urates) were formerly assumed to exist, 
 but it has been shown that these substances are merely mixtures qf 
 uric acid and primary urate. 
 
 In water, in urine, and in the blood we have only to deal with primary 
 urates. It has been recently shown that the primary or mono-sodium 
 urate (CjHgNaN^Og) occurs in two modifications, the a and the p forms. 
 The unstable a-salt is gradually transformed in aqueous solution into 
 the stable j8-salt owing to an intra-molecular change. It is assumed 
 that the two salts correspond to the two 
 tautomeric modifications of uric acid (see 
 p. 201), the lactam form giving rise to the 
 unstable and the lactim form to the stable 
 salt. The solubility of the unstable a form 
 at 37° C. is about 34 per cent, greater than 
 that of the j8 form. These facts have a 
 bearing on the pathological conditions occur- 
 ring in gout ; normally the small amount of 
 urate in the blood is held in solution ; in 
 gout the amount is increased, and the excess 
 is probably in the unstable a form : the con- 
 version of this into the stable /8 form gives 
 rise to the deposition of urates in the tissues p,g 39.— uric add crystals. 
 which occurs during the course of the attack. 
 
 The quantity of uric acid excreted by an adult varies from 7 to 
 lOl grains (0'5 to 0-75 gramme daily). The method used for estimating 
 uric acid is based on the discovery made by Hopkins, that when the 
 urine is saturated with ammonium chloride, all the uric acid is pre- 
 cipitated in the form of ammonium urate ; the precipitate is collected 
 and the uric acid in it is then determined. Details of the Folin-Shaifer 
 method founded on this reaction and of Folin's new colorimetric method 
 with phosphotungstic acid are given in Lesson XXIII. 
 
 Origin of Uric Acid. — Uric acid is not formed in the kidney. When 
 the kidneys are removed uric acid continues to be formed and accumu- 
 lates in the organs, especially in the liver and spleen. The liver has 
 been removed from birds, and uric acid is then hardly formed at all, 
 its place being taken by ammonia and lactic acid. It is therefore 
 probable that in these animals ammonia and lactic acid are normally 
 synthesised in the liver to form uric acid. 
 
200 
 
 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 This synthetic origin of uric acid, which is so important in birds 
 and snakes, does not, however, occur in mammals. In mammals 
 uric acid is the chief end-product of the katabolism of cell nuclei or 
 of nucleic acid, the principal constituent of the nuclei (see p. 64). 
 This therefore leads us next to study : — 
 
 Purine Substances. — Emil Fischer has shown that among the 
 decomposition products of nuclein are derivatives of a substance he 
 has named purine. The empirical formulse for purine, the purine bases, 
 and uric acid are as follows : — 
 
 Purine 
 
 C5H4H, 
 
 
 
 Hypoxanthine 
 
 C^H.N.O 
 
 Monoxypurine 
 
 Xanthine . 
 
 C,H,N,0, 
 
 Dioxypurine 
 
 Purine 
 
 Adenine . 
 
 C.HgN^.NH, 
 
 Amino-purine 
 
 bases. 
 
 Guanine . 
 
 C.HgN.O.NH, 
 
 Amino-oxypurine . 
 
 
 Uric acid . 
 
 CaH.NA 
 
 Trioxypurine 
 
 
 There are a vast number of purine derivatives, but many of them 
 have at present no physiological importance. Others in addition to 
 those already enumerated are theophylline (dimethyl -xanthine), 
 theobromine (also a dimethyl-xanthine), caffeine (trimethyl-xanthine) ; 
 these are of interest, as they occur in tea, cocoa, and coffee. A few 
 words more may be added in respect to those in our list. 
 
 Purine itself has never been discovered in the body. It has the 
 following structural formula : — 
 
 N=C-H 
 
 I I 
 H-C C-NH 
 
 II II 
 N-C-N 
 
 C-H 
 
 The purine nucleus is depicted in the next formula, and its atoms 
 have been empirically numbered as shown, for convenience of 
 description : — 
 
 I I 
 
 2C 5C-7N 
 
 I I 
 
 >«c 
 
 Hypoxanthine is found in the body tissues and fluids, and in the 
 urine. It may be termed 6-oxypurine, as the oxygen is attached to 
 the atom numbered 6 in the purine nucleus. 
 
 Xanthine is found with hypoxanthine in the body. It is 2, 6-dioxy- 
 
URIC ACID 201 
 
 •purine, its Oxygen atoms "being attached to the atoms numbered 2 and 
 6 in the purine nucleus. 
 
 HN - C=0 HN - C=0 
 
 II II 
 
 H-C C-NH, 0=C C-NH. 
 
 II II >C-H I II >C-H 
 
 N - 0- N^ HN - C- N^ 
 
 [hypoxanthine] [xanthine] 
 
 Adenine is found in the tissues, blood, and urine. It is 6-amino- 
 purine. 
 
 Guanine is also a decomposition product of nucleins. Combined 
 with calcium it gives the brilliancy to the seales of fishes, and is also 
 found in the bright tapetum of the eyes in these animals. It is a con- 
 stituent of guano, and here is probably derived from the fish eaten by 
 marine birds. It is 2-amino-6-oxypurine. 
 
 N = C - NH, HN - = 
 
 II II 
 
 H-C C - NH, H„N-C C - NH, 
 
 II II >C-H II II >C-H 
 
 IN - C - N-^ N - C - N^ 
 
 [adenine] [guanine] 
 
 Uric acid (2, 6, 8-trioxypurine) offers an example of tautomerism 
 (see p. 31). E. Fischer showed that it exists in two modifications 
 according to the following formulae : — 
 
 H.N - C : O N - C.OH 
 
 II II II 
 
 O : C C - NH. HO.C C - NH 
 
 I II >C0 I I >C.OH 
 
 H.N - C - NH^ N = C - N ^ 
 
 [lactam modiiication forming [lactim modification forming 
 
 unstable a-uratesj stable ^-urates] 
 
 The close chemical relationship of uric acid to the purine bases is 
 obvious from a study of the formulae just given. Just as in the "case 
 of urea, uric acid, however, may be exogenously or endogenously 
 formed. Certain kinds of food increase uric acid because they contain 
 nuclein (for instance, sweetbreads) in abundance, or purine bases (for 
 instance, hypoxanthine in meat) ; the uric acid which originates in this 
 way is termed exogenous. Certain diets, on the other hand, increase uric 
 acid formation by leading to an increase of leucocytes, and consequently 
 to an increase in the metabolism of their nublei; in other cases the 
 leucocytes may increase from other causes, as in the disease named 
 leucocythaemia. The uric acid that arises from nuclear katabolism 
 is termed endogeTWus. Although special attention has been directed to 
 
202 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 the nuclei of leucocytes because they can be 'readily examined during 
 life, it must be remembered that nuclein metabolism of all cells may 
 contribute to uric acid formation. 
 
 A study of uric acid formation forms a useful occasion on which 
 to allude to enzyme actions in metabolism generally. Enzymes of a 
 digestive kind are not confined to the interior of the alimentary canal ; 
 but most of the body cells are provided with enzymes to assist them 
 either in utilising the nutrient materials brought to them by the blood- 
 stream, or in breaking them down previously to expelling them as 
 waste substances. The enzyme which enables the liver cells to turn 
 glycogen into sugar is the one which has been known longest. The 
 enzyme called arginase (see p. 188), which leads to the hydrolysis of 
 arginine into urea and ornithine, is one of the more recently discovered. 
 Other examples which may be mentioned are proteolytic and peptolytic 
 enzymes (tissue erepsin, etc.) found in many organs. 
 
 The formation of uric acid from nuclein is perhaps the best instance 
 of all, for here we have to deal with numerous enzymes acting one after 
 another. These are present to an almost negligible extent in the juices 
 of the alimentary canal, and have been studied in the extracts of 
 different organs ; their distribution varies a good deal in diiFerent 
 animals, and in the different organs of the same animal; speaking 
 generally, they are most abundant in liver and spleen. The general 
 term nuclease is given to the whole group, and a dozen or more have 
 been described which deal with different steps in the cleavage of the 
 nucleic acid complex. They are classified into nucleinases which resolve 
 the molecule into mononucleotides, i.e. compounds of carbohydrate, 
 phosphoric acid, and one base ; nucleotidases which liberate phosphoric 
 acid, leaving the carbohydrate still united to the base : nucleosidases 
 which hydroly tically cleave the base and carbohydrate apart ; deaminases 
 which remove the amino-group from the purine bases so set free ; one 
 of these, called adenase, converts adenine into hypoxanthine, and another, 
 called guanase, converts guanine into xanthine. Finally, oxidases step 
 in, which convert hypoxanthine into xanthine, and xanthine into uric 
 acid. But even that does not bring the list to a conclusion, for in some 
 organs (especially the liver) there is a capacity to destroy uric acid after 
 it is formed, and so animals are protected from a too great accumulation 
 of this substance. What exactly happens to the uric acid is not certain, 
 although it is clear that the products of its breakdown (probably 
 allantoin and urea) are not so harmful as uric acid itself. The enzyme 
 responsible for uric acid destruction is called the uricolytic enzyme. 
 The uric acid which ultimately escapes as urates (normally) in the urine 
 
URINARY DEPOSITS 203 
 
 IS the undestroyed residue. The.uricolytic enzyme, however, is not 
 present to any marked extent in the human subject. 
 
 HIPPURIC ACID 
 Hippuric acid (CjjHgNOg), combined with bases to form hippurates, 
 is present in small quantities in human urine, but in large quantities in 
 that of herbivora. This is due to the food of herbivora containing 
 substances belonging to the aromatic group — the benzoic acid series. 
 If benzoic acid is given to a man, it unites with glycine with the 
 elimination of a molecule of water, and is excreted as hippuric acid : — 
 CH^NHg CHjNH.CO.CeHj 
 CBH5COOH+ I = I +H2O 
 
 COOH COOH 
 
 [benzoic acid] [glycine] * [hippuric acid] [water] 
 
 This is a well-marked instance of synthesis carried out in the animal 
 body, and experimental investigation shows that it is accomplished by 
 the living cells of the kidney itself ; for if a mixture of glycine, benzoic 
 acid, and deflbrinated blood is injected through the kidney (or mixed 
 with a minced kidney just removed from -the body of an animal), their 
 place is found to have been taken by hippuric acid. 
 
 It may be crystallised from horse's urine by evaporating to a syrup and 
 saturating with hydrocliloric acid. The crystals are dissolved in boiling 
 water, filtered, and on cooling the acid again crystallises out. It melts at 
 186° C, and on further heating gives rise to the odour of bitter almond oil. 
 
 URINARY DEPOSITS 
 
 The dififerent substances that may occur in urinary deposits are 
 formed elements and chemical substances. 
 
 The formed or histological elements may consist of blood corpuscles, 
 pus, mucus, epithelial cells, spermatozoa, casts of the urinary tubules, 
 fungi, and entozoa. All of these, with the exception of a small 
 quantity of mucus, which forms a flocculent cloud in the urine (and 
 spermatozoa), are pathological, and the microscope is chiefly employed 
 in their detection. 
 
 The chemical substances are uric acid, urates, calcium oxalate, 
 calcium carbonate, and phosphates. Rarer forms are leucine, tyrosine, 
 xanthine, and cystine. We shall, however, here only consider the 
 commoner deposits, and for their identification the microscope and 
 chemical tests must both be employed. 
 
 Deposit of Uric Acid. — This is a sandy reddish deposit resembling 
 cayenne pepper. It may be recognised by its crystalline form 
 (fig. 39, p. 199) and by the murexide reaction. The presence of 
 
204 ESSEiSTTIALS OF CHEMICAL i>HVSIOL0GV 
 
 these crystals generally indicates an increased formation of uric acid, 
 and if excessive, may lead to the formation of stones or calculi in 
 the urinary tract. 
 
 Deposit of Urates. — This is much commoner, 
 and may, if the urine is concentrated, occur in 
 normal urine when it cools. It is generally found 
 in the concentrated urine of fevers; and there 
 appears to be a kind of fermentation, called the 
 acid fermentation, which occurs in the urine after 
 it has been passed, and which leads to the same 
 result The chief constituent of the deposit is the 
 Fig. 40.— Mono-sodmm primary or mono-sodium urate. 
 
 urate. ^ •' 
 
 This deposit may be recognised as follows : — 
 
 1. It has a pinkish colour ; the pigment called uro-eri/tkrin is one 
 of the pigments of the urine, but its relationship to the other urinary 
 pigments is not known (see further. Lesson XXV). 
 
 2. It dissolves upon warming the urine. 
 
 Microscopically it is usually amorphous, but crystalline forms 
 similar to those depicted in figs. 40 and 41 may occur. 
 
 ^^ 
 
 Fio. 41.— Mono- Pig. 42.— Envelope crystals Fig. 43.— Cystine crystals, 
 
 ammonium urate. of calcium oxalate. 
 
 Crystals of calcium oxalate may be mixed with this deposit (see 
 fig. 42). 
 
 Deposit of Calcium Oxalate. — This occurs in envelope crystals 
 (octahedra) or dumb-bells. 
 
 It is insoluble in ammonia and in acetic acid. It is soluble with 
 diflBculty in hydrochloric acid. 
 
 Deposit of Cystine. — Cystine (CgHi2N2S20^) is recognised by its 
 colourless six-sided crystals (fig. 43). These are rare : they occur only 
 in acid urine, and they may form concretions or calculi. The constitu- 
 tion of cystine is given on p. 49 ; by reduction a substance called 
 cysteine (amino-thio-propionic acid) is obtained from it. Cysteine yields 
 on oxidation cysteic acid, and this splits into taurine and carbon dioxide. 
 
URINARY DEPOSITS 
 
 There is a good deal of evidence that the taurine of the bile is the source 
 of the cystine of the urine ; this anomalous course of metabolism runs 
 in families. The following formulae indicate the relationship of cysteine, 
 cysteic acid, and taurine : — 
 
 CH„.SH 
 
 CH,.SO,.OH 
 
 CH„.S0,.OH 
 
 CH.NH„ 
 
 CH.NH„ 
 
 CH5.NH2 
 
 COOH COOH 
 
 [cysteine] loysteio aoidj (taurine] 
 
 Deposit of Phosphates. — These occur in alkaline urine. The urine 
 may be alkaline when passed, due to fermentative changes occurring in 
 
 Fig. 44. — Triple phosphate crystals. 
 
 Fig. 45.— Crystals of phosphate of lime 
 (stellar phosphate). 
 
 the bladder. All urine, however, if exposed to the air (unless the air is 
 perfectly pure, as on the top of a snow mountain), will in time become 
 alkaline owing to an enzyme formed during the growth of the Micrococcus 
 urem. This forms ammonium carbonate from the urea. 
 
 CON2H4 + 2H2O = (NH^)2C03 
 
 [urea] [water] [ammonium 
 
 carbonate] 
 
 The ammonia renders the urine alkaline, and precipitates the 
 earthy phosphates. The chief forms of phosphates that occur in 
 urinary deposits are : — 
 
 1. Calcium phosphate, Ca3(P04)2 : amorphous. 
 
 2. Triple or ammonium-magnesium phosphate, MgNH^PO^ + BHgO ; 
 coffin-lids and feathery stars (fig. 44). 
 
 3. Crystalline phosphate of calcium, CaHPO^, in rosettes of prisms, 
 in spherules, or in dumb bells (fig. 45)- 
 
206 
 
 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 4. Magnesium phosphate, Mg3(P04)2 + 2H2O, occurs occasionally and 
 crystallises in long plates. 
 
 All these phosphates are dissolved by acids, such as acetic acid, 
 without effervescence. 
 
 They do not dissolve on heating the urine ; in fact, the amount of 
 precipitate may be increased by heating. 
 
 A solution of ammonium carbonate (1 in 5) eats magnesium phos- 
 phate away from the edges ; it has no effect on the triple phosphate. A 
 phosphate of calcium (CaHPO^ + SHjO) may occasionally be deposited 
 in acid urine. Pus in urine is apt to be mistaken for phosphates, but 
 can be distinguished by the microscope. 
 
 Deposit of calcium carbonate, CaCOj, appears but rarely as whitish 
 balls or biscuit-shaped bodies. It is commoner in the urine of 
 herbivora -(see p. 194). It dissolves in acetic or hydrochloric acid 
 with effervescence. 
 
 The following is a summary of the chemical sediments that may 
 occur in urine : — 
 
 CHEMICAL SEDIMENTS IN URINE 
 
 In Acid Urine 
 
 Uric Acid. — Whetstone, dumb- 
 bell, or sheaf-like aggregations of 
 crystals deeply tinged by pigment 
 (fig. 39). 
 
 Urates. — Generally amorphous. 
 The primary urate of sodium (tig. 40) 
 and of ammonium (fig. 41) may some- 
 times occur in star-shaped clusters of 
 needles or spheroidal clumps with 
 projecting spines. Tinged brick-red. 
 Soluble on warming. 
 
 Calcium Oxalate. — Octahedra, 
 so-called envelope crystals (fig. 42). 
 Insoluble in acetic acid. 
 
 Cystine. — Hexagonal plates (fig. 
 43). Rare. 
 
 Leucine and Tyrosine. — Rare. 
 
 Calcivm Phosphate. 
 
 CaHPOj -I- 2H2O.— Rare. 
 
 In Alkaline Urine 
 
 Phosphates. — Calcium phosphate, 
 Ca3(POj)2. Amorphous. 
 
 Triple phosphate, 
 MgNH^PO^H-eHgO. Coffin-lids or 
 feathery stars (figs. .38 and 44). 
 
 Calcium hydrogen phosphate, 
 CaHPOj. Rosettes, spherules, or 
 dumb-bells (fig. 45). 
 
 Magnesium phosphate, 
 Mg3(P04)2+2H20. Long plates. 
 
 All soluble in acetic acid with- 
 out effervescence. 
 
 Calcium Carbonate, CaCO^. — 
 Biscuit-shaped crystals. Soluble in 
 acetic acid with effervescence. 
 
 Ammonium Urate. — "Thorn- 
 apple '' spherules. 
 
 Leucine and Tyrosine. — Very rare, 
 
LESSON XII 
 PATHOLOGICAL URJNE 
 
 1. URINE A is pathological urine containing albumin. It gives 
 the usual protein tests. The following two are most frequently 
 used in clinical work. 
 
 If the urine is cloudy, it should be filtered before applying these 
 tests. 
 
 (a) Boil the top of a long column of urine in a test-tube. If the 
 urine is acid, the albumin is coagulated. If the quantity of albumin 
 is small, the cloudiness produced is readily seen, as the unboiled urine 
 below it is clear. This is insoluble in a few drops of acetic acid, and 
 so may be distinguished from phosphates. If the urine is alkaline, it 
 should be first rendered acid with a little dilute acetic acid. 
 
 (b) Heller's Nitric-Acid Test. — Pour some of the urine gently 
 on to the surface of some nitric acid in a test-tube. A ring of 
 
 Pig. 46. — Albuminometer of Ksbach. 
 
 white precipitate occurs at the junction of the two liquids. This 
 test is used for small quantities of albumin.^ 
 
 2. ESTIMATION OF ALBUMIN BY ESBACH'S ALBUMINO- 
 METER. — Esbach's reagent for precipitating the albumin is made by 
 dissolving 10 grammes of picric acid and 20 grammes of citric acid 
 in 800 or 900 c.c. of boiling water, and then adding sufS.cient 
 water to make up to a litre (1000 c.c). 
 
 Pour the uxine into the tube (fig. 46) up to the mark U ; then the 
 reagent up to the mark R. Close the tube with a cork, and to ensure 
 complete mixture tilt it to and fro a dozen times without shaking. 
 Allow the corked tube to stand upright twenty-four hours ; then read 
 off on the scale the height of the precipitate. The figures indicate 
 
 ' In very concentrated normal urine, a white ring of urea nitrate may form 
 under these conditions ; this is obviously crystalline. Uric acid may also 
 separate out if large excess of urates is present. This can be obviated by 
 previous dilution of the urine with water. 
 
 207 
 
208 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 grammes of dried albumin in a litre of urine. The percentage is 
 obtained by dividing by 10. Thus, if the sediment stands at 3, the 
 amount of albumin is 3 grammes per litre, or 03 gr. in 100 c.c. 
 When the albumin is so abundant that the sediment is above 4, a 
 more accurate result is obtained by first diluting the urine with 
 one or two volumes of water, and then multiplying the resulting 
 figure by 2 or 3, as the case may be. If the amount of albumin is 
 less than 0'05 per cent., it cannot b^ accurately estimated by this 
 method. ' 
 
 3. UBINE B is a diabetic urine : It has a high specific gravity. 
 The presence of sugar is shown by the reduction (yellow precipitate 
 of cuprous oxide) that occurs on boiling with Fehling's and similar 
 solutions (see p. 20, also p. 197). 
 
 4. ESTIMATION OF GLUCOSE.— The following four methods 
 are available for the quantitative determination of glucose : (a) The 
 polarimetric method (see p. 223, also Appendix) ; (b) the fermenta- 
 tion method; (c) the gravimetric, and (d) volumetric methods by 
 means of Fehling's or similar solutions. 
 
 The fermentation method is less accurate than the other methods. 
 It is carried out in a fermentation saccharimeter, such as Einhom's. 
 This consists of a U-shaped tube, the longer limb of which is closed 
 and carries an empirical graduation, indicating the percentage of 
 glucose, corresponding to the amount of carbonic acid gas developed. 
 Ten c.c. of the urine, mixed with some yeast, are taken, and the 
 apparatus is filled Avith this miiture, care being taken to remove all 
 air-bubbles. After twenty -four hours' fermentation at room tempera- 
 ture, the percentage of glucose is read off. 
 
 The gravimetric method is the most accurate of all, and of the 
 many modifications of it the Kjeldahl-AUihn method is now con- 
 sidered the best. In this the reduction of Fehling's solution, taken 
 in excess, is carried out in an atmosphere of hydrogen (or coal gas), 
 the precipitate of cuprous oxide is filtered through a Soxhlet's 
 asbestos tube and finally reduced to metallic copper by heating the 
 tube in a current of hydrogen. From the amount of copper found 
 by weighing the tube before and after the experiment, the quantity 
 of glucose is calculated by means of a table compiled for this 
 purpose. 
 
 Of the volumetric methods those of Ling and Bendle and of 
 Benedict are given in this place; others will be found in Lesson 
 XIII. 
 
 LING AND EENDLE'S VOLUMETBIC METHOD. -The sugar 
 
PATHOLOGICAL URINE 209 
 
 solution (diabetic urine) is allowed to run from a burette into a 
 known volume of boiling Fehling's solution. The end point, i.e., the 
 complete reduction of the cupric salt, is recognised by means of a 
 solution of ferrous thiocyanate. When a drop of this indicator is 
 placed on a slab, and a drop of a solution containing a cupric salt 
 brought in contact with it, oxidation of the ferrous salt occurs, 
 with the immediate production of the well-known red colour of 
 ferric thiocyanate. 
 
 Preparation of the Indicator. — One gramme of ferrous ammonium 
 sulphate and 1.5 grammes of ammonium thiocyanate are dissolved in 
 10 c.c. of water at a moderate temperature and immediately cooled ; 
 2.5 c.c. of concentrated hydrochloric acid are then added. The 
 solution so obtained has invariably a brownish-red colour, due to the 
 presence of ferric salt, which latter must therefore be reduced. 
 This is effected by shaking with a small guantity of zinc dust. 
 
 Preparation of Fehling's Solution. — Solution No. 1. — 69.278 
 grammes of crystallised copper sulphate are dissolved in water, 
 and the solution made up to 1 litre. 
 
 Solution No. ^.—346 grammes of crystallised potassium-sodium 
 tartrate (Bochelle salt) are dissolved in hot water, mixed with 142 
 grammes of caustic soda, also dissolved in w^ter, and after cooling 
 made up to 1 litre. 
 
 Equal volumes of these two solutions are accurately measured 
 out and mixed in a dry flask before use. 10 c.c. of the mixed 
 solution are equivalent to 0.05 gramme of glucose. 
 
 Analysis. — Freshly mixed Fehling's solution (10 c.c.) is accurately 
 measured into a 200 c.c. boiling flask, diluted with about an equal 
 quantity of water, and raised to boiling point. The urine is diluted 
 with water and placed in a burette ; the dilution should be adjusted 
 so that 20 to 30 c.c. of the diluted urine are required to reduce the 
 10 c.c. of Fehling's solution. In the first experiment the urine should 
 be diluted with nine times its volume of water. This diluted urine is 
 then run into the boiling liquid in small amounts, commtocing with 
 5 c.c. After each addition of sugar solution the mixture is boiled, 
 the liquid being kept moving. About a dozen drops of the indicator 
 are placed on a porcelain slab, and when it is judged that the precipi- 
 tation of cuprous oxide is complete (that is, when the blue colour of 
 the solution is disappearing), a drop of the liquid is withdrawn by a 
 clean glass rod or by a capillary tube, and brought in contact with the 
 middle of one of the drops of the indicator on the slab. The end 
 point is reached when the mixture ceases to give a red colour with a 
 drop of the indicator. It is essential to perform the titration as 
 ■14 
 
210 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 rapidly as possible, as an atmosphere of steam is then kept in the neck 
 of the flask and the influence of atmospheric oxygen avoided. At the 
 final point the liquid is boiled for about ten seconds. As in all volu- 
 metric methods the first titration may only give approximate results 
 and a second will then be necessary to establish accurately the end 
 point. Each titration should take from two to three minutes. 
 
 Eocample. — Suppose that the urine has been diluted tenfold with 
 water, and that 20 c.c. of the diluted urine are found necessary to 
 reduce the 10 c.c. of Fehling's solution; this will be ectuivalent to 
 2 c.c. of the original urine, and that amount will therefore contain 
 
 0'05 gramme of sugar; 1 c.c. will contain —-5, and 100 c.c. will 
 
 contain V^ ^ 100=2*5 grammes of sugar. 
 
 Pavy's modification of Fehling's solution is sometimes used. Here 
 ammonia holds the cuprous oxide in solution, so that no precipitate 
 forms in boiling Pavy's solution with a reducing sugar. The 
 reduction is complete when the blue colour disappears. 10 c.c. 
 of Pavy's solution ^1 c.c. of Fehling's solution =0 "005 gramme of 
 glucose. 
 
 5. BENEDICT'S METHOD.— Benedict's quantitative reagent' is an 
 alkaline solution of copper sulphate containing potassium thiocyanate. 
 This is kept boiling, and the sugar solution is run into it from a burette 
 until the blue colour disappears ; the thiocyanate forms a white pre- 
 cipitate with the cuprous oxide formed, so that no red cuprous 
 oxide obscures the blue tint. 
 
 Preparation of the Solution. — Sodium citrate, 200 grammes, 
 sodium carbonate (crystalline), 200 grammes (or anhydrous sodium 
 carbonate, 75 grammes), and potassium thiocyanate, 125 grammes, 
 are dissolved in hot water ; this when cool is made up with distilled 
 water to 800 c.c. and filtered. 
 
 18 grammes of pure copper sulphate are then dissolved in 100 c.c. 
 of water, and poured slowly with constant stirring into the fixst 
 solution. 5 c.c. of a 5 per-cent. solution of potassium ferrocyanide 
 are then added as an additional precaution to prevent any deposi- 
 tion of cuprous oxide; finally the total volume of the mixture is 
 made up to 1000 c.c. with distilled water. 25 c.c. of this solution 
 are reduced by 0'05 gramme of glucose. 
 
 Analysis. — 3 or 4 grammes of anhydrous sodium carbonate are 
 placed in a 4-oz. flask, then 25 c.c. of the above solution. This is 
 kept boiling over a small flame, and the sugar solution run in from a 
 
 ' Benedict's qualitative reagent (p. 20) contains sodium citrate, 175 grammes ; 
 anhydrous sodium carbonate, 100 grammes in about 150 c.c. of water; to this is 
 added 17'3 grammes of CUSO4 in 100 o.c. of water, the whole being then made up 
 to 1 litre. 
 
PATHOLOGICAL URINE 211 
 
 burette until the last trace of this blue colour disappears. The 
 amount used for this purpose is then read off. 
 
 Calculation. — This may be illustrated by an example. If 
 the sugar solution had been diluted l-in-5 (that is, 10 c.c. 
 with 40 c.c. of water), and the reading of the burette was 10 c.c, 
 then 
 
 10 c.c. of the diluted solution =2 c.c. of the original solution. 
 2 c.c. of the original solution contain 0*05 gramme of glucose. 
 
 1 c.c. ,, ,, contains-^ „ 
 
 and 100 c.c. „ „ contain °'°^ ^ ^°° 
 
 = 2 "5 glucose per cent. 
 ESTIMATION OF OTHER REDUCING SUGARS.— The same 
 two methods may be used for the estimation of other reducing 
 sugars : the only difference is in the final calculation : — 
 
 10 c.c. of Fehling's solution =0'05 gramme of glucose, 
 (or 25 c.c. of Benedict's solution) =0 "053 „ fructose. 
 
 =0-0676 „ lactose 
 =0'074 „ maltose. 
 
 ESTIMATION OF SUCROSE.— Boil 40 c.c. of the sucrose Solution 
 with 30 c.c. of half-normal hydrochloric acid for one minute. Cool, 
 neutralise with 30 c.c. of half-normal sodium hydroxide, cool and 
 make up the total volume to 100 c.c. The reducing sugars so 
 formed are then estimated as before, and the results calculated from 
 the fact that 10 c.c. of Fehling's solution (or 25 c.c. of Benedict's 
 solution) = '0475 sucrose. 
 
 6. ACETO-ACETIC ACID AND ACETONE are freauently found 
 in diabetic urine, and may be detected as follows : — 
 
 (a) To 3 c.c. of the urine add a few drops of 10 percent, solution 
 of ferric chloride as long as a precipitate (ferric phosphate) continues 
 to be formed, Filter this off and add to the filtrate a few more drops 
 of the ferric chloride solution. A claret-like colour (which disappears 
 on heating) is developed if aceto-aoetic acid is present.^ This test 
 may also be carried out by pouring a few c.c. of the urine on to the 
 top of some 10 per-cent. ferric chloride solution ; the claret-like 
 colour appears at the zone of contact. 
 
 (b) Acidulate the urine with sulphuric acid and shake up with 
 ether; the ether on standing floats on the top; it contains the 
 
 1 Carbolic acid, salicylic acid, and phenaoeturic acid, all of which may occur 
 in urine after drug treatment, give a similar colour reaction in both fresh and 
 previously boiled urine ; whilst aceto-acetic acid does not give the reaction 
 if the urine has been previously boiled. 
 
212 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 aceto-acetic acid in solution. Four it off into another test-tube 
 and shake with ferric chloride solution. A red colour is produced 
 if aceto-acetic acid is present. , 
 
 (c) Heat 250 c.c. of the urine with dilute acid or alkali ; the aceto- 
 acetic acid is converted into acetone. Distil the mixture, and collect 
 the first 20 c.c. of the distillate, and examine this for acetone. 
 
 (d) Acetone in the urine itself or in the distillate just obtained 
 may be detected by the following tests :— 
 
 i. Legal's test. Add a dilute freshly prepared solution of sodium 
 nitro-prussid« and a little 20 percent, caustic potash. A red colour 
 is produced. Acidify with strong acetic acid ; the colour disappears 
 at once in the absence of acetone, but remains or is intensified into 
 a purple in its presence. This test is also given by aceto-acetic acid. 
 
 ii. Bothera's modification is useful. Saturate 10 c.c. of urine with 
 (NHJzSOi, add a few drops of dilute sodium nitro-prusside and 2-3 c.c. 
 of strong ammonia. A purple colour dev^ops above the layer of 
 undissolved crystals, usually only after standing some minutes. This 
 test is also given by aceto-acetic acid, but is not interfered with by 
 the presence of creatinine. 
 
 iii. Acetone does not reduce Fehling's solution or ammoniacal 
 silver solution as aldehyde does. 
 
 7. UBINE C is from a case of jaundice and contains bile. 
 
 (a) Bilepigment maybe detected by Gmelin's test (see p. 108), or by 
 Cole's test, which is performed as follows : — Boil 15 c.c. of the suspected 
 urine, add two drops of a saturated solution of MgSOj, then a 10 
 per-cent. solution of BaCl^ drop by drop, boiling between each addition ; 
 continue until no further precipitate is obtained. Allow the tube 
 to stand for a minute, and pour off the supernatant fluid. To the 
 precipitate add 3 to 5 c.c. of 97 per-cent. alcohol, 2 drops of strong 
 sulphuric acid, and 2 drops of 5 percent, aqueous solution of 
 potassium chlorate. Boil for half a minute and allow the barium 
 sulphate to settle ; the presence of bile pigments is indicated by the 
 alcoholic solution being coloured a greenish blue. If the alcoholic 
 fluid is poured off into a dry tube, mixed with a third of its volume 
 of chloroform, and then an equal volume of water added, the tube 
 may be inverted once or twice ; the chloroform which contains the 
 bluish pigment in solution then separates out. 
 
 (b) BUe salts may be detected by Hay's sulphur test (p. 108). 
 Pettenkofer's test may be performed in the following way : Warm 
 a thin film of the urine and cane-sugar solution in a flat porcelain 
 dish; then dip a glass rod in strong sulphuric acid and draw it 
 across the film ; its track is marked by a purplish line. 
 
PROTEINS AND SUGAR IN THE URINE 213 
 
 PROTEINS IN THE URINE 
 
 There is no protein matter in normal urine, and the most common 
 cause of the appearance of albumin in the urine is disease of the 
 kidney (Bright's disease). The best methods of testing for and' esti- 
 mating the albumin are given in the practical heading to this lesson. 
 The term "albumin" is the one used by clinical observers. Properly 
 speaking, it is a mixture of serum albumin and serum globulin. 
 
 A condition called " peptonuria," or peptone in the urine, is observed 
 in certain pathological states, especially in diseases where there is a 
 formation of pus, and particularly if the pus is decomposing owing to 
 the action of a bacterial growth called staphylococcus ; one of the 
 products of disintegration of pus cells appears to be peptone ; and this 
 leaves the body by the urine. The term " peptone," however, is in 
 the strict sense incorrect ; the protein present is deutero-proteose. In 
 certain diseases of bone, a proteose (Bence-Jones protein) may be 
 found in the urine, which more nearly resembles hetero-proteose in 
 its characters. 
 
 SUGAR IN THE URINE 
 
 Normal urine contains no sijgar, or so littlis that for clinical pur- 
 poses it may be considered absent. It occurs in the disease called 
 diabetes mellitus, and can be artificially produced by the methods 
 briefly referred to on p. 119. 
 
 The methods usually adopted for detecting and estimating the 
 sugar are ^ven at the head of this, lesson. The sugar present is glucose. 
 Lactose may occur in the urine of nursing mothers. The blood of 
 diabetic persons often contains y8-hydroxy butyric acid; some of this 
 passes into the urine, but in the body it is largely converted into aceto- 
 acetic acid and acetone, in which form it is passed in the urine 
 ■ (see p. 120); 
 
 j8-hydroxybutyric acid may be detected by fermenting the urine 
 completely with yeast, and then examining it with the polarimeter ; 
 the /8-hydroxybutyric acid is not affected by yeast, and its presence is 
 indicated by Issvo-rotation. 
 
 Pehling's test is not absolutely trustworthy. Often a normal urine will 
 decolorise Fehling's solution, although seldom a red precipitate is formed. 
 This is due to excess of urates and creatinine. Another substance, called 
 glycuronic acid (O6H10O7), is also likely to be confused with sugar by Fehling's 
 test ; the cause of its appearance is sometimes the administration of drugs 
 (chloral, camphor, etc.) ; but in rare cases it appears independently of drug 
 treatment in normal urine. It is frequently found in diabetic urine. This 
 
214 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 acid is produced bv the oxidation of glucose, the H2 in the CHgOH group 
 being replaced by O. 
 
 Then, too, in the rare condition caUed alcaptonuria, confusion may similarly 
 arise. Alcapton is a substance which originates from tyrosine by an unusual 
 form of metabolism. It gires the urine a brown tint, which darkens on 
 exposure to the air. It is an aromatic substance, and the researches of 
 Bauman and Wolkow identified it as homogentisic acid [CgH3(OH)2CH.2. 
 COOH]. 
 
 The best confirmatory tests for sugar are the phenyl-hydrazine test 
 (see Lesson XIII), and ^e fermentation test. 
 
 BILE IN THE TJEINE 
 
 This occurs in jaundice, the conunonest cause of which is obstruc- 
 tion of the bile duct. The urine is dark brown, greenish, or in extreme 
 cases almost black in colour. Excess of urobilin should not be mistaken 
 for bile pigment. 
 
 BLOOD AND BLOOD PIGMENT IN THE UEINE 
 
 When hsemorrhage occurs in any part of the urinary tract, blood 
 appears in the urine. - It is found in the acute stage of Bright" s disease. 
 If a large quantity is present, the urine is deep red. Microscopic 
 examination then reveals the presence of blood corpuscles, and on 
 spectroscopic examination the bands of oxyhsemoglobin are seen. 
 
 If only a small quantity of blood is present, the secretion — especially 
 if acid — has a characteristic reddish-brown colour, which physicians 
 term "smoky." 
 
 The blood pigment may, under certain conditions, appear in 
 the urine without the presence of any blood corpuscles at all. This 
 is produced by a disintegration of the corpuscles occurring in the 
 circulation. The condition so produced is called hcemoglobinuria, 
 and it occurs in several pathological states, as, for instance, in the 
 tropical disease known as " Black-water fever." The pigment is in 
 the condition of methaemoglobin mixed with more or less oxyhsemo- 
 globin, and the spectroscope is the means used for identifying these 
 substances. 
 
 PUS IN THE UEINE 
 
 Pus occurs in the urine as the result of suppuration in any part 
 of the urinary tract. It forms a white sediment resembling that of 
 phosphates, and, indeed, is alwavs mixed with phosphates. The 
 pus corpuscles may be seen with the microscope ; their nuclei are 
 rendered evident by treatment with 1 per-cent. acetic acid, and the 
 
AMINO-ACIDS IN URINE 215 
 
 pus corpuscles are seen to resemble white blood corpuscles, which, 
 in fact, they are in origin. Some of the protein constituents of the 
 pus cells — and the same is true for blood — pass into solution, so that 
 the urine pipetted off from the surface of the deposit gives the tests 
 for albumin. On the addition of caustic potash to the deposit of pus 
 cells a ropy gelatinous mass is obtained. This is distinctive. Mucus 
 treated in the same way is dissolved. 
 
 AMINO-ACIDS IN URINE 
 
 Normal urine contains traces of glycine. Leucine, tyrosine, and 
 other amino-acids may be present after extensive disintegration of 
 tissue protein, such as occurs in acute atrophy of the liver (p. 186). 
 Cystine may occur as a rare anomaly of metabolism. Associated with 
 cystinuria one often finds diaminuria, that is, the passage of diamines 
 into the urine ; these are known as cadaverine and putrescine, and are 
 the result of the removal of COj from the diamino-adds lysine and 
 ornithine respectively (see p. 116). Homogentisic acid, found in 
 alcaptonuria (see preceding page), is another somewhat similar anomaly; 
 it arises from tyrosine. 
 
216 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 DETECTION OF SUBSTANCES OF PHYSIOLOGICAL 
 IMPORTANCE 
 
 Subsequent lessons may be very usefully employed by the class in testing 
 for the various substances the properties of which have been previously 
 , studied. If the substance is in solution, the following scheme will form a 
 guide to the tests to be employed. If the substance under examination is 
 solid, test its solubility in water ; if it is soluble, the same scheme can then 
 be applied to the solution. " If the substance is insoluble in water, test its 
 solubility in alkali and other reagents ; insolubility in water will suggest such 
 substances as uric acid. If the substance is a mixture part of which is soluble 
 in water, filter off the solution and examine that ; then test the solubility in 
 alkali, etc., of the residue. 
 
 1. Note reaction, colour, clearness or opalescen(;e, taste, smell. Coloured 
 liquids suggest blood, bile, urine, etc. Opalescent liquids suggest starch, 
 glycogen, or certain proteins. 
 
 2. Add iodine. A colour is produced : 
 
 If blue : Starch. Confirm by converting into a reducing sugar by saliva 
 at 40° C, or by boiling with dilute sulphuric acid. 
 
 If reddish-brown : Glycogen or dextrin, the distinctions between which 
 are given on p. 30. The iodine test for starch, etc^ fails in alkaline solutions. 
 
 .3. Add copper sulphate and caustic potash. 
 
 (a) Blue solution : boil ; yellow or red precipitate. Glucose, fructose, 
 maltose, lactose, and other reducing sugars (for distinguishing tests see 
 Lesson XIII). 
 
 (6) Blue solution : no reduction on boiling ; boil some of the original 
 solution with 25 per-cent. sulphuric acjd, and then boil with copper sulphate 
 and excess of caustic potash ; abundant yellow or red precipitate : Sucrose. 
 Confirm by HCl test (see p. 21). 
 
 (c) Violet solution : Proteins (albumins, globulins, metaproteins). In 
 presence of magnesium sulphate, the potash causes also a white precipitate 
 of magnesia. 
 
 (d) Pink solution ; biuret reaction. Peptones or proteoses. In presence 
 of ammonium sulphate very largo excess of potash is necessary for this test. 
 Only a trace of copper sulphate must be used. 
 
 4. When proteins are present proceed as follows : Boil the original solu- 
 tion (after adding a trace of 2 per-cent. acetic acid), 
 (a) Precipitate produced : Albumins or globulins. 
 (6) No precipitate : Metaproteins, proteoses, or peptones. 
 
 5. ' If albumin, or globulin, or both are present, saturate a fresh portion 
 with magnesium sulphate or half saturate with ammonium sulphate ; filter ; ■ 
 the precipitate contains the globulin, the filtrate the albumin. 
 
 6. If proteins are present, but albumin or globulin absent ; 
 (a) Neutralisation causes a precipitate soluble in excess^ of weak acid or 
 alkali. Acid or alkali metaprotein, according as the reaction of the original 
 
SUBSTANCES OF PHYSIOLOGICAL IMPORTANCE 217 
 
 liquid is acid or alkaline respectively. If the original liquid is neutral, meta- 
 proteins must be absent. 
 
 (6) Neutralisation produces no such precipitate : Proteose or peptone. 
 
 7. If proteose, or peptone, or both are present, saturate a fresh portion 
 with ammonium sulphate : 
 
 (a) Precipitate : Proteose. (6) No precipitate : Peptone. 
 If both are present, the precipitate contains the proteose, and the filtrate 
 the peptone. 
 
 8. To a fresh portion add nitric acid (if proteins are present). 
 
 (a) No precipitate, even though excess of sodium chloride is also added : 
 Peptone. 
 
 (5) No precipitate, until excess of sodium chloride is added : Deutero- 
 proteose. 
 
 (c) Precipitate which disappears on heating and reappears on cooling : 
 Proteoses. This is a distinctive test for proteoses, and is given by all of 
 them. For one of them, however (deutero-proteose), excess of sodium 
 chloride must be added also. 
 
 (d) Precipitate little altered by heating : Albumin or globulin. 
 
 In all four cases nitric acid plus heat causes a yellow colour turned 
 orange by ammonia (xantho-proteic reaction). 
 
 9. Confirmatory tests for proteins : — 
 (a) Millon's test (see p. 41). 
 
 (6) Adamkiewicz's reaction or, better, the modification of this test intro- 
 duced by Rosenheim (see p. 41). 
 
 (c) To test for fibrinogen : — 
 
 i. It coagulates by heat at 56° C. 
 
 ii. It is changed into fibrin by fibrin-ferment and calcium chloride. 
 
 (d) To test for caseinogen : — 
 i. It is not coagulated by heat. 
 
 ii. It is changed into casein by rennet and calcium chloride. 
 
 10. If blood (or derivatives of hajmoglobin) is suspected : — 
 
 (a) Examine spectroscopically, diluting if necessary. Before doing so, 
 note colour (red or brown) and reaction to litmus paper. Then proceed 
 according to following scheme : — 
 
 /Acid — hsematoporphyrin (two bands): 
 
 I /two bands between D ("Bands remain — CO hsemoglobin. 
 
 Red{ Neutral-! and E lines ; add a-j One band replaces the t^o — 
 I reducing agent. >■ — oxyhsemoglobin. 
 
 ^Alkaline — reduced hsematin (two bands) unaltered by reducing agen|. 
 /-Acid — acid oxyhsematin (band in red). 
 
 Neutral — methsemoglobin .(gives HbO.2, then reduced Hb on re- 
 duction with (NH4)2S. 
 Alkaline — alkaline oxyhsematin (gives reduc-ed hsematin on 
 reduction). 
 
 V 
 
 Brown- 
 
218 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 The best reducing agent to employ in the foregoing tests is sodium 
 hydrosulphite (see p. 135), except in the case of methcemoglobin ; here 
 warming with ammonium sulphide is the best. 
 
 (b) Dry : boil with glacial acetic acid and a crystg.1 of sodium chloride on 
 a glass slide under a cover glass. When cold, hsemin crystals are seen. 
 
 (c) Try guaiacum test and Adler's test (p. 135). 
 
 (d) If the blood is old and dry, and its haemoglobin converted into hsematin : 
 i. Try hsemin test. 
 
 ii. Try guaiacum test and Adler's test. 
 
 iii. Dissolve it in potash : add reducing agent, and examine for spectrum 
 of reduced hsematin. 
 
 11. If bile is suspected : 
 
 (a) Try Gmelin's test for bile pigments (see p. 108) ; also Cole's test 
 (p. 212). 
 
 (b) Try Pettenkofer's and also Hay's tests for bile salts (see p. 108). 
 
 12. Miscellaneous substances. 
 
 (o) Mucin. Precipitated by acetic acid or by alcohol. The precipitate 
 is soluble in lime water. By collecting the precipitate and boiling it with 25 
 per-cent. sulphuric acid a reducing sugar-like substance is obtained. Mucin 
 gives the protein colour tests. 
 
 (b) Nucleo-protein. Precipitated by acetic acid or by alcohol. The 
 precipitate is often viscous. It is soluble in dilute alkalis such as 1 per-cent. 
 sodium carbonate. This solution causes intravascular clotting. If th^ pre- 
 cipitate is collected and subjected to gastric digestion, an insoluble deposit 
 of nuclein is left, which is rich in phosphorus. Nucleo-protein gives the 
 protein colour tests. 
 
 (c) Gelatin. This also gives some of the protein colour tests, but not 
 those of Millon or Adamkiewicz. It is not coagulated, but dissolved in hot 
 water. The solution gelatinises when cold. 
 
 (d) Urea. Very soluble in water. The solution effervesces when sodium 
 hypobromite or fuming nitric acid is added. Concentrate a fresh portion, 
 add nitric acid, and examine for crystals of urea nitrate. Solid urea heated 
 in a dry test-tube gives off ammonia, and the residue is called biuret, which 
 gives a rose-red colour with copper sulphate and caustic potash. 
 
 (e) Uric acid. Very insoluble in water ;. soluble in potash, and precipi- 
 tated from this solution in crystals by hydrochloric acid. Uric acid crystals 
 from human urine are deeply pigmented red. Try murexide, Folin's and 
 Schiff's tests (see p. 19iS). 
 
