Columbia (Mntoersrttp tntljpCitpuflfttigork College of ipfjpatctansi anb gmrgeona Htbrarp t resented by ^DR. WILLIAM J. GI to enrich the library resourcei available to holders of the Us GIES FELLOWSHIP in Biological Chemistry PRACTICAL PHYSIOLOGICAL CHEMISTRY HAWK Absorption Spectra. PLATE I. & V £ /■■ Oxy haemoglobin. Haemoglobin. Carboxy- haemoglobin. Neutral Met- haemoglobin. Alkaline Met- haemoglobin. Alkali Haeaatin. Absorption Spectra. PLATE II. n '<■ / / Reduced Alkali Haematm or HaemiK hromoeen. Acid Haematin in ethereal solution. Acid Haemato- porphyrln. Alkaline Haematopor- phyrin. Urobilin or Hydro- bllirubin in acid solution. Urobilin or Hydro- bilirubin in alkaline solution after the addition of zinc chloride solution. Bilicyanin or Cholecyanin in alkaline solution. PRACTICAL PHYSIOLOGICAL CHEMISTRY A Book UNSIGNED FOR USE IN COURSES IN PRACTICAL PHYSIOLOGICAL CHEMISTRY IN SCHOOLS OF MEDICINE AND OF SCIENCE BY PHILIP B. HAWK. M.S., Ph.D. DBMONSTRATOR "l PHYSIOLOGII A I. I HBMISTRV IN THB DEPARTMENT OF Ml I.I' INI l II I INI\ BR! I NSYLYANIA WITH Tiro FULL PAGE PLATES OF ABSORPTION SPECTRA IN COLORS. :■: ADDITIONAL FULL PACE COLOR PLATES AND ONE HUN- DRED AND TWENTY-SIX FIGURES OF WHICH TWELVE ARE IN COLORS PHILADELPHIA P. BLAKISTON'S SON & CO. IOI2 WALNUT STREET 1907 Copyright 1907, By P. Blakiston's Son & Co. ?s\4 Press of The new Era Printing Compam/ Lancaster, Pa. THESE PAGES ARE AFFECTIONATELY DEDICATED TO MY MOTHER PREFACE The plan followed in the presentation of the subject of this volume is rather different, so far as the author is aware, from that set forth, in any similar volume. This plan. how< he feels to be a logical one and has followed it with satis tory results during a period of three years in his own classes at the University of Pennsylvania. The main point in which the plan of the author differs from those previously pro], is in the treatment of the food stuffs and their digestion. In Chapter IV the "Decomposition Products of Proteids" has been treated although it is impracticable to include the study of this topic in the ordinary course in practical physio- logical chemistry. For the specimens of the decomposition products, the crystalline forms of which are reproduced by original drawings or by micro-photographs, the author is in- debted to Dr. Thomas B. Osborne, of Xew Haven, Conn. Because of the increasing importance attached to the ex- amination of feces for purposes of diagnosis, the author has devoted a chapter to this subject. He feels that a careful study of this topic deserves to he included in the courses in practical physiological chemistry, of medical schools in par- ticular. The subject of solid tissues (Chapters XIII. XIV and XV) has also been somewhat more fully treated than has generally been customary in books of this character. The author is deeply indebted to Professor Lafayette B. Mendel, of Yale University, for his careful criticism of the manuscript and to Professor John Marshall, of the University of Pennsylvania, for his painstaking revision of the proof. He also wishes to express his gratitude to Dr. David L. Edsall for his criticism of the clinical portion of the volume ; to Dr. Otto Folin for suggestions regarding several of his quantitative methods, and to Mr. John T. Thomson for assist- ance in proof-reading. Y11I PHYSIOLOGICAL CHEMISTRY. For the micro-photographs of oxyhemoglobin and haemm reproduced in Chapter XI the author is indebted to Professor E. T. Reichert, of the University of Pennsylvania, who, in collaboration with Professor A. P. Brown, of the University of Pennsylvania, is making a very extended investigation into the crystalline forms of biochemic substances. The micro- photograph of allantoin was kindly furnished by Professor Mendel. The author is also indebted for suggestions and assistance received from the lectures and published writings of numerous authors and investigators. The original drawings of the volume were made by Mr. Louis Schmidt whose eminently satisfactory efforts are highly appreciated by the author. Philip B. Hawk. Philadelphia, March 27, 1907. CONTENTS. CHAPTER I. ( Iarbohydrates I CHAPTER II. Salivary Digestion 32 CHAPTER III. 1 *R( ITEIDS 42 CHAPTER IV. Decomposition Products of Proteids 65 CHAPTER V. Gastric Digestion 83 CHAPTER VI. Fats 96 CHAPTER VII. Pancreatic Digestion 106 CHAPTER VIII. Bile 116 CHAPTER IX. Putrefaction Products 129 CHAPTER X. Feces 139 CHAPTER XI. Blood 148 CHAPTER XII. Milk 187 ix X CONTENTS. CHAPTER XIII. Epithelial and Connective Tissues 197 CHAPTER XIV. Muscular Tissue 206 CHAPTER XV. Nervous Tissue • 220 CHAPTER XVI. Urine: General Characteristics of Normal and Path- ological Urine 226 CHAPTER XVII. Urine : Physiological Constituents 237 CHAPTER XVIII. Urine : Pathological Constituents 282 CHAPTER XIX. Urine : Organized and Unorganized Sediments 318 CHAPTER XX. Urine : Calculi 340 CHAPTER XXI. Urine : Quantitative Analysis 344 CHAPTER XXII. Quantitative Analysis of Milk, Gastric Juice and Blood 3 8 ° LIST OF ILLUSTRATIONS. l'l Ml. I Absorption Spectra. , . . Frontispiece 1 1. AbsorptK m bpectra. 1 1 1 . ( Isazons. .- Opp< »site page 5 l\. Normal Erythrocytes and Leucocytes .... Opposite page 151 V. Uric Acid Crystals Opposite page 247 VI. Ammonium I Irate Opposite page 324 Fig. Pagi 1. Dialyzing Apparatus for Students' Use 6 2. Einhorn Saccharometer 10 3. One Form of Laurent Polariscope 12 4. Diagrammatic Representation of the course of the Light through the Laurent Polariscope 13 5. Polariscope | Schmidt and I lausch Model ) 14 6. Iodoform 21 7. PotaP > Starch 23 8. I '.can Starch 23 i). Arrowroot Starch 23 10. Rye Starch 23 1 1 . I larley Starch 23 12. ( >at Starch 23 13. Buckwheat Starch 23 14. Mai/.e Starch 23 1 5. Rice Starch 27, 16. Pea Starch 23 17. Wheat Starch 23 18. Microscopical Constituents of Saliva 36 19. Coagulation Temperature Apparatus 50 20. Edestin 54 21. Excelsin, the Proteid of the Brazil Nut 55 22. Fischer Apparatus ' v 23. 1 yrosin 24. Leucin "9- 25. Aspartic Acid 7° Xll LIST OF ILLUSTRATIONS. 26. Glutamic Acid 71 27. Glycocoll Ester Hydrochloride 72 28. Phenylalanin y^ 29. L'sevo-a-Prolin 74 30. Copper Salt of Prolin 75 3 1 - Serin 75 32. Cystin j6 33. Lysin Picrate 78 34. Histidin Hydrochloride 79 35. Beef Fat 96 36. Mutton Fat 99 37. Pork Fat 101 38. Palmitic Acid 102 39. Melting-Point Apparatus 103 40. Bile Salts 118 41. Bilirubin (Hsematoidin) 119 42. Cholesterin 125 43. Taurin 126 44. Glycocoll 127 45. Ammonium Chloride 133 46. Microscopical Constituents of Feces 139 47. Hsematoidin Crystals from Acholic Stools 140 48. Charcot-Leyden Crystals 141 49. Boas' Sieve 143 50. Oxyhemoglobin Crystals from Blood of the Guinea Pig 152 51. Oxyhsemoglobin Crystals from Blood of the Rat.... 152 52. Oxyhsemoglobin Crystals from Blood of the Horse. ... 153 53. Oxyhsemoglobin Crystals from Blood of the Squirrel. . 153 54. Oxyhsemoglobin Crystals from Blood of the Dog 154 55. Oxyhsemoglobin Crystals from Blood of the Cat 154 56. Oxyhsemoglobin Crystals from Blood of the Necturus. . 155 57. Effect of Water on Erythrocytes 162 58. Hsemin Crystals from Human Blood 164 59. Hsemin Crystals from Sheep Blood 164 60. Sodium Chloride 167 61. Direct-vision Spectroscope 170 62. Angular-vision Spectroscope Arranged for Absorption Analysis 170 LIST OF [LLUSTRATIONS. xiii 63. Diagram of Angular-vision Spectroscope 171 64. Fleischl's Haemometer [75 65. Pipette of Fleischl's 1 [aem< imeter 1 75 66. Colored Glass Wedge of Fleischl's Haemometer [76 6y. Dare's Haemoglobinometer 17s 68. Horizontal Section of Dare's Haemoglobinometer [79 69. Method of Filling the Capillary Observation Cell of I )are's I [aemoglobinometer 180 70. Tin ima-Zeiss Counting Chamber t8i 71. Thoma-Zeiss Capillary Pipettes 1X2 7_\ ( >rdinary Ruling of Thoma-Zeiss Counting Chamber. . [83 73. Zappert's Modified Ruling of Thoma-Zeiss Counting Chamber [84 74. Normal Milk and Colostrum 188 j^. Lactose [89 76. Calcium Phosphate. 193 yy. Crcatin 209 78. Xanthin 210 79. Hypoxanthin Silver Nitrate 216 80.. Xanthin Silver Nitrate 218 81. Deposit in Ammoniacal Fermentation 229 82. Deposit in Acid Fermentation 22y> 83. Urinometer and Cylinder 231 84. Beckmann-Heiderihain Freezing-Point Apparatus 233 85. Urea 231 ) 86. Urea Nitrate 242 87. Melting- Point Tubes Fastened to Bulb of Thermometer. 243 88. Urea Oxalate 244 89. Pure Uric Acid 241 > 90. Creatinin 251 iji . Creatinin-Zinc Chloride 252 92. Hippuric Acid 93. Allantoin from Cat's Urine 94. Benzoic Acid 264 95. Calcium Sulphate 274 96. " Triple Phosphate " 278 97. The Purdy Electric Centrifuge 318 98. Sediment Tube for the Purdy Electric Centrifuge 318 99. Calcium Oxalate 3 2 ° XIV LIST OF ILLUSTRATIONS. ioo. Calcium Carbonate 321 101. Various Forms of Uric Acid $23 102. Acid Sodium Urate 324 103. Cystin 325 104. Crystals of Impure Leucin 326 105. Epithelium from Different Areas of the Urinary Tract. 329 106. Pus Corpuscles 330 107. Hyaline Casts 331 108. Granular Casts 332 109. Granular Casts 333 1 10. Epithelial Casts 333 111. Blood, Pus. Hyaline and Epithelial Casts 334 1 1 2. Fatty Casts 335 113. Fatty and Waxy Casts 335 1 14. Cylindroids 336 115. Crenated Erythrocytes 337 1 16. Human Spermatozoa 338 117. Esbach's Albuminometer 345 118. Marshall's Urea Apparatus 352 1 19. Hiifner's Urea Apparatus 354 120. Doremus-Hinds Ureometer 355 121. Folin's Urea Apparatus 356 122. Folin's Ammonia Apparatus 358 123. Folin Absorption Tube 359 124. Berthelot-Atwater Bomb Calorimeter 366 125. Soxhlet Apparatus 380 126. Feser's Lactoscope 381 PHYSIOLOGICAL CHEMISTRY. CHAPTER I. CARBOHYDRATES. Tiif: name carbohydrates is given to a class of bodies which are an especially prominent constituent of plants and which are found also in the animal body either free or as an integral part of various proteids. They are called carbohydrates be- cause they contain the elements C, H and O ; the H and O being present in the proportion to form water. The term is not strictly appropriate inasmuch as there are bodies such as acetic acid, lactic acid and inosit which have H and O present in the proportion to form water, but which are not carbohydrates, and there are also true carbohydrates which do not have H and O present in this proportion, e. g., rhamnose, C 6 H 12 O b . Chemically considered, the carbohydrates are aldehyde or ketone derivatives of complex alcohols. Treated from this standpoint the aldehyde derivatives are spoken of as aldoses, and the ketone derivatives are spoken of as ketoses. The carbohydrates are also frequently named according to the number of carbon atoms present in the molecule, e. g., trioses, pentoses and hexoses. The more common carbohydrates may be classified as follows : I. Monosaccharides. i. Hexoses, C 6 H 12 O fi . (a) Dextrose. (b) Lsevulose. 2 PHYSIOLOGICAL CHEMISTRY. (c) Galactose. 2. Pentoses, C 5 H 10 O 5 . (a) Arabinose. (b) Xylose. (c) Rhamnose (Methyl-pentose), C 6 H 12 5 . II. Disaccharides, C^H^On. i. Maltose. 2. Saccharose. 3. Iso-Maltose. 4. Lactose. III. Trisaccharides, C 18 H 32 16 . 1. Raffinose. IV. Polysaccharides, (C 6 H 10 O 5 )x. 1. Starch Group. (a) Starch. (b) Inulin. (c) Glycogen. (d) Lichenin. 2. Gums and Vegetable Mucilage Group. (a) Dextrin. (b) Vegetable Gums. 3. Cellulose Group. (a) Cellulose. (b) Hemi-Cellulose. Each member of the above carbohydrate classes, except the members of the pentose group, may be supposed to con- tain the group C c H 10 O 5 called the saccharide group. The polysaccharides consist of this group alone taken a large num- ber of times, whereas the disaccharides may be supposed to contain two such groups plus a molecule of water, and the monosaccharides to contain one such group plus a molecule of water. Thus, (C H 10 O 5 ) x — polysaccharide, (C 6 H 10 O 5 ) 2 -j- H 2 = disaccharide, C 6 H 10 O 5 + H 2 = monosaccharide. In a general way the solubility of the carbohydrates varies with the number of saccharide groups present, the substances containing the largest number of these groups being the least MONOSACCHARIDES. 3 soluble. This means simply that, as a class, the monosac- charides (hexoses) are the most soluble and the polysac- charides (starches and cellulose) are the least soluble. MONOSACCHARIDES. Hexoses, C 6 Hi 2 O e . The hexoses are monosaccharides containing- six carbon atoms to the molecule. They are the most important of the simple sugars, and two of the principal hexoses. dextrose and lawulose, occur widely distributed in plants and fruits. These two hexoses also result from the hydrolysis of starch and cane sugar. Galactose, which with dextrose results from the hydrolysis of lactose, is also an important hexose. These three hexoses are fermentable by yeast, and yield laevulinic acid upon heating with dilute mineral acids. They reduce metallic oxides in alkaline solution, are optically active, and extremely soluble. With phenylhydrazin they form charac- teristic osazons. CH 2 OH I DEXTROSE, (CHOH) 4 . I CHO Dextrose, also called glucose, grape sugar, or diabetic sugar, is present in the blood in small amount and may also occur in traces in normal urine. After the ingestion of large amounts of saccharose, lactose or dextrose an alimentary gly- cosuria occasionally arises. In diabetes mellitus very large amounts of dextrose are excreted in the urine. The fol- lowing structural formula has been suggested by Victor Meyer for (/-dextrose: 4 PHYSIOLOGICAL CHEMISTRY. COH I H — C — OH HO — C — H I H — C — OH H — C — OH I CH 2 OH (For further discussion of dextrose see section on Hexoses, page 3.) Experiments on Dextrose. 1. Solubility. — Test the solubility of dextrose in the " ordi- nary solvents " and in alcohol. (In the solubility tests throughout the book we shall designate the following solvents as the "ordinary solvents": H 2 0; 10 per cent NaCl; 0.5 per cent Na 2 C0 3 ; 0.2 per cent HC1 ; concentrated KOH ; concentrated HC1.) 2. Molisch's Reaction. — Place approximately 5 c.c. of concentrated H 2 S0 4 in a test-tube. Incline the tube and slowly pour down the inner side of it approximately 5 c.c. of the sugar solution to which 2 drops of a-naphthol solu- tion (about 15 per cent alcoholic solution) has been added, so that the sugar solution will not mix with the acid. A reddish-violet zone is produced at the point of contact. The reaction is due to the formation of furfurol, HC — CH II II HC C -CHO, \ / by the acid. The test is given by all bodies containing a car- bohydrate group and is therefore of very little practical im- portance. PLATE III. OSAZONS. Upper form, dextrosazon ; central form, maltosazon ; lower form, lactosazon. MONOSACCHARIDES. 5 3. Phenylhydrazin Reaction.— -Test according to one of the following methods: (a) To a small amount of phenvl- hydrazin mixture, furnished by the instructor, 1 add 5 c.c. of the sugar solution, shake well and heal on a boiling water- hath for "iK- halt" to three-quarters of an hour. Allow the tube to cool slowly and examine the crystals microscopically ( Plate 111. opposite). If the solution has become too concen- trated in the boiling process it will be light-red in color and no crystals will separate until it is diluted with water. Yellow crystalline bodies called osazons are formed from certain sugars under these conditions, each individual sugar giving rise to an osazon of a definite crystalline form which is typical for that sugar. Each osazon has a definite melting- point and as a further and more accurate means of identifica- tion it may be recrystallized and identified by the determination of its melting-point and nitrogen content. The reaction tak- ing place in the formation of phciiyhlcxtrosazon is as follows: C 6 H 12 O c + 2(H 2 N-NH-C 6 H 5 ) = Dextrose. Phenylhydrazin. C H 10 O 4 (N-NH-C 6 H 5 ) 2 + 2H 2 + H 2 . Phenyldextrosazon. (b) Place 5 c.c. of the sugar solution in a test-tube, add 1 c.c. of the phenylhydrazin-acetate solution furnished by the in- structor," and heat on a boiling water-bath for one-half to three-quarters of an hour. Allow the liquid to cool slowly and examine the crystals microscopically (Plate III, opposite). The phenylhydrazin test has been so modified by Cipollina as to be of use as a rapid clinical test. The directions for this test are given in the next experiment. 1 This mixture is prepared by combining one part of phenylhydrazin- hydrochloride and two parts of sodium acetate, by weight. These are thoroughly mixed in a mortar. 2 This solution is prepared by mixing one part by volume, in each case, of glacial acetic acid, one part of water and two parts of phenylhydrazin (the base). 6 PHYSIOLOGICAL CHEMISTRY. 4. Cipollina's Test. — Thoroughly mix 4 c.c. of dextrose solution, 5 drops of phenylhydrazin (the base) and ]/ 2 c.c. of glacial acetic acid in a test-tube. Heat the mixture for about one minute over a low flame, shaking the tube con- tinually to prevent loss of fluid by bumping. Add 4-5 drops of sodium hydroxide (sp. gr. 1.16), being certain that the fluid in the test-tube remains acid, heat the mixture again for a moment and then cool the contents of the tube. Ordi- narily the crystals form at once, especially if the sugar solu- tion possesses a low specific gravity. If they do not appear immediately allow the tube to stand at least 20 minutes before deciding upon the absence of sugar. Examine the crystals under the microscope and compare them with those shown in Plate III, opposite page 5. 5. Precipitation by Alcohol. — To 10 c.c. of 95 per cent alcohol add about 2 c.c. of dextrose solution. Compare the result with that obtained under Dextrin, 7, page 28. 6. Iodine Test. — Make the regular iodine test as given under Starch, 5, page 24, and compare this result with the results obtained with starch and with dextrin. 7. Diffusibility of Dextrose. — Test the diffusibility of dex- trose solution through animal membrane, or parchment paper, Fig. 1. Dialyzixg Apparatus for Students' Use. making a dialyzer like one of the models shown in Fig. 1, above. MONOSACCHARIDES. 7 8. Moore's Test. — To 2 3 c.c. of sugar solution in a ; tube add an equal volume of concentrated K'MI <>r NaOH, and boil. The solution darkens and finally assumes a brown color. This is an exceedingly crude tesl and is of little prac- tical value. 9. Reduction Tests. — To their aldehyde or ketone struc tine many sugars owe the property of readily reducing alka- line solutions of the oxides of metals like copper, bismuth and mercury ; they also possess the property of reducing ammo- niacal silver solutions with the separation of metallic silver. Upon this pmperty of reduction the most widely used tests for sugars are based. When whitish-blue cupric hydroxide in suspension in an alkaline liquid is heated it is converted into insoluble black cupric oxide, but if a reducing agent like certain sugars be present the cupric hydroxide is reduced to insoluble yellow cuprous hydroxide, which in turn on further heating may be converted into brownish-red or red cuprous oxide. These changes are indicated as follows : OH / Cu m+ Cu=0 + H 2 0. \^ Cupric oxide. OH (black). Cupric hydroxide, (whitish-blue). OH / Cu \ OH =->- 2Cu-OH + H 2 + 0. OH Cuprous hydroxide.. / (yellow). Cu \ OH PHYSIOLOGICAL CHEMISTRY. Cu-OH Cu-OH Cu \ + H 2 / Cu Cuprous hydroxide (yellow). Cuprous oxide, (brownish-red). The chemical equations here discussed are exemplified in Trommer's and Fehling's tests. (a) Trommer's Test. — To 5 c.c. of sugar solution in a test-tube add one-half its volume of KOH or NaOH. Mix thoroughly and add, drop by drop, a very dilute solution of cupric sulphate. Continue the addition until there is a slight permanent precipitate of cupric hydroxide and in consequence the solution is slightly turbid. Heat, and the cupric hydroxide is reduced to yellow cuprous hydroxide or to brownish-red cuprous oxide. If the solution of cupric sulphate used is too strong a small brownish-red precipitate produced in a weak sugar solution may be entirely masked. On the other hand, particularly in testing for sugar in the urine, if too little cupric sulphate is used a light-colored precipitate formed by uric acid and purin bases may obscure the brownish-red pre- cipitate of cuprous oxide. The action of KOH or NaOH in the presence of an excess of sugar and insufficient copper will produce a brownish color. Phosphates of the alkaline earths may also be precipitated in the alkaline solution and be mistaken for cuprous hydroxide. Trommer's test is not very satisfactory. (b) Fehling's Test. — To about 1 c.c. of Fehling's solution 1 fehling's solution is composed of two definite solutions — a cupric sulphate solution and an alkaline tartrate solution, which may be prepared as follows : Cupric sulphate solution = 34.64 grams of cupric sulphate dissolved in water and made up to 500 c.c. Alkaline tartrate solution = 125 grams of potassium hydroxide and 173 grams of Rochelle salt dissolved in water and made up to 500 c.c. These solutions should be preserved separately in rubber-stoppered bot- tles and mixed in equal volumes when needed for use. This is done to prevent deterioration. MONOSACCHARIDES. 9 in a test-tube add about 4 c.c. of water, and boil. This is done to determine whether the solution will of it-elf cause the formation of a precipitate of brownish-red cuprous oxide. If such a precipitate forms, the Fehling's solution must not lie- used. Add sugar solution to the warm Fehling's solution a few drops at a time and heat the mixture after each addition. The production of yellow cuprous hydroxide or brownish-red cuprous oxide indicates that reduction has taken place. The yellow precipitate is more likely to occur if the sugar solution is added rapidly and in large amount, whereas with a less rapid addition of smaller amounts of sugar solution the brownish-red precipitate is generally formed. This is a much more satisfactory test than Trommer's, but even this test is not entirely reliable when used to detect sugar in the urine. Such bodies as conjugate glycuroiiates, uric- acid, nuclco-proteid and homogentisic acid when present in sufficient amount may produce a result similar to that pro- duced by sugar. Phosphates of the alkaline earths may be precipitated by the alkali of the Fehling's solution and in appearance may be mistaken for cuprous hydroxide. Cupric hydroxide may also be reduced to cuprous oxide and this in turn be dissolved by crcatinin, a normal urinary constituent. This will give the urine under examination a greenish tinge and may obscure the sugar reaction even when a considerable amount of sugar is present. (c) Boettger's Test. — To 5 c.c. of sugar solution in a test- tube add 1 c.c. of KOH or NaOH and a very small amount of bismuth subnitrate, and boil. The solution will gradually darken and finally assume a black color due to reduced bis- muth. If the test is made on urine containing albumin this must be removed, by boiling and filtering, before applying the test, since with albumin a similar change of color is produced (bismuth sulphide). (d) Nylandcr's Test (Almens Test). — To 5 c.c. of sugar solution in a test-tube add one-tenth its volume of Nylander's IO PHYSIOLOGICAL CHEMISTRY. Fig. 2. reagent 1 and boil two or three minutes. The solution will darken if reducing sugar is present and upon standing for a few moments a black color will appear. This color is due to the precipitation of bismuth. If the test is made on urine containing albumin this must be removed, by boiling and filtering, before applying the test. It is claimed by Bechold that Nylander's and Boettger's tests give a negative reaction with solutions containing sugar when mercuric chloride or chloroform is present, a claim which Zeidlitz has very recently shown to be incorrect. A positive Nylander or Boettger test is probably due to the following reactions : (a) Bi(OH) 2 N0 3 + KOH = Bi(OH) 3 + KN0 3 . (b) 2Bi(OH) 3 — 30 = Bi 2 + 3H 2 0. 10. Fermentation Test. — "Rub up " in a mortar about 20 c.c. of the sugar solution with a small piece of compressed yeast. Transfer the mixture to a saccharometer (shown in Fig. 2) and stand it aside in a warm place for about twelve hours. If the sugar is fermentable, alcoholic fermentation will occur and carbon dioxide will collect as a gas in the upper portion of the tube. On the completion of fermentation introduce a little potassium hydrox- ide solution into the graduated por- tion by means of a bent pipette, place the thumb tightly over the opening in the apparatus and invert the saccharometer. Explain the re- sult. 1 Nylander's reagent is prepared by digesting 2 grams of bismuth snb- nitrate and 4 grams of Rochelle salt in 100 c.c. of a 10 per cent potas- sium hydroxide solution. The reagent is then cooled and filtered. Einiiorn Saccharometer. MONOSACCHARIDES. II ii. Barfoed's Test. — To -> 3 cc. of Barfoed's solution 1 in a test-tube add a few drops of dextrose solution, and boil. Allow to stand a few moments and examine. Observe tbe red precipitate. What is it? 12. Formation of Caramel. — Gently heat a small amount of pulverized dextrose in a test-tube. After the sugar has melted and turned brown, allow the tube to cool, add water and warm. The coloring matter produced is known as caramel. 13. Demonstration of Optical Activity. — A demonstra- tion of the use of the polariscope, by the instructor, each stu- dent being required to take readings and compute the " spe- cific rotation." Use of the Polariscope. For a detailed description of the different forms of polari- scopes, the method of manipulation and the principles in- volved the student is referred to any standard text-book of physics. A brief description follows : An ordinary ray of light vibrates in every direction. If such a ray is caused to pass through a " polarizing " Nicol prism it is resolved into tzvo rays, one of which vibrates in every direction as before and a second ray which vibrates in one plane only. This latter ray is said to be polarized. Many organic substances (sugars, proteids, etc.) have the power of twisting or rotating this plane of polarized light, the extent to which the plane is rotated depending upon the number of molecules which the polarized light passes. Substances which possess this power are said to be " optically active." The specific rotation of a substance is the rotation expressed in degrees which is afforded by one gram of substance dissolved in 1 cc. of water in a tube one decimeter in length. The specific rotation, (a)„, may be calculated by means of the following formula, 1 Barfoed's solution is prepared as follows: Dissolve 4 grams of copper acetate in 100 cc. of water and acidify with acetic acid. 12 PHYSIOLOGICAL CHEMISTRY. («)»- ^•/' in which -o = sodium light. a = observed rotation in degrees. p = grams of substance dissolved in I c.c. of liquid. / = length of the tube in decimeters. If the specific rotation has been determined and it is desired to ascertain the per cent of the substance in solution, this may be obtained by the use of the following formula, The value of p multiplied by 100 will be the percentage of the substance in solution. An instrument by means of which the extent of the rota- tion may be determined is called a polariscope or polarimeter. Such an instrument designed especially for the examination of sugar solutions is termed a saccharimeter or polarising sac- cliarimeter. The form of polariscope shown in Fig. 3, below, Fig. 3. One Form of Laurent Polariscope. B, Microscope for reading the scale ; C, a vernier ; E, position of the analyzing Nicol prism ; H, polarizing Nicol prism in the tube below this point. MONOSACCIIAUIIH.S. 13 consists essentially of a long barrel provided with a Nicol prism at either end (Fig. 4. below). The solution under examination is contained in a tube which is placed between these two prisms. At the front end of the instrument is an adjusting" eye-piece for focusing and a large recording disc which registers in degrees and fractions of a degree. The light is admitted into the far end of the instrument and is polarized by passing - through a Nicol prism. This polarized ray then traverses the column of liquid within the tube men- tioned above and if the substance is optically active the plane of the polarized ray is rotated to the right or left. Bodies rotating the ray to the right are called dextro-rotatory and those rotating it to the left lecvo-rotatory. Fig. 4. (D / 1 Diagrammatic Representation of the Course of the Light through the Laurent Polariscope. (The direction is reversed from that of Fig. 3, p. 12.) a, Bichromate plate to purify the light ; b, the polarizing Nicol prism ; c, a thin quartz plate covering one-half the field and essential in producing a second polarized plane ; d, tube to contain the liquid under examination ; e, the analyzing Nicol prism ; f and g, ocular lenses. Within the apparatus is a disc which is so arranged as to be without lines and uniformly light at zero. Upon placing the optically active substance in position, however, the plane of polarized light is rotated or turned and it is necessary to rotate the disc through a certain number of degrees in order to secure the normal conditions, i. c " without lines and uniformly light." The difference between this reading and the zero is a or the observed rotation in degrees. Polarizing saccharimeters are also constructed by which the percentage of sugar in solution is determined by making an observation and multiplying the value of each division on a H PHYSIOLOGICAL CHEMISTRY. horizontal sliding- scale by the value of the division expressed in terms of dextrose. The value, in terms of dextrose, of each of the divisions on the scale of the Laurent saccharimeter used in the laboratory of physiological chemistry at the University Fig. s Polariscope (Schmidt and Hansch Model). of Pennsylvania is 0.2051. This factor may vary according to the instrument. qjj q-jj I LJEVULOSE, (CHOH) 3 . I CO I CH 2 OH As already stated, lsevulose, sometimes called fructose or fruit sugar, occurs widely disseminated throughout the plant MONOSACCHARIDES. 1 5 kingdom in company with dextrose. Its reducing- power is somewhat weaker than that of dextrose. Lawulose does not ordinarily occur in the urine in diabetes mellitus-, lmt has been found in exceptional cases. With phenylhydrazin it forms the same osazon as dextrose. With methylphenylhydrazin, laevu- lose forms a characteristic lsevulose-methylphenylosazon. (For a further discussion of kevulose see the section on Hexoses, p. 3.) Experiments on L.evulose. 1. Seliwanoff's Reaction. — If a solution of resorcin in dilute HC1 (1 vol. concentrated 1 1 CI to 2 vols. H 2 0), be warmed with lsevulose the liquid will become red and a pre- cipitate will separate. The precipitate may be dissolved in alcohol to which it will impart a striking red color. 2. Phenylhydrazin Test. — Make the test according to di- rections under Dextrose, 3 or 4, pages 5 and 6. CH 2 OH I GALACTOSE, (CHOH) 4 . CHO Galactose occurs with dextrose as one of the products of the hydrolysis of lactose. It is dextro-rotatory, forms an osazon with phenylhydrazin and ferments slowly with yeast. Experiments on Galactose. 1. Tollens' Reaction. — To 5 c.c. of hydrochloric acid. having a specific gravity of 1.09, add a slight excess of phloroglucin, the acid being kept on a boiling water-bath during the addition. A few cubic centimeters of galactose solution should now be added and the heating continued. A red color is produced. Compare this color with that given by pentoses (see page 16). 2. Phenylhydrazin Test. — Make the test according to di- rections given under Dextrose, 3 or 4, pages 5 and 6. 1 6 PHYSIOLOGICAL CHEMISTRY. Pentoses, C 5 H 10 O 5 . In plants and more particularly in certain gums, very com- plex carbohydrates, called pentosans, occur. These pentosans through hydrolysis by acids may be transformed into pentoses. Pentoses do not ordinarily occur in the animal organism, but have been found in the urine of morphine habitues and others, their occurrence sometimes being a persistent condition with- out known cause. They are non-fermentable, have strong reducing power, and form osazons with phenylhydrazin. Pentoses are an important constituent of the dietary of her- bivorous animals. Glycogen is said to be formed after the ingestion of these sugars containing five carbon atoms. On distillation with strong hydrochloric acid pentoses and pen- tosans yield furfurol, which can be detected by its character- istic red reaction with aniline-acetate paper. CH,OH I ARABINOSE, (CHOH) 3 . I CHO Arabinose, one of the most important pentoses, may be ob- tained from gum arabic, plum or cherry gum by boiling for several hours with 1-2 per cent sulphuric acid. It is dextro- rotatory, forms an osazon and has reducing power. Experiments on Arabinose. 1. Tollens' Reaction. — To equal volumes of arabinose solution and hydrochloric acid (sp. gr. 1.09) add a little phlor- oglucin and heat the mixture on a boiling water-bath. Galac- tose, laevulose, pentose or glycuronic acid will be indicated by the appearance of a red color. To differentiate between these bodies make a spectroscopic examination and look for the absorption band between D and E given by pentoses and gly- curonic acid. Differentiate between the two latter bodies by the melting-points of their osazons. DISACCIIARIDES. 1 7 Compare the reaction with that obtained with galactose i page 15 ). 2. Orcin Test. — Repeat i, using orcin instead of pliloro- glucin. A succession of colors from red through reddish- blue to green is produced. A green precipitate is formed which is soluble in amyl alcohol and has absorption hand- between C and D. 3. Phenylhydrazin Test. — Make this test on the arabinose solution according to directions given under Dextrose, 3 or 4. pages g and 6. CHoOH I XYLOSE, (CHOH) 3 . I CHO Xylose, or wood sugar, is obtained by boiling wood gums with dilute acids as explained under Arabinose, page 16. It is dextro-rotatory and forms an osazon. Experiments on Xylose. 1-3. Same as for arabinose (see page 16). RHAMNOSE, C ( ,H ]2 5 . Rhamnose or methyl-pentose is an example of a true carbo- hydrate which does not have the H and O atoms present in the proportion to form water. Its formula is C c H 12 5 . It has been found that rhamnose when ingested by rabbits or hens has a positive influence upon the formation of glycogen in those organisms. DISACCHARIDES, C 12 H 22 O n . The disaccharides as a class may be divided into two rather distinct groups. The first group would include those disac- charides which are found in nature as such. e. £., saccharose and lactose, and the second group would include those disac- 3 l8 PHYSIOLOGICAL CHEMISTRY. charides formed in the hydrolysis of more complex carbohy- drates, c. g.j maltose and iso-maltose. The disaccharides have the general formula C 12 H 22 11 , to which, in the process of hydrolysis, a molecule of water is added causing the single disaccharide molecule to split into two monosaccharide (hexose) molecules. All of the more common disaccharides except saccharose have the power of reducing certain metallic oxides in alkaline solution, notably those of copper and bismuth. This reducing power is due to the presence of the aldehyde group ( — CHO) in the sugar molecule. MALTOSE, CisHssOu. Maltose or malt sugar is formed in the hydrolysis of starch through the action of a ferment, diastase, contained in sprouting barley or malt. Certain enzymes in the saliva and in the pancreatic juice may also cause a similar hydrolysis. Maltose is also an intermediate product of the action of dilute mineral acids upon starch. It is strongly dextro-rotatory, re- duces metallic oxides in alkaline solution and is fermentable by yeast after being inverted (see Polysaccharides, page 21) by the enzyme maltase of the yeast. In common with the other disaccharides, maltose may be hydrolyzed with the for- mation of two molecules of monosaccharide. In this instance the products are two molecules of dextrose. With phenylhy- drazin maltose forms an osazon, maltosazon. Experiments on Maltose. 1— II. Repeat these experiments as given under Dextrose, pages 4-1 1. iso-maltose, 12 H 2 20 11 . Iso-maltose, an isomeric form of maltose, is formed, along with maltose, by the action of diastase upon starch paste, and also by the action of hydrochloric acid upon dextrose. It also occurs with maltose as one of the products of salivary diges- tion. It is dextro-rotatory and with phenylhydrazin gives an DISACCIIARIDES. 1 9 osazon which is characteristic, [so-maltose is very soluble and reduces the oxides of bismuth and copper ill alkaline solu- tion. Pure iso-maltose is probably only slightly fermentable. LACTOSE, CjjgHajOx!. Lactose or milk sugar occurs ordinarily only in milk, but has often been found in the urine of women during pregnancy and lactation. It may also occur in the urine of normal per- sons after the ingestion of unusually large amounts of lactose in the food. It has a strong reducing power, is dextro- rotatory and forms an osazon with phenylhydrazin. Upon hydrolysis lactose yields one molecule of dextrose and one molecule of galactose. In the souring of milk the bacterium lactis and certain other micro-organisms bring about lactic acid fermentation by transforming the lactose of the milk into lactic acid, H OH I I H — C — C — COOH, I I H H and alcohol. This same reaction may occur in the alimentary canal as the result of the action of putrefactive bacteria. In the preparation of kephyr and koumyss the lactose of the milk undergoes alcoholic fermentation, through the action of fer- ments other than yeast, and at the same time lactic acid is produced. Lactose is not fermentable by pure yeast. Experiments on Lactose. I— II. Repeat these experiments as given under Dextrose, pages 4-1 1. SACCHAROSE, C^EUoOn. Saccharose, also called sucrose or cane sugar, is one of the most important of the sugars and occurs very extensively 20 PHYSIOLOGICAL CHEMISTRY. distributed in plants, particularly in the sugar cane, sugar beet, sugar millet and in certain palms and maples. Saccharose is dextro-rotatory and upon hydrolysis, as be- fore mentioned, the molecule of saccharose takes on a mole- cule of water and breaks down into two molecules of mono- saccharide. The monosaccharides formed in this instance are dextrose and laevulose. This is the reaction : C 12 H 22 O n + H 2 = C n H 12 6 + C 6 H 12 O fi . Saccharose. Dextrose. Laevulose. This process is called inversion and may be produced by weak acids, ferments and bacteria. After this inversion the pre- viously strongly dextro-rotatory solution may be lsevo- rotatory. Saccharose does not reduce metallic oxides in alkaline solu- tion and forms no osazon with phenylhydrazin. It is not fer- mentable directly by yeast, but must first be inverted by the ferment invertin contained in the yeast. Experiments on Saccharose. I— II. Repeat these experiments according to the directions given under Dextrose, pages 4-1 1. 12. Inversion of Saccharose. — To 25 c.c. of saccharose solution in a beaker add 5 drops of concentrated HC1 and boil one minute. Cool the solution,, render alkaline with solid KOH and upon the resulting fluid repeat experiments 3 (or 4) and 9 as given under Dextrose, pages 5, 6 and 7. Explain the results. 13. Production of Alcohol by Fermentation. — Prepare a strong (10-20 per cent) solution of saccharose, add a small amount of egg albumin or commercial peptone and introduce the mixture into a bottle of appropriate size. Add yeast, and by means of a bent tube inserted through a stopper into the neck of the bottle, conduct the escaping gas into water. As fermentation proceeds readily in a warm place the escaping POLYSACCHARIDES. 21 Fic. 6. gas may be collected in a eudiometer tube and examined. When the activity of the yeast lias practically ceased, filter the contents of the bottle into a flask and distil the mixture. Catch the first portion of the distillate separately and test for alcohol by one of the following reactions: (a) Iodoform Test. — Render J v } c.c. of the distillate alkaline with potassium hydroxide solution and add a few drops of iodine so- lution. Heat gently and note the formation of iodoform crystals. Examine these crystals under the microscope and compare them with those in Fig. 6. (b) Aldehyde Test. — Place 5 c.c. of the distillate in a test- tube, add a few drops of potassium dichromate solution, K 2 Cr 2 7 , and render it acid with dilute sulphuric acid. Boil the acid solution and note the odor of aldehyde. Iodoform. (Autenrieth. 1 TRISACCHARIDES, C 18 H 32 lc . RAFFINOSE. This trisaccharide, also called melitose or melitriose, occurs in cotton seed, Australian manna and in the molasses from the preparation of beet sugar. It is dextro-rotatory, does not reduce Fehling's solution and is only partially fermentable by yeast. Raffinose may be hydrolyzed by weak acids the same as the polysaccharides are hydrolyzed, the products being dextrose and melibiose; further hydrolysis of the melibiose yields dex- trose and galactose. POLYSACCHARIDES, (C 6 H 10 5 )x. In general the polysaccharides are amorphous bodies, a few, however, are crystallizable. Through the action of certain 2 2 PHYSIOLOGICAL CHEMISTRY. enzymes or weak acids the polysaccharides may be hydrolyzed with the formation of monosaccharides. As a class the poly- saccharides are quite insoluble and are non-fermentable until inverted. By inversion is meant the hydrolysis of disaccharide or polysaccharide sugars to form monosaccharides, as indi- cated in the following equations : (a) C 12 H 22 O n +H 2 = 2(C 6 H 12 6 )- (b) C 6 H 10 O 5 + H 2 O = C 6 H 12 O G . STARCH, (C G H 10 O 5 ) x . Starch is widely distributed throughout the vegetable king- dom, occurring in grains, fruits and tubers. It occurs in granular form, the microscopical appearance being typical for each individual starch. The granules, which differ in size according to the source, are composed of alternating concen- tric rings of granulose and cellulose. Ordinary starch is in- soluble in cold water, but if boiled with water the cell walls are ruptured and starch paste results. Starch is acted upon by diastatic enzymes, e. g., ptyalin and a my I opsin, with the formation of soluble starch, erythro- dextrin, achroo-dextrin, malto-dextrin, maltose, iso-maltose and dextrose (see Salivary Digestion, page 34). Maltose is the principal end-product of this enzyme action. Upon boil- ing a starch solution with a dilute mineral acid a series of similar bodies is formed, but under these conditions dextrose is the principal end-product. Experiments on Starch. 1. Preparation of Potato Starch. — Pare a raw potato, comminute it upon a fine grater, mix with water, and " whip up" the pulped material vigorously before straining it through cheese cloth or gauze to remove the coarse particles. The starch rapidly settles to the bottom and can be washed by re- peated decantation. Allow the compact mass of starch to drain thoroughly and spread it out on a watch glass to dry in Pea. Wheat. Starch Granules from Various Sources. (LefFmann and Ream.) 24 PHYSIOLOGICAL CHEMISTRY. the air. If so desired this preparation may be used in the ex- periments which follow. 2. Microscopical Examination. — Examine microscopic- ally the granules of the various starches submitted and compare them with those shown in Figs. 7-17, page 23. 3. Solubility. — Try the solubility of one form of starch in each of the ordinary solvents (see page 4). If uncertain regarding the solubility in any reagent, filter and test the fil- trate with iodine solution as given under 5 below. The pro- duction of a blue color would indicate that the starch had been dissolved by the solvent. 4. Iodine Test. — Place a few granules of starch in one of the depressions of a porcelain test-tablet and treat with a drop of a dilute solution of iodine in potassium iodide. The gran- ules are colored blue due to the formation of so-called iodide of starch. The cellulose of the granule is not stained as may be seen by examining microscopically. 5. Iodine Test on Starch Paste. — Repeat the iodine test using the starch paste. Place 2-3 c.c. of starch paste 1 in a test-tube, add a drop of the dilute iodine solution and observe the production of a blue color. Heat the tube and note the disappearance of the color. It reappears on cooling. In similar tests note the influence of alcohol and of alkali upon the so-called iodide of starch. The composition of the iodide of starch is not definitely known. 6. Fehling's Test. — On starch paste (see page 8). 7. Hydrolysis of Starch. — Place about 25 c.c. of starch paste in a small beaker, add 10 drops of concentrated HC1, and boil. By means of a small pipette, at the end of each minute, remove a drop of the solution to the test-tablet and make 1 Preparation of Starch Paste. — Grind 2 grams of starch powder in a mortar with a small amount of cold water. Bring 200 c.c. of water to the boiling-point and add the starch mixture from the mortar with continuous stirring. Bring again to the boiling-point and allow it to cool. This makes an approximate 1 per cent starch paste which is a very satisfactory strength for general use. POLYSACCHARIDES. 25 the regular iodine test. As the testing proceeds the blue color should gradually fade and finally disappear. At this point, after cooling and neutralizing with solid KOH, Fehl- ing's test (see p. 8) should give a positive result due to the formation of a reducing sugar from the starch. Make the phenylhydrazin test upon some of the hydrolyzed starch. Try also Barfoed's test (see p. 11). What sugar has been formed? 8. Influence of Tannic Acid.— Add an excess of tannic acid solution to a small amount of starch paste in a test-tube. The liquid will become strongly opaque and ordinarily a yel- lowish-white precipitate is produced. Compare this result with the result of the similar experiment on dextrin (p. 28). 9. Diffusibility of Starch Paste. — Test the diffusibility of starch paste through animal membrane or parchment paper, making a dialyzer like one of the models shown in Fig. 1, page 6. INULIN, (C 6 H 10 O B ) x Inulin is a polysaccharide which may be obtained as a white, odorless, tasteless powder from the tubers of the arti- choke, elecampane or dahlia. It has also been prepared from the roots of chicory, dandelion and burdock. It is very slightly soluble in cold water and quite easily soluble in hot water. In cold alcohol of 60 per cent or over it is practically insoluble. Inulin gives a negative reaction with iodine solution. The "yellow" color reaction with iodine mentioned in many books is doubtless merely the normal color of the iodine solution. It is very difficult to prepare inulin which does not reduce Fehling's solution slightly. This reducing power may be due to an impurity. Practically all commercial preparations of inulin possess considerable reducing power. Inulin is laevo-rotatory and upon hydrolysis by acids or by the enzyme inulase it yields the monosaccharide laevulose which readily reduces Fehling's solution. The ordinary amylolytic enzymes occurring in the animal body do not digest inulin. 26 physiological chemistry. Experiments on Inulin. i. Solubility. — Try the solubility of inulin powder in each of the ordinary solvents. If uncertain regarding- the solu- bility in any reagent, filter and neutralize the filtrate if it is alkaline in reaction. Add a drop of concentrated hydrochloric acid to the filtrate and boil it for one minute. Render the solution neutral or slightly alkaline with solid KOH and try Fehling's test. AYhat is the significance of a positive Fehling's test in this connection? 2. Iodine Test. — (a) Place 2-3 c.c. of the inulin solution in a test-tube and add a drop of dilute iodine solution. What do you observe? (b) Place a small amount of inulin powder in one of the depressions of a test-tablet and add a drop of dilute iodine solution. Is the effect any different from that observed above? 3. Molisch's Reaction. — Repeat this test according to di- rections given under Dextrose. 2, page 4. 4. Fehling's Test. — Make this test on the inulin solution according to the instructions given under Dextrose, page 8. Is there any reduction ? 1 5. Hydrolysis of Inulin. — Place 5 c.c. of inulin solution in a test-tube, add a drop of concentrated hydrochloric acid and boil it for one minute. Xow cool the solution, neutralize it with concentrated KOH and test the reducing action of 1 c.c. of the solution upon 1 c.c. of diluted (1:4) Fehling's solution. Explain the result. 2 GLYCOGEN, (C H 10 O 5 ) x . (For discussion and experiments see Muscular Tissue, page 206.) 3 See the discussion of the properties of inulin. page 25. 2 If the inulin solution gave a positive Fehling test in the last experi- ment it will be necessary to check the hydrolysis experiment as follows : To 5 c.c. of the inulin solution in a test-tube add one drop of concentrated hydrochloric acid, neutralize with concentrated KOH solution and test the reducing action of 1 c.c. of the resulting solution upon 1 c.c. of diluted (1:4) Fehling's solution. This will show the normal reducing power of the inulin solution. In case the inulin was hydrolyzed, the Fehling's POLYSACCHARIDES. 27 LICHENIN, (C H 10 O.,) x . Lichenin may be obtained from Cetraria islandica ( Iceland moss). It forms a difficultly soluble jelly in cold water and an opalescent solution in hot water. It is optically inactive and gives no color with iodine. Upon hydrolysis with dilute mineral acids lichenin yields dextrins and dextrose. It is said to be most nearly related chemically to starch. Saliva, pan- creatic juice, malt diastase and gastric juice have no noticeable action on lichenin. DEXTRIN, (C 6 H 10 O 5 ) x . The dextrins are the bodies formed midway in the stages of the hydrolysis of starch by weak acids or an enzyme. They are amorphous bodies which are easily soluble in water, acids and alkalis but are insoluble in alcohol or ether. Dextrins are dextro-rotatory and are not fermentable by yeast. The dextrins may be hydrolyzed by dilute acids to form dex- trose. With iodine one form of dextrin (ervthro-dextrin) gives a red color. Their power to reduce Fehling's solution is questioned. Experiments ox Dextrix. 1. Solubility. — Test the solubility of pulverized dextrin in the ordinary solvents (see page 4). 2. Iodine Test.- — Place a drop of dextrin solution in one of the depressions of the test-tablet and add a drop of a dilute solution of iodine in potassium iodide. A red color results. If the reaction is not sufficiently pronounced make a stronger solution from the pulverized dextrin and repeat the test. The solution should be slightly acid to secure the best results. 3. Fehling's Test. — See if the dextrin solution will reduce Fehling's solution. 4. Hydrolysis of Dextrin. — Take 25 c.c. of dextrin solu- tion in a small beaker, add 5 drops of dilute HC1, and boil. test in the hydrolysis experiment should show a more pronounced reduction than that observed in the check experiment. 28 PHYSIOLOGICAL CHEMISTRY. By means of a small pipette, at the end of each minute, remove a drop of the solution to one of the depressions of the test- tablet and make the iodine test. The power of the solution to produce a color with iodine should rapidly disappear. When a negative reaction is obtained cool the solution and neutralize it with solid KOH. Try Fehling's test (see page 8). This reaction is now strongly positive, due to the forma- tion of a reducing sugar. Determine the nature of the sugar by means of the phenylhydrazin test (see pages 5 and 6). 5. Influence of Tannic Acid. — Add an excess of tannic acid solution to a small amount of dextrin solution in a test- tube. No precipitate forms. This result differs from the result of the similar experiment upon starch (see Starch, 8, page 25). 6. Diffusibility of Dextrin. — (See Starch, 9, page 25.) 7. Precipitation by Alcohol. — To about 50 c.c. of 95 per cent alcohol in a small beaker add about 10 c.c. of a concen- trated dextrin solution. Dextrin is thrown out of solution as a gummy white precipitate. Compare the result with that ob- tained under Dextrose, 5, page 6. CELLULOSE, (C e H 10 O 5 ) x . This complex polysaccharide forms a large portion of the cell wall of plants. It is extremely insoluble and its molecule is much more complex than the starch molecule. The best quality of filter paper and the ordinary absorbent cotton are good types of cellulose. Experiments on Cellulose. 1. Solubility. — Test the solubility of cellulose in the ordi- nary solvents (see page 4). 2. Iodine Test. — Add a drop of dilute iodine solution to a few shreds of cotton on a test-tablet. Cellulose differs from starch and dextrin in giving no color with iodine. 3. Formation of Amyloid. 1 — Add 10 c.c. of dilute and 5 'This body derives its name from amylum (starch) and is not to be confounded with amyloid, the gluco-proteid (page 62). REVIEW OF CARBOHYDRATES. 2 9 c.c. of concentrated H 2 S0 4 to some absorbent cotton in a tot tube. When entirely dissolved (without heating) pour one-half of the solution into another test-tube, cool it and dilute with water. Amyloid forms as a gummy precipitate and gives a brown or blue coloration with iodine. After allowing the second portion of the acid solution of cotton to stand about 10 minutes dilute it with water in a small beaker and boil for 15-30 minutes. Now cool, neutral- ize with solid KOH and test with Fehling's solution. Dex- trose has been formed from the cellulose by the action of the acid. 4. Schweitzer's Solubility Test. — Heat some absorbent cotton in a test-tube with Schweitzer's reagent. 1 When com pletely dissolved acidify the solution with acetic acid. An amorphous precipitate of cellulose is produced. Schweitzer's reagent is the only solvent for cellulose. REVIEW OF CARBOHYDRATES. In order to facilitate the student's review of the carbo- hydrates, the preparation of a chart similar to the appended MODEL CHART FOR REVIEW PURPOSES. >% Carbohydrate. >< 3 "5 8 H V a '■S V H "u •s H 9 s 6 ? V H "so c 2 V H "u if 1) H V -0 a a >- H V H -0 Molisch's Reaction. recipitation b Alcohol. Osazon. Rotation. D ffusibility I c 1 u Remarks. H b. « 55 aa Oh Dextrose. Maltose. Lactose. Saccharose. Starch. — Inulin. Dextrin. Cellulose. 1 Schweitzer's reagent is made by adding potassium hydroxide to a solution of cupric sulphate which contains some ammonium chloride. A precipitate of cupric hydroxide forms and this is filtered off, washed and brought into solution in 20 per cent ammonium hydroxide. 30 PHYSIOLOGICAL CHEMISTRY. model is recommended. The signs -f- and — may be con- veniently used to indicate positive and negative reaction. Only those carbohydrates which are of greatest importance from the standpoint of physiological chemistry have been in- cluded in the chart. " Unknown " Solutions of Carbohydrates. At this point the student will be given several " unknown " solutions, each solution containing one or more of the carbohy- drates studied. He will be required to detect, by means of the tests on the preceding pages, each carbohydrate constituent of the several " unknown " solutions and hand in, to the instruc- tor, a written report of his findings, on slips furnished by the laboratory. The scheme given on page 31 may be of use in this con- nection. REVIEW OF CARBOHYDRATES. 31 c (« a. Ih ■O £ u «*H in cs w H r, < P< H Q >H d ffi x O PQ Xj Pi < '£ u ."2 "S fe rt O J>, J5 .5 O 1— 1 H U rt w E H u. W Q 5 n U '_ .2-3 ' r - *j rt P B 3 '-> re 2 ~ S ~ o 3: K « «| . - - 7 . c ►2 re • .2 '-' ■— w *" ■Sof ^ .J. ° "3 u 3 p o 3 re 3 ■**■** i- a - 43 KOU 3 Z 1; 10 •- "> 2 .3 E-= re re P. Ph£ « « 2 pq H 2 ~ oS5 Z ta 1 T3 1) u- 3 ^ rt-5 ° 2 re *^ # ^ a> „ 3 re rt £ > , n C •_n o _o re o> P •*- u 3 *> £ 33 vm te c § >. Q-— O. <_. +j « 5 rt Ph o - O - Q< b; C O ^ 2 re M * •— 3 u . a -p re i: "" « ■- c h o re n: ty 3 5 ■ c i>2§ u Ih 60 «tj •3 3 W Ifl ■^ rt 3 O X S U — 3 rt -0 J5t • 2 3 3 c/i 3 ^-3 3 '-^ — rt «j_ O .2 S 2 % hop '~ .300 *-. U en U -,, bo i. -. Ph a z -p p >^ .3t3 CHAPTER II. SALIVARY DIGESTION. The saliva is secreted by three pairs of glands, the sub- maxillary, sublingual and parotid, reinforced by numerous small glands called buccal glands. The saliva secreted by each pair of glands possesses certain definite characteristics peculiar to itself. For instance, in man, the parotid glands ordinarily secrete a thin, watery fluid, the submaxillary glands secrete a somewhat thicker fluid containing mucin, while the product of the sublingual glands has a more mucilaginous character. The saliva as collected from the mouth is the com- bined product of all the glands mentioned. The saliva may be induced to flow by many forms of stimuli, such as chemical, mechanical, electrical, thermal and psychical, the nature and amount of the secretion depending, to a limited degree, upon the particular class of stimuli employed as well as upon the character of the individual stimulus. For example, in experiments upon dogs it has been found that the mechanical stimulus afforded by dropping several pebbles into the animal's mouth caused the flow of but one or two drops of saliva, whereas the mechanical stimulus afforded by sand in the mouth induced a copious flow of a thin watery fluid. Again, when ice-water or snow was placed in the animal's mouth no saliva was seen, while an acid or anything possessing a bitter taste, which the dog wished to reject, caused a free flow of the thin saliva. On the other hand, when articles of food were placed in the dog's mouth the animal secreted a thicker saliva hav- ing a higher mucin content — a fluid which would lubricate the food and assist in the passage of the bolus through the oesophagus. It was further found that by simply drawing the attention of the animal to any of the substances named above. 32 SALIVARY DIGESTION. 33 results were obtained similar to those secured when the sub- stances were actually placed in the animal's mouth. For ex- ample, when a pretense was made of throwing sand into the dog's mouth, a watery saliva was secreted, whereas food under the same conditions excited a thicker and more slimy secretion. The exhibition of dry food, in which the dog had no particular interest (dry bread) caused the secretion of a large amount of saliva, while the presentation of moist food, which was eagerly desired by the animal, called forth a much smaller secretion. These experiments show it to be rather difficult to differentiate between the influence of physiological and psy- chical stimuli. The amount of saliva secreted by an adult in 24 hours has been variously placed, as the result of experiment and obser- vation, between 1000 and 1500 c.c, the exact amount de- pending, among other conditions, upon the character of the food. The saliva ordinarily has a weak, alkaline reaction to litmus, but becomes acid 2-3 hours after a meal or during fasting. The alkalinity is due principally to di-sodium hy- drogen phosphate (Na 2 HP0 4 ) and its average alkalinity may be said to be equivalent to 0.08 — 0.1 per cent sodium carbo- nate. The saliva is the most dilute of all the digestive fluids, having an average specific gravity of 1.005 an d containing only 0.5 per cent of solid matter. Among the solids are found al- bumin, globulin, mucin, urea, the enzyme ptyalin, phosphates and other inorganic constituents. Potassium sulphocyanide, KSCN, is also generally present in the saliva. It has been claimed that this substance is present in greatest amount in the saliva of habitual smokers. The significance of sulpho- cyanide in the saliva is not known; it may come from the breaking down of proteid. The so-called tartar formation on the teeth is composed al- most entirely of calcium phosphate with some calcium carbo- nate, mucin, epithelial cells and organic debris derived from the food. The calcium salts are held in solution as acid salts, 4 34 PHYSIOLOGICAL CHEMISTRY. and are probably precipitated by the ammonia of the breath. The various organic substances just mentioned are carried down in the precipitation of the calcium salts. The saliva contains an enzyme known as ptyalin. This is an amylolytic enzyme, so-called because it possesses the prop- erty of transforming complex carbohydrates such as starch and dextrin into simpler bodies. The so-called ferments were formerly divided into two general groups, (i) true ferments or so-called organized ferments such as yeast and certain bac- teria, which were supposed to act by virtue of vital processes ; and (2) enzymes such as ptyalin, which are non-living, un- organized bodies of a chemical nature. Recently this distinc- tion between true ferments and enzymes has been proven to be incorrect since it has been shown that certain of the bodies formerly supposed to derive their ferment activity by virtue of their vital processes in reality secrete certain definite enzymes which are solely responsible for their ferment activity. In no sense is it a vital process since the ferment activity is entirely independent of the vital processes of the cell. We may define an enzyme as an unorganized, soluble ferment which is elabor- ated by an animal or vegetable cell and whose activity is en- tirely independent of any of the life processes of such a cell. The more important enzymes may be classified, according to the character of their action, as follows: (1) amylolytic (starch transforming), (2) proteolytic (proteid transform- ing), (3) adipolytic or lipolytic (fat splitting), (4) inverting (possesses inverting power), (5) oxidative (possesses oxidiz- ing power), and (6) proteid coagulating. The action of ptyalin is one of hydrolysis and through this action a series of simpler bodies are formed from the complex starch. The first product of the action of the ptyalin of the saliva upon starch paste is soluble starch (amidulin) and its formation is indicated by the disappearance of the opalescence of the starch solution. This body resembles true starch in giving a blue color with iodine. Next follows the formation, in succession, of a series of dextrins, called erythro-dextrin, SALIVARY DIGESTION. 35 achroo-dextrin and malto-dextrin, the erythro-dextrin being formed directly from the soluble starch and later being itself transformed into achroo-dextrin from which in turn is pro- duced malto-dextrin. Accompanying each dextrin a small amount of maltose is funned, the quantity of maltose growing gradually larger as the process of transformation progre Erythro dextrin gives a w<\ color with iodine, the other dex- trins give no color. The next stage is the transformation of the malto-dextrin into maltose the latter being the principal end-pmduct of the salivary digestion of starch. At this point small amounts of iso-maltose and dextrose are formed from the maltose, the dextrose being produced through the action of the enzyme maltose. Ptyalin acts in alkaline or neutral solutions. It will also act in the presence of relatively strong combined HO (see page 84), whereas a trace (0.003 P er cent t() 0006 per cent) of ordinary free hydrochloric acid will not only prevent the ac- tion but will destroy the enzyme. By sufficiently increasing the alkalinity of the saliva the action of the ptyalin is inhibited. It has recently been shown, by Cannon, to be strongly probable that salivary digestion may proceed for a considerable period after the food reaches the stomach, owing to the slowness with which the contents are thoroughly mixed with the acid gastric juice and the consequent tardy destruction of the enzyme. Microscopical examination of the saliva reveals salivary corpuscle^, bacteria, food debris, epithelial cells, mucus and fungi. In certain pathological conditions of the mouth, pus cells and blood corpuscles may be found in the saliva. Experiments on Saliva. A satisfactory method of obtaining the saliva necessary for the experiments which follow is to chew a small piece of pure paraffin wax thus stimulating the flow of the secretion, which may be collected in a small beaker. Filtered saliva is to be used in every experiment except for the microscopical ex- amination. 36 PHYSIOLOGICAL CHEMISTRY. 1. Microscopical Examination. — Examine a drop of un- filtered saliva microscopically and compare with Fig. 18 below. 2. Reaction. — Test the reaction to litmus. Fig. i 8. * i •*?>'& Microscopical Constituents of Saliva. a, Epithelial cells ; b, salivary corpuscles ; c, fat drops ; d, leucocytes ; e, f and g, bacteria ; h, i and k, fission-fungi. 3. Specific Gravity. — Partially fill a urinometer cylinder with saliva, introduce the urinometer (see Fig. 83, page 232), and observe the reading. 4. Test for Mucin. — To a small amount of saliva in a test- tube add 1-2 drops of dilute acetic acid. Mucin is precipitated. 5. Biuret Test. 1 — Render a little saliva alkaline with an equal volume of KOH and add a few drops of a very dilute (2-5 drops in a test-tube of water) cupric sulphate solution. The formation of a purplish-violet color is due to mucin. 6. Millon's Reaction. 2 — Add a few drops of Millon's re- agent to a little saliva. A light yellow precipitate formed by the mucin gradually turns red upon being gently heated. 7. Preparation of Mucin. — Pour 15 c.c. of saliva into 100 c.c. of 95 per cent alcohol, stirring constantly. Cover the vessel and allow the precipitate to stand at least 12 hours. Pour off the supernatant liquid, collect the precipitate on a filter and wash it, in turn, with alcohol and ether. Finally dry the precipitate, remove it from the paper and make the follow- ing tests on the mucin: (a) Test its solubility in the ordinary 1 The significance of this reaction is pointed out on page 45. 2 The significance of this reaction is pointed out on page 44. SALIVARY DIGESTION. 37 solvents (see page 4), ( /> ) Millon's reaction, (c) dissolve a small amount in KOH, and try the biuret test on the solution, (d) boil the remainder, with 10-25 c.c. of water to which 5 c.c. of dilute I1C1 has been added, until the solution becomes brownish. Cool, render alkaline with solid KOH, and test by Fehling's solution. A reduction should take place. Mucin is what is known as a compound proteid or glucoproteid (see p. 6] ) and upon boiling with the acid the carbohydrate group in the molecule has been split off from the proteid portion and its presence is indicated by the reduction of Fehling's solution. 8. Inorganic Matter. — Test for chlorides, phosphates, sulphates and calcium. For chlorides, acidify with HN0 3 and add AgNO a . For phosphates, acidify with HXO a , heat and add molybdic solution. 1 For sulphates, acidify with HC1 and add BaCl 2 and warm. For calcium, acidify with acetic acid, CH :i COOH. and add ammonium oxalate, (NH 4 ) 2 C 2 4 . 9. Filtration Experiment. — Place filter papers in two fun- nels, and to each add an equal quantity of starch paste (5 c.c). Add a few drops of saliva to one lot of paste and an equivalent amount of water to the other. Note the progress of filtration in each case. Why does one solution filter more rapidly than the other? 10. Test for Nitrites. — Add 1-2 drops of dilute H 2 S0 4 to a little saliva and thoroughly stir. Now add a few drops of- a potassium iodide solution and some starch paste. Nitrous acid is formed which liberates iodine causing the formation of the blue iodide of starch. 11. Sulphocyanide Tests. — (a) Ferric Chloride Test. — To a little saliva in a small porcelain crucible, or dish, add a few drops of dilute ferric chloride and acidify slightly with HC1. Red ferric sulphocyanide forms. To show that the red coloration is not due to iron phosphate add a drop of HgCl 2 when colorless mercuric sulphocyanide forms. 1 Molybdic solution is prepared as follows, the parts being by weight : 1 part, molybdic acid. 4 parts, ammonium hydroxide (Sp. gr. 0.96). 15 parts, nitric acid (Sp. gr. 1.2). 38 PHYSIOLOGICAL CHEMISTRY. (b) Solera's Reaction. — This test depends upon the libera- tion of iodine through the action of sulphocyanide upon iodic acid. Moisten a strip of starch paste-iodic acid test paper 1 with a little saliva. If sulphocyanide be present the test paper will assume a blue color, due to the liberation of iodine and its subsequent formation of the so-called iodide of starch. 12. Digestion of Starch Paste. — To 25 c.c. of starch paste in a small beaker, add 5 drops of saliva and stir thoroughly. At intervals of a minute remove a drop of the solution to one of the depressions in a test-tablet and test by the iodine test. If the blue color with iodine still forms after 5 minutes, add another 5 drops of saliva. The opalescence of the starch solu- tion should soon disappear, indicating the formation of sol- uble starch which gives a blue color with iodine. This body should soon be transformed into erythro-dextrin which gives a red color with iodine and this in turn should pass into achroo- dextrin which gives no color with iodine. This is called the achromic point. When this point is reached test by Fehling's test to show the production of a reducing body. A body formed coincidently with erythro-dextrin may yield a slight response to Fehling's test. What body is it? How long did it take for a complete transformation of the starch? 13. Digestion of Dry Starch. — In a test-tube shake up a small amount of dry starch with a little water. Add a few drops of saliva, mix well and allow to stand. After 10-20 minutes filter and test the filtrate by Fehling's test. What is the result and why ? 14. Digestion of Inulin. — To 5 c.c. of inulin solution in a test-tube add 10 drops of saliva and place the tube in the water-bath at 40 C. After one-half hour test the solution by Fehling's test. 2 Is any reducing substance present? What do you conclude regarding the salivary digestion of inulin? 1 This test paper is prepared as follows : Saturate a good quality of filter paper with 0.5 per cent starch paste containing a little iodic acid and allow the paper to dry in the air. Cut it in strips of suitable size and preserve for use. * If the inulin solution gives a reduction before being acted upon by the saliva it will be necessary to determine the extent of this original reduc- tion by means of a "check" test (see page 26). SALIVARY DIGESTION. 39 15. Influence of Temperature. — In each of four tubes place about 5 c.c. of starch pa^te. Immerse one tube in cold water from the faucet, keep a second at room temperature and place a third <>n the water-bath at 40 ('. Now add to the con- tents of each of these three tubes two drops of saliva and shake well : to the contents of the fourth tube add two drops of boiled saliva. Test frequently by the iodine tot. using the test- tablet, ami note in which tube the most rapid digestion occurs. Explain the results. 16. Influence of Dilution. — Take a series of 6 test-tubes each containing 9 c.c. of water. Add 1 c.c. of saliva to tube 1 and shake thoroughly. Remove 1 c.c. of the solution from tube 1 to tube 2 and after mixing thoroughly remove 1 c.c. from tube 2 to tube 3. Continue in this manner until you have 6 saliva solutions of gradually decreasing strength. Now add starch paste in equal amounts to each tube, mix very thor- oughly and place on the water-bath at 40 C. After 10-20 minutes test by both the iodine and Fehling's tests. In how great dilution does your saliva act? 17. Influence of Acids and Alkalis. — (a) Influence of Free Acid. — Prepare a series of 6 tubes in each of which is placed 4 c.c. of one of the following strengths of free II CI : 0.2 per cent. o. 1 per cent. 0.05 per cent, 0.025 per cent, 0.0125 per cent and 0.006 per cent. Xow add 2 c.c. of starch paste to each tube and shake them thoroughly. Complete the solutions by adding 2 c.c. of saliva to each and repeat the shaking. The total acidity of this series would be as follows: 0.1 per cent, 0.05 per cent. 0.025 per cent. 0.0125 per cent, 0.006 per cent and 0.003 P er cent - Place these tubes on the water-bath at 40 C. for 10-20 minutes. Divide the contents of each tube into two parts, testing one part by the iodine test and testing the other, after neutralization, by Fehling's test. What do you rind ? (b) Influence of Combined Acid. — Repeat the first three experiments of the above series using combined hydrochloric acid 1 see page 84) instead of the free acid. How does the action of the combined acid differ from that of the free acid/ 40 PHYSIOLOGICAL CHEMISTRY. (c) Influence of Alkali. — Repeat the first four experiments under (a) replacing the HC1 by 2 per cent, 1 per cent, 0.5 per cent and 0.25 per cent Na 2 C0 3 . Neutralize the alkalinity before trying the iodine test (see Starch, 5, page 24). (d) Nature of the Action of Acid and Alkali. — Place 2 c.c. of saliva and 2 c.c. of 0.2 per cent HC1 in a test-tube and leave for 15 minutes. Neutralize the solution, add 4 c.c. of starch paste and place the tube on the water-bath at 40 ° C. In 10 minutes test by the iodine and Fehling's tests and ex- plain the result. Repeat the experiment replacing- the 0.2 per cent HC1 by 2 per cent Na 2 CO s . What do you deduce from these two experiments? 18. Influence of Metallic Salts, etc. — In each of a series of tubes place 4 c.c. of starch paste and ^ c.c. of one of the solutions named below. Shake well, add y 2 c.c. of saliva to each tube, thoroughly mix, and place on the water-bath at 40 C. for 10-20 minutes. Show the progress of digestion by means of the iodine and Fehling tests. Use the following chemicals : Metallic salts, 10 per cent plumbic acetate, 2 per cent cupric sulphate, 5 per cent ferric chloride, 8 per cent mercuric chloride; Neutral salts, 10 per cent sodium chloride, 3 per cent barium chloride, 10 per cent Rochelle salt. Also try the influence of 2 per cent carbolic acid, 95 per cent alcohol, and ether and chloroform. What are your con- clusions? 19. Excretion of Potassium Iodide. — Ingest a small dose of potassium iodide (0.2 gram) contained in a gelatin cap- sule, quickly rinse out the mouth with water and then test the saliva at once for iodine. This test should be negative. Make additional tests for iodine at 2 minute intervals. The test for iodine is made as follows : Take 1 c.c. of NaN0 2 and 1 c.c. of dilute H 2 SCV in a test- tube, add a little saliva directly from the mouth, and a small amount of starch paste. If convenient, the urine may also 1 Instead of this mixture a few drops, of HN0 3 possessing a yellowish or brownish color due to the presence of HNO2 may be employed. SALIVARY DIGESTION. 4 1 be tested. The formation of a blue color signifies that the potassium iodide is being excreted through the salivary glands. Note the length of time elapsing between the ingestion of the potassium iodide and the appearance of the first traces of the substance in the saliva. The chemical reactions taking place in this experiment are indicated in the following equations : (a) 2NaN0 2 +H 2 S0 4 = 2HN0 2 + Na 2 S0 4 . (b) 2KI + H 2 S0 4 = 2HI + K 2 S0 4 . (c) 2HN0 2 +2HI = I 2 + 2H 2 + 2NO. 20. Qualitative Analysis of the Products of Salivary Digestion. — To 25 c.c. of the products of salivary digestion (saved from Experiment 12 or furnished by the instructor), add 100 c.c. of 95 per cent alcohol. Allow to stand until the white precipitate has settled. Filter, evaporate the filtrate to dryness, dissolve the residue in 5-10 c.c. of water and try Fehling's test (page 8) and the phenylhydrazin reaction (see Dextrose, 3, page 5). On the dextrin precipitate try the iodine test (page 24). Also hydrolyze the dextrin as given under Dextrin, 4, page 27. CHAPTER III. PROTEIDS. Proteids are a group of very complex organic substances, constituting the most important class of food stuffs and are widely distributed in animal and vegetable tissues. Every proteid contains carbon, hydrogen, nitrogen, oxygen and sul- phur, and a relatively small number contain phosphorus and iron in addition. The percentage composition of the more important members of the group would fall within the fol- lowing limits: C (51 per cent to 55 per cent), H (6 per cent to 7.3 per cent), N (15 per cent to 19 per cent), O (21 per cent to 23 per cent), S (0.3 per cent to 2.5 per cent), and P (0.4 per cent to 0.8 per cent when present) : Fe occurs only in traces. The most important element of the proteid mole- cule is the nitrogen. The human body needs nitrogen for the continuation of life, but it cannot use the nitrogen of the air or that in various other combinations such as we find in nitrites, etc. However, in the proteid molecule the nitrogen is present in a form which is utilizable by the body. No definite knowledge has yet been secured regarding the constitutional formula or the molecular weight of proteid material. The molecular weight of tgg albumin has been placed at about 15,000 and the formula for the crystallized product has been calculated as C 2 39H 3S6 N 58 S 2 7S . Many im- portant and valuable investigations have been promoted re- cently on the subject of the constitution of the proteid mole- cule and our knowledge has been largely increased. The proteids may be classified as follows : I. SIMPLE PROTEIDS. 