COLUMBIA LIBRARIES OFFSITE HEALTH SCIENCES STANDARD HX64141187 QP51 4 .H1 5 1 899 The essentials of ch iffiiMffifiimmitmTmunnu^u; CSC3KHBK1SCJC5J l?.WB'ftffiUaaafl'J 1 i Essentials QF ;hemical physi ri^T ■MKM U ■ mm maim mffim G(Pfl+ H 15 COLLEGE OF PHYSICIANS AND SURGEONS LIBRARY Presented oy g, DR. WILLIAM J. Or fo enrich the library resource's avaz'/a6/e to holders of the G1ES FELLOWSHIP *'/2 Biological Chemistry I " 7 . °. ** ^mm • v;y--yV-.:.-:- V CHEMICAL PHYSIOLOGY By the same Author. A TEXT-BOOK OF CHEMICAL PHYSIOLOGY AND PATHOLOGY. With 104 Illustrations. 8vo. 28s. LONGMANS, GREEN, & CO., 39 Paternoster Row, London, New York, and Bombay. THE ESSENTIALS OF CHEMICAL PHYSIOLOGY FOR THE USE OF STUDENTS BY W. D. HALLIBURTON, M.D., F.E.S. FELLOW OS THE ROYAL COLLEGE OF PHYSICIANS PROFESSOR OF PHYSIOLOGY IX KING'S COLLEGE, LONDON AUTHOR OF 'TEXT-BOOK OF CHEMICAL PHYSIOLOGY AND PATHOLOGY' THIRD EDITION LONGMANS, GREEN, AND CO. 39 PATEBNOSTEB BOW, LONDON NEW YORK AND BOMBAY 1899 All rlgbti reserved mi PBEFACE TO THE THIKD EDITION The rapid advances which Chemical Physiology has made during the past few years has rendered a good deal of alteration necessary in the present edition. The practical exercises are, however, but little modified; the changes introduced there are principally in arrangement ; still it has been thought necessary to amplify a certain number of these, particularly in the advanced course. The lessons on the urinary pigments and crystallisation of egg-albumin are entirely new. The main changes in the book will be found in the large text ; this has been considerably extended, and the sections relating to the proteids have been entirely re-written. I am indebted to numerous friends for various suggestions ; I would particularly thank Prof. Schafer for allowing me to borrow several illustrations from his recently published Text-book of Physiology ; Prof. Gamgee, who has allowed me to reproduce some of his figures illustrating the Photographic Spectrum ; Mr. John Murray, for permission to use some of the illustrations in ' Kirkes' Physiology ' ; Dr. F. Gowland Hopkins, for help in the preparation of the exer- cises on the Crystallisation of Albumin, and Estimation of Uric Acid ; and Dr. H. Willoughby Lyle, who has assisted me in reading the proof-sheets, and preparing the Index. W. D. HALLIBUKTON. King's CoxAdtGB : January 1899. PBEEACE TO THE FIBST EDITION This book is written with the object of supplying the student with directions for examining practically the most important of the subjects included under the heading Chemical Physiology, or Physiological Chemistry. At the same time it is intended to serve as an elemen - tary text-book of the subject. The plan of the work is the same as that adopted by Professor Schafee, in his ' Essentials of Histology.' The practical lessons are followed by a brief description of the matter of which they treat. Each of the elementary lessons may be supposed to occupy a class for one hour. The advanced lessons will take more time — usually two hours. The practical lessons are those which I have been using for many years past in my own classes. Their original source is the ' Syllabus of Lectures ' published by Professor J. Buedon Sandeeson in 1879, and although some few of the lessons are but little altered, the greater number have been very considerably modified to include the results of recent researches. The illustrations have for the most part been transferred from my text book of ' Chemical Physiology and Pathology.' A few additional figures have been expressly drawn for this work. Figs. 4, 10, and 15 have been taken from Yeo's ' Physiology ' ; figs. 16 and 17 from McKendeick's ' Physiology ' ; figs. 51 and 52 from Beyant's ' Surgery ' ; and figs. 76 and 77 from Wallee's ' Physiology.' For leave to use these I beg to tender my thanks to the respective authors arid publishers. The duty of reading the proof-sheets has been greatly lightened for me by my friend Dr. T. G. Beodie, and I have to thank him for many valuable suggestions and alterations. W. D. HALLIBUETON. King's College : July 1893. CONTENTS Introduction 1 ELEMENTARY COURSE I. The Carbohydrates and Fats II. The Proteids III. The Peoteids (continued) IV. Foods .... V. Saliva VI. Peptic Digestion VII. Pancreatic Digestion VIII. Bile .... IX. The Blood X. Urine XI. Urine (continued) XII. Pathological Urine Scheme fob Detecting Physiological Proximate Principles 9 19 20 32 43 oO 58 66 78 101 114 124 129 ADVANCED COURSE Introduction ............. 133 XIII. Cabbohtdbates 134 XIV. A. HON of Malt dpos Starch 137 XV. Ci;. Egg-Albumln 138 XVI. Coaqulatiok o Milk 139 XVII. Ti Ml viii ESSENTIALS OF CHEMICAL PHYSIOLOGY Lesson paue XVIII. Digestion 143 XIX. HEMOGLOBIN AND ITS DERIVATIVES 145 XX. Serum . .149 XXI. Coagulation of Blood 151 XXII. Muscle 153 XXIII. Urea and Chlorides in Urine 157 XXIV. Estimation of Phosphate and Sulphates in Urine . . . 162 XXV. Uric Acid and Creatinine . . .'..'. . . .165 XXVI. Pigments of the Urine 168 APPENDIX Hemacytometers ............ 172 Hemoglobinometers ........... 174 Polarimeters 177 The Spectro-polarimeter ' . . . . . 180 Sir George Johnson's Picro-saccharometer 181 Mercurial Air-pumps ........... 182 Analysis of Gases 185 Kjeldahl's Method of Estimating Nitrogen . . . . . . 186 INDEX 189 LIST OF ILLUSTRATIONS PIG. PAI i K 1. Dextrose Crystals .... Frey 11 2. 12 3. Milk-Sugar Crystals Frey 13 4. Section of Pea, showing Starch Grain s . . Yeo, after Sachs 15 5. Fat Cells . Schafer 16 6. 20 7. 23 8. DlALYSKK ...... 23 9. Diagram of a Cell . . . . . Schafer 29 10. Yeo 35 11 35 12. 45 13. . Langley 45 14. Submaxillary Gland . . . . Heidenhain 45 15. Yeast Cells ...... Yeo's Physiology 46 16. Sf/HIZOMYCETES .... After Zopf 47 17. Koch 48 18. Cardiac Gland . Klein 51 11). Pyloric Gland . Ebstein 51 20. Cu;i>i\< Gland . . Langley 52 21. Al.VKoi.i s of Pamcri IB . Kiihne and Lea 69 22. I.m ■< inj: Crystals . Kilhne 64 28. 64 24. HitM.vioiDiN Crystal* Frey 67 36. -terin Crystals 1 rni 70 X ESSENTIALS OF CHEMICAL PHYSIOLOGY FIG. PAGE 26. Villus of Rat Killed dubing Fat Absorption . . . Schafer 75 27. Mucous Membrane of Frog's Intestine during Fat Absorption Schafer 75 28. Fibrin Filaments and Blood Tablets Schafer 80 29. Action of Eeagents on Blood Corpuscles .... Schafer 84 30. Oxyhemoglobin Crystals Quain's Anatomy 86 31. Hemin Crystals Preyer 87 32. Diagram of Spectroscope 89 33. Figure of Spectroscope and Accessories . . . McKendrick 89 34. Arrangement of Prisms in Direct Vision Spectroscope Gscheidlen 90 35. Stand for Direct Vision Spectroscope 91 36. Absorption Spectra Bollett 91 37. Absorption Spectra .......... 92 38. Dupre's Urea Apparatus ' Gamgee 102 39. Urinometer McKendrick 104 40. Urea Crystals Frey 104 41. Urea Nitrate and Oxalate Frey 106 42. Triple Phosphate Crystals . . . . . . Frey 113 43. Uric Acid Crystals Frey 115 44. Hippuric Acid Crystals Frey 117 45. Creatine Crystals Frey 119 46. Creatinine Crystals Frey 119 47. Acid Sodium Urate Frey 12 48. Acid Ammonium Urate Frey 120 49. Envelope Crystals of Calcium Oxalate .... Frey 121 50. Cystin Crystals . Frey 121 51. Triple Phosphate Crystals Bryant's Surgery 122 52. Calcium Phosphate Crystals .... Bryant's Surgery 122 53. Albuminometer of Esbach 124 54. Two Burettes on Stand . . . . . . . Sutton 125 55. Hot-air Oven with Gas Begulator .... Gscheidlen 134 56. Osazone Crystals Coloured plate to face 134 57. Absorption Spectra of Haemoglobin, &g. ...... 146 58. Photographic Spectrum of Hemoglobin and Oxyhemoglobin Gamgee , 147 LIST OF ILLUSTRATIONS XI Gscheidlen After Hopkins ">V». Photographic Spectrum of Oxyhemoglobin and Methemo- globin Gamqee 60. Centrifrugal Machine .... 61. Absorption Spectra of Myohematin 62. A Desiccator 63. Absorption Spectra of Urinary Pigments 1)4. Gowers' Hemacytometer 65. Oliver's Hemacytometer tit',. Gowers' Hemoglobinometer . 67. Fleischl's Hemometer . 68. Oliver's Hemoglobinometer . 69. Soleil'8 Saccharimeter 70. Diagram of Optical Arrangements in Soleil's Saccharimeter . 71. Laurent's Polarimeter ......... 72. Spectropolarimeter of von Fleischl 73. Sir G. Johnson's Picro-saccharometer . . Sir G. Johnson 74. Diagram of Pfluger's Pump ......... 75. L. Hill's Air Pump 76. Waller's Apparatus for Gas Analysis .... Waller 77. K.iei.dahi/s Method Waller, after Argutinsky 147 151 154 155 170 172 173 174 175 176 177 178 179 180 182 183 184 185 186 ESSENTIALS OF CHEMICAL PHYSIOLOGY INTRODUCTION Chemical Physiology, or Physiological Chemistry, as it is some- times termed, deals with the chemical composition of the body, and also of the food which enters, and the excretions which leave, the body. When a chemist examines living things he is placed at a dis- advantage when compared with an anatomist; for the latter can with the microscope examine cells, organisms, and structures in the living condition. The chemist, on the other hand, cannot at present state anything positive about the chemical structure of living matter,. because the reagents he uses will destroy the life of the tissue he is examining. There is, however, no such disadvantage when he examines non-living matter, like food and urine, and it is therefore in the analysis of such substances that chemical physiology has- made its most important advances, and the knowledge so obtained is of the greatest practical interesl to the student and practitioner of medicine. The animal organism is in its earliest embryonic state a single cell; as development progresses it becomes an adherent mass of simple cells. In the Later stages various tissues become differen- tiated from each other by the cells becoming grouped in different by alterations in the shape of the cells, by deposition of inter- cellular matter between the cells, and by chemical changes in the living matter of the cells themselves. Thus in some situations the sells are grouped into the various epithelial linings; in others the \ 2 ESSENTIALS OF CHEMICAL PHYSIOLOGY cells become elongated, and form muscular fibres ; in the connective tissues we have a preponderating amount of intercellular material, which may become permeated with fibres, or be the seat of the deposi- tion of calcareous salts, as in bone. Instances of chemical changes in the cells themselves are seen on the surface of the body, where the superficial layers of the epidermis become horny (i.e. filled with the chemical substance called keratin) ; in the mucous salivary glands, where the cells become filled with mucin, which they subsequently extrude ; and in adipose tissue, where they become filled with fat. In spite of these changes, the variety of which produces the great complexity of the adult organism, there are many 'cells which still retain their primitive structure ; notable among these are the white corpuscles of the blood. A cell may be defined as a mass of living material containing in its interior a more solid structure called the nucleus. The nucleus exercises a controlling influence over the nutrition and subdivision of the cell. Living material is called protoplasm, and protoplasm is charac- terised by (1) its power of movement (seen in amoeboid movement, ciliary movement, muscular movement) ; (2) its power of assimila- tion, that is, it is able to convert into protoplasm the nutrient material or food which is ingested ; (3) its power of growth — this is a natural consequence of its power of assimilation ; (4) its power of reproduction — this is a variety of growth ; and (5) its power to excrete, to give out waste materials, the products of its other activities. The chemical structure of protoplasm can only be investigated after the protoplasm has been killed. The substances it yields are (1) Water; protoplasm is semifluid, and at least three-quarters of its weight, often more, are due to water. (2) Proteids. These are the most constant and abundant of the solids. A proteid or albu- minous substance consists of carbon, hydrogen, nitrogen, oxygen, with sulphur and phosphorus in small quantities only. In nuclein, a proteid-like substance obtained from the nuclei of cells, phosphorus is more abundant. The proteid obtained in greatest abundance from the cell-protoplasm is nucleo -proteid, that is a compound of proteid with varying amounts of mtclein. White of egg is a familiar instance of an albuminous substance or proteid, and the fact (which is also familiar) that tbis sets into a solid on boiling will serve as a reminder that the greater number of the proteids found in nature have a similar tendency to coagulate under the influence of heat and other INTRODUCTION 3 agencies. (3) Various other substances occur in smaller proportions, the most constant of which are lecithin, a phosphorised fat ; choles- terin, a monatomic alcohol ; and inorganic salts, especially phos- phates and chlorides of calcium, sodium, and potassium. It will be seen from this rapid survey of the composition of the b< el v how many are the substances which it is necessary we should study ; the food from which it is built up is also complex, for animals do not possess to such an extent as plants do the power of building up complex from simple materials. We may now proceed to an enumeration of the chemical constituents of the animal body, and group them in a systematic way. The substances out of which the body is built consist of chemical elements and of chemical compounds, or unions of these elements. The elements found in the body are carbon, hydrogen, nitrogen, oxygen, sulphur, phosphorus, fluorine, chlorine, iodine, silicon, sodium, potassium, calcium, magnesium, lithium, iron, and oc- casionally manganese, copper, and lead. Of these very few occur in the free state. Oxygen (to a small extent) and nitrogen are found dissolved in the blood-plasma ; hydrogen is formed by putrefaction in the alimentary canal. With some few ex- ceptions such as these, the elements enumerated above are found combined with one another to form compounds. The compounds, or, as they are generally termed in physiology, the proximate principles, found in the body are divided into — (1) Mineral or inorganic compounds. (2) Organic compounds, or compounds of carbon. The inorganic compounds present are water, various acids (such as hydrochloric acid in the gastric juice), ammonia (as in the urine), and numerous salts, such as calcium phosphate in bone, sodium chloride in blood and urine, and many others. The organic compounds are more numerous ; they may be sub- divided into — 1. Various groups of alcohols and organic acids, and their derivatives, such as the fats and carbohydrates. 2. Various derivatives of ammonia, amides, amines, urea, itc. 3. Aromatic bodies, or derivatives of benzene. 4. Proteids, the most important of all, and substances allied to ide Like the albuminoids, pigments, and ferments. A more convenient practical method of grouping the prozimati principles of the body and of Ux,d is the following : B 2 Organic 4 ESSENTIALS OF CHEMICAL PHYSIOLOGY ( Water. Inorganic -j Salts — e.<7.chloridesandphosphatesofsodiuni [ and calcium. , / Proteids — e.g. albumin, ray o sin. I -vfj., J Albuminoids — e.g. gelatin, keratin. ° I Simpler nitrogenous bodies — e.g. lecithin, I creatine, urea. i Fats — e.g. butter, fats of adipose tissue. ->r . J Carbohydrates — e.g. sugar, starch. ° I Simple organic bodies — e.g. alcohol, choles- I terin, vegetable acids, and salts, lactic acid. Many of the substances enumerated above only occur in small quantities. The most important are the inorganic substances, water and salts ; and the organic substances, proteids, carbohydrates, and fats. It is necessary in our subsequent study of the principles of chemical physiology that we should always keep in mind this simple classification ; the subdivision of organic substances into proteids,. fats, and carbohydrates forms the starting point, the A B C, as one might say, of chemical physiology. I will conclude this introductory chapter by giving a list of the apparatus and reagents necessary for a practical study of the subject,, and some tables which it will be often found convenient to refer to. The following set of reagents conveniently contained in 4 to 6 oz. glass, stoppered bottles should be provided for each two students : — Sulphuric acid, concentrated. ,, ,, 25 per cent. „ 0-1 „ Nitric acid, concentrated. Fuming nitric acid. Hydrochloric acid, concentrated. „ „ 02 per cent.1 Acetic acid, glacial. ,, ,, 20 per cent. » _ )j 2 ,, Caustic potash. 20 per cent. ,. . „ 0-1 „ Ammonia. Sodium carbonate, 1 per cent. Ammonium sulphide solution. Ammonium sulphate, saturated solution. Silver nitrate, 1 per cent. Barium chloride, saturated solution. Ammonium molybdate solution. Millon's reagent.2 Solution of feiTOcyanide of potassium. ,, litmus. 1 Made by adding 994 c.c. of water to 6 c.c. of the concentrated hydrochloric acid of the British Pbarmacopceia. - Mercury is dissolved in its own weight of strong nitric acid. The solution so obtained is diluted with twice its volume of water. The decanted clear liquid is Millon's reagent. INTRODUCTION 5 Solution of sodium phosphate. „ iodine in potassium iodide. Methylated spirit. Ether. Esbach's reagent.1 Solution of copper sulphate, 1 per cent. The following additional reagents will be required by those taking the advanced course : — Solution of mercuric chloride. „ potassium ferricyanide. Sodium carbonate, saturated solution. „ chloride, saturated solution. ,, ,, 10 per cent, solution. Magnesium sulphate, saturated solution. Lime water. Baryta mixture. - Sodium acetate solution.3 Phosphoric acid, OS per cent. Absolute alcohol. In addition to these, there should be kept in stock in the laboratory, to be given out for the lessons in which they are used, the following : — Solid sodium chloride. ., magnesium sulphate. ,, ammonium ,, sodio-magnesium sulphate. Standard solution of uranium acetate or nitrate for estimating phosphates.' Standard solution of mercuric nitrate for estimating urea. ,, ,, „ .. ,, chlorides. „ „ _ silver „ „ „ Fehling's solution. Caustic soda, 40 per cent. Bromine. Solution of potassium bichromate. Phenyl hydrazine hydrochloride. Solid sodium acetate. Phospho-tungstic acid. Glacial phosphoric acid. Each student should be provided with — A Jninsen burner. 1 dozen test-tubes in test-tube stand. 2 or 8 4-oz. flasks. 2 flat porcelain dishes. 2 or 4 4-oz. beakers. 2 small glass funnels and a funnel stand. A glass stirring-rod and a small pipette. 1 bn. 1 Ten grammes of picric acid and 20 grammes of citric acid are dissolve BOO to '.(00 c.O. of boiling water, and then sufficient water added to ma! < up - .Made by mixing 1 volume of barium nitrate solution with 2 of barium hyd Solution, both saturated in the cold. '■ Prepared as follows:— Sodium acetate, 100 grammes; water, 900 c.c. ; glacial acetic acid, loo c.c. ' Instructions liow to make standard solutions will be given in the l< where they are d ed. 6 ESSENTIALS OF CHEMICAL PHYSIOLOGY An iron tripod with wire gauze. Filter papers and litmus papers. A 100-c.c. cylindrical measuring glass. A thermometer marked in degrees Centigrade. A urinometer. A tin can on a stand to be used as a water-bath. Apparatus which is not so frequently used, such as that employed in generating carbonic anhydride, carbonic oxide, or sulphuretted hydrogen, may be given out as required. The laboratory should also possess a good balance, with its accessories, water and air baths, kept at various temperatures, retorts, and analytical apparatus generally. The microscope, polarimeter, spectroscope, dialyser, are also frequently employed in chemico-physiological investigations. WEIGHTS AND MEASUEES The weights and measures usually employed in science are those of the metric system, but as in this country the practical physician still largely uses English grains and ounces, we may compare the two systems in the following way : — Weights (English System.) 1 grain = 0*0648 gramme 1 ounce = 437*5 grains = 28*3595 grammes 1 lb. = 16 oz. = 7,000 grains = 453*5925 The scruple = 20 grains = 1*296 gramme, and the drachm = 60 grains = 3*888 grammes, are retained in use, but neither is an aliquot part of the ounce; though for practical purposes an ounce is considered to consist of 8 drachms. (Metric System.) 1 milligramme = 0*001 gramme = 0*015432 grain 1 centigramme = 001 ' „ 0*154323 „ 1 decigramme = 0*1 „ = 1*543235 „ 1 gramme = 15*43235 grains 1 decagramme = 10 grammes = 154*3235 ,, 1 hectogramme = 100 „ = 1543*235 1 kilogramme = 1000 „ = 15432*35 = 2 lb. 3 oz. 119*8,, Measures of Length (English System.) 1 inch = 25*4 millimetres 1 foot = 12 inches = 304*8 millimetres (Metric System.) The standard of length is the metre ; subdivisions and multiples of which, with the prefixes milli-, centi-, and deci- on the one hand, and deca-, hecto- and kilo- on the other, have the same relation to the metre as the subdivi- sions and multiples of the gramme, in the table just given, have to the gramme, thus : 1 millimetre = 0*001 metre = 0*03937 inch 1 centimetre = 0*01 „ = 0*3937 „ 1 decimetre =0*1 „ = 3*93707 inches 1 metre = 39*37079 „ INTRODUCTION J Measures of Capacity (English System.) 1 minim = 0*059 cubic centimetre 1 fluid drachm = 60 minims = 3*549 cubic centimetres 1 fluid ounce = 8 fluid drachms = 28-398 „ 1 pint = 20 fluid ounces = 567-936 1 gallon = 8 pints = 4*54837 litres (Metric System.) In the metric system the measures of capacity are intimately connected with the measures of length ; we thus have cubic millimetres, cubic centi- metres, and so forth. The standard of capacity is the litre, which is equal to 1.000 cubic centimetres ; and each cubic centimetre is the volume of 1 gramme of distilled water at 4° C.1 1 cubic centimetre (generally written c.c.) = 16-931 minims. 1 litre = 1,000 c.c = 1 pint 15 oz. 2 drs. 11 m. = 35-2154 fluid ounces. 1 cubic inch = 16*386 c.c. THERMOMETEIC SCALES The scale most frequently used in this country is the Fahrenheit scale ; in this the freezing point of water is 32°, and the boiling point 212°. On the Continent the Reaumur scale is largely employed, in which the freezing point is 0°, and the boiling pomt 80°. In scientific work the Centigrade scale has almost completely taken the place of these ; in this system the freezing point is 0°, and the boiling point 100°. To convert degrees Fahrenheit into degrees Centigrade, subtract 32 and multiply by §, or C = (F — 32) f. Conversely, degrees Centigrade may be con- verted into degrees Fahrenheit by the following formula : F = |C + 32. TENSION OF AQUEOUS VAPOUR IN MILLIMETRES OF MERCURY FROM 10° TO 25° C. 10°. 9*126 11°. 9*751 12°. 10*421 13°. 11*130 14°. 11*882 15°. 12*677 16°. 13-519 17°. 14-409 18°. 15-351 19°. 16*345 20°. 17-396 21°. 18-505 22°. 19-675 23°. 20909 24°. 22-211 25°. 23-582 TABLE OF THE DENSITY OF WATER AT TEMPERATURES BETWEEN 0° AND 30° C. 0°. 0*99988 1°. 0-99993 2°. 0*99997 3°. 0-99999 4°. 1*00000 5°. 0-9999!) 6 . 0-99997 7 . 0-99994 b°. 0*99988 9°. 0*99982 10°. 0*99974 11°. 0-99065 12°. 0-99955 13°. 0-99942 14°. 0-99980 15°. 0-99915 16°. 0-99900 17°. 0-99884 18°. 0-99866 19°. 0-99847 20°. 0-99827 21°. 0-99806 22°. 0-99785 23°. 0-99762 24°. 0-99738 25°. 0-99714 26°. 0-99689 27°. 0-99662 28°. 099635 29°. 0-99607 30°. 0-99579 1 4° C. is the temperature at which water lias the greatest density. For prac- ■■ oses measures are more often constructed so that a cubic oentimetre holds ter at 16° C, which is about th<- average temperature of rooms. '1 he true cubic centimetre contains only 0*999 gramme at 16° C. ESSENTIALS OE CHEMICAL PHYSIOLOGY SYMBOLS AND ATOMIC WEIGHTS OF THE PRINCIPAL ELEMENTS Aluminium Al 27-02 Fluorine F 19-1 Antimony Sb 120-0 Gold Au 197-0 Arsenic As 74-9 Hydrogen H 1-0 Barium Ba 136-8 Iodine I 126-53 Bismuth Bi 208-0 Iron Fe 55-9 Boron B 10-9 Lead Pb 206-4 Bromine Br 79-75 Magnesium Mg 24-0 Cadmium Cd 112-0 Manganese Mn 55-0 Calcium Ca 39-9 Mercury Hg 199-8 j Carbon C 11-97 Nickel Ni 58-6 Chlorine CI 35-37 Nitrogen N 14-01 Copper Cu 63-2 Oxygen O 15-96 Phosphorus ?P 30-96 Platinum Pt 194-3 Potassium K 39-04 Silver Ag 107-66 Silicon Si 28-3 Sodium Na 23-0 Strontium Sr 87-3 Sulphur S 31-98 Tin Sn 118-8 Tungsten W 183-6 Zinc Zn 117-8 ELEMENTABY COURSE LESSON I THE CABBOHYDBATES AND FATS 1. Note the general appearance of the specimens of grape sugar or dex- trose, cane sugar, dextrin, and starch which are given round. 2. Put some of each into cold water. Starch is insoluble ; dextrose, cane sugar, and dextrin dissolve after a time, but more readily in hot water. 3. Trommer's test for dextrose. — Put a few drops of copper sulphate solu- tion into a test-tube, then solution of dextrose, and then strong caustic potash. On adding the caustic potash a precipitate is first formed, which, on adding more potash, redissolves, forming a blue solution. On boiling this a yellow or red precipitate (cuprous hydrate or oxide) forms. Fettling' s test for dextrose. — Fehling's solution is a mixture of copper sulphate, caustic soda, and Rochelle salt of a certain strength. Ttis used for estimating dextrose quantitatively (see Lesson XII.). 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 dextrose and boil again. Pteduction, resulting in the formation of cuprous hydrate or oxide. takes place as in Trommer's test. £4. Cane Sugar. — (a) The solution of cane sugar when mixed with copper sulphate and caustic potash gives a blue solution. But on boiling no reduc- tion 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 dextrose and levulose. It then gives Trommer's or Fehling's test in the typical way. (c) Boil some of the cane sugar solution with an equal volume of con- centrated hydrochloric acid. A deep red solution is formed. J text rose, lactose, and maltose do not give this test. :>. Starch. — (a) Examine microscopically the scrapings from the surfac el ■ freshly cut potato. Note the appearance of the starch grains with their concentric markings. (o) On boiling starch with water an opalescent solution is formed, which, if strong, gelatinises on cooling. (c) Add iodine solution. An intense blue colour is produced, which appears on heating, and if not heated too long reappears on cooling. N.I'..- -Prolonged heating drives off the iodine, and consequently no Kim rur returns after cooling. into I' : t ti ) i and dextrose. To some staid: solution in a flask add a few drops of 25-per-cent. sulphuric acid, and hoi] for 16 minutes. Take some of'fhe liquid, which is now clear, and show the and < I < ■ ■; 1 1 10 ESSENTIALS OF CHEMICAL PHYSIOLOGY 6. Dextrin. — Add iodine solution to solution of dextrin, and a reddish- brown solution is produced. The colour disappears on heating and reappears on cooling. 7. Glycogen. — Solution of glycogen is given round : (a) it is opalescent like that of starch. (6) With iodine it gives a brown colour very like dextrin. The colour disappears on heating and reappears on cooling, (c) By boiling with 25-per-cent. sulphuric acid for 15 to 20 minutes it is converted into grape sugar. 8. Lard is given round as an example of a fat. (a) It is insoluble in water. (b) By boiling with potash it yields a solution of soap. (c) Add to this solution a few drops of 25-per-cent. sulphuric acid. On heating a layer of fatty acid collects on the surface. (d) Shake up some lard with ether. It dissolves, leaving little or no residue. CARBOHYDRATES 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 ; dextrose ; and lactose, or milk sugar. They may be for the greater part arranged into three groups, according to their empirical formulae. The names and formulae of these groups, and the most important members of each, are as follows : — 1. Monosaccharides or Glucoses, C6H120G 2. Disaccharides, Sucroses, or Saccharoses, C^H^O^ 3. Polysaccharides or Amyloses, (C6H1005)„. + Dextrose — Levulose + Galactose + Cane sugar + Lactose + Maltose + Starch + Glycogen + Dextrin Cellulose Gums The + and — signs in the above list indicate that the substances to which they are prefixed are dextro- and levo-rotatory respectively as regards polarised light (see Appendix). 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. This definition is, however, only a rough one, and if pushed too far would include several substances like acetic acid, lactic acid, and inosite, which are not carbohydrates. The formulae given above are merely empirical ; and there is no doubt that the quantity n in the starch group is variable, and often THE CARBOHYDRATES AND FATS 11 large ; hence the name 'polysaccharides that is given to the group. Research has, moreover, shown that the glucoses are either aldehydes or ketones of hexatomic alcohols having the general formula C6H8(OH),j. Thus dextrose is the aldehyde of sorbite, levulose the ketone of mannite, and galactose the aldehyde of dulcite. The amyloses may be regarded as the anhydrides of the glucoses [»G6H, 20G — »H.20 = (C0H,nO^),,]. The sucroses are condensed glucoses — i.e. they are formed by the combination of two molecules of glucose with the loss of one molecule of water (CGH120G + C6H120G — H20 = C12H22Ou) ; hence the term disaccharide. The following are the chief facts in relation to each of the principal carbohydrates : — Pig. I.— Dextrose .crystals MONOSACCHARIDES Dextrose or Grape Sugar. — This carbohydrate is found in fruits, honey, and in minute quantities in the blood (012 per cent.) and numerous tissues, organs, and fluids of the body. It is the form of sugar found in large quantities in the blood and urine in the disease known as diabetes. Dextrose is soluble in hot and cold water and in alcohol. It is crystalline (see fig. 1), 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 solu- tions dextrose reduces salts of silver, bismuth, mercury, and copper. The reduction of cupric to cuprous oxide constitutes Trommer's test, which has been already described at the head of the lesson. On boiling it with an alkaline solution of picric acid, a dark red opaque solution due to reduction of the picric to picramic acid is produced. Another important property of grape sugar is that under the influence of yeast it is converted into alcohol and carbonic acid (C6H12Ofl = 2C2HG0 + 2C0,). Dextrose may be estimated by the fermentation test, by the polarimeter, and by the use of Fehling's solution. 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.). Levulose. When cane sugar is treated with dilute mineral aeids it undergoes a process known as inversion /'.'■. it takes upwater ami 12 ESSENTIALS OE CHEMICAL PHYSIOLOGY is converted into equal parts of dextrose and levulose. The pre- viously dextro-rotatory solution of cane sugar then becomes levo- rotatory, the levo-rotatory power of the levulose being greater than the dextro-rotatory power of the dextrose formed. Hence the term inversion. Similar hydrolytic changes are produced by certain ferments, such as the invert ferment of the intestinal juice. Pure levulose can be crystallised, but so great is the difficulty of obtaining crystals of it that one of its names was uncrystallisable sugar. Small quantities of levulose have been found in blood, urine, and muscle. It has been recommended as an article of diet in diabetes in place of ordinary sugar ; in this disease it does not appear to have the harmful effect that other sugars produce. Levulose gives the same general reactions as dextrose. Galactose is formed by the action of dilute mineral acids or invert- ing ferments on lactose or milk sugar. It resembles dextrose in being dextro-rotatory, 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 (C6H10O8), which is only sparingly soluble in water. Dextrose when treated this way yields an isomeric acid — i.e. an acid with the same empirical formula, called saccharic acid, which is readily soluble in water. ^s^) // X^C^^n. Inosite, or muscle sugar, is found ^#i\ % in muscle, kidney, liver, and other parts of the body in small quanti- ties. It is also largely found in the vegetable kingdom. It is a crystallisable substance (see fig. 2) and has the same formula (C6H12O0) as the glucoses. It is, however, not a sugar. It gives none of the sugar tests, and careful analysis has shown it has quite a different chemical constitution from the true sugars. It belongs to the aromatic series, and is only included here for convenience. Eict. 2. —Inosite crystals. DISACCHARIDES Cane Sugar. — This 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 THE CARBOHYDRATES AND FATS 13 great importance as a food. After abundant ingestion of cane sugar traces may appear in the urine, but the greater part undergoes inversion in the alimentary canal. Pure cane sugar is crystalline and dextro-rotatory. 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 inverting ferments, such as that occurring in the succus entericus or intestinal juice. It then takes up water and is split into equal parts of dextrose and levulose (see p. 11). Cl2H„On + H20 = C6H1206 + C6H120„ [cane sugar] [dextrose] [levulose] With yeast, cane sugar is first inverted by means of a special soluble ferment produced by the yeast cells, and then there is an alcoholic fermentation of the glucoses so formed. Lactose, or milk sugar, occurs in milk. It has also been described as occurring in the urine of women in the early days of lactation or after weaning. It crystallises in rhombic prisms (see fig. 3). It is much less soluble in water than cane sugar or dextrose, and has only a slightly sweet taste. It is in- soluble in alcohol and ether ; aqueous solutions are dextro-rotatory. 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 dextrose. If it required seven parts of a solution of dextrose 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 hydrolysed by the same agencies as those already enumerated in connection with cane sugar. The glucoses formed are dextrose and galactose. C,,H22On + H20 = C6H1206 + C6H,a06 [l».-t. [dextnwe] With yeast it is fust inverted, and then alcohol is formed. This, however, occurs slowly. With the lactic-acid organisms which bring about the souring of / / Milk-sugar orystals. 14 ESSENTIALS OF CHEMICAL PHYSIOLOGY milk the lactic-acid fermentation is produced. 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) 012H22O„ + H20 = 4C3H603 [lactose] [lactic acid] (2) 4C3H603 = 2C4H802 + 4C02 + 4H2 [lactic acid] [butyric acid] 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 su°'ar formed from starch by the diastatic ferments contained in the saliva (ptyalin) and pancreatic juice (amylopsin).1 It can be ob- tained in the form of acicular crystals ; it is strongly dextro-rotatory. It gives Trommer's test.; but its reducing power, as measured by Fehling's solution, is one-third less than that of dextrose. By prolonged boiling with water, or, more readily, by boiling with a dilute mineral acid, or by means of an inverting ferment, such as occurs in the intestinal juice, it is converted into dextrose. C12H,2On + H20 = 2C6H1206 [maltose] [dextrose] It undergoes readily the alcoholic fermentation. The three important physiological sugars (dextrose, lactose, and maltose) may be distinguished from one another by their relative reducing action on Fehling's solution, or by the phenyl-hydrazine test described in Lesson XIII. POLYSACCHARIDES. Starch is widely diffused through the vegetable kingdom. It occurs in nature in the form of microscopic grains, varying in size and appearance, 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. On heating starch with dilute mineral acids, dextrose is formed. By the action of diastatic ferments, maltose is the chief end product. In both cases dextrin is an intermediate stage in the process. 1 An isomeric sugar called iso-maltose (see Lesson XIII.) is also formed under these circumstances. THE CARBOHYDRATES AND FATS 15 F;p. 4.— Section of pea showing starch and aleurone grains embedded in the protoplasm of the cells : a, aleurone grains ; st, starch grains ; i, intercel- lular spaces. ( Y'.i. after Sachs.) give Trommer's test, nor By hydrating Before the formation of dextrin the starch solution loses its opal- escence, a substance called soluble starch or amidulin being formed. This, like native starch, gives a blue colour with iodine. Although the molecular weight of starch is unknown, the formula for soluble starch is probably 5(C1.2H.i0O10)2n- Equations that repre- sent the formation of sugars and dextrins from this are very complex, and are at present hypothetical. Dextrin is the name given to the intermediate products in the hydration of starch, and two chief varieties are distinguished : — erythro-dcxtrin, which gives a reddish-brown colour with iodine ; and achroo-dextrin, which does not. It is readily soluble in water, but insoluble in alcohol and ether. It is gummy and amorphous. It does not does it ferment with yeast. It is dextro-rotatory. agencies it is converted into glucose. Glycogen, or animal starch, is found in liver, muscle, and colour- less blood corpuscles. It is also abundant in all embryonic 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 it gives a reddish or port-wine colour, very similar to that given by erythro-dextrin. Dextrin may be distinguished 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. 