 (/) Cholesterol. Characteristic flat crystalline plates. Por various 
 colour tests see pp. 108, 109. 
 
 , 13. Urine. Normal constituents. 
 
 (a) Chlorides. Acidulate with nitric acid ; add silver nitrate ; white 
 precipitate. 
 
 (b) Sulphates. Acidulate with nitric acid or hydrochloric acid ; add 
 barium chloride ; • white precipitate. 
 
 (c) Phosphates. Acidulate with nitric acid ; add ammonium molybdate ; 
 
SUBSTANCES OF PHYSIOLOGICAL IMPORTANCE 219 
 
 boil ; and a yellow crystalline precipitate forms. To another portion add 
 ammonia ; earthy {i.e. calcium and magnesium) phosphates are precipitated. 
 
 (d) Urea (see p. 218). 
 
 (e) Uric acid. To 100 c.c. of urine add 5 c.c. of hydrochloric acid ; leave 
 for twenty-four hours, and pigmented crystals of uric acid are formed. For 
 tests see above. A more rapid test is to saturate with (NH4)2 SO4, collect the 
 precipitate on a filter, and test it for uric acid. 
 
 (/) Hippuric acid. Evaporate the urine with nitric acid, and heat the 
 residue in a dry test-tube. A smell of oil of bitter almonds is given off. 
 (gr) Creatinine. For colour tests see p. ] 81. 
 
 14. Urine. Abnormal constituents. 
 
 (a) Blood. Microscope (blood corpuscles). Spectroscope (for oxyhsemo- 
 globin or methsemoglobin). Hsemin test. 
 
 (6) Blood pigment may be present without blood cbrpuscles. Spectroscope. 
 
 (c) Bile. Gmelin's, Cole's, Hay's, and Pettenkofer's tests. 
 
 (d) Pus. White deposit. Microscope (pus cells). Add potash ; it 
 becomes stringy. 
 
 (e) Albumin, (i) Precipitated, if acid, by boiling ; precipitate insoluble 
 in acetic acid, so distinguishing it from phosphates, (ii) Precipitated by 
 nitric acid in the cold, (iii) Precipitated by picric acid. 
 
 (/) Sugar, (i) Brown colour with potash and heat (Moore's test), (ii) 
 Ferments with yeast, (iii) Reduces Fehling's solution, (iv) Urine has a 
 high specific gravity, (v) Add picric acid, potash, and boil ; the urine 
 becomes a dark opaque red ; the similar slight coloration in normal urine is 
 due to creatinine. 
 
 {g) Aceto-acetic acid avd acetone. For tests see p. 211. 
 
 (A) Miicus. Flocculent cloud ; may be increased by acetic acid ; soluble 
 in alkalis. A little mucus in urine is not abnormal. 
 
 (i) Deposits. 
 
 i. Examine microscopically for blood corpuscles, pus cells, crystals, etc. 
 
 ii. Phosphates. White deposit often mixed with mucus or pus. In- 
 soluble on heating ; soluble in acetic acid. Urine generally alkaline. Examine 
 microscopically for coffln-lids of triple phosphate and star-like clusters of 
 stellar (calcium) phosphate. 
 
 iii. Urates. Pink deposit, usually amorphous ; may be mixed with 
 envelope crystals of calcium oxalate. Deposit soluble on heating urine. 
 Murexide test. 
 
 iv. Uric acid. Deposit like cayenne pepper. Microscope. Tests as above. 
 
ADVANCED COURSE 
 
 INTRODUCTION 
 
 It -will be presupposed that students who take the following lessons have 
 already been through the elementary course. The order in which the subjects 
 are treated is the same as that already adopted. The instructions given will 
 be mainly practital ; theoretical matter on which they depend, or to which 
 they lead, is, as a rule, too lengthy to be discussed in a short manual like the 
 present volume. The Appendix contains a description of various instruments 
 which are not generally contained in sufficient numbers in a physiological 
 laboratory to admit of each student being able to use them in a class. It also 
 contains a description of certain methods of research which should always be 
 shown in demonstratfons, though there maybe practical difficulties in allowing 
 each member of the class to perform the experiment. The few experiments 
 in which living animals are employed will also necessarily be of the nature of 
 demonstrations. 
 
 220 
 
LESSON XIII 
 
 CAEBOHYDEATES 
 
 * 
 
 1. Glycogen. — A rabbit which has been fed five or six hours previously 
 on carrots is killed by bleeding. The chest and abdomen are opened' quickly 
 and a cannula inserted into the portal vein, and another into the vena cava 
 inferior. A stream of salt solution is then allowed to pass through the liver 
 until it is uniformly pale. The washings are collected in three beakers 
 labelled a, h, and c. 
 
 The liver is cut out quickly, chopped into small pieces, and thrown into 
 boiling water acidulated with acetic acid. The acidulated water extracts a 
 small quantity of glycogen. The pieces of scalded liver are then ground up 
 in a mortar with hot water, and thoroughly extracted with boiling water. 
 Filter. A strong solution of glycogen is thus obtained ; but hot water will 
 not extract glycogen thoroughly. 
 
 Test the solution when cold with iodine. 
 
 To separate the glycogen evaporate the solution to a small bulk on the 
 water-bath and then add excess of alcohol ; the glycogen is precipitated as a 
 flocculent ppwder, which is collected on a filter and dried in an oven at the 
 temperature of 100°. 
 
 2. Examine the washings of the liver in the beakers a, h, and c, for sugar. 
 This may be done in a rough quantitative manner as follows : — Take equal 
 quantities of a, h, and c in three test-tubes : to each add an equal amount of 
 Fehling's solution, and boil : a will give a heavy precipitate of cuprous oxide, 
 b one not so heavy, and c least of all^ or none at all. 
 
 3. Pfliiger's Method of Estimating Glycogen. — 20 to 100 grammes, 
 of the finely chopped liver are boiled' for two or three hours with 100 c.c. 
 of 60 per-cent. potash. After cooling, wash the contents of the flask into a 
 beaker, and add 200 c.c. of water, and then 400 c.c. of 94 per-cent. alcohol, 
 and the mixture is allowed to stand overnight. The glycogen is thus 
 precipitated free from protein. Collect the precipitate on a filter, and wash 
 once with 1 vol. 15 per-cent. potash and 2 vols, of alcohol ; then wash with 
 66 per-cent. alcohol. After this transfer the precipitate and filter^paper to a 
 large beaker and boil thoroughly with water. Neutralise the solution and 
 filter. Dilute the filtrate to 500 c.c. and add 25 c.c. of hydrochloric acid of 
 specific gravity 1'19. Heat for three houis on the boiling water-bath ; this 
 converts the glycogen into glucose. After cooling, neutralise with 20 per-cent. 
 potash and filter ; the filtrate is brought up to 250 c.c. ; estimate the glucose 
 in this, either polarimetrically or by a good volumetric method. The 
 percentage of glucose multiplied by 0'927 gives the amount of glycogen. 
 
 221 
 
222 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 4. Microchemical Detection of Glycogen. — A thin piece of the liver is 
 hardened in 90 per-cent. alcohol. Sections are cut by the free hand, or after 
 embedding in paraffin. If paraffin is used, this is got rid of by means of 
 turpentine ; and the sections prepared by either method are treated with 
 chloroform in which iodine is dissolved, and mounted in chloroform balsam 
 containing some iodine. The glycogen is stained brown, and is most abundant 
 in the cells around the radicals of the hepatic vein. 
 
 5. Phenyl- Hydrazine Test for Sugars. — To 5 c.c. of the suspected fluid 
 {e.g. diabetic urine) add 1 decigramme of phenyl-hydrazine hydrochloride, 
 2 decigrammes of sodium acetate, and heat on the water-bath at 100° C. for 
 thirty to sixty minutes. On cooling, if not before, a crystalline or amorphous 
 precipitate separates out. If amorphous, dissolve it in hot alcohol ; dilute 
 the solution with water, and boil to expel the alcohol, whereupon the osazone 
 separates out in yellow crystals. Examine the crystals with the microscope 
 (see accompanying plate). 
 
 Glucose gives a precipitate of phenyl-glucosazone, C6Hii,Oj(N2H.C6H5)2, 
 which crystallises in yellow needles (melting-point 205° C). 
 
 Fructose yields an osazone identical with this. 
 
 Galactose yields a very similar osazone (phenyl-galactosazone). It difi'ers 
 from phenyl-glucosazone by melting at 190-193°, and in being optically 
 inactive when dissolved in glacial acetic acid. 
 
 Sucrose does not form a compound with phenyl-hydrazine. 
 
 Lactose yields phenyl-lactosazone, Ci2H2o09(N2H.C6H5)2. It crystallises 
 in needles, usually in clusters (melting-point 200° C). It is soluble in 80-90 
 parts of boiling water. Lactose in urine does not give tiiis test readily. 
 
 Maltose yields phenyl-maltosazone (CojHjgN^Og). It crystallises in yellow 
 needles much wider than those yielded by glucose oi' lactose (melting-point 
 206° C). Unlike phenyl -glticosazone, it dissolves in 75 parts of boiling water 
 and is still more soluble in hot alcohol. 
 
 The chemistry of the phenyl-hydrazine reaction is represented in the 
 following equations, glucose being taken as an example of the sugar used : — 
 
 I. CH30H[CH(OH)]3CH(OH)COH -f- H2N.NH(C6H5) 
 
 [glucose] [phenyl-hydrazine] 
 
 CH20H[CH(OH)]3CH(OH)CH 
 = II -I- H.,0 
 
 • N— NH(C6H,,) 
 
 [hydrazone] [water] 
 
 II. CH20H[CH(OH)]3CH(OH)CH 
 
 II -)-2CeH5NH— NH„ 
 
 N— NH(C6H5) [phenyl-hydrazine] 
 
 =CH30H[CH(OH)]3C— CH + CeHs.NHg -)- NHg H- Hp 
 
 CsH^NH— N N— NH.CgHs 
 
 [osazone] [aniline] [ammonia] [water] 
 
 6. To determine the melting-point of the osazones (or other organic sub- 
 stances) place a small quantity of the powder in a capillary thin-walled tube, 
 strapped on to a thermometer by an indiarubber' band. Place this in a 
 sulphuric acid bath which is gradually heated, and note the temperature at 
 which the powder melts. 
 
c. 
 
 Fig. 47. — Plate of osazone crystals highly magnified. 
 A, phenyl-glucomzone. B, phenyl-maltosazone. G, plumjl-lactosazone. 
 
CARBOHYDRATES 223 
 
 7. The Folarimeter. — Estimate the strength of a, solution of glucose by 
 means of the polarimeter (see Appendix). " The polarimetric method is a 
 rapid one. When used for the estimation of glucose in urine, 50 c.c. of the 
 urine are mixed with 5 c.c. of a 10 per-cent. solution of lead acetate in order 
 to precipitate pigments, etc. Filter through a dry filter, and fill the tube of 
 the polarimeter with the clear filtrate. The dilution due to the lead acetate 
 solution must be taken into account when calculating the percentage of 
 glucose from the rotation observed. 
 
 8. Formation of Mucic Acid. — Take 1 gramme of lactose and heat it in 
 a porcelain capsule with 12 c.c. of nitric acid on a water-bath until the 
 fluid is reduced to one-third of its original volume. Cool overnight, and 
 a crystalline precipitate of mucic acid separates out. Cane sugar, maltose, 
 glucose, dextrin, and starch, treated in the same way, yield an isomeric acid 
 called saccharic acid, which, being soluble, does not separate out. Lactose 
 yields both acids ; galactose, mucic acid only. 
 
 9. Pentoses give the ordjnary reduction tests for sugar and yield osazones, 
 but do not ferment with yeast. ' They give the two following characteristic 
 tests ; they may be performed with gum arabic (which contains arabinose) 
 or pine-wood shavings (which contain xylose). 
 
 (a) Phloroglucin reaction. "Warm some distilled water with an equal 
 volume of concentrated hydrochloric acid in a test-tube and add phloroglucin 
 until a little remains undissolved. Add a small quantity of gum arabic, and 
 keep the mixture warm in the water-bath at 100° C. The solution becomes 
 cherry-red, and a precipitate settles out, which is soluble in amyl alcohol. 
 This solution gives an absorption band between the D and E lines. 
 
 (6) Orciu reaction. Substitute orcin for phloroglucin in the foregoing 
 experiment. The solution becomes violet on warming, then blue, red, and 
 finally green. A bluish-green precipitate settles out, soluble in amyl alcohol. 
 This solution gives an absorption band between C and D. 
 
 10. Glycuronic acid (see p. 213) gives all the above reactions : it may be 
 distinguished as follows : — 
 
 (a) Take 50 c.c. of glycuronic acid solution in a dish ; add 1 gramme of 
 ^-bromphenyl-hydrazine and rather more than the same amount of sodium 
 acetate. Keep the mixture in the water-bath at 100° C. for a quarter of an 
 • hour, when yellow crystals of ^-bromphenyl-hydrazone of glycuronic acid 
 separate out. After cooling filter off the crystals and wash them with 
 absolute alcohol, in which they are insoluble. Under the same conditions 
 carbohydrates yield p-bromphenyl-osazones, but these are soluble in absolute 
 alcohol. The jo-bromphenyl-hydrazone is soluble in absolute alcohol to 
 which pyridine has been added ; the rotatory power of this solution is greater 
 than that of any of the osazbnes. 
 
 (6) Tolleni Test. — To 5 c.c. of urine add 0'5 c.c. of a 1 per-cent. naphthol- 
 resorcin solution in alcohol, and 5 c.c. of hydrochloric acid (sp. gr.- 1'19). 
 Raise the mixture to boiling point and boil for one minute over a small flame. 
 Let it stand for four minutes, and then cool under the t^p. Shake it with an 
 equal volume of ether. If glycuronic acid is present, the ether becomes blue 
 or violet, and shows an absorption band near the D line. From 60 normal 
 urines, 40 gave the reaction ; it is especially strong after the administration of 
 
224 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 camphor, chloral, salicylic acid, creosote, etc. The reaction is not absolutely 
 distinctive for glycuronic acid, since it is also given by glyoxylic acid and by 
 all acids which contain both a carbonyl and a carboxyl group ; but none, of 
 these substances is likely to occur in urine. 
 
 Bang's Volumetric Method for the Estimation of Glucose 
 
 Principle of the Method. — A copper solution, containing carbonates and 
 sulphocyanide of potassium, is boiled with a quantity of the sugar solution 
 which is not sufficient to reduce all the cupric salts. Under these circum- 
 stances the cupro-thiocyanate, formed by the reduction, is kept in solution. 
 The excess of the cupric salt is then titrated back in the cold by means of a 
 standard hydroxylamine solution, the end point being a change from a blue 
 to a colourless solution. 
 
 Bertrand's Volumetric Method for the Estimation of Glucose 
 
 This method has recently come into vogue for the estimation of glucose 
 in blood, etc. It is not so suitable for urine as Benedict's method, owing to 
 the fact that in many urines cuprous oxide does not easily settle, but 
 remains partially in colloidal suspension. 
 
 Principle of the Method. — The precipitate of cuprous oxide formed on 
 boiling an excess of Fehling's solution with a glucose solution is filtered off, 
 washed and dissolved in a solution of ferric sulphate in sulphuric acid. The 
 ferrous salt formed is titrated with a standard permanganate solution. 
 
 Solutions required. — (1) A solution which contains 40 grammes of pure 
 copper sulphate in one litre. (2) A solutjon wl}ich contains 200 grammes of 
 Eochelle salt and 120 grammes sodium hydroxide in one litre. (3) A solution 
 which contains 50 grammes ferric sulphate (free from ferrous sulphate), and 
 200 c.c. concentrated sulphuric acid in one litre. (4) A solution which contains 
 5 grammes potassium permanganate in one litre. This solution is standardised 
 in the following way : — Weigh out 250 milligrammes ammonium oxalate, dis- 
 solve it in 50 c.c. water, add 2 c.c. concentrated sulphuric acid, warm to 60- 
 80° C, and titrate with the permanganate solution, until a pink colour persists. 
 
 Analysis. — Measure 20 c.c. of the sugar solution' into a flask of 150 c.c. 
 capacity, and add 20 c.c. each of the copper sulphate and Eochelle salt solu- 
 tions. Heat to boiling and keep on the boil for three minutes. Filter through- 
 art asbestos filter, leaving most of the precipitate in the flask. Wash the 
 precipitate in the flask with a little distilled water and decant through the 
 same filter. Dissolve the precipitate in the flask in about 20 c.c. of the ferric 
 sulphate solution and pour the solution through the filter. A green solution 
 results which is titrated at once with permanganate until the green colour 
 changes sharply into pink. 
 
 Calculation. — The amount of copper, precipitated as cuprous oxide, is 
 calculated first from the following equations taking place during the reaction : — 
 
 CusO + FeaCSOj), + HeSOj = 2CUSO4 + SFeSOj + H2O 
 
 lOFeSOj + 2KMn04 + 8H2SO, = SFeaCSOj)^ 4- K^SO^ + 2MnS0, + 8H2O 
 
 SCaH^Oj 4- 2KMn04 ■+ 3H2SO4 = lOCC, -I- 2MnS04 -)• K^SOj + 8H2O. 
 
 ^ The solution must not contain more than 100 milligrammes of glucose. 
 
ACTION OF DIASTASE UPON STARCH 22;:i 
 
 One molecule of ammonium oxalate corresponds to 2Fe and therefore 
 to 2Cu. The amount of ammonium oxalate taken multiplied by 0'8951 
 
 ( = -r-.w- "^ ) gi'^Bs, therefore, the amount of copper corresponding to the 
 
 number of c.c. of permanganate used. From the amount of copper found the 
 quantity of sugar is calculated by means of the following table : — 
 
 Glucose Cu in 
 
 Glucose Ou in 
 
 Glucose Cu in 
 
 Glucose Cu in 
 
 Glucose Cu in 
 
 in mg. 
 
 mg. 
 
 in mg. 
 
 mg. 
 57-2 
 
 in mg. 
 
 mg. 
 
 in mg. 
 
 mg. 
 
 in mg. 
 
 mg. 
 
 10 
 
 20-4 
 
 29 
 
 47 , 
 
 90-0 
 
 65 
 
 121 -3 
 
 83 
 
 150-9 
 
 11 
 
 22-4 
 
 30 
 
 59-1 
 
 48 
 
 91-8 
 
 66 
 
 123-0 
 
 84 
 
 152-5 
 
 12 
 
 24-3 
 
 31 
 
 60-9 
 
 49 
 
 93-6 
 
 67 
 
 124-7 
 
 85 
 
 154-0 
 
 13 
 
 26-3 
 
 32 
 
 62-8 
 
 50 
 
 95-4 
 
 68 
 
 126-4 
 
 86 
 
 155-6 
 
 14 
 
 28-3 
 
 33 
 
 64-6 
 
 51 
 
 97-1 
 
 69 
 
 128-1 
 
 87 
 
 157-2 
 
 15 
 
 30-2 
 
 34 
 
 66-5 
 
 52 
 
 98-9 
 
 70 
 
 129-8 
 
 88 
 
 158-8 
 
 lb 
 
 32-2 
 
 35 
 
 68-3 
 
 53 
 
 100-6 
 
 71 
 
 131-4 
 
 89 
 
 160-4 
 
 17 
 
 34-2 
 
 36 
 
 70-1 
 
 54 
 
 102-3 
 
 72 
 
 133-1 
 
 90 
 
 162-0 
 
 18 
 
 36-2 
 
 37 
 
 72-0 
 
 55 
 
 104-1 
 
 73 
 
 134-7 
 
 91 
 
 163-6 
 
 19 
 
 381 
 
 38 
 
 73-8 
 
 56 
 
 105-8 
 
 74 
 
 136-3 
 
 92 
 
 165-2 
 
 20 
 
 401 
 
 39 
 
 75-7 
 
 57 
 
 107-6 
 
 75 
 
 137-9 
 
 93 
 
 166-7 
 
 21 
 
 42-0 
 
 40 
 
 77-5 
 
 58 
 
 109-3 
 
 76 
 
 139-6 
 
 94 
 
 168-3 
 
 22 
 
 43-9 
 
 41 
 
 79-3 
 
 59 
 
 111-1 
 
 77 
 
 141-2 
 
 93 
 
 169-8 
 
 •2S 
 
 4.3-8 
 
 42 
 
 81-1 
 
 60 
 
 112-8 
 
 78 
 
 142-3 
 
 96 
 
 171-4 
 
 24 
 
 47-7 
 
 43 
 
 82-9 
 
 61 
 
 114-5 
 
 79 
 
 144-5 
 
 97 
 
 173-1 
 
 25 
 
 49-6 
 
 44 
 
 84-7 
 
 62 
 
 116-2 
 
 80 
 
 146-1 
 
 98 
 
 174-8 
 
 26 
 
 51-5 
 
 4.5 
 
 86-4 
 
 63 
 
 117-9 
 
 81 
 
 147-/ 
 
 99 
 
 176-2 
 
 27 
 
 53-4 
 
 46 
 
 88-2 
 
 64 
 
 119-6 
 
 82 
 
 149-3 
 
 100 
 
 177-8 
 
 28 
 
 55-3 
 
 
 
 
 
 
 
 
 
 LESSON XIV 
 
 CARBOHYDRATES: ACTION OF DIASTASE UPON STARCH 
 
 1. Prepare a 0-5 per-cent. solution of stai'ch. 
 
 2. Prepare some malt extract by digesting 10 grammes of powdered malt 
 with 50 c.c. of water at 50° C. for three hours, and subsequently straining. 
 This extract contains the diastatic or malting enzyme. 
 
 Solutions 1 and 2 may be conveniently prepared beforehand by the 
 demonstrator. 
 
 3. To the starch solution add one-tenth of its volume of malt extract, and 
 place the mixture in a water-bath at 40° C. From time to time test portions 
 of the liquid by mixing a drop with a drop of iodine solution on a testing slab. 
 The blue colour at first seen is soon replaced by violet (mixture of blue and 
 red), and then by a red reaction (due to erythro-dextrin), which gradually 
 vanishes {achromic point). Alcohol added to the liquid when all starch and 
 erythro-dextrin have gone still causes a precipitate of a dextrin, which, as it 
 
 15 
 
226 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 t ' 
 
 gives no colour with iodine, is called aehroo-dextrin. The liquid also con- 
 tains a reducing sugar, maltose. 
 
 4. Take 50 c.c. of a solution of maltose and determine how much of it is 
 necessary to reduce 10 c.c. of Fehling's solution. 
 
 5. Take another 50 c.c. and boil it with 1 c.c. of strong sulphuric acid for 
 half an hour in a flask. This converts it into glucose. After cooling bring 
 the liquid to its original volume (50 c.c.) by adding water, and again determine 
 its increased reducing power with Fehling's solution. If a; = c.c. of maltose 
 
 solution are necessary to reduce 10 c.c. of Fehling's solution, then - = c.c. 
 
 o 
 
 of glucose solution are approximately necessary for the same purpose. 
 Benedict's method (see pp. 210-211) may be employed instead of Fehling's 
 solution in these estimations. 
 
 6. Wohlgemuth' s method for the quantitative determination of diastatic 
 enzymes. A series of test-tubes of equal size, each containing 5 c.c. of a 
 1 per-oent. starch solution, are kept in a vessel filled with ice water, and 
 decreasing quantities of the enzyme solution to be examined are added. By 
 the use of ice water the action of the enzyme is prevented until all the tubes 
 are prepared. They are now transferred into a water-bath kept at 40°, by 
 which means the action of the enzyme begins in all the tubes simultaneously. 
 They are kept at this temperature for thirty to sixty minutes and then again 
 transferred into ice water, in order to stop the action. 
 
 All the tubes are then filled with distilled water and one drop of decinormal 
 iodine solution is added to each. After shaking one observes various colours, 
 such as dark blue, blue violet, reddish brown and yellow, according to the 
 quantity and activity o{ the enzyme. Those tubes which show a yellow to 
 a reddish colour contain aehroo-dextrin or erythro-dextrin, those with a blue 
 violet a mixture of erythro-dextrin arid starch. The last tube, in which a violet 
 colour is produced, is taken as the limit of activity. In the one immediately 
 preceding this all the starch has been converted into dextrin, and from it 
 the strength of the enzyme solution is calculated by estimating the number 
 of c.c. of a 1 per-cent. starch solution, which has been converted into dextrin 
 by 1 CO. of the enzyme solution during the time of the experiment. An 
 example will make this clear. The tube immediately preceding the one which 
 shows a violet colour contained 0'02 c.c. of saliva. The time of experiment was 
 thirty minutes. Therefore, 0'02 c.c. saliva was able to convert & c.c. of a 1 per- 
 cent, starch solution into dextrin within thirty minutes, and therefore 1 c.c. 
 
 of saljva would have produced the same change in — — = 250 c.c. of the starch 
 
 solution. If D designates the diastatic strength, we obtain, therefore, 
 
 D — - =250. In another set of experiments with a different enzyme, 
 
 the index tube contained 0'0125 c.c. saliva. We find, therefore, that 
 0'0125 c.c. in 30' digests 6 c.c. 1 per cent, of a starch solution 
 I'Oc.c. „ „ 400 c.c. of the starch solution. 
 
 Therefore, D ~ = 400. The diastatic power of this saliva was, therefore, 
 
 nearly twice that of the former. The same method and mode of notation 
 may be used for any diastatic enzyme. 
 
CRYSTALLISATION OF PROTEINS 227 
 
 LE8S0N XV 
 CRYSTALLISATION OF PROTEINS 
 
 1. Egg- Albumin. — Fresh egg-white is mixed with an equal bulk of fully 
 saturated, filtered, neutral ammonium sulphate solution. 100 c.c. of the 
 former are measured into a porcelain basin or strong beaker, and 100 c.c. of 
 ammonium sulphate solution are added in successive quantities of 10 or 
 15 c.c, the mixture being thoroughly churned with an egg whisk after each 
 addition. The whole should be finally so thoroughly beaten up as to form a 
 large proportion of light froth. After the greater part of the froth has broken 
 down, the mixture is thrown on a folded filter-paper, moderately rapid filtra- 
 tion being obtained without the use of a filter-pump. The filtrate is strongly 
 alkaline to litmus, and smells of ammonia. To the filtrate, or to as much 
 of it as can be obtained in a convenient time of filtration, further ammo- 
 nium sulphate solution is very cautiously added (best, drop by drop from a 
 burette) until a slight permanent precipitate remains, and this precipitate is 
 afterwards just redissolved by the equally cautious addition of water. Dilute 
 acetic acid (10 per cent.) from a burette is now added drop by drop until such 
 a stage of reaction is reached that a precipitate forms and only just redis- 
 solves. Finally one or two drops (not more) of acid are added in excess of 
 this, whereupon a bulky white precipitate falls. The flask is now corked and 
 allowed to stand. In twenty-four hours or less the precipitate, which will 
 have increased in quantity, will be found to consist entirely of acicular 
 crystals. Small portions should be examined under a ^th objective, avoiding 
 pressure on the cover slip. (F. G. Hopkins.) 
 
 2. Sertuu-Albumin. — Crystals of this protein may be obtained by the 
 same method. Horse's serum is the best to use. 
 
 3. Edestin. — This may be taken as a type of the crystallisable vegetable 
 globulins. One kilogramme of hemp seed is ground, or pressed in an oil press. 
 The remainder of the fat is then removed by extracting with light petroleum. 
 When free from this solvent, the seeds are digested at 60° with 1 litre of 5 per- 
 cent, solution of sodium chloride. The liquid is then filtered off from the 
 residue through calico and allowed to cool. A precipitate forms and settles 
 at the bottom of the vessel. The supernatant liquid is then decanted off 
 and the- precipitate washed by deoantation with distilled water. It is then 
 redissolved in 500 c.c. of 5 per-cent. salt solution, and the solution filtered 
 through a warm filter. On cooling, beautiful crystals of the regular system 
 separate. These are washed with cold 5 per-cent. salt solution, distilled 
 water, alcohol, and ether. The yield is about 100 grammes from a kilogramme 
 of ground hemp seed. A method which gives a larger yield, and is more 
 rapid, has recently been described by Reeves, in which the fact has been 
 made use of that these globulins are much more soluble in solutions of salts 
 like sodium benzoate, which lower the surface tension of waiter, than they 
 are in solutions of sodium chloride (Sohryver). 
 
228 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 LESSON XVI 
 MILK 
 
 1. Caseinogen in milk exists in the form of a salt (calcium caseinogenate). 
 Add acetic acid to milk, and this salt is decomposed, and free caseinogen 
 (with entangled fat) is precipitated. Collect the precipitate so produced from 
 about 500 c.c. of milk on a filter, and wash thoroughly with distilled water : 
 grind it up with calcium carbonate in a mortar, and add about 500 c.c. 
 of distilled water ; allow the mixture to stand for about an hour. The fat 
 rises to the top : the excess of calcium carbonate falls to the bottom. The 
 intermediate fluid contains the caseinogen in a very opalescent, colloidal 
 solution. Take some of this solution and divide it into three parts, A, B, and C. 
 
 To A add calcium-free rennet. 
 
 To B add a few drops of 2 per-cent. solution of calcium chloride. 
 To C add both rennet and calcium chloride. 
 
 Put all three in the water-bath at 40° C. A clot of casein forms in C, but 
 not in A if all calcium salts have been successfully washed away, nor in B. 
 
 2. The formation of casein from caseinogen is a double process ; the first 
 action is that of the enzyme, which converts the caseinogen into what may 
 be called soluble casein ; the second action is that of the calcium salt, which 
 precipitates the casein in an insoluble form, or curd. This may be shown by 
 taking some of the caseinogen solution and adding rennet. Warm to 40° C. ; • 
 no visible change occurs, but nevertheless soluble casein and not caseinogen 
 is now present. Then boil this mixture to destroy the enzyme, cool and add 
 calcium chloride. A formation of insoluble curd now occurs. 
 
 3. The two stages of this process may also be shown as follows : — Warm to 
 40° C. some oxalated milk with rennet ; no curdling occurs, then boil to destroy 
 the rennet ; after cooling add calcium chloride, and a curd is produced. 
 
 Caseinogen may be precipitated as a, salt from milk by the addition of 
 alcohol. This reagent also precipitates the other milk proteins. 
 
 4. The method of salting out described in Lesson VI., Exercise 10 (p. 69), 
 may also be used. Add to some milk an equal volume of saturated solution 
 of ammonium sulphate. Caseinogen as a salt is thus precipitated, and en- 
 tangles the fat with it. Filter off the precipitate and examine the filtrate 
 as follows : — Saturate it with sodium chloride ; a small amount of precipitate 
 comes down. This is the so-called lacto-globulin. This contains only a 
 trace of true globulin ; it is mostly caseinogen previously left in solution 
 together with calcium sulphate. Filter it oft' ; acidify the filtrate with a few 
 drops of 2 per-cent. acetic acid and heat it in a water-bath gradually. About 
 77° C. the remaining protein (lactalbumin) is coagulated. 
 
 5. Fat Estimation in Milk (Gerber's Acido-hutyrovietric Method). 
 Principle of the Method.— 'She proteins of milk and the other solids not 
 
 fat are dissolved in concentrated sulphuric acid, and the fat is subsequently 
 separated by centrifugal force. The separation is helped by the addition of 
 amylic alcohol. The whole process is carried out in a special simple centri- 
 fuge, holding two or more acido-butyrometer tubes, which allow the direct 
 reading oif of the fat percentage on the graduated narrow stem of the tube. 
 Analysis.— By means of the special pipettes supplied with the apparatus 
 
THE PROTEOSES 229 
 
 measure into the tubes 10 c.c. of concentrated sulphuric acid, then 11 c.c. 
 of the sample of milk, and finally 1 c.c. of amylic alcohol. Insert the rubber 
 cork and shake the tube with an up-and-down motion until the curd is dis- 
 solved. Ppsh up the cork, if necessary, so that the graduated neck is full, 
 and place the tubes into the cups of the centrifuge, screw on the cover, and 
 spin the centrifuge for two or three minutes. If the fat is not in a clear 
 limpid layer in the neck, or if the upper portion is frothy, the rotation has 
 not been sufficient and must be repeated. Eead off the percentage of fat by 
 adjusting, by slight pressure on the cork, the bottom layer to one of the 
 larger lines on the scale and count up the number of divisions between this 
 and the lowest curved line at the top. Each of the larger divisions is equal 
 to 1 per cent, of faC, and the smaller Ol per cent, of fat. 
 
 LESSON XVII 
 THE PEOTEOSES 
 
 1. Commercial peptone contains a variable amount of true peptone, but 
 usually consists chiefly of proteoses, which are soluble, like peptone, in 
 neutral saline solutions. 
 
 2. Make a solution of this substance in 10 per-cent. sodium chloride solu- 
 tion, and filter.^ Very little residue is left on the filter. This consists of 
 dysproteose, an insoluble form of hetero-proteose, formed during the pro- 
 cess of preparing the substance. If hot saline solution is used instead of cold 
 as a solvent, this amount of insoluble residue is increased, hetero-proteose 
 •being to a slight extent precipitated by heat. 
 
 3. The solution gives the following tests : — 
 
 (a) It does not coagulate on heating. 
 
 (b) Biuret reaction (due both to peptone and proteoses). 
 
 (c) A drop of nitric acid, best added by a glass rod, gives a precipitate 
 which dissolves upon heating and reappears on cooling. (This is due to the 
 proteoses present.) 
 
 (d) The precipitate produced by the addition of acetic acid and a drop of 
 potassium ferrocyanide is also soluble on heating and reappears on cooling. 
 
 4. For the separation of the proteoses and peptone proceed as follows : — 
 
 (a) Saturate the solution with ammonium sulphate, and filter. The 
 filtrate contains the peptone, and the precipitate the proteoses. The peptone 
 is not precipitated by nitric acid, nor by many of the reagents that precipitate 
 other proteins. It is precipitated completely by alcohol, tannin, and potassio- 
 mercuric iodide ; imperfectly by phosphotungstic and phosphomolybdic acid. 
 
 It gives the biuret reaction, but in the presence of ammonium sulphate a 
 large excess of caustic potash is necessary. 
 
 (b) Dialyse another portion of the solution ; hetero-proteose is precipitated. 
 
 (c) Saturate another portion of the solution with sodium chloride (or half 
 saturate with ammonium sulphate) after faintly acidulating with acetic acid. 
 Proto-proteose and hetero-proteose are precipitated. Filter. The filtrate 
 contains the deutero-proteose and peptone. 
 
230 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 The proto- and hetero-proteoae may be redissolved by adding distilled 
 water, and may be separated from each other by dialysis (see 6). 
 
 Deutero-proteose may be separated from the peptone by saturation with 
 ammonium sulphate, or by the addition of a crystal of phosphoric acid. These 
 reagents precipitate the deutero-proteose, but not the peptone. 
 
 Deutero-proteose gives the nitric acid reaction (see 3, c) characteristic of 
 the proteoses only in the presence of excess of salt. If the salt is removed 
 by dialysis, nitric acid then causes no precipitate. 
 
 5. Another delicate test introduced by MoWilliam may here be mentioned. 
 Salicyl-sulphonic acid precipitates albumins and globulins ; on heating, the 
 precipitate is coagulated. The same reagent precipitates proteoses. On 
 heating, the precipitate dissolves and reappears on cooling. It does not 
 precipitate peptones. 
 
 ■ 6. The use of trichloracetic acid for the separation of various proteins 
 may be illustrated by the following experiment : — Take some blood and add 
 to it some solution of commercial peptone {i.e. proteoses and peptone). Add 
 to this mixture an equal volume of a 10 per-cent. solution of trichloracetic 
 acid. There is an abundant precipitate. Boil rapidly and filter hot. The 
 filtrate contains the proteoses and peptones, all the other proteins being 
 contained in the precipitate. On cooling, the filtrate deposits some of the 
 proteose. The proteose and peptone in the filtrate may be detected in the 
 usual way. 
 
 LESSON XVIII 
 DIGESTION 
 
 Numerous methods have been devised for the purpose of comparing the 
 proteoclastic activity of digestive enzymes, and for determining their rate of 
 action. These methods may be conveniently grouped into two classes : — 
 
 {a) Methods in which the rate of solution of a solid protein is used as the 
 index of the action of the enzyme (Griitzner's, Eoaf's, and Mett's methods). 
 
 (6) Methods in which the rate of formation of the products (amino-acids) 
 serves as the index (Sorensen's, Van Slyke's,. and the ninhydrin methods). 
 
 1. Boaf's Method. — This is a modification of Griitzner's method. 
 Grutzner used fibrin stained with carmine, and when the fibrin is dissolved 
 the carmine is set free, and from the depth of colour the amount of fibrin 
 digested can be estimated. The disadvantage of the method is that it can 
 only be used for gastric juice, for when alkali is present, as in pancreatic 
 fluid, the carmine is dissolved out by the alkali before digestion sets in. 
 This was overcome by Roaf by using Congo-red instead of carmine. 
 
 Preparation of the Stained Fihrin. — Clean fibrin is minced, and placed in a 
 0'5 per-cent. solution of Congo-red solution for twenty-four hours (50 grammes 
 of moist fibrin per 100 c.c. of staining solution). This is then poured into 
 excess of water and heated to 80° C. for five minutes. The fibrin is then 
 collected on a cloth and washed under the tap. It is squeezed as dry as possible 
 
DIGESTION 231 
 
 and kept in equal parts of glycerol and water, a little toluol being added to 
 prevent tlie growth of moulds. As instances of the way in which experi- 
 ments may be performed, the following may be taken : — 
 
 (a) Put an equal weighed quantity of the stained fibrin into two test-tubes ; 
 add to each an equal volume of one of two artificial pancreatic fluids. At 
 the end of a given time (say fifteen minutes) remove the tubes, and filter ; the 
 fluid will be more deeply coloured which contained the more active enzyme ; 
 dilute this until it has the same tint as the lighter fluid, and the amount of 
 dilution necessary will measure the relative efficiency of the two preparations. 
 
 (b) Repeat the experiment, using two specimens of artificial gastric juice. 
 Their relative efficiency is determined in the same waj', except that as the 
 acid of the juice has turned the red into a bluish colour, the reaction should 
 be rendered just alkaline by the addition of a few crystals of sodium carbonate. 
 It is easier to determine the relative depth of tint in red than in blue fluids. 
 When comparing the depth of colour of an acid digest with that resulting from 
 digestion in an alkaline medium, the neutralisation of the former is carried 
 out in the same way, and the depth of the two red solutions can then be 
 directly compared. 
 
 2. Mett's Method. — ^A method which is now very generally employed for 
 estimating the proteolytic activity of a digestive juice is one originally intro- 
 duced by Mett. Pieces of capillary glass tubing of known length are filled 
 with white of egg. This is set into a solid by heating to 95° C. They are 
 then placed in the digestive fluid at 36° C, and the coagulated egg-white is 
 digested. After a given time the tubes are removed ; and if the digestive 
 process has not gone too far, only a part of the little column of coagulated 
 protein will have disappeared ;. the length of the remaining column is easily 
 measured, and the length that has been digested is a measure of the digestive 
 strength of the fluid.' This forms a very convenient method to use in experi- 
 ments on velocity of reaction. Schiitz's Law states that the amount of action 
 is proportional to the square root of the amount of pepsin. In most other 
 cases of enzyme activity the rapidity of action is directly proportional to the 
 amount of enzyme present (see pp. 92-95). 
 
 3. The Method of Gross and Fuld. — In this method a solution of 
 caseinogen is used as the substrate. As caseinogen is soluble in dilute hydro- 
 chloric acid as well as in alkali, the method can be used for observations 
 on both peptic and tryptic enzymes. In peptic digestion, the caseinogen 
 which is still undigested is precipitated by sodium acetate, while the cleavage 
 products remain in solution. In tryptic digestion the end point is estimated 
 by precipitating the undigested caseinogen with alcohol and acetic acid. 
 
 (a) Caseinogen Method for Pepsin Estimations. — The caseinogen solution 
 is prepared by dissolving 1 gramme of caseinogen- (commercial casein) in 
 
 ' Hamburger has used the same method in investigating the digestive action o£ 
 juices on gelatin. The tubes are filled with warm gelatin solution, and this jellies 
 on cooling. They are placed as before in the digestive mixture, and the length of 
 the column that disappears can be easily measured. These experiments must, 
 however, be performed at room temperature, for the temperature (36-40° C. ) at 
 which artificial digestion is usually carried out would melt the gelatin. He has 
 also used the same method for estimating amylolytic activity, by filling the tubes 
 with thick starch paste. 
 
232 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 1 litre of dilute hydrochloric acid (16 c.c. of HCl, specific gravity 1'124, and 
 986 c.c. of water). A series of test-tubes are charged with 10 c.c. of the 
 oaseinogen solution, and decreasing amounts of the gastric juice. The tubes" 
 are incubated for fifteen minutes at body temperature, and then a few drops of 
 a concentrated solution of sodium acetate are added to each. Those tubes in 
 which all the caseinogen is digested will show no precipitate ; those tubes in 
 which much caseinogen remains undigested will show a heavy precipitate ; the 
 first tube in which a mere cloud is observed is taken as containing the amount 
 of enzyme just sufficient for digestion, and this amount is taken as the unit. 
 Normal gastric juice by this method shows 33 units. 
 
 (b) Caseinogen Method for Trypsin Estimations. — One gramme of case- 
 inogen is dissolved in 10 c.c. of decinormal soda, neutralised with decinormal 
 hydrochloric acid, and made up to a litre with distilled water. Again a series 
 of test-tubes are charged with 2 c.c. of the solution and decreasing amounts 
 of the pancreatic fluid. These are incubated for an hour at body temperature, 
 and then a few drops of acid alcohol (1 c.c. acetic acid, 50 c.c. alcohol, 49 c.c. 
 water) added to each. The tube which shows only the faintest cloud is taken 
 as before as the unit. Human pancreatic juice shows by this method on the 
 average 2.50 units, that of the dog 125-2.j0 units. 
 
 4. Sorensen's Method. — This very simple method for the estimation of 
 amino-acids depends on the action of formaldehyde on these substances. 
 Amino-acids combine with formaldehyde to form methylene compounds : — 
 
 E.CH.NH2 + O :CH2=R.CH.N :CH2 + H2U 
 
 I I 
 
 COOH COOH 
 
 The basic character of the amino-acid thus being destroyed, the carboxyl 
 (COOH) or acid group may be titrated in the usual way. The method is 
 carried out by adding an excess of a neutral formaldehyde solution to the 
 digested fluid, and titrating the acid set free with decinormal alkali, as 
 described under estimation of ammonia in urine (Lesson XXII). 
 
 5. Estimation of Amino-Nitrogen by Van Slyke's Method. — The 
 principle of this method is based on the well-known reaction of nitrous acid 
 on aliphatic substances which contain an amino-group. The reaction 
 proceeds according to the formula — 
 
 E.NH2 -h HNO2 = E.OH -I- HgO + N2 
 
 R being the fatty radical. It will be seen that the amount of nitrogen 
 evolved is double that contained in the amino-compound. Therefore the 
 final result must be divided by 2. 
 
 Fig. 48 shows the apparatus designed by Van Slyke for obtaining and 
 measuring the nitrogen evolved. 
 
 The apparatus is first filled with nitric oxide in order to displace the air ; 
 this gas is also used to wash the evolved nitrogen into the eudiometer (F), 
 which is filled with a 1 per-cent. sulphuric acid solution. The excess of 
 nitric oxide is removed by permanganate sollition contained in a Hempel 
 pipette (H). The tube A serves for the supply of nitrous 'acid (sodium 
 nitrite and glacial acetic acid). The reaction between the amino-substance 
 and nitrous acid is carried out in D. The amino-substance in solution is run 
 
DIGESTION 
 
 233 
 
 into D from the graduated tube B. The description of the determination 
 may be divided into three stages : — 
 
 (1) The Displdfcment of Air hy Xitrir Oxide. — The acidulated water in F 
 fills the capillary tube leading to the Hempel pipette (H), and also the capillary 
 tube as far as o. Glacial acetic acid is poured into A up to the mark ; this is 
 run into D, the stopcock c being turned so as to let the air escape from D. 
 Through A one next pours sodium nitrite solution (30 grammes of sodium 
 nitrite per 100 c.c. of water) until D is full of solution. The gas exit from D is 
 now closed by the stopcock c, and a being open, I) is shaken for a few seconds. 
 The nitric oxide which instantly 
 
 collects is let out at c, and the c 
 
 shaking repeated. The second 
 amount of nitric oxide then 
 evolved, which washes out the 
 last portions of air, is also let 
 out through c. D is now shaken 
 until all but 20 c.c. of the solu- 
 tion have been displaced by 
 nitric oxide, and driven back 
 into A. A mark on D indicates 
 the 20 c.c. point. One then 
 closes a, and turns c and / so 
 that D and F are connected. 
 
 (2) Decmnposition of the 
 Amdno-suhstance. — Ten c.c. (or 
 less) are measured off in B, and 
 a known amount of this is run 
 into D, and D is shaken for three 
 to five minutes. With a-amino- 
 acids, proteins, or partially or 
 completely hydrolysed proteins, 
 five minutes' vigorous shaking 
 is sufficient. (A small motor 
 may be arranged for the pur- 
 pose of shaking D and H.) In cases where the solution is viscid, and the 
 liquid threatens to froth over into F, B is rinsed out, and a little caprylic 
 alcohol added through it. 
 
 (3) Absorption of Nitric. ( ).ride and Measurement of Nitrogen. — The reaction 
 being completed, all the gas in D is displaced into F by liquid from A, and 
 this gaseous mixture of nitrogen and nitric oxide is drawn from F into the 
 absorption pipette H. The latter, which is filled with permanganate solution 
 (50 grammes potassium permanganate and 25 grammes caustic potash per 
 litre), is then shaken for a minute, and thus the nitric oxide is absorbed. 
 The remaining gas (which is pure nitrogen) is then returned to F and 
 measured. This amount divided by 2 (see equation) gives the amount of 
 amino-nitrogen, from which the amount of the amino-substance analysed is 
 calculated in the usual way. 
 
 6. TheNinhydrin Beaction. — This reaction was discovered by Euhemann, 
 
 Fig. 48. — Van Slyke's apparatus. 
 
234 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 who found that all acids containing a free amino-group in the a position 
 react with triketohydrindene hydrate (ninhydrin) with the production of 
 an intense blue colour. The reaction is very delicate and will detect, for 
 instance, one part of glycine in 65,000 parts of water. The reaction is only 
 characteristic in the absence of ammonium salts and of aliphatic amines. It 
 has been applied by Abderhalden for the detection of products of protein 
 hydrolysis in tests for pregnancy and cancer, but its value as a specific test 
 in these cases appears to be very doubtful. Eecently Harding and MacLean 
 have shown that the ninhydrin reaction in presence of pyridine can be used 
 as a means of estimating a-amino-acids colorimetrically, and have applied 
 the method to the estimation of amino-apids during protein hydrolysis by 
 acids, or by pancreatic enzymes. The method is a most sensitive one and is 
 relatively simple, and its results agree with those obtained by the Van 
 Slyke method. 
 