1. NATIVE SIMPLE PROTEIDS. (a) Albumins — egg albumin, serum albumin and vege- table albumins. 42 PROTEIDS. 43 ( b) Globulins — scrum globulin, ovoglobulin, edestin and other vegetable globulins. (c) Phospho-proteids (nucleo-albumins) caseinogen and vitellin. 2. DERIVED SIMPLE PROTEIDS. (a) Albuminates — acid albuminate and alkali albuminate. I /> ) Proteoses (or albumoses ) and peptones — proto- proteose, heteyoproteose and deuteroproteose; amphopeptone and antipeptone. (c) Coagulated Proteids — fibrin, and the products of heat coagulation, etc. II. COMPOUND PROTEIDS. (a) Glucoproteids — mucins (from fluids and secretions); mucoids, c. g., osseomucoid and iendomucoid ; amyloid. (b) Nucleo-proteids. (c) Haemoglobins. III. ALBUMINOIDS, ALBUMOIDS OR PRO- TEOIDS (PROTEID-LIKE BODIES). (a) Chondroalbumoid — isolated from cartilage. (b) Collagen — constituent of connective tissue and par- ticularly abundant in tendinous tissue. ( c) Elastin — constituent of connective tissue and particu- larly abundant in ligament. (d) Gelatin — product of the hydrolysis of collagen. (aCl._, solution. A white precipitate forms if sulphur is present. What is this precipitate? GLOBULINS. Globulin^ are simple proteids especially predominant in the vegetable kingdom. They are closely related to the albumins and in common with them give all the ordinary proteid tests. Globulins differ from the albumins in being insoluble in water. Most globulins are precipitated from their solutions by satura- tion with solid sodium chloride or magnesium sulphate. As a class they are much less stable than the albumins, a fact shown by the increasing difficulty with which a globulin dissolves during the course of successive reprecipitations. We have used an albumin of animal origin (egg albumin) for all the proteid tests thus far, whereas the globulin to be studied will be prepared from a vegetable source. There being no essential difference between animal and vegetable proteids, the vegetable globulin we shall study may be taken as a true type of all globulins, both animal and vegetable. Experiments on Globulin. Preparation of the Globulin. — Extract 20-30 grams (a handful) of crushed hemp seed with a 5 per cent solution of XaCl for one-half hour at 6o° C. Filter while hot through a paper moistened with 5 per cent NaCl solution and allow the filtrate to cool slowly. The globulin is soluble in hot 5 per cent NaCl solution and is thus extracted from the hemp seed, but upon cooling this solution much of the globulin separates in crystalline form. This particular globulin is called edestin. It crystallizes in several different forms, chiefly octahedra (see Fig. 20, page 54). (The crystalline form of excelsin. a proteid obtained from the Brazil nut, is shown in Fig. 2 1 , page 55. This vegetable proteid crystallizes in the form of hex- agonal plates.) Filter off the edestin and make the follow- 54 PHYSIOLOGICAL CHEMISTRY. ing tests on the crystalline body and on the filtrate which still contains some of the extracted globulin. Tests on Crystallized Edestin. — (i) Microscopical ex- amination (Fig. 20, below). (2) Solubility. — Try the solubility in the ordinary solvents (see page 4). (3) MiUon's Reaction. (4) Coagulation Test. — Place a small amount of the glo- bulin in a tube, add a little water and boil. Now add dilute HC1 and note that the proteid no longer dissolves. It has been coagulated. Fig. 20. Edestin. Tests on Edestin Filtrate. — (1) Influence of Proteid Precipitants. — Try a few proteid precipitants such as nitric acid, tannic acid, picric acid and mercuric chloride. (2) Biuret Test. (3) Coagulation Test. — Boil some of the filtrate in a test- tube. What happens ? (4) Saturation with Sodium Chloride. — Saturate some of the filtrate with solid NaCl. How does this result differ from that obtained upon saturating egg albumin solution with solid XaCl? PROTEIDS. 55 (5) Precipitation by Dilution. — Dilute some of the filtrate with 10-15 volumes of water. Why does the globulin pre- cipitate? DERIVED SIMPLE PROTEIDS. These bodies are obtained from native simple proteids by various means, e. g., through the action of acids, alkalis, heat or enzymes, the method of treatment to which the native proteid is subjected depending upon the particular class of derived proteid desired. These modified bodies are classified as albuminates, proteoses (or albumoses), peptones and coagu- lated proteids. Albuminates. The albuminates are derived proteids and are produced by the action of acids or alkalis upon the native simple proteids, albumins and globulins. There are two classes of albuminates, Fig. 21. EXCELSIN, THE PROTEID OF THE BRAZIL NUT. (Drawn from crystals furnished by Dr. Thomas B. Osborne, New Haven, Conn.) i. e., acid albuminate and alkali albuminate. They differ from the native simple proteids principally in being insoluble in NaCl solution and in not being coagulated except when sus- 56 PHYSIOLOGICAL CHEMISTRY. pended in neutral fluids. Both forms of albuminate are pre- cipitated upon the neutralization of their solutions. They are precipitated by saturation with (NH 4 ) 2 S0 4 , and by saturation with NaCl also if they are dissolved in an acid solution. Acid albuminate contains a higher percentage of nitrogen and sul- phur than the alkali albuminate from the same source since some of the nitrogen and sulphur of the original proteid is liberated in the formation of the latter. Because of this fact it is impossible to transform an alkali albuminate into an acid albuminate, while it is possible to reverse the process and trans- form the acid albuminate into the alkali modification. ACID ALBUMINATE. Preparation. — Take 25 grams of hashed lean beef, washed free from the major portion of blood and inorganic matter, and place it in a medium-sized beaker with 100 c.c. of 0.2 per cent HC1. Place it on a boiling water-bath for one-half hour, filter, cool and divide the filtrate into two parts. Neutralize the first part with dilute KOH solution, filter off the precipi- tate of acid albuminate and make the following tests : (1) Solubility. — Solubility in the ordinary solvents (see page 4). (2) Milton's Reaction. (3) Coagulation Test. — Suspend a little of the albuminate in water (neutral solution) and heat to boiling for a few moments. Now add 1-2 drops of KOH solution to the water and see if the albuminate is still soluble in dilute alkali. What is the result and why? (4) Test for Loosely Combined Sulphur (see page 52). Subject the second part of the solution to the following tests : (1) Coagulation Test. — Heat some of the solution to boil- ing in a test-tube. Does it coagulate? (2) Biuret Test. (3) Influence of Proteid Precipitant s. — Try a few proteid precipitants such as picric acid and mercuric chloride. How PROTEIDS. 5 7 do the results obtained compare with those from the experi- ments on e.y:g' albumin? (See page 47.) ALKALI ALBUMINATE. Preparation. — Carefully separate the white- from the yolk of a hen's egg and place the former in an evaporating dish. Add concentrated KOH solution, drop by drop, stirring con- tinuously. The mass gradually thickens and finally assumes the consistency of jelly. This is solid alkali albuminate or " Lieberkiihn's jelly." Do not add an excess of KOH or the jelly will dissolve. Cut it into small pieces, place a cloth or wire gauze over the dish and by means of running water wash the pieces free from adherent alkali. Now add a small amount of water, which forms a weak alkaline solution with the alkali within the pieces, and dissolve the jelly by gentle heat. Cool the solution and divide it into two parts. Proceed as follows with the first part: Neutralize with dilute HC1, noting the odor of the liberated H 2 S as the alkali albuminate precipitates. Filter off the precipitate and test as for acid albuminate, page 56, noting particularly the sulphur test. How does this test compare with that given by the acid albuminate? Make tests on the second part of the solution the same as for acid albu- minate, page 56. Proteoses (or Albumoses) and Peptones. Proteoses are intermediate products in the digestion of pro- teids by proteolytic enzymes, as well as in the decomposition of proteids by hydrolysis and the putrefaction of proteids through the action of bacteria. Peptones are formed after the proteoses and are the last products of the above mentioned processes which still possess true proteid characteristics. In other words, the proteid nature of the end-products of the cleavage of the proteid molecule ceases with the peptones, and the simpler bodies formed from peptones are bodies of a dif- ferent type (see page 65). There are several proteoses (protoproteose, heteroproteose and deuteroproteose), and at least two peptones (amphopep- 58 PHYSIOLOGICAL CHEMISTRY. tone and antipeptone), which result from proteolysis. The differentiation of the various proteoses and peptones at present in use is rather unsatisfactory. These compounds are classi- fied according to their varying solubilities, especially in (NH 4 ) 2 S0 4 solutions of different strengths. The exact dif- ferences in composition between the various members of the group remains to be more accurately established. Because of the difficulty attending the separation of, these bodies, pure proteose and peptone are not easy to procure. The so-called peptones sold commercially contain a large amount of proteose. As a class the proteoses and peptones are very soluble, dif- fusible bodies which are non-coagulable by heat. Peptones differ from proteoses in being more diffusible, non-precipitable by (NH 4 ) 2 S0 4 , and by their failure to give any reaction zvith potassium ferrocyanide and acetic acid, potassio-mer curie iodide and HC1, picric acid, and trichloracetic acid. The so- called primary proteoses are precipitated by HN0 3 and are the only members of the proteose-peptone group which are so precipitated. Some of the more general characteristics of the proteose- peptone group may be noted by making the following simple tests on a proteose-peptone powder : (1) Solubility. — Solubility in the ordinary solvents (see page 4). (2) Milton's Reaction. Dissolve a little of the powder in water and test the solu- tion as follows : (1) Precipitation by Picric Acid. — To 5 c.c. of proteose- peptone solution in a test-tube add picric acid until a perma- nent precipitate forms. The precipitate disappears on heating and returns on cooling. (2) Precipitation by a Mineral Acid. — Try the precipita- tion by nitric acid. (3) Coagulation Test. — Heat a little proteose-peptone solu- tion to boiling. Does it coagulate like the other simple pro- teids studied? PROTEIDS. 59 SEPARATION OF PROTEOSES AND PEPTONES. Place 50 c.c. of proteose-peptone solution in an evaporating dish or casserole, and half-saturate it with (NH 4 ) 2 S0 4 solu- tion, which may be accomplished by adding an equal volume of saturated (NH 4 ) 2 S0 4 solution. At this point note the appearance of a precipitate of the primary proteoses (proto- proteose and-heteroproteose). Now heat the half -saturated solution and its suspended precipitate to boiling and saturate the solution with solid (NH 4 ).,S0 4 . At full saturation the secondary proteoses (deuteroproteoses) are precipitated. The peptones remain in solution. Proceed as follows with the precipitate of proteoses : Col- lect the sticky precipitate on a rubber-tipped stirring rod or remove it by means of a watch glass to a small evaporating dish and dissolve it in a little water. To remove the (NH 4 ) 2 S0 4 , which adhered to the precipitate and is now in solution, add BaCO a , boil, and filter off the precipitate of BaS0 4 . Concentrate the proteose solution to a small volume 1 and make the following tests : ( 1 ) Biuret Test. (2) Precipitation by HN0 3 . — What would a precipitate at this point indicate? (3) Precipitation by Trichloracetic Acid. — This precipitate dissolves on heating and returns on cooling. (4) Precipitation by Picric Acid. — This precipitate also disappears on heating and returns on cooling. (5) Precipitation by Potassio-mercuric Iodide and HC1. (6) Coagulation Test. — Boil a little in a test-tube. Does it coagulate? (7) Acetic Acid and Potassium Ferrocyanide Test. The solution containing the peptones should be cooled and *If the proteoses are desired in powder form, this concentrated proteose solution may now be precipitated by alcohol, and this precipitate, after being washed with absolute alcohol and with ether, may be dried and powdered. 60 PHYSIOLOGICAL CHEMISTRY. filtered, and the (NH 4 ) 2 S0 4 in solution removed by boiling with BaC0 3 as described on page 59. After filtering off the BaS0 4 precipitate, concentrate the peptone filtrate to a small volume 1 and repeat the tests as given under the proteose solu- tion, page 59. In the biuret test the solution should be made very strongly alkaline with solid KOH. Coagulated Proteids. These derived proteids are produced from unaltered proteid materials by heat, by long standing under alcohol, or by the continuous movement of their solutions such as that produced by rapid stirring or shaking. In particular instances, such as the formation of fibrin from fibrinogen (see page 157), the coagulation may be produced by ferment action. Ordinary soluble proteids after having been transformed into the coag- ulated modification are no longer soluble in the ordinary sol- vents. Upon being heated in the presence of strong acids or alkalis, coagulated proteids are converted into albuminates. Many proteids coagulate at an approximately fixed tem- perature under definite conditions (see page 50). This char- acteristic may be applied to separate different coagulable pro- teids from the same solution by fractional coagulation. The coagulation temperature frequently may serve in a measure to identify proteids in a manner similar to the melting-point or boiling-point of many other organic substances. The separation of proteids by fractional coagulation thus is analogous to the separation of volatile substances by means of fractional distillation. The nature of the process in- volved in the coagulation of proteids by heat is not well understood, but it is probable that in addition to the altered arrangement of the component atoms in the molecule, there is a mild hydrolysis which is accompanied by the liberation of minute amounts of hydrogen, nitrogen and sulphur. The presence of a neutral salt or a trace of a mineral acid may facilitate the coagulation of a proteid solution (see page 50), 1 See note on preparation of proteose powder, page 59. PROTEIDS. 6 1 whereas any appreciable amount of acid or alkali will retard or entirely prevent such coagulation. Experiments on Coagulated Proteid. Ordinary coagulated egg-white may be used in the following tests : i. Solubility. — Try the solubility of small pieces of the coagulated proteid in each of the ordinary solvents (see page 4). _'. Millon's Reaction. 3. Xanthoproteic Reaction. — Partly dissolve a medium- sized piece of the proteid in concentrated HXO. ; . Cool the solution and add an excess of NH 4 OH. Both the proteid solution and the undissolved proteid will be colored orange. 4. Biuret Test. — Partly dissolve a medium-sized piece of the proteid in concentrated KOH solution. If the proper dilu- tion of CuS0 4 solution is now added the white coagulated proteid, as well as the proteid solution, will assume the char- acteristic purplish-violet color. 5. Hopkins-Cole Reaction. — Conduct this test according to the modification given on page 51. COMPOUND PROTEIDS. Compound proteids consist of a simple proteid combined with some non-proteid material, and they are named accord- ing to the nature of this combining body. Thus we have glucoprotcids. nucleo proteids and Jiccmoglobins as three classes of compound proteids. The glucoprotcids yield, upon decomposition, proteid and carbohydrate derivatives, notably glucosamine, CH 2 OH ■ - (CHOH) 3 • CH(NH 2 ) • CHO, and galactosamine, OHCH 2 ■ - (CHOH) 3 -CH(NH 2 )-CHO. The principal glucoproteids are mucoids, mucins and chondroprotcids. By the term mucoid we may designate those glucoproteids which occur in tissues, such as tendomucoid from tendinous tissue and 62 PHYSIOLOGICAL CHEMISTRY. osseomucoid from bone. The elementary composition of these typical mucoids is as follows : N. s. C. H. 0. Tendomucoid ..11.75 2-33 48.76 6-53 30.60 (Chittenden and Gies) Osseomucoid . . 12.22 2.32 47-43 6.63 31.40 The term mucins may be said to include those forms of glucoproteids which occur in the secretions and fluids of the body. Chondroproteids are so named because chondromncoid, the principal member of the group, is derived from cartilage (chondrigen). Amyloid, which appears pathologically in the spleen, liver and kidneys is also a chondroproteid. The nucleoproteids occur principally in animal and vegetable cells, and following the destruction of these cells they are found in the fluids of the body. These proteids are discharged into the tissue fluids by the activity or disintegration of cells. Combined with the simple proteid in the nucleoproteid mole- cule we find nucleic acid, a body which contains phosphorus and which yields pitrin bases upon decomposition. The so- called nucleins are formed in the gastric digestion of nucleo- proteids. The hemoglobins are those compound proteids which are composed of a simple proteid and a pigment. The haemoglobin of the blood (see page 156) upon decomposition yields a pro- teid termed globin and a modified pigment called hccmatin. For experiments upon a compound proteid see page 199. ALBUMINOIDS, ALBUMOIDS OR PROTEOIDS. These bodies are closely related in character to the proteids, from which class of substances they are derived. They differ ordinarily from true proteids in the character of their decom- position products, in being very resistant to the ordinary pro- teid solvents, and in being unable alone to support life. They generally occur in an insoluble form in some portion of the animal organism. The albuminoids may be divided into sev- eral classes such as keratins, elastins, collagcns, gelatins and I'KOTKIDS. 63 skeletons, and in general the members of each group differ fundamentally in certain characteristics from the member- of any other group. For discussion of and experiments on each of the several groups see the chapter on Epithelial and Con- nective Tissues, pages \ny to 205. REVIEW OF PROTEIDS. In order to facilitate the student's review of the proteids, the preparation of a chart similar to the appended model is recommended. The signs -f- and — may be conveniently used to indicate positive and negative reactions. MODEL CHART FOR REVIEW PURPOSES. Troteid. Solubility. 8 H u "o U !2 '5 u ft. Precipitation Tests. SaltinR- ..111 Tests. 1 5 ~ -5 x ' z a 1 a - X >■ ja a .0 a 1 U V s u Z X*. u x 6 0" U Z •*. m d D x 6 c ■J tn O ui c U !2 IS 3 s "0 X u 5 V ■a . "52 O_o 1+ 3^ E + u 3,2 04 •6 < V H Albumin. Globulin. Acid albuminate. Alkali albuminate. Proteose. Peptone. Coagulated proteid. " Unknown " Mixtures and Solutions of Proteids. At this point the student's knowledge of the characteristics of the various proteids studied will be tested by requiring him to examine several " unknown " proteid mixtures or solutions and make full report upon the same. The scheme given on page 64 may be used in this examination. 6 4 PHYSIOLOGICAL CHEMISTRY. oti u S. w H O p4 Oh o o U W H W O w H O a 2 w U a «>22 "3— JJ 3~ Z wiE g « a" V yj U Jgjj -° o M 2 = « O tj U - «-. ~ 11 at c 5 = '=-€ — 3 * rt ~ ^ ^■5.5 * h a - = a 5 j! 2 c _ ote" "•> ■- o o 3s 2j= 2~ ■Eg Id .2* 3 'S ft i -^ >.. §£ CO o -a o .So. 3 D-S -".§-§ « - >£> ■» 3 §-9 o.« d be •3 fc =§ ^ S-C.t! u ~ ft -s s s « _"o*22 ffl a rt q « 5j s; p ft ^ t) •*! 3 1>4) 5* 4! e '■a & ■a o "■V c gs u S •o-S.*' " — u ' — 6 o o beg o-~ o-o o Soft 5 u g " a rt us O «,^ u ||| g g .S ^ g -a - £ J3 q 3 s u 1)2 ^ O >• < j3 he a <"CQ -2-o e 5" « c ? S • S S*8i° -«■ °£ v rt S o>Ea E? o v % tflJ3 U.2 (o .•«&< BO < SCfl-; o ^.: i— .is i '15 S i 3ft i 2.St3 ? &) o rt ft 0) ' *J 'ft^ o !-§ s 3 'v* •« -oe<3 -J< g« §" U2 K U 0) S-^S225 g a ^ -^ O *j -^ -^ — ..« X - o o „, . ^7 «c^ ■§.« m ?Jc « M - -G 2 g •^2'"„- « * s 2 « A 1 -^ u'O.Su fe-S be ^ ft— n n *- l_u ft IT S-Sag ft tfl O ft cj ■*-> j2 •,-; rt S 5 2 2 « -2 « .g rt ov. P h — a .- ~ f 2 "5 ^ " >> S rt u under Salivary Digestion, page 40. If the experiment was carried out under salivary digestion it will not be necessary to repeat it here. 8. Influence of Bile. — Prepare five tubes as follows : (a) Five c.c. of pepsin-hydrochloric acid solution -f- J4— I c.c. of bile. (b) Five c.c. of pepsin-hydrochloric acid solution -j- 1—2 c.c. of bile. (c) Five c.c. of pepsin-hydrochloric acid solution -f- 2-3 c.c. of bile. (d) Five c.c. of pepsin-hydrochloric acid solution + 5 c.c. of bile. (c) Five c.c. of pepsin-hydrochloric acid solution. Introduce into each tube a small piece of fibrin. Keep the tubes at 40 C. and note the progress of digestion. Does the bile exert any appreciable influence? How? 9. Influence of Rennin on Milk. — Prepare a series of five tubes as follows : (a) Five c.c. of fresh milk + 0.2 per cent HC1 (add slowly until precipitate forms). (b) Five c.c. of fresh milk -f- 5 drops of rennin solution. (c) Five c.c. of fresh milk -f- 10 drops of 0.5 per cent XaX0 3 solution. (d) Five c.c. of fresh milk -f- 10 drops of 5 per cent am- monium oxalate solution. (e) Five c.c. of fresh milk -(- 5 drops of 0.2 per cent HC1. Xow to each of the tubes (c), (d) and (e) add 5 drops of 94 PHYSIOLOGICAL CHEMISTRY. rennin solution. Place the whole series of five tubes at 40 C. and after 10-15 minutes note what is occurring in the different tubes. Give a reason for each particular result. 10. Tests for Lactic Acid, (a) Uffelmann's Reaction. — To a small quantity of Uffelmann's reagent 1 in a test-tube add a few drops of a lactic acid solution. The amethyst-blue color of the reagent is displaced by a straw yellow. Other organic acids give a similar reaction. Mineral acids such as HC1 dis- charge the blue coloration leaving a colorless solution. (b) Ferric Chloride Test. — Place 10 c.c. of very dilute ferric chloride in each of five tubes. To the first add 2 c.c. of 0.2 per cent HC1, to the second 2 c.c. of 10 per cent alcohol, to the third 2 c.c. of 2 per cent saccharose, to the fourth 2 c.c. of lactic acid and to the fifth 2 c.c. of peptone solution. It is evident from the results obtained that neither of the tests given above is satisfactory for the detection of lactic acid in the presence of other substances such as we find in the gastric contents. A satisfactory deduction regarding the presence of lactic acid can only be made after extracting the gastric contents with ether, evaporating the ether extract to dryness and dis- solving the residue in water. This residue will not contain any of the contaminations which interfered with the simple tests as tried above, and therefore if either of the tests is now tried on the dissolved residue of the ether extract we may form an accurate conclusion regarding the presence of lactic acid. 11. Qualitative Analysis of Stomach Contents. — Take 100 c.c. of stomach contents and analyze it according to the following scheme : 1 Uffelmann's reagent is prepared by adding ferric chloride solution to a 1 per cent solution of carbolic acid until an amethyst-blue color is obtained. GASTRIC DIGESTION. 95 Stomach Contents. Filter and test the filtrate for free HC1. I Filtrate I. Divide into two parts. I Residue. Discard after making a micro- scopical examination. Filtrate II. One-fifth portion. Test for: (a) Pepsin. (b) Bile (see p. (c) Starch. (d) Dextrin. Filtrate III. Four-fifths portion. Neutralize carefully; any precipitate is acid albuminate. If a precipitate -). forms filter and divide the filtrate into tzvo parts. If no precipitate forms divide the solution into two parts without filtering. Filtrate IV. Two-thirds portion. Heat to boiling to remove coagulable proteids. If any precipitate forms filter it off; if there is no precipitate pro- ceed directly with the tests. Test for: (a) Sugar. {b) Proteoses, (c) Peptones. Filtrate V. One-third portion. Test for : (a) Lactic acid. (&) Rennin. (c) Ptyalin. CHAPTER VI. FATS. Fats occur very widely distributed in the plant and animal kingdoms, and constitute the third general class of food stuffs. In plant organisms they are to be found in the seeds, roots and fruit, while each individual tissue and organ of an animal organism contains more or less of the substance. In the animal organism fats are especially abundant in the bone marrow and adipose tissue. They contain the same elements as the carbohydrates, i. e., carbon, hydrogen and oxygen, but the oxygen is present in smaller percentage than in the carbohydrates and the hydrogen and oxygen are not present in the proportion to form water. According to the observa- Fig. 35- Beef Fat. {Long.) tions of Benedict and Osterberg human fat contains 76.08 per cent of carbon and 11.78 per cent of hydrogen. They found the heat of combustion of human fat to be 9.523 calories per gram'. 96 FATS. 97 Chemically considered the fats are esters 1 of the tri-atomic alcohol, glycerin, and the mono-basic fatty acids. The II of each of the OH groups of glycerin is replaced by a fatty acid radical (see page 65). For instance CH 2 -OH 1 CH -OH I CH 2 -OH is the formula for glycerin and by replacing the hydrogen of the hydroxyls by hydrocarbon radicals R, R.' and R" we obtain, as the typical formula for an ordinary neutral fat, CH 2 -OOC-R I CH -O-OC-R' I CH 2 - O-OC-R". The positions occupied by R, R' and R" in the above formula may be filled by three radicals of the same fatty acid or by the radicals of three different fatty acids. By hydrolysis of a neutral fat, i. c, by the addition to the molecule of those elements which are eliminated in the forma- tion of the fat from glycerin and fatty acid, it may be resolved into its component parts, i. c, glycerin and fatty acid. In the case of tri-palmitin the following would be the reaction : C 3 H 5 (0-C 15 H 31 CO) 3 + 3H 2 = TH- Palmiti , C 3 H 5 (OH) 3 + 3(C 15 H 31 COOH). Glycerin. Palmitic acid. This process is called saponification and may be produced by boiling with alkalis; by the action of steam under pressure; by long-continued contact with air and light; by the action of certain bacteria and by fat-splitting enzymes or lipases, c. g., 1 An ester is an ethereal salt consisting of an organic radical united with the residue of an inorganic or organic acid. 98 PHYSIOLOGICAL CHEMISTRY. steapsin (see page 109). The cells forming the walls of the intestines evidently possess the peculiar property of synthesiz- ing the glycerin and fatty acid thus formed so that after absorption these bodies appear in the blood not in their in- dividual forms but as neutral fats. This synthesis is similar to that enacted in the absorption of proteid material where the peptones are synthesized into albumin in the act of absorption. The principal animal fats with which we have to deal are stearin, palmitin, olein and butyrin. Such less important forms as laurin and myristin may occur abundantly in plant organisms. The generally accepted system of nomenclature for these fats is to apply the prefix " tri " in each case (e. g., fn-palmitin) since three fatty acid radicals are contained in the neutral fat molecule. Fats occur ordinarily as mixtures of several individual fats. For example, the fat found in animal tissues is a mixture of tri-olein, tri-palmitin and tri-stearin, the percentage of any one of these fats present depending upon the particular species of animal from whose tissue the fat was derived. Thus the ordinary mutton fat contains more tri-stearin and less tri- olein than the pork fat. The crystalline forms of some of the more common fats are reproduced in Figs. 35, 36 and 37 on pages 96, 99 and 101. . Pure neutral fats are odorless, tasteless and generally color- less. They are insoluble in the ordinary proteid solvents such as water, sodium chloride and dilute acids and alkalis but are very readily soluble in ether, benzene, chloroform and boiling alcohol. Each individual fat possesses a specific melting- or boiling-point (according to whether the body is solid or fluid in character) and this property of melting or boiling at a definite temperature may be used as a means of differentiation in the same way as the coagulation temperature (see page 60) is used for the differentiation of coagulable proteids. When shaken with water, or a solution of albumin, soap or gum FATS. 99 arable, the fats are finely divided and assume a condition known as an emulsion. The emulsion with water is transitory, while the emulsions with soap or albumin solution are per- manent. The fat ingested continues essentially unaltered until it reaches the intestines where it is acted upon by steapsin the fat-splitting enzyme of the pancreatic juice (see page 109), and glycerin and fatty acid are formed from a large portion of the fat. Part of the fatty acid thus formed is dissolved in the bile and absorbed while the remainder unites with the alkalis of the pancreatic juice and forms soluble soaps. These soaps may further act to produce an emulsion of the remaining fat and thus aid in its absorption. That bile is of assistance in the absorption of fat is indicated by the increase of fat in the feces when for any reason bile does not pass into the in- testines. The fat distributed throughout the animal body is formed partly from the ingested fat and partly from carbohydrates Fig. 36. Mutton Fat. (Long.) and the " carbon moiety " of proteid material. The formation of adipoccre and the occurrence of fatty degeneration are sometimes given as proofs of the formation of fat from pro- IOO PHYSIOLOGICAL CHEMISTRY. teid. This is questioned by many investigators. Rather more satisfactory and direct proof of the formation of fat from proteid material has been obtained by experimentation upon fix-maggots. The normal content of fat in a number of maggots was determined and later the fat content of others which had developed in blood (84 per cent of the solid matter of blood plasma is proteid material) was determined. The fat content was found to have increased 700 to 11 00 per cent as a result of the diet of blood proteids. Some investigators are not inclined to accept these data as conclusive. Experiments on Fats. 1. Solubility. — Test the solubility of olive oil in each of the ordinary solvents (see page 4) and in cold alcohol, hot alcohol, chloroform and ether. 2. Formation of a Transparent Spot on Paper. — Place a drop of olive oil upon a piece of ordinary writing paper. Note the transparent appearance of the paper at the point of contact with the fat. 3. Reaction. — Try the reaction of fresh olive oil to litmus. Repeat the test with rancid olive oil. What is the reaction of a fresh fat and how does this reaction change upon allowing the fat to stand for some time? 4. Formation of Acrolein. — To a little olive oil in a mortar add some potassium bisulphate, KHS0 4 , and rub up thor- oughly. Transfer to a dry test-tube and cautiously heat. Note the irritating odor of acrolein. The glycerin of the fat has been dehydrolyzed and acrylic aldehyde or acrolein has been produced. This is the reaction which takes place : CH 2 -OH CH • OH -» CH 2 = CH • CHO + 2H 2 0. Acrolein. CH 2 -OH Glycerin. FATS. IOI 5. Emulsification. — (a) Shake up a drop of neutral 1 olive oil with a little water in a test-tube. The fat becomes finely divided, forming an emulsion. This is not a permanent emul- sion since the fat separates and rises to the top upon standing. (b) To 5 c.c. of water in a test-tube add 2 or 3 drops of 0.5 per cent NaXO.j. Introduce into this faintly alkaline solution a drop of neutral olive oil and shake. The emulsion while not permanent is not so transitory as in the ease of water free from sodium carbonate. (c) Repeat (b) using rancid olive oil. What sort of an emulsion do you get and why? ( t> given below. 8. Palmitic Acid. — (a) Examine the crystals under the microscope and compare them with those shown in Fig. 38, opposite. (b) Solubility. — Try the solubility of palmitic acid in the same solvents as used on fats (see p. 100). (cr) Melting-Point. — Determine the melting-point of palmitic acid by <»ne of the methods given on pages 104 and 105. (d) Formation of Transparent Spot on Paper, — Melt a little of the fatty acid and allow a drop to fall upon a piece of ordinary writ- ing paper. How does this com- pare with the action of a fat under similar circumstances ? (c) Acrolein Test. — Apply the test as given under 4, page 100. Explain the result. 9. Saponification of Lard. — To (wgj 25 grams of lard in a flask add 75 c.c. of alcoholic-potash solution and warm upon a water-bath until saponification is complete. (This point is indicated by the complete solubility of a drop of the solution when allowed to fall into a little water.) Now transfer the solu- tion from the flask to an evapo- rating dish containing about ioo c.c. of water and heat on a water- bath until all the alcohol has been driven off. Precipitate the fatty acid with HC1 and cool the solution. Remove the fatty acid which rises to the surface, M 1:1. ting- Point Apparatus. 104 PHYSIOLOGICAL CHEMISTRY. neutralize the solution with Na 2 CO s and evaporate to dryness. Extract the residue with alcohol, remove the alcohol by evapo- ration upon a water-bath and on the residue of glycerin thus obtained make the tests as given below. 10. Glycerin, (a) Taste. — What is the taste of glycerin? (b) Solubility. — Try the solubility of glycerin in water, alcohol and ether. (c) Acrolein Test. — Repeat the test as given under 4, p. 100. (d) Borax Fusion Test. — Fuse a little glycerin on a plat- inum wire with some powdered borax and note the charac- teristic green flame. This color is due to the glycerin ester of boric acid. (e) Fehlings Test. — How does this result compare with the results on the sugars ? (/) Solution of Cu(OH) 2 .— Form a little Cu(OH) 2 by mixing CuS0 4 and KOH. Add a little glycerin to this sus- pended precipitate and note what occurs. 11. Melting-Point of Fat. First Method. — Insert one of the melting-point tubes, furnished by the instructor, into the liquid fat and draw up the fat until the bulb of the tube is about one-half full of the material. Then fuse one end of the tube in the flame of a bunsen burner and fasten the tube to a thermometer by means of a rubber band in such a manner that the bottom of the fat column is on a level with the bulb of the thermometer (Fig. 39, p. 103). Fill a beaker of medium size about two-thirds full of water and place it within a second larger beaker which also contains water, the two vessels being separated by pieces of cork. Immerse the bulb of the ther- mometer and the attached tube in such a way that the bulb is about midway between the upper and the lower surfaces of the water of the inner beaker. The upper end of the tube being open it must extend above the surface of the surround- ing water. Apply gentle heat, stir the water, and note the temperature at which the fat first begins to melt. This point is indicated by the initial transparency. For ordinary fats, raise the temperature very cautiously from 30 C. To deter- FATS. 105 mine the congealing-poittt remove the flame and note the tem- perature at which the fat begins to solidify. Record the melt- ing - - and congealing-points of the various fats submitted by the instructor. Second Method. — Fill a small evaporating dish about one- half full of mercury and place it on a water-bath. Put a small drop of the fat under examination on an ordinary cover glass and place this upon the surface of the mercury. Raise the temperature of the water-bath slowly and by means of a thermometer whose bulb is immersed in the mercury note the melting-point of the fat. Determine the congealing-point by removing the flame and leaving the fat drop and cover glass in position upon the mercury. How do the melting- points as determined by this method compare with those as determined by the first method? Which method is the more accurate, and why? CHAPTER VII. PANCREATIC DIGESTION. As soon as the food mixture leaves the stomach it comes into intimate contact with the bile and the pancreatic juice. Since these fluids are alkaline in reaction there can obviously be no further peptic activity after they have become intimately mixed with the chyme and have neutralized the acidity pre- viously imparted to it by the hydrochloric acid of the gastric juice. The pancreatic juice reaches the intestine through the duct of Wirsung which opens into the intestine near the pylorus. Normally the secretion of pancreatic juice is brought about by the stimulation produced by the acid chyme as it enters the duodenum. This secretion is probably not due to a nervous reflex, but is dependent upon the presence, in the epithelial cells of the duodenum, of a body known as prosecretin. This body is changed into secretin through the hydrolytic action of the acid present in the chyme. The secretin is then ab- sorbed by the blood, passes to the pancreas and stimulates the pancreatic cells, causing a flow of pancreatic juice. The quan- tity of juice secreted under these conditions is proportional to the amount of secretin present. The activity of secretin solu- tions is not diminished by boiling, hence the body does not react like an enzyme. Further study of the body may show it to be a definite chemical individual of relatively low molec- ular weight. It has not been possible thus far to obtain secre- tin from any tissues except the mucus membrane of the duo- denum and jejunum. The juice as obtained from a permanent fistula differs greatly in its properties from the juice as obtained from a temporary fistula, and neither form of fluid possesses the prop- erties of the normal fluid. Pancreatic juice collected from 1 06 PANCREA1 [C DIGESTION. 107 a natural fistula has been found to be a colorless, clear, strongly alkaline fluid which foams readily. It is further characterized by containing albumin and globulin and by the absence of proteoses and peptone. The average daily secretion ol pan- creatic juice is C50 c.c. and its specific gravity is 1.008. The fluid contain^ [.3 per cent of solid matter and the freezing- point is — 0.47 ° C. The normal pancreatic secretion contains at least four distinct enzymes. They are trypsin, a proteolytic enzyme; amylopsin, an amylolytic enzyme; steapsin, a fat- splitting enzyme; and pancreatic rennin, a milk-coagulating enzyme. The most important of the four enzymes of the pancreatic juice is the proteolytic enzyme trypsin. This enzyme resem- bles pepsin in so far as each has the power of breaking down proteid material, but the trypsin has much greater digestive power and is able to cause a more complete decomposition of the complex proteid molecule. In the process of normal diges- tion the proteid constituents of the diet are for the most part transformed into proteoses (albumoses) before coming in con- tact with the enzyme trypsin. This is not absolutely essential however, since trypsin possesses digestive activity sufficient to transform unaltered native proteids and to produce from their complex molecules comparatively simple fragments. Among the products of tryptic digestion are alkali albuminate, pro- teoses (albumoses), peptone, leucin, tyrosin, aspartic acid, glutamic acid, lysiu, histidin, arginin, tryptophan and am- monia. (The crystalline forms of many of these products are reproduced in Chapter IV.) Trypsin does not occur pre- formed in the gland, but exists there as a zymogen called trypsinogen which bears the same relation to trypsin that pepsinogen does to pepsin. Trypsin has never been obtained in a pure form and therefore very little can be stated definitely as to its nature. The enzyme is the most active in alkaline solution but is also active in neutral or slightly acid solutions. Trypsin is destroyed by mineral acids and may also be de- stroyed by comparatively weak alkali {2 per cent sodium car- 108 PHYSIOLOGICAL CHEMISTRY. bonate) if left in contact for a sufficiently long time. Tryp- sinogen, on the other hand, is more resistant to the action of alkalis. The pancreatic juice which is collected by means of a fistula possesses practically no power to digest proteid matter. A body called enterokinase occurs in the intestinal juice and has the power of converting trypsinogen into trypsin. This proc- ess is known as the " activation " of trypsinogen and through it a juice which is incapable of digesting proteid may be made active. Enterokinase is not always present in the intestinal juice since it is secreted only after the pancreatic juice reaches the intestine. It resembles the enzymes in that its activity is destroyed by heat, but differs materially from this class of bodies in that a certain quantity of intestinal juice is capable of activating only a definite quantity of trypsinogen. Entero- kinase has been detected in the higher animals, and a kinase possessing similar properties has been shown to be present in bacteria, fungi, impure fibrin, lymph glands and snake-venom. The activation of trypsinogen into trypsin may be brought about in the gland as well as in the intestine of the living organism (Mendel and Rettger). The manner of the activa- tion in the gland and the nature of the body causing it are unknown at present. Amylopsin, the second of the pancreatic enzymes, is an amylolytic enzyme which possesses somewhat greater digestive power than the ptyalin of the saliva. As its name implies, its activity is confined to the starches, and the products of its amylolytic action are dextrins and sugars. The sugars are principally iso-maltose and maltose and these by the further action of an inverting enzyme are partly transformed into dextrose. It is probable that the saliva as a digestive fluid is not abso- lutely essential. The ptyalin is destroyed by the hydrochloric acid of the gastric juice and is therefore inactive when the chyme reaches the intestine. Should undigested starch be present at this point however, it would be quickly transformed PANCREATIC DIGESTION. 109 by the active amylopsin. This enzyme is not present in the pancreatic juice of infants during- the first few weeks of life, thus showing very clearly that a starchy diet is not normal for this period. It has been claimed that amylopsin has a slight digestive action upon unboiled starch. The third enzyme of the pancreatic juice is called steapsin and is a fat-Splitting enzyme. It has the power of splitting the neutral fats of the food by hydrolysis, into fatty acid and glycerin. A typical reaction would be as follows : C 3 H 5 (0-C 15 H, 1 CO), + 3H 2 = Tri-Palmitin. 3(C 15 H 31 COOH) + C 3 H 5 (OH),. Palmitic acid. Glycerin. Recent researches make it probable that fats undergo saponi- fication to a very large extent prior to their absorption. The fatty acids formed, in part unite with the alkalis of the pan- creatic juice and intestinal secretion to form soluble soaps ; in part they are doubtless absorbed dissolved in the bile. Some observers believe that the fats may also be absorbed in emul- sion — a condition promoted by the presence of the soluble soaps. After absorption the fatty acids are re-synthesized to form neutral fats with glycerin. Steapsin is very unstable and is easily rendered inert by the action of acid. For this reason it is not possible to prepare an extract having a satisfactory fat-splitting power from a pancreas which has been removed from the organism for a sufficiently long time to have become acid in reaction. The fourth enzyme of the pancreatic juice is called pan- creatic rennin. It is a milk-coagulating enzyme whose action is very similar to that of the enzyme rennin found in the gastric juice. It is supposed to show its greatest activity at a tempera- ture varying from 50 to 6o° C. IIO PHYSIOLOGICAL CHEMISTRY. PREPARATION OF AN ARTIFICIAL PANCREATIC JUICE. After removing the fat from the pancreas of a pig or sheep, finely divide the organ by means of scissors and grind it in a mortar. If convenient, the use of an ordinary meat chopper is a very satisfactory means of preparing the pancreas. When finely divided as above the pancreas should be placed in a 500 c.c. flask, about 150 c.c. of 30 per cent alcohol added and the flask and contents shaken frequently for twenty-four hours. (What is the reaction of this alcoholic extract at the end of this period, and why?) Strain the alcoholic extract through cheese cloth, filter, nearly neutralize with KOH solu- tion and then exactly neutralize it with 0.5 per cent Na 2 CO s . Products of Pancreatic Digestion. Take about 200 grams of lean beef which has been freed from fat and finely ground and place it in a large-sized beaker. Nearly fill the beaker with pancreatic extract prepared as above, add 5 c.c. of an alcoholic solution of thymol to prevent putrefaction, and place the beaker in an incubator at 40 C. Stir the contents of the beaker frequently and add more thymol if it becomes necessary. Allow digestion to proceed for from 2 to 5 days and then separate the products formed as follows : Strain off the undissolved residue through cheese cloth, nearly neutralize the solution with dilute hydrochloric acid and then exactly neutralize it with 0.2 per cent hydro- chloric acid. A precipitate at this point would indicate alkali albuminate. Filter off any precipitate and divide the filtrate into two parts, a one-fourth and a three-fourth portion. Transfer the one-fourth portion to an evaporating dish and make the separation of proteoses and peptones as well as the final tests upon these bodies according to the directions given on page 59. Place about 5 c.c. of the three-fourth portion in a test-tube and add about 1 c.c. of bromine water. A violet coloration PANCREATIC DIGESTION. HI indicates the presence of tryptophan (see page 77). Concen- trate 1 the remainder of the three-fourth portion to a thin syrup and make the separation of teuein and tyrosin according to the directions given oil page 81. GENERAL EXPERIMENTS ON PANCREATIC DIGESTION. Experiments on Trypsin, t. The Most Favorable Reaction for Tryptic Digestion. — Prepare seven tubes as follows : (a) 2-3 c.c. of neutral pancreatic extract -f 2 ~3 cc - of water. (b) 2-3 c.c. of neutral pancreatic extract -f- 2-3 c.c. of 1 per cent Na 2 CO :i . (c) 2-3 c.c. of neutral pancreatic extract + 2_ 3 cc - °f °-5 per cent Na 2 C0 3 . (d) 2-3 c.c. of neutral pancreatic extract + 2-3 c.c. of 0.2 per cent HC1. (e) 2-3 c.c. of neutral pancreatic extract + 2-3 c.c. of 0.2 per cent combined HC1. ('/) 2-3 c.c. of neutral pancreatic extract + 2-3 c.c. of 0.4 per cent boric acid. (g) 2-3 c.c. of neutral pancreatic extract + 2-3 c.c. of 0.4 per cent acetic acid. Add a small piece of fibrin to the contents of each tube and keep them at 40 C. noting the progress of digestion. In which tube do we find the most satisfactory digestion, and why? How do the indications of the digestion of fibrin by trypsin differ from the indications of the digestion of fibrin by pepsin? 2. The Most Favorable Temperature. — (For this and the following series of experiments under tryptic digestion use the neutral extract plus an equal volume of 0.5 per cent 1 If the solution is alkaline in reaction, while it is being concentrated, the amino acids will be broken down and ammonia will be liberated. 112 PHYSIOLOGICAL CHEMISTRY. Xa 2 C0 3 .) In each of four tubes place 5 c.c. of alkaline pan- creatic extract. Immerse one tube in cold water from the fau- cet, keep a second at room temperature and place a third on the water-bath at 40 C. Boil the contents of the fourth for a few moments, then cool and also keep it at 40 C. Into each tube introduce a small piece of fibrin and note the progress of digestion. In which tube does the most rapid digestion occur? What is the reason? 3. Influence of Metallic Salts, etc. — Prepare a series of tubes and into each tube place 6 volumes of water, 3 volumes of alkaline pancreatic extract and 1 volume of one of the chemicals listed in Experiment 18 under Salivary Digestion, page 40. Introduce a small piece of fibrin into each of the tubes and keep them at 40 ° C. for one-half hour. Shake the tubes fre- quently. In which tubes do we get the least digestion? 4. Influence of Bile. — Prepare five tubes as follows : (a) Five c.c. of pancreatic extract + j4-i c.c. of bile. (b) Five c.c. of pancreatic extract -j- 1-2 c.c. of bile. (c) Five c.c. of pancreatic extract -j- 2-3 c.c. of bile. (d) Five c.c. of pancreatic extract + 5 c.c. of bile. (e) Five c.c. of pancreatic extract. Introduce into each tube a small piece of fibrin and keep them at 40 C. Shake the tubes frequently and note the prog- ress of digestion. Does the presence of bile retard tryptic digestion? How do these results agree with those obtained under gastric digestion? Experiments on Amylopsin. 1. The Most Favorable Reaction. — Prepare seven tubes as follows : (a) One c.c. of neutral pancreatic extract -j- 1 c.c. of starch paste + 2 c.c. of water. (b) One c.c. of neutral pancreatic extract -j- 1 c.c. of starch paste -f- 2 c.c. of 1 per cent Na 2 CO s . (c) One c.c. of neutral pancreatic extract + 1 c - c - °f starch paste -f- 2 c.c. of 0.5 per cent Na 2 C0 3 . PANCREATIC DIGESTION. 1 13 (d) One c.c. of neutral pancreatic extract -\- J cc - °f starch paste + 2 c.c. of 0.2 per cent HC1. (e) One c.c. of neutral pancreatic extract -f- 1 c.c. of starch paste -f- 2 c.c. of 0.2 per cent combined HC1. (/) One c.c. of neutral pancreatic extract -\- 1 c.c. of starch paste + 2 c.c. of 0.4 per cent boric acid. (g) One c.c. of neutral pancreatic extract -(- 1 c.c. of starch paste -j- 2 c.c. of 0.4 per cent acetic acid. Shake each tube thoroughly and place them on the water- bath at 40 C. At the end of a half-hour divide the contents of each tube into two parts and test one part by the iodine test and the other part by Fehling-'s test. Where do you find the most satisfactory digestion? How do the results here com- pare with those obtained from the similar series under Trypsin, page in. 2. The Most Favorable Temperature. — (For this and the following series of experiments upon amylopsin use the neutral extract plus an equal volume of 0.5 per cent Xa 2 CO ;! .) In each of four tubes place 2-3 c.c. of alkaline pancreatic ex- tract. Immerse one tube in cold water from the faucet, keep a second at room temperature and place a third on the water- bath at 40 C. Boil the contents of the fourth for a few moments, then cool and also keep it at 40 C. Into each tube introduce 2-3 c.c. of starch paste and note the progress of digestion. At the end of one-half hour divide the contents of each tube into two parts and test one part by the iodine test and the other part by Fehling's test. In which tube do you find the most satisfactory digestion? How does this result compare with the result obtained in the similar series of experi- ments under Trypsin (see page 11 1) ? 3. Influence of Metallic Salts, etc. — Prepare a series of tubes and into each tube place 3 volumes of water, 3 volumes of alkaline pancreatic extract, 1 volume of one of the chemicals listed in Experiment 18 under Salivary Digestion, page 40. and 3 volumes of starch paste. Be sure to introduce the. starch paste into the tube last. Why? Shake the tubes well and 9 114 PHYSIOLOGICAL CHEMISTRY. place them on the water-bath at 40 C. At the end of a half- hour divide the contents of each tube into two parts and test one part by the iodine test and the other part by Fehling's test. What are your conclusions? 4. Influence of Bile. — Prepare five tubes as follows : (a) 2-3 c.c. of pancreatic extract + 2-3 c.c. of starch paste + y 2 -i c.c. of bile. (b) 2-3 c.c. of pancreatic extract + 2_ 3 c -c of starch paste -f- 1-2 c.c. of bile. (c) 2-3 c.c. of pancreatic extract -\- 2-3 c.c. of starch paste -f- 2-3 c.c. of bile. (d) 2-3 c.c. of pancreatic extract -\- 2-3 c.c. of starch paste + 5 c.c. of bile. (e) 2-3 c.c. of pancreatic extract -j- 2-3 c.c. of starch paste. Shake the tubes thoroughly and place them on the water- bath at 40 ° C. Note the progress of digestion frequently and at the end of a half-hour divide the contents of each tube into two parts and test one part by the iodine test and the other part by Fehling's test. What are your conclusions regarding the influence of bile upon the action of amylopsin? 5. Digestion of Dry Starch. — To a little dry starch in a test-tube add about 5 c.c. of pancreatic extract and place the tube on the water-bath at 40 C. At the end of a half hour filter and test separate portions of the fil.trate by the iodine and Fehling tests. What do you conclude regarding the action of amylopsin upon dry starch? Compare this result with that obtained in the similar experiment under Salivary Digestion (page 38). 6. Digestion of Inulin. — To 5 c.c. of inulin solution in a test-tube add 10 drops of pancreatic extract and place the tube on the water-bath at 40 C. After one-half hour test the solution by Fehling's test. 1 Is any reducing substance present? What do you conclude regarding the digestion of inulin by amylopsin? 1 If the inulin solution gives a reduction before being acted upon by the pancreatic juice, it will be necessary to determine the extent of the original reduction by means of a "check" test (see page 26). pancreatic digestion. 1 15 Experiments on Steapsix. i. "Litmus-Milk" Test. — Into each of two test-tubes in- troduce 10 c.c. of milk and a small amount of litmus powder. To the contents of one tube add 3 c.c. of neutral pancreatic extract and to the contents of the other tube add 3 c.c. of water or of boiled neutral pancreatic extract. Keep the tubes at 40 C. and note any changes which may occur. What is the result and how do you explain it? 2. Ethyl Butyrate Test. — Into each of two test-tubes intro- duce 4 c.c. of water, 2 c.c. of ethyl butyrate, C a H 7 COO" C 2 H 5 , and a small amount of litmus powder. To the contents of one tube add 4 c.c. of neutral pancreatic extract and to the contents of the other tube add 4 c.c. of water or bailed neutral pan- creatic extract. Keep the tubes at 40° C. and observe any changes which may occur. What is the result and how do you explain it? Write the equation for the reaction which has taken place. Experiments on Pancreatic Rennin. Prepare two test-tubes as follows : (a) Five c.c. of milk + 10 drops of pancreatic extract. (b) Five c.c. of milk + 20 drops of pancreatic extract. Place the tubes at 40°-45° C. for a half hour without shaking. Xote the formation of a clot. 1 How does the action of pancreatic rennin compare with the action of the gastric rennin ? 'This reaction will not always succeed, owing to conditions which are not well understood. CHAPTER VIII. BILE. The bile is secreted continuously by the liver and passes into the intestine through the common bile duct which opens near the pylorus. Bile is not secreted continuously into the intes- tine. In a fasting animal no bile enters the intestine, but when food is taken the bile begins to flow ; the length of time elapsing between the ingestion of the food and the secretion of the bile as well as the qualitative and quantitative character- istics of the secretion depending upon the nature of the food ingested. Fats, the extractives of meat and the end-products of gastric digestion (acid albuminate, proteoses and peptones) cause a copious secretion of bile, whereas such substances as water, acids and boiled starch paste fail to do so. In general a rich proteid diet is supposed to increase the amount of bile secreted, whereas a carbohydrate diet would rather tend to decrease the amount. We may look upon the bile as an excretion as well as a secretion. In the fulfillment of its excretory function it passes such bodies as lecithin, metallic substances and cholesterin into the intestine and in this way aids in removing them from the organism. The bile assists materially in the absorption of fats from the intestine by its solvent action on the fatty acids formed by the action of the pancreatic juice. The bile is a very thick, viscid substance which is alkaline in reaction to litmus and ordinarily possesses a decidedly bitter taste. It varies in color in the different animals, the principal variations being yellow, brown and green. Fresh human bile ordinarily has a green or golden-yellow color. Post-mortem bile is variable in color. It is very difficult to determine accurately the amount of normal bile secreted dur- ing any given period. For an adult man it has been variously 116 BILE. I I 7 estimated at from 500 c.c. to cioo c.c. for twenty four hours. The specific gravity of the bile varies bet \\ een [.010 and 1 .040, and the freezing-point is about — 0.56 C. As secreted by the liver, the bile is a clear, limpid fluid which contains a rela- tively low contenl of solid matter. Such bile would ha specific gravity of approximately r.oio. After it reaches the gall Madder, however, it becomes mixed with mucous material from the walls of the gall-bladder, and this pro* npled with the continuous absorption of water from the bile has a tendency to concentrate the secretion. Therefore the bile, as we find it in the gall-blaader, ordinarily possesses a higher specific gravity than that of the freshly secreted fluid. The specific gravity under these conditions may run as high as 1.040. The principal constituents of the bile are the salts of the bile acids, bile pigments, neutral fats, lecitJiin and cholesterin, besides the salts of iron, copper, calcium and magnesium. Zinc has also frequently been found in traces. The bile acids, which are elaborated exclusively by the hepatic cells, may be divided into two groups, the glycocholic acid group and the taurocholic acid group. In human bile glycocholic acid predominates, while taurocholic acid is the more abundant in the bile of carnivora. The bile acids are conjugate amino-acids, the glycocholic acid yielding glycocoll, CPL • NH„ I COOH, and cholic acid upon decomposition, whereas taurocholic acid gives rise to tauriu, CHo • NH 2 I CH 2 • S0 2 • OH, and cholic acid under like conditions. Glycocholic acid con- tains some nitrogen but no sulphur, whereas taurocholic acid contains both these elements. The sulphur of the taurocholic n8 PHYSIOLOGICAL CHEMISTRY. acid is present in the taurin (amino-ethyl-sulphonic acid), of which it is a characteristic constituent. There are several varieties of cholic acid and therefore we have several forms of glycocholic and taurocholic acids, the variation in consti- tution depending upon the nature of the cholic acid which enters into the combination. The bile acids are present in the bile as salts of one of the alkalis, generally sodium. The sodi- um glycocholate and sodium taurocholate may be isolated in crystalline form, either as balls or rosettes of fine needles or in the form of prisms having ordinarily four or six sides (Fig. 40, below). The salts of the bile acids are dextro-rotatory. Among other properties these salts have the power of holding the cholesterin and lecithin of the bile in solution. Fig. 40. Bile Salts. The bile pigments are important and interesting biliary con- stituents. The following have been isolated : bilirubin, bili- verdin, bilifuscin, biliprasin, bilihumin, bilicyanin and chole- telin. Of these, bilirubin and biliverdin are the most important and predominate in normal bile. Bilirubin may be isolated as a reddish-yellow powder and biliverdin may be obtained in the form of a green powder. The colors possessed by the various BILE. 119 varieties of normal bile are due almosl entirely to these two pigments, the biliverdin being the predominant pigment in greenish bile and the bilirubin being the principal pigment in lighter colored bile. The pigments, other than the two just mentioned, have been found almost exclusively in biliary cal- culi or in altered bile obtained at post-mortem examinations. Bilirubin, which is perhaps the most important of the bile pigments, is apparently derived from the blood pigment, the iron freed in the process being held in the liver. Bilirubin has the same percentage composition as haematoporphyrin, which may be produced from haematin. It is a specific product of the liver cells but may also be formed in other parts of the body. The pigment may be obtained, in part, in the form of reddish-yellow rhombic plates (Fig. 41, below) upon the Fig. 41. Bilirubin (H^matoidin). (Ogdcn.) spontaneous evaporation of its chloroform solution. The crystalline form of bilirubin is practically the same as that of hsematoidin. Tt is easily soluble in chloroform, somewhat less soluble in alcohol and only slightly soluble in ether and benzene. Bilirubin has the power of combining with certain metals, particularly calcium, to form combinations which are no longer soluble in the solvents of the unaltered pigment. Upon long standing in contact with the air, the reddish-yellow bilirubin is oxidized with the formation of the green biliverdin. Bilirubin occurs in animal fluids as soluble bilirubin-alkali. 120 PHYSIOLOGICAL CHEMISTRY. Solutions of bilirubin exhibit no absorption-bands. If an ammoniacal solution of bilirubin-alkali in water is treated with a solution of zinc chloride, however, it shows bands similar to those of bilicyanin (Absorption Spectra, Plate II), the two bands between C and D being rather well defined. Biliverdin is particularly abundant in the bile of herbivora. It is soluble in alcohol and glacial acetic acid and insoluble in water, chloroform and ether. Biliverdin is formed from bilirubin upon oxidation. It is an amorphous substance, and in this differs from bilirubin which may be at least partly crystallized under proper conditions. In common with bili- rubin, it may be converted into hydrobilirubin by nascent hydrogen. The neutral solution 'of bilicyanin or cholecyanin is bluish- green or steel-blue and possesses a blue fluorescence, the alka- line solution is green with no appreciable fluorescence and the strongly acid solution is violet-blue. The alkaline solution exhibits three absorption-bands, the first a dark, well-defined band between C and D somewhat nearer C; the second a less sharply-defined band extending across D and the third a rather faint band between E and F, near E (Absorption Spectra, Plate II). The strongly acid solution exhibits two absorption bands, both lying between C and E and separated by a narrow space near D. A third band, exceedingly faint, may ordinarily be seen between b and F. Biliary calculi, otherwise designated as biliary concretions or gall stones, are frequently formed in the gall-bladder. These deposits may be divided into three classes, cholcsterin calculi, pigment calculi and calculi made up almost entirely of inorganic material. This last class of calculus is formed prin- cipally of the carbonate and phosphate of calcium and is rarely found in man although quite common to cattle. The pigment calculus is also found in cattle, but is more common to man than the inorganic calculus. This pigment calculus ordinarily consists principally of bilirubin in combination with calcium; biliverdin is sometimes found in small amount. The BILE. I 2 I cholesterin calculus is the one found most frequently in man. These may be formed almost entirely of cholesterin, in which event the color of the calculus is very light, or they may con- tain more or less pigment and inorganic matter mixed with the cholesterin, which tends to give us calculi of various colors. For discussion of cholesterin see page 222. Experiments on Bile. 1. Reaction. — Test the reaction of fresh ox bile to litmus. 2. Nucleo-proteid. — Acidify a small amount of hile with dilute acetic acid. A precipitate of nucleo-proteid forms. 3. Inorganic Constituents. — Test for chlorides, sulphates and phosphates (see page 37). 4. Tests for Bile Pigments, (a) Gwielin's Test. — To about 5 c.c. of concentrated nitric acid in a test-tube add 2-3 c.c. of diluted bile carefully so that the two fluids do not mix. At the point of contact note the various colored rings, green, blue, violet, red and reddish-yellow. Repeat this test with different dilutions of bile and observe its delicacy. (b) Rosenbach's Modification of Gmelin's Test. — Filter 5 c.c. of diluted hile through a small filter paper. Introduce a drop of concentrated nitric acid into the cone of the paper and note the succession of colors as given in Gmelin's test. (c) Ilitppcrt's Reaction. — Thoroughly shake equal volumes of undiluted bile and milk of lime in a test-tube. The pig- ments unite with the calcium and are precipitated. Filter off the precipitate, wash it with water and transfer to a small beaker. Add alcohol acidified slightly with hydrochloric acid and warm upon a water-bath until the solution becomes col- ored an emerald green. (d) Hainmarstcn's Reaction. — To about 5 c.c. of Hammar- sten's reagent 1 in a small evaporating dish add a few drops of diluted bile. A green color is produced. If more of the rea- gent is now added the play of colors as observed in Gmelin's test may be obtained. Hammarsten's reagent is made by mixing 1 volume of 25 per cen! nitric acid and ig volumes of 2$ per cent hydrochloric acid and then adding I volume of this acid mixture to 4 volumes of 95 per cent alcohol. 122 PHYSIOLOGICAL CHEMISTRY. (e) Smith's Test. — To 2-3 c.c. of diluted bile in a test- tube add carefully about 5 c.c. of dilute tincture of iodine ( 1 : 10) so that the fluids do not mix. A play of colors, green, blue and violet, is observed. In making this test upon the urine ordinarily only the green color is observed. Try the test upon various dilutions of bile and note its delicacy as compared with that of Gmelin's test. Which test do you consider the more delicate ? 5. Tests for Bile Acids, (a) Pettenkofer's Test. — To 5 c.c. of diluted bile in a test-tube add 5 drops of a 5 per cent solution of saccharose. Now run about 2-3 c.c. of con- centrated sulphuric acid carefully down the side of the tube and note the red ring at the point of contact. Upon slightly agitating the contents of the tube the whole solution gradu- ally assumes a reddish color. As the tube becomes warm, it should be cooled in running water in order that the tempera- ture of the solution may not rise above 70 C. (b) Mylius's Modification of Pettenkofer's Test. To ap- proximately 5 c.c. of diluted bile in a test-tube add 3 drops of a very dilute (1 : 1,000) aqueous solution of furfurol, HC — CH II II HC C • CHO. \/ Now run about 2-3 c.c. of concentrated sulphuric acid care- fully down the side of the tube and note the red ring as above. In this case also, upon shaking the tube, the whole solution is colored red. Keep the temperature of the solution below 70 ° C. as before. ( c) Ncukomm's Modification of Pettenkofer's Test. — To a few drops of diluted bile in an evaporating dish add a trace of a dilute saccharose solution and one or more drops of dilute sulphuric acid. Evaporate on a water-bath and note the development of a violet color at the edge of the evaporating BILE. 123 mixture. Discontinue the evaporation as soon as the color is observed. (d) v. Udrdnsky's Test. — To 5 c.c. of diluted bile in a test- tube add 3 4 drops of a very dilute ( 1 :i,ooo) aqueous solution of furfurol. Place the thumb over the top of the tube and shake the tube until a thick foam is formed. By means O'f a small pipette add 2—3 drops of concentrated sulphuric acid to the foam and note the dark pink coloration produced. (e) Hay's Test. — Cool about 10 c.c. of diluted bile in a test-tube to \J° C. or lower and sprinkle a little finely pulver- ized sulphur upon the surface of the fluid. The presence of bile acids is indicated if the sulphur sinks to the bottom of the liquid, the rapidity with which the sulphur sinks depending upon the quantity of bile acids present in the mixture. The test is said to react with bile acids when they are present in the proportion 1 : 120,000. Some investigators claim that it is impossible to differentiate between bile acids and bile pigments by this test. 6. Crystallization of Bile Salts. — To 25 c.c. of undiluted bile in an evaporating dish add enough animal charcoal to form a paste and evaporate to dryness on a water-bath. Re- move the residue, grind it in a mortar and transfer it to a small flask. Add about 50 c.c. of 95 per cent alcohol and boil on a water-bath for 20 minutes. Filter, and add ether to the filtrate until there is a slight permanent cloudiness. Cover the vessel and stand it away until crystallization is complete. Examine the crystals under the microscope and compare them with those shown in Fig. 40, page 118. Try one of the tests for bile acids upon some of the crystals. I2 4 PHYSIOLOGICAL CHEMISTRY. 7. Analysis of Biliary Calculi. — Grind the calculus in a mortar with 10 c.c. of ether. Filter. Filtrate I. Allow to evaporate and examine for cholesterin crystals (Fig. 42, P- 125). (For further tests see experi- ment 8, below.) Residue I. (On paper and in mortar.) Treat with dilute HC1 and filter. Filtrate II. Test for calcium, phos- phates and iron. Evapo- rate remainder of filtrate to dryness in porcelain crucible and ignite. Dis- solve residue in dilute HC1 and make alkaline with NH4OH. Blue color indicates copper. Residue II. (On paper and in mortar.) Wash with a little water. Dry the filter paper. Treat with 5 c.c. chloroform and filter. Filtrate III. Bilirubin. (Apply test for bile pigments.) Residue III. (On paper and in mortar.) Treat with 5 c.c. of hot alcohol. Biliverdin. 8. Tests for Cholesterin. (a) Microscopical Examination. — Examine the crystals under the microscope and compare them with those shown in Fig. 42, page 125. (b) Iodine-Sulphuric Acid Test. — Place a few crystals of cholesterin in one of the depressions of a test-tablet and treat with a drop of concentrated sulphuric acid and a drop of a very dilute solution of iodine. A play of colors consisting of violet, blue, green and red results. (c) The Liebermann-Bnrchard Test. — Dissolve a few crys- tals of cholesterin in 2 c.c. of chloroform in a dry test-tube. Now add 10 drops of acetic anhydride and 1-3 drops of con- centrated sulphuric acid. The solution becomes red, then blue, and finally bluish-green in color. BILE. I 2 (r- ating dish add a few drops of a mixture of 3 volumes of con- centrated sulphuric acid and 1 volume of 10 per cent ferric chloride. Evaporate to dryness over a low flame and observe the reddish-violet residue which changes to a bluish-violet. 9. Preparation of Taurin. — To 300 c.c. of bile in a casse- role add 100 c.c. of hydrochloric acid and heat until a sticky mass (dyslysin) is formed. This point may be determined by drawing out a thread-like portion of the mass by means of a glass rod, and if it solidifies immediately and assumes a brittle character we may conclude that all the taurocholic and glyco- cholic acid has been decomposed. Decant the solution and concentrate it to a small volume on the water-bath. Filter the hot solution to remove sodium chloride and other substances which may have separated, and evaporate the filtrate to dry- 126 PHYSIOLOGICAL CHEMISTRY. ness. Dissolve the residue in 5 per cent hydrochloric acid and precipitate with ten volumes of 95 per cent alcohol. Filter off the taurin and recrystallize it from hot water. (Save the alcoholic filtrate for the preparation of glycocoll, page 127.) Make the following tests upon the taurin crystals : (a) Examine them under the microscope and compare with Fig. 43, below. (b) Heat a crystal upon platinum foil. The taurin at first melts, then turns brown and finally carbonizes as the tempera- ture is raised. Note the suffocating odor. What is it? (c) Test the solubility of the crystals in water and in alcohol. (d) Grind up a crystal with four times its volume of dry sodium carbonate and fuse on platinum foil. Cool the residue, transfer it to a test-tube and dissolve it in water. Add a little Taurin. dilute sulphuric acid and note the odor of hydrogen sulphide. Hold a piece of filter paper, moistened with a small amount of lead acetate, over the opening of the test-tube and observe the formation of lead sulphide. BILK. 127 10. Preparation of Glycocoll. — Concentrate the alcoholic filtrate from the last experiment (9) until no more alcohol remains. The glyci >e« ill is present here in the form of an hydro- chloride and may be liberated from this combination by the addition of freshly precipitated lead hydroxide or by lead hydroxide solution. Remove the lead by H 2 S. Filter and decolorize the filtrate by animal charcoal. Filter again, con- centrate the filtrate and set it aside for crystallization. Gly- cocoll separates as colorless crystals | Fig. 44, below). Fig. 44. /7 & ' Glycocoll. 11. Synthesis of Hippuric Acid. — To some of the glycocoll prepared in the last experiment or furnished by the instructor, add a little water, about 1 c.c. of benzoyl chloride and render alkaline with potassium hydroxide solution. Stopper the tube and shake it until no more heat is evolved. Now render strongly alkaline with potassium hydroxide and shake the mixture until no odor of benzoyl chloride can be detected. Cool, acidify with HC1, add an equal volume of petroleum ether (ethyl ether may be substituted) and shake thoroughly to remove the benzoic acid. (Evaporate this solution and note the crystals of benzoic acid. Compare them with those shown 128 PHYSIOLOGICAL CHEMISTRY. in Fig. 94, page 264. ) Decant the ethereal solution into a por- celain dish and extract again with ether. The hippuric acid remains in the aqueous solution. Filter it off and wash it with a small amount of cold water while still on the filter. Remove it to a small shallow vessel, dissolve it in a small amount of hot water and set it aside for crystallization. Examine the crystals microscopically and compare them with those in Fig. 92, page 256. The chemistry of the synthesis is represented thus : CH 2 -NH 2 C0C1 OC-NH-CH 2 -COOH. /\ /\ + 11 =11 COOH \/ \/ Glycocoll. Benzoyl chloride. Hippuric acid. CHAPTER IX. PUTREFACTION PRODUCTS. The putrefactive processes in the intestine are the result of the action of bacteria upon the proteid material present. This bacterial action which is the combined effort of many forms of micro-organisms is confined almost exclusively to the large intestine. Some of the products of the putrefaction of proteids are identical with those formed in tryptic digestion although the decomposition of the proteid material is much more ex- tensive when subjected to putrefaction. Some of the more important of the putrefaction products are the following: Indol, skatol, paracresol, phenol, para-ox y phenyl propionic acid, para-oxyphenylacetic acid, volatile fatty acids, hydrogen sul- phide, methane, methyl mereaptan, hydrogen, and carbon diox- ide, beside proteoses, peptones, ammonia and amino acids. Of these the indol, skatol and phenol appear in part in the urine as ethereal sulphuric acids, whereas the oxyacids mentioned pass unchanged into the urine. The potassium indoxyl sul- phate (page 130) content of the urine is a rough indicator of the extent of the putrefaction within the intestine. The portion of the indol which is excreted in the urine is first subjected to a series of changes within the organism and is subsequently eliminated as indican. These changes may be represented thus : /\ CH /\ C(OH) I I II +0 = 1 I II \/\/CH \/\/CB. NH NH Jndol. Indoxyl. /\ ('(OH) /\ C(0-S0 3 H) I I II +HoS0 4 H I II +H 2 \/\/CH \/\/CH NH NH Indoxyl. Indoxyl sulphuric acid. IO 129 13° PHYSIOLOGICAL CHEMISTRY. In the presence of potassium salts the indoxyl sulphuric acid is then transformed into potassium indoxyl sulphate (or indi- can), _C(0-S0 3 K), \/\/CH NH and eliminated as such in the urine. Indican may be decomposed by treatment with concentrated hydrochloric acid (see tests on page 255) into sulphuric acid and indoxyl. The latter body may then be oxidized to form indigo-blue thus : /\ C(OH) /\ CO OC /\ 2 I I || +20=1 || || I +2H 2 \/\/CH \/\/C=C\/\/ NH NH NH ' Indoxyl. Indigo-blue. Skatol is likewise changed within the organism and elimi- nated in the form of a chromogenic substance. Experiments on Putrefaction Products. In many courses in physiological chemistry the instructors are so limited for time that no extended study of the products of putrefaction can very well be attempted. Under such con- ditions the scheme here submitted may be used profitably in the way of a demonstration. Where the number of students is not too great, a single large putrefaction may be started, and, after the initial distillation, both the resulting distillate and residue may be distributed to the members of the class for in- dividual manipulation. Preparation of Putrefaction Mixture. — Place a weighed mixture of coagulated egg albumin and ground lean meat in a flask or bottle and add approximately 2 liters of water for every kilogram of proteid used. Sterilize the vessel and con- tents, inoculate with the colon bacillus and keep at 40 C. for two or three weeks. If cultures of the colon bacillus are not PUT R 1. 