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 ferments 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 bo allows the digestive juice al the 16 ESSENTIALS OF CHEMICAL PHYSIOLOGY stai'ch proper. Cellulose is found in a few animals, as in the test or outer investment of the Tunicates. For farther information regarding the carbohydrates see Lesson XIII. THE FATS 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 postponed to Lesson IV. Pig. 5. — A. few cells from the margin of a fat lobule : fff, fat globule distending fat cell ; n, nucleus ; m, membranous envelope of the cell : c r, bunch of crystals within a fat cell ; c, capillary vessel "r v, venule ; et, connective tissue cell. The fibres of the connective tissue are not represented. 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 melts at —5° C, palmitin at 45° C, and stearin at 53-66° C. It is thus 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 glycerin, and may be termed glycerides or glyceric ethers. The term hydrocarbon, applied to them by some authors, is wholly incorrect. THE CARBOHYDRATES AND FATS 17 The fatty acids form a series of acids derived from the monatomic alcohols by oxidation. Thus, to take ordinary ethyl alcohol, C.2Hr).HO, the first stage in oxidation is the removal of two atoms of hydrogen to form aldehyde, CH3.COH ; on further oxidation an atom of oxygen is added to form acetic acid, CH3.COOH. A similar acid can be obtained from all the other alcohols, thus : — From methyl alcohol CH3.HO, ,, ethyl „ propyl formic C2H5.HO, acetic C3H7.HO, propionic C4H(J.HO, butyric C5Hn.HO, valeric C6H13.HO, caproic acid H.COOH is obtained „ CH3.COOH C2H5.COOH C3H7.COOH C4H9.COOH „ butyl „ amyl „ hexyl „ C6H13.HO, caproic „ C5Hu.COOH and so on. Or in general terms :— *■ From the alcohol with formula C„H2„+i.HO the acid with formula CViH^.CO.OH is obtained. The sixteenth term of this series has the formula C|5H31.CO.OH, and is called palmitic acid; the eighteenth has the formula C17H35.CO.OH, and is called stearic acid. Each acid, as will be seen, consists of a radicle, C^H^^CO, united to hydroxyl (HO). Oleic acid, however, is not a member of the fatty acid series proper, but belongs to a somewhat similar series of acids known as the acrylic series, of which the general formula is C^Ha^COOH. It is the eighteenth term of the series, and its formula is C, 7H33.CO.OH. Glycerin or Glycerol is a triatomic alcohol, C3H5(OH)3 — i.e. three atoms of hydroxyl united to a radicle glyceryl (C3Hr,). The hydrogen in the hydroxyl atoms is replaceable by other organic radicles. As an example take the radicle 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 :— C.H, (OH J OH (OH C,IlJOH 0 0 j [OH /O.CH,.CO 03HS- O.CH3.CO C3HJO.CH3.CO lOB (0.CH3.C0 O.CH3.CO (O.CH..CO [mono ;diacetin] [trlaeetln] Triacetin is a type of a neutral fat ; stearin, pahnitin, and olein ought more properly to be called tristearin, tripalmitin, and triolein Bctively. Bach consists of glycerin in which the three atoms of hydrogen in the bydroxyls are replaced by radicles of the fatty acid. This is r< ed in the following formula : — c 18 ESSENTIALS OE CHEMICAL PHYSIOLOGY Acid Radicle Fat Palmitic acid CrH31.COOH Palruityl C15H31.CO PalmitinC3H5(OC]5H31.CO)3 Stearic acid Cj.H^.COOH Stearyl C17H35.CO Stearin C3H5(OC17H35.CO)3 Oleic acid C17H33-COOH Oleyl C,7H33.CO Olein C3H5(OC17H33.CO)3 Decomposition Products of the Fats.— The fats split up into the substances out of which they are built up. Under the influence of superheated steam, mineral acids, and in the body by means of certain ferments (for instance, the fat-splitting ferment steapsin of the pancreatic juice), a fat combines with water and splits into glycerin and the fatty acid. The following equation represents what occurs in a fat, taking tripalmitin as an example : — C3H5(O.C1.5H31CO)3 + 3H20=C3H5(OH)3 + 3C15H31CO.OH [palmitm— a fat] [glycerin] [palmitic acid — a fatty acid] In the process of saponification much the same sort of reaction occurs, the final products being glycerin 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 — C3H5(O.C15H31CO)3 + 3KHO=C3H.3(OH)3 + 3C15H31CO.OK [palmitin — a fat] [glycerin] [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 is seen in the natural emulsion — milk. 19 LESSON. II THE PROTEIDS 1. Tests for Proteids. — 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 gobulin.) [a) Heat Coagulation. — Faintly acidulate with a few drops of 2-per-cent. acetic acid and boil. The proteid is rendered insoluble (coagulated proteid). (6) Precipitation with Nitric Acid. — The addition of strong nitric acid to the original solution also produces a white precipitate. (c) Xanthoproteic Reaction. — On boiling the white precipitate produced by nitric acid it turns yellow ; after cooling add ammonia ; the yellow becomes orange. (d) Millon's Test. — Millon's reagent (which is a mixture of the nitrates of mercury containing excess of nitric acid ; see p. 4) gives a white precipitate, which turns brick-red on boiling. (e) After the addition of acetic acid, potassium ferrocyanide gives a white precipitate (/) PiotrowskVs test. — Add a drop of a 1-per-cent. solution of cupric sulphate to the original solution and then caustic potash, and a violet solution is obtained. Eepeat experiment {/) with a solution of commercial peptone, and note that a rose-red solution is obtained. This is called the biuret reaction. 2. Action of Neutral Salts. — (a) Saturate the solution of egg-white with lnajmeshun sulphate by adding crystals of the salt and shaking in a flask. In order to saturate proteid solutions with a salt, excess of the finely powdered salt should be added, and the flask shaken for a considerable time ; a machine is frequently employed for this purpose. Too violent shaking residts in the formation of froth. Another good method consists in grinding the salt up with the solution 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. (e) Completely saturate another portion with ammonium sulphate by adding crystals of the salt and shaking — a precipitate is produced of both the globulin and albumin. Filter. The filtrate contains no proteid. id< Repeat the last experiment (c) with a solution of commercial peptone. A precipitate is produced of the albumoses or 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 sulphate precipitates nil j, mi' ids except peptone. C 1 20 ESSENTIALS OF CHEMICAL PHYSIOLOGY LESSON III THE PRO TE IDS {continued) 1. 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, similar to that used in the last lesson. To A add a few drops of Ol-per-cent. solution of caustic potash. To B add the same amount of Ol-per-cent. solution of caustic potash. To C add a rather larger amount of Ol-per-cent. sulphuric acid. Put all three into the warm bath l at about the temperature of the body (36-40° C.) After five minutes remove test-tube A, and boil. The proteid is no Fig. 6. — Simple warm bath, as described iu footnote. longer coagulated by heat, having been converted into alkali- albumin. After cooling, colour with litmus solution and neutralise, with Ol-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-albumin. Add to it a few drops of sodium phosphate, colour with litmus, and neutralise as before. Note that the alkali -albumin now requires more acid for its precipitation 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 acts as a warm stage as in the figure. The stage is kept warm by a small gas flame. Such a warm bath may be placed between every two or three students. THE PROTEIDS 21 than in A. the acid which is first added converting the sodium phosphate into acid sodium phosphate. Now remove C from the bath. Boil it. Again there is no coagulation, the proteid having been converted into iiciil-albuDiiii, or sijntonin. After cooling colour with litmus and neutralise with OT-per-cent. alkali. At the neutral point a precipitate is formed, soluble in excess of acid or alkali. (Acid-albumin is formed more slowly than alkali-albumin, so it is best to leave this experiment to the last.) 2. Take some gelatin and dissolve it in hot water. On cooling, the solu- tion sets into a jelly (gelatinisation). 3. Add a few drops of acetic acid to some saliva. A stringy precipitate of mucin is formed. 4. 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 solu- tion of the interstitial or ground substance by the lime water. Take some of the lime-water extract and add acetic acid. A precipitate of mucin is obtained. The fibres themselves consist of collagen, which yields gelatin on boding. Vitreous humour or the Whartonian jelly of the umbilical cord is much richer in ground substance than tendon, anil, if treated in the same way. a much larger yield of mucin is obtained. The Proteids are the most important substances that occur in animal and vegetable organisms ; none of the phenomena of life occur without their presence ; and though it is impossible to state positively that they occur as such in living protoplasm, they are invariably obtained by subjecting living structures to analytical processes. Proteids 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 differences in chemical and physical properties. They all possess, however, certain common chemical reactions, and are united by a close genetic relationship. The various proteids differ a good deal in elementary composition Hoppe-Seyler gives the following percentages : — C H N S O From 51-5 69 152 0-3 20-9 To 54-5 73 17-0 20 23-5 We are, however, not acquainted with the constitutional formula of proteid substances. There have been many theories on the subject, but practically all that is known with certainty is that many different substances may be obtained by the decomposition of pro- teids. How they are built up into the proteid molecule is unknown. The decompositions that occur in the body are, moreover, differenl from those which can be made to occur in the laboratory; hence the conclusion that living protoplasm differs somewhat from the non-liv- ing proteid materia] obtainable from it. 22 ESSENTIALS OF CHEMICAL PHYSIOLOGY (1) In the body. Carbonic acid, water, and urea1 are the chief final products. Glycocine, leucine, creatine, uric acid, ammonia &c> are probably intermediate products. Carbohydrates (glycogen) and fats may also originate from proteids. (2) Outside the body. Various strong reagents break up proteids into ammonia, carbonic acid, amines, fatty acids, amido-acids like leucine, arginine, and glycocine, and aromatic compounds like tyrosine. TESTS FOR PROTEIDS Solubilities. — All proteids are insoluble in alcohol and ether. Some are soluble in water, others insoluble. Many of the latter are soluble in weak saline solutions. Some are insoluble, others soluble in concentrated saline solutions. It is on these varying solubilities that proteids are classified. All proteids are soluble with the aid of heat in concentrated mineral acids and alkalis. Such treatment, however, decomposes as well as dissolves the proteid. Proteids are also soluble in gastric and pancreatic juices ; but here, again, they undergo a change, being converted into a hydrated variety of proteid of smaller molecular weight called peptone. The intermediate substances formed in this process are called proteoses or albumoses. Commercial peptone con- tains a mixture of proteoses and true peptone. Heat Coagulation. — Many of the proteids which are soluble in water or saline solutions are rendered insoluble when those solutions are heated. This is true for most of the proteids that occur in nature. The solidifying of white of egg when heated is a familiar instance of this. The temperature of heat coagulation differs in different pro- teids : thus myosinogen and fibrinogen coagulate at about 56° C. ; serum albumin and serum globulin at about 75° C. The proteids which are coagulated by heating their solutions come for the most part under two classes — the albumins and the globulins. The full distinction between these we shall see immediately. We may, however, state here that the albumins are soluble in distilled water; the globulins are not, but require salts to hold them in solution. Indiffusibility. — The proteids (peptones excepted) 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 very largely used (see figs. 7 and 8). 1 Eecent research has shown that urea can be also obtained from proteids by analytical methods outside the body (see under Ueinb). THE PROTEIDS 2S Proteids may thus be separated from diffusible (crystalloid) sub- stances like salts, but the process is a somewhat tedious one. If some serum or white of egg is placed in a dialyser, and distilled water outside, the greater amount of the salts passes into the water through the membrane ; the two proteids, albumin and globulin, remain inside. The globulin is, however, precipitated, as the salts which previously kept it in solution have been removed. The terms diffusion and osmosis should be distinguished from each other. If water is carefully poured on the surface of a solution of any substance, this substance gradually spreads through the water, and the composition of the mixture becomes uniform in time. The time occupied is short for substances like sodium chloride, and long for substances like albumin. The Fn;. 7. — Dialyser. The lower opening of the bell jar sus- pended in water is tightly covered with parchment paper. The fluid to be dialysed is placed within this vessel ; the crystalloids pass out into the distilled water outside through the parchment paper. FIG. 8.— In this form of dialyser the substance to be dialysed i- placed within the piece of tubing suspended in the larger vessel of water. The tubing is made of parchment paper. phenomenon is called diffusion. If the solutions are separated by a membrane the term osmosis is employed. Crystallisation. — Haemoglobin, the red pigment of the blood, is a proteid substance, and is crystallisable (for further details, see The Blood). Like other proteids it has an enormously large molecule ; though crystalline, it is not crystalloid in Graham's sense of that term. Blood pigment is not the only crystallisable proteid. Long ago crystals of proteid (globulin or vitellin) were observed in the aleurone grains of many seeds, and in the similar proteid occur- ring in the egg-yolk of some fishes and amphibians. By appropriate methods these have been separated and re-crystallised. Further, egg-albtimin itself has been crystallised. If a solution of white of egg 24 ESSENTIALS OF CHEMICAL PHYSIOLOGY is diluted with half its volume of saturated solution of ammonium sulphate, the globulin present is precipitated and is removed by nitration. 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, either aggregated or separate, make their appearance (Hofmeister). Crystallisation is much more rapid and perfect if a little acetic acid is added (Hopkins). Serum albumin has also been similarly crystal- lised (Givrber). Action on Polarised Light. — All the proteids are levo-rotatory, but the amount of rotation they produce varies with the kind of proteid. Colour Reactions. — The principal colour reactions : (1) the xanthoproteic ; (2) Millon's ; (3) the violet colour with copper sulphate and caustic potash, have been already given in the heading to this lesson. The first two colour reactions depend on the presence of an aromatic radicle in the proteid molecule. Peptones behave differently from the native proteids in this last test. They give a rose-red colour instead of a violet, if only a trace of copper sulphate is used. The albumoses act in this respect like the peptones. This rose-red colour is also given by the substance called biuret ; ] hence the test is called the biuret reaction. Precipitants of Proteids. — Proteids are precipitated by a large number of reagents ; the peptones and albumoses are exceptions in many cases, and will be considered separately afterwards (see Lesson VI.) Solutions of the proteids are precipitated by — 1. Strong acids, like nitric acid. 2. Picric acid. 3. Acetic acid and potassium ferrocyanide. 4. Acetic acid and excess of neutral salts like sodium sulphate. 5. Salts of the heavy metals, like copper sulphate, mercuric chloride, lead acetate, silver nitrate, &c. 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 proteids, be carefully distinguished. The term 1 Biuret is formed by heating solid urea ; ammonia passes off and leaves biuret, thus : — 2CON2H4- NH? =C,0.,N3H5 [urea] [ammonia] [biuret] THE PROTEIDS 25 coagulation is used when an insoluble proteid (coagulated proteid) is formed from a soluble one. This may occur — 1. When the proteid is heated — heat coagulation. 2. Under the influence of a ferment : for instance, when a curd is formed in milk by rennet or a clot in shed blood by the fibrin ferment — ferment coagulation. 3. When an insoluble precipitate in produced by the addition of certain reagents (nitric acid, picric acid, tannin, &c.) There are, however, other precipitants of proteids in which the precipitate formed is readily soluble in suitable reagents, like saline solution, and the proteid continues to show its typical reactions. This precipitation is not coagulation. Such a precipitate is produced by saturation with ammonium sulphate. Certain proteids, 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 proteids but peptone. The globulins are precipitated by certain salts, like sodium chloride and magnesium sulphate, which do not precipitate the albumins. The precipitation produced by alcohol is peculiar in that after a time it becomes a coagulation. Proteid freshly precipitated by alcohol is readily soluble in water or saline media ; but after it has been allowed to stand some weeks under alcohol it becomes more and more insoluble. Albumins and globulins are most readily rendered insoluble by this method ; albumoses and peptones are apparently never rendered insoluble by the action of alcohol. This fact is of value in the separation of these proteids from others. CLASSIFICATION OF PROTEIDS Proteids may be of animal or vegetable origin, and both animal and vegetable proteids may be subdivided in the same way. We shall, however, be chiefly concerned with the animal proteids. If we use the term proteid in the widest sense, the first main sub- division of these substances is into A. The simple proteids. B. The compound proteids. c. The albuminoids. D. The protamines. These substances are regarded by Kossel as the simplest proteids, and though it is doubtful whether this view will be ultimately accepted, it will be convenient to consider them ■ We may now take these four classes one by one. 26 ESSENTIALS OF CHEMICAL PHYSIOLOGY A. The Simple Proteids Class I. Albumins. — These are soluble in water, in dilute saline solutions, and in saturated solutions of sodium chloride and mag- nesium sulphate. They are, however, precipitated by saturating their solutions with ammonium sulphate. Their solutions are co- agulated by heat, usually at 70-73° C. The following are instances : — (a) Serum albumin. Not precipitated by ether. (&) Egg albumin. Precipitated by ether if the solution is not alka- line. For the ether test, dilute 5 c.c. with 2 or 3 times its bulk of 01 per cent, sulphuric acid, add ether and shake briskly. (c) Lact- albumin (see Milk). Class II. Globulins. — These are insoluble in water, soluble in dilute saline solutions, and insoluble in concentrated solutions of neutral salts like sodium chloride, magnesium sulphate, and am* monium sulphate. A globulin dissolved in a dilute saline solution may therefore be precipitated — 1. By removing the salt — by dialysis (see p. 23). 2. By increasing the amount of salt. The best salts to employ are ammonium sulphate (half saturation) or magnesium sulphate (complete saturation). The globulins are coagulated by heat; the temperature of heat coagulation varies considerably. The following are instances : — (a) Fibrinogen , ,-,\ a i i r i i i r \ - m blood plasma. (b) Serum globulin (paraglobuim) ) L (c) Egg globulin in white of egg. (d) Myosinogen in muscle. (e) Crystallin in the crystalline lens. If we compare together these two important classes of proteids, we find that they all give the same general tests, that all are coagu- lated by heat, but that they differ in solubilities. This difference in solubility may be stated in tabular form as follows : — Reagent Albumin Globulin Water ...... soluble insoluble Dilute saline solution soluble soluble Saturated solution of magnesium sul- phate or sodium chloride soluble insoluble Half saturated solution of ammo- nium sulphate .... soluble insoluble Saturated solution of ammonium sulphate ..... insoluble insoluble THE PEOTEIDS 27 Glass III. Proteoses ) These products of digestion will be studied Class IV. Peptones I in Lessons VI. and VII. Class V. Coagulated Proteids. — There are two subdivisions of these : — (a) Proteids in which coagulation has been produced by heat ; they are insoluble in water, saline solutions, weak acids, and weak alkalis ; soluble after prolonged boiling in concentrated mineral acids ; dissolved by gastric and pancreatic juices, they give rise to peptones. (b) Proteids in which coagulation has been produced by fer- ments : — i. Fibrin (see Blood). ii. Myosin (see Muscle). hi. Casein (see Milk). Tables illustrating the methods of testing for and separating pro- teids will be found at the end of this elementary course. Albuminates Albuminates are compounds of proteid (albumin or globulin) with mineral substances. Thus, if a solution of copper sulphate is added to a solution of albumin, a precipitate of copper albuminate is obtained. Similarly, by the addition of other salts of the heavy metals, other metallic albuminates are obtainable. The albuminates which are obtained by the action of dilute acids and alkalis on either albumins or globulins are of considerable physiological interest because they are formed during digestion, and it is to these we shall chiefly confine our attention. The general properties of the acid -albumin, or syntonin, and the alkali-albumin, which are thereby respectively formed will be gathered from the practical exercise which stands at the head of this lesson. They are insoluble in pure water, but are soluble in either acid or alkali, and are precipitated by neutralisation unless disturbing influences like the presence of sodium phosphate are present. It may also be added that, like globulins, they are precipitated by saturation with such neutral salts as sodium chloride and magnesium sulphate. If dis- solved in acid or alkali, they are not coagulated by heat. A variety of alkali albumin (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 Lieberkiihn's jell)'. A similar jelly is formed by adding strong acetic acid to un- diluted egg-white. The halogens (chlorine, bromine and iodine) also form albumi- -., and may be used for the precipitation of proteids. 28 ESSENTIALS OF CHEMICAL PHYSIOLOGY B. The Compound Proteids The compound proteids are compounds of albuminous substances with other organic materials, which are as a rule also of complex nature. They may be divided into the following groups : — 1. Haemoglobin and its allies. These are compounds of proteid with an iron-containing pigment, and will be fully considered under Blood. 2. Gluco-proteids. These are compounds of proteids with members of the carbohydrate group. This class includes the mucins, and substances allied to the mucins, called mucoids. Mucin is a widely-distributed substance, occurring in epithelial cells, or shed out by them (mucus, mucous glands, goblet cells) ; and in connective tissue where it forms the chief constituent of the ground substance or intercellular material. The mucin obtained from different sources varies in composition and reactions. There are probably several mucins : they all agree in the following points : — (a) Physical character. Viscid and tenacious. (b) Precipitability from solutions by acetic acid : they all dissolve in dilute alkalis like lime water. (c) They are all compounds of a proteid with a carbohydrate called animal gum, which by treatment with dilute mineral acid can be hydrated into a reducing but non-fermentable sugar, the nature of which is at present uncertain. The mucoids differ from the mucins either in being non-precipitable from alkaline solutions by acetic acid, or in being readily soluble in excess of acetic acid. One of these, called ovo -mucoid, is found in white of egg, and others called pseuclo-mucin and para-mucin are sometimes found in the fluid of ovarian cysts, and dropsical effusions. Dr. Pavy has shown that a small quantity of a similar carbo- hydrate can be split off from various other proteids, which we have already classified as simple proteids. 3. Nucleins and nucleo-proteids. — These are compounds of proteid with a complex organic acid called nucleic acid, which contains phosphorus. Nucleo-proteids. — Compounds of proteids with nuclein. They are found in the nuclei and protoplasm of cells. Caseinogen of milk and vitellin of egg-yolk are similar substances. In physical charac- THE PEOTEIDS 29 ters they often closely simulate mucin ; in fact, the substance called mucin in the bile is in some animals a nucleo-proteid. They may be distinguished from mucin by the fact that they yield on gastric digestion not only peptone but also an insoluble residue of nuclein which is soluble in alkalis, is precipitable by acetic acid from such a solution, and contains a high percentage (10-11) of phosphorus. Some of tbe nucleo-proteids also contain iron, and it is probable that the normal supply of iron to the body is contained in the nucleo- proteids, or hsematogens (Bunge), of plant and animal cells. The relationship of nucleo-proteids to the coagulation of the blood is described under Blood. Nucleo-proteids may be prepared from cellular structures like testis, thymus, kidney, &c, by two methods : — 1. Woold ridge's method. — The organ is minced and soaked in water for twenty-four hours. Acetic acid added to the aqueous extract precipitates the nucleo-proteid, or, as Wooldridge called it tissue fibrinogen. 2. Sodium chloride method. — The minced organ is ground up in a mortar with solid sodium chloride ; the resulting viscous mass is poured into excess of distilled water, and the nucleo-proteid rises in strings to the _.. ^ Z7^ top of the water. ^'-k'*'vx^1^i^ S1^ The solvent usually employed for a a ^y£.J$pfr<%%££ nucleo-proteid, whichever method it is ^^^Itr^-^'^^^^y^ prepared by, is a 1 per cent, solution of f &^***\^"n^S "{h sodium carbonate. ^ta^vS^W^^ Nuclein is the chief constituent of ^ilsShK cell-nuclei. Its physical characters are ,, • i-i -i ., -,.«. FlG.9. — Diagram of a cell : p. proto- sometnmg like mucin, but it differs plasm composed of spongiopiasm i • n • l ■ • i • i a"'l hyaloplasm ; //, nucleus with chemically in containing a high percentage intranuclear network of chroma- of phosphorus. Nuclein is identical with Safer? " : and "'' nucleolu& the chromatin of histologists (see fig. 9). On decomposition nuclein yields a complex organic acid called nucleic acid, together with a variable amount of proteid. Nucleic acid on decomposition yields phosphoric acid and various bases of the xanthine group. Some forms of nuclein, called pseudo-nuclein, such as are obtained from casein and vitellin, differ from the true nucleins in not yielding these xanthine or, as they are sometimes termed, alloxuric bases. Further particulars concerning the alloxuric bases will be given under Uric Acid, to which they are closely related. The following diagrammatic way of representing the decomposi- 30 ESSENTIALS OF CHEMICAL PHYSIOLOGY tion of nucleo-proteid will assist the student in remembering the relationships of the substances we have just been considering : — Nucleo-proteid Proteid Nuclein Proteid Nucleic acid .j- I i i P205 Alloxuric bodies The nuclein obtained from the nuclei or heads of the spermatozoa consists of nucleic acid without any proteid admixture. In fishes' spermatozoa, however, the nucleic acid is united to protamine, the chemical properties of which we shall be considering immediately. C. The Albuminoids The albuminoids form a heterogeneous group of substances which, though similar to the proteids in many particulars, differ from them in certain other points. The principal members of the group are the following : — 1. Collagen, 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.1 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. It gives most of the proteid colour tests. Many observers state, however, that it contains no sulphur. On digestion it is like proteid converted into peptone-like substances, and is readily absorbed. Though it will replace in diet a certain quantity of proteid, acting as what is called a ' proteid- sparing ' food, it cannot altogether take the place of proteid as a food. Animals fed on gelatin instead of proteid waste rapidly. Chondrin, 1 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, lacuna?, and canaliculi, and proteids 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. THE PROTEIDS 31 the very similar substance obtained from hyaline cartilage, appears to be a mixture of gelatin with mucinoid materials. 4. Elastin. — This is the substance of which the yellow or elastic fibres of connective tissue are composed. It is a very insoluble material. The sarcolemma of muscular fibres and certain basement membranes are very similar. 5. 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 proteids in its high percentage of sulphur. A similar substance, called neurokeratin, is found in neuroglia and nerve fibres. In this connection it is inter- esting to note that the epidermis and the nervous system are both formed from the same layer of the embryo — the epiblast. 6. Chitin and similar substances found in the exo-skeleton of many invertebrates. D. The Protamines Protamines. —These are basic substances which are combined with nuclein in the heads of the spermatozoa of certain fishes (salmon, sturgeon, &c). They resemble proteids in many of their characters — e.g., they give Piotrowski's reaction and some of the other tests for proteids. They are regarded by Kossel as the simplest proteids. By decomposition in various ways they yield bases containing six atoms of carbon, and called in consequence the hexone bases ; the bases are named lysine, arginine, and histidine. The following equation shows the way in which salmine (the protamine from the salmon) breaks up. C H„N17O6+4H2O=C6H9N3O2+3C6HI4N4O2+06H14N2O2 d ine] [Histidine] [Arginine] [Lysine] Sturine (the protamine from the sturgeon) has a somewhat different composition, but yields the same three bases. C86H69N1907+5H20=C6H9N302+3C6H14N402 + 2G6HUN202 [Histidine] [Arginine] [Lysine] The more complex proteids and albuminoids yield these bases also, and so Kossel considers that all these substances contain a protamine nucleus. The more complex proteids, however, yield . other products of decomposition in addition to these bases, such as leucine (C^H^NCX) and tyrosine (C0HnXO8). It is interesting to note how many of these decomposition products contain six carbon atoms, ;md it reminds one that in the sugars ob- tained from starch there are also six atoms of carbon. 32 ESSENTIALS OF CHEMICAL PHYSIOLOGY LESSON IV FOODS A. Milk. 1. Examine a drop of milk with the microscope. 2. Note the specific gravity of fresh milk with the lactometer ; compare 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 usually neutral or slightly alkaline. 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 from the con- version of caseinogen, the chief proteid in milk, into casein. The casein entangles the fat globules. The liquid residue is termed ivliey. No curdling is produced if the rennet solution is previously boiled, because heat kills ferments. 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 sugar by Trommer's test (see Lesson I.) ; for lactalbumin by boiling, or by Millon's reagent (see Lesson II.) ; and for earthy (that is, calcium and magnesium) phosjjhates by ammonia, which precipitates these phosphates. 8. Fat {butter) may be extracted from the precipitate 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 may also be demonstrated by the black colour produced by the addition of osmic acid to the milk. 9. Caseinogen, like globulins, is precipitated by saturating milk with sodium chloride or magnesium sulphate, and by half saturation with ammonium sulphate, but differs from the globulins hi 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 little water into a stiff dough. Wrap this up in a piece of muslin and knead it under a tap or in a capsule 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 proteid called gluten (identify by xanthoproteic or Millon's reaction). C. Bread contains the same constituents as flour, except that some of the starch has been converted into dextrin and dextrose during baking (most flours, however, contain a small quantity of sugar). Extract bread with cold FOODS 33 water, and test the extract for dextrin (iodine test) and for dextrose (Trommer's test). If hot water is used, starch also passes into solution. D. Meat. — This is our main source of proteid food. Cut up some lean meat into fine shreds and grind these up with salt solution. Filter and test for proteids. THE PRINCIPAL FOOD-STUFFS We can now proceed to apply the knowledge we have obtained of the proteids, carbohydrates, and fats to the investigation of some im- portant foods. We do not actually use as food the various organic proximate principles in the pure condition ; it is necessary that in a suitable diet these should be mixed in certain proportions, and in nature we find them already mixed for us. The chief proximate principles in food are : — 1. Proteids \ 2. Carbohydrates [organic. 3. Fats J 4. Water ) 5. Salts ,- inorganic. In milk and in eggs, which form the exclusive food-stuffs of young animals, all varieties of these proximate principles are present in suit- able proportions. Hence they are spoken of as perfect foods. Eggs, though a perfect food for the developing bird, contain too little carbo- hydrate for a mammal. In most vegetable foods carbohydrates are in excess, while in animal food, like meat, the proteids are predomi- nant. In a suitable diet these should be mixed in proper proportions which must 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 charac- ters : — 1. It must contain the proper amount and proportion of the various proximate principles. 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 proxi- mate principles, but these must be present in a digestible form. As an instance of this many vegetables (peas, beans, lentils) contain even more proteid than beef and mutton, but are not so nutritious, as they are less digestible, much passing off in the fueces unused. The nutritive value of a diet depends chiefly on the amount of carbon and nitrogen it contains. A man doing a moderate amount of work will eliminate, chiefly from the lungs in the form of carbonic acid, from 250 to 280 grammes of carbon per diem. During the same D 34 ESSENTIALS OF CHEMICAL PHYSIOLOGY time he will eliminate, chiefly in the form of urea in the urine, about ] 5 to 18 grammes of nitrogen. These substances are derived from the metabolism of the tissues, and various forms of energy, work and heat being the chief, are simultaneously liberated. During muscular exercise the output of carbon greatly increases ; the increased ex- cretion of nitrogen is not nearly so marked. Taking, then, the state of moderate exercise, it is necessary that the waste of the tissues should be replaced by fresh material in the form of food ; and the proportion of carbon to nitrogen should be the same as in the excre- tions : 250 to 15, or 16-6 to 1. The proportion of carbon to nitrogen in proteid is, however, 53 to 15, or 3-5 to 1. The extra supply of carbon must come from non-nitrogenous foods — viz. fat and carbohydrate. Moleschott gives the following daily diet : — Proteid 120 grammes. Fat 90 Carbohydrate 333 ,, Eanke's diet closely resembles Moleschott's ; it is — Proteid 100 grammes. Fat 100 Carbohydrate 250 ,, In preparing diet tables, such adequate diets as those just given should be borne in mind. The following dietary (from G. N. Steward) 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. Quantity Grammes of Food-stuff " Nitro- gen Cai-- bon Pro- teids Fats Carbo- hydrates Salts Metric English system weights Lean meat 250 grammes 9 oz. 8 33 55 8-5 0 4 Bread 500 18 „ 6 112 40 7-5 245 6-5 Milk 500 f pint 3 35 20 20 25 3-5 Butter 30 „ 1 oz. 0 20 0 27 0 0-5 Fat with meat . 30 1 „ 0 22 0 30 0 0 Potatoes . 450 16 „ 1-5 47 10 0 95 4-5 Oatmeal . 75 3 „ 1-7 20-2 30 10 4 48 2 21 299 135 97 413 FOODS 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 insure the proper supply of nitrogen and carbon. Moreover for adults it is relatively too rich in proteid 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 oil globules, varying in diameter from 0'0015 to 0005 milli- metre. 