 7. The Acid of Gastric Juice. — The digestive powers of the acid are 
 proportional to their dissociation and the number of H ions liberated. The 
 anions, however, modify this by having different powers of retarding the 
 action. The greater suitability of hydrochloric over lactic acid, for instance, 
 in gastric digestion is due to the fact that the former acid more readily under- 
 goes dissociation. 
 
 . Hydrochloric add is absent or diminished in some diseases of the stomach, 
 especially in cancer ; this is true for cancer in general even when the stomach 
 is not involved ; the best colour tests for it are the following : — 
 
 (a) Giinzburg's reagent consists of 2 parts of phloroglucinol, 1 part of 
 vanillin, and 30 parts of rectified spirit. A drop of filtered gastric juice is 
 evaporated with an equal quantity of the reagent. Charring must be avoided. 
 Eed crystals form, or, if much peptone is present^ there will be a red paste. 
 The residue is of a bright red colour, even when only 1 part of hydrochloric 
 acid in 10,000 is present. The organic acids do not give the reaction. 
 
 (6) Tropseolin test. Drops of a saturated solution of tropseolin-00 in 94 
 per-cent. methylated spirit are allowed to dry on a porcelain slab at 40° C. 
 A drop of the fluid to be tested is placed on the tropseolin drop, still at 40° C. : 
 and if hydrochloric acid is present a violet spot is left when the fluid has 
 evaporated. A drop of 0'006 per-cent. hydrochloric acid leaves a distinct mark. 
 
 (c) Topfer's test. A drop of dimethyl-amino-azo-benzene is spread in a 
 thin film on a white plate. A drop of dilute hydrochloric acid (up to 1 in 
 10,000) strikes with this in the cold a bright red colour. 
 
 Tropseolin and Topfer's reagent are two of many aniline dyes which can 
 be used for the purpose. 
 
 Lactic acid is sometimes present in the gastric contents, being derived by 
 fermentative processes from the food. It is soluble in ether, and is generally 
 detected by making an ethereal extract of the stomach contents, and evapo- 
 rating the ether. If lactic acid is present in the residue it may Idc identified 
 by'Uffelmann's reaction in the following way : — 
 
 A solution of dilute ferric chloride and carbolic acid is made as follows : 
 
 10 c.c. of a 4 per-cent. solution of carbolic acid. 
 
 20 c.c. of distilled water. 
 
 1 drop of ferric chloride solution. 
 
DIGESTION . 235 
 
 On mixing a solution containing a mere trace (up to 1 part in 10,000) of 
 lactic acid with this violet solution, it is instantly turned yellow. Larger 
 percentages of other acids — for instance, more than 02 per cent, of hydro- 
 chloric acid — are necessary to decolorise the solution, but the yellow colour 
 produced by lactic acid is not obtained. 
 
 Hopkins's Reaction, for Lactic Acid. — Place 3 drops of a 1 per-cent. alcoholic 
 solution of lactic acid in a clean, dry test-tube, add 5 c.c. of concentrated 
 sulphuric acid and 3 drops of a saturated solution of copper sulphate. Mix 
 thoroughly and place the test-tube in a beaker of boiling water for five minutes. 
 Then cool thoroughly under the tap, and add 2 drops of a 0-2 percent, alcoholic 
 solution of thiophene and shake. Replace the tube in the boiling water ; as 
 the mixture gets warm a cherry-red colour develops. The reaction is due to 
 the production of formaldehyde and acetaldehyde by the oxidising agent 
 used ; the thiophene interacts with the aldehydes^ 
 
 8. Demonstration of Pancreatic Secretion. — In an ansesthetised dog in- 
 sert a cannula into the main pancreatic duct, and collect the juice in a suitable 
 vessel. Inject some 0"4 per-cent. hydrochloric acid into the duodenum, and 
 note after some minutes the abundant flow of pancreatic juice. Next ligature 
 off and remove two or three feet of the upper part of the small intestine, wash 
 out the contents and slit it open ; scrape off the mucous membrane with the 
 back of a scalpel ; preserve a small quantity of the scrapiiigs for future use 
 and label this A. Grind up the remainder in a mortar with clean sand or 
 powdered glass, and add 0'4 per-cent. hydrochloric acid. Transfer the mixture 
 to a flask, boil, and when cool neutralise with a little caustic soda solution. 
 Filter ; the filtrate contains secretin, which has been formed by the acid from 
 the prosecretin of the intestinal epithelium. Inject some of this solution 
 through a cannula into the external jugular vein of the dog, and an abundant 
 flow of pancreatic juice is an almost immediate result. 
 
 Characters of the juice so obtained : — 
 
 (a) It is a clear, colourless fluid, and very strongly alkaline. 
 
 (b) Mixed with starch solution and kept at 40° C. dextrin and maltose are 
 rapidly formed. 
 
 (c) Mixed with milk there may be some curdling produced, but the most 
 marked effect is that the milk rapidly becomes acid, and a smell of fatty 
 acids is noticeable. 
 
 (d) Added to fibrin and kept at 40° protein digestion occurs very slowly ; 
 next day, however, the fibrin will be in some measure digested. 
 
 (e) Mix some of the pancreatic juice with the scraping of the intestine 
 which was preserved and labelled A. Then add fibrin. The fluid is now 
 strongly proteolytic, and at 40° C. the fibrin rapidly dissolves ; trypsin has 
 been liberated from the trypsinogen of the juice by the intestinal entero- 
 kinaae. 
 
 9. Products of Pancreatic Digestion of Proteins.— A pancreatic digest 
 should be prepared beforehand by the demonstrator. This may be done by 
 digesting a quantity of protein with artificial pancreatic juice, if the natural 
 juice prepared by the action of secretin is not available ; in the latter case the 
 addition of intestinal epithelium (entero-kinase) should not be forgotten- 
 Unless an antiseptic has been added putrefaction will also occur, and the 
 
236 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 consequent odour will be very perceptible after the mixture has been placed 
 in the warm chamber for some time. 
 
 A very good mixture for the purpose will be found to be the follow- 
 ing :— 
 
 100 grammes of commercial casein. 
 10 grammes of sodium carbonate. 
 
 1 litre of water. 
 25 c.c. of Benger's liquid pancreaticus. 
 0'5 gramme sodium fluoride. 
 3 c.c. chloroform. 
 
 The last two items on the list are added to prevent putrefaction. 
 
 After digestion has progressed for one or two days another 10 c.c. of liquor 
 pancreaticus may be added. 
 
 The products of digestion in one case should be examined, say, after six 
 hours' digestion, and in another case after thirty-six hours or more. The 
 digestive products should then be searched for ; the early products of digestion 
 (alkali meta-protein, deutero-proteose, etc.) will become less abundant with 
 the length of time that digestion has been allowed to progress, and the later 
 products (peptone, leucine, tyrosine, tryptophane, etc.) will become more 
 abundant. 
 
 (a) Tryptophane. — Add a few drops of bromine water ; a violet colour is 
 produced. Add 2 or 3 c.c. of amyl alcohol and shake. The alcohql rises on 
 standing and contains the pigment in solution. 
 
 (6) Leucine and Tyrosine. — i. Examine microscopical specimens of these. 
 The deposit generally found in rather old specimens of Benger's liquid pan- 
 creaticus will be a convenient source of these crystals. 
 
 ii. To some of the pancreatic digest add acetic acid and Millon's reagent 
 and filter off the precipitated protein. Boil the filtrate, and the presence of 
 tyrosine is indicated by a red colour. If tyrosine is abundant the red colour 
 appears without boiling. Leucine does not give this test. 
 
 iii. Faintly acidify another portion of the filtered digest with acetic acid 
 and boil ; if any protein matter is still undigested it will be thus coagulated 
 and can be filtered ofi'. Reduce the filtrate to a small bulk until it begins to 
 become syrupy. Leave overnight in a cool place, and crystals mainly of 
 tyrosine will separate out. Filter these off through fine muslin, and evaporate 
 down the filtrate to the consistency of a thick syrup ; leave this overnight 
 again, and a second crop of crystals, forming a scum on the surface and con- 
 sisting mainly of leucine, will have separated out. 
 
 iv. M&rner's Test for Tyrosine. — The following reagent is used : — 1 c.c. 
 formalin, 45 c.c. distilled watei-, 55 c.c. concentrated sulphuric acid. If a 
 portion of this solution is boiled with a little tyrosine (in the solid form or in 
 solution), an emerald-green colour appears. This test often fails in the presence 
 of organic impurities. 
 
 10. In a digest in which putrefaction has occurred, test for indole as 
 follows : — Add a little sulphuric acid and a few drops of a dilute solution of 
 sodium nitrite ; a bright red colour is produced (Cholera-red reaction). 
 
 11. Zymogen Granules. — Examine microscopically, mounting in aqueous 
 humor or serum (or in glycerol after treatment with osmic acid vapour), 
 
THE BLOOD 237 
 
 small pieces of the pancreas, parotid, and submaxillary glands in a normal 
 guinea-pig,i and ajso in one in which profuse secretion had been produced by 
 the administration of pilocarpine. 
 
 Note that zymogen granules are abundant in the former, and scarce in 
 the latter, being situated chiefly at the free border of the cells. 
 
 LESSON XIX 
 THE BLOOD 
 
 1. Effect of Decalcifying Agents in hindering Coagulation.— From an 
 anaesthetised dog collect samples of blood from the carotid artery, into which 
 a suitable cannula should have been previously inserted. 
 
 • (a) Collect the first sample in an equal volume of 0'4 per cent, solution 
 of potassium oxalate made with physiological salt solution. 
 
 (6) Collect the second sample in a tenth of its volume of a 3 per-cent. solu- 
 tion of sodium fluoride. 
 
 (c) Collect the third sample in a quarter of its volume of 10 per-cent. 
 solution of sodium citrate. 
 
 In all three cases mix thoroughly, and coagulation is hindered owing to 
 decalcification, as explained on p. 139. 
 
 The separation of the plasma from the corpuscles may be most readily 
 carried out by a centrifugal machine ; the corpuscles settle and the super- 
 natant plasma can be then pipetted off. Sedimentation is specially rapid in 
 the case of citrate blood, and a well-marked layer of colourless cor- 
 puscles and platelets may usually be seen on the top of the mass of red 
 corpuscles. 
 
 Oxalate plasma and citrate plasma coagulate on the restoration of the 
 calcium by adding a few drops of calcium chloride solution, as we have already 
 seen in the elementary course (p. 133). Fluoride plasma does not coagulate 
 unless fibrin-ferment(orsome fluid such as serum which contains fibrin-ferment 
 or thrombin) is added as well as the calcium salt. Fluoride plasma thus forms 
 a convenient test fluid for fibrin-ferment. 
 
 If in either case the plasma is previously heated to 60° C. and filtered, 
 coagulation — that is to say, fibrin formation — can never be produced, because 
 its mother substance, fibrinogen, which is coagulated by heat at 56° C, has 
 been coagulated and removed. 
 
 2. Influence of Leech Extract on Coagulation. — The same dog still 
 under the anaesthetic may be next used for the following experiments : — 
 
 (a) Draw off a sample of blood into a clean test-tube, and note the time 
 it takes to clot. 
 
 ' The guinea-pigs should be killed by bleeding, and the blood collected and 
 defibrinated, and utilised for the preparation of oxyhsemoglobin crystals. This 
 will give students an opportunity of seeing the exceptional form (tetrahedra) in 
 which the blood pigment of this animal crystallises. 
 
 The three methods of obtaining crystals described on p. 145 all give good results. 
 If amyl-nitrite is used instead of el her in the third method, crystals of methaemo- 
 globin are obtained. 
 
238 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 (6) Draw off a second sample into about half its volume of leech extract 
 made by grinding up the heads of about twenty leeches in 20 c.c. of salt solu- 
 tion and filtering. This remains unclotted for hours or days. 
 
 (o) Inject 10 c.c. of- the extract into the jugular vein of the animal, and 
 draw off samples of blood from time to time, coniparing the coagulation time 
 (which gradually lengthens) with that of a specimen n. 
 
 (d) Having obtained a specimen which does not clot at all, dilute it, with 
 salt solution and pass a stream of carbon dioxide through it. Clotting is not 
 produced as it is in " peptone " blood (see 3 g, below). In order to produce 
 clotting, excess of serum, or some fluid containing thrombin, must be added. 
 The action of leech extract is mainly due to the fact that it contains anti- 
 thrombin. 
 
 (e) The experiments described under d may be repeated with leech extract 
 plasma, obtained from the blood by centrifugalising. 
 
 (/) Instead of leech extract, a solution of its active principle (hirudin) may 
 be used. This produces little or no fall of blood pressure, and so contrasts 
 with what occurs in " peptone " injection. Leech extract produces a very 
 small fall of arterial pressure. 
 
 3. Influence of Commercial Peptones (Proteoses) on Coagulation. — For 
 the purpose of the following experiments another dog must be employed. 
 
 The animal having been anaesthetised a cannula is placed in the external 
 jugular vein for the injection of the " peptone.'' 
 
 The carotid artery is connected to a mercurial manometer for the registra- 
 tion of arterial pressure. 
 
 Another convenient artery must be exposed and a cannula inserted into it 
 for collection of sample's of blood. 
 
 (a) First draw off a sample of blood and note its coagulation time. 
 
 (6) Draw a second sample into a strong solution of commercial peptone. 
 The coagulation time is somewhat longer than in a. 
 
 (c) Draw off a small sample and make a blood film, staining it with methy- 
 lene blue ; count the colourless corpuscles. 
 
 (d) Then in j ect the peptone q uickly, so that the animal receives 0'3 gramme 
 per kilo of body- weight. Note during and for some time after the injection a 
 great fall in arterial blood pressure. This has been shown by the oncometer 
 to be due to vascular dilatation. 
 
 (e) After the injection draw off successive samples, and note the great 
 prolongation of the coagulation time which is soon produced. 
 
 (/) Make a stained blood film from one sample as before, and note the 
 great scarcity of colourless corpuscles. 
 
 (g) Dilute some of the blood which does not clot with twice its volume of 
 salt solution, and pass a stream of carbonic acid through the mixture ; coagula- 
 tion soon occurs. 
 
 (A) The same experiment may be repeated with the same result, if 
 " peptone " plasma obtained by centrifugalising is used instead of the whole 
 blood. 
 
 (i) Finally bleed the animal to death, collecting the blood in three successive 
 glass cylinders. Place them in the ice chest, and examine them a few days or 
 a week later. 
 
THE BLOOD 
 
 2^9 
 
 The first lot of blood collected will show sedimentation of corpuscles, and 
 a slight clot at the junction of the corpuscles and supernatant plasma — that 
 is, at the place where the white corpuscles and platelets lie. 
 
 The last lot of blood collected shows less sedimentation, and will probably 
 have clotted throughout. This is because the blood removed last has been 
 diluted by tissue lymph, which has passed into the blood-stream in an attempt 
 to increase the volume of the blood, which has been lessened by the previous 
 bleeding ; the clot produced is probably due to the action of thrombo- 
 kinase. 
 
 The middle sample will show something intermediate between the two 
 extremes, the usual state of things being clot through the sediment, and the 
 plasma above it still fluid. 
 
 4. Intravascular Coagulation. — A solution of nucleo-protein from the 
 thymus, testis, lymphatic glands, or kidney has been prepared beforehand 
 by the demonstrator. It may be prepared in one of two ways. 
 
 (a) Wooldridge^s Method. — The gland is cut up small and exti-acted with 
 water for twenty-four hours. Weak acetic acid (0'5 c.c. of the acetic acid of 
 the " Pharmacopoeia " diluted with twice its volume of water for every 100 c.c. 
 of extract) is then added to the decanted liquid. After some hours the pre- 
 cipitated nucleo-protein (called tissue-fibrinogen by Wooldridge) falls to the 
 bottom of the vessel. This is collected and dissolved in 1 per-cent. sodium 
 carbonate solution. i 
 
 (b) The Sodium Chloride Method. — The finely divided gland is ground up 
 in a mortar with about an equal quantity of solid sodium chloride and a, 
 small quantity of water. The resulting viscous mass is poured into excess 
 of distilled water. The nucleo-protein rises to the surface of the water, where 
 it may be collected and dissolved as before. 
 
 A rabbit is anaesthetised, and a cannula inserted into the external jugular 
 vein. The solution is injected into the circulation through this. The animal 
 soon dies from cessation of respiration ; the eyeballs protrude, and the pupils 
 are widely dilated. On opening the animal the heart will be found still 
 beating, and its cavities (especially on the right side) distended with clotted 
 blood. The vessels, especially the veins, are also full of clot. The blood of 
 the portal vein is usually clotted most. If a dog is employed instead of a 
 rabWt in this experiment, coagulation is usually confined to the portal area. 
 This is related to the greater venosity of the blood in this situation. If 
 venosity is increased in any other area, as by tetanising the muscles of one 
 leg, clotting will be found also in the veins of this region. 
 
 5. The Estimation of Glucose in Blood and Blood Serum.— The 
 estimation of glucose necessitates the complete removal of proteins. Of the 
 many methods suggested for this purpose that of Michaelis and Rona is the 
 best. It depends on the property which proteins possess of being adsorbed 
 by other colloids, and they are thus carried down with them when they come 
 into contact with electrolytes. Colloidal iron (ferric hydrate) is generally 
 used for this purpose. 
 
 (a) In Serum. — 00 c.c. serum are diluted to 500 c.c. with distilled water, 
 and 40 c.c. of the colloidal iron solution are added slowly with constant 
 shaking. On filtering, a water-clear filtrate is obtained, free from iron and 
 
240 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 protein. After exactly measuring its volume, it is slightly acidified with 
 acetic acid and concentrated in vacuo or on a water-bath, and made up to 
 50 c.c. in a measuring flask. Glucose is estimated polarimetrically, and by 
 any of the reduction methods described (see Lessons XII and XIII). 
 
 (b) In the whole Blood. — A measured or a weighed quantity of blood (30- 
 40 c.c. or 30-40 grammes) is diluted to 1 litre with distilled water and the iron 
 solution added as before. For 1 gramme of blood about 3 c.c. iron solution 
 are required. The mixture is left standing, with frequent shaking, for about 
 ten minutes. Now one adds 1-1 5 grammes finely powdered magnesium 
 sulphate and shakes for one to two minutes. Filter and proceed as before. 
 Although the addition of magnesium sulphate is necessary in the whole 
 blood, such an addition can be dispensed with in the case of serum, the salts 
 of which act as the electrolytes in the reaction. The reason why the addi- 
 tion of a salt to the whole blood should be necessary is not clear. 
 
 (c) For the purpose of estimating glucose in 1 or 2 c.c. of blood a great 
 many so-called microehemical methods have been brought forward. For 
 clinical purposes, in cases of diabetes or for systematic investigations, pro- 
 longed for a considerable period, such methods are obviously of considerable 
 value. A quick and accurate method is : — 
 
 Lewis and Benedict's Colorimetric Method, in which the red colour 
 (picramic acid) obtained by heating a glucose solution with picric acid 
 is employed as the basis for the determination of blood sugar. The 
 blood protein is removed by precipitation with picric acid, which is itself 
 one of the reagents of the colour reaction. The colour is compared in a 
 colorimeter (see p. 258) with a standard solution of glucose treated with 
 picric acid, or directly with a standard solution of picramic acid. The 
 protein-free filtrate may also be used directly for the estimation of creatinine 
 (see p. 258). 
 
 Analysis. — Two c.c. of blood are aspirated through a hypodermic needle 
 into a pipette, a little powdered potassium oxalate in the tip of the pipette 
 preventing clotting. The blood is discharged at once into a 25 c.c. volumetric 
 flask containing 5 c.c. of water. The contents of the flask are shaken, to 
 ensure hiemolysis of the blood. Then 15 c.c. of a, saturated solution of 
 picric acid are added, as well as a drop or two of alcohol to dispel any foam, 
 and the flask is filled /ip to the mark with water. After filtration 8 c.c. are 
 measured out into a large test-tube. Two c.c. of saturated picric acid solu- 
 tion and 1 c.c. of 10 per-cent. sodium carbonate are added, and the contents 
 of the tube are evaporated rapidly over a free flame until precipitation 
 occurs. About 3 c.c. of water are added, the tube is again heated to boiling 
 to dissolve the precipitate, the contents of the tube are transferred quanti- 
 tatively to a 10 c.c. volumetric flask, cooled, made up to the mark, shaken, 
 and then filtered into the colorimeter vessel. The colour is compared at 
 once with that obtained from 0'64 milligramme of glucose, 5 c.c. of saturated 
 picric acid, and 1 c.c. of 10 per -cent, sodium carbonate, treated and diluted 
 to 10 c.c. as was the unknown solution. A permanent standard solution may 
 be prepared by dissolving 0'064 gramme of picramic acid and O'l gramme 
 acid and 0"1 gramme sodium carbonate in 1 litre of water. 
 
 Calculation : as the amount of blood taken for one estimation is 0'64 c.c. 
 
HAEMOGLOBIN AND ITS DERIVATIVES 241 
 
 and the amount of glucoae in the standard is 0'64 mg., we find the contents 
 
 ., ri,i J • -ii- J 1 • reading of standard 
 
 of 1 C.C. of blood in milhgrammes of glucose is reading of unknown solution. 
 
 Such observations should always be made in duplicate, and the nieaij of the 
 two taken as the correct result. 
 
 LESSON XX 
 
 H.'EMOGLOBIN AND ITS DERIVATIVES 
 
 Defibrinated ox-blood suitaibly diluted may be used in the following 
 experiments as in those described in Lesson IX. 
 
 1 . Place some in a vessel with flat sides in front of the large spectroscope. 
 Note the position of the two characteristic bands of oxyhsemoglobin ; these 
 af e replaced by the single band of reduced heemoglobin after reduction (see 
 p. 135). By means of a small rectangular prism a comparison spectrum 
 showing the bright sodium line (in the position of the dark line named 
 D in the solar spectrum) may be obtained, and focused with the absorption 
 spectrum. 
 
 2. Obtain similar comparison spectra by the use of the microspectroscope. 
 For this purpose a cell containing a small quantity of oxyhajmoglobin 
 solution may be placed on the microscope stage, and a test-tube containing 
 carbonic oxide haemoglobin in front of the slit in the side of the instru- 
 ment. Notice that the two bands of carbonic oxide haemoglobin are very 
 like those of oxyhsemoglobin, but are a little nearer to the violet end of the 
 spectrum (fig. 49, spectrum 4). 
 
 CaAonic oxide haemoglobin may be readily prepared by passing a stream 
 of coal gas through the diluted blood. It has a cherry-red colour, and is not 
 reduced by the addition of reducing agents. 
 
 3. Methsemoglobin. — Add a few drops of ferricyanide of potassium to 
 dilute blood, and warm gently. The colour changes to mahogany-brown. 
 Place the test-tube in front of the small direct vision spectroscope. Note the 
 characteristic band in the red (fig. 49, spectrum 5). On dilution, other bands 
 appear (fig. 49, spectrum 6). Treat with ammonium sulphide, and the band 
 of hsemoglobin appears. 
 
 4. Acid Oxyhaematin. — (a) Prepare the following mixture :— 150 c.c. of 90 
 per-cent. alcohol and 6 c.c. of concentrated sulphuric acid ; take about 5 c.c. 
 of this mixture and boil it in a test-tube. While still hot, drop into it a few 
 drops of undiluted defibrinated blood, and filter. Note the brown colour 
 of the filtrate. Compare the position of the absorption band in the red with 
 that of methaemoglobin ; that of acid oxyhsematin is further from the D line 
 (fig. 49, spectrum 7). 
 
 (6) Add some glacial acetic acid to undiluted defibrinated blood. Extract 
 
 this with ether by gently agitating it with that fluid. The ethereal extract 
 
 should be then poured off and examined spectroacopically. The band in the 
 
 red is seen, and on further diluting with ether, three additional bands appear. 
 
 16 
 
242 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 C D ES F ■ G 
 
 ). 4i). — 1, Solar spectrum. 2, vSpectrum of oxylitemot^lobin (0-37 ]^.<:. solution). First Ijaiid, 
 \Cit<U-5i\i; second band, x,555-r)l7, 8, Spectrum of reduced hieniojjfloliiii. Band, \ 697-5:^5. 4, Spec- 
 trpm of 00-hiemojrlobin. First band, \ 583-504; second band, \ 547-521. 5, Spectrum of 
 metba;mo}^lobin (concentrated solution), (i, Spectrum of meth^emo^dobin (dilute yolution). 
 First band, \ 647-032; second band, \ 5S7-571 ; tbird band, \ .^52-532; fourth band, 
 X r.l4-41H). 7, Spectrum of acid oxyhaBmatin (ethereal solution) First band, x. (i50-6I5; 
 second Itand, x 507-577 ; third band, \ 557-529 ; fourth band, \ 517-488. 8, Spectrum of 
 alkaline oxyhtcmatin. Band from X 63U-B81. 9, Spectrum of reduced haematin. 
 ?'iryt band, '\ 5(if)-542 ; second band, x .53.5-534. 10, Spectrum of acid h;ematoporphyrin. 
 First band, \ ()(l7-593 ; second band, x .585-530. 11, Spectrum of alkaline haamatoporphyrin. 
 First band, x 633-012 ; .second band, x 589-564 ; third band, \ 549-529 ; fourth band, \ 518-488. 
 The above measurements (after MacMunn) are in millionths of a millimetre. The liquid was 
 examined in a layer 1 centimetre thick. The eiig-es of ill-defined bands vary a g-reat deal with 
 tlie concentration of tlie solutions. 
 
H^EMOGLOBIN AND ITS DERIVATIVES 
 
 243 
 
 5. Alkaline Oxyhsematin.— («) Add to diluted blood a small quantity 
 of strong caustic fjotash, and boil. The colour changes to brown, and with the 
 spectrosco]ie a faint shading on the left side of the 1) line is seen (tig. 49, 
 spectrum W), 
 
 h HK L M N 
 
 Fin. 60.— The photographic spectrum of reduced hasmoglobiu .and oxyhemoglobin. (Gamgee.) 
 
 HK LM NO 
 
 Flu. rd. — Tlie photographic wpei'trutu of o\yha:imogloiiiri and methamioglobin. {(-Jamgee.) 
 
 {/>) The band is much better seen in an alcoholic solution. Prepare the 
 following jui.xtore : — l-lOc.c. of !J0 per-cent. alcohol, and 18 c.c. of .'lO per- 
 cent, jjotash. Take about 5 c.c. of this mi.xture in a test-tube and Ixiil it. 
 While still hot, drop into it a few drojjs of undiluted delibiinated blood. The 
 fiuid shows the spectrum of alkaline oxyhsematin. This may then be used 
 for the next experiment. 
 
244 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 6. Beduced hsematin.' — Add a reducing agent to the solution of alkaline 
 oxylisematin ; the colour changes to red, and two bands are seen, one between 
 D and E, and the other nearly coinciding with E and 6 (fig. 49, spectrum 9). 
 The spectrum of alkaline oxyhsematin reappears after a short time after 
 vigorous s'haking with air. 
 
 7. Hsematoporpliyrin. — ^To some strong sulphuric acid in a test-tube add 
 a few drops of undiluted blood, and observe the spectrum of acid haemato- 
 porphyrin (iron-free haematin) (fig. 49, spectrum 10). 
 
 Map out all the spectra you see on a chart. 
 
 8. The Photographic Spectrum. — Haemoglobin and its compounds also 
 show absorption bands in the ultra-violet portipn of the spectrum. This 
 portion of the spectrum is not visible to the eye, but can be rendered visible 
 by allowing the spectrum to fall on n fluorescent screen, or on a sensitive 
 photographic plate. In order to show absorption bands in this part of the 
 spectrum very dilute solutions of the pigment must be used. 
 
 In order to demonstrate these bands, the telescope of a large spectroscope 
 is removed, and a beam of sunlight or of light from the positive pole of an 
 arc lamp is allowed to fall on the slit of the collimator. The spectrum is 
 focused on a fluorescent screen.^ The slit is then opened very widely, and 
 the coloured solution is interposed on the path of the beam falling on the slit. 
 
 Oxyhsemoglobin shows a band (Soret's band) between the lines G and H. 
 In reduced haemoglobin, carbonic oxide haemoglobin, and nitric oxide 
 haemoglobin, this band is rather nearer G. Methaemoglobin and haemato- 
 porphyrin show similar bands. 
 
 The two preceding figures show the " photographic spectra " of reduced 
 haemoglobin, oxyhaemoglobin, and methaemoglobin, and will serve as ex- 
 amples of the results obtained. I am greatly indebted to the late Professor 
 Gamgee, to whom we owe most of our knowledge on this subject, for per- 
 mission to reproduce these two specimens of his numerous photographs. 
 
 9. Preparation of Pure Oxyhaemoglobin. — Centrifugalise dog's defibrin- 
 ated blood, and pour off the serum. Centrifugalise again with physiological 
 saline solution repeatedly until the supernatant fluid contains only traces 
 of protein. Mix the magma of corpuscles with two or three volumes of water 
 saturated with acid-free ether ; the solution becomes clear. Then add a 
 few drops of 1 per-eent. solution of acid sodium sulphate till the mixture 
 looks tinted like fresh blood, owing to the precipitation of the stromata. 
 These can be separated by filtration. Pour ofl" the clear red fluid : cool it to 
 0° C, add one-fourth of its volume of absolute alcohol previously cooled to 
 0° 0. Shake well, and then let the mixture stand a,t .3-15° C. for twenty-four 
 hours. As a rule the whole passes into a glittering crystalline mass. Filter 
 at 0° C. and wash with ice-cold 25 per-cent. alcohol. Redissolve the crystals 
 in a small quantity of water, and recrystallise as before. The crystals may 
 then be spread on plates of porous porcelain, and dried in a vacuum over 
 sulphuric acid. 
 
 1 Called hsemochromogen by Hoppe Seyler ; this name is a misleading one. 
 
 2 Fluorescent screens, similar to those in conunon use in observations made 
 with Rontgen rays, may be made by coating white cardboard with barium platino- 
 cyanide. 
 
MUSCLE AND NERVOUS TISSUES 245 
 
 LESSON XXI 
 
 MUSCLE AND NERVOUS TISSUE^ 
 
 1. Hopkins's Lactic Acid Test (see p. 235) may be applied as follows. 
 Remove one hind limb of a pithed frog. Stimulate the sacral plexus of the 
 other side for ten minutes with a strong Faradic current. Then amputate 
 the other hind limb. Skin both legs, and chop up the muscles of the two sides 
 separately. Pound each in a mortar with clean sand and then with 15 c.c. 
 of 95 per-cent. alcohol. Transfer the mixture to a beaker, and warm in the 
 water-bath for a few minutes. Filter, and evaporate the filtrate to dryness 
 in a water-bath. Extract the residue with about 5 c.c. of cold water, rubbing 
 it up thoroughly with a glass rod. Filter and boil the filtrate in a test-tube 
 for about a minute with as much animal charcoal as will lie on a threepenny- 
 piece. Filter again and evaporate the filtrate to dryness in a water-bath. 
 Allow the residue to cool, and dissolve it by shaking in 5 c.c. of concentrated 
 sulphuric acid. Transfer this to a dry test-tube ; add 3 drops of saturated 
 solution of copper sulphate, and place the tube in boiling water for five 
 minutes. Cool and add 2 drops of 0'2 per-cent. solution of thiophene in 
 alcohol ; replace the tube in the boiling water. A cherry -red colour 
 develops in the tube containing the extract from tetanised muscle, but 
 not in the other. 
 
 2. A rabbit has been killed and its muscles washed free from blood by a 
 stream of salt solution injected through the aorta. The muscles have been 
 quickly removed, chopped up small, and extracted with 5 per-cent. solution 
 of magnesium sulphate. This extract is given out. It will probably be 
 faintly acid. The acid is sarcolactic acid. It may be identified by Ufi'el- 
 mann's or Hopkins's reaction (p. 235). 
 
 3. The coagulation of muscle somewhat resembles that of blood. This 
 may be shown with the salted muscle plasma (the extract given out) as follows ; 
 Dilute some of it with four, times its volume of water ; divide it into two 
 parts ; keep one at 40° C. and the other at the ordinary 'temperature. Co- 
 agulation —that is, formation of a clot of myosin — occurs in both, but earliest 
 in that at 40° C. 
 
 4. Remove the clot of myosin from 3 ; observe it is soluble in 10 per-cent. 
 sodium chloride, and also in 0'2 per-cent. hydrochloric acid, forming acid 
 metaprotein. 
 
 5. Make an extract of muscle in the same way, using a small quantity 
 of physiological salt solution instead of the strong solution of magnesium 
 sulphate employed in the foregoing experiments. Such an extract contains 
 the two principal proteins, viz., paramyosinogen and myosinogen, the two 
 precursors of the muscle-clot or myosin. Small quantities of other proteins 
 also present are mainly due to unavoidable mixture with small amounts of 
 blood and lymph. 
 
 These two proteins differ in temperature of heat coagulation. Take the 
 extract and heat it in a test-tube within- a water-bath ; at 47° C. paramyosin- 
 
246 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 ogen is coagulated ; filter this off, and heat the filtrate ; at 56° C. flocculi of 
 the coagulated myosinogen separate out. 
 
 6. Paramyosinogen is precipitable by dialysis, and is a true globulin. 
 Myosinogen is what is called an atypical globulin, and corresponds to the 
 pseudo-globulin of blood serum and egg-white. Though readily salted out 
 of solution like paramyosinogen it is not precipitable by dialysis. 
 
 7. In the process of clotting, such as occurs in rigor-mortis, paramyosino- 
 gen is directly converted into myosin ; whereas myosinogen first passes into 
 a soluble modification (coagulable by heat at the remarkably low temperature 
 of 40° C.) before myosin is formed. This is shown in a diagrammatic way 
 in the following scheme : — 
 
 Proteins of the Uving muscle 
 
 Paramyosinogen Myosinogen 
 
 I 
 Soluble myosin 
 
 Myosin 
 (the protein of the musole-clot). 
 
 8. When a muscle is gradually heated, at a certain temperature it con- 
 tracts permanently and loses its irritability. This phenomenon is known as 
 heat-rigor, and is due to the coagulation of the proteins in the muscle". If 
 a tracing is taken off the shortening, it is found that the first shortening occurs 
 at the coagulation temperature of paramyosinogen (47-50° C), and if the 
 heating is continued a second shortening occurs at 56° C, the coagulation 
 temperature of myosinogen. If frog's muscles are used there are three 
 shortenings — namely, at 40°, 47°, and 56° C. ; frog's muscle thus contains 
 an additional protein which coagulates at 40° C. This additional protein 
 may be the soluble myosin alluded to above, some of which, in the muscle 
 of cold-blooded animals, is present before rigor-rrwrtis occurs ; at any rate, it 
 has the same coagulation temperature. 
 
 In addition to the proteins mentioned, there is a small quantity of nucleo- 
 protein. 
 
 9. Involuntary Muscle. — The main facts just described for voluntary 
 are true also for involuntary muscle. The chief distinction lies in the quantity 
 of nucleo-protein, which is more abundant in those forms of muscles the fibres 
 of which are least different from the mesoblastic cells from which all ultimately 
 originate. This may be readily shown by the following simple experiment. 
 
 Take equal parts of voluntary muscle, heart muscle, and plain muscle 
 (say from the stomach wall), and extract each for the same time with equal 
 amounts of 0'15 per-cent. solution of sodium carbonate. Filter and add to 
 
MUSCLE 
 
 247 
 
 each filtrate acetic acid, drop by drop. The extract of voluntarj' muscle 
 gives an opalescence only ; in the case of the plain muscle there is sn 
 abundant precipitate ; the heart muscle gives a result intermediate between 
 the other two. 
 
 10. Pigments of Muscle :— 
 
 (n) Notice the difference between the red and pale muscles of a rabbit 
 from which all the blood has been washed out previouslj'. 
 
 (6) Examine a piece of red muscle (e.f/. the diaphragm) spectroscopically 
 for osyheemoglobin (or it may be more convenient to make an aqueous 
 extract of the muscle and examine that). 
 
 (c) A piece of the pectoral muscle of a pigeon has been soaked in glycerol. 
 Press a small piece between two glass slides, and place it in front of the 
 s]jecfcroscope. Observe and map out the bands of myohaematin. This pig- 
 ment is doubtless a derivative of liEemoglobin. 
 
 3 
 
 
 '.>^J.:- >■':*' 
 
 ':;^; 
 
 Fig. 52. — 1, Absorption spectrum of m_ l . II ired transparent 
 
 by glycerol. 2, Absorption spectrum of modified myohsematin. 
 
 (d) Pieces of the same muscles have been placed in ether for twenty-four 
 hours. The ether dissolves out a yellow lipochrome from the adherent fat. 
 A watery fluid below contains modified myohsematin. Filter it ; compare 
 its spe'ctrum with that of reduced hsematin. The mj'ohsematin bands are 
 rather nearer the violet end of the spectrum (fig. 52, spectrum 2) than those 
 of reduced hsematin (fig. 49, spectrum 9). 
 
 11. Creatine : — 
 
 (a) Take some of the red fluid described in 10 d, and let it evaporate to 
 dryness in a desiccator over sulphuric acid. 
 
 In a day or two, crystals of creatine tinged with myohaematin separate 
 out. 
 
 (6) Take an aqueous extract of muscle, such as Liebig's extract of beef- 
 tea ; add baryta water to precipitate the phosphates, and filter. Remove 
 excess of baryta by a stream of carbonic acid ; filter off the barium carbonate 
 Und evaporate the filtrate on the water-bath to a thick syrup. Set it aside 
 
248 
 
 t;SSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 to cool, and in a few days a crystalline deposit of creatine will be found at 
 the bottom of the vessel. This is washed with alcohol and dissolved in hot 
 water. On concentrating the aqueous solution, crystals once more separate 
 out, which may be still further purified by recrystallisation. 
 
 NERVOUS TISSUES 
 
 The chemical investigation of nervous tissues is not well adapted to class 
 exercises; still it may not be uninteresting to state briefly the principal 
 known facts in relation to this subject. The most important points which 
 any table of analyses will show are: (1) the large percentage o/ water, 
 especially in the grey matter ; (2) the large percentage of protein. In grey 
 matter, where the cells are prominent structures, this is most marked, and of 
 the solids, protein material here comprises about half of the total. The 
 following are some analyses which give the mean of a number of observations 
 on the nervous tissues of human beings, monkeys, dogs, and cats : — 
 
 
 
 
 Percentage of 
 
 
 Water. 
 
 Solids. 
 
 Proteins in 
 Solids. 
 
 CeVebral grey matter 
 
 83-5 
 
 16-5 
 
 51 
 
 ,, white ,, 
 
 69-9 
 
 •301 
 
 33 
 
 Cerebellum .... 
 
 79-8 
 
 20-2 
 
 42 
 
 Spinal cord as a whole 
 
 71-6 
 
 28-4 
 
 31 
 
 Cervical cord 
 
 72-5 
 
 27 -.5 
 
 31 
 
 Dorsal cord 
 
 69-8 
 
 30-2 
 
 28 
 
 Lumbar cord 
 
 72-6 
 
 27-4 
 
 33 
 
 Sciatic nerves . 
 
 65-1 
 
 34-9 
 
 29 
 
 The most important protein is nucleo-protein ; there is also a certain 
 amount of globulin, which, like the paramyosinogen of muscle, is coagulated 
 by heat at the low tempei-ature of 47° C. A certain small amount of neuro- 
 keratin (especially abundant in white matter) is included in the foregoing 
 table with the proteins. The granules in nerve cells (Nissl's bodies) are 
 nucleo-protein in nature. 
 
 Heat Contraction in JNerve. — A nerve, when heated, shortens ; this shorten- 
 ing occurs in a series of steps, which, as in the case of muscle, take place at 
 the coagulation temperatures of the proteins present! The first step in the 
 shortening occurs in the frog at about 40°, in the mammal at about 47°, and 
 in the bird at about 52° C. The nerve is killed at the same tenyjeratures. 
 
 Lipoids.— Alter the proteins, the next most abundant substances present 
 are the lipoids. A fuller consideration of these substances is given in Lesson 
 IV (see pp. 37-40). They comprise :— 
 
 1. Phosphatides ; of these, lecithin is the best known ; kephalin and 
 
 sphingomyelin are others. 
 
 2. Galactosides ; these are nitrogenous glucosides free from phosphorus ; 
 
 they yield on hydrolysis the reducing sugar galactose. 
 
Medullated 
 Nerve. 
 
 25 
 
 Non-medullated 
 Nerve. 
 
 47-0 
 
 2-9 
 
 9-8 
 
 ■ 12-4 
 
 23-7 
 
 18-2 
 
 6-0 
 
 NERVOUS TISSUES 249 
 
 3. Cholesterol ; a crystalline monohydrio alcohol free from both nitrogen 
 
 and phosphorus. 
 The following are some analyses of nerve by Falk ; the numbers given 
 are percentages of the total solids : — 
 
 Cholesterol 
 Lecithin 
 Kephalin 
 Galactosides 
 
 Fresh nervous tissues are alkaline, but, like most other living structures, 
 they turn acid after death. The change is particularly rapid in grey matter. 
 The acidity is due to sarcolacftic acid. 
 
 Finally, there are smaller quantities of other extractives and a small pro- 
 portion of mineral salts (about 1 per cent, of the solids). Potassium salts, as 
 in muscle, are stated to be the most abundant salts. Macallum uses for the 
 micro-chemical detection of potassium the following reagent : — Cobalt nitrite 
 20 gr., sodium nitrite 35 gr., glacial acetic acid 10 c.c, water up to 100 c.c. 
 This precipitates in situ the yellow hexanitrite of cobalt, sodium and potas- 
 sium, which is turned black on the addition of ammonium sulphide. His 
 principal results are : Potassium is found in cell protoplasm, but more abun- 
 dantly in intercellular material ; in striped muscle it is limited to the dark 
 bands, and in pancreatic cells to the granular zo^e. It is not discoverable in 
 any nuclei, nor in nerve cells, but in nerve-fibres it is found in patches external 
 to the axis cylinder. Macdonald points out that these are spots which have 
 been injured, and it is apparently only on injury that the potassium is liberated 
 in a form which renders it detectable by Macallum's reagent. Macdonald 
 attributes many of the phenomena of nervous action to electrolytic changes 
 in the potassium salts of the nerve-fibres, and which are present in large 
 amounts, possibly in combination with the colloid materials of the axon. 
 
 Very little is known of the chemical changes nervous tissues undergo 
 during activity. We know that oxygen is very essential, especially for the 
 activity of grey matter ; cerebral anaemia is rapidly followed by logs of 
 consciousness and death. Similar respiratory exchanges, though less in 
 amount, occur in peripheral nerves. It can hardly be doubted that the 
 lipoids, and especially the phosphatides, which are extremely labile substances, 
 participate in metabolism. 
 
 Cerebro-spinal Fluid. — This is secreted by the epithelium which covers 
 the choroid plexuses (choroid gland), and plays the part of the lymph of the 
 central nervous system. It is a very watery fluid, containing, besides some 
 inorganic salts similar to those of the blood, a trace of protein matter (globulin) 
 and a small amount of sugar. It contains no choline or cholesterol normally, 
 and is practically free from cells, except in disease. 
 
 Chemistry of Nerve Degeneration. — In Wallerian degeneration of nerve 
 several investigators have attempted to discover how the degenerated nerve 
 differs from a healthy nerve. Little or no change in the peripheral end can 
 be detected, up to about three days after a nerve has been divided, and the 
 nerve-fibres remain excitable up to that time. They then show a progressive 
 
250 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 increase in the quantity of water, and a corresponding decrease in the pro- 
 portion of solids. The percentage of phosphorus also decreases, and it 
 entirely disappears in a little more than three weeks after the nerve is cut. 
 When regeneration occurs, the nerves returi; approximately to their previous 
 composition. 
 
 It has also been shown that in spinal cords in which a unilateral degenera- 
 tion of the pyramidal tract has been produced by a lesion in the opposite 
 cerebral hemisphere there is a similar increase of water and diminution of 
 phosphorus on the degenerated side. Further, in a divided nerve Noll has 
 shown that the phosphorised material also diminishes somewhat in the 
 central end, due to " disuse atrophy." 
 
 The disappearance of phosphorus must be due to the break-up of phos- 
 phatides, and the liberation of phosphoric acid which is carried away as 
 phosphates by the lymph and blood. 
 
 The staining reactions of a degenerated nerve also indicate that the appear- 
 ances are not only due to a breakdown in an anatomical sense, but in a chemical 
 sense also. Of these staining reactions the one most often employed is that 
 which is associated with the name of Marchi. This is the black staining which 
 the medullary sheaths of degenerated nerve-fibres show when, after being 
 hardened in Miiller's fluid, they are treated with Marchi's reagent, a mixture of 
 Muller's fluid, and osmic acid. HeS,lthy nerve-fibres are not blackened by this 
 reagent, because the more rapidly penetrating chromic acid of the Muller's 
 fluid has already supplied the unsaturated oleic acid radical in the lecithin and 
 other phosphatides with all the oxygen it can take up. But when the nerve ' 
 is degenerated, the oleic acid is either increased in amount, or so liberated from 
 its previous combination in the lecithin molecule, that it is then able also to 
 take oxygen from osmic acid and reduce it to a lower black oxide. In the later 
 stages of degeneration the Marchi reaction is not obtained, because the fat 
 globules have been absorbed. 
 
 In certain diseases of the central nervous system, such as General Paralysis 
 of the insane, degeneration occurs on a large scale, and the products of the 
 chemical disintegration of the cerebral tissue have been sought for in the blood 
 but with more profitable results in the cerebro-spinal fluid. This fluid under 
 these circumstances shows an excess of protein which is mainly nucleo-protein ; 
 cholesterol can also be usually detected in the fluid, and so also can choline 
 or similar bases which originate from the decomposition of phosphatides. 
 Although many physiologists have taken up the choline question and the 
 methods for identifying this base, it must be admitted that the tests hitherto 
 devised are not absolutely conclusive, for sufficient of the base cannot be 
 collected for a, complete analysis. The base which is present if not choline is 
 a nearly related substance, perhaps a derivative of choline, and according to 
 the latest view the questionable material is trimethylamine, which we have 
 already seen is a cleavage product of choline. 
 