1 \ci [ON PRODUCTS. 131 available, add 60 c.c. of a cold saturated solution of sodium carh' mate for every liter of water previously added and inocu- late with sf putrefac- tion. It also serves to diminish the odor arising from the putrefying material. Place the putrefaction mixture at 40° C. for two or three weeks and at the end of that time make a separation of the products of putrefaction according to the following directions: Subject the mixture to distillation until the distillate and residue are approximately equal in volume. 1 Putrefying proteid may be prepared by treating 10 grams of finely ground lean meat with ioo c.c. of water and 2 c.c. of a saturated solution of sodium carbonate and keeping the mixture at 40 C. for twenty-four hours. 2 Concentrated sulphuric acid containing a small amount of isatin may be used as a substitute for mercuric cyanide. When this modification is employed it is necessary to use calcium chloride tubes to exclude moisture from the isatin solution. 132 PHYSIOLOGICAL CHEMISTRY, PART I. MANIPULATION OF THE DISTILLATE. Acidify with hydrochloric acid and extract with ether. Ether Extract No. i. Add an equal volume of water, make alkaline with potassium hy- droxide and shake thoroughly. Residue No. i. Allow the ether to volatilize. Evaporate and detect ammonium chloride crystals (Fig. 45, p. 133). Ether Extract No. 2. Evaporate spontaneously. Indol and skatol remain. Try proper re- actions (see pages 136 and 137). Alkaline Solution No. 1. Acidify with hydrochloric acid, add sodium carbonate and extract with ether. Ether Extract No. 3. Evaporate. Detect phenol and cresol (paracresol). See p. 138. Alkaline Solution No. 2. Acidify with hydrochloric acid, and extract with ether. Ether Extract No. 4. Evaporate. Volatile fatty acids remain. Final Residue. (Discard.) DETAILED DIRECTIONS FOR MAKING THE SEPARATIONS INDICATED IN THE SCHEME. Preliminary Ether Extraction. — This extraction may be conveniently conducted in a separatory funnel. Mix the fluids for extraction in the ratio of two volumes of ether to three volumes of the distillate. Shake very thoroughly for a few moments, then draw off the extracted fluid and add a new por- tion of the distillate. Repeat the process until the entire dis- tillate has been extracted. Add a small amount of fresh IT | KI.I \CTION PRODUCTS. 133 ether at each extraction to replace that dissolved by the water in the preceding extraction. Residue No. 1. — Unite the portions of the distillate extracted as above and allow the ether to volatilize spontaneously. Fig. 45- A M M N I U M CH 1-OR I DE. Evaporate until crystallization begins. Examine the crystals under the microscope. Ammonium chloride predominates. Explain its presence. Ether Extract No. 1. — Add an equal volume of water, ren- der the mixture alkaline with potassium hydroxide and shake thoroughly by means of a separator}- funnel as before. The volatile fatty acids, contained among the putrefaction products, would be dissolved by the alkaline solution (Xo. 1) whereas any indol or skatol would remain in the ethereal solution (No. 2). Alkaline Solution No. 1. — Acidify with hydrochloric acid and add sodium carbonate solution until the fluid is neutral or slightly acid from the presence of carbonic acid. At this point a portion of the solution, after being heated for a few moments, should possess an alkaline reaction on cooling. Extract the whole mixture with ether in the usual way, using care in the 134 PHYSIOLOGICAL CHEMISTRY. manipulation of the stop cock to relieve the pressure due to the evolution of carbon dioxide. The ether (Ether Extract No. 3) removes any phenol or cresol which may be present while the volatile fatty acids will remain in the alkaline solu- tion (No. 2) as alkali salts. Ether Extract No. 2. — Drive off the major portion of the ether at a low temperature on a water-bath and allow the resi- due to evaporate spontaneously. Indol and skatol should be present here. Prove the presence of these bodies. For tests for indol and skatol see p. — . Alkaline Solution No. 2. — Make strongly acid with hydro- chloric acid and extract with a small amount of ether, using a separatory funnel. As carbon dioxide is liberated here, care must be used in the manipulation of the stop cock of the funnel in relieving" the pressure within the vessel. The volatile fatty acids are dissolved by the ether (Ether Extract No. 4). Ether Extract No. 5. — Evaporate this ethereal solution on a water-bath. The oily residue contains phenol and cresol. The cresol is present for the most part as paracresol. Add some water to the oily residue and heat it in a flask. Cool and prove the presence of phenol and cresol. For tests for these bodies see page 138. Ether Extract No. 4. — Evaporate on a water-bath. The volatile fatty acids remain in the residge. PUTREFACTION PRODUCTS. 135 PART II. MANIPULATION OF THE RESIDUE. Kaporate, filter and extract with ether. Ether Extract. Evaporate, -extract the residue with warm water and filter. Aqueous Solution. Evaporate until crystals begin to form. Stand in a cold place until crystallization is complete. Filter. Crystalline Deposit. Consists of a mixture of leiicin and tyrosin crystals. (Figs. 23, 24 and 104, pages 68, 69 and 326.) Filtrate No. 1. Contains proteose, pep- tone, aromatic acids and tryptophan. Filtrate No. 2. Contains oxy acids and skatol-carbonic acid. Residue. Contains non-volatile fatty acids. DETAILED DIRECTIONS FOR MAKING THE SEPARATIONS INDICATED IN THE SCHEME. Preliminary Ether Extraction. — This extraction may be conducted in a separatory funnel. In order to make a satis- factory extraction the mixture should be shaken very thor- oughly. Separate the ethereal solution from the aqueous por- tion and treat them according to the directions given below. Ether Extract. — Evaporate this solution on a safety water- bath until the ether has been entirely removed. Extract the residue with warm water and filter. Aqueous Solution. — Evaporate this solution until crystalli- zation begins. Stand the solution in a cold place until no more crystals form. This crystalline mass consists of impure leucin and tyrosin. Filter off the crystals. 136 PHYSIOLOGICAL CHEMISTRY. Crystalline Deposit. — Examine the crystals under the micro- scope and compare them with those reproduced in Figs. 23, 24 and 104, pages 68, 69 and 326. Do the forms of the crystals of leucin and tyrosin resemble those previously exam- ined? Make a separation of the leucin and tyrosin and apply typical tests according to directions given on pages 81 and 82. Filtrate No. 1. — Make a test for tryptophan with bromine water (see page no), and, also with the Hopkins-Cole rea- gent (see page 45). Use the remainder of the filtrate for the separation of proteoses and peptones. Make the separa- tion according to the directions given on page 5.9. Filtrate No. 2. — This solution contains para-oxyphenylacetic acid, para-oxyphenylpropionic acid and skatol-carbonic acid. Prove the presence of these bodies by appropriate tests. Tests for oxyacids and skatol-carbonic acid are given on page 138. TESTS FOR VARIOUS PUTREFACTION PRODUCTS. Tests for Indol. 1. Herter's Naphthaquinone Reaction. — (a) To a dilute aqueous solution of indol (1 150,000) add one drop of a 2 per cent solution of naphthaquinone sodium-monosulphonate. No reaction occurs. Add a drop of a 10 per cent solution of potassium hydroxide and note the gradual development of a blue or blue-green color which fades to green if an excess of the alkali is added. Render the green or blue-green solution acid and note the appearance of a pink color. Heat facilitates the development of the color reaction. One part of indol in one million parts of water may be de- tected by means of this test if carefully performed. (b) If the alkali be added to the indol solution before the introduction of the naphthaquinone the course of the reaction is different, particularly if the indol solution is somewhat more concentrated than that mentioned above and if heat is used. Under these conditions the blue indol compound ultimately forms as fine acicular crystals which rise to the surface. PUTREI \( i [ON PRODUCT S. I 37 If we do not wait for the production of the crystalline liody but as soon as the blue color forms, shake the aqueous solution with chloroform, the blue color disappears from the solution and the chloroform assumes a pinkish-red hue. This is a distinguishing feature of the indol reaction and facilitates the differentiation of indol from other bodies which yield a similar blue color. 2. Cholera-red Reaction. — To a little of the residue in a test-tube add one-tenth its volume of a 0.02 per cent solution of potassium nitrite and mix thoroughly. Carefully run con- centrated sulphuric acid down the side of the tube so that it forms a layer at the bottom. Note the purple color. Neutra- lize with potassium hydroxide and observe the production of a bluish-green color. 3. Legal's Reaction. — To a small amount of the residue in a test-tube add a few drops of a freshly prepared solution of sodium nitroprusside, Na 2 Fe(CN) 5 NO + 2ILO. Render alkaline with potassium hydroxide and note the production of a violet color. If the solution is now acidified with glacial acetic acid the voilet is transformed into a blue. 4. Pine Wood Test. — Moisten a pine splinter with con- centrated hydrochloric acid and insert it into the residue. The wood assumes a cherry-red color. 5. Nitroso-indol Nitrate Test. — Acidify some of the resi- due with nitric acid, add a few drops of a potassium nitrite solution and note the production of a red precipitate of nitroso- indol nitrate. If the residue contains but little indol simply a red coloration will result. Compare this result with the result of the similar test on skatol. Tests for Skatol. 1. Herter's Naphthaquinone Reaction. — The same pro- cedure may be used here as in the similar test under indol, page 136. The distinctive feature of dilute solutions of skatol when treated with the naphthaquinone compound is that they yield a violet or purple instead of a blue. Concentrated solu- 138 PHYSIOLOGICAL CHEMISTRY. tions of skatol yield the blue color as noted with indol. This reaction possesses relatively the same delicacy as the indol reaction. 2. Color Reaction with HC1. — Acidify some of the resi- due with concentrated hydrochloric acid. Note the production of a violet color. 3. Acidify some of the residue with nitric acid and add a few drops of a potassium nitrite solution. Note the white turbidity. Compare this result with the result of the similar test on indol. Tests for Phenol and Cresol. 1. Color Test. — Test a little of the solution with Millon's reagent. A red color results. Compare this test with the simi- lar one under Tyrosin (see page 82). 2. Ferric Chloride Test. — Add a few drops of neutral ferric chloride solution to a little of the residual fluid. A dirty bluish-gray color is formed. 3. Formation of Bromine Compounds. — Add some bro- mine water to a little of the fluid under examination. Note the crvstalline precipitate of tribromphenol and tribromcresol. Tests for Oxyacids. 1. Color Test. — Test a little of the solution with Millon's reagent. A red color results. 2. Bromine Water Test. — Add a few drops of bromine water to some of the filtrate. A turbidity or precipitate is observed. Test for Skatol-carbonic Acid. Ferric Chloride Test. — Acidify some of the filtrate with hydrochloric acid, add a few drops of ferric chloride solution and heat. Compare the end-reaction with that given by phenol. CHAPTER X. FECES. The feces is the residual mass of material remaining in the intestine after the full and complete exercise of the digestive and absorptive functions and is ultimately expelled from the body through the rectum. The amount of this fecal discharge varies with the individual and the diet. Upon an ordinary mixed diet the daily excretion by an adult male will aggregate 1 10-170 grams with a solid content ranging between 25 and 45 grams ; the fecal discharge of such an individual upon a Fig. 46. Microscopical Coxstitlknts of Feces. ( v. Jaksch.) a, Muscle fibers ; b, connective tissue ; c, epithelium ; d, leucocytes ; e, spiral cells ; /, g, h, i, various vegetable cells ; k, " triple phosphate " crystals ; /. woody vegetable cells ; the whole interspersed with innumerable micro- organisms of various kinds. vegetable diet will be much greater and may even be a* great as 350 grams and possess a solid content of 75 grams. The variation in the normal daily output being so great ren- ders this factor of very little value for diagnostic purposes, except where the composition of the diet is accurately known. •39 140 PHYSIOLOGICAL CHEMISTRY. Lesions of the digestive tract, a defective absorptive function or increased peristalsis as well as an admixture of mucus, pus, blood and pathological products of the intestinal wall may- cause the total amount of excrement to be markedly increased. The fecal pigment of the normal adult is hydrobilirubin (urobilin or stercobilin). Neither bilirubin nor biliverdin occurs normally in the fecal discharge of adults, although the former may be detected in the excrement of nursing infants. The most important factor, however, in determining the color of the fecal discharge is the diet. A mixed diet for instance produces stools which vary in color from light to dark brown, an exclusive meat diet gives rise to a brown- ish-black stool, whereas the stool resulting from a milk- diet is invariably light col- ored. Certain pigmented foods such as the chloro- phyllic vegetables, and var- ious varieties of berries, each Fig. 47- H^matoidin Crystals from Acholic Stools. (v. Jaksch.) Color of crystals same as the color of those in Fig. 41, p. 119. afford stools having a char- acteristic color. Certain drugs ?ct in a similar way to color the fecal discharge. This is well illustrated by the occurrence of green stools following the use of calomel and of black stools after bismuth ingestion. The green color of the calomel stool is generally believed to be due to biliverdin. v. Jaksch however claims to have proven this view to be in- correct since he was able to detect hydrobilirubin (or urobilin) but no biliverdin in stools after the administration of calomel. The bismuth stool derives its color from the black sulphide which is formed from the subnitrate of bismuth. In cases of biliary obstruction the grayish-white acholic stool is formed. Under normal conditions the odor of feces is due to skatol and indol, two bodies formed in the course of putrefac- I ECES. 141 tive processes occurring within the intestine (sec page 129). Such bodies as methane, methyl mercaptan and hydrogen sul- phide may also add to the disagreeable character of the odor. The intensity of the odor depends to a large degree upon the character of the diet, being very marked in stools from a meat diet, much less marked in stools from a vegetable diet and fre- quently hardly detectable in stools from a milk diet. Thus the stool of the infant is ordinarily nearly odorless and any decided odor may generally be readily traced to some patho- logical source. A neutral reaction ordinarily predominates in normal stools although slightly alkaline or even acid stools are met with. The acid reaction is encountered much less frequently than the alkaline and then commonly only following a vegetable diet. The form and consistency of the stool is dependent, in large measure, upon the nature of the diet and particularly upon the quantity of water ingested. Under nor- Fig. 48. ma i con( ]itions the consistency may vary from a thin, pasty discharge to a firmly formed stool. Stools which are exceedingly thin and watery ordinarily have a pathological significance. In general the feces of the car- nivorous animals is of a firmer consistency than that of the herbivora. Charcot-Leyden Crystals. Among- the macroscopical constituents of the feces may be mentioned the following: Intestinal parasites, undigested food particles, gall stones, pathological products of the intestinal wall, enteroliths, intes- tinal sand and objects which have been accidentally swallowed. The fecal constituents which at various times and under different conditions may be detected by the use of the micro- scope are as follows : Constituents derived from the food, such as muscle fibers, connective tissue shreds, starch granules and fat; formed elements derived from the intestinal tract, such as epithelium, erythrocytes and leucocytes; mucus; pus corpuscles; parasites and bacteria. In addition to the consti- I4 2 PHYSIOLOGICAL CHEMISTRY. tuents named, the following crystalline deposits may be de- tected: Cholestcrin, fatty acid, fat, bismuth sulphide, hcema- toidin, " triple phosphate;'' Charcot-Leydcn crystals and the oxalate, carbonate, phosphate, sulphate and lactate of calcium. The detection of minute quantities of blood in the feces ("occult blood") has recently become a recognized aid to a correct diagnosis of certain disorders. In these instances the hemorrhage is ordinarily so slight that the identification by means of macroscopical characteristics as well as the micro- scopical identification through the detection of erythrocytes are both unsatisfactory in their results. Of the tests given for the detection of " occult blood " the aloin-turpentine test (page 144) is probably the most satisfactory. Since "occult blood " occurs with considerable regularity and frequency in gastrointestinal cancer and in gastric and duodenal ulcer, its detection in the feces is of especial value as an aid to a correct diagnosis of these disorders. For diagnostic purposes the macroscopical and microscopical examinations of the feces ordinarily yield much more satis- factory data than are secured from its chemical examination. Experiments on Feces. 1. Macroscopical Examination. — If the stool is watery pour it into a shallow dish and examincdirectly. If it is firm or pasty it should be treated with water and carefully stirred before the examination for macroscopical constituents is attempted. The macroscopical constituents may be collected very satis- factorily by means of a Boas sieve (Fig. 49, page 143). This sieve is constructed of two easily detachable hemispheres which are held together by means of a bayonet catch. In using the apparatus the feces is spread out upon a very fine sieve con- tained in the lower hemisphere and a stream of water is allowed to play upon it through the medium of an opening in the upper hemisphere. The apparatus is provided with an orifice in the upper hemisphere through which the feces may be I KCKS. ' [3 Fig. -i" stirred by means of a glass rod during the washing proo After [5—30 minutes washing nothing but the coarse Fecal constituents remain upon the sieve. 2. Microscopical Examination. — Watery stools should he placed in a shallow dish, thoroughly mixed and a small amount removed to a slide for examination. Stools of a firm or pasty consistency should he rubbed up in a mortar with water and a small portion of the resulting- mixture trans- ferred to a slide for examination. In nor- mal feces look for food particles, bacteria and crystalline bodies. In pathological stools, in addition to these substances, look for animal parasites and pathological prod- ucts of the intestinal wall. 3. Reaction. — Thoroughly mix the feces and apply moist red and blue litmus papers to the surface. If the stool is hard it should be mixed with water before the reaction is taken. Examine the stool as soon after defecation as is convenient, since the reac- tion may change very rapidly. The reaction of the normal stools of adult man is ordinarily neutral or faintly alkaline to litmus, hut seldom acid. Infants' stools are generally acid in reaction. 4. Starch. — If any imperfectly cooked starch-containing food has been ingested it will be possible to detect starch granules by a microscopical examination of the feces. If the granules are not detected by a microscopical examination, the feces should be placed in an evaporating dish or casserole and boiled with water for a few minutes. Filter and test the filtrate by the iodine test in the usual way (see page 24). 5. Cholesterin and Fat. — Extract the dry feces with ether in a Soxhlet apparatus (see Chapter XXII). If this apparatus is not available transfer the dry feces to a tlask. add ether and shake frequently for a few hours. Filter and remove the Boas' Sieve. 144 PHYSIOLOGICAL CHEMISTRY. ether by evaporation. The residue contains cholesterin and the mixed fats of the feces. For every gram of fat add about iy 2 grams of solid potassium hydroxide and 25 c.c. of 95 per cent alcohol and boil in a flask on a water-bath for one- half hour maintaining the volume of alcohol constant. This alcoholic-potash has saponified the mixed fats and we now have a mixture of soaps and cholesterin. Add sodium chloride, in substance, to the mixture and extract with ether to dissolve out the cholesterin. Remove the ether by evaporation and ex- amine the residue microscopically for cholesterin crystals. Try any of the other tests for cholesterin as given on page 124. 6. Blood. — Undecomposed blood may be detected macro- scopically. If uncertain, look for erythrocytes under the microscope, and spectroscopically for the spectrum of oxyhe- moglobin (see Absorption Spectra, Plate I). In case the blood has been altered or is present in minute amount ("occult blood"), and cannot be detected by the means mentioned above, the following tests may be tried : (a) Aloin-Turpentine Test. — Mix the stool very thoroughly and take about 5 grams of the mixture for the test. Reduce this sample to a semi-fluid mass by means of distilled water and extract very thoroughly with an equal volume of ether to remove any fat which may be present. Now treat the ex- tracted feces with one-third its volume, of glacial acetic acid and 10 c.c. of ether and extract very thoroughly as before. The acid-ether extract will rise to the top and may be removed. Introduce 2-3 c.c. of this acid-ether solution into a test-tube, add an equal volume of a dilute solution of aloin in 70 per cent alcohol and 2-3 c.c. of ozonized turpentine and shake the tube gently. If blood is present the entire volume of fluid ordinarily becomes pink and finally cherry red. In some in- stances the color will be limited to the aloin solution which sinks to the bottom. This color reaction should occur within fifteen minutes in order to indicate a positive test for blood, since the aloin will turn red of itself if allowed to stand for a longer period. The color is ordinarily light yellow in a nega- FECES. I }5 tive test. Hydrogen peroxide is not a satisfactory substitute for turpentine in this test. { b) Weber's Guaiac , Test. — Mix a little feces with 30 per cent acetic acid to form a fluid mass. Transfer to a test-tube and extract with ether. If blood is present the ether will assume a brownish-red color. Filter off the ether extract and, to a portion of the filtrate, add an alcoholic solution of guaiac (strength about 1 :6b), 1 drop by drop, until the fluid becomes turbid. Xow add hydrogen peroxide or old turpentine. In the presence of blood a blue color is produced (see page 158). (c) Acid-Hccmatin. — Examine some of the ethereal extract from the last experiment (b) spectroscopically. Note the typical spectrum of acid-haematin (see Absorption Spectra, Plate II). 7. Hydrobilirubin. — Rub up a small amount of feces in a mortar with a concentrated aqueous solution of mercuric chloride. Transfer to a shallow flat-bottomed- dish and allow to stand several hours. The presence of hydrobilirubin will be indicated by a deep red color imparted to the feces. This red color is due to the formation of hydrobilirubin-mercury. If unaltered bilirubin is present the feces will be green in color. Another method for the detection of hydrobilirubin is the following: Treat the dry feces with absolute alcohol acidified with sulphuric acid and shake thoroughly. The acidified alco- hol extracts the pigment and assumes a reddish color. Ex- amine a little of this fluid spectroscopically and note the typical spectrum of hydrobilirubin (Absorption Spectra Plate II). 8. Bilirubin, (a) Gmeliris Test. — Place a few drops of concentrated nitric acid in an evaporating dish or on a porce- lain test-tablet and allow a few drops of feces and water to mix with it. The usual play of colors of Gmeliirs test is pro- duced, i. e.j green, blue, violet, red and yellow. If so desired, this test may be executed on a slide and observed under the microscope. 1 Buckmaster advises the use of an alcoholic solution of guaiaconic acid instead of an alcoholic solution of guaiac resin. I46 PHYSIOLOGICAL CHEMISTRY. (b) Hupperfs Test. — Treat the feces with water to form a semi-fluid mass, add an equal amount of milk of lime, shake thoroughly and filter. Wash the precipitate with water, then transfer both the paper and the precipitate to a small beaker or flask, add a small amount of 95 per cent alcohol acidified slightly with sulphuric acid and heat to boiling on a water- bath. The presence of bilirubin is indicated by the alcohol assuming a green color. 9. Bile Acids. — Extract a small amount of feces with alco- hol and filter. Evaporate the filtrate on a water-bath to drive off the alcohol and dissolve the residue in water made slightly alkaline with potassium hydroxide. Upon this aqueous solu- tion .try any of the tests for bile acids given on page 122. 10. Caseinogen. — Extract the fresh feces first with a dilute solution of sodium chloride, and later with water acidified with dilute acetic acid, to remove soluble proteids. Now extract the feces with 0.5 per cent sodium carbonate and filter. Add dilute acetic acid to the filtrate to precipitate the caseinogen, being careful not to add an excess of the reagent as the casein- ogen would dissolve. Filter off the caseinogen and test it according to directions given on page 192. Caseinogen is found principally in the feces of children who have been fed a milk diet. Mucin would also be extracted by the dilute alkali, if present in the feces. What test cotild you make on the newly precipitated body to differentiate between mucin and caseinogen ? 11. Nucleoproteid. — Mix the stool thoroughly with water, transfer to a flask, and add an equal amount of saturated lime water. Shake frequently for a few hours, filter, and precipi- tate the nucleoproteid with acetic acid. Filter off this precipi- tate and test it as follows : (a) Phosphorus. — Test for phosphorus by fusion (see page 223). (b) Solubility. — Try the solubility in the ordinary solvents. (c) Proteid Color Test. — Try any of the proteid color tests. What proof have you that the above body was not mucin? FECES. 147 What other test can you use to differentiate between nucleo- proteid and mucin?' 12. Albumin and Globulin. — Extract the fresh feces with a dilute solution of sodium chloride. (The preliminary extract from the preparation of caseinogen, page 146, may be utilized here). Filter, and saturate a portion of the filtrate with sodium chloride in substance. A precipitate signifies globulin. Filter off the precipitate and acidify the filtrate slightly with dilute acetic acid. A precipitate at this point signifies albumin. Make a proteid color test on each of these bodies. 13. Proteose and Peptone. — Heat to boiling the portion of the sodium chloride extract not used in the last experiment. Filter off the coagulum, if any forms. Acidify the filtrate slightly with acetic acid and saturate with sodium chloride in substance. A precipitate here indicates proteose. Filter it off and test it according to directions given on page 59. Test the filtrate for peptone by the biuret test. 14. Inorganic Constituents. — Prepare a dilute aqueous solution of dry feces and decolorize it by means of purified animal charcoal. Make the following tests upon the clear solution : (a) Chlorides. — Acidify with nitric acid and add silver nitrate. (b) Phosphates. — Acidify with nitric acid, add molybdic solution and warm gently. (c) Sulphates. — Acidify with hydrochloric acid, add barium chloride and warm. CHAPTER XI. BLOOD. Blood is composed of three types of form-elements (ery- throcytes or red blood corpuscles, leucocytes or white blood corpuscles and blood plates or plaques) held in suspension in a fluid called blood plasma. These form-elements compose about 60 per cent of the blood, by weight. Ordinarily blood is a dark red, opaque fluid due to the presence of the red blood corpuscles, but through the action of certain substances such as water, ether or chloroform it may be rendered transparent. Blood so altered is said to be laked. The laking process is simply a liberation of the haemoglobin from the stroma of the red blood corpuscle. Normal blood is alkaline in reaction 1 to litmus, the alkalinity being due principally to sodium carbonate. The specific gravity of the blood of adults ordinarily varies between 1.045 an( ^ l -°7S- It varies somewhat with the sex, the blood of males having a rather higher specific gravity than that of females. Under pathological conditions also the density of the blood may be very greatly altered. The freez- ing-point (A) of normal blood is about — 0.56 C. Varia- tions between — 0.51 ° and — 0.62 ° C. may be due entirely, to dietary conditions, but if any marked variation is noted it can, in most cases, be traced to a disordered kidney function. The total amount of blood in the body has been variously estimated at from one-twelfth to one-fourteenth of the body weight. Perhaps 1/13.5 is the most satisfactory figure. Among the most important constituents of blood plasma are the four proteid bodies, fibrinogen, nucleoproteid, scrum globulin (euglobulin and pseudo-globulin) and serum albumin. Plasma contains about 8.2 per cent of solids of which the 1 Recently it has been shown by physico-chemical methods that the blood is in reality neutral in reaction. 148 BLOOD. I f9 proteid constituents named above constitute approximately 84 per cent and the inorganic constituents (mainly chlorides, phosphates and carbonates) approximately 10 per cent. Among the inorganic constituents sodium chloride predomi- nates. To prevent coagulation, blood plasma is ordinarily studied in the form of an oxalated or suited plasma. The former may he obtained by allowing the blood to flow from an opened artery into an equal volume of 0.2 per cent ammonium oxalate solution, whereas in the preparation of a sailed plasma 10 per cent sodium chloride solution may be used as the dilut- ing tin id. Fibrinogen is perhaps the most important of the proteid constituents of the plasma. It is also found in lymph and chyle as well as in certain exudates and transudates. Fibrinogen possesses the general properties of the globulins, but differs from serum globulin in being precipitated upon half-saturation with sodium chloride. In the process of coagulation of the blood the fibrinogen is transformed into fibrin. This fibrin is one of the principal constituents of the ordinary blood clot. The nucleo-proteid of blood possesses many of the charac- teristics of serum globulin. In common with this body it is easily soluble in sodium chloride, and is completely precipi- tated from its solutions upon saturation with magnesium sul- phate. It is much less soluble in dilute acetic acid than serum globulin and its solutions coagulate at 65°-69° C. The body formerly called serum globulin is probably not an individual substance. Recent investigations seem to indicate that it may be resolved into two individual bodies called euglobulin and pseudo globulin. The euglobulin is practically insoluble in water and may be precipitated in the presence of 28-36 per cent of saturated ammonium sulphate solution. The pseudoglobulin, on the contrary, is soluble in water and is only precipitated by ammonium sulphate in the presence of from 36 to 44 per cent of saturated ammonium sulphate solution. In common with serum globulin the body known as serum albumin seems also to consist of more than a single individual I50 PHYSIOLOGICAL CHEMISTRY. substance. The so-called serum albumin may be separated into at least two distinct bodies, one capable of crystallization, the other an amorphous body. The solution of either of these bodies in water gives the ordinary albumin reactions. The coagulation temperature of the serum albumin mixture as it occurs in serum or plasma varies from 70 ° to 85 ° C. according to the reaction of the solution and its content of inorganic material. Serum albumin differs from Qgg albumin in being more laevorotatory, in being rendered less insoluble by alcohol, and in the fact that when precipitated by hydrochloric acid it is more easily soluble in an excess of the reagent. When blood coagulates and the usual clot forms, a light yel- lcw fluid exudes. This is blood serum. It differs from blood plasma in containing a large amount of fibrin ferment, a body of great importance in the coagulation of the blood, and also in possessing a lower proteid content. The proteid material present in plasma and not found in serum is the fibrinogen which is transformed into fibrin in the process of coagulation and removed. The specific gravity of the serum of human blood varies between 1.026 and 1.032. Beside the proteid constituents already mentioned, other bodies which are found in both the plasma and serum are the following: Sugar (dextrose), fat, enzymes, lecithin, choles- terin and its esters, gases, coloring-matter (lutein or lipo- chrome) and mineral substances. In addition to these bodies the following substances have been detected in normal human blood: Creatin, carbamic acid, hippuric acid, paralactic acid, area and uric acid. Some of the pathological constituents of blood are, proteoses, leucin, tyrosin and other amino acids, biliary constituents and purin bodies. There has recently been considerable controversy regarding the form of the erythrocytes or red blood corpuscles of human blood. It is claimed by some investigators that the cells are bell-shaped or cup-shaped. As the erythrocytes occur nor- mally in the circidation, however, they are probably thin, non- nucleated, biconcave discs. When examined singly, under PLATE IV. Nokmal Erythrocytes and Leucocytes. BLOOD. 151 the microscope, they possess a pale greenish-yellow color (see Plate IV, opposite), whereas when grouped in large masses a reddish tint is noted. The blood of most mammals contains erythrocytes similar in form to those of human blood. In the blood of a few mammals, however, such as the llama and camel as well as in the blood of birds, fishes, amphibians and reptiles the ery- throcytes are ordinarily more or less elliptical, biconvex and possess a nucleus. The erythrocytes vary in size with the different animals. The average diameter of the erythrocytes of blood from various species is given in the following table :* Elephant 3 fa ^ of an inch. Guinea-pig jj'ij of an inch. Man JT3JJ of an inch. Monkey 7 jjt of an inch. Dog ttoVt of an inch. Rat xfan OI an inch. Rabbit jrfo of an inch. Mouse g yVr of an inch. Lion 4"ttt of an inch. Ox T$tv °f an inch. Horse T j TT of an inch. Pig T yjj of an inch. Cat T jVj of an inch. Sheep ■• 4 yV? of an inch. Goat ^xV? of an inch. Musk-deci ti 1 !;? of an inch. The erythrocytes from whatever source obtained, consist essentially of two parts, the stroma or protoplasmic tissue and its enclosed pigment, Jiccmoglobin. For human blood the number of erythrocytes present in the fluid as obtained from well-developed males in good physical condition is about 5,500,000 per cubic millimeter. 2 The normal content of the blood of adult females is from 4,000,000 to 4,500,000 per cubic millimeter. The number of erythrocytes varies greatly 1 Wormley's Micro-Chemistry of Poisons, second edition, p. 733. 2 This statement is based upon observations made upon the blood of athletes in training. It is generally stated in text-books that the blood of males contains about 5,000,000 per cubic millimeter. 152 PHYSIOLOGICAL CHEMISTRY. Fig. 50. ^8?«* * Oxyhemoglobin Crystals from Blood of the Guinea Pig. Reproduced from a micro-photograph furnished by Prof. E. T. Reichert of the University of Pennsylvania. Fig. 51. Oxyhemoglobin Crystals from Blood of the Rat. Reproduced from a micro-photograph furnished by Prof. E. T. Reichert of the University of Pennsylvania. JU.OOD. '53 Oxyhemoglobin Crystals from Blood of the Horse. Reproduced from a micro-photograph furnished by Prof. E. T. Reichert of the University of Pennsylvania. Fig. :3. ftd ^ & Oxyhemoglobin Crystals from Blood of the Squirrel. Reproduced from a micro-photograph furnished by Prof. E. T. Reichert of the University of Pennsylvania. x 54 PHYSIOLOGICAL CHEMISTRY, Fig. 54. Oxyhemoglobin Crystals from Blood of the Dog. Reproduced from a micro-photograph furnished by Prof. E. T. Reichert of the University of Pennsylvania. Fig. 55- Oxyhemoglobin Crystals from Blood of the Cat. Reproduced from a micro-photograph furnished by Prof. E. T. Reichert of the University of Pennsylvania. BLOOD. Fig. 56. ■D3 Oxyhemoglobin Crystals from Blood of thk Nectlrus. Reproduced from a micro-photograph furnished by Prof. E. T. Reichert of the University of Pennsylvania. 1 under different conditions. For instance the number may be increased after the transfusion of blood of the same species of animal; by residing in a high altitude; or as a result of strenuous physical exercise continued over a short period of time. An increase is also noted in starvation; after partaking of food; after cold or hot baths; after massage, as well as after the administration of certain drugs and accompanying certain diseases such as cholera, diarrhoea, dysentery and yel- low atrophy of the liver. A decrease in the number occurs in the different forms of anaemia. The number has been known to increase to 7,040,000 per cubic millimeter as a result of physical exercise, while 11,000,000 per cubic millimeter have been noted in cases of polycythemia and increases nearly as great in cyanosis. The number has been known to decrease to 500,000 per cubic millimeter or lower in pernicious anaemia. Oxyhemoglobin the coloring matter of the blood is a com- 'The micro-photographs of oxyhemoglobin (see pages 152-155) and haemin (see page 164) are reproduced through the courtesy of Profes- sors E. T. Reichert and Amos P. Brown, of the University of Pennsyl- vania, who are investigating the crystalline forms of biochemic substances. is6 PHYSIOLOGICAL CHEMISTRY pound proteid. Through treatment with hydrochloric acid it may be split into a proteid body called globin, and hcemo- chromogcn an iron-containing pigment. The latter body is rapidly transformed into hcematin in the presence of oxygen and this in turn gives place to hsematin-hydrochloride or Juoniii ( Figs. 58 and 59, page 164). The pigment of arterial blood is for the most part loosely combined with oxygen and is termed oxyhemoglobin, whereas the pigment of venous blood is principally haemoglobin (so-called reduced haemoglo- bin). Oxyhaemoglobin is the oxygen-carrier of the body and belongs to the class of bodies known as respiratory pigments. The reduction of oxyhaemoglobin to form haemoglobin (so- called reduced haemoglobin) occurs in the capillaries. Oxy- haemoglobin may be crystallized and a specific form of crystal obtained from the blood of each individual species (see Figs. 50 to 56, pages 152 to 155). This fact seems to indicate that there are many varieties of oxyhaemoglobin. The pig- ment is held within the stroma of the erythrocyte. The fol lowing bodies may be derived from haemoglobin, and each possesses a specific spectrum which serves as an aid in its detection and identification : Oxyhaemoglobin, methaemoglo- bin, carbon-monoxide haemoglobin, nitric-oxide haemoglobin, haemochromogen, haematin, acid-haematin, alkali-haematin and haematoporphyrin (see Absorption Spectra, Plates I and II). The white corpuscles (or leucocytes) of human blood differ from the red corpuscles (or erythrocytes) in being somewhat larger in size, in containing at least a single nucleus and in possessing amoeboid movement (see Plate IV, opposite page 151). They are typical animal cells and therefore contain the following bodies which are customarily present in such cells : Proteids, fats, carbohydrates, lacithin, cholcsterin, inorganic salts and water. The normal number of leucocytes in human blood varies between 5,000 and 10,000 per cubic millimeter. The ratio between the leucocytes and erythrocytes is about 1 : 350-500. A leucocytosis is said to exist when the number of leucocytes is increased for any reason. Leucocytoses may BLOOD. ! 57 be divided into two general classes, the physiological and the pathological. Under the physiological form would be classed those leucocytoses accompanying pregnancy, parturition and digestion, as well as those due to mechanical and thermal influences. The leucocytoses spoken of as pathological are the inflammatory, infectious, post-haemorrhagic, toxic and experimental forms as well as the type of leucocytosis which accompanies malignant disease. The blood plates (platelets or plaques) arc round or oval, colorless discs which possess a diameter about one-third as great as that of the erythrocytes. Upon treatment with certain reagents, c. g., artificial gastric juice, they may be separated into a homogeneous, non-refractive portion and a granular, refractive portion. The blood plates are probably associated in some way with the coagulation of the blood. This relation- ship is not well understood at present. The processes involved in the coagulation of the blood are not fully understood. Several theories have been advanced and each has its adherents. The theory which appears to be fully as firmly founded upon experimental evidence as any is the following - : Blood contains a zymogen called prothrombin which combines with the calcium salts present to form an enzyme known as thrombin or fibrin-ferment. When freshly drawn blood comes in contact with the air the fibrin- ferment at once acts upon the fibrinogen present and gives rise to the formation of fibrin. This fibrin forms in shreds throughout the blood mass and, holding the form elements of the blood within its meshes, serves to produce the typical blood clot. The fibrin shreds gradually contract, the whole clot assumes a jelly-like appearance and the yellowish serum exudes. If, immediately upon the withdrawal of blood from the body, the fluid be rapidly stirred or thoroughly " whipped " with a bundle of coarse strings, twigs or a specially constructed beater, the fibrin shreds will not form in a network through- out the blood mass but instead will cling to the device used in beating. In this way the fibrin may be removed and the 158 PHYSIOLOGICAL CHEMISTRY. remaining fluid is termed defibrinatcd blood. The above theory of the coagulation of the blood may be stated briefly as follows : I. Prothrombin -f- Calcium Salts = Thrombin (or Fibrin- ferment). II. Thrombin (or Fibrin-ferment) + Fibrinogen = Fibrin. Among the medico-legal tests for blood are the following: (1) Microscopical identification of the erythrocytes, (2) spec- troscopic identification of blood solutions, (3) the guaiac test, (4) preparation of hsemin crystals. Of these four tests the last named is generally considered to be the most satisfactory. It gives equally reliable results with fresh blood and with blood from clots or stains of long standing, provided the latter have not been exposed to a high temperature, or to the rays of the sun for a long period. The technique of the test is simple (see page 163) and the formation of the dark brown or chocolate colored crystals of haemin (Figs. 58 and 59, page 164) is indisputable proof of the presence of blood in the fluid, clot or stain examined. The weak point of the test, medico-legally, lies in the fact that it does not differentiate between human blood and that of certain other species of animal. The guaiac test (see page 163), although generally con- sidered less accurate than the hsemin test, is really a more deli- cate test than the hsemin test if properly performed. One of the most common mistakes in the manipulation of this test is the use of a guaiac solution which is too concentrated and which, when brought into contact with the aqueous blood solu- tion, causes the separation of a voluminous precipitate of a resinous material which may obscure the blue coloration; this is particularly true of the test when used for the examination of blood stains. A solution of guaiac made by dissolving 1 gram of the resin in 60 c.c. of 95 per cent alcohol is very satisfactory for general use. The test is frequently objected to upon the ground that various other substances, e. g., milk, pus, saliva, etc., respond to the test and that it cannot therefore BLOOD. 159 Ik.- considered a specific tc-t for bl 1 and is of value only in a negative sense We have demonstrated to our own satis- faction, however, that milk many times gives the blue color upon the addition of an alcoholic solution of guaiac resin without the addition of hydrogen peroxide or old turpentine. Buckmaster has very recently advocated the use of an alco- holic solution of guaiaconic acid instead of an alcoholic solu- tion of guaiac resin. He claims that he was able to produce the blue color upon the addition of the guaiaconic acid to milk only when the sample of milk tested was brought from the country in sterile bottles, and further, that no sample of London milk which he examined responded to the test. In the application of the guaiac test to the detection of blood, he states that he was able to detect laked blood when present in the ratio 1 : 5,000,000 and unlaked blood when present in the ratio 1 : 1,000,000. This author considers the guaiac test to be far more trustworthy than is generally believed. Up to within very recent times it has been impossible to make an absolute differentiation of human blood. Recently however the so-called " biological " blood test has made such a differentiation possible. This test, known as the Bordet reaction, is founded upon the fact that the blood serum of an animal into which has been injected the blood of another ani- mal of different species develops the property of agglutinating and dissolving erythrocytes similar to those injected, but ex- erts this influence upon blood from no other species. The antiserum used in this test is prepared by injecting rabbits with 5-10 c.c. of human defibrinated blood, at intervals of about four days until a total of between 50 and 80 c.c. has been injected. After a lapse of one or two weeks the animal is bled, the serum collected, placed in sterile tubes and preserved for use as needed. In examining any specific solution for human blood it is simply necessary to combine the antiserum and the solution under examination in the proportion of 1 : 100 and place the mixture at 37 C. If human blood is present in the solution a turbidity will be noted and this will change l6o PHYSIOLOGICAL CHEMISTRY. within three hours to a distinctly flocculent precipitate. This antiserum will react thus with no other known substance. Experiments on Blood. I. Defibrinated Ox-blood. 1. Reaction. — Moisten red and blue litmus papers with 10 per cent sodium chloride solution and test the reaction of the defibrinated blood. 2. Microscopical Examination. — Examine a drop of defi- brinated blood under the microscope. Compare the objects you observe with Plate IV, opposite page 151. Repeat the test with a drop of your own blood. 3. Specific Gravity. — Determine the specific gravity of defibrinated blood by means of an ordinary specific gravity spindle. Compare this result with the specific gravity as de- termined by Hammerschlag's method in the next experiment. 4. Specific Gravity by Hammerschlag's Method. — Fill an ordinary urinometer cylinder about one-half full of a mixture of chloroform and benzene, having a specific gravity of approximately 1.050. Into this mixture allow a drop of the blood under examination to fall from a pipette or directly from the finger in case fresh blood is being examined. Care must be taken not to use too large a drop of blood and to keep the drop from coming in contact with the walls of the cylinder. If the blood drop sinks to the bottom of the vessel, thus show- ing it to be of higher specific gravity than the surrounding fluid, add chloroform until the blood drop remains suspended in the mixture. Stir carefully with a glass rod after adding the chloroform. If the blood drop rises to the surface upon being introduced into the mixture, thus showing it to be of lower specific gravity than the surrounding fluid, add benzene until the blood drop remains suspended in the mixture. Stir with a glass rod after the benzene is added. After the blood drop has been brought to a suspended position in the mixture by means of one or more additions of chloroform and benzene this final mixture should be filtered through muslin and its BLOOD. l6l specific gravity accurately determined. What is the specific gravity of the blood under examination? 5. Tests for Various Constituents. — Place 10 c.c. of de- fibrinated blood in an evaporating dish, dilute with 100 c.c. of water and heat to boiling. Is there any coagulation, and if so what bodies form the coagulum? At the boiling-point acidu- late slightly with dilute acetic acid. Filter. The filtrate should be clear and the coagulum dark brown. Reserve this coag- ulum. What body gives the coagulum this color? Evapo- rate the filtrate to about 25 c.c, filtering off any precipitate which may form in the process. Make the following tests upon the filtrate : (a) Fe tiling's Test. — Test for sugar according to directions given on page 8. (b) Chlorides. — To a small amount of the filtrate in a test- tube add a few drops of nitric acid and a little argentic nitrate. In the presence of chlorides, a white precipitate of argentic chloride will form. (c) Phosphates. — Test for phosphates by nitric acid and molybdic solution according to directions given on page 37. ((/) Proteose and Peptone. — Test a small amount of the solution for proteose and peptone by saturating with ammo- nium sulphate according to directions given on page 59. (e) Crystallization of Sodium Chloride. — Place the re- mainder of the filtrate in a watch glass and evaporate it on a water-bath. Examine the crystals under the microscope and compare them with those in Fig. 60, page 167. 6. Test for Iron. — Incinerate a small portion of the coagu- lum from the last experiment ( 5 ) in a porcelain crucible. Cool, dissolve the residue in dilute hydrochloric acid and test for iron by potassium ferrocyanide or ammonium sulphocyanide. Which of the constituents of the blood contains the iron? 7. Laky Blood. — Note the opacity of ordinary defibrinated blood. Place a few cubic centimeters of this blood in a test- tube and add water, a little at a time, until the blood is ren- dered transparent. It is now laky blood. How does the water 1 62 PHYSIOLOGICAL CHEMISTRY. act in causing this transparency? Examine a drop of laky- blood under the microscope. How does its microscopical ap- pearance differ from that of unaltered blood? What other agents may be used to render blood laky? Fig. 57- \ Effect of Water on Erythrocytes. 8. Osmotic Pressure. — Place a few cubic centimeters of blood in each of three test-tubes. Lake the blood in the first tube according to directions given in the last experiment (7) : add an equal volume of isotonic (0.9 per cent) sodium chlo- ride to the blood in the second tube, and an equal volume of 10 per cent sodium chloride to the blood in the third tube. Mix thoroughly by shaking and after a few moments examine a drop from each of the three tubes under the microscope (see Figs. 57 and 115. pages 162 and 337). What do you find and what is your explanation from the standpoint of osmotic pressure? 9. Diffusion of Haemoglobin. — Prepare some laky blood, thus liberating the haemoglobin from the erythrocytes. Test the diffusion of the haemoglobin by preparing a dialyzer like one of the models shown in Fig. 1, page 6. How does haemo- globin differ from other well-known crystallizable bodies? BLOOD. [63 10. Guaiac Test. — To 5 c.c. of water in a tesl tube add two drops of blood. By means of a pipette drop an alcoholic solution of guaiac (strength about [:6b) 1 into the resulting mixture until a turbidity is observed and add old turpentine or hydrogen pert ixide, dr< >p by drop, until a blue c< dor is obtained. Do any other substances respond in a similar manner to this test? Is a positive guaiac test a sure indication of the p ence of blood? 11. Haemin Test. — (a) Teichmanris Method. — Place a very small drop of blood on a microscopic slide, add a minute grain of sodium chloride 2 and carefully evaporate to dryness over a low flame. Put a cover glass in place, run underneath it a drop of glacial acetic acid and warm gently until the for- mation of gas bubbles is noted. Cool the preparation, exam- ine under the microscope and compare the crystals with those shown in Figs. 58 and 59, page 164. The haemin crystals result from the decomposition of the haemoglobin of the blood. What are the steps involved in this process? The haemin crystals are also called Teichmann's crystals. Is this an absolute test for blood? Is it possible to differentiate be- tween human blood and the blood of other species by means of the haemin test? (b) Zeynek and Nencki's Method. — To 10 c.c. of defibri- nated blood add acetone until no more precipitate forms. Filter off the precipitated proteid and extract it with 10 c.c. of acetone made acid with 2-3 drops of hydrochloric acid. Place a drop of the resulting colored extract on a slide, immediately place a cover glass in position and examine under the micro- scope. Upon the evaporation of the acetone, crystals of haemin will form. Larger crystals may be obtained by evaporating the acetone extract about one-half, transferring it to a stop- pered vessel and allowing it to remain over night. (c) Schalfijew's Method. — Place 20 c.c. of glacial acetic 1 Buckmaster advises the use of an alcoholic solution of guaiaconic acid instead of an alcoholic solution of guaiac resin. J Buckmaster considers the use of potassium chloride preferable. 164 PHYSIOLOGICAL CHEMISTRY. Pic. 58. Hjemin Crystals from Human Blood. Reproduced from a micro-photograph furnished by Prof. E. T. Reichert, of the University of Pennsylvania. Fig. 59. H;emin Crystals from Sheep Blood. Reproduced from a micro-photograph furnished by Prof. E. T. Reichert, of the University of Pennsylvania. BLOOD. 165 acid in a small beaker and heat to 8o c C. Add 5 c.c. of strained defibrinated blood, again bring the temperature to 80 C, remove the flame and allow the mixture* to cool. Examine the crystals under the microscope and compare them with those reproduced in Figs. 58 and 59, page 1^4. 12. Catalytic Action. — To about 10 drops of blood in a test-tube add twice the volume of hydrogen peroxide, without shaking. The mixture foams. What is the cause of this phenomenon ? [3. Preparation of Haematin. — Place 100 c.c. of laked bli k m1 in a beaker and add 95 per cent alcohol until precipitation ceases. What bodies are precipitated? Transfer the precipi- tate to a flask and boil with 95 per cent alcohol previously acidulated with sulphuric acid. Through the action of the acid the haemoglobin is split into haematin and a proteid body called globin*. Later the "sulphuric acid ester of haematin" is formed, which is soluble in the alcohol. Continue heating until the precipitate is no longer colored, then filter. Partly saturate the filtrate with sodium chloride and warm. In this process the " hydrochloric acid ester of haematin " is formed. Filter and dissolve on the filter paper by sodium carbonate. Save this alkaline solution of haematin and make a spectro- scopic examination later after becoming familiar with the use of the spectroscope. How does the spectrum of oxyhemo- globin differ from that of the derived alkali hcematin? 14. Variation in Size of Erythrocytes. — Prepare two small funnels with filter papers such as are used in quantitative analysis. Moisten each paper with normal (isotonic) salt solution. Into one funnel introduce a small amount of defibri- nated ox blood and into the other funnel allow blood to drop directly from a decapitated frog. Note that the filtrate from the ox blood is colored whereas that from the frog blood is colorless. What deduction do you make regarding the relative size of the erythrocytes in ox and frog blood? Does either filtrate clot? Why? 1 66 PHYSIOLOGICAL CHEMISTRY. II. Blood Serum. 1. Coagulation Temperature. — Place 5 c.c. of undiluted serum in a test-tube and determine its temperature of coagula- tion according to the method described on page 50. Note the temperature at which a cloudiness occurs as well as the tem- perature at which coagulation is complete. 2. Precipitation by Alcohol. — To 5 c.c. of serum in a test-tube add twice the amount of 95 per cent alcohol and thoroughly mix by shaking. What is this precipitate? Make a confirmatory test. Test the alcoholic filtrate for proteid. Explain the result. 3. Proteids of Blood Serum. — Place about 20 c.c. of un- diluted serum in a small evaporating dish, heat to boiling and at the boiling-point acidify slightly with dilute acetic acid. Of what does this coagulum consist? Filter off the coagulum (reserve the filtrate) and test it as follows: (a) Millons Reaction. — Make the test according to direc- tions given on page 44. (b) Xanthoproteic Test. — Make the test according to di- rections given on page 44. 4. Sugar in Serum. — Test a little of the filtrate from Ex- periment 3 by Fehling's test. What do you conclude ? 5. Detection of Sodium Chloride. — (a) Test a little of the filtrate from Experiment 3 for chlorides, by the use of nitric acid and argentic nitrate, (b) Evaporate 5 c.c. of the filtrate from Experiment 3 in a watch glass on a water-bath. Examine the crystals and compare them with those reproduced in Fig. 60, page 167. 6. Separation of Serum Globulin and Serum Albunin. — Place 10 c.c. of blood serum in a small beaker and saturate with magnesium sulphate. What is this precipitate? Filter it off and acidify the filtrate slightly with acetic acid. What is this second precipitate? Filter this precipitate off and test the filtrate by the biuret test. What do you conclude ? 167 £#30 Sodium Chloride. III. Blood Plasma. i. Preparation of Oxalated Plasma. — Allow arterial blood to run into an equal volume of 0.2 per cent ammonium oxalate solution. 2. Preparation of Fibrinogen. — To 25 c.c. of oxalated plasma add an equal volume of saturated sodium chloride solu- tion. Note the precipitation of fibrinogen. Filter off the pre- cipitate (reserve the filtrate) and test it by a proteid color test I see page 44). 3. Effect of Calcium. — Place a small amount of oxalated plasma in a test-tube and add a few drops of a 2 per cent calcium chloride solution. What occurs? Explain it. 4. Preparation of Salted Plasma. — Allow arterial blood to run into an equal volume of a saturated solution of sodium sulphate or a 10 per cent solution of sodium chloride. Keep the mixture in a cold place for about twenty-four hours. 5. Effect of Dilution. — Place a few drops of salted plasma in a test-tube and dilute it with 10-15 volumes of water. What do you observe? Explain it. 6. Crystallization of Oxyhemoglobin. — RcicJicrt's Meth- od, — Allow the blood of the dog or horse to Mow into an equal 1 68 PHYSIOLOGICAL CHEMISTRY. volume of 7 per cent ammonium oxalate solution. Place a small amount of this oxalated blood in a test-tube and lake it with ether, being careful to avoid an excess of the reagent. By means of a pipette transfer a drop of this laked blood to a slide, and when the edges of the drop begin to dry place a cover glass in position. Examine under the microscope and compare the crystals with those in Figs. 50 to 56, pages 152 t0 J 55- IV. Fibrin. 1. Preparation of Fibrin. — Allow blood to flow directly from the animal into a vessel and rapidly whip it by means of a bundle of twigs, a mass of strong cords or a specially con- structed beater. If a pure fibrin is desired it is not best to attempt to manipulate a large volume of blood at one time. After the fibrin has been collected it should be freed from any adhering blood clots and washed in water to remove fur- ther traces of blood. The pure product should be very light in color. It may be preserved under glycerin, dilute alcohol or chloroform water. 2. Solubility. — Try the solubility of small shreds of freshly prepared fibrin in the usual solvents. 3. Millon's Reaction. — Make the test according to direc- tions given on page 44. 4. Xanthoproteic Test. — Make the test according to di- rections given on page 44. 5. Biuret Test. — Make the test according to directions given on page 45. V. Detection of Blood in Stains on Cloth, etc. i. Identification of Corpuscles. — If the stain under ex- amination is on cloth a portion should be extracted with a few drops of glycerin or normal (0.9 per cent) sodium chloride solution. A drop of this solution should then be examined under the microscope to determine if corpuscles are present. 2. Tests on Aqueous Extract. — A second portion of the stain should be extracted with a small amount of water and the following tests made upon the aqueous extract: BLOOD. 169 (a) HtBtnochrotnogen. — Make a small amount of the ex tract alkaline by potassium hydroxide or sodium hydroxide, and heat until a brownish-green color results. Cool and add a few drops of ammonium sulphide or Stokes' reagent (see page 170) and make a spectroscopic examination. Compare the spectrum with that of haemochromogen (see Absorption Spectra, Plate II). (/;) Ilccmiii Test. — Make this test upon a small drop of the aqueous extract according to the directions given on page 163. (c) Guaiac Test. — Make this test on the aqueous extract according to the directions given on page 163. The guaiac solution may also be applied directly to the stain without pre- vious extraction in the following manner: Moisten the stain with water, and after allowing it to stand several minutes, add an alcoholic solution of guaiac (strength about 1:60) and a little hydrogen peroxide or old turpentine. The customary blue color will be observed in the presence of blood. (d) Acid Hcematin. — If the stain fails to dissolve in water extract with acid alcohol and examine the spectrum for ab- sorption bands of acid haematin 1 sec Absorption Spectra, Plate II). VI. Spectroscopic Examination of Blood. (For Absorption Spectra see Plates I. and II.). Either the awgtt/ar-vision spectroscope (Figs. 62 and 63, pages 170 and 171) or the direct-vision spectroscope (Fig. 61, page 170) may be used in making the spectroscopic examina- tion of the blood. For a complete description of these instru- ments the student is referred to any standard text-book of physics. 1. Oxyhaemoglobin. — Examine dilute (1:50) defibrinated blood spectroscopically. Note the broad absorption-band be- tween D and E. Continue the dilution until this single broad band gives place to two narrow bands, the one nearer the D line being the narrower. These are the typical absorption- bands of oxyhemoglobin obtained from dilute solutions of 170 PHYSIOLOGICAL CHEMISTRY. blood. Now dilute the blood very freely and note that the bands gradually become more narrow and, if the dilution is sufficiently great, they finally entirely disappear. Fig. 61. Direct-vision Spectroscope. 2. Haemoglobin (so-called Reduced Haemoglobin). — To blood which has been diluted sufficiently to show well defined oxyhemoglobin absorption-bands add a small amount of Fig. 62. Angular-vision Spectroscope Arranged for Absorption Analysis. Stokes' reagent. 1 The blood immediately changes in color from a bright red to a violet-red. The oxyhemoglobin has been reduced through the action of Stokes' reagent and 1 Stokes' reagent is a solution containing 2 per cent ferrous sulphate and 3 per cent tartaric acid. When needed for use a small amount should be placed in a test-tube and ammonium hydroxide added until the precipitate which forms on the first addition of the hydroxide has entirely dissolved. This produces ammonium ferrotartrate which is a reducing agent. BLOOD. I 7 I haemoglobin (so called reduced haemoglobin) has been formed. This has been brought about by the removal of some of the loosely combined oxygen from the oxyhemoglobin. Examine this haemoglobin spectroscopically. Note that in place of the two absorption hands of oxyhemoglobin we now have a single broad hand lying almost entirel) between 1) and E. This is Fig. 63. Diagram of Angular-vision Spectroscope. (Long.) The white light F enters the collimator tube through a narrow slit and passes to the prism P, which has the power of refracting and dispersing the light. The rays then pass to the double convex lens of the ocular tube and are deflected to the eyepiece E. The dotted lines show the magnified virtual image which is formed. The third tube contains a scale whose image is reflected into the ocular and shown with the spectrum. Between the light F, and the collimator slit is placed a cell to hold the solution undergoing examination. the typical spectrum of hemoglobin. If the solution showing this spectrum be shaken in the air for a few moments it will again assume the bright red color of oxyhemoglobin and show the characteristic spectrum of that pigment. 3. Carbon Monoxide Haemoglobin. — The preparation of this pigment may be easily accomplished by passing ordinary illuminating gas 1 through defibrinated ox-blood. Blood thus treated assumes a brighter tint (carmine) than that imparted by oxyhemoglobin. In very dilute solution oxyhemoglobin 1 The so-called water gas with which ordinary illuminating gas is diluted contains usually as much as 20 per cent of carbon monoxide (CO). 172 PHYSIOLOGICAL CHEMISTRY. appears yellowish-red whereas carbon monoxide haemoglobin under the same conditions appears bluish-red. Examine- the carbon monoxide haemoglobin solution spectroscopically. Ob- serve that the spectrum of this body resembles the spectrum of oxyhemoglobin in showing two absorption-bands between D and E. The bands of carbon monoxide haemoglobin, however, are somewhat nearer the violet end of the spectrum. Add some Stokes' reagent to the solution and again examine spec- troscopically. Note that the position and intensity of the absorption bands remain unaltered. 4. Neutral Methaemoglobin. — Dilute a little defibrinated blood (1 :io) and add a few drops of a freshly prepared 10 per cent solution of potassium ferricyanide. Shake this mix- ture and observe that the bright red color of the blood is dis- placed by a brownish-red. Now dilute a little of this solution and examine it spectroscopically. Note the single, very dark absorption-band lying to the left of D and, if the dilution is sufficiently great, also observe the two rather faint bands lying between D and E in somewhat similar positions to those occu- pied by the absorption-bands of oxyhaemoglobin. Add a few drops of Stokes' reagent to the methaemoglobin solution while it is in position before the spectroscope and note the imme- diate appearance of the oxyhaemoglobin spectrum which is quickly followed by that of haemoglobin. 5. Alkaline Methaemoglobin. — Render a neutral solution of methaemoglobin, such as that used in the last experiment (4), slightly alkaline with a few drops of ammonia. The solu- tion becomes redder in color, due to the formation of alkaline methaemoglobin and shows a spectrum different from that of the neutral body. In this case we have a band on either side of D, the one nearer the red end of the spectrum being much the fainter. A third band, darker than either of those men- tioned lies between D and E somewhat nearer E. 6. Alkali Haematin. — Observe the spectrum of the alkali haematin prepared in Experiment 13 on page 165. Also make a spectroscopic examination of a freshly prepared alkali BLOOD. [73 haematin. 1 The typical spectrum of alkali haematin shows a single absorption band lying across D and mainly toward the red end of the spectrum. 7. Reduced Alkali Haematin or Haemochromogen. — Dilute the alkali 1 1 a * n 1 a t i 1 1 solution used in the last experiment (6) to such an extent that it shows no absorption hand. Now add a few drops of Stokes' redgent and note that the greenish- brown color of the alkali haematin solution is displaced by a bright red color. This is due to the formation of haemochro- mogen or reduced alkali haematin. Examine this solution spectroscopically and observe the narrow, dark, absorption- band lying midway between D and E. If the dilution is not too great a faint band may be observed in the green extend- ing across E and b. 8. Acid Haematin. — To some defibrinated blood add half its volume of glacial acetic acid and an equal volume of ether. Mix thoroughly. The acidified ethereal solution of haematin rises to the top and may be poured off and used for the spec- troscopic examination. If desired it may be diluted with acidified ether in the ratio of one part of glacial acetic acid to two parts of ether. A distinct absorption-band will be noted in the red between C and D and lying somewhat nearer C than the band in the metbsemoglobin spectrum. Between D and F may be seen a rather indistinct broad band. Dilute the solution until this band resolves itself into two bands. Of these the more prominent is a broad, dark absorption-band lying in the green between b and F. The second, a narrow band of faint outline, lies in the light green to the red side of E. A fourth very faint band may be observed lying on the violet side of D. 9. Acid Haematoporphyrin. — To 5 c.c. of concentrated sulphuric acid in a test-tube add two drops of blood mixing 1 Alkali haematin may be prepared by mixing one volume of a con- centrated potassium hydroxide or sodium hydroxide solution and two vol- umes of dilute (1:5) defibrinated blood. This mixture should be heated gradually almost to boiling, then cooled and shaken for a few moments in the air before examination. 174 PHYSIOLOGICAL CHEMISTRY. thoroughly by agitation after the addition of each drop. A wine-red solution is produced. Examine this solution spectro- scopically. Acid haematoporphyrin gives a spectrum with an absorption-band on either side of D, the one nearer the red end of the spectrum being the narrower. 10. Alkaline Haematoporphyrin. — Introduce the acid haematoporphyrin solution just examined into an excess of distilled water. Cool the solution and add potassium hydrox- ide slowly until the reaction is but slightly acid. A colored precipitate forms which includes the principal portion of the haematoporphyrin. The presence of sodium acetate facili- tates the formation of this precipitate. Filter off the precipi- tate and dissolve it in a small amount of dilute potassium hydroxide. Alkaline haematoporphyrin prepared in this way forms a bright red solution and possesses four absorption- bands. The first is a very faint, narrow band in the red, midway between C and D; the second is a broader, darker band lying across D, principally to the violet side. The third absorption-band lies principally between D and E, extending for a short distance across E to the violet side, and the fourth band is broad and dark and lies between b and F. The first band mentioned is the faintest of the four and is the first to disappear when the solution is diluted. VII. Instruments Used in the Clinical Examination of the Blood. i. Fleischl's Haemometer (Fig. 64, p. 175). — This is an instrument used quite extensively clinically, for the quantitative determination of haemoglobin. The instrument consists of a small cylinder which is provided with a fixed glass bottom and a movable glass cover, and which is divided, by means of a metal septum, into two compartments of equal capacity. This cylinder is supported in a vertical position by means of a mech- anism which resembles the base and stage of an ordinary mi- croscope. Underneath the stage is placed a colored glass wedge (see Fig. 66, p. 176), so arranged as to run immediately beneath the glass bottom of one of the compartments of the BLOOD. 175 Fig, ''I cylinder and ground in such a manner that each part of the wedge corresponds in color to a solution of haemoglobin of some definite percentage. The glass wedge is held in a metal frame and may be moved backward or forward by means of a rack and pinion arrangement. A scale along the side ol this frame indicates the percentage of the normal amounl of haemoglo- bin which each particular varia- tion in the depth of color of the ground wedge represents, tak- ing the normal haemoglobin con- tent as ioo. 1 In a position corresponding to the position of the mirror on the ordinary mi- croscope is attached a light- colored opaque plate which serves to reflect the light up- ward through the colored wedge and the cylinder to the eye of the observer. In making a determination of the percentage of haemo- globin by this instrument the procedure is as follows : Fill each compartment about three-fourths full of distilled water. Punc- ture the finger-tip or lobe of the ear of the subject by means of a sterile needle or scalpel and, as soon as a drop of blood appears, place one end of the capillary pipette (Fig. 65), which accompanies the instrument, against the drop and allow it to fill by capillary attraction. To prevent the blood from adhering to the exterior of the tube, and so render the deter- mination inaccurate, it is customary to apply a very thin coating of mutton fat to the outer surface before using or to 1 The scale of the ordinary instrument is usually too high. Fl.l ! S< III.'S H.VMOMF/I ER. (Da Costa.) Fig. 65. Pipette of Fleischl's HiEMOMETER. 