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 m © (M) e Fin. 11. — a, b, colostrum corpuscles with fine and coarse fat globules respectively ; c, d, e, pale cells iluvoiil of Eat. (lleiileuhaiu.) CoOuc'tG^00 Wio. 10. — Microscopic appearance of milk in the early stage of lactation, snowing colostrum corpuscles. (Yeo.) mammary gland are seen, which contain fat globules in their interior ; they are called colostrum coi'puscles. Reaction and Specific Gravity. — The reaction of fresh cow's milk and of human milk is generally neutral or slightly alkaline. In carnivora the milk is acid. All milk readily turns acid or sour as the result of fermentative change, part of its lactose being transformed into lactic acid (see p. 37). The specific gravity of milk is usually Mined with the hydrometer. That of normal cow's milk varies from 1028 to 1034. When the milk is skimmed the specific gravity owing to the removal of the light constituent, the Eat, to 1033 to L037. in all cases the specific gravity of water with which other knees are compared is taken us 1000. i) 2 36 ESSENTIALS OF CHEMICAL PHYSIOLOGY Composition. — Frankland gives the following table, contrasting the milk of woman, ass, and cow : — Butter (fat) Lactose Salts Woman Ass Cow Per cent. Per cent. Per cent. caseinogen) 2-7 1-7 4-2 ■■■ i . • 3-5 1-8 3-8 5-0 4-5 3-8 0-2 0-5 0-7 Hence, in feeding infants on cow's milk, it will be necessary to dilute it, and add sugar to make it approximately equal to natural human milk. The Proteids of Milk. — The principal proteid in milk is called caseinogen ; this is the one which is coagulated by rennet to form casein. Cheese consists of casein with the entangled fat. The other proteid in milk is an albumin. It is present in small quantities only ; it differs in some of its properties (specific rotation, coagulation temperature, and solubilities) from serum-albumin ; it is called lact- albumin. The Coagulation of Milk. — Eennet is the agent usually employed for this purpose : it is a ferment 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 albumin of the milk. There is also a small quantity of a new proteid called whey- proteid, which differs from caseinogen by not being convertible into casein. It is produced by the decomposition of the caseinogen molecule during the process of curdling. The curd formed in human milk is more finely divided than that in cow's milk ; hence it is more digestible. In feeding children and invalids on cow's milk, the lumpy condition of the curd may be obviated by the addition of lime water or barley water to the milk. Caseinogen itself may be precipitated by acids such as acetic acid; or by saturation with neutral salts like sodium chloride. This, how- ever, 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. The addition of rennet produces coagulation in milk, provided that a sufficient amount of calcium salts is present. If the calcium salts are precipitated by the addition of potassium oxalate, rennet causes no formation of casein. The process of curdling in milk is a double foods 37 one ; the first action due to rennet is to produce a change in easeino- gen ; the second action is that of the calcium salt which precipitates the altered caseinogen as casein. In blood also, calcium salts are neces- sary for coagulation, but there they act in a different way, namely, in the production of fibrin-ferment (see Coagulation of Blood). Caseinogen is often compared to alkali-albumin. The latter, however, does not clot with rennet, and is, unlike caseinogen, readily soluble in excess of acids. Caseinogen is not a globulin, though it is, like globulins, precipitated by neutral salts. It differs from a globulin in not being coagulated by heat. It is a nucleo-proteid ; that is, a compound of a proteid, with the proteid-like but phosphorous rich material called nude in (see p. 28). 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 relation between these varies somewhat, but the proportion is roughly as follows : — Olein, i ; palmitin, £ ; stearin, £ • butyrin, caproin, and caprylin, ,'4. The old statement that each fat globule is surrounded by a film of caseinogen is not now regarded as true by most authorities. Milk also contains small quantities of lecithin, a phosphorised fat ; of cholesterin, an alcohol which resembles fat in its solubilities (see Bile), and a yellow fatty pigment or lipochrome. Milk Sugar or Lactose. — This is a saccharose (C12H2,Ou). Its properties have already been described in Lesson I. p. 13. Souring of Milk. — When milk is allowed to stand, the chief change which it 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 closed sterilised vessels. Equa- tions showing the change produced are given on p. 14. When souring occurs, the acid which is formed precipitates a portion of the casei- nogen. This must not be confounded with the formation of casein from caseinogen which is produced by rennet. There are, however, some bacterial growths which produce true coagidation like rennet. 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 is first inverted, that is dextrose and galactose are formed from e p. 13), and it is from these sugars that alcohol and carbonic acid originate. 38 ESSENTIALS OF CHEMICAL PHYSIOLOGY The Salts of Milk.— The chief salt present is calcium phosphate ; a small quantity of magnesium phosphate is also present. The other salts are chiefly chlorides of sodium and potassium. EGGS In this country the eggs of hens and ducks are those particularly selected as food-stuffs. The shell is made of calcareous matter, especially calcium carbonate. The zvhite 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 proteid in nature. The proteids are albumin, with smaller quantities of egg- globulin (see p. 19) and ovo-mucoid (p. 28). The remainder is made up of sugar (05 per cent.), traces of fats, lecithin and cholesterin, and 06 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 lipochrome), and the other smaller, transparent and almost colourless : these are proteid in nature, con- sisting of the nucleo-proteid called vitellin (pp. 28, 29). Small quanti- ties of sugar, lecithin, cholesterin and inorganic salts are also present. The nutritive value of eggs is high, as they are so readily diges- tible ; but the more an egg is cooked the more insoluble do its proteid 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 cases 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 proteid, and the principal proteid 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 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 : — FOODS 39 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 Proteids and gelatin . 20-0 19-4 19-9 21-6 22-7 18-3 Fat . 1-5 2-9 6-2 2-5 4-1 0-7 Carbohydrate 0-6 0-8 0-6 0-6 1-8 0-9 Salts . 14 1-3 1-1 1-0 1-1 0-8 The large percentage of water in meat should he particularly noted ; if a man wished to take his daily minimum of 100 grammes of proteid entirely in the form of meat, it would be necessary for him to consume about 500 grammes (i.e. a little more than lib.) of meat per diem. FLOUR 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 proteid. Whole flour is made from the whole grain minus the husk, and thus contains not only the white interior but also the harder and browner outer portion of the grain. This outer region contains a somewhat larger proportion of the proteids of the grain. Whole flour contains 1 to 2 per cent, more proteid than the best white flour, but it has the disadvantage of being less readily digested. 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, &c). Gluten does not exist in the flour as such, but is formed on the addition of water from the pre-existing soluble proteids (e.g. glohulins) in the flour. The following table contrasts the composition of some of the more important vegetable foods : — Constituent* HTheat Hice Lentils Peas Potatoes Water 13-6 13-8 12-4 13-1 12-5 14-8 76-0 Proteid 12-4 11-1 10-4 7-9 24-8 23-7 2-0 . 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 20-6 Cellulose • '2-:. 5-8 11-2 o-C) 3-6 7-5 0-7 Mineral salts L-8 2-7 3-0 1-0 2-4 3-1 l-o . 40 ESSENTIALS OF CHEMICAL PHYSIOLOGY 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. 3. Proteid, except in potatoes, is pretty abundant, and especially so in the pulses (lentils, peas, &c). The proteid in the pulses is not gluten, but consists of vitellin and globulin-like substances. In the mineral matters in 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. A ferment 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 dough. White bread contains, in 100 parts, 7 to 10 of proteid, 55 of carbo- hydrates, 1 of fat, 2 of salts, and the rest water. COOKING OF FOOD The cooking of foods is a development of civilisation, and much relating to this subject is a matter of education and taste rather than of physiological necessity. Cooking, however, serves many useful ends : — 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 trichinae. 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 granulose. 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 tbe binding material, the more important elements of the food, such as muscular fibres, are FOODS 41 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 off. Of the two chief methods of cooking, roasting and boiling, the former is the more economical, as by its means the meat is first surrounded with a coat of coagulated proteid on its exterior, which keeps in the juices to a great extent, letting little else escape but the dripping (fat). Whereas in boiling, unless both bouillon and bouilli are used, there is considerable waste. Cooking, especially boiling, renders the proteids more insoluble than they are in the raw state ; but this is counterbalanced by the other advantages that cooking possesses. 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 boiling 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 beef tea and similar meat extracts should not be regarded as foods. They are valuable as pleasant stimulating chunks 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, creatinine, lactic acid, &c.) 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 different ways to avoid monotony. Some can take koumiss ; but a less expensive variation may be introduced in the shape of junkets, which, although well known in the West of England, are comparatively unknown in other parts. The prepara- tion of a junket consists in adding to warm milk in a bowl or dish a small quantity of essence of rennet (Clark's Essence is very good for this purpose) and flavouring material according to taste. The mix- ture 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 m. Soup contains the extractives of meat, a small proportion of the proteids, and the principal part of the gelatin. The gelatin is usually sed by adding bones and fibrous tissue to the stock. It is the 42 ESSENTIALS OF CHEMICAL PHYSIOLOGY presence of this substance which causes the soup when cold to gela- tinise. ACCESSORIES 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, &c.) 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 pro- perty to an alkaloid called theine or caffeine (C8H10N4O2) ; cocoa to the closely related alkaloid, theobromine (C7H8N402) ; coca to coQctine. 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 a valuable food in addition to its stimulating properties ; it contains about 50 per cent, of fat, and 12 per cent, of proteid. 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 proteid, 2 to 4 carbohydrates, and 1 to 1*5 cellulose per cent. The small amount of nutriment in most green foods ac- counts for the large meals made by, and the vast capacity of the •.alimentary canal of, herbivorous animals. 43 LESSON V SALIVA 1. To a little saliva in a test-tube add acetic acid. Mucin is precipitated in stringy flakes. 2. Filter some fresh saliva to separate cells and mucus, and apply the xanthoproteic or Millon's test to the filtrate ; the presence of proteid is shown. 3. Put some 0-5-per-cent. starch solution into two test-tubes. Add some filtered saliva to one of them, and put both in the water-bath at 40° C. After five minutes remove them and test both fluids with iodine and Trornrner's test. The saliva will be found to have converted the starch into dextrin and sugar (maltose). 4. The presence of potassium sulphocyanide (KCNS) in saliva may be shown bj' the red colour given by a drop of ferric chloride. This colour is discharged by mercuric chloride. 5. The reaction of saliva is alkaline to litmus paper. The saliva is the first digestive juice to come in contact with the food : it is secreted by three pairs of salivary glands, the parotid, submaxillary, and sublingual. The secretions from these differ somewhat in composition, but they are mixed in the mouth, the secretion of the minute mucous glands of the mouth and a certain number of epithelial cells and debris being added to it. The so- called ' salivary corpuscles ' are derived either from the glands them- selves or from the tonsils. 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, and in a branch of the glossopharyngeal 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 secretion the cells of the acini are swollen out with granules : after "ion has occurred the cells shrink, owing to the granules having shed out to con tribute to the secretion (see fig. 12). 44 ESSENTIALS OF CHEMICAL PHYSIOLOGY The submaxillary and sublingual glands are called mucous glands ; their secretion contains mucin. Mucin is absent from parotid saliva. The granules in the cells are larger than those of the parotid gland ; tbey are composed of mucinogen, the precursor of mucin (see fig. 13) In 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. 14). COMPOSITION OF SALIVA On microscopic examination of mixed saliva a few epithelial scales from the mouth and salivary corpuscles from the salivary glands are seen. The liquid is transparent, slightly opalescent, of slimy consistency, 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 05 per cent.), and contains no mucin. Mixed saliva contains in man an average of about 05 per cent, of solids : it is alkaline in reaction, due to the salts in it ; and has a specific gravity of 1,002 to 1,006. The solid constituents dissolved in saliva may be classified thus : — / a. Mucin : this may be precipitated by acetic acid. \ b. Ptyalin : an amylolytic ferment. " I c. Proteid : of the nature of a globulin. d. Potassium sulphocyanide. e. Sodium chloride : the most abundant salt. , /. Other salts : sodium carbonate, calcium phosphate ° and carbonate ; magnesium phosphate ; potassium I chloride THE ACTION OF SALIVA The action of saliva is twofold, physical and chemical. The physical use of saliva consists in moistening the mucous membrane of the mouth, assisting the solution of soluble substances SALIVA 45 in the food, and in virtue of its mucin lubricating the bolus of food to facilitate swallowing. Fig. 12. — Alveoli of serous gland : A, loaded before secretion ; B, after a short period of active- secretion ; C, after a prolonged period. (Langley.) Tig. 13. — Mucous cells from a fresh submaxillary gland of dog : a, loaded with mucinogen granules before secretion ; 6, after secretion : the granules are fewer, especially at the attached border of the cell ; a' and V 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 ni.i;; of mucin traversed by a network of protoplasmic eel '.-substance. (Foster, after Langley.) :. -Section of ],;irt of the human submaxillary gland. fHeidenhain.) To the right is a group of mucous alveoli, to the left a group of Beroue ;il\ eoli. The chemical action of saliva is due to its active; principle, ptyalin. This substance belongs to the class of u/norgarrised ferments 46 ESSENTIALS OF CHEMICAL PHYSIOLOGY or enzymes, and to that special class of unorganised ferments which are called amylolytic (starch splitting) or diastatic (resembling diastase, the similar ferment in germinating barley and other grains). A general description of ferments will be found at the end of this lesson. The starch is first split into dextrin and maltose ; the dextrin is subsequently converted into maltose also : this occurs more quickly with erythro-dextrin, which gives a red colour with iodine, than in the other variety of dextrin called achroo-dextrin, which gives no colour with iodine. Brown and Morris give the following equa- tion : — 10(C6H10O5)„ + 4«H20 [starch] [water] = 4^C12H22Ou + (C6H10O6). + (C6H10O5)„ [maltose] [achroo-dextrin] [erythro-dextrin] 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 conversion of starch into sugar by saliva in the stomach con- tinues for 15 to 30 minutes ; the hydrochloric acid which is poured out by the gastric glands first neutralises the saliva and combines with the proteids 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. FERMENTS 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 'fermentation.' The agent yeast which produces this is called the ferment. Microscopic investigation shows that yeast is composed of minute rapidly growing unicellular organisms (torulae) belonging to the ""SSSS2S35S fungus group of plants, ding, between which rp^g souring 0f milk, the transformation of are some bacteria <-> ' CYeo's ' Physiology.') urea into ammonium carbonate in decomposing urine, and the formation of vinegar (acetic acid) from alcohol are pro- duced by the growth of very similar organisms. The complex series SALIVA 47 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 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 tem- '• — Typical form- 'if Schizomycetes (after Zopf) : a, Micrococcus; 6, macrococcus ; c, bao- •ium ; aneta£ or oxyntic {i.e. acid forming) cells. The pyloric glands, in the pyloric half of the stomach, have long ducts and short tubules lined with cubical cells. There are no parietal cells. The central cells of the cardiac glands and the cells of the pyloric glands are loaded with granules. During secretion they discharge their granules, those that remain being chiefly situated near the lumen, leaving in each cell a clear outer zone (see fig. 20). These are the cells that 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 discharged into the lumen of Flg. 20. — A cardiac gland of simple form from the bat's stomach. Osmic acid preparation (Lang- ley) : r, Columnar epithelium of the surface ; n, neck of the gland, with central and parietal cells ;/,base or fundus, occupied only by principal or central cells, which exhibit the granules ac- cumulated towards the lumen of the gland. PEPTIC DIGESTION 53 the gland. The most important substance in a digestive secretion is the ferment. In the case of the 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 shall find a similar ferment precursor in the cells of the pancreas, and the term zymogen is applied to these ferment precursors. The zymogen in the gastric cells is called pepsinogen. The rennet-fer- ment or rennin that causes the curdling of milk is distinct from pepsin, and is preceded by another zymogen ; it is, however, formed by the same cells. The parietal cells undergo merely a change of size during secre- tion ; at first they are somewhat enlarged, and after secretion they shrink. They are also called oxyntic cells, because it is believed that they secrete the hydrochloric acid of the juice. Heidenham succeeded in making in one dog a cid-de-sacoi the fundus, in another of the pyloric region of the stomach ; the former secreted a juice containing both acid and pepsin ; the latter, parietal cells being absent, secreted a viscid alkaline juice containing pepsin. The for- mation 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 many theories advanced as to how this is done, Maly's is, on the whole, the most satisfactory. He considers that the acid originates by the interac- tion of the calcium chloride with the disodium hydrogen phosphate of the blood, thus : — 2Na2HP04 + 3CaCl2= Ca,(P04)2 + 4NaCl + 2HC1 [disodium [calcium [calcium [sodium [hydro- hydrogen chloride] phosphate] chloride] chloric phosphate] acid] or more simply by the interaction of sodium chloride and sodium di- hydrogen phosphate, as is shown in the following equation : — NaIi2P04 + NaCl=Na2HP04 + HCl nadi- [sodium [disodium [hydro- liydrogen chloride] hydrogen chloric phosphate] phospl acid] The sodium dihydrogen phosphate in the above equation is pro- bably derived from the interaction of the disodium hydrogen phos- pl i ate and the carbonic acid of the blood, thus : — Na2HPO , + C02 + H,0=NaHCO, + NaH.PO ., But as Prof* ' i»mgee has pointed out, these reactions can hardly be considered to occur in the bio ally but rather in 54 ESSENTIALS OF CHEMICAL PHYSIOLOGY the oxyntic cells, which possess the necessary selective powers in reference to the saline constituents of the blood, and the hydro- chloric acid, as soon as it is formed, passes into the secretion of the gland in consequence of its high power of diffusion . COMPOSITION OF GASTRIC JUICE The following table gives the percentage composition of the gastric juice of man and the dog : — Constituents Human Dog Water .... 99-44 97-30 Organic sul pepsin) HCl . )stanc ,es (c rieny 0-32 0-02 1-71 0-30 CaCl, . 0-006 0-06 NaCl . 0-14 0-25 KOI . 0-05 0-11 NH4C1 — 0-05 Ca3(P04), Mg3(P04)2 FeP04 1 j>0-01 0-17 0-02 0-008 One sees from this how much richer in all constituents the gastric juice of the dog is than that of man. Carnivorous animals have always a more powerful gastric juice than other animals ; they have more work for it to do ; but the great contrast seen in the table is, no doubt, partly due to the fact that the persons from whom it has been possible to collect gastric juice have been invalids. 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. Pepsin stands apart from nearly all other ferments by requiring an acid medium in order that it may act. Probably a compound of the two substances called pepsin-hydrochloric 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 appears to be derived by fermentative processes from the food. Some recent very valuable work performed by Pawlow on dogs has enabled him not only to show that the secretory fibres for the gastric glands are contained in the vagus or pneumo-gastrie nerves, but also to obtain a gastric juice free from any admixture with saliva or food. The main facts in relation to this pure juice are as PEPTIC DIGESTION 55 follows : — It is clear and colourless ; it has a specific gravity of 1003 to 1006. It is feebly dextro-rotatory, gives no biuret reaction, but gives the ordinary proteid reactions. It contains from 04 to 06 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 ; 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 (Kuhne). Elementary analysis gave the following results : — Pepsin precipitated by cold — ■ Precipitated by Am.SOj Carbon . 50*73 per cent. 50-37 Hydrogen . 7-23 6-88 Chlorine . . 1-01 to 1-17 0-89 Sulphur . . 0-98 1-34 Nitrogen . not estimated 14-55 to 15-0 Oxygen . . the remainder. the remainder. THE ACTION OF GASTRIC JUICE The principal actions of the gastric juice have been already practically studied : the action of pepsin in converting the proteids of the food into the diffusible peptones is its chief action. The curdling of milk by rennet will be found described in Lesson IV. There is a still further action — that is, the gastric juice is anti- septic ; putrefactive processes do not normally occur in the stomach, and the organisms that produce such processes, many of which are swallowred with the food, are in great measure destroyed, and thus the body is protected from them. The acid is the agent in the juice that possesses this power. The formation of peptones is a process of hydrolysis ; peptones be formed by other hydrating agencies like superheated steam and heating with dilute mineral acids. There are certain inter - mediate steps in this pi'ocess ; the intermediate substances are called propeptones or proteoses. The word ' proteose ' is the best to em- ploy : it includes the albumoses (from albumin), globuloses (from globulin), vitelloses (from vitellin), &c. Similar substances are also formed from gelatin (^elatoses) and elastin (elastoses). Another intermediate step in gastric digestion is called para- peptone : this is acid-albumin or syntonin ; it also, though with somo difficulty, is converted into peptone. In classifying the products of bion it will bi ient to take albumin as our example, but we must remember that globulin, myosin, and all the other 56 ESSENTIALS OF CHEMICAL PHYSIOLOGY proteids form corresponding products. The products of digestion may be classified, according to their solubilities, as follows : — 1. Parapeptone or acid-albumin, | (a) Proto-albumose 1 ^^^^^"^'^ 2. Propeptone •] Cb) Hetero-albumose j J first. 1(c) Deutero-albumose 3. Peptone. The albumin molecule is considered by Kuhne to be made up of two parts, called respectively hemi-albumin and anti-albumin. The former yields ultimately hemipeptone, the latter antipeptone. The intermediate albumoses have similar names : — Albumin Hemi-albumin Anti-albumin Hemi-albumose Anti-albumose and acid-albumin Hemipeptone Antipeptone The hemipeptone differs from the antipeptone in the manner it is affected by the prolonged action of the next digestive juice, the pan- creatic secretion ; hemipeptone yields leucine and tyrosine, antipeptone does not. This theory will be fully discussed in our next lesson. Peptones. — These are the final products of the action of gastric juice on native proteids. They are soluble in water, are not coagulated by heat, and are not precipitated by nitric acid, copper sulphate, ammonium sulphate, and a number of other precipitants of proteids. They are precipi- tated but not coagulated by alcohol. They are also precipitated by tannin, picric acid, potassio-mercuric iodide, phospho-molybdic acid, and phospho-tungstic acid. They give the biuret reaction (rose-red, with a trace of copper sulphate and caustic potash or soda). Peptone is readily diffusible through animal membranes. The utility of the formation of diffusible substances during digestion is obvious. Proteoses. — These are the intermediate products in the hydration of native proteids into peptones. They are not coagulated by heat ; they are precipitated but not coagulated by alcohol ; like peptone they give the biuret reaction. They are precipitated by nitric acid, the precipitate being soluble on PEPTIC DIGESTION 57 heating, and reappearing when the liquid cools. This last is a distinctive property of proteoses. They are slightly diffusible. The folio-wing table will give us at a glance the chief characters of peptones and proteoses in contrast with those of native proteids like albumin and globulin : — Action of Variety of proteid Action of heat Action of alcohol Action of nitric acid Action of ammonium sulphate copper sulphate and caustic potasli Diffusi- bility Albumin Coagulated Precipitated, then coagu- lated Precipitated in the cold; not readily soluble on heating Precipitated by complete saturation Violet colour Nil Globulin Ditto Ditto Ditto Precipitated by half satu- ration ; also precipitated by satura- tion with MgS04 Ditto Ditto Proteoses ot I Corpuscles J 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 coat 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 (buffy 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. 3. Injury to the vessel walls. 4. Agitation. 5. Addition of calcium salts. 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 like sodium sulphate or magnesium sulphate. 3. Contact with the living vascular walls. 4. Contact with oil. 5. Addition of a soluble oxalate (e.g. potassium oxalate) : this precipitates the calcium necessary for coagulation as insoluble calcium oxalate. THE BLOOD 81 6. Injection of commercial peptone (which consists chiefly of preteoses) into the circulation of the living animal. 7. Addition of leech extract. This acts in virtue of a proteose it contains. The theory generally received which accounts best for the coagu- lation of the blood is that of Hammarsten, and it may be briefly stated as follows : — When blood is within the vessels one of the constituents of the plasma , a proteid of the globulin class called fibrinogen, exists in a soluble form. When the blood is shed the fibrinogen molecule is split into two parts : one part is a globulin, which remains in solution, the other is the insoluble material fibrin. This change is brought about by the activity of a special un- organised ferment called the fibrin ferment. TJiis ferment does not exist in healthy blood contained in health// blood vessels, but is one of the products of the disintegration of the white corpuscles and blood tablets that occurs when the blood leaves the vessels or comes into contact with foreign matter. To this may be added, as the result of recent research, that a soluble calcium salt is essential for the formation of the ferment ; that the fibrin ferment belongs to the class of nucleo-proteids ; that other nucleo-proteids (Wooldridge's tissue-fibrinogens, see p. 29) obtained from most of the cellular organs of the body produce intravascular clotting when injected into the circulation of a living animal. The fibrin ferment may very conveniently be called thrombin. Like other ferments it is preceded by a mother substance or zymogen which may be called prothrombin ; the action of calcium salts is to convert prothrombin into thrombin. We may therefore represent the process of clotting in the following tabular way : In the plasma a proteid substance From the colourless corpuscles a exists, called — nucleo-proteid is shed out, called — Fibrinogen. Prothrombin. By the action of calcium salts prothrombin is converted into I ibrin- ferment, or Thrombin. Thrombin acts on fibrinogen in such a way that two new substances are formed. One of these ie unimportant, viz. The other is important, viz. a globulin which remains in solution. Fibein, which entangles the cor- Its amount is wry small. puscles and so forms the Clot. Q ■'82 ESSENTIALS OF CHEMICAL PHYSIOLOGY THE PLASMA AND SERUM The liquid in which the corpuscles float may be obtained by- employing one or other of the methods already described for pre- venting 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 effected more thoroughly by the use of a centrifugal machine (see fig. 60, p. 151). On counteracting the influence which has prevented the blood from ■coagulating, the plasma then itself coagulates. Thus plasma obtained by the use of cold clots on warming gently ; plasma which has been decalcified by the action of a 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 fibrin ferment being necessary in most cases ; where co- agulation occurs without the addition of fibrin ferment, 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 fibrin ferment or liquids like serum that contain fibrin ferment 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 re- moved 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 1,026 to 1,029. Its chief constituents may be enumerated as follows : — 1,000 parts of plasma contain — ■ Water 902-90 Solids .... Proteids : 1, yield of fibrin 2, other proteids Extractives (including fat) Inorganic salts ... 9710 4-05 78-84 5-66 8-55 In round numbers plasma contains 10 per cent, of solids, of which 8 per cent, are proteid in nature. THE BLOOD 83 Serum contains the same three classes of constituents — proteids, extractives, and salts. The extractives and salts are the same in the two liquids. The proteids differ, as is shown in the following table : — Proteids of Plasma Fibrinogen Serum globulin Serum albumin Proteids of Serum Serum globulin Serum albumin Fibrin ferment 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 Eespiration). We may now consider one by one the various constituents of the plasma and serum. A. Proteids — Fibrinogen. — This is the substance acted on by fibrin ferment. It yields, under this action, an insoluble product called fibrin, and a soluble proteid of the globulin class. Fibrinogen is a globulin. It differs from serum globulin, and may be separated from it by the fact that half-saturation with sodium chloride precipitates 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 proteids of the plasma (see table on preceding page). Serum globulin and serum albumin. — These substances are con- sidered in the practical exercises at the head of this lesson ; see also Lesson II. Fibrin ferment. — Schmidt's method of preparing it is to take serum and add excess of alcohol. This precipitates all the proteids, fibrin ferment included. 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 fibrin ferment, which is not so easily coagulated by alcohol as the other proteids are. B. Extractives. — These are non-nitrogenous and nitrogenous. The non-nitrogenous are sugar (012 per cent.), fats, soaps, cholesterin ; and the nitrogenous are urea (002 to O04 per cent.), and still smaller quantities of uric acid, creatine, creatinine, xanthine, and hypoxanthine. C. Salts. — The most abundant salt is sodium chloride: it consti- tutes between 60 and 90 per cent, of the total mineral matter. sium chloride is present in much smaller amount. It consti- tutes about 4 per cent, of the total ash. The other salts are phos- phates and sulphates. o2 84 ESSENTIALS OF CHEMICAL PHYSIOLOGY Schmidt gives the following table 1,000 parts of plasma yield — Mineral matter Chlorine . S03 . P205 Potassium Sodium Calcium phosphate Magnesium phosphate 8-550 8-640 0-115 0-191 0-323 3-341 0-311 0-222 THE WHITE BLOOD CORPUSCLES These corpuscles are typical animal cells, Their nucleus consists of nuclein, their cell-protoplasm yields proteids belonging to the nucleo-proteid and globulin groups. The nucleo-proteid obtained from them is the zymogen of the fibrin ferment, the addition of a calcium salt converting it into the ferment. The protoplasm, of these cells often contains small quantities of fat 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 corpuscle. The method of enumeration of the corpuscles is described in the Appendix. They vary in size and structure in different groups of vertebrates. In mammals they are biconcave (except in the camel tribe, where they are biconvex) non-nucleated discs, in man averaging g-^ou mch in diameter ; during foetal life nucleated red corpuscles are, however, found. In birds, reptiles, amphibians, and fishes they are biconvex oval discs with a nucleus : they are largest in the amphibia. Water causes the corpuscles to swell up, and dissolves out the red pigment (oxyhemoglobin), leaving a globular colourless stroma. Salt sohttion causes the corpuscles to shrink : they become crenated or wrinkled. The action of water and salt solution suggests the existence of a membrane on !fC~^fL the surface of the corpuscles through which sive effects of osmosis takes place, but the existence of water on a red biood corpuscle; such a membrane is still a matter of dis- /, a red corpuscle crenated by salt solution ; sr, action of tannin on cussion. If there is no actual membrane, a red corpuscle. the outer denser portion of the stroma plays the role of one during osmotic phenomena. Dilute alkalis (0-2 per cent, potash) dissolve the corpuscles. Dilute acids (1 per cent. d O THE BLOOD 85 acetic acid) act like water, and in nucleated corpuscles render the nucleus distinct. Tannic acid causes a discharge of haemoglobin from the stroma, but this is immediately altered and precipitated. It remains adherent to the stroma as a brown globule, consisting probably of hasmatin. Boric acid acts similarly, but in nucleated red corpuscles the pigment collects chiefly round the nucleus, which may then be extruded from the corpuscles. Composition. — 1,000 parts of red corpuscles contain — Water ..... 688 parts organic .... 303 "88 ,, Solids ... inorganic . . . oIJ 100 parts of dried corpuscles contain — Proteid Haemoglobin Lecithin Cbolesterin . 5 to 12 parts 86 „ 94 „ 1-8 01 The proteid present appears to be identical with the nucleo- proteid of white corpuscles. The mineral matter consists chiefly of chlorides of potassium and sodium, and phosphates of calcium and magnesium. In man potassium chloride is more abundant than sodium chloride ; this, however, does not hold good for all animals. Oxygen is contained in combination with the haemoglobin to form oxyhaemoglobin. The corpuscles also contain a certain amount of carbonic acid (see Bespibation, at the end of this lesson). The pigment of the red corpuscles. — The pigment is by far the most abundant and important of the constituents of the red cor- puscles. It is a substance which gives the reactions of a proteid, but differs from most other proteids in containing the element iron ; it is also readily crystallisable. It exists in the blood in two conditions : in arterial blood it is combined loosely with oxygen, is of a bright red colour, and is called oxyhaemoglobin ; the other condition is the deoxygenated or reduced hemoglobin (better called simply haemoglobin). 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 oxyhaemoglobin also. Haemoglobin is the oxygen-cai'rier of the body, and it may be called a respiratory pigment. Crystals of oxyhaemoglobin may be obtained with readiness from the blood of such animals asthe rat, guinea-pig, or dog; with difficulty 86 ESSENTIALS OF CHEMICAL PHYSIOLOGY 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 oxyhaemoglobin crystallises out from the solution so formed. 2. Microscopical preparations may also be made by Stein's method, which consists in using Canada balsam instead of water in the above experiment. 