 The tests employed to detect choline are mainly three : — (1) The fluid is 
 diluted with about five times its volume of absolute alcohol and the precipitated 
 proteins are filtered oti'. The filtrate is evaporated to dryness at 40° C, and 
 the residue dissolved in absolute alcohol and filtered ; the filtrate from this is 
 again evaporated to dryness, and again dissolved in absolute alcohol, and this 
 
THE URINE 251 
 
 should be again repeated. To the final alcoholic solution, an alcoholic solu- 
 tion of platinum chloride is added, and the precipitate so formed is allowed to 
 settle and is washed with absolute alcohol by decantation ; the precipitate is 
 then dissolved in 15 per-cent. alcohol, filtered, and the filtrate is allowed to 
 slowly evaporate in a watch-glass at 40° 0. The crystals can then be seen with 
 the microscope. They are recognised not only by their yellow colour and 
 octahedral form, and by their solubility in water and 15 per-cent. alcohol, but 
 also by the fact that on incineration they yield 31 per cent, of platinum and 
 give off the odour of trimethylamine. There is a danger of mistaking such 
 crystals for those obtained from the chlorides of potassium and ammonium ; 
 but the presence of such contaminations may be minimised by the use of 
 alcohol as water-free as possible. The crystals of choline platinum chloride 
 are doubly refracting, whereas the platinum chlorides of ammonium and 
 potassium are not. (2) The following test is distinctive of choline and 
 leads to no risk of confusion with other substances. The final alcoholic 
 solution prepared as above is evaporated to dryness, and the residue taken up 
 with water, to this is added a strong solution of iodine (2 grammes of iodine 
 and 6 grammes of potassium iodide in 100 c.c. of water). In a few minutes 
 dark-brown prisms of choline periodide are formed. These look very like 
 hsemin crystals. If the slide is allowed to stand so that the liquid gradually 
 evaporates, the crystals slowly disappear, and their place is taken by brown 
 oily droplets, but if a fresh drop of the iodine solution is added the crystals 
 slowly form once more. (3) A physiological test, namely, the lowering of 
 arterial blood-pressure (partly cardiac in. origin, and partly due to dilatation 
 of peripheral vessels), which a saline solution of the residue of the alcoholic 
 extract produces : this fall is abolished, or even replaced by a rise of arterial 
 pressure, if the animal has been atropinised. Such tests hav^ already been 
 shown to be of diagnostic value in the distinction between organic and so-called 
 functional diseases of the nervous system. 
 
 LESSON XXII 
 
 THE UEINE 
 
 TOTAL NITROGEN, UREA, AND AMMONIA 
 
 Kjeldahl's Method of Estimating Total Nitrogen.— This simple method 
 can be used in connection with most substances of physiological importance. 
 Briefly, it consists in converting all the nitrogen present into ammonium 
 sulphate by means of sulphuric acid ; then rendering alkaline with soda, 
 and distilling over the ammonia into standard acid, the diminution in acidity 
 of which measures the amount of ammonia present. 
 
 The following modification of the original method is used in this 
 laboratory. 
 
 About 1 gramme of the substance under investigation (or in the case of 
 urine when one wishes to make an estimation of total nitrogen, 5 or 10 c.c. 
 
252 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 of that fluid) is placed in a round-bottomed Jena flask of about 250 c.c. capacity, 
 and 20 c.c. of pure sulphuric acid are added. Six grammes of potassium 
 sulphate and about half a gramme of copper sulphate are also added. The 
 flask should be provided with a loose balloon stopper, and arranged in a sloping 
 direction over a small flame. The mixture is heated slowly until it boils. In 
 about twenty minutes the fluid becomes nearly colourless ; boiling is continued 
 for another forty -five minutes. By this time all the nitrogen will be in com- 
 bination as ammonia. 
 
 After cooling, the fluid is washed into a litre flask of Jenaglass (fig. 53, A) and 
 water added until the total volume of the fluid is about 400 c.c. Add then an 
 excess of 40 percent, caustic soda solution, a few pieces of granulated zinc to 
 avoid bumping in the subsequent distillation, and immediatelyfitthe glass tube 
 B into the neck of the flask by means of a well-fitting rubber stopper. The 
 other end of B leads into the flask C which contains a measured amount (50 or 
 100 c.c ) of standard sulphuric acid ; i-normal acid is a convenient strength 
 to use. The bulb D shown in the figure guards against regurgitation, and the 
 end of the tube should dip just below the surface of the acid 
 in C. The mixture in the flask is now boiled for about 
 half an hour, when all the ammonia will have distilled 
 over ; the use of a condenser around the tube B is 
 unnecessary. The acidity of the standard acid is then 
 determined by titrating with standard alkali, a few drops 
 of lacmoid being added to act as the indicator of the end 
 of the reaction. ■ This gives a pink colour with acid, blue 
 with alkali. 
 
 Pig. 63.— Kjeldahi's Example. — Suppose 1 gramme of a nitrogenous sub- 
 
 ^pSue'!'^'"''''^ stance is taken, and the ammonia distilled over into 
 100 c.c. of J -normal sulphuric acid. This is then titrated 
 with a corresponding solution of sodium hydrate, and it is found that 
 the neutral point is reached when 60 c.c. of the soda solution have been 
 added. The other 40 c.c. must, therefore, have been neutralised by the 
 ammonia derived from the substance under investigation. This 40 c.c. of 
 acid = 8 c.c. of normal acid = 8 c.c. of normal ammonia = 8x0'017 = 0136 
 gramme of ammonia. One gramme of the substance analysed, therefore, 
 yields O'lSG gramme of ammonia, and this dontains 0'112 gramme of nitrogen ; 
 100 grammes will therefore contain 1 1 '2 grammes of nitrogen. If the strength 
 of the acid is that just recommended each c.c. corresponds to 0"0028 gramme 
 of nitrogen. 
 
 ESTIMATION OF AMMONIA IN UEINE 
 
 In all the exact methods the ammonia is set free by alkali, and absorbed by 
 standard acid. As boiling with strong alkalis splits ofi' ammonia from other 
 urinary constituents, Schlossing originally used lime-water as the alkali and 
 allowed the ammonia to be absorbed by decinormal acid under a bell- jar. This 
 method occupies three or four days for its 'completion, but may be carried out 
 more rapidly by distilling off the ammonia in vacuo (Wurster's, Nencki's, and 
 other methods). Folin's method avoids these drawbacks by driving off the 
 ammonia by means of an aii' current. 
 
ESTIMATION OF AMMONIA IN URINE 
 
 253 
 
 1. Folin's Method. — Twenty-five c.c. of urine are measured into a tall 
 cylinder (fig. 54, B), 1 gramme of anhydrous sodium carbonate and 10 c.c. 
 kerosene oil (to prevent frothing) are added, and air is drawn through the 
 apparatus for two hours. The air current is previously passed through acid 
 in the bottle A in gorder to remove ammonia from it. The ammonia set 
 free from the urine is carried away and absorbed in the absorption bottle C, 
 which contains 20 c.c. of decinormal sulphuric acid. After two hours the 
 ammonia is estimated by titrating the contents of the absorption bottle 
 with decinormal alkali. The number of c.c. decinormal alkali subtracted 
 from 20 gives the number of c.c. of decinormal acid neutralised by ammonia. 
 1 c.c. of decinormal acid = 0'0017 gramme of ammonia. Even with this 
 method at least two houi'S are necessary. In Folin's most recent method 
 the, time is reduced to fifteen minutes by only taking 2 c.c. of urine, and 
 estimating the ammonia colorimetrically by means of Nessler's reagentl' 
 
 2. The Formalin Method. — This method is an adaptt^tion to urine of 
 Sorensen's method for the estimation of 
 
 amino-acids (see p. 232). When neutral 
 solutions of ammonium salts are treated with 
 an excess of formaldehyde, the compound 
 hexamethylene tetramine (urotropine) is 
 formed, and a corresponding amount of 
 acid is set free from the ammonium salt 
 (4NH4CI + 6CH2O = N_(CH2)e + 6H2O + 4HC1) 
 which can be titrated in the usual way. 
 
 This method, when applied to urine, does 
 not give exact results, because the small 
 amounts of amino-acids present in the urine 
 also enter into the reaction. It is, how- 
 ever, of value as a rapid clinical method for the estimation of ammonia. 
 
 Twejity-five c.c. of urine are measured into a flask; 15 grammes finely, 
 powdered potassium oxalate^ and a few drops of phenolphthalein solution (as 
 an indicator) are added. Shake for a few minutes and titrate with decinormal 
 alkali. (The number of c.c, required furnishes at the same time data for 
 calculating the acidity of the urine.) Now add 10 c.c. of formalin ' diluted 
 with 10 c.c. water and neutralised with decinormal alkali (using phenolphthalein 
 as an indicator). The pink colour disappears on mixing the urine and the 
 formalin solution. Run in as much of the decinormal alkali as is necessary 
 to bring back the same tint as before. 
 
 Fig. 54,— Folin's apparatus for 
 estimating ammonia. 
 
 1 C.C. ^■NaOH = 0-0017 gramme NH3, 
 
 ' Nessler's reagent is an alkaline solution of mercuric iodide, which gives a 
 characteristic yellow colour with traces of ammonia. 
 
 ^ Potassium oxalate is added to precipitate calcium salts, which interfere with 
 the end point. 
 
 ^ Formalin is a commercial name for a solution containing 40 per cent, of 
 formaldehyde. 
 
254 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 PREPARATION OF UREA FROM URINE 
 
 Evaporate ahout 50 c.c. of urine to a small bulk and finally to complete 
 dryness on the water-bath at boiling temperature. Turn out the flame and 
 extract the residue with about 10 c.c. of acetone. The dish may be replaced 
 on the water-bath, which will still be quite hot enough to boil the acetone. 
 Pour off the hot acetone extract into a dry watch-glass or evaporating basin. 
 On cooling, urea crystallises out in silky needles. The acetone quickly eva- 
 porates spontaneously, and a crystalline mass of urea is left. Dissolve some 
 of this in water ; place a drop of the aqueous solution on a slide, and allow it 
 to crystallise ; examine the crystals with the microscope. Place another 
 drop on another slide, and add a drop of nitric acid ; examine microscopically 
 the crystals of urea nitrate which separate out. 
 
 ESTIMATION OF UREA 
 
 A great many methods have been proposed for the purpose of urea estima- 
 tion, which have all been modified repeatedly. The enzymatic method, 
 depending on the action of the enzyme urease contained in soy-beans, 
 promises to replace all the older methods. All the methods are indirect, 
 i.e. the urea is decomposed in some way, and the quantity of one of the 
 decomposition products is estimated. According to the nature of this 
 decomposition the methods may be divided into two main groups : — 
 
 1. Methods which are based on the Decomposition of Urea into 
 Nitrogen, Carbonic Acid, and Water. — The hypobromite method (see p. 
 180) is a type of these methods, which have been abandoned for accurate work 
 owing to the fact that (1) even pure urea solutions yield variable amounts of 
 nitrogen, and (2) other urinary constituents (ammonia, uric acid, creatinine, 
 allantoin, etc.) also yield nitrogen under the conditions of the experiment. 
 
 2. Methods which are based on the Decomposition of Ur«a into 
 Ammonia and Carbonic Acid. — This decomposition is brought about in 
 several ways. As the urine contains preformed ammonia, this has to be 
 estimated previously, and deducted from the total ammonia found. 
 
 (a) 'J'/ii Urease Method. — This method was introduced by Marshall, who 
 made use of the discovery by Takeuchi of the enzyme urease in soy-beans. 
 Urease is a specific enzyme which rapidly and quantitatively decomposes 
 urea at 35-40° into ammonia and carbonic acid. Marshall has described two 
 methods of utilising urease for the determination of urea in urine. One 
 method consists in adding the enzyme solution (an aqueous extract of soy- 
 beans) to urine and titrating the increased alkalinity with methyl orange as 
 indicator. The other method, which is more accurate, consists in adding the 
 enzyme to 1 c.c. of urine plus 10 c.c. of water, and driving off the ammonia by 
 Folin's aeration method (see p. 253). 
 
 Instead of an aqueous extract of soy-beans, the finely ground beans as 
 such may be used (Plimmer and Skelton), or preferably the commercial urease 
 powder, which is prepared by precipitating aqueous extracts of the beans with 
 acetone (Van Slyke and Cullen). The ammonium carbonate formed during 
 the reaction retards by its alkalinity the action of the enzyme. This can be 
 prevented by the addition of acid phosphate, which is present in the 
 commercial product in the proper proportions. 
 
ESTIMATION OF UREA 255 
 
 Aiialysis. — The apparatus described in Folin's method of estimating 
 ammonia in urine (fig. 54, p. 253) is used. Measure 5 c.c. of urine into the tall 
 cylinder B, add 50 to 60 c.c. of water, 1 gramme of finely ground soy-bean, and 
 ^bout 2 c.c. of liquid parafiin to prevent frothing. Connect the cylinder with 
 the absorption apparatus C, containing 50 c.c. of decinormal H2SO4, and the 
 wash bottle A (containing acid to remove the ammonia from the air current). 
 The cylinder B is kept at a temperature of 35-40° in a water-bath and an 
 air current is drawn through the series. After about an hour the rubber 
 connections are disjointed and 1 gramme of anhydrous sodium carbonate is 
 dropped into the cylinder in order to set free ammonia which may be present 
 as ammonium salt. The connections are again made, and the air current is 
 then drawn through for another hour. After this time the excess of the 
 acid in the absorption vessel is titrated with decinormal KOH, using alizarin 
 red or methyl orange as indicator. The result includes the anjmonia pre- 
 formed in the urine, and this should be estimated separately and subtracted. 
 
 The urease method is also valuable for the estimation of urea in blood, 
 because its action is so specific that it attacks none of the other constituents, 
 and the blood has to undergo no preliminaiy treatment for the removal of 
 the latter. 
 
 Crilriihitioti.— From the formula C0(NH,)2 -I- HgO = COg -f 2NH., it follows 
 that one molecule of urea furnishes two molecules of ammonia, and that 1 c.c. 
 of decinormal HgSOj corresponds to O'OOS gramme of urea. 
 
 Of the older methods those of Benedict and of Folin seem to be the most 
 satisfactory. 
 
 (6) Benedict's Method. — In this method the urine is heated with potas- 
 sium bisulphate and zinc sulphate to 165° C. for one hour. The fluid is 
 diluted, made alkaline, and the ammonia distilled off as inKjeldahl's method. 
 Five c.c. of urine are introduced into a rather wide Jena test-tube, and about 
 3 grammes of potassium bisulphate and 1-2 grammes zinc sulphate are added. 
 A bit of paraffin and a little powdered pumice are introduced to prevent 
 frothing and the mixture boiled practically to dryness, either over a free 
 flame or in a sulphuric acid bath kept at about 130° C. The tube is then 
 immersed in a sulphuric acid bath maintained at 162° and left there for one 
 hour. The contents are transferred with distilled water into a distilling 
 apparatus, made alkaline with sodium carbonate, and distilled as desci'ibed 
 under Kjeldahl's method. When the distillation is conipleted, the standard 
 acid in the receiver should be boiled to remove CO2 before titration. 
 
 N 
 1 c.c. y- H2S0j = 0028 gramme N = 0'00G gramme urea. 
 
 (c) Folin's Method. — In this method only a very smajl quantity of urine 
 is used (1 c.c. of the ten times diluted uiine, containing from 0'75-r5 milli- 
 grammes of urea nitrogen). The decomposition into ammonia and CO2 is 
 brought about by heating with potassium acetate and acetic acid to 155° C. 
 
 for ten minutes.' Caustic alkali is added and the ammonia is aspirated into — 
 
 ' A temperature indicator is used in this prooegs, which consists of a sealed tube 
 containing powdered chloride-iodine of mercury, a salt which isbright red and melts 
 to a clear dark red fluid at 155° 0. The sealed tube is heated together with the 
 urine and the potassium acetate mixture. 
 
256 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 acid. As the quantity of ammonia is too small to be titrated accurately, it is 
 estimated colorimetrically, as described previously (see Ammonia estimation, 
 p. 253). 
 
 LESSON XXIII 
 
 THE URINE {eontrnned) 
 
 URIC ACID AND CREATINmE 
 
 Preparation of Pure Uric Acid. — This may best be done by taking the 
 solid urine of a bird or a snake (which consists principally of the acid 
 ammonium urate) ; one has not then to separate any pigment. 
 
 The material is boiled with 10 per-cent. caustic soda or ammonia, diluted, 
 and then allowed to stand. The clear iluid is decanted and poured into a large 
 excess of water to which 10 per cent, of hydrochloric acid has been added ; the 
 crystals of uric acid which form are filtered off after standing twenty-four 
 hours. These may be purified by washing, re-solution in soda, and re-pre- 
 cipitation by acid. 
 
 ESTIMATION OF URIC ACID 
 
 In human urine, the normal amount of uric acid (in the form of urates) 
 excreted in the day varies from 0'4 to 8 gramme. If the daily volume of 
 urine is taken as 1.500 c.c, the percentage of uric acid will therefore be from 
 0026 to 0052. 
 
 The Folin-Sliafifer Method. — Hopkins's method referred to in the text 
 (p. 199) is now generally replaced by the modification of it introduced by 
 Folin and Shaffer. The chief alteration introduced is the substitution of 
 ammonium sulphate for ammonium chloride as a precipitant. This is first 
 added in an acid sol'ution ; this precipitates a mucoid from the urine which 
 interferes with the subsequent titration ; the urine is then rendered alkaline 
 and the uric acid comes down as ammonium urate. The Folin-Shaffer 
 reagent also contains ui'anium acetate ; this precipitates the phosphates, 
 and thus facilitates filtration and removal of the mucoid. 
 
 Analysis.— To 200 c.c. of urine add 50 c.c. of the FoKn-Shaffer reagent 
 (500 grammes ammonium sulphate, 5 grammes uranium acetate, 60 c.c. of 
 10 per-cent. acetic acid in 650 c c. of water ; the volume of the solution is 
 then almost exactly 1 litre). Allow the mixture to stand for some minutes 
 and then filter through a double dry filter into a dry flask. Measure 125 c.c. 
 of the filtrate (corresponding to 100 c.c. urine) into a beaker, and add 5 c.c. of 
 concentrated ammonia ; allow this to stand for twenty-four hours. Decant 
 the fluid from the precipitate through a small filter ; wash the precipitate on 
 to the filter with 10 per-cent. solution of ammonium sulphate, and repeat 
 the washing twice more to remove the chlorides. Rinse the precipitate back 
 into the beaker with almost 100 c.c. of hot water ; cool, and then add 15 c.c. 
 of concentrated sulphuric acid, and titrate, at once without cooling with ir'-o- 
 normal potassium permanganate solution (1'581 gramme potassium perman- 
 
ESTIMATION OF URIC ACID 257 
 
 ganate to I litre of water) until a permanent rose colour is obtained. The 
 number of c.c. of the permanganate solution used from the burette, multiplied 
 by 375, gives the quantity of uric acid in milligrammes of 100 c.c. of urine. 
 Three milligrammes should be added to the result to allow for the solubility 
 of ammonium urate in the reagents used. 
 
 Folin's a^d Macallum's Colorimetric Method.— In this method the 
 blue colour which is produced by the action of phosphotungstic acid on uric 
 acid is made use of for the quantitative estimation of the latter by comparing 
 it in a colorimeter with a standard solution of uric acid similarly treated. 
 Since the colour reaction is very delicate, only a very small volume of urine 
 is required for analysis. The method is only applicable to normal urines. 
 For pathological urines (diabetic, albuminous) a modified method has been 
 worked out by Folin and Denis. "With some modifications it can, also be 
 applied for the estimation of uric acid in blood. 
 
 From 2-5 c.c. of urine (depending on the specific gravity), say 3 c.c, are 
 measured into a 100 c.c. beaker, and after adding a drop of saturated oxalic 
 acid solution the whole is evaporated to dryness on the water-bath. To 
 the dry, cool residue, are added 10-15 c.c. of a mixture of 2 parts of dry 
 ether and 1 part of methyl alcohol. After standing for a few minutes, the 
 solution is decanted and the residue extracted once more in the same way. 
 By these means certain substances (polyphenols) are removed, which also 
 give a blue colour with phosphotungstic acid. (For ordinary class work 90 
 per-cent. alcohol can be substituted for the ether-alcohol mixture, although 
 the slight solubility of uric acid in it introduces a small source of eri'or.) 
 
 To the washed residue in the bottom of the beaker is next added water 
 (5-10 c.c.) and a drop of saturated sodium carbonate solution, and the mixture 
 IS shaken so as to secure complete solution. Add 2 c.c. of the phosphotungstic 
 acid solution,' and 20 c.c. of a saturated solution of sodium carbonate. The 
 resulting blue solution is transferred to a 100 c.c. measuring flask, diluted with 
 water up to the mark, and poured into one of the tubes of the colorimeter 
 (see next page). The second tube is filled with the standard solution for 
 comparison, which is obtained by treating 1 milligramme of uric acid in 0'4 
 per-cent. lithium carbonate solution with phosphotungstic acid in exactly the 
 same proportions.^ 
 
 ' The solution is prepared by boiling 100 grammes of sodium tungstate with 
 80 c.c. of 85 per-cent. phosphoric acid and 750 c.c. of water for a couple of hours 
 and then diluting to 1 litre. 
 
 ^ This standard solution must be freshly prepared, and in order to avoid this 
 inconvenience, Folin and Denis have introduced a uric acid formaldehyde 
 solution which keeps. One gramme of uric acid in a litre flask is dissolved 
 in 200 c.c. of 0'4 per-cent. solution of lithium carbonate. To this 40 c.c. of 40 
 per-cent. formaldehyde are added, and the mixture shaken and allowed to stand 
 for a few minutes. It is then acidified with 20 c.c. of normal acetic acid, and the 
 whole diluted with water to 1 litre. Next day it is standardised against a freshly 
 prepared lithium carbonate solution of uric acid. The colour produced by 5 c.c. 
 of the solution coriesponds very nearly with the colour obtained from 1 milli- 
 gramme of uric acid. The colorimetric reading obtained for the solution when 
 thus compared against 1 milligramme of pure uric acid is, of course, therefore to 
 be used as the standard value corresponding to I milligramme of uric acid. Quite 
 recently Folin has recommended a solution of uric acid in 10 per-cent. sodium 
 sulphite as the most trustworthy standard. 
 
 17 
 
258 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 Calculation.— Be.t the standard at a depth of a mm. Equality of tint is 
 obtained, when the depth of the unknown solution is b. Therefore 3 c.c. of 
 
 urine originally taken for analysis contain y milligrammes uric acid, and 
 
 100 c.c. contain — -= — 
 3o 
 
 ESTIMATION OF CREATININE 
 
 The following colorimetric method (Folin's) is now generally employed for 
 the estimation of creatinine ; and with a slight modification it may also 
 be used for the estimation of creatine. It is based on the red colour 
 which has been shown by Jaflfe to develop when an alkaline solution of 
 picric acid is added to a solution of creatinine ; this is compared with the 
 colour of a standard solution of potassium bichromate, the tint of the two 
 fluids being almost identical. If creatine has to be estimated, this is first 
 transformed into creatinine by boiling with hydrochloric acid. The 
 apparatus and reagents necessary are : — 
 
 1. A colorimeter consisting of two tubes, the height of the column in 
 which can be read by graduations in tenths of a millimetre. 
 
 2. A half -normal solution of potassium bichromate (24-5 grammes per litre). 
 
 3. A saturated solution of picric acid. 
 
 4. 10 per-cent. caustic soda. 
 
 To perform the analysis one tube of the colorimeter is filled with the bi- 
 chromate solution up to the height of 8 mm. 10 c.c. of urine are measured 
 into a J-litre flask, 15 c.c. of the picric acid solution and 5 c.c. of the caustic 
 soda solution added ; after standing five minutes water is added until the total 
 volume of the mixture is 500 c.c. This solution is poured into the second tube 
 of the colorimeter to such a height (which is read ofi') that, on looking down 
 through it, the intensity of the colour is the same as that in the standard tube 
 by its side. Folin found that a layer 8 mm. deep of the standard solution 
 has the same colour as a layer 8"! mm. deep of a solution prepared from 10 
 milligrammes of pure creatinine, picric acid, and caustic soda. The calculation 
 for X, the number of milligrammes of creatinine in the urine, is therefore 
 
 8"1 
 a; = 10 X — , where a is the millimetre depth of the unknown solution which' 
 a 
 
 matches 8 mm. of a standard bichromate solution. 
 
 LESSON XXIV 
 
 THE UEINE {continued) 
 
 THE INORGANIC SALTS 
 
 The principal inorganic salts in urine are chlorides, sulphates, and phos- 
 phates. 
 
 The chlorides consist chiefly of those of sodium and potassium, the latter 
 being present in quite small quantities. The amount of sodium chloride in 
 human urine is 10-15 grammes />er diem ; taking the total volume of the dally 
 urine at 1500 c.c, this corresponds to 0"6 to 1 per cent. 
 
ESTIMATION OF CHLORIDES 259 
 
 The phosphoric acid in the urine is combined with sodium, potassium, 
 calcium, and magnesium. The total P2O5 in the twenty-four hours is about 
 3"5 grammes, of 0-24 per cent. In addition to the inorganic phosphates, there 
 are small amounts of organic compounds of phosphorus, such as glycero- 
 phosphates. The following exercises, therefore, contain one for the estimation 
 of total phosphorus. 
 
 The sulphates in the urine are of two kinds : the inorganic sulphates, 
 namely, those of sodium and potassium, and the ethereal sulphates (see p. 193). 
 In human urine about 2 grammes of total sulphuric acid are passed per diem, 
 or 013 per cent. 
 
 Although the major portion of the sulphur in the urineis present as sulphate 
 there is in addition a small quantity combined in organic compounds. It is 
 therefore necessary to add an exercise for the estimation of total' sulphur. 
 
 ESTIMATION OF CHLORIDES 
 
 Volhard!s method adopted for the determination of the total chlorides 
 consists in their precipitation by excess of a standard solution of silver nitrate 
 in the presence of nitric acid. The excess of silver is then estimated in an 
 aliquot part of the filtrate with a solution of potassium or ammonium thio- 
 cyanate, which has been previously standardised against the silver solution, a 
 ferric salt being used as indicator. 
 
 The following solutions are necessary : — 
 
 i. A standard solution of silver nitrate of such a strength that 1 c.c. corre- 
 sponds to O'Ol gramme of sodium chloride (29 075 grammes of fused silver 
 nitrate in a litre of distilled water). 
 
 ii. Solution of potassium thiocyanate (8 grammes to the litre). 
 • iii. Pure nitric acid free from chlorides; 
 
 iv. A saturated solution of iron alum. 
 
 The potassium thiocyanate solution is first standardised in the following 
 way :— 
 
 Place 10 c.c. of the silver solution in a beaker, add 5 c.c. of the nitric acid, 
 5 c.c. of the solution of iron alum, and 80 c.c. of water. Eun into this from a 
 burette the thiocyanate solution until a permanent red tinge is obtained. 
 Note the number of c.c. necessary for this purpose, and call this number x. 
 
 Analysis. — Place 10 c.c. of the urine in a 100 c.c. measuring flask with a 
 pipette ; add about 4 c.c. of pure nitric acid, and 20 c.c. of the standard 
 solution of silver nitrate. Fill up the flask to the 100 c.c. mark with distilled 
 water, mix thoroughly, and filter through a dry filter-paper into a dry vessel. 
 
 Measure out 50 c.c. (that is exactly half) of the filtrate, add 5 c.c. of the 
 iron alum solution, and titrate with the thiocyanate solution until a permanent 
 red colour is obtained. Call the number of c.c. so used a. This must be 
 doubled to represent what the total fluid (100 c.c.) would have required. 
 
 In the previous standardisation of the thiocyanate solution we found that 
 X c.c. of the thiocyanate solution is equivalent to 10 c.c. of the silver solu- 
 tion ; therefore 2a c.c. are equivalent to of the silver solution, and this 
 
 represents the amount of silver solution not used in precipitating the chlorides. 
 
260 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 1 X 2fx 
 Therefore 20- is the number of c.c. of the silver nitrate solution 
 
 X ' 
 
 utilised in the precipitation of the chlorides. 
 
 Ten c.c. of urine (the amount taken for analysis), therefore, contains the 
 
 amount of chlorides which require 20 — — c.c. of the standard silver nitrate 
 
 for their precipitation, and as each c.c. of the standard solution is equivalent 
 to O'Oi gramme of sodium chloride, the total chlorides in the 10 c.c. of urine 
 
 (expressed as sodium chloride) is ( 20 - — ) x O'OI =0'2 grammes. If 
 
 2tt 
 one multiplies this by 10, we obtain 2 - — grammes, which is the amount per 
 
 100 c.c. of urine. If the total urine passed in the day is 1500 cc, we have 
 further to multiply this by 15 to obtain the amount excreted in the twenty- 
 four hours. 
 
 ESTIMATION OF PHOSPHATES 
 
 (a) Estimation of the total phosphates. 
 
 For this purpose the following reagents are necessary : — 
 
 i. A standard solution of uranium nitrate. The uranium nitrate solution 
 contains 35'5 grammes in a litre of water ; 1 c.c. corresponds to 0'005 gramme 
 of phosphoric acid (P2O5). 
 
 ii. Acid solution of sodium acetate. Dissolve 100 grammes of sodium 
 acetate in 900 c.c. of water ; add to this 100 cc. of glacial acetic acid. 
 
 iii. Solution of potassium ferrocyanide. 
 
 Method. — Take 50 c.c. of urine. Add 5 c.c. of the acid solution of sodium 
 acetate.^ Heat the mixture to 80° 0. 
 
 Run into it while hot the standard uranium nitrate solution from a burett* 
 until a drop of the mixture gives a distinct brown colour with a drop of potas- 
 sium ferrocyanide placed on a porcelain slab. Read off the quantity of solution 
 used and calculate therefrom the percentage amount of phosphoric acid in the 
 urine. 
 
 Another indicator which may be used is cochineal tincture, a few drops of 
 which may be added to the mixture. A change of colour from red to green is 
 the sign of the end of the reaction. 
 
 (6) Estimation of the phosphoric acid combined with calcium and magnesium 
 (alkaline earths). 
 
 Take 200 c.c. of urine. Render it alkaline with ammonia. Lay the 
 mixture aside for twelve hours. Collect the precipitated earthy phosphates 
 on a filter ; wash with dilute ammonia (1 in 3). Wash the precipitate off 
 the filter with water acidified by a few drops of acetic acid. Dissolve with the 
 aid of heat, adding a little more acetic acid if necessary. Add 5 c.c. of the 
 acid solution of sodium acetate. Bring the volume up to 50 c.c, and estimate 
 the phosphates in this volumetrically by the standard uranium nitrate as 
 before. Subtract the phosphoric acid combined with the alkaline earths thus 
 
 ' Inusing uraniumnitrateitis imperative that sodium acetate should accompany 
 the titration in order to avoid the possible occurrence of free nitric acid in the 
 solution. If uranium acetate is used, it may be omitted. 
 
ESTIMATION OF TOTAL PHOSPHORUS 261 
 
 obtained from the total quantity of phosphoric acid, and the difference is the 
 amount of acid combined with the alkaline metals, sodium and potassium. 
 
 ESTIMATION OF TOTAL PHOSPHORUS 
 
 The phosphorus of the urine is mainly present in the form of inorganic 
 phosphates ; but there are in addition certain organic compounds of phos- 
 phorus such as glycero-phosphates. In order to estimate the total phosphorus, 
 the following method is the best to employ. 
 
 Estimation of total phosphorus by Neumann's method : — 
 
 This method is now in general use for the estimatioii of phosphorus in 
 organic substances, on account of the ease with which the destruction of the 
 organic matter is carried out. The solution obtained after the first stage of the 
 process serves equally well for the estimation of iron, calcium, magnesium, 
 sbdium, and potassium. An estimation of hydrochloric acid may be carried 
 out by a slight modification of the method. 
 
 Principle of the Method. — The organic matter is oxidised with a mixture of 
 equal parts of nitric and sulphuric acids. The phosphoric acid so formed is 
 then precipitated as ammonium phosphomolybdate, and this precipitate, 
 after washing it free from acid, is dissolved in excess of half -normal alkali 
 and titrated with half -normal acid. The difference multiplied by 0"553 gives 
 the amount of phosphorus in milligrammes. If multiplied by 1'268, the 
 result is expressed in terms of milligrammes of PsOo. 
 
 Analysis. — Measure 5 c.c. of the urine into a Kjeldahl flask, and add 
 20 c.c. of the acid mixture (equal parts of nitric and sulphuric acids). Heat 
 in a fume cupboard over a small flame until the evolution of brown fumes 
 ceases. Allow to cool, and add a small quantity of fuming nitric acid ; heat 
 strongly until a clear solution is obtained. After cooling add 100 c.c. of 
 distilled water, heat on the water-bath, and add 100 c.c. of a molybdic acid 
 solution.' The yellow precipitate of ammoniufn phosphomolybdate is 
 filtered and rapidly washed free from acid,^ transferred into a flask, and a 
 measured quantity of half-normal sodium hydrate added until a colourless 
 fluid results. A slight excess (5 to 6 c.c.) of the half -normal sodium hydrate is 
 added, and the solution boiled for about fifteen minutes, until all the ammonia 
 has been removed. After cooling and adding a few drops of phenolphthalein 
 
 ' The solution is prepared as follows : a solution of 75 gramme.s of ammonium 
 molybdate, in 500 c.c. of water, is poured into 500 c.c. nitric acid (250 c.c. of con- 
 centrated nitric acid and 250 o.e. of water), and 1 litre of ammonium nitrate solu- 
 tion (500 grammes dissolved in 1 litre of distilled water) is added to the mixture. 
 Various formulae for the making of this mixture are given, but the above is the 
 one used in this laboratory. 
 
 ^ It is essential that the filtration and washing should be effected rapidly. In 
 Neumann's original method of filtering through filter-paper several slight modi- 
 fications have been introduced by different authors. In this laboratory the 
 following method is used, which allows the filtration and washing to be carried 
 out within five minutes : Filter-pulp is prepared by shaking up 30 grammes of a, 
 pure filtering paper with 1 litre of water and 50 c.c. of concentrated hydro- 
 chloric acid. The pulp is filtered under pressure, well washed with boiling water 
 and then kept, suspended in 2 litres of water, ready for use. About 30 to 40 c.c. 
 of filter-paper pulp are poured into a funnel containing a small perforated porcelain 
 filter-plate, and the filtration and washing of the molybdate precipitate is carried 
 out by the help of the filter-pump. 
 
262 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 as an indicator, the pink solution is titrated with half -normal sulphuric acid 
 until it is colourless. If a = the c.o. of the sodium hydrate solution actually 
 taken, and 6 = the c.c. of the sodium hydrate solution left when the reaction 
 is over ( = the c.c. of half- normal acid added), then x=a-h, x being the 
 number of c.c. of sodium hydrate used up in the reaction with the phospho- 
 molybdate. Further, .rx0'553 = milligrammes of phosphorus present; and 
 «xl-268 = PA. 
 
 ESTIMATION OF SULPHATES 
 
 1. Gravimetric Method. 
 
 (a) Estimation of total {inorganic and ethereal) sulphates (Folin's modifica- 
 tion) : — 
 
 Twenty-five c.c. of urine and 20 c.c. of dilute hydrochloric acid (1 part of 
 hydrochloric acid to 4 parts of water by volume) are gently boiled in a flask, 
 covered with a small watch-glass, for twenty to thirty minutes. This eflfeets 
 the splitting of the ethereal sulphates and the liberation of sulphuric acid. 
 The flask is cooled for two or three minutes in running water, and the contents 
 are diluted with cold water to about 150 c.c. To this cold solution is then 
 added 10 c.c. of a 5 per-cent. solution of barium chloride without any shaking 
 or stirring during the addition. The barium chloride should be added drop by 
 drop, preferably by means of an automatic dropper. At the end of an hour 
 or later the mixture is shaken up and filtered through a weighed Gooch crucible 
 (a porcelain crucible with a perforated bottom containing a layer of asbestos). 
 The precipitate is washed with water, ignited, and weighed. The increase in 
 weight is the amount of barium sulphate obtained. 
 
 (6) Estimation-of inorganic sulphate (Folin's modification) : — 
 
 About 100 c.c. of water, 10 c.c. of the dilute hydrochloric acid, and 25 c.c. 
 of urine are measured into a flask. Ten c.c. of a 5 percent, solution of 
 barium chloride are added as described above, and the filtration, washing, 
 and weighing carried out as before. 
 
 (c) The ethereal sulphates are represented by the difference Ijetween the 
 total and the inorganic sulphates. They, may, however, also be estimated 
 directly by the following method : — 125 c.c. of urine are diluted with 75 c.c. of 
 water and 30 c.c. of the dilute hydrochloric acid. The solution is precipitated in 
 the cold by the addition of 20 c.c. of 5 per-cent. solution of barium chloride 
 as before, and the mixture is filtered, after one hour's standing, through a 
 dry filter. Inorganic sulphates are thus removed. Half of the filtrate (that 
 is, 125 c.c.) is then qtietly boiled for about thirty minutes. By this means 
 sulphuric acid is liberated from the ethereal sulphates, and this is precipitated 
 by the barium chloride present as barium sulphate, which is collected, washed, 
 ignited, and weighed as before. Double the total thus obtained will give in 
 terms of barium sulphate the amount of ethereal sulphate present in the 
 125 c.c. of urine originally taken, and from this the percentage and amount 
 passed in the twenty-four hours can be calculated. 
 
 Calculation. — 233 parts of barium sulphate correspond to 80 parts of SO3, 
 or 32 parts of S. To calculate the SO3, multiply the weight of barium sulphate 
 by A°j = 0'343; to calculate the S, multiply it by ^'A = 0'137. Example: 
 100 c.c. of urine gave 0"5 gramme of total barium sulphate ; this multiplied by 
 0'343=0'171 of total SOj. Another 100 c.c. of the^same urine gave 0'45 
 
ESTIMATION OP TOTAL SULPHUR 263 
 
 gramme of barium sulphate from inorganic sulphates; this multiplied by 
 0-343 = 0-154 of SO3 in inorganic combination. If this is subtracted from the 
 total SO3 (0'17l -0'154) we see that 0-017 gramme of SO3 was in combination 
 as ethereal sulphate, or about one-tenth of the, total, which is the average pro- 
 portion in normal urine. ' 
 
 2. Volumetric Method (Rosenheim and Drummond's benzidine method). 
 
 Principle of the Method. — The inorganic sulphates are precipitated from 
 the faintly acidified urine as benzidine sulphate by means of a solution of 
 benzidine (NHa.CeHj.CBHj.NHa) in hydrochloric acid. As benzidine is a 
 weak base its salts with acids are readily dissociated and the sulphuric acid 
 contained in benzidine sulphate may be quantitatively titrated with standard 
 alkali solutions, using phenolphthalein as an indicator. 
 
 The total sulphates (inorganic and ethereal) are similarly estimated after 
 hydrolysing the ethereal sulphates by boiling with hydrochloric acid. 
 
 The ethereal sulphates are represented by the difference between the 
 total and inorganic sulphates. 
 
 Ancdysis. — (a) Estimation of inorganic sulphates. — 20 c.c. of urine are 
 measured into a flask and acidified with dilute hydrochloric acid (1 : 4) until 
 the reaction is distinctly acid to Congo-red paper. 100 c.c. of benzidine 
 solution are then run in and the precipitate, which forms in a few seconds, 
 allowed to settle for ten minutes. (The benzidine solution is prepared by 
 rubbing 4 grammes of benzidine into a paste with water, and transferring 
 it with 500 c.c. of water into a 2-litre flask. 5 c.c. of concentrated HCl are 
 added, and the solution made up to 2 litres with distilled water.) The 
 precipitate .is filtered under pressure with the precaution of not allowing 
 at any time the precipitate to be sucked dry on the filter. This is washed 
 .with 10 to 20 c.c. of water saturated with benzidine sulphate. The precipitate 
 and filter-paper are transferred into the original precipitation flask with 
 about 50 c.c. water and titrated with decinormal potassium hydrate solution, 
 after the addition of a f^w drops of a saturated alcoholic solution of 
 phenolphthalein. 1 c.c. of decinormal KOH = 49 mg. H2SO4. 
 
 (6) Estimation of total sulphates. — Add 2 c.c. HCl (1 : 4) to 20 c.c. of 
 urine and boil gently for twenty to thirty minutes. After cooling the flask, 
 proceed exactly as under (a). 
 
 ESTIMATION OF TOTAL SULPHUR 
 Benedict's Method (Wolf and Oesterberg's modification). — In this method 
 the organic matter is destroyed by boiling the substance with fuming nitric 
 acid, and the oxidation of sulphur to sulphuric acid is completed by heat- 
 ing with copper nitrate and potassium chlorate. In the solution finally 
 obtained, sulphuric acid is estimated gravimetrically as barium sulphate. 
 
 Analysis. — Measure 1 c.c. of urine into a Kjeldahlflask of 300 c.c. capacity ; 
 add 20 c.c. fuming nitric acid and heat over a small flame. Continue to boil 
 until the fluid is free from solid particles. Transfer the solution to a porcelain 
 dish and add 20 c.c. of Benedict's solution (200 grammes copper nitrate, and 
 50 grammes potassium chlorate dissolved in 1 litre of water). Evaporate 
 to dryness on a sand-bath ; then heat over the free flame until the residue is 
 blackened by the formation of copper oxide. Eaise the flame and heat to 
 redness for ten minutes. After cooling add 25 c.c. of 10 per cent, hydro- 
 
264 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 chloric a<^d and dissolve the black residue by warming gently. The solution 
 (A) is washed into a flask with about 150 c.c. of water, and the sulphuric acid 
 estimated by means of barium chloride in the way described above (a). The 
 filtrate from the barium sulphate precipitate may be used for the estimation 
 of phosphorus by Neumann's method (see p. 261). The sulphate in solution 
 (A) may also be estimated by the benzidine method. 
 
 LESSON XXV 
 
 THE URINE {continued) 
 
 THE PIGMENTS 
 
 The urinary pigments are numerous, and have from time to time been 
 described under different names by various observers. 
 
 1. Urochrome. — This is the essential yellow pigment of the urine. 
 The word was originally introduced by Thudichum, whose investigations 
 have in the main been confirmed and supplemented by the work of Dom- 
 browski. 
 
 Preparation of Urochronne (Dombrowski). — After removal of sulphates, 
 phosphates, and urates by a mixture of barium and calcium acetates and 
 ammonia (86 grammes of calcium acetate, 5.3 igrammes of barium acetate, 
 and 4.3 c.c. of 21 per-cent. ammonia to 10 litres of urine), the filtered urine 
 is neutralised with acetic acid, and urochrome is precipitated by adding 
 copper acetate. The greenish precipitate is filtered off, well washed, and then 
 suspended in water, and the copper is removed by sulphuretted hydrogen at 
 50° C. Baryta water is added to the filtrate, and excess of barium removed 
 by a stream of carbon dioxide ; the barium salt is then concentrated in vacuo 
 and precipitated by alcohol. The precipitate is dissolved in water and freed 
 from chlorine by silver nitrate. The soluble silver salt so formed is pre- 
 cipitated by alcohol and ether, washed free from silver nitrate, dried, and 
 analysed. The free urochrome can be obtained by removing the silver with 
 sulphuretted hydrogen ; it is amorphous and easily soluble in 90 per-cent. 
 .alcohol, in which it keeps well. 
 
 Estimation. — The copper salt obtained as above is dissolved in ammonia, 
 and the purine bases precipitated with 3,mmoniacal silver solution. Nitrogen 
 is estimated in the copper and silver precipitates ; the difference gives the 
 nitrogen of urochrome. Urochrome contains 11 •! per cent, of nitrogen. 
 
 It shows no absorption bands, but outs off the violet part of the spectrum 
 which urobilin allows to pass. It does not fluoresce with zinc salts as urobilin 
 does. It yields a pyrrol derivative which is not identical with hsemopyrrol, 
 and so probably urochrome is not related to urobilin. It contains 5 per cent, 
 of sulphur, most of which is easily split off as sulphide by cold alkali. Cystine 
 sulphur is not present. It is an acid, and reddens litmus. It is probably 
 derived from protein. 
 
 2. Urobilin. — Urobilin is a derivative of the blood pigment, and is 
 identical with stercobilin (see pp. 124, 182). Probably both reduction and 
 hydration occur in its formation. It is very like the substance named hydro- 
 bilirubin by Maly, which he obtained by the action of sodium amalgam on 
 
THE PIGMENTS 265 
 
 bilirubin. The following formulae show the relationship between these allied 
 pigments : — 
 
 Hfematin CsjHga or ss.OjNiFe 
 
 Bilirubin . . . . CsaHaeNA 
 
 Hydrobilirubin C32H40N4O7 
 
 Normal urine contains but little urobilin ; what is present is chiefly in 
 the form of a colourless chromogen, which by oxidation is converted into 
 urobilin. In numerous pathological conditions urobilin is abundant. 
 
 The following are the two methods introduced by Garrod and Hopkins 
 for its separation from the urine : — 
 
 (a) The urine is first saturated with ammonium chloride, and the urate so 
 precipitated is filtered ofi'. The filtrate is then acidified with' sulphuric acid 
 and saturated with ammonium sulphate. This causes a precipitate of 
 urobilin,, which may be collected and dissolved in water. The aqueous 
 solution is again saturated with ammonium sulphate, and the pigment is 
 thus precipitated in a state of purity. 
 
 (6) The urates are first removed, then the urine is acidified ahd saturated 
 with ammonium sulphate as before. The urobilin is then extracted from the 
 mixture by shaking it with a mixture of chloroform and ether (1 : 2) in a 
 large separating funnel. The ether-chloroform extract is then rendered 
 faintly alkaline and shaken with distilled water, and the urobilin passes into 
 solution in the water. The aqueous solution is once more saturated with 
 ammonium sulphate and slightly acidified ; it then once more yields its pig- 
 ment to ether-chloroform. 
 
 By means of either of these methods urobilin is obtained in a pure con- 
 dition ; even normal urine will give some, for the chromogen is partly 
 converted into the pigment by the acid employed. 
 
 Urobilin dissolved in alcohol exhibits a green fluorescence, which is 
 greatly increased by the addition of zinc chloride and ammonia. It shows a 
 well-marked absorption band between b and Y, slightly overlapping the 
 latter (fig. 55, spectrum 4). 
 
 Urobilin, like most animal pigments, shows acidic tendencies and 
 forms compounds with bases ; it is liberated from such combinations by the 
 addition of an acid. 
 
 If urobilin is dissolved in caustic potash or soda, and suflftcient sulphuric 
 or hydrochloric acid is added to render the liquid faintly acid, a turbidity is 
 produced. This turbid liquid shows an additional band in the region of the 
 E line (fig. 55, spectrum 6), which is probably due to the special light absorption 
 exercised by fine particles of urobilin in suspension. It wholly disappears 
 when the precipitate is filtered ofi', and when it is redissolved the ordinary 
 band alone is visible. 
 
 3. Uroerythrin. — This is the colouring matter of pink urate sediments. 
 It may be separated from the sediment as follows : — The deposit is washed 
 with ice-cold water, dried, and placed in absolute alcohol. The alcohol, 
 though a solvent for uroerythrin, does not extract it from the urates. The 
 alcohol is poured off', and the deposit dissolved in warm water. From this 
 solution the pigment is easily extracted by amylic alcohol. 
 
 Uroerythrin has a great affinity for urates, with which it appears to form 
 
266 
 
 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 a loose compound. Its solutions are rapidly decolorised liy light. Spectro- 
 scopically it shows two rather ill-detined bands (fig. 5."j, spectrum 7). It 
 gives a green colour with cau.stic potash, and red or pink with mineral acids. 
 B c D Ed V <i 
 
 B C II E i) F G 
 
 Fig. .^.5.— Chart of absorption spectra; ], acid ha>niatoporphyrin ; 2, alkaline hiematoporphyrin ; 
 :i, liEematoporphyrin as found sometimes in urate sediments ; 4, acid urobilin, concentrated ; 
 .fj, acid urobilin, dilute ; G, the E band spectrum of urobilin ; 7, uroerythrin ; 8, urorosein con- 
 centrated—on dilution the band shrinks rapidly from redward end. (After F. G. Hopkins.) 
 