176 PHYSIOLOGICAL CHEMISTRY. Fig. 66. Colored Glass Wedge of Fleischl's H^emometer. (Da Costa.) wrap the tube in a piece of oily chamois when not in use. As soon as the tube has been accurately filled with blood it should be dipped into the water of one of the compartments of the cylinder and all traces of the blood washed out with water by means of a small dropper which accompanies the instrument. If the blood is not well dis- tributed throughout the compartment and does not form a homogeneous solu- tion the contents of the com- partment should be mixed thoroughly by means of the metal handle of the cap- illary measuring pipette. When this has been done each compartment should be completely filled with distilled water and the glass cover adjusted, care being taken that the contents of the two compartments do not mix. Now adjust the cylinder so that the compartment con- taining the pure distilled water is immediately above the colored glass wedge. By means of the rack and pinion arrangement, manipulate the colored wedge, until a portion of it is found which corresponds in color with the diluted blood. When this agreement in color has been secured the point on the scale corresponding to this particular color should be read and the actual percentage of haemoglobin computed. For instance, if the scale reading is 90 it means that the blood under examina- tion contains 90 per cent of the normal quantity of haemo- globin, i. e., 90 per cent of 14 per cent. 2. Fleischl-Miescher Haemometer. — The apparatus of Fleischl has recently been modified by Miescher. If all pre- cautions are taken, the margin of error in the absolute quan- tity of haemoglobin determined by this instrument does not exceed 0.15-0.22 per cent by weight of the blood. Detailed directions for the manipulation of the Fleischl-Miescher haemometer accompany the instrument. In brief Miescher modified the instrument as follows : ( 1 ) The scale of each BLOOD. 177 instrument is supplied with a caliber table of absolute haemo- globin values, expressed in milligrams: the ^cale of Fleischl's haemometer shows the percentage of haemoglobin in relation to an average selected somewhat arbitrarily. Thus many errors arising from the irregular coloring of the glass wedge of the older apparatus are avoided in the instrument as modi- fied. (2) Bach instrument is accompanied by a measuring pipette (melangeur) which allows of a more accurate meas- urement of the blood than was possible with the capillary tubes of the older apparatus. (3) With the aid of the measuring pipette mentioned above blood of varying degrees of con- centration may be compared. In this way the individual ex- aminations are controlled and a check upon the accuracy of the graduation in the color of the glass wedge is also afforded. This wedge is much more evenly and accurately colored than in the unmodified apparatus of Fleischl. (4) Before reading the percentage as indicated by the scale, the chamber is covered with a glass and a diaphragm which sharply define the field on all sides without the formation of a meniscus. The measuring pipette is constructed essentially the same as the pipettes which accompany the Thoma-Zeiss Apparatus 1 see p. [82). The capillary portion, however, is graduated I, Yz and l /> which enables the observer to dilute the blood sample in the proportion of 1 :200, 1 1300 or 1 1400 as he may desire. If there is difficulty in drawing in the blood exactly to one of the graduations just mentioned the amount of blood above or below the volume indicated by the graduation may be determined by means of certain delicate cross-lines which are placed directly above and below the graduation. Each cross-line corresponds to T ^ of the volume of the capillary tube from the tip to the 1 graduation. A 0.1 per cent solution of sodium carbonate is used to dis- solve the stroma of the erythrocytes and so render the blood solution perfectly clear. If this is not done the color of the blood solution invariably appears darker in tone than that of the colored glass wedge. A freshly prepared sodium carbo- •3 178 PHYSIOLOGICAL CHEMISTRY nate solution should be used in order that the clearness of the solution may not be marred by the presence of sodium bicarbonate. 3. Dare's Haemoglobinometer (Fig. 67, below). — This instrument, as the name signifies, is used for the determina- tion of haemoglobin. In using either Fleischl's hremometer or the in- strument as modified by Miescher the blood is di- luted for examination whereas with the Dare instrument no dilution is required. This probably allows of rather more accurate determinations than are possible with the old Fleischl appa- ratus. The instrument con- sists essentially of the fol- lowing parts : ( 1 ) A cap- illary observation cell, (2) a semicircular col- ored glass wedge, (3) a milled wheel for manip- ulating the wedge, (4) a candle used to illumi- nate portions of the cap- illary observation cell and the colored wedge, (5) a small telescope used in the examination of the areas illuminated by the candle flame, (6) a scale graduated in per- centages of the normal amount of haemoglobin, (7) a hard rubber case, (8) a movable screen attached to the case. The capillary observation cell is formed of two small, Dare's Haemoglobinometer. (Da Costa.) R, Milled wheel acting by a friction bearing on the rim of the color disc ; S, case inclosing color disc, and provided with a stage to which the blood cham- ber is fitted ; T, movable wing which is swung outward during the observation, to serve as a screen for the observer's eyes, and which acts as a cover to inclose the color disc when the instrument is not in use ; U, telescoping camera tube, in posi- tion for examination ; V, aperture admitting light for illumination of the color disc ; X, capillary blood chamber adjusted to stage of instrument, the slip of opaque glass, W, being nearest to the source of light ; Y, detachable candle-holder ; Z, rectangular slot through which the haemoglobin scale indi- cated on the rim of the color disc is read. lil.OOD. T 79 Fig. 68. polished rectangular plates of glass, one being transparent and the other opaque. When held in position on the instru- ment, by means of a small metal bracket, the opaque portion of the cell is nearer the candle and thus serves to soften the glare of light when an observation is being made. The trans- parent portion of the cell is directly over a circular opening in the case,- through which the blood specimen is viewed by means of the small telescope. The semicircular colored glass wedge is so ground that each particular shade of color corresponds to that possessed by fresh blood which contains some definite percentage of haemoglobin. It is mounted upon a disc which may he manipulated by the milled wheel in such a manner as to bring successive portions of the wedge in position to be viewed through a circular opening contiguous to the opening through which the blood specimen is viewed. For a further descrip- tion of the instrument see Figures 67, 68 and 69, on pages 178, 179, and 180 respectively. In using the Dare hsemoglobi- nometer proceed as follows: Punc- ture the finger-tip or lobe of the ear of the subject by means of a needle or scalpel and. after a drop of blood of good proportions has formed, place the flat capillary observa- tion cell in contact with the drop and allow it to fill by capillary attraction ( Fig. 69, page 180). Replace the cell in its proper place on the instrument. When in position, a portion of this cell may be observed through a small telescope attached to the apparatus. It is viewed through a circular opening and near this circle is a second one through which a portion of a semi- circular colored glass wedge is visible. These two circles are Horizontal Section of D FLemoglobinometer. (Da Costa. ) l8o PHYSIOLOGICAL CHEMISTRY. illuminated simultaneously by means of the flame of a candle. The colored glass may be rotated by means of a milled wheel and the point of agreement of the color of the adjoining discs may be determined in the same way as in Fleischl's haemom- eter. The scale reading gives the percentage of the normal Fig. 69. Method of Filling the Capillary Observation Cell of Dare's H.EMOGLOBINOMETER. (Da Costa.) quantity of haemoglobin which the blood sample under exami- nation contains. Compute the actual haemoglobin content in the same manner as from the scale reading of the Fleischl haemometer (seepage 176). 4. Tallquist's Haemoglobin Scale. — This consists essen- tially of a series of ten colors corresponding to stains produced by blood containing varying percentages of haemoglobin. In using this scale a drop of blood is allowed to fall on a small section of filter paper and the resulting color is compared with the ten colors of the scale. When the color in the scale is found which corresponds to the color of the blood stain the accompanying haemoglobin value is read off* directly. This is a very convenient method for determining haemoglobin at the bedside. There is a possibility of the colors being in- accurately printed, however, and even if originally correct in tint, under the continued influence of air and light they must eventually alter somewhat. 5. Thoma-Zeiss Haemocytometer. — This is an instru- ment used in "blood counting," i. e., in determining the num- ber of erythrocytes and leucocytes. The instrument consists Ill.nni). IM of a microscopic slide constructed of heavy glass and provided with a central counting cell (see Fig. 70, below). This cell, with the cover glass in position, is exactly 0.1 millimeter deep The floor of the cell is divided by delicate lines into squares each of which i> 4( 1 )0 - of a square millimeter in area (see Fig, 7_', p. [83 ). The volume of blood therefore between any par- ticular square and the cover glass above must be ,,/,,,, cubic Thoma-Z iixc; Chamber. (Da Costa.) millimeter. Accompanying each instrument are two capil- lary pipettes (Fig. 71, p. 182), each constructed with a mixing bulb in its upper portion. Each bulb is further provided with an enclosed glass bead which is of great assistance in mixing the contents of the chamber. The stem of each pipette is graduated in tenths from the tip to the bulb. The final grad- uation at the upper end of the bulb is 101 on the pipette used in mixing the blood sample in which the erythrocytes are counted (erythrocytometer, see Fig. 71, p. 182), and 11 on the pipette used in mixing the blood sample for the leucocyte count (leucocytometer, see Fig. 71. p. 182). In making "blood counts" with the haemocytometer it is necessary to use some diluting fluid. Two very satisfactory forms of fluid for this purpose are Toison's and Sherrington's solutions. 1 When 1 Toison's solution has the follow- ing formula : Methyl violet 0.025 gram. Sodium chloride 1 gram. Sodium sulphate Sgrams. Glycerin 30 grams. Distilled water i6ograms. Sherrington's solution ha? the fol- lowing formula: Methylene-blue 0.1 gram. Sodium chloride 1.2 gram. Neutral potassium ox- alate 1.2 gram. Distilled water 300.0 grams. 182 PHYSIOLOGICAL CHEMISTRY. Fig. 71. either of these solutions is used as the diluting fluid it is possi- ble to make a very satisfactory count of both the erythrocytes and leucocytes from the same preparation, since the leucocytes are stained by the methyl violet or methylene-blue. In counting the erythrocytes by means of the hsemocytometer proceed as follows : Thoroughly cleanse the tip of the finger or lobe of the ear of the subject by the use of soap and water, alcohol and ether ap- plied in the sequence just given. Punc- ture the skin by means of a needle or scalpel and allow the blood drop to form without pressure. Place the tip of the pipette in contact with the blood drop, being careful to avoid touching the skin, and draw blood into the pipette up to the point marked 0.5 or 1 according to the desired dilution. Rapidly wipe the tip of the pipette and immediately fill it to the point marked 101 with Toison's or Sherrington's solution. Now thoroughly mix the blood and diluting fluid within the mixing chamber by tapping the pipette gently against the finger or by shaking it while held securely with the thumb at one end and the middle finger at the other. After the two fluids have been thoroughly mixed the diluting fluid con- tained in the capillary-tube below the bulb should be discarded in order to insure the collection of a drop of the thoroughly mixed blood and diluting solution for examina- tion. Transfer a drop from the pipette to the ruled floor of the counting chamber and, after placing the cover-glass firmly in position, 1 allow an 1 If the cover glass is in accurate apposition to the counting cell New- ton's rings may be plainly observed. Thoma-Zeiss Cap- illary Pipettes. A, Erythrocytom- eter ; B, leuco- cytometer. i:i.< )(>!>. I8 3 interval of a few minutes to elapse For the corpuscles to settle before making the count. Now place the slide under the microscope and count the number of erythrocytes in a num- ber of squares, counting- the corpuscles which are in contact with the upper and the righl hand boundaries of the square as belonging to that square. Take the squares in some definite Fig. 72. Ordinary Ruling of Thoma-Zeiss Counting Chamber. (Da Costa.) sequence in order that the recounting of the same corpuscles may be avoided. Of course, all things being equal, the greater the number of squares examined the more accurate the count. It is considered essential under all circumstances, where an accurate count is desired, that the counting chamber shall be filled at least twice and the individual counts made in each instance, as indicated above, before the data are deemed satisfactory. To calculate the number of erythrocytes per cubic milli- meter of undiluted blood proceed as follows : Determine the number of corpuscles in any given number of squares and divide this total by the number of squares, thus obtaining the average number of erythrocytes per square. Multiply this average by 4,000 to obtain the number of erythrocytes per i8 4 PHYSIOLOGICAL CHEMISTRY. cubic millimeter of diluted blood, and multiply this product by ioo or 200, according to the dilution, to obtain the number of erythrocytes per cubic millimeter of undiluted blood. Thus : Average number of ery- throcytes per square X 4,000 X 200 (or 100) Number of erythrocytes per cubic millimeter. Great care should be taken to see that the capillary pipette is properly cleaned. After using, it should be immediately rinsed out with the diluting fluid, then with water, alcohol and ether in the sequence given. Finally dry air should be drawn through the capillary and a horse hair inserted to prevent the entrance of dust particles. In counting leucocytes by means of the hsemocytometer pro- ceed as follows : As mentioned above, if the diluting fluid is either Toison's or Sherrington's solution the leucocytes may Fig. 73. Zappert's Modified Ruling of Thoma-Zeiss Counting Chamber. Costa.) (Da be counted in the same specimen of blood in which the ery- throcytes are counted. When this is done it is customary to use a slide provided with Zappert's modified ruling (Fig. 73, above). This method is rather more accurate than the older BLOOD. i s 5 one of counting the leucocytes in a separate specimen of blood. Furthermore it is obviously preferable to count both the erythrocytes and the leucocytes from the same bl sample. To insure accuracy the number of leucocytes within the whole ruled region should be determined in duplicate blood samples. This includes the examination of an area eighteen times as great as the old style Thoma-Zeiss central ruling. This region then would correspond to 3,600 of the small squares and if duplicate examinations were made the total number of small squares examined would aggregate 7.200. The calculation would be as follows: Number of leucocytes in Number of leucocytes per 7.200 squares X 200 X 4.000 -=- 7,200 = cubic millimeter [f a Zappert slide is not available a good plan to follow is to place a diaphragm in the tube of the ocular of the micro- scope consisting of a circle of black cardboard or metal 1 hav- ing a square hole in its centre of such a size as to allow of the examination of exactly 100 squares or one-fourth of a square millimeter at one time. With this arrangement any portion of the specimen may be examined and counted whether within or without the ruled area. In counting by means of this device it is of course helpful if the microscope is pro- vided with a mechanical stage, but even without this arrange- ment, if the observer is careful to see that the leucocytes at the extreme boundary of one field move to the opposite bound- ary when the position of the slide is changed, the device may be very satisfactorily employed. The leucocytes should be counted in 36 of the diaphragm-fields in duplicate specimens and the calculation made in the same manner as explained above. If the leucocytes are counted in a separate specimen of blood ordinarily the diluting fluid is 0.3-0.5 per cent acetic acid, a fluid in which the leucocytes alone remain visible. Under these conditions the dilution is customarily made in the pipette having 1 1 as the final graduation. The capillary 1 Ehrlich's mechanical eye-piece is also very satisfactory for this purpose. i86 PHYSIOLOGICAL CHEMISTRY. portion is of larger caliber and so requires a greater amount of blood to fill it to the 0.5 or 1 mark than is required in the use of the other form of pipette. In counting the leucocytes ac- cording to this method it is customary to draw blood into the pipette up to the 1 mark and immediately fill the remaining portion of the apparatus to the 11 graduation with the 0.3-0.5 per cent acetic acid. It then remains to count the number of leucocytes in the whole central ruled portion of 400 squares. This should be done in duplicate samples and the calculation made as follows : Number of leucocytes in Number of leucocytes per 800 squares. X ^ 00 ° X K> - 800 = cubk mmimeten CI I \ PTER XII. MILK. Milk is the most satisfactory individual food material elaborated by nature. It contains the three nutrients, proteid, fat and carbohydrate and inorganic salts in such proportion as to render it a very acceptable dietary constituent. It is a specific product of the secretory activity of the mammary gland. It contains, as the principal solids, tri-olcin, tri- palniitiii, Iri-stcariti, tri-butyrin, caseinogen, lactalbumin, lacto-globulin, lactose and calcium phosphate. It also contains at least traces of lecithin, cholesterin, urea, creatin, creatinin and the tri-glycerides of caproic, lauric and myristic acids. Fresh milk is amphoteric in reaction, but upon standing for a sufficiently long time, unsterilized, it becomes acid in reaction, due to the production of fermentation lactic acid, H OH I I H - C - C - COOH, H H from the lactose contained in it. This is brought about through bacterial activity. The white color is imparted to the milk partly through the fine emulsion of the fat and partly through the medium of the caseinogen in solution. The specific gravity of milk varies somewhat, the average being about 1.030. Its freezing-point is about — 0.56 C. Fresh milk does not coagulate on being boiled but a film con- sisting of a combination of caseinogen forms on the surface. If the film be removed, thus allowing a fresh surface to come in contact with the air, a new film will form indefinitely upon the application of heat. Surface evaporation and the presence 187 i88 PHYSIOLOGICAL CHEMISTRY of fat facilitate the formation of the film but are not essential. ( Rettger.) If the milk is acid in reaction, through the incep- tion of lactic acid fermentation, or from any other cause, no film will form when heat is applied, but instead a true coagula- tion will occur. The milk-curdling enzymes of the gastric and the pancreatic juice have the power of splitting the caseinogen of the milk, through a process of hydrolysis, into soluble casein Fig. 74 "• - c I - w v w /-> b Normal Milk and Colostrum. a, Normal milk ; b, Colostrum. and a peptone-like body. This soluble casein then forms a combination with the calcium of the milk and an insoluble curd of calcium casein or casein results. The clear fluid surround- ing the curd is known as whey. The most pronounced difference between human milk and cow's milk is in the proteid content, although there are also differences in the fats and likewise striking biological dif- ferences difficult to define chemically. It lias been shown that the caseinogen of human milk differs from the caseinogen of cow's milk in being more difficult to precipitate by acid or coagulate by rennin. The casein curd also forms in a much looser and more flocculent manner than that from cow's milk and is for this reason much more easily digested than the MILK. 189 latter. Interesting data relative to the composition of milk from various sources, may be gathered from the following table which was compiled mainly from the results of investi- gations by Bunge and by Abderhalden. It will be noted that the composition of the milk varies directly with the length of time needed for the young of the particular species to double in weight. Peiiod in which weight of the new-born is doubled (days). ISO 60 47 22 15 •4 9-5 9 6 100 parts of milk contain Species. • ids. ..6 2.0 3-5 3-7 4-9 5-2 7.0 7-4 10.4 Salts 0.2 0.4 0.7 o.S 0.8 0.8 1.0 i-3 2-5 Calcium. O.O33 O.I24 O. 160 O.I97 O.245 O.249 o-455 0.891 Phosphoric acid M;m Cow Pig D °g Rabbit O.O47 O.I3I O.I97 O.284 O.293 O.308 O.508 O.997 Lactose, the carbohydrate constituent of milk, is an impor- tant member of the disaccharide group. It occurs only in Fig. 75. Lactose. milk, except as it is found in the urine of women during preg- nancy, during the nursing period and sbon after weaning; it also 19° PHYSIOLOGICAL CHEMISTRY. occurs in the urine of normal persons after the ingestion of a very large amount of lactose in the food. It is not derived directly from the blood but is a specific product of the cellular activity of the mammary gland. It has strong reducing power, is dextro-rotatory and forms an osazone with phenylhydrazin. The souring of milk is due to the formation of lactic acid from lactose through the agency of the bacterium lactis. Putrefactive bacteria in the alimentary canal may bring about this same reaction. Lactose is not fermentable by pure yeast. Caseinogen, the principal proteid constituent of milk be- longs to the group of phospho-proteids. It has acidic properties and combines with bases to produce salts. It is not coagulable upon boiling and is precipitated from its neutral solution by certain metallic salts as well as upon saturation with sodium chloride or magnesium sulphate. Its acid solution is precipi- tated by an excess of mineral acid. Lactalbumin and lacto-globulin, the other proteid consti- tuents of milk, closely resemble serum albumin and serum globulin in their general properties. Colostrum is the name given to the product of the mam- mary gland secreted for a short time before parturition and during the early period of lactation (see Fig. 74, p. 188). It is yellowish in color, contains more solid matter than ordinary milk and has a higher specific gravity (1. 040-1. 080). The most striking difference between colostrum and ordinary milk is the high percentage of lactalbumin and lactoglobulin in the former. This abnormality in the proteid content is respon- sible for the coagulation of colostrum upon boiling. Experiments on Milk. 1. Reaction. — Test the reaction of fresh cow's milk to litmus. 2. Biuret Test. — Make the biuret test according to direc- tions given on page 45. 3. Microscopical Examination. — Examine fresh whole milk, skimmed or centrif u gated milk and colostrum under the MILK. I9I microscope. Compare the microscopical appearance with Fig. 74, page 188. 4. Specific Gravity. — Determine the specific gravity of both whole and skimmed milk. Which possesses the higher specific gravity? Explain why this is so. 5. Film Formation. — Place 10 c.c. of milk in a small beaker and boil a few minutes. Note the formation of a film. Remove the film and heat again. Does the film now form? Of what substance is this film composed? The biuret test was positive, why do we not get a coagulation here when we heat to boiling? 6. Coagulation Test. — Place about 5 c.c. of milk in a test- tube, acidify slightly with dilute acetic acid and heat to boil- ing. Do you get any coagulation? Why? 7. Action of Hot KOH. — To a little milk in a test-tube add a few drops of KOH and heat. A yellow color develops and gradually deepens into a brown. To what is the formation of this color due? 8. Test for Chlorides. — To about 5 c.c. of milk in a test- tude add a few drops of very dilute nitric acid to form a pre- cipitate. Filter off this precipitate and test the filtrate for chlorides. Does milk contain any chlorides? 9. Guaiac Test. — To about 5 c.c. of water in a test-tube add 3 drops of milk and enough alcoholic solution of guaiac (strength about 1:6c) 1 to cause a turbidity. Thoroughly mix the fluids by shaking and observe any change which may gradually take place in the color of the mixture. If no blue color appears in a short time, heat the tube gently below 6o° C. and observe whether the color reaction is hastened. In case a blue color does not appear in the course of a few minutes, add hydrogen peroxide or old turpentine, drop by drop, until the color is observed. Fresh milk will frequently give this blue color when treated with an alcoholic solution of 1 Buckmaster advises the use of an alcoholic solution of guaiaconic acid instead of an alcoholic solution of guaiac resin. Guaiaconic acid is a constituent of guaiac resin. I9 2 PHYSIOLOGICAL CHEMISTRY. guaiac without the addition of hydrogen peroxide or old tur- pentine. See discussion on page 158. 10. Saturation with MgS0 4 . — Place about 5 c.c. of milk in a test-tube and saturate with solid magnesium sulphate. What is this precipitate? 11. Influence of Rennin on Milk. — Prepare a series of five tubes as follows : (a) 5 c.c. of fresh milk -f 0.2 per cent HC1 (add drop by drop until a precipitate forms). (b) 5 c.c. of fresh milk + 5 drops of rennin solution. (c) 5 c.c. of fresh milk + 10 drops of 0.5 per cent Na 2 C0 3 . (d) 5 c.c. of fresh milk -|- 10 drops of ammonium oxalate. (e) 5 c.c. of fresh milk -f 5 drops of 0.2 per cent HC1. Xow to each of the tubes (c), (d) and (e) add 5 drops of rennin solution. Place the whole series of five tubes at 40 C. and after 10-15 minutes note what is occurring in the differ- ent tubes. Give a reason for each particular result. 12. Preparation of Caseinogen. — Fill a large beaker one- third full of skimmed (or centrifugated) milk and dilute it with an equal volume of water. Add dilute hydrochloric acid until a flocculent precipitate forms. Stir after each acidifica- tion and do not add an excess of the acid as the precipitate would dissolve. Allow the precipitate to settle, decant the supernatant fluid and reserve it for use in later (13-15) ex- periments. Filter off the precipitate of caseinogen and remove the excess of moisture by pressing it between filter papers. Transfer the caseinogen to a small beaker, add enough 95 per cent alcohol to cover it and stir for a few moments. Filter, and press the precipitate between filter papers to remove the alcohol. Transfer the caseinogen again to a small dry beaker, cover the precipitate with ether and heat on a water- bath for ten minutes, stirring continuously. Filter (reserve the filtrate), and press the precipitate as dry as possible be- tween filter papers. Open the papers and allow the ether to evaporate spontaneously. Grind the precipitate to a powder in a mortar. Upon the caseinogen prepared in this way make the following tests: MILK. 193 (a) Solubility. — Try the solubility ill the ordinary solvents (b) M Moris Reaction. — .Make the test according to the directions given on page 44. 1 c) Biuret Test. — Make- the test according to the directions given on page 45. i d 1 Xanthoproteic Reaction. — Make the test according to the directions given on page 44. (e) Loosely Combined Sulphur. — Test for loosely com- bined sulphur according to the directions given on page 52. (/) Fusion Test for Phosphorus. — Test for phosphorus by fusion according to directions given on page 223. 13. Coagulable Proteids of Milk. — Place the filtrate from the original caseinogen precipitate in a casserole and heat, on a wire gauze, over a free flame. As the solution concentrates, a coagulum consisting of lactalbumin and lactoglobulin will form. Continue to concentrate the solution until the volume is about one-half that of the original solution. Filter off the coagulable proteids (reserve the filtrate) and test them as follows : (a) Milloris Reaction. — Make the test according to the directions given on page 44. (b) Biuret Test. — Make the test according to the direc- tions given on page 45. (c) Xanthoproteic Reaction. — Make the test according to the directions given on page 44. 14. Detection of Calcium Phos- phate. — Evaporate the filtrate from the coagulable proteids, on a water-bath, until crystals begin to form. It may be necessary to concentrate to 15 c.c. before any crystallization will be ob- served. Cool the solution, filter oft the crystals (reserve the filtrate) and test ' calch-m Phosphate. them as follows : (a) Microscopical Examination. — Examine the crystals and compare them with those in Fig. 76, above. 14 194 PHYSIOLOGICAL CHEMISTRY. (b) Dissolve the crystals in nitric acid. Test part of the acid solution for phosphates. Render the remainder of the solution slightly alkaline with ammonia, then acidify with acetic acid and add ammonium oxalate. Examine the crystals under the microscope and compare them with those in Fig. 99, p. 320. 15. Detection of Lactose. — Concentrate the filtrate from the calcium phosphate until it is of a syrup-like consistency. Allow it to stand over night and observe the formation of crystals of lactose. Make the following experiments : (a) Microscopical Examination. — Examine the crystals and compare them with those in Fig. 75, page 189. (&) Fehlings Test. — Try Fehling's test upon the mother liquor. (c) Phenylhydrazin Test. — Apply the phenylhydrazin test to some of the mother liquor according to the directions given on page 5. 16. Milk Fat. — (a) Evaporate the ether filtrate from the caseinogen (Experiment 12) and observe the fatty residue. The milk fat was carried down with the precipitate of case- inogen and was removed when the latter was treated with ether. If centrifugated milk was used in the preparation of the caseinogen the amount of fat in the ether filtrate may be very small. To secure a larger yield of fat proceed accord- ing to directions given under (b) below. (b) To 25 c.c. of whole milk in an evaporating dish add a little sand or filter paper and evaporate the fluid to dryness on a water-bath. Grind or break up the residue after cooling and extract with ether in a flask. Filter and remove the ether from the filtrate by evaporation. How can you identify fats in the ethereal residue? 17. Saponification of Butter. — Dissolve a small amount of butter in alcohol made strongly alkaline with potassium hy- droxide. Place the alcoholic-potash solution in a casserole, add about 100 c.c. of water and boil for 10-15 minutes or until the odor of alcohol cannot be detected. Place the cas- serole in a hood and neutralize the solution with sulphuric MILK. 195 acid. Note the odor of volatile fatty acids particularly butyric acid. [8. Detection of Preservatives. — (a) Formaldehyde. I. Gallic Acid Test.— Acidify 30 c.c. of milk with 2 c.c. of normal sulphuric acid and distil. Add <>._' 0.3 c.c. of a satu- rated alcoholic solution of gallic acid to the first 5 c.c of the distillate, then incline the test-tube and slowly introduce 3 5 c.c. of concentrated sulphuric acid, allowing it to run slowly d< i\\ n the side of the tube. A green ring, which finally changes to blue, is formed at the juncture of the fluids. This is claimed, by Sherman, to he twice as delicate as either the sul- phuric acid or the hydrochloric acid test for formaldehyde. II. Hydrochloric Acid Test. — Mix 10 c.c. of milk and 10 c.c. of concentrated hydrochloric acid containing about 0.002 gram of ferric chloride in a small porcelain evaporating dish or casserole and gradually raise the temperature of the mixture nearly to the boiling-point, with occasional stirring. If formaldehyde is present a violet color is produced, while a brown color develops in the absence of formaldehyde. In case of doubt the mixture, after having been heated nearly to the boiling-point for about one minute, should be diluted with 50-75 c.c. of water, and the color of the diluted fluid carefully noted, since the violet color if present will quickly disappear Formaldehyde may be detected by this test when present in the proportion 1 : 250,000. (b) Salicylic Acid and Salicylates. — Remont's Method. 1 Acidify 20 c.c. of milk with sulphuric acid, shake well to break up the curd, add 25 c.c. of ether, mix thoroughly and allow the mixture to stand. By means of a pipette remove 5 c.c. of the ethereal extract, evaporate it to dryness, boil the residue with 10 c.c. of 40 per cent alcohol and cool the alcohlic solution. Make the volume 10 c.c, filter through a dry paper if necessary to remove fat, and to 5 c.c. of the filtrate, which represents 2 c.c. of milk, add 2 c.c. of a 2 per cent solution of ferric chloride. The production of a purple or violet color indi- cates the presence of salicylic acid. 1 Sherman's Organic Analysis, p. 232. I96 PHYSIOLOGICAL CHEMISTRY. This test may form the basis of a quantitative method by diluting the final solution to 50 c.c. and comparing this with standard solutions of salicylic acid. The colorimetric com- parisons may be made in a Duboscq colorimeter. (c) Hydrogen Peroxide. — Add 2-3 drops of a 2 per cent aqueous solution of paraphenylenediamine hydrochloride to 10-15 c.c. of milk. If hydrogen peroxide is present a blue color will be produced immediately upon shaking the mixture or after allowing it to stand for a few minutes. It is claimed that hydrogen peroxide may be detected by this test when present in the proportion 1 : 40,000. (d) Boric Acid and Borates. — To the ash, obtained accord- ing to the directions given on p. — , add 2 drops of dilute hydrochloric acid and 1 c.c. of water. Place a strip of turmeric paper in the dish and after allowing it to soak for about one minute remove it and allow it to dry in the air. The pres- ence of boric acid is indicated by the production of a deep red color which changes to green or blue upon treatment with a dilute alkali. This test is supposed to show boric acid when present in the proportion 1 : 8000. CHAPTER XIII. EPITHELIAL AND CONNECTIVE TISSUES. EPITHELIAL TISSUE (KERATIN). The albuminoid keratin constitutes the major portion of hair, horn, hoof, feathers, nails and the epidermal layer of the skin. There is a group of keratins the members of which possess very similar properties. The keratins as a group are insoluble in the usual proteid solvents and are not acted upon by the gastric or pancreatic juices. They all respond to the xanthoproteic and Millon reactions and are characterized by containing large amounts of sulphur. Keratin from any of its sources may be prepared in a pure form by treatment, in sequence, with artificial gastric juice, artificial pancreatic juice, boiling alcohol and boiling ether, from twenty- four to forty- eight hours being devoted to each process. Experiments ox Epithelial Tissri:. Keratin. Horn shavings may be used in the experiments which fol- low: i. Solubility. — Test the solubility of keratin in the ordinary solvents (see p. 4). 2. Mill 011's Reaction. 3. Xanthoproteic Reaction. 4. Adamkiewictfs Reaction. 5. Iloplcius-Colc Reaction. 6. Test for Loosely Combined Sulphur. CONNECTIVE TISSUE. I. WHITE FIBROUS TISSUE. The principal solid constituent of white fibrous connective ^en. i97 tis>ue is the albuminoid collagen. This body is also found in 198 PHYSIOLOGICAL CHEMISTRY. smaller percentage in cartilage, bone and ligament, but the collagen from the various sources is not identical in composi- tion. In common with the keratins, collagen is insoluble in the usual proteid solvents. It differs from keratin in con- taining less sulphur. One of the chief characteristics of col- lagen is the property of being hydrolyzed by boiling acid or water with the formation of gelatin. It gives Millon's re- action as well as the xanthoproteic and biuret tests. The form of white fibrous tissue most satisfactory for gen- eral experiments is the tendo Achillis of the ox. According to Buerger and Gies the fresh tissue has the following com- position : Water 62.87% Solids 37-13 Inorganic matter 0.47 Organic matter 36.66 Fatty substance (ether-soluble) . . . , 1.04 Coagulable proteid 0.22 Mucoid 1.28 Elastin 1.63 Collagen 31.59 Extractives, etc 0.90 The mucoid mentioned above is called tendomucoid and is a glucoproteid. It possesses properties similar to those of other connective tissue mucoids, e. g., osseomucoid and chon- dromucoid. Gelatin, the body which results from the hydrolysis of col- lagen, is also an albuminoid. It responds to nearly all the pro- teid tests. It differs from the keratins and collagen in being easily digested and absorbed. Gelatin is not a satisfactory substitute for the proteid constituents of a normal diet how- ever, since a certain portion of its nitrogen is not available for the uses of the organism. Gelatin from cartilage differs from the gelatin from other sources in containing a lower percentage of nitrogen. Experiments on White Fibrous Tissue. The tendo Achillis of the ox may be taken as a satisfactory type of the white fibrous connective tissue. EPITHELIAL AND CONNECTIVE TISSUES. I99 i. Preparation of Tendomucoid. — Dissect away the fascia from about the tendon and cut the clean tendon into small pieces. Wash the pieces in water, changing the wash water often in order to remove as much as possible of the soluble proteid and inorganic salts. Transfer the washed pieces of tendon to a flask and add 300 c.c. of lialf-su titrated lime-water. 1 Shake the-flask at intervals for twenty-four hours. Filter off the pieces of tendon and precipitate the mucoid with dilute hydrochloric acid. Allow the mucoid precipitate to settle, decant the supernatant fluid and filter the remainder. Test the mucoid as follows : (a) Solubility. — Try the solubility in the ordinary solvents (see p. 4). (b) Biuret Test. — First dissolve the mucoid in KOH solu- tion and then add a dilute solution of CuS0 4 . (c) Test for Loosely Combined Sulphur. (d) Hydrolysis of Tendomucoid. — Place the remainder of the mucoid in a small beaker, add about 30 c.c. of water and 2 c.c. of dilute hydrochloric acid and boil until the solu- tion becomes dark brown. Cool the solution, neutralize it with solid KOH and test by Fehling's test. With a reduction of Fehling's solution and a positive biuret test what do you conclude regarding the nature of tendomucoid ? 2. Collagen. — This substance is present in the tendon to the extent of about 32 per cent. Therefore in making the fol- lowing tests upon the pieces of tendon from which the mucoid, soluble proteid and inorganic salts were removed in the last experiment, we may consider the tests as being made upon collagen. (a) Solubility. — Cut the collagen into very fine pieces and try its solubility in the ordinary solvents (see page 4). (b) Millon's Reaction. (c) Biuret Test. (d ) Xanthoproteic Reaction. 1 Made by mixing equal volumes of saturated lime-water and water from the faucet. 200 PHYSIOLOGICAL CHEMISTRY. (e) Hopkins-Cole Reaction. (/) Test for Loosely Combined Sulphur. — Take a large piece of collagen in a test-tube and add about 5 c.c. of KOH solution. Heat until the collagen is partly decomposed, then add 1-2 drops of plumbic acetate and again heat to boiling. (g) Hydrolysis of Collagen. — Transfer the remainder of the pieces of collagen to a casserole, fill the vessel about two- thirds full of water and boil for several hours, adding water at intervals as needed. By this means the collagen is hydrolyzed and a body known as gelatin is formed. 3. Gelatin. — On the gelatin formed from the hydrolysis of collagen in the above experiment (g), or on gelatin fur- nished by the instructor make the following tests : (a) Solubility. — Try the solubility in the ordinary solvents (see page 4) and in hot water. (b) Milton's Reaction. (c) Hopkins-Cole Reaction. — Conduct this test according to the modification given on page 51. (d) Test for Loosely Combined Sulphur. Make the following tests upon a solution of gelatin in hot water : (a) Precipitation by Mineral Acids. — Is it precipitated by strong mineral acids such as concentrated hydrochloric acid? (b) Salting-Out Experiment. — Saturate a little of the solu- tion with solid ammonium sulphate. Is the gelatin precip- itated? Repeat the experiment with sodium chloride. What is the result? (c) Precipitation by Metallic Salts. — Is it precipitated by metallic salts such as cupric sulphate, mercuric chloride and plumbic acetate? (d) Coagulation Test. — Does it coagulate upon boiling? (e) Precipitation by Alkaloidal Reagents. — Is it precipi- tated by such reagents as picric acid, tannic acid and trichlor- acetic acid? (/) Biuret Test. — Does it respond to the biuret test? EPITHELIAL AND CONNECTIVE riSSUES. 201 (g) Precipitation by Alcohol. — Fill a test-tube one-half full of 95 per cent alcohol and pour in a small amount of con- centrated gelatin solution. Do you get a precipitate? How would you prepare pure gelatin from the tendo Achillis of the ox ? [I. YELLOW ELASTIC TISSUE (ELASTIN). The Ligatnentum michcr of the ox may be taken as a satis- factory type of the yellow elastic connective tissue. The principal solid constituent of this tissue is elastin, a member of the albuminoid group. In common with the keratins and collagen, elastin is an insoluble body and gives the proteid color reactions. It differs from keratin principally in the fact that it may be digested by enzymes and that it contains a very small amount of sulphur. Yellow elastic tissue also contains mucoid and collagen but these are present in much smaller amount than in white fibrous tissue, as may be seen from the following percentage composi- tion of the fresh ligamentum nucha: of the ox as determined by Vandegrift and Gies : Water 57-57% Solids 4243 Inorganic matter 0.47 Organic matter 41 .96 Fatty substance (ether-soluble) 1.12 Coagulable proteid 0.62 Mucoid 0.53 Elastin 31.67 Collagen 7.23 Extractives, etc 080 Experiments ox Elastin. 1. Preparation of Elastin (Richards and Gies). — Cut the ligament into fine strips, run it through a meat chopper and wash the finely divided material in cold, running water for 24-48 hours. Add an excess of half-saturated lime-water (see note at bottom of p. 199) and allow the hashed ligament 202 PHYSIOLOGICAL CHEMISTRY. to extract for 48-72 hours. Decant the lime-water, remove all traces of alkali by washing in water and then boil in water with repeated renewals until only traces of proteid material can be detected in the wash water. Decant the fluid and boil the ligament in 10 per cent acetic acid for a few hours. Treat the pieces with 5 per cent hydrochloric acid at room temper- ature for a similar period, extract again in hot acetic acid and in cold hydrochloric acid. Wash out traces of acid by means of water and then thoroughly dehydrolyze by boiling alcohol and boiling ether in turn. Dry in an air-bath and grind to a powder in a mortar. 2. Solubility. — Try the solubility of the finely divided elastin, prepared by yourself or furnished by the instructor, in the ordinary solvents (see page 4). How does its solu- bility compare with that of collagen? 3. Millon's Reaction. 4. Xanthoproteic Reaction. 5. Biuret Test. 6. Hopkins-Cole Reaction. — Conduct this test according to the modification given on page 5 1 . 7. Test for Loosely Combined Sulphur. III. CARTILAGE. The principal solid constituents of the matrix of cartilagi- nous tissue are chondro mucoid, chondroitin-sulphuric acid, chondroalbumoid and collagen. Chondromucoid differs from the mucoids isolated from other connective tissues in the large amount of chondroitin-sulphuric acid obtained upon decom- position. Besides being an important constituent of all forms of cartilage, chondroitin-sulphuric acid has been found in bone, ligament, the mucosa of the pig's stomach, the kidney of the ox, the inner coats of large arteries and in human urine. It may be decomposed through the action of acid and yields a nitrogenous body known as chondroitin and later this body yields chondrosin. Chondrosin is also a nitrogenous body and has the power of reducing Fehling's solution more strongly EPITHELIAL AND CONNECTIVE TISSUES. 203 than dextrose. Sulphuric acid is a by-product in the forma- tion of chondroitin. and acetic arid is a by-product in the for- mation of chondrosin. Chondroalbumoid is similar in some respects to elastin and keratin. It differs from keratin in being soluble in gastric juice and in containing considerably less sulphur than any member of the keratin group. It gives the usual proteid color reactions. Experiments on Cartilage. 1. Preparation of the Cartilage. — Boil the trachea of an ox in water until the cartilage rings may be completely freed frpm the surrounding tissue. Use the cartilage so obtained in the following experiments. _'. Solubility. — Cut one of the rings into very small pieces and try the solubility of the cartilage in the ordinary solvents (see page 4). 3. Millon's Reaction. 4. Xanthoproteic Reaction. 5. Hopkins-Cole Reaction. — Conduct this test according to the modification given on page 51. 6. Test for Loosely Combined Sulphur. 7. Preparation of Cartilage Gelatin. — Cut the remaining cartilage rings into small pieces, place them in a casserole with water and boil for several hours. Filter while the solution is still hot. Observe that the filtrate soon becomes more or less solid. What is the reason for this? Bring a portion of the material into solution by heat and try the following tests : (a) Biuret Test. (b) Test for Loosely Combined Sulphur. (c) To about 5 c.c. of the solution in a test-tube add a few drops of barium chloride. Do you get a precipitate, and if so to what is the precipitate due? (d) To about 5 c.c. of the solution in a test-tube add a few drops of dilute hydrochloric acid and boil for a few moments. Now add a little barium chloride to this solution. Is the pre- cipitate any larger than that obtained in the preceding experi- ment ? Why ? 204 PHYSIOLOGICAL CHEMISTRY. (e) To the remainder of the solution add a little dilute hydrochloric acid and boil for a few moments. Cool the solu- tion, neutralize with solid KOH and try Fehling's test. Ex- plain the result. IV. OSSEOUS TISSUE. Bone is composed of about equal parts of organic and in- organic matter. The organic portion, called ossein, may be obtained by removing the inorganic salts through the medium of dilute acid. Ossein is practically the same body which is termed collagen in the other connective tissues, and in com- mon with collagen may be hydrolyzed with weak acids to form gelatin. In common with the other connective tissues bone contains a mucoid and an albumoid. Because of their origin these bodies are called osseomucoid and osseoalbumoid. Osseo- mucoid, when boiled with hydrochloric acid, yields sulphuric acid and a substance capable of reducing Fehling's solution. The composition of osseomucoid is very similar to that of tendomucoid and chondromucoid (see page 62). Experiment on Osseous Tissue. Analysis of Bone Ash. — Take one gram of bone ash in a small beaker and add a little dilute nitric acid. What does the effervescence indicate? Stir thoroughly and when the major portion of the ash is dissolved add an equal volume of water and filter. To the acid filtrate add ammonium hydroxide to alkaline reaction. A heavy white precipitate of phosphates results. (What phosphates are precipitated here by the ammonia?) Filter and test the filtrate for chlorides, sul- phates, phosphates and calcium. Add dilute acetic acid to the precipitate on the paper and test this filtrate for calcium and phosphates. To the precipitate remaining undissolved on the paper add a little dilute hydrochloric acid and test this last filtrate for phosphates and iron. Reference to the following scheme may facilitate the analysis. EPITHELIAL AND CONNECTIVE TISS1 ES. 205 BONE ASH. Add dilute IINO3, stir thoroughly and after the major portion of the ash lias been brought into solution add a little distilled water and filter. Residue I. Filtrate I. (discard) Add NH 4 OH to alka- line reaction and filter. Residue II. Treat on paper with acetic acid. Residue III. Filtrate III. Treat on paper with Test for: HC1. 1. Phosphates. 2. Calcium. Filtrate IV. Test for: 1. Iron. 2. Phosphates. Filtrate II. Test for: 1. Chlorides. 2. Sulphates. 3. Phosphates. 4. Calcium. V. ADIPOSE TISSUE. For discussion and experiments see the chapter on Fats, page 96. CHAPTER XIV. MUSCULAR TISSUE. The muscular tissues are divided physiologically into the voluntary and the involuntary. In the chemical examination of muscular tissue the voluntary form is generally employed. Muscle contains about 25 per cent of solid matter, of which about four-fifths is proteid material and the remaining one- fifth extractives and inorganic salts. The proteids are the most important of the constituents of muscular tissue. In the living muscle we find two proteids, myosinogen and para-myosinogen. These may be shown to be present in muscle plasma expressed from fresh muscles. In common with the plasma of the blood this muscle plasma has the power of coagulating, and the clot formed in this process is called myosin. In the onset of rigor mortis we have an indication of the formation of this myosin clot within the body. The relation between the proteids of living and dead muscle is represented graphically by Halliburton as follows : Proteids of the living muscle. Para-myosinogen. Myosinogen. Soluble myosin. \/ Myosin. (The proteid of the muscle clot.) Of the total proteid content of living muscle about 75 per cent is made up by the myosinogen and the remaining 25 per cent is para-myosinogen. These proteids may be separated by subjecting the muscle plasma to fractional coagulation in the usual way. Under these conditions the para-myosinogen 206 M USCULAR TISS1 E. -"7 is found to coagulate at 47 C. and the myosinogen to coagu- late at 56 C. It is also claimed by some investigators that it is possible to separate these two proteids by the fractional ammonium sulphate method, but the possibility of making an accurate separation by this method is somewhat doubtful. It is well established that para-myosinogen is a globulin since it responds to .certain of the proteid precipitation tests and is insoluble in water. Myosinogen, on the contrary, is not a typical globulin since it is soluble in water. It has been called a pseudo-globulin. Myosin possesses the globulin character- istics. It is insoluble in water but soluble in the other proteid solvents and is precipitated from its solution upon saturation with sodium chloride. Under the name extractives we class a number of muscle constituents which occur in traces in the tissue and may be extracted by water, alcohol or ether. There are two classes of these extractives, the non-nitrogenous extractives and the nitrogenous extractives. Grouped under the non-nitrogenous bodies we have glycogen, dextrin, sugars, lactic acid, inosit, C 6 H c (OH) 6 , and fat. In the class of nitrogenous extractives we have crcatin, creatinin, xanthin, hypoxanthin, uric acid, urea, carnin, phosphocarnic acid, inosinic acid, carnosin and taurin (see formulas on page 210). Not all of these extrac- tives are present in the muscles of all species of animals. Other extractives besides those enumerated above have been described and there are undoubtedly still others whose presence remains undetermined. A detailed consideration w r ould however be unprofitable in this place. Glycogen is an important constituent of muscle. The con- tent of this polysaccharide in muscle varies and is markedly decreased by intense muscular activity. It is transformed into sugar and used as fuel. The liver is the organ which stores the reserve supply of glycogen and transforms it into dextrose which is passed into the blood stream and so carried to the working muscle where it is synthesized into glycogen. The glycogen thus formed is then changed into dextrose as the working muscle may need it. 208 PHYSIOLOGICAL CHEMISTRY. Glycogen is a polysaccharide and has the same percentage composition as starch and dextrin. It resembles starch in forming an opalescent solution and resembles dextrin in being very soluble, in giving a reddish color with iodine and in being dextro-rotatory. Glycogen may be prepared from muscle by extracting with boiling water and then precipitating the gly- cogen from the aqueous solution by alcohol : dilute or concen- trated KOH may also be used to extract the glycogen. Gly- cogen may be prepared in the form of a white, tasteless, amor- phous powder. It is completely precipitated from its solution by saturation with solid ammonium sulphate, but is notprecipitated by saturation with sodium chloride. It may also be precipitated by alcohol, tannic acid or ammoniacal basic lead acetate. It has the power of holding cupric hydroxide in solution in alkaline fluids but cannot reduce it. It may be hydrolyzed with the formation of dextrose by dilute mineral acids and is readily digested by amylolytic enzymes. The lactic acid occurring in the muscular tissue of verte- brates is paralactic or sarcolactic acid, H OH H — C — C — COOH. H H The reaction of an inactive living muscle is alkaline, but upon the death of the muscle, or after the continued activity of a living muscle, the reaction becomes acid, due to the formation of lactic acid. There is a difference of opinion regarding the origin of this lactic acid. Some investigators claim it to arise from the carbohydrates of the muscle, while others ascribe to it a proteid origin. Among the nitrogenous extractives of muscle, those which are of the most interest in this connection are creatin and the purin bases, xanthin and hypoxanthin. Creatin is found in varying amounts in the muscles of different species, the mus- MUSCII.AK TISSUE. 209 cles of birds having shown the largest amount. It has also been found in the blood, the brain, in transudates and in the thyroid gland. Creatin may be crystallized and forms color- less rhombic prisms (Fig. yj, below) which are soluble in warm water and practically insoluble in alcohol and ether. Fig. 77- Creatin. Upon boiling a solution of creatin with dilute hydrochloric acid it is dehydrolyzed and its anhydride creatinin is formed. The creatin of ingested meat is transformed into creatinin and excreted in the urine. Besides being a normal constituent of muscle, xanthin has been found in the brain, spleen, pancreas, thymus, kidneys, testicles, liver, and in the urine. It may be obtained in crys- talline form (Fig. 78, p. 210) but ordinarily it is amorphous. Xanthin is easily soluble in alkalis, less easily soluble in water and dilute acids, and entirely insoluble in alcohol and ether. Hypoxanthin occurs ordinarily in those tissues and fluids which contain xanthin. It has been found, unaccompanied by xanthin, in bone marrow and in milk. Unlike xanthin it may be easily crystallized in the form of small, colorless needles. '5 2IO PHYSIOLOGICAL CHEMISTRY. I: is readily soluble in alkalis, acids and boiling water, less soluble in cold water and practically insoluble in alcohol and ether. The predominating inorganic salt of muscle is potassium phosphate. Besides this salt we have present sulphates, chlo- rides and salts of sodium, calcium, magnesium and iron. Fig. 78. Xanthin. After the drawings of Horbaczewski, as represented in Xeubauer and Vogel. (Ogden.) Muscular tissue is said to contain a reddish pigment called my ohcc matin, which is a derivative of.. haemoglobin. The ordinary commercial " meat extract " is composed prin- cipally of the water-soluble constituents of muscle and con- tains practically nothing of nutritive value. The proteid mate- rial to which meat owes its value as an article of diet is practically all removed in the preparation of the extract. The structural formulas of the nitrogenous extractives of muscle are as follows : NH, HN- ■00 HN = C HN — C N-CBU-CEL-COOH Creatin, C 4 H t ,N30 2 . Methyl-guanidin acetic acid. N-CH 8 -CH S Creatinin, C|H 7 N 3 0. Creatin anhydride. XH., i 1 XHo Urea, CON 2 H 4 . M rsciT.AU TISSIK. 2 1 I cil-xii CH, • S0 2 • OH TaURIN, C2II7NSO3. A mino-cthylsuJ phonic acid. Camosin, C 9 H 14 X 4 3 . Inosinic acid, C ]0 H 13 X T 4 P0 8 . Phosphocarnic acid, C ln ir i7 X 3 05 ° r C, n II,-X 8 ( ">.-.. The following extractives as a group are called purin bodies. Their formulas, together with that of purin from which they are derived and the hypothetical " purin nucleus " follow : X = CH *X — C° II II HC C — XH 2C C 5 -X 7 CH \n II II /^-n- I I /-s N — C — N 3 X— C 4 — N n Purin, C0H4N4. Purin Nucleus. HX — CO HX — CO II II HC C — XH OC C — XH II II )CH I || )CH N — C — X HX — C — X Hypoxantrin, C«H 4 N 4 0. Xanthin, C5H4N4O2. 6-oxypurin. Uoxypurin. HX — CO X — C-NHo II II" OC C — XH HC C — XH I II >C0 II II )CH HX — C — XH X — C — X Uric Acid, Q.IL.NiO.-,. Adenin, C b H 5 N b . 3-6-S-trioxy purin. 6-aminopurin. 212 PHYSIOLOGICAL CHEMISTRY. HN — CO I I HoN-C C — NH II II )CH N — C — N Guanin, C 5 H 5 N 5 0. z-amino-6-oxypurin. Experiments on Muscular Tissue. I. Experiments on "Living" Muscle. I. Preparation of Muscle Plasma (Halliburton). — Wash out the blood vessels of a freshly killed rabbit with 0.9 per cent sodium chloride. This can best be done by opening the abdomen and inserting a cannula into the aorta. Now re- move the skin from the lower limbs, cut away the muscles and divide them into very small pieces by means of a meat chopper. Transfer the pieces of muscle to a mortar and grind them with clean sand and a little 5 per cent magnesium sul- phate. Filter off the salted muscle plasma and make the fol- lowing tests : (a) Reaction. — Test the reaction to litmus. What is the reaction of this fresh muscle plasma? (b) Fractional Coagulation. — Place a little muscle plasma in a test-tube and arrange the apparatus, .for fractional coagu- lation as explained on page 50. Raise the temperature very carefully from 30 C. and note any changes which may occur and the exact temperature at which such changes take place. When the first proteid (para-myosinogen) coagulates filter it off and then heat the clear filtrate as before, being careful to note the exact temperature at which the next coagulation (myosinogen) occurs. There will probably be a preliminary opalescence in each case before the real coagulation occurs. Therefore do not mistake the real coagulation-point and filter at the wrong time. What are the coagulation temperatures of these two proteids? Which proteid was present in greater amount ? MUSCULAR TISSUE. 213 (c) Formation of the Myosin Clot. — Dilute a portion of the plasma with 3 or 4 times its volume of water and place it on a water-bath or in an incubator at 35° C. for several hours. A typical myosin clot should form. Note the muscle serum surrounding the clot. Now test the reaction. Has the reaction changed, and if so, to what is the change due? Make a test for lactic acid. What do you conclude? 2. Preparation of Muscle Plasma (v. Fiirth). — Remove the blood-free muscles of a rabbit as explained on page 212. Finely divide by means of a meat chopper and grind in a mortar with a little clean sand and some 0.9 per cent sodium chloride. Wrap portions of the muscle in muslin and press thoroughly by means of a tincture press or lemon squeezer. Filter and make the tests according to the directions given in the last experiment. 3. " Fuchsin-Frog " Experiment. — Inject a saturated aqueous solution of Fuchsin "S" into the lymph spaces of a frog three or four times daily for two or three days, in this way thoroughly saturating the tissues with the dye. Pith the animal (insert a heavy wire or blunt needle through the occipito atlan- toid membrane), remove the skin from both hind legs and expose the sciatic nerve in one of them. Insert a small wire hook through the jaws of the frog and suspend the animal from an ordinary clamp or iron ring. Pass electrodes under the exposed sciatic nerve, and after tying the other leg to pre- vent any muscular movement, stimulate the exposed nerve by means of make and break shocks from an induction coil. The stimulated leg responds by pronounced muscular contractions, whereas the tied leg remains inactive. Continue the stimula- tion until the muscles are fatigued. The muscular activity has caused the production of lactic acid and this in turn has reacted with the injected fuchsin to cause a pink or red color to develop. The muscles of the inactive leg still remain unchanged in color. The normal color of the Fuchsin " S " when injected was red, but upon being absorbed it became colorless through the 214 PHYSIOLOGICAL CHEMISTRY. action of the alkalinity of the blood. Upon stimulating the muscles, however, as above explained, lactic acid was formed and this acid reacted with the fuchsin and again produced the original color of the dye. II. Experiments on " Dead " Muscle. i. Preparation of Myosin. — Take 25 grams of finely divided lean beef which has been carefully washed to remove blood and lymph constituents and place it in a beaker with 10 per cent sodium chloride. Stir occasionally for several hours. Strain off the meat pieces b}^ means of cheese cloth, filter the solution and saturate it with sodium chloride in substance. Filter off the precipitate of myosin and make the tests as given below. This filtration will proceed very slowly. Myosin collects as a film on the sides of the filter paper and may be removed and tested before the entire volume of fluid has been filtered. Test the myosin precipitate as follows : (a) Solubility. — Try its solubility in the ordinary solvents. Is myosin an albumin or a globulin? (b) Xanthoproteic Reaction. — See page 44. (c) Coagulation Test. — Suspend a little of the myosin in water in a test-tube and heat to boiling for a few moments. Now remove the suspended material and try its solubility in 10 per cent sodium chloride. What property does this ex- periment show myosin to possess ? Test the filtrate from the original myosin precipitate as follows : (a) Biuret Test. — What does this show? (b) Place a little of the solution in a test-tube and heat to boiling. At the boiling-point add a drop of dilute acetic acid and filter. Test this filtrate for proteose with picric acid. Is any proteose present? Saturate another portion of the filtrate with ammonium sulphate and test for peptone in the usual way (see page 59). Do you find any peptone? From your experiments on " living " and " dead " muscle what are your ideas regarding the proteids of muscle? MUSCULAR TISSUE. 215 2. Preparation of Glycogen. — Grind a few scallops in a mortar with sand. Transfer to an evaporating dish, add water and boil for 20 minutes. At the boiling-point faintly acidify with acetic acid. Why is this acid added? Filter, and divide the nitrate into two parts. Note the opalescence of the solu- tion. Test one portion of the filtrate as follows : ( (/ ) Iodine Test. — To 5 c.c. of the solution in a test-tube add 2-3 drops of iodine solution and 2-3 drops of 10 per cent sodium chloride. Warm this slightly and then allow it to cool. What do you observe? Is this similar to the iodine test upon any other body with which we have had to deal? (b) Reduction Test. — Does the solution reduce Fehling's solution? (c) Hydrolysis of Glycogen. — Add 10 drops of concen- trated hydrochloric acid to 10 c.c. of the solution and boil for 10 minutes. Cool the solution, neutralize with solid potassium hydroxide and test with Fehling's solution. Does it still fail to reduce Fehling's solution? If you find a reduction how can you prove the identity of the reducing substance? (d) Influence of Salizv. — Place 5 c.c. of the solution in a test-tube, add 5 drops of saliva and place on the water-bath at 40 C. for 10 minutes. Does this now reduce Fehling's solution? To the second part of the glycogen filtrate add 3-4 volumes of 95 per cent alcohol. Allow the glycogen precipitate to settle, decant the supernatant fluid, filter the remainder and upon the glycogen make the following tests : (a) Solubility. — Try its solubility in the ordinary solvents. (b) Iodine Test. — Place a small amount of the glycogen in a depression of a test-tablet and add a drop of dilute iodine solution and a trace of a sodium chloride solution. The same wine-red color is observed as in the iodine test upon the glycogen solution. Separation of Extractives from Muscle. 1. Creatin. — Dissolve about 10 gram- of a commercial ex- tract of meat in 200 c.c. of warm water. Precipitate the inor- 2l6 PHYSIOLOGICAL CHEMISTRY. ganic constituents by neutral lead acetate, being careful not to add an excess of the reagent. Write the equations for the reactions taking place here. Allow the precipitate to settle., then filter and remove the excess of lead in the warm filtrate by H 2 S. Filter while the solution is yet warm, evaporate the clear filtrate to a syrup and allow it to stand at least 48 hours in a cool place. Crystals of creatin should form at this point. Examine under the microscope (Fig. yy, page 209). Treat the syrup with 200 c.c. of 88 per cent. alcohol, stir well with a glass rod to bring all soluble material into solution, and then filter. The purin bases have been dissolved and are in the filtrate, whereas the creatin crystals were insoluble in the 88 per cent alcohol and remain on the filter paper. Wash the crystals with 88 per cent alcohol, then remove them and bring Fig. 79. Hypoxaxtiiix Silver Nitrate. them into solution in a little hot water. Decolorize the solu- tion by animal charcoal and concentrate it to a small volume. Allow the solution to cool and note the separation of colorless crystals of creatin. Examine these crystals under the micro- scope and compare them with those reproduced in Fig. yy, page 209. MUSCULAR TISSUE. -17 2. Hypoxanthin. — Evaporate the alcoholic filtrate from the creatin to remove the alcohol. Make the solution annnn- niacal and add ammoniacal silver nitrate until precipitation ceases. The precipitate consists principally of hypoxanthin silver and xanthin silver. Collect these silver salts on a filter paper and wash them with water. Place the precipitate and paper in an evaporating dish and boil for one minute with nitric acid having a specific gravity of t.t. Filter while hot through a double paper, wash with the same strength of nitric acid and allow the solution to cool. By this treat- ment with nitric acid hypoxanthin silver nitrate and xan- thin silver nitrate have been formed. The former is in- soluble in the cold solution and separates on standing. After standing several hours filter off the hypoxanthin silver nitrate and wash with water until the wash-water is only slightly acid in reaction. Examine the crystals of hypoxanthin silver ni trate under the microscope and compare them with those in Fig. 79. page 216. Now wash the crystals from the paper into a beaker with a little water and warm the liquid. Remove the silver by H 2 S and filter. By this means hypoxanthin nitrate has been formed and is present in the filtrate. Concentrate on a water-bath to drive off hydrogen sulphide and render the solution slightly alkaline with ammonia. Warm for a time, to remove the free ammonia, filter, concentrate the filtrate to a small volume and allow r it to stand in a cool place. Hypox- anthin should crystallize in small colorless needles. Examine the crystals under the microscope. 3. Xanthin. — To the filtrate from the above experiment containing the xanthin silver nitrate add ammonia in excess. (The crystalline form of xanthin silver nitrate is shown in Fig. 80, p. 218.) A brownish-red precipitate of xanthin silver forms. Treat this suspended precipitate with H 2 S (do not use an excess of H 2 S), warm the mixture for a few moments and filter while hot. Concentrate the filtrate to a small volume and put away in a cool place for crystallization (Fig. 78, p. 210). To obtain xanthin in crystalline form special precautions are 2l8 PHYSIOLOGICAL CHEMISTRY. generally necessary. Evaporate the solution to dryness. Make the following tests on the crystals or residue : (o) Xanthin Test. — Place about one-half of the crystalline or amorphous material in a small evaporating dish, add a few Fig. 80. Xanthin Silver Nitrate. drops of concentrated nitric acid and evaporate to dryness very carefully on a water-bath. The yellow residue upon moistening with caustic potash becomes red in color and upon further heating assumes a purplish-red hue. Now add a few drops of water and warm. In this way a yellow solution re- sults which yields a red residue upon evaporation. How does this differ from the Murexid test upon uric acid ? (b) WeideVs Reaction. — By gently heating bring the re- mainder of the xanthin crystals or residue into solution in bromine-water. Evaporate the solution to dryness on a water- bath. Remove the stopper from an ammonia bottle and by blowing across the mouth of the bottle direct the fumes of ammonia so that they come in contact with the dry residue. Under these conditions the presence of xanthin is shown by the residue assuming a red color. A somewhat brighter color may be obtained by using a trace of nitric acid with the MI'Mi i. \K TISSUE. 219 bromine-water. By the use of this modification however we may get a positive reaction with bodies other than xanthin. Hurtiile's Ex pi-rim ent. Tease a very small piece of frog's muscle on a microscopical slide. Expose the slide to ammonia vapor for a few moments, then adjust a cover glass and examine the muscle fibers under the microscope. Note the large number of crystals of ammo- nium magnesium phosphate, NH 4 — \ Mg — — P = \ / O distributed everywhere throughout the muscle fiber, thus demonstrating the abundance of phosphates and magnesium in the muscle (Fig. 96, page 278). CHAPTER XV. NERVOUS TISSUE. In common with the other solid tissues of the body, nervous tissue contains a large amount of water. The percentage of water present depends upon the particular form of nervous tissue but in all forms it is invariably greater in the gray matter than in the white. Embryonic nervous tissues also contain a larger percentage of water than the tissues of adult life. The gray matter of. the brain of the foetus, for instance, contains about 92 per cent of water, whereas the gray matter of the brain of the adult contains but 83-84 per cent of the fluid. Among the solid constituents of nervous tissue are proteids, cholesterin, cerebrin, lecithin, kephalin, protagon{?) , nuclein, neuro-keratin, collagen/ extractives and inorganic salts. The proteids are present in the greatest amount and comprise about 50 per cent of the total solids. Three distinct proteids, two globulins and a nucleo-proteid, have been isolated from ner- vous tissue. The globulins coagulate at 47 ° C. and 70-75 ° C. respectively, while the nucleo-proteid coagulates at 56-60 C. This nucleo-proteid contains about 0.5 per cent of phosphorus (Halliburton, Levene). Nervous tissue is composed of a rela- tively large quantity of a variety of compounds which col- lectively may be grouped under the term "lipoid" — substances resembling the fats in some of their physical properties and reactions but distinct in their composition. We will class cerebrin, cholesterin and the phosphorized fats, as "lipoids." The group of phosphorized fats are very important con- stituents of nervous tissue. The best known members of this group arc lecithin, protagon ( ?) and kephalin. Lecithin occurs in larger amount than the other members of the group, has been more thoroughly studied than the others and is apparently of greater importance. Upon decomposition lecithin yields N ERVOUS l ISSUE. 22 1 fatty acid, glycero-phosphoric acid and cholin. Each lecithin molecule contains two Tatty acid radicals which may he those of the same or different fatty acids. Thus we have different lecithins depending upon the particular fatty acid radicals which are present in the molecule. The formula of a typical lecithin would be the following: CHoO — C 17 H, 5 CO CHO — C 17 H 35 CO CHoO — PO — 0-C 2 H 4 (CH 3 )iN OH HO ' This lecithin would be called distearyl-lecithin or cholin-dis- tearyl-glycero-phosphoric acid. Upon decomposition the mole- cule splits according to the following reaction : C 44 H 90 NPO + , 3H 2 0=2(C 18 H 36 2 ) + Lecithin. Stearic acid. C 3 H 9 P0 6 + C 5 H ]5 X0 2 . Glycero-phosphoric Cholin. acid. The lecithins are not confined to the nervous tissues but are found in nearly all animal and vegetable tissues. Lecithin is a primary constituent of the cell. It is soluble in chloro- form, ether, alcohol, benzene and carbon disulphide. The chloroform or alcohol-ether solution may be precipitated by acetone. Lecithin may be caused to crystallize in the form of small plates by cooling the alcoholic solution to a low tem- peratures It has the power of combining with acids and bases, and the hydrochloric acid combination has the power of form- ing a double salt with platinic chloride. Protagon, another nitrogenous phosphorized substance is a body over which there has been much discussion. L'pon de- composition it is said by some investigators to yield cerebrin 222 PHYSIOLOGICAL CHEMISTRY. and the decomposition products of lecithin. It has very recently been shown by Posner and Gies that protagon is a mixture and has no existence as a chemical individual. Kephalin is the third member of the group of phosphorized fats. It is precipitated from its acetone-ether extract by alcohol. It contains about 4 per cent of phosphorus and has been given the formula C 42 H 79 NP0 13 . Kephalin may be a stage in lecithin metabolism. Cerebrin, a substance containing nitrogen but no phos- phorus, is an important constituent of the white matter of nervous tissue. It has also been found in the spleen, pus and in egg yolk. It may be extracted from the tissue by boil- ing alcohol and is insoluble in cold alcohol, cold and hot ether and in water and dilute alkalis. Cerebrin is a mixture con- taining phrenosin (pseudo-cerebrin or cerebron), a body yield- ing the carbohydrate galactose on decomposition. Cholesterin, one of the primary cell constituents, is present in fairly large amount in nervous tissue. It is a mon-atomic alcohol with the formula C 27 H 45 OH. It was formerly called a " non-saponifiable fat " but since it is not changed in any way by boiling alkalis it is not a fat. It is soluble in ether, chloroform, benzene and hot alcohol. It crystallizes in the form of thin, colorless, transparent plates (Fig. 42, p. — ). Cholesterin occurs abundantly in one form of biliary calculus. It has also been found in feces, wool iat, egg yolk, and milk, frequently in the form of its esters of higher fatty acids. Nervous tissue yields about 1 per cent of ash which is made up in great part of alkaline phosphates and chlorides. Experiments on the Lipoids of Nervous Tissue. 1 1. Preparation of Lecithin. — Treat the macerated brain of a sheep with ether and allow it to stand in the cold for 1 Preparation of So-called Protagon. — Macerate the brain of a sheep, treat with 85 per cent alcohol and warm on a water-bath at 45° C. for two hours. Filter hot into a bottle or strong flask and cool to 0° C. for one-half hour by means of a freezing mixture. By this procedure both protagon and choles- terin are caused to precipitate. Filter the cold solution rapidly and treat the precipitate on the paper with ice cold ether to dissolve out the choles- terin. The protagon may now be redissolved in warm 85 per cent alcohol from which solution it will precipitate upon cooling. NERVOUS tissue. 