3. On a larger scale the crystals may be obtained by mixing the blood with one-sixteenth of its volume of ether ; the corpuscles dissolve and the blood assumes a laky appearance. After a period, varying from a few minutes to days, abundant crystals are deposited. The accompanying figures repre- sent 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, fig. 30.— Oxyhemoglobin crystals mngni- hexagonal plates ; and in the hamster, fied : 1, from human blood ; 2, from the r" guinea-pig; 3, squirrel ; 4, hamster. rnombohedra and hexagonal plates. The crystals also contain a varying amount of water of crystallisation : this may in part explain their different crystalline forms and solubilities. Different observers have analysed haemoglobin. They find carbon, hydrogen, nitrogen, oxygen, sulphur, and iron. The percentage of iron is 0-4. The amounts of the other elements are variously given, but, roughly, they are the same as in the proteids. We know at present as little of the chemical structure of haemoglobin as of the proteids generally. Oxyhemoglobin may be estimated in the blood (1) by the amount of iron in the ash, or (2) by certain colorimetric methods which are described in the Appendix. On adding an acid or alkali to haemoglobin, it is broken up into two parts, a proteid called globin, and a brown pigment called hcematin, which contains all the iron of the original substance. Globin is a somewhat unique proteid. It is coagulable by heat, THE BLOOD 87 soluble in dilute acids and precipitable from such solutions by ammonia. It closely resembles a substance previously separated from red corpuscles by Kossel and termed by him histone (Schulz). Hsematin is not crystallisable ; according to Hoppe-Seyler its formula is C34H35N4FeO.T ; other observers give different for- mula?. It presents different spectroscopic appearances in acid and alkaline solutions, which we shall study more fully in the advanced course. It also yields several products under the influence of certain reagents, which we shall also again consider in the advanced course. For the present, we will mention only two of these, haemin and haematoporphyrin . Haemin is of great importance, as the obtaining of this substance in a crystalline form is the best chemical test for blood. Haemin crystals, sometimes called, after their discoverer, Teichmann's crystals, are composed of the hydrochloride of haematin. They may be prepared for microscopic examination by boiling a frag- ment of dried blood with a drop of glacial acetic acid on a slide ; on cooling, dark brown plates and prisms belonging to the triclinic system, often in star-shaped clus- ters and with rounded angles (fig. ^rYj ^=* ^*r 31), separate out. i^fO* ^Xr ** ^1 In the case of an old blood- I0 ~7f ^r «**J, x "^ A r v kf "V v /> * T_ -1 7 '•*-* 31. — Hreniin it; rstals magnified, i Preyer. : stain it is necessary to add a ^ il«. v> \^ crystal of sodium chloride in addi- tion. Fresh blood contains suffi- cient sodium chloride in itself. The action of the acetic acid is (1) to split the haemoglobin into haematin and globin ; and (2) to evolve hydrochloric acid from the sodium chloride. The haematin unites with the hydrochloric acid, and thus haemin is formed. The formula for haemin is C35H35N4FeC104 (Morner). Haematoporphyrin is iron-free haematin ; it may be prepared by mixing blood with strong sulphuric acid : the iron is taken out as ferrous sulphate. This substance is also found sometimes in nature : it occurs in certain invertebrate pigments, and may also be found in certain forma of pathological urine. It shows well-marked spectroscopic bands, and so is not identical with the iron-free derivative of haemoglobin called haematoidin which is formed in extravasations of blood in the body (see p. 67). The two substances are possibly isomeric. 88 ESSENTIALS OF CHEMICAL PHYSIOLOGY COMPOUNDS OF HiEMOGLOBIN WITH GASES Haemoglobin forms at least four compounds with gases : — (1. Oxyhaemoglobin. With oxygen j2< Methamoglobin. With carbonic oxide. 3. Carbonic oxide haemoglobin. With nitric oxide. 4. Nitric oxide haemoglobin. These compounds have similar crystalline forms : each probably consists of a molecule of haemoglobin combined with one of the gas in question. They part with the combined gas somewhat readily, and are arranged in order of stability in the above list, the least stable first. Oxyhemoglobin is the compound that exists in arterial blood. Many of its properties have been already mentioned. The oxygen linked to the haemoglobin, which is removed by the tissues through which the blood circulates, may be called the respiratory oxygen of hemoglobin. 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 like hydrogen through the blood, or by the use of reducing agents like ammonium sulphide or Stokes' reagent.1 One gramme of haemoglobin will combine with 1*6 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 haemoglobin. On once more allowing oxygen to come into contact with the haemoglobin, as by shaking the solution with the air, the bright arterial colour returns. 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. It will be first necessary to describe briefly the instrument used. 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 con- stituent colours, which may be allowed to fall on a screen. The 1 Stokes' reagent 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 BLOOD 89 band of colours beginning witb the red, passing through orange, vellow, green, blue, and ending with violet, is called a spectrum : this is seen in nature in the rainbow. It may be obtained artificially by the glass prism or prisms of a spectroscope. Pig. 32. — Diagram of spectroscope. The spectrum of sunlight is interrupted by numerous dark lines crossing it vertically, called Frauenhofer's lines. These are perfectly constant in position, and serve as landmarks in the spectrum. Pig. 33.— Spectroscope : A, collimator with adjustable slit al one (left) end and colliniating lens al the other (right) end ; B, telescope moving on graduated arc divided Into degrees; 0, prism or combination of prisms; D, tabe for scale; E, mirror for illuminating scale; I', vessel with paraOi down with the flat side towards the reader ; I. long spectro- er of flnid; H, Argand burner; <:, oondenser for concen- iraiint' the light froiu II on the slit, (from a photograph taken by Dr. MacMunn, for McKendrick's ' Physiology.') The more prominent are A, B, and C, in the red; D, in the yellow ; aid P, in the green ; <'< mid If, in the violet. These lines are to certain volatile substances in the solar atmosphere. If the light from binning sodium or its compounds be i 3 spectro- 90 ESSENTIALS OF CHEMICAL PHYSIOLOGY scopically, 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 characteristic lines, but none so simple as sodium. If now the flame of a 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 be 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, occupying 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. Pig. 34. — Arrangement of prisms in direct vision spectroscope. The large form of spectroscope (fig. 32) consists of a tube A, called the collimator, 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 isfocussed by the telescope T. The third tube, D, seen in the next figure (fig. 33), carries a small transparent scale of wave lengths, as in accurate observations the position of any point in the spectrum is given in the 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. 32), or a solution of a coloured substance contained in a vessel with parallel sides (the haematoscope of Herrmann, P in fig. 33), 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 oxyhemoglobin of a certain strength gives two bands between the D and E lines ; haemoglobin gives only THE BLOOD 91 one ; and other red solutions, though to the naked eye similar to oxyhemoglobin, will give characteristic bands in other positions. FIG. 35.— stand for direct vision spectroscope : S, spectroscope ; T, test-tube for coloured substance under investigation. A convenient form of small spectroscope is the direct vision spectroscope, in which, by an arrangement of alternating prisms Pig. 86.— Graphic representations of the amount of absorption of light bj Boiuti 1 1 oi oxyhemo- globin, (II) of bamoglobin, Oi i 'J'lw shading indicates tlio al> orp tion oft ' Qtagi . ( liollett.) ,\mi and flint glass, placed as in fig. 34, the spectrum is observed by the eye in the same line as the tube furnished with 92 ESSENTIALS OF CHEMICAL PHYSIOLOGY the slit — indeed slit and prisms are both contained in the same tube. Such small spectroscopes may be used for class purposes, and may for convenience be mounted on a stand provided with a gas- burner and a receptacle for the test-tube (see fig. 35). In the examination of the spectrum of small coloured objects, a combination of the microscope and direct vision spectroscope, called the micro-spectroscope, is used. Pig. 37.— 1, Solar spectrum ; 2, spectrum of oxyhemoglobin (037 per cent, solution) ; 3, spectrum of haemoglobin ; 4, spectrum of CO-hsemoglobin ; 5, spectrum of methsemoglobin (concentrated solution). Fig. 36 illustrates a method of representing absorption spectra diagrammatically. The solution was examined in a layer 1 cen- timetre thick. The base line has on it at the proper distances the chief Frauenhofer lines, and along the right-hand edges are percentages of the amount of oxyhemoglobin present in I, of haemoglobin in II. The width of the shadings at each level represents the position and amount of absorption corresponding to the per- centages. The characteristic spectrum of oxyhemoglobin, as it actually appears through the spectroscope, is seen in the next figure (fig. 37, spectrum 2). There are two distinct absorption bands between the D and E lines ; the one nearest to D (the a band) is narrower, darker, and has better defined edges than the other (the /3 band). As will be seen on looking at fig. 36, a solution of oxyhemoglobin of THE BLOOD 93 concentration greater than 065 per cent, and less than 085 per cent, (examined in a cell of the usual thickness of 1 centimetre) gives one thick band overlapping both D and E, and a stronger solution only lets the red light through between C and D. A solution which gives the two characteristic bands must therefore be a very dilute one. The one band (y band) of haemoglobin (fig. 37, spectrum 3) is not so- well defined as the « or /3 bands. On dilution it fades rapidly, so that in a solution of such strength that both bands of oxyhemoglobin would be quite distinct the single band of haemoglobin has dis- appeared from view. The oxyhaemoglobin 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 « band is still visible. Methaemoglobin.— This may be produced artificially by adding such reagents as potassium ferricyanide or amyl nitrite to a solution of oxyhaemoglobin ; it may also occur in certain diseased conditions in the urine ; it is therefore of considerable practical importance. It can be crystallised, and is found to contain the same amount of oxygen as oxyhaemoglobin, only combined differently. The oxygen is not removable by the air-pump, nor by a stream of a neutral gas like hydrogen. It can, however, by reducing agents like ammonium sulphide, be made to yield haemoglobin. Methaemoglobin is of a brownish red colour, and gives a characteristic absorption band in the red between the C and D lines (fig 37, spectrum 5). The ferricyanide of potassium or sodium not only causes the conversion of oxyhaemoglobin into methaemoglobin, 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 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 oxyhaemoglobin is discharged. This is at first sight puzzling, because, as just stated, methaemoglobin contains the same amount of oxygen that is present in oxyhaemoglobin. What occurs is that after the oxygen is dis- charged from oxyhaemoglobin, an equal quantity of oxygen takes its place from the reagents added. The oxygen atoms of the methaemo- globin must be attached to a different part of the haematin p from the oxygen atoms of the oxyhaemoglobin, so that the tin group when thus altered loses its power of combining with n and carbonic oxide to form compounds which are dissociable in a vacuum. 94 ESSENTIALS OF CHEMICAL PHYSIOLOGY Dr. Haldane, to whom we owe these interesting results, gives the following provisional equation to represent what occurs : — Hb02 + 4Na3Cy6Fe + 4NaHC03=Hb02 + 4Na4Cy6Fe. [oxyhseino- [sodium ferri- [sodium bicar- [methsemo- [sodium ferro- globin] cyanide] bonate] globin] cyanide] + 4C02 + 2H20 + 02. [carbonic [water] [oxygen] acid] Carbonic Oxide Haemoglobin may be readily prepared by passing a stream of carbonic oxide through blood or through a solution of oxyhemoglobin. It has a peculiar cherry-red colour. Its absorption spectrum is very like that of oxyhemoglobin, but the two bands are slightly nearer the violet end of the spectrum (fig. 37, spectrum 4). Eeducing agents, like ammonium sulphide, do not change it ; the gas is more firmly combined than the oxygen in oxy haemoglobin. CO- hemoglobin forms crystals like those of oxyhemoglobin : 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 : this acts as a powerful poison by combining with the hemoglobin of the blood, and thus interfering with normal respiratory processes. The colour of the blood and its resistance to reducing agents are in such cases charac- teristic. Nitric Oxide Haemoglobin. — When ammonia is added to blood, and then a stream of nitric oxide passed through it, this compound is formed. It may be obtained in crystals isomorphous with oxy- and CO-hemoglobin. It also has a similar spectrum. It is even more stable than CO-hemoglobin ; it has little practical interest, but is of theoretical importance as completing the series. Bohr has advanced a theory that haemoglobin forms a compound with carbonic dioxide, and that there are numerous oxyhemoglobins containing different amounts of oxygen, but his views have not been accepted. Very dilute solutions of haemoglobin and its derivatives show an absorption band in the ultra-violet region in addition to those just described in the visible regions of the spectrum (see more fully Advanced Course). CHEMISTRY 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 lungs consist essentially of numerous little hollow sacs in the walls of which is a close plexus of capillary blood vessels. These air THE BLOOD 95 sacs, or alveoli, communicate with the external air by the trachea, bronchi, and bronchial tubes. Inspiration is due to a muscular effort that enlarges the thorax, the closed cavity in which the lungs are situated. Owing to the atmospheric pressure the lungs become dis- tended. The atmospheric air does not, however, actually penetrate beyond the largest bronchial tubes ; the gases which get into the smaller tubes and air sacs do so by diffusion. Expiration is ordinarily brought about by the elastic rebound of the lungs and chest walls, and is only a muscular effort when forced ; but even the most vigorous expiratory effort is unable to expel the alveolar air. This air and the blood in the 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 contradistinc- tion external respiration. We have already seen that oxyhemo- globin 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 two oxides are formed : they are the chief products of combustion. Certain other products which represent the combustion of nitrogenous material (urea, uric acid, &c.) ultimately leave the body by the urine. All these subtances pass into the venous blood, and the gaseous products, carbonic acid, and a portion of the water find an outlet by the lungs. Inspired and Expired Air. — The composition of the inspired and the expired air may be compared in the following table : — Inspired or atmospheric Air 2096 vols, per cent. 79 „ 0-04 „ variable " Expired Air Oxygen Nitrogen Carbonic acid Watery vapour . I Temperature 16*03 vols, per cent. 79 4-4 „ saturated that of body (87° C.) The nitrogen remains unchanged. The recently discovered gases, i, crypton, A:c. are in the above table reckoned in with the 96 ESSENTIALS OF CHEMICAL PHYSIOLOGY 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^. If the inspired and expired airs are carefully measured at the same tempera- ture 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 not cause any change in the volume of the gas, for a molecule of oxygen (02) would give rise to a molecule of carbonic acid (C02), which would occupy the same volume. 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 proteid are eaten. The quotient _. 2 f = — =- is called 1 02 absorbed 4-5 the respiratory quotient. Normally it is — -= 09, but this varies con- siderably with diet, as just stated. It varies also with muscular exercise, when the output of carbonic acid is much increased both absolutely and relatively to the amount of oxygen used up. Gases of the Blood. — From 100 volumes of blood about 60 volumes of gas can be removed by the mercurial air-pump. The average composition of this gas in dog's blood is — - • Arterial Blood Venous Blood Oxygen Nitrogen Carbonic acid . ■ . 20 1 to 2 40 8 to 12 1 to 2 46 The nitrogen in the blood is simply dissolved from the air just as water would dissolve it : it has no physiological importance. The other two gases are present in much greater amount than can be explained by simple solution ; they are, in fact, chiefly present in loose chemical combinations. Less than one volume of the oxygen and about two of carbonic acid are present in simple solution in the plasma. Oxygen in the Blood. — The • amount of gas dissolved in a liquid varies with the pressure of the gas ; double the pressure and the amount of gas dissolved is doubled. Now this does not occur in the case of oxygen and blood ; very nearly the same amount of oxygen is dissolved THE BLOOD 97 whatever be the pressure. We thus have a proof that oxygen is not merely dissolved in the blood, but is in chemical union : and the fact that the oxygen of oxyhemoglobin can be replaced by equivalent quantities of other gases, like carbonic oxide, is a further proof of the ■ same statement. The tension or partial pressure of oxygen in the air of the alveoli is less than than in the atmosphere, but greater than that in venous blood ; hence oxygen passes from the alveolar air into the blood ; the oxygen immediately combines with the haemoglobin, and thus leaves the plasma free to 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 where the partial pressure of oxygen is lower than in the plasma, or in the lymph that bathes the tissue elements ; the plasma parts with its oxygen to the lymph, the lymph to the tissues ; the oxyhemoglobin then undergoes dissociation to supply more oxygen to the plasma and lymph, and this in turn to the tissues once more. This goes on until the oxyhaemo- globin loses a great portion of its store of oxygen, but even in asphyxia it does not lose all. The following values are given by Fredericq for the tension of oxygen in percentages of an atmosphere. His experiments were made on dogs : — External air 20'96 Alveolar air ...... 18 ^ Arterial blood ...... 14 | Tissues ....... 0 The arrow shows the direction in which the gas passes. The methods of obtaining the gases of the blood and analysing them are described in the Appendix. When the gases are being pumped off from the blood, very little oxygen comes off until the pressure is greatly reduced, and then at a certain point, it is suddenly disengaged. This shows it is not in simple solution, but is united chemically to the haemoglobin as oxyhaemoglobin, which is dissociated when the pressure is extremely low. The avidity of the tissues for oxygen is shown byEhrlich's experi- ments with methylene blue and similar pigments. Methylene blue is more stable than oxyhaemoglobin ; but if it is injected into the circu- lation of a living animal, and the animal killed a few minutes later, the blood is found dark blue, but the organs colourless. On exposure to oxygen the organs become blue. In other words the tissues have removed the oxygen from methylene blue to form a colourless reduc- tion product ; on exposure to the air this once more unites with ii to form methylene blue. 98 ESSENTIALS OF CHEMICAL PHYSIOLOGY Carbonic Acid in the Blood. — What has been said for oxygen holds good in the reverse direction for carbonic acid. Compounds are formed in the tissues where the tension of the gas is high : these pass into the lymph, then into the blood, and in the lungs the compounds undergo dissociation, carbonic acid passing into the alveolar air where the tension of the gas is comparatively low, though it is greater here than in the expired air. The relations of this gas and the compounds it forms are more complex than in the case of oxygen. If blood is divided into plasma and corpuscles, it will be found that both yield carbonic acid, but the yield from the plasma is the greater. If we place blood in a vacuum it bubbles, and gives out all its gases ; addition of a weak acid causes no further liberation of carbonic acid. If plasma or serum is similarly treated the gas comes off, but about 5 per cent, of the carbonic acid is fixed — that is, the addition of some stronger acid, like phosphoric acid, is necessary to displace it. Fresh red cor- puscles will, however, take the place of the phosphoric acid, and thus it has been surmised that oxyhemoglobin has the properties of an acid. One hundred volumes of venous blood contain forty- six volumes of carbonic acid. Whether this is in solution or in chemical combination is determined by ascertaining the tension of the gas in the blood. One hundred volumes of blood plasma would dissolve more than an equal volume of the gas at atmospheric pressure, if its solubility in plasma were equal to that in water.1 If, then, the carbonic acid were in a state of solution, its tension would be very high, but it proves to be only equal to 5 per cent, of an atmosphere. This means that when venous blood is brought into an atmosphere containing 5 per cent, of carbonic acid, the blood neither gives off any carbonic acid nor takes up any from that atmosphere. Hence the remainder of the gas, 95 per cent., is in a condition of chemical combination. The chief compound appears to be sodium bicar- bonate. The carbonic acid and phosphoric acid of the blood are in a state of constant struggle for the possession of the sodium. The salts formed by these two acids depend on their relative masses. If carbonic acid is in excess, we get sodium carbonate (Na2C03), and mono-sodium phosphate (NaH2P04) ; but if the carbonic acid is diminished, the phosphoric acid obtains the greater share of sodium to form disodium phosphate (Na2HP04). In this way, as soon as 1 To be exact, the solubility of carbon dioxide in plasma is a little less than in pure water. THE BLOOD 99 the amount of free carbonic acid diminishes, as in the lungs, the amount of carbonic acid in combination also decreases ; whereas in the tissues, where the tension of the gas is highest, a large amount is taken up into the blood, where it forms sodium bicarbonate. The tension of the carbonic acid in the tissues is high, but one cannot give exact figures ; we can measure the tension of the gas in certain secretions ; in the urine it is 9, in the bile 7 per cent. The tension in the cells themselves must be higher still. The following figures (from Fredericq) give the tension of carbonic dioxide in percentages of an atmosphere : — Tissues . . . . . 5 to 9 ) Venous blood .... 3-8 to 5"4 • in dog. & Alveolar air .... 2-8 i j External air ... . 0-03 The arrow indicates the direction in which the gas passes, namely, in the direction of pressure from the tissues to the atmosphere. In some experiments made by Bohr, also on dogs, the following are the figures given : — Arterial blood ...... 2-H Venous blood ...... "r4 & Alveolar air . . . . . . . 3*56 j Expired air 2-8 It will be seen from these figures that the tension of carbonic acid in the venous blood (5-4) is higher than in the alveolar air (3"56) ; its passage into the alveolar air is therefore inteUigible by the laws of osmosis. Osmosis, however, should cease when the tension of the gas in the blood and alveolar air are equal. But the transference goes beyond the establishment of such an equilibrium, for the tension of the gas in the blood continues to sink until it is, when the blood is arterial, ultimately less (2-8) than in the alveolar air. The whole question is beset with great difficulties and contra- dictions. Bohr's results have been subjected to much criticism; some observers have confirmed his results and others have failed to do so. If Bohr, however, is ultimately found to be correct, we can only explain this apparent reversal of a law of nature by supposing with him that the alveolar epithelium possesses the power of excreting carbonic acid, just as the cells of secreting glands are able to select certain materials from the blood and reject others. Recent work by Bohr and Haldane liown that in all probability the same explanation epithelial activity — must be called in to account for the absorption of oxygen. In the swim-bladder of fishes (which is analogous to the lungs of ii 2 100 ESSENTIALS OF CHEMICAL PHYSIOLOGY mammals) the oxygen is certainly far in excess of anything that can be explained by mere diffusion. Some Continental observers have stated that certain noxious substances are ordinarily contained in expired air which are much more poisonous than carbonic acid, but researches in this country have entirely failed to substantiate this. If precautions be taken by absolute cleanliness to prevent admixture of the air with exhalations from skin and clothes, the expired air only contains one noxious substance, and that is carbonic acid. Tissue-Respiration. — Before the processes of respiration were fully understood the lungs were looked upon as the seat of combustion ; they were regarded as the stove for the rest of the body where effete material was brought by the venous blood to be burnt up. "When it was shown that the venous blood going to the lungs already contained carbonic acid, and that the temperature of the lungs is not greater than that of the rest of the body, this explanation had of necessity to be dropped. Physiologists next transferred the seat of the combustion to the blood ; but since then innumerable facts and experiments have shown that it is in the tissues themselves, and not in the blood, that combustion occurs. The methylene-blue experiments already described (p. 97) show this ; and the following experiment is also quite conclusive. A frog can be kept alive for some time after salt solution is substituted for its blood. The metabolism goes on actively if the animal is kept in pure oxygen. The taking up of oxygen and giving out of carbonic acid must therefore occur in the tissues, as the animal has no blood. 101 LESSON X URINE 1. Test the reaction of urine to litmus paper. 2. Determine its specific gravity by the urinometer. 3. Test for the following 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 being precipitated by the nitrate of silver. ib) Sulphates. — Acidulate with hydrochloric acid and add barium chloride. A white precipitate of barium sulphate is produced. Hydro- chloric acid is again 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 alkalme (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 falls. This test is given by both kinds of phosphates. 4. Urea. — 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. '). Heat some urea crystals in a dry test-tube. Biuret is formed, and ammonia comes off. Add a drop of copper- sulph ate solution and a few drops of potash. A rose-red colour is produced. 6. Quantitative estimation of urea. For this purpose Duprc's apparatus dig. 38) is the most convenient. It consists of a bottle united to a measuring tube by indiarubber tubing. The iring 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 alkaline solution of sodium hypobromite (made by mixing 2 c.c. of bromine with 23 c.c. of a U)-per-cent. solution of caustic soda) into the bottle. ire ■> c.c. of urine into a small tube, and lower it carefully, so that no urine spills, into the bottle. Close the bottle securely with a stopper per- , by a glass tube ; this -lass tube ' is connected to the measuring tube by indiarubber tubing ami a ~f -piece. The third limb of the T-piece is closed by a piece oi indiarubber tubing and a pinch-cock, seen at the top oi the figure. Open tin- pincb cock and lower the measuring tube until the surface 1 The efficiency oi tin- apparatus is increased by having a glass bulb blown on ube to prevent froth passing into the rest of tin- app i This is not n in the fi< 102 ESSENTIALS OF CHEMICAL PHYSIOLOGY of the water with which the outer cylinder is filled is at the zero point of the graduation. Close the pinch -cock, and raise the meastiring tube to ascertain if 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 tempera- ture as that in the cylinder. After two or three minutes raise the measur- ing tube until the surfaces of the water inside and outside it are at the same level. Read off the amount of gas evolved. This is nitrogen. The carbonic acid resulting from the de- composition of urea has been absorbed by the excess of soda in the bottle. 35*4 c.c. of nitrogen are yielded by 0-1 gramme of urea. From this the quantity 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 proportion, the total amount of urea is ascertained. Sometimes the measuring tubes sup- plied with this apparatus are graduated in divisions corresponding to percent- ages of urea. 7. Creatinine. — This substance may be detected by adding a little sodium nitro-prusside and caustic soda to the urine. A red colour develops which fades on boiling. If, while the liquid is boiling, acetic acid is added, Prussian Fig. 38.— Dupre's urea apparatus. blue is formed. The kidney is a compound tubular gland, the tubules of which it is composed differing much in the character of the epithelium that lines them in various parts of their course. The true secreting part of the kidney is the glandular epithelium that lines the convoluted portions of the tubules ; there is in addition to this what is usually termed the filteiing apparatus : tufts of capillary blood vessels called the Malpighian glomeruli are supplied with 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 constituents of the blood, especially water and salts, pass URINE 103 through the thin walls of these vessels into the surrounding Bow- man'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, yet it is no purely mechanical process, but the cells exercise a selective influence, and prevent the albuminous constituents of the blood from escaping. During the passage of the water which leaves the blood at the glomerulus through the rest of the renal tubule, it gains the constituents urea, urates, &c, which are poured into it by the secreting cells of the convoluted tubules-. The term excretion is better than secretion as applied to the kidney, for the constituents of the urine are not actually formed in the kidney itself (as, for instance, the bile is formed in the liver), but they are formed elsewhere ; the kidney is simply the place where they are picked out from the blood and eliminated from the body. GENERAL CHARACTERS OF URINE Quantity. —A man of average weight and height passes from 1,400 to 1,600 c.c, or about 50 oz. daily. This contains about 50 grammes (1^ oz.) of solids. The urine should be collected in a tall glass vessel capable of holding 3,000 c.c, which should have a smooth-edged neck accurately covered by a ground-glass plate to exclude dust and avoid evaporation. The vessel, moreover, should be graduated so that the amount may be easily read off. 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 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. The bile pigment (and bly also the hsematin of the food) is in the intestines converted into stercobilin ; most of the stercobilin leaves the body with the fasces : bul 3ome is reabsorbed and is excreted with the urine as urobilin. Urobilin is very like the artificial reduction product of bilirubin called hydrobilirubin (see p. 70). Normal urine, however, contains very little urobilin. The actual body present is a chromogen her substance called urobilinogen, which by oxidation (such as 3 when tin; urine stands exposed to the air) is converted into 104 ESSENTIALS OF CHEMICAL PHYSIOLOGY the pigment proper. In certain diseased conditions the amount of urobilin is considerably increased. The most abundant urinary pigment is a yellow one called wrochvome. It shows no absorption bands. It is probably an oxi- dation product of urobilin (Eiva, A. E. Garrod). (See Lesson XXVI.) Reaction. — The reaction of normal urine is acid. This is not due to free acid, as the uric and hippuric acids in the urine are combined as urates and hippurates respectively. The acidity is due to acid salts, especially acid sodium phosphate. Under 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 Pig. 39. — Uriuometer floating in urine in a testing glass. Fig. 40. — Crystals of urea : a, four-sided prisms ; b, indefinite crystals, such as are usually formed from alcohol solutions. 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. 2. In herbivorous animals and vegetarians. The food here con- tains excess of alkaline salts of acids like tartaric, citric, malic, &c. These acids are oxidised into carbonates, which passing into the urine give it an alkaline reaction. Specific Gravity. — This should be taken in a sample of the twenty- four hours' urine with a good urinometer (see fig. 39). The specific gravity varies inversely as the quantity of urine passed under normal conditions from 1,015 to 1,025. A specific gravity below 1,010 should excite suspicion of hydruria ; one over 1,030 of a febrile condition, or diabetes, a disease in which it may rise URINE 105 to 1,050. The specific gravity has, however, been known to sink as low as 1,002 (after large potations, urina potus), or to rise as high as 1,035 (after great sweating) in perfectly healthy persons. Composition. — The following table gives the average amounts of the urinary constituents passed by a man in the twenty-four hours : — Water .... I.jOOOO grammes Total solids . 72-00 „ Urea ..... 33-18 »» Uric acid .... 0-55 ?? Hippuric acid 0-40 )? Creatinine .... 0-91 ,, Pigment and other organic substances 10-00 ?» Sulphuric acid '2-01 )) Phosphoric acid 3-16 )> Chlorine .... 7-50 „ Ammonia .... 0-77 5» Potassium .... 2-50 )5 Sodium .... 1109 J) Calcium .... 0-26 ,, Magnesium .... 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, sulphates, phosphates. &c. UREA Urea, or Carbamide, CO(NH.2)2, is isomeric (that is, has the same empirical, but not the same structural formula) with ammonium cyanate (NH4)CNO, from which it was first prepared synthetically by Wohler in 1828. Since then it has been prepared synthetically in other ways. Wohler's observation derives interest from the fact that this was the first organic substance which was prepared synthetically by chemists. It may be crystallised out from the urine, and it is then found to be readily soluble both in water and in alcohol : it has a saltish taste, and is neutral to litmus paper. The form of its crystals is shown in fig. 40. When treated with nitric acid, nitrate of urea (COlSLHpHNOa) is formed; this crystallises in octahedra, lozenge-shaped tablets, or gons (fig. 41, a). When treated with oxalic acid, flat or prismatic crystals of urea oxalate (CON2H.1.H2C2O.1 +H,0) are formed (fig. 41, >>)■ 106 ESSENTIALS OF CHEMICAL PHYSIOLOGY These crystals may be readily obtained in an impure form by adding excess of the respective acids to urine which has been con- centrated to a third or a quarter of its bulk.1 Under the influence of an organised ferment, the torula or micro- coccus ureae, which grows readily in stale urine, urea takes up water, and is converted into ammonium carbonate [CON2H4 + 2H20 = (NH4)2C03]. Hence the ammoniacal odour of putrid urine. By means of nitrous acid, urea is broken up into carbonic acid, water, and nitrogen, CON2H4-f-2HN02=C02 + 3H20 + 2N2. This may be used as a test 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. Fig. 41. — a, nitrate : b, oxalate of urea. Hypobromite of soda decomposes urea in the following way CON2H4 + 3NaBrO = C02 + N2 + 2H20 + 3NaBr [urea] [sodium [carbonic [nitrogen] hypobromite] acid] [water] [sodium bromide] This reaction is important, for on it one of the readiest methods for estimating urea depends. There have been various pieces of appa- ratus invented for rendering the analysis easy ; but the one described in the practical exercise at the head of this lesson appears to be the best. If the experiment is performed as directed, 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 The quantity of urea excreted is somewhat variable, the chief cause of variation being the amount of proteid food ingested. In a man in a ' The preparation of urea nitrate is postponed to the next lesson, when other microscopic crystals will also be under examination. UKINE 107 state of equilibrium the quantity of urea excreted daily averages 33 grammes (500 grains). The normal percentage in human urine is 2 per cent. ; but this also varies, because the concentration of the urine varies considerably in health. In dogs it may be 10 per cent. The excretion of urea is usually at a maximum three hours after a meal, especially after a meal rich in proteids. The urea does not come, however, direct from the food ; the food must be first assimi- lated, and become part of the body before it can break down to form urea. An exception to this rule is to be found in the case of the amido- acids. especially leucine and arginine, which are formed in the intes- tinal canal from proteids during digestion. These substances are carried to the liver and converted into urea ; but only a very small fraction of the urea in the urine is formed in this way. Food increases the elimi- nation of urea because it stimulates the tissues to increased activity ; their waste nitrogenous products are converted into urea, which, passing into the blood, is directly excreted by the kidneys. The greater the amount of proteid food given, the more waste products do the tissues discharge from their protoplasm, in order to make room for the new proteid which is built into its substance. Muscular exercise has little immediate effect on the amount of urea discharged. During intense muscular work there is a slight im- mediate increase of urea, but this is quite insignificant when compared to the increase of work. 