 Uroerythrin appears to be a small but constant constituent of urine. Its 
 origin and relationship to the other pigments are unknown. 
 
 4. Haematoporphyrin. — This alsi> occurs in small quantities in normal 
 urine. In some jiathological conditions, especially after the administration 
 of certain drugs (I'.g. sulphonal), its amount is increased. Its amount is 
 stated to increase when the urine stands ; this points to the existence of a 
 colourless chromogen. It may be separated from the urine as follows : — 
 
THE PIGMENTS 267 
 
 Caustic alkali is added to the urine ; this causes a precipitate of phosphates, 
 which carries down the pigment with it : the pigment may be dissolved out 
 with chloroform. The chloroform is evaporated, the residue washed with 
 alcohol, and finally dissolved in acidified alcohol. Urines rich in the pigment 
 yield it easily to acetic ether or to amylic alcohol. 
 
 When the urine is sufficiently rich in the pigment, the bands shown are 
 those of alkaline haematoporphyrin (fig. 55, spectrum 2). On adding sulphuric 
 acid, the spectrum of acid htematoporphyrin is seen (fig. 55, spectrum 1). 
 Occasionally urate sediments are pigmented with a form of the pigment which 
 shows a two-banded spectrum, very like that of oxyhsemoglobin (fig. 55, 
 spectrum 3) ; by treatment with dilute mineral acids this changes immediately 
 to the spectrum of acid hsematoporphyrin. 
 
 5. Chromogens in Urine. — In addition to the ohromogens of urobilin and 
 hsematoporphyrin alluded to in the foregoing paragraphs there are others, of 
 which the following may be mentioned : — (a) Indoxyl. — The origin of this 
 substance from indole is mentioned on p. 193. It is easily oxidised to indigo- 
 blue or indigo-red. 
 
 2CeH,<(^0^)cH + =CeH,<^^Oj>C : O^g^CeH^ + SH.O. 
 
 [indoxyl] [indigo-blue] 
 
 Indigo-red is isomeric with indigo-blue. It is very rare for the urine to be 
 actually pigmented with indigo, for the urinary indoxyl is excreted as a con- 
 jugated sulphate which resists oxidation. When the urine is mixed with an 
 equal volume of hydrochloric acid, indoxyl is liberated from the sulphate. A 
 solution of a hypochlorite is then added drop by drop, when indigo-blue is 
 formed, and on shaking the mixture with chloroform the indigo-blue passes 
 into the chloroform (Jaffe). This teist, however, is not a very good one, for 
 the hypochlorite solution has always to be freshly prepared, and even then a 
 small excess will cause the colour to disappear owing to oxidation of the indigo, 
 and it is difficult to hit ofl' the exact amount to give the reaction. A better 
 test for indoxyl-sulphurio acid (indican) consists in adding 10 e.c. of a 20 per- 
 cent, solution of lead acetate to 50 c.c. of urine, and filtering the mixture. 
 The filtrate is shaken with an equal volume of hydrochloric acid (containing 
 0'2 to 04 per cent, of ferric chloride) and a few c.c. of chloroform. The 
 indigo-blue passes into the chloroform (Obermayer). (6) Skatoxi/l. — When 
 skatoxyl is given by the mouth it passes into the urine, and yields skatoxyl- 
 red on oxidation, (o) Urorosein is distinct from indigo-red. It is produced 
 from its chromogen by the action of mineral acids. According to Herter, 
 indole-acetic acid is its chromogen. It frequently appears when urine is 
 treated with strong hydrochloric acid and allowed to stand, but it appears 
 more readily when an oxidising agent is added as well. It is readily soluble 
 in amylic alcohol, but not in ether. The colour is destroyed by alkalis. It 
 shows an absorption band between the D and E lines (fig. 55, spectrum 8). 
 
 6. Pathological Pigments. — The most frequently appearing of abnormal 
 pigments are those of blood and bile. The urine may contain accidental pig- 
 ments due to the use of drugs (rhubarb, senna, logwood, santonin) ; in carbolic 
 acid poisoning pyrocatechin and hydrochinon are chiefly responsible for the 
 greenish-brown colour of the urine, which increases on exposure to the air. 
 The black or dark-brown pigment called melanin may pass into the urine in 
 oases of melanotic sarcoma. For alcaptonuria, see p. 214. 
 
APPENDIX 
 
 Hi£MACYTOMETEBS 
 
 Gowers's Hsemacytometer. — The enumeration of the blood corpuscles is 
 readily eflfected by the haemacytometer of Gowers. This instrument consists 
 of a glass slide (fig. 56, C), the centre of which is ruled into t^j millimetre 
 squares and surrounded by a glass rim i millimetre thick. It is provided 
 with measuring pipettes (A and B), a vessel (D) for mixing the blood with a 
 
 Fio. 56. — HEBmacytonieter of Sir W. Gowera. 
 
 saline solution (sulphate of soda of specific gravity 1015), a glass stirrer (E), 
 and a guarded needle (F); 
 
 Nine hundred and ninety-five cubic millimetres of the saline solution are 
 measured out by means of A, and then placed in the mixing jar ; 5 cubic 
 millimetres of blood are then drawn from a {juncture in the finger by means 
 of the pipette B, and blown into the solution. The two fluids are well mixed 
 
 268 
 
H/EMACYTOMETERS 
 
 269 
 
 by the stirrer, and a small drop of this diluted mixture placed in the centre 
 of the slide C ; a cover slip is gently laid on (so as to touch the drop, which 
 thus forms a layer i millimetre thick between the slide and cover slip), and 
 pressed down by two brass springs. In a few minutes the corpuscles have 
 sunk to the bottom of the layer of fluid, and rest on the squares. The number 
 
 mmMy//yy///yA m 
 
 W/M////yy^W^^//^///A 
 
 Fig. 57. — Thoma-Zeiss haemacyfcometer. 
 
 on ten squares is then counted, and this multiplied by 10,000 gives the number 
 in a cubic millimetre of blood. The average number of red corpuscles in each 
 square ought, therefpre, in normal human blood to be 45-50. 
 
 Differential counts to show the relative proportions of the varieties of 
 leucocytes are made in appropriately stained blood films. 
 
 Thoma - Zeiss Haemacytometer.— This instrument 
 consists of a glass slide (s) seen in section in fig. 57. In 
 the centre of the slide is a disc of glass m, the upper surface 
 of which is ruled into squares each of which is lir of a 
 square millimetre in area. This is surrounded by a ring of 
 glass (c) which is of such a height as to project tr of a 
 millimetre above m. A drop of diluted blood is placed in 
 the cell so formed, and covered with a cover slip. The 
 volume of blood between any particular square and the 
 cover slip is therefore -fTfVi! of a cubic millimetre. Accom- 
 panying the instrument are two capillary pipettes, one 
 of which is shown in the accompanying drawing (fig. 58). 
 The finger or ear is pricked, and blood is drawn up in the 
 capillary tube to the line marked 1 (or if twice the dilu- 
 tion is regarded as advisable, to the line marked 0'5) ; a 
 suitable saline solution is drawn up then to the mark 101 ; 
 the blood and diluting solution are then well mixed, the 
 glass bead in the bulb aiding the mixing, and a drop of 
 the mixture transferred to the slide ruled in squares. 
 The corpuscles are allowed to settle, and those oh 20 or 
 more squares are then counted, and the average per square 
 multiplied by 400,000 gives the number of red corpuscles 
 per cubic millimetre of undiluted blood. The number of 
 red corpuscles per square in normal blood is about 12. 
 
 The second pipette provided is like the one just 
 described, but is rather larger, and is similarly employed 
 for counting the leucocytes. In some cases a micrometer 
 slide ruled in larger squares is provided for this purpose. 
 The value of the squares in terms of those provided for the counting of the red 
 corpuscles is known, and so the proportion of coloured and colourless corpuscles 
 can be ascertained. In counting the colourless corpuscles the addition of a 
 dye renders the operation easier. 
 
 Fm. 68.— Pijiette of 
 Thoma - Zeiss hae- 
 macytometer. 
 
270 
 
 ESSENTIALS Of- CHEMICAL PHYSIOLOGV 
 
 Sherrington recommends the following solution : — 
 
 Methylene blue I'O grammes 
 
 Sodium chloride . . . . 1-2 „ 
 
 Potassium oxalate . . . . 1'2 , „ 
 
 Distilled water . . ... 3000 „ 
 
 For differential counts a mixture of dyes is applied to blood films. 
 
 Fig. 59. — Oliver's hBemacytometer. 
 
 Oliver's Haemacytometer. — The following method, devised by Dr George 
 Oliver, is a ready way of determining the total number of corpuscles. It is, 
 
H^MOGLOBINOMETERS 
 
 271 
 
 however, not possible to determine the relative proportion of red and white 
 corpuscles by this means. 
 
 The finger is pricked, and the blood allowed to flow into the small capillary 
 pipette (fig. 59, a) until it is full. This is washed out by the dropping tube b 
 into a graduated flattened test-tube, c, with Hayem's fluid.^ The graduations 
 of the tube are so adjusted that with normal blood confining 5,000,000 
 coloured corpuscles per cubic millimetre, the light of a small wax candle placed 
 at a distance of 9 feet from the eye in a dark room is just transmitted as a flne 
 bright line when looked at through the tube held edgeways between the fingers 
 (rf) and filled up to the 100 mark of the graduation. If the number of cor- 
 puscles is less than normal, less of the diluting solution is required for the 
 
 Fig. 60. — HsBmoglobinometer of Sir W. Gowers. 
 
 light to be transmitted ; if above the normal, more of the Hayem's fluid must 
 be added. The tube is graduated, so as to indicate in percentages the decrease 
 or increase of corpuscles per cubic millimetre as compared with the normal 
 standard of 100 per cent. 
 
 HJEMOGLOBINOMETERS 
 
 Gowers's Haemoglobinometer. — The apparatus consists of two glass 
 tubes, C and D, of the same size. D contains glycerin jelly tinted with carmine 
 to a standard colour — viz. that of normal blood diluted 100 times with distilled 
 water. The finger is pricked and 20 cubic millimetres of blood are measured 
 out by the capillary pipette B. This is blown out into the tube C, and diluted 
 with distilled water, added drop by drop from the pipette stopper of the 
 bottle A, until the tint of the diluted blood reaches the standard colour. The 
 tube O is graduated into 100 parts. If the tint of the diluted blood is the same 
 as the standard when the tube is filled up to the graduation 100, the quantity 
 of oxyhsemoglobin in the blood is normal. If it has to be diluted more largely 
 
 ' Sodium sulphate 5 grammes, sodium chloride 1 grm., mercuric chloride 
 O'S grm., distilled water 200 o.c. 
 
272 
 
 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 the oxyhsemoglobin is in excess ; if to a smaller extent, it is less than normal. 
 If the blood has, for instance, to be diluted up to the graduation 50, the amount 
 of haemoglobin is only half what it ought to be — 50 per cent, of the normal — 
 and so for other percentages. 
 
 Haldane's Hsemoglobinometer is more frequently used. Instead of tinted 
 gelatin, the sta^^ard of comparison is a sealed tube filled with a solution of 
 carbonic oxide haemoglobin of known strength. This keeps unchanged for 
 years. 'A stream of coal gas is passed through the blood to be examined. 
 This converts all the haemoglobin present into carboxyhaemoglobin ; this is 
 then diluted with water to match the standard. In Sahli's instrument, a 
 standard solution of acid hsematin is employed ; this keeps even better. 
 
 Vie. 61.— Von Fleischl's hsomometer. 
 
 Von Fleisclirs Hsemometer. — The apparatus (fig. 61) consists of a stand 
 bearing a white reflecting surface (S) and a platform. Under the platform is 
 a slot carrying a glass wedge stained red (K) and moved by a wheel (R). On 
 the -platform is a small cylindrical vessel divided vertically into two compart- 
 ments, a and a'. 
 
 Fill with a pipette the compartment a' over the wedge with distilled water. 
 Fill about a quarter of the otter compartment (a) with water. 
 
 Prick the finger and fill the short capillary pipette provided with the 
 instrument with blood. Dissolve this in the water in compartment a, and fill 
 it up with distilled water. Having arranged the reflector (S) to throw artificial 
 light vertically through both compartments, look down through them, and 
 move the wedge of glass by the milled head (T) until the colour in the two is 
 identical. Read ofi' the scale, which is so constructed as to give the percentage 
 of haemoglobin. 
 
 Oliver's Hsemoglobinometer.- — This method consists in comparing a speci- 
 men of blood, suitably diluted with water in a shallow white palette, with a 
 number of standard tests very carefully prepared by the use of Lovibond's 
 coloured glasses. The capillary pipette c (fig. 62) is first filled with blood 
 
H^MOGLOBINOMETERS 
 
 273 
 
 obtained by pricking the finger. This is washed by water by the mixing 
 pipette d into the blood cell e ; the ceil is then just filled with water, and the 
 
 Fig. 62. — Oliver's haemoglobinometer : a, standard gradations ; h, lancet ; c, capillary 
 measuring pipette ; d, mixing pipette ; e, blood cell and cover glass. 
 
 blood and water thoroughly mixed by the handle of c being used as a stirrer. 
 The cover glass is then adjusted, when a small bubble should form, a clear 
 sign that the cell has not been overfilled. The cell is then placed by the aide 
 18 
 
274 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 of the standard gradations, and the eye quickly recognises its approximate 
 position on the scale. The camera tube provided with the instrument will 
 more accurately define it. Artificial light should be used. 
 
 If it is proved that the blood solution is matched in depth of colour by 
 one of the standard grades, the observation is at an end ; but if the tint is 
 higher than the one grade, but lower than another, the blood cell is placed 
 opposite to the former, and riders (not shown in the illustration) are added 
 to complete the observation. The standard gradations are marked in per- 
 centages, 100 per cent, being taken as normal. 
 
 Specific Gravity of Blood. — Of the numerous methods introduced for 
 taking the specific gravity of fresh blood, that of Hammerschlag is the 
 simplest. A drop of blood from the finger is placed in a mixture of chloroform 
 tand benzene. If the drop falls, add chloroform till it just begins to rise ; if 
 the drop rises, add benzene till it just begins to fall. The fluid will then be 
 of the same specific gravity as the blood. Take the specific gravity of the 
 mixture in the usual way with an accurate hydrometer. 
 
 Schmalz's capillary pycnometer is more accurate. 
 
 POLARISATION OF LIGHT 
 
 If an object, such as a black dot on a piece of white paper, is looked at 
 through a crystal of Iceland spar, two black dots will be seen; and if the 
 crystal is rotated, one black dot will move round the other, which remains 
 stationary. That is, each ray of light entering such a crystal is split into two 
 rays, which travel through the crystal with different velocities, and con- 
 sequently one is more refracted than the other. One ray travels just as 
 it would through glass ; this is the ordinary ray, the ray which gives the 
 stationary image ; the other ray gives the movable image when the crystal is 
 rotated ; the ordinary laws of refraction do not apply to it, and it is called 
 the extraordinary ray. Both rays are of equal brilliancy. In one direction, 
 however, that of the optic axis of the crystMi .^ ^^J °^ ligj^it is transmitted 
 without double refraction. j.^t,' ,' ; 
 
 Ordinary light, according to the' wave theOTJPj is due to vibrations occurring 
 in all planes, transversely to the direction for the propagation of the wave. 
 Light is said to be plane polarised when the vibrations take place all in one 
 plane. The two rays produced by double refraction are both polarised, one 
 in one plane, the other in a plane at right angles to this one. Doubly refract- 
 ing bodies are called anisotropic ; singly refracting bodies, isotropic. The 
 effect of polarisation may be very roughly illustrated by a model. 
 
 If a string is stretched as in the figure, and then touched with the finger, 
 it can be made to vibrate, and the vibrations will be free to occur from above 
 down, or from side to side, or in any intermediate position. If, however, a 
 disc with a vertical slit be placed on the course of the string, the vibrations 
 will all be obliged to take place in a vertical plane, any side-to-side movement 
 being stopped by the edges of the slit^ (fig. ,63). 
 
 Light can be polarised not only by the action of crystals, but by reflection 
 
 1 Such a model is, of course, imperfect ; it does not, for fnstanee, represent the 
 splitting of the ray into two, and moreover the polarisation takes place on each 
 side of the slit ; whereas, in regard to light, it is only the rays on one side of a 
 polariser, viz. those which have passed through it, which are polarised. 
 
POLARISATION OF LIGHT 
 
 275 
 
 from a surface at an angle which varies for different substances (glass 54° 35', 
 water 52° 45', diamond 68°, quartz 57° 32', etc.). It is also found that certain 
 non-crystalline substances, such as muscle, cilia, etc., are doubly refracting. 
 
 Nicol's Prism is the polariser usually employed in polariscopes ; it con- 
 sists of a rhombohedron of Iceland spar divided into two by a section through 
 its obtuse angles. The cut surfaces are polished and cemented together in 
 their former position with Canada balsam. By this means the ordinary ray 
 is totally reflected through the Canada balsam ; the extraordinary ray passes 
 on and emerges in a direction parallel to the entering ray. In this polarised; 
 ray there is nothing to render its peculiar condition visible to the naked eye ; 
 
 but if the eye is aided by a second Nicol's prism, which is called the analyser, 
 it is possible to detect the fact that it is polarised. 
 
 This may be again illustrated by reference to our model (fig. 64). 
 
 Suppose that the string is made to vibrate, and that the waves travel in 
 the direction of the arrow. From the fixed point c to the disc a, the string 
 is theoreticaliy free to vibrate in any plane ; ^ but after passing through the 
 vertical slit in a, the vibrations must all be vertical also ; if a second similar 
 disc b be placed further on, the vibrations will also pass on freely to the other 
 
 b 
 
 extremity of the string d, if as in the figure (fig. 64) the slit in h is also placed 
 vertically. If, however, 6 is so placed that its slit is horizontal (fig. 65) the 
 vibrations will be extinguished on reaching 6, and the string between h and d 
 will be motionless. 
 
 c here represents a source of light ; the vibrations of the string represent 
 the undulations which by the Nicol's prism a are polarised so as to occur in 
 one plane only ; if the second Nicol's prism or the analyser h is parallel to the 
 first, the vibrations will pass on to the eye, which is represented by d ; but if 
 the planes of the two nicols are at right angles, the vibrations allowed to pass 
 through the first are extinguished by the second, and so no light reaches the eye. 
 In intermediate positions, 6 will allow only some of the light to pass through 
 it. It must be clearly understood that a i^icol's prism contains no actual slits, 
 
 ^ The imperfection of the model has been explained in the preceding footnote. 
 
276 
 
 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 but the arrangement of its molecules is such that their action on the particles 
 of ether may be compared to the action of slits in a diaphragm to vibrations 
 of more tangible materials than ether. 
 
 The Polarising Microscope consists of an ordinary microscope with certain 
 additions; below the stage is the, polarising nicol ; in the eye-piece is the 
 analysing nicol ; the eye-piece is so arranged that it can be rotated ; thus the 
 directions of the two nicols can be made parallel, and then the field is bright ; 
 or crossed, and then the field is dark. The stage of the microscope is arranged 
 so that it can also be rotated. 
 
 The polarising microscope is used to detect doubly refracting substances. 
 Let the two nicols be crpssed, so that the field is dark ; interpose between 
 the two, that is, place upon the stage of the microscope a doubly refracting 
 plate of which the principal plane is parallel to the first prism or polariser ; 
 the ray from the first prism is unaffected by the plate, but will be extinguished 
 by the second ; the field, therefore, still remains dark. If the plate is parallel 
 to the second nicol the field is also dark ; but in any intermediate position 
 the light will be transmitted by the second nicol. In other words, if between 
 
 Pig. 65. 
 
 two crossed nicols, which consequently give a dark field, a substance is inter- 
 posed which in certain positions causes the darkness to give place to illumina- 
 tion, that substance is doubly refracting or anisotropic. 
 
 All crystals except those of the regular system are anisotropic, whilst 
 amorphous substances are isotropic. O. Lehmann has, however, discovered 
 an intermediate stage between a;nisotropic solid crystals and their isotropic 
 fused condition. In this state certain substances, such as cholesterol com- 
 pounds, oleates, etc., are anisotropic, but completely fluid. He called this 
 the liquid crystalline state of matter (see also p. 39). 
 
 Rotation of the Plane of Polarisation. — Certain crystals such as those of 
 quartz, and. certain fluids such as the essence of turpentine, aniseed, etc., 
 and solutions of certain'substances such as sugar and albumin, have the power 
 of rotating the plane of polarised light to the right or left. The polarisation 
 of light which is produced by a quartz crystal is diflerent from that produced 
 by a rhombohedron of Iceland spar. The light which passes through the 
 latter is plane polarised ; the light which passes through the former (quartz) 
 is circularly polarised, i.e. the two sub-rays are made up of vibrations which 
 occur not in a plane, but are curved. The two rays are circularly polarised 
 in opposite directions, one describing circles to the left, the other to the right ; 
 these unite on issuing from the quartz plate ; and the net result is a plane 
 polarised ray with the plane rotated to right or left according as the right 
 circularly polarised ray or the left proceeded through the quartz with the 
 
POLARIMETERS 277 
 
 greater velocity. There are two kinds of quartz, one which rotates the plane 
 to the right (dextro-rotatory), the other to the left (Ijevo-rotatory). 
 
 Gordon explains this by the following mechanical illustration. Ordinary 
 light may be represented by a wheel travelling in the direction of its axle, 
 and the vibrations coiiiposing it are executed along any or all of its spokes {a). 
 If the vibrations all take place in the same direction, i.e. along one spoke, 
 and the spoke opposite to it (6), the light is said to be plane polarised. The 
 two spokes as they travel along in the direction of the arrow will trace out 
 a plane (see fig. 66) between 6 and b'. If this polarised beam is made to 
 travel now through a solution of sugar, the net result is that the plane so 
 traced out is twisted or rotated ; the two spokes, as in 66', do not trace out 
 a plane, but we must consider that they rotate as they travel along, as though 
 guided by a spiral or screw thread cut on the axis, so that after a certain 
 distance the vibrations take place as in 6" ; later in 6'", and so on. This 
 effect on polarised light is due to the molecules in solution, and the amount 
 of rotation will depend on the strength of the solution, and on the length of 
 the column of the solution through which the light passes, or in the case of 
 a quartz plate, on its thickness. 
 
 If a plate of quartz is interposed between two nicols, the light will not be 
 extinguished in any position of the prisms, but will pass through various 
 colours as rotation is continued. The rotation produced for different kinds 
 of light being different, while light is split into its various constituent colours ; 
 and the angle of rotation that causes each colour to disappear is constant for 
 a given thickness of quartz plate, being least for the red and greatest for the 
 violet. These facts are made use of in the construction of polarising instru- 
 ments. 
 
 FOLABIMETEBS 
 
 Polarimeters are insti-uments for determining the strength of solutions of 
 sugar, albumin, etc., by the direction and amount of rotation they produce 
 on the plane of polarised light. They are often called saccharimeters, as 
 they are specially useful in the estimation of sugar. 
 
 The following is the one most frequently employed at the present time. 
 
 Laurent's Folarimeter. — Instead of using daylight, or the light of a lamp, 
 monochromatic light (a sodium flame produced by volatilising common salt 
 in a colourless gas flame) is eniployed ; the amount of rotation varies for 
 different colours ; and observations are usually recorded as having been taken 
 with light corresponding to the D or sodiimi line of thespectrum. The essentials 
 of the instrument are a polariser, a tube for the solution, and an analyser. The 
 polarised light before passing into the solution traverses a quartz plate, which, 
 however, covers only half the field, and retards the rays passing through it 
 
m 
 
 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 by half a wave-length. In the 0° position the two halves of the field appear 
 equally illuminated : in any other position, or if rotation has been produced 
 by the solution when the nicols have been set at zero, the two halves appear 
 unequally illuminated. » The amount of rotation produced by the solution 
 is ascertained by rotating the analyser until the two halves of the field are 
 once more equally illuminated. This is measured in degrees by a scale and 
 vernier attached to the instrument. 
 
 The specific rotatory power of any substance is the amount of rotation in 
 
 Fig. 67.— Laurent's polarimeter. 
 
 degrees of a circle of the plane of polarised light produced by 1 gramme of the 
 substance dissolved in 1 c.c. of liquid examined in a column 1 decimetre long. 
 If a = rotation observed. 
 
 w = weight in grammes of the substance per cubic centimetre. 
 I = length of tube in decimetres, 
 [ajo = specific rotation for light with wave-length corresponding to the 
 D line (sodium flame). 
 
 Then [a]„ = ± -V- 
 
 In this formula -I- indicates that the substance is dextro-rotatory, - that 
 it is Isevo-rotatory. 
 
CIRCULAR POLARISATION AND CHEMICAL CONSTITUTION 279 
 
 If, on the other hand, [ajn is known, and we wish to find the value of w, 
 then 
 
 "'= rn ^ f, and the percentage amount is p= p -, ■ ■ 
 If for example we find the rotation of a urine iu a 2-decimetre tube= + 2-12° 
 and the [a],, of glucose is 53°, then 
 
 2-12xl00_ 
 
 that is, the urine contains 2 per cent, of glucose. 
 
 RELATION BETWEEN CIRCULAR POLARISATION AND 
 CHEMICAL CONSTITUTION 
 
 The first work in this direction was performed by Pasteur, and it was his 
 publications on this subject that brought him into prominence. He found 
 that racemic acid, which is optically inactive, can be decomposed into two 
 isomerides, one of which is common tartaric acid which is dextro-rotatory, 
 and the other tartaric acid differing from the common variety in being laevo- 
 rotatory. The salts of tartaric acid usually exhibit hemihedral faces, while 
 those of racemic acid are holohedral. Pasteur found that, although all the 
 tartrate crystals were hemihedral, the hemihedral faces were situated on some 
 crystals to the right, and on others to the left hand of the observer, so that 
 one formed, as it were, the reflected image of the other. These crystals were 
 separated, purified by recrystallisation, and those which exhibited dextro- 
 hemihedry possessed dextro-rotatory .power, while the others were Isevo- 
 • jotatory. Pasteur further showed that if the mould PeniciUium glaucuvi 
 is grown in a solution of racemic acid, dextro-tartaric acid first disappears, 
 and the Isevo-acid alone remains. The subject remained in this condition 
 for many years ; it was, however, conjectured that probably there is some 
 molecular condition corresponding to the naked-eye crystalline appearances 
 which produces the opposite optical effects of various substances. What this 
 molecular structure is, was pointed out independently by two observers — 
 Le Bel in Paris, and Van't Hofi' in Holland — who published their researches 
 within a few days of each other. They pointed out that all optically active 
 bodies contain one or more asymmetric carbon atoms, i.e. one or more atoms 
 of carbon connected with four dissimilar atoms or groups of atoms, as in the 
 following examples : — 
 
 OoHs CO.OH 
 
 H— f C j— CHs H— ( C )— OH 
 
 I I 
 
 CH2.OH CH,— CO.OH 
 
 [amyl alcohol] [malic acid] 
 
 The question, however, remained — d!o all substances containing such 
 atoms rotate the plane of polarised light ? It was found that they do not ; 
 this is explained by Le Bel by supposing that these, like racemic acid, are 
 compounds of two molecules — one dextro-, the other leevo-rotatory ; that 
 this was the case was demonstrated by growing moulds, the enzymic action 
 
2^0 
 
 ESSEiMtULS OP CllfiMICAL tHYSlOLOGV 
 
 of which is to separate the two molecules in question. Then the other ques- 
 tion — how is it that two isomerides, which in chemical characteristics, in 
 graphic as well as empirical formulae, are precisely alike, differ in optical 
 
 properties? — is explained ingeniously 
 by Van't Hoff. He compares the carbon 
 atom to a tetrahedron with its four 
 dissimilar groups, A, B, C, D, at the 
 four corners. The two tetrahedra 
 represented in fig. 68 appear at first 
 sight precisely alike ; but if one be 
 
 ^ 
 
 ■Mercurial air-pump for obtaining the 
 of the hlood (diagrammatic). 
 
 A A 
 
 superimposed on the other, C in one 
 and D in the other could never be 
 made to coincide. This difierence can- 
 not be represented in any other graphic 
 manner, and probably represents the 
 difference in the way the atoms are 
 grouped in the molecule of r^ght- and 
 left-handed substances respectively. 
 
 THE MERCURIAL AIR-PUMP 
 (BAROROFT'S MODIFICATION) 
 
 The instrument consists essentially 
 of the following parts : — 1, a small bulb 
 or tube for measuring the amount of 
 blood to be analysed ; 2, a vacuous 
 chamber. When the part of the appar- 
 atus from 2 to 4 (fig. 69) is rendered 
 vacuous, the blood is emptied from 1 
 into 2, and is then kept continuously 
 boiling by means of a water-bath (C) 
 around its lower portion. A condenser 
 (D) packed with ice surrounds the 
 upper part. There is always a stream of 
 aqueous vapour carrying the boiled-out 
 gases to the top of the chamber ; the 
 vapour is condensed and returns to the 
 blood as drops of water, while the gases 
 In doing so they pass through 3, the 
 So much of the gases as has 
 
 are free to go into the vacuous pump, 
 
 drying chamber, which contains sulphuric acid. 
 
 expanded into the pump (4) can then be expelled by lifting the mercury bottle 
 
 (A), which is connected by strong rubber tubing to the glass tube F. In the 
 
 figure only the attachments of the rubber tube are shown. The mercury is pre 
 
barcroft's differential apparatus 
 
 281 
 
 vented by a valve (V) from going backwards to the drying chamber ; it expels 
 the gases down the tube B into a eudiometer ^tube (E) in which they are 
 collected for measurement and analysis. After boiling for a considerable 
 time, a few exhaustions with the pump are sufficient to deliver all the gases 
 which have boiled off from the blood into the eudiometer tube. 
 
 BARCROFT'S DIFFERENTIAL APPARATUS FOR 
 ANALYSIS OF BLOOD GASES 
 
 The differential method of blood-gas analysis may be used for the measure- 
 ment either of the quantity of oxygen which the blood givds off when treated 
 with ferricyanide of potassium (see p. 164), or to 
 measure the amount of oxygen which partially 
 reduced blood will absorb. The blood for 
 analysis is placed in one bottle ; a control fluid 
 is placed in the other, and as the two are sub- 
 jected to the same changes of temperature, 
 vapour pressure, etc., these factors are eliminated. 
 
 The apparatus consists of two bottles of equal 
 size connected with one another by means of a 
 manometer filled with clove oil. The bottles 
 should be of about 25 c.c. capacity. The stopper 
 of the bottle is prolonged into a small cup capable 
 of holding potassium ferricyanide. From the 
 stopper a straight tube leads, the bore of which 
 is not so small as to preclude its being cleaned 
 with a pipe cleaner. At the junction of this tube 
 with the manometer is a threeway tap. The 
 student should make himself familiar with the 
 mechanism of the tap ; it contains a T boring ; 
 usually there is a spot on the handle of the 
 tap corresponding to the dependent limb of the T. 
 
 The tap will be spoken of as open when the 
 bottles and the manometer communicate with 
 the external air, and shut when the bottles com- 
 municate with the manometer. The whole is 
 mounted on a stand fuinished with a clip, by fig. 70.- 
 means of which it can hang on the side of a 
 water -bath. The success of the subsequent 
 
 analyses depends upon the cleanness of the glass of the manometer when the 
 oil is put into it. It should be chemically clean and dry. The utmost care 
 must be taken that water does not get into the manometer. 
 
 The oil is best put in with a fine pipette. Tor this purpose the stopper on 
 the left side is removed ; that on the right is turned so as to cut the manometer 
 off from the external air and the air of the bottle. The oil is introduced on the 
 left side. The end of the pipette should penetrate as far as the bend in the 
 tubing at the top of the stand. When as much oil as is judged necessary, or a 
 little more, has been put in, the tap on the right side is opened and the oil 
 allowed to sink into the capillary portion of the manometer till the meniscus 
 
 K^ 
 
 -Barcroft's differential 
 apparatus. 
 
282 esse'^tials of chemical physiology 
 
 reaches the zero on the right side. All superfluous oil should then be removed 
 from the top of the left tube with a little sponge mounted on a wire. The 
 right tap may then be opened again, and the oil allowed to find its own 
 level. 
 
 Calibration of the Apparatus 
 
 The volume of gas given off (or absorbed) in the apparatus bears a direct 
 ratio to the difference of pressure set up in the manometer. Supposing, 
 then, that v is the volume of gas given off, and p the difference of pressure 
 
 p=TS. + v. 
 
 The calibration of the instrument consists in finding the value of the 
 constant K. 
 
 This is best done by liberating a known quantity of gas in the apparatus 
 and, observing the pressure produced ; p and v are then known 
 
 and K=S 
 
 V 
 
 The best way of evolving a known quantity of gas in the apparatus is 
 to liberate oxygen from a titrated solution of pure acidulated hydrogen 
 peroxide by mixing potassium permanganate with it. 
 
 The solutions necessary are : (1) a decinormal solution of permanganate, 
 (2) some " 20- volume " hydrogen peroxide, and (3) sulphuric acid. If the 
 " 20- volume " hydrogen peroxide is of full strength, 5 c.c. of it may be made up 
 to 1000 c.c. (Ultimately 1 c.c. of the diluted peroxide should give off about 
 02 c.c. of gas, but it must be borne in mind that only one-half this quantity 
 comes from the peroxide, the other half from the permanganate.) 
 
 KaMn^Og -I- 3H2SO4 = K2SO4 -1- 2MnS04 + r>afi + 50 
 [316 gr.] 50-)-5H202=5H20 + 502 [111 litres] 
 
 or 1 c.c. (0"00316 gr.) permanganate = I'll c.c. of oxygen. 
 
 Titrate 50 c.c. of the dilute peroxide, mixed with excess of sulphuric acid, 
 with the permanganate. About 8-10 c.c. "of the permanganate should be 
 required for the titration if the peroxide is of suitable strength. If it proves 
 to be too weak, as is often the case, a fresh solution must be made up. 
 
 Suppose 8'7 c.c. of K2Mn20j are necessary to reach the end-point (pink 
 
 colour) in the titration of 50 c.c. of acidulated H2O2, then : 1 c.c. of 11262= 
 
 I'll X 8'7 
 
 =0193 c.c. of oxygen at normal temperature and pressure (N.T.P.). 
 
 ou 
 
 Suppose the barometer to be 763 mm., the thermometer to be 15° C, the gas 
 
 will be somewhat greater in volume than at N.T.P., and will be 
 
 •193x||?xt62 = 0'202c.c. 
 273 763 
 
 [0° 0. is 273° above absolute zero, and 15° C. is therefore 288]. 
 
 If then the gas when liberated from the 1 c.c. of peroxide in the apparatus 
 be called v 
 
 i;=0'202 c.c. 
 
barcroft's differential apparatus 283 
 
 Into each bottle put 1 c.c. of the peroxide and 2 c.c. of — sulphuric acid. 
 
 Into the cup in the left stopper put 0"3 c.c. of potassium permanganate, and a 
 little piece of filter-paper to cover the orifice. 
 
 In the right cup put 0'3 c.c. of water and filter-paper. Put the bottles on 
 the stoppers. 
 
 [Be careful that the taps are always open when the bottles are being put on 
 and taken off.'] 
 
 Hang the apparatus on the water-bath for five minutes, then shut the taps ; 
 read the level of the fiuid on each side (suppose it to be lO'O and 10'05) ; if it 
 remains constant for a minute or so, upset the permanganate into the hydrogen 
 peroxide and shake for one minute. At the end replace the apparatus on the 
 bath, and after two minutes more read again. The levels may be 7'0 and 
 13'05. Shake again for a minute and read two minutes later ; the levels may 
 now be 6"9 and 13"15, at which they remain on subsequent shaking. 
 The side with the permanganate has sunk 
 
 from 10-0 to 69 =3-1 cm. 
 
 The other side has risen from 10'05 to 13"15 = 3'1 cm. 
 
 6-2 cm. 
 . Then ^ = 6-2. t)= 0-202 
 
 .-. K = 2=0-306. 
 
 V 
 
 The fluid in the bottle should be pink, with excess of permanganate at the 
 end. 
 
 Another method of calibration is described by Hoffmann {Journal of 
 Physiology, 1913, vol. xlvii., p. 272). The following three exercises illustrate 
 how the instrument may be employed in physiological work. 
 
 Determination of the Oxygen Capacity of Blood 
 
 Into each bottle put I c.c. of blood which has been thoroughly shaken 
 with air (an ordinary 1 c.c. pipette delivers about 0-96 c.c. of blood) and 2 c.c. 
 of ammonia solution (1 c.c. concentrated ammonia made up to 250 c.c. with 
 distilled water). Shake so as to thoroughly lake the blood. Put 03 c.c. of 
 ferricyanide into the cup of one stopper, and, if necessary, a slip of filter-paper. 
 Grease the stoppers, and put on the bottles — hang the apparatus on the water- 
 bath and proceed as in the above example. Suppose the final dift'erence 
 of pressure to be 5-8 cm., the amount of gas given off will be : 
 
 5-8x0-0306 = 0-177 c.c. at 15° C. and 763 mm. barometric pressure. 
 If this were given off' by 0-96 c.c. of blood, the oxygen capacity would be 
 obtained by correcting for temperature and pressure : — 
 
 ^im X ?23 ,< 763=0-175 C.C. at N.T.P. 
 096 288 760 
 
 Determination of Oxygen in Unsaturated Blood 
 Place 2 C.C. of the dilute ammonia in each bottle. Take 1 c.c. of blood 
 that is venous in colour ^ in a pipette, put the end of the pipette under the 
 
 1 On no account should stale blood be used. 
 
284 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 surface of the ammonia, and deliver the blood gently into the bottle so that 
 it lies as a layer underneath the clear solution of ammonia. Into the other 
 bottle similarly deliver 1 c.c. of blood which has been thoroughly saturated 
 with air. Put the bottles on to the stoppers, and with the taps open hang 
 the apparatus on the bath. After five minutes close the taps, and if the 
 zero remains constant, read, and then sh^ke the apparatus as above. The 
 oil will, of course, rise on the side of the unsaturated blood. The shaking 
 should be carried out so as to dirty the cups as little as possible. 
 
 Suppose the ultiE(iate difference in level is 2"1 cm. Now, remove the bottle, 
 which contains the fully oxygenated blood, put ferricyanide into the cup, 
 replace the bottle, and determine the total oxygen capacity of the blood in it. 
 
 Suppose the final reading with ferricyanide to be as before, 5'8 cm., the 
 student will now have at his disposal two determinations : (1) the percentage 
 
 satiration, which is — ^ — - x 100 = 81 per cent., and (2) the actual quantity 
 
 O'o 
 
 of oxygen that was in 1 c.c. of the unsaturated blood, namely, 81 per cent. 
 
 81 
 of the total oxygen capacity, 0'189x Tf7, = 0'153 c.c. 
 
 Standardisation of the Gowers-Haldanb H^moglobinombtbr 
 
 A Gowers-Haldane hsemoglobinometer should indicate 100 when the 
 oxygen capacity is : 
 
 1 c.c. of blood = 0-185 c.c. oxygen at N.T.P. 
 To test this it is only necessary to measure the oxygen capacity of the blood 
 with the difi'erential apparatus (it is best to use ox or sheep's blood) and at 
 the same time to determine the oxygen capacity of the same blood with the 
 hsemoglobinometer. 
 
 The cheaper forms of Gowers's hsemoglobinometer when old will be found 
 often to differ very much from the theoretical value : it is therefore desirable 
 that they should be tested. 
 
 HALDANE'S AFFABATUS FOB GAS ANALYSIS 
 
 The apparatus consists of an accurately graduated gas-burette A, pro- 
 vided with a threeway tap at the top. This is surrounded by a water jacket. 
 It is connected by rubber tubing with the levelling tube B ; this and the 
 burette contain mercury. When the levelling tube is raised, A is filled with 
 mercury ; when it is lowered the mercury falls in A, and the air to be analysed 
 then is allowed to enter it by one (x) of the connections at the top. 
 The other connections of the tap connect the burette with absorption pipettes, 
 into which the air can be driven by raising B. The absorption pipette E is 
 filled with 20 per-cent. caustic potash, and is connected with a movable 
 reservoir, S, by black rubber tubing. The absorption pipette F is filled with 
 an alkaline solution of pyrogallic acid (10 gr. of pyrogallic acid to 100 
 c.c. of saturated caustic potash). This is introduced through the tube K. 
 G and H are partly filled with strong potash solution, which protects the 
 pyrogallate solution from the air. The pressure in the burette is adjusted 
 by using the potash pipette as a pressure gauge and bringing the potash before 
 every reading of the burette to the mark M. 
 
GAS ANALYSIS 
 
 285 
 
 In order to make the readings of the burette independent of changes in 
 temperature, barometric pressure, and percentage of moisture during the 
 analysis, a control tube N stands beside the burette in the water jacket ; 
 the threeway tap at P makes it possible to equalise the pressure in N with 
 that of the atmosphere ; by means of the T-tube O the potash solution is 
 brought into connection with N, and the potash is adjusted to the mark R 
 by raising or lowering S, P being open to the air. P is then turned, so that 
 the control tube N is connected with the potash tube only, and is not again 
 opened until the analysis is complete. Each time a reading of the burette 
 is made, the potash is brought to the mark R by raising or lowering S. The 
 
 Fig. 71. — Diagram of Haldane's apparatus for air analysis. 
 
 potash in the absorption pipette is then brought to the mark M by adjusting 
 the levelling tube B. In this way variations of temperature and pressure 
 are compensated for by mechanical means. The lower part of the control 
 tube N is kept full of water ; and the gas-burette must have its inner surface 
 wet ; the water used for moistening the inner surface of the burette should 
 be slightly acidulated with sulphuric acid. The acidified water is introduced 
 through the free limb of any tap, excess being expelled by raising the levelling 
 tube B. By this means the air in the burette and in the control tube is 
 always kept saturated with moisture. 
 
 In the complete apparatus, a combustion pipette is added also (to 
 estimate carbon monoxide, methane, etc.), but in air analyses, where one has 
 only to deal with oxygen, nitrogen, and carbonic acid, this is hot necessary.^ 
 
 1 For full details see Methods of Air Analysis, by J. S. Haldane. 
 
286 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 In such an analysis the apparatus must first be filled with nitrogen ; if 
 the apparatus is used for a succession of analyses, a supply of nitrogen is 
 left at the end of each. If not, the air in the apparatus should be freed from 
 oxygen and carbonic acid by passing it into the pyrogallate and potash pipettes 
 respectively. The next step is to bring the pyrogallate and potash to the 
 marks D, R, and M. The tap closing the control tube N is then closed. The 
 sample of the air to be analysed is then introduced into the bui'ette by lower- 
 ing the levelling tube ; and the tap is turned so as to connect the burette 
 with the potash pipette. The opening of the tap will probably slightly dis- 
 turb the level M, at which the potash previously stood, and the potash level 
 at R may also shift a little. The potash level is exactly adjusted to R by 
 raising or lowering S ; and the exact levelling to the mark M is adjusted 
 by raising or lowering B. The burette is then read to give the total volume 
 of air introduced into the apparatus. The air is then driven into the potash 
 pipette by raising B ; it is then brought back again by depressing B, and this 
 is repeated until, on measuring the gas, there is no further diminution 
 in volume ; probably half a dozen times will be sufficient ; the reading must 
 always be taken when the potash levels are at M and R. The diminution 
 of volume gives the amount of carbonic acid. The burette is then con- 
 nected with the pyrogallate pipette, and the air driven over into it several 
 times in the same way ; some oxygen, however, will still be left in the con- 
 nection between M and the tap, so this connection is washed out by passing 
 the gas into the potash pipette and back, and then into the pyrogallate 
 pipette twice. Finally, the levels at D, R, and M are adjusted, and the 
 reading indicates the volume of oxygen absorbed. 
 
 SOLUTIONS— DIFFUSION— DIALYSIS— OSMOSIS 
 
 The investigations of physical chemists during recent years have given us 
 new conceptions of the meaning of the words that stand at the head of this 
 section. I propose to state what these conceptions are, and briefly to indi- 
 cate the bearing they have on the elucidation of physiological problems. 
 
 Solutions. — Water is the fluid in which soluble materials are usually 
 dissolved, and at ordinary temperatures it is a fluid, the molecules of which 
 are in constant movement ; the hotter the water the more active are the 
 movements of its molecules, until, when at last it is converted into ste^m, the 
 molecular movements become much more energetic. Perfectly pure water 
 consists of molecules with the formula H2O, and these molecules undergo 
 practically no dissociation into their constituent atoms, and it is for this 
 reason that pure water is not a conductor of electricity. 
 
 If a substance such as sugar is dissolved in the water, the solution still 
 remains incapable of conducting an electrical current. The sugar molecules 
 in solution are still sugar molecules ; they do not undergo dissociation. 
 
 But if a substance such as salt is dissolved in the water, the solution is then 
 capable of conducting electrical currents, and the same is true for most acids, 
 bases, and salts. These substances do undergo dissociation, and the simpler 
 materials into which they are broken up in the water are called ions. Thus 
 if sodium chloride is dissolved in water, a certain number of its molecules 
 become dissociated into sodium ions, which are charged with positive 
 
SOLUTIONS AND IONS 287 
 
 electricity, and chlorine ions, which are charged with negative electricity. 
 Similarly a solution of hydrochloric acid in water contains free hydrogen ions 
 and free chlorine ions. Sulphuric acid is decomposed into hydrogen ions and 
 ions of SO4. The term ion is thus not equivalent to atom, for an ion may be 
 a group of atoms, like SO4, in the example just given. 
 
 Further, in the case of hydrochloric acid, the negative charge of the 
 chlorine ion is equal to the positive charge of the hydrogen ion f but in the 
 case of the sulphuric acid, the negative charge of the SO4 ion is equal to *he 
 positive charge of two hydrogen ions. We can thus speak of monovalent, 
 divalent, trivalent, etc., ions. 
 
 Ions charged with positive electricity -are called kat-ions because they 
 move towards the kathode or negative pole ; those which are charged with 
 negative electricity are called an-ions because they move towards the anode 
 or positive pole. The following are some examples of each class : — 
 
 Kat-ions. Monovalent : H, Na, K, NH4, etc. 
 
 Divalent : Ca, Ba, Fe (in ferrous salts), etc. 
 Trivalent : Al, Bi, Sb, Fe (in ferric salts), etc. 
 
 An-ions. Monovalent : CI, Br, I, OH, NOa,- etc. 
 Divalent : S, Se, SO4, etc. 
 
 Roughly speaking, the greater the dilution the more nearly complete is 
 the dissociation, and in a very dilute solution of such a substance as sodium 
 chloride we may consider that the number of ions is double the number of 
 molecules of the salt present. 
 
 The ions liberated by the act of dissociation are, as we have seen, charged ' 
 with electricity, and when an electrical current is led into such a solution it 
 is conducted through the solution by the movement of the ions. Substances 
 which exhibit the property of dissociation are known as electrolytes. 
 