223 48-72 hours. The cold ether will extract lecithin and choles- terin. Filter, and t\(](\ acetone to the filtrate to precipitate the lecithin. Filter off the lecithin and test it as follows: (a) Microscopical Examination. — Suspend a small portion in a drop of water on a slide and examine under the micro- scope. (b) Osmic Acid Test. — Treat a small portion with osmic acid. What happens? (c) Acrolein Test. — Make the acrolein test according to directions on page 100. (d) "Fusion" Test for Phosphorus. — Place some of the lecithin prepared above, in a small porcelain crucible, add a suitable amount of a fusion mixture composed of KOH and KN0 3 (5:i) and heat carefully until the resulting mixture is colorless. Cool, dissolve the mass in a little warm water, acidify with HNO :! , heat to boiling and add a few cubic centimeters of molybdic solution. In the presence of phos- phorus a yellow precipitate forms. What is it? 2. Preparation of Cholesterin. — Place a small amount of macerated brain tissue under ether and stir occasionally for one hour. Filter, exaporate the filtrate to dryness on a water- bath and test the cholesterin according to directions given below. (If it is desired, the ether extract from the so-called protagon, or the ether-acetone filtrate from the lecithin may be used for the isolation of cholesterin. In these cases it is simply necessary to evaporate the solution to dryness on a water-bath.) Upon the cholesterin prepared by either of the above methods make the following tests : (a) Microscopical Examination. — Examine the crystals under the microscope and compare them with those in Fig. 42, page 125. (b) Iodine-Sulphuric Acid Test. — Place a few crystals of cholesterin in one of the depressions of a test-tablet and treat with a drop of concentrated sulphuric acid and a drop of a very dilute solution of iodine. A play of colors, consisting of violet, blue, green and red, results. 224 PHYSIOLOGICAL CHEMISTRY. (c) The Liebermann-Bur chard Test. — Dissolve a few crys- tals of cholesterin in 2 c.c. of chloroform in a dry test-tube. Now add 10 drops of acetic anhydride and 1-3 drops of con- centrated sulphuric acid. The solution becomes red, then blue, and finally bluish-green in color. (d) Salkowski's Test. — Dissolve a few crystals of choles- terin in a little chloroform and add an equal volume of con- centrated sulphuric acid. A play of colors from bluish-red to cherry-red and purple is noted in the chloroform, while the acid assumes a marked green fluorescence. (e) Schiff's Reaction. — To a little cholesterin in an evapor- ating dish add a few drops of Schiff's reagent. 1 Evaporate to dryness over a low flame and observe the reddish-violet residue which changes to a bluish-violet. (/) Phosphorus. — Test for phosphorus according to direc- tions given on page 223. Is phosphorus present? 3. Preparation of Cerebrin. — Treat the macerated brain tissue, in a flask, with 95 per cent alcohol and boil on a water- bath for one-half hour, keeping the volume constant by adding fresh alcohol as needed. Filter the solution hot and stand the cloudy filtrate away for twenty-four hours. (If the fil- trate is not cloudy concentrate it upon the water-bath until it is so. ) Filter off the cerebrin and test it as follows : (a) Microscopical Examination. — Suspend a small portion in a drop of water on a slide and examine under the micro- scope. (b) Solubility. — Try the solubility of cerebrin in the usual solvents and in hot and cold alcohol and hot and cold ether. (c) Phosphorus. — Test for phosphorus according to direc- tions on page 223. How does the result compare with that on lecithin. (d) Place a little cerebrin on platinum foil and warm. Note the odor. (nditi< >ns. Variations between — 0.5 1 and — 0.62 ° C. may be due entirely to dietary conditions but if any marked variation is noted it can, in most cases, be traced to a disordered kidney function. Freezing-point determinations may be made by means of the Beckmann- Ileidenhain apparatus (Fig. 84. p. 233 ) or the Zikel Pektoscope. The Beck- mann-Heidenhain apparatus consists of the following parts : A strong bat- ter}- jar or beaker (C) furnished with a metal cover which is provided with a circular hole in its center. This strong glass vessel serves to hold the freezing mixture by means of which the temperature of the fluid under ex- amination is lowered. A large glass tube (B) designed as an air-jacket, and formed after the manner of a test-tube is introduced through the central aperture in the metal cover Fig. 84. 1 Beckmanm - Heiden MAIN Freezing-poj nt Ap- paratus. (Long.) D, a delicate thermom- eter ; C, the containing jar; B, the outside or air mantle tube; A, the tube in which the mixture to be observed is placed. Two stirrers are shown, one for the cooling mix- ture in the jar and one for the experimental mix- ture. 234 PHYSIOLOGICAL CHEMISTRY. and into this air-jacket is lowered a smaller tube (A) con- taining the fluid to be tested. A very delicate thermom- eter (D), graduated in hundredths of a degree is intro- duced into the inner tube and is held in place by means of a cork so that the mercury bulb is immersed in the fluid under examination but does not come in contact with any glass sur- face. A small platinum wire stirrer serves to keep the fluid under examination well mixed while a larger stirrer is used to manipulate the freezing mixture. (Rock salt and ice in the proportion I 13 form a very satisfactory freezing mixture.) In making a determination of the freezing-point of a fluid by means of the Beckmann-Heidenhain apparatus proceed as follows : Place the freezing mixture in the battery jar and add water (if necessary) to secure a temperature not lower than 3° C. Introduce the fluid to be tested into tube A, place the thermometer and platinum wire stirrer in position and insert the tube into the air jacket which has previously been inserted through the metal cover of the battery jar. Manipulate the two stirrers in order to insure an equalizataion of temperature and observe the course of the mercury column of the ther- mometer very carefully. The mercury will gradually fall and this gradual lowering of the temperature will be followed by a sudden rise. The point at which the mercury rests after this sudden rise is the freezing-point. This rise is due to the fact that previous to freezing, a fluid is always more or less over cooled and the thermometer temporarily registers a tem- perature somewhat below the freezing-point. As the fluid freezes however there is a very sudden change in the tem- perature of the liquid and this change is imparted to the ther- mometer and causes the rise as indicated. It occasionally occurs that the fluid under examination is very much over cooled and does not freeze. Under such circumstances a small piece of ice is introduced into it by means of the side tube noted in the figure. This so-called " inoculation " causes the fluid to freeze instantaneously. (For details of the method URINE. 235 of determining tin- freezing-point consult standard works on physical or organic chemistry.) Electrical Conductivity. — The electrical conductivity of the urine is dependent up »n the number of inorganic molecules « »r ions present, and in this differs from the freezing-point which is dependent upon the total number of molecules both inorganic and organic which are in solution. The conductivity of the urine has been investigated but slightly, and this very recently, but from the data secured it seems that the value generally falls below ^ = 0.03. The conductivity of blood serum has been determined as « =0.012. Up to the present time the determination of the electrical conductivity of any of the fluids of the body has been put to very slight clinical use. Experience may show the conductivity value to be a more important aid to diagnosis than it is now considered particu- larly if it is taken in connection with the determination of the freezing-point. By a combination of these two methods the portion of the osmotic pressure due respectively to electrolytes and non-electrolytes may be determined. For a discussion of electrical conductivity, the method by which it is determined and the principles involved consult standard works on physical or electrochemistry. Collection of the Urine Sample. — If any dependable data are desired regarding the quantitative composition of the urine the examination of the mixed excretion for twenty-four hours is absolutely necessary. In collecting the urine the bladder may be emptied at a given hour, say 8 A. M.. the urine dis- carded and all the urine from that hour up to and including that passed the next day at 8 A. M. saved, thoroughly mixed and a sample taken for analysis. Powdered thymol, CH, A OoH ■ CH 3 — CH — CH 3 , 236 PHYSIOLOGICAL CHEMISTRY. is a very satisfactory preservative since the excess may be re- moved by filtration, if desired, and any small amount which may go into solution will have no appreciable influence upon the determination of any of the urinary constituents. It has no reducing power and so may safely be used to preserve dia- betic urines. To insure the preservation of the mixed urine of the twenty- four hour period it is advisable to place a small amount of the thymol powder in the urine receptacle before the first fraction of urine is voided. In certain pathological conditions it is desirable to collect the urine passed during the day separately from that passed during the night. When this is done the urine voided between 8 A. M. and 8 P. M. may be taken as the day sample and that voided between 8 P. M. and 8 A. M. as the night sample. The qualitative testing of urine voided at random, except in a few specific instances, is of no particular value so far as giving us any accurate knowledge as to the exact urinary characteristics of the individual is concerned. In the great majority of cases the qualitative as well as the quantitative tests should be made upon the mixed excretion for a twenty- four hour period. CHAPTER XVII. URINE: PHYSIOLOGICAL CONSTITUENTS. i. Organic Physiological Constituents. Urea. I'ric acid. Creatinin. Ethereal sulphuric acids. Hippuric acid. ( ).\alic acid. Neutral sulphur compounds. Allantoin. Aromatic oxvacids - Phenol- and />-cresol-sulphuric acids. Pyrocatechin-sulphuric acid. Indoxyl-sulphuric acid. Skatoxyl-sulphuric acid. Cystin. Chondroitin-sulphuric acid. Sulphocyanides. Taurin derivatives. Oxyproteic acid. Alloxyproteic acid. Uroferric acid. I \araoxyphenyl-acetic acid. Paraoxyphenyl-propionic acid. Homogentisic acid. Uroleucic acid. Oxymandelic acid. Kvnurenic acid. Benzoic acid. Xucleo-proteid. Oxaluric acid. 1 It is impossible to make any absolute classification of the physiological and pathological constituents of the urine. A substance may be present in the urine in small amount physiologically and be sufficiently increased under certain conditions as to be termed a pathological constituent. Therefore it depends, in some instances, upon the quantity of a constituent present whether it may be correctly termed a physiological or a patholog- ical constituent. ^37 238 PHYSIOLOGICAL CHEMISTRY. Enzymes i?:P sil \ ,. , N [Diastatic enzyme (Amylase), f Acetic acid. Volatile fatty acids -I Butyric acid. L Formic acid. Paralactic acid. Phenaceturic acid. Phosphorized compounds. . . ( Glvcerophosphoric acid. 1 t Phospnocarnic acid. fUrochrome. Pigments 1 Urobilin. L Uroerythrin. Ptomaines and leucomaines. Purin bases ' Adenin. Guanin. Xanthin. Epiguanin. Episarkin. Hypoxanthin. Paraxanthin. Heteroxanthin. . i-Methylxanthin. 2. Inorganic Physiological Constituents. Ammonia. Sulphates. Chlorides. Phosphates. Sodium and potassium. Calcium and magnesium. Carbonates. Iron. Fluorides. Nitrates. Silicates. Hydrogen peroxide. URINE. 239 I UREA, ( j = O. I NH, Urea is the principal end-product of the metabolism of pro- teid bodies. It has been generally believed that about 90 per cent of the total nitrogen of the urine was present as urea. Recently, however, Folin has shown that the distribution of the nitrogen of the urine among urea and the other nitrogen- containing bodies present depends entirely upon the absolute amount of the total nitrogen excreted. He found that a de- crease in the total nitrogen excretion was always accompanied by a decrease in the percentage of the total nitrogen excreted as urea, and that after so regulating the diet of a normal per- Fig. 85. Urea. son as to cause the excretion of total nitrogen to be reduced to 3-4 grams in 24 hours, only about 60 per cent of this nitro- gen appeared in the urine as urea. His experiments also seem to show urea to be the only one of the nitrogenous excretions which is relatively as well as absolutely decreased as a result 24O PHYSIOLOGICAL CHEMISTRY. of decreasing the amount of proteid metabolized. This same investigator reports a hospital case in which only 14.7 per cent of the total nitrogen was present as urea and about 40 per cent was present as ammonia. Morner had previously reported a case in which but 4.4 per cent of the total nitrogen of the urine was present as urea, and 26.7 per cent was present as ammonia. Urea occurs most abundantly in the urine of man and car- nivora and in somewhat smaller amount in the urine of herbi- vora; the urine of fishes, amphibians and certain birds also contains a small amount of the substance. Urea is also found in nearly all the fluids and in many of the tissues and organs of mammals. The amount excreted under normal conditions, by an adult man in 24 hours is about 30 grams ; women excrete a somewhat smaller amount. The excretion is greatest in amount after a diet of meat, and least in amount after a diet consisting of non-nitrogenous foods; this is due to the fact that the last mentioned diet has a tendency to decrease the metabolism of the tissue proteids and thus cause the output of urea under these conditions to fall below the output of urea observed during starvation. The output of urea is also in- creased after copious water- or beer-drinking. This increase is due primarily to the washing out of the tissues of the urea previously formed, but which had not been removed in the nor- mal processes, and, secondarily to a stimulation of proteid catabolism. Urea may be formed in the organism from amino acids such as leucin, glycocoll and aspartic acid: it may also be formed from ammonium carbonate (NH 4 ) 2 C0 3 or ammonium carbamate, H 4 N ■ O ■ CO ■ NH 2 . There are differences of opinion regarding the transforma- tion of the substances just named into urea but there is rather conclusive evidence that at least a part of the urea is formed in the liver; it maybe formed in other organs or tissues as well. Urea crystallizes in long, colorless, four or six-sided, anhy- drous, rhombic prisms (Fig. 85, p. 239), which melt at 132 C. and are soluble in water or alcohol and insoluble in ether or URINE. 241 chloroform. If a crystal of urea is heated in a test-tube, it melts and decomposes with the liberation of ammonia. The residue contains cyanuric acid, A N N II I HO ■ C • OH \// N and biuret, NH 2 C = ^>XH C = I NHo. The biuret may be dissolved in water and a reddish-violet color obtained by treating the aqueous solution with cupric sulphate and potassium hydroxide (see Biuret Test, p. 45). Certain hypochlorites or hypobromites in alkaline solution have the power of decomposing urea into nitrogen, carbon dioxide and water. Sodium hypobromite brings about this decomposition as follows : CO(XH 2 ) 2 + 3XaOBr = 3NaBr + N 2 + C0 2 + 2H 2 0. This property forms the basis for the clinical quantitative determination of urea (see page 35 1 ) . Urea has the power of forming crystalline compounds with certain acids : urea nitrate and urea oxalate are the most im- portant of these compounds. Urea nitrate, CO(NH 2 ) 2 ' HXOo. crystallizes in colorless, rhombic or six-sided tiles (Fig. 86, p. 242), which are easily soluble in water. Urea oxalate, 2 • CO(XH 2 ) 2 - H 2 C 2 4 , crystallizes in the form of rhombic "r six-sided prisms or plates (Fig. 88, p. 244) : the oxalate differs from the nitrate in being somewhat less soluble in water. 17 242 PHYSIOLOGICAL CHEMISTRY. A decrease in the excretion of urea is observed in many- diseases in which the diet is much reduced and in some dis- orders as a result of alterations in metabolism, e. g. } myxce- dema, and in others as a result of changes in excretion, as in Fig. 86. Urea Nitrate. severe and advanced kidney disease. A pathological increase is found in a large proportion of diseases which are asso- ciated with a toxic state. Experiments on Urea. 1. Isolation from the Urine. — Place 800 c.c. of urine in a precipitating jar, add 250 c.c. of baryta mixture 1 and stir thor- oughly. Filter off the precipitate of phosphates, sulphates, urates and hippurates and evaporate the filtrate on a water- bath to a thick syrup. This syrup contains chlorides, creatinin, organic salts, pigments and urea. Extract the syrup with warm 95 per cent alcohol and filter again. The filtrate contains the urea contaminated with pigment. Decolorize the filtrate by boiling with animal charcoal, filter again and stand the 1 Baryta mixture consists of a mixture of one volume of a saturated solution of Ba(N0 3 ) 2 and two volumes of a saturated solution of Ba (OH) 2 . URINE. 2 43 Fig. 87. filtrate away in a cold place for crystallization. Examine the crystals under the microscope and compare them with those shown in Fig. 85, page 239. 2. Solubility. — Test the solubility of urea, prepared by yourself or furnished by the instructor, in the ordinary sol- vent- (see p. 4) and in alcohol and ether. 3. Melting-Point. — Determine the melting point of somepure urea furnished by the instructor. Proceed as follows: Into an ordinary melting-point tube, sealed at one end, introduce a crystal of urea. Fasten the tube to the bulb of a thermometer as shown in Fig. 87, p. 243, and suspend the bulb and its attached tube in a small beaker containing sul- phuric acid. Gently raise the tempera- ture of the acid by means of a low flame, stirring the fluid continually, and note the temperature at which the urea begins to melt. 4. Crystalline Form. — Dissolve a crystal of pure urea in a few drops of 95 per cent alcohol and place 1-2 drops of the alcoholic solution on a microscopic slide. Allow the alcohol to evaporate spontaneously, examine the crystals under the microscope and compare them with those reproduced in Fig. 85, p. 239. Re- crystallize a little urea from water in the same way and compare the crystals with those obtained from the alcoholic solution. 5. Formation of Biuret. — Place a small amount of urea in a dry test-tube and heat carefully in a low flame. The urea melts at 132 C. and liberates ammonia. Continue heating until the fused mass begins to solidify. Cool the tube, dis- solve the residue in dilute potassium hydroxide 'solution and add very dilute cupric sulphate solution (see p. 45). The Melting-point Tubes Fastened to Bui.b of Thermometer. 244 PHYSIOLOGICAL CHEMISTRY. purplish-violet color is due to the presence of biuret which has been formed from the urea through the application of heat as indicated. This is the reaction: NH 2 2 C = NH 2 Urea. NH 2 C = \ NH + NH a / C = NH 2 Biuret. 6. Urea Nitrate. — Prepare a concentrated solution of urea by dissolving a little of the substance in a few drops of water. Place a drop of this solution on a microscopic slide, add a drop Fig. Urea Oxalate. of concentrated nitric acid and examine under the microscope. Compare the crystals with those reproduced in Fig. 86, p. 242. 7. Urea Oxalate. — To a drop of a concentrated solution of urea, prepared as described in the last experiment(6),add a drop URINE. 245 of a saturated solution of oxalic acid. Examine under the microscope and compare the crystals with those shown in Fig. 88. page 244. 8. Decomposition by Sodium-Hypobromite. — Into a mixture of 3 c.c. of concentrated sodium hydroxide solution and 2 c.c. of bromine water in a test-tube introduce a crystal of urea or a small amount of a concentrated solution of urea. Through the influence of the sodium-hypobromite, XaOBr, the urea is decomposed and carbon dioxide and nitrogen are liberated. The carbon dioxide is absorbed by the excess of sodium hydroxide while the nitrogen is evolved and causes the marked effervescence observed. This property forms the basis for one of the methods in common use for the quanti- tative determination of urea. Write the equation showing the decomposition of urea by sodium-hypobromite. 9. Furfurol Test. — To a few crystals of urea in a small porcelain dish add 1-2 drops of a concentrated aqueous solu- tion of furfurol and 1-2 drops of concentrated hydrochloric acid. Note the appearance of a yellow color which gradually changes into a purple. Allantoin also responds to this test (see page 261). HX — CO I I URIC ACID, OC C- NH V I II >C0. HN — C — NET Uric acid is one of the most important of the constituents of the urine. Normally about 0.7 gram is excreted in 24 hours but this amount is subject to wide variations, particularly under certain dietary and pathological conditions. Uric acid is a diureide and consequently upon oxidation yields two molecules of urea. It acts as a weak dibasic acid and forms two classes of salts, neutral and acid. The neutral potassium and lithium urates are the most easily soluble of the alkali salts; the am- monium urate is difficultly soluble. The acid-alkali urates are more insoluble and form the major portion of the sediment 246 PHYSIOLOGICAL CHEMISTRY. which separates upon cooling concentrated urine; the alkaline earth urates are very insoluble. Ordinarily uric acid occurs in the urine in the form of urates and upon acidifying the liquid the uric acid is liberated and deposits in crystalline form. This property forms the basis for one of the older methods for the quantitative determination of uric acid (Heintz Method, p. 350) . Uric acid is very closely related to the purin bases as may be seen from a comparison of its structural formula with those of the purin bases given on page 211. According to the purin nomenclature it is designated 2-6-8-trioxypurin. Uric acid forms the principal end-product of the nitrogenous meta- bolism of birds and scaly amphibians ; in the human organism it occupies the fourth position inasmuch as here urea, am- monia and creatinin are the chief end-products of nitrog- enous metabolism. The relation existing between uric acid and urea in human urine under normal conditions varies on the average from 1 : 40 to 1: 100 and is subject to wider varia- tions under pathological conditions. Because of the high content of uric acid in the urine of new-born infants the ratio may be reduced to 1 : 10 or even lower. In man, uric acid probably results principally from the destruction of nuclein material. It may arise from nuclein or other purin material ingested as food or from the disin- tegrating cellular matter of the organism. The uric acid resulting from the first process is said to be of exogenous origin, whereas the product of the second form of activity is said to be of endogenous origin. As the result of experimen- tation, Siven, and Burian and Schur, and Rockwood claim that the amount of endogenous uric acid formed in any given period is fairly constant for each individual under normal conditions, and that it is entirely independent of the total amount of nitrogen eliminated. Recently Folin has taken ex- ception to the statements of these investigators and claims that, following a pronounced decrease in the amount of pro- teid metabolized, the absolute quantity of uric acid is decreased but that this decrease is relatively smaller than the decrease PLATE V. Uric Acid Crystals. Normal Color. (From Purely, after Peyer.) URINE. -}7 in the total nitrogen excretion and that the per cent of the uric acid nitrogen, in terms of the total nitrogen, is therefore de- cidedly increased. In birds and scaly amphibians the formation of uric acid is analogous to the formation of urea in man. In these or- ganisms it is derived principally from the proteid material of the tissues and the food and is formed through a process of synthesis which occurs for the most part in the liver; a comparatively small fraction of the total uric acid excretion of birds and scaly amphibians may result from nuclein material. When pure, uric acid may be obtained as a white, odorless, and tasteless powder which is composed principally of small transparent crystalline rhombic plates. Uric acid as it sepa- rates from the urine is invariably pigmented, and crystallizes in a large variety of characteristic forms, c. g., dumb-bells, wedges, rhombic prisms, irregular rectangular or hexagonal plates, whetstones, prismatic rosettes, etc. Uric acid is in- soluble in alcohol and ether, soluble with difficulty in boiling water {i : 1800) and practically insoluble in cold water (1:39.480, at i8°C). It is soluble in alkalis, alkali car- bonates, boiling glycerin, concentrated sulphuric acid and in certain organic bases such as ethylamine and piperidin. It is claimed that the uric acid is held in solution in the urine by the urea and di-sodium hydrogen phosphate present. Uric acid possesses the power of reducing cupric hydroxide in alkaline solution and may thus lead to an erroneous conclusion in testing for sugar in the urine by means of Fehling's or Trommer's tests. A white precipitate of cuprous urate is formed if only a small amount of cupric hydroxide is present, but if enough of the copper salt is present the characteristic red or brownish-red precipitate of cuprous oxide is obtained. Uric acid does not possess the power of reducing bismuth in alkaline solution and therefore does not interfere in testing for sugar in the urine by means of Boettger's or Xy lander's tests. In addition to being an important urinary constituent uric acid is normally present in the brain, heart, liver, lungs, pan- 248 PHYSIOLOGICAL CHEMISTRY. creas and spleen; it also occurs in the blood of birds and has been detected in traces in human blood under normal condi- tions. Pathologically, the excretion of uric acid is subject to wide variations but the experimental findings are rather contradic- tory. It may be stated with certainty, however, that in leukaemia the uric acid output is increased absolutely as well as relatively to the urea output; under these conditions the ratio between the uric acid and urea may be as low as 1:9, whereas the normal ratio, as we have seen, is 1 150 or higher. In the study of the influence of X-ray on metabolism Edsall has very recently reached some interesting conclusions. He found that the excretion of uric acid is usually increased and that in some conditions, particularly in leukaemia, it may be greatly increased. The excretion of total nitrogen, phos- phates and other substances may also be considerably increased. Experiments on Uric Acid. 1. Isolation from the Urine. — Place about 200 c.c. of filtered urine in a beaker, render it acid with 2-10 c.c. of con- centrated hydrochloric acid, stir thoroughly and stand the vessel in a cold place for 24 hours. Examine the pigmented crystals of uric acid under the microscope and compare them with those shown in Fig. 101, p. 323 and PI. V. opposite p. 247. 2. Solubility. — Try the solubility of pure uric acid, fur- nished by the instructor, in the ordinary solvents (see p. 4) and in alcohol, ether, concentrated sulphuric acid and in boiling glycerin. 3. Crystalline Form of Pure Uric Acid. — Place about 100 c.c. of water in a small beaker, render it distinctly alkaline with potassium hydroxide solution and add a small amount of pure uric acid stirring continuously. Cool the solution, render it distinctly acid with hydrochloric acid and allow it to stand in a cool place for crystallization. Examine the crystals under the microscope and compare them with those reproduced in Fig. 89, page 249. URINE. 249 4. Murexid Test. — To a small amount of pure uric acid in a small evaporating dish add 2-3 drops of concentrated nitric acid. Evaporate to dryness carefully on a water-bath or over a very low flame. A red or yellow residue remains Fig. 89. Pure Uric Acid. which turns purplish-red after cooling the dish and adding a drop of very dilute ammonium hydroxide. The color is due to the formation of murexid. If potassium hydroxide is used instead of ammonium hydroxide a purplish-violet color due to the production of the potassium salt is obtained. The color disappears upon warming; with certain related bodies (purin bases) the color persists under these conditions. 5. Scruff's Reaction. — Dissolve a small amount of pure uric acid in sodium carbonate solution and transfer a drop of the resulting mixture to a strip of filter paper saturated with argentic nitrate solution. A yellowish-brown or black colora- tion due to the formation of reduced silver is produced. 6. Influence upon Fehling's Solution. — Dilute 1 c.c. of Fehling's solution with 4 c.c. of water and heat to boiling. Xow add slowly, a few drops at a time, 1-2 c.c. of a concen- trated solution of uric acid in potassium hydroxide, heating 250 PHYSIOLOGICAL CHEMISTRY. after each addition. From this experiment what do you con- clude regarding the possibility of arriving at an erroneous decision when testing for sugar in the urine by means of Fehling's test? 7. Reduction of Nylander's Reagent. — To 5 c.c. of a solu- tion of uric acid in potassium hydroxide add about one-half a cubic centimeter of Nylander's reagent and heat to boiling for a few moments. Do you obtain the typical black end- reaction signifying the reduction of' the bismuth? NH CO CREATININ, C = NH I I I N-CH 3 -CH 2 . Creatinin is the anhydride of creatin and is a constituent of normal human urine. It is derived from the creatin of in- gested muscular tissue as well as from the creatin of the muscular tissue of the organism. Under normal conditions about 1 gram of creatinin is excreted by an adult man in 24 hours, the exact amount depending in great part upon the nature of the food and decreasing markedly in starvation. Very little that is ' important is known regarding the excre- tion of creatinin under pathological conditions. The creatinin content of the urine is said to be increased in typhoid fever, typhus, tetanus and pneumonia, and to be decreased in anaemia, chlorosis, paralysis and in advanced degeneration of the kid- neys. The greater part of the data, however, relating to the variation of the creatinin excretion under pathological condi- tions are not of much value since, in nearly every instance, the diet was not sufficiently controlled to permit the collection of reliable data. Creatinin crystallizes in colorless, glistening monoclinic prisms (Fig. 90, p. 251) which are soluble in about 12 parts of cold water; they are more soluble in warm water and in warm alcohol. One of the most important and interesting of the compounds of creatinin is creatinin-zinc chloride, t'RIXK. 251 (C 4 H 7 N 3 0)oZnCl 1 ,, which is formed from an alcoholic solu- tion of creatinin upon treatment with zinc chloride in acid solution. Creatinin has the power of reducing cupric hy- droxide in alkaline solution and in this way may interfere with the determination of sugar in the urine. In the reduction by creatinin the blue liquid is first changed to a yellow and the formation of a brownish-red precipitate of cuprous oxide is brought about only after continuous boiling with an excess of the copper salt. Creatinin does not reduce alkaline bismuth solutions and therefore does not interfere with Nylander's and Boettger's tests. I' ig. 90. Creatinin. It has very recently been shown by Folin that the absolute quantity of creatinin eliminated in the urine on a meat-free diet is a constant quantity different for different individuals, but wholly independent of quantitative changes in the total amount of nitrogen eliminated. Experiments on Creatinin. 1. Separation from the Urine. — Place 250 c.c. of urine in a casserole or beaker, render it alkaline with milk of lime and then add CaCl 2 solution until the phosphates are completely 252 PHYSIOLOGICAL CHEMISTRY. precipitated. Filter off the precipitate, render the filtrate slightly acid with acetic acid and evaporate it to a syrup. While still warm this syrup is treated with about 50 c.c. of 95-97 per cent alcohol and the mixture allowed to stand 8-12 hours in a cool place. The precipitate is now filtered off and the filtrate treated with a little sodium acetate and about one- half c.c. of acid-free zinc chloride solution having a specific gravity of 1.2. This mixture is stirred thoroughly and allowed to stand in a cold place for 48-72 hours. Creatinin-zinc chloride (Fig. 91, below) will crystallize out under these con- Fig. 91. Creatinin-Zinc Chloride. (Salkozi'ski.) ditions. Collect the crystals on a filter paper and wash them with alcohol to remove chlorides. Now treat the zinc chloride compound with a little warm water, boil with lead oxide and filter. The filtrate may now be decolorized by animal charcoal, evaporated to dryness and the residue extracted with strong alcohol. (Creatin remains undissolved under these condi- tions.) The alcoholic extract of creatinin is now evaporated to incipient crystallization and left in a cool place until crys- tallization is complete. If desired the crystals may be purified by recrystallization from water. 2. Weyl's Test. — Take 5 c.c. of urine in a test-tube, add a URINE. 253 few drops of sodium nitro-prusside and render the solution alkaline with potassium hydroxide solution. A ruby red color results which soi in turns yellow. See Legal's test for ace- lour, page 305. 3. Salkowski's Test. — To the yellow solution obtained in WVyl's test above add an excess of acetic acid and apply heat. A green color results and is in turn displaced by a blue color. A precipitate of Prussian blue may form. 4. Jaffe's Reaction. — Place 5 c.c. of urine in a test-tube, add an aqueous solution of picric acid and render the mixture alkaline with potassium hydroxide solution. A red color is produced which turns yellow if the solution be acidified. Dex- trose gives a similar red color but only upon the application of heat. This color reaction observed when creatinin in alka- line solution is treated with picric acid is the basic principle of Folin's colorimetric method for the quantitative determi- nation of creatinin (see page 369). ETHEREAL SULPHURIC ACIDS. The most important of the ethereal sulphuric acids found in the urine are phenol-sulphuric acid, p-crcsol-sulphuric acid, iihiihvyl-sulphitric acid and skatoxyl-sulphuvic acid. Pyro- catechin-sulphuric acid also occurs in traces in human urine. The total output of ethereal sulphuric acid varies from 0.09 to 0.62 gram for 24 hours. In health the ratio of ethereal sul- phuric acid to inorganic sulphuric acid is about 1 : 10. These ethereal sulphuric acids originate in part from the phenol, cresol, indol and skatol formed in the putrefaction of proteid material in the intestine. The phenol passes into the urine directly as the corresponding ethereal sulphuric acid whereas the indol and skatol undergo a preliminary oxidation to form indoxyl and skatoxyl respectively before their elimination. It has generally been considered that each of the ethereal sulphuric acids was formed principally in the putrefaction of proteid material in the intestine and that therefore a determi- nation of the total ethereal sulphuric acid content of the urine was an index of the extent to which these putrefactive proc- 254 PHYSIOLOGICAL CHEMISTRY. esses were proceeding- within the organism. Recently, how- ever, Folin has conducted a series of experiments which seem to show that the ethereal sulphuric acid content of the urine does not afford an index of the extent of intestinal putrefac- tion,, since these bodies arise only in part from putrefactive processes. He claims that the ethereal sulphuric acid excretion represents a form of sulphur metabolism which is more in evidence upon a diet containing a very small amount of pro- teid or upon a diet containing absolutely no proteid. The ethereal sulphuric acid content of the urine diminishes as the total sulphur content diminishes but the percentage decrease is much less. Therefore when considered from the stand- point of the total sulphuric acid content the ethereal sulphuric acid content is not diminished but is increased, although the total sulphuric acid content is diminished. Folin's experi- ments also seem to show that the indoxyl sulphuric acid (potassium indoxyl sulphate or indican) content of the urine does not originate to any degree from the metabolism of proteid material but that it arises in great part from intes- tinal putrefaction and that the excretion of indoxyl sulphuric acid may alone be taken as a rough index of the extent of putrefactive processes within the intestine. Indoxyl sulphuric acid, CH //\ HC C — C(0-SO,H), I II II HC C CH V \/ CH NH therefore, which occurs in the urine as potassium indoxyl sul- phate or indican, CH //\ HC C — C(0-SO,K), I II II HC C CH V \/ CH NH is clinically the most important of the ethereal sulphuric acids. URINE. Ti SI S FOR [NDICAN. i. Jaffe's Test. — Nearly fill a test-tube with a mixture com- posed of equal volumes of concentrated HC1 and the urine under examination. Add 2-3 c.c. of chloroform and a few drops of a calcium hypochlorite solution, place the thumb over the end of the test-tube and shake thoroughly. The chloro- form is colored more or less, according to the amount of indican present. Ordinarily a blue color due to the formation of indigo-blue is produced; less frequently a red color due to indigo-red may be noted. This is the reaction (see also pages 129 and 130) : CH //\ HC C-C-OH 2 I || II -'20 = HC C CH V \/ CH NH Indoxyl. C,H,NO. CH CH //\ /\ HC C — C • • C — C CH I I II I II + 2H 2 HC C C C C CH V \/ \/ \// CH NH XH CH Indigo-blue. C 18 H 10 N 5 2 . 2. Obermayer's Test. — Nearly fill a test-tube with a mix- ture composed of equal volumes of Obermayer's reagent 1 and the urine under examination. Add 2-3 c.c. of chloroform, place the thumb over the end of the test-tube and shake thor- oughly. How does this compare with Jaffe's test? C0NHCH 2 C00H. HIPPURIC ACID, I \/ 1 Obermayer's reagent is prepared by adding 2-4 grams of ferric chlo- ride to a liter of concentrated HC1 (sp. gr. I.19). 256 PHYSIOLOGICAL CHEMISTRY. This acid occurs normally in the urine of both the carnivora and herbivora but is more abundant in the urine of the latter. It is formed by a synthesis of benzoic acid and glycocoll which takes place in the kidneys. The average excretion of an adult man for 24 hours under normal conditions is about 0.7 gram. Hippuric acid crystallizes in needles or rhombic prisms (see Fig. 92. Hippuric Acid. Fig. 92. above), the particular form depending upon the ra- pidity of crystallization. It is easily soluble in alcohol or hot water, and only slightly soluble in ether. The output of hip- puric acid is increased in diabetes owing probably to the inges- tion of much proteid and fruit. It is decreased in fevers and in certain kidney disorders where the synthetic activity of the renal cells is diminished. Experiments on Hippuric Acid. 1. Separation from the Urine. — Render 500-1000 c.c. of urine of the horse or cow 1 alkaline with milk of lime, boil for 1 If urine of the horse or cow is not available human urine may serve the purpose fully as well provided means are taken to increase its content of hippuric acid. This may be conveniently accomplished by ingesting 2 URINE. 257 a few moments and filter while hot. Concentrate the filtrate, over a burner, to a small volume. Cool the solution, acidify it strongly with concentrated hydrochloric acid and stand it in a cool place for 24 hours. Filter off the crystals of hippuric acid which have formed and wash them with a little cold- water. Remove the crystals from the paper, dissolve them in a very small amount of hot water and percolate the hot solution through thoroughly washed animal charcoal, being cue ful to wash out the last portion of the hippuric acid solu- tion with hot water. Filter, concentrate the filtrate to a >mall volume and stand it aside for crystallization. Examine the crystals under the microscope and compare them with those in Fig. 92, page 256. 2. Melting-Point. — Determine the melting-point of the hippuric acid prepared in the above experiment (see p. 243). 3. Solubility. — Test the solubility of hippuric acid in the ordinary solvents (page 4) and in alcohol, and ether. 4. Formation of Nitro-Benzene. — To a little hippuric acid in a small porcelain dish add 1-2 c.c. of concentrated HN0 3 and evaporate to dryness on a water-bath. Transfer the residue to a dry test-tube, apply heat and note the odor of the artificial oil of bitter almonds (nitro-benzene). 5. Sublimation. — Place a few crystals of hippuric acid in a dry test-tube and apply heat. The crystals are reduced to an oily fluid which solidifies in a crystalline mass upon cooling. When stronger heat is applied the liquid assumes a red color and finally yields a sublimate of benzoic acid and the odor of hydrocyanic acid. 6. Formation of Ferric Salt. — Render a small amount of a solution of hippuric acid neutral with dilute potassium hydroxide. Now add 1-3 drops of neutral ferric chloride solution and note the formation of the ferric salt of hippuric acid as a cream colored precipitate. grams of ammonium benzoate at night. The fraction of urine passed in the morning will be found to have a high content of hippuric acid. The ammonium benzoate is in no way harmful. 18 258 PHYSIOLOGICAL CHEMISTRY. COOH OXALIC ACID, I COOH. Oxalic acid is a constituent of normal urine, about 0.02 gram being eliminated in 24 hours. It is present in the urine as calcium oxalate, which is kept in solution through the medium of the acid phosphates. The origin of the oxalic acid content of the urine is not well understood. It is eliminated, at least in part, unchanged when ingested, therefore since many of the common articles of diet, e. g., asparagus, apples, cabbage, grapes, lettuce, spinach, tomatoes, etc., contain oxalic acid it seems probable that the ingested food supplies a por- tion of the oxalic acid found in the urine. There is also ex- perimental evidence that part of the oxalic acid of the urine is formed within the organism in the course of proteid and fat metabolism. It has also been suggested that oxalic acid may arise from an incomplete combustion of carbohydrates, especially under certain abnormal conditions. Pathologically, oxalic acid is found to be increased in amount in diabetes mellitus, in organic diseases of the liver and in various other conditions which are accompanied by a derangement of the oxidation mechanism. An abnormal increase of oxalic acid is termed oxahtria. A considerable increase in the content of oxalic acid may be noted unaccompanied by any other apparent symptom. Calcium oxalate crystallizes in at least two distinct forms, dumb-bells and octahedra (Fig. 99, page 320). Experiments. 1. Preparation of Calcium Oxalate. — Place 200-250 c.c. of urine in a beaker, add 10 drops of a saturated solution of oxalic acid and stand the beaker aside in a cool place for 24 hours. Examine the sediment under the microscope and com- pare the crystalline forms with those shown in Fig. 99, p. 320. 2. Solubility. — Test the solubility of calcium oxalate in the ordinary solvents (page 4) and in acetic and hydrochloric acids. URINE. -59 NEUTRAL SULPHUR COMPOUNDS. Under this head may be classed such bodies as cystin (see p. 76), chondroitin-sulphuric acid, oxyproteic acid, alloxypro- teic acid, uroferric acid, sulphocyanides and taurin derivatives. The sulphur content of the bodies just enumerated is generally termed loosely combined or neutral sulphur in order thai it may not be confused with the acid sulphur which occurs in the in- organic sulphuric acid and ethereal sulphuric acid forms. Ordinarily the neutral sulphur content of normal human urine is 14-20 per cent of the total sulphur content. XII-CH-HN I I ALLANTOIN, 00 00. I I NH-CO XFL Allantoin has been found in the urine of suckling- calves as well as in that of the dog and cat. It has also been detected in the urine of infants within the first eight days after birth, as well as in the urine of adults. It is more abundant in the urine Fig. 93. Allan to in, from Cat's Urine. a and b, Forms in which it crystallized from the urine ; c, re-crystallized allantoin. (Drawn from micro-photographs furnished by Prof. Lafayette B. Mendel of Yale University.) 260 PHYSIOLOGICAL CHEMISTRY. of women during pregnancy. Allantoi'n is formed by the oxi- dation of uric acid and the output is increased by thymus or pancreas feeding. When pure it crystallizes in prisms (Fig. 93, p. 259) and when impure in granules and knobs. Patho- logically, it has been found increased in diabetes insipidus and in hysteria with convulsions (Pouchet). Experiments. 1. Separation from the Urine. 1 — Mcissner's Method. — Precipitate the urine with baryta water. Neutralize the fil- trate carefully with dilute sulphuric acid, filter immediately and evaporate the filtrate to incipient crystallization. Com- pletely precipitate this warm fluid with 95 per cent alcohol (reserve the precipitate). Decant or filter and precipitate the solution by ether. Combine the ether and alcohol precipitates and extract with cold water or hot alcohol ; allantoi'n remains undissolved. Bring the allantoi'n into solution in hot water and recrystallize. Allantoi'n may be determined quantitatively by Loewi's method. 2 2. Preparation from Uric Acid. — Dissolve 4 grams of uric acid in 100 c.c. of water rendered alkaline with potassium hy- droxide. Cool and car ef idly add 3 grams of potassium per- manganate. Filter, immediately acidulate the filtrate with acetic acid and allow it to stand in a cool place over night. Filter off the crystals and wash them with water. Save the wash water and filtrate, unite them and after concentrating to a small volume, stand away for crystallization. Now com- bine all the crystals and recrystallize them from hot water. Use these crystals in the experiments which follow. 3. Microscopical Examination. — Examine the crystals made in the last experiment and compare them with those shown in Fig. 93, page 259. 1 The urine of the dog after thymus, pancreas or uric acid feeding may be employed. 2 Archiv fur Experimented Pathologie und Pharmakologie, 1900, xliv, p. 20. URINE. -' i 4. Solubility. — Test the solubility of allantoin in the ordi- nary solvents ( page 4 ). 5. Reaction. — Dissolve a crystal in water and test the re- action to litmus. 6. Furfurol Test. — Place a few crystals of allantoin on a tesl tablet or in a porcelain dish and add 1 2 drops of a con- centrated aqueous solution of furfurol and 1-2 drops of con- centrated hydrochloric acid. Observe the formation of a yellow color which turns to a light purple if allowed to stand. This tot is given by urea hut not by uric acid. 7. Murexid Test. — Try this test according' to the directions given on page ->4<). Note that allantoin fails to respond. 8. Reduction of Fehling's Solution. — Make this test in the usual way (see p. 286) except that the boiling must he pro- longed and excessive. I'ltimately the allantoin will reduce the solution. Compare with the result on uric acid, page J4<>. AROMATIC OXYACIDS. Two of the most important of the oxyacids are paraoxy- phciiyl-acetic acid, CH 2 -COOH, OH and paraoxyplicnyl-propioiiic acid, CHo-CH.-COOH. /V OH They are products of the putrefaction of proteid material and tyrosin is an intermediate stage in their formation. Both these acids for the most part pass unchanged into the urine where they occur normally in very small amount. The con- tent may be increased in the same manner as the phenol con- 262 PHYSIOLOGICAL CHEMISTRY. tent, in particular by acute phosphorus poisoning. A fraction of the total aromatic oxyacid content of the urine is in com- bination with sulphuric acid, but the greater part is present in the form of salts of sodium and potassium. Homogentisic Acid or di-oxyphenyl-acetic acid. OH j^CHo-COOH, OH is another important oxyacid sometimes present in the urine. Under the name glycosuric acid it was first isolated from the urine by Prof. John Marshall of the University of Pennsylva- nia; subsequently Baumann isolated it and determined its chemical constitution. It occurs in cases of alkaptonuria. A urine containing this oxyacid turns greenish-brown from the surface downward when treated with a little sodium hydroxide or ammonia. If the solution be stirred the color very soon becomes dark brown or even black. Homogentisic acid re- duces alkaline copper solutions but not alkaline bismuth solu- tions. Uroleucic acid is similar in its reactions to homogentisic acid. Oxymandelic Acid or paraoxyphenyl-glycolic acid, ( OH \ CH(OH)-COOH, has been detected in the urine in cases of yellow atrophy of the liver. Kynurenic Acid or y-oxy-/?-quinoline carbonic acid, CH COH /\ / V HC C C-COOH, I II I HC C CH \/ \/ CH N URINE. 263 is. present in the urine of the dog" and has recently been detected by Swain in the urine of the coyote. To isolate it from the urine proceed as follows: Acidify the urine with hydrochloric acid in the proportion 1 125. From this acid fluid both the uric acid and the kynurenic acid separate in the course of 24-48 hours. Filter off the combined crystalline deposit of the two acids, dissolve the kynurenic acid in dilute ammonia (uric acid is insoluble) and reprecipitate it with hydrochloric acid. Kynurenic acid may be quantitatively determined by Capal- di's method. 1 COOII. BENZOIC ACID, | V Benzoic acid has been detected in the urine of the rabbit and dog. It is also said to occur in human urine accompanying renal disorders. The benzoic acid probably originates from a fermentative decomposition of the hippuric acid of the urine. Experiments. 1. Solubility. — Test the solubility of benzoic acid in water, alcohol and ether. 2. Crystalline Form. — Recrystallize some benzoic acid from hot water, examine the crystals under the microscope and com- pare them with those reproduced in Fig. 94, page 264. 3. Sublimation. — Place a little benzoic acid in a test-tube and heat over a flame. Note the odor which is evolved and observe that the acid sublimes in the form of needles. 4. Dissolve a little sodium benzoate in water and add a solu- tion of neutral ferric chloride. Note the production of a brownish-yellow precipitate (Salicylic acid gives a reddish- violet color under the same conditions). Add ammonium hy- droxide to some of the precipitate. It dissolves and ferric hy- droxide is formed. Add a little hydrochloric acid to another portion of the original precipitate and stand the vessel away over night. What do you observe? 1 Zeitschrift fur physiologische Chemie, 1897, xxiii, p. 92. 264 PHYSIOLOGICAL CHEMISTRY, Fig. 94. Benzoic Acid. NUCLEO-PROTEID. The nubecula of normal urine has been shown by one investi- gator to consist of a mucoid containing 12.7 per cent of nitro- gen and 2.3 per cent of sulphur. This body evidently origi- nates in the urinary passages. It is probably slightly soluble in the urine. Some investigators believe that the body form- ing the nubecula of normal urine is nucleo-proteid and not a mucin or mucoid as stated above. A discussion of nucleo- proteid and related bodies occurring in the urine under patho- logical conditions will be found on page 296. NH-CO I OXALURIC ACID, CO NH 2 COOH. Oxaluric acid is not a constant constituent of normal human urine, and when found occurs only in traces as the ammonium salt. Upon boiling oxaluric acid it splits into oxalic acid and urea. URINE. 265 ENZYMES. Various types of enzymes have been isolated from the urine. Pepsin, which probably originates in the stomach, and a diastatic enzyme have been more carefully studied than the • •ther forms. The presence of trypsin and rennin in the urine is questioned. VOLATILE FATTY ACIDS. Acetic, butyric and formic acids have been found under normal conditions in the urine of man and of certain carnivora as well as in the urine of herbivora. Normally they arise prin- cipally from the fermentation of carbohydrates and the putre- faction of proteids. The acids containing the fewest carbon atoms (formic and acetic) are found to be present in larger percentage than those which contain a larger number of such atoms. The volatile fatty acids occur in normal urine in traces, the total output for twenty- four hours, according to different investigators, varying from 0.008 gram to 0.05 gram. Pathologically, the excretion of volatile fatty acids is in- creased in diabetes, fevers, and in certain hepatic diseases in which the parenchyma of the liver is seriously affected. Under other pathological conditions the output may be diminished. These variations, however, in the excretion of the volatile fatty acids possess very little diagnostic value. CH 3 PARALACTIC ACID, CH(OH) COOH. Paralactic acid is supposed to pass into the urine when the supply of oxygen in the organism is diminished through any cause, e. g., after acute yellow atrophy of the liver, acute phosphorus poisoning or epileptic attacks. This acid has also been found in the urine of healthy persons following the physical exercise incident to prolonged marching. Paralactic 266 PHYSIOLOGICAL CHEMISTRY. acid has been detected in the urine of birds after the removal of the liver. CH„ • CO • NH • CHo • COOH. /\ PHENACETURIC ACID, Phenaceturic acid occurs principally in the urine of herbivor- ous animals but has frequently been detected in human urine. It is produced in the organism through the synthesis of glyco- coll and phenylacetic acid. It may be decomposed into its component parts by boiling with dilute mineral acids. The crystalline form of phenaceturic acid (small rhombic plates with rounded angles) resembles one form of uric acid crystal. PHOSPHORIZED COMPOUNDS. Phosphorus in organic combination has been found in the urine in such bodies as glycerophosphoric acid, which may arise from the decomposition of lecithin, and phosphocarnic acid. It is claimed that on the average about 2.5 per cent of the total phosphorus elimination is in organic combination. PIGMENTS. There are at least three pigments normally present in human urine. These pigments are uro chrome, urobilin and uroery- thrin. A. UROCHROME. This is the principal pigment of normal urine and imparts the characteristic yellow color to that fluid. It is apparently closely related to its associated pigment urobilin since the latter may be readily converted into urochrome through evaporation of its aqueous-ether solution. Urochrome may be obtained in the form of a brown, amorphous powder which is readily soluble in water and 95 per cent alcohol. It is less soluble in absolute alcohol, acetone, amyl alcohol and acetic ether and insoluble in benzene, chloroform and ether. Urochrome is said to be a nitrogenous body (4.2 per cent nitrogen), free from iron. URINE. 267 B. UROBILIN. Urobilin, which was at one time considered to be the princi- pal pigment of urine, in reality contributes little toward the pigmentation of this fluid. It is claimed thai no urobilin is present in freshly voided normal urine but that its precursor, a chromogen called urobilinogen, is presenl and gives rise to ur< bilin upon decomposition through the influence of light. It is claimed by some investigators that there are various forms oi urobilin, e. g. } normal, febrile, physiological and patholog- ical. Urobilin is said to be very similar to. if not absolutely identical with, hydrobilirubin (see page 140). Urobilin may be obtained as an amorphous powder which varies in color from brown to reddish-brown, red and reddish- yellow depending upon the way in which it is prepared. It is easily soluble in ethyl alcohol, amyl alcohol and chloroform, and slightly soluble in ether, acetic ether and in water. Its solutions show characteristic absorption-bands (see Absorp- tion Spectra, Plate II). Under normal conditions urobilin is derived from tbe bile pigments in the intestine. Urobilin is increased in most acute infectious diseases such as erysipelas, malaria, pneumonia and scarlet fever. It is also increased in appendicitis, carcinoma of the liver, catarrhal icterus, pernicious aiucmia and in cases of poisoning by anti- febrin, antipyrin, pyridin. and potassium chlorate. In gen- eral it is usually increased when blood destruction is excessive and in many disturbances of the liver. It is markedly de- creased in phosphorus poisoning. Experiments. 1. Spectroscopic Examination. — Acidify the urine with HC1 and allow it to remain exposed to the air for a few moments. By this means if any urobilinogen is present it will be transformed into urobilin. The urine may now be examined by means of the spectroscope. If urobilin is present in the fluid the characteristic absorption-band lying between b and F will be observed (see Absorption Spectra, Plate II). It 268 PHYSIOLOGICAL CHEMISTRY may be found necessary to dilute the urine with water before a distinct absorption-band is observed. This test may be'modi- fied by acidifying 10 c.c. of urine with HC1 and shaking it gently with 5 c.c. of amyl alcohol. The alcoholic extract when examined spectroscopically will show the characteristic urobilin absorption-band. (Note the spectroscopic examination in the next experiment.) 2. Ammoniacal-Zinc Chloride Test. — Render some of the urine ammoniacal by the addition of ammonium hydroxide, and after allowing it to stand a short time filter off the preci- tate of phosphates and add a few drops of zinc chloride solution to the filtrate. Observe the production of a greenish fluorescence. Examine the fluid by means of the spectroscope and note the absorption-band which occupies much the same position as the absorption-band of urobilin in acid solution (see Absorption Spectra, Plate II). 3. Gerhardt's Test. — To 20 c.c. of urine add 3-5 c.c. of chloroform and shake well. Separate the chloroform extract and add to it a few drops of iodine solution (I in KI) . Render the mixture alkaline with a dilute solution of potassium hy- droxide and note the production of a yellow or yellowish-brown color. The solution ordinarily exhibits a greenish fluores- cence. 4. Wirsing's Test. — To 20 c.c. of urine add 3-5 c.c. of chloroform and shake gently. Separate the chloroform ex- tract and add to it a drop of an alcoholic solution of zinc chloride. Note the rose-red color and the greenish fluores- cence. If the solution is turbid it may be rendered clear by the addition of a few c.c. of absolute alcohol. 5. Ether- Absolute Alcohol Test. — Mix urine and pure ether in equal volumes and shake gently in a separatory funnel. Separate the ether extract, evaporate it to dryness and dissolve the residue in 2-3 c.c. of absolute alcohol. Note the greenish fluorescence. Examine the solution spectroscopically and observe the characteristic absorption-band (see Absorption Spectra, Plate II). URI X l 269 6. Ring Test. — Acidify 25 c.c. of urine with -' 3 drops of concentrated HC1, add 5 c.c. of chloroform and shake the mixture. Separate the chloroform, place it in a test-tube and add carefully 3 5 c.c. of an alcoholic solution of zinc acetate. Observe the formation of a green ring at the zone of contact of the two fluids. If the tube is shaken a fluorescence may he observed. C. UROERYTHRIN. This pigment is Frequently present in small amount in nor- mal urine. The red color of urinary sediments is due in great part to the presence of uroerythrin. It is easily soluble in amy! alcohol, slightly soluble in acetic ether, absolute alcohol or chloroform, and nearly insoluble in water. Dilute solutions of uroerythrin are pink in color while concentrated solutions are orange-red or bright red : none of its solutions fluoresce. Uroerythrin is increased in amount after strenuous physical exercise, digestive disturbances, fevers, certain liver disorders and in various other pathological conditions. PTOMAINES AND LEUCOMAINES. These toxic substances are said to be present in small amount in normal urine. It is claimed that five different poisons may be detected in the urine, and it is further stated that each of these substances produces a specific and definite symptom when injected intravenously into a rabbit. The resulting symptoms are narcosis, salivation, mydriasis, paralysis and convulsions The day urine is principally narcotic and is 2-4 times as toxic as the night urine which is chiefly productive of convulsions. PURIN BASES. The purin bases found in human urine are adenin. carnin, epiguanin, episarkin, guanin, xanthin, heteroxanthin, hypo- xanthin, paraxanthin and i-methylxanthin. The main bulk of the purin base content of the urine is made up of paraxanthin, heteroxanthin and i-methvlxanthin which are derived for the 270 PHYSIOLOGICAL CHEMISTRY. most part from the caffein, theobromin and theophyllin of the food. The total purin base content is made np of the products of two distinct forms of metabolism, i. e., metabolism of in- gested nucleins and purins and metabolism of tissue nuclein material. Purin bases resulting from the first form of meta- bolism are said to be of exogenous origin whereas those re- sulting from the second form of metabolism are said to be of endogenous origin. The daily output of purin bases by the urine is extremely small and varies greatly with the individual (16-60 milligrams). The output is increased after the inges- tion of nuclein material as well as after the increased destruc- tion of leucocytes. A well marked increase accompanies leu- kaemia. Edsall has very recently shown that the output of purin bases by the urine is increased as a result of X-ray treat- ment. Experiment. 1. Formation of the Silver Salts. — Add an excess of mag- nesia mixture 1 to 25 c.c. of urine. Filter off the precipitate and add ammoniacal silver solution 2 to the filtrate. A precipi- tate composed of the silver salts of the various purin bases is produced. 2. Inorganic Physiological Constituents. Ammonia. Next to urea, ammonia is the most important of the nitro- genous end-products of proteid metabolism. Ordinarily about 4.6-5.6 per cent of the total nitrogen of the urine is eliminated as ammonia and on the average this would be about 0.7 gram per day. Under normal conditions the ammonia is present in the urine in the form of the chloride, phosphate or sulphate. This is due to the fact that combinations of this sort are not 1 Magnesia mixture may be prepared as follows : Dissolve 175 grams of MgSO., and 350 grams of NH 4 C1 in 1400 c.c. of distilled water. Add 700 grams of concentrated NEUOH, mix very thoroughly and preserve the mixture in a glass-stoppered bottle. 2 Ammoniacal silver solution may be prepared according to directions given on page 377. URINE. -71 oxidized in the organism to form urea, 1 >n t arc excreted as such. This explains the increase in the OUtpUl of ammonia which fol- lows the administration of the ammonium salts of the mineral acids or of the acids themselves. On the other hand when ammonium acetate and many other ammonium salts of certain organic acids are administered no increase in the output of ammonia occurs since the salt is oxidized and its nitrogen ulti- mately appears in the urine as urea. The acids formed during the process of proteid destruction within the body have an influence upon the excretion of am- monia similar to that exerted by acids which have been admin- istered. Therefore a pathological increase in the output of ammonia is observed in such diseases as are accompanied by an increased and imperfect proteid metabolism, and especially in diabetes, in which disease diacetic acid and /8-oxybutyric acid are found in the urine in combination with the ammonia. As the result of recent experiments Folin claims that a pronounced decrease in the extent of proteid metabolism, as measured by the total nitrogen in the urine, is frequently accompanied by a decreased elimination of ammonia. The ammonia elimination is therefore probably determined by other factors than the total proteid catabolism as such. Fur- thermore, he believes that a decided decrease in the total nitrogen excretion is always accompanied by a relative increase in the ammonia-nitrogen, provided the food is of a character yielding an alkaline ash. The quantitative determination of ammonia must be made upon the fresh urine since upon standing the normal urine will undergo ammoniacal fermentation (see page 230). Sulphates. Sulphur in combination, is excreted in two forms in the urine; first, as loosely combined, unoxidized or neutral sulphur and second, as oxidized or acid sulphur. The loosely combined sulphur is excreted mainly as a constituent of such bodies as cystin, cystein, taurin, hydrogen sulphide, ethyl sulphide, sul- 272 PHYSIOLOGICAL CHEMISTRY. phocyanides, sulphonic acids, oxyproteic acid, alloxyproteic acid and uroferric acid. The amount of loosely combined sulphur eliminated is in great measure independent of the extent of pro- teid decomposition or of the total sulphur excretion. In this characteristic it is somewhat similar to the excretion of cre- atinin. The oxidised sulphur is eliminated in the form of sul- phuric acid, principally as salts of sodium, potassium, calcium and magnesium ; a relatively small amount occurs in the form of ethereal sulphuric acid, i. c, sulphuric acid in combination with such aromatic bodies as phenol, indol, skatol, cresol, pyro- catechin and hydroquinone. Sulphuric acid in combination with Na, K, Ca or Mg is sometimes termed inorganic or pre- formed sulphuric acid whereas the ethereal sulphuric acid is sometimes called conjugate sulphuric acid. The greater part of the sulphur is eliminated in the oxidized form but the abso- lute percentage of sulphur excreted as the preformed, ethereal or loosely combined type depends upon the total quantity of sulphur present, i. e., there is no definite ratio between the three forms of sulphur which will apply under all conditions. The preformed sulphuric acid may be precipitated directly from acidified urine with BaCl 2 , whereas the ethereal sulphuric acid must undergo a preliminary boiling in the presence of a mineral acid before it can be so precipitated. The sulphuric acid excreted by the urine arises principally from the oxidation of proteid material within the body; a relatively small amount is due to ingested sulphates. Under normal conditions about 2.5 grams of sulphuric acid is elimi- nated daily. Since the sulphuric acid content of the urine has, for the most part, a proteid origin and since one of the most important constituents of the proteid molecule is nitrogen, it would be reasonable to suppose that a fairly definite ratio might exist between the excretion of these two elements. However, when we appreciate that the percentage content of N and S present in different proteids is subject to rather wide variations, the fixing of a ratio which will express the exact relation existing between these two substances, as they appear in the URINE. 273 urine as end-products of proteid metabolism, is practically impossible. It \ya< lieen suggested that the ratio 5 :i expresses this relation in a general way. Pathologically, the excretion of sulphuric acid by the urine is increased in acute fevers and in all other diseases marked by a stimulated metabolism, whereas a decrease in the sulphuric acid excretion is observed in those diseases which are accom- panied by a loss of appetite and a diminished metabolic activity. Experiments. 1. Detection of Inorganic Sulphuric Acid. — Place about 10 c.c. of urine in a test-tube, acidify with acetic acid and add some barium chloride solution. A white precipitate of barium sulphate forms. 2. Detection of Ethereal Sulphuric Acid. — Filter off the barium sulphate precipitate formed in the above experiment, add 1 c.c. of hydrochloric acid and a little barium chloride solu- tion to the filtrate and heat the mixture to boiling for 1-2 minutes. Xote the appearance of a turbidity due to the pres- ence of sulphuric acid which has been separated from the ethereal sulphates and has combined with the barium of the BaCU to form BaSO,. 3. Detection of Loosely Combined or Neutral Sulphur. — Place about 10 c.c. of urine in a test-tube, introduce a small piece of zinc, add sufficient hydrochloric acid to cause a gentle evolution of hydrogen and over the mouth of the tube place a filter paper saturated with plumbic acetate solution. In a short time the portion of the paper in contact with the vapors within the test-tube becomes blackened due to the formation of lead sulphide. The nascent hydrogen has reacted with the loosely combined or neutral sulphur to form hydrogen sulphide and this gas coming in contact with the plumbic acetate paper has caused the production of the black lead sulphide. Sul- phur in the form of inorganic or ethereal sulphuric acid does not respond to this test. 4. Calcium Sulphate Crystals. — Place 10 c.c. of urine in a 19 274 PHYSIOLOGICAL CHEMISTRY. test-tube, add 10 drops of calcium chloride solution and allow the tube to stand until crystals form. Examine the calcium sulphate crystals under the microscope and compare them with those shown in Fig. 95, p. 274. Fig. 95. Chlorides. Calcium Sulphate. and Weil.) (Hensel Next to urea, the chlorides constitute the chief solid con- stituent of the urine. The prin- cipal chlorides found in the urine are those of sodium, po- tassium, ammonium and mag- nesium, with sodium chloride predominating". The excretion of chlorides is dependent, in great part, upon the nature of the diet, but on the average the daily output is about 10-15 grams, expressed as sodium chlo- ride. Copious water-drinking increases the output of chlorides considerably. Because of their solubility, chlorides are never found in the urinary sediment. Since the amount of chlorides excreted in. the urine is due primarily to the chloride content of the food ingested, it fol- lows that a decrease in the amount of ingested chloride will likewise cause a decrease in the chloride content of the urine In cases of actual fasting the chloride content of the urine may be decreased to a slight trace which is derived from the body fluids and tissues. Under these conditions, however, an exam- ination of the blood of the fasting subject will show the per- centage of chlorides in this fluid to be approximately normal. This forms a very striking example of the care nature takes to maintain the normal composition of the blood. There is a limit to the power of the body to maintain this equilibrium, however, and if the fasting organism be subjected to the influ- ence of diuretics for a time, a point is reached where the com- position of the blood can no longer be maintained and a gradual decrease in its chloride content occurs which finally results in URINE. 275 death. Death is supposed to result not so much because of a lack of chlorine as from a deficiency of sodium. This is shown from the fact that potassium chloride, for instance, cannot re- place the sodium chloride of the blood when the latter is de- creased in the manner above slated. When this substitution is attempted the potassium salt is excreted at once in the urine, and death follows as above indicated. Pathologically, the excretion of chlorides may be decreased in some fevers, chronic nephritis, croupous pneumonia, diar- rhoea, certain stomach disorders and in acute articular rheu- matism. Experiment. Detection of Chlorides in Urine. — Place about 5 c.c. of urine in a test-tube, render it acid with nitric acid and add a few drops of a solution of argentic nitrate. A white precipi- tate, due to the formation of argentic chloride, is produced. This precipitate is soluble in ammonium hydroxide. Phosphates. Phosphoric acid exists in the urine in two general forms: First, that in combination with the alkali metals, sodium and potassium, and the radical ammonium; second, that in combi- nation with the alkaline earths, calcium and magnesium. Phos- phates formed through a union of phosphoric acid with the alkali metals are termed alkaline phosphates, or phosphates of the alkali metals, whereas phosphates formed through a union of phosphoric acid with the alkaline earths are termed earthy phosphates, or phosphates of the alkaline earths. Three series of salts are formed by phosphoric acid : Normal, M...PO4, 1 mono-hydrogen, M 2 HP0 4 , and di-hydro- gen, MH 2 P0 4 . The di-hydrogen salts are acid in reaction and it was generally believed that about 60 per cent of the total phosphate content of the urine was in the form of this type of salt, and that the acidity of the urine was due in great part to the presence of sodium di-hydrogen phosphate, Re- 1 M may be occupied by any of the alkali metals or alkaline earths. 276 PHYSIOLOGICAL CHEMISTRY. cently, however, it has been quite clearly shown that the nor- mal acidity of the urine is not due to the presence of this salt but is due. at least in part, to the presence of various acidic radicals. In this connection Folin believes that the phosphates in clear acid urine are all of the mono-hydrogen type, and that the acidity of the urines of this character is generally greater than the combined acidity of all the phos- phates present; the excess in the acidity above that due to phosphates he believes to be due to free organic acids. In bones the phosphates occur principally in the form of the normal salts of calcium and magnesium. The mono-hydro- gen salts as a class are alkaline in reaction to litmus, and it is to the presence of di-sodium hydrogen phosphate, Na 2 HP0 4 , that the greater part of the alkalinity of the saliva is due. The excretion of phosphoric acid is extremely variable but on the average the total output for 24 hours is about 2.5 grams, expressed as P 2 5 . Ordinarily the total output is distributed between alkaline phosphates and earthy phos- phates approximately in the ratio 2:1. The greater part of this phosphoric acid arises from the ingested food, either from the preformed phosphates or more especially from the phosphorus in organic combination such as we find it in phospho-proteids, nucleo-proteids, nncleins and lecithins; the phosphorus-containing tissues of the body also contrib- ute to the total output of this element. Alkaline phos- phates ingested with the food have a tendency to increase the phosphoric acid content of the urine to a greater extent than the earthy phosphates so ingested. This is due, in a measure, to the fact that a portion of the earthy phosphates, under certain conditions, may be precipitated in the intes- tine and excreted in the feces; this is especially to be noted in the case of herbivorous animals. Since the extent to which the phosphates are absorbed in the intestine depends upon the form in which they are present in the food, under ordi- nary conditions, there can be no absolute relationship be- tween the urinary output of nitrogen and phosphorus. If URINE. 277 the diel is constant, however, from day to day, thus allowing of the preparation of both a nitrogen and a phosphorus bal- ance, 1 a definite ratio may be established. In experiments upon dog-,, which were fed an exclusive meat diet, the ratio of nitrogen to phosphorus, in the urine ami free-, was found to be 8. 1 : 1 . Pathologically the excretion of phosphoric acid is increased in such diseases of the hones as diffuse periostosis, osteoma- lacia and rickets: according to some investigators, in the early stages of pulmonary tuberculosis; in acute yellow atrophy of the liver; in diseases which are accompanied by an extensive decomposition of nervous tissue and after sleep induced by potassium bromide or chloral hydrate (Mendel). It is also increased after copious water-drinking. A decrease in the excretion of phosphates is at times noted in febrile affections, such as the acute infectious diseases; in pregnancy, in the period during which the foetal hones are forming, and i; i diseases of the kidneys, because of non-elimination. Experiments. 1. Formation of " Triple Phosphate." — Place some urine in a beaker, render it alkaline with ammonium hydroxide, add a small amount of magnesium sulphate solution and allow the beaker to stand in a cool place over night. Crystals of ammonium magnesium phosphate, "triple phosphate," form under these conditions. Examine the crystalline sediment under the microscope and compare the forms of the crystals with those shown in Fig. 96, page 278. 