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 the venous blood, which is rapidly discharged by the expired air. Careful research has, however, shown that an increase of nitrogenous waste does occur on muscular exertion, but appears as urea in the urine to only a slight extent on the day of tbe work ; the greater part is excreted during the next day. 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 kidneys the forma- tion of urea goes on, and that it accumulates 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 wramia (or urea in the Mood), and unless the urea be discharged from the body the patient dies. There is no doubt, however, that it is not urea but some antecedent of urea that acts most poisonously, and is the of death, for considerable quantities of urea can be injected into rculation without untoward insults. Where, then, is the seat of urea Eormation? Nitrogenous waste 108 ESSENTIALS OF CHEMICAL PHYSIOLOGY occurs in all the living tissues, and the principal final result of this proteid metabolism is urea. It may not be that the formation of urea is perfected in each tissue, for if we look to the most abundant tissue, the muscular tissue, very little urea is to be found. Yet there can be no doubt that the chief place from which urea ultimately comes is the muscular tissue. Some intermediate step occurs in the muscles ; the final steps occur elsewhere. In muscles we find a substance called creatine in fairly large quan- tities. If creatine is injected into the blood it is discharged as crea- tinine. But there is very little creatinine in normal urine ; what little there is can be nearly all accounted for by the creatine in the food ; if the muscular creatine and creatinine are discharged as urea, they must undergo some further change before they leave the muscle. Similarly, other cellular organs, spleen, lymphatic glands, secreting glands, participate in the formation of urea ; but the most important appears to be the liver : at any rate this is the organ where the final changes take place. The urea is then carried by the blood to the kidney, and is there excreted. 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 forma- tion 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. Under 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 in the urine, and, again, there is considerable increase in the ammonia. In this disease leucine and tyrosine are also found in the urine ; undue stress should not be laid upon this latter fact, for the small amounts of leucine and tyrosine found doubtless 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 proteid and urea. A few years ago Drechsel succeeded in artificially pro- URINE 109 during urea from casein. More recent work has shown that this is true for other proteids also. If a proteicl is decomposed by hydro- chloric acid, a little stannous chloride being added to prevent oxida- tion, a number of products are obtained, such as ammonium salts, leucine, tyrosine, aspartic and glutaminic acids. This was known before, so the chief interest centres round two new substances, precipitable by phosphotungstic acid. One of these is called lysine (CriH14N.,0.2, probably di-amido-caproic acid) ; the other was first called lysatinine. Hedin then showed that lysatinine is a mixture of lysine with another base called arginine (C(;H, ,N402) ; it is from the arginine that the urea comes in the experiment to be next described. Arguing from some resemblances between this substance and creatine, Drechsel expected to be able to obtain urea from it, and his expectation was confirmed by experiment. He took a silver compound of the base, boiled it with barium carbonate, and after twenty-five minutes' boiling obtained urea. Dreschel's comparison of arginine to creatine has turned out to be correct ; on decomposition it breaks up into di-amido-valerianic acid and cyanamide (CN.NH2) from which the urea originates. (Schulze and Winterstein.) Lysine and arginine are two of the hexone bases (see p. 31). It is, however, extremely doubtful whether the chemical decom- positions produced in laboratory experiments on proteids are com- parable to those occurring in the body. Many physiologists consider that the amido-acids are intermediate stages in the metabolic pro- cesses that lead to the formation of urea from proteids. We have already alluded to this question in relation to the creatine of muscle, and we are confronted with the difficulty that injection of creatine into the blood leads to an increase not of urea, but of creatinine in the urine. If creatine is an intermediate step, it must undergo some further charlge before it leaves the muscle. Other amido-acids, such as glycocine (amido-acetic acid) and leucine (amido-caproic acid) and arginine, are to be included in the same category. The facts upon which such a theory depends are (1) that the introduction of glycocine or leucine into the bowel, or into the circulation, leads to an increase of area in the mine; there is, however, no evidence that tyrosine acts in this way; and (2) that amido-acids appear in the urine of patients Buffering from acute yellow atrophy of the liver. Then again it is perfectly true that, in the laboratory, urea can be obtained from •ii;, and also from uric acid, hut, such experiments do not prove that creatine or uric acid are normally intermediate products of urea formation in the body. Still, if we admit for the sake of argument 110 ESSENTIALS OF CHEMICAL PHYSIOLOGY that amido-acids are normally intermediate stages in proteid meta- bolism, and glance at their formulae — Glycocine, C2H5N02 Creatine, C4H9N302 Leucine, C6H13N02 Arginine, C6H]4N402 — we see that the carbon atoms are more numerous than the nitro- gen atoms. In urea, CON2H4, the reverse is the case. The amido- acids must therefore be split into simpler compounds, which unite with one another to form urea. Urea formation is thus in part synthetic. There have been various theories advanced as to what these simpler compounds are. Some have considered that cyanate, others that carbamate, and others still that carbonate of ammonium is formed. Schroder's work proves that ammonium carbonate is one of the urea precursors, if not the principal one. The equation which represents the reaction is as follows : — (NH4)2C03 - 2H20 = CON2H4 [ammonium [water] [urea] carbonate] Schroder's principal experiment was this : a mixture of 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 great abundance. This does not occur when the same experi- ment 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. There is, however, no necessity to suppose that the formation of amido-acids is a necessary preliminary to urea formation. The con- version of the leucine and arginine formed in the intestine into ammonium salts and then into urea does certainly occur, but this only accounts for quite an insignificant fraction of the urea in the urine. If this also occurs in tissue metabolism we ought to find considerable quantities of leucine, glycocine, creatine, arginine, and such substances in the blood leaving the various tissues and entering the liver ; but we do not. We do, however, constantly find ammonia, which, after passing into the blood or lymph, has united with car- bonic acid to form either carbonate or carbamate of ammonium. It is quite probable that the nitrogenous waste that leaves the muscles and other tissues is split off from them as ammonia, and not in the shape of large molecules of amido-acid which are subsequently con- verted into ammonia. The experiments outside the body which most closely imitate those occurring within the body are those of Drechsel, URINE 111 in which he passed strong alternating currents through solutions of proteid-like materials. Such alternating currents are certainly absent in the body, but their effect, which is a rapidly changing series of small oxidations and reductions, is analogous to metabolic pro- cesses ; under such circumstances the carbon atoms are burnt off as carbon dioxide, the nitrogen being split off in the form of ammonia, and by the union of these two substances ammonium carbonate is formed. The following structural formate show the relationship between ammonium carbonate, ammonium carbamate and urea. O _ c■') grammes, of which the earthy phosphates contain about half M tol-5gr.). i 114 ESSENTIALS OP CHEMICAL PHYSIOLOGY LESSON XI UBINE (continued) 1. Urea Nitrate. — Evaporate some urine in a capsule to a qiiarter 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. TJric acid. — Kxamine 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 pepper. 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. 43). Dissolve the crystals in caustic potash and then carefully add excess of hydrochloric acid. Small crystals of uric acid again form. Place a little uric acid, or a urate (for instance, serpent's urine), in a cap- sule ; add a little dilute nitric acid and evaporate to dryness. A yellowish-red residue is left. Add a little ammonia carefully. The residue turns to violet. This is due to the formation of rnurexide or purpurate of ammonia. On the addition of potash the colour becomes bluer. 3. 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 brick-dust ; 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. 4. Deposit of Phosphates.— Another specimen of pathological urine contains excess of phosphates, which have formed a white deposit on the urine be- coming alkaline. This precipitate does not dissolve on heating ; it may be increased. It is, however, soluble in acetic acid. Examine microscopically for coffin-lid crystals of triple phosphate (ammonio-magnesium phosphate), for 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 be- come 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. URINE 115 We have now studied urea, the principal nitrogenous constituent of urine, at some length. There are still left for our consideration a number of other nitrogenous constituents, the most important of which are uric acid, hippuric acid, and creatinine. URIC ACID Uric acid (C5N4H403) 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 form 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 fairly 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 dis- solved 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 sub- stance, requiring for its solution 1,900 parts of hot and 15,000 parts of cold water. The FlG' 43-Uric acia crystel8' 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. 43). The murexide test which has just been described among the ical 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 applic- able as a test) is that on treatment with certain oxidising reaj i 2 116 ESSENTIALS OF CHEMICAL PHYSIOLOGY 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 (see p. 109). Uric acid is dibasic, and thus there are two classes of urates — -the normal urates and the acid urates. A normal urate is one in which two atoms of the hydrogen are replaced by two of a monad metal like sodium ; an acid urate is one in which only one atom of hydrogen is thus replaced. The formulae would be — C5H4N403=uric acid C5H3NaN403=acid sodium urate C5H2Na2N40 3= normal sodium urate The acid sodium urate is the chief constituent of the pinkish deposit of urates, which, as we have already stated, is called the lateritious deposit. The quantity of uric acid excreted by an adult varies from 7 to 10 grains (0*5 to 0*75 gramme) daily. The best method for determining the quantity of uric acid in the urine is that of Hopkins. Ammonium chloride in crystals is added to the urine until no more will dissolve. This saturation completely precipitates all the uric acid in the form of ammonium urate. After standing' for two hours the precipitate is collected on a filter, washed with saturated solution of ammonium chloride, and then dissolved in weak alkali. Prom this solution the uric acid is precipitated by neutralising with hydrochloric acid. The precipitate of uric acid is collected on a weighed filter, dried and weighed, or titration may be performed with potassium permanganate (see Advanced Course). Origin of Uric Acid. — Uric acid is not made by the kidneys. When the kidneys are removed uric acid continues to be formed and accumulates 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 there- fore probable that ammonia and lactic acid are normally synthesised in the liver to form uric acid. The principal conditions which lead to an increase of uric acid in the urine are — 1. Increase of meat diet and diminution of oxidation processes, such as occur in people with sedentary habits. 2. Increase of white corpuseles in the blood, especially in the disease known as leucocythaemia. This latter fact is of great interest, as leucocytes contain large quantities of nuclein. Nuclein yields nitrogenous bases which are closely related to uric acid. URINE 117 These bases (the alio. i uric or ■purine bases, see also pp. 29, 30) may be arranged in two pairs : — Adenine has the formula C5H5N5 ; on heating it with sulphuric acid, NH is replaced by O, and hypoxanthine is formed : — C5H4N4.NH + H20 - C5H4N40 + NH3 [adenine] [water] [hypoxanthine] [ammonia]. Both substances contain a radicle, C5H4N4, called adenyl ; adenine is its imide, hypoxanthine its oxide. The following equation shows a similar relationship between the other pair of bases, guanine and xanthine : — C5H4N4O.NH + H20 = C,H4N40, + NH [guanine] [water] [xanthine] 3 [ammonia]. On comparing the formulae of hypoxanthine and xanthine with uric acid, C3H4N403, we see their close relationship. Leaving aside other possible ways in which uric acid is undoubtedly formed in the organism, we have here a way in which uric acid may arise by oxidation from the nuclein bases and thus ultimately from the nuclei of cells. Certain forms of diet increase uric acid formation by leading to an increase of leucocytes and conse- quently increase in the metabolism of their nuclei ; some investigators think, however, that the increase is chiefly due to nuclein in the food. The ques- tion is'not yet settled. HIPPURIC ACID Hippuric acid (C9H,,N03), com- jf~\ bined with bases to form hippurates, is present in small quantities in human Urine, but in large quantities in the urine 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 glycocine with the elimination of a molecule of water, and is excreted as hippuric acid — ( 1L.NH, CH2NH.CO.C6H6 C„H6.COOH+ | = ! +H20 COOH COOH oclne] [hippuric acid] [water] This is a well-marked instance of synthesis carried out in the animal bods, ;ma2xi4> [normal sodium urate] + Na2C03 [sodium carbonate] Published by Smith, Elder & Co., London, 1892. URINE 121 This deposit may be recognised as follows :— 1. It has a pinkish colour ; the pigment called uro-crythrin is one of the pigments of the urine, but its relationship to the other urinary pigments is not known (see further Lesson XXVI). 2. It dissolves upon warming the urine. 3. Microscopically it is usually amorphous, but crystalline forms similar to those depicted in figs. 47 and 48 may occur. Crystals of calcium oxalate may be mixed with this deposit (see fig. 49). Deposit of Calcium Oxalate. — This occurs in envelope crystals (octahedra) or dumb-bells. * PIG. 49. — Envelope crystals Fig. 50. — Cystin crystals. of calcium oxalate. It is insoluble in ammonia, and in acetic acid. It is soluble with difficulty in hydrochloric acid. Deposit of Cystin. — Cystin (C6H,2N2S204) is recognised by its colourless six-sided crystals (fig. 50). These are rare: they occur only in acid urine, and they may form concretions or calculi. Cystin - uria (cystin in the urine) is hereditary. Deposit of Phosphates. — These occur in alkaline urine. The urine may be alkaline when passed, due to fermentative changes occurring in 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 the growth of the micrococcus urea. This forms ammonium carbonate from the urea. CON2H, + 2H20 = (NH4)2Cp3 [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, Ca:i(PO,)._, ; amorphous. 122 ESSENTIALS OF CHEMICAL PHYSIOLOGY 2. Triple or ammonio-magnesium phosphate, MgNH4P04 ; coffin- lids and feathery stars (fig. 51). 3. Crystalline phosphate of calcium, CaHP04, in rosettes of prisms, in spherules, or in dumb-bells (fig. 52). - 4. Magnesium phosphate, Mg3(P04)2-|-22H20, occurs occasion- ally, 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. Very often neutral or alkaline urine will become cloudy when boiled : this may be due to Fig. 51. — Triple phosphate crystals. Fig. 52. — Crystals of phosphate of lime (stellar phosphate). albumin or to phosphates. It is very important to distinguish be- tween these two, as albuminuria is a serious condition. They may be distinguished by the use of acetic acid, which dissolves phosphates but not albumin. A solution of ammonium carbonate (l-in-5) eats magnesium phosphate away from the edges ; it has no effect on the triple phos- phate. A phosphate of calcium (CaHP04 + 2H20) 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, CaC03, appears but rarely as whitish balls or biscuit-shaped bodies. It is commoner in the urine of herbivora (see p. 112). It dissolves in acetic or hydrochloric acid, with effervescence. The following is a summary of the chemical sediments that may occur in urine : — TRINE 123 CHEMICAL SEDIMENTS IN UEINE In Acid Ueink Uric Acid. — Whetstone, dumb- bell, or sheaf-like aggregations of crystals deeply tinged by pigment (hg. 43). Urates. — Generally amorphous. The acid urate of sodium (fig. 47) and of ammonium (fig. 48) 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. 49). Insoluble in acetic acid. Cystin. — Hexagonal plates (fig. 50). Rare. Leucine and Tyrosine. — Bare. Calcium Phosphate. CaHPO, + 2H,0.— Rare. In Alkaline Ueink Phosphates. — Calcium phosphate, Ca.,(PO().,. Amorphous. Triple phosphate, MgNH4P04 + 6H,0. Coffin-lids or feathery stars (figs. 42 and 51). Calcium hydrogen phosphate, CaHPO ,. Rosettes, spherules, or dumb-hells (fig. 52). Magnesium phosphate, Mg3(PO ,), + 22H,0. Long plates. All soluble in acetic acid without effervescence. Calcium Carbonate, CaC03. — • Biscuit-shaped crystals. Soluble in acetic acid with effervescence. Ammonium Urate. C5H2(NHJ2.N4Os. — ' Thorn-apple ' spherules. Leucine and Tyrosine. — -Very rare. 124 ESSENTIALS OF CHEMICAL PHYSIOLOGY LESSON XII PATHOLOGICAL URINE 1. Urine A is pathological urine containing albumin. It gives the usual proteid tests. The two following are most frequently iised in practice :— (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.^-IPoxxx some of the urine gently on to the surface of some nitric acid in a test-tube. A ring of white precipitate occurs at the junction of the two liquids. This test is used for small quantities of albumin. If the urine is cloudy, it should be filtered before applying these tests. 2. Estimation of Albumin by Esbach's Albuminometer. — 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 sufficient water to make up to a litre (1,000 c.c). Fig. 53. — Albuminometer of Esbach. Pour the urine into the tube 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 coagulum. The figures indicate grammes of dried albumin in a litre of urine. The percentage is obtained by dividing by 10. Thus, if the coagulum stands at 3, the amount of albumin is 3 grammes per litre, or 0-3 gr. in 100 c.c. If the sediment falls between any two figures, the distance \, J, or f from the upper or lower figure can be read off with sufficient accuracy. Thus the surface of the sediment being midway between 3 and 4 would be read as 3'5. "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 3S as the case may be. If the amount of albumin is less than 0-05 per cent., it cannot be accu- rately estimated by this method. 3. Urine B is 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 solution. Fehling's solution is an alkaline solution of copper sulphate to which Eochelle salt has been added. The Eochelle salt (double tartrate of potash and soda) holds the cupric hydrate in solution. Fehling's solution should always be freshly prepared, as, on stand- PATHOLOGICAL URINE 125 ing, racemie acid is formed from the tartaric acid, and this substance itself reduces the cupric to cuprous oxide. Fehling's solution should, therefore, always be tested by boiling before it is used. If it remains unaltered by boiling, it is in good condition. 4. Quantitative Determination of Sugar in Urine. — Fehling's solution is pre- pared as follows : — 34*639 grammes of copper sulphate are dissolved in about 200 c.c. of distilled water ; 173 grammes of Eochelle salt are dissolved in 600 c.c. of a 14-per-cent. solution of caustic soda. The two solutions are mixed and diluted to a litre. Ten c.c. of this solution are equivalent to 0*05 gramme of dextrose. Dilute 10 c.c. of this solution with about 40 c.c. of water, and boil it in a flask. Run into this from the burette (see fig. 54) the urine (which should be previously diluted with nine times its volume of distilled water) until the blue colour of the copper solution disappears — that is, till all the cupric hydrate is reduced.1 The mixture in a flask should be boiled after every addition.'- The quantity of diluted urine used from the burette con- tains 0*05 gramme of sugar. Calculate the percentage from this, remembering that the urine has been diluted to ten times its original volume. The following formula will be found useful in con- verting grammes into grains : — x = number of grains of sugar in the 24 hours. a = number of ounces of urine in the 24 hours. 1 ounce = 28-396 c.c. b = number of c.c. of urine used from the burette to decompose 10 c.c. of Fehling's solution (equivalent to 0-05 gramme = 0-77 grain of sugar). Then x = , x 28-396 0-77 x 21-865. Fig. 54.— Two burettes on stand. (Button.) 5. Picric Acid Test. — The work of Sir George Johnson and G. S. Johnson has shown the value of this reagent in detecting both albumin and sugar in the urine. The same reagent may be employed for the detection of both substances. The method of testing for albumin has been already studied with Esbach's tubes. To test for sugar do the following experiment. Take a drachm (about 4 c.c.) of diabetic urine : add to it an equal volume of saturated aqueous solution of picric acid, and half the volume (i.e. 2 c.c.) of the liquor potass* of the 1 It is somewhat difficult for the unpractised observer to determine accurately the exact point at which the blue disappears. The blue colour, if any remains, will be seen by holding the Mask up to the light. Some prefer a white porcelain basin instead of a flask ; the blue can then be seen against the white of the basin. Pavy's modification of Fehling's solution is sometimes used. Here ammonia holds the copper in solution, and no precipitate forms on boiling with sugar, as ammonia holds the cuprous oxide in solution. 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 grammes of dextrose. In some cases of diabetic urine where there is excess of ammonio-magnesic phosphate, the full reduction is not obtained with Fehling's solution, and when the quantity of sugar is small it may be missed. In such a ids or potash should be first added; the precipitated phosphates • and the filtrate after it has been well boiled may then be titrated with Fehling's solution. - On cooling the blue colon- reappi are, owing to re-oxidation. 126 ESSENTIALS OF CHEMICAL PHYSIOLOGY British Pharmacopoeia. Boil the mixture for about a minute, and it becomes so intensely dark red as to be opaque. Now do the same experiment with normal urine. An orange-red colour appears even in the cold, and is deepened by boiling, but it never becomes opaque, and so the urine for clinical purposes may be considered free from sugar. This reduction of picric acid by normal urine is due to creatinine (see p. 119). The reaction described may be used for quantitative purposes (see Appendix, Sir George Johnson's picro-saccharo- meter). The full significance and cause of pathological urine cannot be appreciated until a theoretical and practical acquaintance with disease is obtained, and we shall briefly consider only those abnormal con- stituents which are most frequently met with. PROTEIDS IN THE URINE There is no proteid 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 estimating 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 ob- served 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, includes the ' proteoses.' Indeed, in most, if not all, cases of so- called peptonuria, true peptone is absent. In the disease called ' osteomalacia ' a proteose is usually found in the urine. SUGAR IN THE URINE Normal urine contains no sugar, or so little that for clinical pur- poses it may be considered absent. It occurs in the disease called diabetes mellitus, which can be artificially produced by puncture of the medulla oblongata, by extirpation of the pancreas, and by the administration of the drug called phloridzin. The disease as it occurs in man may be due to disordered metabolism of the liver, to disease of the pancreas, or to other not fully understood causes.1 The methods usually adopted for detecting and estimating the sugar are given at the head of this lesson. The sugar present is dextrose. Lactose may occur in the urine of nursing mothers. 1 Transitory glycosuria is found in many diseases. PATHOLOGICAL URINE 127 Diabetic urine also contains hydroxybutyric acid, and may contain or yield on distillation acetone and ethyl-diacetic acid. Febling's test is not absolutely trustworthy. Often a normal urine will decolorise Fehlrng's solution, though seldom a red precipitate is formed. This is due to excess of urates and creatinine. Another substance called glycuronic acid (C,;HlM0:) is. however, very likely to be confused with sugar by Febling's test ; the cause of its appearance is sometimes the administration of drugs (chloral, camphor, &c.) ; but sometimes it appears independently of drug treatment. The cause of this is not known, but the condition has not the serious meaning one attaches to diabetes ; hence, for life assurance purposes, it is most necessary to conhrm the presence of sugar by other tests. Then, too, in the rare condition called alcaptonuria, confusion may similarly arise. Alcapton is a substance which probably originates from tyrosine by an unusual form of metabolism. It gives the urine abrown tint, which darkens on exposure to the air. It is an aromatic substance, and the researches of Baumann and Wolkow 1 have identified it with homogenti- sinic acid (C,.Ha.(OH)2CH,COOH). The best confirmatory tests for sugar are the phenyl-hydrazine test (see Lesson XIII), and the fermentation test, which is performed as follows : — Half fill a test tube with the urine and add a little German yeast. Fill up the tube with mercury ; invert it in a basin of mercury, and leave it in a warm place for twenty-four hours. The sugar will undergo fermentation : carbonic acid gas accumulates in the tube, and the liquid no longer gives the tests for sugar, or only faintly, but gives those for alcohol instead. A control experiment should be made with yeast and water in another test tube, as a small yield of carbonic acid is sometimes obtained from impurities in the yeast. Sir W. lioberts introduced a method for estimating sugar in urine, by the diminution in specific gravity which it undergoes on fermentation. Every degree lost in the specific gravity corresponds to one grain of sugar per fluid ounce. Four ounces of urine are placed in a bottle, and a piece of German yeast about the size of a small walnut is added. The bottle is closed with a cork, through which a small hole is bored to allow the carbonic acid to < . This is put in a warm chamber (40° C.i, and beside it is placed another similar bottle containing 4 ounces of the urine without any yeast. After 18 to 24 hours, fermentation is complete, and the specific gravity of both is taken; suppose that the specific gravity of the unfermented urine is 1,040, and that of the urine which has undergone fermentation is 1,030 : the number of degrees lost is 10 ; i.e. the urine contained 10 grains of sugar per ounce. The percentage of sugar may be ascertained by multiplying the degrees of specific gravity lost by 0-22 ; thus the percentage in the example just given will be 0'22 x 10 = 2-2. The method, however, is too rough for trustworthy observations to be made, and has dropped out of use. BILE IN THE URINE This occurs in jaundice. The urine is f tli<- albumoses only in the presence of excess of salt. If the salt is removed by dialysis, nitric acid then causes no precipitate. 142 ESSENTIALS OF CHEMICAL PHYSIOLOGY 5. Among the important reactions of proteids is Piotrowski's reaction— that is, the coloration produced by copper sulphate and a caustic alkali ; the term ' biuret reaction ' is applied to the rose-red colour which proteoses and peptones give with these reagents, because biuret (a derivative of urea) gives a similar colour. It does not, however, prove that biuret is contained in the proteid molecule. Biuret and proteid both contain some radicle to which the colour is due. Gnezda1 thought it was cyanogen, and that the cyanogen was differently combined in the peptones and native proteids (albumins and globulins) respectively ; hence the rose-red given by one group and the violet by the other. More recent work by Pickering,2 however, points to a CONH group rather than cyanogen. Gnezda found that if a dilute solution of nickel sulphate is used instead of copper sulphate, the native proteids give different colours from the peptones and proteoses, and Pickering has found the same with cobalt. Their results may be given in the following table : — Proteid Copper sul- phate and ammonia Copper sul- phate and potash Nickel sul- phate and ammonia Nickel sul- phate and potash Cobalt sul- phate and ammonia Cobalt sul- phate and potash Albumins and ) globulins . j Blue Violet Nil Yellow Nil Heliotrope- purple Proteoses and ) peptones . j Rose-red Rose -red Yellow Orange Nil Red-brown 6. Another delicate test introduced by McWilliam may here be men- tioned : Salicyl-sidphonic 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. 7. The use of trichloracetic acid for the separation of various proteids may be illustrated by the following experiment. Take some blood and add to it some solution of Witte's peptone (i.e. proteoses and ppeptone). 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 peptone, all the other proteids being contained in the precipitate. On cooling, the nitrate deposits some of the proteose. The proteose and peptone may be detected in the usual way. 1 Proc. Roy. Society, vol. xlvii. p. 202. 2 Journal of Physiology, vol. xiv. Most of the other colour reactions of pro- teids depend on the aromatic radicle they contain. 143 LESSON XVIII DIGESTION 1. Activity of pepsin solutions. Examine the comparative digestive power of the glycerin extracts of two stomachs. Take in two test-tubes an equal small weighed quantity of fibrin stained with carmine. Add to each 10 c.c. of 0-2 per cent, hydrochloric acid. Add to one a measured quantity of one glycerin extract, and to the other an equal quantity of the other glycerin extract. As the fibrin is digested the carmine is set free, and colours the liquid ; that which is more deeply stained is that which contains the more active preparation of pepsin. This exercise illustrates the principle of Griitzner's method of comparing the digestive powers of solutions. In the original method the amount of carmine set free is estimated by an artificial scale consisting of ten solutions of carmine of different known strengths. The carmine solution for staining the fibrin is prepared by dissolving 1 gramme of carmine in about 1 c.c. of ammonia ; to this 400 c.c. of water are added, and the mixture is kept in a loosely stoppered bottle till the smell of ammonia has become faint. The fibrin is stained by taking it perfectly fresh and clean. It is chopped finely and placed in the carmine solution for twenty-four hours. The fluid is strained off and the fibrin washed in water till the washings are colourless. It is kept in a stoppered bottle with just enough ether to cover it. 2. Tests for Free Hydrochloric Acid. ( a ) Gunsberg'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. lied crystals form, or if much peptone is present, there will be a red paste. The reaction takes place with 1 part of hydrochloric acid in 10,000. The organic acids do not give the reaction. (6) Tropaeolin test. Drops of a saturated solution of tropaeolin — 00 in 94 nt. 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 tropaeolin drop, still at 40° C. ; and if hydrochloric acid is present, a violet spot is left when the fluid has L A drop of 0-006 per cent, hydrochloric acid leaves a distinct mark. t a very complete account of these and other colour reactions see ■ I' ea i - of the Stomach,' chap. v. By Sidney .Martin. F.B.S. 1895.) 8. Pancreatic Digestion. A finely divided ox-pancreas has been allowed to digest at Uf C. for twenty-four to thirty-sis hours in a litre of 1 per cent. 144 ESSENTIALS OF CHEMICAL PHYSIOLOGY solution of sodium carbonate to which the white of an egg has been added every ten hours. Note the odour due to putrefaction. Another preparation has been similarly made, except that thymol has been added to prevent de- composition. These should be got ready by the demonstrator. Filter some of the extract and examine for leucine and tyrosine as follows : — (a) To some of the liquid add Millon's reagent and filter off the pre- cipitated proteid. Boil the filtrate. The presence of tyrosine is indicated by a red colour. If tyrosine is abundant the red colour appears without boiling. (b) Boil another portion of the filtered extract ; filter off the proteid thus coagulated ; reduce the filtrate to a smaU bulk by evaporation on the water- bath at the boiling temperature. Examine a drop microscopically for crystals of leucine and tyrosine. Treat the remainder with excess of alcohol, to pre- cipitate the albumoses and peptones, and again filter. Concentrate the filtrate on the water-bath till it becomes sticky from the presence of leucine. Examine some of the concentrated fluid with the microscope ; leucine will be found in crystalline spheroidal clumps. Examine microscopic specimens of leucine and tyrosine which have b&en prepared by the demonstrator. k 4. Zymogen granules.- — Examine microscopically, mounting in aqueous humour or serum (or in glycerin after treatment with osmic acid vapour), small pieces of the pancreas, parotid and submaxillary glands in a normal guinea-pig,1 and also 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. Extremely good, though not permanent, microscopic specimens may be obtained by teasing in a 33-per-cent. solution of caustic potash. 1 The guinea-pigs should be killed by bleeding, and the blood collected and defibrinated, and utilised for the preparation of oxyhasmoglobin 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. 86 all give good results. If amyl nitrite is used instead of ether in the third method, crystals of methfemo- globin are obtained. 145 LESSON XIX HEMOGLOBIN AND ITS DERIVATIVES Defibrinated ox-blood suitably diluted may be used in the following experiments as in those described in Lesson IX. 1. Place some in a hasniatoscope (see fig. 33, p. 89) in front of the large spectroscope. Note the position of the two chai'acteristic bands of oxyhemo- globin ; these are replaced by the single band of haemoglobin after reduction by the addition of Stokes' reagent (see footnote, p. 88)) or ammonium sulphide. 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 focussed with the absorption spectrum. 