 The conception of electrolytes, which we owe to Arrhenius, is extremely 
 important in view of the question of osmotic pressure which we shall be 
 considering immediately ; because the act of dissociation increases the number 
 of particles moving in the solution and so increases the osmotic pressure, for 
 in this relation the ion plays the same part as a molecule. 
 
 The liquids of the body contain electrolytes in solution, and it is owing 
 to this fact that they are able to conduct electrical currents. 
 
 Another physiological aspect of the subject is seen in a study of the action 
 of mineral salts in solution on living organisms and parts of organisms. Many 
 years ago Ringer showed that contractile tissues (heart, cilia, etc.) continue 
 to manifest their activity in certain saline solutions. Indeed, as Howell puts 
 it, the cause of such rhythmical action is the presence of these inorganic 
 substances in the blood or lymph which usually bathes them. In the case of 
 the heart, the sinus, or venous end of the heart, is peculiarly susceptible to 
 the stimulus of the inorganic salts, and the rhythmical peristaltic waves so 
 started travel thence over the rest of the heart muscle. 
 
 Loeb and his fellow-workers have confirmed these statements, but inter- 
 pret them now as ionic action. Contractile tissues will not contract in pure 
 solutions of non-electrolytes (such as sugar, urea, albumin). But different 
 contractile tissues differ in the nature of the ions which are most favourable 
 stimuli. Thus cardiac muscle, cilia, amoeboid movement, karyokinesis, cell 
 
288 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 division are all alike in requiring a, proper adjustment of ions in their sur- 
 roundings if they are to continue to act, but the proportions must be different 
 in individual cases. 
 
 In the case of the heart, sodium ions are the most potent in maintaining 
 the osmotic conditions that lead to irritability and contractility ; but a solution 
 of pure sodium chloride finally throws the heart into a condition of relaxation : 
 hence it is necessary to mix with it small amounts of calcium ions to restrain 
 this effect ; potassium chloride, the third salt in Ringer's or Locke's fluids, also 
 favours relaxation during diastole. Calcium is the chief ion which produces 
 contraction, and by itself produces intense tonic contraction (calcium rigor). 
 Loeb at one time considered that the process of fertilisation was mainly 
 ionic action, but his later experiments on artificial parthenogenesis have 
 shown that the-flrst change produced by his reagents on the egg-cells of sea 
 urchins and similar animals isthe separation of a membrane from their surface ; 
 this is caused by fatty acids, saponin and other hemolytic agents : this 
 superficial cytolysis stimulates the egg to commence cleavage, but that process 
 soon ceases ; if, however, oxidation is brought about by immersing the egg 
 in hypertonic sea- water for a short time, cleavage continues and well-formed 
 larvae are produced. The spermatozoon has apparently a similar double 
 action ; it produces membrane formation possibly by a fatty acid it carries, 
 and then, having penetrated the membrane, sets up oxidation changes by 
 means of oxidases. In addition to this, certain changes may be produced by 
 other enzymes in the spermatozoon. 
 
 Qramme- molecular Solutions. — From the point of view of osmotic 
 pressure a convenient unit is the gramme-molecule. A gramme-molecule of 
 any substance is the quantity in grammes of that substance equal to its molec- 
 ular weight. A gramme-molecular solution is one which contains a gramme- 
 molecule of the substance per litre. Thus a gramme-molecular solution of 
 sodium chloride is one which contains 58"46 grammes of sodium chloride (Na = 
 23 '000; Cl = 35'46) in a litre. A gramme-molecular solution of glucose 
 (CeHiaOe) is one which contains 180 grammes of glucose in a litre. A gramme- 
 molecule of hydrogen (Ha) is 2 grammes by weight of hydrogen, and if this 
 were compressed to the volume of a litre it would be comparable to a gramme- 
 molecular solution. It therefore follows that a litre containing 2 grammes of 
 hydrogen contains the same number of molecules of hydrogen in it as a litre 
 of a solution containing 58-46 grammes of sodium chloride, or one containing 
 180 grammes of glucose has in it of salt or sugar molecules respectively. To 
 put it another way, the heavier the weight of a molecule of any substance the 
 more of that substance must be dissolved in the litre to obtain its gramme- 
 molecular solution. Or still another way : if solutions of various substances 
 are made all of the same strength per cent., the solutions of the materials 
 of small molecular weight will contain more molecules of those materials than 
 the solutions of the materials which have heavy molecules. We shall see 
 that the calculation of osmotic pressure depends on these facts. 
 
 Diffusion, Dialysis, Osmosis. — If two gases are brought together within 
 a closed space, a homogeneous mixture of the two is soon obtained. This is 
 due to the movements of the gaseous molecules within the space, and the 
 process is called dijffkmon. In a similar way diffusion will effect in time a 
 homogeneous mixture of two liquids or solutions. If water is carefully poured 
 
OSMOTIC PRESSURE 
 
 289 
 
 N 
 
 on to the surface of a solution of salt, the salt or its ions will soon be equally 
 distributed throughout the whole. If a solution of albumin or any other 
 colloidal substance is used instead of salt in the experiment, diffusion will be 
 found to occur much more slowly. If, instead of pouring water on to the 
 surface of a solution of salt or sugar, the two are separated by a membrane 
 made of such a material as parchment paper, a similar diffusion will occur, 
 though more slowly than in cases where the membrane is absent. In time, 
 the water on each side of the membrane will contain the same quantity of 
 sugar or salt. Substances which pass through such membranes are called 
 crystalloids. Substances which have such large molecules (starch, protein, 
 etc.) that they will not pass through such membranes are called colloids. 
 Diffusion of substances in solution in which we have to deal with an interven- 
 ing membrane is usually called dialysis. The process of filtration {i.e. the 
 passage of materials through the pores of a membrane under the influence of 
 mechanical pressure) may be almost excluded in such experiments by placing 
 the membrane (M) vertically as shown in the diagram (fig. 72), and the two 
 fluids A and B on each side of it. Diffusion through 
 a membrane is not limited to the molecules of water, 
 but it may occur also in the molecules of certain sub- 
 stances dissolved in the water. But very few if any 
 membranes are equally permeable to water and to 
 molecules or ions of the substances dissolved in the 
 water. If in the accompanying diagram the compart- 
 ment A is filled with pure water, and B with a sodium 
 chloride solution, the liquids in the two compart 
 ments will ultimately be found to be equal in bulk as 
 they were at the start, and each will be a solutipn of 
 salt of half the original strength of that in the com- 
 partment B. But at first the volume of the liquid 
 in compartment B increases, because more^ water 
 molecules pass into it from A than salt molecules 
 pass from B into A. The term osiuosis is generally limited to the stream of 
 water molecules passing through a membrane, while the term dialysis is 
 applied to the passage of the molecules in solution in the water. The osmotic 
 stream of water is especially important, and in connection with this it is 
 necessary to explain the term osmotic pressure. At first, then, osmosis (the 
 diffusion of water) is more rapid than the dialysis (the diffusion of the salt 
 molecules or ions). The older explanation of this was that salt attracted the 
 water, but we now express the fact differently by saying that the salt in solu- 
 tion exerts a certain osmotic pressure : the result of the osmotic pressure is 
 that "more water flows from the water side to the side of the solution than in 
 the contrary dirfection. The osmotic pressure varies with the amount of 
 substance in solution, and is also altered by variations of temperature, 
 occurring more rapidly at high than at low temperatures. 
 
 If we imagine two masses of water separated by a permeable membrane, 
 as many water molecules will pass through from one side as from the other, 
 and so the volumes of the two masses of water will remain unchanged. If 
 now we imagine the membrane M is not permeable except to water, and 
 the compartment A contains water, and the compartment B contains a 
 19 
 
 A 
 
 B 
 
 m 
 
 E-JrE 
 
 Fla. 72. 
 
290 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 solution of salt or sugar ; in these circumstances water will pass through 
 into B, and the volume of B will increase in proportion to the osmotic pres- 
 sure of the sugar or salt in solution in B, but no molecules of sugar 
 or salt can get through into A from B, so the volume of fluid in A 
 will continue to decrease, until at last a limit is reached. The deter- 
 mination of this limit, as measured by the height of a column of fluid 
 (e.g. mercury) which it will support, will give us a measurement of the osmotic 
 pressure. Membranes of this nature are called semi-permeable. One of 
 the best kinds of semi-permeable membrane is ferrocyanide of copper. This 
 may be made by taking a cell of porous earthenware, and washing it out first 
 with copper sulphate and then with potassium ferrocyanide. An insoluble pre- 
 cipitate of copper ferrocyanide is thus deposited in the pores of the earthen- 
 ware. If such a cell is filled with a 1 per-cent. solution of sodium chloride, 
 water difiiises in till the pressure registered by a manometer connected to it 
 registers the enormous height of 5000 millimetres of mercury. Theoretically 
 it is possible to measure osmotic pressure by a manometer in this way, but 
 practically it is seldom done, and some of the indirect methods of measurement 
 described later are used instead. The reason for this is that it has been found 
 difiicult to construct a membrane which is absolutely semi-permeable. 
 
 Many explanations of the nature of osmotic pressure have been brought 
 forward, but none is perfectly satisfactory. The following simple explanation 
 IS perhaps the best, and may be rendered more intelligible by an illustration. 
 Suppose we have a solution of sugar separated by a semi-permeable membrane 
 from water : this is, the membrane is permeable to water molecules, but not 
 to sugar molecules. The streams, of water from the two sides will then be 
 unequal ; on one side we have water molecules striking against the mem- 
 brane in what we may call normal numbers, whUe on the other side both water 
 molecules and sugar molecules are striking against it. On this side, therefore, 
 the sugar molecules take up a certain amount of room, and do not allow the 
 water molecules to get to the nlembrane ; the membrane is, as it were, screened 
 against the water by the sugar j therefore fewer water molecules will get through 
 from the screened to the unscreened side than vice versa. This comes to the 
 same thing as saying that the osmotic stream of water is greater from the 
 unscreened water side to the screened sugar side than it is in the reverse 
 direction. The more sugar molecules that are present, the greater will be 
 their screening action, and thus we see that the osmotic pressure is propor- 
 tional. to the number of sugar molecules in the solution : that is, to the con- 
 centration of the solution. 
 
 Osmotic pressure is, in fact, equal to that which the dissolved substance 
 would exert if it occupied the same space in the form of a gas (Van't Kofi's 
 hypothesis).' The nature of the substance makes n,o difference ; it is only 
 the number of molecules which causes osmotic pressure to vary. The osmotic 
 pressure, however, of substances like sodium chloride, which are electrolytes, 
 is greater than what one would expect from the number of molecules present. 
 This is because the molecules in solution are dissociated into their constituent 
 
 ' Moore and Frazer find that the law may be more accurately expressed aS 
 follows : The pressure is that which would be exerted if the substance in solution 
 was rendered gaseous at the same temperature and kept to the volume of the pure 
 solvent used (water) instead of the volume of the entire solution. 
 
OSMOTIC PRESSURE 291 
 
 ions, and an ion plays the same part as a molecule, in questions of osmotic 
 pressure. In diute solutions of sodium chloride ionisation is more complete, 
 and as the total number of ions is then nearly double the number of original 
 molecules, the osmotic pressure is nearly double what would have been 
 calculated from the number of molecules. 
 
 The analogy between osmotic pressure and the partial pressure of gases is 
 complete, as may be seen from the following statements : — 
 
 1. At a constant temperature osmotic pressure is proportional to the 
 concentration of the solution (Boyle-Mariotte's law for gases). 
 
 2. With constant concentration, the osmotic pressure rises with and is 
 proportional to the temperature (Gay-Lussac's law for gases). 
 
 3. The osmotic pressur'e of a solution of different substances is equal to 
 the sum of the pressures which the individual substances would exert if they 
 were alone in the solution (Dalton-Henry law for partial pressure of gases). 
 
 4. The osmotic pressure is independent of the nature of the substance in 
 solution, and depends only on the number of molecules or ions in solution 
 (Avogadro's law for gases). 
 
 Calculation of Osmotic Pressure. — We may best illustrate this by an 
 example, and to simplify matters we will take an example in the case of a 
 non-electrolyte such as sugar. We shall then not have to take into account 
 any electrolytic dissociation of the molecules into ions. We will suppose we 
 want to calculate the osmotic pressure of a 1 per-cent. solution of sucrose. 
 
 One gramme of hydrogen at atmospheric pressure and 0° 0. occupies a 
 volume of 11 '2 litres ; 2 grammes of hydrogen will therefore occupy a 
 volume of 22"4 litres. A gramme-molecule of hydrogen — that is, 2 grammes 
 of hydrogen — when brought to the volume of 1 litre will exert a gas pressure 
 equal to that of 22'4 litres compressed to 1 litre — that is, a pressure of 22 4 
 atmospheres. A gramme-molecular solution of sucrose, since it contains 
 the same number of molecules in a litre, must therefore exert an osmotic 
 pressure of 22'4 atmospheres also. A gramme-molefiular solution of sucrose 
 (C12H22O11) contains 342 grammes of sucrose in a litre. A 1 per-cent. 
 solution of sucrose contains only 10 grammes of sucrose in a litre of 
 water ; hence the osmotic pressure of a 1 per-cent. solution of suoro.se is 
 
 — X 22-4 atmospheres, or 0'65 of an atmosphere, which in terms of a column 
 
 of mercury = 760 x 0"65 = 494 mm. 
 
 It would not be possible to make such a calculation in the case of an electro- 
 lyte, because we should not know how many molecules had been ionised. In 
 the liquids of the body, both electrolytes and non-electrolytes aie present, 
 aijd so a calculation is here also impossible. 
 
 We have seen the difficulty of directly measuring osmotic pressure by a 
 manometer ; we now see that mere arithmetic often fails us ; and so we come 
 to the question to which we have been leading up, viz., how osmotic pressure 
 is actually determined. 
 
 Determination of Osmotic Pressure by means of the Freezing-point.— 
 This is the method which is almost universally employed. A very simple 
 apparatus (Beckmann's differential thermometer) is all that is necessary. The 
 principle on which the method depends is the following : — The freezing- 
 
292 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 point of any substance in solution in water is lower than that of water ; the 
 lowering of the freezing-point is proportional to the moleculaq' concentration 
 of the dissolved substance, and that, as we have seen, is proportional to 
 the osmotic pressure. 
 
 When a gramme-molecule of any substance is dissolved in a litre of water, 
 the freezing-point is lowered by 1"87° C, and the osmotic pressure is, as we 
 have seen, equal to 22'4 atmospheres: that is 22'4x 760 = 17,024 mm. of 
 mercury. 
 
 We can therefore calculate the osmotic pressure of any solution if we 
 know the lowering of its freezing-point in degrees Centigrade ; the lowering 
 of the freezing-point is usually expressed by the Greek letter A. 
 
 Osmotic pressure= — — x 17,024. 
 
 For example, a 1 per-cent. solution of sucrose would freeze at — 0'052° C. ; 
 its osmotic pressure is therefore j^ ' =473 mm., a number approxi- 
 mately equal to that we obtained by calculation. 
 
 Mammalian blood serum gives A=0'56° C. A 0'9 percent, solution of 
 sodium chloride has the same A ; hence serum and 09 per-cent. solution 
 of common salt have the same osmotic pressure, or are isotonic. The osmotic 
 
 pressure of blood serum is ——i = 5000 ram. of mercury approximately, 
 
 1*87 
 
 or a pressure of nearly 7 atmospheres. 
 
 The osmotic pressure of solutions may also be compared by observing 
 their effect on red corpuscles, or on vegetable cells such as those in Tra- 
 descantia. If the solution is hypertonic, i.e. has a greater osmotic pressure than 
 the cell contents, the protoplasm shrinks and loses water, or, if red corpuscles 
 are used, they become crenated. If the solution is hypotonic,-e.g. has a smaller 
 osmotic pressure than the material within the cell-wall, no shrinking of the 
 protoplasm in the vegetable cell occurs, and if red corpuscles are used they 
 swell and liberate their pigment. Isotonic solutions produce neither of these 
 effects, because they have the same molecular concentration and osmbtic 
 pressure as the material within the cell-wall. 
 
 Physiological Applications. — It will at once be seen how important all 
 these considerations are from the physiological standpoint. In the body we 
 have aqueous solutions of various substances separated from one another by 
 membranes. Thus we have the endothelial walls of the capillaries separating 
 the blood from the lymph ; we have the epithelial walls of the kidney tubules 
 separating the blood and lymph from the urine ; we have similar epithelium 
 in all secreting glands ; and we have the wall of the alimentary canal separating 
 the digested food from the blood-vessels and lacteals. In such important 
 problems, then, as lymph-formation, the formation of urine and other excre- 
 tions and secretions, and the absorption of food, we have to take into account 
 the laws which regulate the movements both of water and of substances 
 which are held in solution by the water. In the body osmosis is not the only 
 force at work, but we have also to consider filtration ; that is, the =forcible 
 passage of materials through membranes, due to differences of mechanical 
 pressure. Further complicating these two processes we have to take into 
 
OSMOTIC PRESSURE 293 
 
 account another fcice ; namely, the secretory or selective activity of the 
 living cells of which the membranes in question are composed. This is some- 
 times called by the name vital action, which is an unsatisfactory and unscientific 
 expression. The laws which regulate filtration, imbibition, and osmosis are 
 fairly well known and can be experimentally verified. But we have un- 
 doubtedly some other force, or some other manifestation of force, in the case 
 of living membranes. It probably is some physical or chemical property of 
 living matter which has not yet been bi'ought into line with the known chemical 
 and physical forces which operate in the inorganic world. We cannot deny 
 its existence, for it sometimes operates so as to neutralise the known forces of 
 osmosis and filtration. 
 
 The more one studies the question of lymph-formation, the more con- 
 vinced one becomes that mere osmosis and filtration will not explain it entirely. 
 The basis of the action is no doubt physical, but the living cells do not behave 
 like the dead membrane of a dialyser ; they have a selective action, picking 
 out some substances and passing them through to the lymph, while they 
 reject others. 
 
 The question of gaseous interchanges in the lungs has been another battle- 
 field of a similar kind. Some maintain that all can be explained by the laws 
 of difi'usion of gases ; others have asserted that the action is wholly vital ; 
 but recent research has shown that the main facts are explicable on a 
 physical basis (see p. 171). Take again the case of absorption. The object 
 of digestion is to render the food soluble and difi^usible ; it can hardly be 
 supposed that this is useless ; the readily diffusible substances will pass more 
 easily through into the blood and lymph : but still, as Waymouth Eeid has 
 shown, if the living epithelium of the intestine is removed, absorption comes 
 very nearly to a standstill, although from the purely physical standpoint 
 removal of the thick columnar epithelium would increase the facilities for 
 osmosis and filtration. 
 
 The osmotic pressure exerted by crystalloids is very considerable, but 
 their ready diffusibility limits their influence on the flow of water in the body. 
 Thus, if a strong solution of salt is injected into the blood, the first effect 
 will be the setting up of an osmotic stream from the tissues to the blood. 
 The salt, however, would soon diffuse out into the tissues, and would now 
 exert osmotic pressure in the opposite direction. Moreover, both effects will 
 be but temporary, because excess of salt is soon got rid of by the excretions. 
 
 Osmotic Pressure of Proteins. — It has been generally assumed that proteins, 
 the most abundant and important constituents of the blood, exert little or no 
 osmotic pressure. Starling, however, has claimed that they have a small 
 osmotic pressure ; if this is so, it is of importance, for proteins, unlike salt, do 
 not diffuse readily, and their effect, therefore, remains as an almost permanent 
 factor in the blood. Starling gives the osmotic pressure of the proteins of 
 the blood-plasma as equal to 30 mm. of mercury. By others this is attributed 
 to the inorganic salts with which proteins are always closely associated.^ 
 
 1 Bayliss has shown that the saline constituents in a protein are not mechanically 
 mixed with it, but are in a state intermediate between mechanical admixture and 
 chemical combination, to which the term adsorption is applied. Many dyes used 
 for staining fabrics and histological preparations are also adsorbed. 
 
294 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 Moore, for instance, finds that the purer a protein is, the less is its osmotic 
 pressure ; the same is true for other colloidal substances.' It really does not 
 matter much, if the osmotic force exists, whether it is due to the protein 
 itself, or to the saline constituents which are almost an integral part of a 
 protein. It is merely interesting from the theoretical point of view. We 
 should from the theoretical standpoint find it diflScult to imagine that a pure 
 protein can exert more than a minimal osmotic pressure. It is made up of 
 such huge molecules that, eve^ when the proteins are present to the extent 
 of 7 or 8 per cent., as they are in blood-plasma, there are comparatively few 
 protein molecules in solution, and probably none in true solution. Still, by 
 means of this weak but constant pressure it is possible to explain the fact that 
 an isotonic oi» even a hypertonic solution of a diffusible crystalloid may be 
 completely absorbed from the peritoneal cavity into the blood. 
 
 The functional activity of the tissue elements is accompanied by the 
 breaking down of their constituents into simpler materials. These materials 
 pass into the lymph, and increase its molecular concentration and its csmotic 
 pressure ; thus water is attracted (to use the older way of putting it) from the 
 blood to the lymph, and so the volume of the lymph rises and its flow increases. 
 On the other hand, as these substances accumulate in the lymph they will in 
 time attain there a greater concentration than in the blood, and so they will 
 diffuse towards the blood, by which they are carried to the organs of excretion. 
 
 But, again, we have a difficulty with the proteins ; they are most important 
 for the nutrition of the tissues, but they are practically indiffusible. We 
 must provisionally assume that their presence in the lymph is due to filtration 
 from the blood. The plasma in the capillaries is under a somewhat higher 
 pressure than the lymph in the tissues, and this tends to squeeze the con- 
 stituents of the blood, including the proteins, through the capillary walls. 
 I have, however, already indicated that the exact mechanism of lymph- 
 formation is one of the many physiological problems which await solution by 
 the physiologists of the future. 
 
 COLLOIDS AND COLLOIDAL SOLUTIONS 
 
 The proteins, the polysaccharides, and the soaps may be taken as instances 
 of colloids. They do not pass through the membrane of a dialyser, they can 
 be "salted out" of solution, their solutions are opalescent, they crystallise 
 with difficulty if at all, they have a tendency to form jellies, and they exert 
 a very low osmotic pressure. 
 
 The study of colloids, a class to which so many important physiological 
 substances belong, is therefore important, and the following paragraphs deal 
 briefly with some of the principal facts now known in relation to this branch 
 of physical chemistry. 
 
 A colloid material is spoken of as being capable of assuming two condi- 
 tions, which are respectively named sol and gel. If the colloid is fluid, the 
 term sol is used ; if it is solid like a jelly, the word gel is employed. The 
 two conditions are well illustrated in the case of gelatin ; in warm water 
 gelatin is a sol ; when the solution cools we get a gel. In the case of gelatin 
 the condition is easily reversible, but this is not so in relation to all colloids. 
 
 If water is the fluid medium employed, the terms "hydrosol" and 
 
COLLOIDS 295 
 
 hydrogel " are applied ; if alcohol is used, the words " alcoholsol " and 
 "aleoholgel " are employed, and so with other solvents. 
 
 Colloid material is often obtainable in another condition still, namely, as 
 a flocculent precipitate. , This is seen when proteins are " salted out," or 
 when an albuminous solution is heated beyond its " coagulating point." In 
 some cases an enzyme action will alter the physical and chemical structure of 
 the protein so that it becomes insoluble in the fluid in which it was previously 
 apparently dissolved. This is seen in the familiar examples of the clotting 
 of blood by thrombin, and the curdling of milk by rennin. 
 
 According to this conception the curdling of milk is a physical rather 
 than a chemical change, caseinogen being the sol, and tiasein the gel condi- 
 tion. A similar view has been urged in connection with blood clotting by 
 Hekma (a series of papers in Biochem. Zeiisch., vols. 73 and 74 1916), and 
 by Howell (Amer. Jowrn. Physiol., vol. 40, p. 526, 1916) ; they regard 
 fibrinogen as a fibrin sol, and fibrin as the gel condition. Fibrin is first 
 deposited as ultra-microscopic particles (microns), and then fine needle-like 
 crystals ; these by agglutinating together ultimately lead to the formation 
 of typical fibrin threads. 
 
 There are numerous analogies between these organic colloids and the 
 inorganic colloids. Thus several metals, such as gold, silver, and platinum, 
 are obtainable in colloidal form, and the same is true for certain compounds 
 such as silicic acid. These materials are in an unstable physical condition, 
 passing from the sol to the gel condition under slight provocation. This 
 confers upon them the property of producing what is termed catalysis in 
 chemical substances in contact with them, and the similarities between catalysis 
 and enzyme action are so striking and numerous, that the doctrine that enzyme 
 action is a catalytic one rests on a by no means unstable foundation. 
 
 Supposing now the case of a colloid in the condition of a sol, as for instance 
 the proteins are in the plasma or serum of the blood, does that term imply 
 complete solution in the same way as when we use the term in reference to 
 a solution of salt or sugar ? Or have we, on the other hand, rather a condition 
 of suspension, or a kind of attenuated gel ? 
 
 The microscopic examination of such fluids, even with the highest powers, 
 reveals no visible particles. The particles which are present if they are not 
 in solution are present either in a smaller or in a more diffuse condition than 
 the particles of an ordinary suspension or emulsion. 
 
 An ordinary paper filter has far too large pores to keep back any particles 
 from such fluids. It is necessary to construct a filter of a more efficient 
 character. The kind of filter employed is fashioned on the principle of those 
 used for filtering off small particles such as bacteria from fluids. One of the 
 best is that described by C. J. Martin. The case of the candle of a Pasteur- 
 Ohamberland filter is filled with a hot 10 per-cent. solution of gelatin, and 
 this is forced by air pressure through the pores of the porcelain. The hot 
 solution filters through fairly quickly at first, but as the pores get stopped 
 up it runs through more slowly ; when it is cold, the filter case is removed 
 from the compressed-air cylinder, and the filter detached from its case. The 
 gelatin is then washed off from the outside of the filter, and it is ready for us^. 
 
 Instead of a gelatin filter, one of silicic acid can be made. A stiff solution 
 
296 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 of sodium silicate is filtered under pressure through the candle ; after a few 
 minutes, when the pores are filled with it, the candle can be detached, filled 
 with 3 per-cent. hydrochloric acid and immersed in a bath of the same acid for 
 a day or two. The acid diffuses into the 4)ores, and decomposes the sodium 
 silicate, depositing a gelatinous precipitate of silicic acid. 
 
 If fresh serum or egg-white is placed outside the filter, the filtrate which 
 comes through is clear, colourless, and absolutely free from protein. 
 
 Proteoses and crystalloids pass the membrane easily ; meta-proteins 
 slightly ; caramel, biliverdin, and dextrins partially. But the following pro- 
 teins do not pass it at all : egg-albumin, serum-albumin, egg-globulin, serum- 
 globulin, fibrinogen, caseinogen, nucleo-proteins, and hsemoglobin. The 
 colloid carbohydrates starch and glycogen resemble the proteins. 
 
 In other words, substances with large molecules which do pot pass through 
 membranes by dialysis are also stopped by filtration under pressure through 
 a gelatin or silicic-acid filter, and some are inclined to regard the large size of 
 the molecule as the reason of the non-passage in both cases, and do not agree 
 with Ostwald that the solutions are mechanical mixtures and not true solu- 
 tions. The small osmotic pressure which such substances as protein exert 
 may be regarded as evidence of true solution. But, as we have already seen, 
 we are by no means certain that absolutely pure proteins do not exert osmotic 
 pressure, and further, osmotic pressure appears to be exerted by substances 
 which are admittedly not in true solution. The working hypothesis adopted 
 in this dilemma by the majority of observers is, that in such fluids we are not 
 dealing with true solutions nor with suspensions of fine particles, and the 
 term " colloidal solution " has been invented to express the condition of things, 
 which appears to be something of an intermediate nature. 
 
 The similarity between colloidal solution and fine suspension is a marked one. 
 The well-known migration of obvious suspensions (including bacteria) in an 
 electric field is evident also in colloidal solutions ; and as Hardy has shown in the 
 case of certain proteins, the sign of the charge in the colloidal state or in sus- 
 pension may be reversed by very slight alterations in the reaction of the fluid. 
 
 Further, both suspensions and colloidal solutions give what is known as 
 the Tyndall phenomenon, scattering light ; this test forms the basis of 
 what are termed ultra-microscopic observations. 
 
 As compared with ordinary solutions, a very small expenditure of energy 
 is necessary to separate matter in colloidal solution from its " solvent " ; and 
 the vapour pressure and freezing-point of the " solvent " are only altered to a 
 negligible degree by the incorporation of the colloid in it. Still, this in itself 
 is not characteristic of colloids, for the same is true for certain pairs of liquids 
 (for instance, dichloracetic acid and isopentane) which form true solutions 
 together. 
 
 Precipitation by electrolytes is again a striking feature common to col- 
 loidal solutions and suspensions, and the precipitating ions are carried down in 
 amounts which are proportional to the amount of precipitate. The agglutina- 
 tion of bacteria is possibly a phenomenon of the same order. There are, 
 however, diflFerences between the behaviour of inorganic colloids and proteins 
 mot only to electrolytes, but also to non-electrolytes, which Waymouth Reid 
 considers still require elucidation, and in the case of electrolytes Pauli points 
 out a certain specificity in the ionic actions, the end result being determined by 
 
SURFACE TENSION 297 
 
 the algebraic sum of the antagonistic actions of the precipitant and anti- 
 precipitant properties of the kation and anion of a salt. 
 
 The view that a colloidal solution approaches near to the state of extremely 
 fine suspension is favoured by the facts that both reduce the surface tension 
 of the fluid containing them, and that both readily, form surface films of 
 greater concentration which can be heaped up' mechanically and separated 
 by agitation ; this, for instance, occurs in emulsification. The case of haemo- 
 globin shows that this substance, though non-dialysable and capable of being 
 filtered off by an efficient filter, is not on all-foiu's with other pioteins in other 
 particulars, for it is probably dissolved by water (W. Reid). It is possible 
 as I'esearch proceeds othei' exceptions to the general lule may be found, and 
 that the native proteins, although colloids, nevertheless exhibit gradations 
 from those at one extreme which form true solutions, to those at the other 
 which form obvious suspensions only. 
 
 SURFACE TENSION 
 
 The surface layer of a liquid possesses certain properties which are not 
 shared by the rest of it, for in the interior the arrangement of matter is sym- 
 metrical around any point, whereas on the surface the surroundings consist 
 of liquid on one side only, while on the other side is solid or gas, or it may be 
 another liquid. In a gas the molecules are free from one another's attractive 
 influence, and ily about freely with high velocity, producing pressure on the 
 walls of the containing vessel ; in a liquid, the mutual attraction of the 
 molecules is great enough to keep the substance together in a definite volume. 
 In order to separate the molecules and convert the liquid into gas, a large 
 amount of energy is required — the so-called latent heat of evaporation. The 
 molecular attractions in a liquid are thus very great, so that any molecule 
 of the surface layer is strongly pulled inwards, and this layer constitutes a 
 stretched elastic skin, and the power thus exerted is termed surface tension. 
 The effect of surface tension is most simply seen in a free drop of liquid, such 
 as a raindrop, or a drop of oil immersed in a mixture of alcohol and water of 
 the same density. There is then nothing to prevent the surface layer from 
 contracting as much as possible, and the drop will assume a form in which its 
 volume has the smallest surface, that is, the fqrm of a sphere. 
 
 Animal cells are liquid, and when they are at rest, other forces being absent, 
 they also are spherical, and although they do not possess as a rule a definite 
 wall of harder material, such as one finds in most vsgetable cells, nevertheless 
 the surface film, exercising the force called surface tension, plays the part of 
 an elastic skin, and is termed the plasmatio membrane. This membrane plays 
 an important physiological r61e. In the projection of pseudopodia, for instance, 
 variation in the surface tension occurs in different parts of the circumference 
 of the cell, and at the points where the surface tension is lowered pseudopodia 
 will be thrust out. Protoplasm, however, is not a homogeneous liquid, but 
 contains substances of varying chemical composition ; those substances which 
 have the power of diminishing surface .tension always show a tendency to 
 accumulate at the surface. Hence the fats and lipoids, which are powerful 
 depressants of surface tension, are found (probably in a state of an extremely 
 fine emulsion) more abundantly in the plasmatic membrane than in other parts 
 
29S ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 of the cell. The interstitial spaces between the fat globules are filled up with 
 a watery colloidal (protein) solution. 
 
 The theory of diifusion of dissolved substances through membranes as 
 applied to cells has been profoundly influenced by the discovery of the com- 
 position of the plasmatic membrane. At one time it was believed that dif- 
 fusion of a colloid material was prevented by the pores of the membrane being 
 too small to allow large molecules to get through them ; it was considered to 
 act as a sort of sieve. But this cannot be the whole explanation, and it is now 
 held that solution affinities play the most important part ; that is to say, a 
 membrane is permeable to substances which are soluble in the material of the 
 membrane. Such solubility may imply the formation of actual chemical 
 unions, but more frequently the process is one of adsorption ; this latter process 
 comes specially into play where nutritive materials are assimilated by the 
 cell by means of the protein solution which occupies the interstices between 
 the fat globules. On the other hand, the permeability of the plasmatic mem- 
 brane by substances such as chloroform and ether is mainly determined by the 
 solubility of these mate rials inthefattyor fat-like componentsof the membrane, 
 and this consideration is the foundation of the Meyer-Overton theory of the 
 narcotic effect on cells which these volatile anaesthetics exercise. 
 
 THE DETERMINATION OF THE REACTION OF FLUIDS 
 
 In any aqueous solution, if the concentration of hydrogen ions (CJ) is 
 multiplied by that of hydroxyl ions (C~), the product is constant. In 
 distilled water at 18° C, the two concentrations are equal (lO"''"'). Therefore 
 C„xC„„ = 10-''"'xlO-'-»' = 10-"-". In acid solutions 0„ exceeds lO-'-"' and - 
 in alkaline solutions the reverse obtains, but in all cases the product is 10~"'". 
 
 The amount of ionisation which acids undergo in solution varies greatly. 
 For instance, in decinormal hydrochloric acid, 91 per cent, is ionised ; there- 
 fore C„ is 0'091 times the normal and is equal to lO"''"*. The figure l'04(the 
 negative sign is usually omitted) is the logarithm to the base 10 of the 
 concentration of hydrogen ions in grammes per litre, and is conveniently 
 expressed as P^^, On the other hand, decinormal acetic acid is only dis- 
 sociated into its ions to the extent of 1'3 per cent., and its P^ is 2"89. 
 
 In the testing of the reaction of a fluid, various indicators are employed ; 
 these undergo a change of colour on the addition of an acid or alkali, and at a 
 certain point an intermediate tint shows what is termed the neutral point. 
 The fluid, however, is only neutral to the particular indicator employed, and 
 the neutral tint does not imply that the concentrations of hydrogen and 
 hydroxyl ions are equal. Different indicators give the so-called neutral 
 point when the concentrations of ions are widely different. Thus a solution 
 which is neutral to litmus (P^ = 6"5 approximately) will not be so to methyl 
 orange (P^ = 4 approximately). The range of a few indicators is given below : — 
 
 Methyl orange . . . . 3'1 to 4'4 
 
 Topfer's reagent . . . 2'9 to 4'2 
 
 Litmus . . . . . . 5-Oto 8-0 
 
 ' Tropaeolin 00 .1-4 to 26 
 
 Phenolphthalein .... . . 8-3 to 10-0 
 
microc^emical analysis 299 
 
 The amount of decinormal soda which must be added to an acid fluid to 
 render it neutral is called its " titration acidity " to the indicator employed. 
 Thus normal urine, which owes its acidity partly to acid salts and partly to free 
 acids, has a P„ = 5, so that it will be acid to litmus, but alkaline to methyl orange. 
 
 The true acidity is ascertained by combining observations with a large 
 number of indicators, or, better still, by an electrometric examination of the 
 fluid. It is only necessary to estimate the concentration of hydrogen ions ; 
 the concentration of hydroxyl ions can always be calculated, because, as 
 already stated, the product of the two is a constant. 
 
 The electrometric method we owe to Nernst. It is based on the fact that 
 a metal electrode saturated with hydrogen, immersed in a liquid also saturated 
 with hydrogen, gives rise to a difference of potential between the electrode 
 and the liquid which is dependent on the concentration of hydrogen ions 
 previously present in the liquid. This concentration is calculated from the 
 difference of potential observed. The method presupposes that the saturation 
 with hydrogen referred to produces no change in the hydrogen ion concentra- 
 tion of the original liquid. This assumption is not always correct, especially 
 when one is dealing with biological fluids ; these frequently contain volatile 
 acids or bases which will be partly swept out of solution by the current of 
 hydrogen gas. It will be sufficient to say, without entering into technical 
 details, t£at there are methods for overcoming this difficulty. 
 
 MICKOCHEMICAL QUANTITATIVE ANALYSIS 
 
 Certain physiological problems have so far baffled experimental investiga- 
 tion, owing to the minute quantity in which many important substances occur 
 in the organs and fluids of the body. This fact makes it in many cases im- 
 possible to apply to them the usual quantitative methods of the analytical 
 chemist, or to identify them, when isolated*, by the classical methods of 
 elementary analysis. Within recent years, however, the ingenuity of many 
 workers has been applied to the elaboration of microchemical and micro- 
 physical methods, which rival in exactness the older methods. A few of these 
 may be mentioned here in outline, as the full description of experimental 
 detail is outside the scope of this book. 
 
 1. Quantitative Micro -elementary Analysis (Pregl). — The principle 
 of the method is the same as that mentioned in Lesson I. 3. Its application to 
 very small quantities of material (5-10 mg.) has been made possible by the 
 construction of sensitive micro-balances (Ifernst, Kuhlmann), which allow 
 weighings to be made with an accuracy of ±0"001 mg. Suitable small 
 absorption apparatus for carbon dioxide and water have been constructed by 
 Pregl. The same author has also worked out methods for the estimation of 
 nitrogen in small quantities of organic substances depending on the principle 
 of Dumas's and Kjeldahl's (p. 251) rnethods. Micro-Dumas, micro-Kjeldahl, 
 micro-sulphur, and micro-halogefl estimations have been made possible by the 
 introduction of micro-filtration apparatus made out of capillary tubing or of 
 platinum. 
 
 2. Microchemical Analysis of Blood. — Bang and others have described 
 methods for the volumetric estimation of glucose and sodium chloride in 
 2-3 drops ( = 100 mg.) of blood, and have also indicated methods for the estima- 
 
300 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 tion of albumin, globulin, haemoglobin, urea, uric acid, etc., in blood by the 
 use of suitable micro-methods. By Winterstein's method the oxygen can be 
 estimated in 0'05 c.c. of blood. {See also Barcroft's method, p. 281.) 
 
 3. Microchemical Analysis of Urine. — Methods for this purpose have 
 been developed mainly by Folin, by means of which the total nitiogen, ammonia, 
 and urea may be estimated in 1 c.c. of urine. Urea may also be estimated in 
 the same small quantity by Marshall's method, who makes use of the con- 
 version of urea into ammonium carbonate by the enzyme urease contained in 
 soy beans. Further, a quantitative gravimetric method for the estimation of 
 urea in extremely small quantities has been based by Fosse on the insolubility 
 of its xanthydrol compound. Folin's microchemical method for uric acid 
 has already been described (p. 257). The colorimetrie method for the 
 estimation of creatinine (p. 258) may also be mentioned in this connection. 
 Drymmond also has modified the benzidine method (p. 263) for the estimation 
 of sulphur and sulphates in small amounts of urine. 
 
 4. Van Slyke's Method for the estimation of amino-nitrogen (p. 232) has 
 been converted by him into a microchemical method, which requires only 
 05 mg. of amino-nitrogen for analysis. 
 
 5. Van Slyke's Method for the estimation of COj in blood-plasma (p. 178) 
 has also been converted into a microchemical method. 
 
 6. Spectrometric Methods for the estimation of cholestei'ol (and " oxy- 
 cholesterol ") and its esters have been worked out by Lifschiitz, which appear 
 to be more trustworthy than the older colorimetrie methods. 
 
 7. Micro-polarimetric Method of E. Fischer. — The usual polarimetric 
 method (p. 223) has been converted into a micro-polarimetric method by 
 reducing the diameter of the tubes used to 1'5 mm., and theii' contents to 
 O'l c.c. The specific gravity of the solutions to be examined is estimated 
 by a micro-pycnometer. Only 5-10 mg. of substance are necessary for an 
 observation, 
 
 8. Microscopic Molecular Weight Estimations may be carried out by 
 Barger's method, which depends on the fact that equimolecular solutions of 
 different substances possess the same vapour density. The estimations are 
 carried out in capillary tubes under the microscope, and require only a few 
 milligrammes of substance. 
 
INDEX 
 
 Abiuretic substances, 114 
 Abrin, 157 
 Absorption, 127-132 
 
 and osmotic pressure, 293 
 bands, 150 
 channels of, 127 
 influence of epithelium on, 128 
 of carbohydrates, 128 
 of fats, 131 
 of proteins, 128 
 
 spectra of haemoglobin and its deriva- 
 tives, 152, 242 
 of myohsematin, 247 
 of urinary pigments, 266 
 Acetaldehyde, 11, 12, 15 
 Acetic acid, 13, 23,. 35, 36, 94 
 
 ester, 11, 18 
 Aceto-acetic acid, 120, 213 
 
 tests for, 21 1 
 Acetone, 16, 120, 213 
 
 tests for, 211, 212 
 Acetyl, 36 
 
 Achromia point, 83, 225 
 Achroo-dextrin, 29, 83, 98, 226 
 Acid ammonium urate, 204, 256 
 fermentation of urine, 204 
 haematin, 241 
 
 absorption spectrum of, 242 
 ha;matoporphyrin, 244 
 
 afesorption spectrum of, 242, 266 
 metaprotein, 43, 84, 104, 114, 216 
 sodium phosphate, 183 
 
 urate, 204 
 tide, 183 
 Acidity, Nernst's electrometric method 
 
 for, 299 
 Acidosis, 119, 178 
 Acids, see under initial letters 
 action on albumin, 42 
 digestive power of, 234, 235 
 Acini, effect of activity on, 97 
 Acrolein, 34, 35 
 Acrylic acid, 35 
 
 series, 35 
 Activation of enzymes, 90 
 Active immunity, 156 
 
 Acute yellovir atrophy of liver, 186, 215 
 
 Adamkiewicz's reaction, 42, 47, 56, 61 
 
 Adenase, 202 
 
 Adenine, 64, 200, 201, 202 
 
 Adenosine, 65 
 
 Adipose tissue, 2, 35, 77 
 
 Adjuncts to food, 81 
 
 Adler's test for blood, 135 
 
 Adrenal cortex, fat in, 39 
 
 Adrenaline, 117, 119 
 
 Adsorption, 239, 293, 298 
 
 Aerobic micro-organisms, 86 
 
 Aerotonometer, .163 
 
 Agglutinating action, 158, 296 
 
 Agglutinins, 158 
 
 Agmatine, 117 
 
 Air changes in lungs, 160 
 
 inspired and expired, 160 
 
 -pump, mercurial, 164, 280 
 Alanine, 45, 46, 51, 114 
 
 and sugar, 119 
 /3-Alanine, 118 
 Alanyl, 51 
 
 -leucine, 52 
 
 -leucyl-tyrosine, 52 
 Albumin in urine, 207, 213 
 estimation of, 207 
 tests for, 207 
 
 egg, in urine, 128 
 Albuminates, 66 
 Albuminoids, 60 
 Albuminometer, Esbach's, 207 
 Albumins, 2, 55, 58, 59, 216 
 
 action of acids and alkalis on, 42 
 
 and globulins, differences between, 59 
 
 tests for elements in, 5, 6 
 
 vegetable, 67 
 Albumoses, see Proteoses 
 Alcapton, 214 
 Alcaptonuria, 214, 215 
 Alcohol, 81 
 
 action on proteins, 58 
 
 oxidation of, 15 
 
 primary, 15 
 
 secondary, 15 
 
 tertiary, 16 
 
 301 
 
302 
 
 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 Alcohol, tests for, 11 
 Alcoholgel, 295 
 
 Alcoholic fermentation of milk, 75 
 of sucrose, 27 
 potash, 33, 243 
 Alcohols, see also under initial letters, 14 
 dihydric, 16 
 hexahydric, 16 
 monohydric, 14 
 trihydric, 16 
 Alcoholsol, 295 
 Aldehyde, 11, 15 
 glycoUic, 17 
 group, 24 
 resin, 12 
 tests for, 11, 12 
 Aldehydes, 15, 17 
 Aldehydrol, 31, 32 
 Aldoses, 23 
 
 Aleurone grains, fig. 3, 29, 56 
 Aliphatic series, 18 
 Alkali-metaprotein, 42, 104, 113, 216, 
 
 236 
 Alkaline hsematin, 213 
 
 absorption spectrum, 242 
 hsematoporphyrin, 266 
 absorption spectrum, 242, 266 
 phosphates in urine, 194 
 pyrogallate, 284 
 reserve of blood, 178 
 tide, 183 
 Alkaloid products of bacteria, 86 
 Alkyls, definition, 14 
 AUantoin, 202 
 Ally! alcohol, 35 
 Alveolar air, 170 
 
 carbon dioxide pressure in, 172, 173, 
 
 174 
 collection of, 171 
 oxygen pressure in, 171, 172 
 epithelium and secretion, 172 
 Amboceptor, 157 
 Amide nitrogen in proteins, 52, 54 
 Amines, produced by bacteria, 116 
 Amino-acetic acid, ««a/jo Glycine, 18, 45 
 -acids, 2, 18, 44, 45, 65, 72, 114 
 decarboxylation of, 116 
 estimation of, 53, 232 i 
 
 fate of, 129, 187 
 
 list of, 49 ! 
 
 liver and, 72 
 
 in urine, 215 I 
 
 -butyric acid, 118 1 
 
 -caproic acids, 24, 45, 46 
 -ethyl alcohol, 40 
 -ethyl sulphonic acid, 123 
 -glutaric acid, 46 
 group, 45 
 
 -hydroxypropionic acid, 45 
 -nitrogen, estimation of, 233, 299 
 in proteins, 53 
 
 Amino-oxypurine, 64, 200 
 
 -oxypyrimidine, 50 
 
 -propionic acid, see Alanine, 45 
 
 -purine, 64, 200 
 
 -succinamic acid, 46 
 
 -succinic acid, 46 
 
 thio-propionic acid, 204 
 
 -valeric acid, 45 
 Ammonia, 2, 50, 114 
 
 and urea, 189 
 
 and uric acid, 199 
 
 excretion of, 10 
 
 in urine, 119, 190, 254 
 estimation of, 252 
 
 recognition of, 6 
 
 solution of, 283 
 
 uses of, 190 
 Ammoniacal decomposition of urine, 86 
 Ammonium carbonate and urea, 189 
 
 carbonate and urea, 189, 205 
 
 salts, effect of giving, 190 
 
 sulphate on proteins, 57, 69, 134, 
 230 
 Ammonium - magnesium phosphate, 
 
 194, 195 
 Amoeboid movement, 287 
 Amygdalin, 31 
 Amyl alcohol, 279 
 Amylase, 28, 88, 110, 114 
 
 in infants, 114 
 Amylolytic or amyloclastic enzymes, 83, 
 
 88 
 Anabolism,,95 
 
 Anaerobic micro-organisms, 86 
 Anaesthetics, narcotic effect of, 298 
 Analyser prism, 274, 275 
 Anaphylaxis, 158 
 Animal foods, 70, 
 Anions, 92, 234 
 
 list of, 286 
 Animal starch, 30 
 
 Anisotropic bodies, 39, 273, 275, 276 
 Anode or positive pole, 92 
 Antiabrin, 157 
 Antigens, 156 
 
 and anaphylaxis, 158 
 Antiseptics, 83 
 Anti-enzymes, 95 
 
 -pepsin, 106, 138 
 
 -ricin, 157 
 
 -thrombin, 138, 140, 141 
 
 -toxin, 156 
 
 -toxins, 106 
 
 -trypsin, 106, 138 
 
 -venin, 156 
 Apncea, 173 
 Arabinose, 223 
 Arginase, 91, 188, 202 
 Arginine, 48, 53, 54, 91, 114, 117, 202 
 
 fate of, 188, 189 
 
 intravascular injection of, 188 
 
INDEX 
 
 303 
 
 Aromatic amino-acids, 46, 59 
 
 compounds, 18 
 Arrhenius on electrolytes, 287 
 Arterial blood, 148 
 
 gases of, 170 
 Artificial blood corpuscles, 38 
 
 gastric juice, 100 
 
 pancreatic juice, 236 
 
 parlhenogenesis, 288 
 Ash in meat, 5 
 Asparagine, 46 
 Aspartic acid, 46, 114, 118 
 Asphyxia, 145 
 
 Assimilation and immunity, 157 
 Asymmetric carbon atoms, 31, 32, 279 
 Atmospheric air, 160 
 Atomic weights of elements, 8 
 Atypical globulin, 246 
 Autolysis, 89 
 Autolytic enzymes, 89 
 Avogadro's law, 161, 290 
 
 Bacteria, 86 
 
 pathogenic, 158 . 
 