2. " Triple Phosphate " Crystals in Ammoniacal Fer- mentation. — Stand some urine aside in a beaker for several days. Ammoniacal fermentation will develop and " triple phosphate" crystals will form. Examine the sediment under 1 In metabolism experiments, a statement showing the relation existing between the nitrogen content of the food on the one hand and that of the urine and feces on the other, for a definite period, is termed a nitrogen balance or a " balance of the income and outgo of nitrogen." 278 PHYSIOLOGICAL CHEMISTRY. Fig. 96. " Triple Phosphate." (Ogden.) the microscope and compare the crystals with those shown in Fig. 96, above. 3. Detection of Earthy Phosphates. — Place 10 c.c. of urine in a test-tube and render it alkaline with ammonium hydroxide. Warm the mixture and note the separation of a precipitate of earthy phosphates. 4. Detection of Alkaline Phosphates. — Filter off the earthy phosphates as formed in the last experiment, and add a small amount of magnesia mixture (see page 270) to the filtrate. Now warm the mixture and observe the formation of a white precipitate due to the presence of alkaline phosphates. Note the difference in the size of the precipitates of the two forms of phosphates from this same volume of urine. Which form of phosphates were present in the larger amount, earthy or alkaline? 5. Influence upon Fehling's Solution. — Place 2 c.c. of Fehling's solution in a test-tube, dilute it with 4 volumes of water and heat to boiling. Add a solution of sodium di- hydrogen phosphate, NaH 2 P0 4 , a small amount at a time, and heat after each addition. What do you observe? What does this observation force you to conclude regarding the interference of phosphates in the testing of diabetic urine by means of Fehling's test? URINE. 279 Sodium and Potassium. The elements sodium and potassium arc always present in the urine. Usually they are combined with such acidic radi- cals as CI, CO.j, S0 4 and PO.,. The amount of potassium, ex- pressed as l\j< >. excreted in -'4 hours by an adult, subsisting upon a mixed diet, is on the average 2-3 grams, whereas the amount of sodium, expressed as Na 2 0, under the same condi- tions, is ordinarily 4 6 grams. The ratio of K to Xa is gen- erally about"3:5. The absolute quantity of these elements excreted, depends, of course, in large measure, upon the nature of the diet. Because of the non-ingestion of NaCl and the accompanying destruction of potassium-containing body tis- sues, the urine during fasting contains more potassium salts than sodium salts. Pathologically the output of potassium, in its relation to sodium, may be increased during fever; following the crisis, however, the output of this element may be decreased. It may also be increased in conditions associated with acid intoxication. Calcium and Magnesium. The greater part of the calcium and magnesium excreted in the urine is in the form of phosphates. The daily output, which depends principally upon the nature of the diet, aggregates on the average about 1 gram and is made up of the phosphates of calcium and magnesium in the proportion 1 : 2. The percent- age of calcium salts present in the urine at any one time forms no dependable index as to the absorption of this class of salts, since they are again excreted into the intestine after absorp- tion. It is therefore impossible to draw any satisfactory con- clusions regarding the excretion of the alkaline earths unless we obtain accurate analytical data from both the feces and the urine. Very little is known positively regarding the actual course of the excretion of the alkaline earths under pathological conditions except that an excess of calcium is found in acid intoxication and some diseases of the bones. 28o PHYSIOLOGICAL CHEMISTRY. Carbonates. Carbonates generally occur in small amount in the urine of man and carnivora under normal conditions, whereas much larger quantities are ordinarily present in the urine of herbivora. The alkaline reaction of the urine of herbivora is dependable in great measure upon the presence of carbo- nates. In general a urine containing carbonates in appre- ciable amount is turbid when passed or becomes so shortly after. These bodies ordinarily occur as alkali or alkaline earth compounds and the turbid character of urine contain- ing them is usually due principally to the latter class of sub- stances. The carbonates of the alkaline earths are often found in amorphous urinary sediments. Iron. Iron is present in small amount in normal urine. It prob- ably occurs partly in inorganic and partly in organic combi- nation. The iron contained in urinary pigments or chromo- gens is in organic combination. According to different in- vestigators the iron content of normal urine varies from 0.012 gram to 0.15 gram per day. Experiment. Detection of Iron in Urine. — Evaporate a convenient vol- ume (10-15 c.c.) of urine to dryness. Incinerate and dissolve the residue in a few drops of iron-free hydrochloric acid and dilute the acid solution with 5 c.c. of water. Divide the acid solution into two parts and make the following tests: (a) To the first part add a solution of ammonium sulphocyanide ; a red color indicates the presence of iron, (b) To the second part of the solution add a little potassium ferrocyanide solution; a precipitate of Prussian blue forms upon standing. Fluorides, Nitrates, Silicates and Hydrogen Peroxide. These substances are all found in traces in human urine under normal conditions. Nitrates are undoubtedly intro- URINE. 28] duced into the organism in the water ;mt upon a vegetable diet and smallest upon a meat diet. Nitrites are found only in urine which is undergoing decomposition and are formed from the nitrates in the course of ammoniacal fermentation. Hydrogen per- oxide has been detected in the urine, but its presence is be- lieved to possess no pathological importance. CHAPTER XVIII. URINE: PATHOLOGICAL CONSTITUENTS. 1 Proteids Dextrose. Serum albumin. Serum globulin. f Deutero-proteose. Proteoses -j Hetero-proteose. I " Bence- Jones' proteid." Peptone. Nucleo-proteid. Fibrin. Haemoglobin. Blood {Form elements. L Pigment. Bile (K^ents. Acetone. Diacetic acid. /?-Oxybutyric acid. Conjugate glycuronates. Pentoses. Fat. Hsematoporphyrin. Lactose. Laevulose. Inosit. Laiose. Melanin. Urorosein. Unknown substances. DEXTROSE. Traces of this sugar occur in normal urine, but the amount is not sufficient to be readily detected by the ordinary simple 1 See note at the bottom of page 237. 282 URINE. 283 qualitative tests. There are two distinct types of pathological glycosuria, i. <\, transitory glycosuria and persistent glyco- suria. The transitory type may follow the ingestion of an excess of sugar, causing the assimilation limit to be exceeded, or it may accompany any one of several disorders which cause an impairment of the power of assimilating sugar. In the persistent type large amounts of sugar are excreted daily in the urine for long periods of time. Under such circumstances a condition known as diabetes mellitus exists. Ordinarily, diabetic urine which contains a high percentage of sugar pos- sesses a faint yellow color, a high specific gravity and a volume which is above normal. Experiments. 1. Phenylhydrazin Reaction. — Test the urine according to one of the following methods: (a) To a small amount of phenylhydrazin mixture, furnished by the instructor, 1 add 5 c.c. of the urine, shake well and heat on a boiling water- bath for one-half to three-quarters of an hour. Allow the tube to cool slowly and examine the crystals microscopically (Plate III., opposite page 5). If the solution has become too concentrated in the boiling process it will be light-red in color and no crystals will separate until it is diluted with water. Yellow crystalline bodies called osazons are formed from certain sugars under these conditions, each individual sugar giving rise to an osazon of a definite crystalline form which is typical for that sugar. Each osazon has a definite melting- point, and as a further and more accurate means of identi- fication it may be recrystallized and identified by the determi- nation of its melting-point and nitrogen content. The reac- tion taking place in the formation of phenyldextr osazon is as follows : 1 This mixture is prepared by combining one part of phenylhydrazin- hydrochloride and two parts of sodium acetate, by weight. These are thoroughly mixed in a mortar. 284 PHYSIOLOGICAL CHEMISTRY. C 6 H 12 6 + 2(H 2 N*NH- C 6 H 5 ) = Dextrose. Phenylhydrazin. C 6 H 10 4 (N- NH- C 6 H 5 ) 2 + 2H 2 + H 2 . Phenyl dextrosazon. (b) Place 5 c.c. of the urine in a test-tube, add 1 c.c. of phenylhydrazin-acetate solution furnished by the instruc- tor, 1 and heat on a boiling water-bath for one-half to three- quarters of an hour. Allow the liquid to cool slowly and examine the crystals microscopically (Plate III., opposite p. 5). The phenylhydrazin test has been so modified by Cipollina as to be of use as a rapid clinical test. The directions for this test are given in the next experiment. 2. Cipollina's Test. — Thoroughly mix 4 c.c. of urine, 5 drops of phenylhydrazin (the base) and one-half c.c. of glacial acetic acid in a test-tube. Heat the mixture for about one minute over a low flame, shaking the tube continually to pre- vent loss of fluid by bumping. Add 4-5 drops of potassium hydroxide or sodium hydroxide (sp. gr. 1.16), being certain that the fluid in the test-tube remains acid; heat the mixture again for a moment and then cool the contents of the tube. Ordinarily the crystals form at once, especially if the urine possesses a low specific gravity. If they do not appear imme- diately allow the tube to stand at least 20 minutes before deciding upon the absence of sugar. Examine the crystals under the microscope and compare them with those shown in Plate III., opposite page 5. 3. Reduction Tests. — To their aldehyde or ketone struc- ture many sugars owe the property of readily reducing the alkaline solutions of the oxides of metals like copper, bismuth and mercury; they also possess the property of reducing ammoniacal silver solutions with the separation of metallic silver. Upon this property of reduction the most widel) r used tests for sugars are based. When whitish-blue cupric hydrox- 1 This solution is prepared by mixing one part by volume, in each case, of glacial acetic acid, one part of water and two parts of phenylhydrazin (the base). URINE. J.S. ide in suspension in an alkaline liquid is heated it is convi into insoluble black cupric oxide, but if a reducing agent like certain sugars be present the cupric hydroxide is reduced to insoluble yellow cuprous hydroxide, which in turn on further heating may be converted into brownish-red or red cuprous oxide. These changes are indicated as follows: Cu / i \ OH OH Cupric hydroxide, (whitish-blue). Cu Cu / i \ / i \ OH OH OH OH 0u=0 + IT,0. Cupric oxide, (black). 2Cu-OH + H 2 0-f 0. Cuprous hydroxide, (yellow). Cu-OH Cu-OH Cuprous hydroxide (yellow). Cu \ ( / + H 2 0. Cu Cuprous oxide, (brownish-red). The chemical equations here discussed are exemplified in Trommer's and Fehling's tests. (a) Trommer's Test. — To 5 c.c. of urine in a test-tube add one-half its volume of KOH or NaOH. Mix thoroughly and add, drop by drop, agitating after the addition of each drop, a very dilute solution of cupric sulphate. Continue the addition until there is a slight permanent precipitate of 286 PHYSIOLOGICAL CHEMISTRY. cupric hydroxide and in consequence the solution is slightly turbid. Heat, and the cupric hydroxide is reduced to yellow cuprous hydroxide or to brownish-red cuprous oxide. If the solution of cupric sulphate used is too strong, a small brown- ish-red precipitate produced in the presence of a low percent- age of dextrose may be entirely masked. On the other hand, if too little cupric sulphate is used a light-colored precipitate formed by uric acid and purin bases may obscure the brownish- red precipitate of cuprous oxide. The action of KOH or NaOH in the presence of an excess of sugar and insufficient copper will produce a brownish color. Phosphates of the alkaline earths may also be precipitated in the alkaline solution and be mistaken for cuprous hydroxide. Trommer's test is not very satisfactory. (b) Fehling's Test. — To about i c.c. of Fehling's solu- tion 1 in a test-tube add about 4 c.c. of water, and boil. This is done to determine whether the solution will of itself cause the formation of a precipitate of brownish-red cuprous oxide. If such a precipitate forms, the Fehling's solution must not be used. Add urine to the warm Fehling's solution, a few drops at a time, and heat the mixture after each addition. The production of yellow cuprous hydroxide or brownish- red cuprous oxide indicates that reduction has taken place. The yellow precipitate is more likely to occur if the urine is added rapidly and in large amount, whereas with a less rapid addition of smaller amounts of urine the brownish-red pre- cipitate is generally formed. This is a much more satisfactory test than Trommer's, but 1 Fehling's solution is composed of two definite solutions — a cupric sulphate solution and an alkaline tartrate solution, which may be prepared as follows : Cupric sulphate solution = 34.64 grams of cupric sulphate dissolved in water and made up to 500 c.c. Alkaline tartrate solution = 125 grams of potassium hydroxide and 173 grams of Rochelle salt dissolved in water and made up to 500 c.c. These solutions should be preserved separately in rubber-stoppered bot- tles and mixed in equal volumes when needed for use. This is done to prevent deterioration. URINE. 287 even this test is not entirely reliable when used to detect sugar in the urine. Such bodies as conjugate glycuronates, uric acid, nucleo-proteid and homogentisk acid, when present in suffi- cient ann 'tint, may produce a result similar to that produced by sugar. Phosphates of the alkaline earths may be precipitated by the alkali of the Fehling's solution and in appearance may be mistaken for the cuprous hydroxide. Cupric hydroxide may also be reduced to cuprous oxide and this in turn be dissolved by creatinin, a normal urinary constituent. This will give the urine under examination a greenish tinge and may obscure the sugar reaction even when a considerable amount of sugar is present. (c) Allen's Modification of Fehling's Test. — The fol- lowing procedure is recommended: "From 7 to 8 c.c. of the sample of urine to be tested is heated to boiling in a test-tube, and, without separating any precipitate of albumin which may be produced, 5 c.c. of the solution of cupric sulphate used for preparing Fehling's solution is added. This produces a pre- cipitate containing uric acid, xanthin, hypoxanthin, phos- phates, etc. To render the precipitation complete, however, it is desirable to add to the liquid, when partially cooled, from 1 to 2 c.c. of a saturated solution of sodium acetate having a feebly acid reaction to litmus. 1 The liquid is filtered and to the filtrate, which will have a bluish-green color, 5 c.c. of the alkaline tartrate mixture used for preparing Fehling's solution is added, and the liquid boiled for 15-20 sec- onds. In the presence of more than 0.25 per cent of sugar, separation of cuprous oxide occurs before the boiling-point is reached; but with smaller quantities precipitation takes place during the cooling of the solution, which becomes green- 1 Sufficient acetic acid should be added to the sodium acetate solution to render it feebly acid to litmus. A saturated solution of sodium acetate keeps well, but weaker solutions are apt to become mouldy, and then possess the power of reducing Fehling's solution. Hence it is essential in all cases of importance to make a blank test by mixing equal measures of cupric sulphate solution, alkaline tartrate solution and water, adding a little sodium acetate solution, and heating the mixture to boiling. 2b» PHYSIOLOGICAL CHEMISTRY. ish. opaque, and suddenly deposits cuprous oxide as a fine br< »wnish-red precipitate." (d) Boettger's Test. — To 5 c.c. of urine in a test-tube add 1 c.c. of KOH or NaOH and a very small amount of bis- muth subnitrate, and boil. The solution will gradually darken and finally assume a black color due to reduced bismuth. If the test is made with urine containing albumin this must be re- moved, by boiling and filtering, before applying the test, since with albumin a similar change of color is produced (bismuth sulphide). (e) Nylander's Test (Almen's Test). — To 5 c.c. of urine in a test-tube add one-tenth its volume of Nylander's reagent 1 and boil two or three minutes. The mixture will darken if reducing sugar is present and upon standing for a few mo- ments a black color will appear. This color is due to the precipitation of bismuth. If the test is made on urine con- taining albumin this must be removed, by boiling and filtering, before applying the test. It is claimed by Bechold that Ny- lander's and Boettger's tests give a negative reaction with solutions containing sugar when mercuric chloride or chloro- form is present, a claim which has very recently been contra- dicted by Zeidlitz. A positive Nylander or Boettger test is probably due to the following reactions : (a) Bi(OH) 2 N0 3 + KOH = Bi(0H) 8 + KN0 3 . (b) 2Bi(OH) 3 — 30 = Bi 2 + 3H 2 0. 4. Fermentation Test. — Rub up in a mortar about 15 c.c. of the urine with a small piece of compressed yeast. Trans- fer the mixture to a saccharometer (Fig. 2, p. 10) and stand it aside in a warm place for about 12 hours. If dextrose is present, alcoholic fermentation will occur and carbon dioxide will collect as a gas in the upper portion of the tube. On the 1 Nylander's reagent is prepared by digesting 2 grams of bismuth sub- nitrate and 4 grams of Rochelle salt in 100 c.c. of a 10 per cent potassium hydroxide solution. The reagent is then cooled and filtered. URINE. 289 completion of fermentation introduce, by means of a bent pipette, a little K( )l I solution into the graduated portion, place the thumb tightly over the opening in the apparatus and invert the saccharometer. Explain the result. 5. Barfoed's Test. — To 2-3 c.c. of Barfoed's solution 1 in a test-tube add a few drops of urine and boil. Allow the tube to stand a few minutes ami examine. In the presence of dex- tiose a red precipitate forms. What is it? 6. Polariscopic Examination. — For directions as to the use of the polariscope see page 1 1. PROTEIDS. Normal urine contains a trace of proteid material but the amount present is so slight as to escape detection by any of the simple tests in general use for the detection of proteid Urinary constituents. The following are the more important forms of proteid material which have been detected in the urine under pathological conditions : ( 1 ) Serum albumin. (2) Serum globulin. f Deutero-proteose. (3) Proteoses "> Hetero-proteose. I " Bence- Jones' proteid." (4) Peptone. (5) Nucleo-proteid. (6) Fibrin. (7) Haemoglobin. ALBUMIN. Albuminuria is a condition in which serum albumin or serum globulin appears in the urine. There are two distinct forms of albuminuria, i. c, renal albuminuria and accidental albuminuria. Sometimes the terms " true " albuminuria and barfoed's solution is prepared as follows: Dissolve 4 grams of cupric acetate in 100 c.c. of water and acidify with acetic acid. 20 29O PHYSIOLOGICAL CHEMISTRY. " false " albuminuria are substituted for those just given. In the renal type the albumin is excreted by the kidneys. This is the more serious form of the malady and at the same time is more frequently encountered than the accidental type. Among the causes of renal albuminuria are altered blood pres- sure in the kidneys, altered kidney structure, or changes in the composition of the blood entering the kidneys, thus allow- ing the albumin to diffuse more readily. In the accidental form of albuminuria the albumin is not excreted by the kid- neys as is the case in the renal form of the disorder, but arises from the blood, lymph or some albumin-containing exudate coming into contact with the urine at some point below the kidneys. Experiments. 1. Heller's Ring Test. — Place 5 c.c. of concentrated HN0 3 in a test-tube, incline the tube, and, by means of a pipette allow the urine to flow slowly down the side. 1 The liquids should stratify with the formation of a white zone of precip- itated albumin at the point of juncture. If the albumin is present in very small amount the white zone may not form until the tube has been allowed to stand for several minutes. If the urine is quite concentrated a white zone, due to uric acid or urates, will form upon treatment with nitric acid as indicated. This ring may be easily differentiated from the albumin ring by repeating the test after diluting the urine with 3 or 4 volumes of water, whereupon, the ring, if due to uric acid or urates, will not appear. It is ordinarily pos- sible to differentiate between the albumin ring and the uric acid ring without diluting the urine, since the ring, when due to uric acid, has ordinarily a less sharply defined upper border, is generally broader than the albumin ring and fre- quently is situated in the urine above the point of contact with the nitric acid. Concentrated urines also occasionally ex- hibit the formation, at the point of contact, of a crystalline 1 An apparatus called the albumoscope has been devised for use in this test and has met with considerable favor. URINE. 291 ring with very sharply defined borders. This is urea nitrate and is easily distinguished from the " fluffy " ring of albumin. If there is any difficulty in differentiation a simple dilution of the urine with water, as above described, will remove the diffi- culty. Various colored zones, due either to the presence of indican, bile pigments or to the oxidation of other organic uri- nary constituents, may form in this test under certain condi- tion^. These colored rings should never be confounded with the white ring which alone denotes the presence of albumin. \ fter the administration of certain drugs a white precipi- tate of resin acids may form at the point of contact of the two fluids and may cause the observer to draw wrong conclu- sions. This ring, if composed of resin acids, will dissolve in alcohol, whereas the albumin ring will not dissolve. 2. Roberts' Ring Test. — Place 5 c.c. of Roberts' reagent 1 in a test-tube, incline the tube, and, by means of a pipette, al- low the urine to flow slowly down the side. The liquids should stratify with the formation of a white zone of precipitated albumin at the point of juncture. This test is a modification of Heller's ring test and is rather more satisfactory than that test, since the colored rings never form and the consequent confusion is avoided. 3. Spiegler's Ring Test. — Place 5 c.c. of Spiegler's rea- gent 2 in a test-tube, incline the tube, and, by means of a pipette, allow 5 c.c. of urine, acidified with acetic acid, to flow slowly down the side. A white zone will form at the point of contact. This is an exceedingly delicate test, in fact, too deli- cate for ordinary clinical purposes, since it serves to detect albumin when present in the merest trace ( 1 : 250,000) and hence most normal urines will give a positive reaction for albumin when this test is applied. Roberts' reagent is composed of 1 volume of concentrated HNO3 and 5 volumes of a saturated solution of MgSGv 2 Spiegler's reagent has the following composition: Tartaric acid 20 grams. Mercuric chloride 40 grams. Glycerin 100 grams. Distilled water 1000 grams. 292 PHYSIOLOGICAL CHEMISTRY. Some investigators claim that the delicacy of this test de- pends upon the presence of sodium chloride in the urine, the test losing accuracy if the sodium chloride content be low. 4. Jolles' Reaction. — Shake 5 c.c. of urine with 1 c.c. of 30 per cent acetic acid and 4 c.c. of Jolles' reagent 1 in a test- tube. A white precipitate indicates the presence of albumin. Care should be taken to use the correct amount of acetic acid, since the use of too small an amount may result in the forma- tion of mercury combinations which may cause confusion. In the presence of iodine, mercuric iodide will form but may readily be differentiated from albumin through the fact that it is soluble in alcohol. 5. Coagulation or Boiling Test. — (a) Heat 5 c.c. of urine to boiling in a test-tube. A precipitate forming at this point is due either to albumin or to phosphates. Acidify the urine slightly by the addition of 3-5 drops of very dilute acetic acid, adding the acid drop by drop to the hot solution. If the pre- cipitate is due to phosphates it will disappear under these con- ditions, whereas if it is due to albumin it will not only fail to disappear but will become more flocculent in character, since the reaction of a fluid must be acid to secure the complete pre- cipitation of the albumin by this coagulation process. Too much acid should be avoided since it will cause the albumin to go into solution. Certain resin acids may be precipitated by the acid, but the precipitate due to this cause may be easily differentiated from the albumin precipitate by reason of its solubility in alcohol. (b) A modification of this test in quite general use is as follows : Fill a test-tube two-thirds full of urine and gently heat the upper half of the fluid to boiling, being careful that this fluid does not mix with the lower half. A turbidity indi- cates albumin or phosphates. Acidify the urine slightly by 1 Jolles' reagent has the following composition : Succinic acid 40 grams. Mercuric chloride '. 20 grams. Sodium chloride 20 grams. Distilled water 1000 grams. URINE. 293 the addition of 3 5 drops of dilute acetic acid, when the tur- bidity, it" due to phosphates, will disappear. Nitric acid is often used in place of acetic acid in these t< In case nitric arid is used ordinarily 1 2 drops is sufficient. 6. Acetic Acid and Potassium Ferrocyanide Test. — To 5 C.C. of urine in a test-tube add 5 [O drops of acetic acid. Mix well and add potassium ferrocyanide drop by drop, until a precipitate f< >rms. 7. Tanret's Test. — To 5 c.c. of urine in a test-tube add Tanret's reagent 1 drop by drop until a turbidity or precipitate forms. This is an exceedingly delicate test. Sometimes the urine is stratified upon the reagent as in Heller's or Roberts' ring" tests. 8. Sodium Chloride and Acetic Acid Test. — Mix two volumes of urine and one volume of a saturated solution of sodium chloride in a test-tube, acidify with acetic acid and heat to boiling. The production of a cloudiness or the formation of a precipitate indicates the presence of albumin. The resin acids may interfere here as in the ordinary coagulation test 1 page 292 ) but they may be easily differentiated from albumin by means of their solubility in alcohol. GLOBULIN. Serum globulin is not a constituent of normal urine but frequently occurs in the urine under pathological conditions and is ordinarily associated with serum albumin. In albuminuria globulin in varying amounts often accompanies the albumin, and the clinical significance of the two is very similar. Under certain conditions globulin may occur in the urine unaccom- panied by albumin. Experiments. Globulin will respond to all the tests just outlined under Albumin. If it is desirable to differentiate between albumin 'Tanret's reagent is prepared as follows: Dissolve 1.35 gram of mercuric chloride in 25 c.c. of water, add to this solution 3.32 grams of potassium iodide dissolved in 25 c.c. of water, then make the total solution up to 60 c.c. with water and add 20 c.c. of glacial acid to the mixture. 294 PHYSIOLOGICAL CHEMISTRY. and globulin in any urine the following processes may be employed : i. Saturation With Magnesium Sulphate. — Place 25 c.c. of neutral urine in a small beaker and add pulverized mag- nesium sulphate in substance to the point of saturation. If the proteid present is globulin it will precipitate at this point. If no precipitate is produced acidify the saturated solution with acetic acid and warm gently. Albumin will be precipitated if present. The above procedure may be used to separate globulin and albumin if present in the same urine. To do this filter off the globulin after it has been precipitated by the magnesium sul- phate, then acidify the clear solution and warm gently as directed. Note the formation of the albumin precipitate. 2. Half-Saturation With Ammonium Sulphate. — Place 25 c.c. of neutral urine in a small beaker and add an equal volume of a saturated solution of ammonium sulphate. Globu- lin, if present, will be precipitated. If no precipitate forms add ammonium sulphate in substance to the point of saturation. If albumin is present it will be precipitated upon saturation of the solution as just indicated. This method may also be used to separate globulin and albumin when they occur in the same urine. Frequently in urine which contains a large amount of urates a precipitate of ammonium urate may occur when the am- monium sulphate solution is added to the urine. This urate precipitate should not be confounded with the precipitate due to globulin. The two precipitates may be differentiated by means of the fact that the urate precipitate ordinarily appears only after the lapse of several minutes whereas the globulin generally precipitates at once. PROTEOSE AND PEPTONE. Proteoses, particularly deutero-proteose and hetero-proteose, have frequently been found in the urine under various patho- logical conditions such as diphtheria, pneumonia, intestinal URINE. 29S ulcer, carcinoma, dermatitis, osteomalacia, atrophy of the kid- neys and in sarcomata of the bones of the trunk. " Bence- Jones* proteid," a proteose-like substance, is of interest in this connection and it- appearance in the urine is believed to be of great diagnostic importance in cases of multiple myeloma or myelogenic osteosarcoma. By some investigators this pro- teid is held to be a variety of lietero-proteose whereas others claim that it possesses albumin characteristics. Peptone certainly occurs much less frequently as a constitu- ent of the urine than does proteose, in fact most investigators seriously question its presence under any conditions. There are many instances of peptonuria cited in the early literature but because of the uncertainty in the conception of what really constituted a peptone it is probable that in many cases of so- called peptonuria the proteid present was really proteose. Experiments. 1. Boiling Test. — Make the ordinary coagulation test ac- cording to the directions given under Albumin, page 292. If no coagulable proteid is found allow the boiled urine to stand and note the gradual appearance, in the cooled fluid, of a flaky precipitate of proteose. This is a crude test and should never be relied upon. 2. Schulte's Method. — Acidify 50 c.c. of urine with dilute acetic acid and filter off any precipitate of nucleo-proteid which may form. Now test a few cubic centimeters of the urine for coagulable proteid, by tests 2 and 5 under Albumin, pp. 291- 292. If coagulable proteid is present remove it by coagulation and filtration before proceeding. Introduce 25 c.c. of the urine, freed from coagulable proteid, into 150 c.c. of absolute alcohol and allow it to stand for 12-24 hours. Decant the supernatant fluid and dissolve the precipitate in a small amount of hot water. Xow filter this solution, and after testing again for nucleo-proteid with very dilute acetic acid, try the biuret test. If this test is positive the presence of proteose is indicated. 1 1 If it is considered desirable to test for peptone the proteose may be removed by saturation with (NH^SCX according to the directions given on page 59 and the tilt rate tested for peptone by the biuret test. 296 PHYSIOLOGICAL CHEMISTRY. Urobilin does not ordinarily interfere with this test since it is almost entirely dissolved by the absolute alcohol when the proteose is precipitated. 3. v. Aldor's Method. — Acidify 10 c.c. of urine with hydrochloric acid, add phosphotungstic acid until no more pre- cipitate forms and centifugate 1 the solution. Decant the super- natant fluid, add some absolute alcohol to the precipitate and centrifugate again. This washing with alcohol is intended to remove the urobilin and hence should be continued so long as the alcohol exhibits any coloration whatever. Now suspend the precipitate in water and add potassium hydroxide to bring it into solution. At this point the solution may be blue in color in which case decolorization may be secured by gently heating. Apply the biuret test to the cool solution. A posi- tive biuret test indicates the presence of proteoses. 4. Detection of " Bence-Jones' Proteid." — Heat the sus- pected urine very gently, carefully noting the temperature. At as low a temperature as 40 C. a turbidity may be observed and as the temperature is raised to about 6o° C. a flocculent precipitate forms and clings to the sides of the test-tube. If the urine is now acidified very slightly with acetic acid and the temperature further raised to ioo° C. the precipitate at least partly disappears; it will return upon cooling the tube. This property of precipitating at so low a temperature and of dissolving at a higher temperature is typical of " Bence- Jones' proteid " and may be used to differentiate it from all other forms of proteid material occurring in the urine. NUCLEO-PROTEID. There has been considerable controversy as to the proper classification for the proteid body which forms the " nubecula " of normal urine. By different investigators it has been called mucin, mucoid, phospho-proteid, nuclco-albumin and nucleo- proteid. Of course, according to the modern acceptation of 1 If not convenient to use a centrifuge the precipitate may be filtered off and washed on the filter paper with alcohol. URINE. 297 the meanings of these terms they cannot be synonymous. Mucin and mucoid are glucoproteids and hence contain no phosphorus (see p. 61). whereas phospho-proteids, nucleo- alhnmins and nncleo-proteids are phosphorized bodies. It may possibly be that both these forms of proteid. i. <\, the glucopro- teid and the phosphorized type, occur in the urine under certain conditions (seepage 264). In this connection we will use the term nucleo-proteid. The pathological conditions under which the content of nucleo-proteid is increased includes all affections of the urinary passages and in particular pyelitis, nephritis and inflammation of the bladder. » Experiments. 1. Detection of Nucleo-proteid. — Place 10 c.c. of urine in a small beaker, dilute it with three volumes of water, to prevent precipitation of urates, and make the reaction very strongly acid with acetic acid. If the urine becomes turbid it is an indi- cation that nucleo-proteid is present. If the urine under examination contains albumin the greater portion of this substance should be removed by boiling the urine before testing it for the presence of nucleo-proteid. 2. Ott's Precipitation Test. — Mix 2^ c.c. of the urine with an equal volume of a saturated solution of sodium chloride and slowly add Almen's reagent. 1 In the presence of nucleo- proteid a voluminous precipitate forms. BLOOD. The pathological conditions in which blood occurs in the urine may be classified under the two divisions hematuria and hemoglobinuria. In hematuria we are able to detect not only the haemoglobin but the unruptured corpuscles as well, whereas in hemoglobinuria the pigment alone is present. Hematuria is brought about through blood passing into the urine because of some lesion of the kidney or of the urinary tract below the 1 Dissolve 5 grams of tannin in 240 c.c. of 50 per cent alcohol and add 10 c.c. of 25 per cent acetic acid. 298 PHYSIOLOGICAL CHEMISTRY. kidney. Hemoglobinuria is brought about through haemo- lysis, i. c, the rupturing of the stroma of the erythrocyte and the liberation of the haemoglobin. This may occur in scurvy, typhus, pyemia, purpura and in other diseases. It may also occur as the result of a burn covering a considerable area of the body, or may be brought about through the action of cer- tain poisons or by the injection of various substances having the power of dissolving the erythrocytes. Transfusion of blood may also cause hemoglobinuria. Experiments. 1. Heller's Test. — Render 10 c.c. of urine strongly alkaline with potassium hydroxide solution and heat to boiling. Upon allowing the heated urine to stand a precipitate of phosphates, colored red by the contained haematin, is formed. It is ordi- narily well to make a "control" experiment using normal urine, before coining to a final decision. Certain substances such as cascara sagrada, rhubarb, san- tonin, and senna cause the urine to give a similar reaction. Reactions due to such substances may be differentiated from the true blood reaction by the fact that both the precipitate and the pigment of the former reaction disappear when treated with acetic acid, whereas if the color is due to haematin the acid will only dissolve the precipitate of phosphates and leave the pig- ment undissolved. 2. Teichmann's Haemin Test. — Place a small drop of the suspected urine or a small amount of the moist sediment on a microscopic slide, add a minute grain of NaCl and carefully evaporate to dryness over a loiv flame. Put a cover glass in place, run underneath it a drop of glacial acetic acid and warm gently until the formation of gas bubbles is observed. Cool the preparation, examine under the microscope and compare the form of the crystals with those reproduced in Figs. 58 and 59, page 164. 3. Heller-Teichmann Reaction. — Produce the pigmented precipitate according to directions given in Heller's test on p. URINE. 299 298. It' there Is a copious precipitate of phosphates and but little pigment the phosphates may be dissolved by treatment with acetic acid and the residue used in the formation of the haemin crystals according to directions in Experiment 2, p. 298. 4. Zeynek and Nencki's Haemin Test. — To 10 c.c. of the urine under examination add acetone until no mure precipitate forms. Filter off the precipitate and extract it with 10 c.c. of acetone rendered acid with 2-3 drops of hydrochloric acid. Place a drop of the resulting colored extract on a slide, immedi- ately place a cover glass in position and examine under the microscope. Compare the form of the crystals with those shown in Figs. 58 and 59, page [64. Haemin crystals pro- duced by this manipulation are sometimes very minute, thus rendering- it difficult to determine the exact form of the crystal. 5. Schalfijew's Haemin Test. — Place 20 c.c. of glacial acetic acid in a small beaker and heat to 8o° C. Add 5 c.c. of the urine under examination, raise the temperature to 8o° C. and stand the mixture aside to cool. Examine the crystals under the microscope and compare them with those shown in Figs. 58 and 59, page 164. 6. Guaiac Test. — Place 5 c.c. of urine in a test-tube and by means of a pipette introduce a freshly prepared alcoholic solu- tion of guaiac into the fluid until a turbidity results; then add old turpentine or hydrogen peroxide, drop by drop, until a blue color is obtained. This is a very delicate test when properly performed. Buckmaster has recently suggested the use of guaiaconic acid instead of the solution of guaiac. See discus- sion on page 158 and test on page 163. 7. Spectroscopic Examination. — Submit the urine to a spectroscopic examination according to the directions given on page 169 looking especially for the absorption-bands of oxy- hemoglobin and methaemoglobin (see Absorption Spectra, Plate I.). 300 PHYSIOLOGICAL CHEMISTRY. BILE. Both the pigments and the acids of the bile may be detected in the urine under certain pathological conditions. Of the pig- ments, bilirubin is the only one which has been positively iden- tified in fresh urine; the other pigments, when present, are probably derived from the bilirubin. A urine containing bile may be yellowish-green to brown in color and when shaken foams readily. The staining of the various tissues of the body through the absorption of bile due to occlusion of the bile duct causes a condition known as icterus or jaundice. Bile is always present in the urine under such conditions unless the amount of bile reaching the tissues is extremely small. Experiments. Tests for Bile Pigments, i. Gmelin's Test. — To about 5 c.c. of concentrated nitric acid in a test-tube add an equal volume of urine carefully so that the two fluids do not mix. At the point of contact note the various colored rings, green, bine, violet, red and reddish- yellow. 2. Rosenbach's Modification of Gmelin's Test. — Filter 5 c.c. of urine through a small filter paper. Introduce a drop of concentrated nitric acid into the cone of the paper and observe the succession of colors as given in Gmelin's test. 3. Huppert's Reaction. — Thoroughly shake equal volumes of urine and milk of lime in a test-tube. The pigments unite with the calcium and are precipitated. Filter off the precipi- tate, wash it with water and transfer to a small beaker. Add alcohol acidified slightly with hydrochloric acid and warm upon a water-bath until the solution becomes colored an emerald green. 4. Hammarsten's Reaction. — To about 5 c.c. of Ham- marsten's reagent 1 in a small evaporating dish add a few drops 1 Hammarsten's reagent is made by mixing 1 volume of 25 per cent nitric acid and 19 volumes of 25 per cent hydrochloric acid and then adding 1 volume of this acid mixture to 4 volumes of 95 per cent alcohol. URINE. 301 of urine. A green color is produced. If more of the reagent is now added the play of colors as noted in Gmelin's test may be 1 ibtained. 5. Smith's Test. — To 2-3 c.c. of urine in a test-tube add carefully about 5 c.c. of dilute tincture of iodine (1: 10) so thai the fluids do not mix. A green ring is observed at the point of contact. Tests for Bile Acids. 1. Pettenkofer's Test. — To 5 c.c. of urine in a test-tube add 5 drops of a 5 per cent solution of saccharose. Now in- cline the tube, run about 2-3 c.c. of concentrated sulphuric acid carefully down the side and note the red ring at the point of contact. Upon slightly agitating the contents of the tube the whole solution gradually assumes a reddish color. As the tube becomes warm, it should be cooled in running water in order that the temperature may not rise above 70 C. _'. Mylius's Modification of Pettenkofer's Test. — To ap- proximately 5 c.c. of urine in a test-tube add 3 drops of a very dilute (1 : 1,000) aqueous solution of furfurol. HC CH II II HC C • CHO. \/ Xow incline the tube, run about 2-3 c.c. of concentrated sul- phuric acid carefully down the side and note the red ring as above. In this case also, upon shaking the tube, the whole solution is colored red. Keep the temperature below 70 C. as before. 3. Neukomm's Modification of Pettenkofer's Test. — To a few drops of urine in an evaporating dish add a trace of a dilute saccharose solution and one or more drops of dilute sul- phuric acid. Evaporate on a water-bath and observe the development of a violet color at the edge of the evaporating mixture. Discontinue the evaporation as soon as the color is observed. 302 PHYSIOLOGICAL CHEMISTRY. 4. v. Udransky's Test. — To 5 c.c. of urine in a test-tube add 3-4 drops of a very dilute (1 : 1,000) aqueous solution of furfurol. Place the thumb over the top of the tube and shake until a thick foam is formed. By means of a small pipette add 2-3 drops of concentrated sulphuric acid to the foam and observe the dark pink coloration produced. 5. Salkowski's Test. — Render 5 c.c. of urine alkaline with a few drops of a 10 per cent sodium carbonate solution and add a 10 per cent solution of calcium chloride, drop by drop, until the supernatant fluid exhibits the normal urinary color when the contents of the test-tube are thoroughly mixed. Filter off the precipitate, and after washing it, place it in a second tube with 95 per cent alcohol. Acidify the alcohol with hydro- chloric acid and, if necessary, shake the tube to bring the pre- cipitate into solution. Heat the solution to boiling and observe the appearance of a green color which changes through blue and violet to red; if no bile is present the solution does not undergo any color change. This test will frequently exhibit greater delicacy that Gmelin's test. 6. Hay's Test. — Cool about 10 c.c. of urine in a test-tube to 1 7 C. or lower, and sprinkle a little finely pulverized sul- phur upon the surface of the fluid. The presence of bile acids is indicated if the sulphur sinks to the bottom of the liquid, the rapidity with which the sulphur sinks depending upon the amount of bile acids present in the urine. The test is said to react with bile acids when the latter are present in the propor- tion 1 : 120,000. Some investigators claim that it is impossible to differentiate between bile acids and bile pigments by this test. CH 3 I ACETONE, C = 0. I CH 3 It was formerly very generally believed that acetone appeared URINE. 303 in the urine under pathological conditions because of increased proteid decomposition. It is now generally thought that, in man, the output of acetone arises principally from the breaking down of fatty tissues or fatty foods within the organism. The quantity of acetone eliminated has been shown to increase when the subject is fed an abundance of fat-containing food as well as during fasting, whereas a replacement of the fat with carbohydrates is followed by a marked decrease in the acetone excretion. Conditions are different with certain of the lower animals. With the dog, for instance, the output of acetone is not diminished when the animal is fed upon a carbohydrate diet, is decreased during fasting and increased when the animal is fed upon a diet of meat. Acetone and the closely related bodies, /?-oxybutyric acid and diacetic acid, are generally classified as the acetone bodies. They are all associated with a deranged metabolic function and may appear in the urine together or separately, depending upon the conditions. Acetone and diacetic acid may occur alone in the urine but /8-oxybutyric acid is never found except in con- junction with one or the other of these bodies. Acetone and diacetic acid arise chiefly from the oxidation of /?-oxybutyric acid. The relation existing between these three bodies is shown in the following reactions : (a) CH 3 -CH(OH)-CH 2 -COOH + = -oxvbutyric acid. CH 3 CO • CH 2 • COOH + H 2 0. Diacetic acid. (b) CH 3 CO-CH 2 -COOH=(CH 3 ) 2 CO + C0 2 . Diacetic acid. Acetone. Acetone, chemically considered, is a ketone, di-m ethyl ketone. When pure it is a liquid which possesses a characteristic aro- matic fruit-like odor, boils at 56— 57 C. and is miscible with water, alcohol or ether in all proportions. Acetone is a physiological as well as a pathological constituent of the urine and under normal conditions the daily output is about 0.0 1- 0.03 gram. 3O4 PHYSIOLOGICAL CHEMISTRY. Pathologically, the elimination of acetone is often greatly increased and at such times a condition of acetonuria is said to exist. This pathological acetonuria may accompany diabetes mellitus, scarlet fever, typhoid fever, pneumonia, nephritis, phosphorus poisoning, grave anaemias, fasting' and a deranged digestive function; it also frequently accompanies auto-intoxi- cation and chloroform anaesthesia. The types of acetonuria most frequently met with are those noted in febrile conditions and in advanced cases of diabetes mellitus. Experiments. 1. Isolation from the Urine. — In order to facilitate the de- tection of acetone in the urine, the specimen under examination should be distilled and the tests as given below applied to the resulting distillate. If it is not convenient to distil the urine, the tests may be conducted upon the undistilled fluid. To obtain an acetone distillate proceed as follows : Place 100-250 c.c. of urine in a distillation flask or retort and render it acid with acetic acid. Collect about one-third of the original volume of fluid as a distillate, add 5 drops of 10 per cent hydro- chloric acid and redistil about one-half of this volume. With this final distillate conduct the tests as given below. 2. Gunning's Iodoform Test. — To about 5 c.c. of the urine or distillate in a test-tube add a few drops of Lugol's solution 1 or ordinary iodine solution (I in KI) and enough NH 4 OH to form a black precipitate (nitrogen iodide). Allow the tube to stand (the length of time depending upon the content of acetone in the fluid under examination) and note the formation of a yellowish sediment consisting of iodoform. Examine the sedi- ment under the microscope and compare the form of the crys- tals with those shown in Fig. 6, p. 21. If the crystals are not well formed recrystallize them from ether and examine again. The crystals of iodoform should not be confounded with those of stellar phosphate (Fig. j6, p. 193) which may be formed in 'Lugol's solution may be prepared by dissolving 5 grams of iodine and 10 grams of potassium iodide in 100 c.c. of distilled water. trim:. 305 this test, particularly if made upon the undistilled urine. This test is preferable to Lieben's test (4) since no substance other than acetone will produce iodoform when treated accord- ing to the directions for this test; both alcohol and aldehyde yield iodoform when tested by Lieben's test. Gunning's test is rather the most satisfactory test yet sug- gested for the detection of acetone, and may be used with good results even upon the undistilled urine. In some instances where the amount of acetone present is very small it is neces- sary to allow the tube to stand 24 hours before making the examination for iodoform crystals. This test serves to detect acetone when present in the ratio 1 : 100,000. 3. Legal's Test. — Introduce about 5 c.c. of the urine or distillate into a test-tube, add a few drops of a freshly prepared aqueous solution of sodium nitro-prusside and render the mix- ture alkaline with potassium hydroxide. A ruby red color, clue to creatinin, a normal urinary constituent, is produced (see Weyl's test. p. 2^2). Add an excess of acetic acid and if acetone is present the red color will be intensified, whereas in the ab- sence of acetone a yellow color will result. Make a control test upon normal urine to show that this is so. A similar red color may be produced by paracresol in urines containing no acetone. 4. Lieben's Test. — Introduce 5 c.c. of the urine or distillate into a test-tube, render it alkaline with potassium hydroxide and add 1-2 c.c. of iodine solution, drop by drop. If acetone is present a yellowish precipitate of iodoform will be produced. Identifv the iodoform by means of its characteristic odor and its typical crystalline form (see Fig. 6. p. 21). While fully as delicate as Gunning's test (2) this test is not as accurate, since by means of the procedure involved, either alcohol or aldehyde will yield a precipitate of iodoform. This test is especially liable to lead to erroneous deductions when urines from the advanced stages of diabetes are under examination, because of the presence of alcohol formed from the sugar through fermentative processes. 306 PHYSIOLOGICAL CHEMISTRY. 5. Reynolds-Gunning Test. — This test depends upon the solubility of mercuric oxide in acetone and is performed as fol- lows : To 5 c.c. of the urine or distillate add a few drops of mercuric chloride, render the solution alkaline with potassium hydroxide and add an equal volume of 95 per cent alcohol. Shake thoroughly in order to bring the major portion of the mercuric oxide into solution and filter. Render the clear filtrate faintly acid with hydrochloric acid and stratify some ammonium sulphide, (NH 4 ) 2 S, upon this acid solution. At the zone of contact a blackish-gray ring of precipitated mercu- ric sulphide, HgS, will form. Aldehyde also responds to this test. Aldehyde, however, has never been detected in the urine and could only be present in this instance if the acidified urine was distilled too far. CH 3 I DIACETIC ACID, C = CHo-COOH. Diacetic or acetoacetic acid occurs in the urine only under pathological conditions and is rarely found except associated with acetone. It is formed from /?-oxybutyric acid, another of the acetone bodies, and upon decomposition yields acetone and carbon dioxide. Diaceturia occurs ordinarily under the same conditions as the pathological acetonuria, i. e., in fevers, dia- betes, etc. (see p. 304). If very little diacetic acid is formed it may all be transformed into acetone, whereas if a larger quantity is produced both acetone and diacetic acid may be present in the urine. Diaceturia is most frequently observed in children, especially accompanying fevers and digestive dis- orders; it is perhaps less frequently observed in adults, but when present, particularly in fevers and diabetes, it is fre- quently followed by fatal coma. Diacetic acid is a colorless liquid which is miscible with water, alcohol, and ether, in all proportions. It differs from acetone in giving a violet-red or Bordeaux-red color with a dilute solution of ferric chloride. URINE. 307 Experiments. 1. Gerhardt's Test. — To 5 cc of urine in a test-tube add ferric chloride solution, drop by drop, until no more precipi- tate forms. In the presence of diacetic acid a Bordeaux-red color is produced ; this color may be somewhat masked by the precipitate of ferric phosphate, in which case the fluid should be filtered. A positive result from the above* manipulation simply indi- cates the possible presence of diacetic acid. Before making a final decision regarding the presence of this body make the two following control experiments: 1 (/ ) Place 5 cc. of urine in a test-tube and boil it vigorously for 3-5 minutes. Cool the tube and, with the boiled urine, make the test as given above. As has been already stated, diacetic acid yields acetone upon decomposition and acetone does not give a Bordeaux-red color with ferric chloride. By boiling as indicated above therefore, any diacetic acid present would be decomposed into acetone and carbon dioxide and the test upon the resulting fluid would be negative. If positive the color is due to the presence of bodies other than diacetic acid. I />) Place 5 cc. of urine in a test-tube, acidify with H 2 S0 4 , tc free diacetic acid from its salts, and carefully extract the mixture with ether by shaking. If diacetic acid is present it will be extracted by the ether. Now remove the ethereal solu- tion and add to it an equal volume of dilute ferric chloride; diacetic acid is indicated by the production of the character- istic Bordeaux-red color. This color disappears spontaneously in 24-48 hours. Such substances as antipyrin, kairin, phena- cetin. salicylic acid, salicylates, sodium acetate, sulphocyanides and thallin yield a similar red color under these conditions, but when due to the presence of any of these substances the color does not disappear spontaneously but may remain permanent for days. Many of these disturbing substances are soluble in benzene or chloroform and may be removed from the urine by this means before extracting with ether as above. Diacetic acid is insoluble in benzene or chloroform. 3CS PHYSIOLOGICAL CHEMISTRY. 2. Arnold-Lipliawsky Reaction. — This reaction is some- what more delicate than Gerhardt's test (i) and serves to de- tect diacetic acid when present in the proportion i : 25,000. It is also negative toward acetone, /8-oxybutyric acid and the inter- fering drugs mentioned as causing' erroneous deductions in the application of Gerhardt's test. If the urine under examination is highly pigmented it should be partly decolorized by means of animal charcoal before applying the test as indicated below. Place 5 c.c. of the urine under examination and an equal volume of the Arnold-Lipliawsky reagent 1 in a test-tube, add a few drops of concentrated ammonia and shake the tube vigor- ously. Note the production of a brick-red color. Take 1-2 c.c. of this colored solution, add 10-20 c.c. of hydrochloric acid (sp. gr. 1.19), 3 c.c. of chloroform and 2-4 drops of ferric chloride solution and carefully mix the fluids without shaking. Diacetic acid is indicated by the chloroform assum- ing a violet or blue color; if diacetic acid is absent the color may be yellow or light red. H OH H I I I /3-OXYBUTYRIC ACID, H — C — C — — COOH. I I I H H H This acid does not occur as a normal constituent of urine but is found only under pathological conditions and then always in conjunction with either acetone or diacetic acid. Either of these bodies may be formed from /3-oxybutyric acid under proper conditions. It is present in especially large 1 This reagent consists of two definite solutions which are ordinarily preserved separately and mixed just before using. The two solutions are prepared as follows : (a) One per cent aqueous solution of potassium nitrite. (b) One gram of />-amino-acetophenon dissolved in 100 c.c. of distilled water and enough hydrochloric acid (about 2 c.c.) added, drop by drop, to cause the solution, which is at first yellow, to become entirely colorless. An excess of acid must be avoided. Before using, a and b are mixed in the ratio 1 : 2. URINE. 3°9 amount in severe cases of diabetes and has also been detected in digestive disturbances, continued fevers, scurvy, measles and in starvation. It is probable that, in man. |8-oxybutyric acid, in common with acetone and diacetic acid, arises principally from the breaking down of fatty tissues within the organism. The condition in which large amounts of acetone and diacetic acid, and in severe cases |8-oxybutyric acid also, are excreted in the urine is known as " acidosis." In diabetes the deranged metabolic conditions cause the production of great quantities of these substances which lead to an acid intoxication and ultimately to diabetic coma. Ordinarily /B-oxybutyric acid is an odorless, transparent syrup, which is laevorotatory and easily soluble in water, alco- hol and ether; it may be obtained in crystalline form. Experiments. i. Polariscopic Examination. — Subject some of the urine (free from proteid) to the ordinary fermentation test (see page 288). This will remove dextrose and lsevulose, which would interfere with the polariscopic test. Now examine the fermented fluid in the polariscope and if it is laevorotatory the presence of 0-oxybutyric acid is indicated. This test is not absolutely reliable, however, since conjugate glycuronates are also laevorotatory after fermentation. 2. Kiilz's Test. — Evaporate the urine, after fermenting it as indicated in the last test, to a syrup, add an equal volume of concentrated sulphuric acid and distil the mixture directly without cooling. Under these conditions a-crotonic acid is formed and is present in the distillate. Allow the distillate to cool slowly and note the formation of crystals of a-crotonic acid which are soluble in ether and melt at 72 ° C. In case very slight traces of /?-oxybutyric acid be present in the urine under examination the amount of a-crotonic acid formed may be too small to yield a crystalline product. In this event the distillate should be extracted with ether, the ethereal extract evaporated and the residue washed with water. Under these 3IO PHYSIOLOCxICAL CHEMISTRY. conditions the impurities will be removed and the a-crotonic acid will remain behind as a residue. The melting-point of this residue may then be determined. CONJUGATE GLYCURONATES. Glycuronic acid does not occur free in the urine but is found, for the most part, in combination with phenol. Much smaller quantities are excreted in combination with indoxyl and skatoxyl. The total content of conjugate glycuronates seldom exceeds 0.004 per cent under normal conditions. The output may be very greatly increased as the result of the administration of antipyrin, borneol, camphor, chloral, men- thol, morphine, naphthol, turpentine, etc. The glycuronates as a group are lsevorotatory, whereas glycuronic acid is dextro-rotatory. Most of the glycuronates reduce alkaline metallic oxides and so introduce an error in the examination of urine for sugar. Conjugate glycuronates often occur associated with dextrose in glycosuria, diabetes mellitus and in some other disorders. As a class the glycuronates are non- fermentable. Experiments. 1. Fermentation-Reduction Test. — Test the urine by Fehling's test. If there is reduction try Barfoed's test. If negative this indicates the absence of dextrose. A negative fermentation test would now indicate the presence of conju- gate glycuronates (or lactose in rare cases). If dextrose is present in the urine tested for glycuronates the urine must first be subjected to a polariscopic examina- tion, then fermented and a second polariscopic examination made. The sugar being dextro-rotatory and fermentable and the glycuronates being laevorotatory and non- fermentable the second polariscopic test will show a lsevorotation indica- tive of conjugate glycuronates. 2. Tollens' Reaction. — Make this test according to direct- ions given under Pentoses, page 311. URINE. 311 PENTOSES. We have two distinct types of pentosuria, i. c, alimentary pentosuria, resulting from the ingestion of large quantities of pentose-rich vegetables such as prunes, cherries, grapes or plums, and fruit juices, in which condition the pentoses appear only temporarily in the urine; and the chrome form of pento- suria, in which the output of pentoses bears no relation what- ever to the quantity and nature of the pentose content of the food eaten. In occurring in these two forms, pentosuria resembles glycosuria (see page 283), but it is definitely known that pentosuria bears no relation to diabetes mellitus and there is no generally accepted theory to account for the occur- rence of the chronic form of pentosuria. The pentose de- tected most frequently in the urine is arabinose, the inactive form generally occurring in chronic pentosuria and the lsevo- rotatory variety occurring in the alimentary type of the disorder. Experiments. 1. Tollens' Reaction. — To equal volumes of urine and hydrochloric acid (sp. gr. 1.09) add a little phloroglucin and heat the mixture on a boiling water-bath. Pentose, galactose, laevulose or glycuronic acid will be indicated by the appearance of a red color. To differentiate between these bodies examine by the spectroscope and look for the absorption band between D and E given by pentoses and glycuronic acid, and then differ- entiate between the two latter bodies by the melting-points of their osazons. 2. Orcin Test. — Place equal volumes of urine and hydro- chloric acid (sp. gr. 1.09) in a test-tube, add a small amount of orcin, and heat the mixture to boiling. Color changes from red, through reddish-blue to green will be noted. When the solution becomes green it should be shaken in a separatory funnel with a little amyl alcohol, and the alcoholic extract examined spectroscopically. An absorption band between C and D will be observed. 312 THYSIOLOGICAL CHEMISTRY. FAT. When fat finds its way into the urine through a lesion which brings some portion of the urinary passages into com- munication with the lymphatic system a condition known as chyhtria is established. The turbid or milky appearance of such urine is due to its content of chyle. This disease is encountered most frequently in tropical countries, but is not entirely unknown in more temperate climates. Albumin is a constant constituent of the urine in chyluria. Upon shaking a chylous urine with ether the fat is dissolved by the ether and the urine becomes clearer or entirely clear. HiEMATOPORPHYRIN. Urine containing this body is occasionally met with in various diseases but more frequently after the use of quinine, tetronal, trional and especially sulphonal. Such urines ordi- narily possess a reddish tint, the depth of color varying greatly under different conditions. Experiments. i. Spectroscopic Examination. — To ioo c.c. of urine add about 20 c.c. of a 10 per cent solution of KOH or NH 4 OH. The precipitate which forms consists principally of earthy phosphates to which the haematoporphyrin adheres and is carried down. Filter off the precipitate, wash it and transfer to a flask and warm with alcohol acidified with hy- drochloric acid. By this process the haematoporphyrin is dis- solved and on filtering will be found in the filtrate and may be identified by means of the spectroscope (see page 173, and Absorption Spectra, Plate II). 2. Acetic Acid Test. — To 100 c.c. of urine add 5 c.c. of glacial acetic acid and allow the mixture to stand 48 hours. Haematoporphyrin deposits in the form of a precipitate. URINE. 313 LACTOSE. Lactose is rarely found in the urine except as it is excreted by women during pregnancy, during the nursing period or soon after weaning. It is rather difficult t«> show the pres- ence of lactose in the urine in a satisfactory manner, since the formation of the characteristic lactosazon is not attended with any great measure of success under these conditions. It is, however, comparatively easy to show that it is not dex- trose, for. while it responds to reduction tests, it docs not ferment with pure yeast and does not give a dextrosazon. An absolutely conclusive test, of course, is the isolation of the lactose in crystalline form (Fig. 75, p. 189) from the urine. Experiments. 1. Rubner's Test. — To 10 c.c. of urine in a small beaker add some plumbic acetate, in substance, heat to boiling and add XH 4 OH until no more precipitate is dissolved. In the presence of lactose a brick-red or rose-red color develops, whereas dextrose gives a coffee-brown color, maltose a light yellow color and lsevulose no color at all under the same conditions. 2. Compound Test. — Try the phenylhydrazin test, the fermentation test and Barfoed's test according to directions given under Dextrose, pages 283, 288 and 289. If these are negative, try Xylander's test, page 288. If this last test is positive, the presence of lactose is indicated. L^VULOSE. Diabetic urine frequently possesses the power of rotating the plane of polarized light to the left, thus indicating the presence of a l?evorotatory substance. This kevorotation is sometimes due to the presence of laevulose, although not nec- essarily confined to this carbohydrate, since conjugate glycu- ronates and /?-oxybutyric acid, two other lsevorotatory bodies, 314 PHYSIOLOGICAL CHEMISTRY. are frequently found in the urine of diabetics. Laevulose is invariably accompanied by dextrose in diabetic urine, but Icevulo&uria has been observed as a separate anomaly. The presence of laevulose may be inferred when the percentage of sugar, as determined by the titration method, is greater than the percentage indicated by the polariscopic examination. Experiments. 1. Seliwanoff's Reaction. — If a solution of resorcin in dilute HC1 (1 volume of concentrated HC1 to 2 volumes of H 2 0) be warmed with an equal volume of a urine containing laevulose, the liquid will become red and a precipitate will separate. The precipitate may be dissolved in alcohol to which it will impart a striking red color. 2. Phenylhydrazin Test. — Make the test according to directions under Dextrose, 1 page 283. 3. Polariscopic Examination. — A simple polariscopic ex- amination, when taken in connection with other ordinary tests, will furnish the requisite data regarding the presence of laevulose, provided laevulose is not accompanied by other laevo- rotatory substances, such as conjugate glycuronates and /?-oxybutyric acid. INOSIT. Inosit occasionally occurs in the urine in albuminuria, diabetes mellitus and diabetes insipidus. It is claimed also that copious water-drinking causes this body to appear in the urine. By some investigators inosit is believed to occur in traces in normal urine. Experiment. 1. Detection of Inosit. — Acidify the urine with concen- trated nitric acid and evaporate nearly to dryness. Add a few drops of NH 4 OH and a little CaCl 2 solution to the moist residue and evaporate the mixture to dryness. In the pres- ence of inosit (0.001 gram) a bright red color is obtained. URINE. 315 LAIOSE. This substance is occasionally found in the urine in severe cases of diabetes mellitus. By some investigators laiose is classed with the sugars. It resembles laevulose in that it has the property of reducing certain metallic oxides and is laevoro- tatbry, but differs from laevulose in being amorphous, non- fermentable "and in not possessing a sweet taste. MELANINS. These pigments never occur normally in the urine but are present under certain pathological conditions, their presence being especially associated with melanotic tumors. Ordi- narily the freshly passed urine is clear, but upon exposure to the air the color deepens and may at the last be very dark brown or black in color. The pigment is probably present in the form of a chromogen or melanogen and upon coming in contact with the air oxidation occurs, causing the transfor- mation of the melanogen into melanin and consequently the darkening of the urine. It is claimed that melanuria is proof of the formation of a visceral melanotic growth. In many instances, without doubt, urines rich in indican have been wrongly taken as diagnostic proof of melanuria. The pigment melanin is sometimes mis- taken for indigo and melanogen for indican. It is compara- tively easy to differentiate between indigo and melanin through the solubility of the former in chloroform. In rare cases melanin is found in urinary sediment in the form of fine amorphous granules. Experiments. 1. Zeller's Test. — To 50 c.c. of urine in a small beaker add an equal volume of bromine water. In the presence of melanin a yellow precipitate will form and will gradually darken in color, ultimately becoming black. 6 l6 PHYSIOLOGICAL CHEMISTRY. 2. von Jaksch-Pollak Reaction. — Add a few drops of ferric chloride solution to 10 c.c. of urine in a test-tube and note the formation of a gray color. Upon the further addition of the chloride a dark precipitate forms, consisting of phos- phates and adhering melanin. An excess of ferric chloride causes the precipitate to dissolve. This is the most satisfactory test for the identification of melanin in the urine. UROROSEIN. This is a pigment which is not present in normal urine but may be detected in the urine of various diseases, such as pul- monary tuberculosis, typhoid fever, nephritis and stomach dis- orders. Urorosein, in common with various other pigments, does not occur preformed in the urine, but is present in the form of a chromogen, which is transformed into the pigment upon treatment with a mineral acid. Experiments. i. Robin's Reaction. — Acidify 10 c.c. of urine with about 15 drops of concentrated hydrochloric acid. Upon allowing the acidified urine to stand, a rose-red color will appear if urorosein is present. 2. Nencki and Sieber's Reaction. — To 100 c.c. of urine in a beaker add 10 c.c. of 25 per cent sulphuric acid. Allow the acidified urine to stand and note the appearance of a rose-red color. The pigment may be separated by extraction with amyl alcohol. UNKNOWN SUBSTANCES. Ehrlich's Diazo Reaction. — Place equal volumes of urine and Ehrlich's diazobenzenesulphonic acid reagent 1 in a test- 1 Two separate solutions should be prepared and mixed in definite pro- portions when needed for use. (a) Five grams of sodium nitrite dissolved in 1 liter of ditilled water. (b) Five grams of sulphanilic acid and 50 c.c. of hydrochloric acid in 1 liter of distilled water. URINE. 317 tube, mix thoroughly by shaking and quickly add ammonium hydroxide in excess. The tesl is positive if both the fluid and the foam assume a red color. If the tube is allowed to stand a precipitate fi irms, the upper p< >rtii >n 1 >f which exhibits a blue, green, greenish-black or violet color. Normal urine gives a brownish-yellow reaction with the above manipulation. The exact nature of the substance or substances upon whose presence in the urine this reaction depends is not well under- stood. Some investigators claim that a positive reaction in- dicates an abnormal decomposition of proteid material, whereas others assume it to be due to an increased excretion of alloxyproteic acid, oxyproteic acid or uroferric acid. The reaction may be taken as a metabolic symptom of certain disorders, which is of value diagnostically only when taken in connection with the other symptoms. The reaction appears principally in the urine in febrile disorders and in particular in the urine in typhoid fever, tuberculosis and measles. The reaction has also been obtained in the urine in various other disorders such as carcinoma, chronic rheuma- tism, diphtheria, erysipelas, pleurisy, pneumonia, scarlet fever, syphilis, typhus, etc. The administration of alcohol, chrysa- robin. creosote, cresol, dionin, guaiacol, heroin, morphine, naphthalene, opium, phenol, tannic acid, etc., will also cause the urine to give a positive reaction. The following chemical reactions take place in this test : (a) NaN0 2 + HCl = HN0 2 + NaCl. NH 2 N / / \ (b) C C H 4 +HNOo = C H 4 N + 2PL0. \ \ / HSO3 S0 3 Sulphanilic acid. Diazo-benzenesulphonic acid. Solutions a and b should be preserved in well stoppered vessels and mixed in the proportion 1 : 50 when required. Green asserts that greater delicacy is secured by mixing the solutions in the proportion 1 : 100. The sodium nitrite deteriorates upon standing and becomes unfit for use in the course of a few weeks. CHAPTER XIX. URINE: ORGANIZED AND UNORGANIZED SEDIMENTS. The data obtained from carefully conducted microscopical examinations of the sediment of certain pathological urines are of very great importance, diagnostically. Too little em- phasis is sometimes placed upon the value of such findings. Fig. 97. Fig. 98. The Purdy Electric Centrifuge. Sediment Tube for the Purdy Electric Centrifuge. The sedimentary constituents may be divided into two classes, i. e., organized and unorganized. The sediment is ordinarily collected for examination by means of the centri- fuge (Fig. 97, above). An older method, and one still in 318 urink: si in. m i.n is. 319 vogue in some quarters, is the so-called gravity method. This simply consists in placing the urine in a conical glass and allowing the sediment to settle. The collection of the sedi- ment by means of the centrifuge, however, is much preferable, since the process of sedimentation may be accomplished by the use of this instrument in a few minutes, and far more per- fectly, whereas when the other method is used it is frequently necessary to allow the urine to remain in the conical glass 12-24 hours before sufficient sediment can be secured for the microscopical examination. (a) Unorganized Sediments. Ammonium magnesium phosphate ("Triple phosphate"). Calcium oxalate. Calcium carbonate. Calcium phosphate. Calcium sulphate. Uric acid. Urates. Cystin. Cholesterin. Hippuric acid. Leucin and tyrosin. Hamiatoidin and bilirubin. Magnesium phosphate. Indigo. Xanthin. Melanin. Ammonium Magnesium Phosphate (" Triple Phos- phate "). — Crystals of "triple phosphate" are a characteristic constituent of the sediment when alkaline fermentation of the urine has taken place either before or after being voided. They may even be detected in amphoteric or slightly acid urine provided the ammonium salts are present in large enough quantity. This substance may occur in the sediment in two forms, i. e., prisms and the feathery type. The pris- 320 PHYSIOLOGICAL CHEMISTRY. matic form of crystal (Fig. 96, p. 278) is the one most com- monly observed in the sediment; the feathery form (Fig. 96. p. 278) predominates when the urine is made ammoniacal with ammonia. The sediment of the urine in such disorders as are accom- panied by a retention of urine in the lower urinary tract con- tains " triple phosphate " crystals as a characteristic constit- uent. The crystals are frequently abundant in the sediment during paraplegia, chronic cystitis, enlarged prostate and chronic pyelitis. Calcium Oxalate. — Calcium oxalate is found in the urine in the form of at least two distinct types of crystals, i. e., the dumb-bell type and the octahedral type (Fig. 99, below). Fig. 99. Calcium Oxalate. (Ogden.) Either form may occur in the sediment of neutral, alkaline or acid urine, but both forms are found most frequently in urine having an acid reaction. Occasionally, in alkaline urine, the octahedral form is confounded with " triple phos- phate " crystals. They may be differentiated from the phos- phate crystals by the fact that they are insoluble in acetic acid. The presence of calcium oxalate in the urine is not of itself a sign of any abnormality, since it is a constituent of normal urine. It is increased above the normal, however, in such pathological conditions as diabetes mellitus, in organic dis- eases of the liver and in various other conditions which are accompanied by a derangement of digestion or of the oxida- URINE : SEDIMENTS. 