2. Obtain similar comparison spectra by the use of the rnicrospectroscope. For this purpose a cell containing a small quantity of oxyhsemoglobin 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 instrument. Notice that the two bands of carbonic oxide haemoglobin are very like those i if oxyhemoglobin, but are a little nearer to the violet end of the spectrum. Carbonic 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 ammonium sulphide (fig. 57, spectrum 4). 3. Methaemoglobin. — 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. 57, spectrum o). On dilution other bands appear (fig. 57. spectrum 6). Treat with ammonium sulphide, and the band of hemoglobin appears. 4. Acid Haematin. — Add a few drops of glacial acetic acid to dilute blood ; tin- colour changes to brown. Examine with the spectroscope. Compare tli'- position of the absorption band m the red with that of methaemoglobin ;, that of acid haematin is further from the D line (fig. 57, spectrum 7). Take some undiluted Mood and add glacial acetic acid as before. Extract this with ether by gently agitating it with that fluid. The ethereal extract should then be poured off and examined. The band in the red is seen, and on further diluting with ether three additional bands appi 5. Alkaline Haematin. Add to diluted blood a small quantity of strong caustic potash and warm. The colour changes to brown, and with the speotro- faint shading OS the left side of the 1> line is seen (tig. o7. spectrum 8). 6. Haemochromogen. Add ammonium sulphide to a solution of alkaline L 116 ESSENTIALS OF CHEMICAL PHYSIOLOGY Fig. 57. — 1, Solar spectrum. 2, Spectrum of oxyhemoglobin (037 p.c. solution). First band, A 589- 564 : second band, A 555-517. 3, Spectrum of haemoglobin. Band, A 597-535. 4, Spectrum of C0- lifeuioglobin. First band, A 583-564 : second band, A 547-521. 5, Spectrum of methfemoglobin (concentrated solution). 6. Spectrum of methsemoglobin (dilute solution"). First band, A 647- 622; second baud, A 587-571; third band, A 552-532 ; fourth band, A 514-490. 7, Spectrum of acid hoematin (ethereal solution). First band, A 656-615 ; second band, A 597-577 ; third band. A 557-529 ; fourth band, A 517-488. 8. Spectrum of alkaline haematin. Band from A 630-581. 9, Spectrum of hsemochromogen (reduced hsematin). First band, A 569-542 ; second band, A 535-504. 10, Spectrum of acid hajmatoporphyrin. First band, A 607-593 ; second band, A 585- 536. 11. Spectrum of alkaline hsematoporphyrin. First band, A 633-612 : second band, A 589-564;- third band. A 549-529 ; fourth band, A 518-488. The above measurements (after MacMunn) are in millionths of a millimetre. The liquid was examined in a layer 1 centimetre thick. The edges of ill-defined bands vary a good deal with the concentration of the solutions. HAEMOGLOBIN 14' kit-matin : the colour changes to red. and two bands are seen, one between D and 1". and the other nearly coinciding with E and b (fig. 57, spectrum 9). The spectrum of alkaline hsematin reappears for a short time after vigorous shaking with air. 7. Haeniatoporphyrin. — To some strong sulphuric acid in a test tube add a J.— The photographic spectrum of liicmofilobiu anil oxyhnemo?Iobin. (Gamgee. I Fi'.. '.'.'.--Tiie photographic spectrum at oxyhemoglobin and methtemoglobin. (Gamgee.) few drops of undiluted blood, and observe the spectrum of acid hemato- porphyrin (iron-free heematin) (fig. 57, spectrum 10). Map out all the Bpectra ee on a chart. S. The Photographic Spectrum. Hemoglobin and its compounds also shov. absorption bands in the ultra-violet portion of the Bpectrum. This portion of the spectrum i- not visible to the eye, but can be rendered visible l2 148 ESSENTIALS OF CHEMICAL PHYSIOLOGY "by allowing the spectrum to fall on a 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 focussed on a fluorescent screen.1 The slit is then opened very widely, and the coloured solution is interposed on the path of the beam falling on the slit. Oxyhemoglobin shows a band (Soret's band) between the lines G and H. In haemoglobin, carbonic oxide haemoglobin, and nitric oxide haemoglobin, this band is rather nearer G. Methaemoglobin and haematoporphyrin show similar bands. The two preceding figures show the ' photographic spectra ' of haemo- globin, oxyhaemoglobin, and methaemoglobin, and will serve as examples of the results obtained. I am greatly indebted to Prof. Gamgee, to whom we •owe most of our knowledge on this subject, for permission to reproduce these two specimens of his numerous photographs. 9. Preparation of Pure Oxyhaemoglobin. — The following method is described in Stirling's ' Practical Physiology' (3rd edit. p. 65). Centrifugalise dog's defibrinated blood and pour off the serum. Centrifugalise again with physio- logical saline solution repeatedly until the supernatant fluid contains only traces of proteid. 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-cent. 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 centrifugalising. (I have found that they aggre- gate together and can be easily removed by filtration.) Pour off the clear red fluid : cool it to 0° G, add one-fourth of its volume of absolute alcobol pre- viously cooled to 0° 0. Shake well, and then let the mixture stand at 5°-15° C. for 24 hours. As a rule the whole passes into a glittering crystalline mass. Filter at 09 C. and wash with ice-cold 25-per-cent. alcohol. Eedissolve 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 Fluorescent screens, similar to those in common use in observations made with Kontgen r&ys, may be made by coating white cardboard with barium platino- cyanide. 14J» LESSON XX SEE UM 1. The following methods of precipitating serum globulin (paraglobulin) should be performed : — (a) Panum's Method. — Dilute serum with fifteen times its bulk of water. It becomes cloudy owing to partial precipitation of the serum globulin. Add a few drops of 2-per-cent. acetic acid ; the precipitate becomes more abun- dant, and it dissolves in excess of the acid. It was formerly called ' serum casein." i b ' Alexander Schmidt's Method. — Dilute serum with twenty times its bulk of water and pass a stream of carbonic acid through it. A fairly abundant precipitate of serum globulin falls. Let it settle, and an additional precipitate can be obtained from the decanted liquid by treating it with a trace of acetic acid (the -serum casein' mentioned above). Repeat the carbonic acid method without dilution ; no precipitate forms. (c) By Dialysis. — Put some serum in a dialyser with distilled water in the outer vessel. The water must be frequently changed. In order to prevent decomposition a few crystals of thymol are added. In a day or two the salts have passed out ; the proteids remain behind : of these the serum albumin is still in solution ; the serum globulin is precipitated, as it requires a small quantity of salt to hold it in solution. I-/ 1 By Addition of Salts : — li.i Schmidt's method. {Saturate some serum with sodium chloride. A precipitate of serum globulin is produced. (ii.i Hammarsten's method. Use magnesium sulphate instead of sodium chloride. A more abundant precipitate is produced, because this salt is a more perfect precipitant of serum globulin than sodium chloride. In order to obtain complete saturation with these salts it is necessary to shake the mixture of salt and serum for sonic hours.1 liii.i Lauder's method. Balf saturate serum with ammonium sulphate. This is done by adding to the scrum an equal volume of saturated solution. of ammonium sulphate. This precipitates the globulin. Complete Batura- .. itL the -alt precipitates the albumin also. 2. Heat Coagulation.- -Saturate serum with magnesium sulphate and filter off the precipitate ; preserve tbu filtrate and label it ' B.1 Wash the pitate on the filter with saturated solution of magnesium sulphate until i ooroenientlj don< by a shaking machine before the class meets. 150 ESSENTIALS OF CHEMICAL PHYSIOLOGY the washings do not give the tests for albumin,1 then dissolve the precipitate by adding distilled water. It readily dissolves, owing to the salt adherent to it. The solution is opalescent. Label it ' A.' Kender A faintly acid with a drop of 2-per-cent. acetic acid, and heat in a test-tube. The temperature of the test-tube may be raised bj placing it in a flask of water gradually heated over a flame. A thermometer is placed in the test-tube, and should be kept moving so as to ensure that all parts of the liquid are at the same temperature. The quantity of liquid in the test-tube should be just sufficient to cover the bulb of the thermometer. A flocculent precipitate of coagulated serum globulin separates out at about 75°. Now take the filtrate B. This contains the serum albumin. Dilute it with an equal volume of water ; render it faintly acid as before, testing the reaction with litmus paper. Heat. A flocculent precipitate (a) falls at about 73° C. ; filter this off; note that the filtrate is less acid than that from which the precipitate has separated, or it may even be alkaline. If so, make it faintly acid again, and heat; a precipitate falls at 77-79° C. (S). A third precipitate is similarly obtained at 84-86° C. (y)n In the serum of the ox, sheep , and hcrse the a precipitate is absent : in cold-blooded animals, the H and y varieties are absent. 3. Take a fresh portion of B, and saturate it with sodium sulphate. The serum albumin is precipitated (completely after prolonged shaking). This is due to the formation of sodio-magnesium sulphate. B was already saturated with magnesium sulphate (MgS04 + 7ILO) ; on adding sodium sulphate a double salt ( MgS04.Na.,S04 + GH.,0) is formed. Shake some serum with sodium sulphate alone. A small precipitate of globulin is produced. Saturate another portion of the serum with sodio-magnesium sulphate ; both globulin and albumin are precipitated. Of the methods used for precipitating serum globulin practically only two are used now. These are Hammarsten's and Kauder's. The other methods only precipitate the globulin incompletely. Kauder's method is rapid and efficacious : if the globulin is filtered off, the albumin may be precipitated in the filtrate by complete saturation with the same salt, ammonium sulphate. This method avoids the trouble of using two salts as described under 3. This last method is instructive, but not nearly so quick as Kauder's. With regard to the separation of serum albumin into o, 8 and y varieties by the use of the method of fractional heat coagulation, it must be men- tioned that at present no further difference has been shown to exist between them, and the opinion has been very freely expressed that the results obtained are not trustworthy. I am convinced that the method is a good one, especially as in other cases (see Muscle) theproteids so separated can be shown to possess other differences. In the case of serum, however — and the same is true for egg albumin — the matter must still be considered sub judice. 1 On account of the prolonged nature of these operations they must necessarily be performed by the demonstrator beforehand. 151 LESSON XXI COAGULATION OF BLOOD 1. Salted plasma and oxalate plasma are the two kinds of blood-plasma which are most easily prepared (Lesson IX.). The separation of the plasma and corpuscles may most readily be carried out by the centrifugal machine (one form of this is represented in the next figure) ; the corpuscles settle and the supernatant plasma is pipetted off. The principal properties of these two forms of plasma have already been —Centrifugal oade byRunne of Heidelberg. Glass vessels containing the sub- Used are placed within the six- metallic tubes which hang vertically while icblnery is set going they ii)T out into the horizontal position. A - engine may be used to work the instrument. A small bnt effective hand- Laidkvw & Co., Glasgow. I Lesson IX. ; the following additional experiments may now be ned. 1. Seat a portion of the salted plasma to 60° C. The fibrinogen is < 'I (coagulated by beat) at 56° C. Filter. Dilute the filtrate as in I • won IX. ;;. and add fibrin ferment. No coagulation occurs. ite plasma or decalcified plasma coagulates when a little calcium 152 ESSENTIALS OF CHEMICAL PHYSIOLOGY chloride is added, but not if the oxalate plasma lias been previously heated to 60° C. and filtered because the fibrinogen has been thus removed. 4. Hydrocele Fluid. — This does not clot spontaneously, or only very slowly. Divide it into four parts — A, B, C, and D. To A add an equal volume of serum. To B add a few drops of fibrin-ferment solution. To C add a piece of buffy coat. Put them into the warm bath, and coagulation takes place in each. The serum or the buffy coat supplies the missing fibrin ferment. The serum does not produce its effect in virtue of the serum globulin it contains ; hydrocele fluid contains both fibrinogen and serum globulin, as the following experiment shows : — Take the portion I) and half saturate it with sodium chloride by adding to it an equal bulk of saturated solution of sodium chloride. Fibrinogen is precipitated. The precipitate is a small one, and on standing aggregates together, and so becomes more apparent. Filter, and saturate the filtrate with sodium chloride, or, better, magnesium sulphate ; serum globulin is precipitated. 5. Intravascular coagulation. — A solution of nucleo-proteid from the thymus, testis, lymphatic glands, or kidney has been prepared beforehand by the demonstrator. It rna}' be prepared in one of two ways. (a) Wooldridge's Method. — -The gland is cut up small and extracted with water for 24 hours. Weak acetic acid (05 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-proteid (called tissue-fibrinogen by Wooldridge) falls to the bottom of the vessel. This is collected and dissolved in 1-per-cent. sodium carbonate solution. (b) The Sodium Chloride Method. — The finely divided gland is ground up in a mortar with about an equal volume of sodium chloride. The re- sulting viscous mass is poured into excess of distilled water. The nucleo- proteid 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, also are fall of clot. 153 LESSON XXII MUSCLE 1. 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. 2. The extract will probably be faintly acid. The acid is lactic acid. It may be identified by the following reaction : — 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 the liquor ferri perchloridi of the British Pharmacopoeia. 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 ol other acids (for instance, more than 0-2 per cent, of hydro- chloric acid) are necessary to decolorise the test solution. 6. The coagulation of muscle is very like 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. Coagu- lation, that is. formation of a clot of myosin, occurs in both, but earliest in that at 40 I . 4. Add a few drops of 2-per-cent. acetic acid to some of the extract ; a stringy precipitate of myosinogen is produced. .-,. Remove the clot of myosin from 3; observe it is readily soluble in 10-per-cent. sodium chloride, and also in 0-2 per-cent. hydrochloric acid, forming syntonin, 6. Perform fractional heat coagulation — (a) With the original extract. Coagula are obtained at 47°, 56°, 68°, (b) With the liquid (salted muscle serum) in 3, after separation of the clot. Coagula are obtained at 68° ami 73° C. (c) With muscle extract whicb has been saturated with magnesium sulphate and filtered. The globulins are thus separated. Coa«ulation now a; 78' ' ., but the amount of coagulum is smalL 154 ESSENTIALS OF CHEMICAL PHYSIOLOGY The following table represents these facts concisely Names of proteid 1. Musculin or i paramyosinogen I 2. Myosinogen . 3. Myo- globulin 4. Myo -albumin Coag ala- tion tem- perature 47° c. 56° c. 63° c. , 73° c. Action of UgS04 precipitated not precipitated Is it globulin or albumin ? globulin albumin I These go to form the muscle clot (myosin) These are left in the muscle serum 7. Pigments of Muscle : — (a) Notice the difference between the red and pale muscles of the rabbit. (b) Examine a piece of red muscle (e.g. the diaphragm) spectroscopically for oxyhemoglobin (or it may be more convenient to make an aqueous extract of the muscle and examine that) . cv B D E PIG. 61.— 1, Absorption spectrum of rnyohaamatin, as seen in muscle rendered transparent by glycerin. 2, Absorption spectrum of modified myoha3inatin. (o) A piece of the pectoral muscle of a pigeon has been soaked in glycerin. Press a small piece between two glass slides and place it in front of the spec- troscope. Observe and map out the bands of myohtematin. (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 myohaematin. Filter it ; compare its spectrum with that of hsemochromogen. The myohaematin bands are rather nearer the violet end of the spectrum (fig. 61, spectrum 2) than those of hsemochromogen (fig. 57, spectrum 9). 8. Creatine : — (a) Take some of the red fluid described in 7, d, and let it evaporate to dryness in a desiccator over sulphuric acid (fig. 62). In a day or two crystals of creatine tinged with myohaematin separate out. MUSCLE 155 tb) Take an aqueous extract of niuscle, like Liebig's extract or 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 and evaporate the filtrate on the water-bath to a thick syrup. Set it aside to cool, and in a few days crystalline deposits of creatine will be found at the bottom of the vessel. These are 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. Pig. 62. — A desiccator. (Gscheidlen.1 Creatinine can be obtained from meat extracts by Johnson's method (see pp. 118, 119) : indeed, Johnson states it is usually more abundant than crea- tine, and that the same is true for fresh meat. Note. — The exercises on muscle plasma described in this lesson have been in the main derived from my own investigations on the subject C Journ. of Physiol.,' viii. p. loo, 'Chemical Physiology and Pathol.,' Chap. XX.). The same subject has been recently taken up by v. Fiirth (•Arch. Exp. Path. u. Pharni. 1895,' vol. xxxvi. p. 231). His nomenclature of the proteids is somewhat different from mine, but on the main question we are in substantial agreement — viz. that in the muscle plasma there are two pro- teids which become changed, and contribute to the formation of the muscle clot, or myosin fibrin as he terms it. He uses physiological saline solution to extract the muscle plasma, and this coagulates spontaneously on standing ; paramyosinogen passes directly into the condition of myosin-fibrin ; but myo- ginogen first passes into a soluble condition (coagulable by heat at the remark- ably low temperature of 40° C.) before the myosin fibrin separates out. The main points of difference between us are (1) he looks upon myosinogen ;•- imi being a true globulin, though like a globulin in some of its characters ; (2) Myo-globulin is not a separate proteid, but only some myosinogen which has escaped coagulation. (3) The phenomenon I have termed re-coagulation of muscle plasma, he looks upon as being merely a reprecipitation ol the globulins, and not a true coagulation process. He does not appear to have investigated the question of a specific myosin ferment. When a muscle is gradually heated, at a, certain temperature it contracts permanently and loses its irritability. This phenomenon is known as heat- 156 ESSENTIALS OF CHEMICAL PHYSIOLOGY rigor, and is due to the coagulation of the proteids in the muscle. If a tracing is taken of the shortening, it is found that the first shortening occurs at the coagulation temperature of para-rnyosinogen (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 proteid which coagulates at 40° C. (T. G. Brodie). v. Fiirth regards this additional proteid as the soluble myosin alluded to above, some of which in frog's musclfi is present before rigor mortis occurs. 157 LESSON XXIII UREA AND CHLORIDES IN URINE ESTIMATION OF UREA If albumin is present it must be first separated by boiling after acidulation with acetic acid if necessary, and filtering off the flakes of coagulated proteid. The three chief methods of estimating urea ar< i (a) The mercuric nitrate or Liebig's method. (6) The hypobromite, or Htifher's method. (c) The method of Morner and Sjbqvist. (a) Liebig's Method. — The combination between urea and mercury has the formula (COX.H,) ,Hg(NO.).,(HgO).. It forms a white precipitate, in- soluble in water and weak alkaline solutions. It is therefore necessary to prepare a standard solution of mercuric nitrate, and to have an indicator by which to detect the point when all the urea has entered into combination with the mercury, and the latter slightly predominates. This indicator is sodium carbonate, which tuves a yellow colour with the excess of mercury, owing to the formation of hydrated mercuric oxide. Theoretically. 100 parts of urea should require 720 parts of mercuric oxide but practically 772 of the latter are necessary to remove all the urea, and at the same time show the yellow colour with alkali : consequently the solution of mercuric nitrate must be of empirical strength in order to give accurate results. The following solutions must be prepared — i. Standard mercuric nitrate solution. Dissolve 77-2 grammes of red oxide of mercury (weighed after it lias been dried over a water-bath), or 71-5 gr. of the metal itself, in dilute nitric acid. Expel excess of acid by evapor- ating the liquid to a Byrupy consistence. Makeup to 1,000 c.c. with distilled water, adding the water gradually. Tins solution is of such a strength that 18 c.c. will precipitate 10 c.c. (if a 2-per-cent urea solution. Add o2'6 c.c. of to the litre of the mercuric nitrate solution and shake well ; then 20 c.c. (instead of 19) = 10 c.c. 2-per-cent. urea solution, i.e. 1 c.c. = *01 gr. urea. ii. Baryta mixture. This is a mixture of two volumes of solution of barium hydrate with one of solution of barium nitrate, both saturated in the cold. Analysis. Take 40 c.c. of urine. Add to this 20 c.c. baryta mixture and filter off th< precipitate of barium salts (phosphates and sulphates). Take 15 c.c. of the filtrate (this corresponds to 10 c.c. of urine) in a beaker. Hun into it the mercuric nitrate solution from a burette, until it is found that, on mixing a drop of the mixture with a drop of ,i -nturated solution of sodium 158 ESSENTIALS OF CHEMICAL PHYSIOLOGY carbonate on a white tile, a pale lemon colour is produced. Then read the amount used from the burette, and calculate thence the percentage of urea. Corrections. — This method only approaches accuracy when the quantity of urea present is about 2 per cent., which is about the normal percentage of urea in urine. The chlorine in the urine must also be estimated, and the quantity of urea indicated reduced by the subtraction of 1 gramme of urea for every 1*3 gramme of sodium chloride found. If the urine contains less than 2 per cent, of mea, 0*1 c.c. of mercuric nitrate solution must be deducted for every 4 c.c. used ; if more than 2 per cent, of urea, a second titration must be performed with the urine diluted with half as much water as has been needed of the mercurial solution above 20 c.c. Suppose that 28 c.c. have been used in the first titration, the excess is 8 c.c. ; therefore 4 c.c. of water must be added to the urine before the second titration is made. When ammonium carbonate is present, first estimate the urea in one portion of urine, and the ammonia by titration with normal sulphuric acid in another ; O017 gramme of ammonia = 0"030 of urea. The equivalent of ammonia must be added to the urea found in the first portion of urine. b. The Hypobromite Method. — This is a far easier method. It consists in decomposing urea into water, carbonic acid, and nitrogen by means of an alkaline solution of hypobromite of soda ; the carbonic acid combines with, the soda, and the nitrogen which is evolved is measured, and the quantity of urea calculated from this. There are many kinds of apparatus for per- forming this operation, but the best is that of Dupre (see Lesson X.). Beactions and Corrections. — The reaction by which urea is decomposed in this proceeding may be denoted by the following formula : — CON,!!, + 3NaBrO = C02 + N2 + 2H,0 + 3NaBr. From 1 gramme of urea 0*46 gramme of nitrogen = 372-7 c.c. is obtained. In practice, however, it is found that only 354*3 c.c. are obtained,1 except in diabetic urine, in which the urea yields nearly the normal amount of nitrogen. Moreover, urine contains small quantities of creatinine and urates, which yield some of their nitrogen when acted on by sodium hypo- bromite. When great exactitude is required these must be removed — creatinine by an alcoholic solution of zinc chloride, and the urates by acetate of lead followed by sodium phosphate (Yvon) 5 c.c. of a 2-per-cent. solution of urea in urine yield 35*4 c.c. of nitrogen. This quantity is taken as representing 2 per cent, of urea, and serves as a basis for the graduations of the tubes which are marked in percentages. When exactitude is required, the quantity of nitrogen must be measured in cubic centimetres, and the volume obtained corrected for temperature,, pressure, and tension of aqueous vapour by the formula below.2 1 The cause of this loss of nitrogen has been investigated by Luther, Zeit. physiol. Chem. xiii. p. 500. He finds part is combined as a nitrate, and part in an unknown organic compound which gives off ammonia when distilled with alkali. - V = correct volume ; V = vol. observed ; B = barometric pressure corrected for temperature ; t= temp, in degrees Centigrade; T = tension of aqueous vapour in millimetres of mercury at t° (see table, p. 7). Then Vx(B-T) V' = 760 x (1 + 0-0036650 UREA AND CHLORIDES IN URINE 159 Of the two methods just described, Liebig's is so cumbrous and inexact that it has almost passed out of use. Tlie hypobromite method holds its own, as it is easy and sufficiently exact for clinical purposes. When absolute accuracy is necessary, one of the numerous recently introduced methods must be employed, and of these the method of Morner and Sjoqvist appears to be best. (c) Method of Morner and Sjoqvist.— The following reagents are neces- sary :— i. A saturated solution of barium chloride containing 5 per cent, of barium hydrate. ii. A mixture of ether and alcohol in proportion 1 : 2. iii. The apparatus, etc., necessary for carrying out Ejeldahl's method of estimating nitrogen (see p. 186). Analysis. — Five c.e.-of urine are mixed with 5 c.c. of the barium mixture, and 100 c.c. of the mixture of ether and alcohol. By this means all nitrogenous substances except urea are precipitated. Twenty-four hours later this is filtered off. and the precipitate is washed with 50 c.c. of the ether-alcohol mixture, the filter-pump being used to accelerate the process. The washings are added to the filtrate ; a little magnesia is added to this to drive off ammonia. The alcohol and ether is then driven off at a temperature of 55° C. and evaporation is continued at this temperature until the volume of the residue is 10 — 15 c.c. The nitrogen in this is estimated by Ejeldahl's method. The nitrogen found is multiplied by 2'143, and the result is the amount of urea. PREPARATION OF UREA FROM URINE (1) Evaporate the urine to a small bulk. Add strong pure nitric acid in excess, keeping the mixture cool during the addition of the acid. Pour off the excess of fluid from the crystals of urea nitrate which are formed; strain through muslin and press between filter paper. Add to the dry product barium carbonate in large excess. This forms barium nitrate and sets the urea free. Mix thoroughly with sufficient methylated spirit to form a paste. Dry on a water-bath and extract with alcohol; filter; evaporate the nitrate on a water-bath and set aside. The urea crystallises out, and may be de- colorised by animal charcoal and purified by recrystallisation. (2) The following method is well adapted for the preparation of micro- scopic specimens of urea and urea nitrate : Take 20 c.c. of urine ; add • baryta mixture ' itwo volumes of barium hydrate solution and one volume of barium nitrate solution, both saturated in the cold) until no further preci- pitate is produced; filter, evaporate the filtrate to a thick syrup on the water-bath, and extract with alcohol; pour off and filter the alcoholic extract ; evaporate it to dryness on the water-bath and take up the residue with water. Place a drop of the aqueous solution on a slide and allow it to crystallise; crystals of urea separate out. Place another drop on another and add a drop of nitric acid ; crystals of una nitrate separate out. ESTIMATION OF CHLORIDES '1 In- chlorides in the urine consist of those of sodium and potassium, the latter only in small quantities. The method adopted for the determination 160 ESSENTIALS OF CHEMICAL PHYSIOLOGY of the total chlorides consists in their precipitation by a standard solution of silver nitrate or mercuric nitrate. Mohfs Method. — Precipitation by silver nitrate. The following solutions must be prepared : — Standard silver nitrate solution. Dissolve 29*075 grammes of fused nitrate of silver in a litre (1,000 c.c.) of distilled water ; 1 c.c. = 0*01 gramme of sodium chloride. (a) Saturated solution of neutral potassium chromate. Analysis. — Take 10 c.c. of mine ; dilute with 100 c.c. of distilled water. Add to this a few drops of the potassium chromate solution. Drop into this mixture from a burette the standard silver nitrate solu- tion ; the chlorine combines with the silver to form silver chloride, a white precipitate. When all the chlorides are so precipitated, silver chromate (red in colour) goes down, but not while any chloride remains in solution. The silver nitrate must therefore be added until the precipitate has a pink tinge. From the amount of standard solution used, the quantity of sodium chloride in 10 c.c. of urine, arid thence the percentage may be calculated. Sources of Error and Corrections. — A nigh-coloured urine may give rise to difficulty in seeing the pink tinge of the silver chromate : this is overcome by diluting the urine more than stated in the preceding paragraph. 1 c.c. should always be subtracted from the total number of c.c. of the silver nitrate solution used, as the urine contains small quantities of certain compounds more easily precipitable than the chromate. (6) To obviate such sources of error the following modification of the test, as described by Sutton,1 may be used : — 10 c.c. of urine are measured into a thin porcelain capsule and 1 gramme of pure ammonium nitrate added ; the whole is then evaporated to dryness, and gradually heated over a small spirit lamp to low redness till all vapours are dissipated and the residue becomes white. It is then dissolved in a small quantity of water, and the carbonates produced by the combustion of the organic matter neutralised by dilute acetic acid ; a few grains of pure calcium carbonate to remove all free acid are then added, and one or two drops of potassium chromate. The mixture is then titrated with decinormal silver solution (16"966 gr. of silver nitrate per litre) until the end reaction, a pink colour, appears. Each c.c. of silver solution represents 0*005837 gr. of salt; con- sequently, if 12*5 c.c. have been used, the weight of salt in the 10 c.c. of urine is 0'07296 gr., or 0-7296 per cent. If 5"9 c.c. of urine are taken for titration, the number of c.c. of silver solution used will represent the number of parts of salt per 1,000 parts of urine. (c) Liebig's Method. — Precipitation by mercuric nitrate. The following solutions must be first prepared : — i. Standard mercuric nitrate solution : — Dissolve 20 grammes of pure mercury in boiling nitric acid ; then dilute to nearly a litre. To dilute this to the right strength, preliminary experiments must be performed with a standard solution of pure sodium chloride, 20 grammes to the litre. Take 10 c.c. of the standard sodium chloride solution, add to this 2 c.c of a 4-per cent, solution of urea and 5 c.c. of a saturated solution of sodium sulphate. 1 Volumetric Analysis, p. 309. UREA AND CHLORIDES OF URINE 161 Into this mixture allow the mercuric nitrate solution to now from a burette, stining the mixture the while. A precipitate forms, which redissolves on stirring ; add the mercuric nitrate solution till a permanent precipitate (not an opalescence) forms ; the reaction is then complete. The strength of the mercurial solution is thus determined, and it is then diluted so that 20 c.c. = 0*2 gramme of sodium chloride = 10 c.c. of the standard sodium chloride solution ; 1 c.c, therefore, corresponds to 0-01 gramme of sodium chloride, or 0-006059 gramme of chlorine. ii. Baryta mixture, made by adding two volumes of barium hydrate solution to one of barium nitrate solution, both saturated in the cold. iii. Dilute nitric acid (1 in 20). Analysis. — Take 40 c.c. of urine. Add 20 c.c. of baryta mixture. Filter off the precipitate which forms, which consists of sulphate and phosphate of barium. Take 15 c.c. of the filtrate : this corresponds to 10 c.c. of the original urine. Render this slightly acid with dilute nitric acid. Run in the standard mercuric nitrate solution from a burette, stirring the mixture well until a permanent precipitate appears. Read off the number of c.c. used ; multiply by 0-01. This gives the amount of chloride as sodium chloride contained in 10 c.c. urine. Explanation and Corrections. — This test depends on the fact that when mercuric nitrate and sodium chloride in solution are mixed, sodium nitrate and mercuric chloride, which are both soluble in water, are formed. It is not till all the chloride in the urine is so decomposed that mercuric nitrate begins to combine with the urea present to form a permanent white pre- cipitate. Hence the necessity of estimating the chlorides when using Liebig's method for the determination of urea. In order to obtain the exact point at whicli the precipitate becomes a permanent one, the process must be repeated in another specimen. The advantage of this process is its simplicity : its disadvantage is that the end point is rather obscure. If the urine used is albuminous, the albumin must be first removed by boiling, after the addition of a few drops of acetic acid, and filtering off the precipitated proteid. 162 ESSENTIALS OF CHEMICAL PHYSIOLOGY LESSON XXIV ESTIMATION OF PHOSPHATES AND SULPHATES IN URINE ESTIMATION OF PHOSPHATES The phosphoric acid in the urine is combined with soda, potash, lime, and magnesia. (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 O005 gramme of phosphoric acid (P.205). ii. Acid solution of sodium acetate. Dissolve 100 grammes of sodium acetate in 900 c.c. of water ; add to this 100 c.c. 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.1 Heat the mixture to 80° C. Run into it while hot the standard uranium nitrate solution from a burette until a drop of the mixture gives a distinct brown colour with a drop of potassium 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. (b) Estimation of the phosphoric acid combined with lime and magnesia (alkaline earths). Take 200 c.c. 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 phos- phates in this volumetrically by the standard uranium nitrate as before. Subtract the phosphoric acid combined with the alkaline earths thus obtained from the total quantity of phosphoric acid, and the difference is the amount of acid combined with the alkalis soda and potash. (c) Instead of uranium nitrate a standard solution of uranium acetate may be used. The directions for the making of these standard solutions will be found in Sutton's ' Volumetric Analysis.' As a rule, it is less troublesome, and not much more expensive, to purchase standard solutions ready-made. 1 In using uranium nitrate it is imperative that sodium acetate should ac- company tbe 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 PHOSPHATES AND SULPHATES IN URINE 163 ESTIMATION OF SULPHATES The sulphates in the urine are of two kinds : the pre-formed sulphates — viz. those of soda and potash, and the combined or ethereal sulphates. (a) For the determination of the total amount of sulphuric acid (S03) (i.e. pre-formed and combined sulphuric acid together) in the urine, one of two methods is adopted :— 1. Volumetric method. 2. Gravimetric method. Both methods will be given here ; the former is, however, better suited for class experiments. 1. Volumetric Determination. — This process consists in adding to a given volume of the urine a standard solution of chloride of barium so long as a precipitate of barium sulphate is formed. The following solutions are necessary : — i. Standard barium chloride solution : 305 grammes of crystallised chloride of barium in a litre of distilled water ; 1 c.c. of this solution corre- sponds to O01 gi-amme of sulphuric acid (SO.,). ii. Solution of sulphate of potash : 20 per cent. hi. Pure hydrochloric acid. Method. — 100 c.c. of urine are taken in a flask. This is rendered acid by 5 c.c. of hydrochloric acid, and boiled. The combined sulphates are thus converted into ordinary sulphates, and give a precipitate like them with barium chloride. The chloride of barium solution is allowed to drop into this mixture as long as any precipitate occurs, the mixture being heated before every addition of barium chloride to it. After adding 5 to 8 c.c. of the standard solution, allow the precipitate to settle ; pipette off a few drops of the clear, supernatant fluid into a watch-glass ; add to it a few drops of the standard barium chloride solution. If any precipitate occurs, return the whole to the flask and add more barium chloride ; again allow the precipitate to settle, and test as before ; go on in this way until no more barium sulphate is formed on the addition of barium chloride. Excess of barium chloride must also be avoided ; when only a trace of excess is present a drop of the clear fluid removed from the precipitate gives a cloudiness with a drop of the potassium sulphate solution placed on a glass plate over a black ground. If more than a cloudiness appears, too large a quantity of barium chloride has been added, and the operation must be repeated. From the quantity of barium chloride solution used, the per- centage of sulphuric acid in the urine is calculated. 