 Bacterial action, 115, 116 
 Bactericidal power of blood and lymph, 
 
 154 
 Bacteriolysins, 155, 157, 158 
 Bang's method of glucose estimation, 
 224 
 micro-method of blood analysis, 299 
 Barcroft's blood-gas apparatus, 165, 280, 
 281, 282 
 mercurial pump, 279, 280 
 tonometer, 166 
 Barger on molecular weight estimation, 
 
 300 
 Barley, 66 
 Baryta mixture, 212 
 Basement membranes, 61, 100 
 Basic group, 48 
 Bayliss on adsorption, 293 
 Beans as food , 70 
 
 Beaumont, Dr, on gastric secretion, 100 
 Beckmann's differential thermometer, 291 
 Beef tea, 80, 247 
 Beetroot, 27 
 
 Bence-Jones protein, 213 
 Benedict's method of sugar estimation, 
 210 
 of sulphur estimation, 262 
 of urea estimation, 254 
 qualitative test for sugar, 20 
 solutions for sugar, 210 
 for sulphur, 262 
 Benedict-Lewis method for blood-sugar, 
 
 240 
 Benger's liquor pepticus, 84 
 
 pancreaticus, 107 
 Benzene, 18 
 
 Benzene nucleus, 18, 19 
 
 ring, 46, 47 
 Benzidine test for blood, 135 
 
 test for sulphates, 263, 300 
 Benzoates for extracting vegetable 
 
 protein, 227 
 Benzoic acid, 203 
 
 ester, 11 
 Beri-beri, 82 
 Bernard Claude, diabetic puncture, 119 
 
 on liver glycogen, 128 
 Bertrand's method of glucose estima- 
 tion, 225 
 Bienenfeld on human caseinogen, 76 
 Bile, 38, 120-125, 217 
 
 actions of, 107, 108, 125 
 
 amount of, daily, 121 
 
 characters of, 121 
 
 circulation of, 122, 125 
 
 constituents of, 121, 122 
 
 in meconium, 127 
 
 in urine, 212, 214, 267 
 
 mucin of, 108, 122 
 
 pigments, 121, 123, 182 
 
 salts of, 90, 121, 122 
 
 tests for, 108, 212 
 
 uses of, 115, 124, 132 
 Biliary fistula, 120 
 Bilicyanin, 124 
 Bilipurpurin, 124 
 Bilirubin, 121, 123, 265 
 Biliverdin, 121, 123 
 Bi-molecular reactions, 93 
 Biological test for blood, 76, 154, 159 
 Birds' blood, coagulation of, 139 
 
 muscle, 247 
 
 plasma, 139 
 
 urine, 256 
 Bitter almonds, 31 
 Bi-urates, 198 
 Biuret, 57, 179 
 
 reaction, 41,. 42, 56, 66, 85, 105 
 cause of, 57 
 " Black-water fever," 214 
 Blood, 133-154, 217, 238-240 
 
 agglutinating action of, 188 
 
 amino-acids in, 129 - 
 
 biological test for, 76, 154, 159 
 
 changes in lungs, 160 
 
 clot, 136 
 
 coagulation, 89, 136, 141 
 experiments on, 133 
 
 corpuscles, 136, 268-271 
 
 crystals, 135 
 
 gas analysis, 164, 280 
 
 gases of, 161-170 
 
 laking of, 144 
 
 micro-chemical analysis of, 299 
 
 oxygen capacity of, 283 
 
 oxygen of, 165 
 
 reaction of, 169 
 
304 
 
 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 Blood plasma and serum, 1, 133, 141, 
 142, 143 
 
 platelets, 136 
 
 pressure, peptone on, 239 
 leech extract on, 239 
 hirudin on, 239 
 
 specific gravity of, 274 
 
 spectroscopic examination of, 135, 
 241 
 
 tests for, 135, 154 
 Bone, 9, 60, 81 
 
 composition of, 60 
 
 corpuscles, 60 
 
 marrow, 35 
 Bowman's capsale, 182 
 Boyle-Mariotte's law, 291 
 Brain, 38, 39, 109 
 Bran, 78 
 Bread, 67, 69, 79 
 
 composition of, 79 
 
 tests with, 69 
 Bright's disease, 213, 214 
 Brown flour, 78 
 Brown and Morris on starch hydrolysis, 
 
 98 
 Buchner on yeast enzymes, 88 
 Buckmaster on COg and haemoglobin, 
 
 170 
 Buffers in blood, 170 
 Buffy coat, 136 
 
 Building stones of protein, 130 
 Bunge on haematogens, 63 
 
 on milk, 72, 73 
 Bush tea, 81 
 Butter, 2, 68 
 Butyl alcohol, 16 
 Butyric acid, 28, 115, 116, 119 
 
 fermentation, 23 
 Butyrin in milk, 75 
 
 Cadaverine, 116, 215 
 Cadmium, -8 
 Caffeine, 81, 200 
 Calcium, 1, 8, 9 
 carbonate in saliva, 97 
 
 in urine, 203, 206 
 caseinogenate, 74, 75, 229 
 chloride, 60 
 fluoride, 60 
 
 blood, 139 
 oxalate in blood, 139 
 in urine, 203, 204 
 
 crystals, 197, 204 s 
 
 rigor of heart, 288 
 
 salts and coagulation of blood, 74, 
 138, 139 
 ofmilk, 74, 229 
 soap, 34 
 Camphor and Tollens' test, 213, 224 
 Canaliculi, 60 
 
 Cane sugar, see Sucrose 
 
 Caproic acid, 45 
 
 Caproin, 75 
 
 Caramel, 20 
 
 Carbamate, 189 
 
 Carbamide, 184, see also Urea 
 
 Carbohydrates, 1, 2, 20-32, 70, 221- 
 
 227 
 
 absorption of, 128 
 
 colloidal, 30 
 
 definition o(, 23 
 
 in nucleic acid, 63 
 
 in proteins, 62 
 
 list of, 25 
 
 oxidation of, 161 
 
 tests for, 20 
 
 uses of, 3 
 Carbolic acid, 19 
 
 in urine, 211 
 
 poisoning, urine in, 267 
 
 tests for, 13 
 Carbon, 1, 8 
 
 atoms in albumin, 50 
 
 daily output from lungs, 70 
 
 detection of, 5, 9 
 
 to nitrogen, ratio of, 71 
 Carbonates in blood, 170 
 
 in urine, 194 
 Carbon dioxide in blood, 142, 169, 
 170, 178 
 
 in red corpuscles, 169 
 
 in plasma, 169 
 
 pressure in alveolar air, 172, 173 
 
 solubility in water, 161 
 
 tension in blood, 170, 172 
 in tissues, 172 
 Carbonic oxide, effect of breathing, 153 
 
 haemoglobin, 148, 153, 217, 241, 242, 
 ,271 
 
 haemoglobin absorption spectrum, 
 152, 153, 242 
 Carboxyl, 15, 116, 198 
 Cardiac glands, 100 
 
 muscle, 246 
 Carnivorous bile, 122 
 Carotin, 40 
 Cartilage, 61 
 
 Casein, 61, 68, 73, 89, 229 
 Caseinogen, 7, 50, 53, 61, 68, 69, 73, 
 74, 217 
 
 action of erepsin on, 113 
 
 as a substrate, 231 
 
 cleavage products of, 50 
 
 free, 74, 229 
 
 nitrogen, distribution in, 53 
 
 preparation of, 228 
 Caseinogenales of the alkalis, 75 
 
 of the alkali earths, 75, 89, 229 
 Casts in urine, 203 
 Catalysis and enzyme action, 295 
 Catalysts, inorganic and organic, 91 
 
INDEX 
 
 305 
 
 Catalytic action of enzyir.es, 91 
 Cell, diagram of, fig. 8, 63 
 
 protoplasm, nueleo-proteins of, 62, 63 
 Cellulose, 29, 30, 98 
 
 in animals, SO 
 
 putrefaction of, 89, 115 
 Central cells of fundus glands, 99, 100 
 Cerebral anaemia, 249 
 Cerebrin, 39 
 
 Cerebro-galactosides, 37, 39 
 Cerebron, 39 
 Cerebro-spinal fluid, 249 
 
 in nerve degeneration, 250 
 Cerebrote, 39 
 Charges on ions, 286 
 Cheese, 73 
 
 Chemical constitution and circular 
 polarisation, 279 
 
 nature of enzymes, 90 
 
 physiology, 1 , 
 
 structure of protoplasm, 2 
 Chemistry of nerve degeneration, 249 
 
 of respiration, 160 
 Children, feeding of, 76 
 Chittenden on diet, 71 
 Chitosamine, 62 
 
 Chloral and ToUens' test, 213, 224 
 Chlorides in urine, 192, 193 
 
 amount per diem, 258 
 
 estimation of, 259 
 
 source of, 10 
 
 tests for, 179 
 Chlorine, 1, 8 
 Chlorophyll, 148 
 Cholagogue, 121 
 Cholalic acid, 123, 125 
 Cholera-red reaction for indole, 236 
 Cholesterin, see Cholesterol 
 Cholesterol, 2, 3, 37, 38, 39, 121, 217 
 
 compounds, 276 
 
 crystals, fig. 4, 39 
 
 esters of blood, 143 
 
 in fsces, 125 
 
 in nervous tissue, 249 
 
 micro-method of estimating, 300 
 
 of bile, 124 
 
 of blood, 143 
 
 preparation from brain, 109 
 
 protective action of, 159 
 
 tests for, 108 
 Choletelin, 124 
 Choline, 40 
 
 in nerve degeneration, 250 
 
 on blood pressure, 251 
 
 tests for, 250-251 
 Chondrin, 61 
 Chondro-mucoid, 62 
 Chondroitin sulphuric acid, 62 
 Chondrosamine, 62 
 Chorda tympani nerve, 95 
 
 saliva, 97 
 
 20 
 
 Choroid gland, 249 
 
 plexus, 249 
 Chromatin, 62 
 
 Chromogens in urine, 182, 264, 265, 266 
 Chromo-proteins, 58, 62 
 Chyle, 131, 132 
 
 molecular basis of, 131 
 Chyme, 121 
 Cilia, 287 
 Circular polarisation and chemical con- 
 
 - stitution, 279 
 Circulating protein, 187 
 Circulation of bile, 122, 125 
 Cirrhosis of liver, 186 
 Citrates, action on blood, 139, 238 
 Citric acid, 183 
 
 in milk, 76 
 Clark's essence of rennet, 81 
 Cleavage products of digestion, 44 
 
 of proteins, 2, 44, 45, 50, 51 
 Clotting of blood, 136, 295 
 
 intravascular, 137 
 Clupeine, 59 
 COg haemoglobin,' 170 
 Coagulating point, 295 
 Coagulation and precipitation, 57 
 
 of blood, 136-141, 238-240, 295 
 
 of lymph, 138 
 
 ofmilk, 68, 69, 74, 229 
 
 of muscle, 245, 246 
 
 of proteins, 55, 57 
 
 schema, 141 
 Coagulative enzymes, 89 
 Cobra venom, 38 
 Cocoa, 81, 200 
 
 Coefficient of solubility of a gas, 162 
 Coefficients of oxidation, 176 
 
 table of, 177 
 Co-enzyme of lipase, 115 
 Co enzymes, 90 
 Coffee, 81, 200 
 Coffin-lid crystals of triple phosphate, 
 
 194, 205, 206 
 Cola nut, 81 
 Cole's test for bile, 212 
 Collagen, 43, 60 
 
 effect of cooking on, 80 
 
 pancreatic juice and, 114 ■ 
 
 Collimator of spectroscope, 149 
 Colloid carbohydrates, salting out of, 30 
 Colloidal iron, 240 
 
 solution, 54, 295, 296 
 and surface tension, 296 
 and suspension, 296 
 Colloids, 55, 289, 294 
 
 and diffusion, 289 
 
 and emulsions, 115 
 
 and ionic reactions, 92 
 
 examples of, 294, 2B5 
 
 inorganic, 295 
 
 osmotic pressure of, 293 
 
306 
 
 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 Colostrum, 72 
 
 corpuscles, 72 
 
 microscopical appearance, 73 
 Combustion in body, 10 
 Complement, 157 
 Compounds, aromatic, 18 
 
 found in body, 1, 2 
 
 typical organic, test for, 11, 12, 13 
 Condiments, 81 
 Conductors of electricity, 286 
 Congo-red fibrin, 231 
 Conjugated proteins, 58, 61, 146 
 
 classes of, 62 
 Connective tissue, 77 
 
 ground substance of, 62 
 
 white fibres of, 60 
 Continuous spectrum, 149 
 Contractile tissues, Ringer on, 287 
 Cooking of food, uses of, 79, 80 
 Copper, 1, 8 
 
 albuminate, 66 
 Coprosterol, 125' 
 Couerbe on cerebrote, 39 
 Cream, 68, 73 
 Creatase, 191 
 Creatinase, 191 
 Creatine, 48, 80, 191 
 
 estimation of, 258 
 
 fate on injection, 191 
 
 in muscle, 191 
 
 in urine, 191 
 
 preparation of, 247 
 Creatinine, 44, 187, 191 
 
 and Fehling'stest, 213 
 
 and sugar, 181 
 
 and urine nitrogen, 254 
 
 estimation of, 258 
 
 in urine, 191 
 
 tests for, rSl' 
 Creosote and ToUens' reaction, 224 
 Croft Hill on reversibility of enzymes, 
 
 94 
 Crossed niccils, action of, 275 
 Crystallin, 60- 
 Crystalline lens, 60 
 Crystallisable proteins, 56 
 Crystallisation of proteins, 56, 227 
 Crystalloids, 55, 288, 295 
 Crystals from blood, 135, 145, 146, 147, 
 
 237 
 Cuorin of Erlandsen, 38 • 
 Curative inoculation, 155 
 Curd, 68, 74 
 
 Curding of milk, 68, 73, 295 
 Cyclopterine, 59 
 Cysteic acid, 204 
 Cysteine, 204, 205 
 Cystine, 49, 53, 54 
 
 crystals, 204 
 
 deposit in urine, 203, 204 
 
 in keratin, 61 
 
 Cystine in proteins, 49 
 
 in urine, 215 
 
 source of, 205 
 Cystinuria, 215 
 Cytosine, 50, 64 
 
 Dakin on liver enzymes, 120 
 
 on amino-acids, 52 
 Dalton-Henry law, 162, 291 
 A, meaning of, 292 
 A of serum, 292 
 
 A of '9 per cent, sodium chloride, 292 
 Deaminases, 89, 202 
 Deamination, 130 
 Deaminised bodies, 65, 187 
 Deaminising enzymes, 65 
 Decalcification of blood, 139, 237 
 
 of milk, 74 
 Decarboxylation of amino-acids, 116 
 Decinormal acid, ammonia equivalent 
 
 of, 253 
 Decomposition products of fats, 36 
 Defences of the body, 154 
 Deficiency diseases, 82 
 Dentine, 60 
 
 Deposits in urine, 203-206 
 Desoleolecithin and hzemolysis, 189 
 Deutero-proteose, 104, 105, 113, 216, 
 230, 231, 236 
 
 and salt, 230 
 
 in urine, 213 
 Dextrin, 21, 29, 30, 88, 215 
 
 distinction from glycogen, 30 
 
 tests for, 21 
 Dextro-rotatory bodies, 25 , 
 Dextrose, see Glucose 
 Diabetes mellitus, ,118, 119, 213 
 Diabetic coma, 119 
 
 urine, 208-212 
 Diacetin, 36 
 Dialyser, 55 
 Dialysis, 55, 288 
 
 of proteoses, 230 
 Diamino-acids, 48, 49 
 
 caproic acid, 48 
 
 nitrogen in proteins, 52 
 
 valeric acid, 48 
 Diaminuria, 215 
 Diastase, 23, 88, 225 
 Diastatic enzymes, quantitative deter- 
 mination of, 226 
 Diazine derivative, 48 
 Dicarboxylic acids, 17, 46 
 
 amino-acid, 49 
 Dichloracctic acid, 296 
 Diet, 70, 71, 72 
 
 tables, 71 
 Dietary, 71 
 Diffusion, 55, 288 
 
 in lung, 170 
 
INDEX 
 
 307 
 
 Digestion, 83, 231-237 
 
 gastric, 84, 85 
 
 object of, 293 
 
 pancreatic, 107 
 
 salivary, 83 
 Dihydric alcohols, 16 
 Dimethyl xanthine, 200 
 Dioxypurine, 64, 200 
 Dioxypyrimidine, 50 
 Dipeptides, 51 
 Diphtheria anti-toxin, 156 
 
 toxin, 156 
 Direct-vision spectroscope, 150, 151 
 Disaccharides, 24, 27, 32 
 Disintegration of leucocytes, 138 
 Dissociation, 235, 286 
 
 curve of haemoglobin in blood, 169 
 
 in water, 168 
 Disuse atrophy, chemical changes in, 250 
 Di- (thio-amino-propionic) acid, 49 
 Dombrowski's method for urochrome, 
 
 264 
 Dough, 67, 69, 78, 79 
 Drechsel on jecorin, 40 
 Dropsical effusions, mucin of, 62 
 Drummond's test for sulphates, 263, 300 
 Dulcitol, 23 
 
 Dumb-bell crystals of calcium oxalate, 
 204 
 
 of calcium phosphate, 205 
 
 of uric acid, 198 
 Duptfs's urea apparatus, 164, 180 
 Dysalbumose, 229 
 
 Earthy phosphates in urine, 68, .194 
 Eck's fistula, 186 ■ 
 Edestin, 67 
 
 cleavage products of, 50 
 
 nitrogen distribution in, 53, 54 
 
 preparation of, 228 
 Egg-albumin, 34, 42, 59, 77, 295 
 
 cleavage products of, 50 
 crystallisation of, 56, 227 
 
 ■globulin, 42, 60, 77, 296 
 
 mucoid, 77 
 
 shell, composition of, 77 
 
 white, 2, 41, 77 
 antibody to, 157 
 
 yolk, 77 
 phosphatides of, 38, 40 
 protein crystals in, 56 
 vitamine of, 82 
 Eggs, 70, 77 
 
 Ehrlich's side-chain theory, 157 
 Einhorn's saccharimeter, 208 
 Elastic fibres, 61 
 Elastin, 60 
 
 in bone, 60 
 Elastoses, 104 
 Electrolytes, 92, 2$7 
 
 Electrometric method of determining 
 
 acidity, 299 
 Elements, atomic weights of, 8 
 
 detection of, 9 
 Elements found in body, 1 
 
 symbols of, 8 
 Emulsification, 34, 37, 131, 132 
 Emulsion, 107 
 Enamel, 60 
 Endogenous metabolism, 188, 191 
 
 uric acid, 201 
 Energy from food, 70 
 Entero-kinase, 90,' 113, 236 
 Entozoa in urine, 203 
 Envelope crystals of calcium oxalate, 
 
 197, 204, 206 
 Enzyme action, table of, 88 
 
 coagulation, 57, 60 
 
 milk curdling, 115 
 Enzymes, 17, 37, 86, 87-95 
 
 deaminising, 65 
 
 diastatic, 28 
 
 hydrolytic, 89 
 
 inverting, 29, 88 , 
 . lipolytic or lipodastic, 88 
 
 oxidative, 65 
 
 peptolytic, 113, 202 
 
 proteolytic or proteoclastic, 202, 231 
 
 tissue, 89, 202 
 Epiblast, 60, 61 
 Epidermis, 61 
 Epithelial cells in urine, 203 
 
 mucin in, 62 
 Epsom salts, taste of, 193 
 ' Erepsin, 89, 113 
 
 of tissues, 202 
 Erythrocytes, 136 
 Erythrodextrin, 29, 83, 98, 225 
 
 tests for, 21, 22 
 Esbach's albuminometer, 207 
 
 reagent, 207 
 Esters, 17 
 
 decomposition by. alkali, 93 
 Estimation of albumin , 207 
 
 of ammonia, 252, 253 
 
 of chlorides, 259 
 
 of creatine, 258 
 
 of creatinine, 258 
 
 of glucose, 209, 210, 223, 224, 225, 
 239, 240 
 
 of glycogen, 221 
 
 of lactose, 211 
 
 of maltose, 211, 226 
 
 of nitrogen, 251, 252 
 
 of phosphates, 260 
 
 of phosphorus, 261 
 
 of sucrose, 211 
 
 of sulphates, 262 
 
 of sulphur, 263 
 
 of urea, 100, 254 
 
 of uric acid, 256 
 
308 
 
 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 Estimation of urochrome, 264 
 
 Ethane, 14 
 
 Ether, 15 
 
 Ethereal sulphates in urine, 193 
 
 estimation of, 262 
 Etherification and enzymes, 17 
 Ethers, 17 
 Ethyl, 14 
 
 acetate, II, 18, 94 
 
 alcohol, 11, 14, 94 
 
 chloride, 14 
 
 mercaptan, 11 
 
 nitrite, 18 
 
 sulphuric acid, 17 
 Euglobulin; 134 
 Exogenous metabolism, 187, 201 
 
 urea, 187 
 
 uric acid, 201 
 Expired air, 160 
 External respiration, 160 
 
 secretion, 118 
 Extirpation of pancreas, 118 
 Extractives, 77 
 
 of meat, 81 
 
 of plasma, 143 
 Extraordinary ray, 274 
 
 F^CES, 126, 127 
 
 Falk on nerve chemistry, 249 
 
 Fat, 1, 2, 3, 33, 35-37, 60, 69, 131 
 
 absorption of, 131, 132 
 
 cells, protein envelope of, 103 
 
 constitution of, 35 
 
 decomposition products of, 36, 37 " 
 
 melting-points of, 35 
 
 origin of, in body, 131 
 
 oxidation of, 161 
 
 synthesis, 132 
 
 tests for, 33 
 Fat-splitting by lipase, 33, 37 
 Fital temperature for enzymes, 94 
 Fatigue products on respiratory centre, 
 
 174 
 Fats, 1, 2, 3, 70 
 
 estimation of, 228 
 
 of milk, 69, 75, 228 
 
 uses of, 3 
 Fatty acids, 2, 16, 33, 35, 36 
 
 on egg cells, 288 
 
 from proteins, 44, 116 
 
 solvent for, 132 
 
 tests for, 33, 34 
 Fatty degeneration, 39 
 Fatty or aliphatic series, 18 
 Feathery star crystals of triple phos- 
 phate, 194, 205, 206 
 Feeding of children, 76 
 Fehling's solution, 20, 209 
 
 test, 20, 26, 69 
 value of, 213 
 
 Fermentation, 86 
 
 butyric acid, 28 
 
 lactic acid, 28 
 
 test for glucose, 20, 26, 208, 214 
 Ferments, see Enzymes 
 Ferricyanide of potash on oxyhoemo- 
 
 globin, 153, 164, 281 
 Fertilisation, Loeb on, 288 
 Fibrin, 60, 84, 89, 136, 137, 295 
 
 and calcium, 139 
 
 nitrogen, distribution in, 54 
 Fibrin ferment or thrombin, 57, 74, 98, 
 137 
 
 preparation of, 133 
 
 source of, 137 
 
 test for, 237 
 Fibrin filaments, fig. 22, 166, 295 
 Fibrin, gel and sol, 295 
 Fibrinogen,' 60, 89, 137, 143, 217, 237 
 
 heat coagulation of, 55, 143 
 
 percentage in plasma, 143 
 
 solution, 139 
 Fibrino-globulin, 137 
 Fibrous tissue, 81 
 Filtration, 289, 293, 295 
 Fischer, E., on protein constitution, 
 
 52 
 Fischer's " Lock and Key" simile, 91 
 
 micro-polarimetric method, 300 
 Fish spermatozoa nnclein, 63 
 Fistula bile, 125 
 
 biliary, 120 
 
 gastric, 100 
 Fixed carbon dioxide of blood, 169 
 Fleischl's haemometer, 272 
 Flour, 69, 78, 79 
 
 brown, 78 
 
 tests with, 69 
 
 whole, 78 
 Fluorescent screen, 244 
 Fluoride blood, 238 
 
 action of, on coagulation, 189 
 
 plasma, 139, 237 
 Fluorine, 1, 8 
 Folin on creatinine, 191 
 
 on nitrogen excretion, 187 
 Folin's method of ammonia estimation, 
 252, 253 
 of creatinine estimation, 258 
 of urea estimation, 255 
 for total sulphates, 262 
 
 micro-chemical method for uric acid, 
 256, 300 • 
 
 test for uric acid, 196 
 Folin and Denis, uric acid estimation, 
 
 257 
 Folin and Macallum's method for uric 
 
 acid, 256 
 Folin-Shaffer method of uric acid es- 
 timation, 199, 256 
 
 reagent, 256 
 
INDEX 
 
 309 
 
 Foods, 68-81 
 accessories to, see Vitamines 
 adjuncts to, 81 
 cooking of, 79 
 tests for, 68-69 
 Food-stuiTs, 70-72 
 Formaldehyde, 15, 41, 42, 253 
 
 method of ammonia estimation, 253 
 
 of amino-acid estimation, 232 
 reaction for proteins, 56 
 Formalin, 253 
 Formic acid, 12, 35 
 tests for, 12 
 ester, 12 
 Fractional heat coagulation of muscle, 
 245 
 of nerve, 248 
 Fraunhofer's lines, 134, 149, 151 
 Fredericq on apncea, 173 
 Free caseinogen, 74, 228 
 Free elements in body, 1 
 Freezing-point and osmotic pressure, 
 
 291 
 Frog's intestine and fat absorption', fig. 
 
 20, 131 
 Fructose or Isevulose, 20, 23, 24, 26, 
 32, 88, 94, 112, 215 
 and phenyl-hydrazine test, 222 
 Functional activity and tissue respira- 
 tion, 176 
 Fundus glands, 99, 100 
 
 of stomach, food in, 98 
 Fungi in urine, 203 
 Furfuraldehyde, 123 
 
 Galactose, 23, 24, 26, 28, 39, 75, 88, 
 248 
 and phenyl-hydrazine, 222 
 Galactosides, 37, 248 
 Gall-bladder bile, 121, 122 
 Gall-stones, 38 
 Gamgeej, photographic ' spectra, 243, 
 
 244 
 Garrod and Hopkins on stercobilin, 
 124 
 method for preparing urobilin, 265 
 Gas analysis, Barcroft's apparatus for, 
 280 
 chemical method, 164 
 Haldane's apparatus, 284 
 in fluid, measurement of, 163 
 Gaseous exchange in lung, 170, 293 
 in tissue, 171 
 of organ, 176 
 metabolism of body, 177 
 Oases of blood, 170 
 of intestine, 115 
 of plasma and serum, 142 
 solution of, in water, 161, 165 
 
 Gastric digestion, experiments on, 84 
 of proteins, 104 
 fistula, 100 
 glands, 100 
 juice, 98 
 
 acid of, 154, 234 
 actions of, 103-105 
 antiseptic, 85 
 artificial, 100 
 composition of, 102, 103 
 dog's, 102, 103 
 secretion of, 98, 100 
 
 psychical element in, 103 
 lipase, 103 
 Gastrin, 112 
 
 Gay-Lussac's law for gases, 291 
 Gel, 294 
 
 Gelatin, 2, 60, 61, 80, 81, 218 
 cleavage, products of, 50 
 filter, 295 
 
 nitrogen distribution in, 54 
 tests for, 43 
 Gelatinisation, 43 
 Gelatoses, 104 
 
 General paralysis of the insane, 250 
 Gerber's acido-butyrometric method, 228 
 Germ theory of disease, 86 
 Glands, oxygen pressure in, 175 
 Gliadin, 66, 67 
 
 cleavage products of, 50 
 nitrogen distribution in, 53, 54 
 Gliadins, 67 
 Globin, 69, 146, 147 
 Globulicidal power of blood and serum, 
 
 155 
 Globulin, 55, 58, 59, 60, 217 
 
 distinction from albumins, 59, 60 
 in nervous tissues, 248 
 Globulins, vegetable, 67 
 Globuloses, 104 
 Glossopharyngeal nerve, 95 
 Gluco-proteins, 58, 62 
 Glucosamine, 62 
 
 Glucose, 23, 24, 25, 26, 28, 32, 88, 94, 
 112, 213 
 Bang's method of estimation, 224 
 Bertrand's method of estimation, 224 
 in blood. Bang's micro-method, 299 
 
 estimation of, 240, 299 
 in urine, 213 
 
 polarimetric estimation, 223 
 phenyl-hydrazine test, 222 
 reducing power of, 29, 211 
 tests for, 20 
 Glucosides, 30, 31, 32 
 Glutamic acid, 46, 66, 114, 118 
 Glutelins, 67 
 Gluten, 66, 67, 78 
 
 tests with, 69 
 Glutenin, wheat, 67 
 nitrogen distribution in, 53 
 
310 
 
 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 Glyceric ethers, 35 
 Glycerides, 35 
 Glycerin, see Glycerol 
 Glycero-phosphoric acid, 40 
 Glycerol, 2, 16, 35, 36 
 Glycerol, extract of stomach, 84 
 
 tests for, 34 
 Glyceryl, 36 
 Glycine, 18, 45, 46, 51, 208 
 
 in faeces, 125 
 
 in globulin, 60 
 
 in urine, 215 
 Glycocholate of sodium, 121, 122, 123 
 Glycocholic acid, 123 
 Glycocoll, see Glycine 
 Glycogen, 30, 88, 98, 221 
 
 estimation of, 221 
 
 in liver, 128 
 
 micro chemical detection of, 222 
 
 preparation of, 221 
 
 tests for, 22 
 Glycogenic function of liver, 119, 128 
 Glycol, 16. 17 
 GlyColeucine, 46 
 GlycoUic acid, 17 
 
 aldehyde, 17 
 Glycolysis, 119 
 Glycolytic enzyme, 119 
 Glycosuria, 119 
 Glycuronic acid, 223 
 
 tests for, 223 
 
 Tollens' test for, 223 
 Glycyl, 51 
 
 -glycine, 51 
 
 -leucine, 51 
 Glyoxal, 17 
 Glyoxylic acid, 17, 42 
 
 and Tollens' test, 224 
 Gmelin's test for bile pigments, 123 
 Goblet cells, 62 
 Gooch crucible, 262 
 Gordon on circular polarisation, 277 
 Gout, 199 
 
 Gowers, Sir William, hsemacytometer, 
 268 
 
 haemoglobinometer, 271 
 Gowers - Haldane haemoglobinometer, 
 
 standardisation of, 284 
 Graham on colloids, 55 
 Gramme-molecular solutions, 288 
 Granules, zymogen, 90 
 
 activity of, 97 
 
 in gastric glands, 101 
 Granulose, 29 
 Grape sugar, see Glucose 
 Gravimetric glucose estimation, 208 
 Green vegetables, 81 
 Gross and Fuld's method for comparing 
 
 proteolytic enzymes, 231 
 Ground substance of connective tissue, 62 
 
 of tendon, 43 
 
 Griitzner's method for enzymes, 230 
 Guaiacum test, in blood, 135, 154 
 
 in milk, 154 
 Guanase, 202 
 
 Guanine, 64, 65, 200, 201, 202 
 Guano, 201 
 Guanosine, 65 
 Guanylic acid, 65 
 Guarana, 81 
 Gum and emulsions, 34 
 Gunzberg's reagent, 234 
 
 test for free hydrochloric acid, 234 
 Gurber on serum albumin crystals, 
 56 
 
 H/«MACYTOMETER of Sir William 
 Gowers, 268 
 of Dr George Oliver, 270 
 of Thoma-Zeiss, 269 
 H^matin, 146, 147, 217, 265 
 acid, 147, 241 
 
 absorption spectrum, 242 
 in ether, 241 
 alkaline, 147, 243 
 
 absorption spectrum, 242 
 iron-free, see Hsematoporphyrin 
 Hseraatogen, 63 
 Hsematoidin, 121, 147 
 
 crystals, 121 
 Hsematoporphyrin, 147 
 acid, 244 
 
 spectrum, 242 
 in urine, 147, 266 
 spectrum of, 266 
 Haemin, 147, 154 
 crystals, 135, 147 
 preparation, of, 135, 147 
 Haemochromogen, see Reduced Hiematin 
 Hemoglobin, 9, 56, 62, 134, 145, 146, 
 241 
 absorption spectrum, 150, 152, 242, 
 
 243 
 cleavage products of, 54, 146 
 compounds of, 148, 154 
 crystals, 145, 146 
 derivatives of, 147, 148, 241-244 
 photographic spectrum, 244 
 Hemoglobinometer of Sir William 
 Gowers, 271 
 of Dr George Oliver, 272 
 Haldane's, 272 
 
 standardisation of, 284 
 Sahli's, 272 
 Haemoglobinuria, 214 
 
 and haemolysins, 155 i 
 
 Haemolysins, 155, 157, 159 
 Haemolysis, 144 
 
 Haemometer, von Fleischl's, 272 
 Haemopyrrol, 121, 124. 148 
 Hair, 43, 61 
 
INDEX. 
 
 311 
 
 Haldane's gas analysis apparatus, 284 
 haemoglobinometer, 272 
 on methasmoglobin, 153 i 
 
 Haldane and Priestley on apnoea, 173 
 
 Haldane and Priestley, method for 
 collecting alveolar air, 171 
 
 Halogen estimation, micro-, 299 
 
 Halogens and proteins, 66 
 
 Hamburger on enzymes, 231 
 
 Hammerschlag's method for blood 
 specific gravity, 274 
 
 Haptophor group, 157 
 
 Hardy on colloidal solutions, 296 
 
 Hausmann on protein constitution, 52 
 
 Haversian canals, 60 . 
 " Hayem's fluid, 271 
 
 Hay's test for bile salts, 108 
 
 Head on apnoea, 173 
 
 Heart, action of ions on, 288 
 calcium rigor of, 288 
 
 Heart muscle, 246 
 
 Heat coagulation of muscle, 245 
 of proteins, 57, 24 1, 248 
 contraction in nerve, 248 
 formation and muscle activity, 176 
 rigor, 246 
 
 Heidenhain on secretion of gastric juice, 
 
 Hekma on blood clotting, 295 
 
 Heller's nitric acid test, 207 
 
 Hemp, 67 
 
 Henry-Dalton law, 160, 29l 
 
 Heptoses, 24 
 
 Herbivorous bile, 122 
 
 urine, 183 
 Herring and Simpson on pressure in 
 
 bile duct, 120 
 Heterocyclic compounds, 19 
 
 nitrogen in proteins, 53 
 
 nucleus, 49 
 Hetero-proteose, 104, 105, 230 
 Hexahydric alcohols, 16, 23 
 Hexahydroxy-benzene, 27 
 Hexamethylene tetramine, 253 
 Hexone bases, 49, 58 
 Hexoses, 24, 25 
 
 Hill, Croft, on reversibility of enzymes, 94 
 Hilum, 29 
 
 H-ions in blood, 169, 174 
 Hippuric acid, 19, 183, 203 
 
 preparation of, 203 
 Hirudin, 141, 238 
 
 plasma, 239 
 Histidine, 48, 53, 54, 117 
 Histones, 58, 59, 147 
 Hofmeister on crystallisation of ^gg- 
 
 albumin, 56 
 Homogentisic acid, 214 
 Hoofs, 61 
 
 Hopkins on crystallisation of egg- 
 albumin, 56 
 
 Hopkins' method for uric acid estimation, 
 199 
 
 on vitamines, 82 
 
 test for lactic acid, 235 
 
 test for lactic acid in muscle, 245 
 Hoppe-Seyler on protein composition, 44 
 Hordein, 66 
 
 nitrogen distribution in, 53 
 Hormones, 112 
 Horns, 61 
 Howell on rhythmical action, 287 
 
 on blood clotting, 141, 295 
 Human blood, identification of, 154, 
 
 159 
 Humin nitrogen in proteins, 54 
 Hydrazone, 12, 222 
 Hydrobilirubin, 124, 182, 265 
 Hydrocarbons, 14 
 Hydrocele fluid, 142 
 Hydrochinon in urine, 267 
 Hydrochloric acid, 98 
 
 actions of, 103 
 
 formation of, 101 
 
 of gastric juice, 102 
 
 tests for, 234 
 
 0'2 per cent., 84 
 Hydrogel, 295 i 
 Hydrogen, 1, 8 
 
 detection of, 5, 6 
 
 electrode, 299 
 
 ion concentration on respiration, 174 
 Hydrolysis, 44, 104 
 
 protein, 65, 66 ' 
 
 Hydrosol, 295 
 ;8-Hydroxybutyrase, 120 
 /3-Hydroxybutyric acid, 120 
 
 detection of, 213 
 
 in urine, 213 
 Hydroxyethylamine, 40 
 Hydroxyl, 14, 15 
 Hydroxylamine solution for Bang's 
 
 method, 224 
 a-Hydroxypentacosanic acid, 39 
 Hydroxypropionic acid, 45 
 Hydruria, 183 
 ^Hypertonic solutions, 292 
 Hypobromite of sodium.on urea, 185 
 
 method, defects of, 254 
 Hypotonic solutions, 292 
 Hypoxanthine, 64, 65, 80, 200, 201, 
 202 
 
 Imbibition, 293 
 
 Imidazole-amino-propionic acid, 48 
 
 Imidazolethylamine, 117 
 
 Immune body or amboceptor, 157 
 
 Immunity, 76, 154-159 
 and assimilation, 157 
 side-chain theory of, 157 
 
 Inactive lipase, 115 
 
312 
 
 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 Indican of plants, 31, 193 
 of urine, 193 
 Jaffa's test for, 267 
 Obermayer's test for, 267 
 Indicators, 298 
 
 range of a few, 298 
 Indiffusibility of proteins, 55 
 Indigo, 193 
 blue, 267 
 red, 267 
 Indole, 47, 116 
 ring, 54 
 test for, 237 
 Indole-amino-propionic acid, 47, 114 
 Indoxyl, 31, 193, 194, 267 
 
 -sulphuric acid, 267 
 Inexhaustibility of enzymes, 91 
 Infection, 86 
 
 Inflation and deflation of lung, 173 
 Inoculation, curative and protective, 
 
 155 
 Inorganic catalysts, 91 
 colloids, 294, 296 
 salts in protoplasm, 3 
 salts in urine, tests for, 179 
 
 amount per diem, 259 
 sulphates in urine, estimation of, 262 
 Inosite, see Inositol 
 Inositol, 23, 25, 26, 27 
 Inspired air, 160 
 Intensity of respiration, 177, 178 
 Internal respiration, 160 
 
 secretion, 118 
 Intestinal juice, 27 
 
 reaction, 1 16 
 Intra-cellular enzymes, 89 
 -vascular coagulation, 137, 138, 239 
 injections, 294 
 Inversion, 21, 26, 112 
 Invertase, 89, 94 
 of succus entericus, 26, 29, 88, 112 
 of yeast, 27, 87, 88 
 Invertebrate pigments, 147 
 Involuntary muscle, 246 
 Iodine, 1, 8, 9 , 
 
 Iodoform reaction for alcohol, 11 
 
 for acetone, 211 
 Ionic action, 287, 296 
 concentration, 298 
 reactions, 92 
 lonisation, 101 
 Ions, 92, 101, 286 
 Iron, 1, 8, 9 
 -free hasmatin, 147 
 in bile, 121, 123 
 in milk, 72 
 in haemoglobin, 166 
 Islets of Langerhans, 110, 118 
 Iso-amylamine, 117 
 Isobutyl-a-amino-acetic acid, 46 
 Iso-cholesterol, 38 
 
 Isomerides, 23 
 Isomerism, 15 
 
 stereo-chemical, 24 
 Isothermic actions, 95 
 Isotonic fluids, 292, 294 
 Isotropic bodies, 274 
 
 Jaffa's test for creatinine, 181 
 
 for indican, 267 
 Jaundice, 214 
 Jecorin, 40 
 Jelly, Whartonian, 43 
 Juice, intestinal, 27, 112 
 Junkets, 80 
 
 Karyokinesis, 287 
 Katabolism, 44, 95, 120, 187 
 Katabolites, 187 
 Kathode, 92 
 Kations, 92 
 
 list of, 287 
 Kephalin, 37, 4fl, 248 
 Kerasin, 39 
 Keratin, 9, 43, 49, 50, 54, 61 
 
 cleavage products of, 50 
 
 nitrogen distribution in, 54 
 
 tests for, 43 
 Ketone group, 24 
 Ketones, 16, 17 
 Ketoses, 23 
 Kidney, 10, 182, 186 
 Kjeldahl's method, 52 
 
 of nitrogen estimation, 251 
 
 micro-method, 299 
 Kjeldahl-AUihn method for glucose, 
 
 208 
 Kossel on protamines, 58 
 Koumiss, 75, 80 
 
 Klrogh's aerotonometer, 163, 172 
 KUhne on precipitation of pepsin, 103 
 Kyes, Preston, on amboceptor, 159 
 
 Lacmoid, 252 
 
 Lactalbumin, 59, 68, 74, 229 
 
 Lactam form of uric acid, 199, 201 
 
 Lactase, 88 
 
 Lactation, urine in, 28 
 
 Lacteals, 131 
 
 Lactic acid, 2, 23, 28, 80 
 
 and uric acid formation, 199 
 
 fermentation, 28, 115 
 
 from inositol, 27 
 
 in gastric juice, 102, 234 
 
 in muscle, 245 
 
 Hopkin's test for, 245 
 
 in nervous tissue, 249 
 
 organisms, 33 
 
 tests for, 235, 245 
 
INDEX 
 
 313 
 
 Lactim form of uric acid, 199, 201 
 
 Laclo-globulin, 228 
 
 Lactometerj 68 
 
 Lactose, 23, 24, 26, 28, 29, 32, 68, 75, 
 
 91, 112, 213 
 Lactose, crystals, 28 
 
 in urine, 213 
 
 mucic acid test for, 223 
 
 phenyl-hydrazine test for, 222 
 
 reducing power, 29, 211 
 Lacunas, 60 
 Laevo-rotatory, 25 
 Lsevulose, see Fructose 
 Laking of blood, 144, 146 
 Lanoline, 38 
 Lard, 33 
 
 Latent heat of evaporation, 297 
 Lateritious deposit, 197 
 Laurent's polarimeter, 277 
 Law of Arrhenius, 94 
 
 of mass action, 101 
 Laws of gases, 291 
 Lead, 1, 8 
 Lecithin, 2, 7, 9, 37, 39, 40, 121, 248 
 
 an amboceptor, 159 
 Leech extract on coagulation, 137, 140, 
 
 237 
 
 intra-vascular injection of, 238 
 on blood-pressure, 238 
 Legal's test for acetone, 212 ' 
 Lehmann on the liquid crystalline state, 
 
 38,276 
 Lentils as food, 70 
 Lethal dose, 156 
 
 Leucine, 45, 46, 48, 51, 111, 114, 
 236 
 crystals, 45 
 
 in urine, 187, 203, 215 
 preparation of, 236 
 Leucocytes, 136 
 and uric acid formation, 201, 202 
 in peptone blood, 140, 238 
 Leucocythsemia, 201 
 Leucosin (wheat), 67 
 
 nitrogen distribution in, 53 
 Leucyl, 51 
 -alanine, 52 
 -glycyl-alanine, 52 
 Levene on yeast nucleic acid, 65 
 Lewis and Benedict's method for esti- 
 mating sugar in blood, 280 
 Lieben's reaction for alcohol, 11, 21 
 Lieberkiihn's jelly, 66 
 Liebermann - Burchard reaction for 
 
 cholesterol, 109 
 Liebig's extract, 247 
 Life, a combustion, 9 
 
 characteristic sign of, 44 
 Lifschiitz's spectrometric method for 
 
 cholesterol, 300 
 Lignoceric acid, 39 
 
 Ling and Rendle's indicator, 209 
 
 method for glucose, 208 
 Linoleic series, 40 
 Lipase, 88 
 
 experiments with, 33 
 gastric, 103 
 pancreatic, 37, 90, 111 
 Lipochromes, or Luteins, 40 
 Lipochrome of egg-yolk, 77 
 of fat, 247 
 of milk, 75 
 Lipoids, 2, 37 
 and toxins, 159 
 classification of, 37 
 of cell-membrane, 146 
 of egg, 77 
 of milk, 75 
 
 of nervous tissues, 37, 248, 249 
 Lipolytic or lipoclastic enzymes, 88 
 
 enzyme and haemolysis, 159 
 Liquid crystalline stat^, 38, 276 
 Liquor pancreaticus, 107 
 pepticus, 84 
 sanguinis, 136 
 Lithium, 1, 8 
 Liver, 40 
 and amino-acids, 72 
 and anlithrombin, 140 
 and uric acid, 199 
 enzymes in, 202 
 fat in, 39 
 
 glycogenic function of, 119, 128 
 guanylic acid, 24, 65 
 urea formation in, 186 
 Living material, 2 
 membranes, 292 
 test-tube experiment, 142 
 Locke's fluid, 288 
 Loeb on fertilisation, 288 
 
 on ionic action, 288 
 Logarithmic curve of reaction velocity, 
 92 
 law of enzyme reaction, 95 
 Lung, 10 
 
 carbon dioxide exchange in, 172 
 inflation and deflation of, 1'73 
 oxygen exchange in, 170 
 Luteins, see Lipochromes 
 Lymph formation and osmotic pressure, 
 292 
 partial pressure of oxygen in, 171 
 Lymphocytes and fat absorption, 132 
 Lysine, 48, 53, 54, 215 
 bacterial action on, 116 
 in hsemoglobin, 54 
 