321 tion mechanism, such as occurs in certain diseases of the heart and lungs. Calcium Carbonate. — Calcium carbonate crystals form a typical constituent of the urine of herbivorous animals. They occur less frequently in human urine. The reaction of urine containing these crystals is nearly always alkaline, although they may occur in amphoteric or in slightly acid urine. It gen- erally crystallizes in the form of granules, spherules or dumb- bells (Fig. 100, below). The crystals of calcium carbonate may be differentiated from calcium oxalate by the fact that they dissolve in acetic acid with the evolution of carbon dioxide gas. Calcium Phosphate (Stellar Phosphate). — Calcium phos- phate may occur in the urine in three forms, i. e., amorphous, granular or crystalline. The crystals of calcium phosphate Fig. 100. Calcium Carbonate. are ordinarily pointed, wedge-shaped formations which may occur as individual crystals or grouped together in more or less regularly formed rosettes (Fig. j6, p. 193). Acid sodium urate crystals (Fig. 102, p. 324) are often mistaken for crystals of calcium phosphate. We may differentiate between these 22 322 PHYSIOLOGICAL CHEMISTRY. two crystalline forms by the fact that acetic acid will readily dissolve the phosphate, whereas the urate is much less soluble and when finally brought into solution and recrystallized one is frequently enabled to identify uric acid crystals which have been formed from the acid urate solution. The clinical significance of the occurrence of calcium phosphate crystals in the urinary sediment is similar to that of " triple phosphate " (seepage 319). Calcium Sulphate. — Crystals of calcium sulphate are of quite rare occurrence in the sediment of urine. Their pres- ence seems to be limited in general to urines which are of a decided acid reaction. Ordinarily it crystallizes in the form of long, thin, colorless needles or prisms (Fig. 95, page 274) which may be mistaken for calcium phosphate crystals. There need be no confusion in this respect, however, since the sulphate crystals are insoluble in acetic acid which reagent readily dissolves the phosphate. As far as is known their occurrence as a constituent of urinary sediment is of very little clinical significance. Uric Acid. — Uric acid forms a very common constituent of the sediment of urines which are acid in reaction. It occurs in more varied forms than any of the other crystalline sediments (Plate V, opposite page 247, and Fig. 101, page 323), some of the more common varieties of crystals being- rhombic prisms, wedges, dumb-bells, whetstones, prismatic rosettes, irregular rectangular or hexagonal plates, etc. Crys- tals of pure uric acid are always colorless (Fig. 89, page 249), but the form occurring in urinary sediments is impure and under the microscope appears pigmented, the depth of color varying from light yellow to a dark reddish-brown according to the size and form of the crystal. The presence of a considerable uric acid sediment does not, of necessity, indicate a pathological condition or a urine of increased uric acid content, since this substance very often occurs as a sediment in urines whose uric acid content is diminished from the normal merely as a result of changes in ( ui \ ■ i- : si' hi m i NTS. [23 reaction, etc. Pathologically, uric acid sediments occur in gout, acute febrile conditions, chronic interstitial nephritis, etc. If the microscopical examination is not conclusive, uric acid may be differentiated from Other crystalline urinary sedi- FlG. IOI. Various Forms of Uric Acid. i. Rhombic plates; 2, whetstone forms; 3, 3, quadrate forms; 4, 5, pro- longed into points ; 6, 8. rosettes ; 7. pointed bundles ; 9, barrel forms pre- cipitated by adding hydrochloric acid to urine. ments from the fact that it is soluble in alkalis, alkali carbo- nates, boiling glycerin, concentrated sulphuric acid and in cer- tain organic bases such as ethylamine and piperidin. It also responds to the murexid test (see page 249) and to Schiff's reactiqn (see page 249). Urates. — The urate sediment may consist of a mixture of the urates of ammonium, calcium, magnesium, potassium and sodium. The ammonium urate may occur in neutral, alkaline or acid urine, whereas the other forms of urates are confined to the sediments of acid urines. Sodium urate occurs in sediments more abundantly than the other urates. 324 PHYSIOLOGICAL CHEMISTRY, The urates of calcium, magnesium and potassium are amor- phous in character, whereas the urate of ammonium is crystal- line. Sodium urate may be either amorphous or crystalline. When crystalline it forms groups of fan-shaped clusters or colorless, prismatic needles (Fig. 102, below). Ammonium urate is ordinarily present in the sediment in the burr-like form of the "thorn-apple" crystal, i. e., yellow or reddish- brown spheres, covered with sharp spicules or prisms (Plate VI, opposite page 324). The urates are all soluble in hydro- FlG. 102. Acid Sodium Urate. chloric acid or acetic acid and their acid solutions yield crystals of uric acid upon standing. They also respond to the murexid test. The clinical significance of urate sediments is very simi- lar to that of uric acid. A considerable sediment of amor- phous urates does not necessarily indicate a high uric acid content, but ordinarily signifies a concentrated urine having a very strong acidity. Cystin. — Cystin is one of the rarer of the crystalline uri- nary sediments. It has been claimed that it occurs more often in the urine of men than of women. Cystin crystal- lizes in the form of thin, colorless, hexagonal plates (Fig. 32, PLATE VI. Ammonium Urate, showing Spherules and Thorn vpple-shaped Crystals (From Ogden, alter Peyer.) urine: sediments. 325 p. 76, and Fig. [03, bel<»\v) which arc insoluble in water, alco- hol and acetic acid and soluble in minerals acids, alkalis and especially in ammonia. Cystin may be identified by burn- ing it upon platinum foil under which condition it does not melt but vields a bluish-green flame. o I Fig. 103. • Cvstix. (Ogden.) Cholesterin. — Cholesterin crystals have been but rarely detected in urinary sediments. When present they probably arise from a pathological condition of some portion of the urinary tract. Crystals of cholesterin have been found in the sediment in cystitis, pyelitis, chyluria and nephritis. Or- dinarily it crystallizes in large regular and irregular colorless, transparent plates, some of which possess notched corners (Fig. 42, page 125). Frequently, instead of occurring in the sediment, it is found in the form of a film on the surface of the urine. Hippuric Acid. — This is one of the rarer sediments of human urine. It deposits under conditions similar to those which govern the formation of uric acid sediments. The crystals, which are colorless needles or prisms (Fig. 92. page 256) when pure, are invariably pigmented in a manner similar to the uric acid crystals when observed in urinary sediment and because of this fact are frequently confounded with the rarer forms of uric acid. Hippuric acid may be differen- tiated from uric acid from the fact that it does not respond to the murexid test and is much more soluble in water and in ether. The detection of crystals of hippuric acid in the 326 PHYSIOLOGICAL CHEMISTRY. urine has very little clinical significance, since its presence in the sediment depends in most instances very greatly upon the nature of the diet. It is particularly prone to occur in the sediment after the ingestion of certain fruits as well as after the ingestion of benzoic acid (see page 256). Leucin and Tyrosin. — Leucin and tyrosin have frequently been detected in the urine, either in solution or as a sediment. Neither of them occurs in the urine ordinarily except in asso- ciation with the other, i. e., whenever leucin is detected it is more than probable that tyrosin accompanies it. They have been found pathologically in the urine in acute yellow atrophy of the liver, in acute phosphorus poisoning, in cir- rhosis of the liver, in severe cases of typhoid fever and small- pox, and in leukaemia. In urinary sediments leucin ordi- narily crystallizes in characteristic spherical masses which show both radial and concen- FlG. I04. . ... , 1-11 trie striations and are highly refractive (Fig. 104, p. 326). For the crystalline form of pure leucin obtained as a decompo- sition product of proteicl see Fig. 24, p. 69. Tyrosin crys- tallizes in urinary sediments in the well known sheaf or tuft W formation (Fig. 23, p. 68). Crystals of Impure Leucin. p or ot l ier tests on leucill and (Ogden.) tyrosin see pages 80 to 82. Haematoidin and Bilirubin. — There are divergent opin- ions regarding the occurrence of these bodies in urinary sedi- ment. Each of them crystallizes in the form of tufts of small needles or in the form of small plates which are ordi- narily yellowish-red in color (Fig. 41, p. 119). Because of the fact that the crystalline form of the two substances is identical many investigators claim them to be one and the same body. Other investigators claim, that while the crystal- line form is the same in each case, that there are certain chem- i rink: SEDIMENTS. 327 ical differences which may he brought out very strikingly Im- properly testing. For instance, it has heen claimed that hsematoidin may he differentiated from bilirubin through the fact that it gives a momentary color reaction (hlue) when nitric acid is brought in contact with it. and further, that it is not dissolved on treatment with ether or potassium hydroxide. Pathologically, typical crystals of hsematoidin or bilirubin have been found in the urinary sediment in jaun- dice, acute yellow atrophy of the liver, carcinoma of the liver, cirrhosis of the liver, and in phosphorus poisoning, typhoid fever and scarlatina. Magnesium Phosphate. — Magnesium phosphate crystals occur rather infrequently in the sediment of urine which is neutral, alkaline or feebly acid in reaction. It ordinarily crystallizes in elongated, highly refractive, rhombic plates which are soluble in acetic acid. Indigo. — Indigo crystals are frequently found in urine which has undergone alkaline fermentation. They result from the breaking down of indoxyl-sulphates or indoxyl- glycuronates. Ordinarily indigo deposits as dark blue stel- late needles or occurs as amorphous particles or broken frag- ments. These crystalline or amorphous forms may occur in the sediment or may form a blue film on the surface of the urine. Indigo crystals generally occur in urine which is alkaline in reaction, but they have been detected in acid urine. Xanthin. — Xanthin is a constituent of normal urine but is found in the sediment in crystalline form very infrequently, and then only in pathological urine. When present in the sediment xanthin generally occurs in the form of whetstone- shaped crystals somewhat similar in form to the whetstone va- riety of uric acid crystal. They may be differentiated from uric acid by the great ease with which they may be brought into solution in dilute ammonia and on applying heat. Xanthin may also form urinary calculi. The clinical significance of xanthin in urinary sediment is not well understood. Melanin. — Melanin is an extremelv rare constituent of 328 PHYSIOLOGICAL CHEMISTRY. urinary sediments. Ordinarily in melanuria the melanin re- mains in solution; if it separates it is generally held in sus- pension as fine amorphous granules. (b) Organized Sediments. Epithelial cells. Pus cells. ' Hyaline. Granular. Epithelial. Casts 1 Blood. Fatty. Waxy. Pus. Cylindroids. Erythrocytes. Spermatozoa. Urethral filaments. Tissue debris. Animal parasites. Micro-organisms. Fibrin. Foreign substances clue to contamination.. Epithelial Cells. — The detection of a certain number of these cells in urinary sediment is not, of itself, a patho- logical sign, since they occur in normal urine. However, in certain pathological conditions they are greatly increased in number, and since different areas of the urinary tract are lined with different forms of epithelial cells, it becomes nec- essary, when examining urinary sediments, to note not only the relative number of such cells, but at the same time to carefully observe the shape of the various individuals in order to determine, as far as possible, from what portion of the tract they have been derived. Since the different layers of the epithelial lining are composed of cells different in form from those of the associated layers, it is evident that a careful microscopical examination of these cells may tell us the par- urine: sediments. 329 ticular layer which is being desquamated. It is frequently a most difficult undertaking', however, to make a clear differen- tiation between the various forms of epithelial cells present in a sediment. If skilfully done, such a microscopical differ- entiation may prove to be of very great diagnostic aid. The principal forms of epithelial cells met with in urinary sediments are shown in Fig. 105, below. Fig. 105. Epithelium from Different Areas of the Urinary Tract. a, Leucocyte (for comparison); b, renal cells; c, superficial pelvic cells; d, deep pelvic cells ; e, cells from calices ; /, cells from ureter ; g, g, g, g, g, squa- mous epithelium from the bladder ; /;, h, neck-of-bladder cells ; i, epithelium from prostatic urethra ; k, urethral cells ; /, /, scaly epithelium ; m, in', cells from seminal passages ; n, compound granule cells ; o, fatty renal cell. (Ogden.) Pus Cells. — Pus corpuscles or leucocytes are present in ex- tremely small numbers in normal urine. Any considerable increase in the number, however, ordinarily denotes a patho- logical condition, generally an acute or chronic inflammatory condition of some portion of the urinary tract. The sudden appearance of a large amount of pus in a sediment denotes the opening of an abscess into the urinary tract. Other form elements, such as epithelial cells, casts, etc., ordinarily accom- 33° PHYSIOLOGICAL CHEMISTRY. pany pus corpuscles in urinary sediment and a careful exami- nation of these associated elements is necessary in order to form a correct diagnosis as to the origin of the pus. Proteid is always present in urine which contains pus. Fig. 106. Pus Corpuscles. (After Ultsmann.) i. Normal; 2, showing amoeboid movements; 3, nuclei rendered distinct by acetic acid; 4, as observed in chronic pyelitis; 5, swollen by ammonium carbonate. The appearance which pus corpuscles exhibit under the microscope depends greatly upon the reaction of the urine containing them. In acid urine they generally present the appearance of round, colorless cells composed of refractive, granular protoplasm, and may frequently exhibit amoeboid movements, especially if the slide containing them be warmed slightly. They are nucleated (one or more nuclei), the nuclei being clearly visible only upon treating the cells with water, acetic acid or some other suitable reagent. In urine which has a decided alkaline reaction, on the other hand, the pus corpus- cles are often greatly degenerated. They may be seen as swollen, transparent cells, which exhibit no granular structure and as the process of degeneration continues the cell outline ceases to be visible, the nuclei fade, and finally only a mass urine: sediments. 331 of debris containing isolated nuclei and an occasional cell remains. It is frequently rather difficult to make a differentiation between pus corpuscles and certain types of epithelial cells which arc similar in form. Such confusion may be avoided by the addition <>t" iodine solution (I in KI ), a reagent which stains the pus corpuscles a deep mahogany-brown and trans- mits to the epithelial cells a light yellow tint. The test pro- posed by Vitali often gives very satisfactory results. This simply consists in acidifying- the urine (if alkaline) with acetic acid, then filtering, and treating the sediment on the filter paper with freshly prepared tincture of guaiac. The presence of pus in the sediment is indicated if a blue color is observed. Large numbers of pus corpuscles are present in the urinary sediment in gonorrhoea, leucorrhcea, chronic pyelitis and in abscess of the kidney. Fig. 107. Hyaline Casts. One cast is impregnated with four renal cells. 33* PHYSIOLOGICAL CHEMISTRY, Casts. — These are cylindrical formations, which originate in the uriniferous tubules and are forced out by the pressure of the urine. They vary greatly in size but in nearly every instance they possess parallel sides and rounded ends. The finding of casts in the urine is very important because of the fact that they generally indicate some kidney disorder ; if albu- min accompanies the casts the indication is much accentuated. Casts have been classified according to their microscopical char- acteristics as follows: (a) Hyaline, (b) granular, (c) epi- thelial, (d) blood, (e) fatty, (/) waxy, (g) pus. (a) Hyaline Casts. — These are composed of a basic material which is transparent, homogeneous and very light in color (Fig. 107, p. 331). In fact, chiefly because of these physical Fig. 108. Granular Casts. (After Peyer.) properties, they are the most difficult form of renal cast to detect under the microscope. Frequently such casts are im- pregnated with deposits of various forms such as erythrocytes, epithelial cells, fat globules, etc., thus rendering the form of URINE : SEDIMENTS. 333 the cast more plainly visible. Staining is often resorted to in order to render the shape and character of the casl more easily determined. Ordinary iodine solution (1 in K I ) may be used in this connection; many of the anilin dyes arc also in common use for this purpose, e. g. } gentian-violet, Bismarck-brown, methylene-blue. fuchsin and eosin. Generally, hut not always, albumin is present in urine containing hyaline casts. Hyaline casts are common t<> all kidney disorders, but occur particu- larly in the earliest and recovering stages of parenchymatous nephritis and in interstitial nephritis. (b) Granular Casts. — The common hyaline material is ordi- narily the basic substance of this form of cast. The granular material generally consists of albumin, epithelial cells, fat or Fig. 109. Fig. 1 10. Granular Casts. a, Finely granular ; b, coarsely granular. Epithelial Casts. disintegrated erythrocytes or leucocytes, the character of the cast varying according to the nature and size of the granules (Fig. 108, page 332, and Fig. 109, above). Thus we have casts of this general type classified as finely granular and coarsely granular casts. Granular casts, and in particular the finely granular types, occur in the sediment in practically every kidney disorder but are probably especially characteristic of the sediment in inflammatory- disorders. 334 PHYSIOLOGICAL CHEMISTRY.- (c) Epithelial Casts. — These are casts bearing upon their surface epithelial cells from the lining of the uriniferous tubules (Fig. no, p. 333). The basic material of this form of cast may be hyaline or granular in nature. Epithelial casts are particularly abundant in the urinary sediment in acute nephritis. (d) Blood Casts. — Casts of this type may consist of ery- throcytes borne upon a hyaline or a fibrinous basis (Fig. Ill, below). The occurrence of such casts in the urinary sediment Fig. hi. Blood, Pus, Hyaline and Epithelial Casts. a, Blood casts ; b, pus cast ; c, hyaline cast impregnated with renal cells ; d, epithelial casts. denotes renal haemorrhage and they are considered to be especially characteristic of acute diffuse nephritis and acute congestion of the kidney. (e) Fatty Casts. — Fatty casts may be formed by the deposi- tion of fat globules or crystals of fatty acid upon the surface of a hyaline or granular cast (Fig. 112, p. 335). In order to TRIM: : SEDIMENTS. 335 Fatty Casts. (After Peyer.) Fig. 113. a, Fatty casts ; b, waxy casts. Fatty and Waxy Casts. 336 PHYSIOLOGICAL CHEMISTRY. constitute a true fatty cast the deposited material must cover the greater part of the surface area of the cast. The presence of fatty casts in urinary sediment indicates fatty degeneration of the kidney ; such casts are particularly characteristic of sub- acute and chronic inflammations of the kidney. Fig. 114. Cylindroids. (After Peyer.) (f) Waxy Casts. — These casts possess a basic substance similar to that which enters into the foundation of the hyaline form of cast. In common with the hyaline type they are color- less, refractive bodies but differ from this form of cast in be- ing, in general, of greater length and diameter and possessing sharper outlines and a light yellow color (Fig. 113, p. 335). Such casts occur in several forms of nephritis but do not appear to characterize any particular type of the disorder ex- cept amyloid disease, in which they are rather common. (g) Pus Casts. — Casts whose surface is covered with pus cells or leucocytes are termed pus casts (Fig. m,p. 334. They are frequently mistaken for epithelial casts. The differentia- URINE : SEDIMENTS. 337 tion between these two types is made very simple however by treating - the cast with acetic acid which causes the nuclei of the leucocytes to become plainly visible. The true pus cast is quite rare and indicates renal suppuration. Cylindroids. — These formations may occur in normal or pathological urine and have no particular clinical significance. They are frequently mistaken for true casts, especially the hya- line type, but they are ordinarily flat in structure with a rather smaller diameter than casts, may possess forked or branching ends and are not composed of homogeneous material as are the hyaline casts. Such " false casts " may become coated with urates, in which event they appear granular in structure. The basic substance of cylindroids is often the nucleo-proteid of the urine (see Fig. 114. page 336). Erythrocytes. — These form elements are present in the urinary sediment in various diseases. They may appear as Fig. 11 v Crenated Erythrocytes. the normal biconcave, yellow erythrocyte (Plate IV, opposite page 151) or may exhibit certain modifications in form such as the crenated type (Fig. 115, above) which is often seen in con- 23 338 PHYSIOLOGICAL CHEMISTRY. centrated urine. Under different conditions they may become swollen sufficiently to entirely erase the biconcave appearance and may even occur in the form of colorless spheres having a smaller diameter than the origina 1 disc-shaped corpuscles. Erythrocytes are found in urinary sediment in hemorrhage of the kidney or of the urinary tract, in traumatic hemorrhage, hemorrhage from congestion and in hemorrhagic diathesis. Spermatozoa. — Spermatozoa may be detected in the urinary sediment in diseases of the genital organs, as well as after coitus, nocturnal emissions, epileptic and other convulsive Fig. i i 6. Human Spermatozoa. attacks and sometimes in severe febrile disorders, especially in typhoid fever. In form they consist of an oval body, to which is attached a long, delicate tail (Fig. 116, above). Upon ex- amination they may show motility or may be motionless. Urethral Filaments. — These are peculiar thread-like bodies which are sometimes found in urinary sediment. They may occasionally be detected in normal urine and pathologically are found in the sediment in acute and chronic gonorrhoea and in urethrorrhcea. The ground-substance of these urethral fila- ments is in part, at least, similar to that of the cylindroids (see urine: sediments. 339 page 337)* The urine first voided in the morning is besl adapted for the examination for filaments. These filaments may ordinarily l>e removed l>y a pipette since they are gener- ally macr< >scopic. Tissue Debris. — Masses of cells or fragments of tissue arc frequently found ill urinary sediment. They may be found in the sediment in tubercular affections of the kidney and urinary tract or in tumors of these organs. Ordinarily it is necessary to make a histological examination of such tissue fragments before coming t<» a final decision as to their origin. Animal Parasites. — The cysts, booklets and membrane shreds of echinococci are sometimes found in urinary sedi- ments. Other animal organisms which are more rarely met with in the urine are embryos of the Filaria sanguinis and eggs of the Distoma h< ft V3 -■ g The pow- With a little K< »ll ?o 5" !? rt E i- der when o - s< ' — n < en ft treate n O a. o 3 — g 5 3 rt -■ 3* ft r— p o S. _: — !T i B 3 c a. a p> ft E. M 5 5 3 ft f s ft — 2-S? eg 2 S 1 3 « ►1 ft IB S" 51 p cn o 3 — n En | - < ft w ft < ft orq *" ft 3 _ ft £"* -l 3 P y! 3 3 o 3 c' 3 c » ft 1 i iple pho te" ( mixed wi nown amount hy phosphate ) •a e g ft 3 to n 3" B ,-^ 2. 5 3 B 5' 3 o - p s r n B | E 3 5' C n tr b' X p 3 o" p o 5" ■ 2>Er . 3« ? 'CHAPTER XXI. URINE: QUANTITATIVE ANALYSIS. I. Proteid. i. Scherer's Coagulation Method. — The content of coag- ulable proteid may be accurately determined as follows : Place 50 c.c. of urine in a small beaker and raise the temperature of the fluid to about 40 C. upon a water-bath. Add dilute acetic acid, drop by drop, to the warm urine, to precipitate the proteid which will separate in a flocculent form. Care should be taken not to add too much acid ; ordinarily less than twenty drops is sufficient. The temperature of the water in the water-bath should now be raised to the boiling-point and main- tained there for a few minutes in order to insure the complete coagulation of the proteid present. Now filter the urine through a previously zvashed, dried and zveighed filter paper, wash the precipitated proteid, in turn, with hot water, 95 per cent alcohol and with ether, and dry the paper and precipitate, to constant weight, in an air-bath at no° C. Subtract the weight of the filter paper from the combined weight of the paper and precipitate and calculate the percentage of proteid in the urine specimen. Calculation. — To determine the percentage of proteid pres- ent in the urine under examination, multiply the weight of the precipitate, expressed in grams, by 2. 2. Esbach's Method. — This method depends upon the precipitation of proteid by Esbach's reagent 1 and the appa- ratus used in the estimation is Esbach's albuminometer (Fig. 117, p. 345). In making a determination fill the albuminometer to the point U with urine, then introduce the reagent until the point R is reached. Now stopper the tube, invert it slowly 1 Esbach's reagent is prepared by dissolving 10 grams of picric acid and 20 grams of citric acid in 1 liter of water. 344 urine: quantitative analysis. 345 several times in order to insure the thorough mixing of the fluids and stand the tube aside for 24 hours. Creatinin. resin acids, etc., are precipitated in this method, and for this and other reasons it is n<>t as accurate as the coagulation method. It is, however, ex- tensively used clinically. Calculation.- — The graduations on the albuminometer indicate grams of proteid po- liter of urine. Thus, if the proteid precipi- tate is level with the figure 3 of the gradu- ated scale this denotes that the urine ex- amined contains 3 grams of proteid to the liter. To express the amount of proteid in per cent simply move the decimal point otic place to the left. In the case under consider- ation the urine contains 0.3 per cent of proteid. II. Dextrose. 1. Fehling's Method. — Place 10 c.c. of the urine under examination in a 100 c.c. volumetric flask and make the volume up to 100 c.c. with distilled water. Thoroughly mix this diluted urine, by pouring it into a beaker and stirring with a glass rod, then transfer a portion of it to a burette which is properly supported in a clamp. Xow place 10 c.c. of Fehling's solution 1 in a small beaker, dilute it with approxi- mately 40 c.c. of distilled water, heat to boiling, and observe whether decomposi- tion of the Fehling's solution itself has occurred as indicated by the production of a turbidity. If such turbidity is produced the Fehling's solu- tion is unfit for use. Clamp the burette containing the diluted urine immediately over the beaker and carefully allow from 0.5 1 Directions for the preparation of Fehling's solution are given in a note at the bottom of page 8. Esbach's Albumi- nometer. (Ogden.) 34^ PHYSIOLOGICAL CHEMISTRY. to i c.c. of the diluted urine to flow into the boiling Fehling's solution. Bring the solution to the boiling-point after each addition of urine and continue running in the urine from the burette. 0.5-1 c.c. at a time, as indicated, until the Fehling's solution is completely reduced, i. c, until all the cupric oxide in solution has been precipitated as cuprous oxide. This point will be indicated by the absolute disappearance of all blue color. When this end-point is reached note the number of cubic centimeters of diluted urine used in the process and calculate the percentage of dextrose present, in the sample of urine analyzed, according to the method given on page 347. This is a very satisfactory method, the main objection to its use being the uncertainty attending the determination of the end-reaction, i. e., the difficulty with which the exact point where the blue color finally disappears is noted. Sev- eral means of accurately fixing this point have been suggested but they are practically all open to objection. As good a " check " as any, perhaps, is to filter a few drops of the solu- tion, through a double paper, after the blue color has appar- ently disappeared, acidify the filtrate with acetic acid and add potassium ferrocyanide. If the copper of the Fehling's solu- tion has been completely reduced, there will be no color reac- tion, whereas the production of a brown color indicates the presence of unreduced copper. Harrison has recently sug- gested the following procedure to determine the exact end- point: To about 1 c.c. of a starch iodide solution 1 in a test- tube add 2-3 drops of acetic acid and introduce into the acidified mixture 1-2 drops of the solution to be tested. Un- reduced copper will be indicated by the production of a pur- plish-red or blue color due to the liberation of iodine. It is ordinarily customary to make at least three deter- 1 The starch-ioclide solution may be prepared as follows: Mix 0.1 gram of starch with cold water in a mortar and pour the suspended starch granules into 75-100 c.c. of boiling water, stirring continuously. Cool the starch paste, add 20-25 grams of potassium iodide and dilute the mixture to 250 c.c. This solution deteriorates upon standing, and therefore must be freshly prepared as needed. URINE: QUANTITATIVE ANALYSIS. 347 miriations by Fehling's method In-fore coming to a final conclusion regarding the sugar content of the urine under examination. Calculation. — Ten c.c. of Fehling's solution is completely reduced by 0.05 gram of dextrose, [f y represents the num- ber of cubic centimeters of undiluted urine (obtained by dividing the burette reading by 10) necessary to reduce the 10 c.c. of Fehling's solution, we have the following proportion: v : 0.05 : : 100 : .r ( percentage of dextrose) . 2. Purdy's Method. — I 'inch's solution 1 is a modification of Fehling's solution and is said to possess greater stability than the latter. One of the most satisfactory points about the method as suggested by Purdy is the ease with which the exact end-reaction may be determined. In determining the percentage of dextrose by this method proceed as follows: Place 35 c.c. of Purdy's solution in a 200 c.c. Erlenmeyer flask and dilute the fluid with approximately two volumes of distilled water. Fit a cork, provided with two perforations, to the neck of the flask and through one perforation introduce the tip of a burette and through the second perforation intro- duce a tube bent at right angles in such a manner as to allow the steam to escape and keep the fumes of ammonia away from the face of the operator as completely as possible. 1 N< >w 1 Purdy's solution has the following composition : Cupric sulphate 4/52 grams. Potassium hydroxide 23.5 grams. Ammonia (U. S. P., sp. gr. 0.9) 3500 c.c. Glycerin 38.0 c.c. Distilled water, to make total volume 1 liter. In preparing the solution bring the CuSO< and KOH into solution in separate vessels, mix the two solutions, cool the mixture and add the ammonia and glycerin. After this has been done the total volume should be made up to 1 liter with distilled water. Thirty-five cubic centimeters of Purdy's solution is exactly reduced by 0.02 gram of dextrose. 'This side tube may also be equipped with a simple air-valve, thus insuring the exclusion of air and thereby contributing to the accuracy of 348 PHYSIOLOGICAL CHEMISTRY. bring the solution to the boiling-point and add the urine, drop by drop, until the intensity of the blue color begins to diminish. When this point is reached add the urine somewhat more slozvly until the blue color is entirely dissipated and an absolutely decolorized solution remains. Take the burette reading and calculate the percentage of dextrose in the urine examined according to the method given below. Care should be taken not to boil the solution for too long a period, since, under these conditions, sufficient ammonia might be lost to allow the cuprous hydroxide, CuOH, to precipitate. Some investigators consider it to be advisable to dilute the urine before applying the above manipulation, but ordinarily this is not necessary unless the urine has a high content of dextrose (5 per cent or over). In this event the urine may be diluted with 2-3 volumes of water and the proper correc- tion made in the calculation. Calculation. — Thirty-five c.c. of Purdy's solution is com- pletely reduced by 0.02 gram of dextrose. If y represents the number of cubic centimeters of undiluted urine necessary to reduce 35 c.c. of Purdy's solution, we have the following proportion : y : 0.02 : : 100 : x (percentage of dextrose) . 3. Fermentation Method. — This method consists in the measurement of the volume of C0 2 evolved when the dex- trose of the urine undergoes fermentation with yeast. None of the various methods whose manipulation is based upon this principle is absolutely accurate. The method in which Ein- horn's saccharometer (Fig. 2, page 10) is the apparatus em- ployed is perhaps as satisfactory as any for clinical purposes. The procedure is as follows: Place about 15 c.c. of urine in the determination, inasmuch as the cuprous salts would be reoxidized upon coming in contact with the air. If one is careful to maintain the solution continuously at the boiling-point throughout the entire process, however, there is no opportunity for air to enter and therefore no need of an air-valve. urine: quantitative analysis. 349 a mortar, add about 1 gram of yeast (tV of the ordinary cake of compressed yeasl I and carefully crush the latter by means of a pestle. Transfer the mixture to the saccharometer, being careful to note that the graduated tube is completely tilled and that no air bubbles gather at the top. Allow the apparatus to stand in a warm place (30 C.) for 1 2 hours and Observe the percentage of dextrose as indicated by the graduated scale of the instrument. Both the percentage of dextrose and the number of cubic centimeters of CO.. are indicated by the graduations on the side of the saccharometer tube. 4. Polariscopic Examination. — Before subjecting urine to a polariscopic examination the slightly acid fluid should be decolorized as thoroughly as possible by the addition of a little plumbic acetate. The urine should be well stirred and then filtered through a filter paper which has not been pre- viously moistened. In this way a perfectly clear and almost colorless liquid is obtained. In determining dextrose by means of the polariscope it should be borne in mind that this carbohydrate is often accom- panied by other optically active substances, such as proteids. la-vulose, /8-oxybutyric acid and conjugate glycuronates which may introduce an error into the polariscopic reading; the method is, however, sufficiently accurate for practical purposes. For directions as to the manipulation of the polariscope see page 1 1 . III. Uric Acid. 1. Folin-Shaffer Method. — Introduce 100 c.c. 1 of urine into a beaker, add 25 c.c. of the Folin-Shaffer reagent 2 and allow the mixture to stand. 3 without further stirring,, until the precipitate has settled (5-10 minutes). Filter, transfer 'It is preferable to use more than 100 c.c. of urine if the fluid lias a specific gravity less than 1.020. 2 The Folin-Shaffer reagent consists of 500 grams of ammonium sul- phate. 5 grams of uranium acetate and 60 c.c. of 10 per cent acetic acid in 650 c.c. of distilled water. 3 The mixture should not be allowed to stand for too long a time at this point, since uric acid may be lost through precipitation. 35° PHYSIOLOGICAL CHEMISTRY. ioo c.c. of the filtrate to a beaker, add 5 c.c. of concentrated ammonia and allow the mixture to stand for 24 hours. Transfer the precipitated ammonium urate quantitatively to a filter plant, 1 using" 10 per cent ammonium sulphate to remove the final traces of the urate from the beaker. Wash the precipitate approximately free from chlorides by means of 10 per cent ammonium sulphate solution, remove the paper from the funnel, open it and by means of hot water rinse the precipitate back into the beaker in which the urate was originally precipitated. The volume of fluid at this point should be about 100 c.c. Cool the solution to room temperature, add 15 c.c. of concentrated sulphuric acid and titrate at once with ^5- potassium permanganate, K 2 Mn 2 O s , solution. The first tinge of pink color which extends throughout the fluid after the addition of two drops of the permanganate solution, while stirring with a glass rod, should be taken as the end-reaction. Take the burette reading and compute the percentage of uric acid present in the urine under examination. Calculation. — Each cubic centimeter of ^V potassium per- manganate solution is equivalent to 3.75 milligrams (0.00375 gram) of uric acid. The 100 c.c. from which the ammonium urate was precipitated is equivalent to only four-fifths of the 100 c.c. of urine originally taken, therefore we must take five- fourths of the burette reading in order to ascertain the number of cubic centimeters of the permanganate solution required to titrate 100 c.c. of the original urine to the correct end-point. If y represents the number of cubic centimeters of the permanaganate solution required, we may make the following calculation : y X 0.00375 = weight of uric acid in 100 c.c. of urine. Calculate the quantity of uric acid in the twenty-four hour urine specimen. 2. Heintz Method. — This is a very simple method and was the first one in general use for the quantitative determi- 1 The Schleicher and Schiill hardened papers are the best for this purpose. urine: quantitative analysis. 353 nation of uric acid. It is believed to be somewhat less accu- rate than the method just described. The procedure is as follows: Place too c.c. of filtered urine in a beaker, add 5 c.c. of concentrated hydrochloric acid, stir the fluid thoroughly and stand it away in a cool place for -'4 hours. Filter off the uric acid crystals upon a washed, dried and weighed filter paper and wash them with (•<n dioxide, cool and titrate with an alkali of known strength. In this method, as well as in Folin's method (see p. 355), correction must he made for the ammonia originally present in the urine and in the magnesium chloride. V. Ammonia. 1. Folin's Method. — Place 2^ c.c. of urine in an rerometer cylinder, 30-45 cm. in height (Fig i_'_\ p. 358), add about one gram of dry sodium carbonate and introduce some crude petroleum to prevent foaming-. Insert into the neck of the cylinder a rubber stopper provided with two perforations into each of which passes a glass tube one of which reaches below the surface of the liquid. The shorter tube ( 10 cm. in length | is connected with a calcium chloride tube filled with cotton and this tube is in turn joined to a glass tube extending to the bottom of a 500 c.c. wide mouthed ilad< which is intended to absorb the ammonia. and for this purpose should contain 20 c.c. of y$ sulphuric acid, 200 c.c. of distilled 1 There is some decomposition of urea at 6o° C. 35* PHYSIOLOGICAL CHEMISTRY. water and a few drops of an indicator (" alizarin red "). To insure the complete absorption of the ammonia the absorption Fig. 122. Folin's Ammonia Apparatus. flask is provided with a Folin absorption tube (Fig. 123, p. 359) which is very effective in causing the air passing from the cylinder to come into intimate contact with the acid in the absorption flask. In order to exclude any error due to the presence of ammonia in the air a similar absorption apparatus to the one just described is attached to the other side of the aerometer cylinder, thus insuring the passage of ammonia-free air into the cylinder. With an ordinary filter pump and good water pressure the last trace of ammonia should be removed from the cylinder in about one and one-half hours. 1 The number of cubic centimeters of the jq sulphuric acid neutral- ized by the ammonia of the urine may be determined by direct titration with y^ sodium hydroxide. This is one of the most satisfactory methods yet devised for the determination of ammonia. 1 With any given filter pump a A check " test should be made with urine or better with a solution of an ammonium salt of known strength to de- termine how long the air current must be maintained to remove all the ammonia from 2; c.c. of the solution. urine: QUANTITATIVE ANALYSIS. 359 Fig. 123. Calculation. — Subtract the number of cubic centimeters of idium hydroxide used in the titration from the number of cubic centimeters of , N n sulphuric acid taken. The remainder is the number of cubic centimeters of sulphuric acid neutralized by the -\7/ : . of the urine. 1 cc. of n! sulphuric acid is equivalent to 0.0017 gram of A r //. : . There- it y represents the volume of urine used in the determination and y' the num- ber of cubic centimeters of fV sulphuric acid neutralized by the A7/.. af the urine. we have the following proportion: y : 100 : : v' X 0.00 1 7 : x (percentage of X 1 1 .. in the urine examined). Calculate the quantity of XH. in the twenty-four hour urine specimen. VI. Nitrogen. Kjeldahl Method. 1 — The principle of this method is the conversion of the various nitrogenous bodies of the urine into ammonium sulphate by boiling with concentrated sulphuric acid, the subsequent de- composition of the ammonium sulphate by means of a fixed alkali (XaOH) and the collection of the liberated ammonia in an acid of known strength. Finally, this partly neutralized acid solution is titrated with an alkali of known strength and the nitrogen content of the urine under examination com- puted. The procedure is as follows : Place 5 cc. of urine in a 200-300 cc. long-necked, Jena glass Kjeldahl digestion flask, add 20 cc of concentrated sulphuric acid and about 0.2 gram of cupric sulphate and boil the mixture for some time after 1 There are numerous modifications of the original Kjeldahl method; the one described here, however, has given excellent satisfaction and is recommended for the determination of the nitrogen content of urine. Folin Absorption Tube. 360 PHYSIOLOGICAL CHEMISTRY. it is colorless (about one hour). Allow the flask to cool and transfer 1 the contents, by means of about 200 c.c. of water, to a 750 c.c. Jena glass distillation flask. Add a little more of a concentrated solution of NaOH than is necessary to neutralize the sulphuric acid 2 and introduce into the flask a little coarse pumice stone or a few pieces of granulated zinc, to prevent bumping, and a small piece of paraffin to lessen the tendency to froth. By means of a safety-tube connect the flask with a condenser so arranged that the de- livery-tube passes into a vessel containing a known volume (the volume used depending upon the nitrogen content of the urine) of y\ sulphuric acid, using care that the end of the delivery-tube reaches beneath the surface of the fluid. 3 Mix the contents of the distillation flask very thoroughly by shaking and distil the mixture until its volume has diminished about one-half. Titrate the partly neutralized y\ sulphuric acid solution by means of y\ sodium hydroxide, using congo red as indicator, and calculate the content of nitrogen of the urine examined. Calculation. — Subtract the number of cubic centimeters of yq sodium hydroxide used in the titration from the number of cubic centimeters of y$ sulphuric acid taken. The re- mainder is equivalent to the. number of cubic centimeters of yv sulphuric acid, neutralized by the ammonia of the urine. One c.c. of ^ sulphuric acid is equivalent to 0.0014 gram of nitrogen. Therefore, if y represents the volume of urine used in the determination, and y' the number of cubic centimeters of Yq sulphuric acid neutralized by the ammonia of the urine, we have the following proportion : 'A very satisfactory modification of this procedure includes the use of a 750 c.c. flask for both the digestion and the distillation, thus making unnec- essary any transfer of contents. 2 This concentrated sodium hydroxide solution should be prepared in quantity and " check " tests made to determine the volume of the solution necessary to neutralize the volume (20 c.c.) of concentrated sulphuric acid used. 3 This delivery-tube should be of large caliber in order to avoid the "sucking back" of the fluid. urine: quantitative analysis. 361 y: 100: : y' X 0.0014 \x (percentage of nitrogen in the urine examined ). Calculate the quantity of nitrogen in the twenty-four hour urine specimen. VII. Sulphur. 1. Total Sulphates. — Folin' s Method. — Place 25 c.c. of urine in a 200 -'50 c.c. Erlenmeyer flask, add 20 c.c. of dilute hydrochloric acid 1 (1 volume of concentrated 11(1 to 4 vol- umes of water) and gently boil for 20 30 minutes. To mini- mize the loss of water by evaporation the mouth of the flask should be covered with a small watch glass during the boiling process. Cool the tlask for 2-3 minutes in running water, and dilute the contents to about 150 c.c. by means of cold water. Add ro c.c. of a 5 per cent solution of barium chloride slowly. drop by drop, to the cold solution. 2 The contents of the flask should not be stirred or shaken during the addition of the barium chloride. Allow the mixture to stand at least one hour, then shake up the solution and filter it through a weighed Gooch crucible. 3 Wash the precipitate of BaSO, with about 250 c.c. of cold water, dry it in an air-bath or over a very low flame, then ignite. ' a m >1 and weigh. 1 If it is desired, 50 c.c. of urine and 4 c.c. of concentrated acid may be used instead. "A dropper or capillary funnel made from an ordinary calcium chloride tube and so constructed as to deliver 10 c.c. in 2-3 minutes is recom- mended for use in adding the barium chloride. 3 If a Gooch crucible" is not available the precipitate of BaSOi may be filtered off upon a washed filter paper (Schleicher & Schtill's, Xo. 589, blue ribbon) and after washing the precipitate with about 250 c.c. of cold water the paper and precipitate may be dried in an air-bath, or over a low flame. The ignition may then be carried out in the usual way in the ordinary platinum or porcelain crucible. In this case correction must be made for the weight of the ash of the filter paper used. * Care must be taken in the ignition of precipitates in Gooch crucibles. The flame should never be applied directly to the perforated bottom or to the sides of the crucible, since such manipulation is invariably attended by mechanical losses. The crucibles should always be provided with lids and tight bottoms during the ignition. In case porcelain Gooch crucibles, 362 PHYSIOLOGICAL CHEMISTRY. Calculation. — Subtract the weight of the Gooch crucible from the weight of the crucible and the BaS0 4 precipitate to obtain the weight of the precipitate. The weight of SO3 1 in the volume of urine taken may be determined by means of the following proportion : Mol. wt. Wt. of Mol. Wt. BaS0 4 : BaS0 4 : : S0 3 : x (wt. of S0 3 in grams), ppt. Representing the weight of the BaS0 4 precipitate by 3' and substituting the proper molecular weights, we have the follow- ing proportion : 231.7: 3' :: 79.5 : x (wt. of SO s in grams in the quantity of urine used). Calculate the quantity of S0 3 in the twenty-four hour specimen of urine. To express the result in percentage of S0 3 simply divide the value of x, as just determined, by the quantity of urine used. 2. Inorganic Sulphates. — Folin's Method. — Place 25 c.c. of urine and 100 c.c. of water in a 200-250 c.c. Erlenmeyer flask and acidify the diluted urine with 10 c.c. of dilute hydro- chloric acid (1 volume of concentrated HC1 to 4 volumes of water). In case the urine is dilute 50 c.c. may be used instead of 25 c.c. and the volume of water reduced proportionately. From this point proceed as indicated in the method for the determination of Total Sulphates, page 361. Calculate the quantity of inorganic sulphates, expressed as S0 3 , in the twenty-four hour urine specimen. whose bottoms are not provided with a non-perforated cap, are used, the crucible may be placed upon the lid of an ordinary platinum crucible during ignition. The lid should be supported on a triangle, the crucible placed upon the lid and the flame applied to the improvised bottom. Igni- tion should be complete in 10 minutes if no organic matter is present. 1 It is considered preferable by many investigators to express all sulphur values in terms of S rather than SO3. urine: quantitative analysis. 363 Calculation. — Calculate according to the directions given under Total Sulphates, page 36] . 3. Ethereal Sulphates.— /•'. ride. Allow the mixture to stand at least one hour, then shake up the solution and filter it through a weighed Gooch crucible. Manipulate the precipitate of BaS0 4 accord- ing to directions given under Total Sulphates, page 361. Calculate the quantity of sulphur, expressed as S0 3 or S, present in the twenty-four hour urine specimen. 1 See Sherman's Organic Analysis, p. 19. 2 Only a small amount of urine should he added at one time, it being necessary to make several evaporations before the block contains sufficient urinary residue to proceed with the combustion. 3 The Berthelot-Atwater apparatus (Fig. 124, page 366) is well adapted to this purpose. 4 See note (2) at the bottom of page 361. 3 66 PHYSIOLOGICAL CHEMISTRY. Fig. 124. Berthelot-Atwater Bomb Calorimeter. (Cross-sectiox of Apparatus as Ready for Use.) A, Steel cup or bomb proper ; C, collar of steel ; G, opening through which oxygen is forced into the bomb ; H and I', insulated wires which serve to conduct an electric current for igniting the substance which is held in the small capsule; L, a stirrer which serves to keep the water surrounding the bomb in motion and insures the equalization of temperature ; P, a delicate thermometer which shows the rise in temperature of the water surrounding the bomb. urine: quantitative analysis. v 7 VIII. Phosphorus. i. Total Phosphates. — Uranium Acetate Method. — To 50 c.c. of urine in a small beaker or Erlenmeyer flask add 5 c.c. of a special sodium acetate solution 1 and heat the mixture to the boiling-point. From a burette, run into the hot mixture, drop by drop, a standard solution of uranium acetate 2 until a precipi- tate ceases t<> form and a drop of the mixture when removed by means of a* glass rod and brought in contact with a drop ■ ■I a solution <>f potassium ferrocyanide on a porcelain tesjt- tablet produces instantaneously a brownish-red coloration. 1 Take the burette reading and calculate the P..O- content of the urine under examination. Calculation. — Multiply the number of cubic centimeter^ <<\ uranium acetate solution used by 0.005 t0 determine the num- ber of grams of P 2 O b in the 50 c.c. of urine used. To express the result in percentage of P 2 O r , multiply the value just ob- tained by -'. e. g., if 50 c.c. of urine contained 0.074 gram of PoOj it would be equivalent to 0.148 per cent. Calculate, in terms of P-O-j. the total phosphate content of the twenty-four hour urine specimen. 2. Earthy Phosphates. — To 100 c.c. of urine in a beaker 1 The sodium acetate solution is prepared by dissolving ioo grams of sodium acetate in 800 c.c. of distilled water, adding 100 c.c. of 30 per cent acetic acid to the solution and making the volume of the mixture up to I liter with water. 1 This uranium acetate solution may be prepared by dissolving 35.461 grams of uranium acetate in one liter of water. One c.c. of such a solu- tion should be equivalent to 0.005 gram of P2O5, phosphoric anhydride. This solution may be standardized as follows : To 50 c.c. of a standard solution of disodium hydrogen phosphate, of such a strength that the 50 c.c. contains 0.1 gram of P-Os, add 5 c.c. of the sodium acetate solution, mentioned above, and titrate with the uranium solution to the correct end-reaction as indicated in the method proper. Inasmuch as 1 c.c. of the uranium solution should precipitate 0.005 gram of P2O5, exactly 20 c.c. of the uranium solution should be required to precipitate 50 c.c. of the standard phosphate solution. If the two solutions do not bear this rela- tion to each other they may be brought into proper relation by diluting the uranium solution with distilled water or by increasing its strength. A 10 per cent solution of potassium ferrocyanide is satisfactory. 368 PHYSIOLOGICAL CHEMISTRY. add an excess of ammonium hydroxide and allow the mixture to stand 12-24 hours. Under these conditions the phosphoric acid in combination with the alkaline earths, calcium and mag nesium, is precipitated as phosphates of these metals. Col- . lect the precipitate on a filter paper and wash it with very dilute ammonium hydroxide. Pierce the paper, and remove the precipitate by means of hot water. Bring the phosphates into solution by adding a small amount of dilute acetic acid to the warm solution. Make the volume up to 50 c.c. with water, add 5 c.c. of sodium acetate solution and determine the P 2 5 content of the mixture according to the directions given under the previous method. Calculation. — Multiply the number of cubic centimeters of uranium acetate solution used by 0.005 to determine the num- ber of grams of P 2 5 in the 100 c.c. of urine used. Since 100 c.c. of urine was taken this value also expresses the per- centage of P 2 5 present. Calculate the quantity of earthy phosphates, in terms of P 2 5 , present in the twenty-four hour urine specimen. The quantity of phosphoric acid present in combination with -the alkali metals may be determined by subtracting the content of earthy phosphates from the total phosphates. 3. Total Phosphorus. — Sodium Hydroxide and Potassium Nitrate Fusion Method. — Place 25 c.c. of urine in a large silver crucible and evaporate to a syrup on a water-bath. Add 10 grams of NaOH and 2 grams of KN0 3 to the residue and fuse the mass until all organic matter has disappeared and the fused mixture is clear. Cool the mixture, transfer it to a casserole by means of hot water, acidify the solution slightly with pure nitric acid and evaporate to dryness on a water-bath. Moisten the residue with a few drops of dilute nitric acid, dis- solve it in hot water and transfer to a beaker. Now add an equal volume of mplybdic solution 1 and keep the mixture at 40° C. for twenty-four hours. Filter off the precipitate, wash it with dilute molybdic solution and dissolve it in dilute am- 1 Directions for the preparation of the solution are given on page 2>7- urine: quantitative analysis. 369 monia. Add dilute hydrochloric acid to the solution, being careful to leave the solution distinctly ammoniacal. Magn mixture 2 (10 [5 c.c.) should now be added and after stirring thoroughly and making strongly ammoniacal with concentrated ammonia the solution should l>e allowed to stand in a cool place for twenty-four hours. Filter off the precipitate, wash it free from chlorine by means of dilute ammonia (1:5), dry, incinerate and weigh, as magnesium pyrophosphate, MgJ'-Oj, in the usual manner. In thismethod the phosphoric acid of the urine is precipitated as ammonium magnesium phosphate and in the process of in- cineration this body is transformed into magnesium pxrophos- phate. Calculation. — The quantity of phosphorus, expressed in terms of P 2 5 , in the volume of urine taken may he determined by means of the following proportion : Mol. wt. Wt of Mol. wt Mg 2 P 2 0, : Mg 2 P 2 7 : : P 2 6 : x ( wt. of P 2 6 in grams). ppt. If 31 represents the weight of the Mg 2 P 2 7 precipitate and we make the proper substitutions we have the following pro- portion : 221.1:3?:: 140.9:.!' ( wt. of P 2 5 , in grams, in the quantity of urine used). To express the result in percentage of P 2 5 simply divide the value of x, as just determined, by the quantity of urine used. IX. Creatinin. Folin's Colorimetric Method. — This method is based upon the characteristic property possessed alone by creatinin, of yielding a certain definite color-reaction in the presence of picric acid in alkaline solution. The procedure is as follows : 2 Directions for the preparation of magnesia mixture may be found on page 2.-0. ■ 25 37° PHYSIOLOGICAL CHEMISTRY. Place 10 c.c. of urine in a 500 c.c. volumetric flask, add 15 c.c. of a saturated solution of picric acid and 5 c.c. of a 10 per cent solution of sodium hydroxide and allow the mixture to stand for 5-6 minutes. During this interval pour a little f potas- sium bichromate solution 1 into each of the two cylinders of the colorimeter (Duboscq's) and carefully adjust the depth of the solution in one of the cylinders to the 8 mm. mark. A few preliminary colorimetric readings may now be made with the solution in the other cylinder, in order to insure greater accu- racy in the subsequent examination of the solution of unknown strength. Obviously the two solutions of potassium bichromate are identical in color and in their examination no two readings should differ more than 0.1-0.2 mm. from the true value (8 mm.). Four or more readings should be made in each case and an average taken of all of them exclusive of the first read- ing, which is apt to be less accurate than the succeeding read- ings. In time as one becomes proficient in the technique it is perfectly safe to take the average of the first two readings. At the end of the 5-6 minute interval already mentioned, the contents of the 500 c.c. flask are diluted to the 500 c.c. mark, the bichromate solution is thoroughly rinsed out of one of the cylinders with the solution thus prepared and a number of colorimetric readings are immediately made. Ordinarily 10 c.c. of urine is used in the determination by this method but if the content of creatinin is above 15 mg. or below 5 mg. the determination should be repeated with a volume of urine selected according to the content of creatinin. This variation in the volume of urine according to the content of creatinin is quite essential since the method loses in accuracy when more than 15 mg. or less than 5 mg. of creatinin is pres- ent in the solution of unknown strength. Calculation. — By experiment it has been determined that 10 mg. of pure creatinin, when brought into solution and di- luted to 500 c.c. as explained in the above method, yields a mixture 8.1 mm. of which possesses the same colorimetric J This solution contains 24.55 grams of potassium bichromate to the liter. urine: quantitative analysis. 37 1 value as 8 mm. of a g solution of potassium bichromate. Bearing this in mind the computation is readily made by means of the following proportion in which v represents the number of mm. of the solution of unknown strength equivalent to the 8 mm. of the potassium bichromate solution: >y:S.i :: 10:* (mgs. of creatinin in the quantity of urine used). This proportion may be used for the calculation no matter what volume"of urine (5, 10 or 15 c.c.) is used in the deter- mination. The 10 represents 10 mg. of creatinin, which gives a color equal to 8.1 mm., whether dissolved in 5. 10 or 15 c.c. of fluid. Calculate the quantity of creatinin in the twenty-four hour urine specimen. X. Chlorides. 1. Mohr's Method. — To [OC.c. of urine in a small platinum or porcelain crucible or dish add about 2 grams of chlorine-free potassium nitrate and evaporate to dryness at ioo° C. (The evaporation may be conducted over a low flame provided care is taken to prevent loss by spurting-.) By means of crucible tongs hold the crucible or dish over a free flame until all car- bonaceous matter has disappeared and the fused mass is slightly yellow in color. Cool the residue somewhat and bring it into solution in a small amount (15-25 c.c.) of distilled water acidi- fied with about 10 drops of nitric acid. Transfer the solution to a small beaker, being sure to rinse out the crucible or dish very carefully. Test the reaction of the fluid, and if not already acid in reaction, to litmus, render it slightly acid with nitric acid. Now neutralize the solution by the addition of calcium carbonate in substance. 1 add 2-5 drops of neutral potassium chromate solution to the mixture and titrate with a standard argentic nitrate solution. 2 1 The cessation of effervescence and the presence of some undecomposed calcium carbonate at the bottom of the vessel are the indications of neutral- ization. 3 Standard argentic nitrate solution may be prepared by dissolving 29.060 grams of argentic nitrate in 1 liter of distilled water. Each cubic 37- PHYSIOLOGICAL CHEMISTRY. This standard solution should be run in from a burette. stirring the liquid in the beaker after each addition. The end- reaction is reached when the yellow color of the solution changes to a slight orange-red. At this point take the burette reading and compute the percentage of chlorine and sodium chloride in the urine examined. Calculation.- — Since I c.c. of the standard argentic nitrate solution is equivalent to o.oio gram of sodium chloride, to obtain the weight, in grams, of the sodium chloride in the 10 c.c. of urine used multiply the number of cubic centimeters of standard solution used by o.oio. If it is desired to express the result in percentage of sodium chloride move the decimal point one place to the right. To obtain the weight, in grams, of the chlorine in the 10 c.c. of urine used multiply the number of cubic centimeters of standard solution used by 0.006, and if it is desired to express the result in percentage of chlorine move the decimal point one place to the right. Calculate the quantity of sodium chloride and chlorine in the twenty-four hour urine specimen. 2. Volhard-Arnold Method. — Place 10 c.c. of urine in a 100 c.c. volumetric flask, add 20-30 drops of nitric acid (sp.gr. 1.2) and 2 c.c. of a cold saturated solution of ferric alum. If necessary, at this point a few drops of an 8 per cent solution of potassium permanganate may be added to dissipate the red color. Now slowly run in the standard argentic nitrate 1 solu- tion (20 c.c. is ordinarily used) until all the chlorine has been precipitated and an excess of the argentic nitrate solution is present, continually shaking the mixture during the addition of the standard solution. Allow the flask to stand 10 minutes, then fill it to the 100 c.c. graduation with distilled water and thoroughly mix the contents. Now filter the mixture through a dry filter paper, collect 50 c.c. of the filtrate and titrate it with centimeter of this solution is equivalent to 0.010 gram of sodium chloride or to 0.006 gram of chlorine. ' See note (2) at the bottom of page 371. urine: quantitative analysis. 573 standardized ammonium sulphocyanide solution. 1 The first permanent tinge of brown indicates the end-point. Take the burette reading and compute the weight of sodium chlo- ride in tin.' [O c.c. 1 if urine used. Calculation. — The number of cubic centimeters of am- monium sulphocyanide solution used indicates the excess of standard argentic nitrate solution in the 50 c.c. of filtrate titrated. Multiply this reading by _\ inasmuch as only one- half of the filtrate was employed, and subtracl this product from the number of cubic centimeters of argentic nitrate ( 20 c.c. ) originally used, in order to obtain the actual number of cubic centimeters of argentic nitrate utilized in the precipi- tation of the chlorides in the 10 c.c. of urine employed. To obtain the weight, in grams, of the sodium chloride in the 10 c.c. of urine used multiply the number of cubic centimeters of the standard argentic nitrate solution, actually utilized in the precipitation, by 0.010. If it is desired to express the re- sult in percentage of sodium chloride move the decimal point one place to the right. Jn a similar manner the weight, or percentage of chlorine may be computed using the factor 0.006 as explained in Mohr's method, page 371. Calculate the quantity of sodium chloride and chlorine in the twenty-four hour urine specimen. 'This solution is made of such a strength that i c.c. of it is equal to 1 c.c. of the standard argentic nitrate solution used. To prepare the solu- tion dissolve 12.9 grams of ammonium sulphocyanide, NITSCN, in a little less than a liter of water. In a small flask place 20 c.c. of the standard argentic nitrate solution, 5 c.c. of the ferric alum solution and 4 c.c. of nitric acid (sp. gr. 1.2), add water to make the total volume 100 c.c. and thoroughly mix the contents of the flask. Now run in the ammo- nium sulphocyanide solution from a burette until a permanent brown tinge is produced. This is the end-reaction and indicates that the last trace of argentic nitrate has been precipitated. Take the burette reading and calculate the amount of water necessary to use in diluting the ammonium sulphocyanide in order that 10 c.c. of this solution may be exactly equal to 10 c.c. of the argentic nitrate solution. Make this dilution and titrate again to be certain that the solution is of the proper strength. 374 PHYSIOLOGICAL CHEMISTRY. XI. Acetone. Messinger-Huppert Method. — Place ioo c.c. of urine in a distillation flask and add 2 c.c. of 50 per cent acetic acid. Connect the flask with a condenser, properly arrange a re- ceiver, attach a terminal series of bulbs containing- water and distil over about nine-tenths of the urine mixture. Remove the receiver, attach another and subject the residual portion of the mixture to a second distillation. Test this fluid for acetone and if the presence of acetone is indicated add about 100 c.c. of water to the residue and again distil. Treat the united acetone distillates with 1 c.c. of dilute (12 per cent) sul- phuric acid and redistil, collecting this second distillate in a glass-stoppered flask. During distillation, however, the glass stopper is replaced by a cork with a double perforation, the glass tube from one perforation passing to the condenser, while the bulbs containing' water, before-mentioned, are at- tached by means of the tube in the other perforation. Allow the distillation process to proceed until practically all of the fluid has passed over, then remove the receiving flask and insert the glass stopper. Now treat the distillate carefully with 10 c.c. of a yg- solution of iodine and add sodium hydroxide solution, drop by drop, until the blue color is dissipated and the iodoform precipitates. Stopper the flask and shake it for about one minute, acidify the solution with concentrated hydrochloric acid, and note the production of a brown color if an excess of iodine is present. In case there is no such excess, the solution should be treated with -j-q iodine solution until an excess is obtained. Retitrate this excess of iodine with y$ sodium thiosulphate solution until a light yellow color is observed. At this point a few cubic centimeters of starch paste should be added and the mixture again titrated until no blue color is visible. This is the end-reaction. Calculation. — Subtract the number of cubic centimeters of yq thiosulphate solution used from the volume of j^ iodine solution employed. Since 1 c.c. of the iodine solution is equivalent to 0.967 milligrams of acetone, and since 1 c.c. of urine: quantitative analysis. 375 the thiosulphate solution is equivalent to 1 c.c. of the iodine solution, if we multiply the remainder from the above sub- traction by 0.967 we will obtain the number of milligrams of acetone in the 100 c.c. of urine examined. Calculate the quantity of acetone in the twenty-four hour urine specimen. XII. /3-Oxybutyric Acid. 1. Darmstadter's Method. — This method is based on the fact that crotonic acid is formed from /3-oxybutyric acid under the influence of concentrated mineral acids. The method is as follows : Render 100 c.c. of urine slightly alka- line with sodium carbonate and evaporate nearly to dryness on a water-bath. Dissolve the residue in 150-200 c.c. of 50—55 per cent sulphuric acid, transfer the acid solution to a one liter distillation flask and connect it with a condenser. Through the cork of the flask introduce the stem of a drop- ping funnel containing water. Heat the flask gently until foaming ceases, then use a full flame and distil over about 300-350 c.c. of fluid, keeping the volume of liquid in the dis- tillation flask constant by the addition of water from the dropping funnel as the distillate collects. Ordinarily it will take about 2-2^4 hours to collect this amount of distillate. Extract the distillate two or three times with ether in a sepa- ratory funnel, evaporate the ether and heat the residue at 160 C. for a few minutes to remove volatile fatty acids. Dissolve the residue in 50 c.c. of water, filter and titrate this aqueous solution of crotonic acid with T \ sodium hydroxide solution, using phenolphthalein as indicator. Calculation. — One c.c. of y sodium hydroxide solution equals 0.0086 gram of crotonic acid, 1 part of crotonic acid equals 1.2 1 part of /3-oxybutyric acid, and 1 c.c. of j 6 sodium hydroxide solution equals 0.01041 gram of /3-oxybutyric acid. To compute the quantity of /?-oxybutyric acid, in grams, mul- tiply the number of cubic centimeters of fv sodium hydroxide solution used by 0.01041. 376 PHYSIOLOGICAL CHEMISTRY. 2. Bergen's Method. — Render 100-300 c.c. of sugar-free 1 urine slightly alkaline with sodium carbonate, evaporate the alkaline urine to a syrup on a water-bath, cool the syrup, rub it up with syrupy phosphoric acid (being careful to keep the mixture cool), 20-30 grams of finely pulverized, anhydrous cupric sulphate and 20-25 grams of fine sand. Mix the mass thoroughly, place it in a paper extraction thimble 2 and extract the dry mixture with ether in a Soxhlet apparatus (Fig. 125 page 380). Evaporate the ether, dissolve the residue in about 25 c.c. of water, decolorize the fluid with animal charcoal, if necessary, and determine the content of /?-oxybutyric acid by a polarization test. 3. Boekelman and Bouma's Method. — Place 25 c.c. of urine in a flask, add 25 c.c. of 12 per cent sodium hydroxide and 25 c.c. of benzoyl chloride, stopper the flask and shake it. vigorously for three minutes under cold zvater. Remove the clear fluid by means of a pipette, filter it and subject it to a polarization test. Through the action of the benzoyl chloride all the lasvorotatory substances except /3-oxybutyric acid will have been removed and the lsevorotation now exhibited by the urine will be due entirely to that acid. XIII. Acidity. Folin's Method. — The total acidity of urine may be deter- mined as follows : Place 25 c.c. of urine in a 200 c.c. Erlen- meyer flask and add 15-20 grams of finely pulverized potas- sium oxalate and 1-2 drops of a 1 per cent phenolphthalein solution to the fluid. Shake the mixture vigorously for 1-2 minutes and titrate it immediately with ■%■$ sodium hydroxide until a permanent faint pink coloration is produced. Take the burette reading and calculate the acidity of the urine under examination. Calculation. — If y represents the number of cubic centi- 1 If sugar is present it must be removed by fermentation. 2 The Schleicher and Schiill fat-free extraction thimble is very satis- factory. urine: quantitative analysis. 377 meters of , N (I sodium hydroxide used and y' represents the volume of urine excreted in twenty four hours, the total acidity of the twenty-four hour urine specimen may be cal- culated by means of the following proportion: 25 : v : : v':.r( acidity "I* -\| hour urine expressed in cubic centi- meters of T N U sodium hydroxide). Each cubic centimeter of ,\ sodium hydroxide contains 0.004 gram "of sodium hydroxide and this is equivalent to O.O063 & ram of oxalic acid. 'There fore, in order to express the total acidity of the twenty- four hour urine specimen in equivalent grams of sodium hydroxide, multiply the value of x, as just determined, by 0.004, or multiply the value of x by 0.0063 if it is desired to express the total acidity in grams of oxalic acid. XIV. Purin Bases. Salkowski's Method.— IMace 400-600 c.c. of proteid- free urine in a beaker. Introduce into another beaker 30-50 c.c. of an ammoniacal silver solution 1 with 30-50 c.c. of mag- nesia mixture, 2 add some ammonium hydroxide and if nec- essary some ammonium chloride to clear the solution. Now add this solution to the urine, stirring continually with a glass rod, and allow the mixture to stand for one-half hour. Col- lect the precipitate on a filter paper, wash it with dilute am- monium hydroxide and finally wash it back into the original beaker. Suspend the precipitate in 600-800 c.c. of water, add a few drops of hydrochloric acid and decompose it by means of hydrogen sulphide. Now heat the solution to boiling, filter while hot and evaporate the filtrate to dryness on a water-bath. Extract the residue with 20 30 c.c. of hot 3 per cent sulphuric acid and allow the extract to stand twenty- four hours. Filter off the uric acid, wash it, make the filtrate 1 Prepared by dissolving 26 grams of silver nitrate in about 500 c.c. of water, adding enough ammonium hydroxide to redissolve the precipitate which forms upon the first addition of the ammonia and making the balance of the mixture up to 1 liter with water. 2 Directions for preparation may be found on page -70. 37$ PHYSIOLOGICAL CHEMISTRY. ammoniacal and precipitate the purin bases again with silver nitrate. Collect this precipitate on a small-sized chlorine-free filter paper, wash, dry and incinerate it in the usual manner. Now dissolve the ash in nitric acid and titrate with ammonium sulphocyanide according to the Volhard-Arnold method (seep. 372). Calculate the content of purin bases in the urine exam- ined, bearing in mind that in an equal mixture of the silver salts of the purin bases, such as we have here, one part of sil- ver corresponds to 0.277 gram of nitrogen or to 0.7381 gram of the bases. XV. Oxalic Acid. Salkowski-Autenrieth and Barth Method. — Place the twenty- four hour urine specimen in a precipitating jar, add an excess of calcium chloride, render the urine strongly am- moniacal, stir it well and allow it to stand 18-20 hours. Filter off the precipitate, wash it with a small amount of water and dissolve it in about 30 c.c. of a hot 15 per cent solution of hydrochloric acid. By means of a separatory funnel extract the solution with 150 c.c. of ether which con- tains 3 per cent of alcohol, repeating the extraction four or five times with fresh portions of ether. Unite the ethereal extracts, allow them to stand for an hour in a flask and then filter through a dry filter paper. Add 5 c.c. of water to the filtrate, to prevent the formation of diethyl oxalate when the solution is heated, and distil off the ether. If necessary, decolorize the liquid with animal charcoal and filter. Con- centrate the filtrate to 3-5 c.c, add a little calcium chloride solution, make it ammoniacal and after a few minutes render it slightly acid with acetic acid. Allow the acidified solution to stand several hours, collect the precipitate of calcium oxa- late on a washed filter paper, 1 wash, incinerate strongly (to CaO) and weigh in the usual manner. Calculation. — Since 56 parts of CaO are equivalent to 90 parts of oxalic acid, the quantity of oxalic acid in the volume 1 Schleicher and Schiill, No. 589, is satisfactory. urine: quantitative analysis. 