2. Gravimetric Determination (i.e. by weight).— This method consists in weighing the precipitate of barium sulphate obtained by adding barium chloride to a known volume of urine ; 100 parts of sulphate of barium correspond to 34*33 parts of sulphuric acid (SO;1). Method (Salkowski). — 100 c.c. of urine axe taken in a beaker. This is boiled with 5 c.c. of hydrochloric acid a I "lore. Chloride of barium is added till no more precipitate occurs. collected on a mall filter of known ash, and washed with hot distilled water till no more barium chloride occurs in the lilt rate — 164 ESSENTIALS OY CHEMICAL PHYSIOLOGY i.e. until the filtrate remains clear after the addition of a few drops of hydric sulphate. Then wash with hot alcohol, and afterwards with ether. Remove the filter, and place it with its contents in a platinum crucible. Heat to redness. Cool over sulphuric acid in a desiccator ; weigh, and deduct the weight of the crucible and filter ash .; the remainder is the weight of barium sulphate formed. Error. — When the experiment is carried out as above there is a slight error from the formation of a small quantity of sidphide of barium. This may be corrected as follows : After the platinum crucible has become cool add a few drops of pure sulphuric acid (H,S04). The sulphide is converted into sulphate. Heat again to redness to drive off excess of sulphuric acid. (b) The following is Salkowski's 1 method of estimating the combined sulphuric acid — that is, the amount of SO . in ethereal sulphates : — 100 c.c. of urine are mixed with 100 c.c. of alkaline barium chloride solution, which is a mixture of two volumes of solution of barium hydrate with one of barium chloride, both saturated in the cold. The mixture is stirred, and after a few minutes filtered ; 100 c.c. of the filtrate ( = 50 c.c. of urine) are acidified with 10 c.c. of hydrochloric acid, boiled, kept at 100° C. on the water-bath for an hour, and then allowed to stand till the precipitate has completely settled ; if possible, it should be left in this way for twenty-four hours. The further treatment of this precipitate ( = combined sulphates) is then carried out as in the last case. Calculation. — 233 parts of barium sulphate correspond to 98 parts of H2S04, or 80 parts of SO. or 32 parts of S. To calculate the H,S04, multiply the weight of barium sulphate by = 0*4206 ; to calculate the S03 multiply by ®? = 0-34335 ; to calculate the S multiply by ~ = 0-13734. This method 233 Zoo of calculation applies to the gravimetric estimation both of total sulphates and of combined sulphates. (c) To obtain the amount of pre-formed sulphuric acid subtract the amount of combined SO, from the total amount of SOa. The difference is the pre-forrned SO ... Example : 100 c.c. of urine gave 0-5 gramme of total barium sulphate. 80 This multiplied by - = 0-171 gr. = total S03. Another 100 c.c. of the same Zoo urine gave 0*05 gr. of barium stdphate from ethereal sulphates ; this multi- plied by f80 = 0-017 gr. of combined SO,. Total SO. - combined S03 = 0-171-0-017 = 0-154 gr. of pre-formed S03. 1 Zeit. physiol. Chem. x. p. 346. This method is a modification of Baumann's original method, ibid. i. p. 71. 165 LESSON XXV URIC ACID AND CREATININE 1. Preparation of Pure Uric Acid. — If one wishes to prepare pure uric acid the solid urine of a reptile or bird, which consists principally of the acid ammonium salt, should be selected ; one has not then to separate any pig- ment. It is boiled with 10 per cent, caustic soda or ammonia, diluted, and then allowed to stand. The clear fluid is decanted and poured into a large excess of water to which 10 per cent, of hj-drochloric acid has been added ; after twenty-four hours, crystals of uric acid are deposited. These may be purified by washing, re-solution in soda, and re-precipitation by acid. 2. Estimation of Uric Acid (Hopkins' method). — The following reagents are required : — Pure chloride of ammonium, finely powdered. A wash bottle containing a filtered saturated solution of the same salt. A twentieth normal solution of potassium permanganate made by dissolving 1*581 grammes of permanganate in a litre of water. Separation of uric acid from, the urine. — Measure 100 c.c. of urine into a beaker of about 150 c.c. capacity. Add to this 25 grammes (approximately weighed) of ammonium chloride? stirring briskly till all the salt is dissolved. Now add 2 c.c. of strong ammonium hydrate, and allow the mixture to stand until the precipitate of ammonium urate, which rapidly forms, has wholly settled to the bottom of the beaker ; its subsidence is promoted by occasional brisk stirring. Adjust a small filter paper (7 cm. diam.) in a funnel of such size that only a small margin of glass projects above the edge of the folded paper, and transfer to this the ammonium urate precipitate Filtration should not be commenced until the precipitate has settled satisfactorily, and the supernatant liquid is clear. The latter should be first poured on to the filter, the precipitate being so far as possible retained in the r until the greater part of the clear liquid has filtered through; finally transfer the whole to the filter with the help of a wash bottle containing saturated ammonium chloride solution. After the filter has thoroughly drained, wash the precipitate twice again with the same solution. While the last washings arc running through the paper, distilled water should be heated to boiling in a wash bottle provided with a fine jet. The funnel containing the filter is now held horizontally ovei a small porcelain basin (of about 50 c.o. capacity) ami the precipitate washed into the latter with a jet of hot water, the Sltei il lelf being afterwards opened out mer i he n order that any urate adhering to its folds can lie washed off. Not 166 ESSENTIALS OF CHEMICAL PHYSIOLOGY ruore than 20-30 c.c. of water need be employed in this transference ; if much more has been used the liquid should be concentrated over the water bath at this stage. A little strong HC1 (1 c.c.) is next added to the contents of the basin, and the whole is then heated over a burner until it just reaches the boiling point. It is then set aside for the uric acid to crystallise out. Titration of the uric acid. — If the mixture is artificially cooled all the uric acid will separate out in 2 hours, otherwise it is best allowed to stand over night or longer. The crystals are filtered off through a very small filter paper (4 cm. diam.) ; the filtrate is received into a graduated cylinder so that the amount of mother liquor may be noted (see below). The uric acid is next washed with cold distilled water until free from chlorides. It is unnecessary to transfer the whole to the filter ; the greater part may be washed by decanta- tion. Such of the crystals as are upon the filter are now washed back into the basin (best by the aid of hot water) and the whole quantity is dissolved by heating to boiling with 1 c.c. of 10 per cent, sodium carbonate solution and as much distilled water as the basin will safely hold. The solution is transferred to a j-litre Erlenmeyer flask, which should be marked roughly at 100 c.c. The solution is made up to this mark with dis- tilled water, and cooled to the temperature of the room. The standard permanganate solution being ready in a burette, 20 c.c. of strong sulphuric acid are added to the contents of the flask, and the mixture shaken and titrated. During the addition of the standard solution the liquid in the flask should be kept in vigorous movement. It will be found that at first the disappear- ance of the pink colour is so rapid that each drop as it is added is decolorised before it has time to diffuse through the whole liquid. The firsb instan- taneous appearance of a diffused flush throughout the solution indicates the end point of the reaction. This colour rapidly disappears, but it will be found that the effect of adding further quantities of permanganate after the end point has been passed is quite different from the effect before the end point was reached ; each drop is now able to diffuse throughout the fluid. For each c.c. of the solution necessary to produce the end point just described 0*00375 gramme of uric acid is present. To the value so obtained 1 mgm. must be added for each 15 c.c. of the mother liquor from which the crystals separated. Thus the uric acid from 100 c.c. of a sample of urine used up 18*5 c.c. of the standard permanganate solution. The mother liquor filtered from the crystals measured 25 c.c. 18-5 x -00375 = -0694 gr. nearly •001 x ^ = -0017 15 Total = -0711 The urine contained 71 mgms. uric acid per 100 c.c. 3. Estimation of Creatinine. — The crystalline compound which creatinine forms with zinc chloride is employed in estimating the quantity of creatinine in urine ; 100 parts of the compound corresponds to 62-42 of creatinine. Method. — Take 250 c.c. of urine. Add milk of lime and calcium chloride URIC ACID AND CREATININE 167 in excess to precipitate the phosphates. Filter, and evaporate the filtrate to a small bulk ; to this add 50 c.c. absolute alcohol, and let the mixture stand for six hours. Then add 10 or 15 drops of an alcoholic solution of zinc chloride ; the crystals form, and after two or three days' standing in a dark place rnay be collected on a weighed filter. Wash with 90 per cent, alcohol, dry and weigh, and thence calculate the percentage of creatinine. 4. Estimation of Creatinine. Johnson's Method. — Take 100 c.c. of urine ; add to it 5 c.c. of a saturated solution of sodium acetate, and then 20 c.c. of a saturated solution of mercuric chloride. This produces an abundant precipi- tate of urates, sulphates, and phosphates. Filter. Set the filtrate aside for 24 hours, and the mercury compound of creatinine crystallises out. Examine this deposit with the microscope : note it is composed of spherules. For quantitative purposes, this is collected, washed, dried, and weighed in the usual way. One-fifth of the weight obtained is creatinine. Throughout the processes no heat is used, otherwise the characteristic properties of urinary creatinine are altered ; but if only a quantitative analysis is wanted the method may be hastened by boiling the first filtrate for ten minutes, instead of letting it stand 24 hours. In order to separate the base itself (see pp. 118, 119), much larger volumes of urine must be employed, for there is considerable loss in the later stages of the process. Johnson himself used some hundreds of litres. 168 ESSENTIALS OF CHEMICAL PHYSIOLOGY LESSON XXVI THE PIGMENTS OF THE UBINE The urinary pigments are numerous, and have from time to time been described under different names by various observers. 1. TTrochrome. — -This is the essential yellow pigment of the urine. The word was originally introduced by Thudichum, and the substance he obtained is now recognised to have been a mixture of several pigments, of which, however, the essential yellow pigment formed a large proportion. Garrod's method of separating it from the urine is as follows : — The urine is saturated with ammonium sulphate and filtered. The filtrate contains the pigment ; this is shaken with alcohol. The alcohol separates readily from the saline mixture, and as it does so dissolves out much of the urochrome. By repeated extraction all the pigment passes into solution in the alcohol. The alcoholic solution is diluted with water, and the mixture again saturated with ammonium sulphate. The alcohol con- taining the pigment in solution again separates out. The second alcoholic solution is made faintly alkaline with ammonia and evaporated to dryness. The residue is extracted once or twice with acetic ether, and then again dissolved in strong alcohol. Finally the alcohol is concentrated till it is deep orange in tint, and poured into an equal volume of ether. The pure pigment is by this means precipitated as an amorphous brown powder. TJrochrome shows no absorption bands. As already stated (p. 104) it is probably an oxidation product of urobilin. 2. Urobilin. — As already stated (pp. 70, 103), urobilin is a derivative of the blood-pigment, and is identical with stercobilin. Probably both reduction and hydration occur in its formation. It is a substance very like the sub- stance named hydrobilirubin by Maly, which he obtained by the action of sodium amalgam on bilirubin. The following formulae ' show the relation- ship between these allied pigments : — Hsematin C3oH3ON404Fe Bilirubin . . . . . . C32H36N406 Hydrobilirubin CMH40N4O7 Urobilin is probably a further stage in reduction. Normal urine contains but little urobilin ; what is present is chiefly in the form of a colourless chromdgen, which by oxidation is converted into urobilin. In numerous pathological conditions urobilin is abundant. The so- 1 The formulae given are those of Nencki and Sieber. They differ from those previously given (p. 87) by Hoppe-Seyler. THE PIGMENTS OF THE UEINE 169 called ' pathological urobilin ' described by previous writers is ordinary urobilin incompletely separated from various impurities. The following are the 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 off. 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. (o) The urates are first removed, then the urine is acidified and 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 larj,re separating funnel. The ether-chloroform extract is then rendered faintly alkaline and shaken with distiDed water, and the urobilin passes into solution in the water. The aqueous solution is now once more saturated with ammonium sulphate and slightly acidified ; it then once more yields its pigment 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 con- verted 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 F, slightly overlapping the latter (fig. 63, 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 sufficient sulphuric or hydrochloric acid is added to render the liquid faintly acid, a turbidity is produced. This turbid liquid shows an additional baud in the region of the E line (fig. 63, spectrum 6). which is probably due to the special light absorp- tion exercised by fine particles of urobilin in suspension. It wholly disappears when the precipitate is filtered off, and when it is re-dissolved the ordinary band alone is visible. '■'>. Uroerythrin. — This is the colouring matter of pink urate sediments. It may be separated from the sediment as follows : — The deposit is washed witli 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. I roerythrin has a great affinity for urates, witli which it appears to form a loose compound. Its solutions are rapidly decolorised by light. Spectro- scopically it shows two rather ill-defined hands ifig. 63, spectrum 7). It gives a green colour with caustic potash, and red or pink with mineral acids. • thrin appears to be a small but constant constituent of urine. Its origin and relationship to other pigments are unknown. 1. Haematoporphyrin.— This also occurs in small quantities in normal 170 ESSENTIALS OF CHEMICAL PHYSIOLOGY urine. In some pathological conditions, especially after the administration of certain drugs {e.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 : — Caustic alkali is added to the urine ; this causes a precipitate of phos- O D E b F G- Fig. 63.— Chart of absorption spectra : 1, acid hasmatoporphyrin ; 2, alkaline haematoporphyrin ; 3, hgematoporphyrin as found sometimes in urate sediments ; 4, acid urobilin, concentrated ; 5, acid urobilin, dilute ; 6, the E band spectrum of urobilin ; 7, uroerythrin : 8, urorosein concen- trated— on dilution the band shrinks rapidly from redward end. (After F. G-. Hopkins.) phates, which carries down the pigment with it ; the pigment may be dis- solved 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 mine is sufficiently rich in the pigment, the bands shown are THE PIGMENTS OF THE URINE 171 those of alkaline haematoporphyrin (tig. 63, spectrum 2). On adding sulphuric acid the spectrum of acid haematoporphyrin is seen (fig. 63, spectrum 1). Occasionally urate sediments are pigmented with a form of the pigment which shows a two-banded spectrum, very like that of oxyhemoglobin (fig. 63, spectrum 3i ; by treatment with dilute mineral acids this changes immedi- ately to the spectrum of acid haematoporphyrin. 5. Chromogens in urine. — In addition to the chromogens of urobilin and haematoporphyrin 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. 112. It is easily oxidised to indigo-blue or indigo-red. 2C, H4<£°H>CH + 02 = C, H , <%>C : C <^>C6H4 + 2H ,0. [indoxyl] [indigo-blue] Indigo red is isomeric with indigo-blue, its structural formula being I ,H N. It is very rare for the urine to be actually pigmented with indigo, for the urinary indoxyl is excreted as a conjugated 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.) ib) Skaioxyl. — "When skatoxyl is given by the mouth it passes into the urine, and yields skatoxyl-red on oxidation, (c) Urorosein is distinct from indigo-red. It is produced from its chromogen by the action of mineral acids. 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 chromogen is precipitated by satura- tion with ammonium sulphate. The colour is destroyed by alkalis. It shows an absorption band between the D and E lines (fig. 63, spectrum 8). 6. Pathological pigments. — The most frequently appearing of abnormal pigments are those of blood and bile. The urine may contain accidental pigments due to the use of drugs (rhubarb, senna, logwood, santonin) ; in carbolic acid poisoning pyrocatechin and hydrochinon are chiefly responsible for the brown colour of the urine, which increases on exposure to the air. The black or dark In-own pigment called melanin may pass into the urine in cases of melanotic iptonuria see p. 127. APPENDIX HEMACYTOMETERS Gowers' s Hemacytometer. — The enumeration of the blood corpuscles is readily effected by the hemacytometer of Gowers. This instrument consists of a glass slide (fig. 64, C) , the centre of which is ruled into ^ millimetre squares and surrounded by a glass rim J millimetre thick. It is provided with measuring pipettes (A and B), a vessel (D) for mixing the blood with a saline solution (sulphate of soda of specific gravity 1,015), a glass stirrer (E), and a guarded needle (F). Fig. 64. — Hemacytometer of Sir W. Gowers. 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 puncture in the finger by means of the pipette B, and blown into the solution. The two fluids are well mixed by the stirrer, and a small drop of this diluted mixture placed in the APPENDIX 173 centre of the slide C. a cover glass is gently laid on (so as to touch the drop which thus forms a layer £ millimetre thick between the slide and cover glass), 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 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 therefore in normal human blood to be 45-50. Oliver's Hemacytometer. ' — The following method devised by Dr. George Oliver is a ready way of determining the total number of corpuscles. It is, i"n.. 86. liver' lia macytometer. 1 Tint ompany, 6 Farriogdon Avenue, London, 10. C. 174 ESSENTIALS OF CHEMICAL PHYSIOLOGY 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 65, a) until it is full. This is washed out by the dropping tube b into a graduated flattened test-tube, e, with Hayem's fluid.1 The graduations of the tube are so adjusted that with normal blood con- taining 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 fine bright line when looked at through the tube held edgeways between the fingers (d) and filled up to the 100 mark of the gradua- tion. If the number of corpuscles is less than normal, less of the diluting solution is required for the 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. H.EM0GL0BIN0METERS Gowers's Haemoglobinoineter. — 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 Fig. 66. — HEemoglobmometer of Sir W. Gowers. 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 C 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 1 Sodium sulphate 5 grammes, sodium chloride 1 grm., mercuric chloride 0-5 grm., distilled water 200 c.c. APPENDIX 175 quantity of oxyhemoglobin in the blood is normal. If it has to be diluted more largely, the oxyhemoglobin 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 hemoglobin is only half what it ought to be — 50 per cent, of the normal — and so for other percentages. The instrument only yields approximate results, but is extremely useful in clinical observations. Von Fleischl's Hemometer.— The apparatus (tig. 07) 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 com- partments, a and a'. Fill with a pipette the compartment a' ov^r the wedge with distilled water. Fill about a quarter of the other compartment (a) with distilled water. PlG. UT.— Fleischl's liaemouu-tcr. 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. Bead off the scale which is so constructed as to give the percentage ot ooglobin. Dr. Oliver's Haemoglobinometer. -This method consists in comparing a specimen 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, 68) is first filled with blood obtained by pricking the finger, This is washed with water 176 ESSENTIALS OF CHEMICAL PHYSIOLOGY by the mixing pipette cl into the blood cell e ; the cell is then just filled with water, and the blood and water thoroughly mixed by the handle of c being Fig. 68. — Oliver's bsemoglobiuorneter : a, standard gradations ; b, lancet ; c, capillary measuring pipette ; d, mixing pipette ; e, blood cell and cover glass. 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 APPENDIX 177 then placed by the side 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 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 the normal. ' Worth' of the Corpuscles. — If the percentage of haemoglobin is 100, and the percentage number of corpuscles is 100 also ; then the quotient - = 1 is taken as the normal. This varies in health from 0"95 to 1*05 in men, and from 0'9 to 1 in women. This quotient has been termed the worth of the corpuscle. 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 and 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 picnometer is a more accurate method.' POLARIMETERS Soleil's Saccharimeter. — This instrument (see fig. 69) consists of a Nicol's prism, d, called the polariser : this polarises the light entering it, and the polarised beam then passes through a quartz plate (b in fig. 69), 3*75 mm. thick, one half of which (d in fig. 70) is made out of dextrorotatory, the other half (g in fig. 70) of lsevorotatory quartz. I'i' . 69. Soleil'a saccharimeter. The light then passes through the tube containing the solution in the position of the dotted line in fig. 69, then through a quartz plate cut per- pendicularly to its axis (rj in lig. 70), then through an arrangement called Ik compensator (r in fig. 70), then through a second nicol called the analyser, ;i.nd lastly through a telescope (L in fig. 70). ' hcutsch. Arch. f. klAn. Med. xlvii. p. 1.45. N 178 ESSENTIALS OF CHEMICAL PHYSIOLOGY The compensator consists of two quartz prisms (RE, fig. 70) cut perpen- dicularly to the axis, but of contrary rotation to the plate just in front of them. These are wedge-shaped, and slide over each other, the sharp end of one being over the blunt end of the other. ' By a screw the wedges may be moved from each other, and this diminishes the thickness of quartz inter- posed ; if moved towards each other the amount of quartz interposed is increased. The effect of the quartz plate (d, g) next to the polariser (i in fig. 70) is to give the polarised light a violet tint when the two nicols are parallel to each other. But if the nicols are not parallel, or if the plane of the polarised light has been rotated by a solution in the tube, one half the field will change in colour to the red end, the other to the violet end of the spectrum, because the two halves of the quartz act in the opposite way. The instrument is first adjusted with the compensator at zero, and the nicols parallel, so that the whole field is of one colour. The tube containing the solution is then interposed ; and if the solution is optically inactive the field is still uniformly violet. But if the solution is dextrorotatory the two halves will have different tints ; a certain thickness of the compensating quartz plate which is lsevorotatory must be interposed to make the tint of 7-j a. t er 7 7? --■■■£» 10 - ia~ !G> w Pig. 70. Diagram of optical arrangements in Soleil's saccharimeter. the two halves of the field equal again ; the thickness so interposed can be read off in amounts corresponding to degrees of a circle by means of a vernier and scale (E in fig. 70) worked by the screw which moves the compensator. If the solution is laevorotatory, the screw must be turned in the opposite direction. Zeiss's polarimeter is in principle much the same as Soleil's ; the chief difference is that the rotation produced by the solution is corrected not by a quartz compensator but by actually rotating the analyser in the same direction, the amount of rotation being directly read off in degrees of a circle. Laurent's polarimeter is a more valuable instrument. Instead of using daylight, or the light of a lamp, monochromatic light (a sodium flame pro- duced by volatilising common salt in a colourless gas flame) is employed ; the amount of rotation varies for different colours ; and observations are recorded as having been taken with light corresponding to the D or sodium line of the spectrum. The essentials of the instrument are, as before, 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 by half a wave-length. In the 0° position the two halves of the field appear equally APPENDIX 179 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 un- equally illuminated. This is corrected by means of a rotation of the analyser that can be measured in degrees "by a scale attached to it. Fia. 71. — Laurent's polariraeter. Specific rotatory power of any substance is the amount of rotation in 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 one deci metre long. if " = rotation observed. w = weight in grammes of the substance per cubic centimetre. I = length of tube in decimetres. c rotation for light with wave-length corresponding to the I) line (sodium Same). W7|.,= ± //'/ In this formula + indicates that the substance in dextrorol is laevorotatory. n 2 180 ESSENTIALS OF CHEMICAL PHYSIOLOGY If, on the other hand, (a)o is known, and we wish to find the value of w, then a w = ± (o.)D x I THE SPECTEO-PQLAKIMETEK This instrument is one in which a spectroscope and polarising apparatus are combined for the purpose of determining the concentration of substances which rotate the plane of polarised light. It was invented by E. v. Fleischl for the estimation of sugar in diabetic urine. Its chief advantage is that no difficulty arises of forming a judgment as to the identity of two coloured surfaces, as in Soleil's saccharimeter, or of two shades of the same colour, as in Laurent's instrument. The light enters at the right-hand end of the instrument, is polarised by the Nicol's prism b, and then passes through two quartz plates, cc, placed horizontally over each other. One of these plates is dextro-, the other lsevorotatory, and they are of such a thickness (7"75 mm.) Fig. 72. — Spectropolarimeter of t. Fleischl. that the green rays between the E and b lines of the spectrum are circularly polarised through an angle of 90°, the one set passing off through the upper quartz to the left, the other through the lower to the right. The light then continues through a long tube, ff, which contains 15 cc. of the solution under examination. It then passes through an analysing nicol, d, and finally through a direct vision spectroscope, e. On looking through the instrument, the tube ff being empty, or filled with water or some other optically inert substance, two spectra are seen, one over the other, but each shows a dark band between E and b owing to the extinction of these rays by the circular polarisation, produced by the quartz. The analyser can be rotated : a vernier, g, is attached to, and moves with it, round a circular disc (seen in APPENDIX 181 section at h) graduated in degrees. The two bands in the .spectra coincide when the zeros of vernier and scale correspond. If now the tube / is filled with an optically active substance like sugar, the bands are shifted, one to the right, the other to the left, according to the direction of rotation of the sub- stance in/. The rotation is corrected by rotating the analyse)- into such a position that the two bands exactly coincide once more as to vertical position. The number of degrees through which it is thus necessary to move the analyser measures the amount of rotation produced by the substance in f, and is a measure of the concentration of the solution. The degrees marked on the circular scale are not degrees of a circle, but an arbitrary degree of Buch a length that each corresponds to 1 per cent, of sugar in the given length of the column of fluid uxff (177*2 mm.) SIE GEORGE JOHNSON'S PICEO-SACCHAEOMETEE The following solutions and apparatus are required : — 1. Standard solution of ferric acetate.— This has the same tint as that yielded by a solution of dextrose containing 1 grain per fl. oz. This standard solution is prepared as follows : — E. Liq. ferri perchlor. fort. (P.B., sp. gr. 1-42) . . 1 fl. drachm Acid. acet. glacialis (P.B., sp. gr. P058) . . . 4 ,, drachms Liq. ammonia (P.B., sp. gr. 0*959) . . . 2 „ „ Mix first the iron and the acid; then add the ammonia, and distilled water up to 4 fl. oz. 2. Saturated solution of picric acid. — Prepared by boiling the crystals with distilled water in the proportion of 6 grains to 1 fl. oz., and allowing the excess to crystallise out on cooling. 3. Liq. jjotasna (P. 15.. sp. gr. 1*058). 4. A tube about 12 inches in length, graduated into 100 cubic centimetres, with longer divisions at each 10 cubic centimetres, accurately stoppered and lipped. .">. A tube, half the above length, and of equal diameter, accurately stoppered to hold the standard solution. 0. A boiling tube, 10 inches long, =}-inch in diameter (internal), lipped, and graduated up to 4 fl. drachms. 7. One drachm measure. Measure 1 fl. drachm of urine into the boiling tube. Add 1 fl. drachni of the saturated picric acid solution, and £ fl. drachm of liq. potasste. Make up to the 4 drachm mark on the tube witli distilled water. Heat and keep the liquid boiling for about a minute. Cool by dipping the tube after a minute in cold water, and ascertain that the cold liquid nn actly 4 fl. drachms, [f U up to the 4 drachm mark with distilled water; if more, evaporate down to the l drachm mark. If tli' colour of the boiled liquid is the that of the ferric ad standard, or paler, the urine is either free fron 3 than 1 grain per fl. 02. 182 ESSENTIALS OF CHEMICAL PHYSIOLOGY If the colour is paler than the standard, boil with 2 drachms of urine instead of 1, then divide the indicated reduction by 2. It should be borne in mind that all normal urines reduce picric acid to an extent equivalent to from \ grain to 1*2 grain of dextrose per fl. oz. This reduction is due to creatinine. This should be allowed for, when the quantity of dextrose present is very small. If the colour of the boiled liquid is darker than the standard, introduce it into the graduated tube until it stands at 10 divisions, whilst the stoppered tube at the side is filled with the ferric acetate standard (see fig. 73). Now dilute the dark red liquid in the gradu- ated tube with distilled water, till the colour is the same as that of the standard. Each division above 10 = 0*1 grain per fi. oz. Thus, 13 div. = 1*3 grain, 30 div. = 3 grains per fl. oz., &c. &c. If more than 6 grains per fl. oz. are indicated, dilute the urine 10 times, by pouring urine up to 10 divisions on the graduated tube, and distilled water up to 100. Then analyse the diluted liquid as before. In this case, each division on the saccharometer indicates 1 grain of sugar per fl. oz. Thus, diluting from 10 up to 48 divisions, shows that the urine contains 48 grains of sugar per fl. oz. If the urine, when 10 times diluted, gives a colour paler than the standard, it contains less than 10 grains of sugar per fl. oz. Another portion should thus be diluted 5 times, by filling the graduated tube up to 10 divisions with urine, then up to 50 divisions with distilled water. The dilution of the urine may be more conveniently made by pouring 5 or 10 c.c. into a 50 c.c. flask, and adding water up to the 50 c.c. mark. The analysis is performed as before. The value of the divisions now will be half that with a ten times diluted sample. Thus, 18 divisions would indicate 9 grains per fl. oz. If the urine has a specific gravity of 1*035 or more, it should be at once diluted 5 or 10 times before commencing an analysis. The 'percentage of sugar in the urine may be ascertained by dividing the number of grains per fl. oz. by 4*8. Fig. 73. — Sir G-. Johnson's picro - saccharometer (made by Miiller, 148 High Holborn. W.O.). MERCURIAL AIR-PUMPS Pfliiger's Pump. — I is a large glass bulb filled with mercury ; from its lower end a straight glass tube, m, about 3 feet long, extends, which is connected by an india-rubber tube, n, with a reservoir of mercury, o, which can be raised or lowered as required by a simple mechanical arrangement. From the upper end of the bulb, I, a vertical tube passes ; above the stopcock, h, APPENDIX 183 this has a horizontal branch, which can be closed by the stopcock, /. The vertical part is continued into the bent tube, which dips under mercury in the trough. Ji. A stopcock,^', is placed on the course of this tube. Beyond/ the horizontal tube leads into a large double glass bulb, a b ; a mercurial gauge, c, and a drying-tube, d, tilled with pieces of pumice-stone moistened with sulphuric acid, are interposed. a is called the blood-bulb, and the I'n;. 