 Macallum's cobalt-nitrite test for 
 
 potassium, 249 ' 
 
 Macdonald on potassium in nerve, 249 
 McWilliam's test for proteins, 230 
 
314 
 
 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 Magnesium, 1, 8 
 
 phosphate, in bone, 60 
 in urine, 206 
 
 sulphate, on proteins, 42, 58, 66, 69, 
 134 
 Maize, 66 
 
 Malic acid, 194, 279 
 Mallow, 27 
 
 Malpighian glomeruli, 182 
 Malt, 78 
 
 diastase, 28, 93, 225 
 
 extract, 225 
 Maltase, 88 
 Malting enzyme, 225 
 Maltose, 24, 26, 28, 32, 83, 88, 91, 107, 
 112, 216 
 
 inversion, 29 
 
 phenyl-hydrazine test, 222 
 
 reducing power, 29, 226, 227, 211 
 Maly on hydrochloric acid formation, 
 
 101 
 Mammary gland, 72 
 
 hormone, 112 
 Mandelic nitrate, 31 
 Manganese, 1, 8 
 Mannitol, 23 
 Mannose, 23 
 Maquenne on inositol, 27 
 Marchi method for nerve degeneration, 
 
 250 
 Marchi's fluid, 250 
 Margarine, 83 
 Marrow, 60 
 Marsh gas, 14 
 
 Marshall's urease method of urea esti- 
 mation, 184, 254, 300 
 " Mass action," law of, 101 
 Mat6, 81 
 Meat, 5, 77-78 
 
 tests with, 5, 69 
 Meats, percentage composition of, 
 
 78 
 Meconium, 127 
 Melanin in urine, 267 
 Melanotic sarcoma, 267 
 Mellanby on creatine and creatinine, 
 
 191, 192 
 Melting-point of fats, 35 
 
 of osazones, determination of, 222 
 Membranes, living, 292 
 
 semi-permeable, 290 
 Mercurial air-pump, 280 
 Mercury chloride-iodide temperature 
 
 indicator, 255 
 Mesoblastic tissues, 60 
 Metabolism, 2, 70 
 
 endogenous, 188 
 
 exogenous, 187 
 
 gaseous, of body, 177 
 Metaproteins, 66, 216, 296 
 
 experiments on, 42, 43 
 
 Methjemoglobin, 148, 152, 153, 21?, 
 241 
 
 crystals of, 237 
 in urine, 214 
 
 photographic spectrum, 243 
 spectrum of, 152, 242 
 Mfethane, 14 
 Methyl, 14 
 
 alcohol, 114 
 Methyl glucosides, a and ^, 31 
 glycine, 48 
 
 guanidine acetic acid, 48 
 indole, 47 
 uracil, 50 
 MetschnikofTs view of phagocytosis, 
 
 158 
 Mett's method for proteoclastic enzymes, 
 
 231 
 Meyer and Overton on narcotic effects 
 
 of ether, 298 
 Michaelis and Rona on glucose estima- 
 tion, 239 
 Micro-balances, 300 
 Microchemical analysis of blood, 299 
 of urine, 300 
 detection of glycogen, 222 
 estimation of glucose, 241 
 quantitative analysis, 299, 300 
 Micrococcus ureie, 87, 184, 205 
 Micro-Dumas method of analysis, 299 
 -elementary analysis (Pregl), 299 
 -filtration apparatus, 299 
 -Kjeldahl analysis, 299 
 -organisms, 86, 87 
 
 action on cellulose, 89 
 -polarimetric method of E. Fischer, 
 
 300 
 -pycnometer, 300 
 Microscopic molecular weight estima- 
 tions, 300 
 Micro-spectroscope, 150, 241 
 Milk, 37, 72-77, 228 
 
 alcoholic fermentation of, 75 
 anti-body to, 157 
 citric acid of, 76 
 coagulation of, 74, 228 
 composition of, 73 
 curdling enzyme, 75, 103, 111 
 fat estimation in, 228 
 fats of, 35, 72, 75 
 proteins of, 73, 74 
 skimmed, 68 
 souring of, 73, 75 
 sugar, see Lactose 
 tests for, 68 
 Millon's reaction, 41, 56, 61, 69 
 
 reagent, 41 
 Mineral acids and ammonia excretion, 
 190 
 and fats, 37 
 and proteins, 65 
 
INDEX 
 
 315 
 
 Mineral compounds in body, 1, 9 
 
 salts, action on living organisms, 
 287 
 Mixed saliva, 97 
 Molecular basis of chyle, 131 
 
 reactions, 92 
 
 weight estimation, microscopic, 300 
 Molisch's test for carbohydrates, 21 
 Molybdic acid solution for Neumann's 
 
 method, 261 
 Mono-acetin, 36 
 
 -amino acids, 47, 49 
 dihydroxyalcohol, 39 
 nitrogen in proteins, 52 
 
 -carboxylic acids, 16 
 Monochromatic light, 277 
 Mono-hydric alcohols, 3, 14, 35, 38, 
 
 249 
 Mononijcleotides, 65, 202 
 Monosaccharides, 24, 25-29 
 Mono-sodium urate, 199 
 Mono-urates, 198 
 Monoxypurine, 64, 200 
 Moore on osmetic pressure of proteins, 
 
 294 
 Moore's test for glucose, 20, 25 
 Moore and Frazer on van't Hoft's 
 
 hypothesis, 290 , 
 Morawitz on blood coagulation, 141 
 Morner's test for tyrosine, 236 
 Morris and Brown on starch hydrolysis, 
 
 98 
 Mucic acid, 26, 223 
 Mucin, 62, 217 
 
 in saliva, 83, 97 
 
 tests for, 43 
 Mucinogen granules, 97 
 Mucinoid substance of bile, 121 
 Mucoids, 61, 62 
 
 tests for, 43 
 Mucoitin sulphuric acid, 62 
 Mucous acini, 97 
 
 cells, 96 
 
 glands, 62, 97 
 Mucus, 62 
 
 in urine, 197, 203, 215 
 Munlc on fat synthesis, 132 
 Murex, 198 
 Murexide test for uric acid, 196, 
 
 198 
 Muscle, 77, 245-248 
 
 cardiac, 246 
 
 coagulation of, 245 
 
 involuntary, 246 
 
 pale, 247 
 
 pigments, 247 
 
 plasma, 245 
 
 proteins, heat coagulation of, 245 
 
 red, 24'7 
 
 respiration, 175 
 
 sugar, see Inositol 
 
 Muscular activity ajid heat formation, 
 176 
 
 energy, source of, 185, 186 
 
 exercise on carbon output, 70 
 
 on nitrogen output, 70 
 Muta-rotation, 31 
 Myelin, 37 
 
 forms, 39 
 Myo-hsematin, 247 
 
 spectrum, 247 
 Myosin, 2, 60, 77, 89, 245 
 Myosinogen, 55, 89, 245, 246 
 Myxcedema, 118 
 
 Nail, 61 
 
 Narcotic effect of anaesthetics,, 298 
 
 Natural emulsion, 37 
 
 Negative effect, 137 
 
 electrical charges, 92 
 Nencki on hsemin, 147 
 
 on urea formation, 189 
 Nernst's electrometric method for 
 
 acidity, 299 
 Nerve, chemistry of, 37, 61, 248, 249 
 
 heat contraction in, 248 
 
 degeneration, chemistry of, 249, 250 
 Nervous system, 61 
 
 tissues, chemistry of, 248, 249 
 Nessler's reagent, 253 
 Neumann's method for total phos- 
 phorus, 7, 261 
 Neuroglia, 61 
 Neuro-keratin, 61, 248 
 
 -stearic acid, 39 
 Neutral fat, 36 
 
 point, 298 
 
 salts, action on carbohydrates, 30 
 action on' proteins, 42, 57, 58, 66, 
 69, 134, 230 
 Nickel, 8 
 Nicol's prism, 275 
 Ninhydtin reaction, 233 
 Nissl bodies, nature of, 248 
 Nitrate of urea, 184, 185, 196 
 Nitric oxide hsemoglobin, 148, 154 
 Nitrogen, 1, 8 
 
 amount evolved from urea, 180 
 
 distribution in proteins, 53, 54 
 
 estimation of, 251, 252 
 
 excretion of, 10, 70, 187, 198 
 
 in blood, 170 
 
 solubility in water, 162 
 
 tests for, 5, 6 
 Nitrogenous food, 70-72 
 
 katabolites, 187 
 
 lipoids, 2, 37 
 Nitrous acid, action on amino-group, 
 53, 232 
 
 on urea, 184 
 
316 
 
 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 Non-amino nitrogen in proteins, 53, 
 54 
 
 -electrolytes, 92, 291 
 
 -nitrogenous lipoids, 2, 37 
 residue of amino-acids, uses of, 72, 
 130, 188 
 
 -protein nitrogen in blood, 129 
 Norleucine, 46 
 
 Nuclear metabolism, 200, 201 
 Nucleases, 65, 202 
 Nuclei, 62 
 Nucleic acid, 62, 63 
 
 decomposition of, 63, 64, 65 
 Nuclein, 2, 9, 10, 62, 64 
 Nucleinases, 202 
 
 Nucleo-protein, 2, 7, 9, 50, 58, 62-64, 
 239 
 
 and coagulation, 138, 239 
 
 decomposition schema, 64 
 
 in bile, 122 
 
 in cell protoplasm, 2, 63 
 
 in heart muscle, 246, 247 
 
 in involuntary muscle, 246, 247 
 
 in nervous tissues, 248 
 
 in voluntary muscle, 246, 247 
 
 intra-vascular injection of, 239 
 
 preparation of, 63, 239 
 
 solvent for, 63, 239 
 
 tests for, 217 
 Nucleosidases, 202 
 Nucleosides, 65 
 Nucleotidases, 202 
 Nucleotides, 65 
 Nucleus, benzene, 18, 19 
 
 heterocyclic, 49 
 Nutrition of embryos and young animals, 
 
 61 
 Nylander's reagent and test, 20, 197, 
 208 
 
 Oatmeal, 71 
 
 Obermayer's reagent for indican, 267 
 Oleates and liquid crystals, 276 
 Oleic acid, 35, 36 
 
 in nerve degeneration, 250 
 Olein, 34, 35, 36 
 
 melting-point, 35 
 
 osmic acid test for, 34 
 Oleo-rtiargarine, 83 
 Oleyl, 36, 40 
 Olfactory nerve, 95 
 Olive oil, 33 
 
 Oliver's, Dr George, hsemacytometer, 
 270 
 
 haemoglobinometer, 272 
 Opsonins," 158 
 Optimum diet, 72 
 
 temperature of" enzyme action, 93 
 Orcin reaction for pentoses, 223 
 
 Ordinary ray, 274 
 Organic catalysts, 91 
 
 chemistry, 9 
 
 phosphates in urine, 261 
 
 radicals, 14 
 Organs of excretion, 10, 294 
 Ornithine, 48, 91, 202, 215 
 
 bacterial action on, 116 
 
 fate of, 189, 190 
 Osazones, 222 
 
 Osborne on milk coagulation, 75 
 Osmic acid test for fat, 34, 35, 69, 
 
 132 
 Osmium, 8' 
 Osmosis, 55, 289 
 Osmotic pressure, 144, 287, 289-294 
 
 and freezing-point, 21 
 
 and partial pressure of gases, 291 
 
 calculation of, 291 
 
 determination of, 291 
 
 of 1 per cent, sodium chloride, 292 
 
 of solutions, comparison of, 292 
 
 physiological applications of, 292 
 Ossein, 60 
 
 Ovarian cyst fluid, 62 
 Overton on lipoids, 37 
 Ovo-mucoid, 62, 77 
 Ox bile, 108 
 
 Oxalate of calcium in urine, 197, 204, 
 206 
 
 of calcium crystals, 204 
 
 of urea, 184, 185 
 Oxalated blood, 139, 237 
 
 milk, 68, 74 
 
 plasma, 139, 237 
 Oxalic acid, 17 
 
 tests for, 13 
 Oxidases, 89, 120, 202 
 
 in spermatozoa, 288 
 Oxidative enzymes, 65 
 Oxy - cholesterol, micro - method of 
 
 estimation, 300 
 Oxygen, 1, 8 
 
 capacity of blood, estimation of, 283 
 
 in blood, 142, 165-169 
 
 in solution in blood, 165, 166 
 
 in unsaturated blood, estimation of, 
 283 
 
 interchange in lung, 170, 171 
 
 pressure in alveoli, 170, 171, 172 
 in glands, 175 
 
 solubility in water, 161 
 
 tension in arterial blood, 170, 171 
 in tissues, 169 
 
 want, 172, 174 
 Oxyhsematin, 147, see also Hsematin 
 Oxyhaemoglobin, 145, 148, 241 
 absorption spectrum of, 152, 242 
 crystals, 135, 145, 146, 237, 
 dissociation curve in blood, 169 
 in water, 168 
 
INDEX 
 
 317 
 
 Oxyhsemoglobin, estimation of, 146 
 
 in muscle, 247 
 
 in urine, 214 
 
 photographic spectrum of, 243 
 
 preparation of pure, 244 
 Oxyntic cells of gastric glands, 100-102 
 Oxyph^nyl, 47 
 /-Oxyphenyl-alanine, 47 
 Oxj^henyl-ethylamine, 117 
 Oxyproline, 49, 53, 54 
 
 Pale muscle, 246 
 Palmitic acid, 35, 36, 37 
 Palmitin, 35, 37, 75 
 
 melting-point, 35 
 Palmityl, 36 
 Pancreas, extirpation of, 118 
 
 grafting of, 118 
 
 internal secretion of, 118, 119 
 
 nerves of, 1 12 
 
 structure of, 110 
 Pancreatic digest, 235 i 
 
 digestion, 85, 107, 108 
 
 hormone, 119 
 
 juice, 28, 110, 111, 235, 236 
 actions of, 113-115, 236 
 artificial, 110 
 characters and composition of, 110, 
 
 256 
 regurgitation into stomach, 103 
 secretion of. 111, 112 
 
 lipase, 90, 114 
 
 secretion, demonstration of, 235 
 Para-mucin, 62 
 
 -myosinogen, 60, 245-246 
 Parasites, gastric juice on, 80 
 Parasitic worms and anti-trypsin, 106 
 Parietal or oxyntic cells of fundus glands, 
 
 99, 100, 101 
 Parotid gland, 95 
 
 saliva, 97 
 Partial pressure of a gas, 162 
 
 of oxygen in tissues, 171 
 Passive immunity, 156 
 Pasteur on circular polarisation, 279 
 Pathogenic bacteria, 158 
 Pathological urine, 207 
 
 pigments of, 267 
 Pauli on ionic actions, 296 
 Pavloff on entero-kinase, 90 
 
 on secretory nerves of stomach, 102 
 
 on rennin, 104 
 
 on succus entericus, 112, 113 
 Pavy on fate of liver glycogen, 128 
 
 on carbohydrate radical in proteins, 
 62 
 Pavy's solution for glucose estimation, 
 
 210 
 Peas, 70 
 Penicillium and- circular polarisation, 279 
 
 Pentoses, 24, 64 
 tests for, 223 
 Pepsin, 88,( 89, 90, 98, 101, 102, 103 
 -hydrochloric add, 90, 102 
 on polypeptides, 114 
 precipitation of, 103 
 Pepsinogen, 90, 101 
 Peptic activity, estimation of, 231-233 
 digestion, 103-106 
 exercises on,' 84, 85 
 Peptolytic or peptoclastic enzymes, 89, 
 
 91, 113 
 Peptone, 45, 52, 57, 65, 89, 104, 105, 
 216, 238 
 blood, 140, 238 
 commercial, exercises on, 229 
 effect of intra-vascular injection of, on 
 blood pressure and leucocytes, 238 
 in Urine, 213 
 
 and pus, 213 
 nature of, 66 
 plasma, 140, 238 
 precipitants for, 105, 230 
 tests for, 41, 42 
 Peptonuria, 213 
 Perfect foods, 70, 72 
 Pericardial fluid, coagulation of, 142 
 Peristalsis in stomach, 98 
 Peroxidase of milk, 154 
 Pettenkofer's test for bile salts, 108, 123 
 Pfluger's method of glycogen estimation, 
 
 221 
 Phagocytes and bacteria, 155, 158 
 Phenaceturic acid in urine, 211 
 Phenol, 19, 116, 193 
 -phthalein, 33, 34 
 tests for, 13 
 Phenyl, 46 
 -alanine, 46 
 
 intra-vascular injection of, 131 
 -hydrazine test for sugars, 29, 214, 222 
 -sulphate of potassium, 193 
 Phloridzin, 119 
 diabetes, 119 
 Phloroglucinol reaction for pentoses, 223 
 Phosphate, stellar, 205, 206 
 
 triple, 205, 206 
 Phosphates, alkaline, in urine, tests for, 
 179 
 deposits in urine, 197, 203, 205, 206 
 
 tests for, 206 
 earthy, in urine, estimation of, 206 
 
 tests for, 179 
 in urine, 194, 195 
 daily amount, 195 
 estimation of total, 260 
 origin of, 10, 192, 195 
 Phosphatides, 37, 39, 145, 248 
 Phospho-molybdic acid, 229 
 -proteins, 58, 61 
 importance of, 61 
 
U8 
 
 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 Phosphoias, 1, 7, 8 
 estimation of total, 261 
 in nerve d^eneiation, "250 
 in serum globulin, 61 
 Phospbo-tuDgstic acid, 52, 229 
 
 solution, 256 
 Photc^rapbic spectra, 243, 244 
 Phrenosin, 39 
 Phrenosinic acid, 39 
 Phyllo-poiphyrio, 148 
 Physiolt^cal chemistry, 1 
 
 compounds, tests for elements in, 5, 7 
 tests for, 216-219 
 salt solution, 144 
 Phyto-cholesterols, 38 
 Pbytosterols, 38, 125 
 Picramic acid test for glucose, 25, 26 
 Picric acid, 13, 26 
 Pigment of muscle, 247 
 of urine, 264 
 
 absorption spectra of, 266 
 of red corpuscles, 145 
 Pilocarpine injection on zymogen gran- 
 ules, 236 
 Piotrowski's reaction, 41, 58 
 Placenta, putrefaction of, 116 
 Plain muscle, 246, 247 
 Plane of polarisation, rotation of, 276 
 Plane-polarised light, 274 
 Plasma of muscle, 245 
 
 blood, composition of, 141 
 citrate, 238 
 exercises on, 133 
 fluoride, 237 
 gases of, 142, 152, 169 
 leech extract, 237 
 magnesium sulphate, 133 
 oxalate. 133, 237 
 peptone, 238 
 preparation of pure, 142 
 proteins of, 142, 143 
 sodium sulphate, 133 
 Poisonous protein s, 8 6 
 Polarimeters, 26. ?77 
 
 of Laurent, 277 
 Polarimetric estimation of glucose, 223 
 Polarisation, circular, 276,'278 
 
 of light, 274 
 Polarised light, 25, 274 
 
 action of carbohydrates on, 277 
 proteins on, 56, 277 
 Polariser prism, 274, 275 
 Polarising microscope, 276 
 Polished rice and beri-beri, 82 
 Polynucleotides, 65 
 
 PolypepUdes, 45, 51, 65, 66, 89, 91,114 
 Polyphenols, 257 
 Pol>-saccharides, 24, 29, 83 
 
 tesu for, 21, 22 
 Popielski on pancreatic secretion, 111 
 Pork, indi^estibility of, 77 
 
 Portal vein, 120 
 
 Positive electrical charges, 92 
 
 phase in clotting, 137 
 Potash, alcoholic, 33 
 Potassio-mercuric iodide, 229 
 Potassium, 1, 8 
 
 caseinc^enate, .74 
 
 emission spectmm of, 149 
 
 ferricyanide on oxyhsemoglobin, 164, 
 281 
 
 ferrocyanide in phosphate estimation, 
 260 
 
 in tissues, detection of, 249 
 
 -indoxyl sulphate, 193, 194 
 
 oxalate in ammonia esrimation, 253 
 
 palmitate, 37 
 
 permanganate solution, 256 
 
 thiocyanate, 83, 97 
 
 solution for chlorides, 259 
 Potatoes, 71 
 Potato-starch, 21 
 Precipitants of proteins, 57 
 Predpitation, 57 
 Precipirins, 76, 159 
 
 specificity of, 189 
 Pr^l on micro-elementary analysis, 299 
 Pressor bases in urine, 117 
 Primary alcohol, 15 
 
 oxidation of, 13, 16 
 propyl alcohol, IS 
 proteoses, 104, 105, 113, 229 
 salts of uric acid, 198 
 Principal cells of gastric glands, 100 
 Prisms in direct vision spectroscope, 150 
 Pro-amvlase, 113 
 Proline; 49, 53, 54 
 Prolipase, 113 
 Propeptones, see Proteoses 
 Propionic acid, 45, 46 
 
 aldehyde, 15 
 Propyl alcohol, 15 
 
 ketone, 16 
 Pro-secretin, 111, 235 
 Prosthetic group in proteins, 62 
 Protagon, 39 
 Protamines, 58, 59> 63 
 Protectee inoculation, 155 
 Protein hydrolysis, 65 
 
 metabolism, 44, 187 
 
 -sparing food. 61 
 Proteins, 1, 2, 5, 9, 44-67, 70, 81. 83, 
 216 
 
 absorption of, . 128 
 
 chromo-, 62 
 
 classification of, 58 
 
 cleavage products, 45-50, 72, 129 
 
 coagulated, 57 
 
 colour reactions of, 41, 42, 56 
 
 composition of, 2, 44 
 
 conjugated, 61 
 
 crystallisation of, 36, 227 
 
INDEX 
 
 319 
 
 Proteins, definition of, 44 
 
 digestion, of, by gastric juice, 84, 104 
 by pancreatic juice, 105, 113, 114 
 
 flocculent precipitate of, 295 
 
 gluco-, 62 
 
 heat coagulation of, 41, 55 
 
 in pathological urine, 213 
 
 nitrogen distribution in, 53, 54 
 
 nucleo-, 62 
 
 of blood-plasma, 133, 142, 143 
 
 of milk, 73, 74 
 
 of muscle, 245, 246 
 
 of serum, 133, 142 
 
 osmotic pressure of, 293 
 
 phospho-, 61 
 
 precipitants of, 41, 57 
 
 putrefaction of, 116 
 
 saltfng out, 57, 58 
 
 scleto-, 60 
 
 separation of, 230 
 
 solubilities of, 54 
 
 tests for, 41-43 
 Proteoclastic enzymes, 88 
 
 activity of, 230, 231 
 Proteolytic enzymes, 88 
 Proteoses, 42, 45, 53, 57, 65, 66, 89, 
 104, 216, 229 
 
 in urine, 213 
 
 intra-vascular injection of, 129, 238 
 
 separation of, 229 
 Pro-thrombin, 90, 138 
 Protones, 58 
 Protoplasm, 2 
 
 and surface tension, 297 
 
 chemical structure of, 2 
 substances in, 2 
 Proto-proteose, 104, 105, 229 
 Protrypsinogen, 113 
 Pseudo-globulin, 134, 246 
 
 -mucin, 62 
 Pseudopodia formation, 297 
 Ptomaines, 86 
 
 Ptyalin, 28, 83, 88, 91, 97, 98 
 Pulmonary ventilation, 174 
 Pulses, 79 
 
 Pump, mercurial air-, 280 
 Puncture diabetes, 119 
 Purine, 64, 200 ' 
 
 bases, 29, 50, 61,63, 64, 200 
 Purpurate of ammonia, 196 
 Pus in urine, 203, 206 
 
 tests for, 214, 219 
 Putrefaction, 1,28, 86, 116 
 Putrescine, 116 
 
 in urine, 215 
 Pycnometer, Schmalz's capillary, 274 
 
 micro-, 300 
 Pyloric glands, 99, 100 
 Pyridine, 19 
 Pyrimidine, 82 
 
 bases, 49, 63, 64 
 
 Pyrimidine ring, 49 
 Pyrocatechin in urine, 267 
 Pyrrol, 19 
 
 ring, 47 
 Pyrrolidine-carboxylic acid, 49 
 
 derivatives, 49 
 
 QUADRI-URATES, 199 
 
 Quantitative estimation of albumin, 207 
 
 of chlorides, 259 
 
 of creatine, 258 
 
 of creatinine, 258 
 
 of enzymes, 231-236 
 
 of fats in milk, 228 
 
 of glucose, 209, 210, 223, 224, 225, 239 
 
 of glycogen, 221 
 
 of lactose, 211 
 
 of maltose, 211, 226 
 
 of nitrogen, 251, 252 
 
 of phosphates, 260 
 
 of phosphorus, 261 
 
 of sucrose, 211 
 
 of sulphates, 262 
 
 of sulphur, 263 
 
 of urea, 108, 254 
 
 of uric acid, 256 
 
 of urochrome, 264 
 Quantity and tension of gas in blood, 165 
 Quartz, influence on light, 276 
 
 use in polarimeter, 276, 277 
 
 Racemic acid, 279 
 
 Ramsden on milk fat, 75 
 
 Rancid oil, 34 
 
 Range of indicators, 298 
 
 Ranke's diet, 71 
 
 Reaction of blood, 169 
 
 Reaction of fluids, determination of, 298 
 
 velocity, 92 
 Reactions of first order, 92 
 
 of second order, 93 
 Receptor groups, 157 
 Red blood corpuscles, 144 
 
 composition of, 145 
 Red corpuscles, enumeration of; 268-271 
 
 action of reagents on, 144 
 
 pigment of, 144 
 
 estimation of, 271-273 
 Red muscle, 247 
 Reduced hsematin, 147, 244 
 
 absorption spectrum, 242 
 
 haemoglobin, see Hsenjoglobin, 135 
 Reducing action of unsaturated bodies, 
 35 
 
 agents, 148 
 
 power of sugars, 211 
 
 sugars, estimation of, 210, 211 
 Reductases, 80 
 Reeves on vegetable protein, 227 
 
320 
 
 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 Reflex action (saliva), 95 
 
 Reid, Weymouth, on absorption, 293 
 
 Rennet, 57, 68, 73, 74, 77, 89, 101, 103, 
 
 228 
 Resin, aldehyde, 12 
 Respiration, chemistry of, 160 
 
 external, 160 
 
 intensity of, 177, 178 . 
 
 internal or tissue, 160, 175 
 
 normal chemical stimulus for, 174 
 
 regulation of, 173 
 Respiratory centre, 173 
 
 effect of blood gases on, 173, 174 
 
 effisct of nerves on, 174 
 
 oxygen of haemoglobin, 148 
 
 pigments, 145, 165 
 
 quotient, 161 
 effect of diet on, 161 
 Reversibility of enzyme action, 94 
 Rhythmical action, Howell on, 287 
 i/-Ribose, 64, 65 
 Rice and gliadin, 67 
 Ricin, 157 
 Rigor mortis, 80, 89, 246 
 
 scheme of changes in, 245 
 ^ Ringed amino-acids, 49 
 Ringer on contractile tissues, 287 
 Roafs method of comparing proteolytic 
 
 activity of enzymes, 230 
 RoUett's absorption spectra of hEemo- 
 
 globin, 151 
 Rose's reaction for protein, 41, 58 
 Rosenheim's formaldehyde reaction, 41 
 ' test for sulphates, 263 
 Rotation of plane of polarisation, 276 
 Rotatory power, specific, 278 
 Rothera's test for acetone, 212 
 
 Saccharic acid, 26, 223 
 
 sugars yielding, 223 
 Saccharimeter, Einhorn's, 208 
 
 Laurent's, 277 
 Sahli's haemoglobinometer, 272 
 St Martin, Alexis, fistula of, 100 
 Salicin, 31 
 
 Salicylic acid in urine, 211 
 and Tollens' test, 224 
 
 alcohol, 31 
 Salicyl-sulphonic acid, use of, in separa- 
 tion of proteins, 230 
 Saliva, 28, 43, 95 
 
 action of, 98 
 
 composition of, 97 
 
 tests with, 83' 
 Salivary corpuscles, 97 
 
 glands, 95-97 
 
 nerve supply of, 95 
 Salkowski's reactiori for cholesterol, 109 
 Salmine, 59 
 Salt solution, physiological, 144 
 
 Salted blood-plasma, 130 
 
 muscle plasma, 245 
 
 whey, 69 
 Salting out colloid carbohydrates, 30 
 
 proteins, 30, 58, 66 
 Salts of blood, 143 
 
 of milk, 75 
 Salts of urine, 192 
 Sampling tube, Haldane's, 171 
 Saponification, 33, 37, 114, 131 
 Saponin, 159 
 
 action on egg cells, 288 
 Sarco-lactic acid, 174, 245 
 
 in nerve, 249 
 Sarcolemma, 61 
 Sarcosine, 48, 191 
 Scatole, 47, 116 
 
 Schafer on fat absorption, 130, 131 
 Schiff' on circulation of bile, 122, 125 
 
 on the biuret reaction, 57 
 Schiff's test for uric acid, 196 
 Schizomycetes, types of, 87 
 Schlosing's method of ammonia estima- 
 tion, 252 
 Schmalz's capillary pycnometer, 274 
 Schmidt on salts of plasma, 143 
 Schmidt's method of thrombin pre- 
 paration, 143 
 Schroder's experiments on urea forma- 
 tion, 189 
 Schiitz's law of enzyme action, 95, 231 
 Schryver's method for obtaining vege- 
 table protein, 287 
 Sclero-proteinSj 58, 60 
 Scott on regulation of respiration, 174 
 Scurvy, 82 
 Sebum, 38 
 
 Secondary alcohols, 15 
 oxidation of, 16 
 
 propyl alcohol, 16 
 
 proteose, 104 
 
 salts of uric acid, 198 
 Secretin, 111, 112 
 
 preparation of, 235 
 Secretion, external, 118 
 
 internal, 118 
 
 of gastric juice, 98 
 
 of pancreatic juice. 111 
 
 of oxygen in lung, 172 
 in swim bladder, 172 
 
 of saliva, 95 
 Sediments in urine, table of, 206 
 Seeds, proteins in, 67 
 Semi-normal potassium bichromate, 258 
 
 permeable membranes, 290 
 Serine, 45, 46 
 Serous alveoli, 96 
 
 gland, 95 
 Serpent's urine, 256 
 Serum, 55, 143, 144 
 
 agglutinating action of, 158 
 
INDEX 
 
 321 
 
 Serum albumin, 59, 134, 143 
 cleavage products of, 50 
 crystallisation of, 56, 227 
 heat coagulation of, 55 
 bactericidal action of, 155, 156, 157 
 gases of, 142 
 
 globulicidal acti on of, 155, 156, 157 
 Serum globulin, 60, 134, 143 
 cleavage products of, 50 
 coagulation of, 55 
 phosphorus in, 61 
 tests for, 134 
 glucose in, estimation of, 239 
 ' lutein, 133 
 proteins, 142 
 separation of, 134 
 tests for, 133, 134 
 Sham feeding, 103 
 Sheep's-Wfool fat, 38 
 
 Sherrington's solution for leucocytes, 270 
 Side-chain theory of immunity, 157 
 Silicic-acid filter, 296 
 Silicon, 1, 8 
 Silver, 8 
 nitrate solution for estimating chlo- 
 rides, 258 
 Skatole, 116 
 Skatoxyl, 267 
 
 red, 267 
 Skimmed milk, 68, 73 
 Skin, 10 
 
 Smoky urine, 214 
 Snake poisoning, 156 
 venom, 86 
 nature of, 156 
 Soaps, 34, 37, 111, 114, 121 
 emulsifying action of, 115 
 Sodium hydrosulphite, 135j 148 
 Sodium, 1, 8, 9 
 
 carbonate in blood, 170 
 caseinogenate, 74 
 chloride, action on fibrinogen, 143 
 on deutero-proteose, 230, 231 
 on nucleo-proteins, 63, 240 
 emis^ioif spectrum of, 149 
 hypobromite solution, 180 
 action on urea, 185 
 method for urea, 180 
 defects of, 254 
 phosphate and alkali-metaprotein, 43 
 
 acid in urine, 183 
 sulphate plasma, 133 
 Sol, 294 
 
 Solar spectrum, 149 
 Soluble starch, 29 
 Solution affinities and diffusion, 287 
 Solutions, 286 
 Sorbitol, 23 
 Sorensen's method of comparing 
 
 enzymes, 232 
 Soret's band, 244 
 
 21 
 
 Soup, 81 
 
 Souring of milk, 75, 86 
 Soy beans, 184, 254, 300 
 Specific gravity of blood, estimation of, 
 273 
 
 of milk, 73 
 
 of urine, 183 
 
 oxygen capacity of htemoglobin, 166 
 
 rotatory power, 277, 278 
 Specificity of enzymes, 65, 90 
 Spectra of blood pigments and deriva- 
 tives, 135, 150-154, 241-244 
 
 photographic, of haemoglobin, 243 
 of methsemoglobin, 243 
 of oxyhaemoglobin, 243 
 
 of urinary pigments, 263-266 
 Spectrometric method for cholesterol 
 
 estimation, 300 
 Spectroscope, 149, 150 
 Spermatozoa, 62, 63 
 
 action on egg cell, 287 
 
 in urine, 203 
 Sphsero-crystals, 40 
 Sphingo-myelin, 37, 40, 248 
 Sphingosine, 39 
 Spleen hormone, 113 
 Splitting process, 94 
 Spores, 86 
 
 Staphylococcus and peptonuria, 213 
 Starch, 2, 28, 29, 80, 88, 92, 98, 
 215 
 
 action of diastase (malt) on, 226 
 
 cellulose, 29 
 
 grains, 29 
 
 granulose, 29 
 
 salivary digestion of, 98 
 Starch, soluble, 29 
 
 tests for, 21 
 Starling on osmotic pressure of proteins, 
 
 293 
 Starling and Bayliss on secretin. 111 
 Stearic acid, 34, 35, 36 
 Stearin, 35, 36 
 Stearyl, 36 
 Stein's method for htemoglobin crystals, 
 
 145 
 Stellar phosphates in urine, 194, 205 
 Stercobilin, 124, 182, 263 
 Stereo-chemical isomerism, 24 
 Stewart's dietary, 71 
 Stimulants, 81 
 
 Stokes's reagent, 135, 148, 241 
 Stomach glands, 99, 100 
 
 secretion of, 101 
 Sturine, 59 
 Sublingual gland, 97 
 
 •saliva, 97 
 Submaxillary glandj 96, 97 
 
 saliva, 97 
 Substrate, 88, 91, 94, 233 
 Succus entericus, 27, 90, 112 
 
322 
 
 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 Sucrose, 24, 27, 91, 94, 216, 222 
 
 constitutional formula of, 32 
 
 estimation of, 211 
 
 in urine, 27 
 
 inversion of, 27 
 
 tests for, 21 
 Sudan III., fat stain, 35 
 Sugar of blood, 128, 143 
 estimation of, 239 
 
 of urine, 213 
 
 confirmatory tests for, 214 
 estimation of, 208 
 Sugar-cane, 27 
 Sugar maple, 27 
 Sugars, 24 
 
 phenyl-hydrazine, test for, 222 
 
 reducing, 20 
 Sulphates in urine, 10, 192, 193, 
 262 
 
 amount per diem, 193 
 
 and urea excretion, 192 
 
 estimation of ethereal, 262 
 
 inorganic, 262 
 
 total, 262 
 Sulphonal poisoning, urine in, 266 
 Sulphur, 1, 8, 10 
 
 in urine, estimation of total, 263 
 
 micro-estimation of, 300 
 
 tests for, 6, 7 
 Sulphuric acid, 44 
 Superheated steam, action on fats, 37 
 
 on proteins, 65 
 Suprarenal, removal of, 118 
 Surface tension, 297 
 
 and bile, 132 
 Suspending medium, 34 
 Suspension. 54, 295, 296 
 Sweetbreads and uric acid, 201 
 Swim-bladder, secretion in, 172 
 Symbols of elements, 8 
 Sympathetic nerve, 95 
 
 saliva, 97 
 Synthesis of proteins in intestine, 129 
 
 in tissues, 129 
 Synthetic process (enzyme action), 94 
 
 Tannin, 81, 229 " 
 Tapetum, 201 
 Tapeworms, 80 
 Tartaric acid, 194 
 
 and circular polarisation, 279 
 Taurine, 204, 205 
 
 in faeces, 125 
 Tauro-carbamic acid, 125 
 Taurocholate of sodium, 121, 123 
 Taurocholic acid, 123 
 Tautomerism, 32, 199 
 Tchistovitch's experiments, 159 
 Tea, 81, 200 
 Teichmann's crystals, 147 
 
 Temperature indicator in Folin's 
 
 method, 255 
 Tendo-mucoid, 62 
 Tendon, 43 
 Tension of carbonic dioxide in blood, 172 
 
 of gas in fluid, 162 
 measurement of, 163 
 
 of gases in tissues, 172^ 
 
 of oxygen in blood, 172 
 Terpene series, 38 
 Tertiary alcohol, 16 
 
 alcohols, oxidation of, 16 
 Testis, 239 
 
 removal of, 118 
 Tetra-peptides, 52 
 Tetroses, 24 
 Theine, 81 
 
 Theobromine, 81, 200 
 Theophylline, 200 
 Thoma-Zeiss hzemacytometer, 269 
 Thoracic duct, 131 
 Thrombin, 89, 90, 138-141, 143 
 
 preparation of, 133 
 
 Schmidt's, method of preparation, 143 
 Thrombogen, 90, 138, 139 
 Thrombokinase, 90, 138, 139 
 
 nature of, 141 
 Thrombo-plastin or kinase, 141 
 , Thrombosis,* 138 
 Thudichum on protagon, 39 
 Thymine, 50, 64 
 Thyroid, 9 
 
 removal of, 118 
 Tin, 8 . 
 Tissue enzymes, 65, 191 
 
 fibrinogen, 240 
 
 metabolism, 187-190 
 Tissue protein, 187 
 
 respiration, 160, 175, 176 
 Tissues, functional activity of, 293 
 
 gaseous exchange in, l7l 
 Titration acidity, 299 
 Tollens' test for glycuronic acid, 223, 224 
 Tdraes on enamel, 60 
 Tonometer, Barcroft's, 166 ' 
 Tonsils, 97 
 Tooth, 9 
 
 Topfer's test for hydrochloric acid, 234 
 Torricellian vacuum, 148 
 Toxins, 38, 156, 159 
 
 mode of action, 157 
 Triacetin, 36 
 Tribromo-phenol test, 13 
 Trichinae, 80 
 Trichloracetic acid in the preparation of 
 
 proteins, 230 
 Trihydric alcohols, 16, 36 
 Trimethylamine in nerve degeneration, 
 
 250 
 Trimethyl-xanthine, 200 
 Triolein, 36 
 
INDEX 
 
 323 
 
 Trioses, 24 
 
 Trioxypurine, 64 
 
 Tripalmitin, 36 
 
 Tripeptides, 62 
 
 Triple phosphate, 194, 195, 197, 205 
 
 Tristearin, 36 
 
 Trivalent ions, 286 
 
 Troinmer's test, 20, 25, 69 
 
 Tropseolin test for hydrochloric acid, 234 
 
 True acidity of urine, 299 
 
 Trypsin, 89, 90, HO, 113, 114, 235 
 
 Trypsinogen, 90, 110, 113, 235 
 
 Mellanby and WooUey on, 113 
 Tryptic activity, estimation of, 232, 233 
 Tryptophane, 46, 47, 53, 54, 61, 114 
 
 intra-vascular injection of, 131 
 
 test for, 236 
 Tubulo-racemose gland, 110 
 Tunicates, cellulose in, 30 
 Tyndall phenomenon, 296 
 Tyrosine, 19, 46, 47, 61, HI, 114, 236 
 
 crystals, 47 
 
 in urine, 203, 215 
 
 intra-vascular injection of, 131, 189 
 
 preparation of, 236 
 
 tests for, 236 
 
 Uffelmann's reaction for lactic acid, 
 234 
 reagent, 234 
 Ultra-microscopic observation, 296 
 Umbilical cord, 43 
 Uncooked starch, ptyalin and, 98 
 Unimolecular reactions, 92, 94 
 Unpeptonised proteins, bile on, 108 
 Unsaturated bodies, 35 
 Uracil, 50, 64 
 Uraemia, 186 
 
 Uranium solution, standard, prepara- 
 tion of, 260 
 and phosphoric acid, 260 
 Urate, acid ammonium, deposit of, 203, 
 204, 206 
 add sodium, deposit of, 203, 204, 206 
 Urates, 198, 199 
 
 a and /3, form of, 199 
 and Fehling's test, 213 
 deposit in urine, 197, 203, 204 
 spectrum of, 266 
 Urea, 2, 10, 44, 91, 184-190, 202, 218 
 characters and composition of, 184 
 compounds of, 184 
 de'composition of, 184, 185 
 estimation of, by hypobromite, 180 
 Benedict's method, 255 
 Folin's method, 255 
 urease method, 254, 300 
 mode and site of formation, 186-190 
 nitrate, 184, 185 
 formation of, 196 
 
 Urea oxalate, 184, 185 
 formation of, 196 
 
 preparation of, 254 
 
 quantity excreted, 183, 185 
 
 tests for, 179, 254 
 Urease in soy beans, 184, 254, 300 
 Uric acid, 64, 187, 198-203, 218 
 
 amount excreted, 199 
 
 characters of, 198 
 
 crystals of, 199 
 
 deposit in urine, 196, 203, 204 
 
 estimation of, 199 
 
 Folin and Macallum's method, 257 
 Folin-Shafifer method, 256 
 
 in blood, estimation of, 257 
 
 lactam form, 199, 201 
 
 lactim form, 199, 201 
 
 preparation from urine, 198 
 
 preparation of pure, 256 
 
 primary salts of, 198 
 
 secondary salts of, 198 . 
 
 tests for, 196, 197 
 Uricolytic enzyme, 202, 203 
 Urina potus, 183 
 Urinary calculi, 204 
 
 deposits, 203 
 Urine, 10, 179-215, 217, 251-267 
 
 albumin in, 207 
 estimation of, 207 
 
 amino-acids in, 187, 215 
 in normal, 215 
 
 amount, 182 
 
 bile in, 212, 214, 267 
 
 blood in, 214, 267 
 
 blood pigment in, 214 
 
 characters of, 182 
 
 chlorides of, 192, 259 
 
 chromogens of, 267 
 
 composition of, 183-184 
 
 formation of, 182 
 
 and osmotic pressure, 292 
 
 inorganic constituents, of, 10, 192, 
 258 
 
 micro chemical analysis of, 300 
 
 pathological, 207 
 
 peptone in, 213 
 
 phosphates of, 10, 194, 195, 258, 260 
 
 pigments of, 264-267 
 
 pressor bases of, 117 
 
 sulphates of, 10, 193, 262 
 
 tests for, 179, 196 
 Urinometer, 183 
 Urobilin, 124, 182, 183, 214, 264, 266 
 
 absorption spectrum, 266 
 acid, 265 
 
 absorption spectrum of, 266 
 
 preparation of, 265 
 
 Urobilinogen, 182, 265 
 
 Urochrome, 183, 264 
 
 estimation of, 264 
 
 preparation of, 264 
 
324 
 
 ESSENTIALS OF CHEMICAL PHYSIOLOGY 
 
 Uroerythrin, 197, 204 
 
 absorption spectrum of, 266 
 
 preparation of, 265 
 Urorosein, 267 
 
 absorption spectrum of, 266 
 Urotropine, 253 
 
 Vaccination, 155 
 
 Vagus, on oxygen secretion, 172 
 
 on respiration rate, 174 
 
 on stomach, 102 
 Valeric acid, 45, 116 
 Valine, 45, 46 
 
 Van Slyke's method for CO^ in blood, 
 178, 300 
 
 method of protein analysis, 53 
 
 micro-method for amino-nitrogen, 300 
 Van't Hoff and circular polarisation, 279 
 Van't Hors hypothesis, 290 
 Vegetable acids, 194 
 
 albumins, 67 
 
 foods, 70, 79 
 
 composition of, 79 
 
 globulin, crystallisation of, 227 
 
 globulins, 67 
 
 proteins, 66 
 
 cleavage products of, 66 
 Vegetables, green, composition of, 81 
 Vegetarians, urine of, 183 
 Velocity of reaction, 91 
 Venous blood, 148 
 
 carbon dioxide, tension of, 172 
 
 gases of, 170 
 Villus during fat absorption, 130 
 Vinegar formation, 86 
 Virchow on myelin forms, 39 
 Vital action, 293 
 Vitamines, 72, 82 
 Vitellin, 56, 61, 74, 77, 104 
 Vitelloses, 104 
 Vitreous humour, 43 
 Voit on nitrogen in fffices, 127 
 
 on circulating protein, 187 
 Voit's diet, 71 
 
 Volhard's method of chloride estima- 
 tion, 259 
 Volumetric methods for sugar, 208 
 Voluntary muscle, 245, 246 
 
 Water in food, 70 
 
 in protoplasm, 2 
 Wave theory of light, 274 
 Weaning, lactose in urine, 28 
 
 late, and iron in milk, 72 
 Weinland on antipepsin, 106 
 
 Wertheimer and Le Page on secretin. 111 
 Weyl's test for creatinine, 181 
 Whartonian jelly, 43 
 Wheat, 66 
 flour, 69 
 Whetstones of uric acid, 196, 206 
 Whey, 68, 74 
 protein, 74 
 salted, 69 
 Whipping blood, 136 
 White corpuscles, 144 
 
 enumeration of, 269, 270 
 of egg, 2, 55, 77, 84 
 globulin of, 62, 77 
 ovomucoid of, 62, 77 
 Whole flour, 78 
 
 meal brfead, vitamine in, 82 
 Widal's reaction in typhoid, 158 
 Windaus on cholesterol, 38 
 Winterstein's method of oxygen esti- 
 mation in blood, 299 
 Wohler's synthesis of urea, 184 
 Wohlgemuth's method of estimating 
 
 diastatic enzymes, 226 
 Wooldridge and tissue fibrinogen, 239 
 Wooldridge's method for nucleo-pro- 
 
 teins, 63, 239 
 Wright, Sir A. E. , on opsonins, 158 
 
 Xanthine, 64, 65, 111, 200, 201, 202 
 Xantho-proteic reaction, 41, 56, 69 
 Xanthophyll, 40- 
 
 Xanthydrol compound of urea, 300 
 Xylose in wood shavings, 223 
 
 Yeast, 26, 27, 28, 64, 65, 75, 79, 82, 
 86, 88, 208 
 action in bread-making, 79 
 in testing for sugar, 208, 214 
 Yellow elastic fibres, 61 
 lipochrome of fat, 247 
 Yolk of egg, 77 
 spherules, 77 
 
 Zein, 66 
 
 cleavage products of, 50 
 
 nitrogen distribution in, 53 
 Zinc, 8 
 Zymase, 27, 87, 88, 90, 208 
 
 and phosphates, 90 
 Zymogen, 90, 101, 110, 138 
 
 granules,- 110, 236 
 pilocarpine on, 236 
 Zymogens, 110 
 
 Prinieei in Great Britain at The Darien Press, Edinburgh 
 
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