379 of urine taken may be determined by multiplying the weight 1 >f ( '.i( ) by tin.' factor [.607 1 . XVI. Total Solids. 1. Drying Method. — Place 5 c.c. of urine in a weighed shallow dish, acidify very slightly with acetic acid (1-3 drops) and dry it in vacuo in the presence of sulphuric acid, to constant weight. Calculate the percentage of solids in the urine sample and the total solids for the twenty- four hour period. Practically all the methods the technique of which includes evaporation at an increased temperature, either under atmo- spheric conditions or in vacuo are attended with error. 2. Calculation by Long's Coefficient. — The quantity of solid material contained in the urine excreted for any twenty- four hour period may be approximately computed by multi- plying the second and third decimal figures of the specific gravity by 2.6. This gives us the number of grams of solid matter in one liter of urine. From this value the total solids for the twenty-four hour period may easily be determined. Calculation. — If the volume of urine for the twenty-four hours was 1120 c.c. and the specific gravity 1.018, the calcu- lation would be as follows : (a) 18 X 2.6 = 46.8 grams of solid matter in 1 liter of urine. ,, N 46.8 X 1 120 ,., . , {0) - =52.4 grams solid matter 111 1120 c.c. of urine. Long's coefficient was determined for urine whose specific gravity was taken at 25 ° C. and is probably more accurate, for conditions obtaining in America, than the older coefficient of Haeser, 2.33. CHAPTER XXII QUANTITATIVE ANALYSIS OF MILK, GASTRIC JUICE AND BLOOD. (a) Quantitative Analysis of Milk. I. Specific Gravity. — This may be determined conve- niently by means of a Soxhlet, Veith or Quevenne lactometer. A lactometer reading of 32 ° denotes a specific gravity of Fig. 125. 1-032. The determination should be made at about 60 ° F. and the lactometer reading cor- rected by adding or subtracting o.i° for every degree F. above or below that temperature. 2. Fat. — (a) Adams' Paper Coil Method. — Introduce about 5 c.c. of milk into a small beaker, quickly ascertain the weight to centigrams, stand a fat-free coil 1 in the beaker and incline the vessel and rotate the coil in order to hasten the absorption of the milk. Immediately upon the complete absorption of the milk remove the coil and again quickly ascertain the weight of the beaker. The dif- ference in the weights of the beaker at the two weighings rep- resents the quantity of milk absorbed by the coil. Dry the coil carefully at a temperature below ioo° C. and extract it with ether for 3-5 hours in a 'Very satisfactory coils are manufactured by Schleicher and Schull. 380 SOX II LET Al'I'AKAT US. QUANTITATIVE ANALYSIS OF MILK. 38l Im<; 126. N - - . tOl_ ! c »— 1 — ' Soxhlet apparatus (Fig. [25, p. 380), using a safety water-bath, Heat the flask containing the fat to constant weight at a temperature bel< m roo C. Calculation. — Divide the weight of fat, in grams, by the :u of milk, in grams. The quotient is the percentage of fat contained in the milk examined. > Approximate Determination by Feser's LactOSCOpe. — Milk is opaque mainly because of the suspended fat globules and therefore"by means of the estimation of this opacity we may obtain data as to the approximate content of fat. Feser's lactoscope (Fig. 126, p. 38] 1 may be used for this purpose. Pr as follows: By means of the graduated pipette accompanying the instrument introduce 4 c.c. l>\ milk into the lactoscope. Add water grad- ually, shaking after each addition, and note the point at which the black lines upon the inner white glass cylinder are distinctly visible. Ob- serve the point on the graduated scale of the lactoscope which is level with the surface of the diluted milk. This reading - represents the per- centage of fat present in the undiluted milk. Pure milk should contain at least 3 per cent of fat. 3. Total Solids. 1 — Introduce 2-5 grams of milk into a weighed flat-bottomed platinum dish and quickly ascertain the weight to milligrams. Expel the major portion of the water by heating the open dish on a water-bath and continue the heating in an air-bath or water oven at 97 : -100 C. until the weight is constant. ( This residue may be used in the deter- mination of ash according to the method described on p. 382. 1 1 The percentage of total solids may be calculated from the specific gravity and percentage of fat by means of the following formula which has been proposed by Richmond : S = o.^5 L+1.2 F + 0.14 S = total solids. L = lactometer reading. F= fat content. J Feser's Lactoscope. 382 PHYSIOLOGICAL CHEMISTRY. Calculation. — Divide the weight of the residue, in grams, by the weight of milk used, in grams. The quotient is the per- centage of solids contained in the milk examined. 4. Ash. — Heat the dry solids from 2-5 grams of milk, obtained according to the method just given, over a very low flame 1 until a white or light gray ash is obtained. Cool the dish in a desiccator and weigh. (This ash may be used in testing for preservatives according to directions on page 195.) 5. Proteids. — Introduce a known weight of milk (5-10 grams) into a 200-300 c.c. Kjeldahl digestion flask and add 20 c.c. of concentrated sulphuric acid and about 0.2 gram of cupric sulphate. Expel the major portion of the water by heating over a low flame and finally use a full flame and allow the mixture to boil 1-2 hours. Complete the deter- mination according to the directions given under Kjeldahl Method, page 359. Calculation. — Multiply the total nitrogen content by the factor 6.37 s to obtain the proteid content of the milk ex- amined. 6. Casein. — Mix about 20 grams of milk with 40 c.c. of a saturated solution of magnesium sulphate and add the salt in substance until no more will dissolve. The precipitate consists of casein admixed with a little fat and lacto-globulin. Filter off the precipitate, wash it thoroughly with a saturated solution of magnesium sulphate, 3 transfer the filter paper and precipitate to a Kjeldahl digestion flask and determine the nitrogen content according to the directions given in the pre- vious experiment. Calculation. — Multiply the total nitrogen by the factor 6.37 to obtain the casein content. 1 Great care should be used in this ignition, the dish at no time being heated above a faint redness, as chlorides may volatilize. 2 The usual factor employed for the calculation of proteid from the nitrogen content is 6.25 and is based on the assumption that proteids contain on the average 16 per cent of nitrogen. This special factor of 6.37 is used here to calculate the proteid content from the total nitrogen, since the principal proteid constituents of milk, i. e. } casein and lactalbumin. contain 15.7 per cent of nitrogen. 3 Preserve the filtrate and washings for the determination of lactalbumin. QUANTITATIVE ANALYSIS OF GASTRIC JUICE. 7. Lactalbumin. — To the filtrate and washings from the de- termination of casein, as jusl explained, add Almen's reagent 1 until no more precipitate forms. Filter oft" the precipitate and determine the nitrogen content according to the direc- tions given under Proteids, page 382. Calculation. — Multiply the total nitrogen by the factor 6.37 to obtain the lactalbumin content. 8. Lactose. — To about 350 c.c. of water in a beaker add 20 grams of milk, mix thoroughly, acidify the fluid with about 2 c.c. of 10 per cent acetic acid and stir the acidified mixture continuously until a flocculent precipitate forms. At this point the reaction should be distinctly acid to litmus. Heat the solution to boiling for one-half hour, filter, rinse the beaker thoroughly and wash the precipitated proteids and the adherent fat with hot water. Combine the filtrate and wash water and concentrate the mixture to about 150 c.c. Cool the solution and dilute it to 200 c.c. in a volumetric flask. Titrate this sugar solution according to directions given under Fehling's Method, page 345. Calculation. — Make the calculation according to directions p-iven under Fehling's Method, p. 34s, bearing in mind that 10 c.c. of Fehling's solution is completely reduced by 0.0676 gram of lactose. (b) Quantitative Analysis of Gastric Juice. Topfer's Method. This method is much less elaborate than many others but is sufficiently accurate for ordinary clinical purposes. The method embraces the volumetric determination of (1) total acidity, (2) combined acidity, and (3) free acidity, and the subsequent calculation of (4) acidity due to organic acids and acid salts, from the data thus obtained. Strain the gastric contents and introduce 10 c.c. of the strained material into each of three small beakers or porcelain 1 Almen's reagent may be prepared by dissolving 5 grams of tannin in 240 c.c. of 50 per cent alcohol and adding 10 c.c. of 25 per cent acetic acid. 384 PHYSIOLOGICAL CHEMISTRY. dishes. 1 Label the vessels A, B and C, respectively, and pro- ceed with the analysis according to the directions given below. 1. Total Acidity. 2 — Add 3 drops of a 1 per cent alcoholic solution of phenolphthalein 3 to the contents of vessel A and titrate with j% sodium hydroxide solution until a dark pink color is produced which cannot be deepened by further addi- tion of a drop of y^" sodium hydroxide. Take the burette reading and calculate the total acidity. Calculation. — The total acidity may be expressed in the following ways : 1. The number of cubic centimeters of ^ sodium hydrox- ide solution necessary to neutralize 100 c.c. of gastric juice. 2. The weight (in grams) of sodium hydroxide necessary to neutralize 100 c.c. of gastric juice. 3. The weight (in grams) of hydrochloric acid which the total acidity of 100 c.c. of gastric juice represents, i. c, per- centage of HC1. The forms of expression most frequently employed are 1 and 3, preference being given to the former. In making the calculation note the number of cubic centi- meters required to neutralize 10 c.c. of the gastric juice and multiply it by 10 to obtain the number of cubic centimeters necessary to neutralize 100 c.c. of the fluid. If it is desired to express the acidity of 100 c.c. of gastric juice in terms of hydro- chloric acid, by weight, multiply the value just obtained by 0.00365. 4 2. Combined Acidity. 5 — Add 3 drops of sodium alizarin sulphonate solution 6 to the contents of vessel B and titrate with ys sodium hydroxide solution until a violet color is pro- duced. In this titration the red color, which appears after 1 If sufficient gastric juice is not available it may be diluted with water or a smaller amount, e. g., 5 c.c, taken for each determination. 2 This includes free and combined acid and acid salts. 3 One gram of phenolphthalein dissolved in 100 c.c. of 95 per cent alcohol. * One c.c. of yV hydrochloric acid contains 0.00365 gram of hydrochloric acid. 5 Hydrochloric acid combined with proteid material. 6 One gram of sodium alizarin sulphonate dissolved in 100 c.c. of water. Ql AMITATIVF. ANALYSIS OF GASTRK JUICE. 385 the tinge of yellow duo to the addition of the indicator has disappeared, musl be entirely replaced by a distinct violet color. Take the burette reading and calculate the combined acidity. Calculation. — Since the indicator used reacts to all acidities except combined acidity, in order to determine the number of cubic centimeters of -j* sodium hydroxide necessary to neutralize the combined acidity of 10 c.c. of the gastric juice, we must subtract the burette reading just obtained from the burette reading obtained in the determination of the total acidity. The data for 100 c.c. of gastric juice may be calcu- lated according" to the directions given under Total Acidity, page 384. 3. Free Acidity. 1 — Add 4 drops of di-methyl-amino-azo- benzene (Topfer's reagent) solution 2 to the contents of the ves- sel C and titrate with y sodium hydroxide solution until the initial red color is replaced by lemon yellow. 9 Take the burette reading and calculate the free acidity. Calculation. — The indicator used reacts only to free acid- ity, hence the number of cubic centimeters of t N q sodium hydroxide used indicates the volume necessary to neutralize the free acidity of 10 c.c. of gastric juice. To determine the data for 100 c.c. of gastric juice proceed according to the directions given under Total Acidity, page 384. 4. Acidity due to Organic Acids and Acid Salts. — This value may be conveniently calculated by subtracting the num- ber of cubic centimeters of y 5 sodium hydroxide used in neu- tralizing the contents of vessel C from the number of cubic centimeters of T ^ sodium hydroxide solution used in neutral- izing the contents of vessel B. The remainder indicates the number of cubic centimeters of -£$ sodium hydroxide solution necessary to neutralize the acidity due to organic acids and acid salts present in 10 c.c. of gastric juice. The data for 'Hydrochloric acid not combined with proteid material. 2 One-half gram dissolved in 100 c.c. of 95 per cent alcohol. 1 If the lemon yellow color appears as soon as the indicator is added it denotes the absence of free acid. 26 386 PHYSIOLOGICAL CHEMISTRY. 100 c.c. of gastric juice may be calculated according to direc- tions given under Total Acidity, page 384. (c) Quantitative Analysis of Blood. For the methods involved in the quantitative examination of blood see Chapter XL APPENDIX. Almen's Reagent. 1 — Dissolve 5 grams of tannin in 240 c.c. of 50 per cent alcohol and add i«» c.c. of -'5 per cent acetic acid. Ammoniacal Silver Solution.- — Dissolve 26 grams of silver nitrate in about 500 c.c. of water, add enough ammo- nium hydroxide to redissolve the precipitate which forms upon the first addition of the ammonium hydroxide and make the volume of the mixture up to 1 liter with water. Arnold-Lipliawsky Reagent.'' — This reagent consists of two definite solutions which are ordinarily preserved sepa- rately and mixed just before using. The two solutions are prepared as follows : 1 (/ ) One per cent aqueous solution of potassium nitrite. ) One gram of ^-amino-acetophenon dissolved in 100 c.c. of distilled water and enough hydrochloric acid (about 2 c.c.) added, drop by drop, to cause the solution, which is at first yellow, to become entirely colorless. An excess of acid must be avoided. Barfoed's Solution. 4 — Dissolve 4 grams of cupric acetate in 100 c.c. of water and acidify with acetic acid. Baryta Mixture.' — A mixture consisting of one volume of a saturated solution of barium nitrate and two volumes of a saturated solution of barium hydroxide. Boas' Reagent." — Dissolve 5 grams of resorcin and 3 grams of saccharose in 100 c.c. of 95 per cent alcohol. Congo Red." — Dissolve 0.5 gram of congo red in 90 c.c. of water and add 10 c.c. of 95 per cent alcohol. 1 Ott'> precipitation test, p. 297. Determination of lactalbumin, p. 383. 2 Salkowski's method, page 377. 'Arnold-Lipliawsky reaction, page 308. * Barfoed's test, page 11. 'Isolation of urea from urine, page -'4-'. 'Test fur free acid, page 88. 'Test for free acid, page 88. 3*7 388 PHYSIOLOGICAL CHEMISTRY. Ehrlich's Diazo Reagent. 1 — Two separate solutions should be prepared and mixed in definite proportions when needed for use. (a) Five grams of sodium nitrite dissolved in 1 liter of distilled water. (b) Five grams of sulphanilic acid and 50 c.c. of hydro- chloric acid in 1 liter of distilled water. Solutions a and b should be preserved in well stoppered ves- sels and mixed in the proportion 1 : 50 when required. Green asserts that greater delicacy is secured by mixing the solution in the proportion 1 : 100. The sodium nitrite deteriorates upon standing and becomes unfit for use in the course of a few weeks. Esbach's Reagent. 2 — Dissolve 10 grams of picric acid and 20 grams of citric acid in 1 liter of water. Fehling's Solution. 3 — Fehling's solution is composed of two definite solutions — a cupric sulphate solution and an alkaline tartrate solution, which may be prepared as follows : Cupric sulphate solution = 34.64 grams of cupric sulphate dissolved in water and made up to 500 c.c. Alkaline tartrate solution= 125 grams of potassium hy- droxide and 173 grams of Rochelle salt dissolved in water and made up to 500 c.c. These solutions should be preserved separately in rubber- stoppered bottles and mixed in equal volumes when needed for use. This is done to prevent deterioration. Ferric Alum Solution. 4 — A cold saturated solution. Folin-Shaffer Reagent. 5 — This reagent consists of 500 grams of ammonium sulphate, 5 grams of uranium acetate and 60 c.c. of 10 per cent acetic acid in 650 c.c. of distilled water. Furfurol Solution. — Add 1 c.c. of furfurol to 1000 c.c. of distilled water. 1 Ehrlich's diazo reaction, page 316. 2 Esbach's method, page 344. 3 Fehling's method, page 345. Fehling's test, pages 8 and 286. 4 Volhard-Arnold method, page 372. Folin-Shaffer method, page 349. 8 Mylius's modification of Pettenkofer's test, pages 122 and 301. v. Udransky's test, pages 123 and 302. APPENDIX. 389 Gallic Acid Solution.' — A saturated alcoholic solution. Guaiac Solution."' — Dissolve 0.5 gram of guaiac resin in 30 c.c. 1 »f 95 per cent alo >h< »1. Giinzberg's Reagent. 3 — Dissolve 2 -ranis of phloroglucin and 1 gram of vanillin in ioo c.c. of 95 per cent alcohol. Hammarsten's Reagent.' — Mix 1 voluble of 25 per cent nitric acid and [9 volumes of 25 per cent hydrochloric acid and add 1 vplume of this acid mixture to 4 volumes of 95 per cent alcohol. It is preferable that the acid mixture be prepared in advance and allowed to stand until yellow in color before adding it t<> the alcohol. Hopkins-Cole Reagent.' — To one liter of a saturated solution of oxalic acid add 00 grams of sodium amalgam and allow the mixture t<> stand until the evolution of gas ceases. Filter and dilute with _' 3 volumes of water. Hypobromite Solution. 6 — The ingredients of this solu- tion should be prepared in the form of tzco separate solutions which may he united as needed. (a) Dissolve 125 grams of sodium bromide in water, add 125 grams of bromine and make the total volume of the solu- tion 1 liter. (b) A solution of sodium hydroxide having a specific gravity of 1.25. This is approximately a 22.^ per cent solution. Preserve both solutions in rubber-stoppered bottles and when needed for use mix equal volumes of solution a, solution b, and water. Iodine Solution.' — Prepare a 2 per cent solution of potas- sium iodide and add sufficient iodine to color it a deep yellow. Jolles' Reagent." — This reagent has the following com- posite in : 1 Gallic acid tot. page 195. : Guaiac test, page* 163, 191 and 331. 8 Test for free acid, page 88. * Hammarsten's reaction, pages ui and 300. Hopkins-Cole reaction, page 45. " Methods for determination of urea, page 351. ' Iodine test, page 24. 8 Jolles' reaction, pages 48 and 292. 39° PHYSIOLOGICAL CHEMISTRY. Succinic acid 40 grams. Mercuric chloride 20 grams. Sodium chloride 20 grams. Distilled water 1000 grams. Lugol's Solution. 1 — Dissolve 5 grams of iodine and 10 grams of potassium iodide in 100 c.c. of distilled water. Magnesia Mixture. 2 — Dissolve 175 grams of magnesium sulphate and 350 grams of ammonium chloride in 1400 c.c. of distilled water. Add 700 grams' of concentrated ammo- nium hydroxide, mix thoroughly and preserve the mixture in a glass-stoppered bottle. Millon's Reagent. 3 — Digest 1 part (by weight) of mer- cury with 2 parts (by weight) of HNO s (sp. gr. 1.42) and dilute the resulting solution with 2 volumes of water. Molybdic Solution. 4 — Molybdic solution is prepared as follows, the parts being by weight: Molybdic acid 1 part. Ammonium hydroxide (sp. gr. 0.96) 4 parts. Nitric acid (sp. gr. 1.2) 15 parts. Morner's Reagent 5 — Thoroughly mix 1 volume of for- malin. 45 volumes of distilled water and 55 volumes of con- centrated sulphuric acid. Neutral Olive Oil. 6 — Shake ordinary olive oil with a 10 per cent solution of sodium carbonate, extract the mixture with ether and remove the ether by evaporation. The residue is neutral olive oil. Nylander's Reagent 7 — Digest 2 grams of bismuth sub- nitrate and 4 grams of Rochelle salt in 100 c.c. of a 10 per 1 Gunning's iodoform test, page 304. 2 Sodium hydroxide and potassium nitrate fusion method for determi- nation of total phosphorus, page 368. B Millon's reaction, page 44. 1 Sodium hydroxide and potassium nitrate fusion method for determi- nation of total phosphorus, page 368. ".Morner's test, page 82. Emulsification of fats, page 101. 7 Nylander's test, pages 9 and 288. APPENDIX. 391 cent solution of potassium hydroxide. The reagent should then be ci " iled and filtered* Obermayer's Reagent. 1 — A<1<1 2 4 grams of ferric chlo- ride i" a liter of hydrochloric acid (sp. gr. [.19). Oxalated Plasma.- — Allow arterial blood to run into an equal volume of 0.2 per cent ammonium oxalate solution. Paraphenelenediamine Hydrochloride Solution.'' — Two grams dissolved in 100 c.c. of water. Phenolphthalein/ — Dissolve 1 gram of phenolphthalein in 100 c.c. of 95 per cent alcohol. Phenylhydrazin Mixture.'' — This mixture is prepared by combining 1 part of phenylhydrazin-hydrochloride and 2 parts of -odium acetate by weight. These are thoroughly mixed in a mortar. Phenylhydrazin-Acetate Solution.'' — This solution is pre- pared by mixing 1 volume of glacial acetic acid, 1 volume of water and 2 volumes of phenylhydrazin (the base). Purdy's Solution. 7 — Purdy's solution has the following composition : Cupric sulphate 4752 grams. Potassium hydroxide 23.5 grams. Ammonia (U. S. P.. sp. gr. 0.9) 350.0 c.c. Glycerin 38.0 c.c. Distilled water, to make total volume 1 liter. Roberts' Reagent/ — Mix i volume of concentrated nitric acid and 5 volumes of a saturated solution of magnesium sulphate. Salted Plasma. 1 ' — Allow arterial blood to run into an equal volume of a saturated solution of sodium sulphate or a 10 1 Obermayer's test, page 255. ' Experiments on blood plasma, page 167. 3 Detection of hydrogen peroxide, page 196. 4 Topfer's method, page 383. 6 Phenylhydrazin reaction, pages 5 and 283. '*' Phenylhydrazin reaction, pages 5 and -'84. ' Purdy's method, page 347. v Robert's ring test, pages 48 and 291. ' Experiments on blood plasma, page 167. 39 2 PHYSIOLOGICAL CHEMISTRY. per cent solution of sodium chloride. Keep the mixture in the cold room for about 24 hours. Schweitzer's Reagent. 1 — Add potassium hydroxide to a solution of cupric sulphate which contains some ammonium chloride. Filter off the precipitate of cupric hydroxide, wash it and bring it into solution in 20 per cent ammonium hy- droxide. Sherrington's Solution. 2 — This solution possesses the fol- lowing formula : Methylene-blue 0.1 gram. Sodium chloride 1.2 gram. Neutral potassium oxalate 1.2 gram. Distilled water 300.0 grams. Sodium Acetate Solution. 3 — Dissolve 100 grams of so- dium acetate in 800 c.c. of distilled water, add 100 c.c, of 30 per cent acetic acid to the solution and make the volume of the mixture up to 1 liter with distilled water. Sodium Alizarin Sulphonate/ — Dissolve 1 gram of sodium alizarin sulphonate in 100 c.c. of water. Solera's Test Paper. 5 — Saturate a good quality of filter paper with 0.5 per cent starch paste containing a little iodic acid and allow the paper to dry in the air. Cut it in strips of suitable size and preserve for use. Spiegler's Reagent. 6 — This reagent has the following com- position : Tartaric acid 20 grams. Mercuric chloride 40 grams. Glycerin 100 grams. Distilled water 1000 grams. Standard Ammonium Sulphocyanide Solution. 7 — This solution is made of such a strength that 1 c.c. of it is equal 1 Schweitzer's solubility test, page 29. 2 "Blood counting," page 181. 3 Uranium acetate method, page 367. 4 Topfer's method, page 383. Solera's reaction, page 38. " Spiegler's ring test, pages 48 and 291. 7 Volhard-Arnold method, page 372. APPENDIX. 393 to i c.c. of the standard argentic nitrate solution mentioned below. To prepare the solution dissolve [2.9 grams of am- monium sulphocyanide, NH 4 SCN, in a little less than a liter of water. In a small flask place 20 C.C. of the standard argentic nitrate solution. 5 c.c. of a cold saturated solution of ferric alum and 4 c.c. of nitric acid (sp. gr. i.j ). add water to make the total volume 100 c.c. and thoroughly mix the content^ of the flask. Now run in the ammonium sulpho- cyanide solution from a hurette until a permanent brown tinge is produced. This is the end-reaction and indicates that the last trace of argentic nitrate has been precipitated. Take the burette reading and calculate the amount of water necessary to use in diluting the ammonium sulphocyanide in order that 10 c.c. of this solution may be exactly equal to 10 c.c. of the argentic nitrate solution. Make the dilution and titrate again to be certain that the solution is of the proper strength. Standard Argentic Nitrate Solution. 1 — Dissolve 29.06 grams of argentic nitrate in 1 liter of distilled water. Each cubic centimeter of this solution is equivalent to 0.01 gram of sodium chloride or to 0.006 gram of chlorine. Standard Uranium Acetate Solution." — Dissolve 35461 grams of uranium acetate in 1 liter of water. One c.c. of such a solution should be equivalent to 0.005 gram of P 2 5 , phos- phoric anhydride. This solution may be standardized as follows : To 50 c.c. of a standard solution of disodium hydrogen phosphate, of such a strength that the 50 c.c. contains 0.1 gram of P 2 5 . add 5 c.c. of the sodium acetate solution mentioned on p. 392 and titrate with the uranium solution to the correct end-reaction as indicated in the method proper on p. 367. Inasmuch as 1 c.c. of the uranium solution should precipitate 0.005 g ram of P 2 5 , exactly 20 c.c. of the uranium solution should be required to precipitate the 50 c.c. of the standard phosphate solution. If 1 Volhard-Arnold method, page 372. Mohr's method, page 371. 2 L'ranium acetate method, page 367. 394 rilYSIOLOGICAL CHEMISTRY. the two solutions do not bear this relation to each other they must be brought into the proper relation by diluting the ura- nium solution with distilled water or by increasing its strength. Starch Iodide Solution. 1 — Mix o.i gram of starch powder with cold water in a mortar and pour the suspended starch granules into 75-100 c.c. of boiling water, stirring continu- ously. Cool the starch paste, add 20-25 grams of potassium iodide and dilute the mixture to 250 c.c. This solution deteri- orates upon standing, and therefore must be freshly prepared as needed. Starch Paste. 2 — Grind 2 grams of starch powder in a mortar with a small amount of water. Bring 200 c.c. of water to the boiling-point and add the starch mixture from the mortar with continuous stirring. Bring again to the boiling-point and allow it to cool. This makes an approximate 1 per cent starch paste which is a very satisfactory strength for general use. Stokes' Reagent. 3 — A solution containing 2 per cent fer- rous sulphate and 3 per cent tartaric acid. When needed for use a small amount should be placed in a test-tube and am- monium hydroxide added until the precipitate which forms on the first addition of the hydroxide has entirely dissolved. This produces ammonium fcrrotartrate which is a reducing agent. Tanret's Reagent. 4 — Dissolve 1.35 grams of mercuric chloride in 25 c.c. of water, add to this solution 3.32 grams of potassium iodide dissolved in 25 c.c. of water, then make the total solution up to 60 c.c. with distilled water and add 20 c.c. of glacial acetic acid to the mixture. Tincture of Iodine. 5 — Dissolve 70 grams of iodine and 50 grams of potassium iodide in 1 liter of 95 per cent alcohol. Toison's Solution. 6 — This solution has the following formula : 1 Fehling's method, page 345. 2 Experiments on starch, page 24. 3 Haemoglobin, page 170. Hsemochromogen, page 173. 4 Tanret's test, pages 48 and 293.' Smith's test, pages 122 and 301. ""Blood counting," page 181. APPENDIX. 395 Methyl violel 0.025 gram. Sodium chloride 1.0 gram. Sodium sulphate 8.0 grams Glycerin 30.0 grams. I tistilled water [60.0 Topfer's Reagent.' — Dissolve 0.5 grams of di-methyl- amino-azobenzene in too c.c. of 95 per cent alcohol. Tropseolin OO.-' Dissolve 0.05 gram of tropaeolin OO in 100 c.c. of go per cent alcohol. Uffelmann's Reagent." — Add a 5 per cent solution of ferric chloride to a 1 per cent solution of carbolic acid until an ame- thyst-blue col< >r is obtained. 1 Topfer's methi id, page 383. 1 Tesl For Free acid, page 89. 'Uffelman's reaction, page 04. NDEX. Acetone, 28-', 302 formula for, 302 Gunning's iodoform test for, 304 I 1 gal's tesl for, 305 Lieben's test for, 305 quantitative determination of, 374 Reynolds-Gunning test for, 306 Acholic stool, 140 AchrOO dextrin, 22, 35 Acid, acetic, 238, 265 alloxyproteic, 237, 259, 317 amino-acetic, 71 amino-ethyl-sulphonic, 118, 211 a-amino-/3-hydroxy-propionic, 74 a-amino-/3-imido-azol -propionic, 80 a -ami no-iso-butyl -acetic, 69 a-amino-normal glutaric, 70 o-amino-propionic. 72 amino-succinic, 70 amino-valerianic, 73 a-amino-iso-valerianic, 73 aspartic, 65, 70, 107 benzoic, 127, 237, 263 butyric, 84, 194, 238, 265 caproic, 187 carbamic, 150 cholic, 117 chondroitin-sulphuric, 202, 237, 259 combined hydrochloric, 84, 87 cyanuric, 241 a-e-di-amino-caproic, 78 diazo-benzene-sulphonic, 317 ethereal sulphuric, 129, 237, 253 fatty, 97, 99, 102 formic. 238. 265 free hydrochloric, 84, 87 glutamic. 65, 70, 107 glycocholic, 117 glycuronic, 16 glycerophosphoric, 221, 238, 265 glyoxylic, 45 guaniain-o-amino-valerianic, 79 hippuric. 127, 128, 237, 255 homoeentisic. 9. 237, 262 indol-amino-propionic, 77 indoxyl-sulphuric, 237. 253 inosinic, 207, 211 kynurenic. 237, 262 Acid, lactic, 10. 84, -'"7 lauric. 187 myristic. 187 nucleic, 62 oxalic, 237, 258 oxaluric, 237, 264 oxymandclic. 237, 262 oxyproteic, 237, 259, 317 palmitic, 97, 102, 103 para cresol-sulphuric, 237, 253 para-oxyphenyl-acetic, 129, 136, 237, 261 para oxyphenyl-a amino-propionic, 66 para-oxyphenyl-propionic, 129, 136. 237, 261 paralactic, 208, 238, 265 penaceturic, 238, 265 phenol-sulphuric, 237, 253 phenyl -a -amino-propionic. 73 phosphocarnic. 207, 211, 238, 265 pyrocatechin-sulphuric, 237, 253 a-pyrrolidin-carboxylic. 74 sarcolactic, 208 skatol-carbonic, 135, 136 skatoxyl-sulphuric, 237, 253 sulphanilic, 316. 317 tannic. 25, 28, 47 taurocholic, 117 uric. 9. 207, 237, 245 uro ferric, 237, 259, 317 uroleucic. 237, 262 volatile fatty, 129, 132, 194, 238 Acid albuminate, 55, 56 coagulation of, 55 experiments on, 56 precipitation of, 56 preparation of, 56 solubility of, 56 sulphur content of, 56 Acidity of gastric juice, quantitative determination of, 383 urine, cause of, 228 quantitative determination of. 376 Acidosis, cause of, 309 Acid-haematin, 173 Acrolein, formation of, from olive oil, 100 from glycerin, 104 397 39« INDEX. Adams' paper coil method for deter- mination of fat in milk, 380 Adamkiewicz reaction, 45 Adenin, 21 1, 238 Adipocere, 99 Adipolytic enzymes, 34 Alanin, 65, 72 Albumin, egg, 42, 51 powdered, preparation of, 51 tests on, 51 serum, 42, 52, 148, 149, 282, . 28q . Albumin in urine, 282, 289 acetic acid and potassium ferrocyanide test for, 293 coagulation or boiling test for, 292 Heller's ring test for, 290 Jolles' reaction for, 292 Robert's ring test for, 291 sodium chloride and acetic acid test for, 293 Spiegler's ring test for, 291 Tanret's test for, 293 tests for, 290 Albuminate, 55 acid, 55, 56 alkali, 55, 57 precipitation of, 56 sulphur content of, 56 Albuminoids, 43, 62 Albumoids, 43, 62 Albumoses (see proteoses, pp. 43, 57. 59) Aldehyde, 1, 7 Aldehyde group, 18 Aldehyde test for alcohol, 21 v. Aldor's method of detecting pro- teose in urine, 296 Aldose, 1 Alkali albuminate, 55, 56, 57, 107 experiments on, 57 precipitation of, 57 preparation of, 57 sulphur content of, 56 Alkali-hrematin, 172 Allantoin, 237, 259 crystalline form of, 259 experiments on, 260 formula for, 259 preparation of, from uric acid, 260 separation of, from the urine, 260 Allen's modification of Fehling's test, 287 A linen's reagent, preparation of, 297 Alloxyproteic acid, 237, 259, 317 Aloin-turpentine test for " occult blood," 142, 144 Amide, definition of, 65 Amine, definition of, 65 Amino acids, 65, 129 a-amino-/3-hydroxypropionic acid, 74 a-amino-/3-imido-azol-propionic acid, 80 a-amino-iso-butyl-acetic acid, 69 a-amino-normal-glutaric acid, 70 Amino-succinic acid, 70 Amino-valerianic acid, 7s a-amino-iso-valerianic acid, 73 Ammonia, 65, 107 Ammonia in urine, 238, 270 quantitative determination of, 357 Ammoniacal silver solution, prepara- tion of, 377 Ammoniacal-zinc chloride test for urobilin, 268 Ammonium magnesium phosphate ("Triple phosphate"), 278 in urinary sediments, 319 Amphopeptone, 43 Amyloid, 28, 29, 43, 62 Amylolytic enzymes, 34, 108 Amylopsin, 108 digestion of dry starch by, 109, 114 inulin by, 114 experiments on, 112 influence of bile upon action of, 114 metallic salts, upon action of, 113 most favorable temperature for action of, 113 Animal parasites in feces, 143 in urinary sediments, 339 Anti-albumid, 86, 87 Antipeptone, 43 Appendix, 387 Arabinose, 2, 16 orcin test on, 17 phenylhydrazin test on, 17 Tollens' reaction on, 16 Arginin, 65, 78, 107 Arnold-Lipliawsky reaction for dia- cetic acid, 308 reagent, preparation of, 308 Aromatic oxyacids, 237, 261 Asparagin, 70 Aspartic acid, 65, 70 crystalline form of, 70 formula for, 70 Ash of milk, quantitative determina- tion of, 382 Barfoed's reagent, preparation of, n [NDEX. Barfoed's test For dextrose, u, 289 Baryta mixture, preparation of, 242 Bayberry tallow, saponification of. 102 Beckmand-Heidenhain apparatui " Bence Joins' proteid," detection of, 296 Benzoic acid, u;, 237 crystalline form of, 264 experiments upon, formula for, 263 ilulity of, 263 sublimation of, 263 Berthelot-AtwatCr bomb calorimeter, 366 Bergell's method for determination of /9-oxybutyric acid, 376 Bile. 1 16. 282, 300 constituents of, 1 1 7 daily secretion of, 116 freezing-point of. 11- influence on digestion, gastric, 93 pancreatic. 112, 114 inorganic constituents of, 117, 121 nucleo-proteid of, 121 reaction of, 116, 121 secretion of. 1 16 specific gravity of, 117 Bile acids 1 1 7 Hay's test for, 123 Mylius's test for, 122 {Jeukomm's test for, 122 Pettenkofer's test for, 122 tests for, 122 v. Udransky's test for, 123 Bile acids in feces, detection of, 146 Bile acids in urine, 2S2 Hay's test for, 302 Mylius's test for, 301 Xeukomm's test for. 301 Pettenkofer's test for, 301 Salkowski's test for, 302 tests for, 301 v. Udransky's test for, 302 Bile pigments, 118 Gmelin's test for, 121 Hammarsten's reaction for, 121 Huppert's reaction for, 121 Rosenbach's test for. 121 Smith's test for, 122 tests for, 121 Bile pigments in urine, 282, 300 Gmelin's test for, 300 Hammarsten's reaction for, 300 Bile pigments in urine, rluppert'i reaction for, Rosenba* h'a ti -< for, 300 Smith's test for, 301 300 Bile salts, crystallization of, 123 Biliary calculi. 120 analysis of, 124 Bilicyanin, 118, 120 Bilifuscin, 1 is Bilihumin, 118 Biliprasin, 118 Bilirubin, 118. 119 crystalline form of, 1 tg in urinary sediments, 326 Biliverdin, 1 1 8, 120 "Biological" blood test, 159 Biuret, 46. 241 formation of, from urea. 46. 241, 243 Biuret test. 45 Posner's modification of, 46 Blood, 148, 282, 297 Bordet test for, 159 clinical examination of. 174 coagulation of. 157 constituents of, 148, 150 defihrinated, 160 detection of, 158, 159, 163, 168 erythrocytes of, 150 experiments on, 160 form elements of, 148 guaiac test for, 158, 163 haemin test for, 158, 163 oxyhemoglobin of, 155 "occult," in feces, 142, 144 in urine, 282, 297 leucocytes of, 156 medico-legal tests for, 158 microscopical examination of, 160, 168 nucleo-proteid of, 148, 149 pigment of, 155 plaques, 148 plasma, 148, 149, 167 preparation of hxmatin from, 165 preparation of laky, 161 quantitative analysis of, 386 reaction of, 148, 160 serum, 150, 166 specific gravity of, 148, 160 spectroscopic examination of, 169 test for iron in, 161 total amount of, 148 Zeynek and Xencki's haemin test for, 163 Blood casts in urine, 334 400 INDEX. Blood corpuscles, 148, 150, 156, 168 " counting," 180 Blood in urine, 282, 297 guaiac test for, 299 Teichmann's hremin test for, 298 Heller's test for, 298 Heller-Teichmann reaction for, 298 Schalfijew's hsemin test for, 299 spectroscopic examination of, 299 tests for, 298 Zeynek and Nencki's hsemin test for, 299 Blood plasma, 148, 149, 167 constituents of, 148 crystallization of oxyhemo- globin of, 156, 167 effect of calcium on oxa- lated, 167 experiments on, 167 preparation of fibrinogen from, 167 oxalated, 167 salted, 167 Blood serum, 150, 166 coagulation temperature of, 166 constituents of, 150 experiments on, 166 precipitation of proteids of, 166 separation of albumin and globulin of, 166 sodium chloride in, 166 sugar in, 166 Blood stains, examination of, 168 Boas' reagent, as indicator, 88 preparation of, 88 Boekelman and Bouma's method for determination of /3-oxybutyric acid, 376 Boettger's test for sugar, 9, 288 Bomb calorimeter, Berthelot-Atwater, 366 Bone, constituents of, 204 ossein of, preparation of, 204 Bone ash, scheme for analysis of, 205 Bordet test, detection of human blood by, 159 Boric acid and borates in milk, de- tection of, 196 Buccal glands, 32 Butyric acid, 84, 194 Butyrin, 98, 187 Cadaverin, 78 Calcium and magnesium in urine, 238, 279 carbonate in urinary sediments 319, 321 casein, 188 oxalate, 319 in urinary sediments, 320 phosphate in urinary sediments, 321 in milk, 193 sulphate in urinary sediments, 322 Calculi, biliary, 120 urinary, 340 calcium carbonate in, 341 oxalate in, 341 cholesterin in, 342 cystin in, 342 fibrin in, 342 indigo in, 342 phosphates in, 341 uric acid and urates in, 341 urostealiths in, 342 xanthin in, 342 Cane sugar (see saccharose, p. 19) Caproic acid, 187 Carbamic acid, 150 Carbohydrates, 1 classification of, 1 composition of, 1, 2 review of, 29 scheme for detection of, 31 variation in solubility of, 2 Carbonates in urine, 238, 280 Carbon monoxide haemoglobin, 171 Carnin, 207 Carnosin, 207, 211 Cartilage, 202 constituents of, 202 experiments on, 203 Hopkins-Cole reaction on, 203 loosely combined sulphur in, 203 Millon's reaction on, 203 preparation of gelatin from, 203 solubility of, 203 xanthoproteic test on, 203 Casein, 188 soluble, 188 calcium, 188 quantitative determination of, 382 Caseinogen, 146, 187, 190 action of rennin upon, 188 biuret test on, 193 INDEX. 4OI aogen, Millon'a tesl on, 193 precipitation oi preparation of, 19a solubility of, 193 tesl for loosely combined sulphur in, 193 test for phosphorus in, 193 .!-' x . 333 blood, 334 epithelial, 334 fatty, 334 granular, 333 hyaline. pus, 336 waxj Casts in urinary sediments, 328, 332 Cellulose, 2, 28 action of Schweitzer's reagent on, 29 hydrolysis of, 29 iodine test on, 28 solubility of, 28 Cellulose group, 2 Cerebrin, 220 experiments on, 224 hydrolysis of, 224 microscopical examination of, 224 preparation of, 224 solubility of, 224 Charcot-Leyden crystals, 142 form of, 141 Chlorides in urine, 238, 274 detection of, 275 quantitative determination of, 371 Cholecyanin, 120 Cholera-red reaction for indol. 137 Cholesterin. 121, 124, 222 crystalline form of, 125 formula for, 222 iodine-sulphuric acid test for, 124, 223 isolation of, from biliary calculi, 124 Liebermann-Burchard test for, 124, 224 occurrence of, in urinary sedi- ments. 319, 325 preparation of, from nervous tis- sue, 223 Salkowski's test for, 125, 224 Schiffs reaction for, 125, 224 tests for, 124. 223 Choletelin, 118 Cholin, 221 Chondrigen. 62 27 ( hondroalbumoid, 4.*. 202 1 Ihondromucoid, 6a, 202 ( Ihondroitin, 202 Chondroitia sulphuric acid, 202, 237, 259 Chondrosin, 202 Cipollina's test, 6, 284 Coagulated proteids, 60 biuret test on, 6l formation of, 60 Hopkins-Cole reaction on, 61 Millon's reaction on, 61 solubility of, 61 xanthoproteic reaction on, 61 Coagulation of proteids, 49, 50, 60 changes in composition dur- ing, 60 fractional, 60 Coagulation temperature of proteids, 50 apparatus used in determin- ing, 50 method employed in deter- mining, 50 Collagen, 43, 62, 198, 199 experiments on, 199 hydrolysis of, 200 percentage of, in ligament, 201 in tendon, 198 production of gelatin from, 200 solubility of, 199 Colostrum, 190 microscopical appearance of. 188 Combined hydrochloric acid, 84, 87 tests for, 87-90 Compound proteids, 43, 61 classes of, 61 experiments on, 199 nomenclature of, 61 occurrence of, 62 Compound test for lactose in urine, 3i3 Congealing-point of fat, 105 Congo red, as indicator, 88 preparation of, 88 Conjugate glycuronates, 9, 282, 310 fermentation-reduction test for, 310 ToIIens' reaction on, 310 Connective tissue, 197 Creatin, 150 crystalline form of, 209 formula for, 210 separation of, from meat extract, 215 Creatinin, q, 207. 237, 250 crystalline form of, 251 40: INDEX. Creatinin, daily excretion of. 250 experiments on, 251 formula for. 210, 250 Jaffe's reaction for, 253 quantitative determination of, 369 Salkowski's test for, 253 separation of, from urine, 251 Weyl's test for, J52 Creatinin-zinc chloride, formation of, 250, 252 Cresol, para, 65, 129, 132 tests for, 138 Cryoscopy, 232 Cul-de-sac, 84 Cyanuric acid, 241 formula for. 241 Cylindroids in urinary sediments, 337 Cystin, 65. 76. 237, 324 crystalline form of, 76, 325 detection of, 325 formula for, 76 in urinary sediments, 324 Dare's hjemoglobinometer, 178 description of, 178 determination of haemo- globin by. 179 Darmstadter's method for determina- tion of /3-oxybutyric acid, 375 Decomposition products of proteids, 65 crystalline forms of, 68-79 experiments on, 80 isolation of, 80 Delusive feeding experiments, 83 Detection of preservatives in milk, 195 boric acid and borates, 196 formaldehyde, 195 hydrogen peroxide, 196 salicylic acid and salicylates, 195 Deuteroproteose, 43 Dextrin, 2, 27 achroo-, 22, 35 erythro-. 22, 27, 35 action of tannic acid on, 28 diffusibility of, 28 Fehling's test on, 27 hydrolysis of, 27 iodine test on, 27 solubility of, 27 Dextrosazon, crystalline form of, Plate III, opposite p. 5. Dextrose, 1. 3. 4, 282 Allen's modification of Fehling's test for, 287 Barfoed's test for, 11, 289 Boettger's test on, 9, 288 Dextrose, Cipollina's test on, 6, 284 diffusibility of, 6 Fehling's test on, 8, 2S6 fermentation of. 10, 288 iodine test on. 6 Molisch's reaction on, 4 Moore's test on, 7 Xylander's test on, 9, 288 phenylhydrazin test on, 5, 283 quantitative determination of, 345 reduction tests on, 7, 284 solubility of, 4 Trommer's test on, 8, 285 Diacetic acid, 282, 306 Arnold-Lipliawsky test for, 308 formula for, 306 Gerhardt's test for, 307 a-e-di-amino-caproic acid, 78 Diastase, 18 Diazo-benzene-sulphonic acid. 317 reagent, preparation of, 316 Diazo reaction (Ehrlich's). 316 Differentiation between pepsin and pepsinogen. 92 Digestion, gastric. 83 pancreatic, 106 salivary, 32 Di-methyl-amino-azobenzene (see Top- fer's reagent), 88 Disaccharides, 17 classification of, 2 Doremus-Hinds ureometer, 355 Drying method for determination of total solids in urine, 379 Earthy phosphates in urine, 275 quantitative determination of, 367 Edestin, 53, 54 coagulation of, 54 crystalline forms of, 54 microscopical examination of, 54 Millon's test on, 54 preparation of, 53 solubility of, 54 tests on crystallized. 54 filtrate of, 54 Ehrlich's diazo-benzene-sulphonic acid reagent, preparation of, 316 Ehrlich's diazo reaction, 316 Einhorn's saccharometer, 10 Elastin, 43, 62, 198 201 experiments on, 201 preparation of. 201 solubility of, 201, 202 Electrical conductivity of urine, 235 Enterokinase. 108 403 Enzymes, 3 \. 238 classification ol uanin, 238 Episarkin, Epithelial cells in urinary sediments, casts in urinary sediments, 328, 33*i Epithelial tissue, 107 experiments on, 1 • j r Erythrocytes, [48, 150, 151 counting the, 180 diameter af, 151 form of, 1 so influence of osmotic pressure on, [62 in urinary sediments, .12%, 337 number of, per cubic mm., 15 1 of different species, 1 5 1 stroma of, 151, 156 variation in number of, 151 Erythro-dextrin, 22, 27. 34, 3 s Esbach*s albuminometer, 345 method for determination of albumin, .144 reagent, preparation of, 344 Ester, definition of, 97 hydrochloric acid. 165 sulphuric acid, 165 Ethereal sulphates, 272, 273 quantitative determination of. 363 Ethereal sulphuric acid. 129, 237, 253 Euglobulin, 149 Extractives of muscular tissue, 207 nitrogenous, 207 non-nitrogenous. 207 Fats, 96 absorption of, 99 apparatus for determination of melting-point of, 103 boiling-point of, 98 chemical composition of, 97 congealing-point of, 105 crystallization of, 98, 101 digestion of, 99 emulsification of, 99, 101 experiments on, 100 formation of acrolein from, 100 hydrolysis of, 97 in milk, 187, 194 in urine. 2S2, 312 melting-point of, 98, 104 nomenclature of. 98 occurrence of. 96, 98 permanent emulsions of, 99, 101 quantitative determination of, in milk. 380 Fats, reaction of, i"" nification of, '17. 102, solubility of, 98, transitory emulsions of, 'n, i"i Fatty acid. 97, 99, 1 oa Fatty casts in urinary sediments, 328, 3.U Fatty degeneration, 99 1 39 blood in, 142 daily excretion of, 139 detection of albumin and globulin in, 147 Kill- acids in, 146 bilirubin in, 145 caseinogen in, 146 cholestcrin in, 143 hydrobilirubin in, 145 inorganic constituents of, ' 17 nucleo pr'oteid in. 146 proteose and peptone in, 147 experiments on. 142 form and consistency of, 141 macroscopic constituents of. 141 microscopic constituents of, 14! odor of. 140 pigment of, 140 reaction of. 141 plitting enzymes, 86, 97, 99, 109 Fehling's method for determination of dextrose. 345 solution, preparation of, 8, 2S6 test, 8, 286 Allen's modification of. 287 Ferments, classification of. 34 Fermentation test, 10, 288 Fermentation method for determina- tion of dextrose, 3 (.8 Fermentation-reduction test for con- jugate glycuronatcs. 310 Ferric chloride test for sulphocyanide in saliva. 37 Fibrin, 1411, 168, 282 in urinary sediments, 328, 339 separation of, from blood, 168 solubility of, 168 Fibrin ferment, 150. 158 Fibrinogen, 1 4.8, 149, 158 Fischer apparatus, photograph of, 67 1 'leischl's haemometer, 1 74 description of. 174 determination of haemoglobin by. 175 Fleischl-Miescher ha?mometer, 176 Fluorides in urine. 23S. 280 Fly-maggots, experiments on, 100 Folin's method of determination of acidity of urine. 376 404 INDEX. Folin's method of determination of acidity of ammonia. 357 creatinin, 369 ethereal sulphates, 363 inorganic sulphates. 362 total sulphates, 361 urea, 355 Folin-Shaffer method for determina- tion of uric acid, 349 Foreign substances in urinary sedi- ment. 328, 339 Form elements of blood, 148 Formic acid, 238, 265 Fractional coagulation of proteids, 60 Free hydrochloric acid, 84, 87 tests for, 87-90 Freezing-point of bile, 117 blood, 148 milk, 187 pancreatic juice, 107 urine, 232 Fuchsin-frog experiment, 213 Fundus glands, 83 Furfurol solution, preparation of, 122 Fusion mixture, preparation of, 52 Galactose. 2, 15 experiments on, 15 Gallic acid test for formaldehyde, 195 Gastric digestion, 83 conditions essential for, 85, 91 general experiments on, 91 influence of bile on, 93 influence of different tem- peratures on, 91 most favorable acidity for, 91 power of different acids in, 92 products of, 85, 87 Gastric fistula, 83 Gastric juice, 83-86 acidity of, 84, 85 artificial, preparation of, 86 composition of, 84 enzymes of, 84 quantitative analysis of, 383 quantity of. 83 reaction of, 84 specific gravity of. 84 lactic acid in, tests for, 94 Gelatin, 43, 62, 68, 198, 200 coagulation of, 200 experiments on, 200 formation of, 200 Hopkins-Cole reaction on, 200 Millon's reaction on, 200 precipitation of. by alcohol, 201 alkaloidal reagents, 200 metallic salts, 200 Gelatin, precipitation of, by mineral acids, 200 preparation of, from cartilage, 203 from collagen, 200 salting-out of, 200 solubility of, 200 Gerhardt's test for diacetic acid, 307 Gerhardt's test for urobilin. 268 Gliadin, 68, 69, 71, 73, 74, 76 Globulin, 43, 53 experiments on, 53 preparation of, 53 serum, 43, 148, 149, 282 in urine, 282, 289. 293 tests for, 293 Glucoproteid, 43, 61, 199 experiments on, .199 hydrolysis of, 199 Glucose (see Dextrose, p. 3) Glutamic acid, 65, 70 formula for, 70 Glutenin, 70, 72 Glycerin, 97, 99, 104 borax fusion test on, 104 experiments on, 104 formula for, 97, 100 Glycerin extract of pig's stomach, preparation of, 87 Glycerophosphoric acid, 221, 238, 266 Glycocholic acid, 117 Glycocholic acid group, 117 Glycocoll, 65, 71, 117 formula for, 71, 117 preparation of, 127 Glycocoll ester hydrochloride, crys- talline form of, 72 Glycogen, 2, 26, 207, 208 experiments on, 215 hydrolysis of, 215 influence of saliva on, 215 iodine test on, 215 preparation of. 215 Glycosuria, alimentary, 3 Glycuronates, conjugate, 9, 282, 310 Glycuronic acid, 16 Glyoxylic acid, 45 formula for, 45 Gmelin's test for bile pigments, 121, 300 Rosenbach's modification of, 121, 300 Granular casts in urinary sediments, 328, 333 Granulose, 22 Green stools, cause of, 140 Guaiac solution, preparation of, 163 Guaiac test on blood, 158, 163 milk, 191 IX'iKX. 40 5 Guaiac tesl on pus, 3.*' Guanidin-a-amino-valerianic acid, 79 ( rtianin, -■ i . tliiius and vegetable mucilage group i>\ carbohydrati Gunning's iodoform test i"r acetone, Gunzberg's reagent, as indicator, 88 preparation of, 88 Hsematin, 156 acid-. 173 alkali-. 172 preparation of, 165 reduced .alkali-. 173 Hsematoidin, 1 19, 142 crystalline form of, 119. 140 in urinary sediments Hematuria, 297 toporphyrin, [56, 17.?. 174, 282 iti urine. 282, 312 Ila-niin crystals, form of, 164 test, 163 Haemochromogen, 156, 173 Haemoglobin, 151, 156, 170, 282 carbon monoxide, 156, 171 decomposition of, 156 diffusion of, 162 met, 156, 172 oxy, 156, 169 quantitative determination of, 174. 1/8 reduced, 1 70 Hemoglobinuria, 297 Hammerschlag's method for deter- mination of specific gravity of blood. 160 Hammarsten's reaction, ijr. 300 reagent, preparation of, 121, 300 Heintz method for determination of uric acid, 350 Heller's test for blood in urine, 298 Heller-Teichmann reaction for blood in urine. 298 Heller's ring test for proteid. 47, 290 Hemi-cedulose. 2 Herter's naphthaquinone reaction for indol and skatol. 136. 137 Heteroproteose. 43 Heteroxanthin, 238 Hexoses, 1, 3 Hippuric acid. 127, 128, 237, 25s crystalline form of. 256 experiments on, 127, 256 formula for, 128, 255 in urinary sediments. 325 melting-point of, 257 separation of, from urine. 256' solubility of. 257 Hippuric acid, sublimation of, 257 synthesis of, 1 27 llistidin. 65, 79, 107 hydrochloride, crystalline form of, 79 tion for tyrosit ;entisic acid. •>, 262 formula for, 262 I [opkins Cole reaction, 45 on solutions, 45 on solids, 51 Hopkins-Cole reagent, preparation of, 45 Hufner*s urea apparatus. 354 Human fat, composition of Huppert's reaction for bile pigments, 1 -• 1 . 300 Hurthle's experiment, 219 ll>. lime casts in urinary sediments, 328, 332 Hydrobilirubin, detection of, in feces, 1 45 extraction of, 145 Hydrochloric acid test for formalde- hyde, 195 Hydrogen peroxide in urine. 238, 280 detection of, in milk, 196 1 [ydrolysis of cellulose, 29 cerebrin, 224 collagen, 200 dextrin, 27 glycogen, 215 inulin, 26 saccharose, 20 starch. 24 Hyperacidity, 84 Hypoacidity, 84 Hypobromite solution, preparation of, 35i Hypoxanthin. 207, 217, 238 formula for, 21 1 Hypoxanthin silver nitrate, crystal- line form of, 216 Indican. 120, 130, 254 formula for, 130. 254 TafTe's test for. 255 Obermayer's test for. 25; origin of, 129, 130 Indigo-blue, 130, 255 formula for, 130, 255 Indigo in urinary sediments, 318, 327 Indol. 65, 77- 129, 132, MO formula for. 129 origin of, 120. 140 tests for, 136 Indol-amino-propionic acid. 77 Indoxyt, 129 formula for. 120 406 INDEX. lndoxyl, origin of, 129, 130 potassium sulphate (see Indican, pp. 129-130, 254.) Indoxyl-sulphuric acid. 129, 237, 253 formula for, 129 Inorganic physiological constituents of urine, 238 Inosinic acid, 207, 211 Inosit, 1, 207, 282 in urine, 282, 314 Inulase, 25 Inulin, 2, 23 action of amylolytic enzymes on, 25 Fehling's test on. 26 hydrolysis of, 26 iodine test on, 26 reducing power of, 25 solubility of, 25, 26 sources of, 25 Invertin, 20 Inverting enzymes, 34 Iodine test, 24 Iodine-sulphuric acid test for choles- terin, 124, 223 Iodoform test for alcohol, 21 Iron in blood, 156, 161 detection of, 161 in bone ash, 204, 205 detection of, 204, 205 Iron in proteid, 42 Iron in urine, 238, 280 detection of, 280 Isomaltose, 2, 18 Jaffe's reaction for creatinin, 253 Jaffe's test for indican, 255 v. Jaksch-Pollak reaction for mel- anin, 316 Jolles' reaction for proteid, 48, 292 reagent, preparation of, 48, 292 Juice, gastric, 83-86 pancreatic, 106-109 Kephalin, 220, 222 Kephyr, 19 Keratin, 43, 62, 197 experiments on, 197 solubility of, 197 sources of, 197 sulphur content of, 197 Ketone, 1, 7 Ketose, 1 Kjeldahl method for determination of nitrogen, 359 Knop-Hiifner hypobromite method for determination of urea, 351, 353 Koumyss, 19 Kulz's test for /3-oxybutyric acid, 309 Kynurenic acid, 23-, 262 formula for, 262 isolation of, from urine, 263 Lactalbumin, 187, 190 quantitative determination of, 383 Lactic acid, 19, 84, 207 ferric chloride test for, 94 in muscular tissue, 207, 208 in stomach contents, 94, 95 tests for, 94 Uffelmann's test for, 94 Lacto-globulin, 187, 190 Lactometer, determination of specific gravity of milk by, 380 Lactosazon, crystalline form of, Plate III, opposite p. 5 Lactose, 2, 19, 187, 189, 313 experiments on, 19 fermentation of, 19 in urine, 282, 313 quantitative determination of, 383 Lffivo-a-prolin, 74 Lxvulose, 3, 14 in urine, 282, 313 methyl-phenylhydrazin test for, IS phenylhydrazin test on, 15 Seliwanoff's reaction for, 15 Laiose in urine, 282, 315 Laked blood, 148, 159 Laky blood, 161 Laurie acid, 187 Laurin, 98 Lecithin, 116, 117, 150, 220 acrolein test on, 223 decomposition of, 220 experiments on, 223 formula for, 221 microscopical examination of, 223 osmic acid test on, 223 preparation of, 222 test for phosphorus in, 223 Legal's reaction for indol, 137 Legal's test for acetone, 305 Legumin, 78 Leucin, 65, 107. 150 crystalline form of impure, 326 pure, 69 experiments on, 82 formula for, 69 in urinary sediments, 318, 326 microscopical examination of. 82 separation of, from tyrosin, 81 solubility of, 82 sublimation of, 82 INDEX. 407 11 ytes, 1 (8, 1 56 tinting the, 180, (84 number of, per cubic mm., 156 size of, 156 variation in number of, 156, 157 I ,euco< vtosis, 1 56 urn, 2, 37 Lieben'a test for acetone, 305 Lieberkuhn's jelly (see Alkali al- buminate, p. 57 I Liebermann-Burchard test for choles- terin, 1 -• t. aa 1 Lieberrnann'a reaction, 46 I .ipase, 84. 86- Lipoids of nervous tissue, 220, 222 Lipolytic enzymes, 34 "Litmus-milk" tesl for steapsin, 115 Lugol's solution, preparation of, 304 Lysin, 65, 78, 107 Lysin picrate, crystalline form of, ~^ Magnesia mixture, preparation of, 270 Magnesium in urine, 238 phosphate in urinary sediments, 327 Maltase, 18 Malto-dextrin, 35 Maltosazon, crystalline form of, Plate III, opposite p. 5 Maltese, 2. 18 experiments on, 18 Marshall's urea apparatus, 352 Melanin in urine, 282, 315 urinary sediments, 327 Melting-point apparatus. 103 of fats, determination of, 104 Messinger-Huppert method for de- termination of acetone, 374 Methaemoglobin, 156, 172 Methyl-mercaptan, 120. 131 Methyl-pentose (see Rhamnose, p. 2) i-methylxanthin, 238 Micro-organisms in urinary sedi- ments, 328. 339 Milk. 187 detection of calcium phosphate in. 193 lactose in, 194 preservatives in, 195 difference between human and Cow's, 188 experiments on, 190 formation of film on, 187, 191 freezing-point of, 187 guaiac test on, 19.1 influence of rennin on, 188, 192 isolation of fat from, 194 Mill., microscopical appearance of, IMS. [90 preparation of ca rom, 19a pi "i" rtii 1 inogen of, 190 quantitative analysis of, 380 iction of, 187, 190 separation of coagulable proteids of. I93 Specific i;ra\ ity of, 187, 191 Millon 11, 44 reagent, preparation of, 44 Mohr's method lor determination of chlorides, ;;i Molisch's reaction, 4 Molybdic solution, preparation of, 37 Monosaccharides, 1, 2, 3 classification of, 1 Morner-Sjoqvist-Folin method for de termination of urea. 356 Morner's reagent, preparation of, 82 test for tyrosin, 82 Motor and functional activities of the stomach, 93 Mucin, 33, 36, 43, 61, 62 biuret test on, 36 hydrolysis of, 37 isolation of, from saliva, 36 Millon's reaction on, 36 Mucoid, 43, 61, 62, 198 experiments on, 199 hydrolysis of, 199 in urine. 264. 296 preparation of, from tendon, 199 Murexid test, 249 Muscle plasma, 206, 212, 213 formation of myosin clot in, 206 fractional coagulation of, 206 preparation of 212, 213 reaction of. 212 Muscular tissue, 206 commercial extracts of, 210 experiments on " dead," 214 " living," 212 extractives of, 207. 215 formulas of nitrogenous ex- tractives of, 210 glycogen in. 207, 208 lactic acid in, 207, 208 pigment of, 210 preparation of glycogen from, 215 muscle plasma from, 2\ 2. 213 proteids of, 206 reaction of living, 208 separation of extractives from. 215 406 INDEX. Myoh r ■ ■ ■ • Pettenkoper's test for bile acids, uj, 301 Mylius's modifica- tion Of, 122. 301 Neukomm's modifi- cation of, 122, 301 Phenaceturic acid, 238, 266 Phenol, 65, 129 tests for, 138 Phenolphtnalein as indicator, 89 preparation of, 89 Phenol-sulphuric acid, 237, 253 Phenyl-a-amino-propionic acid, 73 Phenylalanin, 65, 73 Phenyldextrosazon, 5 crystalline form of, Plate III. op- posite p. 5 Phenylhydrazin, 5 acetate solution, preparation of, 5 mixture, preparation of, 5 reaction, 5 Cipollina's modification of, 6 Phenyllactasazon. crystalline form of, Plate III. opposite p. 5. Phenylmaltosazon crystalline form of, Plate III. opposite p. 5 Phosphates in urine, 238. 275 detection of, 278 experiments on. 277 quantitative determination of, 367 Phosphocarnic acid. 207. 211. 238, 266 Phospho-proteid, 43, 190 Phosphorized compounds in urine. 238 Physiological constituents of urine, 237 Pigments of urine, 238, 266 Pine wood test for indol. 137 Piria's test for tyrosin. 82 Polariscope, use of, in detection of conjugate glycuronates, 310 in determination of 13. 340 /3-oxybutyric acid, 376 Polysaccharides, 2. 21 410 INDEX. Polysaccharides, classification of, 2 properties of, 21 Posner's modification of biuret test, 46 Potassium in urine, 238 Potassium indoxyl-sulphate (see Indi- can, pp. 129, 130, 254) formula for, 130, 254 origin of, 129, 130 tests for, 255 Primary proteoses, 58, 59 Prolin, 65, 74 crystalline form of laevo-a-, 74 crystalline form of copper salt of, 75 Prosecretin, 106 Protagon, 220, 221 preparation of, 222 Proteids, 42, 282 acetic acid and potassium ferro- cyanide test for, 49 action of alkaloidal reagents on, 47 action of metallic salts on, 47 mineral acids, alkalis and organic acids on, 47 Adamkiewicz reaction on, 45 biuret test on, 45 chart for use in review of, 63 chemical composition of, 42 classification of, 42 coagulation or boiling test for, 49 color reactions of, 43 decomposition of, 65 by hydrolysis, 65 by oxidation, 65 products of, 65 experiments on, 80 separation of, 80 study of, 65, 80 derived simple, 43, 55 formation of fat from, 99 formulas of, 42 Heller's ring test on, 47 importance of, to life, 42 Hopkins-Cole reaction on, 45 in urine, 282, 289 tests for, 290 Liebermann's reaction on, 46 Millon's reaction on, 44 molecular weights of, 42 native simple, 42 Posner's reaction on, 46 precipitation of, by alcohol, 51 alkaloidal reagents, 47 metallic salts, 47 mineral acids, 47 precipitation reactions of, 46 Proteids, quantitative determination of, in milk, 382 review of, 63 salting-out experiments on, 49 scheme for separation of, 64 xanthoproteic reaction on, 44 Proteids, coagulated, 60 biuret test on, 61 formation of, 60 Hopkins-Cole reaction on, 61 Millon's reaction on, 61 solubility of, 60, 61 xanthoproteic reaction on, 61 Proteid-coagulating enzymes, 34 Proteids, compound, 43, 61 classes of, 43, 61 experiments on, 199 nomenclature of, 61 occurrence of, 62 Proteid-cystin, 7y Proteids of milk, 187, 190 quantitative determination of, 382 Proteoids, 43, 62 Proteolytic enzymes, 34 Proteolysis, peptic, 85 tryptic, 86, 107 Proteose, 43, 65, 107, 282, 294 v. Aldor's method for detection of, 296 biuret test on, 59 coagulation test on, 59 deutero, 43, 57, 282 differentiation of, from peptone, 58 experiments on, 59, 295 hetero, 43, 57, 282 in urine, 282, 295 tests for, 295 potassium ferrocyanide and ace- tic acid test on, 59 powder, preparation of, 59 precipitation of, by nitric acid, 59 by picric acid, 59 by potassio mercuric iodide, 59 by trichloracetic acid, 59 primary, 59 proto, 43, 57 Schulte's method for detection of, 295 secondary, 59 separation of, from peptones, 59 Protoproteose, 43, 57 Proteoses and peptones, 57 separation of, 59 tests on, 59 Proteose-peptone, 58 INDEX. 411 ne peptone, coagulation u si on, 58 experiment! on, 5s Millon'a reaction on, 58 precipitation of, by nitric acid, 58 by pi< ric acid, 5K Prothrombin, 157, 158 Pseudo globulin, [49, 207 aines and leucomaines in urine, 338, 369 Ptyalin, 33, 34 activity of, in stomach, 35 inhibition of acth ity of, 35 nature of action of, 34 products of action of, 34 Purdy's method for determination of dextrose, 347 solution, preparation of, 347 Turin bases, 62, 238, 269 in urine quantitative determina- tion of, 377 Pus casts in urinary sediments, 328, 336 Pus cells in urinary sediments, 328, 329 Putrefaction, indican as an index of, 129 Putrefaction mixture, preparation of a, 130 Putrefaction products, 129 experiments on, 130 most important, 129 tests for, 136 Pyloric glands, 83 Pyrocatechin-sulphuric acid, 237, 253 a-pyrrolidin-carboxylic acid (see Pro- lin, pp. 65, 67) Qualitative analysis of the products of salivary digestion, 41 stomach contents, 95 Quantitative analysis of blood, 386 of gastric juice, 383 of milk, 380 of urine, 344 Quantitative determination of am- monia in urine, 357 acetone in urine. 374 acidity of urine, 376 ash of milk, 382 casein of milk, 382 chlorides in urine, 371 creatinin, 369 dextrose in urine, 345 fat in milk, 380 lactalbumin in milk, 383 lactose in milk, 383 nitrogen in urine, 359 oxalic acid in urine, 378 j8-oxybutyric acid in urine, 3/5 Quantitative determination of phos phoniS in urine. proteid in milk, 382 proteid in urine, 344 purin bases in urine, 377 sulphur in urine, .t'.i total solids in milk. 381 total solids in urin< 1111 .1 111 urine. 33 1 uric acid in urine, Quevcnnc lactometer, determination of specific gravity of milk by, 380 Raffinose, 2, 21 Reaction of tile urine, 228 Reduced alkali -lnematin, 173 Reduced haemoglobin, 170 Reichet's method for crystallization of oxyhemoglobin, 167 Remont's method for detection of salicylic acid and salicylates, 195 Rennin, 84, 86 action of, upon caseinogen, 86, 188 experiments on, 93, 95 influence of, upon milk, 93, 192 in gastric juice, absence of, 86 nature of action of, 86 occurrence of, 86 Rennin, pancreatic, 107, 109 experiments on. 115 Reticulin. 43 Reynolds-Gunning test for acetone, 306 Rhaninose. 2, 17 Ring test for urobilin. 269 Robin's reaction for urorosein, 316 Robert's ring test for proteid, 48, 291 reagent, preparation of, 48, 291 Rubner's test for lactose in urine, 313 Saccharide group, 2 Saccharose, 2. 19 experiments on, 20 inversion of, 20 production of alcohol by fermen- tation of, 20 Saliva. 32 alkalinity of, 33 amount of, 33 bacteria in, 35 biuret test on. 36 calcium in. 33, 37 chlorides in, 37 constituents of, 33 digestion of dry starch by, 38 digestion of inulin by, j8 digestion of starch paste by, 38 enzyme contained in. 34 412 INDEX. Saliva, excretion of potassium iodide in, 40 inorganic matter in, tests for, 37 Millon's reaction on, 36 mucin from, preparation of, 36 nitrites in, test for, 37 phosphates in, test for, 37 potassium sulphocyanide in, 37 reaction of, 36 secretion of, 32 sulphocyanide in, tests for, 37 specific gravity of, 36 sulphates in, test for, 37 Salivary digestion, 32 influence of acids and alka- lis on, 35, 39 dilution on, 39 metallic salts on, 40 temperature on, 39 nature of action of acids and alkalis on, 40 qualitative analysis of pro- ducts of, 41 Salivary digestion in stomach, 35 Salivary glands, 32 Salivary stimuli, 32 Salkowski-Autenrieth-Barth method for determination of oxalic acid in urine, 378 Salkowski's method for determination of purin bases, 377 Salkowski's test for cholesterin, 125 creatinin, 253 Salted plasma, preparation of, 167 Salting-out experiments on proteids, 49 Sarcolactic acid, 208 Schalfijew's method for preparation of hffimin, 163 Scheme for analysis of biliary calculi, 124 bone ash, 205 stomach contents, 95 urinary calculi, 343 separation of carbohydrates, 31 proteids, 64 Scherer's coagulation method for de- termination of albumin in urine, 344 Schiff's reaction for cholesterin, 125, 224 uric acid, 249 Schulte's method for detection of proteose in urine, 295 Schweitzer's reagent, action of, on cellulose, 29 preparation of, 29 Secondary proteoses, 59 Secretin, 106 Serin, 65, 74 crystalline form of, 75 formula for, 75 Serum albumin, 42, 52, 148, 149, 282 289 in urine, 282, 289 tests for, 290 Serum globulin, 43, 148, 149, 282 in urine, 282, 289, 293 tests for, 293 Sherman's compressed oxygen method for determination of total sulphur in urine, 365 Sherrington's solution, preparation of, 181 Silicates in urine, 238, 280 Skatol, 65, 129, 132, 140 tests for, 137 Skatol-carbonic acid, 135, 136 test for, 138 Skatol-sulphuric acid, 237, 253 Skeletins, 63 Smith's test for bile pigments, 122, 301 Sodium and potassium in urine, 238, 279 Sodium alizarin sulphonate as indi- cator, 90 preparation of, 90 Sodium hydroxide and potassium ni- trate fusion method for determina- tion of total sulphur and phos- phorus in urine, 364, 368 Sodium hypobromite solution, prepar- ation of, 351 Solera's reaction for detection of sulphocyanide in saliva, 38 test paper, preparation of, 38 Soxhlet apparatus for extraction of fat, 380 Soxhlet lactometer, determination of specific gravity of milk by, 380 Spectroscope, use of, in detection of blood, 169 Spermatozoa in urinary sediments, 328, 338 microscopical appearance of hu- man, 338 Spiegler's ring test for proteid, 48, 291 reagent, preparation of, 48, 291 Standard ammonium sulphocyanide solution, preparation of, 373 argentic nitrate solution, prepar- ation of, 371 uranium acetate solution, prepar- ation of, 367 Starch, 2, 22 action of alcohol on iodide of, 24 INDEX. 41 j Starch, action of alkali on iodide of, heal "ii iodide of, 24 dry, digestion of, by amylopsin, 1 09, 1 1 1 dry. digestion of, by ptyalin, 38 experiments on, -•-■ iodine test for, -( microscopical characteristics of, 22 microscopical examination of, 24 potato, preparation of, 22 solubility of, -4 various forms .of. 23 Starch group, 2 Starch paste, action of tannic acid on, 25 diffusibility of, 25 digestion of, by amylopsin, 108, 112 by ptyalin. 34. 38 Fehling's test on, 24 hydrolysis of, 24 preparation of, 24 Steapsin, 107, 100 experiments on. 1 1 5 ethyl-hutyrate test for, 115 "litmus-milk" test for, 115 Stearic acid, 221 Stearin. 98, 187 Stellar phosphate. 19.3, 321 Stokes' reagent, action of. 170 preparation of. 170 Stomach, motor and functional ac- tivities of, 93 Stomach contents, lactic acid in, tests for, 94 qualitative analysis of, 95 Stone-cystin, 77 Sublingual glands, characteristics of saliva secreted by, 32 Submaxilary glands, characteristics of saliva secreted by, 32 Sucrose (see Saccharose, p. 19) Sulphanilic acid. 316. 317 Sulphates in saliva, test for. 37 Sulphates in urine. 238, 271 experiments on. 273 ethereal. 272. 273 quantitative determina- tion of. 363 inorganic. 272 quantitative determina- tion of, 362 total, quantitative determina- tion of. 361 Sulphocyanide in saliva, significance of. 33 ferric chloride test for. 37 Solera's reaction for. 38 Sulphocyanides in urine, 22,7 Sulphur in proteid, 52 loosely combined, test for, 52 in urine, quantitative determina- tion of, 361 aeid. 52 lead blackening, ?-• mercaptan, 52 neutral, 52 oxidized, 52 unoxidized, 52 Tallow, bayberry, saponification of, 102 Tallquist'a haemoglobin scale. mination of haemoglobin by, 180 Tannic acid, influence of, on dextrin, 28 on starch, 25 Tanret's reagent, preparation of, 48, 293 Tanret's test, 48, 293 Tartar, formation of, 33 Taurin, 117, 207. 211 formula for, 117. 211 preparation of, 125 Taurin derivatives, 237 Taurocholic acid, 117 group, 117 Teichmann's crystals, form of Csee hsmin crystals, p. 164) Tendomucoid. 43. 61. 62, 199 biuret test on, 199 chemical composition of, 62 hydrolysis of, 199 loosely combined sulphur in. test for. 199 preparation of, 199 solubility of. 199 Thoma-Zeiss hxmocytometer, 180 Thrombin. 157, 158 Tincture of iodine, preparation of, 394 Tissue, adipose, experiments on, 100, 205 connective, 197 white fibrous, 197 composition of. 198 experiments on. 198 yellow elastic. 201 composition of, 201 experiments on, 201 epithelial. 197 experiments on. tor muscular. 206 experiments on, 212 nervous. 220 experiments on, 222 osseous. 204 experiments on, 204 414 INDEX. Tissue debris in urinary sediments, 328, 339 Toison's solution, preparation of, 181 Tollens' reaction on conjugate gly- curonates, 310 galactose, 15 arabinose, 16 Topfer's method for quantitative analysis of gastric juice, 383 Topfer's reagent, as indicator, 88 preparation of, 88 Total solids of milk, quantitative de- termination of, 381 of urine, quantitative deter- mination of, 379 Total sulphur of urine, quantitative determination of, 363, 364, 365 phosphorus of urine, quantitative determination of, 368 Tri-butyrin. 187 Tri-olein. 98, 187 Tri-palmitin, 98, 187 Tri-stearin, 98, 187 Trichloracetic acid, precipitation of proteid by, 47 Trioses. 1 Triple phosphate, 278 crystalline form of, 278 formation of, 277 Trisaccharides, 2, 21 Trommer's test, 8, 285 Tropreolin 00, as indicator, 89 preparation of, 89 Trypsin, 107 action of, upon proteids, 107 experiments on, 111 influence of alkalis and mineral acids upon, 107 nature of, 107 pure, preparation of, 107 Trypsinogen, 107, 108 activation of, 108 Tryptic digestion, 107 influence of bile on, 112 metallic salts on, 112 most favorable reaction for, in temperature for, in products of, 107, no Tryptic proteolysis, 86, 107 Tryptophan, 45, 65, 77, 107, in bromine water test for, no formula for, 77 group in the proteid molecule, 45 Hopkins-Cole reaction for, 45 occurrence of, as a decomposi- tion product of proteid, 65, 77 occurrence of, as an end-product of pancreatic digestion, 107, 1 1 1 Tyrosin, 65, 66, 107 crystalline form of, 68 experiments on, 81 formula for, 66 Hoffmann's reaction for, 82 in urinary sediments, 319, 326 microscopical examination of, 81 Morner's test for, 82 occurrence of, 67 Piria's test for, 82 salts of, 68 separation of, from leucin, 68, 81 solubility of, 82 sublimation of, 82 Tyrosin-sulphuric acid, 82 v. Udransky's test for bile acids, 123, 302 Uffelmann's reagent, preparation of, 94 Uffelmann's reaction for lactic acid, 94 Unknown substances in urine, 282, 316 Uranium acetate method for deter- mination of total phosphates in urine, 367 Urate, ammonium, crystalline form of, Plate VI, opposite p. 324 sodium, crystalline form of, 324 Urates in urinary sediments, 319, 323 Urea, 237, 239 crystalline form of, 239 decomposition of, by sodium hypobromite, 241 excretion of, 240 experiments on,- 242 formation of, 240 formula for, 239 furfurol test for, 245 isolation of, from the urine, 242 melting-point of, 243 quantitative determination of, 35i Urea nitrate, 241 crystalline form of, 242 formula for, 241 oxalate, 241 crystalline form of, 244 formula for, 241 Urethral filaments in urinary sedi- ments, 328, 338 Uric acid, 9, 207, 2^7, 245 crystalline form of pure, 249 endogenous, 246 exogenous, 246 experiments on, 248 formula for, 245 in leukaemia, 248 IXDK.X. 415 Uric acid in urinary sediment-. crystalline Eorin of, Plate V, opposite p. 347, 333 isolation of, from the urine, 248 murexid tesl for, 349 origin "' quantitative determination of, 349 Folin Schaffer method for, 349 llrintz method for, 350 reducing power of, 0, 249 Schiff's reaction for, 249 Urinary calculi. 3 p> calcium carbonate in, 341 oxalate in. 341 cholesterin in, 342 comnound, 340 cvstin in, 34-' fibrin in. 343 indigo in, 342 phosphates in, 341 scheme for chemical anal- ysis of. 343 simple, 340 uric acid and urates in. 341 urostealiths in, 342 xanthin in, 342 Urinary concrements (see urinary calculi, p. 34°^ Urinary concretions (see urinary calculi, p. 34°^ Urination, frequency of, 228 Urinary sediments. 318 ammonium magnesium phos- phate in. 319 animal parasites in. 339 calcium carbonate in, 321 oxalate in, 320 phosphate in, 321 sulphate in. 322 casts in. 332 cholesterin in, 325 collection of. 318 cylindroids in, 337 cvstin in. 324 epithelial cells in, 328 erythrocytes in. 337 fibrin in. ; ") forei.en substances in. 339 hematoidin and bilirubin in, 326 hipnuric acid in, 325 indisro in. 327 leucin and tvrosin in. 326 magnesium phosphate in, 327 Urinary sediments, melanin in, micro-organisms in. 339 organized, 338 PUS cells in. -p. 1 matozoa in, 338 tissue debris in unorganized, 319 urates in. 3^3 urethral filaments in. 338 uric acid in. 322 xanthin in, 3 2 7 Urine, 326-379 acetone in, 302 acidity of, 228 acid fermentation of, 230 albumin in, 289 alkaline fermentation of, 230 allantoin in, 259 ammonia in, 270 aromatic oxyacids in, 261 benzoic acid in, 263 bile in, 300 blood in, 297 calcium in, 279 carbonates in, 280 chlorides in, 274 collection of. 235 conjugate glycuronates in, 310 color of. 226 creatinin in, 250 dextrose in, 282 diacetic acid in, 306 electrical conductivity of, 235 enzymes in, 265 ethereal sulphuric acid in, 253 fat in, 312 fluorides in 280 freezing-point of, 232 general characteristics of. 226 globulin in, 293 Haser's coefficient for solids in, 379 hxmatoporphyrin in, 312 hippuric acid in. 255 hydrogen peroxide in, 280 inorganic physiological consti- tuents of, 238 inosit in. 314 iron in. 280 lactose in. 313 lrcvulose in, 313 laiose in, 315 leucomaines in, 269 T. one's coefficient for solids in, 379 magnesium in. 279 melanin in. 315 neutral sulphur compounds in, 259 416 INDEX. Urine, nitrates in, 280 nucleoproteid in, 264, 296 odor of, 228 organic physiological constituents of, 237 oxalic acid in, 258 oxaluric acid in, 264 /3-oxybutyric acid in, 308 pathological constituents of, 282 paralactic acid in, 265 pentoses in, 311 peptone in, 294 phenaceturic acid in, 266 phosphates in, 275 phosphorized compounds in, 266 physiological constituents of, 237 pigments of, 266 potassium in, 279 proteids in, 289 proteoses in, 294 ptomaines in, 269 purin bases in, 269 quantitative analysis of, 344 reaction of, 228 silicates in, 280 sodium in, 279 solids of, 232, 379 specific gravity of, 230 sulphates in, 271 transparency of, 228 unknown substances in, 316 urea in, 239 uric acid in, 245 urorosein in, 316 volatile fatty acids in, 265 volume of, 226 Urobilin, i.;8, 266, 267 tests for, 267 Urochrome, 238, 266 Uroerythrin, 238, 266, 269 Uroferric acid, 237, 259, 317 Uroleucic acid, 237. 262 Urorosein. 282, 316 tests for, 316 Vegetable gums, 2 Veith lactometer, determination of specific gravity of milk by, 380 Volatile fatty acids, 132, 133, 195 Volhard-Arnold method for determin- ation of chlorides, 372 Volume of the urine, 226 Waxy casts in urinary sediments, 336 Weber's guaiac test for blood in feces, 145 Weyl's test for creatinin, 252 White fibrous connective tissue, 197 experiments on, 198 Wirsing's test for urobilin, 268 Xanthin, 207, 238 crystalline form of, 210 formula for, 211 in urinary sediments, 327 isolation of, from meat extract, 217 Weidel's reaction for, 218 Xanthin bases (see Purin bases, pp. 62, 238, 269) Xanthin silver nitrate, 217 crystalline form of, 218 Xanthoproteic reaction, 44 Xylose, 2, 17 orcin reaction on, 17 phenylhydrazin reaction on, 17 Tollens' reaction on, 17 Yellow elastic connective tissue, 201 composition of, 201 experiments on, 201 Zappert slide, 184 Zeller's test for melanin, 315 Zeynek and Nencki's hsemin test, 163 299 Zikel pektoscope, 233 Zymogen, 85, 107 Practical physiological chemistry