7i. Diagram of PflUger'a pump. blood is brought into it by the tube, CJ the gases, as they come off, cause the blood to froth, and the bulb, b, is called the froth-chamber, as it intercepts the froth, preventing it from passing into the rest of the apparatus. The pump is used in the following way: I is filled with mercury, the level in i and o being the same; k is closed ; o is then lowered, and when it 184 ESSENTIALS OF CHEMICAL PHYSIOLOGY is 30 inches lower than the stopcock h, the mercury in Z falls also, leaving that bulb empty ; j being closed and / open, Tc is then opened, and the air in a, b, d, &c. rushes into the Torricellian vacuum in I ; / is closed andj opened ; the reservoir, o, is raised ; the mercury in I rises also, pushing the air before it, and it bubbles out into the atmosphere through the mercury (the tube, h, is not at this stage in position). When Z is full of mercury, h and j are once more closed and o is again lowered ; when Z is thus rendered once more a vacuum, h and / are opened and more of the air remaining in a, b, d, &c. rushes into the vacuum ; / is closed, j is opened, and this air is expelled as before. The process is repeated as often as is necessary to make a, b, d, &c. as complete a vacuum as indicated by the mercury in the gauge, e, as is obtainable. a being now empty and the stopcock, _/', closed, blood is introduced by the tube, c ; it froths and gives off all its gases, especially if heated to 40-45° C. In the case of serum, acid has to be added to disengage the more firmly combined carbonic acid.1 The bulb, Z, is once more rendered a vacuum, and ~k and / are opened, j being closed. The gas from a and b rushes into the bulb Z, being dried as it passes through d ; /is then closed and j opened ; the reservoir o is raised, and as the mercury in I rises simultaneously, it pushes the gases into the cylinder, h, which is filled with mercury and inverted oyer the end of the bent tube. This gas can be subseqiiently analysed. By alternately raising and lowering o, and regulating the stopcocks in the manner already described, all the gas from the quantity of blood used can be ultimately expelled into h. . A good grease for the stopcocks is a mixture of two parts of vaseline to one of white wax. Alvergniat's pump has the advantage over Pfliiger's of fewer connections, and all of these are surrounded by mercury, which effectually prevents leakage ; it has the disadvange of a rather small bulb in place of Z, and thus it is more labour to obtain a vacuum. Leonard Hill's Pump. — This is a simpler instrument, and is sufficient for most purposes. It consists of three glass bulbs (B.B. in fig 75), which we will call the blood bulb ; this is closed above by a piece of tubing and a clip, a; this is connected by good india-rubber tubing to another bulb, d. Above d, however, there is a stopcock with two ways cut through it ; one by means of which B.B. and d may be connected, as in the figure ; and another seen in section, which unites d to the tube e, when the stopcock is turned through B.B. Fig. 75. — L. Hill's air-pump. Phosphoric acid is usually employed. APPENDIX 185 a right angle. In intermediate positions the stopcock cuts off all communi- cation from d to all parts of the apparatus above it ; d is connected by tubing to a receiver, R, which can be raised or lowered at will. At first the whole apparatus is filled with mercury, R being raised. Then a being closed, R is lowered, and when it is more than the height of the barometer (30 inches) below the top of B.B. the mercury falls and leaves the blood bulb empty ; by lowering R still further. (7 can also be rendered a vacuum. A few drops of mercury should be left behind in B.B. B.B. is then detached from the rest of the apparatus and weighed, the clips, a and b, being tightly closed. Blood is then introduced into it by connecting the tube with the clip a on it to a cannula filled with blood inserted in an artery or vein of a living animal. Enough blood is withdrawn to fill about half of one of the bulbs. This is defibrinated by shaking it with the few drops of mercury left in the buibs. It is then weighed again ; the increase of weight gives the amount of blood which is being investigated. B.B. is then once more attached to the rest of the apparatus, hanging downwards, as in the side drawing in fig. 75, and the blood gases boiled off; these pass into d, which has been made a vacuum ; and then, by raising R again, the mercury rises in d, pushing the gases in front of it through the tube e (the stopcock being turned in the proper direction) into the eudiometer, E, which has been filled with, and placed over, mercury. The gas can then be measured and analysed. ANALYSIS OF GASES Waller's modification of Zuntz's more complete apparatus will be found very useful in performing gas analysis, say, of the expired air : a 100 c.c. rneasuring-tube J 1^ graduated in tenths of a cubic centimetre lEp between 75 and 100 ; a filling bulb and two gas pipettes are connected up as in the diagram. It is first charged with acidulated water up to the zero mark by raising the filling bulb, tap 1 being open. It is then filled with 100 c.c. of expired air, the filling bulb being lowered till the fluid in the tube has fallen to the 100 mark. Tap 1 is now closed. The amount of earbonic acid in the expired air is next ed; tap 2 ia opened, and the air is expelled into the gas pipette eontaining strong caustic potash solution by raising the filling bulb until the fluid has risen to the zero mark of the measuring tube. Tap 2 is closed, Pig. 7n. \\ allei a apparatu iUI.-il 186 ESSENTIALS OF CHEMICAL PHYSIOLOGY and the air left in the gas pipette for a few minutes, during which the carbonic acid is absorbed by the potash. Tap 2 is then opened and the air drawn back into the measixring tube by lowering the filling bulb. The volume of air (mimes the carbonic acid) is read, the filling bulb being adjusted so that its contents are at the same level as the fluid in the measuring tube. The amount of oxygen is next ascertained in a precisely similar manner, by sending the air into the other gas pipette, which contains sticks of phosphorus in water, and measuring the loss of volume (due to absorption of oxygen) in the air when drawn back into the tube. KJELDAHL'S METHOD OF ESTIMATING NITROGEN This simple and accurate method has very largely replaced the older complicated processes. , I take the following account of the method with the modifications pro- posed by Warington from Sutton's ' Volumetric Analysis.' From Ol to 1 gramme of the dry powdered substance is put into a boiling flask holding about 100-120 c.c. The acid used for the destruction of the organic material is made by mixing 200 c.c. pure oil of vitriol with 50 c.c. Nordhausen oil of vitriol, and 2 grammes of phosphoric acid in sticks ; all these must, of course, be free from ammonia : 10-20 c.c. of this mixture are poured over the substance in the flask and heated on wire gauze over a small Bunsen B3S0, hu.ll> Fig. 77. — Kjeldahl's method. (Waller, after Argutinsky.) flame. The temperature must be kept below boiling ; with prolonged heating the organic matter is gradually destroyed, and the liquid becomes clear and quiet. The nitrogen originally present is thus converted into ammonia, and this may be hastened by adding to the liquid very minute pinches of pure potassium-permanganate. A violent commotion takes place with every addition, but there is no fear of any ammonia being lost. The operation is ended when the mixture becomes permanently greenish (from one to two hours), and moderate heat is continued for a few minutes more. The flask is cooled, some water added, and the contents washed out into a large flask of 700 c.c. capacity with as little water as possible. It is then made alkaline APPENDIX 187 with excess of either pure caustic soda or potash solution (sp. gr. 1*3). A little metallic zinc is added to prevent bumping during the subsequent distUlation. The flask is then rapidly closed with a perforated caoutchouc stopper, through which passes an upright tube with two bulbs about an inch in diameter blown upon it : these arrest and carry back any spray of soda from the liquid. The tube above the bidbs is bent over and connected to a condenser, and the delivery end of the condenser leads into a flask, containing a measured excess of standard acid. The mixture in the flask is then distilled, the ammonia passes over into the acid. Distillation is continued from forty-five to sixty minutes. The amount of acidity is then determined in the distillate by titration with standard potash or soda, methyl orange being used as the indicator of the end of the reaction. (This gives a pink colour with acid, yellow with alkali.) Example. — Suppose 0*15 gramme of a nitrogenous substance is taken, treated with acid, neutralised, and the ammonia distilled over and received by 100 c.e. of a decinormal solution of hydrochloric acid ( = 10 c.c. normal acid). The distillate is then titrated with decinormal soda, and it is found that the neutral point is reached when 60 c.c. of the decinormal soda have beenadded. The other 40 c.c. must therefore have been neutralised by the ammonia derived from the nitrogenous substance under investigation. This 40 c.c. of decinormal acid = 4 c.c. of normal acid = 4 c.c. of normal ammonia = 4 x 0*017 = 0*068 gramme of ammonia ; 0*15 gramme of the substance therefore yields 0-068 gramme of ammonia, and this amount contains 0*050 gramme of nitrogen : 100 grammes of the substance will therefore contain 100 x 0*056 .,„ „ e .L = o7*o grammes of nitrogen. 0*15 Figure 77 represents the apparatus as modified by Willfarth. In this, oxidation is assisted by adding a small quantity of metallic mercury (about vV c.c). To avoid bumping during distillation talc is added instead of zinc. 12 c.c. of strong potassium sulphate solution are also added. Decinormal sulphuric acid is used as the standard acid, and this is contained in the bulbs shown in the figure. When this method is used for determining the total nitrogen hi urine, 5 c.c. of urine and *20 c.c. of the mixed acids are boiled for half an hour in a flask of 800 c.c. capacity. After cooling, this is distilled with soda, and the process completed as already described. INDEX Iu cases where there are several figures after auy subject, the oue in heavy type indicates where the principal matter in relation to the subject is to be found. Absorption, 73 ; of carbohydrates, 73 ; of proteids, 74 ; of fats, 75 Absorption bands, 90 Absorption spectra of hamioglobin and its derivatives, 92, 146; of myohaematin, 154 ; of urinary pigments, 103, 170 Accessories of food, 42 Acetic acid, 17 Acetone, 127 Acetyl, 17 Achroo-dextrin, 15, 40, 137 Acid albumin, 21, 27, 37, 50, 53, 54, 56, 130 Acid hsematin, 145 Acid hasmatoporphyrin, absorption bands of, 170 Acid, lactic, 4, 37 ; test for, 153 Acid sodium phosphate, 21 Acid tide, 4 Acids, vegetable, 4 Acute yellow atrophy of liver, 108 Adenine, 117 Adenyl, 117 Adipose tissue, 2, 4, 16 Aerobic micro-organisms, 49 Air, expired and inspired, 95 Air-pump.s. mercurial, 182, 183, 184 Alanine, 64 Albumin, acid, 21, 27, 50, 55, 130 Albumin, action of acids and alkalis on, 20 Albumin, alkali, 20, 27, 58, 60, 130 Albuminates, 27 Albumin in urine, estimation of, 124 Albumin in urine, tests for, 124 Albumins, 4, 22, 23, 27, 57 Albuminoids, 3, 4. 25, 30, 60 Albumiiio.': i bach, 124 Albumosc, 19, 22, 50, 55, 57, ■ 129, 141 Albumoses and peptone, separation of. 141 Albtunoses, testa tor, 141 Alcapton, 127 Alcaptonuria, 127 Alcohol, action of, on proteid-, 25 Alcoholic fermentation, 13, 14 ; of m ilk, 37 Alcohols, 3, 4, 17, 42 Aldehyde, 11, 17 Aleurone grains, 23 Alexander Schmidt's method of pre- cipitating serum-globulin, 149 Alexis St. Martin, case of, 52 Alkali-albumin, 20, 27, 58, 60. 130 Alkaline hsematin, 145 Alkaline hamiatoporphyrin, absorption bands of, 170 Alkaline tide, 104 Alkaloids, 42 Alloxuric bases, 29, 117 Alvergniat's pump, 184 Amides, 3 Amido-acetic acid, 64 Amido-acids, 22, 64, 109 Amido-caproic acid, 64 Amido-ethyl-sulphonic acid, 69 Amido-iso-butyl-acetic acid, 64 Amido-propionic acid, 64 Amido-pyrotartaric acid, 60 Amido-succinic acid, 60 Amidulin, 15 Amines, 3, 22 Ammonia, 3, 22 Aininoniacal odour of putrid urine, 106 Ammonium carbonate and carbamate as urea precursors, 110 Ammonium cyanate, 105 Ammonium sulphate, action of, on pro- teids, 19, 24, 26, 129, 139, 141 id movement, 2 Amylolytic ferments, 46, 4s ; in blood serum, 135 Amylopsin, 14, 48, 61 Amylases, 10, 11, In Anaerobic micro-organisms, 19 Analysis of gases, 185 Animal gum Animal starch, 10 Anti-albumin, 56, 60 Anti-peptone, 56, 60 190 ESSENTIALS OF CHEMICAL PHYSIOLOGY Antiseptics, 47 Apparatus necessary for practical work, 4 Aqueous vapour, tension of, 7 Arginine, 22, 31, 60, 107, 109, 110 Aromatic compounds, 3, 12, 22 Arterial blood, gases of, 96 Artificial gastric juice, 52 Aspartic acid, 60, 61, 109 Assimilation, 2 Atmosphere, 95 Atomic weights, 8 B Bacilli, 48 Bacteria, 48, 73 Bacterial action, 62 Barfoed's reagent, 136 Bath, warm, 20 Baumann and Wolkow on alcapton, 127 Beaumont, Dr., 52 Beef, 33 Beef tea, 41 Beetroot, 12 Benzene, derivatives of, 3 Benzoic acid, 117 Bernard, Claude, on glycogen, 74 Bile, 58, 62, 67-77 ; amount secreted, 67 ; circulation of, 72 ; characters of, 68 ; constituents of, 67, 68, 72 ; mucin of, 68; pigments, 66, 67, 69; salts of, 66, 67, 69 ; uses of, 71 ; in urine, 127 Biliary fistula, 66 Bilirubin, 67, 69, 168 ; of meconium, 73 Biliverdin, 67, 69 ; of meconium, 73 Biuret, 24, 101 Biuret reaction, 19, 24, 58, 142 Blood, 78-100 ; coagulation of, 79, 80, 81, 151 ; corpuscles, 79 ; detection of, 130; gases of, 96; tablets, 79, 80; pigment of, 85-94, 145-148 ; plasma and serum of, 3, 78, 149 ; in urine, 128 Bohr on absorption of oxygen, 99 ; on haemoglobin, 94 ; on tension of car- bonic dioxide, 99 Bone, composition of, 30 ; marrow, 16 Bowman's corpuscle, 103 Bran, 39 Bread, 32, 40 Bright's disease, 126 Brodie on muscle proteid, 156 Brown and Morris on starch digestion, 46 Buffy coat, 80 . Bunge on hasmatogens, 29 ; on milk, 35 Bush tea, 42 Butter, 32, 36 Butyric acid, 14, 17, 63 Butyrin, 37 C Caffeine, 42 Calcium, 3, 8 ; phosphate, 3 Calcium oxalate in urine, 114, 121 Calcium salts, importance of, in coagu- lation of blood, 78, 81, 151 ; of milk, 32, 36, 140 Cane sugar, 9, 10, 12, 74, 135 ; in urine, 13 ; tests for, 9, 13 Caproic acid, 17 Caproin, 37 Caprylin, 37 Carbamide, 105, 111. See also Urea Carbohydrates, 3, 4, 9-16, 134 ; absorp- tion of, 73, 74 ; classification of, 10 ; definition of, 10 ; tests for, 10-16, 134-136 Carbolic acid poisoning and urine, 171 Carbon, 3, 8 Carbonates in blood, 98 ; in urine, 111, 112, 123 Carbonic acid, in air, 95 ; in blood, 98 Carbonic oxide haemoglobin, 88, 94, 145, 148 Cardiac glands, 51, 52 Carmine-stained fibrin, 143 Carnic acid, 61 Cartilage, hyaline, 31 Casein, 27, 29, 32, 36 Caseinogen, 28, 32, 35, 36 ; preparation of, 139 Cell, definition of, 2 ; diagram of, 29 Cells, differentiation of, 1 Cellulose, 10, 14, 15, 39, 46, 49, 72 Centigrade scale, 7 Central cells of cardiac glands, 52 Centrifugal machine, 151 Cheese, 36 Chemical physiology, 1 Chemical sediments of urine, 123 Chemical structure of protoplasm, 2 Chemistry of respiration, 94 Chitin, 31 Chlorides of urine, 111 ; estimation of, 159, 160 ; tests for, 101 Chlorine, 3, 8 Chlorophyll, 72 Cholalic acid, 69, 72 Cbolesterin, 3, 4, 67, 68, 70, 72, 73, 83 ; tests for, 66, 71, 131 Choletelin, 70 Choline, 63 Chondrin, 30 Chorda Tympani, 43 Chromatin, 29 Chromogens, 103 ; of urine, 171 Chyme, 67 Ciliary movement, 2 INDEX 191 Circulation of bile, 7*2 Cirrhosis of liver, 108 Citric acid, 104 Clark's essence of rennet, 41 Claude Bernard on glycogen. 74 Clotting of blood, 81 Coagulated proteids, 27 Coagulation, 24 Coagulation of blood, 79, 151 ; of hydro- cele fluid, 152 ; of milk, 139-140 ; of muscle, 153 ; of proteids, 2 Coagulative ferments, 49 Cobalt sulphate, action on proteids, 142 Cocaine, 42 Cocoa, 42 Coffee, 42 Cola nut, 42 Collagen, 21, 30. 40 Collimator, 89, 14S Colloids, 22 Colostrum, 35 Colostrum corpuscles, 35 Commercial peptone, 19, 22, 81 Compound proteids, 28 Compounds found in the body, 3 Compounds of carbon. 3 Condiments, 42 Cooking of foods, 40 Copper, 3. 8 Cream, 32 Creatine, 118 : crystals, 119 ; in muscle, 155 ; as a urea precursor, 108, 110 Creatinine, 41, 83, 105, 118; crystals of, 119 ; detection of, 102 ; estima- tion of, 16G ; preparation of, 119 Crypts of Lieberkiihn, 62 Crystallin, 26 Crystalline lens, 26 Crystallisable proteids, 85, 86, 138 Crystallisation of egg albumin, 138 Crystalloids, 23 Crystals from blood, 86, 87, 148 Curds and whey, 32, 36 Cvanamide, 1 18 Cystin, 120, 121. 123 ; crystals of, 121 J) D mii.k, 32, 139, 140 - blood, 78, 151 Decomposition, products of fats, 18 ity of water, 7 Dentine, composition of, 30 I), j, -its in urine, 114, 119-123 Desiccator. 1"" ro-albomose, 56, 141 m. 9, 10. 1 1, 15, 82, 43, 16, 129; for, io, it; rotatory, 10, 12, 13, 14, 15 I, 10, 11, 12, 32, 37, 74, 135; tals, 11; in blood, 11 ; in urine, 1 1 Diabetes mellitus, 11, 65, 126 Diabetic urine, 124 Diaeetin, 17 Dialyser, 22, 23 Dialysis of albumoses, 141 ; of serum, 149 Diastase, 48 Diastatic ferments, 14, 46, 137 Diet, 33 Diet tables, 34 Diffusion, 23 Digestion, 143 ; gastric, 50-57 ; intes- tinal, 62 ; pancreatic, 58-65 ; sali- vary, 44 Direct vision spectroscope, 91 Disaccharides, 10, 11, 12 Dough, 39, 40 Drechsel on urea formation, 108, 110 Dripping, 41 Dropsical effusions, 28 Dulcite, 11 Dupre's urea apparatus, 101, 102 Dysalbumose, 141 E Eck's fistula, 108 Egg-albumin, 19. 26; crystallisation of, 23, 138 ; in mine, 74 Egg-globulin, 19, 26, 38 Eggs, 38 Egg white, 19, 20, 22, 23. 38 Egg yolk, 38 Ehrlich's experiments with methylene blue, 97 Elastic fibres, 31 Elastin, 31, 60 Elastoses, 55 Elements found in the body, 3 ; symbols and atomic weights of, 8 Emulsification, 18 Emulsion, 18, 58, 61 Enamel, comjjosition of, 30 English system of weights and measures, 6 Envelope crystals, 114 Enzymes, 46, 47, 48, 49 Epidermis. 2 Epithelium of intestine, 75 Epsom suit, 112 Erlenmeyer flask, 166 Kry Lino-dextrin, 15, 46, 137 Esbach's albuminometer, 124; rea 5 ; tube, 124 Estimation of chlorides, 159, 160 ; of creatinine, 166, 167 ; of dextrose, 125, 181 ; of lactose, 13 ; of maltose, 14, 1 37 : of nitrogen, 186 ; of phosphates, 162; <>l sulphates, 163; of urea, 101, 157-159; of uric acid, 165 192 ESSENTIALS OF CHEMICAL PHYSIOLOGY Ethereal sulphates in urine, 112 ; esti- mation of, 164 Ethyl alcohol, 17 Ethyl-diacetic acid, 127 Expired air, 95 External respiration, 95 Extirpation of pancreas, 65 Extractives of blood, 83 ; of muscle, 41, 153 F Feces, 72 Fahrenheit scale, 7 Fats, 2, 3, 4, 16-18, 22, 38 Fats, absorption of, 75 ; constitution of, 16 ; melting point of, 16 ; of milk, 37 ; solubility of, 16 ; tests for, 10, 16 Fatty acids, 22 Fehling's solution, 5, 9, 11, 124 Fehling's test, 9, 13, 14, 127 Ferment coagulation, 25 Ferment, invert, 12 Fermentation, 46 Fermentation test, 11, 127 Ferments, 3, 46 ; of gastric juice, 53 ; of pancreatic juice, 59 ; of saliva, 44 ; of succus entericus, 62 ; unorganised, 45, 46 Ferments, classification of, 48 Fibre, elastic, 31 Fibrin, 27, 80, 81, 82 Fibrin ferment, 49, 78, 81, 82, 83 Fibrin filaments, 79, 80 Fibrinogen, 22, 26, 83, 152 Fistula, gastric, 52 Fleischl's hsemometer, 175; spectro- polarimeter, 180 Flour, 32, 39 Fluorescent screens, 148 Fluorine, 3, 8 Food, cooking of, 40 Foods, 32-42 ; perfect, 33, 35 Formic acid, 17 Fractional heat coagulation of muscle proteids, 153, 154 Frauenhofer's lines, 79, 89, 92 Fredericq on oxygen tension, 97 ; on tension of carbon dioxide, 99 Free hydrochloric acid in gastric juice, tests for, 143 Furth, v., on muscle plasma, 155 G Galactose, 10, 11, 12, 37, 135 Gallstones, 70 Gamgee on secretion of hydrochloric acid, 53 ; on photographic spectra, 148 Garrod on urochrome, 104 Garrod and Hopkins on urobilin and stercobilin, 169 Garrod's methods of separating pigments from urine, 168 Gas analysis, 185 Gases of blood, 96 Gastric fistula, 52 Gastric juice, 3, 143 ; action of, 55 ; composition of, 54 ; properties of, 55 ; secretion of, 50 Gelatin, 4, 21, 30, 40, 41 ; tests for, 131 Gelatinisation, 21 Gelatoses, 55 Germ theory of disease, 47 Globin, 86 Globulin, 22, 23, 26, 57 Globulose, 55 Glossopharyngeal nerve, 43 Gluco-proteids, 28 Glucose, 10, 11, 74 Glutamic acid, 60, 109 Glutaminic acid. See Glutamic acid Gluten, 32, 39, 40 Glyceric ethers, 16 Glycerides, 16 Glycerin, 16, 17, 58 Glycerol, 17 Glyceryl, 17 Glycine, see Glycocine Glycocholate of soda, 67, 69 Glycocholic acid, 69 Glycocine, 22, 64, 69, 72, 109, 110 Glycogen, 10, 15, 22, 46, 48, 74, 129 ; microchemical detection of, 135 ; preparation of, 134 ; tests for, 10, 15 Glycosuria, 126 Glycuronic acid, 127 Gmelin's test, 66, 69, 70 Gnezda on biuret reaction, 142 Gowers' hemacytometer, 172 ; hsemo- globinometer, 174 Graham, Thomas, on colloids and crystalloids, 22, 23 Granulose, 14 Grape sugar, see Dextrose Ground substance, 21, 28 Griitzner's method of comparing diges- tive power of solutions of pepsin, 143 Guanine, 117 Guarana, 42 Gums, 10 Gunsberg's reagent, 143 Giirber on serum albumin crystals, 24 H Hemacytometer of Sir William Gowers, 172 ; of Oliver, 173 INDEX 193 Hsraatin, 72, 86, 87, 168 ; acid, 145 ; alkaline. 14-5 ; iron-free, 147 ; of food, 103 Haeniatogen, 29 Haeniatoidin, 67, 87 ; crystals, 67 Hsematoporphyrin, 87, 147, 148, 169; absorption bands of, 170 Haematoseope of Herrmann, 90 Hffimin, 87 ; crystals, preparation of, 79 Haemochromogen. 145 Haemoglobin, 23, 28, 79, 85, 145. 148 ; composition of, 86 ; derivatives of, 145 Hsemoglobinonieter of Sir William Gowers, 174 ; of Oliver, 175 Haemoglobinuria, paroxysmal, 128 Haemometer of von Fleischl, 175 Haldane on niethasmoglobin, 94 ; on ab- sorption of oxygen, 99 Hammarsten on blood coagulation, 81 ; method of precipitating serum-globu- lin, 149, 150 Hammerschlag's method of estimating the specific gravity of blood, 177 Hayem's fluid, 174 Heat coagulation of proteids, 22 ; of serum globulin and serum albumin, 150 Heat rigor, 155 Heidenhainon secretion of gastric juice, 53 ; on pressure in bile duct, 67 Heller's nitric acid test, 124 Hemi-albumin, 56, 60 Hemi-albumose, 56 Hemi-peptone, 56, 60 Herrmann, ha?matoscope of, 90 Hetero-albumose, 56, 141 Hexatomic alcohol, 11 Hexone bases, 31, 61, 109 Hill's air-pump, 184 Hippuric acid, 105, 117 ; crystals of, 117 Histidine, 31, 61 Histone of Kossel, 87 Hofmeister on crystallisation of egg albumin, 24 Homogentisinic acid, 127 Hopkins' method of estimating uric acid, 116, 165; on crystallisation of ;!bumin, 24, 138 Hoppe-Seyler on haomatin, 87 ; on pro- ieids, 21 Burner's method of estimating urea, 158 Hyaline cartilage :;l obilirubin, 70, 72, 103, 168 Hydrocarbon, 16 II rdrooele fluid, 82, 152 11 .'li ochinone, 171 chloric acid, :; ; of gastric juice, lor, 1 1:; ; test for ax, 9 Hydrogen, 3, 8 Hydrolysis, 13 Hydrometer, 35, 177 Hydroxy-butyric acid, 127 Hydroxyl, 17 Hypobromite of soda, action of, urea, 101, 106, 158 Hypoxanthine, 83, 117 Indicax, 112 Indiffusibility of proteids, 22 Indigo, 112 ; blue, 171 ; red, 171 Indole, 63, 72, 112 Indoxyl, 112, 171 Indoxyl sulphate of potassium, 112 Infection, 47 Inorganic compounds, 3 ; salts, 3 Inosite, 10, 12 ; crystals of, 12 Inspired air, 95 Internal respiration, 95 Internal secretion, 65 Interstitial substance, 21, 28 Intestinal juice, 13 ; digestion, 62 Intravascular coagulation, 152 Inversion, 11, 12, 13 Inversion of cane sugar, 49 Inversive ferments, 49 Invert ferment, 12 Invertin, 62 Iodine, 3, 8 Iodine test, 10, 15, 33, 123, 134, 137 Iron, 3, 8, 86 ; in milk, 35 Iron-free haematin, 87, 147 Iso-cholesterin, 71 Iso-maltose, 14, 135 Jaffk on test for indoxyl, 171 Jaundice, 127 Jelly, Lieberkiihn's, 27 Jelly, Whartonian. 21 Johnson, G. S., on creatinine, 118, 167; on sugar in urine, 125 Johnson, Sir G., on picric acid test, 125, 181 ; on picro-saccharometer, 181, 182 Juice, intestinal, 13 Junkets, 41 K Kaudkk'k method of precipitating serum- globulin, 149, 150 Keratin, 2, 4, 31, 72 Ketone, 11 Kidney, 102 Kidneys, removal "I part of, 65 o 194 ESSENTIALS OF CHEMICAL PHYSIOLOGY Kjeldahl's method of estimating nitro- gen, 186 Kossel on protamines, 25, 31 ; on his- tone, 87 Koumiss, 37 Kuhne, on albumin, 56 ; on peptone, 60 ; on precipitation of pepsin, 55 Kiilz's method of extracting glycogen, 134 Kutscher on antipeptone, 61 Lactalbumtn, 26, 32, 139 ; properties of, 36 Lactic acid, 4, 10, 54 ; test for, 153 Lactic acid fermentation, 14, 63 ; in milk, 37 ; in muscle, 41, 153 ; or- ganisms, 13 Lactometer, 32 Lactose, 9, 10, 12, 13, 32, 36, 37, 135 ; in urine, 13, 126, 135 ; tests for, 135 Lsevo-rotatory, 10, 12, 24 Laky blood, 86 Lanoline, 71 Lard, 10 Lateritious deposit, 114, 116 Laurent's polarimeter, 178, 179 Lead, 3, 8 Lecithin, 3, 4, 63, 68, 113 Leech extract, 81 Leucine, 22, 31, 56, 59, 60, 61, 63 ; as a urea precursor, 107-110 ; in urine, 120, 123 ; tests for, 144 Leucine crystals, 64 Levulose, 10, 11, 12, 135 ; in blood, 12 ; in muscle, 12 ; in urine, 12 ; re- actions of, 12 Lieberkuhn's crypts, 62 ; jelly, 27 Liebig's extract, 155; method of esti- mating chlorides, 160 ; urea, 157 Lime water, 21 Lipochrome, 37, 38 ; in muscle, 1 54 Lipolytic ferments, 49 Lithates, see Urates Lithium, 3 Litre, standard of capacity, 7 Liver, function of, in relation to urea, 108 ; uric acid, 116 Living test-tube experiment, 82 Lungs, 94 Luther on nitrogen loss in hypobromite method of estimating urea, 158 Lysatinine, 109 Lysine, 31, 60, 109 M McKendbick on cholestebik, 71 MacMunn on stercobilin, 73 McWilliam's test for proteids, 142 Magnesium, 3, 8 Magnesium sulphate, action of, on pro- teids, 19, 26, 149 Malic acid, 104 Mallow, 12 Malpighian capillaries, 102 Malting ferment, 137 Maltose, 9, 10, 14, 43, 46. 74, 135 Malt upon starch, action of, 137; diastase, 14 Maly on the formation of hydrochloric acid, 53 Manganese, 3, 8 Mannite, 11 Marsh gas, 49 Measures of capacity, 7 ; of length, 6 Meat, 33, 38 ; constituents of, 39 Meconium, 73 Medulla oblongata, diabetic puncture of, 126 Melanin, 171 Mercurial air-pumps, 182, 183, 184 Mercuric chloride creatinine, 119, 132, 167 Mercuric nitrate, method of estimating chlorides, 160 ; urea, 157 Mercury compound of creatinine, 119, 132, 167 Metabolism, 65 Methaunoglobin, 88, 93, 94, 145, 148; crystals of, 144 ; in urine, 128 Methane, 63 Methylene blue experiments, 97 Metric system, 6 Microchemical detection of glycogen,135 Micrococcus urea1, 106, 122 Microspectroscope, 92, 145 Milk, 16, 32, 35-38 ; alcoholic fer- mentation of, 37; coagulation of, 36, 139-140 ; composition of, 36 ; fats of, 37 ; proteids of, 36 ; salts of, 38 ; souring of, 37 Milk-curdling ferment of pancreas, 59 62 ; of stomach, 32, 55 Milk-sugar, 10, 12, 13, 32, 74 ; crystals, 13 Millon's test, 4, 19, 24, 32, 43 Mineral compounds, 3 Mohr's method of estimating chlorides, 160 Moleschott's diet, 34 Monatomic alcohols, 17 Monoacetin, 17' Monochromatic light, 178 Monosaccharides, 10, 11 Moore and Eockwood on fat absorption, 77 Moore's test for sugar, 11 Morner and Sjoqvist method of esti- mating urea, 159 INDEX 195 Morner on haemin, 87 Morris and Brown on starch digestion. a; Mucic acid. 12 Mucin. 2, 21, 28. 72 ; in bile, 29,68; in saliva, 43, 44 ; in urine, 119 ; tests for, 131 Mncinogen, 44 Mucoids, 28 Mucous glands, structure of, 45 Mucous membrane of frog's intestine. 75 Mucous salivary glands, 2 Munk on fat absorption, 76 Murexide test, 114, 115 Muscle, 38, 153 ; pigments of, 154 ; plasma of, 153 ; extractives of, 153 Muscle-sugar, 12 Muscular movement, 2 ; exercise and urea, 107 ; exercise and carbonic- acid. 96 Musculin, 154 Mutton. 33 Myo-albumin, 154 Myo-globulin, 154 Myo-haematin, absorption spectrum of, 154 Myosin, 4, 27, 38, 153 ; ferment. 19 Myosinogen, 22, 26, 153 Myxcedema, 65 >; Nj.ncki and Sieber, on pigments, 168 Nencki's experiments, on urea, 110 Neurokeratin, 31 Neutral salts, action of, on proteids, 19, 25, 26, 32. 30, 139, 141, 149, 150, 152, 153 Nickel sulphate, action of, on proteids, 112 Nitrate of urea, 106, 114 Nitric oxide hemoglobin, 88, 94, 148 Nitrogen, 3 ; estimation of, 186 Nitrogenous foo I Nitrous acid, action of, on urea, 100 Nucleic a:< Nnclein, 2. 28. 29, 30, ■'.!. 113; bases from, 117 Nucleo-proteid, 2,28-30; in bill decomposition of, 30 ; teste for, 131 Nucleus, functions of, 2 O Ouro Af.n.. 17, 18 Olein, 10, 17, 18, 37 Olfactory Ol | :• 13 Oliver's hemacytometer, 173 ; hemo globinometer, 175, 170 Organic compounds, 3 Osazones, 135, 136 Osmosis, 23, 99 Ossein, 30 Osteomalacia, 120 Ovarian cyst fluid, 28 Ovo-mucoid, 28, 38 Oxalate of calcium in milk, 32 ; in plasma, 78, 151 ; in urine, 114, 121 Oxalate of urea, 100 Oxidases, 49 Oxygen, 3, 8 ; in blood. 90 Oxyhemoglobin, 85, 88, 145. 148; in muscle, 154 Oxyhemoglobin crystals, 86, 144 ; pre- paration of, 79, 144 Oxyhemoglobin, pure, preparation of, 148 Oxyntic cells of cardiac glands, 52 Oxyphenyl-amido-propionic acid, 65 Pale muscle, 154 Palmitic acid, 17, 18 Palmitine, 16, 17, 18, 37 Pancreas, extirpation of, 65, 120 ; graf ing the, 65 ; structure of, 58 Pancreatic digestion, 58-65, 143 Pancreatic juice, action of, 60 ; compo- sition of, 59 ; secretion of, 58 Panunrs method of precipitating serum globulin, 149 Paraglobulin, see Serum globulin Para-mucin, 28 Paramyosinogen, 154 Parapeptone, 50, 50 Parietal cells of cardiac glands, 52 Parotid gland, 43, 44 Paroxysmal hemoglobinuria, 128 Partial pressure of gases, 90-100 Pathological urine, 124 ; pigments of, 171 Pavy on composition of proteid, 28 ; on glycogenic function of liver, 74 ; on estimation of sugar, 125 Pawlow on secretory nerve-fibres to the gastric glands, 54 ; to the pancreas, Pepsin, 19, 50, 53, 54, 01 ; -olutions, activity of. If:; Pepsinogen, 53 Peptic digestion, 50-57 Peptone, 19, 22, 27,29, 56,57.58, 74, 129; in mine. 120; test-: for, 141 Peptonuria, 120 Perfect foods, 33 irdiaJ tluids, 82 196 ESSENTIALS OF CHEMICAL PHYSIOLOGY Pettenkofer's test for bile salts, 66, 69, 128 Pfluger's mercurial air-pump, 182, 183 Phenol, 63, 72 Phenol sulphate of potassium, 112 Phenyl-hydrazine test for sugars, 14, 135 Phloridzin, 126 Phosphates of urine, 113, 114, 123 ; es- timation of, 162 ; tests for, 101 Phosphates, stellar, 121 Phosphates, triple, 113 Phosphorus, 3, 8 Photographic spectra, 147 Physiological chemistry, 1 Physiological proximate principles, de- tection of, 129 Pickering on colour reactions of pro- teids, 142 Picramic acid, 11 Picric acid test for sugar, 11, 125, 181 Picro-saecharometer of Sir George John- son, 181, 182 Pigment of red corpuscles, 85 ; of muscle, 154 ; of urine, 168 Pigments, 3 Piotrowski's reaction, 19, 24, 142 Plasma, constituents of, 83 ; gases of, 83 ; proteids of, 83 Plasma of blood, 78, 80, 82 ; of muscle, 153 Poisonous alkaloids, 63 Polarimeter of Laurent, 178, 179 ; of Soleil, 177 ; of Zeiss, 178 Polarised light, action of carbohydrates on, 10-14 ; of proteids on, 24 Polysaccharides, 10, 11, 14 Pork, 38 Portal vein, 66 Potash, method of extracting glycogen, 134 ; of showing zymogen granules, 144 Potassium, 3, 8 Potassium f erricyanide in preparation of methaemoglobin, 145 Potassium ferrocyanide in the estima- tion of phosphates, 162 Potassium permanganate in estimation of uric acid, 165 Potassium sulphocyanide in saliva, 43 Precipitants of proteids, 24, 25 Precipitation, 24 Prevost and Dumas on formation of urea, 107 Primary albumoses, 56 Principal cells of stomach, 52 Prisms in direct vision spectroscope, 90 Propeptone, 50, 55, 56 Propionic acid, 17 Protamines, 25, 31 Proteids, 2, 4, 19-31, 38, 39, 41; ab- sorption of, 74 ; classification of, 25 ; coagulation of, 2, 22, 27 ; composition of, 2, 21 ; compound, 25 ; crystalli- sation of, 23 ; definition of, 21 ; di- gestion of, by gastric juice, 55-57 ; digestion of, by pancreatic juice, 60, 61 ; molecule, 21 ; of blood plasma, 83 ; of milk, 36 ; of muscle, 154 ; of serum, 83 ; in urine, 126 ; precipi- tants of, 24, 25 ; simple, 25, 26 ; so- lubilities of, 22, 26 ; tests for, 19, 22, 24, 129 ' Proteid-sparing ' food, 30 Proteolytic ferments, 49, 50 Proteoses, 19, 22, 27, 55, 56, 57, 74, 129 ; in urine, 126 Prothrombin, 81 Proto-albumose, 56, 141 Protoplasm, chemical structure of, 2 ; properties of, 2 Proximate principles, classification of, 3, 4 ; of food, 33 ; scheme for detect- ing, 129 Pseudo-mucin, 28 Pseudo-nuclein, 29 Ptomaines, 47 Ptyalin, 14, 44, 46, 48 Pulses, 40 Pumps, mercurial air, 182, 183, 184 Purine bases, 117 Purpurate of ammonia, 114 Pus in urine, 128 ; tests for, 128 Putrefaction, 47 Pyloric glands, 51, 52 Pyrocatechin, 171 Q Quantitative estimation of albumin, 124; of chlorides, 159-161 ; of creatinine, 166, 167; of dextrose, 125, 181; of glycogen, 134; of lactose, 13, 125 ; of maltose, 14, 125, 137; of nitrogen, 186 ; of phosphates, 162 ; of sul- phates, 163; of sugar, 125, 181; of urea, 101, 157, 158, 159 ; of uric acid, 116, 165 Bacemic acid, 125 Eanke's diet, 34 Eeagents necessary for practical work, 4 Reaumur's scale, 7 Eed blood corpuscles, 84 Eed muscle, 154 Reflex action, 43 Eennet, 32, 49, 53, 139 Eennin, 53 Eeproduction, 2 INDEX 197 Respiration, chemistry of, 94 Respiratory quotient, 96 Rigor mortis, 41 Ringer on caseinogen, 139 Riva on urochrome, 104 Rockwood and Moore on fat absorption, s Sacchakic acid, 12 Saccharimeters, 177 Saccharoses, 10 Salicylsulphonic acid, action of, on proteids, 142 Saliva. 21, 43-49; action of, 44 ; com- position of, 44 ; secretion of, 43 Salivary corpuscles, 43 ; glands, 43 Salkowski's reaction, 71 ; method of estimating sulphates, 163, 164 Salmine, 31 Saponification, 61 Sarcolemma, 31 Sarcosine, 118 Schiifer on internal secretion, 65 Schiff on bile circulation, 68-72 Schizomycetes, forms of, 47 Schmalz's capillary picnometer, 177 Schmidt on precipitating serum globu- lin, 149; on salts of plasma, 84 ; on preparation of fibrin-ferment, 83 Schroder's work on urea, 110 Schwann, white substance of, 70 Sebum, 71 Secretion, internal, 65 ; of bile, 67 Sediments in urine, 119-123 Serous glands, 45 Serum albumin, 22, 26, 78 ; heat coagu- lation of, 150 Serum casein, 149 Serum globulin, 22, 26, 78, 7!) ; heat co- agulation of, 150 Serum lutein, 78 Serum of blood, 23, 78, 79, 82, 149; proteids of, 83 ; of muscle, 153 Serum proteids, separation of, 78 Sheep's-wool fat, 71 Shell of eggs, 38 Siegfried on antipeptone, 61 Silicon, 3, 8 Silver nitrate, method of estimating ■.i ides, 160 Sir William Gowers' hemacytometer, 172: hsmoglobinometer, 174 Sir William Roberts on estimation of ai in urine, 127 Bkatole, 63, 72 Skatoxyl, 171 ; red, 171 Skimmed milk, 92, 96 Smoky urin<-. ] 28 renom, 18 Soap, 41, 59, 62, 83 Sodio-magnesium sulphate, action of, on proteids, 150 Sodium, 3, 8 Sodium bicarbonate in blood, 98, 99 Sodium chloride, 3 ; action on proteids, 26, 119; on nucleo-proteids, 152 Sodium hypobromite method of estimat- ing urea, 101 Sodium phosphate, 21 ; acid, 21 Sodium sulphate plasma, 78, 151 Solar spectrum, 89 SoleiPs saccharimeter, 177 Soluble starch, 15 Sorbite, 11 Soret's band, 148 Specific gravity of blood, 171 ; of milk, 35 ; of urine, 104 Specific rotatory power, 179 Spectra, 89, 92 ; of haemoglobin and its derivatives, 146 ; photographic, of methasmoglobin, oxyhamioglobin and haemoglobin, 147 ; of myohamatin, 154, of urinary pigments, 103, 170 Spectro-polarimeter, 180 Spectroscope, 79, 88, 89 Spermatozoa, 30 Starch, 4, 9, 10, 14, 39, 129; soluble, 15 ; action of malt on, 137 ; digestion of, 43, 48, 61 ; test for, 9, 14 Steapsin, 49, 58 ; action of, 59, 61 Stearic acid, 17, 18 Stearin, 16, 17, 18, 37 Steatolytic ferments, 49 Stellar phosphates, 121 Stercobilin, 70, 72, 73, 103, 168 Steward, G. N., dietary, 34 Stirling on preparation of pure oxy- hemoglobin, 148 Stokes' fluid, 88 Stomach, glands of, 51 Sturine, 31 Sublingual gland, 43, 44 Submaxillary gland, 43, 44 Succus entericus, 13, 62, 74 Sucroses, 10, 11 Sugar, 4, 48 ; cane, 12 ; in blood, 83 ; in urine, 126 ; muscle, 12 ; tests for, 9-14, 129 Sugar in urine, quantitative determina- tion of, 125, 181 Sugar maple, 12 Sulphates of urine, 112 ; estimation of, L63 ; tests for, 101 Sulphur, 3, 8 Suprarenal gland, removal of, 96 Sutton's modification of Mohr's method of estimating chlorides, 160 Swim-bladder of lishes, 9!) Symbols and atomic weights, 8 Syntonin, 21, 27,60, 55, 153 198 ESSENTIALS OF CHEMICAL PHYSIOLOGY Tartaric acid, 104 Taurine, 42, 69, 72 Tauro-carbamic acid, 72 Tauro-cholate of soda, 67, 69 Tauro-cholic acid, 67, 69 Tea, 42 Teichmann's crystals, 87 Tendon, 21 Tension of aqueous vapour, 7 ; of gases, 97-99 Testis, removal of, 65 Theine, 42 Theobromine, 42 Thermometric scales, 7 Thrombin, 81 Thudichum on urochrome, 168 Thyroid gland, removal of, 65 Tissue-fibrinogens, 29, 152 Tissue respiration, 95, 100 Tomes on enamel, 30 Tonsils, 43 Torula urese, 106 Torulse, 46, 48 Torricellian vacuum, 88, 184 Triacetin, 17 Triatomic alcohol, 17 Trichloracetic acid as a precipitant of proteids, 142 Triolein, 17 Tripalmitin, 17 Triple phosphate, 113, 121, 123 Tristearin, 17 Trammer's test, 9, 11-15, 32, 43 Tropaeolin test, 143 Trypsin, 49, 58, 59 ; action of, 60 Trypsinogen, 59 Tryptic enzyme, 61 Tryptophan, 60 Tunicates, 16 Tyrosine, 22, 31, 56, 59-61, 64, 127 ; in urine, 108, 109, 120, 123 ; tests for, 144 U Umbilical cord, 21 Uncrystallisable sugar, 12 Unorganised ferments, 45 Uraemia, 107 Uranium acetate in estimation of phos- phates, 162 Uranium nitrate in estimation of phos- phates, 162 - — Urate, acid ammonium, deposit of, 120 ; acid sodium, deposit of, 120 Urates, 114, 120, 123 Urea, 3, 4, 22, 46, 65, 72, 83, 101, 105- 111 ; composition and compounds, 105 ; crystals of, 104 ; decomposition of, 106 ; estimation of, 101, 157 ; mode and site of formation, 107 ; pre- paration of, 159 ; quantity excreted, 107 ; tests for, 131 ; where formed, 107 Urea nitrate, 105 ; preparation of, 114 Urea oxalate, 105 Uric acid, 22, 72, 83, 115, 123 ; crystals of, 115 ; estimation of, 116, 165 ; pre- paration of, 114, 115, 165 ; origin of, 116 ; tests for, 131 Urina potus, 105 Urinary deposits, 119 Urine, 101-128 ; composition of, 105 ; inorganic constituents of, 111 ; tests for abnormal constituents of, 132 ; tests for constituents of, 131 Urinometer. 101, 104 Urobilin, 70, 72, 103, 168 ; absorption bands of, 169, 170 Urobilinogen, 103 Urochrome, 104, 168 Uroerythrin, 114, 120, 169 : absorption bands of, 169, 170 Urorosein, 171 ; absorption bands of, 170 Valeric acid, 17, 63 Vegetable acids, 4 ; foods, composition of, 39 ; parchment, 22 Vegetables, composition of, 42 ; green, 42 Venous blood, gases of, 96 Villus, section of, 75 Vitellin, 23, 28, 29, 38, 40 Vitellose, 55 Vitreous humour, 21 W Waller's modification of Zuntz's gas apparatus, 185 Warm bath, 20 Water in protoplasm, 2, 4 ; density of, 7 Weights and measures, 6 Whartonian jelly, 21 Whey, 32, 36 Whey, proteid, 36 White blood corpuscles, 2, 84 White of egg, 2, 19, 20, 50, 58 Whole flour, 39 Witte's peptone, 141 Wohler, preparation of urea, 105 Wooldridge on tissue-fibrinogen, 29, 81, 152 | ' Worth ' of corpuscles, 177 INDEX 199 x Xahthdjb, 59, 83, 117, 120 Xanthine group, 29 Xanthoproteic test, 19, 43 Y Yeast, action of, 11, 12, 13; cells, 46; in bread-making, 40 ; in testing for sugar, 11, 127 Yvon on removal of creatinine and urates, 158 Z Zeiss' polarimeter. 178 Zinc chloride ci'eatinine, 118, 167 Zuntz's gas apparatus, 185 Zymogens, 53, 84 ; granules, 144 imi.vimi i;v BPOTTIBWOODJ :.\V-.-l 1:1:1.1 SQUABS DOS A LIST OF WORKS ON MEDICINE, SURGERY, AND GENERAL SCIENCE, PUBLISHED BY LONGMANS, GREEN & CO., 39 PATERNOSTER ROW, LONDON, E.C. 91 AND 93 FIFTH AVENUE, NEW YORK, and 32 HORNBY ROAD, BOMBAY. 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